Kalyan Ghatak

The pericyte as a cellular regulator of penile erection and a novel therapeutic target for erectile dysfunction

Pericytes are known to play critical roles in vascular development and homeostasis. However, the distribution of cavernous pericytes and their roles in penile erection is unclear. Herein we report that the pericytes are abundantly distributed in microvessels of the subtunical area and dorsal nerve bundle of mice, followed by dorsal vein and cavernous sinusoids. We further confirmed the presence of pericytes in human corpus cavernosum tissue and successfully isolated pericytes from mouse penis. Cavernous pericyte contents from diabetic mice and tube formation of cultured pericytes in high glucose condition were greatly reduced compared with those in normal conditions. Suppression of pericyte function with anti-PDGFR-β blocking antibody deteriorated erectile function and tube formation in vivo and in vitro diabetic condition. In contrast, enhanced pericyte function with HGF protein restored cavernous pericyte content in diabetic mice, and significantly decreased cavernous permeability in diabetic mice and in pericytes-endothelial cell co-culture system, which induced significant recovery of erectile function. Overall, these findings showed the presence and distribution of pericytes in the penis of normal or pathologic condition and documented their role in the regulation of cavernous permeability and penile erection, which ultimately explore novel therapeutics of erectile dysfunction targeting pericyte function.

Establishment of in vitro model of erectile dysfunction for the study of high‐glucose‐induced angiopathy and neuropathy

Penile erection requires complex interaction between vascular endothelial cells, smooth muscle cells, pericytes, and autonomic nerves. Diabetes mellitus is one of the most common causes of erectile dysfunction (ED) and multiple pathogenic factors, such as cavernous angiopathy and autonomic neuropathy, are associated with diabetic ED. Although a variety of animal models of diabetic ED play an important role in understanding pathophysiologic mechanisms of diabetes‐induced ED, these animal models have limitations for addressing the exact cellular or molecular mechanisms involved in ED. Therefore, we established an in vitro model of ED for the study of high‐glucose‐induced angiopathy and neuropathy. We successfully isolated and cultivated mouse cavernous endothelial cells (MCECs) and mouse cavernous pericytes (MCPs). The cells were exposed to the normal‐glucose (5 mmoL) or high‐glucose (30 mmoL) condition for 48 h. In vitro matrigel assay revealed impairments in tube formation in primary cultured MCECs or MCPs exposed to high‐glucose condition. To study cellular interaction between MCECs and MCPs, co‐culture systems including indirect contact, indirect non‐contact, and direct mixed co‐culture system, were established. We observed impaired tube formation and increased permeability in MCECs‐MCPs co‐culture exposed to high‐glucose condition. To evaluate the effect of high‐glucose on neurite sprouting, the mouse major pelvic ganglion (MPG) tissue was harvested and cultivated in matrigel. Neurite outgrowth and nNOS‐positive nerve fibers were significantly lower in MPG tissues exposed to the high‐glucose condition than in the tissues exposed to the normal‐glucose condition. We believe that in vitro model of ED will aid us to understand the role of each cellular component in the pathogenesis of diabetic ED, and also be a useful tool for determining the efficacy of candidate therapeutics targeting vascular or neuronal function. This model would present a new avenue for drug discovery and development of novel therapeutic modalities for erectile dysfunction.

Pericyte-Derived Dickkopf2 regenerates damaged penile neurovasculature through an angiopoietin-1-Tie2 pathway

Penile erection requires well-coordinated interactions between vascular and nervous systems. Penile neurovascular dysfunction is a major cause of erectile dysfunction (ED) in patients with diabetes, which causes poor response to oral phosphodiesterase-5 inhibitors. Dickkopf2 (DKK2), a Wnt antagonist, is known to promote angiogenesis. Here, using DKK2-Tg mice or DKK2 protein administration, we demonstrate that the overexpression of DKK2 in diabetic mice enhances penile angiogenesis and neural regeneration and restores erectile function. Transcriptome analysis revealed that angiopoietin-1 and angiopoietin-2 are target genes for DKK2. Using an endothelial cell-pericyte coculture system and ex vivo neurite sprouting assay, we found that DKK2-mediated juxtacrine signaling in pericyte-endothelial cell interactions promotes angiogenesis and neural regeneration through an angiopoietin-1-Tie2 pathway, rescuing erectile function in diabetic mice. The dual angiogenic and neurotrophic effects of DKK2, especially as a therapeutic protein, will open new avenues to treating diabetic ED.

Penile erection induces angiogenic, survival, and antifibrotic signals: molecular events associated with penile erection induced by cavernous nerve stimulation in mice

To determine the molecular events related to penile erection in the corpus cavernosum tissue of mice after electrical stimulation of the cavernous nerve.

Calorie restriction reverses age‐related alteration of cavernous neurovascular structure in the rat

Calorie restriction (CR) refers to a reduction of calorie intake without compromising essential nutrients to avoid malnutrition. CR has been established as a non‐genetic method of altering longevity and attenuating biological changes associated with aging. Aging is also an important risk factor for erectile dysfunction. The aim of this study was to examine whether CR diet can reverse the age‐related alterations of erectile tissue in the aged rat. Four groups of rats were used: young rats (7 months) + ad libitum, aged rats (22 months) + ad libitum, young rats + CR diet, and aged rats + CR diet. The ad libitum group had free access to both food and water, and CR groups were fed 60% of the food intake of their ad libitum littermates, starting from 6 weeks before sacrifice. The penis was harvested and stained with antibodies to von Willebrand factor, smooth muscle α‐actin, platelet‐derived growth factor receptor‐β, phospho‐eNOS, nNOS, and neurofilament. We also performed Masson trichrome staining and TUNEL assay. The blood samples were collected for the measurement of serum total testosterone level. The contents of endothelial cells, smooth muscle cells, pericytes, and neuronal cells as well as serum testosterone levels were significantly lower in the penis of aged rats than in their young littermates. CR significantly restored cavernous endothelial cells, smooth muscle cells, pericytes, and neuronal cell contents and decreased cavernous endothelial cell apoptosis and fibrosis in both young and aged rats. CR also increased serum testosterone level in aged rats, but not in young rats. CR successfully improved age‐related derangements in penile neurovascular structures and hormonal disturbance. Along with a variety of lifestyle modifications, our study gave us a scientific rationale for CR as a non‐pharmaceutical strategy to reprogram damaged erectile tissue toward neurovascular repair in aged men.

Optimizing in vivo gene transfer into mouse corpus cavernosum by use of surface electroporation

Purpose Electroporation is known to enhance the efficiency of gene transfer through a transient increase in cell membrane permeability. The aim of this study was to determine the optimal conditions for in vivo electroporation-mediated gene delivery into mouse corpus cavernosum. Materials and Methods Diabetes was induced in C57BL/6 mice by intraperitoneal injections of streptozotocin. After intracavernous injection of pCMV-Luc (100 µg/40 µL), different electroporation settings (5-50 V, 8-16 pulses with a duration of 40-100 ms) were applied to the penis to establish the optimal conditions for electroporation. Gene expression was evaluated by luciferase assay. We also assessed the undesired consequences of electroporation by visual inspection and hematoxylin-eosin staining of penile tissue. Results Electroporation profoundly induced gene expression in the corpus cavernosum tissue of normal mice in a voltage-dependent manner. We observed electrical burn scars in the penis of normal mice who received electroporation with eight 40-ms pulses at a voltage of 50 V and sixteen 40-ms pulses, eight 100-ms pulses, and sixteen 100-ms pulses at a voltage of 30 V. No detectable burn scars were noted in normal mice stimulated with eight 40-ms pulses at a voltage of 30 V. Electroporation also significantly induced gene expression in diabetic mice stimulated with 40-ms pulse at a voltage of 30 V without injury to the penis. Conclusions We have established the optimal electroporation conditions for maximizing gene transfer into the corpus cavernosum of mice while avoiding damage to the erectile tissue. The electroporation-mediated gene delivery technique will be a valuable tool for gene therapy in the field of erectile dysfunction.

Silencing Histone Deacetylase 7 Alleviates Transforming Growth Factor-β1-Induced Profibrotic Responses in Fibroblasts Derived from Peyronie's Plaque

Purpose Epigenetic modifications, such as histone acetylation/deacetylation and DNA methylation, play a crucial role in the pathogenesis of inflammatory disorders and fibrotic diseases. The aim of this study was to study the differential gene expression of histone deacetylases (HDACs) in fibroblasts isolated from plaque tissue of Peyronies disease (PD) or normal tunica albuginea (TA) and to examine the anti-fibrotic effect of small interfering RNA (siRNA)-mediated silencing of HDAC7 in fibroblasts derived from human PD plaque. Materials and Methods For differential gene expression study, we performed reverse-transcriptase polymerase chain reaction for HDAC isoforms (1–11) in fibroblasts isolated from PD plaque or normal TA. Fibroblasts isolated from PD plaque were pretreated with HDAC7 siRNA (100 pmol) and then stimulated with transforming growth factor-β1 (TGF-β1, 10 ng/mL). Protein was extracted from treated fibroblasts for Western blotting. We also performed immunocytochemistry to detect the expression of extracellular matrix proteins and to examine the effect of HDAC2 siRNA on the TGF-β1-induced nuclear translocation of Smad2/3 and myofibroblastic differentiation. Results The mRNA expression of HDAC2, 3, 4, 5, 7, 8, 10, and 11 was higher in fibroblasts isolated from PD plaque than in fibroblasts isolated from normal TA tissue. Knockdown of HDAC7 in PD fibroblasts inhibited TGF-β1-induced nuclear shuttle of Smad2 and Smad3, transdifferentiation of fibroblasts into myofibroblasts, and abrogated TGF-β1-induced production of extracellular matrix protein. Conclusions These findings suggest that specific inhibition of HDAC7 with RNA interference may represent a promising epigenetic therapy for PD.

MP43-06 INHIBITION OF PRONGF PATHWAY RESTORES ERECTILE FUNCTION THROUGH DUAL ANGIOGENIC AND NEUROTROPHIC EFFECTS IN THE DIABETIC MOUSE

TUNEL, and immunohistochemical analysis for cleaved caspase-3 (apoptosis indicator), -8, -9, SHH and its receptor Patched. RESULTS: Cleaved caspase-3 was present in normal pelvic plexus and increased in a time dependent manner in all nerves of the pelvic plexus when the CN was crushed. Caspase 3 cleaved was identified primarily in glial cells, rather than neurons. Caspase 9 increased in glial cells of all nerves of the pelvic plexus, while very little caspase 8 was observed. SHH was abundant in neurons and glia of the CN, PN, HYG and ANC, while PTCH1 was identified only in neurons. CONCLUSIONS: Interruption of CN innervation, as occurs in the majority of prostatectomy patients, results in induction of apoptosis in other regions of the pelvic plexus, thus affecting continence. Apoptosis occurred primarily through the intrinsic pathway in response to CN injury. Identification of HYG and PN contribution to SUI, and involvement of the SHH pathway, identifies novel treatment avenues for intervention.

PD49-03 EFFECT OF INTRACAVERNOUS ADMINISTRATION OF EMBRYONIC STEM CELL-DERIVED EXOSOME ON ERECTILE FUNCTION IN THE STREPTOZOTOCIN-INDUCED DIABETIC MOUSE

INTRODUCTION AND OBJECTIVES: Exosome contains a variety of protein, mRNA, and miRNA and is known to play an important role in intercellular communication as a bio-nanoparticle with a diameter of 40 to 100 nm. Recent studies have demonstrated the therapeutic potential of exosome in a variety of animal models for cardiovascular diseases and neuropathies. The aim of this study was to investigate effectiveness of embryonic stem cell (ESC)-derived exosome in restoring erectile function in diabetic mice. METHODS: Diabetes was induced by intraperitoneal injection of streptozotocin into 8-week-old C57BL/6 male mice. At 8 weeks after the induction of diabetes, the animals were distributed into 3 groups: control nondiabetic mice and diabetic mice receiving two successive intracavernous injections of PBS (days -3 and 0; 20 mL) or ESC-derived exosome (days -3 and 0; 1 mg in 20 mL of PBS). Twoweek after treatment, we measured erectile function by electrical stimulation of the cavernous nerve. The penis was harvested and stained with antibodies to PECAM1, smooth muscle a-actin, NG2, and bIII-tubulin. We also determined angiogenic potential of ESC-derived exosome in an ex vivo aortic ring assay and in primary cultured mouse cavernous endothelial cell (MCEC) and pericyte (MCP) mono-culture or co-culture system in vitro. RESULTS: Intracavernous injections of ESC-derived exosome significantly improved erectile function in diabetic mice, which reached up to 90% of control values. ESC-derived exosome induced significant restoration of cavernous contents of endothelial cells, smooth muscle cells, pericytes, and neuronal cells in diabetic condition. Moreover, ESC-derived exosome promoted microvascular sprouting from aortic ring and accelerated tube formation in primary cultured MCEC and MCP mono-culture or co-culture system in vitro. CONCLUSIONS: ESC-derived exosome successfully restored erectile function through enhanced cavernous angiogenesis and neural regeneration in diabetic mice. Further studies are needed to clarify mechanism by which ESC-derived exosome induces neurovascular repair.

MP45-11 DICKKOPF2 PROMOTES ANGIOGENESIS AND NEURAL REGENERATION THROUGH AN ANGIOPOIETIN-1-TIE2 PATHWAY AND RESCUES ERECTILE FUNCTION IN THE DIABETIC MOUSE

penile morphology in normotensive and hypertensive rats after 5-areductase inhibitors treatment. METHODS: Sixty male rats were assigned into 6 groups as following: WKY e group composed by untreated Wistar Kyoto rats (normotensive strain); WKYþD Wistar Kyoto rats treated with dutasteride (0.5 mg/Kg/day); WKYþF Wistar Kyoto rats treated with finasteride (5 mg/Kg/day); H group composed by the strain of spontaneously hypertensive rats (SHR); HþD SHR treated with dutasteride; HþF SHR treated with finasteride. All treatments were given by gavage during 40 days after what the animals were killed and their penis were collected and processed for histomorphometrical analysis. Sections stained with hematoxylin and eosin were used to study the cross-sectional penile area, while Masson’s trichrome was used for study the surface density of smooth muscle fibers, connective tissue, and sinusoidal spaces of the corpus cavernosum. The surface density of elastic system fibers was studied in Weigert’s resorcin fucsin stained section. The results were compared by one-way-ANOVA with Bonferroni’s post test, considering p>0.05 as significant. RESULTS: The cross-sectional penile area of normotensive animals that received dutasteride or finasteride was reduced by 39.9% and 40% in comparison to untreated normotensive animals. The connective tissue of H group was 13.7% higher than WKY, and HþD animals had an increase of 12.9% of connective tissue in comparison to untreated hypertensive animals. The sinusoidal space was reduced by 33.7% in H in comparison to WKY. In respect to the smooth muscle surface density, WKYþD showed a reduction of 26.1% in comparison to WKY, while both HþD and HþF showed reductions of 29.4 and 32.5% in comparison to untreated H. Despite no difference in the elastic system fibers surface density was observed between H and WKY, groups WKYþD, WKYþF, HþD, and HþF had an increase of 35,7%, 41,1% 82,6%, and 31,5% in comparison to WKY. Also, HþD showed a 45,8% increase in comparison to H. CONCLUSIONS: Hypertension promoted important modifications on penile structure. Both 5-a-reductase inhibitors (dutasteride and finasteride) promoted modifications in penile morphology of normotensive and hypertensive rats, although these modifications were more prominent in hypertensive animals. Dutasteride was the drug that most affected the corpus cavernosum in this rodent model.