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Background: Protein Kinase C (PKC) is a promiscuous serine/threonine kinase regulating vasodilatory responses in vascular endothelial cells. Calcium-dependent PKCbeta (PKCβ) and calcium-independent PKCeta (PKCη) have both been implicated in the regulation and dysfunction of endothelial responses to shear stress and agonists., Objective: We hypothesized that PKCβ and PKCη differentially modulate shear stress-induced nitric oxide (NO) production by regulating the transduced calcium signals and the resultant eNOS activation. As such, this study sought to characterize the contribution of PKCη and PKCβ in regulating calcium signaling and endothelial nitric oxide synthase (eNOS) activation after exposure of endothelial cells to ATP or shear stress., Methods: Bovine aortic endothelial cells were stimulated in vitro under pharmacological inhibition of PKCβ with LY333531 or PKCη targeting with a pseudosubstrate inhibitor. The participation of PKC isozymes in calcium flux, eNOS phosphorylation and NO production was assessed following stimulation with ATP or shear stress., Results: PKCη proved to be a robust regulator of agonist- and shear stress-induced eNOS activation, modulating calcium fluxes and tuning eNOS activity by multi-site phosphorylation. PKCβ showed modest influence in this pathway, promoting eNOS activation basally and in response to shear stress. Both PKC isozymes contributed to the constitutive and induced phosphorylation of eNOS. The observed PKC signaling architecture is intricate, recruiting Src to mediate a portion of PKCη's control on calcium entry and eNOS phosphorylation. Elucidation of the importance of PKCη in this pathway was tempered by evidence of a single stimulus producing concurrent phosphorylation at ser1179 and thr497 which are antagonistic to eNOS activity., Conclusions: We have, for the first time, shown in a single species in vitro that shear stress- and ATP-stimulated NO production are differentially regulated by classical and novel PKCs. This study furthers our understanding of the PKC isozyme interplay that optimizes NO production. These considerations will inform the ongoing design of drugs for the treatment of PKC-sensitive cardiovascular pathologies., (Copyright © 2021. Published by Elsevier Inc.)

Objectives: The effect of NO on smooth muscle cell contractility is crucial in regulating vascular tone, blood flow, and O 2 delivery. Quantitative predictions for interactions between the NO production rate and the myogenic response for microcirculatory blood vessels are lacking., Methods: We developed a computational model of a branching microcirculatory network with four representative classes of resistance vessels to predict the effect of endothelium-derived NO on the microvascular pressure-flow response. Our model links vessel scale biotransport simulations of NO and O 2 delivery to a mechanistic model of autoregulation and myogenic tone in a simplified microcirculatory network., Results: The model predicts that smooth muscle cell NO bioavailability significantly contributes to resting vascular tone of resistance vessels. Deficiencies in NO seen during hypoxia or ischemia lead to a decreased vessel diameter for all classes at a given intravascular pressure. At the network level, NO deficiencies lead to an increase in pressure drop across the vessels studied, a downward shift in the pressure-flow curve, and a decrease in the effective range of the autoregulatory response., Conclusions: Our model predicts the steady state and transient behavior of resistance vessels to perturbations in blood pressure, including effects of NO bioavailability on vascular regulation., (© 2018 John Wiley & Sons Ltd.)

Interactions between cardiac myoglobin (Mb), nitrite, and nitric oxide (NO) are vital in regulating O 2 storage, transport, and NO homeostasis. Production of NO through the reduction of endogenous myocardial nitrite by deoxygenated myoglobin has been shown to significantly reduce myocardial infarction damage and ischemic injury. We developed a mathematical model for a cardiac arteriole and surrounding myocardium to examine the hypothesis that myoglobin switches functions from being a strong NO scavenger to an NO producer via the deoxymyoglobin nitrite reductase pathway. Our results predict that under ischemic conditions of flow, blood oxygen level, and tissue pH, deoxyMb nitrite reduction significantly elevates tissue and smooth muscle cell NO. The size of the effect is consistent at different flow rates, increases with decreasing blood oxygen and tissue pH and, in extreme pathophysiological conditions, NO can even be elevated above the normoxic levels. Our simulations suggest that cardiac deoxyMb nitrite reduction is a plausible mechanism for preserving or enhancing NO levels using endogenous nitrite despite the rate-limiting O 2 levels for endothelial NO production. This NO could then be responsible for mitigating deleterious effects under ischemic conditions., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Nitrite infusion into the bloodstream has been shown to elicit vasodilation and protect against ischemia-reperfusion injury through nitric oxide (NO) release in hypoxic conditions. However, the mechanism by which nitrite-derived NO escapes scavenging by hemoglobin in the erythrocyte has not been fully elucidated, owing in part to the difficulty in measuring the reactions and transport on NO in vivo. We developed a mathematical model for an arteriole and surrounding tissue to examine the hypothesis that dinitrogen trioxide (N 2 O 3 ) acts as a stable intermediate for preserving NO. Our simulations predict that with hypoxia and moderate nitrite concentrations, the N 2 O 3 pathway can significantly preserve the NO produced by hemoglobin nitrite reductase in the erythrocyte and elevate NO reaching the smooth muscle cells. Nitrite retains its ability to increase NO bioavailability even at varying flow conditions, but there is minimal effect under normoxia or very low nitrite concentrations. Our model demonstrates a viable pathway for reconciling experimental findings of potentially beneficial effects of nitrite infusions despite previous models showing negligible NO elevation associated with hemoglobin nitrite reductase. Our results suggest that additional mechanisms may be needed to explain the efficacy of nitrite-induced vasodilation at low infusion concentrations., (Copyright © 2016 Elsevier Inc. All rights reserved.)

We developed a mass transport model for a parallel-plate flow chamber apparatus to predict the concentrations of nitric oxide (NO) and adenine nucleotides (ATP, ADP) produced by cultured endothelial cells (ECs) and investigated how the net rates of production, degradation, and mass transport for these three chemical species vary with changes in wall shear stress (τw). These simulations provide an improved understanding of experimental results obtained with parallel-plate flow chambers and allows quantitative analysis of the relationship between τw, adenine nucleotide concentrations, and NO produced by ECs. Experimental data obtained after altering ATP and ADP concentrations with apyrase were analyzed to quantify changes in the rate of NO production (RNO). The effects of different isoforms of apyrase on ATP and ADP concentrations and nucleotide-dependent changes in RNO could be predicted with the model. A decrease in ATP was predicted with apyrase, but an increase in ADP was simulated due to degradation of ATP. We found that a simple proportional relationship relating a component of RNO to the sum of ATP and ADP provided a close match to the fitted curve for experimentally measured changes in RNO with apyrase. Estimates for the proportionality constant ranged from 0.0067 to 0.0321 μM/s increase in RNO per nM nucleotide concentration, depending on which isoform of apyrase was modeled, with the largest effect of nucleotides on RNO at low τw (<6 dyn/cm(2))., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Topical delivery of nitric oxide (NO) through a wound dressing has the potential to reduce wound infections and improve healing of acute and chronic wounds. This study characterized the antibacterial efficacy of an ointment containing NO-loaded, zinc-exchanged zeolite A that releases NO upon contact with water. The release rate of NO from the ointment was measured using a chemiluminescence detection system. Minimum bactericidal concentration assays were performed using five common wound pathogens, including Gram-negative bacteria (Escherichia coli and Acinetobacter baumannii), Gram-positive bacteria (Staphylococcus epidermidis and meticillin-resistant Staphylococcus aureus) and a fungus (Candida albicans). The time dependence of antimicrobial activity was characterized by performing log-reduction assays at four time points after 1-8 h ointment exposure. The cytotoxicity of the ointment after 24 h was assessed using cultured 3T3 fibroblast cells. Minimum microbicidal concentrations (MMCs) for bacterial organisms (5×10(7) c.f.u.) ranged from 50 to 100 mg ointment (ml media)(-1); the MMC for C. albicans (5×10(4) c.f.u.) was 50 mg ointment (ml media)(-1). Five to eight log reductions in bacterial viability and three log reductions in fungal viability were observed after 8 h exposure to NO-zeolite ointment compared with untreated organisms. Fibroblasts remained viable after 24 h exposure to the same concentration of NO-zeolite ointment as was used in antimicrobial tests. In parallel studies, full-thickness cutaneous wounds on Zucker obese rats healed faster than wounds treated with a control ointment. These data indicate that ointment containing NO-loaded zeolites could potentially be used as a broad-spectrum antimicrobial wound-healing dressing.

People with diabetes suffer from early accelerated atherosclerosis, which contributes to morbidity and mortality from myocardial infarction, stroke, and peripheral vascular disease. Atherosclerosis is thought to initiate at sites of endothelial cell injury. Hyperglycemia, a hallmark of diabetes, leads to non-enzymatic glycosylation (or glycation) of extracellular matrix proteins. Glycated collagen alters endothelial cell function and could be an important factor in atherosclerotic plaque development. This study examined the effect of collagen glycation on endothelial cell response to fluid shear stress. Porcine aortic endothelial cells were grown on native or glycated collagen and exposed to shear stress using an in vitro parallel plate system. Cells on native collagen elongated and aligned in the flow direction after 24 h of 20 dynes/cm(2) shear stress, as indicated by a 13% decrease in actin fiber angle distribution standard deviation. However, cells on glycated collagen did not align. Shear stress-mediated nitric oxide release by cells on glycated collagen was half that of cells on native collagen, which correlated with decreased endothelial nitric oxide synthase (eNOS) phosphorylation. Glycated collagen likely inhibited cell shear stress response through altered cell-matrix interactions, since glycated collagen attenuated focal adhesion kinase activation with shear stress. When focal adhesion kinase was pharmacologically blocked in cells on native collagen, eNOS phosphorylation with flow was reduced in a manner similar to that of glycated collagen. These detrimental effects of glycated collagen on endothelial cell response to shear stress may be an important contributor to accelerated atherosclerosis in people with diabetes., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

We developed a mathematical model to simulate shear stress-dependent nitric oxide (NO) production and transport in a 3D microcirculatory network based on published data. The model consists of a 100 μm × 500 μm × 75 μm rectangular volume of tissue containing two arteriole-branching trees, and nine capillaries surrounding the vessels. Computed distributions for NO in blood, vascular walls, and surrounding tissue were affected by hematocrit (Hct) and wall shear stress (WSS) in the network. The model demonstrates that variations in the red blood cell (RBC) distribution and WSS in a branching network can have differential effects on computed NO concentrations due to NO consumption by RBCs and WSS-dependent changes in NO production. The model predicts heterogeneous distributions of WSS in the network. Vessel branches with unequal blood flow rates gave rise to a range of WSS values and therefore NO production rates. Despite increased NO production in a branch with higher blood flow and WSS, vascular wall NO was predicted to be lower due to greater NO consumption in blood, since the microvascular Hct increased with redistribution of RBCs at the vessel bifurcation. Within other regions, low WSS was combined with decreased NO consumption to enhance the NO concentration.

Several apparent paradoxes are evident when one compares mathematical predictions from models of nitric oxide (NO) diffusion and convection in vasculature structures with experimental measurements of NO (or related metabolites) in animal and human studies. Values for NO predicted from mathematical models are generally much lower than in vivo NO values reported in the literature for experiments, specifically with NO microelectrodes positioned at perivascular locations next to different sizes of blood vessels in the microcirculation and NO electrodes inserted into a wide range of tissues supplied by the microcirculation of each specific organ system under investigation. There continues to be uncertainty about the roles of NO scavenging by hemoglobin versus a storage function that may conserve NO, and other signaling targets for NO need to be considered. This review describes model predictions and relevant experimental data with respect to several signaling pathways in the microcirculation that involve NO.

Recent evidence in the literature suggests that tissues play a greater role than blood in reducing nitrite to NO under ischemic or hypoxic conditions. Our previous mathematical model for coupled NO and O(2) transport around an arteriole, modified to include superoxide generation from dysfunctional endothelium, was developed further to include nitrite reductase activity in blood and tissue. Steady-state radial and axial NO and pO(2) profiles in the arteriole and surrounding tissue were simulated for different blood flow rates and arterial blood pO(2) values. The resulting computer simulations demonstrate that nitrite reductase activity in blood is not a very effective mechanism for conserving NO due to the strong scavenging of NO by hemoglobin. In contrast, nitrite reductase activity in tissue is much more effective in increasing NO bioavailability in the vascular wall and contributes progressively more NO as tissue hypoxia becomes more severe.

Nitric oxide (NO) produced by the endothelium is involved in the regulation of vascular tone. Decreased NO production or availability has been linked to endothelial dysfunction in hypercholesterolemia and hypertension. Shear stress-induced NO release is a well-established phenomenon, yet the cellular mechanisms of this response are not completely understood. Experimental limitations have hindered direct, real-time measurements of NO under flow conditions. We have overcome these challenges with a new design for a parallel-plate flow chamber. The chamber consists of two compartments, separated by a Transwell® membrane, which isolates a NO recording electrode located in the upper compartment from flow effects. Endothelial cells are grown on the bottom of the membrane, which is inserted into the chamber flush with the upper plate. We demonstrate for the first time direct real-time NO measurements from endothelial cells with controlled variations in shear stress. Step changes in shear stress from 0.1 dyn/cm(2) to 6, 10, or 20 dyn/cm(2) elicited a transient decrease in NO followed by an increase to a new steady state. An analysis of NO transport suggests that the initial decrease is due to the increased removal rate by convection as flow increases. Furthermore, the rate at which the NO concentration approaches the new steady state is related to the time-dependent cellular response rather than transport limitations of the measurement configuration. Our design offers a method for studying the kinetics of the signaling mechanisms linking NO production with shear stress as well as pathological conditions involving changes in NO production or availability., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The goal of this study was to investigate the complex co-transport of nitric oxide (NO) and oxygen (O2) in a paired arteriole-venule, surrounded by capillary-perfused tissue using a computer model. Blood flow was assumed to be steady in the arteriolar and venular lumens and to obey Darcy's law in the tissue. NO consumption rate was assumed to be constant in the core of the arteriolar and venular lumen and to decrease linearly to the endothelium. Average NO consumption rate by capillary blood in a unit tissue volume was assumed proportional to the blood flux across the volume. Our results predict that: (1) the capillary bed, which connects the arteriole and venule, facilitates the release of O2 from the vessel pair to the surrounding tissue; (2) decreasing the distance between arteriole and venule can result in a higher NO concentration in the venular wall than in the arteriolar wall; (3) in the absence of capillaries in the surrounding tissue, diffusion of NO from venule to arteriole contributes little to NO concentration in the arteriolar wall; and (4) when capillaries are added to the simulation, a significant increase of NO in the arteriolar wall is observed.

The purpose of this investigation was to study the effect of the presence of red blood cells (RBCs) in the plasma layer near the arteriole wall on nitric oxide (NO) and oxygen (O2) transport. To this end, we extended a coupled NO and O2 diffusion-reaction model in the arteriole, developed by our group, to include the effect of the presence of RBCs in the plasma layer and the effect of convection. Two blood flow velocity profiles (plug and parabolic) were tested. The average hematocrit in the bloodstream was assumed to be constant in the central core and decreasing to zero in the boundary layer next to the endothelial surface layer. The effect of the presence or absence of RBCs near the endothelium was studied while varying the endothelial surface layer and boundary layer thickness. With RBCs present in the boundary layer, the model predicts that 1) NO decreases significantly in the endothelium and vascular wall; 2) there is a very small increase in endothelial and vascular wall Po2; 3) scavenging of NO by hemoglobin decreases with increasing thickness of the boundary layer; 4) the shape of the velocity profile influences both NO and Po2 gradients in the bloodstream; and 5) the presence of RBCs in the boundary layer near the endothelium has a much larger effect on NO than on O2 transport.

NO and PO(2) microelectrodes were used to quantify the effects of increased availability of L-arginine in an exteriorized rat mesentery and small intestine microcirculatory preparation in n = 16 rats. During short periods of elevated L-arginine added to the superfusion bath, transient changes in perivascular NO or PO(2) were measured at 171 perivascular sites near intestinal arterioles and venules, simultaneously with tissue perfusion using laser Doppler flowmetry (LDF). Excess L-arginine increased perivascular NO over twofold, by 411 +/- 42 nM above the baseline of 329 +/- 30 nM (P < 0.0001), and increased tissue perfusion by 35.5 +/- 7.5% (P < 0.0001). No difference between arterioles and venules was observed in the magnitude or time course of the NO responses. Both increases and decreases in perivascular PO(2) were observed after excess L-arginine, with a similar increase in tissue perfusion by 42.0 +/- 12.3% (P < 0.0001). Our NO measurements confirm that increased bioavailability of L-arginine causes a significant increase in NO production throughout the microcirculation of this preparation, with increased tissue perfusion, and provides direct in vivo evidence for the L-arginine paradox.

Nitric oxide (NO) generated from nitrite through nitrite reductase activity in red blood cells has been proposed to play a major role in hypoxic vasodilation. However, we have previously predicted from mathematical modeling that much more NO can be derived from tissue nitrite reductase activity than from red blood cell nitrite reductase activity. Evidence in the literature suggests that tissue nitrite reductase activity is associated with xanthine oxidoreductase (XOR) and/or aldehyde oxidoreductase (AOR). We investigated the role of XOR and AOR in nitrite-mediated vasodilation from computer simulations and from in vivo exteriorized rat mesentery experiments. Vasodilation responses to nitrite in the superfusion medium bathing the mesentery equilibrated with 5% O2 (normoxia) or zero O2 (hypoxia) at either normal or acidic pH were quantified. Experiments were also conducted following intraperitoneal (IP) injection of nitrite before and after inhibiting XOR with allopurinol or inhibiting AOR with raloxifene. Computer simulations for NO and O2 transport using reaction parameters reported in the literature were also conducted to predict nitrite-dependent NO production from XOR and AOR activity as a function of nitrite concentration, PO2 and pH. Experimentally, the largest arteriolar responses were found with nitrite >10mM in the superfusate, but no statistically significant differences were found with hypoxic and acidic conditions in the superfusate. Nitrite-mediated vasodilation with IP nitrite injections was reduced or abolished after inhibiting XOR with allopurinol (p < 0.001). Responses to IP nitrite before and after inhibiting AOR with raloxifene were not as consistent. Our mathematical model predicts that under certain conditions, XOR and AOR nitrite reductase activity in tissue can significantly elevate smooth muscle cell NO and can serve as a compensatory pathway when endothelial NO production is limited by hypoxic conditions. Our theoretical and experimental results provide further evidence for a role of tissue nitrite reductases to contribute additional NO to compensate for reduced NO production by endothelial nitric oxide synthase during hypoxia. Our mathematical model demonstrates that under extreme hypoxic conditions with acidic pH, endogenous nitrite levels alone can be sufficient for a functionally significant increase in NO bioavailability. However, these conditions are difficult to achieve experimentally. [ABSTRACT FROM AUTHOR]

The purpose of this investigation was to study the effect of the presence of red blood cells (RBCs) in the plasma layer near the arteriole wall on nitric oxide (NO) and oxygen (O2) transport. To this end, we extended a coupled NO and O2 diffusion-reaction model in the arteriole, developed by our group, to include the effect of the presence of RBCs in the plasma layer and the effect of convection. Two blood flow velocity profiles (plug and parabolic) were tested. The average hematocrit in the bloodstream was assumed to be constant in the central core and decreasing to zero in the boundary layer next to the endothelial surface layer. The effect of the presence or absence of RBCs near the endothelium was studied while varying the endothelial surface layer and boundary layer thickness. With RBCs present in the boundary layer, the model predicts that 1) NO decreases significantly in the endothelium and vascular wall; 2) there is a very small increase in endothelial and vascular wall Po2; 3) scavenging of NO by hemoglobin decreases with increasing thickness of the boundary layer; 4) the shape of the velocity profile influences both NO and Po2 gradients in the bloodstream; and 5) the presence of RBCs in the boundary layer near the endothelium has a much larger effect on NO than on O2 transport. [ABSTRACT FROM AUTHOR]

Endothelial dysfunction, characterised by impaired nitric oxide (NO) bioavailability, arises in response to a variety of cardiovascular risk factors and precedes atherosclerosis. NO is produced by tight regulation of endothelial nitric oxide synthase (eNOS) activity in response to vasodilatory stimuli. This regulation of eNOS is mediated in part by store-operated calcium entry (SOCE). We hypothesized that both ATP- and flow-induced eNOS activation are regulated by SOCE derived from Orai1 channels and members of the transient receptor potential canonical (TRPC) channel family. Bovine aortic endothelial cells (BAECs) were pre-treated with pharmacological inhibitors of TRPC channels and Orai1 to examine their effect on calcium signaling and eNOS activation in response to flow and ATP. The peak and sustained ATP-induced calcium signal and the resulting eNOS activation were attenuated by inhibition of TRPC3, which we found to be store operated. TRPC4 blockade reduced the transient peak in calcium concentration following ATP stimulation, but did not significantly reduce eNOS activity. Simultaneous TRPC3 & 4 inhibition reduced flow-induced NO production via alterations in phosphorylation-mediated eNOS activity. Inhibition of TRPC1/6 or Orai1 failed to lower ATP-induced calcium entry or eNOS activation. Our results suggest that TRPC3 is a store-operated channel in BAECs and is the key regulator of ATP-induced eNOS activation, whereas flow stimulation also recruits TRPC4 into the pathway for the synthesis of NO. [ABSTRACT FROM AUTHOR]