Staining For Endothelial Nitric Oxide Synthase Research Articles

Adventitial Collagen Cross-Linking by Glutaraldehyde Reinforcing Human Saphenous Vein - Implication for Coronary Artery Bypass Grafting.

IntroductionA weak venous wall is one of the major reasons contributing to vein graft failure after coronary artery bypass grafting (CABG). We investigated whether adventitial collagen cross-linking by glutaraldehyde reinforces venous wall, preserving the endothelium of veins during high-pressure distention.MethodsHuman saphenous veins (SVs) were collected from 40 patients undergoing CABG, and adventitia cross-linking was performed with 0.3% glutaraldehyde for five minutes. The cross-linked SVs were accessed by biodegradation assay, immunofluorescent staining, and tensile test. Native SVs and cross-linked SVs from another 20 patients received the 200 mmHg pressure distention for two minutes. Pressure-induced injury of SVs were accessed by immunohistochemistry and electron microscopy.ResultsTime to digestion was 97±13 minutes for native SVs and 720±0 minutes for cross-linked SVs (P<0.05). After adventitial cross-linking, the collagen I fibres of the vein remarkably presented with compact and nonporous arrangement. In the high-stretch region (stretch ratio 1.4-1.8), the Young’s elastic modulus of stress-stretch ratio curve in cross-linked SVs was larger than that in native SVs (13.88 vs. 5.83, P<0.05). The cross-linked SVs had a lower extent of endothelial denudation without fibre fracture during high-pressure distension than native SVs. Comparing with the non-cross-linked SVs, the percentage of endothelial nitric oxide synthase staining length on the endothelium of cross-linked SVs was significantly preserved after high-pressure distension (85.2% vs. 64.7%, P<0.05).ConclusionAdventitial collagen cross-linking by glutaraldehyde reinforced venous wall by increasing stiffness and decreasing extensibility of SVs and mitigated the endothelial damage under high-pressure distension.

Potential Vasculoprotective Effects of Blackcurrant (Ribes nigrum) Extract in Diabetic KK-Ay Mice

Polyphenols are bioactive compounds found naturally in fruits and vegetables; they are widely used in disease prevention and health maintenance. Polyphenol-rich blackcurrant extract (BCE) exerts beneficial effects on vascular health in menopausal model animals. However, the vasculoprotective effects in diabetes mellitus (DM) and atherosclerotic vascular disease secondary to DM are unknown. Therefore, we investigated whether BCE is effective in preventing atherosclerosis using KK-Ay mice as a diabetes model. The mice were divided into three groups and fed a high-fat diet supplemented with 1% BCE (BCE1), 3% BCE (BCE2), or Control for 9 weeks. The mice in the BCE2 group showed a considerable reduction in the disturbance of elastic lamina, foam cell formation, and vascular remodeling compared to those in the BCE1 and Control groups. Immunohistochemical staining indicated that the score of endothelial nitric oxide synthase staining intensity was significantly higher in both BCE2 (2.9) and BCE1 (1.9) compared to that in the Control (1.1). Furthermore, the score for the percentage of alpha-smooth muscle actin was significantly lower in the BCE2 (2.9%) than in the Control (2.1%). Our results suggest that the intake of anthocyanin-rich BCE could have beneficial effects on the blood vessels of diabetic patients.

L-Arginine and tetrahydrobiopterin, but not sodium nitrite partially restored erectile dysfunction in aged rats

Aging is associated with erectile dysfunction (ED), in which nitric oxide synthase (NOS) activity and NO bioavailability are reduced due to deficiencies of NOS cofactor (tetrahydrobiopterin, BH4) and substrate (l-arginine). We determined whether the prolonged treatment with sodium nitrite (NaNO2) as a storage form of NO ameliorates ED in aged rats. Male Sprague–Dawley rats were divided: younger, aged and NaNO2-treated (20 mg/kg per day) aged groups. The erectile (intracavernosal pressure [ICP]/mean arterial pressure [MAP]) and corpus cavernous (CC) responses were evaluated after 12 weeks. The ICP/MAP in aged rats was lower than in young controls, which was not improved by the NaNO2 treatment. Immunohistochemical (IHC) staining for endothelial NOS and collagen deposition was performed. We assayed NO indirectly by measuring the level of its stable end products, nitrite/nitrate, using the Griess reagent. The relaxations to ACh and EFS in the aged group were considerably less than in the younger group, which were normalized by acute incubations of l-arginine or BH4 of aged CC. In conclusion, NaNO2 treatment did not restore erectile response while nitrate levels were enhanced in aged penis. The cofactor or substrate administrations, but not chronic exogenous modulation of NO system may be beneficial in aged men with ED.

Surgical vein graft preparation promotes cellular dysfunction, oxidative stress, and intimal hyperplasia in human saphenous vein

Human saphenous vein (HSV) is the most widely used bypass conduit for peripheral and coronary vascular reconstructions. However, outcomes are limited by a high rate of intimal hyperplasia (IH). HSV undergoes a series of exvivo surgical manipulations prior to implantation, including hydrostatic distension, marking, and warm ischemia in solution. We investigated the impact of surgical preparation on HSV cellular function and development of IH in organ culture. We hypothesized that oxidative stress is a mediator of HSV dysfunction. HSV was collected from patients undergoing vascular bypass before and after surgical preparation. Smooth muscle and endothelial function were measured using a muscle bath. Endothelial preservation was assessed with immunohistochemical staining. An organ culture model was used to investigate the influence of surgical preparation injury on the development of IH. Superoxide levels were measured using a high-performance liquid chromatography-based assay. The influence of oxidative stress on HSV physiologic responses was investigated by exposing HSV to hydrogen peroxide (H2O2). Surgical vein graft preparation resulted in smooth muscle and endothelial dysfunction, endothelial denudation, diminished endothelial nitric oxide synthase staining, development of increased IH, and increased levels of reactive oxygen species. Experimental induction of oxidative stress in unmanipulated HSV by treatment with H2O2 promoted endothelial dysfunction. Duration of storage time in solution did not contribute to smooth muscle or endothelial dysfunction. Surgical vein graft preparation causes dysfunction of the smooth muscle and endothelium, endothelial denudation, reduced endothelial nitric oxide synthase expression, and promotes IH in organ culture. Moreover, increased levels of reactive oxygen species are produced and may promote further vein graft dysfunction. These results argue for less injurious means of preparing HSV prior to autologous transplantation into the arterial circulation.

Placental endothelial nitric oxide synthase in multiple and single dose betamethasone exposed pregnancies

To compare endothelial nitric oxide synthase expression and capillary density (CDS) in placentas exposed to single or multiple courses of betamethasone. Placental specimens exposed to single vs repeat courses of betamethasone were analyzed through immunohistochemistry and digital image quantification for endothelial nitric oxide synthase and CD34. Quantified endothelial nitric oxide synthase staining, calculated capillary density, ratio of endothelial nitric oxide synthase to capillary density, and clinical characteristics were compared. Linear regression was performed with these as dependent variables. Mean and maximum capillary density were increased (P = .013 and .005) and the ratio of endothelial nitric oxide synthase to capillary density decreased (P = .016) in specimens exposed to 4 courses of betamethasone compared with 1 to 3 courses. Exposure to 4 courses of betamethasone was associated with increased capillary density, but not with endothelial nitric oxide synthase expression. Exposure to 4 courses of betamethasone is associated with increased placental capillary density. The placental effects of multiple courses of betamethasone are unrelated to endothelial nitric oxide synthase expression.

Coronary nitric oxide production controls cardiac substrate metabolism during pregnancy in the dog

The aim of this study was to examine the role of nitric oxide (NO) in the control of cardiac metabolism at 60 days of pregnancy (P60) in the dog. There was a basal increase in diastolic coronary blood flow during pregnancy and a statistically significant increase in cardiac output (55 +/- 4%) and in cardiac NOx production (44 +/- 4 to 59 +/- 3 nmol/min, P < 0.05). Immunohistochemistry of the left ventricle showed an increase in endothelial nitric oxide synthase staining in the endothelial cells at P60. NO-dependent coronary vasodilation (Bezold-Jarisch reflex) was increased by 20% and blocked by N(G)-nitro-l-arginine methyl ester (l-NAME). Isotopically labeled substrates were infused to measure oleate, glucose uptake, and oxidation. Glucose oxidation was not significantly different in P60 hearts (5.4 +/- 0.5 vs. 6.2 +/- 0.4 micromol/min) but greatly increased in response to l-NAME injection (to 19.9 +/- 0.9 micromol/min, P < 0.05). Free fatty acid (FFA) oxidation was increased in P60 (from 5.3 +/- 0.6 to 10.4 +/- 0.5 micromol/min, P < 0.05) and decreased in response to l-NAME (to 4.5 +/- 0.5 micromol/min, P < 0.05). There was an increased oxidation of FFA for ATP production but no change in the respiratory quotient during pregnancy. Genes associated with glucose and glycogen metabolism were downregulated, whereas genes involved in FFA oxidation were elevated. The acute inhibition of NO shifts the heart away from FFA and toward glucose metabolism despite the downregulation of the carbohydrate oxidative pathway. The increase in endothelium-derived NO during pregnancy results in a tonic inhibition of glucose oxidation and reliance on FFA uptake and oxidation to support ATP synthesis in conjunction with upregulation of FFA metabolic enzymes.

Overview of the 2007 Food and Drug Administration Circulatory System Devices Panel Meeting on the Endeavor Zotarolimus-Eluting Coronary Stent

Initial trials comparing stenting with balloon angioplasty demonstrated improved angiographic and clinical outcomes with the former.1–3 The main clinical problem with bare metal stents (BMS) became the development of in-stent restenosis.4–6 Early data suggested that drug-eluting stents (DES) could mitigate, if not abolish, this problem.7–9 Since the approval of DES, these devices have become the predominant stents used in percutaneous coronary interventions, regardless of indication. As of September 2007, the Cypher sirolimus-eluting stent (SES) (Cordis Johnson & Johnson, Miami Lakes, Fla) had been deployed in >3 million patients worldwide,10 and the millionth Taxus paclitaxel-eluting stent (PES) (Boston Scientific, Natick, Mass) was implanted as of January 2005.11 Over the past 2 years, numerous reports of very late stent thrombosis (ST) with first-generation DES have surfaced. This highly morbid complication and data suggesting that death and myocardial infarction (MI) may be more common with DES12,13 have altered the focus of trials and registries working to determine optimal DES use. There is a hope that second-generation DES will be less prone to ST as a result of differences in drug, stent design, and polymer. Medtronic, Inc (Minneapolis, Minn) presented safety and efficacy data to a public meeting of the US Food and Drug Administration (FDA) Circulatory System Devices Panel (CSDP) in October 2007 on its Endeavor zotarolimus-eluting stent (ZES), seeking approval for the indication of treating de novo native coronary lesions ≤27 mm with reference vessel diameters of 2.5 to 3.5 mm. The FDA asked the CSDP to determine whether the data presented demonstrated a reasonable level of safety and effectiveness, with clinical benefits clearly outweighing short- and long-term risks of ZES use. Important efficacy end points included ischemia-driven target lesion revascularization (TLR) or target vessel revascularization (TVR); safety end points included death, MI, and ST. The …

Simvastatin preserves myocardial function by enhancing endothelial nitric oxide production in heart failure

Background: Endothelial nitric oxide synthase (ENOS) is reduced in heart failure (HF). Statins upregulate NO production independent of lipid-lowering. We evaluated the ability of simvastatin (Sim) lo reverse ventricular remodeling and dysfunction by restoring coronary NO production and NO mediated regulation of apoptosis. Methods: Five month old cardiomyopathic (CMP) hamsters were divided into 2 groups Sim (n=5), and Untreated (n=5). Age-matched normal hamsters ware used as controls (n=6). Sim group received 20 “g/kg Sim daily by oral gavage for 6 weeks. Serial 2D echocardiograms were obtained at baseline and 2 week intervals. LV function and dimensions indexed lo body surface area were compared between groups. Myocardial apoptosis was evaluated by immunostaining and caspasa 3 activity assay. Myocardial ENOS expression was measured by Western blot and immunohistochemistry. Results: At baseline, CMP hamsters had lower LV shortening fraction (SF) compared to controls (17*2% vs.59+2%), higher LV diastolic volume (30*2 vs.6iO.5 ml/m*) and lower LV mass/volume ratio (0.5iO.02 vs. 0.72t0.02 “g/ml, p<O.OOl). Untreated CMP hamsters demonstrated a progressive decrease in LVSF (12*1%, ~~0.05) and increase in LV volume from baseline (37*2 ml/m2, ~~0.05). The Sim group showed no deterioration in LV function (SF 18+.1%) or LV dilatation (LV volume, 26*2 “!I”‘, p<O.O5) compared to the untreated group. lmmunostainina revealed a hiaher percentaae of apoptotic nuclei in _ _ the HF group compared to controls (&096*0.04% vs.O.O32+0.02%, ~‘0.002). Caspase 3 activity was lower in the Sim cornoared to the untreated orouo f1~0.001). lnhibitina -... , ” endogenous NO increased caspase 3 activity in the Sim (p=O.O5) but not in the untreated group. Myocardial ENOS staining revealed increased capillary density per 3 high power fields in the Sim (llOil2. pqO.05) compared to controls (47*9) and untreated hamsters (56ill). Conclusions: Sim administration preserves myocardial function, decreases LVremodeling and retards the progression of HF. This may be related to an increase in coronary microvasculature resulting in increased endothelial NO availability and reduced cardiomyocyte apoptosis.

Human sperm endothelial nitric oxide synthase expression: correlation with sperm motility

Objective: To characterize the pattern of endothelial nitric oxide synthase (eNOS) expression on human spermatozoa and to determine whether sperm eNOS expression correlates with sperm function. Design: Prospective, observational study. Setting: University infertility clinic. Patient(s): Twelve nonazoospermic infertile men. Intervention(s): Semen samples (n = 12) obtained from nonazoospermic infertile men were fractionated on discontinuous Percoll gradients. Endothelial nitric oxide synthase staining on spermatozoa was correlated with sperm motility in Percoll gradient–fractionated spermatozoa. Endothelial nitric oxide synthase protein was detected with the use of a previously characterized monoclonal antibody. Control slides were incubated with preabsorbed antibody or mouse immunoglobulin G. Main Outcome Measure(s): Localization of eNOS on human spermatozoa and correlation between the pattern of sperm eNOS expression and sperm motility. Result(s): Morphologically normal spermatozoa exhibited postacrosomal and equatorial eNOS immunostaining. However, abnormally shaped spermatozoa often exhibited aberrant staining (in the midpiece and/or head region). A significant negative correlation was observed between the percentage of sperm with aberrant eNOS immunostaining and the percentage of motile sperm ( r = −.46). Conclusion(s): The specific localization of eNOS to human spermatozoa suggests that nitric oxide may be involved in normal sperm physiology. However, aberrant patterns of sperm eNOS expression are associated with decreased sperm motility, possibly through the generation of excessive cytotoxic oxidants.

Role of nitric oxide and cGMP system in regulation of ductus arteriosus tone in ovine fetus.

Although endogenous nitric oxide (NO) modulates basal tone in the fetal pulmonary and systemic circulations, little is known about its role in regulating ductus arteriosus (DA) tone. Immunostaining of DA tissue from late-gestation fetal lambs demonstrated strong staining for endothelial NO synthase (eNOS) in DA endothelium. To study the physiological role of the NO and guanosine 3',5'-cyclic monophosphate (cGMP) system in the DA in vivo, we measured the hemodynamic effects of NG-nitro-L-arginine (L-NNA; 30 mg), a NOS inhibitor, methylene blue (40 mg), a guanylate cyclase inhibitor, and indomethacin (0.8 mg), a cyclooxygenase inhibitor, in 10 chronically prepared late-gestation fetal lambs. L-NNA increased main pulmonary artery (MPA) and aortic pressures (P < 0.05 vs. baseline) but did not change the pressure gradient between the MPA and the aorta. L-NNA caused a small decrease in DA flow and a slight rise in resistance across the DA. Methylene blue increased both MPA pressure and the pressure gradient between the MPA and the aorta from 0.3 +/- 0.2 (baseline) to 7.0 +/- 2.7 mmHg (P < 0.05). Indomethacin increased both MPA pressure and the pressure gradient between the MPA and the aorta from 1.1 +/- 0.4 (baseline) to 6.3 +/- 1.5 mmHg (P < 0.05) after 40 min. Indomethacin decreased DA flow and increased DA resistance. We conclude that eNOS is in fetal DA endothelial cells and that NOS inhibition causes constriction of the DA in vivo. DA constriction after NOS inhibition is minimal, especially in comparison with cyclooxygenase inhibition. Methylene blue also constricts the DA, suggesting that guanylate cyclase activity contributes to DA relaxation. We speculate that, although the NO and cGMP system modulates DA tone, prostaglandins may play a greater role.