International Journal of Urology (2016) 23, 614--622
doi: 10.1111/iju.13105
Original Article: Laboratory investigation
Penile erection induces angiogenic, survival, and antifibrotic signals: molecular events associated with penile erection induced by cavernous nerve stimulation in mice Mi-Hye Kwon,1,† Soo-Hwan Park,1,† Kang-Moon Song,1 Kalyan Ghatak,1 Anita Limanjaya,1 Dong-Soo Ryu,2 Jiyeon Ock,1 Soon-Sun Hong,3,4 Ji-Kan Ryu1,4 and Jun-Kyu Suh1 1
National Research Center for Sexual Medicine and Department of Urology, Inha University School of Medicine, Incheon, Department of Urology, Sungkyunkwan University School of Medicine, Samsung Changwon Hospital, Changwon, 3Department of Medicine, and 4Inha Research Institute for Medical Sciences, Inha University School of Medicine, Incheon, Korea 2
Abbreviations & Acronyms Ang1 = angiopoietin-1 ED = erectile dysfunction ES = electrical stimulation HGF = hepatocyte growth factor ICP = intracavernous pressure MMP-2 = matrix metalloproteinase 2 MMP-9 = matrix metalloproteinase 9 MSBP = mean systolic blood pressure NG2 = neural/glial antigen 2 PAI-1 = plasminogen activator inhibitor-1 PDGFR-b = platelet-derived
growth factor receptor-b PECAM-1 = platelet/endothelial cell adhesion molecule-1 PI3K = phosphatidylinositol 3kinase RT–PCR = reverse
transcription polymerase chain reaction SMA = smooth muscle a-actin TGF-b = transforming growth factor-b
Objectives: To determine the molecular events related to penile erection in the corpus cavernosum tissue of mice after electrical stimulation of the cavernous nerve. Methods: Twelve-week-old male C57BL/6 mice were used in this study. Electrical stimulation of the cavernous nerve was carried out to induce penile erection. Corpus cavernosum tissues were then harvested to determine the effect of nerve-induced penile erection on signaling pathway involved in angiogenesis (vascular endothelial growth factor, hepatocyte growth factor, angiopoietin-1, matrix metalloproteinase 2, and matrix metalloproteinase 9), cell survival and proliferation (phosphatidylinositol 3-kinase, phospho-Akt/Akt, and phospho-ERK/ERK), and tissue fibrosis (phospho-Smad2/Smad2, phospho-Smad3/Smad3, and plasminogen activator inhibitor-1). Results: Cavernous nerve stimulation enhanced the expression of factors involved in angiogenesis (vascular endothelial growth factor, hepatocyte growth factor, angiopoietin1, matrix metalloproteinase 2, and metalloproteinase 9), and activated intracellular signaling mediators related to cell survival and proliferation (phosphatidylinositol 3-kinase, phospho-Akt/Akt, and phospho-ERK/ERK), while suppressing the pathways involved in tissue fibrosis (phospho-Smad2/Smad2, phospho-Smad3/Smad3, and plasminogen activator inhibitor-1). Conclusions: Penile erection in mice is accompanied by the activation of a cascade of signaling pathways involved in angiogenesis, cell survival and proliferation, and antifibrosis. The present results might provide a theoretical and molecular basis for understanding the importance of penile rehabilitation and subsequent restoration of nocturnal or sexually-mediated penile erections.
Key words: angiogenesis, antifibrosis, cell survival, molecular pathway, penile erection, penile rehabilitation.
VE = vascular endothelial VEGF = vascular endothelial growth factor
Introduction Correspondence: Jun-Kyu Suh M.D., Ph.D., National Research Center for Sexual Medicine and Department of Urology, Inha University School of Medicine, 7206 Third Street, ShinheungDong, Jung-Gu, Incheon 400-711, Korea. Email:
[email protected] †These authors contributed equally to this work. Received 1 July 2015; accepted 17 March 2016. Online publication 24 April 2016 614
Penile erection is a well coordinated neurovascular phenomenon that requires functional interaction between vascular endothelial cells, smooth muscle cells and neuronal cells.1 Nitric oxide, mainly derived from non-adrenergic and non-cholinergic nerves, and from cavernous endothelium, diffuses into the adjacent cavernous smooth muscles and reacts with guanylylate cyclases, which catalyze the conversion of guanosine triphosphate to guanosine monophosphate. The increase in intracellular cyclic guanosine monophosphate leads to smooth muscle relaxation, and induces penile erection by regulating intracellular calcium concentrations.2 Despite great advances in our understanding of the molecular mechanisms involved in the physiology of penile erection, the molecular events related to penile erection and their physiological implications are as yet largely unknown. Why do sexually potent men have cyclic penile erection during sleep? What are the clinical implications of nocturnal penile erection? Nocturnal penile erections induce oxygenation of the corpus cavernosum, and have an important role in maintaining normal erectile function. During the flaccid status, cavernous oxygen tension is similar to that of the venous blood (mean PO2, 34 mmHg), and increases © 2016 The Japanese Urological Association
Molecular events related to penile erection
Methods Animals and nerve-induced penile erection Twelve-week-old male C57BL/6 mice were used, and fed a normal diet. The experiments were approved by the Institutional Animal Care and Use Subcommittee of Inha University, Incheon, Korea. All experimental procedures and animal care were carried out in accordance with the guidelines of the same institution. Mice were randomly assigned to the cavernous nerve-stimulated group or to the unstimulated control group. The mice from each group were anesthetized with ketamine (100 mg/kg) and xylazine (5 mg/kg) intramuscularly. Bipolar platinum wire electrodes were placed around the cavernous nerve. The stimulation parameters were 5 V at a frequency of 12 Hz and a pulse width of 1 ms. ES of the cavernous nerve was carried out for 60 s to induce penile erection, immediately after which the corpus cavernosum tissues were harvested for western blot analysis and histological examination. We also harvested corpus cavernosum tissue 10 min after 60 s duration of cavernous nerve stimulation to test whether molecular changes persist after discontinuation of the penile erection. In the cavernous nerve-stimulated group, penile erection was confirmed by measurement of ICP during ES of the cavernous nerve. The unstimulated control group underwent exposure of the prostate to enable visualization of the cavernous nerve without ES and baseline ICP was determined in this group (Fig. 1). Systemic blood pressure was measured with a non-invasive tail-cuff system (Visitech Systems, Apex, NC, USA).
Western blot analysis Proteins were extracted from the corpus cavernosum tissue of cavernous nerve-stimulated or unstimulated mice (n = 4 per group). Equal amounts of protein (50 lg per lane) were electrophoresed on sodium dodecyl sulfate-polyacrylamide gels (8% or 12%), transferred to nitrocellulose membranes and © 2016 The Japanese Urological Association
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to arterial blood levels during erection (mean PO2, 99 mmHg).3 A decrease in nocturnal penile tumescence is noted in a variety of pathological conditions, such as diabetes and radical prostatectomy.4,5 Recently, much attention has focused on penile rehabilitation through the use of oral phosphodiesterase type 5 inhibitors or intracavernous injection of vasoactive agents in patients with ED after radical prostatectomy, because the prolonged hypoxia resulting from the neurogenic elimination of nocturnal erections might lead to cavernous endothelial and smooth muscle apoptosis and fibrosis.6,7 Daily sildenafil administration was reported to improve nocturnal penile tumescence and rigidity, and erectile function in men who underwent nerve-sparing radical prostatectomy for prostate cancer.8 However, the exact molecular mechanisms by which the restoration of nocturnal penile erection affects erectile function remain unexplored. The aim of the present study was to determine the molecular events related to penile erection in the corpus cavernosum tissue of mice by use of ES of the cavernous nerve followed by western blot analysis and histological examination of harvested corpus cavernosum tissues.
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Fig. 1 ICP elicited by ES of the cavernous nerve. (a) Representative ICP responses for mice stimulated at 5 V at a frequency of 12 Hz, a pulse width of 1 ms and a duration of 60 s. The stimulus interval is shown by a solid bar (60 s). (b) Mean basal ICP and MSBP were determined in unstimulated control mice (ES). Maximal ICP and MSBP were calculated for cavernous-nerve stimulated mice (ES+). Each bar depicts the mean values (SE) from n = 16 animals per group. *P < 0.01 compared with the unstimulated control group.
then probed with antibodies to VEGF (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA), HGF (1:500; Santa Cruz Biotechnology), Ang1 (1:500; NOVUS Biologicals, Littleton, CO, USA), MMP-2 (1:500; Santa Cruz Biotechnology), MMP-9 (1:500; Santa Cruz Biotechnology), PI3K (1:250; Cell Signaling, Beverly, MA, USA), phospho Akt (Ser473, 1:500; Cell Signaling), Akt (1:1000; Cell Signaling), phospho ERK (Thr202/Tyr204, 1:500; Cell Signaling), ERK (1:1000; Cell Signaling), phospho Smad2 (Ser465/467, 1:300; Cell Signaling), phospho Smad3 (Ser423/425, 1:300; Cell Signaling), Smad2/3 (1:500; Cell Signaling), PAI-1 (1:300; Santa Cruz Biotechnology), VE-cadherin (1:500; Millipore, San Francisco, CA, USA), NG2 (1:1000; Millipore), PDGFR-b (1:500; Santa Cruz Biotechnology), SMA (1:1000; Santa Cruz Biotechnology) or b-actin (Abcam, Cambridge, UK). Results were quantified by densitometry.
Quantitative real-time RT–PCR VEGF, HGF, Ang1, MMP-2 and MMP-9 mRNA expression was determined by quantitative real-time RT–PCR in a fluorescent temperature cycler (StepOnePlus Real-Time PCR System Thermal Cycling Block; Applied Biosystems, Foster City, CA, USA). The following primer was used for sequence reactions: VEGF, Mm00437306_m1 (TaqMan gene expression assay; Applied Biosystems); HGF, Mm01135184_m1 (TaqMan gene expression assay; Applied Biosystems); Ang1, Mm00456503_m1 (TaqMan gene expression assay; Applied 615
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frozen tissue sections (7-lm thick) were incubated overnight with antibodies against VEGF (1:50; Santa Cruz Biotechnology), HGF (1:50; Santa Cruz Biotechnology), Ang1 (1:500; NOVUS Biologicals), NG2 (1:50; Millipore), PDGFR-b (1:50; Santa Cruz Biotechnology), SMA (1:100; Santa Cruz Biotechnology) or PECAM-1 (an endothelial cell marker; Chemicon, Temecula, CA, USA; 1:50 at 4°C overnight. After several washes with phosphate-buffered saline, the sections were incubated with tetramethyl rhodamine isothiocyanate- or fluorescein isothiocyanate-conjugated secondary antibodies (1:100; Zymed Laboratories, South San Francisco, CA, USA) for 2 h at room temperature. Signals were visualized and
Histological examinations For fluorescence microscopy (n = 4 per group), the penis tissue was fixed in 4% paraformaldehyde for 24 h at 4°C, and ES
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Biosystems); MMP-2, Mm00439498_m1 (TaqMan gene expression assay; Applied Biosystems); and MMP-9, Mm00442991_ m1 (TaqMan gene expression assay; Applied Biosystems). The relative abundance of transcripts was normalized to the constitutive expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Mm99999915_g1 TaqMan gene expression assay; Applied Biosystems) (n = 4 per group).
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Fig. 2 Effects of nerve-induced penile erection on the expression of angiogenic factors and matrix metalloproteinase. (a) Representative western blot for VEGF, HGF, Ang1, MMP-2 and MMP-9 in the corpus cavernosum tissue of cavernous nerve-stimulated (ES+) or unstimulated mice (ES). Corpus cavernosum tissues were harvested immediately after ES of the cavernous nerve for 60 s. (b) Data are presented as the relative density of each protein compared with that of b-actin. The relative ratio of the unstimulated control group was arbitrarily set equivalent to 1. Each bar depicts the mean values (SE) from n = 4 animals per group. *P < 0.01 compared with the unstimulated control group. (c) Time-dependent protein expression of Ang1. Corpus cavernosum tissues were harvested immediately after ES of the cavernous nerve for 5, 10, 30 and 60 s. (d) Data are presented as the relative density of Ang1 protein compared with that of b-actin. The relative ratio of the unstimulated control group was arbitrarily set equivalent to 1. Each bar depicts the mean values (SE) from n = 4 animals per group and per each time point. *P < 0.05 compared with the unstimulated control group. (e) Measurement of VEGF, HGF, Ang1, MMP-2 and MMP-9 gene expression by quantitative real-time RT–PCR. The gene expression levels are expressed as arbitrary units normalized to glyceraldehyde 3-phosphate dehydrogenase. The relative ratio measured in the control group is arbitrarily presented as 1. Each bar depicts the mean values (SE) from n = 4 animals per group. *P < 0.01 compared with the unstimulated control group.
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Molecular events related to penile erection
digital images were obtained with a confocal microscope (FV1000; Olympus, Tokyo, Japan). The intensity of fluorescence in the stained area of the corpus cavernosum tissue was measured by image analysis (National Institutes of Health Image J 1.34, http://rsb.info.nih.gov/ij/index.html).
Statistical analysis Results are expressed as means standard errors. Group comparisons of data were carried out by use of Mann–Whitney rank-sum tests. We carried out statistical analysis with SigmaStat 3.5 Software (Systat Software Inc., Richmond, CA, USA). P-values
