Available online 27 December 2005

Combined Angiopoietin-1 and Vascular Endothelial Growth Factor Gene Transfer Restores Cavernous Angiogenesis and Erectile Function in a Rat Model of Hypercholesterolemia Ji-Kan Ryu, 1 Chung-Hyun Cho, 2 Hwa-Yean Shin, 1 Sun U. Song, 3 Seung-Min Oh, 1 Minhyung Lee, 3 Shuguang Piao, 1 Jee-Young Han, 4 In-Hoo Kim, 5 Gou Young Koh, 2 and Jun-Kyu Suh 1,3, * 1 Department of Urology, 3 Clinical Research Center, and 4 Pathology, Inha University School of Medicine, Incheon 400-711, Republic of Korea 2 Biomedical Research Center and Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Republic of Korea 5 Research Institute, National Cancer Center, Goyang, Gyeonggi 411-760, Republic of Korea *To whom correspondence and reprint requests should be addressed at the Department of Urology, Inha University School of Medicine, 7-206 3rd Street, Shinheung-Dong, Jung-Gu, Incheon 400-711, Republic of Korea. Fax: +82 32 890 3097. E-mail: jksuh@inha.ac.kr. Available online 27 December 2005 Hypercholesterolemia-related endothelial cell dysfunction and decreased endothelium-derived nitric oxide formation may account for impaired angiogenesis and subsequent erectile dysfunction. Angiopoietin-1 (Ang1) is a critical angiogenic factor for vascular maturation and enhances vascular endothelial growth factor (VEGF)-induced angiogenesis in a complementary manner. We hypothesized that combined adenovirus-delivered human Ang1 (ad-ang1) and VEGF165 (ad- VEGF165) gene transfer might promote angiogenesis cooperatively in a rat model of hypercholesterolemic erectile dysfunction and result in a recovery of erectile function. Ad-Ang1 and ad- VEGF165 were injected either alone or in combination into the corpus cavernosum of the penis. Combined gene transfer of both ad-ang1 and ad-vegf165 significantly increased cavernous angiogenesis, enos phosphorylation, and cgmp expression compared with that in the groups treated with either therapy alone. Erectile function, as evaluated by electrical stimulation of the cavernous nerve 2 and 8 weeks after treatment, was completely restored in the combined treatment group, whereas intracavernous injection of either ad-ang1 or ad-vegf165 alone elicited partial improvement. The results indicate that combined application of angiogenic factors may enhance cavernous angiogenesis cooperatively by reinforcing the endothelium both structurally and functionally, which results in an additive effect on erectile function in hypercholesterolemic rats. Key Words: erectile dysfunction, hyperlipidemia, angiogenesis, gene therapy, adenoviral vector, angiopoietin-1, vascular endothelial growth factor INTRODUCTION The penis has a specialized vascular bed, and not surprisingly, erectile dysfunction (ED) has a predominantly vasculogenic origin. In a retrospective study, Virag and colleagues [1] assessed 440 men with ED and found hypercholesterolemia, diabetes mellitus, hypertension, and smoking to be independent risk factors for vasculogenic ED. Penile vascular impairment significantly increases as the number of vascular risk factors increases [2]. Of those risk factors, hypercholesterolemia is well known to have a powerful effect on the development of both angiopathy and ED induced by other risk factors. The association between hypercholesterolemia and ED is attributed to the impairment of endotheliumdependent relaxation in the smooth muscle cells of the corpus cavernosum [3]. A functional impairment of endothelial nitric oxide synthase (enos) activity may lead to impairment of cavernous smooth muscle relaxation in response to endothelium-mediated stimuli in hypercholesterolemic rabbits [4]. It was also shown in a rat model of hind-limb ischemia that hypercholesterolemia inhibits ischemia-induced angiogenesis by inhibit- 705 1525-0016/$30.00

doi:10.1016/j.ymthe.2005.10.016 ing endothelium-derived nitric oxide (EDNO) [5]. Recent reports showed that EDNO is a critical modulator for angiogenesis [6 8]. For example, angiogenesis induced by a potent angiogenic factor, vascular endothelial growth factor (VEGF), is attenuated by inhibitors of NOS [6,7]. Additionally, angiogenesis and collateral vessel formation after operatively induced hind-limb ischemia are severely impaired in mice lacking the gene for enos [8]. Several clinical trials are currently evaluating angiogenic factors for their ability to induce neovascularization in ischemic tissues [9,10], and the intracellular signaling pathways that mediate the proangiogenic effects of these growth factors are being extensively investigated. Local intracavernous delivery of VEGF gene or protein has been shown to recover erectile function in rat and rabbit models of vasculogenic ED induced by castration [11], traumatized iliac arteries [12], or hyperlipidemia [13,14]. Increased endothelial content after therapy was not consistently shown in these studies, however, although physiologic improvement, such as transient and partial recovery of erectile function, was noted. Furthermore, it was reported that VEGF administration can initiate vessel formation in adult animals, but by itself promotes the formation of only leaky, immature, and unstable vessels [15 17]. Because blood vessel formation requires a cascade of growth factors, their receptors, and intracellular signals, such therapies may require the application of more than a single growth factor. Angiopoietin-1 (Ang1), the ligand of the Tie-2 receptor, is an angiogenic factor that plays important roles in the stabilization and maturation of blood vessels in preand postnatal angiogenesis [18,19]. Ang1 also counteracts VEGF-induced inflammation in endothelial cells while having an additive effect on vessel formation [20,21]. Therefore, combined treatment with Ang1 and VEGF may be better than treatment with either factor alone for enhancing therapeutic vascularization and angiogenesis while avoiding inflammation. Several recent studies have shown that administration of Ang1 together with VEGF strongly promotes revascularization in ischemic animal models [21 25]. The primary goal of the present study was to investigate in a rat model the effectiveness of intracavernous delivery of the human Ang1 (approved gene symbol TM7SF2) and VEGF165 (approved gene symbol VEGF) genes for the treatment of hypercholesterolemic ED. We hypothesized that combined adenovirus-delivered Ang1 (ad-ang1) and VEGF165 (ad-vegf165) gene transfer to the corpus cavernosum might promote angiogenesis cooperatively and result in recovery of erectile function. RESULTS AND DISCUSSION Lipid Profiles The serum total cholesterol and low-density-lipoprotein (LDL) cholesterol concentrations of animals fed the diet containing 4% cholesterol with 1% cholic acid for 3 months were significantly higher than concentrations in the age-matched controls. Increases in total and LDL cholesterol and decreases in high-density-lipoprotein (HDL) cholesterol are known to be associated with a high risk of developing ED [26 28]. In the present study, however, there was no significant difference in HDL cholesterol concentrations between rats fed the cholesterol diet and those fed the normal diet. This finding may be attributable to similar physical activity in both groups, because HDL cholesterol concentrations can be improved by exercise [29]. Also, no significant difference in triglyceride concentrations was found between groups. All the animals fed the high-cholesterol diet had similar lipid profiles, regardless of the treatment given (Table 1). In Vivo Gene or Protein Expression in Rat Corpora Cavernosa We detected exogenous human Ang1 and VEGF165 mrna in the corpus cavernosum from hypercholesterolemic rats 3, 7, 14, and 21 days after injection of ad- Ang1, ad-vegf165, or a combination of both. We observed virtually no exogenous Ang1 or VEGF165 mrna expression in animals injected with ad-lacz. The level of exogenous human Ang1 and VEGF165 transcript peaked at the earliest time point assayed (3 days), and expression was still detectable at 21 days (Fig. 1A). These results were typical of four independent observations. TABLE 1: Serum lipid profiles in rats fed a control diet or a high-cholesterol diet and injected with adenovirus-delivered angiogenic growth factors Cholesterol diet Am-C LacZ Ang1 VEGF Ang1 + VEGF Co-C TC 77.5 F 3.0* 245.7 F 11.9 234.1 F 18.3 261.2 F 18.5 252.6 F 25.4 255.2 F 19.6 LDL 40.8 F 2.1* 186.3 F 12.3 191.6 F 17.6 219.3 F 19.2 205.1 F 13.9 208.5 F 18.8 TG 37.5 F 3.8 56.4 F 10.7 55.4 F 9.1 46.7 F 6.2 44.0 F 6.6 48.8 F 8.9 HDL 29.1 F 0.5 32.3 F 1.9 31.4 F 1.8 32.6 F 1.2 27.7 F 2.9 31.2 F 2.4 Values are the means F SE (mg/dl) for n = 12 animals per group. Am-C, age-matched control; Ang1, angiopoietin-1; VEGF, vascular endothelial growth factor; Co-C, cholesterol-only control; TC, total cholesterol; LDL, low-density-lipoprotein cholesterol; TG, triglycerides; HDL, high-density-lipoprotein cholesterol. * P b 0.01 vs the LacZ, Ang1, VEGF165, Ang1 + VEGF165, and Co-C groups by ANOVA. 706

FIG. 1. In vivo expression of Ang1 and VEGF gene and protein. (A) Detection of human Ang1- and VEGF-specific mrna in the corpus cavernosum from hypercholesterolemic rats. PCR was performed with primers specific for human Ang1 and VEGF165. Tissue RNA was extracted from the corpus cavernosum 3, 7, 14, and 21 days after intracavernous administration of ad-ang1 (1 10 10 parts/0.1 ml), ad-vegf165 (1 10 10 parts/0.1 ml), or a combination of both (1 10 10 parts, respectively/0.1 ml). Densitometric analyses are presented as the relative ratio of human Ang1 or VEGF165 mrna to h-actin mrna. The relative ratio measured 3 days after injection of ad-ang1 or ad-vegf165 is arbitrarily presented as 1. Bars represent the mean F SE from four experiments. (B) Western blot analysis of the protein expression of Ang1 and VEGF in cavernous tissues from hypercholesterolemic rats 3, 7, 14, and 21 days after intracavernous administration of ad-ang1, ad-vegf165, or a combination of both. The relative ratio of Ang1 or VEGF to h-actin measured in the control group is arbitrarily presented as 1. Bars represent the mean F SE from four experiments. (C, D) Immunohistochemical staining of cavernous tissue performed with antibody to Ang1 (C) or VEGF (D) in hypercholesterolemic rats 7 days after intracavernous injection of ad-lacz (1 10 10 parts/0.1 ml), ad-ang1 (1 10 10 parts/0.1 ml), or ad-vegf165 (1 10 10 parts/0.1 ml). 2 nd Ab, secondary antibody control; arrowheads, endothelial cells; arrows, smooth muscle cells; asterisks, nerve bundles. Bars indicate 50 Am. (E) Localization of h-galactosidase in the whole penis from hypercholesterolemic rats 3 days after administration of virus vehicle alone or of ad-lacz (1 10 10 parts/ 0.1 ml). Arrows denote positive endothelial cells (E), smooth muscle cells (SM), and nerve bundles (N). Tissues were counterstained with nuclear fast red. Bars indicate 10 Am. (F) h-galactosidase activity in the corpus cavernosum from hypercholesterolemic rats 3, 7, 14, and 21 days after intracavernous administration of vehicle or ad-lacz (1 10 10 parts/0.1 ml). Each bar depicts the mean F SE for n = 4 animals in each group. *P b 0.01, yp b 0.05 vs control by ANOVA. 707

doi:10.1016/j.ymthe.2005.10.016 We also evaluated Ang1 and VEGF protein expression in the corpus cavernosum 3, 7, 14, and 21 days after injection of ad-ang1, ad-vegf165, or a combination of both by Western blot. Ang1 and VEGF protein expression was highest 7 days after injection, whereas trace amounts, corresponding to endogenous Ang1 or VEGF, were present in hypercholesterolemic rats injected with ad-lacz as a control. At 14 and 21 days after injection, protein levels of Ang1 and VEGF returned to the control level (Fig. 1B). To localize each transgene postdelivery, we performed immunohistochemical staining for Ang1 or VEGF 7 days after intracavernous injection of ad-lacz, ad-ang1, or ad-vegf165. Immunohistochemical staining produced only a faint staining of endothelial cells, indicating little endogenous Ang1 or VEGF protein expression in the ad-lacz-treated rats. In the ad-ang1- or ad-vegf165-treated animals, Ang1 or VEGF staining showed strong expression of each protein in endothelial cells and also in some smooth muscle cells and nerve fibers (Figs. 1C and 1D). X-gal histochemistry in fixed sagittal blocks of the whole penis 3 days after injection of ad-lacz showed uniform staining along the shaft and glans, whereas we observed virtually no endogenous h-galactosidase activity in animals injected with virus vehicle alone (Fig. 1E, top). We used frozen transverse sections from the penis of rats 3 days after injection of ad-lacz to determine its cellular distribution. We observed the X-gal staining in different cell types, mainly cavernous smooth muscle cells and endothelial cells lining the cavernous space and also some nerve fibers (Fig. 1E, bottom). At 3 to 21 days after intracavernous administration of vehicle or ad-lacz, the magnitude of h-galactosidase activity was measured by chemiluminescence. Cavernous tissue from hypercholesterolemic rats treated with FIG. 2. Combined gene therapy with Ang1 and VEGF165 completely restores intracavernous pressure (ICP) elicited by electrical stimulation of the cavernous nerve. (A) Representative ICP responses for age-matched control (Am-C) or hypercholesterolemic rats stimulated 14 and 56 days after intracavernous injection of ad-lacz (1 10 10 parts/0.1 ml), ad-ang1 (1 10 10 parts/0.1 ml), ad-vegf165 (1 10 10 parts/0.1 ml), ad-ang1 + ad-vegf165 (1 10 10 parts, respectively/0.1 ml), or cholesterol only (Co-C). The stimulus interval is indicated by a solid bar. SAP, systemic arterial pressure. (B D) Ratio of mean maximal ICP to mean arterial pressure (MAP), total ICP (area under the curve), and tumescence slope calculated for each group. The slope and area under the curve were calculated from ICP recordings normalized to MAP. Each bar depicts the mean F SE for n = 12 animals per group. (B) Ratio of ICP to MAP. *P b 0.01 vs ad-lacz and Co-C groups, yp b 0.05 vs Am-C group, zp b 0.01 vs ad-lacz, ad-ang1, ad-vegf165, and Co-C groups by ANOVA. (C) Total ICP (area under the curve). *P b 0.01 vs ad-lacz and Co-C groups, yp b 0.01 vs ad-lacz, ad-ang1, and Co-C groups, zp b 0.05 vs ad-vegf165 group, **P b 0.01 vs ad-lacz, ad- Ang1, ad-vegf165, and Co-C groups. (D) Slope. *P b 0.01 vs ad-lacz and Co-C groups, yp b 0.01 vs ad-lacz, ad-ang1, ad-vegf165, and Co-C groups. 708

vehicle alone showed minimal h-galactosidase activity, whereas cavernous tissue from ad-lacz-transfected cholesterol rats had significantly higher h-galactosidase activity than did that from the vehicle control group up to 14 days, and the expression of h-galactosidase peaked 3 days after injection (Fig. 1F). Effect of Adenoviral Gene Transfer of Angiogenic Growth Factors on Erectile Function We measured the effect of cavernous nerve stimulation on erectile function in vivo 14 and 56 days after treatment to evaluate the physiologic relevance of the injection of angiogenic growth factor genes. A representative intracavernous tracing after stimulation of the cavernous nerve at 5 V, 12 Hz, for 1 min in age-matched control (Am-C) or hypercholesterolemic rats transfected with ad- LacZ, ad-ang1, ad-vegf165, ad-ang1 + ad-vegf165, or cholesterol only (Co-C) is depicted in Fig. 2A. During electrical stimulation of the cavernous nerve, the ratio of maximal intracavernous pressure (ICP) to mean arterial pressure (MAP), total ICP, and slope were significantly lower in hypercholesterolemic rats treated with ad-lacz or cholesterol only than in age-matched controls. Combined ad-ang1 and ad-vegf165 injection completely normalized all erection parameters, whereas intracavernous injection of either ad-ang1 or ad-vegf165 alone partially restored erectile function (Figs. 2B 2D). No detectable difference was found in resting ICP or MAP among the six experimental groups (data not shown). Effect of Adenovirus-Mediated Gene Transfer of Angiogenic Growth Factors on Cavernous Blood Flow We also evaluated the effect of cavernous nerve stimulation on cavernous blood flow in vivo 14 days after treatment. A representative blood flow tracing after stimulation of the cavernous nerve at 1 V, 12 Hz, for 1 min in each group is depicted in Fig. 3A. During electrical stimulation of the cavernous nerve, all blood flow variables, such as maximal perfusion unit (PU), total PU, and slope, were significantly lower in hypercholesterolemic rats treated with ad-lacz or cholesterol only than in age-matched controls. Combined ad-ang1 and ad-vegf165 injection FIG. 3. Combined gene therapy with Ang1 and VEGF165 completely restores cavernous blood flow elicited by electrical stimulation of the cavernous nerve. (A) Representative blood flow responses for age-matched control (Am-C) or hypercholesterolemic rats stimulated 14 days after intracavernous injection of ad-lacz (1 10 10 parts/0.1 ml), ad-ang1 (1 10 10 parts/0.1 ml), ad-vegf165 (1 10 10 parts/0.1 ml), ad-ang1 + ad-vegf165 (1 10 10 parts, respectively/0.1 ml), or cholesterol only (Co-C). The stimulus interval is indicated by a solid bar. (B D) Maximal perfusion unit (PU), total PU, and slope calculated for each group. Each bar depicts the mean F SE for n = 4 animals per group. *P b 0.05 vs ad-lacz and Co-C groups by ANOVA. 709

doi:10.1016/j.ymthe.2005.10.016 completely normalized all blood flow parameters, whereas intracavernous injection of either ad-ang1 or ad-vegf165 alone partially restored blood flow (Figs. 3B 3D). Quantification of Endothelial Area, enos and neuronal (n) NOS Immunoreactivity, and Smooth Muscle Area We performed immunohistochemical staining of cavernous tissue with antibody to factor VIII, enos, nnos, or smooth muscle a-actin (a-sma) in age-matched control and hypercholesterolemic rats 14 and 56 days after intracavernous injection of ad-lacz, ad-ang1, ad-vegf165, ad- Ang1 + ad-vegf165, or cholesterol only. Representative photomicrographs of each group are presented in Figs. 4A and 5A. We quantified immunohistochemical endothelial area, which represents cavernous angiogenesis; enospositive area; nnos-positive area; and smooth muscle area with an image analyzer. We found a significantly smaller number of endothelial cells in the hypercholesterolemic rats transfected with ad-lacz or cholesterol only FIG. 4. Quantification of endothelial area, enos and nnos immunoreactivity, and smooth muscle area. (A) Immunohistochemical staining of cavernous tissue performed with antibody to factor VIII, enos, nnos, or a-sma in agematched control (Am-C) or hypercholesterolemic rats 14 days after intracavernous injection of ad-lacz (1 10 10 parts/0.1 ml), ad-ang1 (1 10 10 parts/0.1 ml), ad-vegf165 (1 10 10 parts/0.1 ml), ad-ang1 + ad-vegf165 (1 10 10 parts, respectively/0.1 ml), or cholesterol only (Co-C). Bars indicate 200 Am for factor VIII, enos, and a-sma and 50 Am for nnos. (B E) Quantitative analysis of endothelium, enos- and nnos-positive area, and smooth muscle content in cavernous tissue was done with an image analyzer. Each bar depicts the mean F SE for n = 12 animals per group. (B) Endothelial area. *P b 0.05 vs ad-lacz and Co-C groups, yp b 0.01 vs ad- LacZ, ad-ang1, and Co-C groups, zp b 0.05 vs ad-vegf165 group by ANOVA. (C) enos-positive area. (D) nnos-positive area. *P b 0.01 vs ad-lacz and Co-C groups. (E) Smooth muscle area. *P b 0.05, yp b 0.01 vs Am-C group. 710

FIG. 5. Quantification of endothelial area, enos and nnos immunoreactivity, and smooth muscle area. (A) Immunohistochemical staining of cavernous tissue performed with antibody to factor VIII, enos, nnos, or a-sma in age-matched control (Am-C) or hypercholesterolemic rats 56 days after intracavernous injection of ad-lacz (1 10 10 parts/0.1 ml), ad-ang1 (1 10 10 parts/0.1 ml), ad-vegf165 (1 10 10 parts/ 0.1 ml), ad-ang1 + ad-vegf165 (1 10 10 parts, respectively/ 0.1 ml), or cholesterol only (Co-C). Bars indicate 200 Am for factor VIII, enos, and a-sma and 50 Am for nnos. (B E) Quantitative analysis of endothelium, enos- and nnos-positive area, and smooth muscle content in cavernous tissue was done with an image analyzer. Each bar depicts the mean F SE for n = 12 animals per group. (B) Endothelial area. *P b 0.05 vs ad-lacz and Co-C groups, yp b 0.01 vs ad-lacz, ad-ang1, and Co-C groups, zp b 0.05 vs ad-vegf165 group by ANOVA. (C) enospositive area. (D) nnos-positive area. *P b 0.05 vs Am-C, ad- LacZ, and Co-C groups. (E) Smooth muscle area. *P b 0.01 vs Am-C group. than in age-matched control rats. Reported mechanisms for the impaired angiogenesis in hypercholesterolemia are endothelial dysfunction and decreased EDNO formation [30,31], degradation of nitric oxide by superoxide anion production [32], and inhibition of endothelial cell migration mediated by oxidized LDL and lysophosphatidylcholine, which are major atherogenic molecules in the arterial wall [33]. The additive effect of ad-ang1 and ad-vegf165 in cavernous angiogenesis was confirmed by the marked increase in factor VIII-positive endothelial density in the corpus cavernosum, which was comparable with that in the age-matched control. Endothelial content was approximately 50% higher in hypercholesterolemic rats injected with ad-vegf165 than in the ad-lacz or cholesterol-only control group, but did not reach the level of the age-matched control or combined gene therapy group. Such an increase was not apparent, however, after intracavernous administration of ad-ang1 alone (Figs. 4A, 4B, 5A, and 5B), although previous studies showed that Ang1 has a proliferative effect on endothelial cells in vivo 711

doi:10.1016/j.ymthe.2005.10.016 [34,35]. This disparity may result from a different organ or different dosage of Ang1 being used in different studies. Total enos protein expression did not change in control or hypercholesterolemic rats regardless of treatment (Figs. 4A, 4C, 5A, and 5C). We also investigated the potential direct effect of angiogenic factors on nerve cells with nnos immunohistochemistry. We found a significantly smaller nnospositive area in the hypercholesterolemic rats transfected with ad-lacz or cholesterol only than in age-matched control rats. nnos-positive area was restored to the level of the age-matched controls in hypercholesterolemic rats 14 days after injection of either combined or single angiogenic factor genes. At 56 days after injection, nnos content was decreased compared to the age-matched controls, but it was still significantly higher than that in rats treated with either ad-lacz or cholesterol only. However, we found no significant difference in nnos content between hypercholesterolemic rats treated with combined ad-ang1 + VEGF165 and those treated with either ad-ang1 or ad-vegf165 alone (Figs. 4A, 4D, 5A, and 5D). Interestingly, cavernous smooth muscle content was significantly higher in hypercholesterolemic rats than in the age-matched controls. It appears that increased cavernous smooth muscle content is a manifestation of the diseased state of hypercholesterolemia, because vascular smooth muscle cell proliferation is a pathologic consequence of hypercholesterolemia [14,36]. This increase was not reversible in response to the treatments (Figs. 4A, 4E, 5A, and 5E), which suggests that angiogenic growth factors do not play a definite role in cavernous smooth muscle content, although these factors are involved in cavernous smooth muscle relaxation and increased blood flow through a nitric oxide/ cgmp-dependent mechanism. In Vivo Expression of enos/phospho-enos Protein and cgmp We evaluated the expression of phospho-enos/enos in the corpus cavernosum of age-matched control and hypercholesterolemic rats 7, 14, and 56 days after transfection with ad-lacz, ad-ang1, ad-vegf165, ad-ang1 + ad-vegf165, or cholesterol only. In hypercholesterolemic rats treated with ad-lacz or cholesterol only, basal levels of phospho-enos (Ser1177) were significantly lower than in the age-matched controls as determined by the ratio of phospho-enos to total enos. At 7 days after intracavernous gene transfer of ad-ang1 + ad-vegf165, endogenous enos phosphorylation increased dramatically by approximately 4.0-fold compared with that in the controls as determined by immunoblot analysis. Hypercholesterolemic rats injected intracavernously with either ad-ang1 or ad-vegf165 also exhibited 2.1- or 2.9-fold increases, respectively, in phospho-enos expression compared with that in age-matched controls. At 14 and 56 days after injection, the expression of FIG. 6. Western blot analysis demonstrating the relative protein abundance of phospho-enos (Ser1177) in age-matched control (Am-C) and hypercholesterolemic rat penis 7, 14, and 56 days after transfection with ad-lacz (1 10 10 parts/0.1 ml), ad-ang1 (1 10 10 parts/0.1 ml), ad-vegf165 (1 10 10 parts/ 0.1 ml), ad-ang1 + ad-vegf165 (1 10 10 parts, respectively/0.1 ml), or cholesterol only (Co-C). (A) Representative Western immunoblot of phosphoenos (Ser1177) and total enos in each group. (B) Quantitative analysis of phospho-enos (Ser1177). Data are representative of four densitometric values from independent experiments with separate rats for each experiment. Each bar represents the mean F SE of the ratio of phospho-enos/enos expressed relative to the control result. *P b 0.05 vs Am-C group, **P b 0.01 vs Am-C group, yp b 0.01 vs Am-C, ad-lacz, and Co-C groups, zp b 0.01 vs Am- C, ad-lacz, ad-ang1, ad-vegf165, and Co-C groups, ***P b 0.01 vs ad-lacz, ad-ang1, ad-vegf165, and Co-C groups by ANOVA. 712

phospho-enos in hypercholesterolemic rats treated with combined ad-ang1 + ad-vegf165 returned to the control level, but was still significantly higher than that in rats treated with either ad-ang1 or ad-vegf165 alone (Fig. 6). Similar to the results of immunohistochemistry, immunoblot analysis of penile homogenates showed that total enos protein, normalized to total proteins, remained unchanged 7, 14, and 56 days after treatment (Fig. 6). This finding is similar to those of Musicki et al. [37], who showed a change in phosphoenos but not total enos protein expression in the penis of castrated mice after intracavernous injection of ad-vegf145. One explanation for that finding, as discussed by those authors, is that the increase in phospho-enos was not caused by changes in the total enos protein expression level but rather represented changes in the phosphorylation status of the enzyme. We also measured cavernous tissue concentrations of cgmp in age-matched control rats and in hypercholesterolemic rats 7, 14, and 56 days after intracavernous injection of ad-lacz, ad-ang1, ad-vegf165, ad-ang1 + ad-vegf165, or cholesterol only. Cavernous cgmp decreased significantly in hypercholesterolemic rats treated with ad-lacz or cholesterol only compared with that in the age-matched controls. However, cgmp concentrations increased approximately 1.7- to 2.0-fold in the cavernous tissue of hypercholesterolemic rats transfected with ad-ang1 + ad-vegf165 compared with concentrations in the age-matched controls. This increased formation of cgmp, which is a messenger in the downstream signal transduction of nitric oxide, is clear evidence of excellent erectile response to cavernous nerve stimulation in the hypercholesterolemic rats. Cavernous cgmp concentrations were significantly higher in hypercholesterolemic animals treated with ad-ang1 or ad- VEGF165 than in hypercholesterolemic rats treated with ad-lacz or cholesterol only, but were still lower than the concentrations of the age-matched controls (Fig. 7). Despite the introduction of the novel oral phosphodiesterase 5 (PDE5) inhibitors in the treatment of ED [38 40], new therapeutic strategies, such as gene therapy, are warranted. The PDE5 inhibitors may fail to raise cgmp to the required level if the endogenous nitrergic mechanism is insufficient, which may explain the failure of these drugs in some forms of organic impotence [41]. A direct and stable increase in cgmp production in the penis during sexual stimulation is therefore an attractive alternative to current medical treatments for ED. Gene therapy can restore physiologic erections to the normal endogenous signals in the absence of any other form of therapy. Moreover, the penis is a convenient organ for gene therapy because of its external location, ubiquity of endothelial-lined spaces, slow circulation in the flaccid state, and gap junctions between smooth muscles, which ensure wide distribution of injected genes. Our results clearly show that adenoviral combined gene delivery of FIG. 7. Changes in cavernous cgmp concentrations in age-matched control (Am-C) or hypercholesterolemic rats 7, 14, and 56 days after transfection with ad-lacz (1 10 10 parts/0.1 ml), ad-ang1 (1 10 10 parts/0.1 ml), ad- VEGF165 (1 10 10 parts/0.1 ml), ad-ang1 + ad-vegf165 (1 10 10 parts, respectively/0.1 ml), or cholesterol only (Co-C). Each bar depicts the mean F SE for n = 4 animals per group. *P b 0.01 vs Am-C group, yp b 0.01 vs ad-lacz and Co-C groups, **P b 0.05 vs ad-lacz and Co-C groups, zp b 0.01 vs Am-C, ad-lacz, ad-ang1, ad-vegf165, and Co-C groups by ANOVA. Ang1 and VEGF165 can increase the expression of their mrnas and proteins, the ratio of phospho-enos to enos, and cgmp as well as cavernous angiogenesis cooperatively, which results in physiologically relevant changes in erectile function as evidenced by cavernous nerve stimulation up to 8 weeks after treatment. However, new vector systems that offer longer gene-transfer efficiency, a higher level of transgene expression, and little or no immunogenic reactions are required for safe application in the future treatment of human ED. In summary, the results of the present study show that adenoviral combined gene delivery of Ang1 and VEGF165 into the corpus cavernosum of hypercholesterolemic rats produces an additive effect on erectile function through remodeling of cavernous angiogenesis (namely, bcavernogenesisq) compared with either therapy alone. To our knowledge, this is the first study showing the efficacy of treatment with Ang1 and VEGF combined in the field of ED. We expect that combined angiogenic growth factor gene therapy will be a curative treatment modality in vasculogenic ED. MATERIALS AND METHODS Adenoviral vectors. Two replication-deficient adenoviruses encoding human Ang1 and VEGF165 were generated by homologous recombination. Gene expression was driven by a cytomegalovirus promoter/ enhancer. Ad-LacZ was used as a parallel control during gene transfection. All viruses were propagated in 293 cells, which were purified and titered by use of a standard method and then stored at 808C until used. 713

doi:10.1016/j.ymthe.2005.10.016 Amplification and purification were performed by the National Cancer Center (Division of Basic Science) in Republic of Korea. In vivo gene delivery to the corpora cavernosa. Two-month-old male Sprague Dawley rats were used in this study. The experiments performed were approved by the Institutional Animal Care and Use Subcommittee of our university. The control animals were fed a normal diet and the experimental animals were fed a diet containing 4% cholesterol and 1% cholic acid (TD01408; Harlan Teklab, Madison, WI, USA) for 3 months. The hypercholesterolemic rats were anesthetized with chloral hydrate (20 mg/kg) intraperitoneally and placed in a supine position on a thermoregulated surgical table. The penis was exposed using sterile technique. Ad-LacZ, ad-ang1, ad-vegf165, or a mixture of ad-ang1 and ad-vegf165 was administered at 1 10 10 viral particles per injection in 0.1 ml phosphate-buffered saline (PBS) (parts/0.1 ml). Each gene was injected into the mid portion of the corpus cavernosum with a 30-gauge insulin syringe as previously described [42]. The incision was closed with 6-O Vicryl sutures. After evaluation of erectile function (n = 12 per group) and cavernous blood flow (n = 4 per group) by cavernous nerve electrical stimulation 14 or 56 days after treatment, each group of animals was sacrificed and corpus cavernosum tissue was harvested for histologic examination. Cavernous specimens from a separate group of animals were used for biochemical study [reverse transcriptase polymerase chain reaction (RT-PCR), Western blot, or cgmp measurement]. Blood was extracted from the carotid artery or by direct cardiac puncture and lipid profiles were determined with commercially available kits (Boehringer Mannheim GmbH, Mannheim, Germany) and an automatic analyzer (Hitachi 7600; Hitachi Koki Co., Hitachinaka, Japan). Measurement of erectile function. The rats from each cholesterol group and their age-matched controls were anesthetized with chloral hydrate, and a carotid artery was cannulated to measure systemic arterial pressure. Bipolar platinum wire electrodes were placed around the cavernous nerve for electrical stimulation, and a 26-gauge needle filled with 250 U/ml heparin was inserted into one side of the corpus cavernosum for monitoring of intracavernous pressure (ICP). Each rat was subjected to electrical field stimulation at 5 V, a frequency of 12 Hz, a pulse width of 1 ms, and duration of 1 min. Three electrostimulations were replicated at intervals of 10 min. The ratio of maximal ICP to mean arterial pressure at the peak erectile response was determined to control for variations in systemic blood pressure. The total erectile response or total ICP was determined by the area under the curve in cm H 2 O/s from the beginning of cavernous nerve stimulation until ICP returned to baseline or prestimulation pressure, and tumescence slope was calculated. Measurement of cavernous blood flow. Cavernous blood flow was measured by laser Doppler flow probe (Periflux 5000; Perimed AB, Stockholm, Sweden) after cavernous nerve stimulation (1 V, 1 ms, 12 Hz for 1 min). Three electrostimulations were replicated at intervals of 10 min, and the values are presented as an arbitrary perfusion unit. Maximal PU, total PU (area under the curve), and slope during the cavernous nerve stimulation were calculated. Immunohistochemistry and immunofluorescence. For immunohistochemistry, frozen tissue sections (20 Am) were incubated with antibody to Ang1 (Fitzgerald Industries, Inc., Concord, MA, USA) or VEGF (Santa Cruz Biotechnology, Santa Cruz, CA, USA), followed by Histostain-SP kit (Invitrogen Co., Carlsbad, CA, USA). The sections were subsequently stained with hematoxylin. For fluorescence microscopy, frozen tissue sections (20 Am) were incubated with antibody to factor VIII (DakoCytomation, Glostrup, Denmark), enos (Santa Cruz Biotechnology), nnos (Transduction Laboratories, Inc., Lexington, KY, USA), or FITC-conjugated antibody to a-sma (Sigma Aldrich, St. Louis, MO, USA) at 48C overnight. After several washes with PBS, the sections were incubated with Cy3-conjugated mouse antibody to IgG or Cy3-conjugated rabbit antibody to IgG for 1 h at room temperature. Signals were visualized and digital images were obtained with an Apotome microscope (Zeiss, Gfttingen, Germany). The extent of staining for factor VIII, enos, nnos, and a-sma was evaluated by calculating the labeled area (total labeled pixels) and the staining intensity (degree of labeling within the labeled area). For factor VIII, enos, and a-sma, each area was expressed as a percentage of the total cavernous area [(positive area/total cavernous area) 100]; for nnos, each area was expressed as a percentage of the high-power fields [(positive area/high-power fields) 100]. Detection of human Ang1- and human VEGF165-specific mrna in rat corpus cavernosum. The expression of human Ang1 and human VEGF165 mrna in the corpus cavernosum was detected by RT-PCR 3, 7, 14, and 21 days after intracavernous gene injection. First-strand cdna was synthesized by using reverse transcriptase with oligo(dt) primers, and PCR was performed with a human VEGF165-specific forward primer (5V-GATGAGATCGAGTACATCTT-3V) and reverse primer (5V-CACCG- CCTCGGCTTGTCACAT-3V) and a human Ang1-specific forward primer (5V-CAGGAGGATGGTGGTTTGATGCTT-3V) and reverse primer (5V- GCTTCCATCAAACGAGTTGGTGCT-3V). The set of primers for h-actin included the following: forward primer (5V-TCTACAATGAGCTGCGTG- TG-3V) and reverse primer (5V-AATGTCACGCACGATTTCCC-3V). All signals were visualized and analyzed by densitometry. Western blot. The expression of Ang1 and VEGF protein in the corpus cavernosum was detected by Western blot at 3, 7, 14, and 21 days after intracavernous gene injection. We also evaluated the expression of enos and phospho-enos in control and hypercholesterolemic rat penis 7, 14, and 56 days after injection (n = 4, respectively). Equal amounts of protein (60 Ag/lane) were electrophoresed on 8% SDS polyacrylamide gels, transferred to nitrocellulose membranes, and probed with antibody to Ang1 (Chemicon, Temecula, CA, USA; 1:50), VEGF (Santa Cruz Biotechnology; 1:200), enos (Transduction Laboratories, Inc.; 1:1000), phosphoenos (Ser1177, Cell Signaling, Beverly, MA, USA; 1:1000), or h-actin (Abcam, Cambridge, UK). Results were quantified by densitometry. Expression of b-galactosidase in cavernous tissue. h-galactosidase expression was evaluated in cavernous tissue samples by using a h- galactosidase reporter gene assay system (Galacto-Light Plus; Tropix, Bedford, MA, USA) at 3, 7, 14, or 21 days after intracavernous injection of virus vehicle alone or the ad-lacz construct (1 10 10 parts/0.1 ml, n =4at each time point). We also performed h-galactosidase histochemistry as previously described [43]. Measurement of cavernous tissue cgmp levels. At 7, 14, and 56 days after intracavernous gene injection (n = 4 per each time point), the penile tissue was removed and rinsed with PBS, quick frozen in liquid nitrogen, and stored at 708C until determination of cgmp levels. The samples were then processed according to the instructions provided with the kit (R&D Systems, Inc., Minneapolis, MN, USA). Data are expressed as pmol/ mg wet wt tissue. Statistical analysis. Results are expressed as means F SE. Statistical analysis was performed using one-way ANOVA followed by Scheffé multiple-comparison tests. Probability values less than 5% were considered significant. All statistical analyses were performed using the Statistical Package for Social Sciences, version 10.0, for Windows (SPSS, Inc., Chicago, IL, USA). 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