Anita Limanjaya

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.

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.

MP45-13 INTRACAVERNOUS DELIVERY OF DICKKOPF3 GENE OR PEPETIDE RESCUES ERECTILE FUNCTION THROUGH ENHANCED CAVERNOUS ANGIOGENESIS IN THE DIABETIC MOUSE

INTRODUCTION AND OBJECTIVES: Patients with diabetic erectile dysfunction (ED) often have severe endothelial dysfunction, which results in poor response to oral phosphodiesterase-5 inhibitors. Dickkopf-3 (DKK3), originally reported to interact Wnt signaling pathway during embryonic development, is known to involve in the endothelial cell proliferation. However, the role of DKK3 in ED is as yet not reported. The aim of this study was to investigate whether and how DKK3 gene or peptide restores erectile function in diabetic mice. METHODS: Eight-week-old C57BL/6 mice were used, and diabetes was induced by intraperitoneal injection of streptozotocin. At 8 weeks after the diabetes induction, the efficacy of DKK3 peptide or gene were determined by three independent experiments: Experiment 1 (DKK2 peptide; Control, DM þ PBS, DM þ DKK3 peptide [5 mg/20 mL]); Experiment 2 (DKK2 plasmid DNA with electroporation; Control, DM þ empty vector (100 mg/20 mL), DM þ DKK3 plasmid (10 mg, 40 mg, or 100 mg/20 mL, respectively); and Experiment 3 (DKK3 adenovirus; Control, DM þ PBS, DM þ Ad-GPF (1 x 10 vp/20 mL), DM þ Ad-DKK3 (1 x 10, 1 x 10, 1 x 10 vp/20 mL, respectively). One (peptide) or two weeks (gene) after treatment, we measured erectile function by electrical stimulation of the cavernous nerve. The penis was then harvested for histologic examination. We also determined angiogenic activity of DKK3 in primary cultured mouse cavernous endothelial cells (MCECs). RESULTS: The protein expression of DKK3 was significantly lower in cavernous tissue of diabetic mice than in controls. Intracavernous injection of DKK3 peptide or gene partially restored erectile function in diabetic mice, which reached up to 70-80% of the control values. DKK3 significantly restored cavernous endothelial cell content and endothelial cell-cell junction proteins (ZO-1 and claudin-5) in diabetic mice. Treatment of MCECs with DKK3 peptide significantly increased the expression of basic fibroblast growth factor and angiopoietin-1, which accelerated tube formation and endothelial migration, and restored integrity of the endothelial cellcell junction. CONCLUSIONS: DKK3 restored erectile function in diabetic mouse through the restoration of cavernous endothelial cells and the integrity of endothelial cell-cell junction. Therapeutic cavernous endothelial regeneration by use of DKK3 may provide a good opportunity for treating ED from vascular causes.

MP45-18 CALORIE RESTRICTION REVERSES THE AGE-RELATED ALTERATION OF CAVERNOUS NEUROVASCULAR STRUCTURE IN THE RAT

INTRODUCTION AND OBJECTIVES: Four groups of rats were used: young rats (7 months) þ ad-libitum, old rats (22 months) þ ad-libitum, young ratsþCRdiet, andold ratsþCRdiet. Thead-libitumgrouphad freeaccess toboth foodandwaterandCRgroupswere fed60%of the food intake of their ad-libitum littermates, for 6 weeks before decapitation. The penis was harvested and stained with antibodies to vWF, smooth muscle a-actin, PDGFR-b, neurofillament, and S100. We also performedMasson trichrome staining and TUNNELassay. Theblood sampleswere collected for the measurement of plasma testosterone level. METHODS: Four groups of rats were used: young rats (7 months) þ ad-libitum, old rats (22 months) þ ad-libitum, young rats þ CR diet, and old rats þ CR diet. The ad-libitum group had free access to both food and water and animals in the CR groups were fed 60% of the food intake of their ad-libitum littermates, for 4 weeks. The penis was harvested and stained with antibodies to vWF, smooth muscle a-actin, PDGFR-b, neurofillament, and S100. We also performed Masson trichrome staining and TUNNEL assay. The blood samples were collected for the measurement of plasma testosterone level. RESULTS: The contents of endothelial cell, smooth muscle cells, pericytes, and neuronal cells as well as serum testosterone levels were significantly lower in old rats than in their young littermates. In old rats, but not in young rats, CR diet significantly restored cavernous endothelial cells, smooth muscle cells, pericytes, and neuronal cell contents; increased plasma testosterone level; and decreased endothelial cell apoptosis and cavernous fibrosis as determined by the ratio of collagen to smooth muscle contents. CONCLUSIONS: CR diet successfully improved age-related derangements in penile neurovascular structures. Along with a variety of life style modifications, our study gave us a scientific rationale to use CR diet as a non-pharmaceutical strategy for treating or preventing erectile dysfunction in aged men.

Dickkopf2 rescues erectile function by enhancing penile neurovascular regeneration in a mouse model of cavernous nerve injury

Penile erection is a neurovascular event and neurologic or vascular disturbances are major causes of erectile dysfunction (ED). Radical prostatectomy for prostate cancer not only induces cavernous nerve injury (CNI) but also results in cavernous angiopathy, which is responsible for poor responsiveness to oral phosphodiesterase-5 inhibitors. Dickkopf2 (DKK2) is known as a Wnt signaling antagonist and is reported to promote mature and stable blood vessel formation. Here, we demonstrated in CNI mice that overexpression of DKK2 by administering DKK2 protein or by using DKK2-Tg mice successfully restored erectile function: this recovery was accompanied by enhanced neural regeneration through the secretion of neurotrophic factors, and restoration of cavernous endothelial cell and pericyte content. DKK2 protein also promoted neurite outgrowth in an ex vivo major pelvic ganglion culture experiment and enhanced tube formation in primary cultured mouse cavernous endothelial cells and pericytes co-culture system in vitro. In light of critical role of neuropathy and angiopathy in the pathogenesis of radical prostatectomy-induced ED, reprogramming of damaged erectile tissue toward neurovascular repair by use of a DKK2 therapeutic protein may represent viable treatment option for this condition.

Penile neurovascular structure revisited: immunohistochemical studies with three-dimensional reconstruction

Penile erection is a neurovascular phenomenon that requires well coordinated and functional interaction between penile vascular and nervous systems. In order to provide a useful tool to examine pathologic changes in the erectile tissue, mainly focusing on penile neurovascular dysfunction, we established the technique to determine the differential distribution of endothelial cells, smooth muscle cells, pericytes, and nerve fibers in the mouse penis using immunohistochemical staining with three‐dimensional reconstruction. Immunofluorescent staining of penile tissue was performed with antibodies against CD31 (an endothelial cell marker), smooth muscle α ‐actin (SMA, a smooth muscle cell marker), NG2 (a pericyte marker), or βIII‐tubulin (a neuronal marker). We reconstructed three‐dimensional images of penile vascular or neurovascular system from stacks of two‐dimensional images, which allows volume rendering and provides reliable anatomic information. CD31‐positive endothelial cells, SMA‐positive smooth muscle cells, and NG2‐positive pericytes were evenly distributed and composed sinusoidal or venous wall. However, the endothelial layer of the cavernous artery or dorsal artery was mainly covered with smooth muscle cells and rarely associated with pericytes. The reconstructed three‐dimensional images clearly visualized typical wavy appearance of nerve fibers that evenly innervate to cavernous sinusoids, cavernous artery, dorsal vein, and dorsal artery. We observed a significant decrease in CD31‐positive endothelial cells, NG2‐positive pericytes, and βIII‐tubulin‐positive nerve fibers in the penis of diabetic mice compared with those in normal condition. Our protocol for immunofluorescent staining with three‐dimensional reconstruction will allow a better understanding of the penile neurovascular anatomy and may constitute a standard technique to determine the efficacy of candidate therapeutics targeting therapeutic angiogenesis or neural regeneration.