Shear Stress-induced NO Research Articles

Effect of spatial heterogeneity and colocalization of eNOS and capacitative calcium entry channels on shear stress-induced NO production by endothelial cells: A modeling approach.

Colocalization of endothelial nitric oxide synthase (eNOS) and capacitative Ca2+ entry (CCE) channels in microdomains such as cavaeolae in endothelial cells (ECs) has been shown to significantly affect intracellular Ca2+ dynamics and NO production, but the effect has not been well quantified. We developed a two-dimensional continuum model of an EC integrating shear stress-mediated ATP production, intracellular Ca2+ mobilization, and eNOS activation to investigate the effects of spatial colocalization of plasma membrane eNOS and CCE channels on Ca2+ dynamics and NO production in response to flow-induced shear stress. Our model examines the hypothesis that subcellular colocalization of cellular components can be critical for optimal coupling of NO production to blood flow. Our simulations predict that heterogeneity of CCE can result in formation of microdomains with significantly higher Ca2+ compared to the average cytosolic Ca2+. Ca2+ buffers with lower or no mobility further enhanced Ca2+ gradients relative to mobile buffers. Colocalization of eNOS to CCE channels significantly increased NO production. Our results provide quantitative understanding for the role of spatial heterogeneity and the compartmentalization of signals in regulation of shear stress-induced NO production.

Effects of Physiological Oxygen Tension on In Vitro Fluid Shear Stress-Induced endothelial NO Synthase Activation

Effects of Physiological Oxygen Tension on In Vitro Fluid Shear Stress-Induced endothelial NO Synthase Activation

Dysregulated flow-mediated vasodilatation in the human placenta in fetal growth restriction.

Increased vascular resistance and reduced fetoplacental blood flow are putative aetiologies in the pathogenesis of fetal growth restriction (FGR); however, the regulating sites and mechanisms remain unclear. We hypothesised that placental vessels dictate fetoplacental resistance and in FGR exhibit endothelial dysfunction and reduced flow-mediated vasodilatation (FMVD). Resistance was measured in normal pregnancies (n = 10) and FGR (n = 10) both in vivo by umbilical artery Doppler velocimetry and ex vivo by dual placental perfusion. Ex vivo FMVD is the reduction in fetal-side inflow hydrostatic pressure (FIHP) following increased flow rate. Results demonstrated a significant correlation between vascular resistance measured in vivo and ex vivo in normal pregnancy, but not in FGR. In perfused FGR placentas, vascular resistance was significantly elevated compared to normal placentas (58 ± 7.7 mmHg and 36.8 ± 4.5 mmHg, respectively; 8 ml min−1; means ± SEM; P < 0.0001) and FMVD was severely reduced (3.9 ± 1.3% and 9.1 ± 1.2%, respectively). In normal pregnancies only, the highest level of ex vivo FMVD was associated with the lowest in vivo resistance. Inhibition of NO synthesis during perfusion (100 μm l-NNA) moderately elevated FIHP in the normal group, but substantially in the FGR group. Human placenta artery endothelial cells from FGR groups exhibited increased shear stress-induced NO generation, iNOS expression and eNOS expression compared with normal groups. In conclusion, fetoplacental resistance is determined by placental vessels, and is increased in FGR. The latter also exhibit reduced FMVD, but with a partial compensatory increased NO generation capacity. The data support our hypothesis, which highlights the importance of FMVD regulation in normal and dysfunctional placentation.Key points A correlation was found between in vivo umbilical artery Doppler velocimetry and resistance to fetal-side flow in the human ex vivo dually perfused placenta, highlighting that the fetoplacental vascular bed is a key site of resistance to umbilico-placental flow in pregnancy. We discovered high resistance and poor flow-mediated vasodilatory responses in placentas from pregnancies associated with fetal growth restriction (FGR). Endothelial cells isolated from the FGR placentas and grown in static and flow culture showed a dysregulated phenotype, with biochemical signalling demonstrating a failed compensatory response to high blood-flow resistance.

Acute laminar shear stress reversibly increases human glomerular endothelial cell permeability via activation of endothelial nitric oxide synthase

Laminar shear stress is a key determinant of systemic vascular behavior, including through activation of endothelial nitric oxide synthase (eNOS), but little is known of its role in the glomerulus. We confirmed eNOS expression by glomerular endothelial cells (GEnC) in tissue sections and examined effects of acute exposure (up to 24 h) to physiologically relevant levels of laminar shear stress (10–20 dyn/cm2) in conditionally immortalized human GEnC. Laminar shear stress caused an orientation of GEnC and stress fibers parallel to the direction of flow and induced Akt and eNOS phosphorylation along with NO production. Inhibition of the phophatidylinositol (PI)3-kinase/Akt pathway attenuated laminar shear stress-induced eNOS phosphorylation and NO production. Laminar shear stress of 10 dyn/cm2 had a dramatic effect on GEnC permeability, reversibly decreasing the electrical resistance across GEnC monolayers. Finally, the laminar shear stress-induced reduction in electrical resistance was attenuated by the NOS inhibitors l-NG-monomethyl arginine (l-NMMA) and l-NG-nitroarginine methyl ester (l-NAME) and also by inhibition of the PI3-kinase/Akt pathway. Hence we have shown for GEnC in vitro that acute permeability responses to laminar shear stress are dependent on NO, produced via activation of the PI3-kinase/Akt pathway and increased eNOS phosphorylation. These results suggest the importance of laminar shear stress and NO in regulating the contribution of GEnC to the permeability properties of the glomerular capillary wall.

AMP-Activated Protein Kinase Is Involved in Endothelial NO Synthase Activation in Response to Shear Stress

The regulation of AMP-activated protein kinase (AMPK) is implicated in vascular biology because AMPK can phosphorylate endothelial NO synthase (eNOS). In this study, we investigate the regulation of the AMPK-eNOS pathway in vascular endothelial cells (ECs) by shear stress and the activation of aortic AMPK in a mouse model with a high level of voluntary running (High-Runner). By using flow channels with cultured ECs, AMPK Thr172 phosphorylation was increased with changes of flow rate or pulsatility. The activity of LKB1, the upstream kinase of AMPK, and the phosphorylation of eNOS at Ser1179 were concomitant with AMPK activation responding to changes in flow rate or pulsatility. The blockage of AMPK by a dominant-negative mutant of AMPK inhibited shear stress-induced eNOS Ser1179 phosphorylation and NO production. Furthermore, aortic AMPK activity and level of eNOS phosphorylation were significantly elevated in the aortas of High-Runner mice. Our results suggest that shear stress activates AMPK in ECs, which contributes to elevated eNOS activity and subsequent NO production. Hence, AMPK, in addition to serving as an energy sensor, also plays an important role in regulating vascular tone.

Free fatty acid elevation impairs insulin-mediated vasodilation and nitric oxide production.

The effect and time course of free fatty acid (FFA) elevation on insulin-mediated vasodilation (IMV) and the relationship of FFA elevation to changes in insulin-mediated glucose uptake was studied. Two groups of lean insulin-sensitive subjects underwent euglycemic-hyperinsulinemic (40 mU x m(-2) x min(-1)) clamp studies with and without superimposed FFA elevation on 2 occasions approximately 4 weeks apart. Groups differed only by duration of FFA elevation, either short (2-4 h, n = 12) or long (8 h, n = 7). On both occasions, rates of whole-body glucose uptake were measured, and changes in leg blood flow (LBF) and femoral vein nitric oxide nitrite plus nitrate (NOx) flux in response to the clamps were determined. Short FFA infusion did not have any significant effect on the parameters of interest. In contrast, long FFA infusion decreased rates of whole-body glucose uptake from 47.7 +/-2.8 to 32.2 +/- 0.6 micromol x kg(-1) x min(-1) (P < 0.01), insulin-mediated increases in LBF from 66 +/- 8 to 37 +/- 7% (P < 0.05), and insulin-induced increases in NOx flux from 25 +/- 9 to 5 +/- 9% (P < 0.05). Importantly, throughout all groups, FFA-induced changes in whole-body glucose uptake correlated significantly with FFA-induced changes in insulin-mediated increases in LBF (r = 0.706, P < 0.001), which indicates coupling of metabolic and vascular effects. In a different protocol, short FFA elevation blunted the LBF response to NG-monomethyl-L-arginine (L-NMMA), which is an inhibitor of NO synthase. LBF in response to L-NMMA decreased by 17.3 +/- 2.4 and 9.0 +/- 1.4% in the groups without and with FFA elevation, respectively (P < 0.05), which indicates that FFA elevation interferes with shear stress-induced NO production. Thus, impairment of shear stress-induced vasodilation and IMV by FFA elevation occurs with different time courses, and impairment of IMV occurs only if glucose metabolism is concomitantly reduced. These findings suggest that NO production in response to the different stimuli may be mediated via different signaling pathways. FFA-induced reduction in NO production may contribute to the higher incidence of hypertension and macrovascular disease in insulin-resistant patients.

Inhibition of nitric oxide synthase augments the positive inotropic effect of nitric oxide donors in the rat heart.

1. In this investigation we studied the effects of nitric oxide on contractility and heart rate in normal saline-perfused rat hearts where shear stress-induced endothelial NO synthesis substantially contributes to total cardiac NO production. In addition, we sought to estimate the concentrations of exogenous NO producing inotropic effects. 2. We investigated the effects of glyceryl trinitrate (GTN), S-nitroso-d,l-penicillamine (SNAP), sodium (Z)-1-(N, N-diethylamino)diazen-1-ium-1,2-diolat (DEA/NO), and DEA/NO in the presence of the NO synthase inhibitor Nomega-nitro-L-arginine (L-NA) in constant-flow-perfused spontaneously beating rat Langendorff hearts and in rat working hearts. 3. In Langendorff hearts, GTN (10 nM to 100 microM, n = 32) induced a positive inotropic response that plateaued at 1 microM GTN with a maximal rate of increase of left ventricular pressure during ventricular contraction (+dP/dtmax) of 6. 33 +/- 2.56 % (n = 11, P < 0.5). Similarly, both spontaneous NO donors (0.1 nM to 1 microM, corresponding to approximately 0.03-0.3 microM NO) induced a positive inotropic response of 10.6 +/- 3.1 % (SNAP; n = 15, P < 0.05) and 11.5 +/- 2.7 % (DEA/NO, n = 15, P < 0. 05). 4. The positive inotropic effect of SNAP and DEA/NO progressively declined from 1 microM to 100 microM of the NO donors (corresponding to approximately 0.3-30 microM NO). 5. In the isolated working rat heart, 0.1 microM DEA/NO induced an increase of +dP/dtmax of 7.5 +/- 2.5 % (n = 9, P < 0.05). Inhibition of NO synthase by L-NA produced a 4-fold increase in this effect of DEA/NO. 6. We suggest that physiological NO concentrations support myocardial performance. In normal rat hearts the positive inotropic effect of NO appears to be almost maximally exploited by the endogenous NO production.