Nitric oxide is an important mediator of renal tubular epithelial cell death in vitro and in murine experimental hydronephrosis

Macrophages play a pivotal role in tissue injury and fibrosis during renal inflammation. Although macrophages may induce apoptosis of renal tubular epithelial cells, the mechanisms involved are unclear. We used a microscopically quantifiable co-culture assay to dissect the cytotoxic interaction between murine bone marrow-derived macrophages and Madin-Darby canine kidney cells and primary murine renal tubular epithelial cells. The induction of tubular cell apoptosis by cytokine-activated macrophages was reduced by inhibitors of nitric oxide synthase whereas tubular cell proliferation was unaffected. Furthermore, cytokine-activated macrophages derived from mice targeted for the deletion of inducible nitric oxide synthase were noncytotoxic. We then examined the role of nitric oxide in vivo by inhibiting inducible nitric oxide synthase in the model of murine experimental hydronephrosis. l-N(6)-(1-iminoethyl)-lysine was administered in the drinking water between days 5 and 7 after ureteric obstruction. Macrophage infiltration was comparable between groups, but treatment significantly inhibited tubular cell apoptosis at day 7. Tubular cell proliferation was unaffected. Inducible nitric oxide synthase blockade also reduced interstitial cell apoptosis and increased collagen III deposition. These data indicate that nitric oxide is a key mediator of macrophage-directed tubular cell apoptosis in vitro and in vivo and also modulates tubulointerstitial fibrosis.     

当归、川芎、红花、人参萃取液对离体培养牛血管内皮细胞一氧化氮合酶的影响

目的：研究以当归、川芎、红花、人参为主要成份的CO2超临界萃取液对低切应力条件下培养的内皮细胞一氧化氮合酶(eNOS)表达的影响;探讨该萃取液改善血管功能、防止动脉粥样硬化的机理。方法：采用Westem blot印迹免疫检测,研究该萃取液对低切应力环境下体外培养的牛血管内皮细胞eNOS表达的影响。结果：低切应力水平下,内皮细胞eNOS表达降低,给予0.5％萃取液1ml(以萃取原液为100％),eNOS的表达明显增加,并接近在高切应力水平下内皮细胞eNOS的正常表达水平。结论：当归、川芎、红花、人参萃取液可上调低切应力环境下内皮细胞eNOS的表达。     

Elevated glucose attenuates agonist- and flow-stimulated endothelial nitric oxide synthase activity in microvascular retinal endothelial cells.

Impaired vasoactive release of opposing vasodilator and vasoconstrictor mediators due to endothelial dysfunction is integral to the pathogenesis of diabetic retinopathy. The aim of this study was to determine the effect of hyperglycemia on the expression of endothelial nitric oxide synthase (eNOS) and the release of nitric oxide (NO) in bovine microvascular retinal endothelial cells (BRECs) under both static (basal and acetylcholine stimulated) and flow (laminar shear stress [10 dynes/cm2 and pulsatile flow 0.3 to 23 dynes/cm2) conditions using a laminar shear apparatus and an in vitro perfused transcapillary culture system. The activity and expression of eNOS, measured by nitrate levels and immunoblot, respectively, were determined following exposure of BRECs to varying concentrations of glucose and mannitol (0 to 25 mM). Under static conditions the expression of eNOS decreased significantly following exposure to increasing concentrations of glucose when compared to osmotic mannitol controls and was accompanied by a significant dose-dependent decrease in nitrate levels in conditioned medium. The acetylcholine stimulated increase in NO release (2.0 +/- 0.3-fold) was significantly reduced by 55% +/- 5% and 65% +/- 4.5% following exposure to 16 and 25 mM glucose, respectively, when compared to osmotic controls. In parallel studies, glucose significantly inhibited both laminar shear stress and pulsatile flow-induced activity when compared to mannitol. We conclude that hyperglycemia impairs agonist- and flow-dependent release of NO in retinal microvascular endothelial cells and may thus contribute to the vascular endothelial dysfunction and impaired autoregulation of diabetic retinopathy.     

Ultrastructural localisation of nitric oxide synthase, endothelin and binding sites of lectin (from Bandeirea simplicifolia) in the rat carotid artery after balloon catheter injury

An immunocytochemical and cytochemical study has been made on the ultrastructural localisation of type III (endothelial) nitric oxide synthase, endothelin-1 and the binding sites of lectin from Bandeirea simplicifolia to the endothelium surface-associated glycoproteins in the rat left common carotid artery at 1 and 28 d after Fogarty embolectomy balloon catheter-induced injury. Controls were carotid arteries from sham operated rats. In the controls, the immunoreactivity to nitric oxide synthase-III and endothelin-1 was localised in different proportions in vascular endothelial cells (36·9%±4·3 and 7·6%±2·7, respectively); immunoreactivity was confined to the cytoplasm and the membranes of intracellular organelles and structures. In contrast, staining with lectin was localised on the luminal surface of all endothelial cells. 1 d after injury, platelets were adherent to the endothelium-denuded intima. Some of the platelets displayed immunoreactivity to nitric oxide synthase-III and endothelin-1 and were stained with lectin. 28 d after injury, a neointimal thickening of substantial size was present. Subpopulations of the regrown endothelial cells covering the luminal surface of the neointima showed positive immunoreactivity to nitric oxide synthase-III and endothelin-1 but there was a significant decrease in the proportion of nitric oxide synthase-III-containing endothelial cells (17·2%±1·9; P < 0·001) and a significant increase in the proportion of endothelin-1-containing endothelial cells (36·9%±4·7; P < 0·001) compared with the controls. Staining with lectin was associated with the cell membrane of all endothelial cells and in addition with cells located ‘deeper’ in the neointima which showed lectin-positive plasmalemma, Golgi complex and multivesicular bodies/lysosomes. In conclusion, regenerated endothelial cells of the neointima showed reduced population (2–fold) of nitric oxide synthase-III- and increased population (5–fold) endothelin-1-positive cells. The subendothelial location of some lectin-stained cells after balloon catheter injury indicates the heterogeneity of the neointima and suggests that some of these cells are involved in early angiogenesis. 24 h and 28 d after injury some platelets showed positive immunoreactivity for nitric oxide synthase-III and endothelin-1.     

Effects of pulsatile shear stress on signaling mechanisms controlling nitric oxide production, endothelial nitric oxide synthase phosphorylation, and expression in ovine fetoplacental artery endothelial cells.

During gestation, placental blood flow, endothelial nitric oxide (NO) production, and endothelial cell nitric oxide synthase (eNOS) expression are elevated dramatically. Shear stress can induce flow-mediated vasodilation, endothelial NO production, and eNOS expression. Both the activity and expression of eNOS are closely regulated because it is the rate-limiting enzyme essential for NO synthesis. The authors adapted CELLMAX artificial capillary modules to study the effects of pulsatile flow/shear stress on ovine fetoplacental artery endothelial (OFPAE) cell NO production, eNOS expression, and eNOS phosphorylation. This model allows for the adaptation of endothelial cells to low physiological flow environments and thus prolonged shear stresses. The cells were grown to confluence at 3 dynes/cm2, then were exposed to 10, 15, or 25 dynes/cm2 for up to 24 h and NO production, eNOS mRNA, and eNOS protein expression were elevated by shear stress in a graded fashion (p < .05). Production of NO by OFPAE cells exposed to pulsatile shear stress was de novo; i.e., inhibited by L-NMMA (N(G)-monomethyl-L-arginine) and reversed by excess NOS substrate L-arginine. Rises in NO production at 25 dynes/cm2 (8-fold) exceeded (p < .05) that seen for eNOS protein (3.6-fold) or eNOS mRNA (1.5-fold). Acute rises in NO production with shear stress occurred by eNOS activation, whereas prolonged NO rises were via elevations in both eNOS expression and enzyme activation. The authors therefore used Western analysis to investigate the signaling mechanisms underlying pulsatile shear stress-induced increases in eNOS phosphorylation and protein expression by "flow-adapted" OFPAE cells. Increasing shear stress from 3 to 15 dynes/cm2 very rapidly increased eNOS Ser1177, ERK1/2 (extracellular signal-regulated kinase 1 and 2) and Akt, but not p38 MAPK (p38 mitogen-activated protein kinase) phosphorylation by Western analysis. Phosphorylation of eNOS Ser1177 under shear stress was elevated by 20 min, a response that was blocked by PI-3K (phosphatidylinositol 3-kinase) inhibitors wortmannin and LY294002, but not the MEK (MAPK kinase) inhibitor UO126. Basic fibroblast growth factor (bFGF) enhanced eNOS protein levels in static culture via a MEK-mediated mechanism, but it could not further augment the elevated eNOS protein levels induced by 15 dynes/cm2 shear stress. Blocking of either signaling pathways or p38 MAPK did not change the shear stress-induced increase in eNOS protein levels. Therefore, shear stress induced rapid eNOS phosphorylation on Ser1177 in OFPAE cells through a PI-3K-dependent pathway. The bFGF-induced rise in eNOS protein levels in static culture was much less than those observed under flow and was blocked by inhibiting MEK. Prolonged shear stress-stimulated increases in eNOS protein levels were not affected by inhibition of MEK- or PI-3K-mediated pathways. In conclusion, pulsatile shear stress greatly induces NO production by OFPAE cells through the mechanisms of both PI-3K-mediated eNOS activation and elevations in eNOS protein levels; bFGF does not further stimulate eNOS expression under flow condition.     

Role of nitric oxide in the ethylcholine aziridinium model of delayed apoptotic neurodegeneration in vivo and in vitro

The involvement of nitric oxide in neurodegenerative processes still remains incompletely characterized. Although nitric oxide has been reported to be an important mediator in neuronal degeneration in different models of cell death involving NMDA-receptor activation, increasing evidence for protective mechanisms has been obtained. In this study the role of nitric oxide was investigated in a model of NMDA-independent, delayed apoptotic cell death, induced by the neurotoxin ethylcholine aziridinium ethylcholine aziridinium both in vivo and in vitro. For the in vivo evaluation rats received bilateral intracerebroventricular injections of ethylcholine aziridinium (2nmol/ventricle) or vehicle. In the hippocampus a transient decrease in nitric oxide synthase activity occurred, reaching its lowest levels three days after ethylcholine aziridinium treatment (51.7+/-9.8% of controls). The decrease coincided with the maximal reduction in choline acetyltransferase activity as marker for the extent of cholinergic lesion. The effect of pharmacological inhibition of nitric oxide synthase was tested by application of various nitric oxide synthase inhibitors with different selectivity for the nitric oxide synthase-isoforms. Unspecific nitric oxide synthase inhibition resulted in a significant potentiation of the loss of choline acetyltransferase activity in the hippocampus measured seven days after ethylcholine aziridinium application, whereas the specific inhibition of neuronal or inducible nitric oxide synthase was ineffective. These pharmacological data are suggestive for a neuroprotective role of nitric oxide generated by endothelial nitric oxide synthase. In vitro experiments were performed using serum-free primary neuronal cell cultures from hippocampus, cortex and septum of E15-17 Wistar rat embryos. Ethylcholine aziridinium-application in a range of 5-80microM resulted in delayed apoptotic neurodegeneration with a maximum after three days as confirmed by morphological criteria, life-death assays and DNA laddering. Nitric oxide synthase activity in harvested cells decreased in a dose- and time-dependent manner. Nitric oxide production as determined by measurement of the accumulated metabolite nitrite in the medium was equally low in controls and in ethylcholine aziridinium treated cells (range 0.77-1.86microM nitrite). An expression of inducible nitric oxide synthase messenger RNA could not be detected by semiquantitative RT-PCR 13h after ethylcholine aziridinium application.The present data indicate that in a model of delayed apoptotic neurodegeneration as induced by ethylcholine aziridinium neuronal cell death in vitro and in vivo is independent of the cytotoxic potential of nitric oxide. This is confirmed by a decrease in nitric oxide synthase activity, absence of nitric oxide production and absence of inducible nitric oxide synthase expression. In contrast, evidence for a neuroprotective role of nitric oxide was obtained in vivo as indicated by the exaggeration of the cholinergic lesion after unspecific nitric oxide synthase inhibition by N-nitro-L-arginine methylester.     

Effect of streptavidin-biotin on endothelial vasoregulation and leukocyte adhesion

The current study examines whether the adhesion promoting arginine-glycine-aspartate-streptavidin mutant (RGD-SA) also affects two important endothelial cell (EC) functions in vitro: vasoregulation and leukocyte adhesion. EC adherent to surfaces via fibronectin (Fn) or Fn plus RGD-SA were subjected to laminar shear flow and media samples were collected over a period of 4h to measure the concentration of nitric oxide (NO), prostacyclin (PGI(2)), and endothelin-1 (ET-1). Western blot analysis was used to quantify the levels of endothelial-derived nitric oxide synthase (eNOS) and cyclooxygenase II (COX II). In a separate set of experiments, fluorescent polymorphonuclear leukocyte (PMN) adhesion to EC was quantified for EC with and without exposure to flow preconditioning. When cell adhesion was supplemented with the SA-biotin system, flow-induced production of NO and PGI(2) increased significantly relative to cells adherent on Fn alone. Previous exposure of EC to shear flow also significantly decreased PMN attachment to SA-biotin supplemented EC, but only after 2h of exposure to shear flow. The observed decrease in PMN-EC adhesion was negated by NG-nitro-L-arginine methyl ester (L-NAME), an antagonist of NO synthesis, but not by indomethacin, an inhibitor to PGI(2) synthesis, indicating the induced effect of PMN-EC interaction is primarily NO-dependent. Results from this study suggest that the use of SA-biotin to supplement EC adhesion encourages vasodilation and PMN adhesion in vitro under physiological shear-stress conditions. We postulate that the presence of SA-biotin more efficiently transmits the shear-stress signal and amplifies the downstream events including the NO and PGI(2) release and leukocyte-EC inhibition. These results may have ramifications for reducing thrombus-induced vascular graft failure.     

Nitric oxide and prostacyclin mediate coronary shear force-induced alterations in cardiac electrophysiology

Previous studies have shown that an increase in coronary flow or shear force enhances the oxygen consumption and contractility of myocardium (the "Gregg effect"). Recent studies have indicated that an increase in coronary shear force also affects atrioventricular conduction and ventricular repolarization. The mechanisms of "Gregg effect" and the shear force-induced alterations in cardiac electrophysiology are unclear. It has been proposed that the distension of the coronary arteries and the stretch of the surrounding myocytes may play a key role but this is not supported by the latest studies. Intracoronary perfusion increases coronary endothelial synthesis of nitric oxide and prostacyclin. Emerging evidence has indicated that both nitric oxide and prostacyclin prolong the action potential duration or effective refractory period in the in vitro and in vivo animal heart. We therefore hypothesized that shear force-induced alterations in cardiac function and electrophysiology are largely attributed to the enhanced endothelial release of nitric oxide and prostacyclin.     

The effect of myocardial bridging of the coronary artery on vasoactive agents and atherosclerosis localization

The relationship between alterations in the immunohistochemical expression of three vasoactive agents [endothelial nitric oxide synthase (eNOS), endothelin-1 (ET-1), and angiotensin-converting enzyme (ACE)] and the occurrence human atherosclerosis was investigated in relation to the myocardial bridge (MB) of the left anterior descending coronary artery (LAD), an anatomical site that experiences increased shear stress. Five millimetre cross-sections of LADs with MB from 22 autopsied cases were taken from the left coronary ostium to the cardiac apex and were immunohistochemically stained with antibodies against eNOS, ET-1, and ACE. The extent of atherosclerosis in each section was calculated using the atherosclerosis ratio (intimal cross-sectional area/medial cross-sectional area) determined by histomorphometry. The results were analysed according to their anatomical location relative to the MB, either proximal, beneath, or distal. The extent of atherosclerosis was significantly lower beneath the MB, compared with proximal and distal segments. The expression of eNOS, ET-1, and ACE was also significantly lower beneath the MB. The expression of these agents correlated significantly with the extent of atherosclerosis. Because nitric oxide, after its production by eNOS, is believed to be degraded by superoxide radicals, the effect of eNOS expression on atherosclerosis remains controversial. However, the present findings clearly indicate that the expression of ET-1 and ACE is directly related to the development of human coronary atherosclerosis in vivo through shear stress.     

Lipopolysaccharide plus 12-o-tetradecanoylphorbol 13-acetate induction of migration and invasion of glioma cells in vitro and in vivo: Differential inhibitory effects of flavonoids

In an earlier study, we reported that nitric oxide is involved in lipopolysaccharide plus 12-o-tetradecanoylphorbol 13-acetate-induced malignant transformation via increases in metalloproteinase 9 enzyme activity and inducible nitric oxide synthase gene expression in rat glioma C6 cells, however the mechanism has remained undefined. Lipopolysaccharide plus 12-o-tetradecanoylphorbol 13-acetate, but not lipopolysaccharide or 12-o-tetradecanoylphorbol 13-acetate alone, induced transformation in glioma C6 cells (but not in human glioblastoma cells GBM-8401 cells) without affecting their viability. An increase in inducible nitric oxide synthase protein expression, nitric oxide production, and metalloproteinase 9 enzyme activity is identified lipopolysaccharide/12-o-tetradecanoylphorbol 13-acetate-treated C6 cells, however lipopolysaccharide/12-o-tetradecanoylphorbol 13-acetate and 12-o-tetradecanoylphorbol 13-acetate (but not lipopolysaccharide) addition shows the similar inductive pattern on metalloproteinase 9 enzyme activity without affecting inducible nitric oxide synthase protein expression and nitric oxide production in GBM-8401 cells. Treatment of C6 cells with lipopolysaccharide/12-o-tetradecanoylphorbol 13-acetate increases the expression of phosphorylated extracellular regulated protein kinases and Jun N-terminal kinases, but not p38, proteins, and an addition of the extracellular regulated protein kinases inhibitor PD98059 or Jun N-terminal kinases inhibitors SP600125, but not the p38 inhibitor SB203580, significantly blocked lipopolysaccharide/12-o-tetradecanoylphorbol 13-acetate-induced inducible nitric oxide synthase protein expression and metalloproteinase 9 enzyme activity accompanied by blocking morphological transformation in C6 cells. Among 19 structurally related flavonoids, kaempferol and wogonin exhibit significant inhibitory effects on lipopolysaccharide/12-o-tetradecanoylphorbol 13-acetate-induced morphological transformation and colony formation, and attenuation of inducible nitric oxide synthase, phosphorylated extracellular regulated protein kinases protein expression, and metalloproteinase 9 enzyme activity was observed. 2'-OH flavone at a dose of 100 microM inhibition of lipopolysaccharide/12-o-tetradecanoylphorbol 13-acetate-induced events via apoptosis induction is identified. Furthermore, lipopolysaccharide/12-o-tetradecanoylphorbol 13-acetate, but not lipopolysaccharide or 12-o-tetradecanoylphorbol 13-acetate, induces tumoral invasion and migration in vitro and in vivo, and those are blocked by kaempferol and wogonin addition. These data suggest that combination of lipopolysaccharide and 12-o-tetradecanoylphorbol 13-acetate promotes tumoral progression via activating metalloproteinase 9 enzyme activity and inducible nitric oxide synthase gene expression, which is located downstream of mitogen-activated protein kinases activation, in rat glioma cells C6. Kaempferol and wogonin exhibit effective inhibitory effects on lipopolysaccharide/12-o-tetradecanoylphorbol 13-acetate-induced events, and thus possess the potential for further development.     

切应力通过Pim1/eNOS途径调节人脐静脉内皮细胞NO分泌

目的:探讨层流切应力是否可通过Pim1调节内皮型一氧化氮合酶(eNOS)活性,从而调节血管内皮细胞一氧化氮(NO)分泌。方法:体外原代培养人脐静脉内皮细胞(HUVECs),运用平行平板流动腔系统给HUVECs加载层流切应力(15 dyn/cm²)。采用Western blot法检测Pim1蛋白表达及eNOS-Ser1177磷酸化水平;硝酸还原酶法检测NO分泌量;利用特异性小干扰RNA(siRNA)转染技术沉默Pim1基因后再检测上述指标的变化。结果:切应力作用HUVECs 15 min,可以显著上调Pim1蛋白表达(P<0.05),同时显著增强eNOS-Ser1177磷酸化水平(P<0.05),伴随HUVECs NO分泌显著增多(P<0.05)。转染siPim1可以抑制切应力诱导的Pim1表达(P<0.05),同时抑制eNOS-Ser1177磷酸化(P<0.05),NO分泌随之显著降低(P<0.05)。结论:流体切应力可能通过Pim1/eNOS途径调节血管内皮细胞NO分泌。     

Adenovirus mediated endothelial nitric oxide synthase gene transfer prevents restenosis of vein grafts

The possibility of endothelial nitric oxide synthase (eNOS) gene transfer, which prevents restenosis of vein grafts, was explored in 16 goats. The recombinant adenoviral vector coding endothelial nitric oxide synthase (AdCMVeNOS) and adenoviral vector (AdCMV) were constructed. A total of 6 cm jugular vein was removed, cut into two equal lengths for vein grafts, and infected with AdCMVeNOS or AdCMV in vitro. One segment (2 cm) of each carotid artery was removed. The vein graft that had been infected with AdCMVeNOS was anastomosed to the right carotid artery, and the vein graft that had been infected with AdCMV was anastomosed to the left. The functional expression of eNOS in vein grafts was assessed by the immunohistochemical staining and measurement of NO concentration. The inhibition of intimal hyperplasia in vein grafts was evaluated by the assay of 3H-TDR incorporation, histologic analysis, measurement of intimal thickness, and percent area stenosis. Adenovirus mediated eNOS gene transfer to goat vein grafts resulted in functional transgene expression with increased NO release. Increased local NO production could inhibit intimal hyperplasia and increase the patent rate of vein grafts.     

How mental stress affects endothelial function

Mental stress is an important factor contributing to recognized mechanisms underlying cardiovascular events. Among these, stress-related endothelial dysfunction is an early risk factor that predicts future development of severe cardiovascular disorders. Acute mental stress by a variety of tests impairs endothelial function in humans, although the opposite results have been reported by some investigators. Chronic stress always deteriorates endothelial function in humans and experimental animals. Stress hormones, such as glucocorticoids and pro-inflammatory cytokines, and endothelin-1 liberated in response to mental stress participate in endothelial dysfunction possibly via downregulation of endothelial nitric oxide synthase (eNOS) expression, eNOS inactivation, decreased nitric oxide (NO) actions, and increased NO degradation, together with vasoconstriction counteracting against NO-induced vasodilatation. Catecholamines do not directly affect endothelial function but impair its function when blood pressure elevation by the amines is sustained. Endogenous opioids favorably affect endothelial function, which counteract deteriorating effects of other stress hormones and mediators. Inhibition of cortisol and endothelin-1 production, prevention of pro-inflammatory mediator accumulation, hypnotics, mirthful laughter, humor orientation, and lifestyle modification would contribute to the prevention and treatment for stress-related endothelial dysfunction and future serious cardiovascular disease.     

Hemodynamic Regulation of Inflammation at the Endothelial–Neutrophil Interface

Arterial shear stress can regulate endothelial phenotype. The potential for anti-inflammatory effects of shear stress on TNFα-activated endothelium was tested in assays of cytokine expression and neutrophil adhesion. In cultured human aortic endothelial cells (HAEC), arterial shear stress of 10 dyne/cm² blocked by >80% the induction by 5 ng/mL TNFα of interleukin-8 (IL-8) and IL-6 secretion (50 and 90% reduction, respectively, in the presence of nitric oxide synthase antagonism with 200 μM nitro-l-arginine methylester, l-NAME). Exposure of TNFα-stimulated HAEC to arterial shear stress for 5 h also reduced by 60% (p < 0.001) the conversion of neutrophil rolling to firm arrest in a venous flow assay conducted at 1 dyne/cm². Also, neutrophil rolling lengths at 1 dyne/cm² were longer when TNFα-stimulated HAEC were presheared for 5 h at arterial stresses. In experiments with a synthetic promoter that provides luciferase induction to detect cis interactions of glucocorticoid receptor (GR) and NFκB, shear stress caused a marked 40-fold induction of luciferase in TNFα-treated cells, suggesting a role for GR pathways in the anti-inflammatory actions of fluid shear stress. Hemodynamic force exerts anti-inflammatory effects on cytokine-activated endothelium by attenuation of cytokine expression and neutrophil firm arrest.     

Inhibition of TGF-beta1 signaling by eNOS gene transfer improves ventricular remodeling after myocardial infarction through angiogenesis and reduction of apoptosis.

INTRODUCTION: Endothelial nitric oxide synthase and nitric oxide have been implicated in protection against myocardial ischemia injury. However, the angiogenic effect of endothelial nitric oxide synthase on infarcted myocardium and the role of tumor growth factor beta1 signaling in cardiac remodeling mediated by endothelial nitric oxide synthase/nitric oxide have not yet been elucidated. METHODS: Human endothelial nitric oxide synthase gene in an adenovirus vector was delivered locally into rat heart 4 days prior to the induction of myocardial infarction by left anterior descending coronary artery ligation. Cardiomyocyte apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, and neovascularization was identified immunohistochemically. RESULTS: Endothelial nitric oxide synthase gene transfer significantly reduced infarct size and improved cardiac contractility and left ventricular diastolic function at 24 h after myocardial infarction. In addition, endothelial nitric oxide synthase significantly reduced myocardial-infarction-induced cardiomyocyte apoptosis. Activation of tumor growth factor beta1 and Smad-2 after myocardial infarction was also dramatically reduced by endothelial nitric oxide synthase. Moreover, the deterioration of both systolic and diastolic functions, in conjunction with thin left ventricular remodeling at 7 days after myocardial infarction, was prevented by endothelial nitric oxide synthase. Capillary density, as identified by alpha-smooth muscle actin immunostaining, was significantly increased in the infarcted myocardium after endothelial nitric oxide synthase transfer compared with myocardial infarction control. All cardioprotective effects of endothelial nitric oxide synthase were blocked by N(omega)-nitro-l-arginine methyl ester administration, indicating a nitric-oxide-mediated event. CONCLUSION: These results demonstrate that the endothelial nitric oxide synthase/nitric oxide system provides cardiac protection after myocardial infarction injury through inhibition of cardiac apoptosis, stimulation of neovascularization, and suppression of tumor growth factor beta1/Smad-2 signaling.     

The cardiovascular response in sepsis: proposed mechanisms of the beneficial effect of adrenomedullin and its binding protein (review)

Sepsis and its complications are leading causes of morbidity and mortality. A better understanding of the mechanisms responsible for the shift from the early, hyperdynamic phase of sepsis to the late hypodynamic phase could lead to novel therapies that might improve the outcome of the septic patient. Adrenomedullin is a vasodilatory peptide which shows sustained elevation starting early in sepsis and is important in initiating the hyperdynamic response. As sepsis progresses, however, the vascular response to adrenomedullin is blunted and this decreased sensitivity is important in producing the shift to the late, hypodynamic phase. The decline in the vascular response to adrenomedullin is related to a sepsis-induced decrease in the binding protein for adrenomedullin (i.e., adrenomedullin binding protein-1) rather than a change in gene expression of the components of adrenomedullin receptors. Treatment of septic animals with the combination of adrenomedullin and its binding protein prevents the transition to the late phase of sepsis, maintains cardiovascular stability, and reduces sepsis-induced mortality. We propose that the mechanisms responsible for the beneficial effect of adrenomedullin and adrenomedullin binding protein-1 in sepsis are associated with downregulation of proinflammatory cytokines (TNF-alpha, IL-1beta, IL-6), maintainence of endothelial constitutive nitric oxide synthase, and reduction of vascular endothelial cell apoptosis.     

Pro-atherosclerotic disturbed flow disrupts caveolin-1 expression,localization, and function via glycocalyx degradation

Background

Endothelial-dependent atherosclerosis develops in a non-random pattern in regions of vessel bending and bifurcations, where blood flow exhibits disturbed flow (DF) patterns. In contrast, uniform flow (UF), normal endothelium, and healthy vessel walls co-exist within straight vessels. In clarifying how flow protectively or atherogenically regulates endothelial cell behavior, involvement of the endothelial surface glycocalyx has been suggested due to reduced expression in regions of atherosclerosis development. Here, we hypothesized that pro-atherosclerotic endothelial dysfunction occurs as a result of DF-induced reduction in glycocalyx expression and subsequently impairs endothelial sensitivity to flow. Specifically, we propose that glycocalyx degradation can induce pro-atherosclerotic endothelial dysfunction through decreased caveolin-1 and endothelial nitric oxide synthase expression and localization.

Methods

We studied endothelial cells in atherosclerotic-prone DF and atherosclerotic-resistant UF conditions in parallel plate flow culture and in C57Bl/6 mice. The effects of flow conditioning on endothelial cell behavior were quantified using immunocytochemistry. The glycocalyx was fluorescently labeled for wheat germ agglutinin, which serves as a general glycocalyx label, and heparan sulfate, a major glycocalyx component. Additionally, mechanosensitivity was assessed by immunocytochemical fluorescence expression and function of caveolin-1, the protein that forms the mechanosignaling caveolar invaginations on the endothelial surface, total endothelial-type nitric oxide synthase (eNOS), which synthesizes nitric oxide, and serine 1177 phosphorylated eNOS (eNOS-pS1177), which is the active form of eNOS. Caveolin function and eNOS expression and activation were correlated to glycocalyx expression. Heparinase III enzyme was used to degrade a major glycocalyx component, HS, to identify the role of the glycocalyx in caveoin-1 and eNOS-pS1177 regulation.

Results

Results confirmed that DF reduces caveolin-1 expression and abolishes most of its subcellular localization preferences, when compared to the effect of UF. DF down-regulates caveolin-1 mechanosignaling, as indicated by its reduced colocalization with serine 1177 phosphorylated endothelial-type nitric oxide synthase (eNOS-pS1177), a vasoregulatory signaling molecule whose activity is regulated by its residence in caveolae. As expected, DF inhibited glycocalyx expression compared to UF. In the absence of heparan sulfate, a major glycocalyx component, UF-conditioned endothelial cells exhibited near DF-like caveolin-1 expression, localization, and colocalization with eNOS-pS1177.

Conclusions

This is the first demonstration of a flow-defined role of the glycocalyx in caveolae expression and function related to vasculoprotective endothelial mechanosensitivity that defends against atherosclerosis. The results suggest that a glycocalyx-based therapeutic targeted to areas of atherosclerosis development could prevent disease initiation and progression.

     

Shear stress increases endothelial hyaluronan synthase 2 and hyaluronan synthesis especially in regard to an atheroprotective flow profile

Recent studies revealed that in vivo the inner blood vessel surface is lined with an endothelial surface layer at least 0.5 μm thick, which serves as an aegis, protecting the vessel wall from arteriosclerosis. Hyaluronan seems to be a constitutive component in regard to the atheroprotective properties of this surface structure. It has been shown that arterial pulsatile laminar blood flow increases the thickness of this surface layer in vivo, while it is significantly reduced at atheroprone regions with disturbed flow. This study was undertaken to reveal whether endothelial hyaluronan synthesis via hyaluronan synthase 2 (HAS2) can be changed by different shear stress conditions in vitro, especially in regard to an undisturbed, arterial-like pulsatile flow profile. Human umbilical vein endothelial cells, exposed to constant or pulsatile shear stress in a cone-and-plate system, were analysed for HAS2 expression by real-time RT-PCR and immunoblotting, and for hyaluronan by ELISA. Hyaluronan synthase 2 mRNA and protein were found to be transiently increased in a shear stress-dependent manner via the phosphatidylinositol 3-kinase-Akt pathway. Especially pulsatile, arterial-like shear stress conditions induced enzyme and hyaluronan effectively, while lower shear stress that continuously changed its direction did not induce any differences in comparison with control cultures not exposed to shear stress. These experiments provide a link between the production of a constitutive component of the endothelial surface layer by endothelial cells and blood flow.     

Molecular mechanism of T-cell control of Chlamydia in mice: role of nitric oxide in vivo.

T-cell-mediated immunity is crucial for the control of Chlamydia in mice. Recent evidence from studies in an in vitro model of the mucosal epithelium, the polarized epithelial-lymphocyte co-culture (PELC) system, indicated that protective murine T cells mediated intracellular inhibition of the Chlamydia trachomatis agent of mouse pneumonitis (MoPn) at least partly by activating the interferon-gamma (IFN-gamma)-inducible nitric oxide synthase (iNOS) pathway. To investigate whether nitric oxide played a role in controlling chlamydial infection in vivo, the protective capacity of a chlamydial-specific T-cell clone (clone 2.14-0) was analysed in mice in the presence of a specific inhibitor of iNOS. The results revealed that the ability of this clone to clear Chlamydia in vivo is in part mediated by induction of nitric oxide (NO) production. The L-arginine analogue and iNOS inhibitor, NG-monomethyl-L-arginine monoacetate (MLA), increased the chlamydial burden in infected mice and inhibited the ability of clone 2.14-0 to clear genital MoPn infection in vivo. The results are consistent with the working hypothesis that the IFN-gamma-inducible iNOS pathway is involved in the control of Chlamydia by T lymphocytes in mice.