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Nitric oxide (NO) production by endothelial nitric oxide synthase (eNOS) inhibits platelet and leukocyte adhesion while promoting vasorelaxation in smooth muscle cells. Dysfunctional regulation of eNOS is a hallmark of various vascular pathologies, notably atherosclerosis, often associated with areas of low shear stress on endothelial cells (ECs). While the link between EC morphology and local hemodynamics is acknowledged, the specific impact of EC morphology on eNOS regulation remains unclear. Morphological differences between elongated, aligned ECs and polygonal, randomly oriented ECs correspond to variations in focal adhesion and cytoskeletal organization, suggesting differing levels of cytoskeletal prestress. However, the functional outcomes of cytoskeletal prestress, particularly in the absence of shear stress, are not extensively studied in ECs. Some evidence suggests that elongated ECs exhibit decreased immunogenicity and enhanced NO production. This study aims to elucidate the signaling pathways governing VEGF-stimulated eNOS regulation in the aligned EC phenotype characterized by elongated and aligned cells within a monolayer. Using anisotropic topographic cues, bovine aortic endothelial cells (BAECs) were elongated and aligned, followed by VEGF treatment in the presence or absence of cytoskeletal tension inhibitors. Phosphorylation of eNOS ser1179, AKT ser437 and FAK Tyr397 in response to VEGF challenge were significantly heightened in aligned ECs compared to unaligned ECs. Moreover this response proved to be robustly tied to cytoskeletal tension as evinced by the abrogation of responses in the presence of the myosin II ATPase inhibitor, blebbistatin. Notably, this work demonstrates for the first time the reliance on FAK phosphorylation in VEGF-mediated eNOS activation and the comparatively greater contribution of the cytoskeletal machinery in propagating VEGF-eNOS signaling in aligned and elongated ECs. This research underscores the importance of utilizing appropriate vascular models in drug development and sheds light on potential mechanisms underlying vascular function and pathology that can help inform vascular graft design., (© 2024 Wiley Periodicals LLC.)

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.)

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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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.)

Introduction: Colocalization of endothelial nitric oxide synthase (eNOS) and capacitative Ca 2+ entry (CCE) channels in microdomains such as cavaeolae in endothelial cells (ECs) has been shown to significantly affect intracellular Ca 2+ 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 Ca 2+ mobilization, and eNOS activation to investigate the effects of spatial colocalization of plasma membrane eNOS and CCE channels on Ca 2+ 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 Ca 2+ compared to the average cytosolic Ca 2+ . Ca 2+ buffers with lower or no mobility further enhanced Ca 2+ 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., Competing Interests: Disclosures: Kenneth Barbee, Jaimit Parikh, Yien Liu, Donald Buerk, and Dov Jaron declare that they have no conflicts of interest.

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% O 2 (normoxia) or zero O 2 (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 O 2 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, PO 2 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.

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.)

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 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.)

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.

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.

Thermal plasma is a valued tool in surgery for its coagulative and ablative properties. We suggested through in vitro studies that nonthermal plasma can sterilize tissues, inactive pathogens, promote coagulation, and potentiate wound healing. The present research was undertaken to study acute toxicity in porcine skin tissues. We demonstrate that floating electrode-discharge barrier discharge (FE-DBD) nonthermal plasma is electrically safe to apply to living organisms for short periods. We investigated the effects of FE-DBD plasma on Yorkshire pigs on intact and wounded skin immediately after treatment or 24h posttreatment. Macroscopic or microscopic histological changes were identified using histological and immunohistochemical techniques. The changes were classified into four groups for intact skin: normal features, minimal changes or congestive changes, epidermal layer damage, and full burn and into three groups for wounded skin: normal, clot or scab, and full burn-like features. Immunohistochemical staining for laminin layer integrity showed compromise over time. A marker for double-stranded DNA breaks, γ-H2AX, increased over plasma-exposure time. These findings identified a threshold for plasma exposure of up to 900s at low power and <120s at high power. Nonthermal FE-DBD plasma can be considered safe for future studies of external use under these threshold conditions for evaluation of sterilization, coagulation, and wound healing., (Copyright © 2013 Elsevier Inc. All rights reserved.)

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.)

Diffuse axonal injury (DAI), a major component of traumatic brain injury, is characterized by a sequence of neurochemical reactions initiated at the time of trauma and resulting in axonal degeneration and cell death. Calcium influx through mechanically induced axolemmal pores and subsequent activation of calpains are thought to be responsible for the cytoskeletal damage leading to impaired axonal transport. Focal disruption of cytoskeleton accompanied by the accumulation of transported membranous cargo leads to axonal beading which is the characteristic morphology of DAI. By applying fluid shear stress injury on cultured primary neurons, acute calcium (Ca(2+)) and calpain responses of axons to mechanical trauma were investigated. Intracellular Ca(2+) concentration ([Ca(2+)](i)) shows a steady increase following injury that can be blocked by sealing membrane pores with Poloxamer 188 and by chelating intra- or extracellular Ca(2+). Calpain activity increases in response to mechanical injury and this increase depends on Ca(2+) availability and on axolemmal permeability. Both the [Ca(2+)](i) increase and calpain activity exhibit focal peaks along the axons which co-localize with mitochondria and predict future axonal bead locations. These findings suggest that mechanoporation may be the initiating mechanism resulting in ensuing calcium fluxes and subsequent calpain activity and that post-injury membrane repair may be a valid therapeutic approach for acute intervention in DAI.

Focal axonal beading and focal disruption of microtubule structure are characteristic to traumatic axonal injury. We have recently reproduced these morphological and structural changes in our in vitro model system [D. Kilinc, G. Gallo, K.A. Barbee, Mechanically induced membrane poration causes axonal beading and localized cytoskeletal damage, Exp. Neurol. 212 (2008) 422-430]. In order to measure bead formation objectively, an observer-independent quantification of beading was necessary. In addition, a quantitative measure for the extent of co-localization of axonal beads and microtubule disruptions was required to establish a causal relationship between focal cytoskeletal damage and bead formation. In this paper we describe Matlab-based, interactive image analysis programs for axonal beading quantification and co-localization analysis. Injury-induced increases in the axonal beading could be successfully detected using the bead analysis program.

Diffuse axonal injury (DAI), a major component of traumatic brain injury, is a manifestation of microstructural cellular trauma and various ensuing neurochemical reactions that leads to secondary neuronal death. DAI is suggested to result from the initial increase in the membrane permeability caused by the mechanical forces acting on the axons. Permeability increases disturb ion balance and lead to cytoskeletal disruption resulting in the impairment of axonal transport. We present an in vitro model that reproduces important features of in vivo DAI such as membrane permeability changes, focal disruption of microtubules, impaired axonal transport, and focal accumulation of organelles. We induced fluid shear stress injury (FSSI) on cultured primary chick forebrain neurons and characterized the resulting structural and morphological changes. In addition, we tested the effect of Poloxamer 188 (P188), a tri-block co-polymer that is known to promote resealing membrane pores. We found that FSSI induces mechanoporation that leads to axonal bead formation, the "hallmark" morphology of DAI. Beads contained accumulated mitochondria and co-localized with focal microtubule disruptions, also a characteristic of DAI. Post-injury P188 treatment prevented FSSI-induced membrane permeability changes and reduced axonal beading to control levels. These results indicate that acute mechanoporation of axons in response to injury is a necessary condition for subsequent axonal pathology, suggesting that membrane integrity is a potential target for therapeutic interventions. P188 provides neuroprotection via resealing the plasma membrane following injury and prevents focal disruption of microtubules and axonal bead formation.

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.

The drug-eluting stent's increasingly frequent occurrence late stage thrombosis have created a need for new strategies for intervention in coronary artery disease. This paper demonstrates further development of our minimally invasive, targeted drug delivery system that uses induced magnetism to administer repeatable and patient specific dosages of therapeutic agents to specific sites in the human body. Our first aim is the use of magnetizable stents for the prevention and treatment of coronary restenosis; however, future applications include the targeting of tumors, vascular defects, and other localized pathologies. Future doses can be administered to the same site by intravenous injection. This implant-based drug delivery system functions by placement of a weakly magnetizable stent or implant at precise locations in the cardiovascular system, followed by the delivery of magnetically susceptible drug carriers. The stents are capable of applying high local magnetic field gradients within the body, while only exposing the body to a modest external field. The local gradients created within the blood vessel create the forces needed to attract and hold drug-containing magnetic nanoparticles at the implant site. Once these particles are captured, they are capable of delivering therapeutic agents such as antineoplastics, radioactivity, or biological cells.

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 cell adhesion process and the molecular interactions that determine its kinetics were investigated using a thickness shear mode (TSM) sensor. The goal of this study was to correlate sensor readings with the progression of cell adhesion. In particular, the specific effects of receptor-mediated adhesion, the glycocalyx, and surface charge on initial cell-surface attachment and steady-state adhesion of endothelial cells were investigated. We found a strong correlation between resistance changes (DeltaR) and the development of cell adhesion strength by comparing the sensor readings with independently assessed cell adhesion. The result showed that integrin binding determines the kinetics of initial cell attachment while heparan sulfate proteoglycan (HSPG) modulates steady-state adhesion strength. Coating the sensor surface with the positively charged poly-d-lysine (PDL) enhanced the initial interaction with substratum. These data confirm our current understanding of the contribution of these three phenomena to the adhesion process. The real-time monitoring capability of this technique with high temporal resolution provides more detailed information on the kinetics of the different stages of the adhesion process. This technique has the potential to facilitate the evaluation of biomaterials and surface treatments used for implants and tissue-engineering scaffolds for their bioactive effects on the cell adhesion process.

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.

The tissues of the body are continually subjected to mechanical stimulation by external forces, such as gravity, and internally generated forces, such as the pumping of blood or muscle contraction. Within a physiological range, the forces elicit adaptive responses acutely (to rapidly alter function) and chronically (to remodel tissue structure to optimize load-bearing capabilities). When the forces exceed certain thresholds, injury results. To understand the mechanisms of mechanical injury at the cellular level, we must analyze the structural response of the cell to various modes of deformation and examine the biological consequences of the structural alterations caused by the trauma. This chapter reviews the mechanics of cell membrane deformation and failure. Evidence for the strain-rate-dependent, transient disruption of cell membranes, or mechanoporation, is presented for a variety of cell types. The complex interactions between the structural damage and the biological sequelae are illustrated using clinically relevant forms of cell injury. Finally, novel therapeutic approaches targeting membrane integrity are described.

A method is described for the manufacture of a microelectrode array for chronic, multichannel, single neuron recording. The ceramic-based, multisite electrode array has four recording sites patterned onto a ceramic shaft the size of a single typical microwire electrode. The sites and connecting wires are applied to the ceramic substrate using a reverse photolithographic procedure. Recording sites (22 x 80 microm) are separated by 200 microm along the shaft. A layer of alumina insulation is applied over the whole array (exclusive of recording sites) by ion-beam assisted deposition. These arrays were capable of recording single neuron activity from each of their recording sites for at least three weeks during chronic implantation in the somatosensory cortex of rats, and several sites had recordings that lasted for more than 8 weeks. The vertical arrangement of the recording sites on these electrodes is ideal for simultaneously recording across the different layers of brain areas such as the cerebral cortex and hippocampus in chronic preparations.

The endothelium of blood vessels presents a wavy surface to the flowing blood. The subcellular distribution of shear stress depends on the shape and orientation of the cells and on their spatial arrangement within the monolayer. By studying details of the distribution of stress at this scale and the morphological responses that serve to modify the distribution, we can gain insight into the physical mechanisms by which the cell senses its fluid mechanical environment. The rapidly growing body of evidence indicates that endothelial cells discriminate between subtle variations in the exact loading conditions including differences in temporal and spatial gradients of shear stress, steady and pulsatile laminar flow, and laminar and turbulent flows. While in a few studies the effects of these individual flow characteristics have been carefully isolated, it is difficult to assess the relative importance of any one parameter. To interpret the relationships between isolated flow characteristics or the integrated effects of combined loading conditions and the biochemical signaling events that mediate the cell response, a full stress analysis of the cell is needed. The microscopic distribution of shear stress acting upon the cell surface provides the boundary condition for such an analysis. Experimental and analytical tools are being developed to assess the stress distribution throughout the cellular structures that might be involved in mechanotransduction.