Patent application title: METHODS AND COMPOSITIONS FOR PREVENTING AND TREATING MALE ERECTILE DYSFUNCTION AND FEMALE SEXUAL AROUSAL DISORDER
Tom F. Lue (Hillsborough, CA, US)
Ching Shwun Lin (San Mateo, CA, US)
Yuet W. Kan (San Francisco, CA, US)
Peter Carroll (San Francisco, CA, US)
THE REGENTS OF THE UNIVERSITY OF CALIFORNIA
IPC8 Class: AA61K3818FI
Class name: Designated organic active ingredient containing (doai) peptide containing (e.g., protein, peptones, fibrinogen, etc.) doai 25 or more peptide repeating units in known peptide chain structure
Publication date: 2009-04-16
Patent application number: 20090099070
The invention provides a method for preventing or treating male erectile
dysfunction or female sexual arousal disorder by administering an
effective amount of one or more factors from a group of factors including
vascular endothelial growth factor, brain-derived neurotrophic factor,
basic fibroblast growth factor, neurotrophin-3, neurotrophin-4, or
angiopoietin-1, wherein the factor is a full length protein or a nucleic
acid encoding the factor, or a functional derivative or fragment thereof,
or an agent that enhances production and/or male erection or female
sexual arousal stimulating function of the factor(s). Combinations, kits,
and combinatorial methods are also provided.
1. A method for preventing or treating male erectile dysfunction or female
sexual arousal disorder induced or secondary to nerve injury, comprising
administering to a mammal to whom such prevention or treatment is needed
or desirable, a combination comprising:a) an effective amount of vascular
endothelial growth factor (VEGF), wherein the VEGF is a full length
protein or a functional derivative or fragment thereof; andb) an
effective amount of a factor selected from the group consisting of
brain-derived growth factor (BDNF), neurotrophin-3 (NT-3), and
neurotrophin-4 (NT-4), wherein the factor is a full length protein or a
functional derivative or fragment thereof;thereby preventing or treating
the male erectile dysfunction or the female sexual arousal disorder in
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. application Ser. No. 11/040,947, filed Jan. 21, 2005, now U.S. Pat. No. 7,625,091, issued Sep. 4, 2007, which is a divisional of U.S. application Ser. No. 10/155,785, filed May 23, 2002, now U.S. Pat. No. 7,223,406, issued May 29, 2007, which is a continuation-in-part of U.S. patent application Ser. No. 09/909,544, filed Jul. 19, 2001, now U.S. Pat. No. 6,852,323, issued Feb. 8, 2005, which claims the benefit of the priority date of U.S. provisional patent application Ser. No. 60/220,031, filed Jul. 21, 2000, under 35 U.S.C. § 119(e). The disclosures of the above-described applications are incorporated herein by reference in their entirety.
REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB
This application is being filed electronically via the USPTO EFS-WEB server, as authorized and set forth in MPEP §1730 II.B.2(a)(A), and this electronic filing includes an electronically submitted sequence (SEQ ID) listing. The entire content of this sequence listing is herein incorporated by reference for all purposes. The sequence listing is identified on the electronically filed .txt file as follows:
TABLE-US-00001 File Name Date of Creation Size (bytes) 22002201601seqlist.txt Sep. 4, 2007 16,924 bytes
FIELD OF THE INVENTION
This invention relates generally to the field of urology. In particular, the invention provides a method for preventing or treating male erectile dysfunction or female sexual arousal disorder in a mammal in need of such treatment, comprising administering an effective amount of a factor from a group of factors including vascular endothelial growth factor (VEGF), brain-derived neurotrophic factor (BDNF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), or angiopoietin-1 (Ang-1), wherein the factor is a full length protein or a functional derivative or fragment thereof, or a nucleic acid encoding said factor or functional derivative or fragment thereof, or an agent that enhances production and/or male erection or female sexual arousal stimulating function of the factor, thereby preventing or treating male erectile dysfunction or female sexual arousal disorder in the mammal. Combinations, kits, and combinatorial methods for preventing or treating male erectile dysfunction or female sexual arousal disorder are also provided.
BACKGROUND OF THE INVENTION
VEGF is a family of proteins that were discovered on the basis of their ability to stimulate VEC (vascular endothelial cell) growth (angiogenesis). It now comprises five members, namely, VEGF-A, VEGF-B, VEGF-C, VEGF-D, and PLGF (placenta growth factor) that are encoded from distinct genes. Achen, et al., Proc. Nat'l. Acad. Sci. USA, 95: 548 (1998), Joukov, et al., EMBO J., 15: 1571 (1996), Maglione, et al., Oncogene, 8: 925 (1993), Olofsson, et al., Proc. Nat'l. Acad. Sci. USA, 93: 2576 (1996), Yamada, et al., Genomics, 42: 483 (1997). Each of the five members in turn comprises two or more isoforms that arise by the splicing of their respective pre-mRNAs. For example, the VEGF-A family includes VEGF206, VEGF189, VEGF183, VEGF165, VEGF145, VEGF121, and VEGF111. Anthony, et al., Placenta, 15: 557 (1994), Neufeld, et al., FASEB J., 13: 9 (1999), Lei, et al., Biochim. Biophys. Acta, 1443: 400 (1998), Jingjing, et al., Ophthamol. Vis. Sci., 40: 752 (1999), Cheung, et al., Am. J. Obstet. Gynecol., 173: 753 (1995), Burchardt, et al., Biol. Reprod., 60: 398 (1999). Among all VEGF proteins and isoforms, VEGF165 is by far the most frequently used form of VEGF both in basic and clinical studies.
It has been shown that, among different vascular cell types (endothelial, smooth muscle cells (SMC), and fibroblasts), SMC is the principal source for the secreted VEGF. Pueyo, et al., Exp. Cell Res., 238: 354 (1998). Expression of VEGF in SMC is upregulated by multiple factors including phorbol esters (Tischer, et al., J. Biol. Chem., 266: 11947 (1991)), cAMP (Claffey, et al., J. Biol. Chem., 267: 16317 (1992)), and hypoxia (Goldberg, et al., J. Biol. Chem., 269: 4355 (1994), Shweiki, et al., Proc. Nat'l Acad. Sci. USA, 92: 768 (1995)). The secreted VEGF acts on VEC principally through two different cell surface receptors, VEGFR-1 and VEGFR-2. Activation of VEGFR-1 results in VEC migration, while activation of VEGFR-2 VEC migration and proliferation. Waltenberger, et al., J. Biol. Chem., 269: 26988 (1994), Neufeld, et al., FASEB J., 13: 9 (1999), Ortega, et al., Front. Biosci., 4: D141 (1999). Although VEGFR-1 and VEGFR-2 have long been considered endothelium-specific, they have both been detected in human uterine and bovine aorta SMC. Grosskreutz, et al., Microvasc. Res., 58: 128 (1999), Brown, et al., Lab. Invest., 76: 254 (1997). Cultured uterine SMC responded to VEGF in the form of cell proliferation and cultured aorta SMC cell migration. Cultured human colon SMC, however, did not express VEGF receptors, nor did they respond to VEGF treatment. Brown, et al., Lab. Invest., 76: 254 (1997).
Angiogenesis is a complex process that includes activation, migration and proliferation of endothelial cells and formation of new blood vessels. D'Amore, et al., Ann. Rev. Physiol., 49(9-10): 453-64 (1987). VEGF has been shown to be intimately involved in the entire sequence of events leading to growth of new blood vessels. Gross, et al., Proc. Nat'l. Acad. Sci., 80(9): 2623-27 (1983), Folkman, et al., Proc. Nat'l. Acad. Sci., 76(10): 5217-21 (1979). Five human VEGF isoforms of 121, 145, 165, 189 and 206 amino acids have been isolated. Gross, et al., Proc. Nat'l. Acad. Sci., 80(9): 2623-27 (1983), Leung, et al., Science, 246: 1306-09 (1989), Poltorak, et al., J. Biol. Chem., 272(11): 7151-78 (1997). Among the isoforms, VEGF 165 seems to be the most effective and most commonly used. The effect of VEGF 165 in augmenting perfusion and in stimulating formation of collateral vessels has been shown in animal models Hopkins, et al., J. Vascular Surgery, 27(5): 886-94 (1998), Asahara, et al., Circulation, 91(11): 2793-801 (1995), Hariawala, et al., J. Surg. Res., 63(1): 77-82 (1996), Bauters, et al., Circulation, 91(11): 2802-9 (1995), Bauters, C., et al., Am. J. Physiol., 267(4 Pt 2): H1263-71 (1994), Takeshita, et al., J. Clin. Invest., 93(2): 662-70 (1994), Takeshita, et al., Circulation, 90(5 Pt 2): 11228-34 (1994), Takeshita, et al., Am. J. Path., 147(6): 1649-60 (1995), Banai, et al., Circulation, 89(5): 2183-9 (1994). In clinical trials, successful induction of collateral blood vessels in ischemic heart disease and critical limb ischemia by VEGF have also been reported. Baumgartner, et al., Circulation, 97(12): 1114-23 (1998), Losordo, et al., Am. Heart J., 138(2 Pt 2): 132-41 (1999).
Platelet-derived growth factor (PDGF) is a potent mitogen for cells of mesenchymal origin, stimulating both connective tissues and neuroglial cells. PDGF also acts as a potent chemoattractant for mesenchymal cells, mononuclear cells, and neutrophils. PDGF is stored in platelet granules and released with platelet activation. Other cell types also produce PDGF, including endothelial cells, monocytes/macrophages, vascular smooth muscle cells, fibroblasts, and cytotrophoblasts. PDGF consists of disulfide-linked dimers of αα, αβ, or ββ configuration. PDGF has a short half-life and usually produces only local effects. Two distinct PDGF receptors have been identified that are structurally related and have an intracellular protein kinase domain.
Neurotrophins are a class of structurally related growth factors that promote neural survival and differentiation. They stimulate neurite outgrowth, suggesting that they can promote regeneration of injured neurons, and act as target-derived neurotrophic factors to stimulate collateral sprouting in target tissues that produce the neurotrophin. Korsching, J. Neurosci., 13: 2739 (1993). Recently, local synthesis and autocrine mechanisms of action have been reported. Lewin and Barde, Ann. Rev. Neurosci., 19: 289 (1996). In vivo overexpression of a neurotrophic factor, through gene transfer, would ensure local and continuous neurotrophin production in a manner resembling the physiologic, as these proteins are usually produced and secreted by target and glial cells surrounding neurons.
Neurotrophin-3 (NT-3) is a member of the neurotrophin class of structurally related growth factors. NT-3 is a 27 kDa homodimer that supports the growth and survival of sympathetic neurons as well as sensory neurons. NT-3 is highly conserved across species and is primarily expressed in kidney, spleen, and heart with lower expression levels found in the skin, skeletal muscle, lung, thymus, and ovaries. NT-3 binds the low affinity NGF receptor, p75NTR, and may initiate apoptosis through this receptor. NT-3 also binds and induces signaling through the TrkC receptor.
Neurotrophin-4 (NT-4) is yet another member of this class of neurotrophins. NT-4 is a homodimer that supports the growth and survival of sympathetic neurons, dorsal root ganglion neurons, nodose ganglion neurons, basal forebrain cholinergic neurons and neurons of the locus coeruleus. NT-4 is less highly conserved between species, unlike other neurotrophins. NT-4 expression is widespread in brain and peripheral tissues. NT-4 induces cellular signaling through the p75NTR receptor as well as the TrkB receptor.
Brain-derived neurotrophic factor (BDNF), another member of the neurotrophins, was initially characterized as a basic protein present in brain extracts and capable of increasing the survival of dorsal root ganglia. Leibrock, et al., Nature, 341: 149 (1989). When axonal communication with the cell body is interrupted by injury, Schwann cells produce neurotrophic factors such as nerve growth factor (NGF) and BDNF. Neurotrophins are released from the Schwann cells and dispersed diffusely in gradient fashion around regenerating axons, which then extend distally along the neurotrophins' density gradient. Ide, Neurosci. Res., 25: 101 (1996). Local application of BDNF to transected nerves in neonatal rats has been shown to prevent the massive death of motor neurons that follows axotomy. DiStefano, et al., Neuron, 8:983 (1992), Oppenheim, et al., Nature, 360: 755 (1992), Yan, et al., Nature, 360: 753 (1992). The mRNA titer of BDNF increases to several times the normal level 4 days after axotomy and reaches its maximum at 4 weeks. Meyer, et al., J. Cell Biol., 119: 45 (1992). Moreover, BDNF has been reported to enhance the survival of cholinergic neurons in culture. Nonomura, et al., Brain Res., 683: 129 (1995).
Angiopoietin-1 (Ang-1) is a member of a family of endothelium growth factors. Ang-1 is a ligand for the Tie-2 receptor, a receptor tyrosine kinase with immunoglobulin and epidermal growth factor homology domains expressed primarily on endothelial cells and very early hematopoietic cells. Ang-1 promotes chemotaxis, cell survival, cell sprouting, vessel growth and stabilization of Tie-2-expressing endothelial cells. Ang-1 is thought to have a distinct angiogenic role from that of VEGF involving the recruitment of peri-endothelial cells that will become pericytes and smooth muscle tissue of the blood vessel, thereby maintaining the stability of the blood vessels. See, e.g., Hanahan, D., Science, 277: 48-50.
Basic fibroblast growth factor (bFGF) is a member of the fibroblast growth factor family. bFGF stimulates the proliferation of all cells of mesodermal origin including smooth muscle cells, neuroblasts, and endothelial cells. bFGF stimulates neuron differentiation, survival, and regeneration. In vitro functions suggest that bFGF modulates angiogenesis, wound healing and tissue repair, and neuronal function in vivo. bFGF, a heparin-binding growth factor, is capable of inducing functionally significant angiogenesis in models of myocardial and limb ischemia. Zheng, et al., Am. J. Physiol. Heart Circ. Physiol., 280: H909-17 (2001), Laham, et al., J. Am. Coll. Cardiol., 36: 2132-39 (2000), Laham, et al., Curr. Interv. Cardiol. Rep., 1: 228 (1999), Unger, et al., Am. J. Cardiol., 85: 1414-19 (2000), Kawasuji, et al., Ann. Thorac. Surg., 69: 1155 (2000), Rajanayagam, et al., J. Am. Coll. Cardiol., 35: 519 (2000), Kornowski, et al., Circulation, 101: 545-48 (2000), Ohara, et al., Gene Ther., 8: 837 (2001), Lazarous, et al., J. Am. Coll. Cardiol., 36: 1239 (2000), Rakue, et al., Japan Circ. J., 62: 933-39 (1998), Baffour, et al., J. Vasc. Surg., 16: 181 (1992).
Erectile function is a hemodynamic process of blood in-flow and pressure maintenance in the cavernosal spaces. Christ, Urol. Clin. North Am., 22: 727 (1995). Following sexual arousal and the release of nitric oxide to the erectile tissue, three processes occur to achieve an erection. These are relaxation of the trabecular smooth muscle, arterial dilation and venous compression. Id. During this final stage, arterial flow fills sinusoidal spaces, compressing subtunical venules thereby reducing venous outflow. Blood flows into the cavernous spaces of the penis, thus expanding and stretching the penis into a rigid organ. The flow of blood in and out of the cavernous spaces is controlled by cavernous smooth muscle cells (CSMC) embedded in the trabeculae of the cavernous spaces. With normal erectile function, a high intracavernous pressure (ICP) is maintained with a low inflow rate. Karadeniz, et al., Urol. Int., 57: 85 (1996).
As such, the penis is a predominantly vascular organ, and vascular or penile arterial insufficiency is the most common etiology of erectile dysfunction (ED). Sinusoidal smooth muscle atrophy and collagen deposition is a common finding in men with long standing ED of various etiologies, whether due to hormonal, neurological or vascular causes. Karadeniz, et al., Urol. Int., 57: 58 (1996). Such degradation in smooth muscle quantity and quality leads to veno-occlusive dysfunction. This represents an end-stage muscular degeneration akin to myocardial changes with congestive heart failure or dilated cardiomyopathy for which no treatment currently exists with hope of reversing the underlying pathologic process.
Veno-occlusive disease is a common finding among patients with erectile dysfunction (ED). Following radical prostatectomy, for example, approximately 30% of patients may have vasculogenic ED in addition to neurogenic ED and at least half of these men may have venous leak. Regardless of the etiology of organic ED (neurogenic, traumatic, hormonal, and vascular, etc.), venous leakage is a common final condition resulting from smooth muscle atrophy. Mersdorf, et al., J. Urol., 154: 749 (1991). Veno-occlusive dysfunction is the most common etiology of ED among non-responders to medical management of ED. None of the medical therapy currently exists is curative for this condition. Patients with veno-occlusive dysfunction exhibit a poor response to intracavernous injection with vasoactive agents (papavarine, prostaglandin E1, phentolamine, or combinations, for example), despite good arterial flow demonstrated by duplex ultrasound. The diagnosis of veno-occlusive disease may be confirmed with specific findings on cavernosometry and cavernosography. Nehra, et al., J. Urol., 156: 1320 (1996).
Atherosclerotic or traumatic arterial occlusive disease of the pudendal-cavernous-helicine arterial tree can decrease the perfusion pressure and arterial flow to the sinusoidal spaces, thus decreasing the rigidity of the erect penis. Common risk factors associated with generalized arterial insufficiency include hypertension, hyperlipidemia, cigarette smoking, diabetes mellitus, and pelvic irradiation. Goldstein, et al., JAMA, 251: 903-910 (1984), Rosen, M. P., et al., Radiology, 174(3 Pt 2): 1043-48 (1990), Levine, F. J., et al., J. Urology, 144(5): 1147-53 (1990). Epidemiological studies have shown a high incidence of ED in patients with coronary arterial disease. Heaton, J. P., et al., Int'l J. Impotence Res., 8(1): 35-39 (1996). Focal lesion of the common penile or cavernous artery is most often seen in young patients who have sustained blunt pelvic or perineal trauma such as in cases of biking accidents. Levine, F. J., et al., J. Urology, 144(5): 1147-53 (1990).
Because of the close proximity of the cavernous nerves to the capsule of the prostate, ED is a frequent complication after radical prostatectomy or cystectomy and prostatic cryosurgery. Although the nerve-sparing prostatectomy technique developed by Walsh, et al., Br. J. Urol., 56: 694 (1984) has significantly reduced the postoperative impotence rate, a large number of patients still suffer from inadequate penile rigidity. Peripheral nerve regeneration is a slow process, and the fact that most patients do not recover potency for 6 months to 2 years indicates substantial axonal damage, even with preservation of the neural sheath. An anatomic study of the cavernous nerves by Paick et al., Urology, 42: 145 (1993) revealed both a medial and a lateral bundle of cavernous nerves at the level of the prostate, suggesting that in some cases the lateral bundle can be saved, even in non-nerve-sparing prostatectomy.
The sprouting of the remaining nerves in penile tissue appears to be more important in regeneration than re-growth of nerves through the damaged and fibrotic tissues. The importance of sprouting in the remaining nerves was confirmed in an animal study that revealed regeneration of the cavernous nerves after unilateral resection. Carrier, et al., J. Urol., 153: 1722 (1995). In addition, a previous study in our laboratory showed that systemic growth hormone injection significantly enhanced cavernous nerve regeneration after unilateral injury. Jung, et al., J. Urol., 160: 1899 (1998).
Methods for treating erectile dysfunction have included from the administration of prostaglandin E (U.S. Pat. No. 5,942,545), local administration of vascular muscle relaxants and vasoactive pharmaceutical agents. See, for example, U.S. Pat. Nos. 5,942,545, 6,056,966; and 5,646,181.
Advancement in molecular biology has brought improved understanding of pathophysiology on the gene and molecular level, and offers promise of treatment possibilities aimed at a specific pathologic molecular mechanism. As in other vasculopathies such as limb claudication (Baumgartner, et al., Circulation, 97: 1114 (1998) and coronary artery disease (Symes, et al., Ann. Thorac. Surg., 68: 830 (1999), treatment with VEGF in either protein or gene form has increased neovascularity in animal models and improved symptomatic angina and wound healing in humans with inoperable heart disease and critical limb ischemia, respectively. The penis represents a convenient tissue target for gene or growth factor therapy due to the penis' external location on the body, ubiquity of endothelial-lined spaces and low-level blood flow in the flaccid state. In addition, the penis is filled with billions of endothelial and smooth muscle cells both are rich in VEGF receptors. Liu, et al., J. Urol., 166: 354-360 (2001).
Recently, we have established an animal model in which CSMC was seen decreased following internal iliac artery ligation that restricted blood supply to the penis. However, rats treated with intracavernous injection of vascular endothelial growth factor (VEGF) shortly after internal iliac artery ligation had nearly normal CSMC. The protective effects of VEGF on CSMC could be due to partial restoration of blood supply as VEGF is expected to stimulate vascular endothelial cell (VEC) proliferation. Lin, et al., Proc. Nat'l Acad. Sci. USA, 97: 10242-47 (2000). Alternatively, VEGF might act directly on CSMC, as we will present evidence that CSMC express one of the two principal VEGF receptors. Sondell, et al., Eur. J. Neurosci., 12: 4243-54 (2000); Liu, et al., J. Urol., 166: 354-360 (2001).
Females can also have sexual dysfunction, and this dysfunction can increase with age. It is usually associated with the presence of vascular risk factors, genital smooth muscle atrophy, and onset of menopause. Some of the vascular and muscular mechanisms that contribute to penile erection in the male are believed to be similar vasculogenic factors in the female genital response. It is known that in women sexual arousal is accompanied by arterial inflow which engorges the vagina and increases vaginal lubrication, and that the muscles in the clitoris and the perineum assist in achieving clitoral erection.
In the female patient, sexual arousal disorder can arise from organic and pyschogenic causes, or from a combination of the foregoing. Female sexual arousal disorder is classified into five categories: 1) hypoactive sexual desire disorder, 2) sexual aversion disorder, 3) sexual arousal disorder, 4) orgasmic disorder, and 5) sexual pain disorder. The present invention applies to sexual arousal disorder. Sexual arousal disorder is the persistent or recurring inability to attain or maintain adequate sexual excitement, causing personal distress. It may be experienced as the lack of subjective excitement or the lack of genital lubrication or swelling or other somatic responses. Organic female sexual arousal disorder is known to be related in part to vasculogenic impairment resulting in inadequate blood flow, vaginal engorgement insufficiency and clitorial erection insufficiency. Animal studies have demonstrated the dependence of vaginal vascular engorgement and clitoral erection on blood flow. See, for example, Park et al., "Vasculogenic female sexual dysfunction: the hemodynamic basis for vaginal engorgement insufficiency and clitoral erectile insufficiency, "Int'l J. Impotence Res., 9(1), 27-37 (March 1997).
Female sexual dysfunction has been treated with pharmacological intervention to stimulate blood flow as well as with prostaglandins. See, for example, U.S. Pat. Nos. 6,193,992 B1; 5,945,117; 6,031,002; and 5,891,915.
SUMMARY OF THE INVENTION
The present invention provides for methods, combinations, and kits for the treatment and prevention of male erectile dysfunction or female sexual arousal disorder. These methods, combinations, and kits involve the administration of vascular endothelial growth factor (VEGF), brain-derived growth factor (BDNF), basic fibroblast growth factor (bFGF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), platelet-derived growth factor (PDGF), angiopoietin-1 (Ang-1), or a combination thereof.
In one aspect, the present invention provides for a method for preventing or treating male erectile dysfunction or female sexual arousal disorder, which method comprises administering to a mammal to whom such prevention or treatment is needed or desirable, an effective amount of a factor, wherein the factor is VEGF, BDNF, bFGF, NT-3, NT-4, PDGF, Ang-1, an agent that enhances production and/or male erection or female sexual arousal stimulating function of the factor, or a combination thereof, thereby preventing or treating the male erectile dysfunction or the female sexual arousal disorder in the mammal. Preferably, the factor is a full length protein or a functional derivative or fragment thereof or a nucleic acid encoding the factor or functional derivative or fragment thereof.
In a specific embodiment, the mammal is a human and the factor, or a functional derivative or fragment thereof, or the nucleic acid encoding the factor, or a functional derivative or fragment thereof, is of human origin.
Preferably, the factor protein or nucleic acid, or a functional derivative or fragment thereof, is administered by intracavernous injection, subcutaneous injection, intravenous injection, intramuscular injection, intradermal injection, or topical administration.
In a specific embodiment, the factor nucleic acid, or a functional derivative or fragment thereof, is administered via a gene therapy vector, preferably the gene therapy vector is an adenovirus associated vector, a retroviral vector, an adenovirus vector, or a lentivirus vector. More preferably, the gene therapy vector is an adenovirus associated vector.
In yet another specific embodiment, the factor protein, or a functional derivative or fragment thereof, is administered via a liposome.
In another specific embodiment, the factor nucleic acid, or a functional derivative or fragment thereof, is administered via a liposome.
Preferably, the male erectile dysfunction to be treated or prevented is erectile dysfunction induced by or secondary to nerve dysfunction, arterial insufficiency, venous leakage, severe vascular insufficiency, mild vascular disease, hormonal insufficiency, drug use, surgery, chemotherapy, or radiation.
Preferably, the female sexual arousal disorder to be treated or prevented is sexual dysfunction induced by or secondary to nerve dysfunction, arterial insufficiency, severe vascular insufficiency, mild vascular disease, hormonal insufficiency, drug use, surgery, chemotherapy, or radiation.
In a specific embodiment, the factor protein or a functional derivative or fragment thereof, or a nucleic acid encoding the factor or functional derivative or fragment thereof, or an agent that enhances production and/or female sexual arousal stimulating function of the factor, is administered in an amount sufficient to improve blood flow and regenerate nerve and smooth muscle in the clitoris and vaginal wall.
In another specific embodiment, the factor protein or a functional derivative or fragment thereof, or a nucleic acid encoding the factor or functional derivative or fragment thereof, or an agent that enhances production and/or female sexual arousal stimulating function of the factor, is administered in a cream or via injection to the clitoris and vaginal wall of the patient.
In yet another specific embodiment, the factor protein or a functional derivative or fragment thereof, or a nucleic acid encoding the factor or functional derivative or fragment thereof, or an agent that enhances production and/or the male erection or female sexual arousal stimulating function of the factor, is administered by intracavernous injection.
In a preferred embodiment, the male erectile dysfunction or female sexual arousal dysfunction is induced by or secondary to nerve injury, and the combination of factors administered are: a) VEGF and NT-3, b) VEGF and NT-4, or c) VEGF and BDNF.
In another preferred embodiment, the male erectile dysfunction or female sexual arousal disorder is induced by or secondary to severe vascular insufficiency and the combination of factors administered are: a) VEGF and PDGF, b) VEGF and bFGF, or c) VEGF and Ang-1.
In yet another preferred embodiment, the male erectile dysfunction or female sexual arousal dysfunction is induced by or secondary to mild vascular disease and the factor administered is VEGF.
In another aspect, the present invention provides for a combination for preventing or treating male erectile dysfunction or female sexual arousal disorder, which combination comprises: a) an effective amount of an agent that stimulates male erectile or female sexual function; and b) an effective amount of a factor, wherein the factor is a full length protein or a functional derivative or fragment thereof, or a nucleic acid encoding said factor or functional derivative or fragment thereof, an agent that enhances production and/or male erection or female sexual arousal stimulating function of said factor, or a combination thereof, and the factors include VEGF, BDNF, bFGF, NT-3, NT-4, PDGF, and Ang-1.
In yet another aspect, the present invention provides a combination for preventing or treating male erectile dysfunction or female sexual arousal disorder induced by or secondary to nerve injury, which combination comprises: a) an effective amount of VEGF, wherein the VEGF is a full length protein or a functional derivative or fragment thereof, or a nucleic acid encoding the VEGF or functional derivative or fragment thereof; and b) an effective amount of a factor selected from the group consisting of NT-3, NT-4, and BDNF, wherein the factor is a full length protein or a functional derivative or fragment thereof, or a nucleic acid encoding the factor or functional derivative or fragment thereof, or an agent that enhances production and/or male erection or female sexual arousal stimulating function of the factor.
In another aspect, the present invention provides a combination for preventing or treating male erectile dysfunction or female sexual arousal disorder induced by or secondary to severe vascular insufficiency, which combination comprises: a) an effective amount of VEGF, wherein the VEGF is a full length protein or a functional derivative or fragment thereof, or a nucleic acid encoding the VEGF or functional derivative or fragment thereof; and b) an effective amount of a factor selected from the group consisting of PDGF, bFGF, and Ang-1, wherein the factor is a full length protein or a functional derivative or fragment thereof, or a nucleic acid encoding the factor or functional derivative or fragment thereof, or an agent that enhances production and/or male erection or female sexual arousal stimulating function of the factor.
In yet another aspect, the present invention provides for the above-described combinations, preferably in the form of a pharmaceutical composition, that can be used for preventing or treating male erectile dysfunction or female sexual arousal disorder. The above-described combinations can further comprise a pharmaceutically acceptable carrier or excipient.
In another aspect, the present invention provides a method for preventing or treating male erectile dysfunction or female sexual arousal disorder, which method comprises administering an effective amount of the above described combinations to a mammal in need thereof, thereby preventing or treating the male erectile dysfunction or the female sexual arousal disorder in the mammal.
In one aspect, the present invention provides for a kit comprising the above-described combinations and an instruction for using the combination in treating or preventing male erectile dysfunction or female sexual arousal disorder.
In yet another aspect, the present invention provides a method of promoting sprouting of new nerve fibers from blood vessel explants, which method comprises the steps of: a) isolating a blood vessel; b) attaching the blood vessel to a media-coated coverslip; and c) incubating with a growth-stimulating compound. In one preferred embodiment, the growth-stimulating compound is VEGF. The present invention also provides for a method of identifying a compound for promoting sprouting of new nerve fibers from blood vessel explants, which method comprises assaying candidate compounds for nerve growth promoting activity ex vivo using the above described method and identifying a compound that promotes nerve growth in a blood vessel explant as indicative of a compound that promotes nerve growth.
In another aspect, the present invention provides for a method of inducing angiogenesis, which method comprises the steps of: a) co-culturing an isolated blood vessel and an isolated muscle explant; and b) incubating with a growth-stimulating compound. In a preferred embodiment, the compound is a combination of VEGF and PDGF. The present invention also provides for a method of identifying a compound for inducing angiogenesis, which method comprises assaying candidate compounds for angiogenic activity ex vivo using the method described above, and identifying a compound that promotes angiogenesis in a blood vessel cell as indicative of a compound that promotes angiogenesis.
In yet another aspect, the present invention provides for a method for promoting growth of cavernous nerves from major pelvic ganglia (MPG), which method comprises contacting the MPG with an effective amount of a factor, wherein the factor is selected from the group consisting of vascular endothelial growth factor (VEGF), brain-derived growth factor (BDGF), basic fibroblast growth factor (bFGF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), platelet-derived growth factor (PDGF), and angiopoietin-1 (Ang-1), thereby promoting growth of the cavernous nerves from the MPG.
In another aspect, the present invention provides for a method of identifying a compound that promotes growth of cavernous nerves from major pelvic ganglia (MPG), which method comprises: a) in vitro culturing MPG; b) measuring growth of cavernous nerves from the MPG in the presence and absence of a candidate compound; and c) identifying a compound that promotes nerve growth as indicative of a compound that promotes growth of cavernous nerves from MPG.
Other aspects of the invention are described throughout the specification.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. Electrostimulation of the cavernous nerve at 8 weeks: A) sham operation group; B) LacZ group; and C) BDNF group. Note higher maximal intracavernous pressure in the BDNF than in the LacZ group. Scan rate=10/sec.
FIG. 2. Quantification of VEGF secreted by CSMC. Equal number (4×105) of CSMC from different-aged rats were seeded in each well of 6-well plates and allowed to grow for three days in medium containing 10% of FBS. The medium from each well was then assayed for the concentration of rat-specific VEGF (panel B) and the cell number was determined (panel A). The calculated amount of VEGF in each well was then divided by the number of cells in each well to derive the data shown in panel C.
FIG. 3. Effects of VEGF on the growth rates of VSMC and CSMC. Equal numbers (5,000) of VSMC (from 16-weeks-old rats, panel A) and CSMC (from rats of indicated ages, panels B to I) were seeded in each well of 96-well plates and allowed to grow for three days in media containing the indicated concentrations of VEGF. The final numbers of cells were determined with a proliferation assay kit and expressed as optical density (OD490nm) values. These values were converted into % of control (shown on the left of panels A through J) with the value of the growth rate in medium containing no added VEGF being referred to as control (i.e., 100%). Panel J compares the growth rates of CSMC from rats of the indicated ages in media containing the optimal concentration (12.5 ng/ml) of VEGF.
FIG. 4. Effects of VEGF on the mobility of VSMC and CSMC. Equal numbers (8,000) of VSMC (from 16-weeks-old rats, panel A) and CSMC (from rats of indicated ages, panels B to I) were loaded in each well of 24-well Transwell plates and allowed to migrate through a membrane toward a medium containing the indicated concentration of VEGF. Four hours later, the numbers of cells that had migrated through the membrane were counted under a microscope. These numbers are indicated on the left of the panels. Panel J compares the numbers of CSMC (from rats of the indicated ages) that had migrated through the membrane toward media containing the optimal concentration (10 ng/ml) of VEGF.
FIG. 5. Identification of VEGFR-1 and VEGFR-2 mRNA expression. RNAs of the following cells and tissues (lanes 1-15) were subjected to RT-PCR with primer pair VEGFR-1s and VEGFR-1a, primer pair VEGFR-2s and VEGFR-2a, and primer pair β-actin-s and β-actin-a (Table 1). The reaction products were electrophoresed in a 1.5% agarose gel and stained with ethidium bromide. The RT-PCR product in each lane was derived from 25 ng (for VEGFR-1 and VEGFR-2) or 1 ng (for β-actin) of total cellular or tissue RNAs. M, 100-bp size marker. Lane 1, CSMC from 1-week-old rats; lane 2, CSMC from 2-weeks-old rats; lane 3, CSMC from 3-weeks-old rats; lane 4, CSMC from 4-weeks-old rats; lane 5, CSMC from 6-weeks-old rats; lane 6, CSMC from 11-weeks-old rats; lane 7, CSMC from 16-weeks-old rats; lane 8, CSMC from 28-months-old rats; lane 9, aorta SMC from 16-weeks-old rats; lane 10, heart of a 16-weeks-old rat; lane 11, aorta of a 16-weeks-old rat; lane 12, corpus cavemosum of a 16-weeks-old rat.
FIG. 6. Identification of VEGFR-1 protein expression. Protein extracts of CSMC from rats of the following ages (lanes 1-8) were electrophoresed in 7.5% SDS-PAGE and then transferred to PVDF membrane. Detection of VEGFR-1 protein on the membrane was performed by the ECL procedure using an anti-VEGFR-1 rabbit serum. Lane 1, 1-week-old; lane 2, 2-weeks-old; lane 3, 3-weeks-old; lane 4, 4-weeks-old; lane 5, 6-weeks-old; lane 6, 11-weeks-old; lane 7, 16-weeks-old; lane 8, 28-months-old.
DETAILED DESCRIPTION OF INVENTION
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this invention belongs. All patents, patent applications and other publications and sequences from GenBank and other databases referred to herein are incorporated by reference in their entirety. If a definition set forth in this section is contrary to or otherwise inconsistent with a definition set forth in patents, patent applications and other publications and sequences from GenBank and other databases that are herein incorporated by reference, the definition set forth in this section prevails over the definition that is incorporated herein by reference.
As used herein, "a" or "an" means "at least one" or "one or more."
As used herein, an "erectile dysfunction (or impotence)" refers to the inability of a male mammal, e.g., a man, to achieve and maintain penile erection for satisfactory sexual intercourse.
As used herein, a "female sexual arousal disorder" refers to the persistent or recurring inability to attain or maintain adequate sexual excitement causing personal distress. It may be experienced as lack of subjective excitement or lack of genital (lubrication and swelling) or other somatic responses.
As used herein, "vascular endothelial growth factor (VEGF)" or "brain-derived neurotrophic factor (BDNF)" or "basic fibroblast growth factor (bFGF)" or "platelet-derived growth factor (PDGF)" or "neurotrophin-3 (NT-3)" or "neurotrophin-4 (NT-4)" or "angiopoietin-1 (Ang-1)" includes those variants with conservative amino acid substitutions that do not substantially alter their male erection- or female sexual arousal-stimulating activity. Suitable conservative substitutions of amino acids are known to those of skill in this art and may be made generally without altering the biological activity of the resulting molecule. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Bejacmin/Cummings Pub. co., p. 224).
As used herein, a "functional derivative or fragment of a factor" refers to a derivative or fragment of the factor that still substantially retains its function as an erection or sexual arousal stimulant. Normally, the derivative or fragment retains at least 50% of its erection or sexual function stimulating activity. Preferably, the derivative or fragment retains at least 60%, 70%, 80%, 90%, 95%, 99% and 100% of its erection or sexual function stimulating activity.
As used herein, an "agent that enhances production of the factor" refers to a substance that increases transcription and/or translation of a factor gene, or a substance that increases post-translational modification and/or cellular trafficking of a factor precursor, or a substance that prolongs half-life of a factor protein.
As used herein, an "agent that stimulates erection or female sexual arousal function activity of a factor" refers to a substance that increases potency of the factor's erection or sexual arousal stimulating activity, or a substance that increases sensitivity of a factor's natural ligand in an erection or sexual arousal stimulation signaling pathway, or a substance that decreases potency of a factor's antagonist. Such an agent is not VEGF, BDNF, bFGF, PDGF, NT-3, NT-4, or Ang-1.
As used herein, a "combination" refers to any association between two or among more items.
As used herein, a "composition" refers to any mixture of two or more products or compounds. It may be a solution, a suspension, liquid, powder, a paste, aqueous, non-aqueous or any combination thereof.
As used herein, a "nerve dysfunction" refers to the inability of the penis to hold the blood during erection or the persistent or recurring inability to attain or maintain adequate sexual excitement in a male or female mammal causing personal distress including, but not limited to dysfunction caused by diabetes mellitus, hypertension, hyperlipidemia, penile injury, aging, pelvic surgery or irradiation.
As used herein, an "arterial insufficiency" refers to reduced perfusion pressure and arterial flow associated with trauma or disease including, but not limited to, that associated with hypertension, hyperlipidemia, cigarette smoking, diabetes mellitus, and pelvic irradiation.
As used herein, a "venous leakage" refers to the inability of the penis to hold the blood during erection caused by a disorder including, but not limited to diabetes mellitus, hypertension, hyperlipidemia, penile injury, aging, pelvic surgery or irradiation.
As used herein, a "hormonal insufficiency" refers to a group comprising, but not limited to, perimenopausal-, post menopausal-, cancer-related-, hypogonadism-, and osteoporosis-hormonal insufficiencies.
As used herein, a "drug use" includes pharmaceutical drug use and substance abuse.
As used herein, a "surgery" refers to the performance of an operation including reconstructive, cosmetic, and restorative procedures and removal of an organ or tissue or some portion thereof.
As used herein, a "radiation" refers to treatment by photons, electrons, neutrons or other ionizing radiation.
As used herein, a "chemotherapy" refers to the administration of any agent that mediates the regression of malignant growth by inducing cell death or retarding cell growth through DNA damaging and non-DNA damaging mechanisms.
As used herein, a "nerve injury" refers to damage of the somatic nerve or autonamic nerve that controls sensation and blood flow to the penis, vagina or the clitoris. The injury can occur as a result of blunt or penetrating trauma to the pelvis, perineum or the penis as well as surgery or irradiation of the pelvic organs or the external genitalia.
As used herein, a "severe vascular insufficiency" refers to near complete cessation of blood flow to the penis, vagina, or clitoris due to damage of both internal pudendal or penile arteries.
As used herein, a "mild vascular insufficiency" refers to mild to moderate restriction of blood flow to the penis, vagina, or clitoris and thus impairs erectile function or female sexual arousal due to partial damage to the pudenal or penile arteries.
For clarity of disclosure, and not by way of limitation, the detailed description of the invention is divided into the subsections that follow.
B. Methods to Prevent or Treat Male Erectile Dysfunction or Female Sexual Arousal Disorder
In one aspect, the present invention provides for a method for preventing or treating male erectile dysfunction or female sexual arousal disorder, which method comprises administering to a mammal to whom such prevention or treatment is needed or desirable, an effective amount of a factor, wherein the factor is VEGF, BDNF, bFGF, NT-3, NT-4, PDGF, or Ang-1, thereby preventing or treating the male erectile dysfunction or the female sexual arousal disorder in the mammal. Preferably, the factor is a full length protein or a functional derivative or fragment thereof, or a nucleic acid encoding the factor or functional derivative or fragment thereof, or an agent that enhances production and/or male erection or female sexual arousal stimulating function of the factor.
Any mammal can be treated with the present method. Preferably, male erectile dysfunction or female sexual arousal disorder in humans are treated or prevented. When human patients are treated, any suitable factor protein, or a functional derivative or fragment thereof, or any suitable factor nucleic acid, or a functional derivative or fragment thereof, can be used. Preferably, the factor protein, or a functional derivative or fragment thereof, or factor nucleic acid, or a functional derivative or fragment thereof, is of human origin. But, suitable factor protein, or a functional derivative or fragment thereof, or factor nucleic acid, or a functional derivative or fragment thereof, of non-human origin can also be used when the non-human factor binds and stimulates the cell through the human factor receptor through cross-reactivity.
VEGF, BDNF, bFGF, PDGF, NT-3, NT-4, or Ang-1 proteins or functional derivatives or fragments thereof, or a nucleic acid encoding VEGF, BDNF, bFGF, PDGF, NT-3, NT-4, Ang-1, or functional derivatives or fragments thereof, can be prepared by any methods known in the art, e.g., synthetic methods, recombinant methods or a combination thereof. Any suitable DNA construct encoding VEGF, BDNF, bFGF, PDGF, NT-3, NT-4, or Ang-1 can be used in the present invention. Such constructs include, but are not limited to VEGF-GenBank accession number M32977 (SEQ ID NO:9), BDNF-GenBank accession number M61176 (SEQ ID NO:10), bFGF-GenBank accession number E02544 (SEQ ID NO:11), NT-3-GenBank accession number M37763 (SEQ ID NO:12), NT-4-GenBank accession number M86528 (SEQ ID NO:13), and PDGF-GenBank accession number X02811 (SEQ ID NO:14) (PDGF-α) and X03795 (SEQ ID NO:15) (PDGF-β), and Ang-1-GenBank accession number U83508 (SEQ ID NO:16). Further contemplated for use in the present invention are the DNA sequences and resultant proteins described in U.S. Pat. No. 5,607,918, U.S. Pat. No. 5,438,121, U.S. Pat. No. 5,229,500, U.S. Pat. No. 5,180,820, U.S. Pat. No. 5,387,673, U.S. Pat. No. 5,155,214, U.S. Pat. No. 5,026,839, and U.S. Pat. No. 6,037,320.
Any suitable method for the production or the stabilization of the VEGF, BDNF, bFGF, PDGF, NT-3, NT-4, or Ang-1 protein known to a skilled artisan may be used in this invention. Formulations that may be used in these inventions include, but are not limited to those described in U.S. Pat. No. 5,217,954, U.S. Pat. No. 5,235,043, U.S. Pat. No. 5,986,070, U.S. Pat. No. 6,077,829, U.S. Pat. No. 5,130,418, U.S. Pat. No. 5,188,943, and U.S. Pat. No. 5,770,228.
The formulation, dosage and route of administration of the above-described compositions, combinations, preferably in the form of pharmaceutical compositions, can be determined according to the methods known in the art (see e.g., Remington: The Science and Practice of Pharmacy, Alfonso R. Gennaro (Editor) Mack Publishing Company, April 1997; Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Banga, 1999; and Pharmaceutical Formulation Development of Peptides and Proteins, Hovgaard and Frkjr (Ed.), Taylor & Francis, Inc., 2000; Medical Applications of Liposomes, Lasic and Papahadjopoulos (Ed.), Elsevier Science, 1998; Textbook of Gene Therapy, Jain, Hogrefe & Huber Publishers, 1998; Adenoviruses: Basic Biology to Gene Therapy, Vol. 15, Seth, Landes Bioscience, 1999; Biopharmaceutical Drug Design and Development, Wu-Pong and Rojanasakul (Ed.), Humana Press, 1999; Therapeutic Angiogenesis: From Basic Science to the Clinic, Vol. 28, Dole et al. (Ed.), Springer-Verlag New York, 1999). The compositions, combinations or pharmaceutical compositions can be formulated for oral, rectal, topical, inhalational, buccal (e.g., sublingual), parenteral (e.g., subcutaneous, intramuscular, intradermal, intracavemous, or intravenous), transdermal administration or any other suitable route of administration. The most suitable route in any given case will depend on the nature and severity of the condition being treated and on the nature of the particular composition, combination or pharmaceutical composition which is being used.
Any form of male erectile or female sexual arousal disorder can be treated by the present method. The present invention provides a method for the treatment or prevention of male erectile dysfunction induced by or secondary to nerve dysfunction, nerve injury, arterial insufficiency, venous leakage, severe vascular insufficiency, mild vascular disease, hormonal insufficiency, drug use, surgery, chemotherapy or radiation. In another aspect, the present invention provides a method for the treatment and prevention of female sexual arousal disorder induced by or secondary to nerve dysfunction, nerve injury, arterial insufficiency, severe vascular insufficiency, mild vascular disease, hormonal insufficiency, drug use, surgery, chemotherapy or radiation.
In a preferred embodiment, the factor protein or a functional derivative or fragment thereof, or a nucleic acid encoding the factor or functional derivative or fragment thereof, or an agent that enhances production and/or female sexual arousal stimulating function of the factor, is administered in an amount sufficient to improve blood flow and regenerate nerve and smooth muscle in the clitoris and vaginal wall. A preferred route of administration is topically in a cream or via injection to the clitoris and vaginal wall of the patient.
In another preferred embodiment, the present invention provides for a method of treatment for the treatment of male erectile dysfunction or female sexual arousal disorder by administering the factor protein or a functional derivative or fragment thereof, or a nucleic acid encoding the factor or functional derivative or fragment thereof, or an agent that enhances production and/or the male erection or female sexual arousal stimulating function of the factor, by intracavernous injection.
C. Combinations and Kits
In another aspect, the present invention provides for a combination for preventing or treating male erectile dysfunction or female sexual arousal disorder, which combination comprises: a) an effective amount of an agent that stimulates male erectile or female sexual function; and b) an effective amount of one or more factors, wherein the factor is a full length protein or a functional derivative or fragment thereof, or a nucleic acid encoding said factor or functional derivative or fragment thereof, or an agent that enhances production and/or male erection or female sexual arousal stimulating function of said factor and said factors are selected from the group consisting of vascular endothelial growth factor (VEGF), brain-derived growth factor (BDNF), basic fibroblast growth factor (bFGF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), platelet-derived growth factor (PDGF), and angiopoietin-1 (Ang-1).
The instant invention may further comprise the coadministration of an agent that enhances, complements, or is synergistic with the erectile stimulating or female sexual arousal stimulating activity of VEGF, BDNF, bFGF, PDGF, NT-3, NT-4, or Ang-1, or some combination thereof. The agent may comprise an agent with independent pharmacologic activity or one that prolongs the functional or structural half-life of VEGF, BDNF, bFGF, PDGF, NT-3, NT-4, or Ang-1, or some combination thereof. This invention also contemplates the administration of such VEGF, BDNF, bFGF, PDGF, NT-3, NT-4, or Ang-1, a combination thereof, with or without an accompanying agent simultaneously or separately to maximize the male erectile or female sexual arousal stimulating activity.
The present invention provides for administration of a combination of factors to treat or prevent male erectile dysfunction or female sexual arousal disorder. The combinations of VEGF and NT-3, VEGF and NT-4, or VEGF and BDNF are contemplated as useful combinations when the dysfunction or disorder is induced by or secondary to nerve injury. The combinations of VEGF and PDGF, VEGF and bFGF, and VEGF and Ang-1 are contemplated as useful combinations when the dysfunction or disorder is induced by or secondary to severe vascular insufficiency. The administration of VEGF alone is contemplated as useful when the dysfunction or disorder is induced by or secondary to mild vascular disease.
In a further embodiment of the invention, the VEGF, BDNF, bFGF, PDGF, NT-3, NT-4, or Ang-1, or some combination thereof may be delivered to said mammal using a gene therapy vector. The exemplary vectors include, but are not limited to retroviruses, adenoviruses, adeno-associated viruses, lentiviruses, herpesviruses, and vaccinia viruses vectors. Replication-defective recombinant adenoviral vectors can be produced in accordance with known techniques. See, Quantin, et al., Proc. Natl. Acad. Sci. USA, 89:2581-2584 (1992); Stratford-Perricadet, et al., J. Clin. Invest., 90:626-630 (1992); and Rosenfeld, et al., Cell, 68:143-155 (1992).
The vector may include an expression construct of VEGF, BDNF, bFGF, PDGF, NT-3, NT-4, or Ang-1 nucleic acid, a functional derivative fragment thereof under the transcription control of a promoter. In a preferred embodiment, the gene therapy vector may be administered by intracavemous injection of about 0.5 to 2 ml of AAV-VEGF, AAV-BDNF, AAV-bFGF, AAV-PDGF, AAV-NT-3, AAV-NT-4, or AAV-Ang-1 at a concentration of about 1010 virus titer. An example of a suitable promoter that may be used is the 763-base-pair cytomegalovirus (CMV) promoter. Viral promoters, cellular promoters/enhancers and inducible promoters/enhancers that could be used in combination with the nucleic acid contemplated for use in this invention include those listed in Jin, et al., U.S. Pat. No. 6,251,871. The expression construct may be inserted into a vector, such as pUC118, pBR322, or other known plasmid vectors, that includes an origin of replication. See, for example, Current Protocols in Molecular Biology, Ausubel, et al. eds., John Wiley & Sons, Inc. (2000), Sambrook, et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory press, (1989). The plasmid vector may also include a selectable marker such as the β-lactamase gene for ampicillin resistance, provided that the marker polypeptide does not adversely effect the metabolism of the organism being treated.
The preferred embodiment of the VEGF, BDNF, bFGF, PDGF, NT-3, NT-4, or Ang-1, or agent nucleic acid may be delivered via an adeno-associated viral (AAV) vector. These viruses are single-stranded DNA, nonautotomous parvoviruses that are able to integrate efficiently into the genome of nondividing cells of a very broad host range. Although ubiquitous in nature, AAV has not been shown to be associated with any known human disease and does not elicit an immune response in an infected human host. GOODMAN & GILMAN, PHARMACOLOGICAL BASIS OF THERAPEUTICS, 9th ed., McGraw-Hill Press (1996), p. 77-101. The method to produce purified replication deficient recombinant adeno-associated virions is described in Dwarki, et al., U.S. Pat. No. 6,221,646 B1, and its contents are incorporated in their entirety herein.
The present invention contemplates the use of AAV vectors that are known to those of skill in the art. For example, the vectors and vector production methods described in U.S. Pat. Nos. 5,589,377; 5,753,500; and 5,693,531. In another embodiment of this invention, the nucleic acid may be delivered in a retroviral vector, using vector and production method known in the art. See, for example, U.S. Pat. No. 5,830,725. A further embodiment would deliver the nucleic acid in an adenovirus vector, using vectors and vector production methods known in the art. See, for example, U.S. Pat. Nos. 6,063,622; 6,083,750; 5,994,128; and 5,981,225.
In a further embodiment of the invention, factors used in the present method and the encoding nucleic acids may be delivered in a liposome. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). For a review of the procedures for liposome preparation, targeting and delivery of contents, see Mannino and Gould-Fogerite, Bio Techniques, 6:682 (1988). See also, Current Protocols in Molecular Biology, Ausubel, et al. eds., John Wiley & Sons Press (2000), Chapters 9 and 16. The process of making and loading the liposomes with nucleic acid or protein may employ techniques known in the art. See, for example, U.S. Pat. Nos. 6,007,838; 6,197,333 B1; 6,133,026; 6,120,798; 5,939,096; 5,662,931; 5,552,157; and 5,270,053.
According to the present invention, the VEGF, BDNF, bFGF, PDGF, NT-3, NT-4, or Ang-1 peptides, proteins, polynucleotides, nucleic acids, or agent that enhances production and/or erection or sexual arousal stimulating function of said factor may be formulated for intracavemous injection, subcutaneous injection, intravenous injection, intramuscular injection, intradermal injection, or topical administration. The method may employ formulations for injectable administration in unit dosage form, in ampules or in multidose containers, with an added preservative. The formulations may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, sterile pyrogen-free water or other solvents, before use. Topical administration in the present invention may employ the use of a foam, gel, cream, ointment, transdermal patch, or paste.
Pharmaceutically acceptable compositions and methods for their administration that may be employ for use in this invention include, but are not limited to those described in U.S. Pat. Nos. 5,736,154; 6,197,801 B1; 5,741,511; 5,886,039; 5,941,868; 6,258,374 B1; and 5,686,102.
One preferred embodiment is the intracavemous administration of about 0.5 to 2.0 ml of the AAV-VEGF, AAV-BDNF, AAV-bFGF, AAV-PDGF, AAV-NT-3, AAV-NT-4, or AAV-Ang-1 gene therapy vector at a concentration of about 1010 virus titer.
The magnitude of a therapeutic dose in the acute or chronic treatment of erectile dysfunction or female sexual arousal disorder will vary with the severity of the condition to be treated and the route of administration. The dose, and perhaps dose frequency, will also vary according to age, body weight, condition and response of the individual patient. A preferred dosage for the treatment or prevention of male erectile dysfunction and/or female sexual arousal disorder is about 10-200 mcg/70 Kg body about once every two to six months.
One preferred embodiment of the present invention contemplates the administration by the application of a cream or by an injection to the clitoris and vaginal wall of a patient with female sexual arousal disorder in an amount sufficient to improve blood flow and regenerate nerve and smooth muscle in the clitoris and vaginal wall.
It should be noted that the attending physician would know how to and when to terminate, interrupt or adjust therapy to lower dosage due to toxicity, or adverse effects. Conversely, the physician would also know how to and when to adjust treatment to higher levels if the clinical response is not adequate (precluding toxic side effects).
Any suitable route of administration may be used. Dosage forms include tablets, troches, cachet, dispersions, suspensions, solutions, capsules, patches, and the like. See, Remington's Pharmaceutical Sciences.
In practical use, VEGF, BDNF, bFGF, PDGF, NT-3, NT-4, or Ang-1 peptides, proteins, polynucleotides, nucleic acids, or agent that enhances production and/or erection or sexual arousal stimulating function of said factor may be combined as the active in intimate admixture with a pharmaceutical carrier or incipient according to conventional pharmaceutical compounding techniques. The carrier may take a wide form of preparation desired for administration, topical or parenteral. In preparing compositions for parenteral dosage form, such as intravenous injection or infusion, similar pharmaceutical media may be employed, water, glycols, oils, buffers, sugar, preservatives, liposomes, and the like known to those of skill in the art. Examples of such parenteral compositions include, but are not limited to dextrose 5% w/v, normal saline or other solutions. The total dose of VEGF, BDNF, bFGF, PDGF, NT-3, NT-4, or Ang-1 to be administered may be administered in a vial of intravenous fluid, ranging from about 1 ml to 2000 ml. The volume of dilution fluid will vary according to the total dose administered.
The invention also provides for kits for carrying out the therapeutic regimens of the invention. Such kits comprise in one or more containers therapeutically effective amounts of the VEGF, BDNF, bFGF, PDGF, NT-3, NT-4, Ang-1, or an agent stimulating the production and/or function of VEGF, BDNF, bFGF, PDGF, NT-3, NT-4, Ang-1, or some combination thereof in pharmaceutically acceptable form. Preferred pharmaceutical forms would be in combination with sterile saline, dextrose solution, or buffered solution, or other pharmaceutically acceptable sterile fluid. Alternatively, the composition may be lyophilized or dessicated; in this instance, the kit optionally further comprises in a container a pharmaceutically acceptable solution, preferably sterile, to reconstitute the complex to form a solution for injection purposes. Exemplary pharmaceutically acceptable solutions are saline and dextrose solution.
In another embodiment, a kit of the invention further comprises a needle or syringe, preferably packaged in sterile form, for injecting the composition, and/or a packaged alcohol pad. Instructions are optionally included for administration of composition by a physician or by the patient.
In yet another embodiment, a kit of the invention further comprises in a container a pharmaceutically acceptable composition suitable for topical administration. Instructions are optionally included for administration of composition by a physician or by the patient.
The following examples are intended to illustrate but not to limit the invention.
The Effect of Vascular Endothelial Growth Factor (VEGF) on a Rat Model of Traumatic Arteriogenic Erectile Dysfunction
Fifty 3-months-old male Sprague Dowley rats weighing 350-400 grams were anesthetized with intraperitoneal pentobarbital (35 mg/kg) after being sedated with inhalation of methoxyflurane. Midline laparotomy was performed to identify the iliac vessels. Under operating microscope, the iliac veins were carefully dissected to expose the internal iliac arteries that vary from 2 to 4 in number. Rats in the experimental group (n=44) underwent bilateral ligation of internal iliac arteries at their origin. A separate incision was made in the perineum to identify one of the crura. A 23-gauge scalp vein needle was inserted to the crus and connected to a pressure monitor for intracavernous pressure monitoring during electrostimulation of the cavernous nerve. Additional ligation of arterial branches was performed until minimal or no intracavernous pressure rise during electrostimulation. Intracavernous injection of 4 μg of VEGF in PBS (phosphate-buffered saline) with 0.1% BSA (bovine serum albumin); 2 μg of VEGF in PBS with 0.1% BSA; or PBS solution with 0.1% BSA was then administered through the same needle to 3 groups of rats (n=16, 12 and 16 respectively). The wound was closed in layers and the animals were closely monitored for up to 6 weeks. At week 1, 2 and 6 roughly one-third of rats from each group underwent electrostimulation of the cavernous nerve to assess erectile function and then sacrificed. Penile tissues of 3 rats randomly chosen from each of the three subgroups at 2 and 6 weeks were obtained for immunohistochemical staining and electron microscopic examination.
Bipolar platinum-wire electrodes were used to stimulate the cavernous nerve. The exposed end of the electrodes were hooked around the nerve to be stimulated, with the positive electrode being positioned proximally and the negative electrode two to three mm distally. Stimulus parameters were 1.5 volts, frequency of 20 pulses per second, pulse width of 0.2 msec, and the duration of 50 seconds. Intracavernous pressure was monitored and recorded by inserting a number 23 scalp vein needle to one of the crura and connected to a pressure monitor.
Penile tissue were fixed for 3 hours in a cold, freshly prepared solution of 2% formaldehyde, 0.002% picric acid in 0.1 M. phosphate buffer, pH 8.0. Tissues were cryoprotected for 24 hours in cold 30% sucrose in 0.1 M. phosphate buffer, pH 8.0. They were then embedded in O.C.T. compound (Tissue-Tek, Miles Laboratory), frozen in liquid nitrogen, and stored at -70° C. After freezing, Cryostat tissue sections were cut at 10 μm., adhered to charged slides, air-dried, and hydrated for 5 min. with 0.05 M. sodium phosphate buffer (PBS, pH 7.4). Sections were treated with hydrogen peroxide/methanol to quench endogenous peroxidase activity. After rinsing with water, sections were washed twice in PBS for 5 min. followed by 30 min. of room-temperature incubation with 3% horse serum/PBS/0.3% triton X-100. After draining solution from sections, tissues were incubated for 60 min. at room temperature with mouse monoclonal anti-nNOS (Transduction Laboratories, Lexington, Ky.) at a 1:500 dilution. After washing for 5 min. with PBS/TX and then for 5 min. twice with PBS alone, sections were immunostained with the avidin-biotin-peroxidase method (Elite ABC, Vector Labs, Burlingame Calif.), with diaminobenzidine as the chromagen, followed by counterstaining with hematoxylin.
The penis was dissected, thinly sliced (˜1 mm thick) and placed in Karnovsky's fixative (1% para-formaldehyde/3% glutaraldehyde/0.1 M sodium cacodylate buffer, pH 7.4) at room temperature for 30 minutes and then stored at 4° C. The fixed tissue was then rinsed in buffer, post-fixed in 2% aqueous OsO4, and stained en bloc with uranyl acetate before being dehydrated in ethanol, cleared in propyline oxide, and embedded in eponate12 (Ted Pella Co., Redding, Calif.). Thick sections were cut and stained with toludine blue, examined under light microscope to select the area to be thin-sectioned. Thin sections were cut by Leica ultracut E microtome (Bannockburn, Ill.), stained with uranyl acetate and Reynold's Lead to enhance contrast and examined under Philips Tecnai 10 electron microscope (Eidhoven, Netherlands).
Data were evaluated with Mann-Whitney rank-sum test. Significance was defined as p<0.05.
The peak sustained intracavernous pressure during electrostimulation of the cavernous nerve is shown in Table I. There is no difference in intracavernous pressure between the sham operated and the normal rats. After bilateral ligation of the internal iliac arteries, the intracavernous pressure immediately dropped to around 20 cm H2O and produced no or minimal pressure increase in response to neurostimulation in all rats. In the PBS-treated group, poor erectile response persisted at weeks 1 and 2 and slight recovery of erectile function was noted at week 6.
In the VEGF-treated rats, at weeks 1 and 2, moderate recovery of erectile function was noted in the 4-μg group but not the 2-μg group. At week 6, statistically significant improvement in intracavernous pressure was seen in both the 2-μg and 4-μg groups as compared with the PBS-treated group. The intracavernous pressure of the 4-μg group was also significantly higher than that of the 2-μg group.
To identify the new source of blood flow in the 4-μg VEGF-treated group, we noted a decrease in erectile response after clamping one external iliac artery and no erectile response at all after clamping both external iliac arteries. This strongly suggests that the collateral vessels were derived from the external iliac arteries.
TABLE-US-00002 TABLE 1 Peak sustained intracavernous pressure (cm H20) during electrostimulation of the cavernous nerves in saline- and VEGF-treated rats Saline treated Group VEGF-treated Group PBS + 0.1% BSA PBS + 2 μg VEGF PBS + 4 μg VEGF Week 1 20.33 ± 3.45 23.50 ± 2.38 71.17 ± 16.89 (n = 6) (n = 4) (n = 6) Week 2 27.75 ± 9.70 43.00 ± 8.37 86.25 ± 8.18 (n = 4) (n = 4) (n = 4) Week 6 46.75 ± 14.85 69.00 ± 8.83 96.67 ± 13.50 (n = 6) (n = 4) (n = 6) Sham operated group (n = 6): 98 ± 8.50
There was a trend of decreased nNOS-immunoreactive in both dorsal and intracavernous nerves two weeks after arterial ligation in all subgroups. At week 6, moderate recovery of nNOS-positive nerve fibers was noted in both dorsal and intracavernous nerves in both 2 & 4 μg VEGF-treated groups but not in the PBS-treated group. However, the differences were not statistically significant by computer-assisted image analysis.
In sham-operated rats, no difference was noted between specimens obtained from the 2 and 6-week groups. The dorsal nerve in these rats was filled with both myelinated and non-myelinated nerve bundles. The mean diameter of the individual myelinated nerve axon was 4.42±1.36 μm (excluding myelin sheath). The mean thickness of the myelin sheath was 0.58±0.21 μm. The mean diameter of the non-myelinated nerve fibers was 0.96±0.37 μm. The nuclei of Schwann cells were seen occasionally near the nerve fibers.
In ligated +PBS treated rats, dramatic changes were noted at week 2. There was an increase in the number of Schwann cells and many of which contained vacuous within the cytoplasm. There was also a decrease in the number of both non-myelinated and myelinated nerve fibers. Overall the size of the axons was smaller than that of the controls, and many of the non-myelinated nerve fibers were smaller and less discrete. At week 6, varying degree of regeneration of both myelinated and non-myelinated nerve fibers was apparent. However, the nerve fibers of both the myelinated and non-myelinated nerve fibers were still smaller than the control groups. (mean myelinated axon 3.17±1.01 μm, myelin sheath 0.46±0.11 μm, non-myelinated axon 0.81±0.38 μm, p=0.062, 0.189, and 0.069 respectively compared with those of the sham group.) Many of the non-myelinated fibers were less than one third of the size of the larger ones. There was also an increase in the number of nucleated Schwann cells.
At week 6, in ligated rats treated with 4 μg of VEGF, the mean diameter of the myelinated axons (6.19±2.38 μm) was larger than the ligated +PBS treated ones (3.17±1.01 μm) with a p=0.008. The mean diameter of the nonmyelinated axons was 0.82±0.45 μm which was similar to that of the ligated +PBS treated rats (0.81±0.38 μm). However, the non-myelinated nerve fibers appeared more even in size.
Intracavernosal Smooth Muscle:
In sham-operated rats, no difference was noted between the specimens from the 2 and 6-week groups. The smooth muscle cells, most of which were arranged in clusters, were embedded in fine strands of fibro-connective tissue. The cytoplasm of these myocytes contained abundant contractile myofilaments and dense bodies. Occasionally, small aggregates of organelles, including mitochondria, rough endoplasmic reticulum and Golgi apparatus, were found adjacent to the nucleus. The cell membrane (sarcolemma) was consisted of many alternating dense and light bands with the latter containing numerous pinocytotic vesicles (caveolae). The intercellular spaces were narrow and many cell-cell contacts (gap junctions) were visible. Nerve terminal varicosities were seen occasionally near clusters of smooth muscle cells.
At week 2, in ligated +PBS-treated rats, many atrophic smooth muscle cells separated by large amounts of collagen fibers were noted. These atrophic smooth muscle cells appeared scattered in a sheet of connective tissues while normal smooth muscle cells were separated only by minimal amounts of connective tissues. At week 6, the number of normal-appearing smooth muscle cells increased, although many of them still showed significant loss of myofilaments.
In ligated +4 μg of VEGF-treated rats (both 2 and 6-week groups), most of the smooth muscle cells appeared normal with large amount of myofilaments and narrow intercellular spaces.
In sham-operated rats, the cavernous sinusoids were lined with intact endothelium, the cytoplasm of which contained numerous pinocytotic vesicles (caveolae), mitochondria, rough endoplasmic reticulum, and Golgi apparatus. The nuclei of the endothelial cells were sparsely seen and appeared flattened. In ligated +PBS-treated rats, (both 2 and 6-week groups) the capillary and cavernous sinusoidal endothelium appeared normal, although increase in the number of endothelial cells could be seen in some fields. In ligated +4 μg of VEGF-treated rats, (both 2 and 6-week groups) striking differences were noted when compared with other groups of rats. Many reactive endothelial cells with plump nuclei could be seen lining the sinusoids and capillaries. These endothelial cells were larger and more numerous indicative of endothelial cell hypertrophy and hyperplasia. Lee M C, et al., J. Urol. 167:761-7 (2002).
1. Goldstein, L., et al., Radiation-associated impotence. A clinical study of its mechanism. Jama, 1984. 251(7): p. 903-10. 2. Rosen, M. P., et al., Arteriogenic impotence: findings in 195 impotent men examined with selective internal pudendal angiography. Young Investigator's Award. Radiology, 1990. 174(3 Pt 2): p. 1043-8. 3. Levine, F. J., A. J. Greenfield, and I. Goldstein, Arteriographically determined occlusive disease within the hypogastric-cavernous bed in impotent patients following blunt perineal and pelvic trauma. Journal of Urology, 1990. 144(5): p. 1147-53. 4. Heaton, J. P., et al., Coronary artery bypass graft surgery and its impact on erectile function: a preliminary retrospective study. International Journal of Impotence Research, 1996. 8(1): p. 35-9. 5. D'Amore, P. A. and R. W. Thompson, Mechanisms of angiogenesis. Annual Review of Physiology, 1987. 49(9-10): p. 453-64. 6. Folkman, J., C. C. Haudenschild, and B. R. Zetter, Long-term culture of capillary endothelial cells. Proceedings of the National Academy of Sciences of the United States of America, 1979. 76(10): p. 5217-21. 7. Gross, J. L., D. Moscatelli, and D. B. Rifkin, Increased capillary endothelial cell protease activity in response to angiogenic stimuli in vitro. Proceedings of the National Academy of Sciences of the United States of America, 1983. 80(9): p. 2623-7. 8. Leung, D. W., et al., Vascular endothelial growth factor is a secreted angiogenic mitogen. Science, 1989. 246(4935): p. 1306-9. 9. Poltorak, Z., et al., VEGF145, a secreted vascular endothelial growth factor isoform that binds to extracellular matrix. Journal of Biological Chemistry, 1997.272(11): p. 7151-8. 10. Hopkins, S. P., et al., Controlled delivery of vascular endothelial growth factor promotes neovascularization and maintains limb function in a rabbit model of ischemia. Journal of Vascular Surgery, 1998. 27(5): p. 886-94; discussion 895. 11. Asahara, T., et al., Local delivery of vascular endothelial growth factor accelerates reendothelialization and attenuates intimal hyperplasia in balloon-injured rat carotid artery [see comments]. Circulation, 1995. 91(11): p. 2793-801. 12. Hariawala, M. D., et al., VEGF improves myocardial blood flow but produces EDRF-mediated hypotension in porcine hearts. Journal of Surgical Research, 1996. 63(1): p. 77-82. 13. Bauters, C., et al., Recovery of disturbed endothelium-dependent flow in the collateral-perfused rabbit ischemic hindlimb after administration of vascular endothelial growth factor [see comments]. Circulation, 1995. 91(11): p. 2802-9. 14. Bauters, C., et al., Physiological assessment of augmented vascularity induced by VEGF in ischemic rabbit hindlimb. American Journal of Physiology, 1994. 267(4 Pt 2): p. H1263-71. 15. Takeshita, S., et al., Therapeutic angiogenesis. A single intraarterial bolus of vascular endothelial growth factor augments revascularization in a rabbit ischemic hind limb model. Journal of Clinical Investigation, 1994. 93(2): p. 662-70. 16. Takeshita, S., et al., Intramuscular administration of vascular endothelial growth factor induces dose-dependent collateral artery augmentation in a rabbit model of chronic limb ischemia. Circulation, 1994. 90(5 Pt 2): p. II228-34. 17. Takeshita, S., et al., Time course of increased cellular proliferation in collateral arteries after administration of vascular endothelial growth factor in a rabbit model of lower limb vascular insufficiency. American Journal of Pathology, 1995. 147(6): p. 1649-60. 18. Banai, S., et al., Angiogenic-induced enhancement of collateral blood flow to ischemic myocardium by vascular endothelial growth factor in dogs. Circulation, 1994.89(5): p. 2183-9. 19. Baumgartner, I., et al., Constitutive expression of phVEGF165 after intramuscular gene transfer promotes collateral vessel development in patients with critical limb ischemia [see comments]. Circulation, 1998. 97(12): p. 1114-23. 20. Losordo, D. W., P. R. Vale, and J. M. Isner, Gene therapy for myocardial angiogenesis. American Heart Journal, 1999. 138(2 Pt 2): p. 132-41. 21. Azadzoi, K. M. and I. Saenz de Tejada, Hypercholesterolemia impairs endothelium-dependent relaxation of rabbit corpus cavernosum smooth muscle. Journal of Urology, 1991. 146(1): p. 238-40. 22. Kim, J. H., et al., Experimental hypercholesterolemia in rabbits induces cavernosal atherosclerosis with endothelial and smooth muscle cell dysfunction. Journal of Urology, 1994. 151(1): p. 198-205. 23. Neufeld, G., et al., Vascular endothelial growth factor (VEGF) and its receptors. Faseb Journal, 1999. 13(1): p. 9-22. 24. Liu, X*, Lin, C-S*, Graziottin, T, Resplande, J, and Lue, TF: Vascular Endothelial Growth Factor Promotes Proliferation and Migration of Cavernous Smooth Muscle Cells. J Urol., 2001. 166: 354-360. 25. Magovern, C. J., et al., Regional angiogenesis induced in nonischemic tissue by an adenoviral vector expressing vascular endothelial growth factor. Human Gene Therapy, 1997. 8(2): p. 215-27. 26. Folkman, J. and Y. Shing, Angiogenesis. Journal of Biological Chemistry, 1992. 267(16): p. 10931-4. 27. Ferrara, N., The role of vascular endothelial growth factor in pathological angiogenesis. Breast Cancer Research and Treatment, 1995. 36(2): p. 127-37. 28. Walder, C. E., et al., Vascular endothelial growth factor augments muscle blood flow and function in a rabbit model of chronic hindlimb ischemia. Journal of Cardiovascular Pharmacology, 1996. 27(1): p. 91-8. 29. Harada, K., et al., Vascular endothelial growth factor administration in chronic myocardial ischemia. 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Intracavernosal Vascular Endothelial Growth Factor (VEGF) and Adeno-Associated Virus Mediated VEGF Gene Therapy Prevents and Reverses Venogenic Erectile Dysfunction in Rats
Male Sprague-Dawley rats age 3-6 months (wt 350-450 grams) were used in this study. They were housed in our animal care facility with rat chow and water available ad libitum on a 12 hr light/dark cycle. All animal care, treatments and procedures were performed in compliance with requirements of the Committee on Animal Research at our institution. Rats were randomly divided for the animal model of vasculogenic ED (Experiment 1) and the VEGF prevention trial (Experiment 2). For the VEGF treatment trial (Experiment 3), the animals underwent castration and then were treated with VEGF after venous leak was demonstrated, to evaluate the efficacy of VEGF treatment in reversing established venogenic ED.
To determine normal values for rodent pharmacologic cavernosometry and validate the model of venogenic ED in the rat, vasculogenic ED was induced. Arterial insufficiency was produced after performing a bilateral ligation of the internal iliac arteries. The acute and chronic effects of arterial insufficiency were evaluated 7 days and 30 days after bilateral iliac artery ligation (n=7). Venogenic ED was induced by castration, and pharmacologic cavernosometry was performed 6 weeks after surgery (n=10). Control animals underwent a sham laparotomy and studied 6 weeks later (n=13).
For the prevention trial of VEGF in rats with venogenic erectile ED, adult males were castrated and immediately treated with hormone, intracavernosal VEGF. Hormone replacement was accomplished using a subcutaneously placed testosterone-filled silastic implant (n=6), as previously described.17 A therapeutic testosterone serum titer was confirmed in this animal group by testosterone radioimunoassay performed by the biomedical core lab at our institution.18 Intracavernous treatment with VEGF was administered using either recombinant VEGF protein (n=5) or an adeno-associated virus vector expressing the VEGF gene (AAV-VEGF, n=14). Control animals received an silastic implant containing saline (n=3), an intracavernous injection of normal saline (n=3) or an adenovirus transfection vector expressing lacZ reporter gene without the VEGF gene (AAV-LacZ, n=11).
The trial of VEGF treatment was performed in castrated animals that were shown, 6-8 weeks following castration, to develop venogenic erectile ED by pharmacologic cavernosometry. These animals (n=8) were treated with intracavemous VEGF protein and then after one month cavernosometry repeated to measure the effect of VEGF treatment.
Prior to all surgical procedures, animals received anesthesia consisting of isoflurane inhalation as pre-anesthetic followed by an intraperitoneal injection of sodium pentobarbital (40 mg/kg). After the animal was asleep, electric clippers were used to trim the ventral abdominal hair and the skin was prepped with clorhexidine scrub. Antiseptic technique was maintained for all procedures. Following surgery, the anterior abdominal fascia and skin were approximated with 4-0 silk suture, analgesic buprenorphine (0.5 mg/kg SC) was administered and the animal allowed to awaken covered with a heating pad. Euthanasia was accomplished by an intraperitoneal injection of sodium pentobarbital (200 mg/kg) followed by bilateral thoracotomy when the animal was fully asleep.
Arterial Ligation Surgery:
A 2 cm midline longitudinal low abdominal incision was made and a wheatlander retractor placed so that the plane between the prostate and sigmoid colon could be bluntly opened. A dissecting microscope with 2.5-10× objectives was essential to safely performing this procedure. Using sterile cotton-tipped swabs, the iliac artery bifurcation was identified and the common iliac exposed to the external iliac take-off. The internal iliac arteries were identified as those medial branches off the common iliac between the iliac bifurcation and the take-off of the external iliac. These were doubly ligated using 7-0 nylon sutures. After this was performed on both the right and left side, the incision was closed and the animal recovered as noted above.
A 2 cm midline longitudinal low abdominal incision was made and each testicle was grasped using forceps and brought into the incision. Each gubernaculum was divided using electrocautery and then the spermatic cords ligated with 4-0 silk suture and divided. After confirming hemostasis, the abdomen was closed and the rat recovered as above.
A 1.5 cm oblique incision was made in the lower abdominal skin extending from the midline just above the penile hilum to below the level of the glans about 1 cm lateral to the midline. The skin was sharply dissected from the anterior surface of the penis and then the penis was retracted anteriorly using a towel clamp placed around it atraumatically with the foreskin left intact. Using blunt dissection the penile base and crura were exposed. The ischiocavernosus muscles were sharply dissected off the anterior surface of the crus until the white of the tunica albuginea of the corpora cavernosa was identified. The crus was then gently cannulated using a 23 gage butterfly needle, and saline flush with a visual erectile response was used to confirm that the needle tip was truly intracavernosal. The intracavernosal injections were then administered with either VEGF protein (Calbiochem, La Jolla, Calif. #676472) at the dosage 4 ug/injection (in 0.1 cc PBS with 0.1% BSA), AAV-VEGF (1010 viral particles in 0.1 cc NS), AAV-LacZ (1010 viral particles in 0.1 cc NS), or 0.1 ml NS alone. The AAV-VEGF and AAV-LacZ constructs were a generous gift from Dr. Yuet W. Kan (Howard Hughes Medical Research Institute, San Francisco, Calif.).19,20 Following injection, the needle was left in place for 5 minutes and then removed to allow the medication to diffuse throughout the cavernosal space. Immediately thereafter, pinpoint electrocautery was applied to the needle hole for hemostasis and then the wound was closed and the animal recovered as above.
Following castration, testosterone- or saline-filled silastic implants were placed in the subcutaneous tissue of the anterior abdominal wall, as previously described.17 Implants were prepared using sterile silastic tubing (Dow Corning, Midland, Mich. #602-265, inner diameter 0.062'') that was filled with testosterone propionate powder (Sigma Chemical, St. Louis, Mo.) with the aid of wall suction. By radioimmunoassay,18 serum testosterone titer was found to be undetectable in the castrated animals and in the normal range for animals given testosterone implants.
Pharmacologic Cavernosometry in the Rat:
To perform pharmacologic cavernosometry, both the right and left crura were separately cannulated using 23 gauge butterfly needles as described above. One cannula was flushed with sterile heparinized saline (100 U heparin/ml NS) and attached to a pressure detector for continuous intracorporal pressure (ICP) monitoring as previously described.11 The contralateral cannula was attached to an infusion pump (Harvard Pump, Southwick, MA #55-2222), filled with sterile dilute heparinized saline (20 U heparin/ml NS). The baseline ICP was recorded (flaccid ICP) and then a dose of papavarine (1 mg in 0.1 cc NS) was administered through the infusion cannula. Five minutes was allowed for the papavarine to diffuse throughout the corpora and then the infusion cannula was flushed with heparinized saline and the pressure monitor cannula vented to normalize ICP after flushing. After 5 minutes more, the ICP was again recorded (ICP after papavarine) and the infusion started. An infusion rate of 0.05 ml/min was started and increased (by 0.05 ml/min every 10 seconds) until the ICP started to rise. Subsequent increases in inflow rate were made only after the ICP reached a plateau pressure. By slowly adjusting the inflow rate, an intracorporal pressure of 100 cm H2O (erectile pressure) was reached and the infusion rate required to maintain this pressure recorded (the maintenance rate). After this pressure was steady for 20 seconds, the infusion was terminated and the change in ICP over the subsequent 60 seconds was recorded (the drop rate).
After pharmacologic cavernosometry was performed, the penis was amputated at the crural bony attachments and immediately placed in ice-cold saline. The Y-shaped crura was sharply cut from the penile base and then a 1 mm thick slice cut for electron microscopy and placed in Karnofsky's solution (3% gluteraldehyde, 1% para-formaldehyde, 0.1 M sodium cacodylate buffer, pH 7.4). A 3 mm thick section of the distal penile shaft was then cut and placed in 10% normal buffered formalin for paraffin sections, and the balance of the penile shaft was flash frozen using dry ice in OCT compound (Sakura Finetek USA, Torrance, Calif.) for frozen sectioning and immunohistochemistry.
Frozen sections were cut at 10 microns, adhered to charged slides, air dried for 15 minutes then rehydrated with 0.05M PBS for 5 minutes. Sections were treated with hydrogen peroxide/methanol to quench endogenous peroxidase activity. After rinsing, sections were washed twice in PBS for 5 minutes then incubated with 3% horse serum and 0.3% triton X-100 at room temperature for 30 minutes. The serum solution was drained and then sections were incubated for 60 minutes with mouse monoclonal anti-alpha-smooth muscle actin (Sigma, St. Louis, Mo.) at a dilution of 1:4000 in PBS. After washing, sections were immunostained using the avidin-biotin-peroxidase method (Elite ABC-Vector Labs, Burlingame, Calif.), with diaminobenzidine as the chromogen, followed by counterstaining with hematoxylin. Immunochemistry was performed in penile tissues from 4 rats randomly chosen from each subgroup.
Enzyme-Linked Immuno-Sorbent Assay:
Serum samples from both systemic and penile blood were collected after whole-blood centrifugation. Solid phase enzyme-linked immuno-sorbent assay for VEGF was performed using the Quantikine M mouse VEGF Immunoassay Kit (R&D Systems, Minneapolis, Minn.) as previously described.21 Briefly, samples were diluted and added to micro plate strip wells that were then treated with the enzyme-labeled immunoreactant VEGF conjugate. After incubation for 2 hours and washing, the substrate solution was added and incubated for 30 minutes. The stop solution was added and then the optical density in each well determined using a micro plate reader set to 450 nm. Sample results were plotted on a curve generated by the optical density of standard samples ranging in concentration (0-500 pg/ml VEGF).
Transmission Electron Microscopy:
After fixing in Karnofsky's solution for 30 minutes at room temperature, 1 mm sections at the penile base were stored at 40 C until processed, as previously described.22 Briefly, samples were rinsed in PBS, post-fixed in 2% aqueous OsO4 and stained en bloc with uranyl acetate. They were then dehydrated in ethanol, cleared in propyline oxide and embedded in Eponate 12 (Ted Pella Co., Redding, Calif.). Thick sections were cut and stained with toluidine blue to select specific areas for thin sectioning. Thin sections were cut, stained with both uranyl acetate and Reynold's Lead, and examined under the Phillips Tecna±10 transmission electron microscope. Penile tissues from 4 randomly chosen rats in each subgroups were subjected to electron microscopic examinations.
Data in the present studies was analyzed using the student's t-test (homoscedastic, 2-tailed) when 2 means were compared (Experiment 1 and 2). A paired, 2-tailed t-test was used where values represent findings before and after treatment in the same animals (Experiment 3).
The first goal was to determine normal values for pharmacologic cavernosometry in a rat model of vasculogenic ED. As shown in Table 2, flaccid ICP were comparable, in the range of 30 cm H2O. After papavarine injection, however control animals had a steep rise in ICP to >100 cm H2O while the castrated and ligated animals had less response. Only a minimal increase in ICP (5-10 cm H2O) was noted in the animals following either castration or chronic internal iliac ligation, characteristic of vasculogenic ED. Acute ligation animals had a better response to papavarine yet significantly less than seen in normal animals. After the infusion was started, both the control and acute ligation group promptly achieved erectile pressure with minimal inflow required. When the infusion was stopped, these animals had a minimal pressure drop, evidencing their intact veno-occlusive mechanism. The castration and chronic ligation groups, on the other hand, required a significantly higher infusion rate to maintain erectile pressure and experienced a steep pressure drop when the infusion was terminated. These findings are characteristic of venous leakage in the chronic ligation and castration groups.
TABLE-US-00003 TABLE 2 (Experiment 1) Cavernosometric findings in rat model of vasculogenic ED. ICP After Maintenance Drop Rate in Flaccid ICP Papavarine Rate 1 min. (cm H2O) (cm H2O)* (ml/min)* (cm H2O)* Control 35.4 (+/-9.3) 104 (+/-59) 0.024 9 (+/-13) (+/-0.3) Castration 22.0 (+/-5.2) 35.0 (+/-5.0) 1.14 75 (+/-5.4) (+/-0.5) Acute 28.7 (+/-7.6) 73.3 (+/-10) 0.06 13.2 (+/-13.8) Ligation (+/-0.12) Chronic 29.3 (+/-7.6) 38.3 (+/-19) 1.9 45.8 (+/-19) Ligation (+/-1.8)
Cavernosometry was performed 6 weeks after castration, 7 days after internal iliac ligation in the acute ligation group, and 30 days after ligation in the chronic ligation group. (* p<0.05, for comparison of castration vs. control groups, and castration vs. acute ligation groups)
Our second goal was to perform a prevention trial using intracavernosal VEGF either in the form of recombinant protein or virus-directed gene expression vector in an attempt to prevent the development of venogenic erectile ED in castrated animals. As shown in Table 3, flaccid ICP was again in the range of 30 cm H2O in each of the animal groups. After papavarine administration, however, both control groups (castration only and castration with LacZ injection) exhibited only a weak rise in ICP (5-10 cm H2O), required a significant infusion rate to sustain an erectile ICP of 100 cm H2O, and had a steep pressure drop after the inflow was terminated. In contrast, the 3 treatment groups exhibited nearly normal erectile function with high ICP in response to papavarine, a very low maintenance rate to sustain erectile ICP and minimal pressure drop when the infusion was stopped. Of note, the VEGF gene-treated animals showed marginally less erectile function with a lesser response to papavarine and a higher drop rate than the animals treated with either testosterone replacement or intracavernosal VEGF protein.
TABLE-US-00004 TABLE 3 (Experiment 2) Cavernosometric findings in castrated animals after prevention trial of testosterone replacement (C + Testosterone), VEGF protein treatment (C + VEGF), AAV-VEGF gene therapy (C + VEGF gene), or LacZ control (C + LacZ control). ICP After Flaccid ICP Papavarine Maintenance Drop Rate in 1 min. (cm H2O) (cm H2O)* Rate (ml/min)* (cm H2O)* C + saline 23.4 (+/-5.3) 29.4 (+/-15) 0.51 (+/-0.26) 56.1 (+/-15) C + Testosterone 28.9 (+/-7.5) 87.7 (+/-26) 0.09 (+/-0.1) 11.7 (+/-16) C + VEGF 27.8 (+/-5.2) 85.0 (+/-28) 0.04 (+/-0.09) 9.0 (+/-20) C + VEGF gene 23.4 (+/-7.1) 61.4 (+/-36) 0.04 (+/-0.03) 27.8 (+/-18) C + LacZ 27.0 (+/-6.7) 36.0 (+/-12.8) 0.25 (+/-0.31) 58.6 (+/-8.1)
Pharmacologic cavernosometry was performed 9 weeks after castration. (* p<0.05, comparing castration only to the treatment groups, excluding the LacZ control group.)
The third phase was to perform a treatment trial using intracavernosal VEGF in animals with venous leak. The goal was to assess the efficacy of VEGF at reversing established venogenic erectile dysfunction in an animal model. Animals were castrated and then 4-6 weeks later underwent cavernosometry. As shown in Table 4, before VEGF treatment, this animal group displayed a weak response to papavarine with intracorporal pressure reaching 33 cm H2O compared to normal animals who attain nearly 100 cm H2O with such treatment (see Table 2). Also, these castrates required a relatively high maintenance rate (0.19 ml/min.) to achieve erectile pressure and a steep drop rate when the infusion was terminated (45.1 cm H2O in 60 seconds), evidencing venous leak. After these animals received intracorporal VEGF treatment, however, nearly normal erectile function returned with a prompt rise in intracorporal pressure after papavarine (to 84 cm H2O), a low maintenance rate (0.08 ml/min.) to achieve erectile pressure and a minimal drop in intracorporal pressure (17.4 cm H2O in 60 seconds) after the infusion was terminated.
TABLE-US-00005 TABLE 4 (Experiment 3) Cavernosometric findings in VEGF treatment trial. ICP After Flaccid ICP Papavarine Maintenance Drop Rate in 1 min. (cm H2O) (cm H2O)* Rate (ml/min)* (cm H2O)* Before VEGF 22.4 (+/-6.9) 33.0 (+/-12.3) 0.19 (+/-0.18) 45.1 (+/-18) treatment (6 wks after castration) 1 month 25.3 (+/-8.5) 83.9 (+/-31) 0.08 (+/-0.15) 17.4 (+/-24) following VEGF treatment
Animals were castrated and then shown to have venous leak (after approximately 6 weeks) by pharmacologic cavernosometry. They were then treated with intracavernosal VEGF and one month later underwent repeat cavernosometry. (* p<0.05)
Cross-sectional micrographs of the rat penis at the proximal shaft were examined after immunohistochemistry for alpha actin. Alpha actin, a marker for penile smooth muscle, stains brown and can be seen surrounding the sinusoidal spaces. Qualitatively, we see decreased smooth muscle content 6 weeks after castration compared to the normal control. In castrates treated with either testosterone replacement or intracavernosal VEGF protein, the quantity of smooth muscle returns to normal morphology. Computerized image analysis with Adobe Photoshop was used to quantify the area of immunostaining by counting the number of digitized pixels corresponding to the area of brown staining, thereby providing some numerical comparison of the quantity of smooth muscle in each specimen. This analysis shows the following pixel count: sham (43518), castrated (37214), AAV-VEGF treated (51690), VEGF protein treated (52990).
Transmission Electron Microscopy
In sham-operated rats, the dorsal nerve was filled with both myelinated and non-myelinated nerve bundles. The mean diameter of the individual myelinated axon (excluding myelin sheath) was 2.54±1.04 μm. The mean thickness of the myelin sheath was 0.74±0.21 μm. The mean diameter of the non-myelinated axon was 0.97±0.35 μm. The cytoplasm and nuclei of Schwann cells were seen occasionally near the nerve fibers.
In castrated rats, with or without LacZ injection, the diameter of both the myelinated and non-myelinated axons appeared smaller than those of the sham-operated rats. Mean diameters were the following: myelinated axon 1.64±1.0 μm; myelin sheath 0.49±0.13 μm; non-myelinated axon 0.64±0.32 μm. Comparing the castrated rats to the sham-operated rats, the p values were 0.06, 0.004 and 0.001 respectively. Many non-myelinated nerve fibers became indistinct and smaller. There was also an increase in the number of nucleated Schwann cells.
Although many small myelinated nerve fibers were still present in castrated rats treated with VEGF or AAV-VEGF, larger fibers with thick myelin sheaths were also noted. The mean diameter of the myelinated nerve and myelin sheath were 2.36±0.92 μm and 0.93±0.44 μm respectively. The non-myelinated nerve fibers were more clearly defined but were not as abundant as the sham group. The mean diameter of non-myelinated axons was 0.96±0.33 μm. Comparing the VEGF-treated group to the castrated +Lac Z group, the p values of myelinated axon, myelin sheath and nonmyelinated axon were 0.113, 0.05 and 0.000 respectively. The nerve fibers and myelin sheath in the testosterone replacement group appeared similar to the sham group.
Intracavernous Smooth Muscle Cells:
In sham-operated rats, the smooth muscle cells (myocytes) were usually arranged in clusters and were separated by fine strands of fibroconnective tissue. The cytoplasm of these myocytes contained abundant contractile myofilaments and dense bodies. Occasionally, small aggregates of organelles, including mitochondria, rough endoplasmic reticulum and Golgi apparatus, were found adjacent to the nucleus. The cell membrane (sarcolemma) consisted typically of alternating dense bands and light bands. The light bands contain numerous pinocytotic vesicles (caveolae). The intercellular spaces among myocytes were usually quite narrow with many gap junctions connecting individual cells. Nerve terminal varicosities were frequently seen located near clusters of smooth muscle cells. In low power micrographs (6,500×) of castration with or without LacZ rats, the smooth muscle cells appeared scattered in a field of connective tissues. The major differences between the castrated and castrated+testosterone-treated rats were the increase in cytoplasmic myofilaments and the decrease in intercellular spaces in the latter group of rats. The myocytes in testosterone-treated rats appeared packed in clusters rather than scattered.
Striking differences were noted when comparing the AAV-VEGF and VEGF protein-treated rats to the castrated +Lac Z rats. The smooth muscles were arranged in clusters with minimal intercellular spaces. Under high power (9,400×), we noted the following: an increase in myofilaments and dense bodies, a decrease in dense bands, and an increase in the number of caveolae within the light bands of the sarcolemma.
In sham-operated rats, the cavernous sinusoids were lined by intact endothelium, the cytoplasm of which contained numerous pinocytotic vesicles (caveolae), mitochondria, rough endoplasmic reticulum, and Golgi apparatus. The nuclei of the endothelial cells were occasionally seen and appeared oval-shaped or elongated. In castration with or without LacZ rats, the appearance of the capillaries and cavernous sinusoidal endothelium was similar to the sham operated group. In AAV-VEGF and VEGF protein-treated rats, the nuclei of the endothelial cells lining most of the capillaries and sinusoids were plump and more numerous, indicative of endothelial hypertrophy and hyperplasia.
Enzyme-Linked Immuno-Sorbent Assay:
The goal of using an adenovirus vector for delivering the VEGF gene is to transfect the penile tissue such that VEGF protein expression may be increase in the penis. To document that the AAV-VEGF treated animals had increased VEGF expression in the penile blood, samples were taken from the penis (penile bleed following glans amputation) and compared with a sample of systemic blood (from the abdominal aorta) for animals groups treated with both AAV-VEGF (n=7) and AAV-LacZ (n=7) (Table 5). While the mean VEGF titer in the systemic serum of animals that did not receive the VEGF gene (AAV-LacZ group) is 9.5±12.6 pg/ml, the AAV-VEGF treated animals demonstrated a marked increase in VEGF titer at 23.3±9.6 pg/ml (p=0.04). Similarly, serum from the penile blood in the AAV-LacZ group had a VEGF titer of 13.6±11.4 pg/ml compared to a mean of 29.7±14.4 pg/ml in the group receiving the intracavernosal VEGF gene (p=0.039). This difference is statistically significant suggesting that increased VEGF expression is occurring in the penile tissue after treatment with AAV-VEGF.
TABLE-US-00006 TABLE 5 Results of ELISA for VEGF protein (mean VEGF in pg/ml) in serum samples from animals treated with intracorporal AAV-VEGF (p = 0.03) and AAV-LacZ (p = 0.07) AAV-VEGF-treated AAV-LacZ-treated animals animals Penile bleed 29.7 (+/-14.4) 13.6 (+/-11.4) Systemic blood 23.3 (+/-9.6) 9.5 (+/-12.6)
To test our hypothesis, we first developed an animal model of venogenic erectile ED (Experiment 1: Model validation). Mills et al10, 11, 26 have previously shown that rats develop venogenic erectile ED within 6-8 days following castration. Animals receiving testosterone repletion maintain an intact veno-occlusive mechanism after castration. These studies were performed using ganglionic electro-stimulation to generate an erection and the penile response gauged with ICP monitoring during either cavernosometry11 or a penile arterial inflow measurement using a laser Doppler flow.10 Our goal was to devise a technique for evaluating venous leak in animals similar to the technique used in humans. For this reason, erection was generated using pharmacologic agents (papavarine) instead of ganglionic electro-stimulation. The physiologic parameters (maintenance inflow rate and ICP drop rate) used to diagnose venous leak in humans27,28 were reproduced in a rat model. Using this technique, pharmacocavernosometric findings were determined in normal animals and animals with venogenic and arteriogenic ED. This method was found to be a sensitive and reproducible technique to evaluate penile arterial insufficiency and venous leak in a rat model.
This model was then used to evaluate the efficacy of VEGF, administered intracorporally as recombinant protein or adeno-associated virus gene vector, to prevent the development of venogenic erectile ED (Experiment 2: Prevention trial). It has been previously shown that castration induces an involution of the prostate gland and its vasculature.13 Furthermore, after testosterone replacement, endothelial cell proliferation is stimulated and both blood flow and vascular volumes are normalized. After castration, prostatic VEGF synthesis is down regulated, as determined by RT-PCR, western blot and immunohistochemical analysis.12 Also, testosterone induces VEGF synthesis, suggesting that VEGF may be a tissue mediator of androgenic effects on the prostate. The goal of Experiment 2 was to determine if VEGF could prevent the development of venous leak in the rat model. Both testosterone replacement and VEGF treatment maintained erectile function when administered immediately after castration. Animal groups receiving no testosterone replacement or intracorporal AAV-LacZ showed persistent venogenic erectile ED after castration. Histological examination of smooth muscle content and morphology revealed deterioration in both the quality and quantity of penile smooth muscle after castration. Electron microscopic examination also revealed alteration of cell membrane and widening of intercellular spaces. Smooth muscle content as measured by alpha actin staining was normalized in animals receiving either testosterone or VEGF, evidence of preserved smooth muscle integrity with such preventative treatment.
The final phase was a treatment trial (Experiment 3) in which animals were first documented to have venous leak, 6 weeks after castration, and then treated with intracorporal VEGF protein. One month after such treatment, cavernosometry was repeated and restoration of near normal erectile function was found. We believe that this is the first experimental evidence of any medical therapy improving venogenic erectile ED. The exact mechanism by which VEGF improves erectile function is unknown. Nevertheless, we observed clear evidence of restoration of neural and smooth muscle integrity as well as hyperplasia and hypertrophy of endothelial cells after VEGF treatment. Conceivably, increased cavernosal neovascularity may lead to functional or structural changes in the nerve and smooth muscles. Alternatively, the direct effect of VEGF on the nerve and smooth muscle may also play a role since VEGF has been reported to have a direct trophic effect on the penile smooth muscle cells and spinal neurons in culture 14-16.
Nerve function and NOS expression is depressed with androgen ablation, and this may be another target for VEGF action in the penis. Further studies are underway to study the mechanism of VEGF action in the penis.
The technique presented here for pharmacologic cavernosometry is a simple and reproducible method to evaluate vasculogenic ED in a rat model. Normal erectile function and ED due to arterial insufficiency or venous leak may be diagnosed by characteristic cavernosometric findings. Using this technique, the presence of veno-occlusive disease may be diagnosed in animals 6 weeks after either castration or ligation of the internal iliac arteries.
Animals treated with testosterone replacement at the time of castration retain normal erectile function while those without testosterone replacement develop venous leak. If these animals are treated with intracavernosal recombinant VEGF protein or AAV-VEGF at the time of castration, their erectile function is maintained and venous leak is prevented. The mechanism for this is not known at present, but we find a decrease in penile smooth muscle content in the castrated group compared with either the testosterone replacement group or the group receiving intracavernosal VEGF or AAV-VEGF. Penile smooth muscle morphology is uniformly degenerated after castration. Animals treated with the intracorporal AAV-VEGF transfection vector demonstrate significantly more VEGF protein in their penile serum compared to the systemic serum, and markedly more that control animals, indicating an increased expression of penile VEGF in these animals.
When rats with established venogenic ED are treated with one dose of intracavernosal recombinant VEGF protein, their erectile function returns to nearly normal, with reversal of the veno-occlusive defect. Electronmicroscopy revealed endothelial cell hyperplasia and hypertrophy as well as restoration of smooth muscle and neural integrity in the penile tissue after VEGF treatment. Since impairment of erectile nerve, endothelial cell, and the cavernous smooth musculature is the final common pathway of various type of organic ED, VEGF therapy may hold the key to prevention and cure of many forms of ED.
1. Christ, G. J.: The penis as a vascular organ. The importance of corporal smooth muscle tone in the control of erection. Urol Clin North Am, 22: 727, 1995. 2. Karadeniz, T., Topsakal, M., Ariman, A. et al.: Judgment of color Doppler ultrasound with respect to cavernous artery occlusion pressure in dynamic infusion cavernosometry when evaluating arteriogenic impotence. Urol Int, 57: 85, 1996. 3. Nehra, A., Goldstein, I., Pabby, A. et al.: Mechanisms of venous leakage: a prospective clinicopathological correlation of corporeal function and structure. J Urol, 156: 1320, 1996. 4. Mersdorf, A., Goldsmith, P. C., Diederichs, W. et al.: Ultrastructural changes in impotent penile tissue: a comparison of 65 patients. J Urol, 145: 749, 1991. 5. Garban, H., Marquez, D., Cai, L. et al.: Restoration of normal adult penile erectile response in aged rats by long-term treatment with androgens. Biol Reprod, 53: 1365, 1995. 6. Meisel, R. L., O'Hanlon, J. K. & Sachs, B. D.: Differential maintenance of penile responses and copulatory behavior by gonadal hormones in castrated male rats. Horm Behav, 18: 56, 1984. 7. Zvara, P., Sioufi, R., Schipper, H. M. et al.: Nitric oxide mediated erectile activity is a testosterone dependent event: a rat erection model. Int J Impot Res, 7: 209, 1995. 8. Lekas, E., Johansson, M., Widmark, A. et al.: Decrement of blood flow precedes the involution of the ventral prostate in the rat after castration. Urol Res, 25: 309, 1997. 9. Haggstrom, S., Wikstrom, P., Bergh, A. et al.: Expression of vascular endothelial growth factor and its receptors in the rat ventral prostate and Dunning R3327 PAP adenocarcinoma before and after castration. Prostate, 36: 71, 1998. 10. Mills, T. M., Lewis, R. W. & Stopper, V. S.: Androgenic maintenance of inflow and veno-occlusion during erection in the rat. Biol Reprod, 59: 1413, 1998. 11. Mills, T. M., Stopper, V. S. & Wiedmeier, V. T.: Effects of castration and androgen replacement on the hemodynamics of penile erection in the rat. Biol Reprod, 51: 234, 1994. 12. Haggstrom, S., Lissbrant, I. F., Bergh, A. et al.: Testosterone induces vascular endothelial growth factor synthesis in the ventral prostate in castrated rats. J Urol, 161: 1620, 1999. 13. Franck-Lissbrant, I., Haggstrom, S., Damber, J. E. et al.: Testosterone stimulates angiogenesis and vascular regrowth in the ventral prostate in castrated adult rats [see comments]. Endocrinology, 139: 451, 1998. 14. Liu X, Lin C--S, Graziottin T, Resplande J and Lue T F: Vascular endothelial growth factor promotes proliferation and migration of cavernous smooth muscle cells. J Urol; 166: 354-360, 2001. 15. Jin K L, Mao X O, Greenberg D A.: Vascular endothelial growth factor: direct neuroprotective effect in vitro ischemia. Proc Natl Acad Sci USA. 97:10242-7, 2000. 16. Sandal M, Sunder F, Kanji M. Vascular endothelial growth factor is a neurotrophic factor which stimulates axonal outgrowth through the flk-1 receptor. Eur J Neurosci: 2000 12:4243-54, 2000. 17. Verdonck, A., De Ridder, L., Kuhn, R. et al.: Effect of testosterone replacement after neonatal castration on craniofacial growth in rats. Arch Oral Biol, 43: 551, 1998. 18. Parker, C. R., Jr., Ellegood, J. O. & Mahesh, V. B.: Methods for multiple steroid radioimmunoassay. J Steroid Biochem, 6: 1, 1975. 19. Maeda, Y., Ikeda, U., Ogasawara, Y. et al.: Gene transfer into vascular cells using adeno-associated virus (AAV) vectors. Cardiovasc Res, 35: 514, 1997. 20. Dong, J. Y., Fan, P. D. & Frizzell, R. A.: Quantitative analysis of the packaging capacity of recombinant adeno-associated virus. Hum Gene Ther, 7: 2101, 1996. 21. Ferrara, N., Houck, K., Jakeman, L. et al.: Molecular and biological properties of the vascular endothelial growth factor family of proteins. Endocr Rev, 13: 18, 1992. 22. Stenberg, P. E., Shuman, M. A., Levine, S. P. et al.: Redistribution of alpha-granules and their contents in thrombin-stimulated platelets. J Cell Biol, 98: 748, 1984. 23. Karadeniz, T., Topsakal, M., Aydogmus, A. et al.: Correlation of ultrastructural alterations in cavernous tissue with the clinical diagnosis vasculogenic impotence. Urol Int, 57: 58, 1996. 24. Baumgartner, I., Pieczek, A., Manor, O. et al.: Constitutive expression of phVEGF165 after intramuscular gene transfer promotes collateral vessel development in patients with critical limb ischemia [see comments]. Circulation, 97: 1114, 1998. 25. Symes, J. F., Losordo, D. W., Vale, P. R. et al.: Gene therapy with vascular endothelial growth factor for inoperable coronary artery disease. Ann Thorac Surg, 68: 830, 1999. 26. Mills, T. M., Dai, Y., Stopper, V. S. et al.: Androgenic maintenance of the erectile response in the rat. Steroids, 64: 605, 1999. 27. Hatzichristou, D. G., Saenz de Tejada, I., Kupferman, S. et al.: In vivo assessment of trabecular smooth muscle tone, its application in pharmaco-cavernosometry and analysis of intracavemous pressure determinants [see comments]. J Urol, 153: 1126, 1995. 28. Karadeniz, T., Ariman, A., Topsakal, M. et al.: Value of color Doppler sonography in the diagnosis of venous impotence. Urol Int, 55: 143, 1995.
The Effect of Adeno-Associated Virus-Mediated Brain-Derived Neurotrophic Factor (BDNF) in an Animal Model for Neurogenic Impotence
Male Sprague-Dawley rats (N=34; age, 3 months; weight, 350 to 400 gm.) were divided into two groups: sham (N=10) and experimental (N=24). The rats in the sham group underwent periprostatic dissection and identification of bilateral cavernous nerves without other manipulation; those in the experimental groups underwent bilateral cavernous nerve freezing. Several minutes after surgery, half of the experimental animals (LacZ group, N=12) received intracavernous AAV-LacZ injection, and the remainder (BDNF group, N=12) received AAV-BDNF. Half of the rats in each group were sacrificed at week 4 and the rest at week 8 for collection of penile tissue. In all animals, erectile function was assessed by electrostimulation of the cavernous nerves before sacrifice.
Surgical Procedure and Transfection Technique
Under intraperitoneal pentobarbital sodium anesthesia (50 mg./kg.), each animal was placed on a heating pad to maintain its body temperature at 37° C. Through a lower abdominal midline incision, the area posterolateral to the prostate was explored. The major pelvic ganglia and the cavernous nerve were identified with an operating microscope (Olympus, 10-40×). In the experimental group, the cavernous nerve was frozen bilaterally for 1 min. with a thermocouple used to control the temperature (5 mm. diameter, Omega HH21 handheld microprocessor digital thermometer). (Before surgery, the thermocouple had been placed in a 15-ml. disposable centrifuge tube filled with ground dry ice and kept in a thermo-flask [Lab-Line Instruments Inc.] filled with dry ice.) The temperature of the probe at the beginning of the procedure was -80° C., increasing to -50° C. at 1 min. To prevent disruption of the nerve, 0.2 ml. saline was used to disengage the tip of the probe from the nerve before removal.
After the freezing procedure, the right side of the proximal crus was exposed, and 0.05 ml. of either 1010 AAV-LacZ or 1010 AAV-BDNF was injected into 12 rats each through a tuberculin syringe with a 30 G needle.
Preparation of AAV-BDNF
Cloning of BDNF cDNA:
We used RT-PCR to identify BDNF expression in a human neuroblastoma cell line, SK-N-BE(2). We then used a primer pair, 5'-CCCTACAGGTCGACCAGGTGA-3' (SEQ ID NO:1) and 5'-CTATACAACATGGATCCACTA-3' (SEQ ID NO:2), to amplify the coding sequence of BDNF from SK-N-BE(2) cDNA (underlined sequences are designed XhoI and BamHI restriction sites, respectively). After digestion with XhoI and BamHI, the amplified product was cloned into pBluescript (Stratagene Inc., La Jolla, Calif.) and fully sequenced. The BDNF cDNA was then re-cloned into pcDNA4, a modified version of pcDNA3 plasmid (Invitrogen, Inc., Carlsbad, Calif.) that contains the cytomegalovirus (CMV) promoter for driving the expression of BDNF in mammalian cells.
Construction of rAAV-BDNF:
The above pcDNA4BDNF was digested with Sal1 to release the expression cassette containing cytomegalovirus immediate-early (CMVie) promoter, BDNF gene and bovine growth hormone (BGH) poly-A signal. This expression cassette was inserted into an AAV vector, pAV53, resulting in the construction of rAAV-BDNF.
Virus Production and Titration: rAAV-BDNF was produced by a three-plasmid co-transfection method. Twenty 15-cm plates of 293 cells (50 to 60% confluent) were maintained in Dulbecco modified Eagle medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (Hyclone) and 25 mM. HEPES and co-transfected by the calcium phosphate method with a total of 45 μg. DNA, 15 μg. of AAV-BDNF vector, and 15 μg. each of pLHP19 (AAV helper plasmid) and pLadeno5 (adenovirus helper plasmid), kindly provided by AVIGEN. Six hours after transfection, the medium was replaced with fresh DMEM containing 1% fetal bovine serum. The cells were harvested at 48 h posttransfection by centrifugation (1000 g for 10 min.), and the cell pellets were re-suspended in 0.1 M. Tris-HCL, 0.15 M. NaCl solution (pH 8.0) and subjected to four cycles of freeze-thaw and removal of cell debris. Large-scale rAAV CsCl purification was carried out as described previously.12 AAV-BDNF vector titer was determined by quantitative dot blot hybridization of DNase-treated stocks. The AAV vector titer used in the experiment refers to the particle number of AAV vector genomes in the sample as determined by the quantitative dot blot assay.
Functional Evaluation and Tissue Procurement
At weeks 4 and 8 postoperatively, rats in each group were re-explored for direct electrostimulation of the cavernous nerves before tissue collection. The skin overlying the penis was incised and the ischiocavernous muscle was partly removed to expose both penile crura. A 23 G butterfly needle connected to PE-50 tubing was inserted in the right crus for pressure measurement. Electrostimulation was performed with a delicate stainless-steel bipolar hook electrode attached to a multi-jointed clamp. (Each pole was 0.2 mm. in diameter; the two poles were separated by 1 mm.) Short wave pulses were generated by a Macintosh computer with a custom-built constant current amplifier. Stimulus parameters were 1.5 mA., frequency 20 Hz., pulse width 0.2 m sec., duration 50 sec. Each cavernous nerve was stimulated and intracavernous pressures were measured and recorded with a Macintosh computer programmed with LabVIEW 4.0 software (National Instruments, Austin, Tex.). The pressure for each animal was determined by the mean of both sides.
NADPH Diaphorase Staining
After sacrifice, samples of major pelvic ganglia and penile tissue were fixed for 3 hours in a cold, freshly prepared, solution of 2% formaldehyde, 0.002% picric acid in 0.1 M. phosphate buffer, pH 8.0. Tissues were cryoprotected for 24 hours in cold 30% sucrose in 0.1 M. phosphate buffer, pH 8.0. They were then embedded in O.C.T. compound (Tissue-Tek, Miles Laboratory), frozen in liquid nitrogen, and stored at -70° C. Cryostat tissue sections were cut at 10 Mm., adhered to charged slides, air-dried for 5 min., and hydrated for 5 min. with 0.1 M. PO4, pH 8.0. Sections were incubated with 0.1 mM. NADPH, 0.2 mM. nitroblue tetrazolium, 0.2% Triton X-100 in 0.1 M. PO4, pH 8.0, for 60 min. at room temperature. The reaction was terminated by washing in buffer. Slides were then coverslipped with buffered glycerin as the mounting medium.13
The presence of NADPH diaphorase-positive nerves was evidenced as a blue stain in the major pelvic ganglia, dorsal nerves and cavernous tissue, and the staining pattern was assessed by counting the number of positive neurons in 4 random fields (magnification 400×). The percentage of darkly and lightly stained cells in the major pelvic ganglia was calculated by dividing the number of these cells by the total number of positive cells.
Nitric Oxide Synthase Antibody Staining
Tissue fixation was the same as with NADPH-diaphorase specimens. After freezing, 10-μm. cryostat tissue sections were adhered to charged slides, air-dried, and hydrated for 5 min. with 0.05M. sodium phosphate buffer (PBS, pH 7.4). Sections were treated with hydrogen peroxide/methanol to quench endogenous peroxidase activity. After rinsing with water, sections were washed twice in PBS for 5 min. followed by 30 min. of room-temperature incubation with 3% horse serum/PBS/0.3% triton X-100. After draining solution from sections, tissues were incubated for 60 min. at room temperature with mouse monoclonal anti-nNOS (Transduction Laboratories, Lexington, Ky.) at a 1:500 dilution. After washing for 5 min. with PBS/TX and then for 5 min. twice with PBS alone, sections were immunostained with the avidin-biotin-peroxidase method (Elite ABC, Vector Labs, Burlingame Calif.), with diaminobenzidine as the chromagen, followed by counterstaining with hematoxylin.
Statistical Analysis: The nonparametric Mann-Whitney U and ANOVA tests with Statview 4.02 software were used to compare results. Values were considered significant at p<0.05.
In both the LacZ and BDNF groups, maximal intracavernous pressure in response to bilateral cavernous nerve electrostimulation was less than in the sham group. However, the pressure in the BDNF group was significantly higher than in the LacZ group at both 4 and 8 weeks (FIG. 1 and Table 6).
TABLE-US-00007 TABLE 6 Maximal intracavernous Pressure In Response to Electrostimulation 4 and 8 Weeks After Bilateral Cavernous Nerve Freezing Time Sham Operation LacZ BDNF (Weeks) (n = 10) (n = 12) (n = 12) 4 105 ± 10.5* 28.4 ± 5.5 585 ± 11.7† 8 115.5 ± 7.7 37.7 ± 7.9 61.3 ± 12.5† *All values are expressed as cm H2O; mean ± SD †p < 0.05 v. LacZ.
NADPH Diaphorase Staining
At week 4, the LacZ group showed significantly fewer NADPH diaphorase-positive nerve fibers than did the BDNF group. At week 8, the number had increased in both groups, but there were still significantly fewer in the LacZ group When compared with the sham group, both experimental groups showed fewer positive nerve fibers (Table 7).
TABLE-US-00008 TABLE 7 NADPH Diaphorase-positive Nerve Fibers In the Dorsal Nerve and Cavernous Tissue Time Sham Operation LacZ BDNF (Weeks) (N = 10) (N = 12) (N = 12) Dorsal Nerve 4 Weeks 138.5 ± 10.5* 45.7 ± 5.8 65.5 ± 15.5.sup.† 8 Weeks 140.2 ± 9.8 55.6 ± 8.4 86.4 ± 12.2.sup.† Cavernous Tissue 4 Weeks 103 ± 9.8 25.5 ± 3.6 45.5 ± 10.5.sup.† 8 Weeks 110.2 ± 10.5 35.6 ± 10.4 66.1 ± 15.2.sup.† *All values expressed as Mean ± S.D. .sup.†p < 0.05 vs. LacZ
Histologic evaluation showed significantly fewer NADPH diaphorase-positive nerve fibers in the trabecular smooth muscle of the LacZ group than in the BDNF group at both 4 and 8 weeks. When compared with the sham group, both experimental groups had significantly fewer (Table 7).
Major Pelvic Ganglia:
At week 4, most of the neurons in the major pelvic ganglia in the LacZ group exhibited a lighter staining pattern than that seen in the BDNF group. In addition, at 8 weeks most of these LacZ neurons had irregular cell contour and multiple vacuoles in the cytoplasm. The percentage of darkly stained cells in the BDNF group at both time points was significantly higher than in the LacZ group, and their appearance (i.e. smooth contour and very few vacuoles in the cytoplasm) was similar to that in the sham group. In both experimental groups, the percentage of darkly stained cells was significantly less than in the sham group (Table 8).
TABLE-US-00009 TABLE 8 NADPH Diaphorase-positive Neurons in the Major Pelvic Ganglion Time Group Dark Stain Light Stain Total % Dark Stain % Light Stain 4 Weeks Sham 102 ± 0.5* 21.7 ± 3.5 123.5 ± 4.5† 82.2 ± 5.3 17.3 ± 6.8 LacZ 32.4 ± 15.2 52.5 ± 20.4 85 ± 20.1 38.5 ± 17.2 60.7 ± 17.2 BDNF 61.8 ± 23.7 33.8 ± 9 95.7 ± 22.4 62.1 ± 13.l.dagger-dbl. 38.6 ± 11.8 8 Weeks Sham 101 ± 12.3 25.5 ± 5.5 136.7 ± 11.3† 80.7 ± 9.8 19.2 ± 4.5 LacZ 45 ± 12.7 53.3 ± 9.5 98.5 ± 14.3 45.8 ± 0.8 54.1 ± 0.8 BDNF 69.4 ± 21.1 38.4 ± 14 107.8 ± 23.58 64.3 ± 10.7.dagger-dbl. 35.6 ± 10.7 *All values expressed as Mean ± S.D. †p < 0.05 vs. either experimental group .dagger-dbl.p < 0.05 vs. LacZ group
Immunostaining of penile tissue and the major pelvic ganglia for nNOS revealed positive staining in the same nerve fibers and neurons as with NADPH diaphorase. The neurons of the major pelvic ganglia of the LacZ group showed lighter staining patterns and many more vacuoles in the cytoplasm than did the neurons in the BDNF and sham groups.
The aim of the present study was to investigate the feasibility of using AAV-BDNF gene transfer to facilitate recovery of potency after bilateral cavernous nerve injury. Our past studies have led us to believe that bilateral cavernous nerve freezing in the rat is a suitable model for such injury because the neural sheath is preserved, as it is in patients undergoing nerve-sparing prostatectomy or cryoablation. In addition, the course and extent of functional recovery have been well documented in this mode14 We used maximal intracavernous pressure in response to cavernous nerve electrostimulation to assess recovery of erectile function. Although apomorphine-induced erection may be more physiologic, we do not believe it can reliably differentiate partial from full erection in rats with cavernous nerve injury.
In this study, the number of nNOS-containing neurons in the major pelvic ganglia of both experimental groups was less than in the sham group. However, the percentage of darkly stained neurons was significantly greater in the BDNF group than in the LacZ group at both 4 and 8 weeks. Moreover, most neurons of the BDNF and sham groups did not show the cytoplasmic vacuoles and irregular cell contour seen in the LacZ group. These findings suggest that the production of BDNF protein in penile tissue can be retrogradely transported to the major pelvic ganglia to prevent neuronal damage and preserve nNOS enzymes in the neurons. This in turn facilitates the recovery of erectile function, as evidenced by the more numerous nNOS-positive nerve fibers in the erectile tissue and higher intracavernous pressure in the BDNF group.
A previous study has shown a significantly increased survival of motoneurons 1 week after axotomy in animals pretreated with adenovirus-encoding BDNF or glial cell line-derived neurotrophic factor (GDNF).17 However, because of the disadvantages of the adenovirus, we used adeno-associated virus (AAV), a unique member of the non-enveloped, single-stranded-DNA Parvovirus that possesses several properties that distinguish it from other gene-transfer vectors. Its advantages include stable and efficient integration of viral DNA into the host genome,18 lack of associated human disease,19 broad host range, ability to infect growth-arrested cells,20 and ability to carry non-viral regulatory sequences without interference from the viral genome.21 In addition, no superinfection inhibition is associated with AAV vectors.18 The infected cells are spread several millimeters around the needle tract. AAV is able to infect axon terminals and is retrogradely transported. Injection of AAV vector expressing LacZ into several brain regions has shown the presence of transgene expression as early as 24 hours,22 lasting (at significantly decreased levels) as long as 6 months.
The penis is a convenient organ for gene therapy because of its external location and slow circulation in the flaccid state. In addition, its sinusoidal structure and the gap junctions between smooth muscles ensure wide distribution of injected vectors. To our knowledge, this is the first demonstration of gene therapy with AAV-BDNF used to facilitate the recovery of nNOS-containing nerves and neurons and consequent erectile function. Because our previous studies have shown that 3 to 6 months may be required for more complete regeneration of cavernous nerves and erectile function in both unilateral resection and unilateral freezing models,3,16 we are presently conducting a further study to examine the effects of higher AAV-BDNF titer and longer follow-up in this model.
Our results showed that intracavernous injection of AAV-BDNF after freezing of bilateral cavernous nerves had the following effects: 1) facilitated the recovery of erectile function, 2) enhanced the regeneration of the intracavernous and dorsal nerves, and 3) prevented neuronal degeneration in the major pelvic ganglia. If further studies confirm its effectiveness and safety, intracavernous injection of neurotrophins or other growth factors has the potential to be a curative therapy for neurogenic erectile dysfunction after cryoablation or radical pelvic surgery. Bakircioglu M E, et al., J. Urol. 165:2103-9 (2001).
1. Walsh, P. C., and Mostwin, J. L. Radical prostatectomy and cystoprostatectomy with preservation of potency. Results using a new nerve-sparing technique. Br. J. Urol., 56: 694, 1984. 2. Paick, J. S., Donatucci, C. F., and Lue, T. F. Anatomy of cavernous nerves distal to prostate: microdissection study in adult male cadavers. Urology, 42: 145, 1993. 3. Carrier, S., Zvara, P., Nunes, L., Kour, N. W., Rehman, J., and Lue, T. F. Regeneration of nitric oxide synthase-containing nerves after cavernous nerve neurotomy in the rat. J. Urol. 153: 1722, 1995. 4. Jung, G. W., Spencer, E. M., and Lue, T. F. Growth hormone enhances regeneration of nitric oxide synthase-containing penile nerves after cavernous nerve neurotomy in rats. J. Urol. 160: 1899, 1998. 5. Leibrock, J., Lottspeich, F., Hohn, A., Hofer, M., Hengerer, B., Masiakowski, P., Thoenen, H., and Barde, Y. A. Molecular cloning and expression of brain-derived neurotrophic factor. Nature. 341: 149, 1989. 6. Ide, C.: Peripheral nerve regeneration. Neurosci. Res. 25: 101, 1996. 7. DiStefano, P. S., Friedman, B., Radziejewski, C., Alexander, C., Boland, P., Schick, C. M., Lindsay, R. M., and Wiegand, S. J. The neurotrophins BDNF, NT-3, and NGF display distinct patterns of retrograde axonal transport in peripheral and central neurons. Neuron. 8: 983, 1992. 8. Oppenheim, R. W., Yin, Q. W., Prevette, D., and Yan, Q. Brain-derived neurotrophic factor rescues developing avian motoneurons from cell death. Nature. 360: 755, 1992. 9. Yan, Q., Elliott, J., and Snider, W. D. Brain-derived neurotrophic factor rescues spinal motor neurons from axotomy-induced cell death. Nature. 360: 753, 1992. 10. Meyer, M., Matsuoka, I., Wetmore, C., Olson, L., and Thoenen, H. Enhanced synthesis of brain-derived neurotrophic factor in the lesioned peripheral nerve: different mechanisms are responsible for the regulation of BDNF and NGF mRNA. J. Cell Biol. 119: 45, 1992. 11. Nonomura, T., Nishio, C., Lindsay, R. M., and Hatanaka, H. Cultured basal forebrain cholinergic neurons from postnatal rats show both overlapping and non-overlapping responses to the neurotrophins. Brain Res. 683: 129, 1995. 12. Matsushita, T., Elliger, S., Elliger, C., Podsakoff, G., Villarreal, L., Kurtzman, G. J., Iwaki, Y., and Colosi, P. Adeno-associated virus vectors can be efficiently produced without helper virus. Gene Ther. 5: 938, 1998. 13. Alm, P., Larsson, B., Ekblad, E., Sundler, F., and Andersson, K. E. Immunohistochemical localization of peripheral nitric oxide synthase-containing nerves using antibodies raised against synthesized C- and N-terminal fragments of a cloned enzyme from rat brain. Acta Physiol. Scand. 148: 421, 1993. 14. Korsching, S. The neurotrophic factor concept: a reexamination. J. Neurosci. 13: 2739, 1993. 15. Lewin, G. R. and Barde, Y. A. Physiology of the neurotrophins. Annu. Rev. Neurosci. 19: 289, 1996. 16. E1-Sakka, A. I., Hassan, M. U., Selph, C., Perinchery, G., Dahiya, R., and Lue, T. F. Effect of cavernous nerve freezing on protein and gene expression of nitric oxide synthase in the rat penis and pelvic ganglia. J. Urol. 160: 2245, 1998. 17. Gimenez y Ribotta, M., Revah, F., Pradier, L., Loquet, I., Mallet, J., and Privat, A. Prevention of motoneuron death by adenovirus-mediated neurotrophic factors. J. Neurosci. Res. 48: 281, 1997. 18. McLaughlin, S. K., Collis, P., Hermonat, P. L., and Muzyczka, N. Adeno-associated virus general transduction vectors: analysis of proviral structures. J. Virol. 62: 1963, 1988. 19. Berns, K. I., Cheung, A., Ostrove, J., and Lewis, M.: Adeno-associated virus latent infection. In: B. W. J. Mahy, A. C. Minson, and G. K. Darby (eds.), Virus Persistence. Cambridge, UK: Cambridge University Press, 1982. 20. Podsakoff, G., Wong, K. K., Jr., and Chatterjee, S. Efficient gene transfer into nondividing cells by adeno-associated virus-based vectors. J. Virol. 68: 5656, 1994. 21. Miller, J. L., Walsh, C. E., Ney, P. A., Samulski, R. J., and Nienhuis, A. W. Single-copy transduction and expression of human gamma-globin in K562 erythroleukemia cells using recombinant adeno-associated virus vectors: the effect of mutations in NF-E2 and GATA-1 binding motifs within the hypersensitivity site 2 enhancer [published erratum appears in Blood 1995 Feb. 1; 85(3):862]. Blood. 82: 1900, 1993. 22. During, M. J. and Leone, P. Adeno-associated virus vectors for gene therapy of neurodegenerative disorders. Clin. Neurosci. 3: 292, 1995.
Vascular Endothelial Growth Factor Promotes Proliferation and Migration of Cavernous Smooth Muscle Cells
Male Wistar rats were obtained from Charles River Laboratories (Wilmington, Mass.). Young rats were 1, 2, 3, 4, 6, 11, and 16 weeks of age. Old rats were 28 months of age.
All chemicals were from Sigma-Aldrich Co. (St. Louis, Mo.) unless noted otherwise. Recombinant human VEGF165 was from Calbiochem Biosciences Inc. (La Jolla, Calif.). Fetal bovine serum (FBS) and Trypsin-EDTA were from Life Technologies, Inc. (Grand Island, N.Y.). All other cell culture regents were obtained from Cell Culture Facility, University of California, San Francisco.
Each primary culture of CSMC was prepared from the corpora cavernosa of 2-3 rats by the following procedure. The penis was cleared of the urethra, blood vessels, fat and connective tissue. The remaining smooth muscle tissue was washed 3 times in sterile PBS (phosphate-buffered saline) and cut into 2-3 mm3 segments. The segments were placed evenly onto a 100-mm cell culture dish (Falcon-Becton Dickinson Labware, Franklin Lakes, N.J.) inside a cell culture hood. Approximately 10 min later, 10 ml of Dulbecco's Modified Eagle Medium (DMEM) containing penicillin (100 units/ml), streptomycin (100 μg/ml), and 10% FBS was carefully pipetted into the dish. The dish was then kept undisturbed in a humidified 37° C. incubator with 5% CO2. Five days later, tissue segments that have detached from the dish were removed, and the medium was replaced with fresh medium. Another 5 days later, all tissue segments were removed and the medium was again replaced with fresh medium. When small islands of cells were noticeable, they were trypsinized and transferred to a fresh culture dish. Expansion of each cell strain was continued with change of medium every 3 days and passages (trypsinization and seeding) approximately every 10 days. All cells used in the following experiments were from passages 4 through 10. Primary aorta SMC cultures were prepared similarly with aortas isolated from 16-week-old male rats. All cell cultures were confirmed for their smooth muscle identity by an indirect immunofluorescence staining with an anti-smooth muscle myosin heavy chain antibody (Sigma-Aldrich Co. St. Louis, Mo.).
Quantification of VEGF
Each CSMC from rats of different ages was seeded at 4×105 cells per well in 3 ml of DMEM with 10% FBS in 6-well culture plates. Seventy-two hr later, the medium was removed for quantification of VEGF and the cells were trypsinized for the determination of cell number. For quantifying VEGF in the medium, the Mouse VEGF Immunoassay Kit (R&D Systems, Minneapolis, Minn.), which reacts with rat VEGF but not with bovine VEGF, was used. All assays were performed in duplicate in each experiment and all data presented in the Results section are the average of three independent experiments.
Cell proliferation assays were performed with the CellTiter-96 kit from Promega Inc. (Madison, Wis.). Each SMC strain from different-aged rats was assayed in one flat-bottom 96-well cell culture plate. The plate was divided into 12 rows that contained VEGF at concentrations from 0 to 100 ng per ml of serum-free DMEM (supplemented with 0.1% BSA). Each well in the same row received 50 μl of the medium containing the same concentration of VEGF. Thereafter, SMC that were grown to 70% confluence were rinsed twice with PBS, trypsinized, and resuspended in serum-free DMEM (supplemented with 0.1% BSA) at 100,000 cell per ml. Aliquots of 50 μl of the cell suspension were then transferred to the 96-well plate so that each well contained 5,000 cells in a final volume of 100 μl. The plate was incubated in a 37° C., humidified incubator with 5% CO2. Three days later, 201 of CellTiter 96® AQueous One Solution Reagent was added to each well. After 4 hr of further incubation at 37° C. in the humidified, 5% CO2 incubator, color development, which reflects cell numbers, was recorded with a plate reader (Molecular Devices Corp., Sunnyvale, Calif.) at 490-nm absorbance. For proliferation assays concerning concentrations of FBS in the growth medium, the assay procedure was the same except that different amounts of FBS, instead of VEGF, were added to the medium. All assays were performed in duplicate in each experiment and all data presented in the Results section are the average of three independent experiments.
Cell migration assays were performed in 6.5-mm Transwell chambers of Corning Costar Corporation (Cambridge, Mass.). The Transwell inserts (upper chambers) were bathed in a solution containing 13.4 μg/ml fibronectin in PBS at 37° C. for 1 hr and allowed to air-dry. The dried upper chambers were then placed in the lower chambers, each of which contained 7001 of serum-free DMEM supplemented with 0.1% BSA and 0 to 500 ng/ml of VEGF. SMC that had been grown to 70% confluence were further conditioned in serum-free DMEM (containing 0.1% BSA) for 2 hr and then trypsinized. The trypsinized cells were washed in PBS and resuspended at a concentration of 80,000 cells per ml in serum-free DMEM (containing 0.1% BSA). One hundred 1 of the cell suspension was then added to each upper chamber. After 4 hr of incubation at 37° C., all liquid in the upper and lower chambers was removed by aspiration. The membranes in the upper chambers were subsequently fixed in 1% buffered formalin for 5 min and stained with 2% crystal violet. Non-migratory cells on the upper side of the membranes were scraped off with cotton swabs. Well locations were marked on the membranes and total cells per well were counted visually in a masked fashion. Well locations were then correlated with the concentrations of VEGF in the wells. All assays were performed in duplicate in each experiment and all data presented in the Results section are the average of three independent experiments.
Cultured cells and rat tissues were homogenized in Tri-Reagent RNA extraction solution (Molecular Research Center, Cincinnati, Ohio). Following the recommended procedure by the supplier, RNAs were further treated with DNase I to remove traces of contaminating DNA. Quantity and integrity of RNAs were examined by spectrophotometry and agarose gel electrophoresis, respectively. Human heart RNAs were purchased from Clontech Laboratories, Inc. (Palo Alto, Calif.).
RT-PCR (reverse transcription-polymerase chain reaction) was performed in an RT step and a PCR step. In the RT step, the cellular mRNAs were reverse-transcribed into a "library" of complementary DNAs (cDNAs). This cDNA library was then used for the analysis of various genes in the PCR step. The RT procedure was performed with the SuperScript reverse transcriptase (Life Technologies, Inc., Gaithersburg, Md.) and its accompanying reagents. Briefly, 2.5 μg of each tissue RNA was annealed to 0.4 μg of oligo-dT primer in a 12 μl volume. Four μl of 5× buffer, 2 μl of 0.1 M DTT, 1 μl of 10 mM dNTP, and 1 μl of SuperScript reverse transcriptase were then added to bring the final reaction volume to 20 μl. After one hour of incubation at 42° C., the RT mixture was incubated at 70° C. for 10 min to inactivate the reverse transcriptase. Eighty μl of TE buffer was then added to make a 5× diluted library. A portion of this library was further diluted to various concentrations (up to 100× dilution). One μl of each dilution was then used in a 10 μl PCR to identify the optimal input within the linear amplification range. In addition to the 1 μl diluted library, the PCR mixture consisted of 10 ng of each of a primer pair and reagents supplied with the Taq polymerase (Life Technologies, Inc., Gaithersburg, Md.). PCR was performed in the DNA Engine thermocycler (MJ Research, Inc., Watertown, Mass.) under calculated temperature control. The cycling program was set for 35 cycles of 94° C., 5 sec; 55° C., 5 sec; 72° C., 10 sec, followed by one cycle of 72° C., 5 min. The PCR products were electrophoresed in 1.5% agarose gels in the presence of ethidium bromide, visualized by UV fluorescence, and recorded by a digital camera connected to a computer.
TABLE-US-00010 TABLE 9 Oligonucleotide primers Size of Primer PCR Gene name Sequence product β-Actin Actin-s 5'-TCTACAATGAGCTGCGTGTG-3' 368 bp (SEQ ID NO: 3) Actin-a 3'-AATGTCACGCACGATTTCCC-5' (SEQ ID NO: 4) VEGFR- 1 VEGFR-1s 5'-ATGCTGGATTGCTGGCACA-3' 323 bp (SEQ ID NO: 5) VEGFR-1a 3'-TCAAACATGGAGGTGGCATT-5' (SEQ ID NO: 6) VEGFR-2 VEGFR-2s 5'-GCCTTTGGCCAAGTGATTGA-3' 479 bp (SEQ ID NO: 7) VEGFR-2a 3'-TCCAAGGTCAGGAAGTCCTT-5' (SEQ ID NO: 8)
Primer pairs for RT-PCR analysis of VEGFR-1, VEGFR-2 and β-actin genes are listed in Table 9. They were designed to recognize the respective mRNAs in both humans and rats.
Western Blot Analysis
Cultured cells were lysed in a buffer containing 1% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 1×PBS. Cell lysates containing indicated amounts of protein were electrophoresed in 7.5% SDS-PAGE and then transferred to PVDF membrane. The membrane was stained with Ponceau S to verify the integrity of the transferred proteins and to monitor the unbiased transfer of all protein samples. Detection of VEGFR-1 protein on the membrane was performed with the ECL kit (Amersham Life Sciences Inc., Arlington Heights, Ill.) using an anti-VEGFR-1 rabbit serum from Santa Cruz Biotech, Inc. (Santa Cruz, Calif.).
Growth Rates of CSMC from Different-Aged Rats
Like most other cultured cells, our rat CSMC were maintained in a medium supplemented with 10% FBS. However, because certain experiments may require the use of media containing lower concentrations of FBS, we wished to know the differences between different age groups in their growth rates under different concentrations of FBS. All cells, regardless of the ages of rats from which they were derived, grew at increasing rates with increasing FBS concentrations. At lower concentrations of FBS (0 and 2.5%), the growth rates showed little differences between different age groups. However, at higher concentrations of FBS (5 and 10%), differences in growth rates became more pronounced. In particular, cells from 4-week-old rats seemed to respond best to higher concentrations of FBS and cells from 28-month-old rats least well.
VEGF Secretion by CSMC from Different-Aged Rats
VSMC have been shown to be the principal source of secreted VEGF in the vascular (aorta) system. Pueyo, et al., Exp. Cell Res., 238: 354 (1998). We therefore examined our rat CSMC for their ability to produce VEGF. We chose 72 hr after seeding the cells as the point of time to assay for the secreted VEGF. Because CSMC from different age groups grew at different rates, we also determined the cell numbers for each of the tested cells. When the concentrations of the secreted VEGF were adjusted for the numbers of cells at the time of assay (FIG. 2A), it is apparent that, within the young rat groups (ages 1 to 16 weeks), CSMC from more matured rats secreted more VEGF than CSMC from less matured rats (FIG. 2C). However, CSMC from old rats (28 months old) produced similar amounts of VEGF as those from the very young rats (1 and 2 weeks old).
Effects of VEGF on Cell Growth
It has been reported that VEGF did not stimulate growth of VSMC. Grosskreutz, et al., Microvasc. Res., 58: 128 (1999). This observation was confirmed with VSMC from 16-weeks-old rats (FIG. 3A). However, CSMC from all ages of rats responded to VEGF in the form of cell proliferation. Their growth rates increased with increasing concentrations of VEGF up to 12.5 ng/ml, and after which, the cell growth rate started to decline with increasing concentrations of VEGF (FIG. 3B-I). When compared at the optimal dosage (12.5 ng/ml) of VEGF, all cells from young rats (1 to 16 weeks old) outgrew cells from the old rats (28 months) and the peak growth occurred with cells from 11-weeks-old rats (FIG. 3J).
Effects of VEGF on Cell Motility
It has been reported that VEGF stimulated migration of VSMC. Grosskreutz, et al., Microvasc. Res., 58: 128 (1999). This observation was confirmed with VSMC from 16-weeks-old rats (FIG. 4A). Similarly, VEGF stimulated migration of CSMC from both young and old rats in a dose-dependent manner up to the 10 ng/ml point (except the 1-week-old, which peaked at 1 ng/ml). At higher concentrations (100 and 500 ng/ml) of VEGF, the mobility of all tested cells started to decline (FIG. 4B-I). When compared at the optimal dosage (10 ng/ml) of VEGF, all cells from young rats (1 to 16 weeks old) out-migrated cells from the old rats (28 months) and the peak migration rate occurred with cells from 4-weeks-old rats (FIG. 4J).
Identification of VEGFR-1 and VEGFR-2 mRNA Expression
The above cell proliferation and migration assay results suggested the presence of functional VEGF receptors in CSMC. To verify this, we use RT-PCR to examine the CSMC for the expression of VEGFR-1 and VEGFR-2 mRNAs. As shown in FIG. 5A, CSMC of different-aged rats expressed VEGFR-1 mRNA at different levels, being very low for the young ones (1 to 3 weeks of age) and the very old (28 months of age) and high for the adolescent (4 and 6 weeks of age) and the matured (11 and 16 weeks of age). This pattern of VEGFR-1 mRNA expression is similar to that of VEGF-induced cell proliferation (FIG. 3).
On the other hand, CSMC, regardless of the ages of rats from which they were derived, did not express VEGFR-2 (Lanes 1 to 8, FIG. 5B). This negative result could not have been due to improper RT-PCR conditions or improper primer design because rat heart, aorta, and penis all produced positive results under the same experimental conditions (lanes 10, 11, and 12, FIG. 5B). The positive VEGFR-2 expression in heart, aorta, and penis was most likely derived from vascular endothelial cells that were included in the preparation of these tissue RNAs. It should be pointed out that our rat VSMC were also negative for VEGFR-2 expression (Lane 9, FIG. 5B). This is in agreement with a previous study which reported that SMC of rat carotid arteries express VEGFR-1 but not VEGFR-2. Couper, et al., Circ. Res., 81: 932 (1997).
Identification of VEGFR-1 Protein Expression
To ascertain that VEGFR-1 protein was indeed expressed in CSMC, we performed immunoblotting experiments using a VEGFR-1-specific antibody. As shown in FIG. 6, VEGFR-1 was detected in CSMC from rats of all ages, and the levels of its expression was very similar to those seen in the results of RT-PCR experiments (FIG. 5A). Therefore, both the expression of VEGFR-1 mRNA and VEGFR-1 protein correlated well with the VEGF-regulated growth rate of CSMC.
The erectile function of the penis is that of a vascular organ. Like those of other vascular organs, the development and growth of the penile vasculature are expected to be governed by angiogenic growth factors such as VEGF. Surprisingly, reports concerning VEGF expression in the penis have been scant. In two separate studies, Burchardt et al. reported the identification of novel VEGF splice variants in the penis. Burchardt et al., Biol. Reprod., 60: 398 (1999), Burchardt, et al., IUBMB Life, 48: 405 (1999). In another study concerning the expression of various growth factors in the penis, Jung et al. reported the identification of VEGF189 mRNA in the penis. Jung, et al., Int. J. Impotence Res., 11: 247 (1999). Our ischemia rat model clearly demonstrated the beneficial effects of VEGF on the restoration of the erectile function following surgical procedures that restricted blood supply to the penis.
We have conducted several experiments (RT-PCR, western blots, immunohistochemical stainings) on the expression of various VEGF forms and their receptors in the penis (unpublished). In trying to interpret the results of those experiments, we were confronted with the question which cell types (smooth muscle, endothelium, nerve, etc.) were the source of a positive gene expression. By using cultures of a single cell type, we were able to show in the present study that CSMC expressed VEGF and VEGFR-1 but not VEGFR-2. We also showed that both the secretion of VEGF and the expression of VEGFR-1 increased with the age of young rats (from 1 to 16 weeks of age) but declined in very old rats (28 months of age).
We are aware of only three previously published reports that studied the effects of VEGF on SMC or expression of VEGF receptors in SMC. First, Brown et al. showed that cultured human uterine SMC expressed both VEGFR-1 and VEGFR-2 and responded to VEGF stimulation in the form of cell proliferation. Brown, et al., Lab. Invest., 76: 245 (1997). These authors also showed that human colon SMC did not express VEGF receptors and did not respond to VEGF stimulation. Secondly, Couper et al. observed high levels of VEGFR-1, but no VEGFR-2, expression in SMC of rat carotid arteries following balloon injury. Couper, et al., Circ. Res., 81: 932 (1997). Thirdly, Grosskreutz et al. showed that cultured bovine aorta SMC expressed both VEGFR-1 and VEGFR-2 and responded to VEGF stimulation in the form of cell migration (but not cell proliferation). Grosskreutz, et al., Microvasc. Res., 58: 128 (1999). In the present study, we used rat aorta SMC for comparison with rat CSMC. We found that, like bovine aorta SMC, rat aorta SMC responded to VEGF stimulation in the form of cell migration but not cell proliferation. However, we could only identify VEGFR-1 but not VEGFR-2 expression in rat aorta SMC. Whether the discrepancy regarding VEGFR-2 expression is due to species difference (bovine verses rat) or other factors (age) needs to be clarified in future studies.
The significance of the proliferative and migratory responses of CSMC toward VEGF is not known. Brown et al. speculated that alterations in expression of VEGF or VEGF receptors may play a role in the pathogenesis of smooth muscle tumors in the uterus. Brown, et al., Lab. Invest., 76: 245 (1997). Grosskreutz et al. proposed that VEGF might have a chemoattractant role in the recruitment of smooth muscle cells during the formation of a blood vessel wall. Grosskreutz, et al., Microvasc. Res., 58: 128 (1999). Because the proliferative effect of VEGF occurred mainly with CSMC of young adult rats (11 weeks of age), it is possible that VEGF at least play a role in maintaining a healthy population of CSMC. As for the migratory effect of VEGF, which occurred mainly with even younger rats (4 weeks of age), we propose that VEGF might play a role in recruiting and/or locating CSMC to the proper sites in the cavernous spaces during adolescence.
In conclusion, we believe our present study has made the following novel observations: (1) CSMC secreted VEGF, (2) CSMC expressed a VEGF receptor, (3) CSMC exhibited migratory and proliferative responses to VEGF, and (4) CSMC from different-aged rats expressed different levels of VEGF and VEGFR-1 and responded to VEGF at different rates. Liu, X., et al., J. Urol. 166:354-360 (2001).
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The Effect of a Vascular Endothelial Growth Factor (VEGF) and Adeno-Associated Brain Derived Neurotrophic Factor (AAV-BDNF) for the Treatment of Erectile Dysfunction Induced by Hypercholesterolemia in a Rat Model
Twenty-one Sprague-Dawley rats were used. All rats were fed a 2.5% cholesterol diet with added lard starting at 2 weeks of age for 6 months. The rats were divided into three groups. All groups after two months of a high cholesterol diet underwent serum evaluation of cholesterol and intracavernous injection of saline or treatment. Group 1, the control group, received intracavernous injection of saline, group 2, the VEGF group, received intracavernous injection of 4 ug of VEGF, and group 3, the AAV-BDNF group received 15 ug of BDNF. After six months of cholesterol diet and 4 months of treatment, all rats were subjected to cavernous nerve electrostimulation, cavernosometry with intracavernous papaverine injection, and infusion cavernosometry with heparinized saline to measure erectile function.
Serum cholesterol levels were significantly higher in animals fed the high cholesterol diet. Systemic arterial pressure was not significantly different among the different groups. During electrostimulation of the cavernous nerve, peak sustained intracavernous pressure was significantly lower in the cholesterol only group (50+/-23 cm. H2O) compared to the control and the VEFG and AAV-BDNF groups. During the pharmacologic erection phase of the cavernosometry, the VEGF and BDNF treated groups had significantly higher sustained intracavernous pressures in comparison to the high cholesterol controls. No difference was noted with respect to the infusion cavernosometry assessing venous leak when comparing the controls and the treated groups.
Rats developed erectile dysfunction after being fed a high cholesterol diet (2.5% with lard) for 6 months. VEGF and AAV-BDNF seem to reverse the erectile dysfunction caused by high cholesterol diet.
The Effect of Intracavernous Vascular Endothelial Growth Factor (VEGF) on a Rodent Model of Neurogenic Erectile Dysfunction
To test the hypothesis that intracavernous injection of VEGF can facilitate regeneration of the cavernous nerve and restore erectile function after cavernous nerve injury in rats
Materials and Methods
Seventeen 3 months old Sprague Dowry rats underwent bilateral freezing of the cavernous nerves with a thermocouple immersed in liquid nitrogen. Through an abdominal incision both cavernous nerves were isolated lateral to the prostate and frozen with thermocouple for one minute twice (temperature cycle -130° C. to -3° C.). Minutes later, intracavernous injection of saline (n=7) or VEGF, 4 ug, (n=9) was given. Three months later, all rats underwent re-exploration and electrostimulation of the cavernous nerves to assess erectile function. Eight additional rats underwent exploration only (sham group)
The maximal intracavernous pressure in the rats underwent sham operation was 90±8.15 cm H2O. The maximal intracavernous pressure of both the saline treated group (39.29±5.02 cm H2O) and the VEGF treated group (72.78±10.87 cm H2O) were lower than the sham group. Nevertheless, the intracavernous pressure in the VEGF treated group was significantly higher than the saline treated group (p=0.0389).
Although VEGF is known as an angiogenetic factor, previous reports have suggested that it may also be neuroprotective and neurotrophic. Our study shows that intracavernous injection of VEGF significantly facilitated recovery of erectile function after bilateral cavernous nerve injury. If further studies confirmed that VEGF enhances both angiogenesis and neural regeneration, intracavernous VEGF therapy may be the treatment of choice in helping patient recover potency after radical prostatectomy or cryoablation of the prostate.
Synergistic Neurotrophic Effects of Vascular Endothelial Growth Factor and Brain-Derived Neurotrophic Factor
Neurogenic impotence due to cavernous nerve injury occurs frequently in patients having undergone radical surgeries for prostate, bladder and rectal cancer. To investigate the feasibility of an in vitro assay system for nerve regeneration, we devised a model in which the major pelvic ganglia (MPG), from which the cavernous nerves are originated, were isolated and grown in culture.
Materials and Methods
Each freshly isolated MPG was cut into 8 pieces of approximately equal size and each piece was then attached to a Matrigel-coated coverslip. The MPG pieces were cultured in serum-free medium supplemented with phosphate-buffered saline (PBS, control), 50 ng/ml of vascular endothelial growth factor (VEGF), 20 ng/ml of brain-derived neurotrophic factor (BDNF), or VEGF+BDNF. After 2 days of incubation, the ganglial tissues with their outgrowing nerve fibers were stained for the expression of NADPH diaphorase and acetylcholinesterase (AchE).
The average lengths of the outgrowing nerve fibers were determined to be 16±7 nm, 89±21 nm, 75±11 nm and 124±23 nm, for tissues treated with PBS, VEGF, BDNF, and VEGF+BDNF, respectively (Table 10). The levels of NADPH diaphorase expression, calculated as integrated density value (IDV), were 2,010±82, 10,126±187, 9,376±111 and 13,616±210 for tissues treated with PBS, VEGF, BDNF and VEGF+BDNF, respectively. The levels of AchE expression were 29±2, 8,470±199, 1,102±211 and 11,006±198 for tissues treated with PBS, VEGF, BDNF and VEGF+BDNF, respectively.
TABLE-US-00011 TABLE 10 Growth Average fiber NADPH staining AchE staining factor length, in nm intensity* intensity* VEGF 89 ± 21 10,126 ± 187 8,470 ± 199 BDNF 75 ± 11 9,376 ± 111 1,102 ± 211 VEGF + 124 ± 23 13,616 ± 210 11,006 ± 198 BDNF Control 16 ± 7 2,010 ± 82 29 ± 2 (PBS) *Expressed in Integrated Density Value (IDV).
These results indicate that both VEGF and BDNF had neurotrophic effects and were synergistic in combination. In addition, it appears that BDNF preferentially promoted the growth of NOS-containing nerves (positive for NADPH diaphorase), as opposed to cholinergic nerves (AchE-positive).
Synergistic Neurotrophic Effects of Vascular Endothelial Growth Factor and Neurotrophins
We have been using several experimental systems to test the efficacy of combination growth factor therapies for impotence. One system employs cultured rat major pelvic ganglia (MPG) that are known to innervate all urogenital organs, including the penis. We discovered that the cultured MPG are able to sprout new nerve fibers (nerve regeneration) from the axonal ends during the first 3 days of culture, especially when treated with VEGF, BDNF, NT-3, or NT-4. The average lengths (in nM) of the growing fibers 48 hours after culturing with various treatments are listed in Table 11.
TABLE-US-00012 TABLE 11 GROWTH FACTORS FIBER LENGTH VEGF 89 BDNF 75 NT-3 112 NT-4 81 VEGF + BDNF 124 VEGF + NT-3 132 VEGF + NT-4 101 BDNF + NT-3 88 BDNF + NT-4 81 Control (treated with saline) 16
Compared with an average length of 16 nm in control MPG, it is clear that VEGF, BDNF, NT-3, and NT-4 all have neurotrophic effects. More importantly, when VEGF is combined with each of the three neurotrophins, there was a synergistic effect.
VEGF-Enhanced Neurotrophin Therapy (VENT)
This example shows the testing of the hypothesis that intracavernous injection of vascular endothelial growth factor (VEGF) enhances the effect of brain derived neurotrophic factor (BDNF) and thus facilitate neural and erectile function recovery after cavernous nerve injury.
Materials and Methods
Three months old Sprague-Dawley rats (N=30) were used: 6 underwent a sham operation; 24 underwent bilateral cavernous nerve freezing and then randomly divided into four groups of 6 each. The four groups of rats received intracavernous injection of one of the following: 1) phosphate buffered saline (PBS), 2) VEGF protein (4 μg), 3) adeno-associated virus (AAV)-BDNF, and 4). VEGF+AAV-BDNF. Erectile function was assessed by cavernous nerve electrostimulation 3 months later and samples of penile tissue were obtained for electron microscopy and immunohistochemical studies.
The maximal intracavernous pressure (MICP) of the rats (mean±SEM cm H2O) in the sham, PBS, VEGF, BDNF and VEGF+BDNF groups were 100.67±6.55, 29.25±3.90, 37.33±3.29, 69.75±8.54, 87.17±6.00 respectively. The differences were statistically significant between the sham and all other groups but not between sham and the VEGF+BDNF group. The blood vessel+capillary count was decreased in the PBS group (3±1.6 vs. 6±1.3 in sham group) but was significant higher in the VDGF+BDNF group (9.8±2.3, p=0.01 vs. PBS group). Image analysis of nNOS staining revealed significantly less nNOS positive nerve fibers in the dorsal nerves in the PBS groups as compared to the sham group. There was an increased number of positive nerve fibers in VEGF, BDNF and VEGF+BDNF groups as compared to PBS group. Electron microscopy revealed that the VEGF+BDNF group had the best overall morphology of both myelinated and nonmyelinated nerve fibers in the penis.
Intracavernous injection of VEGF and AAV-BDNF improved the regeneration of nerve fibers in the penis and the recovery of erectile function. The best results were obtained with intracavernous injection of both VEGF and AAV-BDNF. The increased endothelial permeability of VEGF may facilitate the transport of itself and AAV-BDNF to the erectile tissue and thus enhance the neurotrophic and angiogenic effect.
A New Model for Angiogenesis Studies
Penile dorsal arteries were isolated from rats and cut into 1-mm segments under a 10× microscope. Each segment was attached to a Matrigel-coated coverslip, which was then put in a 60-mm culture dish. Serum-free medium with or without VEGF was added and the arterial culture was incubated for 3 days. The results demonstrate the budding of new blood vessels that were significantly longer in VEGF-treated than in untreated culture. The budding vessels were stained positive with RECA antibody, indicating the endothelial identity. To our best knowledge, this is the first demonstration of angiogenesis from blood vessel explants. The ease of this experimental procedure in comparison with existing methods should greatly facilitate future angiogenesis studies.
Artery-Muscle Co-Culture Systems
We extended our above angiogenesis model to include muscles in a co-culture system. The source of muscle was corpus cavernosum, urethra sphincter, or bladder. In each case, the outgrowing blood vessels showed attraction to and seemingly entering the muscle. It also appears that VEGF encourage the growth of endothelial cells and VEGF+PDGF stimulated the growth of both endothelial and smooth muscle cells. In addition, the combination of VEGF and PDGF encouraged more extensive contacts between the outgrowing vessels with the muscle. More studies with either single or a combination of other growth factors and the molecular mechanism of vessel growth are currently underway. Once the best growth factor or combinations are identified, we will perform in vivo studies in rats with severe vasculogenic ED to see if the in vitro assay helps identify the best angiogenins.
The examples set forth above are provided to give those of ordinary skill in the art with a complete disclosure and description of how to make and use the preferred embodiments of the compositions, and are not intended to limit the scope of what the inventors regard as their invention. Modifications of the above-described modes for carrying out the invention that are obvious to persons of skill in the art are intended to be within the scope of the following claims. All publications, patents, and patent applications cited in this specification are incorporated herein by reference as if each such publication, patent or patent application were specifically and individually indicated to be incorporated herein by reference.
16121DNAArtificial SequencePrimer 1ccctacaggt cgaccaggtg a 21221DNAArtificial SequencePrimer 2ctatacaaca tggatccact a 21320DNAArtificial SequencePrimer Actin-s 3tctacaatga gctgcgtgtg 20420DNAArtificial SequencePrimer Actin-a 4ccctttagca cgcactgtaa 20519DNAArtificial SequencePrimer VEGFR-1s 5atgctggatt gctggcaca 19620DNAArtificial SequencePrimer VEGFR-1a 6ttacggtgga ggtacaaact 20720DNAArtificial SequencePrimer VEGFR-2s 7gcctttggcc aagtgattga 20820DNAArtificial SequencePrimer VEGFR-2a 8ttcctgaagg actggaacct 209990DNAArtificial SequenceSynthetic construct 9cagtgtgctg gcggcccggc gcgagccggc ccggccccgg tcgggcctcc gaaaccatga 60actttctgct gtcttgggtg cattggagcc tcgccttgct gctctacctc caccatgcca 120agtggtccca ggctgcaccc atggcagaag gaggagggca gaatcatcac gaagtggtga 180agttcatgga tgtctatcag cgcagctact gccatccaat cgagaccctg gtggacatct 240tccaggagta ccctgatgag atcgagtaca tcttcaagcc atcctgtgtg cccctgatgc 300gatgcggggg ctgctgcaat gacgagggcc tggagtgtgt gcccactgag gagtccaaca 360tcaccatgca gattatgcgg atcaaacctc accaaggcca gcacatagga gagatgagct 420tcctacagca caacaaatgt gaatgcagac caaagaaaga tagagcaaga caagaaaatc 480cctgtgggcc ttgctcagag cggagaaagc atttgtttgt acaagatccg cagacgtgta 540aatgttcctg caaaaacaca gactcgcgtt gcaaggcgag gcagcttgag ttaaacgaac 600gtacttgcag atgtgacaag ccgaggcggt gagccgggca ggaggaagga gcctccctca 660gggtttcggg aaccagatct ctcaccagga aagactgata cagaacgatc gatacagaaa 720ccacgctgcc gccaccacac catcaccatc gacagaacag tccttaatcc agaaacctga 780aatgaaggaa gaggagactc tgcgcagagc actttgggtc cggagggcga gactccggcg 840gaagcattcc cgggcgggtg acccagcacg gtccctcttg gaattggatt cgccatttta 900tttttcttgc tgctaaatca ccgagcccgg aagattagag agttttattt ctgggattcc 960tgtagacaca ccgcggccgc cagcacactg 990101574DNAArtificial SequenceSynthetic construct 10gttccccaac tgctgtttta ttgtgctatt catgcctaga catcacatag ctagaaaggc 60ccatcagacc cctcaggcca ctgctgttcc tgtcacacat tcctgcaaag gaccatgttg 120ctaacttgaa aaaaattact attaattaca cttgcagttg ttgcttagta acatttatga 180ttttgtgttt ctcgtgacag catgagcaga gatcattaaa aattaaactt acaaagctgc 240taaagtggga agaaggagaa cttgaagcca caatttttgc acttgcttag aagccatcta 300atctcaggtt atatgctaga tcttgggggc aaacactgca tgtctctggt ttatattaaa 360ccacatacag cacactactg acactgattt gtgtctggtg cagctggagt ttatcaccaa 420gacataaaaa aaccttgacc ctgcagaatg gcctggaatt acaatcagat gggccacatg 480gcatcccggt gaaagaaagc cctaaccagt tttctgtctt gtttctgctt tctccctaca 540gttccaccag gtgagaagag tgatgaccat ccttttcctt actatggtta tttcatactt 600tggttgcatg aaggctgccc ccatgaaaga agcaaacatc cgaggacaag gtggcttggc 660ctacccaggt gtgcggaccc atgggactct ggagagcgtg aatgggccca aggcaggttc 720aagaggcttg acatcattgg ctgacacttt cgaacacgtg atagaagagc tgttggatga 780ggaccagaaa gttcggccca atgaagaaaa caataaggac gcagacttgt acacgtccag 840ggtgatgctc agtagtcaag tgcctttgga gcctcctctt ctctttctgc tggaggaata 900caaaaattac ctagatgctg caaacatgtc catgagggtc cggcgccact ctgaccctgc 960ccgccgaggg gagctgagcg tgtgtgacag tattagtgag tgggtaacgg cggcagacaa 1020aaagactgca gtggacatgt cgggcgggac ggtcacagtc cttgaaaagg tccctgtatc 1080aaaaggccaa ctgaagcaat acttctacga gaccaagtgc aatcccatgg gttacacaaa 1140agaaggctgc aggggcatag acaaaaggca ttggaactcc cagtgccgaa ctacccagtc 1200gtacgtgcgg gcccttacca tggatagcaa aaagagaatt ggctggcgat tcataaggat 1260agacacttct tgtgtatgta cattgaccat taaaagggga agatagtgga tttatgttgt 1320atagattaga ttatattgag acaaaaatta tctatttgta tatatacata acagggtaaa 1380ttattcagtt aagaaaaaaa taattttatg aactgcatgt ataaatgaag tttatacagt 1440acagtggttc tacaatctat ttattggaca tgtccatgac cagaagggaa acagtcattt 1500gcgcacaact taaaaagtct gcattacatt ccttgataat gttgtggttt gttgccgttg 1560ccaagaactg aaaa 157411444DNAArtificial SequenceSynthetic construct 11atgccagcat tgcccgagga tggcggcagc ggcgccttcc cgcccggcca cttcaaggac 60cccaagcggc tgtactgcaa aaacgggggc ttcttcctgc gcatccaccc cgacggccga 120gttgacgggg tccgggagaa gagcgaccct cacatcaagc tacaacttca agcagaagag 180agaggagttg tgtctatcaa aggagtgtgt gctaaccgtt acctggctat gaaggaagat 240ggaagattac tggcttctaa atgtgttacg gatgagtgtt tcttttttga acgattggaa 300tctaataact acaatactta ccggtcaagg aaatacacca gttggtatgt ggcactgaaa 360cgaactgggc agtataaact tggatccaaa acaggacctg ggcagaaagc tatacttttt 420cttccaatgt ctgctaagag ctga 444121020DNAArtificial SequenceSynthetic construct 12taacacagac tcagctgcca gagcctgctc ttaacacctg tgtttccttt tcagatctta 60caggtgaaca aggtgatgtc catcttgttt tatgtgatat ttctcgctta tctccgtggc 120atccaaggta acaacatgga tcaaaggagt ttgccagaag actcgctcaa ttccctcatt 180attaagctga tccaggcaga tattttgaaa aacaagctct ccaagcagat ggtggacgtt 240aaggaaaatt accagagcac cctgcccaaa gctgaggctc cccgagagcc ggagcgggga 300gggcccgcca agtcagcatt ccagccggtg attgcaatgg acaccgaact gctgcgacaa 360cagagacgct acaactcacc gcgggtcctg ctgagcgaca gcaccccctt ggagcccccg 420cccttgtatc tcatggagga ttacgtgggc agccccgtgg tggcgaacag aacatcacgg 480cggaaacggt acgcggagca taagagtcac cgaggggagt actcggtatg tgacagtgag 540agtctgtggg tgaccgacaa gtcatcggcc atcgacattc ggggacacca ggtcacggtg 600ctgggggaga tcaaaacggg caactctccc gtcaaacaat atttttatga aacgcgatgt 660aaggaagcca ggccggtcaa aaacggttgc aggggtattg atgataaaca ctggaactct 720cagtgcaaaa catcccaaac ctacgtccga gcactgactt cagagaacaa taaactcgtg 780ggctggcggt ggatacggat agacacgtcc tgtgtgtgtg ccttgtcgag aaaaatcgga 840agaacatgaa ttggcatctc tccccatata taaattatta ctttaaatta tatgatatgc 900atgtagcata taaatgttta tattgttttt atatattata agttgacctt tatttattaa 960acttcagcaa ccctacagta tataagcttt tttctcaata aaatcagtgt gcttgccttc 1020131404DNAArtificial SequenceSynthetic construct 13cttgtcaccc aggtggcagg ggagtggtgc actctctgct cactgcaacc tcggcctcct 60gggttcgagt gattctccta cctcagccta ctgagtagct gggattacag gcgtgcagca 120ctatgcccgg ttaattttgg tatttttggt agagatgagg tttcaccatg ttgaccagct 180gctctggaac tcctgacctc aagtcatcca cctgcctcag cctcccagag tgctgggatt 240agaggtgtgg ggcacagtgc ctggcctgta gtagttgaat atttattatt aatctacaag 300ttgcgcatta cgcaagccct agatataggg tcccccaaac ttctagaaca agggcttccc 360cacaatcctg gcaggcaagc ctcccctggg gttcccaact tctttcccca ctgaagtttt 420tacccccttc tctaatccca gcctccctct ttctgtctcc aggtgctccg agagatgctc 480cctctcccct catgctccct ccccatcctc ctccttttcc tcctccccag tgtgccaatt 540gagtcccaac ccccaccctc aacattgccc ccttttctgg cccctgagtg ggaccttctc 600tccccccgag tagtcctgtc taggggtgcc cctgctgggc cccctctgct cttcctgctg 660gaggctgggg cctttcggga gtcagcaggt gccccggcca accgcagccg gcgtggggtg 720agcgaaactg caccagcgag tcgtcggggt gagctggctg tgtgcgatgc agtcagtggc 780tgggtgacag accgccggac cgctgtggac ttgcgtgggc gcgaggtgga ggtgttgggc 840gaggtgcctg cagctggcgg cagtcccctc cgccagtact tctttgaaac ccgctgcaag 900gctgataacg ctgaggaagg tggcccgggg gcaggtggag ggggctgccg gggagtggac 960aggaggcact gggtatctga gtgcaaggcc aagcagtcct atgtgcgggc attgaccgct 1020gatgcccagg gccgtgtggg ctggcgatgg attcgaattg acactgcctg cgtctgcaca 1080ctcctcagcc ggactggccg ggcctgagac ccatgcccag gaaaataaca gagctggatg 1140ctgagagacc tcagggatgg cccagctgat ctaaggaccc cagtttggga actcatcaaa 1200taatcacaaa atcacaattc tctgattttg agctcaatct ctgcaggatg ggtgaaacca 1260catggggttt tggaggttga ataggagttc tcctggagca acttgagggt aataatgatg 1320atgatataat aataatagcc actatttact gagtgtttac tgtttcttat ccctaataca 1380taactcctca gatcaactct catg 1404142137DNAArtificial SequenceSynthetic construct 14ccctgcctgc ctccctgcgc acccgcagcc tcccccgctg cctccctagg gctcccctcc 60ggccgccagc gcccattttt cattccctag atagagatac tttgcgcgca cacacataca 120tacgcgcgca aaaaggaaaa aaaaaaaaaa aagcccaccc tccagcctcg ctgcaaagag 180aaaaccggag cagccgcagc tcgcagctcg cagcccgcag cccgcagagg acgcccagag 240cggcgagcgg gcgggcagac ggaccgacgg actcgcgccg cgtccacctg tcggccgggc 300ccagccgagc gcgcagcggg cacgccgcgc gcgcggagca gccgtgcccg ccgcccgggc 360ccgccgccag ggcgcacacg ctcccgcccc cctacccggc ccgggcggga gtttgcacct 420ctccctgccc gggtgctcga gctgccgttg caaagccaac tttggaaaaa gttttttggg 480ggagacttgg gccttgaggt gcccagctcc gcgctttccg attttggggg cctttccaga 540aaatgttgca aaaaagctaa gccggcgggc agaggaaaac gcctgtagcc ggcgagtgaa 600gacgaaccat cgactgccgt gttccttttc ctcttggagg ttggagtccc ctgggcgccc 660ccacacggct agacgcctcg gctggttcgc gacgcagccc cccggccgtg gatgctgcac 720tcgggctcgg gatccgccca ggtagcggcc tcggacccag gtcctgcgcc caggtcctcc 780cctgcccccc agcgacggag ccggggccgg gggcggcggc gccgggggca tgcgggtgag 840ccgcggctgc agaggcctga gcgcctgatc gccgcggacc cgagccgagc ccacccccct 900ccccagcccc ccaccctggc cgcgggggcg gcgcgctcga tctacgcgtt cggggccccg 960cggggccggg cccggagtcg gcatgaatcg ctgctgggcg ctcttcctgt ctctctgctg 1020ctacctgcgt ctggtcagcg ccgaggggga ccccattccc gaggagcttt atgagatgct 1080gagtgaccac tcgatccgct cctttgatga tctccaacgc ctgctgcacg gagaccccgg 1140agaggaagat ggggccgagt tggacctgaa catgacccgc tcccactctg gaggcgagct 1200ggagagcttg gctcgtggaa gaaggagcct gggttccctg accattgctg agccggccat 1260gatcgccgag tgcaagacgc gcaccgaggt gttcgagatc tcccggcgcc tcatagaccg 1320caccaacgcc aacttcctgg tgtggccgcc ctgtgtggag gtgcagcgct gctccggctg 1380ctgcaacaac cgcaacgtgc agtgccgccc cacccaggtg cagctgcgac ctgtccaggt 1440gagaaagatc gagattgtgc ggaagaagcc aatctttaag aaggccacgg tgacgctgga 1500agaccacctg gcatgcaagt gtgagacagt ggcagctgca cggcctgtga cccgaagccc 1560ggggggttcc caggagcagc gagccaaaac gccccaaact cgggtgacca ttcggacggt 1620gcgagtccgc cggcccccca agggcaagca ccggaaattc aagcacacgc atgacaagac 1680ggcactgaag gagacccttg gagcctaggg gcatcggcag gagagtgtgt gggcagggtt 1740atttaatatg gtatttgctg tattgccccc atggggcctt ggagtagata atattgtttc 1800cctcgtccgt ctgtctcgat gcctgattcg gacggccaat ggtgcctccc ccacccctcc 1860acgtgtccgt ccacccttcc atcagcgggt ctcctcccag cggcctccgg ctcttgccca 1920gcagctcaag aagaaaaaga aggactgaac tccatcgcca tcttcttccc ttaactccaa 1980gaacttggga taagagtgtg agagagactg atggggtcgc tctttggggg aaacgggttc 2040cttcccctgc acctggcctg ggccacacct gagcgctgtg gactgtcctg aggagccctg 2100aggacctctc agcatagcct gcctgatccc tgaaccc 2137151308DNAArtificial SequenceSynthetic construct 15tccgcaaata tgcagaatta ccggccgggt cgctcctgaa gccagcgcgg ggaggcagcg 60cggcggcggc cagcaccggg aacgcaccga ggaagaagcc cagcccccgc cctccgcccc 120ttccgtcccc acccccatcc cggcggccca ggaggctccc cgcgctggcg cgcactccct 180gtttctcctc ctcctggctg gcgctgcctg cctctccgca ctcactgctc gccgggcgcc 240gtccgccagc tccgtgctcc ccgcgccacc ctcctccggg ccgcgctccc taagggatgg 300tactgatttt cgccgccaca ggagaccggc tggagcgccg ccccgcggcc tcgcctctcc 360tccgagcagc cagcgcctcg ggacgcgatg aggaccttgg cttgcctgct gctcctcggc 420tgcggatacc tcgcccatgt tctggccgag gaagccgaga tcccccgcga ggtgatcgag 480aggctggccc gcagtcagat ccacagcatc cgggacctcc agcgactcct ggagatagac 540tccgtaggga gtgaggattc tttggacacc agcctgagag ctcacggggt ccatgccact 600aagcatgtgc ccgagaagcg gcccctgccc attcggagga agagaagcat cgaggaagct 660gtccccgctg tctgcaagac caggacggtc atttacgaga ttcctcggag tcaggtcgac 720cccacgtccg ccaacttcct gatctggccc ccgtgcgtgg aggtgaaacg ctgcaccggc 780tgctgcaaca cgagcagtgt caagtgccag ccctcccgcg tccaccaccg cagcgtcaag 840gtggccaagg tggaatacgt caggaagaag ccaaaattaa aagaagtcca ggtgaggtta 900gaggagcatt tggagtgcgc ctgcgcgacc acaagcctga atccggatta tcgggaagag 960gacacgggaa ggcctaggga gtcaggtaaa aaacggaaaa gaaaaaggtt aaaacccacc 1020taaagcagcc aaccagatgt gaggtgagga tgagccgcag ccctttcctg ggacatggat 1080gtacatggcg tgttacattc ctgaacctac tatgtacggt gctttattgc cagtgtgcgg 1140tctttgttct cctccgtgaa aaactgtgtc cgagaacact cgggagaaca aagagacagt 1200gcacatttgt ttaatgtgac atcaaagcaa gtattgtagc actcggtgaa gcagtaagaa 1260gcttccttgt caaaaagaga gagagagaaa agaaaaaaaa aggaattc 1308162149DNAArtificial SequenceSynthetic construct 16cagctgactc aggcaggctc catgctgaac ggtcacacag agaggaaaca ataaatctca 60gctactatgc aataaatatc tcaagtttta acgaagaaaa acatcattgc agtgaaataa 120aaaattttaa aattttagaa caaagctaac aaatggctag ttttctatga ttcttcttca 180aacgctttct ttgaggggga aagagtcaaa caaacaagca gttttacctg aaataaagaa 240ctagttttag aggtcagaag aaaggagcaa gttttgcgag aggcacggaa ggagtgtgct 300ggcagtacaa tgacagtttt cctttccttt gctttcctcg ctgccattct gactcacata 360gggtgcagca atcagcgccg aagtccagaa aacagtggga gaagatataa ccggattcaa 420catgggcaat gtgcctacac tttcattctt ccagaacacg atggcaactg tcgtgagagt 480acgacagacc agtacaacac aaacgctctg cagagagatg ctccacacgt ggaaccggat 540ttctcttccc agaaacttca acatctggaa catgtgatgg aaaattatac tcagtggctg 600caaaaacttg agaattacat tgtggaaaac atgaagtcgg agatggccca gatacagcag 660aatgcagttc agaaccacac ggctaccatg ctggagatag gaaccagcct cctctctcag 720actgcagagc agaccagaaa gctgacagat gttgagaccc aggtactaaa tcaaacttct 780cgacttgaga tacagctgct ggagaattca ttatccacct acaagctaga gaagcaactt 840cttcaacaga caaatgaaat cttgaagatc catgaaaaaa acagtttatt agaacataaa 900atcttagaaa tggaaggaaa acacaaggaa gagttggaca ccttaaagga agagaaagag 960aaccttcaag gcttggttac tcgtcaaaca tatataatcc aggagctgga aaagcaatta 1020aacagagcta ccaccaacaa cagtgtcctt cagaagcagc aactggagct gatggacaca 1080gtccacaacc ttgtcaatct ttgcactaaa gaaggtgttt tactaaaggg aggaaaaaga 1140gaggaagaga aaccatttag agactgtgca gatgtatatc aagctggttt taataaaagt 1200ggaatctaca ctatttatat taataatatg ccagaaccca aaaaggtgtt ttgcaatatg 1260gatgtcaatg ggggaggttg gactgtaata caacatcgtg aagatggaag tctagatttc 1320caaagaggct ggaaggaata taaaatgggt tttggaaatc cctccggtga atattggctg 1380gggaatgagt ttatttttgc cattaccagt cagaggcagt acatgctaag aattgagtta 1440atggactggg aagggaaccg agcctattca cagtatgaca gattccacat aggaaatgaa 1500aagcaaaact ataggttgta tttaaaaggt cacactggga cagcaggaaa acagagcagc 1560ctgatcttac acggtgctga tttcagcact aaagatgctg ataatgacaa ctgtatgtgc 1620aaatgtgccc tcatgttaac aggaggatgg tggtttgatg cttgtggccc ctccaatcta 1680aatggaatgt tctatactgc gggacaaaac catggaaaac tgaatgggat aaagtggcac 1740tacttcaaag ggcccagtta ctccttacgt tccacaacta tgatgattcg acctttagat 1800ttttgaaagc gcaatgtcag aagcgattat gaaagcaaca aagaaatccg gagaagctgc 1860caggtgagaa actgtttgaa aacttcagaa gcaaacaata ttgtctccct tccagcaata 1920agtggtagtt atgtgaagtc accaaggttc ttgaccgtga atctggagcc gtttgagttc 1980acaagagtct ctacttgggg tgacagtgct cacgtggctc gactatagaa aactccactg 2040actgtcgggc tttaaaaagg gaagaaactg ctgagcttgc tgtgcttcaa actactactg 2100gaccttattt tggaactatg gtagccagat gataaatatg gttaatttc 2149
Patent applications by Ching Shwun Lin, San Mateo, CA US
Patent applications by Peter Carroll, San Francisco, CA US
Patent applications by Tom F. Lue, Hillsborough, CA US
Patent applications by Yuet W. Kan, San Francisco, CA US
Patent applications by THE REGENTS OF THE UNIVERSITY OF CALIFORNIA
Patent applications in class 25 or more peptide repeating units in known peptide chain structure
Patent applications in all subclasses 25 or more peptide repeating units in known peptide chain structure