Mechanotransduction in endothelial cell migration

The migration of endothelial cells (ECs) plays an important role in vascular remodeling and regeneration. EC migration can be regulated by different mechanisms such as chemotaxis, haptotaxis, and mechanotaxis. This review will focus on fluid shear stress-induced mechanotransduction during EC migration. EC migration and mechanotransduction can be modulated by cytoskeleton, cell surface receptors such as integrins and proteoglycans, the chemical and physical properties of extracellular matrix (ECM) and cell-cell adhesions. The shear stress applied on the luminal surface of ECs can be sensed by cell membrane and associated receptor and transmitted throughout the cell to cell-ECM adhesions and cell-cell adhesions. As a result, shear stress induces directional migration of ECs by promoting lamellipodial protrusion and the formation of focal adhesions (FAs) at the front in the flow direction and the disassembly of FAs at the rear. Persistent EC migration in the flow direction can be driven by polarized activation of signaling molecules and the positive feedback loops constituted by Rho GTPases, cytoskeleton, and FAs at the leading edge. Furthermore, shear stress-induced EC migration can overcome the haptotaxis of ECs. Given the hemodynamic environment of the vascular system, mechanotransduction during EC migration has a significant impact on vascular development, angiogenesis, and vascular wound healing.     

Role of cell surface heparan sulfate proteoglycans in endothelial cell migration and mechanotransduction

Endothelial cell (EC) migration is critical in wound healing and angiogenesis. Fluid shear stress due to blood flow plays an important role in EC migration. However, the role of EC surface heparan sulfate proteoglycans (HSPGs) in EC adhesion, migration, and mechanotransduction is not well understood. Here, we investigated the effects of HSPG disruption on the adhesion, migration, and mechanotransduction of ECs cultured on fibronectin. We showed that disruption of HSPGs with heparinase decreased EC adhesion rate by 40% and adhesion strength by 33%. At the molecular level, HSPG disruption decreased stress fibers and the size of focal adhesions (FAs), increased filopodia formation, and enhanced EC migration. Under flow condition, heparinase treatment increased EC migration speed, but inhibited shear stress-induced directionality of EC migration and the recruitment of phosphorylated focal adhesion kinase in the flow direction, suggesting that HSPGs are important for sensing the direction of shear stress. In addition, decreasing cell adhesion by lowering fibronectin density enhanced EC migration under static and flow condition, but did not affect the directional migration of ECs under flow. Based on our results, we propose that HSPGs play dual roles as mechanotransducer on the EC surface: (1) HSPGs-matrix interaction on the abluminal surface regulates EC migration speed through an adhesion-dependent manner, and (2) HSPGs without binding to matrix (e.g., on the luminal surface) are involved in sensing the direction of flow through an adhesion-independent manner.     

Effects of morphological patterning on endothelial cell migration

The migration of vascular endothelial cells (ECs) plays an important role in vascular remodeling. Here we studied the effects of cell morphology on the migration of bovine aortic ECs by culturing cells on micropatterned strips of collagen matrix (60-, 30-, and 15-microm wide). The spreading areas of the cells on 15- and 30-microm wide strips were 30% lower than those on 60-microm wide strips and unpatterned collagen. The cells on 15-microm wide strips completely aligned in the direction of the strip, and had significantly lower shape index than those in all other groups. On strips of all widths, ECs tended to migrate in the direction of strips. ECs on 15-microm wide strips had highest speed, particularly in the direction of the strip. Vinculin staining showed that the leading edge of ECs on 15-microm wide strips had focal adhesions that were oriented with their lamellipodial protrusion and the direction of cell migration; this arrangement of the focal adhesions may promote EC migration. The present study provides direct evidence on the role of cell morphology in EC migration, and will help us to understand the mechanisms of EC migration during angiogenesis and wound healing.     

Rac1 mediates laminar shear stress-induced vascular endothelial cell migration

The migration of endothelial cells (ECs) plays an important role in vascular remodeling and regeneration. ECs are constantly subjected to shear stress resulting from blood flow and are able to convert mechanical stimuli into intracellular signals that affect cellular behaviors and functions. The aim of this study is to elucidate the effects of Rac1, which is the member of small G protein family, on EC migration under different laminar shear stress (5.56, 10.02, and 15.27 dyn/cm²). The cell migration distance under laminar shear stress increased significantly than that under the static culture condition. Especially, under relative high shear stress (15.27 dyn/cm²) there was a higher difference at 8 h (P < 0.01) and 2 h (P < 0.05) compared with static controls. RT-PCR results further showed increasing mRNA expression of Rac1 in ECs exposed to laminar shear stress than that exposed to static culture. Using plasmids encoding the wild-type (WT), an activated mutant (Q61L), and a dominant-negative mutant (T17N), plasmids encoding Rac1 were transfected into EA.hy 926 cells. The average net migration distance of Rac1Q61L group increased significantly, while Rac1T17N group decreased significantly in comparison with the static controls. These results indicated that Rac1 mediated shear stress-induced EC migration. Our findings conduce to elucidate the molecular mechanisms of EC migration induced by shear stress, which is expected to understand the pathophysiological basis of wound healing in health and diseases.     

A model for studying the effect of shear stress on interactions between vascular endothelial cells and smooth muscle cells

Vascular endothelial cells (ECs) are constantly subjected to blood flow-induced shear stress and the influences of neighboring smooth muscle cells (SMCs). In the present study, a coculture flow system was developed to study the effect of shear stress on EC-SMC interactions. ECs and SMCs were separated by a porous membrane with only the EC side subjected to the flow condition. When ECs were exposed to a shear stress of 12 dynes/cm2 for 24 h, the cocultured SMCs tended to orient perpendicularly to the flow direction. This perpendicular orientation of the cocultured SMCs to flow direction was not observed when ECs were exposed to a shear stress of 2 dynes/cm2. Under the static condition, long and parallel actin bundles were observed in the central regions of the cocultured SMCs, whereas the actin filaments localized mainly at the periphery of the cocultured ECs. After 24 h of flow application, the cocultured ECs displayed very long, well-organized, parallel actin stress fibers aligned with the flow direction in the central regions of the cells. Immunostaining of platelet endothelial cell adhesion molecule-1 confirmed the elongation and alignment of the cocultured ECs with the flow direction. Coculture with SMCs under static condition induced EC gene expressions of growth-related oncogene-alpha and monocyte chemotactic protein-1, and shear stress was found to abolish these SMC-induced gene expressions. Our results suggest that shear stress may serve as a down-regulator for the pathophysiologically relevant gene expression in ECs cocultured with SMCs.     

Shear-stress effect on mitochondrial membrane potential and albumin uptake in cultured endothelial cells

Endothelial cells (ECs) that line the inner surface of blood vessels are continuously exposed to shear stress induced by blood flow in vivo, and shear stress affects ATP-dependent macromolecular transport in ECs. However, the relationship between the ATP production and shear stress is still unclear. We, therefore, evaluated mitochondrial ATP synthesis activity in cultured endothelial cells exposed to shear stress, using a confocal laser scanning microscope (CLSM) and a mitochondrial membrane potential probe (5,5',6,6'-tetrachloro-1,1',3, 3'-tetraethyl-benzimidazolycarbocyanine iodide, JC-1). Low shear stress (10 dyn/cm(2)) increased mitochondrial membrane potential by 30%. On the contrary, high shear stress (60 dyn/cm(2)) decreased it by 20%. This observation was consistent with the ATP-dependent albumin uptake into endothelial cells. Our results indicate that ATP synthetic activity is related to the albumin uptake into endothelial cells.     

CXCR1 and CXCR2 are novel mechano-sensors mediating laminar shear stress-induced endothelial cell migration

The migration of endothelial cells (ECs) plays critical roles in vascular physiology and pathology. The receptors CXCR1 and CXCR2, known as G protein-coupled receptors which are essential for migratory response of ECs toward the shear stress-dependent CXCL8 (interleukin-8), are potential mechano-sensors for mechanotransduction of the hemodynamic forces. In present study, the mRNA and protein expression of CXCR1 and CXCR2 in EA.hy926 cells was detected by RT-PCR and Western blot analysis under three conditions of laminar shear stress (5.56, 10.02 and 15.27 dyn/cm(2)) respectively. Using a scratched-wound assay, the effects of CXCR1 and CXCR2 were assessed by the percentage of wound closure while CXCR1 and CXCR2 were functional blocked by the CXCL8 receptor antibodies. The results showed that the mRNA and protein expression of CXCR1 and CXCR2 was both upregulated by 5.56 dyn/cm(2) laminar shear stress, but was both downregulated by 15.27 dyn/cm(2). The wound closure was inhibited significantly while cells were treated with those antibodies in all the conditions. It was suggested that CXCR1 and CXCR2 are involved in mediating the laminar shear stress-induced EC migration. Taken together, these findings indicated that CXCR1 and CXCR2 are novel mechano-sensors mediating laminar shear stress-induced EC migration. Understanding this expanded mechanism of laminar shear stress-induced cell migration will provide novel molecular targets for therapeutic intervention in cancer and cardiovascular diseases.     

Effect of fluid force on vascular cell function

Endothelial cells (ECs) that line the inner surface of blood vessels are continuously exposed to fluid frictional force (shear stress) induced by blood flow, and shear stress affects the intracellular calcium ([Ca2+]i), which initiates cellular responses. Here, we studied the effect of long-term exposure of shear stress on [Ca2+]i responses in cultured ECs by using a confocal laser microscope and calcium indicator. At the initiation of shear stress of 20 dyn/cm2 (0 hr), 27% of the cells exhibited [Ca2+]i responses. This percentage gradually decreased with increasing exposure time, reaching about 4% after 24 hr of exposure. These data indicate that long-term shear-stress exposure affects [Ca2+]i responses in cultured ECs. Furthermore, we studied the effect of magnitude of shear stress on macromolecule uptake. For the low shear-stress, the uptake was enhanced, whereas the uptake was inhibited for higher shear-stress.     

Microtopography and flow modulate the direction of endothelial cell migration

The migration of vascular endothelial cells under flow can be modulated by the addition of chemical or mechanical stimuli. The aim of this study was to investigate how topographic cues derived from a substrate containing three-dimensional microtopography interact with fluid shear stress in directing endothelial cell migration. Subconfluent bovine aortic endothelial cells were seeded on fibronectin-coated poly(dimethylsiloxane) substrates patterned with a combinatorial array of parallel and orthogonal microgrooves ranging from 2 to 5 microm in width at a constant depth of 1 microm. During a 4-h time-lapse observation in the absence of flow, the majority of the prealigned cells migrated parallel to the grooves with the distribution of their focal adhesions (FAs) depending on the groove width. No change in this migratory pattern was observed after the cells were exposed to moderate shear stress (13.5 dyn/cm(2)), irrespective of groove direction with respect to flow. After 4-h exposure to high shear stress (58 dyn/cm(2)) parallel to the grooves, the cells continued to migrate in the direction of both grooves and flow. By contrast, when microgrooves were oriented perpendicular to flow, most cells migrated orthogonal to the grooves and downstream with flow. Despite the change in the migration direction of the cells under high shear stress, most FAs and actin microfilaments maintained their original alignment parallel to the grooves, suggesting that topographic cues were more effective than those derived from shear stress in guiding the orientation of cytoskeletal and adhesion proteins during the initial exposure to flow.     

Arterial shear stress regulates endothelial cell-directed migration, polarity, and morphology in confluent monolayers

Hemodynamic regulation of directional endothelial cell (EC) migration implies an essential role of shear stress in governing EC polarity. Shear stress induces reorientation of the microtubule organizing center toward the leading edge of migrating cells in a Cdc42-dependent manner. We have characterized the global patterns of EC migration in confluent monolayers as a function of shear stress direction and exogenous pleiotropic factors. Results demonstrate the presence of mitogenic factors significantly affects the flow-induced dynamics of movement by prolonging the onset of monolayer quiescence up to 4 days, but not shear stress-induced morphology. In conjunction with increased motility, exogenous growth factors contributed to the directed migration of ECs in the flow direction. ECs exposed to arterial flow in serum/growth factor-free media and then supplemented with growth factors rapidly increased directional migration to 85% of cells migrating in the direction of flow and induced an increase in the distance traveled with the flow direction. This response was modulated by the directionality of flow and inhibited by the expression of dominant-negative Par6, a major downstream effector of Cdc42-induced polarity. Shear stress-induced directed migratory polarity is modulated by exogenous growth factors and dependent on Par6 activity and shear stress direction.     

Laminar high shear stress up-regulates type IV collagen synthesis and down-regulates MMP-2 secretion in endothelium. A quantitative analysis

Remodeling of endothelial basement membrane is important in atherogenesis. Since little is known about the actual relationship between type IV collagen and matrix metalloprotease−2 (MMP-2) in endothelial cells (ECs) under shear stress by blood flow, we performed quantitative analysis for type IV collagen and MMP-2 in ECs under high shear stress. The mRNA of type IV collagen from ECs exposed to high shear stress (10 and 30 dyn/cm²) had a higher expression compared to ECs exposed to a static condition or low shear stress (3 dyn/cm²) (P < 0.01). ³H-proline uptake analysis and fluorography revealed a remarkable increase of type IV collagen under high shear stress (P < 0.01). In contrast, zymography revealed that exposing to high shear stress, however similar positivity was leveled in the intracellular MMP-2 in the control and high shear stress-exposed ECs, reduced the secretion of MMP-2 in ECs. The results of Northern blotting, gelatin zymography and monitoring the intracellular trafficking of GFP-labeled MMP-2 revealed that MMP-2 secretion by ECs was completely suppressed by high shear stress, but the intracellular mRNA expression, protein synthesis, and transport of MMP-2 were not affected. In conclusion, we suggest that high shear stress up-regulates type IV collagen synthesis and down-regulates MMP-2 secretion in ECs, which plays an important role in remodeling of the endothelial basement membrane and may suppress atherogenesis.     

Nitric oxide-dependent stimulation of endothelial cell proliferation by sustained high flow

Little is understood about endothelial cell (EC) responses to high flow, which mediate adaptive outward remodeling as well as cerebral aneurysm development. Opposite EC behaviors have been reported in vivo including cell loss during aneurysm initiation and cell proliferation during adaptive outward remodeling. This study aims at elucidating the EC growth response to elevated wall shear stress (WSS) and determining if nitric oxide (NO) is involved. A confluent EC monolayer was subjected to steady-state, laminar flow with WSS ranging from 15 to 100 dyn/cm(2) for 24 and 48 h. Cells oriented to the direction of the flow with a time course that varied with WSS. At 48 h, all cells were aligned with the flow. EC proliferation was examined using bromodeoxyuridine (BrdU) incorporation. The percentage of proliferating ECs rose linearly from 15 to 50 dyn/cm(2) to more than sixfold at 50-100 dyn/cm(2) compared with the accepted physiological baseline of 15-20 dyn/cm(2). In addition, terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling (TUNEL) staining revealed that apoptosis decreased with increasing WSS. These results demonstrate that high WSS stimulates EC proliferation and suppresses apoptosis. Furthermore, immunostaining revealed increased endothelial nitric oxide synthase (eNOS) production with increasing WSS. NOS inhibition with N(omega)-nitro-l-arginine methyl ester (l-NAME) drastically reduced the WSS-stimulated proliferation, indicating a critical role of NO production in the stimulation of EC proliferation by high WSS.     

Cooperative Effects of Matrix Stiffness and Fluid Shear Stress on Endothelial Cell Behavior

Arterial hemodynamic shear stress and blood vessel stiffening both significantly influence the arterial endothelial cell (EC) phenotype and atherosclerosis progression, and both have been shown to signal through cell-matrix adhesions. However, the cooperative effects of fluid shear stress and matrix stiffness on ECs remain unknown. To investigate these cooperative effects, we cultured bovine aortic ECs on hydrogels matching the elasticity of the intima of compliant, young, or stiff, aging arteries. The cells were then exposed to laminar fluid shear stress of 12 dyn/cm². Cells grown on more compliant matrices displayed increased elongation and tighter EC-cell junctions. Notably, cells cultured on more compliant substrates also showed decreased RhoA activation under laminar shear stress. Additionally, endothelial nitric oxide synthase and extracellular signal-regulated kinase phosphorylation in response to fluid shear stress occurred more rapidly in ECs cultured on more compliant substrates, and nitric oxide production was enhanced. Together, our results demonstrate that a signaling cross talk between stiffness and fluid shear stress exists within the vascular microenvironment, and, importantly, matrices mimicking young and healthy blood vessels can promote and augment the atheroprotective signals induced by fluid shear stress. These data suggest that targeting intimal stiffening and/or the EC response to intima stiffening clinically may improve vascular health.     

Vascular endothelial wound closure under shear stress: role of membrane fluidity and flow-sensitive ion channels.

Sufficiently rapid healing of vascular endothelium following injury is essential for preventing further pathological complications. Recent work suggests that fluid dynamic shear stress regulates endothelial cell (EC) wound closure. Changes in membrane fluidity and activation of flow-sensitive ion channels are among the most rapid endothelial responses to flow and are thought to play an important role in EC responsiveness to shear stress. The goal of the present study was to probe the role of these responses in bovine aortic EC (BAEC) wound closure under shear stress. BAEC monolayers were mechanically wounded and subsequently subjected to either "high" (19 dyn/cm(2)) or "low" (3 dyn/cm(2)) levels of steady shear stress. Image analysis was used to quantify cell migration and spreading under both flow and static control conditions. Our results demonstrate that, under static conditions, BAECs along both wound edges migrate at similar velocities to cover the wounded area. Low shear stress leads to significantly lower BAEC migration velocities, whereas high shear stress results in cells along the upstream edge of the wound migrating significantly more rapidly than those downstream. The data also show that reducing BAEC membrane fluidity by enriching the cell membrane with exogenous cholesterol significantly slows down both cell spreading and migration under flow and hence retards wound closure. Blocking flow-sensitive K and Cl channels reduces cell spreading under flow but has no impact on cell migration. These findings provide evidence that membrane fluidity and flow-sensitive ion channels play distinct roles in regulating EC wound closure under flow.     

Endothelial cell response to biomechanical forces under simulated vascular loading conditions

In vivo, endothelial cells (EC) are constantly exposed to the haemodynamic forces (HF) of pressure, wall shear stress and hoop stress. The main aim of this study was to design, create and validate a novel perfusion bioreactor capable of delivering shear stress and intravascular pressure to EC in vitro and to characterise their morphology, orientation and gene expression. Here we report the creation and validation of such a simulator and the dual application of pressure (120/60 mmHg) and low shear stress (5 dyn/cm(2)) to a monolayer of EC established on a non-compliant silicone tube. Under these conditions, EC elongated and realigned obliquely to the direction of applied shear stress in a time-dependent manner. Furthermore, randomly distributed F-actin microfilaments reorganised into long, dense stress fibres crossing the cells in a direction perpendicular to that of flow. Finally, combinatorial biomechanical conditioning of EC induced the expression of the inflammatory-associated E-selectin gene.     

Flow-induced focal adhesion remodeling mediated by local cytoskeletal stresses and reorganization

Cells respond to fluid shear stress through dynamic processes involving changes in actomyosin and other cytoskeletal stresses, remodeling of cell adhesions, and cytoskeleton reorganization. In this study we simultaneously measured focal adhesion dynamics and cytoskeletal stress and reorganization in MDCK cells under fluid shear stress. The measurements used co-expression of fluorescently labeled paxillin and force sensitive FRET probes of α-actinin. A shear stress of 0.74 dyn/cm² for 3 hours caused redistribution of cytoskeletal tension and significant focal adhesion remodeling. The fate of focal adhesions is determined by the stress state and stability of the linked actin stress fibers. In the interior of the cell, the mature focal adhesions disassembled within 35-40 min under flow and stress fibers disintegrated. Near the cell periphery, the focal adhesions anchoring the stress fibers perpendicular to the cell periphery disassembled, while focal adhesions associated with peripheral fibers sustained. The diminishing focal adhesions are coupled with local cytoskeletal stress release and actin stress fiber disassembly whereas sustaining peripheral focal adhesions are coupled with an increase in stress and enhancement of actin bundles. The results show that flow induced formation of peripheral actin bundles provides a favorable environment for focal adhesion remodeling along the cell periphery. Under such condition, new FAs were observed along the cell edge under flow. Our results suggest that the remodeling of FAs in epithelial cells under flow is orchestrated by actin cytoskeletal stress redistribution and structural reorganization.     

Fluid shear stress‐induced JNK activity leads to actin remodeling for cell alignment

Fluid shear stress (FSS) exerted on endothelial cell (EC) surfaces induces actin cytoskeleton remodeling through mechanotransduction. This study was designed to determine whether FSS activates Jun N‐terminal kinase (JNK), to examine the spatial and temporal distribution of active JNK relative to the actin cytoskeleton in ECs exposed to different FSS conditions, and to evaluate the effects of active JNK on actin realignment. Exposure to 15 and 20 dyn/cm² FSS induced higher activity levels of JNK than the lower 2 and 4 dyn/cm² flow conditions. At the higher FSS treatments, JNK activity increased with increasing exposure time, peaking 30 min after flow onset with an eightfold activity increase compared to cells in static culture. FSS‐induced phospho‐JNK co‐localized with actin filaments at cell peripheries, as well as with stress fibers. Pharmacologically blocking JNK activity altered FSS‐induced actin structure and distribution as a response to FSS. Our results indicate that FSS‐induced actin remodeling occurs in three phases, and that JNK plays a role in at least one, suggesting that this kinase activity is involved in mechanotransduction from the apical surface to the actin cytoskeleton in ECs. J. Cell. Physiol. 226: 110–121, 2010. © 2010 Wiley‐Liss, Inc.     

Flow-induced focal adhesion remodeling mediated by local cytoskeletal stresses and reorganization

Cells respond to fluid shear stress through dynamic processes involving changes in actomyosin and other cytoskeletal stresses, remodeling of cell adhesions, and cytoskeleton reorganization. In this study we simultaneously measured focal adhesion dynamics and cytoskeletal stress and reorganization in MDCK cells under fluid shear stress. The measurements used co-expression of fluorescently labeled paxillin and force sensitive FRET probes of α-actinin. A shear stress of 0.74 dyn/cm² for 3 hours caused redistribution of cytoskeletal tension and significant focal adhesion remodeling. The fate of focal adhesions is determined by the stress state and stability of the linked actin stress fibers. In the interior of the cell, the mature focal adhesions disassembled within 35-40 min under flow and stress fibers disintegrated. Near the cell periphery, the focal adhesions anchoring the stress fibers perpendicular to the cell periphery disassembled, while focal adhesions associated with peripheral fibers sustained. The diminishing focal adhesions are coupled with local cytoskeletal stress release and actin stress fiber disassembly whereas sustaining peripheral focal adhesions are coupled with an increase in stress and enhancement of actin bundles. The results show that flow induced formation of peripheral actin bundles provides a favorable environment for focal adhesion remodeling along the cell periphery. Under such condition, new FAs were observed along the cell edge under flow. Our results suggest that the remodeling of FAs in epithelial cells under flow is orchestrated by actin cytoskeletal stress redistribution and structural reorganization.     

Short-Term Shear Stress Induces Rapid Actin Dynamics in Living Endothelial Cells

Hemodynamic shear stress guides a variety of endothelial phenotype characteristics, including cell morphology, cytoskeletal structure, and gene expression profile. The sensing and processing of extracellular fluid forces may be mediated by mechanotransmission through the actin cytoskeleton network to intracellular locations of signal initiation. In this study, we identify rapid actin-mediated morphological changes in living subconfluent and confluent bovine aortic endothelial cells (ECs) in response to onset of unidirectional steady fluid shear stress (15 dyn/cm²). After flow onset, subconfluent cells exhibited dynamic edge activity in lamellipodia and small ruffles in the downstream and side directions for the first 12 min; activity was minimal in the upstream direction. After 12 min, peripheral edge extension subsided. Confluent cell monolayers that were exposed to shear stress exhibited only subtle increases in edge fluctuations after flow onset. Addition of cytochalasin D to disrupt actin polymerization served to suppress the magnitude of flow-mediated actin remodeling in both subconfluent confluent EC monolayers. Interestingly, when subconfluent ECs were exposed to two sequential flow step increases (1 dyn/cm² followed by 15 dyn/cm² 12 min later), actin-mediated edge activity was not additionally increased after the second flow step. Thus, repeated flow increases served to desensitize mechanosensitive structural dynamics in the actin cytoskeleton.     

Mitochondrial fission in endothelial cells after simulated ischemia/reperfusion: role of nitric oxide and reactive oxygen species

Ischemia (I)/reperfusion (RP)-induced endothelial cell (EC) injury is thought to be 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. Because 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 10dyn/cm(2)). By exposing ECs to shear stress, H, H/reoxygenation (RO), or simulated I/RP and employing MitoTracker staining, we assessed the differential effects of changes in mechanical forces and/or O(2) levels on the mitochondrial network. 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.