Effect Of Laminar Shear Stress Research Articles

Laminar shear stress-induced activation of Raf/MEK/ERK1/2 signaling pathway contributes to upregulation of t-PA expression in human endothelial progenitor cells

BackgroundShear stress has been proved to enhance the expression of tissue-type plasminogen activator (t-PA) in endothelial progenitor cells (EPCs), which may contribute to maintenance of vascular homeostasis. However, the underlying...

In Vitro Shear Stress Responses of Endothelin-1 in African American and Caucasian HUVECs

Higher levels of plasma Endothelin-1 (ET1), a potent vasoconstrictor, have been reported in African Americans (AA) when compared to Caucasians (CA). However, this racial difference has never been considered when investigating the endothelin system at the cell level. PURPOSE: To determine the effects of laminar shear stress (LSS), which mimics the exercise shear stress on the endothelium, in vitro and the effects of aerobic exercise training on the endothelin system in vivo. METHODS: AA (n=2) and CA (n=2) human umbilical vein endothelial cells (HUVECs) were cultured and underwent LSS using a cone and plate system at both 5 dyne/cm2 and 20 dyne/cm2 for 24 hours. Western Blots were performed to determine Endothelin Converting Enzyme 1 (ECE1) expression. Cell media ET1 content was measured using an ELISA Assay (Assay Designs Inc.). AA subjects (n=15) ages 41-62, who were sedentary, non-diabetic, non-smoking, and free of cardiovascular disease underwent 6 months of aerobic exercise training (3days/week, 65% of VO2max, 40 minutes/day). Plasma ET1 levels were determined before and after exercise training via ELISA assay (Assay Designs Inc.). RESULTS: AA HUVECs had higher amounts of secreted ET1 (267.9±53.6 vs. 132.5±13.0 pg/μg, p=.133) and higher ECE1 expression (0.812±0.065 vs. 0.638±0.02 relative density, p=.127) than Caucasian HUVECs at LSS levels of 5 dyne/cm2. After high physiological LSS (20 dyne/cm2) ET1 was reduced in both African American (267.9±53.5 vs. 120.0±18.2 pg/μg, p=.149) and Caucasian (132.5±13.0 vs. 94.1±30.1 pg/μg, p=.537) HUVECs. ECE1 expression was increased in African American HUVECs (0.812±0.065 vs. 1.085±0.295 relative density, p=.587) and unchanged in Caucasian HUVECs (0.638±0.02 vs. 0.695±0.093 relative density, p=.578) after high LSS (20 dyne/cm2). Additionally, aerobic exercise training significantly (p≤0.05) lowered plasma ET1 levels in African Americans (2.78 pg/ml vs. 0.565 pg/ml, p=.024). CONCLUSION: African American HUVECs present higher levels of ECE1 expression and higher amounts of secreted ET1 than Caucasian HUVECs at resting levels of LSS. Both shear stress and aerobic exercise can lower levels of ET1 in vitro and in vivo respectively. Further research with a larger sample size will be needed to confirm these findings.

Laminar shear stress delivers cell cycle arrest and anti-apoptosis to mesenchymal stem cells

Biomechanical forces are emerging as critical regulators of cell function and fluid flow is a potent mechanical stimulus. Although the mechanisms of osteoblasts and osteocytes responding to fluid flow are being elucidated, little is known about how the osteoprogenitors, mesenchymal stem cells (MSCs), respond to fluid flow. Here, we examined the effects of laminar shear stress (LSS) on MSCs in vitro. MSCs from bone marrow of Sprague-Dawley rats were isolated, purified, and subjected to physiological levels of LSS. DNA synthesis and cell cycle were measured through [(3)H]thymidine and by flow cytometry, respectively, to detect the cellular proliferation. Annexin V immunostaining and Bcl-2/Bax mRNA expression were evaluated to determine the effect of LSS on MSCs apoptosis. Results showed that fluid shear stress caused a dose-related reduction of MSCs' proliferation rate with the majority of cells being arrested in the G(0) or G(1) phase. Moreover, it was found that physiological levels of LSS exerted a potent suppression effect on MSC apoptosis. In summary, these data revealed a critical role of LSS in maintaining the quiescence of MSCs.

Effect of simvastatin on Kruppel-like factor2, endothelial nitric oxide synthase and thrombomodulin expression in endothelial cells under shear stress

Aims Statins (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors) and fluid wall shear stress have been reported to modulate the expression of genes related to inflammation, blood coagulation, thrombosis, and vascular constriction in cultured endothelial cells. In this study, we investigated the combined effect of laminar shear stress (LSS) and statins on endothelial cell gene expression. Main methods Kruppel-like factor 2 (KLF2), endothelial nitric oxide synthase (eNOS), and thrombomodulin (TM) mRNA and protein expression were evaluated in human abdominal aortic endothelial cells (HAAEC) treated with simvastatin (0.1, 1 or 10 µM) at various levels of LSS (0, 1.25, 12.5 or 25 dynes/cm 2). Key findings As expected, simvastatin and LSS separately enhanced KLF2, eNOS, and TM mRNA expressions. The combination of simvastatin and LSS resulted in significantly higher mRNA levels of all three genes compared to cells treated with LSS only. The highest KLF2, eNOS, and TM mRNA levels were detected at 10 µM simvastatin and 25 dynes/cm 2. Under these conditions, eNOS and TM protein levels were also elevated. Combining LSS and simvastatin produced an overall additive increase in KLF2, eNOS, and TM mRNA. Treatment of the endothelial cells with 10 µM simvastatin and 200 µM mevalonate completely eliminated the effect of simvastatin. Significance Our results suggest an additive increase in KLF2, eNOS, and TM expressions when simvastatin and LSS are combined. These results may help to explain the proposed non-lipid lowering benefits of statins observed in the clinic.

Laminar Shear Stress Modulates Phosphorylation and Localization of RNA Polymerase II on the Endothelial Nitric Oxide Synthase Gene

In endothelial cells exposed to unidirectional laminar shear stress, endothelial nitric oxide synthase transcription (eNOS), mRNA stability, and protein levels are enhanced. We have previously demonstrated that these changes are associated with increased 3' polyadenylation of eNOS mRNA. Here, we investigated the effect of laminar shear stress on the phosphorylation and localization of RNA polymerase (Pol) II, the enzyme primarily responsible for coordinating transcription and posttranscriptional processing. Using Western and chromatin immunoprecipitation analyses, Pol II phosphorylation and localization on the eNOS gene were assessed in bovine aortic endothelial cells exposed to laminar shear stress. Total Pol II (phosphorylated and unphosphorylated) levels were increased 65% in response to laminar shear stress. This was associated with an increase in Pol II phosphoserine 2, but no change in levels of the unphosphorylated or phosphoserine 5 isoforms. Quantitative chromatin immunoprecipitation analysis showed that laminar shear stress enhanced binding of Pol II phosphoserine 2 to the 3' end of the eNOS gene, particularly exon 26, which encodes the 3'UTR. Treatment of cells with DRB attenuated laminar shear stress-induced Pol II phosphorylation, eNOS 3' polyadenylation, and eNOS expression. These data suggest that laminar shear stress enhances eNOS mRNA 3' polyadenylation by modulating phosphorylation and localization of Pol II.

Molecular Mechanisms Responsible for the Atheroprotective Effects of Laminar Shear Stress

The endothelium lining the inner surface of blood vessels of the cardiovascular system is constantly exposed to hemodynamic shear stress. The interaction between endothelial cells and hemodynamic shear stress has critical implications for atherosclerosis. Regions of arterial narrowing, curvatures, and bifurcations are especially susceptible to atherosclerotic lesion formation. In such areas, endothelial cells experience low, or oscillatory, shear stress. Corresponding changes in endothelial cell structure and function make them susceptible to the initiation and development of atherosclerosis. In contrast, blood flow with high laminar shear stress activates signal transductions as well as gene and protein expressions that play important roles in vascular homeostasis. In response to laminar shear stress, the release of vasoactive substances such as nitric oxide and prostacyclin decreases permeability to plasma lipoproteins as well as the adhesion of leukocytes, and inhibits smooth muscle cell proliferation and migration. In summary, different flow patterns directly determine endothelial cell morphology, metabolism, and inflammatory phenotype through signal transduction and gene and protein expression. Thus, high laminar shear stress plays a key role in the prevention of atherosclerosis through its regulation of vascular tone and long-term maintenance of the integrity and function of endothelial cells.

Enhancement of Mesenchymal Stem Cell Attachment to Decellularized Porcine Aortic Valve Scaffold byIn VitroCoating with Antibody Against CD90: A Preliminary Study on Antibody-Modified Tissue-Engineered Heart Valve

The importance of cell adhesion to the scaffold in the tissue-engineered heart valve remains to be determined. The current study examined the feasibility of conjugating antibody against CD90 to a decellularized porcine aortic valve scaffold and binding mesenchymal stem cells to that scaffold through interaction with a cell surface antigen. After decellularization, the porcine aortic valve was reacted with biotin, avidin, and biotinylated anti-rat CD90 antibody sequentially and inserted into a laminar flow system used to test the effect of laminar shear stress. Rat bone mesenchymal stem cells (BMSC) were injected and circulated in a flow system to study the ability of anti-CD90 antibody to trap and immobilize cells on the valve surface. The results demonstrated that anti-CD90 antibody on the valve surface remains bound, even under high shear conditions. Compared with the control valve (no antibody), the modified (antibody-coated) valve immobilized significantly more rat BMSC (p < 0.05). Thus, the avidin-biotin system can be used to attach anti-CD90 antibody to these valves, and the bound antibody can immobilize rat BMSC in a flow chamber, suggesting that antibody-modified scaffolds might be used to fabricate shear stress-resistant, tissue-engineered heart valves.

Laminar shear stress acts as a switch to regulate divergent functions of NF‐κB in endothelial cells

Regions of the arterial tree exposed to laminar flow, which exerts high shear stress, are protected from inflammation, endothelial cell (EC) death and atherosclerosis. TNFalpha activates NF-kappaB transcription factors, which potentially exert dual functions by inducing both proinflammatory and cytoprotective transcripts. We assessed whether laminar shear stress protects EC by modulating NF-kappaB function. Human umbilical vein EC (HUVEC) were cultured under shear stress (12 dynes/cm2 for 16 h) using a parallel-plate flow chamber or were maintained in static conditions. Comparative real-time PCR revealed that preshearing significantly alters transcriptional responses to TNFalpha by enhancing the expression of cytoprotective molecules (Bcl-2, MnSOD, GADD45beta, A1) and suppressing proinflammatory transcripts (E-selectin, VCAM-1, IL-8). We demonstrated using assays of nuclear localization, NF-kappaB subunit phosphorylation, DNA-binding, and transcriptional activity that NF-kappaB is activated by TNFalpha in presheared HUVEC. Furthermore, a specific inhibitor revealed that NF-kappaB is essential for the induction of cytoprotective transcripts in presheared EC. Finally, we observed that NF-kappaB can be activated in vascular endothelium exposed to laminar shear stress in NF-kappaB-luciferase reporter mice, thus validating our cell culture experiments. We conclude that shear stress primes EC for enhanced NF-kappaB-dependent cytoprotective responsiveness while attenuating proinflammatory activation. Thus modulation of NF-kappaB function may underlie the atheroprotective effects of laminar shear stress.

44 KRUPPEL-LIKE FACTOR 4 REGULATES ENDOTHELIAL INFLAMMATION.

<h3>Background</h3> The vascular endothelium plays a critical role in vascular homeostasis. A limited proinflammatory, prothrombotic state is appropriate in the context of infection or wound healing, but sustained endothelial activation leads to deleterious conditions such as atherosclerosis and pathologic thrombosis. Inflammatory cytokines and nonlaminar blood flow induce endothelial dysfunction and confer a proadhesive and prothrombotic phenotype. Therefore, identification of factors that mediate the effects of these stimuli on endothelial function is of considerable interest. Kruppel-like factors (KLFs) are a subclass of the zinc-finger family of transcription factors. Previous studies demonstrate that KLF proteins typically regulate critical aspects of cellular differentiation and tissue development. Studies from our laboratory and others demonstrate an emerging role for this family of transcriptional regulators in vascular biology. KLF4/GKLF (gut-enriched Kruppel-like factor) was first identified as being highly expressed in epithelial cells, and subsequent work has verified a critical role in skin and intestinal development. KLF4 has also been identified in endothelial cells, yet its function in vessel biology has yet to be elucidated. <h3>Methods and Results</h3> Immunohistochemical analysis of mouse and human vascular tissues demonstrates the expression, in vivo, of KLF4 in human and mouse endothelial cells. Northern blot analysis of total mRNA harvested from primary endothelial cell lines derived from various human arterial vascular beds (aorta, pulmonary artery, and umbilical artery) shows expression of endothelial KLF4 in a variety of arterial and venous cell lines. Furthermore, we demonstrate that endothelial KLF4 is induced by proinflammatory stimuli and shear stress. Overexpression of KLF4 induces expression of multiple anti-inflammatory and antithrombotic factors, including eNOS and thrombomodulin, and inhibits basal and cytokine-mediated expression of a diverse set of proinflammatory factors, including MCP-1, RANTES, CRP, PAI-1, IL-6, and IL-8. The significance of endogenous expression of KLF4 on target genes was assessed in experiments using siRNA-mediated knockdown of KLF4. These experiments demonstrate that KLF4 depletion leads to enhancement of TNF-α-induced VCAM-1 and tissue factor expression. In addition to the determination of target genes, we have verified the functional importance of KLF4 by demonstrating that KLF4 expression markedly decreases inflammatory cell adhesion to the endothelial surface and prolongs clotting time under inflammatory states. As a first step toward understanding the molecular basis of KLF4-mediated regulation of endothelial target genes, we assessed the effect of KLF4 on target gene promoters. KLF4 differentially regulates the promoter activity of pro- and anti-inflammatory genes in a manner consistent with its anti-inflammatory function. <h3>Conclusion</h3> Inflammatory cytokines and the biomechanical effects of laminar shear stress are the most potent effectors of endothelial homeostasis identified to date. Perturbation of endothelial function by proinflammatory cytokines or nonlaminar flow has been documented in most vascular disease states, including vasculitis, aneurysm formation, pathologic thrombosis, and atherosclerosis. The observations described above implicate KLF4 as a novel regulator of endothelial activation in response to proinflammatory stimuli. A greater understanding of KLF4 function is thus of considerable scientific and potentially therapeutic interest.

NO‐mediated regulation of NAD(P)H oxidase by laminar shear stress in human endothelial cells

The flowing blood generates shear stress at the endothelial cell surface. In endothelial cells, NAD(P)H oxidase complexes have been identified as major sources of superoxide anion (.O(2)(-)) formation. In this study, we analysed the effect of laminar shear stress on .O(2)(-) formation by cytochrome c reduction assay and on NAD(P)H oxidase subunit expression by standard calibrated competitive reverse transcription-polymerase chain reaction and Western blot in human endothelial cells. Primary cultures of human umbilical vein endothelial cells were exposed to laminar shear stress in a cone-and-plate viscometer for up to 24 h. Short-term application of shear stress transiently induced .O(2)(-) formation. This was inhibited by NAD(P)H oxidase inhibitor gp91ds-tat, but NAD(P)H oxidase subunit expression was unchanged. Long-term arterial laminar shear stress (30 dyne cm(-2), 24 h) down-regulated .O(2)(-) formation, and mRNA and protein expression of NAD(P)H oxidase subunits Nox2/gp91(phox) and p47(phox). In parallel, endothelial NO formation and eNOS, but not Cu/Zn SOD, protein expression was increased. Down-regulation of .O(2)(-) formation, gp91(phox) and p47(phox) expression by long-term laminar shear stress was blocked by l-NAME. NO donor DETA-NO down-regulates .O(2)(-) formation, gp91(phox) and p47(phox) expression in static cultures. In conclusion, our data suggest a transient activation of .O(2)(-) formation by short-term shear stress, followed by a down-regulation of endothelial NAD(P)H oxidase in response to long-term laminar shear stress. NO-mediated down-regulation by shear stress preferentially affects the gp91(phox)/p47(phox)-containing NAD(P)H oxidase complex. This mechanism might contribute to the regulation of endothelial NO/.O(2)(-) balance and the vasoprotective potential of physiological levels of laminar shear stress.

15-deoxy-delta 12,14-prostaglandin J2 and laminar fluid shear stress stabilize c-IAP1 in vascular endothelial cells.

Laminar shear stress strongly inhibits vascular endothelial cell apoptosis by unknown mechanisms. We reported that shear stress stimulates endothelial cells to produce 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) by elevating the expression level of lipocalin-type prostaglandin D synthase. To investigate the role of 15d-PGJ2 produced in the vascular wall, we examined the effect of 15d-PGJ2 on endothelial cell apoptosis. We induced apoptosis in human umbilical vein endothelial cells (HUVECs) by growth factor deprivation. 15d-PGJ2 strongly inhibited DNA ladder formation, nuclear fragmentation, and caspase-3-like activity in HUVECs. To elucidate the mechanism by which 15d-PGJ2 inhibits endothelial cell apoptosis, we examined expression of the inhibitor of apoptosis proteins (IAP) cellular-IAP1 (c-IAP1), c-IAP2, x-linked IAP, and survivin in HUVECs. In parallel with the inhibition of apoptosis, 15d-PGJ2 elevated the expression level of c-IAP1 protein in a dose- and time-dependent manner without changing the mRNA level. Laminar shear stress also induced c-IAP1 expression. Chase experiments with the use of cycloheximide revealed that 15d-PGJ2 and shear stress both inhibited the proteolytic degradation of c-IAP1 protein. These results suggested that 15d-PGJ2 inhibits endothelial cell apoptosis through, at least in part, c-IAP1 protein stabilization. This mechanism might be involved in the antiapoptotic effect of laminar shear stress.

Shear Stress-Dependent Expression of Apoptosis-Regulating Genes in Endothelial Cells

Laminar shear stress exerts potent anti-apoptotic effects. Therefore, we analyzed the influence of laminar shear stress on the expression of apoptosis-regulating genes in human umbilical vein endothelial cells (HUVEC). Application of high levels of laminar shear stress (15 and 30 dyn/cm2) decreased the susceptibility of HUVEC to undergo apoptosis, whereas low shear stress (1 dyn/cm2) had no effect. These diminished signs of apoptosis were accompanied by a decreased mRNA expression of apoptosis-inducing Fas receptor. Furthermore, mRNA and protein expression of anti-apoptotic, soluble Fas isoform FasExo6Del and anti-apoptotic Bcl-xL were induced. Surprisingly, high shear stress also elevated mRNA and protein expression of pro-apoptotic Bak. The shear stress-induced up-regulation of Bcl-xL and Bak mRNA can be abrogated by inhibition of the endothelial NO synthase. We propose that altered expression of Bcl-xL and the Fas system is involved in the protective effect of laminar shear stress against apoptosis in human endothelial cells.

Shear stress modulates expression of Cu/Zn superoxide dismutase in human aortic endothelial cells.

A major determinant of the level of cellular superoxide anion (O2-.) is the dismutation of O2-. to hydrogen peroxide by the enzyme superoxide dismutase (SOD). Three forms of SOD exist, but in endothelial cells, the major form outside of the mitochondria is the cytosolic copper/zinc-containing superoxide dismutase (Cu/Zn SOD). Since fluid shear stress is an important determinant of the function and structure of endothelial cells in vivo, we examined the effect of laminar shear stress on the expression of Cu/Zn SOD in cultured human aortic endothelial cells. Laminar shear stress of 0.6 to 15 dyne/cm2 increased Cu/Zn SOD mRNA in a time- and dose-dependent manner in human aortic endothelial cells. Shear stress also increased both Cu/Zn SOD protein content and the enzyme activity. Nuclear runon assays showed that nuclei from human aortic endothelial cells exposed to laminar shear stress had a 1.6-fold greater transcriptional activity of the Cu/Zn SOD gene compared with cells not exposed to shear, indicating that an increase in Cu/Zn SOD mRNA induced by laminar shear stress is at least in part mediated by increased transcription. In contrast, shear stress had no effect on Cu/Zn SOD mRNA levels in human aortic smooth muscle cells. These findings show that physiological levels of shear stress increase expression of Cu/Zn SOD in the endothelium. This adaptation to shear stress might augment the effect of locally produced NO. and thereby promote the antiatherogenic and anti-inflammatory properties of the endothelial cell.

Flow-dependent cytosolic acidification of vascular endothelial cells.

Hemodynamic shear stress affects endothelial cell structure and function, but little is known about the signal transduction mechanisms involved in these processes. The effect of laminar shear stress on cytosolic pH (pHi) was examined in rat aortic endothelial cells cultured in glass capillary tubes. Shear stress forces led to a rapid decrease in pHi (maximal effect 0.09 pH unit at 13.4 dynes per square centimeter). Removal of specific ions or addition of exchange inhibitors suggests that in vascular endothelial cells shear stress forces activate both an alkali extruder, sodium ion-independent chloride-bicarbonate ion exchange, and an acid extruder, sodium-hydrogen ion exchange; the net effect in physiologic buffer with the bicarbonate ion is a decrease in pHi.