Coordinated regulation of endothelial calcium signaling and shear stress-induced nitric oxide production by PKCß and PKCη.

BACKGROUND: Protein Kinase C (PKC) is a promiscuous serine/threonine kinase regulating vasodilatory responses in vascular endothelial cells. Calcium-dependent PKCbeta (PKCß) 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 PKCß 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 PKCß 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 PKCß 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. PKCß 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.

TRPC channel-derived calcium fluxes differentially regulate ATP and flow-induced activation of eNOS.

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.

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.

INTRODUCTION: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: Our results provide quantitative understanding for the role of spatial heterogeneity and the compartmentalization of signals in regulation of shear stress-induced NO production.

A dynamic computational network model for the role of nitric oxide and the myogenic response in microvascular flow regulation.

OBJECTIVES: The effect of NO on smooth muscle cell contractility is crucial in regulating vascular tone, blood flow, and O2 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 O2 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.

Nitric oxide release by deoxymyoglobin nitrite reduction during cardiac ischemia: A mathematical model.

Interactions between cardiac myoglobin (Mb), nitrite, and nitric oxide (NO) are vital in regulating O2 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 O2 levels for endothelial NO production. This NO could then be responsible for mitigating deleterious effects under ischemic conditions.

Cholesterol Enrichment Impairs Capacitative Calcium Entry, eNOS Phosphorylation & Shear Stress-Induced NO Production.

Endothelial dysfunction, characterized by decreased production or availability of nitric oxide (NO), is widely believed to be the hallmark of early-stage atherosclerosis. In addition, hypercholesterolemia is considered a major risk factor for development of atherosclerosis and is associated with impaired flow-induced dilation. However, the mechanism by which elevated cholesterol levels leads to decreased production of NO is unclear. NO is released in response to shear stress and agonist-evoked changes in intracellular calcium. Although calcium signaling is complex, we have previously shown that NO production by endothelial nitric oxide synthase (eNOS) is preferentially activated by calcium influx via store-operated channels. We hypothesized that cholesterol enrichment altered this signaling pathway (known as capacitive calcium entry; CCE) ultimately leading to decreased NO. Our results show that cholesterol enrichment abolished ATP-induced eNOS phosphorylation and attenuated the calcium response by the preferential inhibition of CCE. Furthermore, cholesterol enrichment also inhibited shear stress-induced NO production and eNOS phosporylation, consistent with our previous results showing a significant role for ATP autocrine stimulation and subsequent activation of CCE in the endothelial flow response.

Nitrite-Mediated Hypoxic Vasodilation Predicted from Mathematical Modeling and Quantified from in Vivo Studies in Rat Mesentery.

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 >10 mM 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.

A mathematical model for the role of N2O3 in enhancing nitric oxide bioavailability following nitrite infusion.

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 (N2O3) acts as a stable intermediate for preserving NO. Our simulations predict that with hypoxia and moderate nitrite concentrations, the N2O3 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.

Mathematical model for shear stress dependent NO and adenine nucleotide production from endothelial cells.

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)).

Shear stress-induced NO production is dependent on ATP autocrine signaling and capacitative calcium entry.

Flow-induced production of nitric oxide (NO) by endothelial cells plays a fundamental role in vascular homeostasis. However, the mechanisms by which shear stress activates NO production remain unclear due in part to limitations in measuring NO, especially under flow conditions. Shear stress elicits the release of ATP, but the relative contribution of autocrine stimulation by ATP to flow-induced NO production has not been established. Furthermore, the importance of calcium in shear stress-induced NO production remains controversial, and in particular the role of capacitive calcium entry (CCE) has yet to be determined. We have utilized our unique NO measurement device to investigate the role of ATP autocrine signaling and CCE in shear stress-induced NO production. We found that endogenously released ATP and downstream activation of purinergic receptors and CCE plays a significant role in shear stress-induced NO production. ATP-induced eNOS phophorylation under static conditions is also dependent on CCE. Inhibition of protein kinase C significantly inhibited eNOS phosphorylation and the calcium response. To our knowledge, we are the first to report on the role of CCE in the mechanism of acute shear stress-induced NO response. In addition, our work highlights the importance of ATP autocrine signaling in shear stress-induced NO production.

Nitric oxide signaling in the microcirculation.

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.

Modeling O2-dependent effects of nitrite reductase activity in blood and tissue on coupled NO and O2 transport around arterioles.

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.

3D network model of NO transport in tissue.

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.

Direct, real-time measurement of shear stress-induced nitric oxide produced from endothelial cells in vitro.

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.

Food security and population health and well being.

Food security is an important element in the multi-factorial systems analysis of health and well being. The interaction between food supply and other important factors making up the system can shed light on individual and population health. A critical analysis of the health system must also include consideration of disparity in food security since it represents one of the most dramatic indicators of economic and health inequality. A large fraction of the world's population--particularly in Sub-Saharan Africa and in South East Asia--is chronically hungry. Distributing food commodities alone does not appear to significantly reduce global food insecurity. In addition, promoting agricultural development, economic growth, and education assistance is needed in order to mitigate the underlying causes of chronic hunger, and in turn improve health and well being.

A Food in Health Security (FIHS) platform in the Asia-Pacific Region: the way forward.

The advent of multiple global crises, especially those of climate change, economics, energy, water, food and health evident in 2008, is of considerable moment to those who are suffering their consequences and for those with responsibility and interest in the systems affected. A coalition of parties in the Asia Pacific Region who work in the food and health systems met in August, 2009 in Taiwan and instigated a Food in Health Security (FIHS) Network which might join with other like-minded networks in and beyond the region. Sustainable health has many dimensions, among which food and nutrition is often neglected; there is a wide spectrum of nutritionally-related disorders. Malnutrition remains the global concern for agricultural research and development scientists and linkage with the health sector is key to progress. The disconnect between agricultural and health sectors negatively impacts consumer nutrition and health. Ethical and equity affect food and health systems. Food and health security is attainable only when the underlying social inequities are addressed; it is an ethical issue as reflected in the UN Universal declaration of Human Rights which includes the right to food for health and well-being. Food and health security are part of the larger security agenda and merit corresponding attention. Policy recommendations with immediacy are greater investment in combined food and health research; an Asia Pacific security agenda which emphasizes planetary, human, health and food security as relevant to traditional defence security; and community and household security measures which include maternal literacy, communication technology and entrepreneurial opportunity.

Transport-dependent calcium signaling in spatially segregated cellular caveolar domains.

We developed a two-dimensional model of transport-dependent intracellular calcium signaling in endothelial cells (ECs). Our purpose was to evaluate the effects of spatial colocalization of endothelial nitric oxide synthase (eNOS) and capacitative calcium entry (CCE) channels in caveolae on eNOS activation in response to ATP. Caveolae are specialized microdomains of the plasma membrane that contain a variety of signaling molecules to optimize their interactions and regulate their activity. In ECs, these molecules include CCE channels and eNOS. To achieve a quantitative understanding of the mechanisms of microdomain calcium signaling and the preferential sensitivity of eNOS to calcium entering the cell through CCE channels, we constructed a mathematical model incorporating the cell morphology and cellular physiological processes. The model predicts that the spatial segregation of calcium channels in ECs can create transport-dependent sharp gradients in calcium concentration within the cell. The calcium concentration gradient is affected by channel density and cell geometry. This transport-dependent calcium signaling specificity effect is enhanced in ECs by increasing the spatial segregation of the caveolar signaling domains. Our simulation significantly advances the understanding of how Ca2+, despite its many potential actions, can mediate selective activation of signaling pathways. We show that diffusion-limited calcium transport allows functional compartmentalization of signaling pathways based on the spatial arrangements of Ca2+ sources and targets.