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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.

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

Endothelial cell dysfunction is the hallmark of every cardiovascular disease/condition, including atherosclerosis and ischemia/reperfusion injury. Fluid shear stress acting on the vascular endothelium is known to regulate cell homeostasis. Altered hemodynamics is thought to play a causative role in endothelial dysfunction. The dysfunction is associated with/preceded by mitochondrial oxidative stress. Studies by our group and others have shown that the form and/or function of the mitochondrial network are affected when endothelial cells are exposed to shear stress in the absence or presence of additional physicochemical stimuli. The present review will summarize the current knowledge on the interconnections among intracellular Ca2+ - nitric oxide - mitochondrial reactive oxygen species, mitochondrial fusion/fission, autophagy/mitophagy, and cell apoptosis vs. survival. More specifically, it will list the evidence on potential regulation of the above intracellular species and processes by the fluid shear stress acting on the endothelium under either physiological flow conditions or during reperfusion (following a period of ischemia). Understanding how the local hemodynamics affects mitochondrial physiology and the cell redox state may lead to development of novel therapeutic strategies for prevention or treatment of the endothelial dysfunction and, hence, of cardiovascular disease.

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.

The mitochondrial network is dynamic with conformations that vary between a tubular continuum and a fragmented state. The equilibrium between mitochondrial fusion/fission, as well as the organelle motility, determine network morphology and ultimately mitochondrial/cell function. Network morphology has been linked with the energy state in different cell types. In this study, we examined how bioenergetic factors affect mitochondrial dynamics/motility in cultured vascular endothelial cells (ECs). ECs were transduced with mitochondria-targeted green fluorescent protein (mito-GFP) and exposed to inhibitors of oxidative phosphorylation (OXPHOS) or ATP synthesis. Time-lapse fluorescence videos were acquired and a mathematical program that calculates size and speed of each mitochondrial object at each time frame was developed. Our data showed that inner mitochondrial membrane potential (ΔΨ(m)), ATP produced by glycolysis, and, to a lesser degree, ATP produced by mitochondria are critical for maintaining the mitochondrial network, and different metabolic stresses induce distinct morphological patterns (e.g., mitochondrial depolarization is necessary for "donut" formation). Mitochondrial movement, characterized by Brownian diffusion with occasional bursts in displacement magnitude, was inhibited under the same conditions that resulted in increased fission. Hence, imaging/mathematical analysis shed light on the relationship between bioenergetics and mitochondrial network morphology; the latter may determine EC survival under metabolic stress.

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.

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.

Antibody-mediated rejection is characterized by deposits of complement (C) C4 and C3 split products on endothelial cells (ECs). C3 split products are critical mechanistically and diagnostically because they are deposited in amplified quantities, bind covalently to ECs and act as ligands for leukocytes. This study was designed to determine whether cultured vascular human ECs could clear covalently bound C3 split products from their surface. An immunoglobulin M (IgM) antibody against beta(2)-microglobulin of major histocompatibility complex class I antigens was used to activate C in human serum. Some cells were exposed to hypoxia/reoxygenation and/or interleukin 1beta (IL-1beta) prior to incubation with antibody. C3b/iC3b and C3d deposition on the cell surface was measured by flow cytometry. Incubation with antibody followed by human serum caused a dose-dependent deposition of C3b/iC3b and C3d. Over half of deposited C3b/iC3b and one-third of C3d were cleared from the cell surface during a 3-7-h incubation period with human serum. Neither hypoxia/reoxygenation nor IL-1beta further increased the deposition of C3b/iC3b and C3d, and only slightly modulated their rates of clearance. In summary, human ECs rapidly clear iC3b and C3d from their surface. This finding may have important diagnostic and mechanistic implications to transplantation because C3d is used as a marker of antibody-mediated rejection.

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

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.

The presence of platelets increases the fibrinolytic resistance of thrombi. When activated, platelets secrete PAI-1 and /spl alpha//sub 2/-antiplasmin and induce clot retraction. The search for potent antiplatelet agents has led to inhibitors of the platelet receptor GP IIb/IIIa, one of which is ReoPro (c7E3 Fab; abciximab). The effect of ReoPro on rt-PA-induced fibrinolysis was studied using an in vitro whole blood reperfusion model. When ReoPro was administered simultaneously with rt-PA at a wall shear rate of 500 s/sup -1/, it had no effect on the rate of fibrinolysis. rt-PA was found to lyse preformed gel-filtered platelet-fibrin substrates containing fixed platelets quicker than those containing normal platelets. ReoPro was shown to block platelet-fibrin interactions under arterial flow. Hence, the authors hypothesized (and are currently testing) that a combination of a GP IIb/IIIa inhibitor preincubated with the substrate, and a fibrinolytic drug injected with the flow, may hasten lysis of platelet-fibrin substrates under well-defined hemodynamic conditions. ReoPro may prove a necessary adjunctive treatment during thrombolytic therapy of platelet-rich thrombi.

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.

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.