Phosphorylated Endothelial Nitric Oxide Synthase Mediates Vascular ...

BIOLOGY OF REPRODUCTION 70, 282–289 (2004) Published online before print 1 October 2003. DOI 10.1095/biolreprod.103.021113

Phosphorylated Endothelial Nitric Oxide Synthase Mediates Vascular Endothelial Growth Factor-Induced Penile Erection1 Biljana Musicki,2 Michael A. Palese, Julie K. Crone, and Arthur L. Burnett Department of Urology, The Johns Hopkins Hospital, Baltimore, Maryland 21287 ABSTRACT

Reports from several laboratories indicate the role of angiogenic growth factors, such as vascular endothelial growth factor (VEGF), in the modulation of penile vascularity and the improvement of erectile response. Both human and rat VEGF exist in several isoforms of 121, 145, 165, 189, and 206 amino acids (human) as a result of differential splicing [5]. Previous studies have shown that all known VEGF isoforms are abundantly expressed in rat and human penile tissues [6–8]. Local intracavernosal delivery of VEGF has been shown to recover erectile function in rat and rabbit models of vasculogenic erectile dysfunction induced by castration [9], traumatized iliac arteries [10], or hyperlipidemia [11–13]. However, the mechanisms by which VEGF stimulates erection are complex and not fully understood. The VEGF promotes endothelial cell proliferation and migration in vitro and angiogenesis in vivo, and it enhances microvascular permeability. These effects of VEGF are largely dependent on eNOS activation, as shown with in vitro models and verified using animal models. The VEGFstimulated endothelial cell proliferation, migration, and angiogenesis can be blocked by NO synthase inhibition [14, 15]. In eNOS knockout (eNOS2/2) mice, angiogenesis is impaired following ischemic challenge and is not restored by VEGF treatment [16, 17]. However, the role of NO in angiogenesis is controversial, and the mechanism by which VEGF induces eNOS activation also remains unclear. Treatment of endothelial cells with VEGF promotes the activation of eNOS by a transient increase in intracellular calcium as well as by activation of diverse protein tyrosine kinase and serine/threonine kinase pathways [18]. The VEGF stimulates protein kinase B (Akt), which directly phosphorylates eNOS at serine (Ser) 1177 [19–21]. This posttranslational modification accelerates electron flux through the enzyme, reducing the enzyme’s calcium requirement and causing increased and prolonged production of NO [22]. This pathway is responsible for both shear stress- and growth factor-enhanced blood flow. Previous studies in our laboratory have shown that phosphorylation of eNOS at Ser1177 in the penis mediates blood flow-induced erection by shear stress stimulation [23]. In rat and rabbit models of vasculogenic erectile dysfunction, intracavernosal delivery of VEGF restores erectile function within weeks and is associated with increased cavernosal endothelial cell content and restoration of neural and smooth muscle integrity [10, 11]. Cavernosal smooth muscle cells express VEGF receptor 1 and respond in vitro to VEGF stimulation by proliferation and migration [8, 24]. These findings would suggest angiogenesis and the resulting restoration of blood supply and, possibly, trophic effects on smooth muscle cells as likely mechanisms for the observed VEGF-induced recovery in erectile function, at least in the long term. Also, VEGF induces eNOS mRNA and protein levels in penile homogenates [25], providing a

The objective of the present study was to evaluate whether vascular endothelial growth factor (VEGF)-induced penile erection is mediated by activation of endothelial nitric oxide synthase (eNOS) through its phosphorylation. We assessed the role of constitutively activated eNOS in VEGF-induced penile erection using wild-type (WT) and eNOS-knockout (eNOS2/2) mice with and without vasculogenic erectile dysfunction. Adult WT and eNOS2/2 mice were subjected to sham operation or bilateral castration to induce vasculogenic erectile dysfunction. At the time of surgery, animals were injected intracavernosally with a replication-deficient adenovirus expressing human VEGF145 (109 particle units) or with empty virus (Ad.Null). After 7 days, erectile function was assessed in response to cavernous nerve electrical stimulation. Total and phosphorylated protein kinase B (Akt) as well as total and phosphorylated eNOS were quantitatively assessed in mice penes using Western immunoblot and immunohistochemistry. In intact WT mice, VEGF145 significantly increased erectile responses, and in WT mice after castration, it completely recovered penile erection. However, VEGF145 failed to increase erectile responses in intact eNOS2/ 2 mice and only partially recovered erectile function in castrated eNOS2/2 mice. In addition, VEGF145 significantly increased phosphorylation of eNOS at Serine 1177 by approximately 2fold in penes of both intact and castrated WT mice. The data provide a molecular explanation for VEGF stimulatory effect on penile erection, which involves phosphorylated eNOS (Serine 1177) mediation.

growth factors, male reproductive tract, nitric oxide, penis, signal transduction

INTRODUCTION

Penile erection is a hemodynamic process involving increased arterial inflow and restricted venous outflow. The role of nitric oxide (NO), generated by both endothelial NO synthase (eNOS) and neuronal NO synthase, as the main mediator of penile erection is well documented [1]. Nitric oxide enters smooth muscle cells and activates soluble guanylyl cyclase, leading to increased intracellular cGMP accumulation and activation of cGMP-dependent protein kinase, with the end result of increased smooth muscle relaxation. Penile vascular insufficiency drastically suppresses erectile capability and decreases NO synthase activity and NO production [2–4]. Supported by U.S. Public Health Service Grant DK 02568. Correspondence: Biljana Musicki, The Johns Hopkins Hospital, Department of Urology, 600 North Wolfe Street, Marburg 405, Baltimore, MD 21287-2411. Fax: 410 614 3695; e-mail: [email protected]

1 2

Received: 10 July 2003. First decision: 29 July 2003. Accepted: 23 September 2003. Q 2004 by the Society for the Study of Reproduction, Inc. ISSN: 0006-3363. http://www.biolreprod.org

282

PHOSPHO-eNOS MEDIATES VEGF-INDUCED ERECTION

mechanism for prolonged production of NO, which may support recovery of erectile function. However, evidence linking eNOS activity in the penis in response to both longand short-term VEGF treatment is lacking. In addition, angiogenesis is unlikely to have a role in the short-term effect of VEGF on penile erection. The present investigation was undertaken to evaluate a postulated mechanism of VEGF action in the penis associated with activation of eNOS through its phosphorylation. We assessed the role of eNOS in VEGF-induced penile erection using wild-type (WT) and eNOS2/2 mice both with and without vasculogenic erectile dysfunction induced by castration. We further evaluated whether VEGF, similar to shear stress, promoted penile erection by phosphorylation/ activation of Akt (Ser473) and eNOS (Ser1177). MATERIALS AND METHODS

283

FIG. 1. Representative Western blot analysis of VEGF in the mouse penis 3 days after vehicle or AdVEGF145 treatment of intact WT mice. Recombinant VEGF121 was included as a positive control, and the arrow points at the position of VEGF121 band. Using tissue from different mice, the same results were obtained for replicate experiments.

Reagents Polyclonal rabbit anti-phospho-Akt (Ser473), rabbit anti-Akt, and rabbit anti-phospho-eNOS (Ser1177) antibodies were from Cell Signaling Technology (Beverly, MA). Rabbit polyclonal anti-eNOS antibody was from BD Transduction Laboratories (San Diego, CA). Rabbit polyclonal anti-human VEGF antibody (against amino-terminal peptide) and VEGFblocking peptide were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Recombinant human VEGF121 was from Cell Sciences, Inc. (Norwood, MA). Replication-deficient adenovirus vector containing cDNA for human VEGF145 (AdVEGF145; driven by the cytomegalovirus promoter) and empty adenovirus (AdCMV.Null; used as a control vector) were supplied by GenVec, Inc. (Gaithersburg, MD). The 2959-ADP sepharose and enhanced chemiluminescence kit were purchased from Amersham Pharmacia Biotech (Piscataway, NJ). Elite Vectastain kit was obtained from Vector Laboratories, Inc. (Burlingame, CA). Diaminobenzidine was obtained from DAKO, (DakoCytomation, Carpinteria, CA).

lulose membrane. The membranes were stained with Ponceau Red to ascertain equal loading of proteins (except for purified eNOS), and probed with antibodies against phospho-eNOS (Ser1177) at 1:400, phospho-Akt (Ser473) at 1:1000, and VEGF at 1:400 dilution. Membranes used for phospho-protein analysis were then stripped and reprobed for eNOS (1: 1000) or Akt (1:1000). Band densities were quantified using NIH Image 1.29 (http://rsb.info.nih.gov/ij). Phospho-Akt and phospho-eNOS densities were normalized relative to those of total Akt and eNOS signals, respectively, and the ratio of phospho-protein to total protein was determined in terms of arbitrary units and expressed relative to the ratio for sham-treated, unstimulated animals prepared and blotted at the same time. For analysis of total eNOS expression by Western immunoblotting, a separate set of penile homogenates was used without purification. Molecular weight of VEGF was determined with recombinant VEGF121 and verified with the blocking peptide.

Animal Models

Immunohistochemistry and Quantitative Histomorphometry of Phospho-eNOS and eNOS

Adult male C57BL6 WT and eNOS2/2 mice, both from The Jackson Laboratories (Bar Harbor, ME), were used. To induce vasculogenic erectile dysfunction in mice [26], animals were castrated under anesthesia with 40 mg/kg of pentobarbital sodium. Immediately after castration or sham operation, the penis was exposed, and using a 30-gauge needle, 30 ml of AdVEGF145 (109 particle units) or vehicle (AdCMV.Null) were injected into the corpus cavernosum. The animals were used in the studies after 3–7 days. All experiments were conducted in accordance with the Johns Hopkins University School of Medicine Guidelines for the Care and Use of Animals.

Physiologic Erection Studies To monitor intracavernosal pressure (ICP), the shaft of the penis was denuded of skin and fascia, and the left corpus cavernosum was perforated with a 25-gauge needle connected via PE-50 tubing to a pressure transducer (DI-190; Dataq Instruments, Akron, OH). Because of concerns about the viability of mice with extensive procedures, cannulations of the right carotid artery to monitor systemic blood pressure (mean arterial pressure [MAP]) were performed only in pilot studies. For electrically stimulated penile erections, a bipolar electrode attached to a Grass Instruments S48 stimulator (Quincy, MA) was placed around the cavernous nerve as described previously [27]. Stimulation parameters were between 1 and 4 V at a frequency of 16 Hz with square-wave duration of 5 msec for 1 min. Response parameters were calculated using MATLAB software (Mathworks, Natick, MA).

Western Blot Analysis and Protein Band Densitometry For immunoblot studies, penes were excised at baseline and after electrical stimulation (4 V, 16 Hz, 5 msec, 1-min duration) of the cavernous nerve and processed for phosphoprotein and total protein analysis. Minced penile tissue was homogenized and partially purified for eNOS as described previously [23]. Purified eNOS (for phospho-eNOS analysis) or 50 mg of crude homogenate (for phospho-Akt and total Akt, total eNOS, and VEGF analyses) were resolved on 7.5%, 12%, or 15% Tris gels under reducing conditions and transferred to polyvinylidene fluoride or nitrocel-

Frozen transverse sections (thickness, 6 mm) of the whole penes were fixed in 4% paraformaldehyde and quenched in 1.5% hydrogen peroxide in PBS for 15 min. All slides were subsequently incubated in PBS containing 1% goat serum for 1 h and then at 48C overnight in PBS with 0.2% BSA with the indicated antibodies: eNOS at 1:250 dilution and phospho-eNOS (Ser1177) at 1:200 dilution [23]. Staining was performed with the Elite Vectastain kit using diaminobenzidine as the chromagen. Sections were subsequently counterstained with hematoxylin. The staining was analyzed using equipment and software of the AutoCyte Pathology Work Station (Tripath Imaging, Inc., Burlington, NC) running QUICK IMMUNO Version 1.1 software. Kohler Illumination was obtained, and the camera was white balanced before measurement fields were captured. Each slide contained the test samples (vehicle- and VEGF-treated) as well as the negative control (without the primary antibody), which was used for immunohistochemical calibration. For each tissue section, 10 fields of view were captured at 4003 magnification using a Zeiss Axioscope with a phased alternating line 3 CCD color camera [28]. The extent of staining for phospho-eNOS and eNOS was evaluated by calculating the labeled area (total labeled pixels) and the staining intensity (degree of labeling within the labeled area).

Statistical Evaluation Statistical analysis was performed by using one-way ANOVA followed by Newman-Keuls multiple comparison test or by t-test when appropriate. The data are expressed as the mean 6 SEM. A value of P , 0.05 was considered to be significant.

RESULTS

Determination of VEGF Transfer in AdVEGF145-Treated Mice

In preliminary dose-response studies, we established that the dose of 109 particle units per mouse was sufficient to

284

MUSICKI ET AL.

TABLE 1. Erectile response (ICPmax : MAP) to electrical stimulation of the cavernous nerve at increasing voltage in vehicle- and AdVEGF145-treated WT mice. Treatmenta Voltage 1 2 4

Vehicle

VEGF

0.38 6 0.10 0.52 6 0.16 0.58 6 0.04

0.51 6 0.06 0.89 6 0.04b 0.80 6 0.09bc

Data are presented as the mean 6 SEM (n 5 3–7). P , 0.05 compared with AdVEGF-treated mice at 1 V. c P , 0.05 compared with vehicle-treated mice at 4 V. a

b

induce penile erection. We confirmed VEGF transfection by Western immunoblot analysis. Three days after intracavernosal injection of AdVEGF145, VEGF protein was abundantly expressed in the penis of AdVEGF145-transfected WT mice, whereas trace amounts, corresponding to endogenous VEGF, were present in vehicle-treated controls (Fig. 1). Seven-days posttransfection, a somewhat less intense VEGF signal was observed in AdVEGF145-treated mice, but the levels of VEGF were still greater than in vehicle-treated controls. Effect of AdVEGF145 Treatment on Erectile Responses in WT and eNOS2/2 Mice

Erectile response, expressed as the ratio of maximal ICP (ICPmax) to MAP, increased with increasing voltage during

FIG. 2. Effect of AdVEGF145 treatment on maximal ICP in intact and castrated WT and eNOS2/2 mice after electrical stimulation of the cavernous nerve. Each bar represents the mean 6 SEM (n 5 6). a, P , 0.05 vs. intact 1 vehicle; b, P , 0.05 vs. castrated 1 vehicle.

FIG. 3. Western blot analysis demonstrating the relative protein abundance of phospho-eNOS (Ser1177) in the mouse penis 7 days after AdVEGF145 or vehicle treatment of intact and castrated WT mice. A) Representative Western immunoblot of phospho-eNOS (Ser1177) and total eNOS at baseline in the penis of intact 1 vehicle (lane 3), castrated 1 vehicle (lane 4), intact 1 VEGF (lane 5), and castrated 1 VEGF (lane 6) mice. Controls were obtained from HEK293 cells transfected with a plasmid expressing eNOS and starved of serum for 24 h (negative control, lane 1) or starved and then stimulated with 50 ng/ml of IGF-1 for 5 min (positive control, lane 2). B) Quantitative analysis of phospho-eNOS (Ser1177) levels in the mouse penis 7 days after intracavernosal AdVEGF145 or vehicle treatment of intact and castrated WT mice at baseline and after electrical stimulation (ES) of the cavernous nerve for 1 min. Bands were quantified in terms of arbitrary units by densitometry. Data are representative of five to eight densitometric values from independent experiments with separate mice for each experiment and are presented as the mean 6 SEM of the ratio of phospho-protein/total protein expressed relative to results obtained with intact animals treated with vehicle at baseline. a, P , 0.05 vs. intact 1 vehicle at baseline; b, P , 0.05 vs. castrated 1 vehicle at baseline.

electrical stimulation of the cavernous nerve in vehicletreated WT mice, with a maximum ICPmax:MAP ratio obtained at 4 V (Table 1). In AdVEGF145-treated WT mice, the ICPmax:MAP ratio reached maximal levels by 2 V. However, at 2 V, the ICPmax:MAP ratio in vehicle- and AdVEGF-treated WT mice did not differ statistically. At 4 V, the ICPmax:MAP ratio in AdVEGF145-treated mice was significantly (P , 0.05) higher than in vehicle-treated mice (Table 1). All subsequent studies were performed with 4 V. Figure 2 shows that the VEGF145 gene, administered intracavernosally into intact WT mice, significantly (P , 0.05) increased the ICP response to electrical stimulation of the cavernous nerve compared with vehicle-treated controls. Castration of WT mice decreased ICP by 70% (P , 0.05), and this decreased ICP was completely reversed by intracavernosal injection of AdVEGF145. However, VEGF145 gene administered intracavernosally into eNOS2/2 mice did not affect the ICP response to electrical stimulation of the cavernous nerve. Castration of eNOS2/2 mice decreased ICP by 50% (P , 0.05). Intracavernosal injection of AdVEGF145 in castrated eNOS2/2 mice only partially reversed ICP (P , 0.05 vs. castrated 1 vehicle, P , 0.05 vs. intact 1 vehicle) (Fig. 2). Notably, consistent with our previous work [29], we observed a rather sub-

285


FIG. 4. Immunohistochemical localization of phospho-eNOS (Ser1177) and eNOS in frozen serial sections of penile tissue obtained from WT mice treated with vehicle and AdVEGF145. The negative control section processed without the antibodies did not stain (data not shown). Sections represent the dorsal aspect of penis. Arrows indicate phospho-eNOS (Ser1177) and eNOS staining. A, artery; N, nerve bundle; V, vein. Magnification 340 (objective) and 310 (eye piece).

stantial erectile response to cavernous nerve stimulation in eNOS2/2 mice compared with WT mice (P , 0.05). Effect of AdVEGF145 Treatment on Phosphorylation of eNOS in Penes of WT Mice

Intracavernosally administered AdVEGF145 dramatically increased endogenous eNOS (Ser1177) phosphorylation in mice penes as determined by immunoblot analysis (Fig. 3). In sham-operated mice, basal levels of phospho-eNOS (Ser1177) were significantly (P , 0.05) increased by AdVEGF145 by approximately 2.5-fold as determined by the ratio of phospho-eNOS to total eNOS. Although castration did not result in significant changes in basal phospho-eNOS levels compared to those of unstimulated intact mice, castrated mice injected intracavernosally with VEGF145 gene exhibited significant (P , 0.05) increases in basal phospho-eNOS expression, approximating levels found in VEGF-treated intact animals. Electrical stimulation of the cavernous nerve of both intact and castrated WT mice treated with vehicle significantly (P , 0.05) increased

the levels of phospho-eNOS in the penis by approximately 2-fold. In contrast, electrical stimulation of the cavernous nerve of intact and castrated WT mice treated with AdVEGF145 did not produce any further increases in phospho-eNOS levels. We localized phospho-eNOS (Ser1177) immunohistochemically in serial sections of penes after intracavernosal delivery of vehicle or AdVEGF145 to intact and castrated WT mice. The phospho-eNOS was localized in the endothelial layers of the dorsal penile arteries and vein and in the sinusoids of the corpora cavernosa (Fig. 4). Similar to the results of Western blot analysis, immunohistochemical study also demonstrated that intracavernosally administered AdVEGF145 significantly (P , 0.05) increased endogenous eNOS (Ser1177) phosphorylation in mice penes (Table 2 and Fig. 4). Effect of AdVEGF145 Treatment on Total eNOS Protein in Penes of WT Mice Treatment with AdVEGF145 did not change the expression of total eNOS protein in the penis of intact and cas-

TABLE 2. The extent of staining for phospho-eNOS (Ser1177) and eNOS in penes from AdVEGF145-treated intact and castrated WT mice.a Phospho-eNOS (Ser1177) Treatment Intact 1 vehicle Intact 1 VEGF Castrated 1 vehicle Castrated 1 VEGF

Total eNOS

Labeled area

Staining intensity

Labeled area

Staining intensity

100 237.6 6 33.7b 100 157.4 6 18.9b

100 151.3 6 16.8b 100 127.0 6 5.1b

100 98.4 6 19.6 100 92.3 6 11.2

100 99.6 6 9.1 100 92.4 6 8.2

Data are representative of 10 fields from independent experiments with three to four mice for each treatment. Data are presented as the mean 6 SEM relative to results obtained with vehicle-treated intact and castrated control mice, respectively. b P , 0.05 compared with vehicle-treated control. a

286

MUSICKI ET AL.

FIG. 5. Western blot analysis demonstrating the relative protein abundance of phospho-Akt (Ser473) in the mouse penis 7 days after AdVEGF145 or vehicle treatment of intact and castrated WT mice. A) Representative Western immunoblot of phospho-Akt (Ser473) and total Akt as detailed in the legend to Figure 3. Controls are obtained from HEK293 cells transfected with a plasmid expressing Akt and starved of serum for 24 h (negative control, lane 1) or starved and then stimulated with 50 ng/ ml of IGF-1 for 5 min (positive control, lane 2). B) Quantitative analysis of phospho-Akt (Ser473) levels as described in the legend to Figure 2. a, P , 0.05 vs. intact 1 vehicle at baseline; b, P , 0.05 vs. castrated 1 vehicle at baseline.

trated mice as determined by Western blot analysis and immunohistochemistry. Western immunoblot analysis of nonpurified penile homogenates showed that eNOS protein, normalized to total proteins, remained unchanged with the treatments: intact 1 VEGF, 96.6% 6 12.2%; castrated 1 vehicle, 96.0% 6 12.5%; castrated 1 VEGF, 107.9% 6 12.9% (all compared to vehicle-treated intact control [100%]). Immunohistochemical analysis also showed that total eNOS protein did not change with AdVEGF145 treatment (Table 2 and Figure 4). In addition, eNOS was confirmed to be localized in the sinusoidal and vascular endothelium (Fig. 4). Effect of AdVEGF145 Treatment on Phosphorylation of Akt in Penes of WT Mice

The VEGF145 gene, when administered intracavernosally into intact and castrated WT mice, did not have any effect on either basal or stimulated levels of phospho-Akt (Ser473) in the penis (Fig. 5). Electrical stimulation of the cavernous nerve of intact and castrated WT mice significantly (P , 0.05) increased the levels of phospho-Akt. Castration resulted in a significant (P , 0.05) decrease in phospho-Akt levels compared to those of intact mice. DISCUSSION

The major finding of the present study is that eNOS constitutively activated by Ser1177 phosphorylation mediates the erectogenic actions of VEGF. This conclusion is based on several observations. First, VEGF improved and restored erections in WT mice during intact and castrated conditions, respectively, but it did not do so in mice lacking

eNOS. Second, eNOS was phosphorylated in the penis following VEGF treatment of both intact and castrated WT mice. We additionally observed that eNOS2/2 mice display rather substantial erectile function compared with WT mice after cavernous nerve stimulation, which is consistent with our previous report [29], although the exact mechanism for this phenomenon is not known. Thus, although we confirmed that VEGF exerts potent erectogenic effects, consistent with several previous reports of VEGF-induced erection in animal models of vasculogenic erectile dysfunction [9–13], we have gained new understanding about the mechanisms of VEGF action in the penis. Originally, eNOS was thought to be expressed in endothelial cells as a constitutive enzyme. Recent studies, however, have demonstrated that eNOS is expressed in diverse cell types, and its activity is dynamically regulated. In addition to being controlled by calcium/calmodulin [30], the activity of eNOS is regulated by complex signal transduction pathways involving a direct interaction of the enzyme with cellular proteins (e.g., heat shock protein 90 and caveolin-1), by subcellular localization, and by posttranslational modifications, including N-myristoylation, cysteine palmitoylation, and phosphorylation [31]. Phosphorylation of eNOS regulates activation of the enzyme by various stimuli, including shear stress [19, 32, 33] and several growth factors and hormones [19–21, 34]. We have recently demonstrated the importance of Akt (Ser473) and eNOS (Ser1177) phosphorylation in blood flow-induced penile erection [23]. Our present study demonstrates that VEGF, similar to shear stress, induced penile erection by phosphorylation of eNOS (Ser1177). Apparently, VEGF induced maximal phosphorylation of eNOS, which could not be further increased by electrical stimulation of the cavernous nerve. Maximal VEGF activation and phosphorylation of eNOS in cultured endothelial cells consistently have been reported to be only approximately 2-fold [20, 21]. In addition, VEGF had no effect on the total amount of eNOS as determined by both Western blot analysis (in a separate set of penile homogenates) and by immunohistochemistry. This finding differs from those of Lin et al [25], who demonstrated up-regulation of eNOS expression in the penis after a single intracavernosal injection of VEGF protein. This disparity may result from a different form and a different dose of VEGF being used in different studies. Our result demonstrates that the increase in phospho-eNOS was not caused by changes in the eNOS protein expression level. Rather, it represents changes in the phosphorylation status of the enzyme. Based on our previous finding that the phospho-Akt/ phospho-eNOS pathway mediates shear stress-induced penile erection in the rat [23] and on studies showing that VEGF mainly phosphorylates eNOS (Ser1177) in endothelial cells via Akt [31], we considered the possibility that a similar paradigm might lead to VEGF-induced penile erection. However, we were unable to detect increased phosphorylation of Akt in the penis of AdVEGF145-treated mice. The use of VEGF gene delivery did not allow us to perform short-term functional studies with phosphatidylinositol 3-kinase (PI3-kinase)/Akt inhibitors to assess the functional role of Akt in VEGF-stimulated penile erection. Thus, we cannot exclude the possible mediatory action of PI3-kinase/Akt in VEGF action in the penis. At the same time, the possibility that VEGF signaling to phospho-eNOS in the penis may involve pathways not including Akt is worthy of consideration. Michell et al. [21] reported that whereas both insulin-like growth factor (IGF)-


1 and VEGF activated and phosphorylated Akt and eNOS in bovine aortic endothelial cells, IGF-1 was a more potent activator of Akt, and VEGF promoted greater phosphorylation of eNOS at Ser1177. The same study also demonstrated that inhibition of PI3-kinase, an upstream activator of Akt, completely inhibited VEGF-induced Akt activation but only partially inhibited VEGF-induced phosphorylation of eNOS at Ser1177. One of the explanations, as discussed by those authors, was that VEGF signals more strongly by an alternative pathway, perhaps by increasing intracellular calcium through the activation of phospholipase Cg. On eNOS, Ser1177 is the target of multiple protein kinases, in addition to Akt, including AMP-activated protein kinase [35], cAMP- and cGMP-dependent protein kinases [32, 33, 36], and protein kinase C [37]. Moreover, eNOS can be phosphorylated at several other sites in addition to Ser1177 (human)/Ser1179 (bovine), including threonine (Thr)495 (human)/Thr497 (bovine) [37, 38], Ser114 (human)/Ser116 (bovine) [39], Ser633 (human)/Ser635 (bovine) [32, 40], and Ser615 (human)/Ser617 (bovine) [40]. Depending on the vascular origin of endothelial cells, VEGF may promote eNOS activation not only through phosphorylation of Ser1177 but also through phosphorylation of other positive regulatory sites, such as Ser635 [32, 40] and Ser617 [40), and dephosphorylation of Thr495 [37, 41] and Ser116 [39] (negative regulatory sites). Although the molecular pathway leading to eNOS (Ser1177) phosphorylation by VEGF in the mouse penis is not known at present, it appears that eNOS (Ser1177) can be phosphorylated by different protein kinases depending on the cellular context and that the phosphorylation of Ser1177 may be affected by the state of phosphorylation/dephosphorylation of other residues on eNOS [42]. Further functional studies using specific protein kinase inhibitors may provide clarification regarding the cellular pathway that mediates the VEGF stimulatory effect on eNOS phosphorylation in the penis. Sustained NO production in response to VEGF-induced eNOS phosphorylation apparently increases the erection potential of the penis and enhances the functional response of the penis to neurostimulation. However, the exact mechanism by which VEGF improves penile vascular homeostasis and erection physiology is not known. Angiogenesis is unlikely to have a role in the action of VEGF on penile erection within 7 days; long-term VEGF exposure may involve angiogenic tissue proliferative effects. Increased production of NO may change the balance between pro- and antierectile mechanisms that operate physiologically in the penis. The balance between vasorelaxation and vasoconstriction pathways appears to control the degree of contraction of the smooth muscle of the corpora cavernosa and determines the functional state of the penis [43]. Vasorelaxation in the penis is mostly mediated by the NO-cGMPprotein kinase G pathway. Vasoconstriction (evoked by noradrenaline, endothelins, and angiotensins), which maintains the penis in the nonerect state, is mediated, in part, by the RhoA/Rho-kinase pathway. During erection, this pathway is inhibited, most likely by NO [44]. Thus, increased production of NO may overcome the inhibitory effects of antierectile mechanisms, leading to full erection. We found that castration caused a decrease in phosphoAkt levels in the mouse penis. Testosterone, in contrast to estrogen, has been shown to have no effect on androgen receptor-associated PI3-kinase activity [34]. Thus, it appears that in the castration model used in the present study to induce vasculogenic erectile dysfunction, withdrawal of testosterone may, in addition to decreasing erectile func-

287

tion, have some nonspecific effect on penile tissue. At this point, the finding cannot be explained and awaits further clarification. A mouse model of vasculogenic erectile dysfunction induced by castration, which we used in the present study, was established by Palese et al [26]. Those authors demonstrated that 7 days after castration, mice exhibited a maximal reduction in erectile response most likely associated with a defect in veno-occlusion, which is consistent with similar observations in rats [45, 46]. This time period was also chosen in the present study based on the characteristics of adenoviral delivery of VEGF. We used replication-deficient adenovirus vector containing cDNA for human VEGF145 to deliver VEGF directly into the corpora cavernosa. The VEGF145 is present in the penis [6], and it has been shown to induce the proliferation of vascular endothelial cells and to promote angiogenesis in vivo [47] similar to VEGF121 and VEGF165, the most predominant VEGF isoforms in the penis. Studies in experimental animals have shown that replication-deficient, recombinant adenovirus gene transfer provides high transfection efficiency and results in highly localized increases in protein expression within a few days after infection, which returns to the control level at approximately Day 14 [48]. We measured VEGF expression in penile tissue to confirm that VEGF was elevated appropriately after AdVEGF145 treatment. Our findings that castrated eNOS2/2 mice did exhibit a minimal degree of erectile recovery with VEGF treatment suggest that the VEGF effect on erection may involve an alternative signaling pathway in eNOS2/2 mice, as well as in WT mice, that is distinct from its effect on eNOS. It has been shown that rat [24] and human [8] cavernosal smooth muscle cells express VEGF receptor 1 and respond in vitro to VEGF stimulation by proliferation and migration. Thus, the trophic effect of VEGF on smooth muscle cells may be an additional mechanism of action by VEGF, leading to improvement in erectile function. In conclusion, the present study offers a molecular mechanism involving eNOS phosphorylation that accounts for short-term, VEGF-induced penile erection that is consistent with the physiologic events of penile erection. These findings establish a specific basis whereby VEGF promotes erectile function, although they do not exclude other mechanisms for the erection-promoting effects of VEGF over a long-term period. ACKNOWLEDGMENTS We would like to thank Dr. Robert W. Veltri and Mr. Cameron van Rootselaar for assistance with the AutoCyte Pathology Work Station and histomorphometric analysis. We also thank Dr. Imre Kovesdi for helpful discussion regarding adenoviral construct.

REFERENCES 1. Burnett AL, Lowenstein CJ, Bredt DS, Chang TS, Snyder SH. Nitric oxide: a physiologic mediator of penile erection. Science 1992; 257: 401–403. 2. Traish AM, Park K, Dhir V, Kim NN, Moreland RB, Goldstein I. Effects of castration and androgen replacement on erectile function in a rabbit model. Endocrinology 1999; 140:1861–1868. 3. Park KH, Kim SW, Kim KD, Paick JS. Effects of androgens on the expression of nitric oxide synthase mRNA in rat corpus cavernosum. BJU Int 1999; 83:327–333. 4. Marin R, Escrig A, Abreu P, Mas M. Androgen-dependent nitric oxide release in rat penis correlates with levels of constitutive nitric oxide synthase isoenzymes. Biol Reprod 1999; 61:1012–1016. 5. Robinson CJ, Stringer SE. The splice variants of vascular endothelial

288

6.

7.

8. 9.

10. 11.

12.

13.

14.

15. 16.

17.

18.

19. 20.

21.

22.

23.

24. 25.

MUSICKI ET AL.

growth factor (VEGF) and their receptors. J Cell Sci 2001; 114:853– 865. Burchardt T, Burchardt M, Chen MW, Buttyan R, de la Taille A, Shabsigh A, Shabsigh R. Expression of VEGF splice variants 144/ 145 and 205/206 in adult male tissues. IUBMB Life 1999; 49:405– 408. Burchardt M, Burchardt T, Chen MW, Shabsigh A, de la Taille A, Buttyan R, Shabsigh R. Expression of messenger ribonucleic acid splice variants for vascular endothelial growth factor in the penis of adult rats and humans. Biol Reprod 1999; 60:398–404. Rajasekaran M, Kasyan A, Allilain W, Monga M. Ex vivo expression of angiogenic growth factors and their receptors in human penile cavernosal cells. J Androl 2003; 24:85–90. Rogers RS, Graziottin TM, Lin CS, Kan YW, Lue TF. Intracavernosal vascular endothelial growth factor (VEGF) injection and adeno-associated virus-mediated VEGF gene therapy prevent and reverse venogenic erectile dysfunction in rats. Int J Impot Res 2003; 15:26–37. Lee MC, El-Sakka AI, Graziottin TM, Ho HC, Lin CS, Lue TF. The effect of vascular endothelial growth factor on a rat model of traumatic arteriogenic erectile dysfunction. J Urol 2002; 167:761–767. Byrne RR, Henry GD, Rao DS, Huynh TT, Pippen Am, Annex BH, Hagen PO, Donatucci CF. Vascular endothelial growth factor restores corporeal smooth muscle function in vitro. J Urol 2001; 165:1310– 1315. Henry GD, Byrne R, Hunyh TT, Abraham V, Annex BH, Hagen PO, Donatucci CF. Intracavernosal injections of vascular endothelial growth factor protects endothelial dependent corpora cavernosal smooth muscle relaxation in the hypercholesterolemic rabbit: a preliminary study. Int J Impot Res 2000; 12:334–339. Gholami SS, Rogers R, Chang J, Ho HC, Grazziottin T, Lin CS, Lue TF. The effect of vascular endothelial growth factor and adeno-associated virus mediated brain derived neurotrophic factor on neurogenic and vasculogenic erectile dysfunction induced by hyperlipidemia. J Urol 2003; 169:1577–1581. Papapetropoulos A, Garcia-Cardena G, Madri JA, Sessa WC. Nitric oxide production contributes to the angiogenic properties of vascular endothelial growth factor in human endothelial cells. J Clin Invest 1997; 100:3131–3139. Kon K, Fujii S, Kosaka H, Fujiwara T. Nitric oxide synthase inhibition by N(G)-nitro-L-arginine methyl ester retards vascular sprouting in angiogenesis. Microvasc Res 2003; 65:2–8. Murohara T, Asahara T, Silver M, Bauters C, Masuda H, Kalka C, Kearney M, Chen D, Chen D, Symes JF, Fishman MC, Huang PL. Nitric oxide synthase modulates angiogenesis in response to tissue ischemia. J Clin Invest 1998; 101:2567–2578. Fukumura D, Gohongi T, Kadambi A, Izumi Y, Ang J, Yun CO, Buerk DG, Huang PL, Jain RK. Predominant role of endothelial nitric oxide synthase in vascular endothelial growth factor-induced angiogenesis and vascular permeability. Proc Natl Acad Sci U S A 2001; 98:2604– 2609. Gelinas DS, Bernatchez PN, Rollin S, Bazan NG, Sirois MG. Immediate and delayed VEGF-mediated NO synthesis in endothelial cells: role of PI3K, PKC, and PLC pathways. Br J Pharmacol 2002; 137:1021–1030. Dimmeler S, Fleming I, Fisslthaler B, Hermann C, Busse R, Zeiher AM. Activation of nitric oxide synthase in endothelial cells by Aktdependent phosphorylation. Nature 1999; 399:601–605. Fulton D, Gratton JP, McCabe TJ, Fontana J, Fujio Y, Walsh K, Franke TF, Papapetropoulos A, Sessa WC. Regulation of endothelium-derived nitric oxide production by the protein kinase Akt. Nature 1999; 399: 597–601. Michell BJ, Griffiths JE, Mitchelhill KI, Rodriguez-Crespo I, Tiganis T, Bozinovski S, de Montellano PR, Kemp BE, Pearson RB. The Akt kinase signals directly to endothelial nitric oxide synthase. Curr Biol 1999; 9:845–848. McCabe TJ, Fulton D, Roman LJ, Sessa WC. Enhanced electron flux and reduced calmodulin dissociation may explain ‘‘calcium-independent’’ eNOS activation by phosphorylation. J Biol Chem 2000; 275: 6123–6128. Hurt KJ, Musicki B, Palese MA, Crone JK, Becker RE, Moriarity JL, Snyder SH, Burnett AL. Akt-dependent phosphorylation of endothelial nitric-oxide synthase mediates penile erection. Proc Natl Acad Sci U S A 2002; 99:4061–4066. Liu X, Lin CS, Graziottin T, Resplande J, Lue TF. Vascular endothelial growth factor promotes proliferation and migration of cavernous smooth muscle cells. J Urol 2001; 166:354–360. Lin CS, Ho HC, Chen KC, Lin G, Nunes L, Lue TF. Intracavernosal

26. 27. 28. 29. 30. 31. 32.

33.

34. 35.

36.

37.

38.

39.

40.

41.

42.

43. 44.

45.

46.

47.

injection of vascular endothelial growth factor induces nitric oxide synthase isoforms. BJU Int 2002; 89:955–960. Palese MA, Crone JK, Burnett AL. A castrated mouse model of erectile dysfunction. J Androl 2003; 24:699–703. Sezen S, Burnett AL. Intracavernosal pressure monitoring in mice: responses to electrical stimulation of the cavernous nerve and to intracavernosal drug administration. J Androl 2000; 21:311–315. Bishop JW, Marcelpoil R, Schmid J. Machine scoring of Her2/neu immunohistochemical stains. Anal Quant Cytol Histol 2002; 24:257– 262. Burnett AL, Chang AG, Crone JK, Huang PL, Sezen SE. Noncholinergic penile erection in mice lacking the gene for endothelial nitric oxide synthase. J Androl 2002; 23:92–97. Busse R, Mulsch A. Calcium-dependent nitric oxide synthesis in endothelial cytosol is mediated by calmodulin. FEBS Lett 1990; 265: 133–136. Fleming I, Busse R. Molecular mechanisms involved in the regulation of the endothelial nitric oxide synthase. Am J Physiol Regul Integr Comp Physiol 2003; 284:R1–R12. Boo YC, Hwang J, Sykes M, Michell BJ, Kemp BE, Lum H, Jo H. Shear stress stimulates phosphorylation of eNOS at Ser 635 by a protein kinase A-dependent mechanism. Am J Physiol Heart Circ Physiol 2002; 283:H1819–H1828. Boo YC, Sorescu G, Boyd N, Shiojimas I, Walsh K, Du J, Jo H. Shear stress stimulates phosphorylation of endothelial nitric oxidesynthase at Ser 1179 by Akt-dependent mechanisms. Role of protein kinase A. J Biol Chem 2002; 277:3388–3396. Simoncini T, Hafezi-Moghadam A, Brazil DP, Ley K, Chin WW, Liao J. Interaction of estrogen receptor with the regulatory subunit of phosphatidylinositol-3-OH kinase. Nature 2000; 407:538–541. Chen ZP, Mitchelhill KI, Michell BJ, Stapleton D, Rodriguez-Crespo I, Witters LA, Power DA, de Montellano PR, Kemp BE. AMP-activated protein kinase phosphorylation of endothelial NO synthase. FEBS Lett 1999; 443:285–289. Butt E, Bernhardt M, Smolenski A, Kotsonis P, Fro¨hlich LG, Sickmann A, Meyer HE, Lohmann SM, Schmidt HH. Endothelial nitricoxide synthase (type III) is activated and becomes calcium independent upon phosphorylation by cyclic nucleotide-dependent protein kinases. J Biol Chem 2000; 275:5179–5187. Harris MB, Ju H, Venema VJ, Liang H, Zou R, Michell BJ, Chen ZP, Kemp BE, Venema RC. Reciprocal phosphorylation and regulation of endothelial nitric oxide-synthase in response to bradykinin stimulation. J Biol Chem 2001; 276:16587–16591. Fleming I, Fisslthaler B, Dimmeler S, Kemp BE, Busse R. Phosphorylation of Thr(495) regulates Ca(21)/calmodulin-dependent endothelial nitric oxide synthase activity. Circ Res 2001; 88:E68–E75. Kou R, Greif D, Michel T. Dephosphorylation of endothelial nitric oxide-synthase by vascular endothelial growth factor. Implications for the vascular response to cyclosporine A. J Biol Chem 2002; 277: 29669–29673. Michell BJ, Harris MB, Chen ZP, Ju H, Venema VJ, Blackstone MA, Huang W, Venema RC, Kemp BE. Identification of regulatory sites of phosphorylation of the bovine endothelial nitric-oxide synthase at serine 617 and serine 635. J Biol Chem 2002; 277:42344–42351. Michell BJ, Chen Z, Tiganis T, Stapleton D, Katsis F, Power DA, Sim AT, Kemp BE. Coordinated control of endothelial nitric-oxide synthase phosphorylation by protein kinase C and cAMP-dependent protein kinase. J Biol Chem 2001; 276:17625–17628. Bauer PM, Fulton D, Boo YC, Sorescu GP, Kemp BE, Jo H, Sessa WC. Compensatory phosphorylation and protein-protein interactions revealed by loss of function and gain of function mutants of multiple serine phosphorylation sites in endothelial nitric oxide synthase. J Biol Chem 2003; 278:14841–14849. Andersson KE. Pharmacology of penile erection. Pharmacol Rev 2001; 53:417–450. Mills TM, Chitaley K, Lewis RW, Webb RC. Nitric oxide inhibits RhoA/Rho-kinase signaling to cause penile erection. Eur J Pharmacol 2002; 439:173–174. Dai Y, Stopper V, Lewis R, Mills T. Effects of castration and testosterone replacement on veno-occlusion during penile erection in the rat. Asian J Androl 1999; 1:53–59. Mills TM, Lewis RW, Stopper VS. Androgenic maintenance of inflow and veno-occlusion during erection in the rat. Biol Reprod 1998; 59: 1413–1418. Poltorak Z, Cohen T, Sivan R, Kandelis Y, Spira G, Vlodavsky I, Keshet E, Neufeld G. VEGF145, a secreted vascular endothelial

PHOSPHO-eNOS MEDIATES VEGF-INDUCED ERECTION growth factor isoform that binds to extracellular matrix. J Biol Chem 1997; 272:7151–7158. 48. Gowdak LH, Poliakova L, Wang X, Kovesdi I, Fishbein KW, Zacheo A, Palumbo R, Straino S, Emanueli C, Marrocco-Trischitta M, Lakatta

289

EG, Anversa P, Spencer RG, Talan M, Capogrossi MC. Adenovirusmediated VEGF(121) gene transfer stimulates angiogenesis in normoperfused skeletal muscle and preserves tissue perfusion after induction of ischemia. Circulation 2000; 102:565–571.