The TGF-β1/Smad/CTGF pathway and corpus cavernosum fibrous-muscular alterations in rats with streptozotocin-induced diabetes

Diabetes-associated erectile dysfunction is associated with increased extracellular matrix deposition and reduced smooth muscle content in the corpus cavernosum. The mechanisms of these processes are not well understood. In this study, we investigated fibromuscular changes in the corpus cavernosum of rats with streptozotocin-induced diabetes to determine the mechanisms underlying pathologic changes in penile structure and function. Forty 8-week-old Sprague-Dawley rats were randomly distributed into control and diabetic groups. Diabetes was induced by a one-time intraperitoneal injection of streptozotocin 60 mg/kg. Twelve weeks later, erectile function was measured by cavernous nerve electrostimulation with real-time intracorporal pressure assessment. The penis was harvested for histologic examination (Masson trichrome stain, picrosirius red stain, Hart elastin stain, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling, and immunohistochemistry) and Western blot. Diabetes significantly attenuated erectile response to cavernous nerve electrostimulation. Diabetic animals exhibited a decreased smooth muscle/collagen ratio in the corpus cavernosum. The ratio of collagen I to II fibers was significantly lower in the corpora of diabetic rats compared with controls. Cavernous elastic fibers were fragmented in diabetic rats. There was up-regulation of the transforming growth factor β1/Smad/connective tissue growth factor signaling pathway in diabetic rats. Phospho-Smad2 expression was higher in smooth muscle cells and fibroblasts of diabetic rats, as was the apoptotic index. The up-regulation of the transforming growth factor β1/Smad/connective tissue growth factor signaling pathway might play an important role in diabetes-induced fibrous-muscular structural changes and deterioration of erectile function.     

Reduction of endothelial and smooth muscle density in the corpora cavernosa of the streptozotocin induced diabetic rat

PURPOSE: Erectile dysfunction is one of the most prevalent complications of diabetes in males. Because adequate vascular perfusion is needed for appropriate erectile tissue function a likely reason for the high incidence of this complication in diabetics is a pathological change associated with the disease in vascularization of erectile tissues. We investigate whether chronic diabetes may induce changes in vascularization of the corpora cavernosa using a computerized image analysis system to quantify changes in the smooth muscle and endothelial cell content of the corpora cavernosa of diabetic rats induced by streptozotocin 6 months previously, and compare these changes to those associated with aging. MATERIALS AND METHODS: We studied 3 groups of rats, including 10-week-old untreated controls, diabetic rats treated with streptozotocin for 6 months starting at age 10 weeks and 18-month-old rats (aged). Penile shafts from these groups were excised, fixed, sectioned and immunostained with anti-smooth muscle actin to identify smooth muscle cells and anti-CD31 to identify endothelial cells. Computerized image analysis was used to quantify the percent area within the corpora cavernosa occupied by smooth muscle cells or endothelial cells, and the data were compared among the groups. RESULTS: We identified a highly significant decrease in the percentage of smooth muscle and endothelial cells within the cavernosa areas of diabetic rats compared to control or aged rats. Mean cavernous smooth muscle cell content was 15.28 +/- 2.54% in control rats and 9.83 +/- 1.21% in diabetic rats (p = 0.0001). Likewise, cavernous endothelial cell content was 6.93 +/- 0.86% in the control group and 4.01 +/- 1.08% in the diabetic group (p = 0. 0001). However, no statistical difference of smooth muscle or endothelial cell content was found between control and aged rats. CONCLUSIONS: Using the streptozotocin treated rat as a model for diabetes, we showed that smooth muscle and endothelial cell density is significantly decreased in diabetic corpora cavernosa but not in normal aged rats. This observation is a further step toward the understanding of the pathomechanisms for diabetic related erectile dysfunction.     

Potassium outward currents in freshly dissociated rabbit corpus cavernosum myocytes

PURPOSE: Cavernous smooth muscle cells have a key role in the control of penile erection and detumescence. In this study the types of smooth muscle cells and currents present in isolated rabbit corpus cavernosum myocytes were characterized. MATERIALS AND METHODS: Immunohistochemical methods were used to identify cavernous smooth muscle cells. Currents were recorded from freshly dissociated myocytes using the whole cell and amphotericin perforated patch clamp techniques. RESULTS: Cavernous myocytes were identified by alpha-smooth muscle actin and smooth muscle myosin immunoreactivity. Based on electrical properties at least 2 types of myocytes were present. Type I cells showed more depolarized membrane potentials, lower capacitance, higher input resistance and increased current densities at positive potentials than type II cells. In types I and II cells at voltages positive to 30 mV, maxi K+ channel (Ca2+ activated large conductance K+ channel or BK) blockade with iberiotoxin or charybdotoxin reduced outward currents by approximately 40% to 80% at 80 mV. Maxi K+ channel blocking did not affect cell membrane potential. Type II cells showed delayed rectifier K+ channel-type outward currents that were not detected in type I cells. Delayed rectifier K+ channel-type currents were resistant to iberiotoxin or charybdotoxin, activated at approximately -50 to -40 mV. and inactivated weakly. CONCLUSIONS: The data suggest that cavernous smooth muscle cells are heterogeneous with at least 2 subtypes identified based on membrane potential, capacitance, input resistance, current density and delayed rectifier K+ channel expression. The activation threshold suggests that delayed rectifier K+ channels are open at the resting membrane potential and, therefore, contribute to control and regulation of the cavernous myocyte excitability.     

Effect of a Chinese herbal medicine mixture on a rat model of hypercholesterolemic erectile dysfunction

PURPOSE: We examine the effect of a Chinese herbal medicine mixture on erectile function in a rat model of hypercholesterolemic erectile dysfunction. MATERIALS AND METHODS: In this study 32, 3-month-old Sprague-Dawley rats were used. The 8 control animals were fed a normal diet and the remaining 24 were fed 1% cholesterol diet for 4 months. After 2 months herbal medicine was added to the drinking water of the treatment group of 16 rats but not the cholesterol only group of 8. Of the 16 rats 8 received 25 mg./kg. per day (group 1) and 8 received 50 mg./kg. per day (group 2) of Chinese herbal medicine mixture. Serum cholesterol levels were measured at 2 and 4 months. At 4 months erectile function was evaluated with cavernous nerve electrostimulation in all animals. Penile tissues were collected for electron microscopy, and to perform Western blot for endothelial nitric oxide synthase, neuronal nitric oxide synthase, basic fibroblast growth factor (bFGF) and caveolin-1. RESULTS: Serum cholesterol levels were significantly higher in animals fed the 1% cholesterol diet compared to controls at 2 and 4 months. Nevertheless, there was no significant difference among group 1 (145 +/- 30 mg./dl.), group 2 (157 +/- 20) and the cholesterol only group (143 +/- 15). Systemic arterial pressure was not significantly different between the animals that were fed the 1% cholesterol diet and the controls. During electrostimulation of the cavernous nerve peak sustained intracavernous pressure was significantly lower in the cholesterol only group (50 +/- 23 cm. H2O) compared to the control group. Conversely erectile function was not impaired in the herbal medicine treated rats. Electron microscopy showed many caveolae with fingerlike processes in the cavernous smooth muscle and endothelial cell membranes in control and treated rats but not in the cholesterol only group of rats. Western blot did not show a difference among groups in protein expression for endothelial nitric oxide synthase and neuronal nitric oxide synthase in penile tissue but caveolin-1 and bFGF protein expression was significantly higher in groups 1 and 2 than in the cholesterol only and control groups. CONCLUSIONS: Rats developed erectile dysfunction after being fed a 1% cholesterol diet for 4 months. Although serum cholesterol levels were similar in the cholesterol only rats and those treated with Chinese herbal medicine mixture, erectile response was significantly better in the treated group. The mechanism of the herbal medicine is unknown. High levels of bFGF and caveolin-1 expression in the treated group may protect the cavernous smooth muscle and endothelial cells from the harmful effect of high serum cholesterol.     

Vascular endothelial growth factor stimulates a novel calcium-signaling pathway in vascular smooth muscle cells

BACKGROUND: Vascular endothelial growth factor (VEGF) is a potent vascular mitogen that selectively stimulates vascular smooth muscle cell (VSMC) migration through an unknown mechanism while having no effect on VSMC proliferation. It is known that VSMC migration and proliferation are dependent on the second messenger Ca2+ and, in particular, mitogen-stimulated Ca2+ influx. We hypothesized that the selective effect of VEGF on VSMC migration versus proliferation was a result of differential VEGF-stimulated Ca2+ signaling pathways. METHODS: Primary cultured human aortic smooth muscle cells (VSMCs) were grown to subconfluency and assigned to the following experimental groups: no stimulation, stimulation with platelet-derived growth factor-BB (PDGF-BB) (20 ng/mL) as positive control, and stimulation with VEGF165 (40 ng/mL). Total increase in [Ca2+]cyt and intracellular calcium release was quantified with the use of a fura-2 fluorescence assay. Assays for the following receptors VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-1) and PDGFR-beta were performed by immunoprecipitation, while PLCgamma1, Akt 1/2, and phospholamban B phosphorylation were assessed with Western immunoblotting. RESULTS: VSMCs stimulated with VEGF165 exhibited no intracellular Ca2+ release, compared with a 75 +/- 30 nmol/L intracellular calcium release after PDGF-BB stimulation, (P < .02) VEGF165-stimulated VSMCs in Ca2+-containing media exhibited 192 +/- 26 nmol/L increase in [Ca2+]cyt, compared with 354 +/- 54 nmol/L increase after PDGF-BB stimulation (P < .02). VEGF165 did not phosphorylate PLCgamma1 after 1, 5, or 10 minutes of treatment. VEGF165 treatment did not result in PI3-K/Akt activation at 1-, 5-, or 10-minute time points. Calmodulin-dependent kinase II (CaMKII) was activated by both VEGF165 and PDGF-BB after 1 and 5 minutes of stimulation. The presence of the receptors VEGFR-1, VEGFR-2, and PDGFR-beta was confirmed in all experimental groups. CONCLUSIONS: VEGF induces extracellular calcium influx but no intracellular calcium release in VSMCs. This lack of intracellular Ca2+ release stems from the inability of VEGF165 to activate the PLCgamma1 cascade and IP3 receptor-mediated Ca2+ release. The lack of PI3-K/Akt activation at these time points indicates a novel extracellular Ca2+ influx pathway sufficient to activate CaMKII. A paradigm relating extracellular Ca2+ influx to CaMKII activation and migration is suggested and may account for the selective effects of VEGF on VSMC migration.     

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

PURPOSE: We examined neurogenic and vasculogenic erectile dysfunction associated with hypercholesterolemia and evaluated vascular endothelial growth factor (VEGF) and adeno-associated virus (AAV) mediated, brain derived neurotrophic factor (BDNF) for potential treatment. MATERIALS AND METHODS: A total of 21, 2-month-old male rats were fed a 2% cholesterol diet and another seven were fed a normal diet. Two months later serum cholesterol levels were measured and test agents were given intracavernously. Those on normal diet (controls) received phosphate buffered saline (PBS). Those on cholesterol diet were randomly divided into 3 groups receiving PBS, VEGF (4 microg.) or AAV-BDNF (10 viral particles). Four months later erectile function was evaluated and cavernous tissues were collected for erectile dysfunction and immunohistochemical staining. RESULTS: Serum cholesterol levels were higher in rats fed the high fat diet than in controls. Intracavernous pressure was lower in cholesterol plus PBS treated rats than in rats of the other 3 groups. All hypercholesterolemic rats had less nerve content, fewer endothelial cells and higher smooth muscle content than rats with normal cholesterol levels. In cholesterol plus PBS treated rats electron microscopy showed hypermyelination and severe atrophy of axons, a remarkable decrease in the number and size of nonmyelinated axons, disarray of the smooth muscle cells with scant myofilaments and foamy cytoplasm, and denuded endothelial lining of the sinusoids covered by numerous platelets. VEGF and AAV-BDNF appeared to alleviate partially these changes. CONCLUSIONS: A high fat diet caused erectile dysfunction with accompanying neurological and vascular changes. VEGF and AAV-BDNF seemed to alleviate these problems.     

Midazolam stimulates vascular endothelial growth factor release in aortic smooth muscle cells: role of the mitogen-activated protein kinase superfamily

BACKGROUND: Intravenous anesthetics used during perioperative periods affect the vascular signaling molecules and the vascular reactivity. Vascular endothelial growth factor (VEGF), an angiogenesis factor produced in and secreted from aortic smooth muscle cells, is a specific mitogen for vascular endothelial cells. This study investigated the effects of various intravenous anesthetics on VEGF release, and the underlying mechanism, in a rat aortic smooth muscle cell line, A10 cells. METHODS: Intravenous anesthetics (midazolam and propofol) were continuously administered to rats by infusion. Cultured A10 cells were stimulated by intravenous anesthetics (midazolam, propofol, and ketamine). VEGF was evaluated by immunoassay. The phosphorylation of mitogen-activated protein (MAP) kinases was evaluated by Western blotting. RESULTS: Continuous infusion of midazolam, but not propofol, increased the VEGF concentration in rat plasma. In cultured cells, midazolam stimulated VEGF release, but propofol and ketamine did not. Midazolam induced phosphorylation of p44/p42 MAP kinase and stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK), without affecting p38 MAP kinase. PD98059 and U0126, specific inhibitors of MAP kinase kinase, significantly reduced the midazolam-stimulated release of VEGF. SP600125, a specific inhibitor of SAPK/JNK, significantly reduced midazolam-stimulated VEGF release. Applied together, PD98059 and SP600125 produced an additive reduction in midazolam-stimulated VEGF release. Moreover, a bolus injection of PD98059 truly inhibited the midazolam-increased VEGF concentration in rat plasma in vivo. CONCLUSIONS: Midazolam, but not propofol or ketamine, stimulates VEGF release in aortic smooth muscle cells. Its effect is mediated at least in part via activation of p44/p42 MAP kinase and SAPK/JNK.     

增龄对原代培养的阴茎海绵体平滑肌细胞分泌胰岛素样生长因子-1的影响

目的:探讨年龄对大鼠原代培养的阴茎海绵体平滑肌细胞(CCSMCs)分泌胰岛素样生长因子-1(IGF-1)水平的影响,及其与老年性ED的关系。方法:体外原代培养不同年龄段(4月龄、12月龄和24月龄)的大鼠CCSMCs,免疫组化染色鉴定;定量培养CCSMCs,采用酶免疫分析法(EIA)定量检测CCSMCs分泌IGF-1含量的变化,分析比较不同年龄对CCSMCs分泌IGF-1含量的影响。结果:成功原代培养CCSMCs,随着月龄的增加,CCSMCs分泌的IGF-1含量减少,与4月龄(7.1 ng/105个细胞)比较、12月龄(2.2 ng/105个细胞)和24月龄(1.9ng/105个细胞)CCSMCs分泌的IGF-1含量明显减少,有统计学意义(P<0.01)。结论:年龄是影响原代培养CCSMCs分泌IGF-1水平的因素之一,IGF-1的低表达可能和老年性ED的发生有关。     

Midazolam Stimulates Vascular Endothelial Growth Factor Release in Aortic Smooth Muscle Cells: Role of the Mitogen-activated Protein Kinase Superfamily

Background: Intravenous anesthetics used during perioperative periods affect the vascular signaling molecules and the vascular reactivity. Vascular endothelial growth factor (VEGF), an angiogenesis factor produced in and secreted from aortic smooth muscle cells, is a specific mitogen for vascular endothelial cells. This study investigated the effects of various intravenous anesthetics on VEGF release, and the underlying mechanism, in a rat aortic smooth muscle cell line, A10 cells.

Methods: Intravenous anesthetics (midazolam and propofol) were continuously administered to rats by infusion. Cultured A10 cells were stimulated by intravenous anesthetics (midazolam, propofol, and ketamine). VEGF was evaluated by immunoassay. The phosphorylation of mitogen-activated protein (MAP) kinases was evaluated by Western blotting.

Results: Continuous infusion of midazolam, but not propofol, increased the VEGF concentration in rat plasma. In cultured cells, midazolam stimulated VEGF release, but propofol and ketamine did not. Midazolam induced phosphorylation of p44/p42 MAP kinase and stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK), without affecting p38 MAP kinase. PD98059 and U0126, specific inhibitors of MAP kinase kinase, significantly reduced the midazolam-stimulated release of VEGF. SP600125, a specific inhibitor of SAPK/JNK, significantly reduced midazolam-stimulated VEGF release. Applied together, PD98059 and SP600125 produced an additive reduction in midazolam-stimulated VEGF release. Moreover, a bolus injection of PD98059 truly inhibited the midazolam-increased VEGF concentration in rat plasma in vivo.     

Expression of TGF-beta1 in smooth muscle cells regulates endothelial progenitor cells migration and differentiation

OBJECTIVE: Endothelial angiogenesis in the intima of the arterial wall is one of key events in the pathogenesis of arteriosclerosis. The molecular mechanisms by which transforming growth factor beta 1 (TGFbeta1) and endothelial progenitor cells may be responsible for angiogenesis of arteriosclerosis lesions are poorly understood. MATERIALS AND METHODS: Primary culture smooth muscle cells were transfected with pMAMneoTGFbeta1. ELISA checked VEGF expression in smooth muscle cells. Human EPCs (CD34+ cells) were cultured in pMAMneoTGFbeta1 or pMAMneo transfected smooth muscle cells conditional medium. After 21 days, differentiated endothelial colonies were confirmed by immunofluorescence for von Willebrand factor (vWF) and vascular-endothelial (VE)-cadherin. The VEGFR-1 expression in differentiated endothelial colonies was detected by ELISA. Cells migration and adhesion toward pMAMneoTGFbeta1 and pMAMneo transfected smooth muscle cells were also measured in parallel flow chamber. RESULTS: Abundant TGFbeta1 stable expressed in smooth muscle cells. TGFbeta1 transfected smooth muscle cells expressed significantly higher level VEGF than pMAMneo group. As judged by positive staining for endothelial markers vWF and VE-cadherin, the combination of TGFbeta1 transfected smooth muscle cells conditional medium produced significantly more endothelial colonies (P<0.05) than did pMAMneo group. The adhesion force between endothelial progenitor cells and smooth muscle cells in TGFbeta1 group was higher than control. CONCLUSION: TGFbeta1 expressed smooth muscle cells can be helpful for increasing endothelial progenitor cells adhesion and differentiation. It may be responsible for angiogenesis of arteriosclerosis lesions and useful for blood vessel tissue engineering.     

Transdifferentiation of rat fetal brain stem cells into penile smooth muscle cells

OBJECTIVES

To evaluate whether rat fetal brain stem cells can be induced to acquire cell fates outside the nervous system, hypothesising that cell‐based replacement therapy with stem cells can aid in the regeneration of penile smooth musculature and might help to attenuate organic erectile dysfunction (ED), as the degeneration of penile smooth muscle cells leading to subsequent impairment of function is important in organic ED.

MATERIALS AND METHODS

Fetal brain stem cells (FBSCs) from embryonal 12‐day Fisher 344 rats were isolated and characterized. For in vitro studies, undifferentiated FBSCs were cultured for 21 days in either N2 media (control) or N2 media conditioned in rat penile smooth muscle cell culture. These were then subjected to immunocytochemistry for specific markers of neural stem cells (nestin) and penile smooth muscle cells, i.e. α‐smooth muscle actin (αSMA), penis‐specific myosin light chain (MLC) desmin, calponin, vimentin, phosphodiesterase‐5 (PDE5) and connexin. For in vivo studies, male adult Fisher 344 rats had an intracavernous injection with saline (five rats, control) or FBSCs that were labelled genetically by an expression construct for green fluorescent protein (GFP, nine rats, experimental) and maintained for 6 weeks. The rats were then killed and penile tissue was harvested and subjected to immunocytochemistry for markers of neural stem cells, smooth muscle cells, and sinusoidal endothelium (vascular endothelial growth factor, VEGF).

RESULTS

Undifferentiated cells exposed to N2 media continued to maintain the characteristic morphological and protein marker features of FBSCs, while the cells exposed to the conditioned media acquired the morphological features of smooth muscle cells. In addition, the differentiated cells (30–40%) expressed smooth muscle markers. Rats implanted with FBSCs had cells that showed double‐labelling for GFP/αSMA, GFP/calponin and GFP/VEGF. The control group had no evidence of such double‐labelling.

CONCLUSIONS

These results suggest the transdifferentiation of FBSCs into penile smooth muscle cells. Such transdifferentiated cells showed long‐term survival when injected into the cavernous tissue, thus raising the possibility of a novel therapeutic option for organic ED.     

Androgens modulate the balance between VEGF and angiopoietin expression in prostate epithelial and smooth muscle cells

BACKGROUND: The vasculature of the prostate responds to androgens. Androgens most likely affect the vasculature indirectly by modulating the expression of angiogenic factors in the cells of the prostate. Most studies to date have examined the production of angiogenic factors by the prostate luminal epithelium. Here we examine the effects of androgen on production of three angiogenic factors, vascular endothelial growth factor (VEGF), angiopoietin-1, and angiopoietin-2, by the three major cell types in the prostate. METHODS: The ability of androgen to modulate VEGF, angiopoietin-1, and angiopoietin-2 production in cultured mouse prostate luminal epithelial, basal epithelial, and smooth muscle cells (SMCs) was assessed by Western blot and RT-PCR. RESULTS: The production of VEGF was modulated by androgens in both luminal epithelial and prostate SMCs but not in basal epithelial cells. However, in prostate luminal epithelial cell cultures, VEGF was predominately secreted apically, suggesting that in vivo most of the epithelium-derived VEGF is unavailable to the underlying blood vessels. In addition, prostate luminal epithelial cells produced angiopoietin-2, an angiogenesis inhibitor. In contrast, prostate SMCs produced angiopoietin-1, a positive modulator of angiogenesis. Synthesis of the angiopoietins did not respond to androgen treatment. CONCLUSIONS: Prostate smooth muscle may play an important role in regulating vascular responses to androgen.     

Rat and human aortic smooth muscle cells display differing migration and matrix metalloproteinase activities in response to dexamethasone

OBJECTIVE: The steroid dexamethasone inhibits neointimal hyperplasia development in rats but not in humans. This study investigates the differential effects of dexamethasone on rat and human smooth muscle cell migration and matrix metalloproteinase (MMP) activity. METHODS: Rat aortic smooth muscle cells were harvested from Sprague-Dawley rats. Human aortic smooth muscle cells were obtained from Clonetics. Boyden chamber migration assays were performed with chemoattractant (platelet-derived growth factor) and varying concentrations of dexamethasone (10(-9) to 10(-5) mol/L). Zymography of culture media was used to assess MMP activity, and Western blot analysis was used for quantification of MMP-2 and tissue inhibitor of MMP-2 (TIMP-2) secretion. RESULTS: Dexamethasone inhibits rat aortic smooth muscle cell migration in a dose-dependent fashion. An increase in concentrations of dexamethasone does not effect human aortic smooth muscle cell migration. Rat aortic smooth muscle cell MMP-2 activity is inhibited with dexamethasone in a dose-dependent fashion, and human aortic smooth muscle cell MMP-2 activity is unchanged with dexamethasone. MMP-2 secretion is inhibited with dexamethasone in rat aortic smooth muscle cells but remains unaltered in human aortic smooth muscle cells. Dexamethasone increases rat aortic smooth muscle cell TIMP-2 secretion, and human aortic smooth muscle cell TIMP-2 secretion remains constant. CONCLUSION: Dexamethasone inhibits rat aortic smooth muscle cell migration, MMP-2 activity, and MMP-2 secretion and increases TIMP-2 secretion. These effects are not observed in human aortic smooth muscle cells. These findings may explain why dexamethasone inhibits neointimal hyperplasia in animal models but is ineffective in humans. Inhibition of human smooth muscle cell migration in vitro may be useful in predicting the effectiveness of future therapeutic agents for treatment of neointimal hyperplasia in humans.     

Islet microvasculature in islet hyperplasia and failure in a model of type 2 diabetes

Gene expression profiling of islets from pre-diabetic male Zucker diabetic fatty (ZDF) rats showed increased expression of hypoxia-related genes, prompting investigation of the vascular integrity of the islets. The islet microvasculature was increased approximately twofold in young male ZDF rats by both morphometric analysis and quantifying mRNA levels of endothelial markers. ZDF rats at 12 weeks of age showed a significant reduction in the number of endothelial cells, which was prevented by pretreatment with pioglitazone. Light and electron microscopy of normoglycemic 7-week-old ZDF rats showed thickened endothelial cells with loss of endothelial fenestrations. By 12 weeks of age, there was disruption of the endothelium and intra-islet hemorrhage. Islets from 7- and 12-week-old ZDF rats showed an approximate three- and twofold increase in vascular endothelial growth factor (VEGF)-A mRNA and VEGF protein secretion, respectively, compared with lean controls. Thrombospondin-1 mRNA increased in 7- and 12-week-old rats by 2- and 10-fold, respectively, and was reduced by 50% in 12-week-old rats pretreated with pioglitazone. Islets from young male control rats induced migration of endothelial cells in a collagen matrix only after pretreatment with matrix metalloproteinase (MMP)-9. Islets from 7-week-old ZDF rats showed a fivefold increase in migration score compared with wild-type controls, even without MMP-9 treatment. Islets from 15-week-old ZDF rats did not induce migration; rather, they caused a significant rounding up of the duct-derived cells, suggesting a toxic effect. These data suggest that in the ZDF rat model of type 2 diabetes, an inability of the islet to maintain vascular integrity may contribute to beta-cell failure.     

Vascular endothelial growth factor (VEGF) gene therapy using a nonviral gene delivery system improves erectile function in a diabetic rat model

Erectile dysfunction (ED) is a cause of decreased quality of life in more than 70% of diabetic men. Vascular endothelial growth factor (VEGF) has shown to improve overall endothelial and smooth muscle cell dysfunction in models of ED. We describe a novel technique for nonviral, in vivo gene transfection of VEGF in the rat corpus cavernosum. Diabetic rats were transfected with DNA encoding a fusion VEGF/green fluorescent protein (GFP) complex and fluorescence microscopy was used to monitor the expression of VEGF-GFP fusion protein. Western blot and PCR analyses confirmed the expression of the GFP-VEGF fusion protein and mRNA. Functional studies using cavernous nerve stimulation revealed maximal intracavernous pressures (ICPs) of 63.1 mm Hg, and 30.7 mm Hg in the normal and diabetic control groups, respectively, and 47.4 mm Hg in VEGF-GFP-transfected diabetic group. Immunohistochemical analysis of the cavernosal tissue from transfected rats showed increased smooth muscle content compared with the diabetic control group. We show for the first time in our animal model that expression of the transfected VEGF in cavernosal tissue leads to an overall improvement of maximal ICP and smooth muscle content. On the basis of these results, it is tempting to speculate that our nonviral vector system offers an excellent system for gene delivery into cavernosal tissue, and that VEGF gene therapy using this system could be useful in improving erectile function in diabetic men.     

Plasma levels of cavernous and systemic norepinephrine and epinephrine in men during different phases of penile erection

PURPOSE: Knowledge of the functional anatomy, hemodynamics, neurophysiology and pharmacology of penile erection has improved tremendously during the last 2 decades. However, only few in vivo studies on human peripheral neurotransmission have been carried out up until now. Therefore, we conducted a study to examine plasma levels of catecholamines norepinephrine (NE) and epinephrine (E) in the peripheral and cavernous blood of healthy men during penile flaccidity and in different phases of erection. MATERIALS AND METHODS: Blood samples were drawn simultaneously from the corpus cavernosum (CC) and the cubital vein (P) in 53 healthy volunteers with normal erectile function, in four different functional states of the cavernous erectile tissue (flaccidity = 1, tumescence = 2, rigidity = 3, detumescence = 4). Penile erections were induced by audiovisual and tactile stimulation and the plasma concentrations of NE and E were determined by means of a radioimmunoassay (RIA). RESULTS: A significant (p <0.001) reduction of NE in CC plasma was found from flaccidity (362 + or - 173 pg./ml.) to rigidity (248 + or - 122 pg./ml.), followed by an increase in the detumescence phase (336 + or - 199 pg./ml.), (p <0.001). In contrast, changes in NE levels in the peripheral plasma were less pronounced from 1P (202 + or - 102 pg./ml.) to 3P (229 + or - 118 pg./ml.), (p = 0.006) and from 3P to 4P (222 + or - 127 pg./ml.), respectively (p = 0.370). The most pronounced increase in cavernous E levels were observed from flaccidity (47 + or - 41 pg. /ml.) to tumescence (130 + or - 106 pg./ml.) (p <0.001). Cavernous E levels dropped significantly from 113 + or - 67 pg./ml. during rigidity to 76 + or - 57 pg./ml. + or - during detumescence (p <0.001). The course of peripheral plasma levels of E was similar to that in the cavernous blood. Mean peripheral E level was 69 + or - 55 pg./ml. in the state of penile flaccidity, reaching 98 + or - 78 pg./ml. in tumescence and 82 + or - 64 pg./ml. in rigidity (p <0.001), respectively, and finally decreasing to 62 + or - 46 pg./ml. in detumescence. CONCLUSION: Penile erection, based on the relaxation of cavernous and arterial smooth muscle, is accompanied by a significant reduction of NE in cavernous blood, while E levels rose in peripheral and cavernous blood during developing erection.     

Growth potential of orbital cavernous hemangioma suggested by vascular endothelial growth factor and its receptor flk-1

Orbital cavernous hemangiomas (CHs) manifest as slowly developing symptoms indicative of slow growth. The present study investigated the involvement of angiogenic factors and their receptors in the growth of orbital CHs. Surgical specimens of orbital CHs were obtained from nine patients. Formalin-fixed, paraffin-embedded specimens were stained immunohistochemically using antibodies against Ki-67, CD31, alpha-smooth muscle actin (alpha-SMA), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and VEGF receptors (flt-1 and flk-1). CD31 was expressed in the single layer of endothelial cells lining the vascular cavity. The thick vascular walls were positive for alpha-SMA, indicating that the vascular walls were smooth muscle cells. Ki-67 antigen immunostaining was mostly positive in the vascular walls and the staining index ranged from 0% to 6.8% (mean +/- standard deviation, 2.7 +/- 1.9%). VEGF and bFGF immunostaining were positive in all specimens. Flt-1 immunostaining was negative in all specimens, but flk-1 immunostaining was positive in both endothelial cells and smooth muscle cells. These results suggest that both VEGF and its receptor flk-1 are important in the growth of orbital CH.     

Autocrine and paracrine functions of vascular endothelial growth factor (VEGF) in renal tubular epithelial cells

BACKGROUND: VEGF secreted by organ parenchymal cells controls vascularization by recruiting endothelial cells and supporting their proliferation. In the developing kidney VEGF-expressing epithelial cells also express VEGF receptors. We showed that VEGF stimulates tubulogenesis in addition to promoting vascularization in metanephric explants. Since explants are grown in serum-free media and are not perfused, we hypothesized that VEGF secreted by renal epithelia may induce their proliferation in an autocrine manner and chemoattract endothelial cells. METHODS: To test these hypotheses, we analyzed VEGF-mediated responses in vitro using several renal epithelial cell lines [immortalized rat proximal tubular cells (IRPT), transformed mouse proximal tubular cells (tsMPT), and normal rat kidney cells (NRK-52E)] expressing VEGF receptors (VEGFR). RESULTS: We demonstrated that VEGFR-2 phosphorylates upon human recombinant VEGF (rhVEGF) exposure, indicating that VEGFR-2 is the signaling receptor. All three cell lines secreted VEGF into the media as indicated by enzyme-linked immunosorbent assay (ELISA) and Western blotting. We showed that these tubular epithelial cells chemoattract endothelial cells when cocultured in vitro and that the chemoattraction is abolished by anti-VEGF neutralizing antibody. rhVEGF (10 ng/mL) induced a mitogenic effect similar to 10% fetal bovine serum (FBS) as assessed by H(3)-thymidine incorporation and elicited 30% decrease in apoptosis as determined by annexin V-fluorescein isothiocyanate (FITC) staining. CONCLUSION: These in vitro studies indicate that (1) tubular epithelial cells chemoattract endothelial cells in a paracrine fashion by secreting VEGF, and (2) VEGF stimulates proliferation and promotes survival of renal epithelial cells in an autocrine manner via VEGFR-2. Taken together, our results suggest that VEGF supports the growth of renal epithelia in addition to mediating kidney vascularization.     

Characterization of corpus cavernosum smooth muscle cell phenotype in diabetic rats with erectile dysfunction

Phenotypic modulation from a contractile to a proliferative state within vascular smooth muscle cells has a critical role in the pathogenesis of a variety of cardiovascular diseases. To investigate the characterization of corpus cavernosum smooth muscle cell phenotype in diabetic rats with erectile dysfunction, a group of Sprague-Dawley rats (n=30) were induced by intraperitoneal injection of streptozotocin (60?mg?kg(-1)) and screened by subcutaneous injection of apomorphine (100?μg?kg(-1)) for the measurement and comparison of the penile erections, and then three different groups were defined. Primary corpus cavernosum smooth muscle cells were cultured and passaged. The cavernous tissue segments were subjected to quantitative real-time polymerase chain reaction to determine the expressions of smooth muscle α-actin (SMA), SM myosin heavy chain (SMMHC), smoothelin, calponin and myocardin. Cell contractility in vitro and western blot analysis of SMA and SMMHC in the cavernous tissues and cells were determined. Compared with the control group (n=8) and the diabetes mellitus group (n=5), the expressions of SMA, calponin, SMMHC, smoothelin and myocardin mRNA were decreased in the cavernous tissues in rats of the diabetic erectile dysfunction group (n=15; P=0.001 and 0.02, P=0.014 and 0.012, both P<0.001, P=0.005 and <0.001, P=0.003 and 0.035, respectively). The levels of SMA and SMMHC proteins showed a significant decrease in cavernous tissues and cultured cells in rats of the diabetic erectile dysfunction group. Cells of the diabetic erectile dysfunction group exhibited significantly less contractility compared with those of other groups (P<0.001). Corpus cavernosum SM cell possesses the ability to modulate the phenotype under hyperglycemic conditions, which could have a key role in the pathogenesis of diabetic erectile dysfunction.