Search

Calcium (Ca2+) signaling is critical for cell function and cell survival. Mitochondria play a major role in regulating the intracellular Ca2+ concentration ([Ca2+]i). Mitochondrial Ca2+ uptake is an important determinant of cell fate and governs respiration, mitophagy/autophagy, and the mitochondrial pathway of apoptosis. Mitochondrial Ca2+ uptake occurs via the mitochondrial Ca2+ uniporter (MCU) complex. This review summarizes the present knowledge on the function of MCU complex, regulation of MCU channel, and the role of MCU in Ca2+ homeostasis and human disease pathogenesis. The channel core consists of four MCU subunits and essential MCU regulators (EMRE). Regulatory proteins that interact with them include mitochondrial Ca2+ uptake 1/2 (MICU1/2), MCU dominant-negative β-subunit (MCUb), MCU regulator 1 (MCUR1), and solute carrier 25A23 (SLC25A23). In addition to these proteins, cardiolipin, a mitochondrial membrane-specific phospholipid, has been shown to interact with the channel core. The dynamic interplay between the core and regulatory proteins modulates MCU channel activity after sensing local changes in [Ca2+]i, reactive oxygen species, and other environmental factors. Here, we highlight the structural details of the human MCU heteromeric assemblies and their known roles in regulating mitochondrial Ca2+ homeostasis. MCU dysfunction has been shown to alter mitochondrial Ca2+ dynamics, in turn eliciting cell apoptosis. Changes in mitochondrial Ca2+ uptake have been implicated in pathological conditions affecting multiple organs, including the heart, skeletal muscle, and brain. However, our structural and functional knowledge of this vital protein complex remains incomplete, and understanding the precise role for MCU-mediated mitochondrial Ca2+ signaling in disease requires further research efforts.

Upon inositol trisphosphate (IP3) stimulation of non-excitable cells, including vascular endothelial cells, calcium (Ca2+) shuttling between the endoplasmic reticulum (ER) and mitochondria, facilitated by complexes called Mitochondria-Associated ER Membranes (MAMs), is known to play an important role in the occurrence of cytosolic Ca2+ concentration ([Ca2+]Cyt) oscillations. A mathematical compartmental closed-cell model of Ca2+ dynamics was developed that accounts for ER-mitochondria Ca2+ microdomains as the µd compartment (besides the cytosol, ER and mitochondria), Ca2+ influx to/efflux from each compartment and Ca2+ buffering. Varying the distribution of functional receptors in MAMs vs. the rest of ER/mitochondrial membranes, a parameter called the channel connectivity coefficient (to the µd), allowed for generation of [Ca2+]Cytoscillations driven by distinct mechanisms at various levels of IP3 stimulation. Oscillations could be initiated by the transient opening of IP3 receptors facing either the cytosol or the µd, and subsequent refilling of the respective compartment by Ca2+ efflux from the ER and/or the mitochondria. Only under conditions where the µd became the oscillation-driving compartment, silencing the Mitochondrial Ca2+ Uniporter led to oscillation inhibition. Thus, the model predicts that alternative mechanisms can yield [Ca2+]Cyt oscillations in non-excitable cells, and, under certain conditions, the ER-mitochondria µd can play a regulatory role.

Bovine aortic endothelial cells (ECs) respond to nitric oxide (NO) donors by activating the redox-sensitive NF-E2-related factor 2/antioxidant response element pathway and up-regulating heme oxygenase (HO)-1 expression. EC exposure to steady laminar shear stress causes a sustained increase in NO, a transient increase in reactive oxygen species (ROS), and activation of the HO-1 gene. Because steady laminar flow increases the mitochondrial superoxide (\documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{O}}_{2}^{\overline{.}}\end{equation*}\end{document}) production, we hypothesized that mitochondria-derived ROS play a role in shear-induced HO-1 expression. Flow (10 dynes/cm2, 6 h)-induced expression of HO-1 protein was abolished when BAECs were preincubated and sheared in the presence of either NG-nitro-l-arginine methyl ester or N-acetyl-l-cysteine, suggesting that either NO or ROS up-regulates HO-1. Ebselen and diphenylene iodonium blocked HO-1 expression, and uric acid had no effect. The mitochondrial electron transport chain inhibitors, myxothiazol, rotenone, or antimycin A, and the mitochondria-targeted antioxidant peptide, Szeto-Schiller (SS)-31, which scavenges \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{O}}_{2}^{\overline{.}}\end{equation*}\end{document}, hydrogen peroxide (H2O2), peroxynitrite, and hydroxyl radicals, markedly inhibited the increase in HO-1 expression. These data collectively suggest that mitochondrial H2O2 mediates the HO-1 induction. MitoSOX and 2′,7′-dichlorofluorescin (DCF) fluorescence showed that mitochondrial \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{O}}_{2}^{\overline{.}}\end{equation*}\end{document} levels and intracellular peroxides, respectively, are higher in sheared ECs compared with static controls and, in part, dependent on NO. SS-31 significantly inhibited both the shear-induced MitoSOX and DCF fluorescence signals. Either phosphatidylinositol 3-kinase or mitogen-activated protein kinase cascade inhibitors blocked the HO-1 induction. In conclusion, under shear, EC mitochondria-derived H2O2 diffuses to the cytosol, where it initiates oxidative signaling leading to HO-1 up-regulation and maintenance of the atheroprotective EC status.

Shear stress is known to stimulate an intracellular free calcium concentration ([Ca2+]i) response in vascular endothelial cells (ECs). [Ca2+]i is a key second messenger for signaling that leads to vasodilation and EC survival. Although it is accepted that the shear-induced [Ca2+]i response is, in part, due to Ca2+ release from the endoplasmic reticulum (ER), the role of mitochondria (second largest Ca2+ store) is unknown. We hypothesized that the mitochondria play a role in regulating [Ca2+]i in sheared ECs. Cultured ECs, loaded with a Ca2+-sensitive fluorophore, were exposed to physiological levels of shear stress. Shear stress elicited [Ca2+]i transients in a percentage of cells with a fraction of them displaying oscillations. Peak magnitudes, percentage of oscillating ECs, and oscillation frequencies depended on the shear level. [Ca2+]i transients/oscillations were present when experiments were conducted in Ca2+-free solution (plus lanthanum) but absent when ECs were treated with a phospholipase C inhibitor, suggesting that the ER inositol 1,4,5-trisphosphate receptor is responsible for the [Ca2+]i response. Either a mitochondrial uncoupler or an electron transport chain inhibitor, but not a mitochondrial ATP synthase inhibitor, prevented the occurrence of transients and especially inhibited the oscillations. Knockdown of the mitochondrial Ca2+ uniporter also inhibited the shear-induced [Ca2+]i transients/oscillations compared with controls. Hence, EC mitochondria, through Ca2+ uptake/release, regulate the temporal profile of shear-induced ER Ca2+ release. [Ca2+]i oscillation frequencies detected were within the range for activation of mechanoresponsive kinases and transcription factors, suggesting that dysfunctional EC mitochondria may contribute to cardiovascular disease by deregulating the shear-induced [Ca2+]i response.

Both in vivo models of ischemia/reperfusion and in vitro models of hypoxia (H)/reoxygenation (R) have demonstrated the crucial role of the Rac1-regulated NADPH oxidase in the production of injurious reactive oxygen species (ROS) by vascular endothelial cells (ECs). Since membrane lipid peroxidation has been established as one of the mechanisms leading to cell death, we examined lipid peroxidation in H/R-exposed cultured human umbilical vein ECs (HUVECs) and the role of Rac1 in this process. H (24 h at 1% O2)/R (5 min) caused an increase in intracellular ROS production compared to a normoxic control, as measured by dichlorofluorescin fluorescence. Nutrient deprivation (ND; 24 h), a component of H, was sufficient to induce a similar increase in ROS under normoxia. Either H(24 h)/R (2 h) or ND (24 h) induced increases in lipid peroxidation of similar magnitude as measured by flow cytometry of diphenyl-1-pyrenylphosphine-loaded HUVECs and Western blotting analysis of 4-hydroxy-2-nonenal-modified proteins in cell lysates. In cells infected with a control adenovirus, H (24 h)/R (2 h) and ND (24 h) resulted in increases in NADPH-dependent superoxide production by 5- and 9-fold, respectively, as measured by lucigenin chemiluminescence. Infection of HUVECs with an adenovirus that encodes the dominant-negative allele of Rac1 (Rac1N17) abolished these increases. Rac1N17 expression also suppressed the H/R- and ND-induced increases in lipid peroxidation. In conclusion, ROS generated via the Rac1-dependent pathway are major contributors to the H/R-induced lipid peroxidation in HUVECs, and ND is able to induce Rac1-dependent ROS production and lipid peroxidation of at least the same magnitude as H/R.

Production of reactive oxygen species (ROS) by ischemic tissue after ischemia-reperfusion (I/RP) is an important factor that contributes to tissue injury. The small GTPase Rac1 mediates the oxidative burst, and ROS act on signaling pathways involved in expression of inflammatory genes. Because there is evidence implicating monocytes in the pathogenesis of I/RP injury, our objective was to determine the molecular mechanisms that regulate adhesive interactions between monocytes and hypoxia-reoxygenation (H/RO)-exposed cultured endothelial cells (ECs). When U937 cells were perfused over human umbilical vein ECs at 1 dyn/cm2, H (1 h at 1% O2)/RO (13 h) significantly increased the fluxes of rolling and stably adherent U937 cells. Either EC treatment with the antioxidant pyrrolidine dithiocarbamate (PDTC) or infection with AdRac1N17, which results in expression of the dominant-negative form of Rac1, abolished H/RO-induced ROS production, attenuated rolling, and abolished stable adhesion of U937 cells to H/RO-exposed ECs. Infection with AdRac1N17 also abolished H/RO-induced upregulation of vascular cell adhesion molecule (VCAM)-1. In turn, blocking VCAM-1 abolished U937 cell stable adhesion and slightly increased rolling. We concluded that the Rac1-dependent ROS partially regulate rolling and exclusively regulate stable adhesion of monocytic cells to ECs after H/RO and that stable adhesion, but not rolling, is mediated by ROS-induced expression of VCAM-1.

Calcium (Ca 2+ ) transport by mitochondria is an important component of the cell Ca 2+ homeostasis machinery in metazoans. Ca 2+ uptake by mitochondria is a major determinant of bioenergetics and cell fate. Mitochondrial Ca 2+ uptake occurs via the mitochondrial Ca 2+ uniporter (MCU) complex, an inner mitochondrial membrane protein assembly consisting of the MCU Ca 2+ channel, as its core component, and the MCU complex regulatory/auxiliary proteins. In this review, we summarize the current knowledge on the molecular nature of the MCU complex and its regulation by intra- and extramitochondrial levels of divalent ions and reactive oxygen species (ROS). Intracellular Ca 2+ concentration ([Ca 2+ ] i ), mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) and mitochondrial ROS (mROS) are intricately coupled in regulating MCU activity. Here, we highlight the contribution of MCU activity to vascular endothelial cell (EC) function. Besides the ionic and oxidant regulation, ECs are continuously exposed to haemodynamic forces (either pulsatile or oscillatory fluid mechanical shear stresses, depending on the precise EC location within the arteries). Thus, we also propose an EC mechanotransduction-mediated regulation of MCU activity in the context of vascular physiology and atherosclerotic vascular disease.

During thrombolytic therapy and after recanalization is achieved, reduction in the volume of mural thrombi is desirable. Mural thrombi are known to contribute to rethrombosis and reocclusion. The lysis rate of mural thrombi has been demonstrated to increase with fluid flow in different experimental models, but the mechanisms responsible are unknown. An experimental and a theoretical study were developed to determine the contribution of outer convective transport to the lysis of mural fibrin clots. Normal human plasma containing recombinant tissue-type plasminogen activator (tPA; 0.5 μg/mL) was (re)perfused over mural fibrin clots with fluorescently labeled fibrin at low arterial, arterial, or higher wall shear stresses (4, 18, or 41 dyn/cm2, respectively) and lysis was monitored in real time. Flow accelerated lysis, but significantly only at the highest shear stress: The average lysis front velocity was 3 × 10−5 cm/s at 41 dyn/cm2 vs. almost half of that at the lower shear stresses. Confocal microscopy showed fibrin fibers dissolving only in a narrow region close to the surface when permeation velocity was predicted to be low. A heterogeneous transport-reaction finite element model was used to describe mural fibrinolysis. After scaling the effects of outer and inner convection, inner diffusion, and chemical reactions, a simplified inner diffusion/reaction model was used. Correlation to fibrin lysis data in purified systems dictated higher rates of plasmin(ogen) and tPA adsorption onto fibrin and a decreased catalytic rate of plasmin-mediated fibrin degradation, compared with published parameters. At each shear stress, the model predicted a temporal pattern of lysis of mural fibrin (similar to that observed experimentally), and protease accumulation in a narrow fibrin region and significant lysis inhibition by plasma α2-antiplasmin (according to the literature). Increasing outer convection accelerated mural fibrinolysis, but the model did not predict the big increase in lysis rate at the highest shear stress. At higher than arterial flows, additional mechanisms not accounted for in the current model, such as fibrin collapse at the fibrin front, may regulate the lysis of mural clots and determine the outcome of thrombolytic therapy. © 2002 John Wiley & Sons, Inc. Biotechnol Bioeng 77: 405–419, 2002; DOI 10.1002/bit.10127

Laminar shear stress activates NADPH oxidase in vascular endothelial cells (ECs), and the generated superoxide radicals (O2–·) are known to be involved in intercellular adhesion molecule (ICAM)-1 expression. In this study, the role of a glycosphingolipid (GSL), lactosylceramide (LacCer), as a second messenger in the shear-induced O2–· generation and ICAM-1 expression was examined. It is known that glucosylceramide synthase (GlcT-1) catalyzes the synthesis of glucosylceramide (GlcCer) from ceramide, and subsequently lactosylceramide synthase (GalT-2) synthesizes LacCer from GlcCer. We observed that exposing cultured human umbilical vein ECs (HUVECs) to fluid shear stress (20 dyn/cm2 for 30 min) activated GalT-2. Shear stress also increased EC O2–· generation, that peaked at 30 min, and surface ICAM-1 protein expression at 6 h post-shear. EC preincubation with the antioxidant N-acetylcysteine (NAC; 20 mM for 2 h) completely abolished the shear-induced O2–· production and significantly inhibited ICAM-1 expression. EC preincubation with D-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), an inhibitor of the GSL glycosyltransferases GlcT-1 and GalT-2, abrogated the shear-induced activation of GalT-2. D-PDMP also abolished the shear-induced O2–· production and ICAM-1 expression. We conclude that laminar shear stress activates GalT-2 to produce LacCer. In turn, LacCer activates NADPH oxidase, which produces O2–·, and O2–· mediates the shear-induced increase in ICAM-1 expression. Thus, LacCer may play an important role in hemodynamic force-induced pathological conditions, such as atherosclerosis and ischemia/reperfusion injury.

The shear-induced intracellular signal transduction pathway in vascular endothelial cells involves tyrosine phosphorylation and activation of mitogen-activated protein (MAP) kinase, which may be responsible for the sustained release of nitric oxide. MAP kinase is known to be activated by reactive oxygen species (ROS), such as H2O2, in several cell types. ROS production in ligand-stimulated nonphagocytic cells appears to require the participation of a Ras-related small GTP-binding protein, Rac1. We hypothesized that Rac1 might serve as a mediator for the effect of shear stress on MAP kinase activation. Exposure of bovine aortic endothelial cells to laminar shear stress of 20 dyn/cm2for 5–30 min stimulated total cellular and cytosolic tyrosine phosphorylation as well as tyrosine phosphorylation of MAP kinase. Treating endothelial cells with the antioxidants N-acetylcysteine and pyrrolidine dithiocarbamate inhibited in a dose-dependent manner the shear-stimulated increase in total cytosolic and, specifically, MAP kinase tyrosine phosphorylation. Hence, the onset of shear stress caused an enhanced generation of intracellular ROS, as evidenced by an oxidized protein detection kit, which were required for the shear-induced total cellular and MAP kinase tyrosine phosphorylation. Total cellular and MAP kinase tyrosine phosphorylation was completely blocked in sheared bovine aortic endothelial cells expressing a dominant negative Rac1 gene product (N17rac1). We concluded that the GTPase Rac1 mediates the shear-induced tyrosine phosphorylation of MAP kinase via regulation of the flow-dependent redox changes in endothelial cells in physiological and pathological circumstances.

An in vitro whole blood reperfusion model was employed to quantify: (a) initial rates of lysis of mural platelet deposits from flowing blood onto fibrin-coated surfaces and (b) plasmin-mediated consumption of plasma plasminogen and fibrinogen, by recombinant tissue-type plasminogen activator (rt-PA) and two t-PA variants, KHRR 296–299 AAAA (K-tPA) and T103N, N117Q, KHRR 296–299 AAAA (TNK-tPA), at wall shear rates of either 500 or 1000 s−1. K- and TNK-tPA are more fibrin-specific than rt-PA, and are also resistant to inactivation by plasminogen activator inhibitor−1 (PAI−1). At 500 s−1, no agent showed significant lysis of mural platelet deposits on fibrin, even at concentrations as high as 10 μ g/ml of blood. At 1000 s−1, each agent demonstrated a dose-dependent lysis of mural platelet deposits, due to plasmin-mediated lysis of the fibrin substrate (fibrinolysis). The local concentration of thrombolytic agents close to the fibrin-coated surface is probably higher than the concentration of released PAI−1 from the adherent and activated platelets. Hence, the initial rates of lysis achieved by K- and TNK-tPA were not significantly different from that by rt-PA, when each agent was tested at either 1 or 10 μ g/ml of blood. However, TNK-tPA, at 1 μ g/ml,> caused the most extensive lysis at the end of the 50 min reperfusion period (50% vs 29% and 17% by rt-PA and K-tPA, respectively). K- and TNK-tPA, at concentrations as high as 10 μ g/ml of blood, caused plasminogen activation that was controlled by the natural plasmin inhibitors, and, thus, no proteolytic degradation of plasma fibrinogen (fibrinogenolysis). On the contrary, rt-PA at 1 μ g/ml revealed slight fibrinogenolysis that became extensive at 10 μ g/ml. This study demonstrates the potential use of an in vitro model, that mimics the in vivo hemodynamic environment, in evaluating the performance of thrombolytic agents. The data suggest that: (a) adequate flow must accompany fibrinolysis for successful embolization, and (b) the TNK variant may lyse annular thrombi after recanalization, at least as efficiently as rt-PA does, while causing lesser defect of systemic hemostasis. © 1998 Biomedical Engineering Society. PAC98: 8745Hw, 8790+y, 8380Lz, 8715Kg

The effect of fibrinolytic agents on platelet adhesion onto insolubilized collagen type I was evaluated. Normal human whole blood samples were incubated with agents and perfused over collagen-coated surfaces in a parallel-plate flow chamber. Platelet adhesion and aggregation were analyzed by video microscopy and image processing. When blood was perfused at 1500/s, both streptokinase and urokinase, each at 500 U/ml, caused a significantly less normalized platelet deposition, compared with controls. At 480/s, platelet deposition was not different between controls and test samples. Inhibition of platelet deposition at high flow rates was partly due to inhibition of platelet adhesion. Both ristocetin- and ADP-induced platelet aggregation were inhibited in test samples. The agents caused proteolytic degradation of plasma fibrinogen, but no degradation of platelet glycoproteins Ib and IIb-IIIa (GPIb and GPIIb-IIIa) and of plasma von Willebrand factor in test samples prior to perfusion. Post-perfusion von Willebrand factor degradation was not found. Plasmin may cause functional changes to plasma proteins and/or platelet receptors, altering their adhesive properties under flow. At high shear, fibrinogen degradation products may interfere with GPIIb-IIIa binding to insolubilized von Willebrand factor, leading to decreased platelet adhesion. Inhibition of platelet adhesion by thrombolytic agents could help maintain vessel patency after recanalization in stenosed arteries. Publishers.

Ischemia (I)/reperfusion (RP)-induced endothelial cell (EC) injury is thought to occur due to mitochondrial reactive oxygen species (mtROS) production. MtROS have been implicated in mitochondrial fission. We determined whether cultured EC exposure to simulated I/RP causes morphological changes in the mitochondrial network, and the mechanisms behind those changes. Since shear stress results in nitric oxide (NO)-mediated endothelial mtROS generation, we simulated I/RP as hypoxia (H) followed by oxygenated flow over the ECs (shear stress of 10 dyne/cm2). By exposing ECs to shear stress, H, H/reoxygenation (RO) or simulated I/RP and employing mitotracker staining, the differential effects of changes in mechanical forces and/or O2 levels on the mitochondrial network were assessed. Static or sheared ECs maintained their mitochondrial network. H- or H/RO-exposed ECs underwent changes, but mitochondrial fission was significantly less compared to that in ECs exposed to I/RP. I/RP-induced fission was partially inhibited by antioxidants, a NO synthase inhibitor or an inhibitor of the fission protein dynamin-related protein 1 (Drp1), and was accompanied by Drp1 oligomerization and phosphorylation (Ser616). Hence, shear-induced NO, ROS (including mtROS), and Drp1 activation are responsible for mitochondrial fission in I/RP-exposed ECs, and excessive fission may be an underlying cause of EC dysfunction in postischemic hearts.

Two likely mechanisms for the initiation of arterial platelet thrombus formation under conditions of elevated fluid shear stresses are: (1) excessive adhesion and aggregation of platelets from rapidly flowing blood onto the exposed sub-endothelium of injured, atherosclerotic arteries; or (2) direct, fluid shear stress-induced aggregation of platelets in constricted arteries with intact endothelial cells. Mechanism (1) was simulated using a parallel plate flow chamber, fibrillar collagen type I-coated slides, and mepacrine-labeled (fluorescent) platelets in whole blood anticoagulated with citrate, hirudin, unfractionated porcine heparin, or low molecular weight heparin flowing for 1 to 2 minutes at wall shear rates of 100 to 3,000 seconds-1 (4 to 120 dynes/cm2). The precise sequence of interactions among von Willebrand factor (vWF), glycoprotein (GP)Ib, and GPIIb-IIIa during platelet adhesion and subsequent aggregation were resolved by direct real-time observation using a computerized epifluorescence video microscopy system. Adhesion at high shear rates was the result of the adsorption of large vWF multimers onto collagen and the binding of platelet GPIb to the insolubilized vWF. Aggregation occurred subsequently and required the binding of ligands, including vWF via its RGD binding domain, to GPIIb-IIIa. Mechanism (2) was modeled by producing shear stresses of 90 to 180 dynes/cm2 in a rotational cone and plate viscometer, which aggregates platelets from platelet-rich- plasma (PRP) anti-coagulated with citrate, hirudin, or either type of heparin in reactions that require large vWF multimers, Ca2+, adenosine diphosphate, and both GPIb and GPIIb-IIIa. Both vWF-mediated shear- aggregation in PRP and platelet-collagen adhesion in flowing whole blood (anticoagulated with citrate and hirudin) are inhibited by two potentially useful anti-arterial thrombotic agents: polymeric aurin tricarboxylic acid (ATA; 28.5 to 114 micrograms/mL), which binds to vWF and inhibits its attachment of GPIb, and a recombinant vWF fragment (rvWF445–733; 30 to 200 micrograms/mL) that binds to platelet GPIb (in the absence of any modulator) and blocks attachment of vWF multimers. Unfractionated heparin, but not low molecular weight heparin, apparently binds to rvWF445–733 and counteracts the inhibitory effects of the vWF fragment in vitro on shear-aggregation and platelet-collagen adhesion.

The effect of flow-induced shear stress on membrane K+ permeability was investigated by measuring86Rb+ efflux in cultured calf pulmonary artery endothelial cells. Cells were subjected to step changes in shear stress from 1 dyn/cm2 to 2.4, 4.8, or 10 dyn/cm2 in a parallel-plate flow chamber. Increasing shear stress produced a graded, transient increase in86Rb+ efflux which peaked within 1 min and subsequently declined rapidly toward pre-stimulus levels. Upon returning shear stress to 1 dyn/cm2,86Rb+ efflux initially decreased, but returned slowly to basal values. In contrast, application of bradykinin at a constant shear stress of 1 dyn/cm2 produced a transient increase in86Rb+ efflux that was followed by a sustained elevated phase during which time efflux gradually returned to pre-stimulus levels. In order to exclude the possibility that the transient increase in86Rb+ efflux with shear stress simply reflects a flow-dependent change in the washout of radiotracer, the transient convection-diffusion equation was solved using finite element simulation. When the flux of86Rb+ from the cell monolayer was assumed to be constant with time, the mathematical model predicted an increase in efflux rate coefficients upon step increases in flow that were only 7–19% of that observed experimentally. The numerical predictions correlated well with the experimentally obtained peaks when the flux of86Rb+ from the cell monolayer was simultaneously increased with flow to a new steady value. These simulations however, could not predict the transient nature of the response to increased shear stress. The results from the computer modeling suggest that the transient increase in86Rb+ efflux does not reflect a washout phenomenon and supports the hypothesis that shear stress produces a graded, transient increase in the K+ permeability of vascular endothelial cells.

Study of the mechanisms of monocyte adhesion initiating atheroslerotic lesions has engaged investigators for decades. Single-phase computational fluid dynamics (CFD) analyses fail to account for particulate migration. Consequently, inconsistencies arise when correlating adhesion with wall shear stress (WSS). The purpose of this paper is to present, to our knowledge, the first computational analysis of in vitro U937 monocyte-like human cell adhesion data using a coupled multiphase CFD-population balance adhesion model. The CFD model incorporates multiphase non-Newtonian hemodynamic models to compute the spatial distributions of freely flowing monocytes and WSSs in control volumes adjacent to the wall. Measurements of monocyte adhesion onto an E-selectin-coated flow model that included an idealized stenosis and an abrupt expansion were available from the literature. In this study, we develop a new monolayer population balance adhesion model, based on the widely accepted mechanism of ligand-receptor binding, coupled to the CFD results. The monolayer population balance model accounts for the interactions of freely flowing, rolling, and adhering monocytes with surfaces via first-order reactions, transport of rolling cells in the monolayer, and the concept of a WSS detachment threshold, clearly evident in the adhesion experiments. The new paradigm of coupling the multiphase hemodynamic CFD model with the proposed adhesion model is illustrated by determining and interpreting the model parameters for experimental datasets having Reynolds numbers of 100 and 140. The coupled multiphase CFD adhesion model is able to simultaneously predict the spatial variations in freely flowing monocytes, their adherent number density, and carrier fluid WSSs adjacent to ligand-coated flow cell surfaces.

Cultured vascular endothelial cell (EC) exposure to steady laminar shear stress results in peroxynitrite (ONOO−) formation intramitochondrially and inactivation of the electron transport chain. We examined whether the “hyperoxic state” of 21% O2, compared with more physiological O2tensions (Po2), increases the shear-induced nitric oxide (NO) synthesis and mitochondrial superoxide (O2·−) generation leading to ONOO−formation and suppression of respiration. Electron paramagnetic resonance oximetry was used to measure O2consumption rates of bovine aortic ECs sheared (10 dyn/cm2, 30 min) at 5%, 10%, or 21% O2or left static at 5% or 21% O2. Respiration was inhibited to a greater extent when ECs were sheared at 21% O2than at lower Po2or left static at different Po2. Flow in the presence of an endothelial NO synthase (eNOS) inhibitor or a ONOO−scavenger abolished the inhibitory effect. EC transfection with an adenovirus that expresses manganese superoxide dismutase in mitochondria, and not a control virus, blocked the inhibitory effect. Intracellular and mitochondrial O2·−production was higher in ECs sheared at 21% than at 5% O2, as determined by dihydroethidium and MitoSOX red fluorescence, respectively, and the latter was, at least in part, NO-dependent. Accumulation of NO metabolites in media of ECs sheared at 21% O2was modestly increased compared with ECs sheared at lower Po2, suggesting that eNOS activity may be higher at 21% O2. Hence, the hyperoxia of in vitro EC flow studies, via increased NO and mitochondrial O2·−production, leads to enhanced ONOO−formation intramitochondrially and suppression of respiration.

Exposure of vascular endothelial cells (ECs) to steady laminar shear stress activates the NF-E2-related factor 2 (Nrf2) which binds to the antioxidant response element (ARE) and upregulates the expression of several genes. The onset of shear is known to increase the EC reactive oxygen species (ROS) production, and oxidative stress can activate the ARE. ARE-regulated genes include phase 2 enzymes, such as glutathione-S-transferase (GST) and NAD(P)Hquinone oxidoreductase 1 (NQO1), and antioxidants, such as glutathione reductase (GR), glutathione peroxidase (GPx) and catalase. We examined how shear stress affects the antioxidant/phase 2 enzyme activities and whether ROS mediate these effects. ROS production, measured by dichlorofluorescin fluorescence, depended on level and time of shear exposure and EC origin, and was inhibited by either an endothelial nitric oxide synthase (eNOS) inhibitor or a superoxide dismutase (SOD) mimetic and peroxynitrite (ONOO-) scavenger. Shear stress (10 dynes/cm2, 16 h) significantly increased the NQO1 activity, did not change significantly the glutathione (GSH) content, and significantly decreased the GR, GPx, GST and catalase activities in human umbilical vein ECs. Either eNOS inhibition or superoxide radical (O 2 •- )/ONOO- scavenging differentially modulated the shear effects on enzyme activities suggesting that the intracellular redox status coordinates the shear-induced expression of cytoprotective genes. © Biomedical Engineering Society 2007.

There is evidence that nitric oxide (NO), superoxide (O2•−), and their associated reactive nitrogen species (RNS) produced by vascular endothelial cells (ECs) in response to hemodynamic forces play a role in cell signaling. NO is known to impair mitochondrial respiration. We sought to determine whether exposure of human umbilical vein ECs (HUVECs) to steady laminar shear stress and the resultant NO production modulate electron transport chain (ETC) enzymatic activities. The activities of respiratory complexes I, II/III, and IV were dependent on the presence of serum and growth factor supplement in the medium. EC exposure to steady laminar shear stress (10 dyn/cm2) resulted in a gradual inhibition of each of the complexes starting as early as 5 min from the flow onset and lasting up to 16 h. Ramp flow resulted in inhibition of the complexes similar to that of step flow. When ECs were sheared in the presence of the NO synthase inhibitor NG-nitro-l-arginine methyl ester (l-NAME; 100 μM), the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO; 100 μM), or the peroxynitrite (ONOO−) scavenger uric acid (UA; 50 μM), the flow-inhibitory effect on mitochondrial complexes was attenuated. In particular, l-NAME and UA abolished the flow effect on complex IV. Increased tyrosine nitration was observed in the mitochondria of sheared ECs, and UA blocked the shear-induced nitrotyrosine staining. In summary, shear stress induces mitochondrial RNS formation that inhibits the electron flux of the ETC at multiple sites. This may be a critical mechanism by which shear stress modulates EC signaling and function.

when flowing blood leukocytes interact with activated endothelium, the initial rolling event is mediated by selectins, which bind to their carbohydrate-based ligands, and the later firm adhesion event is mediated by integrins, which bind to cell adhesion molecules (CAMs). Specifically, neutrophils

A sharp increase in shear stress followed by steady laminar shear stress (step flow) imposed on the surface of macrovascular endothelial cells (ECs) causes changes to the metabolism, gene expression, protein synthesis, as well as to the reactive oxygen species (ROS) production. In this study, we monitored the shear-induced cytosolic ROS production in human umbilical vein ECs (HUVECs) and in human dermal microvascular ECs (HMVECs) using the oxidant-sensitive fluorescent probe 2,7-dichlorodihydrofluorescein diacetate (DCF-DA). HMVECs increased their ROS levels in response to shear stress in a time- and dose-dependent manner, when the shear stress was in the range of those encountered in postcapillary venules (2 or 4 dynes/cm/sup 2/). whereas HUVECs responded only to a higher shear stress tested (10 dynes/cm/sup 2/). It is known that many oxidants induce DCF-DA oxidation (hydra peroxides, ONOO/sup -/, NO/sub 2/ radical). In agreement to that, either an ecNOS inhibitor or a ONOO scavenger/SOD mimetic inhibited the shear-induced DCF signal. We are currently establishing a method to monitor lipid peroxidation (formation of lipid hydroperoxides) in the membranes of sheared ECs. Our plan is to investigate the contribution of cytosolic ROS to membranous lipid peroxidation and to intracellular shear-induced signaling.

Adhesion of platelets to the exposed extracellular matrix proteins at sites of vascular injury is partly regulated by the local fluid shear stress. Because the Leu33Pro (Pl(A)) polymorphism of integrin beta(3) confers only a modest increase in adhesion under static conditions, we used CHO and 293 cells expressing the Leu33 or Pro33 isoform of beta(3) in flow chamber experiments to test whether shear forces would alter the Pl(A) adhesive phenotype. We found that shear force augmented the Pro33-mediated enhanced adhesion to fibrinogen. This Pro33-dependent enhancement was aspirin-sensitive and was also observed on immobilized von Willebrand factor and cryoprecipitate, but not fibronectin. Thus, shear stress enhances the adhesive phenotype of the Pro33 cells to multiple physiologic substrates.

A model of tissue-type plasminogen activator (t-PA) mediated mural clot lysis has been developed. The model treats the clot as a porous two-phase mixture, and includes mechanisms of kinetics and fluid transport of chemical species, and mechanical property degradation as the fibrin dissolves. The model is consistent with experimental lysis of platelet-fibrin substrates in an in vitro whole blood reperfusion model. Lysis time is predicted to decrease with increasing wall shear stress, and increase with increasing plasminogen activator inhibitor (PAI)-1 concentration.

Monocyte adhesion to the endothelium depends on concentrations of receptors/ligands, local concentrations of chemoattractants, monocyte transport to the endothelial surface and hemodynamic forces. Monocyte adhesion to the inert surface of a three-dimensional perfusion model was shown to correlate inversely with wall shear stress, but was also affected by flow patterns which influenced the near-wall cell availability. We hypothesized that (a) under the same flow conditions, insolubilized E-selectin on the model's surface may mediate adhesive interactions at higher wall shear stresses, compared to an uncoated model, and (b) pulsatile flow may modify the adhesion profile obtained under steady flow. An axisymmetric flow model with a stenosis and a sudden expansion produced a range of wall shear stresses and a separated flow with recirculation and reattachment. Pre-activated U937 cells were perfused through the model under either steady ( Re =100, 140) or pulsatile ( Re mean =107) flow. The velocity field was characterized through computational fluid dynamics and validated by inert particle tracking. Surface E-selectin greatly increased cell adhesion in all regions at Re =100 and 140, compared to an uncoated model under the same flow conditions. In regions where the cells near the wall were abundant (taper and stenosis), adhesion to E-selectin correlated with the reciprocal of local wall shear stress when flow was steady. Pulsatile flow distributed the adherent cells more evenly throughout the coated model. Hence, characterizing both the local hemodynamics and the biological activity on the vessel wall is important in leukocyte adhesion.

Although the Fab fragment of the mouse-human chimeric anti-alphaIIbbeta3 (GP IIb/IIIa) monoclonal antibody (MoAb) c7E3 facilitates recombinant tissue-type plasminogen activator (rt-PA)-mediated thrombolysis, it is not clear whether this is due to inhibition of new clot formation and/or a direct effect on the lysis rate. We employed an in vitro flow (re)circulation model to investigate how c7E3 Fab affected (a) platelet adhesion to clotted fibrin substrates under laminar flow at wall shear rates of 100 or 500 s(-1) and (b) rt-PA-induced lysis of preformed mural platelet-fibrin substrates at 500 s(-1). c7E3 Fab dose-dependently (0.5-5 microg/ml) inhibited platelet adhesion from flowing whole blood onto fibrin substrates ( approximately 14 microm thick) at each wall shear rate. When at 5 min after the onset of flow, c7E3 Fab (0.1-10 microg/ml) and rt-PA (1 microg/ml) were coinjected in flowing blood, it was found that modest fibrinolysis caused major platelet release from fibrin substrates and there was no difference in the lysis rate in the presence of rt-PA + c7E3 Fab compared to rt-PA alone. Platelet pretreatment with c7E3 Fab (10 microg/ml) had no effect on the lysis rate of thin ( approximately 40 microm), and slightly delayed the lysis rate of thick (< 250 microm), platelet-fibrin substrates containing evenly dispersed platelets (10(9)/ml). When the platelets within thick platelet-fibrin substrates were organized in platelet-rich regions ("residual thrombi"), these substrates followed a nonuniform lysis pattern, where fibrin between the thrombi lysed first and the residual thrombi lysed at a slower rate. Platelet pretreatment with c7E3 Fab (10 microg/ml) abolished the formation of the lytic-resistant residual thrombi and the associated platelet-protected fibrin zones. Hence, treatment with c7E3 Fab has no direct effect on the rate of rt-PA-mediated lysis, but is expected to block platelet-fibrin interactions that lead to clot retraction, thus maintaining a fibrin architecture that is more susceptible to lysis.

The inhibitory effect of two novel antiplatelet agents, 1,10-bis[3-(N,N-diethylcarbamoyl)piperidino]decane dihydrobromide (G-110) and 1,6-bis[3-(N,N-diethylcarbamoyl)piperidino]hexane dihydrobromide (G-112), on platelet adhesion and subsequent aggregation on collagen-coated surface was evaluated under controlled flow. Glass coverslips coated with bovine fibrillar collagen type I were exposed to heparinized human whole blood that had been preincubated with either aqueous solutions of one of the two carbamoylpiperidine congeners or corresponding amounts (1.0-4.0 microliters/ml blood) of distilled water, at a wall shear rate of 1000 s-1, in a parallel-plate perfusion chamber. Epifluorescence video microscopy with a microphotometric measurement technique was used to visualize and quantify deposition of fluorescently-labeled platelets from flowing whole blood onto the collagen-coated surface. At concentrations of 100 and 200 microM, G-110 inhibited platelet accumulation by 30 +/- 9% (+/- S.E.) and 63 +/- 3% (+/- S.E.), respectively; while G-112 reduced platelet deposition by 19 +/- 3% (+/- S.E.) and 31 +/- 2% (+/- S.E.) at concentrations of 200 and 400 microM, respectively. Digital image processing techniques were used to analyze the dynamics of thrombus growth on the collagen-coated surfaces. It was found that the compounds reduced the rate of thrombus growth by impeding both surface coverage and the number of platelets per thrombus in a concentration-dependent manner. This study, together with others on related compounds GT-12, BPAT-117 and BPAT-143, corroborates the nature of pivotal features in the molecular structure of carbamoylpiperidine and nipecotoylpiperazine derivatives which enhance desirable antithrombotic effects, e.g. intramolecular distance between two tertiary amines (ring nitrogens) and levels of hydrophobicity.

Mitochondria-derived oxygen-free radical(s) are important mediators of oxidative cellular injury. It is widely hypothesized that excess NO enhances O 2 •− generated by mitochondria under certain pathological conditions. In the mitochondrial electron transport chain, succinate–cytochrome c reductase (SCR) catalyzes the electron transfer reaction from succinate to cytochrome c . To gain the insights into the molecular mechanism of how NO overproduction may mediate the oxygen-free radical generation by SCR, we employed isolated SCR, cardiac myoblast H9c2, and endothelial cells to study the interaction of NO with SCR in vitro and ex vivo. Under the conditions of enzyme turnover in the presence of NO donor (DEANO), SCR gained pro-oxidant function for generating hydroxyl radical as detected by EPR spin trapping using DEPMPO. The EPR signal associated with DEPMPO/ • OH adduct was nearly completely abolished in the presence of catalase or an iron chelator and partially inhibited by SOD, suggesting the involvement of the iron–H 2 O 2 -dependent Fenton reaction or O 2 •− -dependent Haber–Weiss mechanism. Direct EPR measurement of SCR at 77 K indicated the formation of a nonheme iron–NO complex, implying that electron leakage to molecular oxygen was enhanced at the FAD cofactor, and that excess NO predisposed SCR to produce • OH. In H9c2 cells, SCR-dependent oxygen-free radical generation was stimulated by NO released from DEANO or produced by the cells following exposure to hypoxia/reoxygenation. With shear exposure that led to overproduction of NO by the endothelium, SCR-mediated oxygen-free radical production was also detected in cultured vascular endothelial cells.