Physiological Levels Of Shear Stress Research Articles

Hydrodynamic Regulation of Monocyte Inflammatory Response to an Intracellular Pathogen

Systemic bacterial infections elicit inflammatory response that promotes acute or chronic complications such as sepsis, arthritis or atherosclerosis. Of interest, cells in circulation experience hydrodynamic shear forces, which have been shown to be a potent regulator of cellular function in the vasculature and play an important role in maintaining tissue homeostasis. In this study, we have examined the effect of shear forces due to blood flow in modulating the inflammatory response of cells to infection. Using an in vitro model, we analyzed the effects of physiological levels of shear stress on the inflammatory response of monocytes infected with chlamydia, an intracellular pathogen which causes bronchitis and is implicated in the development of atherosclerosis. We found that chlamydial infection alters the morphology of monocytes and trigger the release of pro-inflammatory cytokines TNF-α, IL-8, IL-1β and IL-6. We also found that the exposure of chlamydia-infected monocytes to short durations of arterial shear stress significantly enhances the secretion of cytokines in a time-dependent manner and the expression of surface adhesion molecule ICAM-1. As a functional consequence, infection and shear stress increased monocyte adhesion to endothelial cells under flow and in the activation and aggregation of platelets. Overall, our study demonstrates that shear stress enhances the inflammatory response of monocytes to infection, suggesting that mechanical forces may contribute to disease pathophysiology. These results provide a novel perspective on our understanding of systemic infection and inflammation.

Dynamic Adhesion of Umbilical Cord Blood Endothelial Progenitor Cells under Laminar Shear Stress

Late outgrowth endothelial progenitor cells (EPCs) represent a promising cell source for rapid reendothelialization of damaged vasculature after expansion ex vivo and injection into the bloodstream. We characterized the dynamic adhesion of umbilical-cord-blood-derived EPCs (CB-EPCs) to surfaces coated with fibronectin. CB-EPC solution density affected the number of adherent cells and larger cells preferentially adhered at lower cell densities. The number of adherent cells varied with shear stress, with the maximum number of adherent cells and the shear stress at maximum adhesion depending upon fluid viscosity. CB-EPCs underwent limited rolling, transiently tethering for short distances before firm arrest. Immediately before arrest, the instantaneous velocity decreased independent of shear stress. A dimensional analysis indicated that adhesion was a function of the net force on the cells, the ratio of cell diffusion to sliding speed, and molecular diffusivity. Adhesion was not limited by the settling rate and was highly specific to α5β1 integrin. Total internal reflection fluorescence microscopy showed that CB-EPCs produced multiple contacts of α5β1 with the surface and the contact area grew during the first 20 min of attachment. These results demonstrate that CB-EPC adhesion from blood can occur under physiological levels of shear stress.

Proteomic analysis of shear stress-mediated protection from TNF-alpha in endothelial cells.

Previous studies have shown that physiological levels of shear stress can protect endothelial cells (ECs) from apoptotic stimuli. Here, we differentiate between acute and chronic protection and demonstrate the use of proteomic technologies to uncover mechanisms associated with chronic protection of ECs. We hypothesized that changes in abundance of proteins associated with the TNF-alpha signaling cascade orchestrate shear stress-mediated protection from TNF-alpha when cells are preconditioned with shear prior to the exposure of apoptotic stimuli. Detection of cleaved caspase 3 through Western blot analysis confirmed chronic shear stress-mediated protection from TNF-alpha. In the presence of the nitric oxide synthase inhibitor, LNMA (N(omega)-monomethyl-l-arginine), chronic protection remained. Treatment with a de novo protein synthesis inhibitor, cycloheximide, eliminated this protective effect. Isotopic-labeling experiments, coupled with LC-MS/MS (liquid chromatography-tandem mass spectrometry) of isolated components of the TNF-alpha pathway revealed that CARD9, a known activator of the NF-kappaB pathway, was increased (60%) in sheared cells versus nonsheared cells. This result was confirmed through Western blot analysis. Our data suggest that de novo formation of proteins is required for protection from TNF-alpha in ECs chronically exposed to shear stress, and that CARD9 is a candidate protein in this response.

Shear stress modulates the thickness and architecture of Candida albicans biofilms in a phase‐dependent manner

Biofilm formation plays an integral role in catheter-associated bloodstream infections caused by Candida albicans. Biofilms formed on catheters placed intravenously are exposed to shear stress caused by blood flow. In this study, we investigated whether shear stress affects the ability of C. albicans to form biofilms. Candida biofilms were formed on catheter discs and exposed to physiological levels of shear stress using a rotating disc system (RDS). Control biofilms were grown under conditions of no flow. Tetrazolium (XTT) assay and dry weight (DW) measurements were used to quantify metabolic activity and biofilm mass respectively. Confocal scanning laser microscopy (CSLM) was used to evaluate architecture and biofilm thickness. After 90 min, cells attached under no-flow exhibited significantly greater XTT activity and DW than those under shear. However, by 24 h, biofilms formed under both conditions had similar XTT activities and DW. Interestingly, thickness of biofilms formed under no-flow was significantly greater after 24 h than of those formed under shear stress, demonstrating that shear exposure results in thinner, but denser biofilms. These studies suggest that biofilm architecture is modulated by shear in a phase-dependent manner.

Lowering Caveolin-1 Expression in Human Vascular Endothelial Cells Inhibits Signal Transduction in Response to Shear Stress

Vascular endothelial cells have an extensive response to physiological levels of shear stress. There is evidence that the protein caveolin-1 is involved in the early phase of this response. In this study, caveolin-1 was downregulated in human endothelial cells by RNAi. When these cells were subjected to a shear stress of 15 dyn/cm2 for 10 minutes, activation of Akt and ERK1/2 was significantly lower than in control cells. Moreover, activation of Akt and ERK1/2 in response to vascular endothelial growth factor was significantly lower in cells with low levels of caveolin-1. However, activation of integrin-mediated signaling during cell adhesion onto fibronectin was not hampered by lowered caveolin-1 levels. In conclusion, caveolin-1 is an essential component in the response of endothelial cells to shear stress. Furthermore, the results suggest that the role of caveolin-1 in this process lies in facilitating efficient VEGFR2-mediated signaling.

Differential gene responses in endothelial cells exposed to a combination of shear stress and cyclic stretch

We developed a compliant tube-type flow-loading apparatus that allows simultaneous application of physiological levels of shear stress and cyclic stretch to cultured cells and examined gene responses to a combination of the two forces. Human umbilical vein endothelial cells were exposed to shear stress and/or cyclic stretch for 24 h, and changes in the mRNA levels of endothelin-1 (ET-1), a potent vasoconstrictor, and endothelial nitric oxide synthase (eNOS), which catalyzes the production of a potent vasodilator, NO, were determined by reverse transcriptase/PCR. Cyclic stretch (10%, 1 Hz) alone increased ET-1 mRNA levels approximately 1.6-fold, but had no effect on eNOS mRNA levels. A shear stress of 7 dynes/cm 2 and 15 dynes/cm 2 alone decreased ET-1 mRNA levels to around 83% and 61%, respectively, of the basal level, but increased the eNOS mRNA level to around 2.2-fold and 3.2-fold, respectively. When cyclic stretch and shear stress were applied simultaneously, ET-1 mRNA levels did not change significantly, but the eNOS mRNA level increased to a level equivalent to the increase in response to shear stress alone. These results indicate that the response of endothelial genes to shear stress or cyclic stretch depends on whether the two forces are applied separately or together.

Shear Stress–Mediated Arterial Remodeling in Atherosclerosis

Under physiological conditions, chronic changes in blood flow stimulate compensatory changes in arterial size. This arterial remodeling process occurs during normal growth and development and contributes to the adaptive response to a variety of clinical situations. For example, repetitive increases in blood flow during exercise may stimulate expansive remodeling of conduit arteries in the limbs and heart,1 whereas chronic disuse of the lower extremities as a result of spinal cord injury is associated with constrictive remodeling of the femoral arteries.2 Uterine arteries display expansive remodeling during pregnancy and then undergo constrictive remodeling after delivery. Thus, dynamic remodeling of the arterial tree plays a critical role in maintaining the appropriate balance between tissue demand and blood supply throughout life. Article p 2826 A primary signal for arterial remodeling is shear stress, which is the frictional force at the endothelial surface produced by flowing blood.3,4 Shear stress relates directly to flow and blood viscosity and inversely to the third power of arterial radius.3 A macroscopic increase in blood flow increases local shear stress and stimulates arterial expansion until shear stress has been restored to baseline. Conversely, low shear stress leads to constrictive remodeling. This important homeostatic mechanism maintains shear stress in an appropriate range. When exposed to physiological levels of shear stress (15 to 40 dyne/cm2), endothelial cells appropriately elongate, align in the direction of flow, and maintain barrier function.4 Furthermore, normal shear stress promotes expression of vasodilator and antithrombotic factors, suppresses growth and proinflammatory factors, and generally maintains a state of vascular health. In contrast, low, oscillating, and disordered shear stress promotes the development of atherosclerosis. Expansive remodeling in response to chronic or repetitive increases in flow involves a coordinated sequence of events in the arterial wall, as has been extensively reviewed.3,5,6 Over a …

Trophoblast Migration Under Flow Is Regulated by Endothelial Cells1

During pregnancy, trophoblasts enter the uterine vasculature and are found in spiral arteries far upstream of uterine capillaries. It is unknown whether trophoblasts reach the spiral arteries by migration within blood vessels against blood flow or by intravasation directly into spiral arteries after interstitial migration. We have developed an in vitro system consisting of early gestation macaque monkey trophoblasts cocultured with uterine endothelial cells and have exposed the cells in a parallel plate flow chamber to physiological levels of shear stress. Videomicroscopy followed by quantitative image analysis revealed that the migratory activity (expressed as average displacement and average migration velocity) of trophoblasts cultured on top of endothelial cells remained unchanged between shear stresses of 1-30 dyne/cm(2) whereas activity of trophoblasts alone increased with increasing shear stress. When the direction of migration was assessed at 1 and 7.5 dyne/cm(2), the extent of migration against and with flow was roughly equal for both trophoblasts alone and cocultured trophoblasts. At shear stress levels of 15 and 30 dyne/cm(2), trophoblasts incubated alone showed a significant decrease in migration against flow and corresponding increased migration in the direction of flow. In contrast, trophoblasts cocultured with uterine endothelial cells maintained the same extent of migration against flow at all shear stress levels. Migration against flow was also maintained when trophoblasts were cultured with endothelial cell-conditioned medium or fixed endothelial cells. The results indicate that factors expressed on the surface of uterine endothelial cells and factors released by endothelial regulate trophoblast migration under flow.

The cerebral vasculature as a therapeutic target for neurological disorders and the role of shear stress in vascular homeostatis and pathophysiology

It is widely accepted that vascular mechanisms are involved in the genesis of many neurological disorders. In particular, blood–brain barrier (BBB) dysfunction has been related to the severity of Alzheimer's disease, encephalopathy due to meningitis, multiple sclerosis, HIV-associated encephalopathy, epilepsy, gliomas and metastatic brain tumors. The BBB may constitute an important therapeutic target to protect neurons after CNS diseases. Both in vivo and in vitro, the functional phenotype of vascular endothelium is dynamically responsive to circulating cytokines, growth factors and puslatile blood flow (shear stress). Shear stress can play a critical role in vascular homeostasis and pathophysiology; it is a major regulator of remodeling in developed blood vessels and in blood vessels affected by atherosclerotic lesions. The physiological fluid mechanic stimulus, shear stress, could be considered to be an important 'differentiative' stimulus capable of modulating endothelial phenotype in vivo. Endothelial cells undergo cell cycle arrest after exposure to physiological levels of shear stress. As for mature endothelial cells, in which flow mediated shear stress may play a role in the induction, progression and/or prevention of atherosclerosis by changing their function, stress may play a role in endothelial cell differentiation from hemopoietic stem cells and/or from embryonic stem cells. Stem cells may be used to repair vascular damage, including loss of EC, due to a variety of diseases (e.g. myocardial neovascularization by adult bone marrow derived angioblasts). In the brain, it was proposed that neuron-producing stem cells may be used to treat Alzheimer's disease, paralysis, etc. Surprisingly, very few investigators are exploring the use of endothelial precursors to revert or prevent cerebrovascular disease. This review summarizes the most recent data related to cerebral vasculature as a therapeutic target for neurological disorders and the role of shear stress in blood–brain barrier homeostasis and pathophysiology.

Attachment, morphology and adherence of human endothelial cells to vascular prosthesis materials under the action of shear stress

In an effort to improve the long-term patency of vascular prostheses several groups now advocate seeding autologous endothelial cells (ECs) onto the lumen of the vessel prior to implantation, a procedure that involves pre-treating the prosthesis material with fibrin, collagen and/or other matrix molecules to promote cell attachment and retention. In this study, we examined the degree to which human umbilical venous endothelial cells (HUVECs) adhered to three materials commonly used polymeric vascular prosthesis that had been coated with the same commercial extra cellular matrix proteins, and after exposure to fluid shear stresses representative of femoro-distal bypass in a cone-and-plate shearing device. We quantified cell number, area of coverage and degree of cell spreading using image analysis techniques. The response of cells that adhered to the surface of each material, and following exposure to fluid shear stress, depended on surface treatment, topology and cell type. Whereas collagen coating improved primary cellular adhesion and coverage significantly, the degree of spreading depended on the underlying surface structure and on the application of the shear stress. In some cases, fewer than 30% of cells remained on the surface after only 1-h exposure to physiological levels of shear stress. The proportion of the surface that was covered by cells also decreased, despite an increase in the degree to which individual cells spread on exposure to shear stress. Moreover, the behaviour of HUVECs was distinct from that of fibroblasts, in that the human ECs were able to adapt to their environment by spreading to a much greater extent in response to shear. The quality of HUVEC attachment, as measured by extent of cell coverage and resistance to fluid shear stress, was greatest on expanded polytetrafluoroethylene samples that had been impregnated with Type I/III collagen.

Assembly and Reorientation of Stress Fibers Drives Morphological Changes to Endothelial Cells Exposed to Shear Stress

Fluid shear stress greatly influences the biology of vascular endothelial cells and the pathogenesis of atherosclerosis. Endothelial cells undergo profound shape change and reorientation in response to physiological levels of fluid shear stress. These morphological changes influence cell function; however, the processes that produce them are poorly understood. We have examined how actin assembly is related to shear-induced endothelial cell shape change. To do so, we imposed physiological levels of shear stress on cultured endothelium for up to 96 hours and then permeabilized the cells and exposed them briefly to fluorescently labeled monomeric actin at various time points to assess actin assembly. Alternatively, monomeric actin was microinjected into cells to allow continuous monitoring of actin distribution. Actin assembly occurred primarily at the ends of stress fibers, which simultaneously reoriented to the shear axis, frequently fused with neighboring stress fibers, and ultimately drove the poles of the cells in the upstream and/or downstream directions. Actin polymerization occurred where stress fibers inserted into focal adhesion complexes, but usually only at one end of the stress fiber. Neither the upstream nor downstream focal adhesion complex was preferred. Changes in actin organization were accompanied by translocation and remodeling of cell-substrate adhesion complexes and transient formation of punctate cell-cell adherens junctions. These findings indicate that stress fiber assembly and realignment provide a novel mode by which cell morphology is altered by mechanical signals.

Shear stress mediates tyrosylprotein sulfotransferase isoform shift in human endothelial cells

In this study, we examined expression of tyrosylprotein sulfotransferase (TPST) isoforms TPST1 and TPST2 in primary cultures of human umbilical vein endothelial cells. For the first time coexpression of both isoforms is shown in primary human cells. Application of physiological levels of shear stress regulates expression of TPST isoforms in a time- and dose-dependent manner. Sustained application of arterial laminar shear stress causes downregulation of TPST1 mRNA and protein expression, while TPST2 is upregulated. This TPST isoform shift is mediated by different signaling pathways. Shear stress-dependent downregulation of TPST1 involves protein kinase C, while upregulation of TPST2 is mediated by a tyrosine kinase-dependent pathway.

A chamber to permit invasive manipulation of adherent cells in laminar flow with minimal disturbance of the flow field.

An obstacle to real-time in vitro measurements of endothelial cell responses to hemodynamic forces is the inaccessibility of the cells to instruments of measurement and manipulation. We have designed a parallel plate laminar flow chamber that permits access to adherent cells during exposure to flow. The "minimally invasive flow device" (MIF device) has longitudinal slits (1 mm wide) cut in the top plate of the chamber to allow insertion of a recording, measurement, or stimulating instrument (e.g., micropipette) into the flow field. Surface tension forces at the slit openings are sufficient to counteract the hydrostatic pressure generated in the chamber and thus prevent overflow. The invasive probe is brought near to the cell surface, makes direct contact with the cell membrane, or enters the cell. The slits provide access to a large number (and choice) of cells. The MIF device can maintain physiological levels of shear stress (<1-15 dyn/cm2) without overflow in the absence and presence of fine instruments such as micropipettes used in electrophysiology, membrane aspiration, and microinjection. Microbead trajectory profiles demonstrated negligible deviations in laminar flow near the surface of target cells in the presence of microscale instruments. Patch-clamp electrophysiological recordings of flow-induced changes in membrane potential were demonstrated. The MIF device offers numerous possibilities to investigate real-time endothelial responses to well-defined flow conditions in vitro including electrophysiology, cell surface mechanical probing, local controlled chemical release, biosensing, microinjection, and amperometric techniques.

Shear stress enhances human endothelial cell wound closure in vitro.

Repair of the endothelium occurs in the presence of continued blood flow, yet the mechanisms by which shear forces affect endothelial wound closure remain elusive. Therefore, we tested the hypothesis that shear stress enhances endothelial cell wound closure. Human umbilical vein endothelial cells (HUVEC) or human coronary artery endothelial cells (HCAEC) were cultured on type I collagen-coated coverslips. Cell monolayers were sheared for 18 h in a parallel-plate flow chamber at 12 dyn/cm(2) to attain cellular alignment and then wounded by scraping with a metal spatula. Subsequently, the monolayers were exposed to a laminar shear stress of 3, 12, or 20 dyn/cm(2) under shear-wound-shear (S-W-sH) or shear-wound-static (S-W-sT) conditions for 6 h. Wound closure was measured as a percentage of original wound width. Cell area, centroid-to-centroid distance, and cell velocity were also measured. HUVEC wounds in the S-W-sH group exposed to 3, 12, or 20 dyn/cm(2) closed to 21, 39, or 50%, respectively, compared with only 59% in the S-W-sT cells. Similarly, HCAEC wounds closed to 29, 49, or 33% (S-W-sH) compared with 58% in the S-W-sT cells. Cell spreading and migration, but not proliferation, were the major mechanisms accounting for the increases in wound closure rate. These results suggest that physiological levels of shear stress enhance endothelial repair.

Laminar shear stress upregulates the complement-inhibitory protein clusterin : a novel potent defense mechanism against complement-induced endothelial cell activation.

The complement system is implicated in the pathogenesis of atherosclerosis. Complement has been shown to activate endothelial cells (ECs) by inducing a proinflammatory response. Physiological levels of shear stress exert potent antiatherosclerotic effects. Therefore, we investigated whether shear stress antagonizes the effects of complement on ECs. Incubation of ECs with nonlytic concentrations of complement serum (CS: 0.2 U/mL for 6 hours) resulted in an upregulation of interleukin-8 (IL-8) (165+/-12%) and monocyte chemoattractant protein-1 (MCP-1) mRNA expression (267+/-34%). Preexposure of ECs for 18 hours with laminar shear stress (15 dyne/cm(2)) abrogated CS-induced IL-8 release to 106+/-10% (P<0.001) and reduced CS-induced MCP-1 expression (170+/-31%; P<0.05). To examine the mechanism of the protective effect of shear stress, expression of the complement-inhibitory protein clusterin was analyzed under shear exposure. Shear stress increased clusterin mRNA (225+/-76%, 6 hours) and protein expression (164+/-22%, 18 hours). Specific inhibition of clusterin by transfection with antisense oligonucleotides reversed the protective effect of shear stress on CS-induced MCP-1 and IL-8 upregulation (P<0.05 versus sense-transfected cells). Moreover, clusterin overexpression inhibited CS-induced EC activation. Shear stress abrogates the complement-induced proinflammatory response of ECs by upregulation of the complement-inhibitory protein clusterin. Upregulation of clusterin may contribute to the potent antiatherosclerotic effects of shear stress by preventing endothelial activation through the complement cascade.

Suppression of apoptosis by nitric oxide via inhibition of interleukin-1beta-converting enzyme (ICE)-like and cysteine protease protein (CPP)-32-like proteases.

Physiological levels of shear stress alter the genetic program of cultured endothelial cells and are associated with reduced cellular turnover rates and formation of atherosclerotic lesions in vivo. To test the hypothesis that shear stress (15 dynes/cm2) interferes with programmed cell death, apoptosis was induced in human umbilical venous cells (HUVEC) by tumor necrosis factor-alpha (TNF-alpha). Apoptosis was quantified by ELISA specific for histone-associated DNA-fragments and confirmed by demonstrating the specific pattern of internucleosomal DNA-fragmentation. TNF-alpha (300 U/ml) mediated increase of DNA-fragmentation was completely abrogated by shear stress (446 +/- 121% versus 57 +/- 11%, P <0.05). This anti-apoptotic activity of shear stress decreased after pharmacological inhibition of endogenous nitric oxide (NO)-synthase by NG-monomethyl-L-arginine and was completely reproduced by exogenous NO-donors. The activation of interleukin-1beta-converting enzyme (ICE)-like and cysteine protease protein (CPP)-32-like cysteine proteases was required to mediate TNF-alpha-induced apoptosis of HUVEC. Endothelial-derived nitric oxide (NO) as well as exogenous NO donors inhibited TNF-alpha-induced cysteine protease activation. Inhibition of CPP-32 enzyme activity was due to specific S-nitrosylation of Cys 163, a functionally essential amino acid conserved among ICE/CPP-32-like proteases. Thus, we propose that shear stress-mediated NO formation interferes with cell death signal transduction and may contribute to endothelial cell integrity by inhibition of apoptosis.

Shear stress inhibits apoptosis of human endothelial cells

Physiological levels of shear stress alter the genetic program of cultured endothelial cells and reduce endothelial cell turnover in vivo. To test the hypothesis that shear stress interferes with programmed cell death, apoptosis was induced in human umbilical venous endothelial cells by growth factor withdrawal or incubation with tumor necrosis factor α(TNFα) for 18 h. Apoptosis was quantified by ELISA specific for histone-associated DNA fragments and confirmed by demonstrating the specific pattern of internucleosomal DNA fragmentation detected by electrophoresis and immunohistochemical staining. The TNFα (300 U/ml)-mediated increase in DNA fragmentation was completely abrogated by shear stress. Furthermore, shear stress dose-dependently reduced DNA fragmentation induced by growth factor withdrawal with maximal effect at 45 dyn/cm 2. Inhibition of the CPP32-like proteases with Ac-DEVD-CHO (100 μM) revealed similar anti-apoptotic effects. In contrast, CPP32-independent induction of endothelial cell apoptosis by C 2-ceramide (50 μM) was not prevented by shear stress. Thus, we propose that shear stress interferes with common cell death signal transduction involving the CPP32-like protease family and may contribute to endothelial cell integrity by inhibition of apoptosis.

Role of calcium and calmodulin in flow-induced nitric oxide production in endothelial cells

These experiments demonstrate that exposure of cultured endothelial cells (EC) to well-defined laminar fluid flow results in an elevated rate of NO production. NO production was monitored by release of NOx (NO2- + NO3(2-) and by cellular guanosine 3',5'-cyclic monophosphate (cGMP) concentration. NO synthase (NOS) inhibitor blocked the flow-mediated stimulation of both NOx and cGMP, indicating that both measurements reflect NO production. Exposure to laminar flow increased NO release in a biphasic manner, with an initial rapid production consequent to the onset of flow followed by a less rapid, sustained production. A similar rapid increase in NO production resulted from an increase in flow above a preexisting level. The rapid initial production of NO was not dependent on shear stress within a physiological range (6-25 dyn/cm2) but may be dependent on the rate of change in shear stress. The sustained release of NO was dependent on physiological levels of shear stress. The calcium (Ca2+) or calmodulin (CaM) dependence of the initial and sustained production of NO was compared with bradykinin (BK)-mediated NO production. Both BK and the initial production were inhibited by Ca2+ and CaM antagonists. In contrast, the sustained shear stress-mediated NO production was not affected, despite the continued functional presence of the antagonists. Dexamethasone had no effect on either the initial or the sustained shear stress-mediated NO production. An inducible NOS does not, therefore, explain the apparent Ca2+/CaM independence of the sustained shear stress-mediated NO production.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of shear stress on the uptake and metabolism of arachidonic acid by human endothelial cells

The uptake and metabolism of arachidonic acid (AA) by human umbilical vein endothelial cells was studied for cells in stationary culture and for cells exposed to physiological levels of shear stress. For cells grown in stationary culture, the initial incorporation of arachidonic acid was primarily into diacylglycerol and phospholipids. Cells exposed to flow incorporated labeled arachidonic acid at a similar rate as cells maintained in stationary culture; however, the distribution of the label was altered by flow. The incorporation of arachidonic acid into diacylglycerol and phosphatidylinositol was increased in cells exposed to flow. The largest increase occurred for cells exposed to arterial levels of shear stress for the shortest time period studied, 0.5 h. Prostacyclin (PGI2) and PGF2 alpha were the principal arachidonic acid metabolites formed. Shear stress-stimulated cells preferentially produced PGI2 relative to other eicosanoid products. The initiation of flow caused a burst of AA metabolism which was highly specific for PGI2. This might represent an increase in the turnover of phosphatidylinositol-bound arachidonic acid which is specifically converted to PGI2 as a result of flow-induced membrane stresses.