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The number of men with type II diabetes-associated erectile dysfunction (ED) continues to grow rapidly; however, the majority of basic science studies has examined mechanisms of ED in animal models of type I diabetes. In this study, we first establish an in vivo mouse model of type II diabetic ED using the leptin receptor mutated db/db and wild-type control BKS mouse. Furthermore, we hypothesized that dual mechanistic impairments contribute to the impaired erectile function in the type II diabetic mouse, altered vasoreactivity, and venoocclusive disorder. In vivo erectile function was measured as intracavernosal pressure (ICP) normalized to mean arterial pressure (MAP) following electrical stimulation of the cavernosal nerve. Venoocclusion was assessed by the maintenance of elevated in vivo ICP following intracorporal saline infusion. Vasoreactivity of isolated cavernosum in response to contractile and dilatory stimulation was examined in vitro by myography. Collagen and elastin content were evaluated by quantification of hydroxyproline and desmosine, respectively, as well as by quantitative PCR and histological analysis of isolated cavernosum. Erectile function was significantly decreased in db/db vs. BKS mice in a manner consistent with impairments in venoocclusive ability and decreased inflow. Heightened vasoconstriction and attenuated dilation in cavernosum of db/db vs. BKS mice suggest an overall lowered relaxation ability and thus impaired filling of the cavernosal spaces. A decrease in desmosine and hydroxyproline as well as lowered mRNA levels for tropoelastin, fibrillin-1, and [alpha](I) collagen were detected. These vasoreactive and sinusoidal matrix alterations may alter tissue compliance dispensability, preventing the normal expansion necessary for erection. matrix; elastin; collagen; penis

Relaxation of the smooth muscle cells in the cavernosal arterioles and sinuses results in increased blood flow into the penis, raising corpus cavernosum pressure to culminate in penile erection. Nitric oxide, released from non-adrenergic/non-cholinergic nerves, is considered the principle stimulator of cavernosal smooth muscle relaxation, however, the inhibition of vasoconstrictors (that is, norepinephrine and endothelin-1, refs. 5-9) cannot be ignored as a potential regulator of penile erection. The calcium-sensitizing [rho]-A/Rho-kinase pathway may play a synergistic role in cavernosal vasoconstriction to maintain penile flaccidity. Rho-kinase is known to inhibit myosin light chain phosphatase, and to directly phosphorylate myosin light-chain (in solution), altogether resulting in a net increase in activated myosin and the promotion of cellular contraction. Although Rho-kinase protein and mRNA have been detected in cavernosal tissue, the role of Rho-kinase in the regulation of cavernosal tone is unknown. Using pharmacologic antagonism (Y-27632, ref. 13, 18), we examined the role of Rho-kinase in cavernosal tone, based on the hypothesis that antagonism of Rho-kinase results in increased corpus cavernosum pressure, initiating the erectile response independently of nitric oxide. Our finding, that Rho-kinase antagonism stimulates rat penile erection independently of nitric oxide, introduces a potential alternate avenue for the treatment of erectile dysfunction., Author(s): Kanchan Chitaley (corresponding author) [1]; Christopher J. Wingard [2]; R. Clinton Webb [2]; Heather Branam [2]; Vivienne S. Stopper [2]; Ronald W. Lewis [3]; Thomas M. Mills [2, 3] [...]

Aims We investigated the mechanisms of diabetic bladder dysfunction (BD) through analysis of the roles of L- and T-type voltage-gated calcium channels (VGCCs), with the ultimate goal of identifying potential drug targets for diabetic BD. Methods Bladder function of db/db (type 2 diabetes) and wild type ( Wt) mice was evaluated by behavioral tests and in vivo cystometry. Contractile responses of bladder strips to carbachol were measured with or without pre-treatment with nifedipine (a L-type VGCC blocker) or mibefradil (a T-type VGCC blocker). Furthermore, the effects of mibefradil and nifedipine on the proliferation of human bladder smooth muscle cells (BSMCs) were studied. Results db/db mice had significantly increased voiding frequency, bladder weight, bladder compliance and capacity, and heightened contractile response to carbachol, compared to Wt mice. Nifedipine, but not mibefradil, dramatically suppressed bladder tissue contraction in Wt mice. Whereas nifedipine nearly completely inhibited bladder contraction in db/db mice, mibefradil 'normalized' the heightened bladder contractility of db/db mice to the level of Wt mice. In culture, mibefradil, but not nifedipine, inhibited the proliferation of human BSMCs. Conclusion Our results indicate that while L-type VGCCs play a major role in the contraction of both diabetic and non-diabetic bladders, T-type VGCCs are involved in the contraction of diabetic bladders and mediate BSMC proliferation. This study provides support for further investigations on the effect of blockade of T-type VGCC or combined blockade of both types of VGCCs in the treatment of diabetic BD. Neurourol. Urodynam. 33:147-152, 2014. © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Context: Inhalation of fine particulate matter (PM2.5) is associated with acute pulmonary inflammation and impairments in cardiovascular function. In many regions, PM2.5 is largely derived from diesel exhaust (DE), and these pathophysiological effects may be due in part to oxidative stress resulting from DE inhalation. The antioxidant glutathione (GSH) is important in limiting oxidative stress-induced vascular dysfunction. The rate-limiting enzyme in GSH synthesis is glutamate cysteine ligase and polymorphisms in its catalytic and modifier subunits (GCLC and GCLM) have been shown to influence vascular function and risk of myocardial infarction in humans. Objective: We hypothesized that compromised de novo synthesis of GSH in Gclm−/+ mice would result in increased sensitivity to DE-induced lung inflammation and vascular effects. Materials and methods: WT and Gclm−/+ mice were exposed to DE via inhalation (300 μg/m3) for 6 h. Neutrophil influx into the lungs, plasma GSH redox potential, vascular reactivity of aortic rings and aortic nitric oxide (NO•) were measured. Results: DE inhalation resulted in mild bronchoalveolar neutrophil influx in both genotypes. DE-induced effects on plasma GSH oxidation and acetylcholine (ACh)-relaxation of aortic rings were only observed in Gclm−/+ mice. Contrary to our hypothesis, DE exposure enhanced ACh-induced relaxation of aortic rings in Gclm−/+ mice. Discussion and conclusion: These data support the hypothesis that genetic determinants of antioxidant capacity influence the biological effects of acute inhalation of DE. However, the acute effects of DE on the vasculature may be dependent on the location and types of vessels involved. Polymorphisms in GSH synthesis genes are common in humans and further investigations into these potential gene-environment interactions are warranted. [ABSTRACT FROM AUTHOR]

The proliferation of all nontransformed adherent cells is dependent upon the development of mechanical tension within the cell; however, little is known about the mechanisms by which signals regulated by mechanical tension are integrated with those regulated by growth factors. We show here that Skp2, a component of a ubiquitin ligase complex that mediates the degradation of several proteins that inhibit proliferation, is upregulated when increased mechanical tension develops in intact smooth muscle and that its upregulation is critical for the smooth muscle proliferative response to increased mechanical tension. Notably, whereas growth factors regulate Skp2 at the level of protein stability, we found that mechanical tension regulates Skp2 at the transcriptional level. Importantly, we demonstrate that the calcium-regulated transcription factor NFATc1 is a critical mediator of the effect of increased mechanical tension on Skp2 transcription. These findings identify Skp2 as a node at which signals from mechanical tension and growth factors are integrated to regulate proliferation, and they define calcium-NFAT-Skp2 signaling as a critical pathway in the mechanoregulation of proliferation. [ABSTRACT FROM AUTHOR]

Introduction. Diabetes mellitus (DM) is a major risk factor for the development of erectile dysfunction (ED). Although most diabetic ED cases are in patients with type 2 diabetes (T2DM), the majority of basic science studies examining mechanisms of diabetic ED have been conducted in animal models of type 1 diabetes. Aim. Recently, however, clinical and laboratory-based studies have uncovered some key underlying factors of T2DM-associated ED, which we have compiled in this review of T2DM ED. Main Outcome Measures. The outcomes discussed in this review include major mechanisms underlying T2DM, discussing both clinical and basic science studies. Methods. We conducted an extensive search of pertinent clinical and basic science literature using PUBMED. Results. Mechanisms causing ED in T2DM are multifactorial and often lead to resistance to current therapy. Systemic effects of hyperglycemia and hypogonadism contribute to the development of impaired vasodilatory signaling, smooth muscle cell hypercontractility, and veno-occlusive disorder in T2DM ED. Conclusions. Understanding the different causes for ED in T2DM patients may allow targeted therapy for improved erectile function. Hidalgo-Tamola J, and Chitaley K. Type 2 diabetes mellitus and erectile dysfunction. J Sex Med **;**:**–**. [ABSTRACT FROM AUTHOR]

We previously reported the inhibition of Rho-kinase to result in increased intracavernosal pressure (ICP) in an in vivo rat model of erection. Expression of an upstream activator of Rho-kinase, RhoA, has been demonstrated in the penile vasculature; however, the functional role of RhoA in the regulation of erection remains unknown. We used adeno-associated viral gene transfer of a dominant negative RhoA mutant (T19NRhoA) into rat cavernosum to test the hypothesis that RhoA activation is physiologically important for maintenance of the non-erect state and inhibition of this pathway leads to erection. Anesthetized, male, Sprague–Dawley rats transfected with the T19NRhoA mutant exhibited an elevated baseline ICP/mean arterial pressure (MAP) and nerve stimulation-induced ICP/MAP as compared with β-galactosidase-transfected controls. The novel findings of this study demonstrate a functional role of RhoA in maintaining the flaccid penis and provide support for the inhibition of RhoA as a potential therapy for the enhancement of erectile function. [Copyright &y& Elsevier]

This study tests the hypothesis that microtubule (MT) depolymerization facilitates contraction of rat aorta via activation of Rho-kinase. Aortic rings from Sprague–Dawley rats were placed in a muscle bath for the measurement of isometric force generation. Bath temperature was decreased from 37 to 10–20 °C (30 min), inducing MT depolymerization. Some vessels were treated with nocodazole (10−5 M) or colchicine (10−8–10−5 M) to stabilize the MTs in the depolymerized state, and the remaining vessels were treated with dimethyl sulfoxide (DMSO: vehicle). Warming of vessels to 37 °C induced a significantly greater contraction in nocodazole- and colchicine-treated vessels as compared with controls, and this increase was blocked by pretreatment with taxol (10−5 M; a MT stabilizing agent) [force (mg): NOC 1159±93; COL 1138±69; DMSO 578±14; TAX+NOC 526±43; TAX+COL 538±90]. Following the sustained contraction in response to rewarming, Rho-kinase inhibition with Y-27632 (10−5 M) relaxed nocodazole- and colchicine-treated rings to a significantly greater extent as compared to DMSO-treated vessels (percent relaxation: NOC 64±2; COL 65±5; DMSO 33±5). These results support the hypothesis that MT depolymerization facilitates contraction of rat aorta via activation of Rho-kinase. [Copyright &y& Elsevier]

Erectile dysfunction is a harbinger of cardiovascular disease. The pro-oxidant, copper-transporter, ceruloplasmin (CP), is elevated in diabetic patients and rodents. In this study, we tested the hypothesis that CP impairs penile endothelium-dependent dilation in diabetes. Cp-deficient (Cp-/-) and wild-type (Wt) mice (2-3 mo) were treated with streptozotocin (STZ; 100mg/kg, i.p., 2 days) or citrate buffer (CNT). After 6-8 wks, blood glucose and CP expression were measured, and cavernosal relaxation to acetylcholine (ACh) assessed by myography. In additional studies, CP (3 µg/µl in 50µl) was administered intracorporally into Wt mice. Intracavernosal pressure/mean arterial pressure (ICP/MAP) to electrical stimulation of the cavernous nerve was assessed prior to and after CP injection. ACh relaxation did not change in non-diabetic, Wt-CNT vs. Cp-/--CNT, mice. STZ increased blood glucose in both groups. Penile CP expression was elevated in STZ vs. CNT-treated Wt mice. Maximum ACh relaxation (10µM) was significantly decreased in STZ-treated Wt mice vs. Wt-CNT (50±5 vs. 65±2%), but not in Cp-/--STZ vs. Cp-/--CNT mice (68±3 vs. 66±7%). Further, injection of CP significantly blunted ICP/MAP (mean change in area under the curve after CP or vehicle: 25±6.4 vs. -0.4±3.2, respectively). Altogether, these loss and gain of function data support the hypothesis that CP impairs penile vascular endothelial function in diabetes. [ABSTRACT FROM AUTHOR]

Objective: Vascular gene transfer is a powerful tool for investigating and treating vascular diseases; however, its utility is limited by brevity of transgene expression and vector-associated inflammation. Helper-dependent adenovirus (HDAd), an advanced-generation adenovirus that lacks all viral genes, is superior to first-generation adenovirus (FGAd) in normal rabbit arteries. We compared HDAd to FGAd in arteries of cholesterol-fed rabbits, a model of early atherogenesis in which transgene expression might be decreased, and inflammation increased., Methods and Results: Carotid arteries of chow- and cholesterol-fed rabbits were infused with FGAd, HDAd, or medium. HDAd expressed a transgene at least as well in arteries of cholesterol-fed rabbits as in arteries of chow-fed rabbits and expressed more durably than FGAd. In arteries of cholesterol-fed rabbits, HDAd stimulated less intimal growth, lipid deposition, and inflammation than FGAd. Neither vector affected phenylephrine-induced contraction or nitroprusside-mediated relaxation; however, both vectors decreased maximal acetylcholine-stimulated vasorelaxation. The relative absence of intimal growth in HDAd arteries could interfere with the utility of this model for testing atheroprotective genes; however, both coinfusion of FGAd and extension of cholesterol feeding yielded larger intimal lesions, on which atheroprotective genes could be tested., Conclusion: HDAd is superior to FGAd for expression of transgenes in atherosclerosis-prone arteries.

Urokinase-type plasminogen activator (uPA) is expressed at increased levels in stenotic, atherosclerotic human arteries. However, the biological roles of uPA in the artery wall are poorly understood. Previous studies associate uPA with both acute vasoconstriction and chronic vascular remodeling and attribute uPA-mediated vasoconstriction to the kringle - not the catalytic - domain of uPA. We used an in-vivo uPA overexpression model to test the hypothesis that uPA-induced vasoconstriction is a reversible vasomotor process that can be prevented - and uPA fibrinolytic activity preserved - by: 1) removing the growth factor and kringle domains; or 2) anchoring uPA to the endothelial surface. To test this hypothesis we constructed adenoviral vectors that express: wild-type rabbit uPA (AduPA); a uPA mutant lacking the NH(2)-terminal growth-factor and kringle domains (AduPAdel); a mutant lacking catalytic activity (AduPAS-->A), and a cell-surface anchored mutant (AdTMuPA). uPA mutants were expressed and characterised in vitro and in carotid arteries in vivo. uPAS-->A had no plasminogen activator activity. Activity was similar for uPA and uPAdel, whereas AdTMuPA had only cell-associated activity. AduPAS-->A arteries were not constricted. AduPA, AduPAdel, and AdTM-uPA arteries were constricted (approximately 30% smaller lumens; p< or =0.008 vs. AdNull arteries). Papaverine reversed constriction of AduPA arteries. uPA-mediated arterial constriction is a vasomotor process that is mediated by uPA catalytic activity, not by the NH(2)-terminal domains. Anchoring uPA to the endothelial surface does not prevent vasoconstriction. uPA catalytic activity, generated by artery wall cells, may contribute to lumen loss in human arteries. Elimination of uPA vasoconstrictor activity requires concomitant loss of fibrinolytic activity.

To determine specific molecular features of endothelial cells (ECs) relevant to the physiological process of penile erection we compared gene expression of human EC derived from corpus cavernosum of men with and without erectile dysfunction (HCCEC) to coronary artery (HCAEC) and umbilical vein (HUVEC) using Affymetrix GeneChip microarrays and GeneSifter software. Genes differentially expressed across samples were partitioned around medoids to identify genes with highest expression in HCCEC. A total of 190 genes/transcripts were highly expressed only in HCCEC. Gene Ontology classification indicated cavernosal enrichment in genes related to cell adhesion, extracellular matrix, pattern specification and organogenesis. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed high expression of genes relating to ECM-receptor interaction, focal adhesions, and cytokine-cytokine receptor interaction. Real-time PCR confirmed expression differences in cadherins 2 and 11, claudin 11 (CLDN11), desmoplakin, and versican. CLDN11, a component of tight junctions not previously described in ECs, was highly expressed only in HCCEC and its knockdown by siRNA significantly reduced transendothelial electrical resistance in HCCEC. Overall, cavernosal ECs exhibited a transcriptional profile encoding matrix and adhesion proteins that regulate structural and functional characteristics of blood vessels. Contribution of the tight junction protein CLDN11 to barrier function in endothelial cells is novel and may reflect hemodynamic requirements of the corpus cavernosum.

The proliferation of all nontransformed adherent cells is dependent upon the development of mechanical tension within the cell; however, little is known about the mechanisms by which signals regulated by mechanical tension are integrated with those regulated by growth factors. We show here that Skp2, a component of a ubiquitin ligase complex that mediates the degradation of several proteins that inhibit proliferation, is upregulated when increased mechanical tension develops in intact smooth muscle and that its upregulation is critical for the smooth muscle proliferative response to increased mechanical tension. Notably, whereas growth factors regulate Skp2 at the level of protein stability, we found that mechanical tension regulates Skp2 at the transcriptional level. Importantly, we demonstrate that the calcium-regulated transcription factor NFATc1 is a critical mediator of the effect of increased mechanical tension on Skp2 transcription. These findings identify Skp2 as a node at which signals from mechanical tension and growth factors are integrated to regulate proliferation, and they define calcium-NFAT-Skp2 signaling as a critical pathway in the mechanoregulation of proliferation.

Objective: Vasodilator-stimulated phosphoprotein (VASP) was identified as a substrate for cGMP-dependent protein kinase (PKG) and cAMP-dependent protein kinase (PKA). It is preferentially phosphorylated at serine239 by PKG, whereas serine157 is a preferred phosphorylation site for PKA. In addition, serine157 is phosphorylated by PKC in response to serum. We have investigated the effects of VASP and VASP phosphorylation at serine157 and serine239 on smooth muscle cell (SMC) proliferation and nitric oxide (NO)-mediated growth inhibition., Methods and Results: Aortic SMCs derived from VASP-deficient mice were transduced with retroviral vectors encoding either wild-type VASP or VASP mutants (S157A-VASP and S239A-VASP), in which serine157 and serine239, respectively, were replaced by a nonphosphorylatable amino acid, alanine. Expression of wt-VASP and S239A-VASP significantly increased proliferation, whereas expression of S157A-VASP was inhibitory. Expression of S239A-VASP rendered SMCs less sensitive to growth inhibition by the NO donor, S-nitroso-n-acetylpenicillamine, when compared with cells expressing wt-VASP. Similar effects were observed in cultured rat SMCs in which wt-VASP, S157A-VASP, and S239A-VASP were expressed., Conclusions: Our data suggest that VASP phosphorylation at serine157 is required for the growth-stimulatory effect of VASP in SMCs, whereas VASP phosphorylation at serine239 is involved in the growth inhibitory effects of NO on SMCs.

NO induces vasodilation through cGMP-dependent protein kinase--dependent and --independent mechanisms. A recent study demonstrated that recombinant cGMP-dependent protein kinase can phosphorylate the small G protein, RhoA, thus inhibiting its activity. Additionally, sodium nitroprusside was found to reverse the phenylephrine-induced translocation of RhoA, which is further indicative of the inhibition of RhoA activity. RhoA is known to be involved in the Ca(2+) sensitization of vascular smooth muscle through the actions of one of its downstream effectors, Rho-kinase. This study examined whether NO endogenously induces the relaxation of intact rat aorta via the inhibition of the Rho-kinase--mediated Ca(2+)-sensitizing pathway. Endogenous Rho-kinase inhibitor activity was inhibited by the selective compound Y-27632. Treatment of endothelium-intact rat aorta with Y-27632 (1 micromol/L) resulted in an attenuation of maximal force generated in response to phenylephrine. In endothelium-denuded rings, however, 1 micromol/L Y-27632 was ineffective at inhibiting the phenylephrine-induced contraction. Additionally, 1 micromol/L Y-27632 was significantly less effective at inhibiting the phenylephrine-induced contraction of endothelium-intact rings in the presence of inhibitors of NO synthase or guanylate cyclase (N(omega)-nitro-L-arginine and 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one, respectively). Interestingly, sodium nitroprusside restored the ability of 1 micromol/L Y-27632 to attenuate phenylephrine-induced contraction. Rho-kinase inhibition was also found to increase the sensitivity of the endothelium-denuded aorta to sodium nitroprusside. These data demonstrate that NO inhibits Rho-kinase activity in the intact rat aorta, supporting the hypothesis that endogenous NO-mediated vasodilation occurs through the inhibition of Rho-kinase constrictor activity in the intact rat aorta.