Modifications of Erectile Tissue Components in the Penis during the Fetal Period

Background

The penile erectile tissue has a complex microscopic anatomy with important functions in the mechanism of penile erection. The knowledge of such structures is necessary for understanding the normal physiology of the adult penis. Therefore, it is important to know the changes of these penile structures during fetal development. This study aims to analyze the development of the main components of the erectile tissue, such as collagen, smooth muscle fibers and elastic system fibers, in human fetuses.

Methodology/Principal Findings

We studied the penises of 56 human fetuses aged 13 to 36 weeks post-conception (WPC). We used histochemical and immunohistochemical staining, as well as morphometric techniques to analyze the collagen, smooth muscle fibers and elastic system fibers in the corpus cavernosum and in the corpus spongiosum. These elements were identified and quantified as percentage by using the Image J software (NIH, Bethesda, USA). From 13 to 36 WPC, in the corpus cavernosum, the amount of collagen, smooth muscle fibers and elastic system fibers varied from 19.88% to 36.60%, from 4.39% to 29.76% and from 1.91% to 8.92%, respectively. In the corpus spongiosum, the amount of collagen, smooth muscle fibers and elastic system fibers varied from 34.65% to 45.89%, from 0.60% to 11.90% and from 3.22% to 11.93%, respectively.

Conclusions

We found strong correlation between the elements analyzed with fetal age, both in corpus cavernosum and corpus spongiosum. The growth rate of these elements was more intense during the second trimester (13 to 24 WPC) of gestation, both in corpus cavernosum and in corpus spongiosum. There is greater proportional amount of collagen in the corpus spongiosum than in corpus cavernosum during all fetal period. In the corpus spongiosum, there is about four times more collagen than smooth muscle fibers and elastic system fibers, during all fetal period studied.     

Bariatric Surgery Improves the Cavernosal Neuronal,Vasorelaxation, and Contraction Mechanisms for Erectile Dysfunction As Result of Amelioration of Glucose Homeostasis in a Diabetic Rat Model

Background

Bariatric surgery is an effective treatment option for both obesity and obesity-related type 2 diabetes mellitus (T2DM). However, little is known regarding the effects of bariatric surgery on erectile dysfunction among patients with T2DM. Therefore, we investigated whether bariatric surgery would lead to structural and biochemical changes in the corpus cavernosum.

Material and Method

Twenty-five male Otsuka Long-Evans Tokushima Fatty rats were assigned to either a control group (sham operation, n = 10) or a bariatric surgery group (gastric bypass surgery, n = 15). Four weeks after the operation, each group of rats was evaluated with an oral glucose tolerance test (OGTT). The penile intracavernous pressure was measured for erectile functional analysis. Histologic evaluation of the tissue was performed with Masson''s trichrome staining. Endothelial nitric oxide synthase (eNOS), neuronal nitric oxide synthase (nNOS), Rho kinase, and 8-hydroxy-2-deoxyguanosine (8-OHdG) levels in the corpus cavernosum were assayed by using western blot and ELISA.

Results

The mean body weight of the bariatric surgery group was lower than the control group (p = 0.002). The postoperative OGTT result was lower in the bariatric surgery group than in the control group (p = 0.014), and this was lower than the preoperative value (p = 0.037). The intracavernous pressure/mean arterial pressure ratio was higher in the bariatric surgery group compared to the control group (p = 0.021), and a higher cavernosum smooth muscle/collagen ratio was observed in the bariatric surgery group compared to the control group (p = 0.025). Likewise, the expression of eNOS and nNOS was higher in bariatric surgery group than in the control group (p = 0.027 and p = 0.008, respectively). Decreased expression of Rho kinase and levels of 8-OHdG were observed in the bariatric surgery group (p = 0.032).

Conclusion

In this animal model, bariatric surgery appears to ameliorate T2DM-related metabolic dysfunction leading to structural and biochemical changes in the corpus cavernosum, and thus, results in improvement of erectile dysfunction associated with T2DM.     

Sodium-Coupled Neutral Amino Acid Transporter 1 (SNAT1) Modulates L-Citrulline Transport and Nitric Oxide (NO) Signaling in Piglet Pulmonary Arterial Endothelial Cells

Rationale

There is evidence that impairments in nitric oxide (NO) signaling contribute to chronic hypoxia-induced pulmonary hypertension. The L-arginine-NO precursor, L-citrulline, has been shown to ameliorate pulmonary hypertension. Sodium-coupled neutral amino acid transporters (SNATs) are involved in the transport of L-citrulline into pulmonary arterial endothelial cells (PAECs). The functional link between the SNATs, L-citrulline, and NO signaling has not yet been explored.

Objective

We tested the hypothesis that changes in SNAT1 expression and transport function regulate NO production by modulating eNOS coupling in newborn piglet PAECs.

Methods and Results

A silencing RNA (siRNA) technique was used to assess the contribution of SNAT1 to NO production and eNOS coupling (eNOS dimer-to-monomer ratios) in PAECs from newborn piglets cultured under normoxic and hypoxic conditions in the presence and absence of L-citrulline. SNAT1 siRNA reduced basal NO production in normoxic PAECs and prevented L-citrulline-induced elevations in NO production in both normoxic and hypoxic PAECs. SNAT1 siRNA reduced basal eNOS dimer-to-monomer ratios in normoxic PAECs and prevented L-citrulline-induced increases in eNOS dimer-to-monomer ratios in hypoxic PAECs.

Conclusions

SNAT1 mediated L-citrulline transport modulates eNOS coupling and thus regulates NO production in hypoxic PAECs from newborn piglets. Strategies that increase SNAT1-mediated transport and supply of L-citrulline may serve as novel therapeutic approaches to enhance NO production in patients with pulmonary vascular disease.     

Diazeniumdiolate mediated nitrosative stress alters nitric oxide homeostasis through intracellular calcium and S-glutathionylation of nitric oxide synthetase

Background

PABA/NO is a diazeniumdiolate that acts as a direct nitrogen monoxide (NO) donor and is in development as an anticancer drug. Its mechanism of action and effect on cells is not yet fully understood.

Methodology/Principal Findings

We used HPLC and mass spectrometry to identify a primary nitroaromatic glutathione metabolite of PABA/NO and used fluorescent assays to characterize drug effects on calcium and NO homeostasis, relating these to endothelial nitric oxide synthase (eNOS) activity. Unexpectedly, the glutathione conjugate was found to be a competitive inhibitor of sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA) presumably at the same site as thapsigargin, increasing intracellular Ca²⁺ release and causing auto-regulation of eNOS through S-glutathionylation.

Conclusions/Significance

The initial direct release of NO after PABA/NO was followed by an eNOS-mediated generation of NO as a consequence of drug-induced increase in Ca²⁺ flux and calmodulin (CaM) activation. PABA/NO has a unique dual mechanism of action with direct intracellular NO generation combined with metabolite driven regulation of eNOS activation.     

Dual Role of Endothelial Nitric Oxide Synthase in Oxidized LDL-Induced,p66Shc-Mediated Oxidative Stress in Cultured Human Endothelial Cells

Background

The aging gene p66^Shc, is an important mediator of oxidative stress-induced vascular dysfunction and disease. In cultured human aortic endothelial cells (HAEC), p66^Shc deletion increases endothelial nitric oxide synthase (eNOS) expression and nitric oxide (NO) bioavailability via protein kinase B. However, the putative role of the NO pathway on p66^Shc activation remains unclear. This study was designed to elucidate the regulatory role of the eNOS/NO pathway on p66^Shc activation.

Methods and Results

Incubation of HAEC with oxidized low density lipoprotein (oxLDL) led to phosphorylation of p66^Shc at Ser-36, resulting in an enhanced production of superoxide anion (O₂ ^-). In the absence of oxLDL, inhibition of eNOS by small interfering RNA or L-NAME, induced p66^Shc phosphorylation, suggesting that basal NO production inhibits O₂ ^- production. oxLDL-induced, p66^Shc-mediated O₂- was prevented by eNOS inhibition, suggesting that when cells are stimulated with oxLDL eNOS is a source of reactive oxygen species. Endogenous or exogenous NO donors, prevented p66^Shc activation and reduced O_2- production. Treatment with tetrahydrobiopterin, an eNOS cofactor, restored eNOS uncoupling, prevented p66^Shc activation, and reduced O₂- generation. However, late treatment with tetrahydropterin did not yield the same result suggesting that eNOS uncoupling is the primary source of reactive oxygen species.

Conclusions

The present study reports that in primary cultured HAEC treated with oxLDL, p66^Shc-mediated oxidative stress is derived from eNOS uncoupling. This finding contributes novel information on the mechanisms of p66^Shc activation and its dual interaction with eNOS underscoring the importance eNOS uncoupling as a putative antioxidant therapeutical target in endothelial dysfunction as observed in cardiovascular disease.     

Urotensin-ⅡReceptor Antagonist SB-710411 Protects Rat Heart against Ischemia-Reperfusion Injury via RhoA/ROCK Pathway

Aim

SB-710411 is a rat selective urotensin-II (U-II) receptor antagonist, which can block U-II-induced contraction of the aorta and inhibit U-II-induced myocardial fibrosis in rats. However, the effect of SB-710411 on myocardial ischemia-reperfusion (I/R) injury is unclear. The present study was designed to investigate whether SB-710411 has a protective effect on myocardial I/R injury in rats and the possible mechanisms.

Methods and Results

Myocardial I/R injury was induced by occluding the left anterior descending coronary artery in adult male Sprague-Dawley rats. Hemodynamic parameters, electrocardiogram (ECG), infarct size, histological alteration, lactate dehydrogenase (LDH), creatine phosphokinase-MB (CK-MB), cardiac troponin I (cTnI), RhoA, and the protein expressions of U-II receptor (UTR), ROCK₁ and ROCK₂ were evaluated. Cardiac I/R injury significantly up-regulated the expressions of UTR, ROCK₁ and ROCK₂ proteins in rat myocardium. SB-710411 1.0 and 2.0 μg/kg significantly reduced cardiac I/R-induced the infarct size and histological damage in rat myocardium, markedly inhibited the changes of hemodynamic parameters and the increases of ST-segment in ECG, the serum LDH and CK-MB activities and cTnI level in rats subjected to myocardial I/R injury. Furthermore, SB-710411 obviously prevented myocardial I/R-increased RhoA activity and UTR, ROCK₁ and ROCK₂ protein expressions.

Conclusions

Our results indicate that cardiac I/R injury increases myocardial UTR expression, and SB-710411 has a potent protective effect on myocardial I/R injury in rats. The cardioprotection may be associated with the inhibition of UTR-RhoA/ROCK pathway.     

Urotensin-II activates L-arginine/nitric oxide pathway in isolated rat aortic adventitia

Urotensin-II (U-II), a cyclic peptide widely expressed in blood vessels, has diverse vascular actions that range from potent vasoconstriction to vasodilation. Although, U-II-induced vasodilation has been shown to be partially dependent on nitric oxide (NO), the involvement of vascular adventitia-derived NO, remains unknown. The present study aimed to elucidate the activation of U-II on L-arginine/NO pathway in isolated rat aortic adventitia. In adventitia of thoracic and abdominal aortas, the l-arginine/NO pathway was similarly characterized: the uptake of l-[(3)H]arginine was Na(+)-independent, with the peak occurring over around 40 min incubation; the total NO synthase (NOS) activity was mostly calcium-independent (>90%), and significantly inhibited by a specific iNOS inhibitor AMT; the production of NO metabolites nitrate and nitrite (NO(x)) was stimulated by L-arginine but not by D-arginine. In aortic adventitia exposed to rat U-II (10(-9) and 10(-8)M) for 6 h, the V(max) of l-[(3)H]arginine uptake over 40 min incubation was significantly increased, while the K(m) of l-[(3)H]arginine uptake showed no significant change. Besides, the iNOS mRNA level was up-regulated, the total NOS activity, largely calcium-independent, was significantly induced, and the NO(x) production was significantly stimulated by U-II. According to the same protocol as U-II, the positive control lipopolysaccharide (LPS, 10 microg/ml), which had been established to activate adventitial L-arginine/NO pathway, increased l-[(3)H]arginine uptake, iNOS activity and NO(x) production to a greater extent than U-II. In addition, the total NOS activities induced by 3 and 6h incubation of U-II and LPS were significantly inhibited by a specific inhibitor of protein synthesis, actinomycin D. In conclusion, the results showed that rat U-II activated L-arginine/NOS/NO pathway in rat aortic adventitia, suggesting a potential contributive role of adventitia-derived NO in the vasodilator response of U-II.     

Chronic deficiency of nitric oxide affects hypoxia inducible factor-1α (HIF-1α) stability and migration in human endothelial cells

Background

Endothelial dysfunction in widely diffuse disorders, such as atherosclerosis, hypertension, diabetes and senescence, is associated with nitric oxide (NO) deficiency. Here, the behavioural and molecular consequences deriving from NO deficiency in human umbilical vein endothelial cells (HUVECs) were investigated.

Results

Endothelial nitric oxide synthase (eNOS) was chronically inhibited either by N ^G-Nitro-l-arginine methyl ester (l-NAME) treatment or its expression was down-regulated by RNA interference. After long-term l-NAME treatment, HUVECs displayed a higher migratory capability accompanied by an increased Vascular Endothelial Growth Factor (VEGF) and VEGF receptor-2 (kinase insert domain receptor, KDR) expression. Moreover, both pharmacological and genetic inhibition of eNOS induced a state of pseudohypoxia, revealed by the stabilization of hypoxia-inducible factor-1α (HIF-1α). Furthermore, NO loss induced a significant decrease in mitochondrial mass and energy production accompanied by a lower O₂ consumption. Notably, very low doses of chronically administered DETA/NO reverted the HIF-1α accumulation, the increased VEGF expression and the stimulated migratory behaviour detected in NO deficient cells.

Conclusion

Based on our results, we propose that basal release of NO may act as a negative controller of HIF-1α levels with important consequences for endothelial cell physiology. Moreover, we suggest that our experimental model where eNOS activity was impaired by pharmacological and genetic inhibition may represent a good in vitro system to study endothelial dysfunction.     

Estrogen Receptor Alpha as a Key Target of Red Wine Polyphenols Action on the Endothelium

Background

A greater reduction in cardiovascular risk and vascular protection associated with diet rich in polyphenols are generally accepted; however, the molecular targets for polyphenols effects remain unknown. Meanwhile evidences in the literature have enlightened, not only structural similarities between estrogens and polyphenols known as phytoestrogens, but also in their vascular effects. We hypothesized that alpha isoform of estrogen receptor (ERα) could be involved in the transduction of the vascular benefits of polyphenols.

Methodology/Principal Findings

Here, we used ERα deficient mice to show that endothelium-dependent vasorelaxation induced either by red wine polyphenol extract, Provinols™, or delphinidin, an anthocyanin that possesses similar pharmacological profile, is mediated by ERα. Indeed, Provinols™, delphinidin and ERα agonists, 17-beta-estradiol and PPT, are able to induce endothelial vasodilatation in aorta from ERα Wild-Type but not from Knock-Out mice, by activation of nitric oxide (NO) pathway in endothelial cells. Besides, silencing the effects of ERα completely prevented the effects of Provinols™ and delphinidin to activate NO pathway (Src, ERK 1/2, eNOS, caveolin-1) leading to NO production. Furthermore, direct interaction between delphinidin and ERα activator site is demonstrated using both binding assay and docking. Most interestingly, the ability of short term oral administration of Provinols™ to decrease response to serotonin and to enhance sensitivity of the endothelium-dependent relaxation to acetylcholine, associated with concomitant increased NO production and decreased superoxide anions, was completely blunted in ERα deficient mice.

Conclusions/Significance

This study provides evidence that red wine polyphenols, especially delphinidin, exert their endothelial benefits via ERα activation. It is a major breakthrough bringing new insights of the potential therapeutic of polyphenols against cardiovascular pathologies.     

Biosynthesis of nitric oxide and citrulline from L-arginine by constitutive nitric oxide synthase present in rabbit corpus cavernosum.

The objective of this study was to determine whether a constitutive isoform of nitric oxide (NO) synthase is present in rabbit corpus cavernosum that could account for the involvement of the L-arginine-NO pathway in neurogenically-elicited relaxation of the corpus cavernosum and, therefore, penile erection. Citrulline was determined by monitoring the formation of 3H-citrulline from 3H-L-arginine. NO was determined by monitoring the formation of total NO(x) (NO+nitrite [NO2-]+nitrate [NO3-]) by chemiluminescence after reduction of NO(x) to NO by acidic vanadium (III). Equimolar quantities of NO plus citrulline were generated from L-arginine and the formation of both products was time-dependent at 37 degrees C. NO synthase activity was distributed almost entirely to the cytosolic fraction. Enzymatic activity was completely dependent on NADPH, calmodulin, and calcium. Addition of tetrahydrobiopterin increased NO synthase activity by about 30 percent. The NO synthase inhibitor NG-nitro-L-arginine, abolished enzymatic activity. The Km for L-arginine was 17 microM and the Vmax of the reaction was 18 pmol/min/mg protein. These observations indicate that a cytosolic, constitutive isoform of NO synthase, like that found in brain neuronal tissue, is present in rabbit corpus cavernosum.     

Interaction between Advanced Glycation End Products Formation and Vascular Responses in Femoral and Coronary Arteries from Exercised Diabetic Rats

Background

The majority of studies have investigated the effect of exercise training (TR) on vascular responses in diabetic animals (DB), but none evaluated nitric oxide (NO) and advanced glycation end products (AGEs) formation associated with oxidant and antioxidant activities in femoral and coronary arteries from trained diabetic rats. Our hypothesis was that 8-week TR would alter AGEs levels in type 1 diabetic rats ameliorating vascular responsiveness.

Methodology/Principal Findings

Male Wistar rats were divided into control sedentary (C/SD), sedentary diabetic (SD/DB), and trained diabetic (TR/DB). DB was induced by streptozotocin (i.p.: 60 mg/kg). TR was performed for 60 min per day, 5 days/week, during 8 weeks. Concentration-response curves to acetylcholine (ACh), sodium nitroprusside (SNP), phenylephrine (PHE) and tromboxane analog (U46619) were obtained. The protein expressions of eNOS, receptor for AGEs (RAGE), Cu/Zn-SOD and Mn-SOD were analyzed. Tissues NO production and reactive oxygen species (ROS) generation were evaluated. Plasma nitrate/nitrite (NO_x ⁻), superoxide dismutase (SOD), catalase (CAT), thiobarbituric acid reactive substances (TBARS) and N^ε-(carboxymethyl) lysine (CML, AGE biomarker). A rightward shift in the concentration-response curves to ACh was observed in femoral and coronary arteries from SD/DB that was accompanied by an increase in TBARS and CML levels. Decreased in the eNOS expression, tissues NO production and NO_x ⁻ levels were associated with increased ROS generation. A positive interaction between the beneficial effect of TR on the relaxing responses to ACh and the reduction in TBARS and CML levels were observed without changing in antioxidant activities. The eNOS protein expression, tissues NO production and ROS generation were fully re-established in TR/DB, but plasma NO_x ⁻ levels were partially restored.

Conclusion

Shear stress induced by TR fully restores the eNOS/NO pathway in both preparations from non-treated diabetic rats, however, a massive production of AGEs still affecting relaxing responses possibly involving other endothelium-dependent vasodilator agents, mainly in coronary artery.     

Spironolactone lowers portal hypertension by inhibiting liver fibrosis, ROCK-2 activity and activating NO/PKG pathway in the bile-duct-ligated rat

Objective

Aldosterone, one of the main peptides in renin angiotensin aldosterone system (RAAS), has been suggested to mediate liver fibrosis and portal hypertension. Spironolactone, an aldosterone antagonist, has beneficial effect on hyperdynamic circulation in clinical practice. However, the mechanisms remain unclear. The present study aimed to investigate the role of spionolactone on liver cirrhosis and portal hypertension.

Methods

Liver cirrhosis was induced by bile duct ligation (BDL). Spironolactone was administered orally (20 mg/kg/d) after bile duct ligation was performed. Liver fibrosis was assessed by histology, Masson''s trichrome staining, and the measurement of hydroxyproline and type I collagen content. The activation of HSC was determined by analysis of alpha smooth muscle actin (α-SMA) expression. Protein expressions and protein phosphorylation were determined by immunohistochemical staining and Western blot analysis, Messenger RNA levels by quantitative real time polymerase chain reaction (Q-PCR). Portal pressure and intrahepatic resistance were examined in vivo.

Results

Treatment with spironolactone significantly lowered portal pressure. This was associated with attenuation of liver fibrosis, intrahepatic resistance and inhibition of HSC activation. In BDL rat liver, spironolactone suppressed up-regulation of proinflammatory cytokines (TNFα and IL-6). Additionally, spironolactone significantly decreased ROCK-2 activity without affecting expression of RhoA and Ras. Moreover, spironolactone markedly increased the levels of endothelial nitric oxide synthase (eNOS), phosphorylated eNOS and the activity of NO effector- protein kinase G (PKG) in the liver.

Conclusion

Spironolactone lowers portal hypertension by improvement of liver fibrosis and inhibition of intrahepatic vasoconstriction via down-regulating ROCK-2 activity and activating NO/PKG pathway. Thus, early spironolactone therapy might be the optional therapy in cirrhosis and portal hypertension.     

Phosphomimetic Modulation of eNOS Improves Myocardial Reperfusion and Mimics Cardiac Postconditioning in Mice

Objective

Myocardial infarction resulting from ischemia-reperfusion injury can be reduced by cardiac postconditioning, in which blood flow is restored intermittently prior to full reperfusion. Although key molecular mechanisms and prosurvival pathways involved in postconditioning have been identified, a direct role for eNOS-derived NO in improving regional myocardial perfusion has not been shown. The objective of this study is to measure, with high temporal and spatial resolution, regional myocardial perfusion during ischemia-reperfusion and postconditioning, in order to determine the contribution of regional blood flow effects of NO to infarct size and protection.

Methods and Results

We used myocardial contrast echocardiography to measure regional myocardial blood flow in mice over time. Reperfusion after myocardial ischemia-reperfusion injury is improved by postconditioning, as well as by phosphomimetic eNOS modulation. Knock-in mice expressing a phosphomimetic S1176D form of eNOS showed improved myocardial reperfusion and significantly reduced infarct size. eNOS knock-out mice failed to show cardioprotection from postconditioning. The size of the no-reflow zone following ischemia-reperfusion is substantially reduced by postconditioning and by the phosphomimetic eNOS mutation.

Conclusions and Significance

Using myocardial contrast echocardiography, we show that temporal dynamics of regional myocardial perfusion restoration contribute to reduced infarct size after postconditioning. eNOS has direct effects on myocardial blood flow following ischemia-reperfusion, with reduction in the size of the no-reflow zone. These results have important implications for ongoing clinical trials on cardioprotection, because the degree of protective benefit may be significantly influenced by the regional hemodynamic effects of eNOS-derived NO.     

Sildenafil reduces insulin-resistance in human endothelial cells

Background

The efficacy of Phosphodiesterase 5 (PDE5) inhibitors to re-establish endothelial function is reduced in diabetic patients. Recent evidences suggest that therapy with PDE5 inhibitors, i.e. sildenafil, may increase the expression of nitric oxide synthase (NOS) proteins in the heart and cardiomyocytes. In this study we analyzed the effect of sildenafil on endothelial cells in insulin resistance conditions in vitro.

Methodology/Principal Findings

Human umbilical vein endothelial cells (HUVECs) were treated with insulin in presence of glucose 30 mM (HG) and glucosamine 10 mM (Gluc-N) with or without sildenafil. Insulin increased the expression of PDE5 and eNOS mRNA assayed by Real time-PCR. Cytofluorimetric analysis showed that sildenafil significantly increased NO production in basal condition. This effect was partially inhibited by the PI3K inhibitor LY 294002 and completely inhibited by the NOS inhibitor L-NAME. Akt-1 and eNOS activation was reduced in conditions mimicking insulin resistance and completely restored by sildenafil treatment. Conversely sildenafil treatment can counteract this noxious effect by increasing NO production through eNOS activation and reducing oxidative stress induced by hyperglycaemia and glucosamine.

Conclusions/Significance

These data indicate that sildenafil might improve NOS activity of endothelial cells in insulin resistance conditions and suggest the potential therapeutic use of sildenafil for improving vascular function in diabetic patients.     

Imbalance between Pro and Anti-Oxidant Mechanisms in Perivascular Adipose Tissue Aggravates Long-Term High-Fat Diet-Derived Endothelial Dysfunction

Background

The hypothesis of this study is that long-term high-fat diets (HFD) induce perivascular adipose tissue (PVAT) dysfunction characterized by a redox imbalance, which might contribute to aggravate endothelial dysfunction in obesity.

Methods and Results

C57BL/6J mice were fed either control or HFD (45% kcal from fat) for 32 weeks. Body weight, lumbar and mesenteric adipose tissue weights were significantly higher in HFD animals compared to controls. The anticontractile effect of PVAT in mesenteric arteries (MA) was lost after 32 week HFD and mesenteric endothelial-dependent relaxation was significantly impaired in presence of PVAT in HFD mice (E_max = 71.0±5.1 vs E_max = 58.5±4.2, p<0.001). The inhibitory effect of L-NAME on Ach-induced relaxation was less intense in the HFD group compared with controls suggesting a reduction of endothelial NO availability. Expression of eNOS and NO bioavailability were reduced in MA and almost undetectable in mesenteric PVAT of the HFD group. Superoxide levels and NOX activity were higher in PVAT of HFD mice. Apocynin only reduced contractile responses to NA in HFD animals. Expression of ec-SOD and total SOD activity were significantly reduced in PVAT of HFD mice. No changes were observed in Mn-SOD, Cu/Zn-SOD or catalase. The ratio [GSSG]/([GSH]+[GSSG]) was 2-fold higher in the mesenteric PVAT from HFD animals compared to controls.

Conclusions

We suggest that the imbalance between pro-oxidant (NOX, superoxide anions, hydrogen peroxide) and anti-oxidant (eNOS, NO, ecSOD, GSSG) mechanisms in PVAT after long-term HFD might contribute to the aggravation of endothelial dysfunction.     

阴茎勃起及勃起功能障碍的研究进展

勃起功能障碍的基础研究须近十年来取得了较大进展,一氧化氮－cGMP(NO-cGMP)通路的发现使得阴茎平滑肌松驰的机制进一步阐明。一氧化氮合酶（NOS）、磷酸二酯酶（PDEs）的研究为勃起功能障碍的临床治疗提供了坚实的基础,进而促使了万艾可的问世。目前,勃起功能障碍的基因治疗停留在实验室阶段,但随着分子生物学的深入研究,转基因疗法可能成为临床上治疗勃起功能障碍的有效方法之一。     

Impaired Acetylcholine-Induced Endothelium-Dependent Aortic Relaxation by Caveolin-1 in Angiotensin II-Infused Apolipoprotein-E (ApoE?/?) Knockout Mice

Objective

The angiotensin II (AngII)-infused apolipoprotein E-deficient (ApoE^−/−) mouse model is widely used to study atherosclerosis and abdominal aortic aneurysm. An increase in blood pressure has been reported in this model however the underlying mechanism has not been fully explored. In this study, we investigated whether vasomotor dysfunction develops in AngII-infused ApoE^−/− mice and the underlying mechanism involved.

Methods

ApoE^−/− mice were infused with vehicle (distilled water) or AngII subcutaneously for 14 days. Blood pressure and heart rate were measured using the non-invasive tail cuff method. Aortic vascular reactivity and expression of key proteins (endothelial nitric oxide synthase (eNOS), phospho-eNOS and caveolin-1) were assessed using tension myography and Western blotting respectively. Plasma nitric oxide (NO) level was estimated using a colorimetric assay.

Results

AngII infusion caused a time-dependent increase in blood pressure (P<0.001). Aortas from AngII-infused mice were significantly less responsive to acetylcholine-induced endothelium-dependent relaxation when compared to aortas from mice infused with vehicle control (P<0.05). Contractile responses to phenylephrine (P<0.01) and potassium chloride (P<0.001) were significantly enhanced in aortas from AngII-infused mice. eNOS phosphorylation was significantly decreased in the aorta of AngII-infused mice (P<0.05). Aortic caveolin-1 protein expression was significantly increased in AngII-infused mice (P<0.05). Plasma nitrate/nitrite level was significantly reduced in AngII-infused mice (P<0.05). Pharmacological disruption of caveolae using methyl-β-cyclodextrin (MβCD) in isolated aortas from AngII-infused mice caused a significant leftward shift of the acetylcholine-induced relaxation concentration-response curve when compared to vehicle control (P<0.05).

Conclusion

Upregulation of caveolin-1 protein expression and reduced NO bioavailability contributes to aortic endothelial dysfunction in AngII-infused ApoE^−/− mice.     

PPAR-γ Regulates Carnitine Homeostasis and Mitochondrial Function in a Lamb Model of Increased Pulmonary Blood Flow

Objective

Carnitine homeostasis is disrupted in lambs with endothelial dysfunction secondary to increased pulmonary blood flow (Shunt). Our recent studies have also indicated that the disruption in carnitine homeostasis correlates with a decrease in PPAR-γ expression in Shunt lambs. Thus, this study was carried out to determine if there is a causal link between loss of PPAR-γ signaling and carnitine dysfunction, and whether the PPAR-γ agonist, rosiglitazone preserves carnitine homeostasis in Shunt lambs.

Methods and Results

siRNA-mediated PPAR-γ knockdown significantly reduced carnitine palmitoyltransferases 1 and 2 (CPT1 and 2) and carnitine acetyltransferase (CrAT) protein levels. This decrease in carnitine regulatory proteins resulted in a disruption in carnitine homeostasis and induced mitochondrial dysfunction, as determined by a reduction in cellular ATP levels. In turn, the decrease in cellular ATP attenuated NO signaling through a reduction in eNOS/Hsp90 interactions and enhanced eNOS uncoupling. In vivo, rosiglitazone treatment preserved carnitine homeostasis and attenuated the development of mitochondrial dysfunction in Shunt lambs maintaining ATP levels. This in turn preserved eNOS/Hsp90 interactions and NO signaling.

Conclusion

Our study indicates that PPAR-γ signaling plays an important role in maintaining mitochondrial function through the regulation of carnitine homeostasis both in vitro and in vivo. Further, it identifies a new mechanism by which PPAR-γ regulates NO signaling through Hsp90. Thus, PPAR-γ agonists may have therapeutic potential in preventing the endothelial dysfunction in children with increased pulmonary blood flow.     

Nitric Oxide Synthetic Pathway in Red Blood Cells Is Impaired in Coronary Artery Disease

Background

All the enzymatic factors/cofactors involved in nitric oxide (NO) metabolism have been recently found in red blood cells. Increased oxidative stress impairs NO bioavailability and has been described in plasma of coronary artery disease (CAD) patients. The aim of the study was to highlight a potential dysfunction of the metabolic profile of NO in red blood cells and in plasma from CAD patients compared with healthy controls.

Methods

We determined L-arginine/NO pathway by liquid-chromatography tandem mass spectrometry and high performance liquid chromatography methods. The ratio of oxidized and reduced forms of glutathione, as index of oxidative stress, was measured by liquid-chromatography tandem mass spectrometry method. NO synthase expression and activity were evaluated by immunofluorescence staining and ex-vivo experiments of L-[¹⁵N₂]arginine conversion to L-[¹⁵N]citrulline respectively.

Results

Increased amounts of asymmetric and symmetric dimethylarginines were found both in red blood cells and in plasma of CAD patients in respect to controls. Interestingly NO synthase expression and activity were reduced in CAD red blood cells. In contrast, oxidized/reduced glutathione ratio was increased in CAD and was associated to arginase activity.

Conclusion

Our study analyzed for the first time the whole metabolic pathway of L-arginine/NO, both in red blood cells and in plasma, highlighting an impairment of NO pathway in erythrocytes from CAD patients, associated with decreased NO synthase expression/activity and increased oxidative stress.