Suzanne G. Eskin

DNA microarray reveals changes in gene expression of shear stressed human umbilical vein endothelial cells

Using DNA microarray screening (GeneFilter 211, Research Genetics, Huntsville, AL) of mRNA from primary human umbilical vein endothelial cells (HUVEC), we identified 52 genes with significantly altered expression under shear stress [25 dynes/cm2 for 6 or 24 h (1 dyne = 10 μN), compared with matched stationary controls]; including several genes not heretofore recognized to be shear stress responsive. We examined mRNA expression of nine genes by Northern blot analysis, which confirmed the results obtained on DNA microarrays. Thirty-two genes were up-regulated (by more than 2-fold), the most enhanced being cytochromes P450 1A1 and 1B1, zinc finger protein EZF/GKLF, glucocorticoid-induced leucine zipper protein, argininosuccinate synthase, and human prostaglandin transporter. Most dramatically decreased (by more than 2-fold) were connective tissue growth factor, endothelin-1, monocyte chemotactic protein-1, and spermidine/spermine N1-acetyltransferase. The changes observed suggest several potential mechanisms for increased NO production under shear stress in endothelial cells.

Bradykinin-induced increases in cytosolic calcium and ionic currents in cultured bovine aortic endothelial cells.

The goal of the present study was to determine if voltage-sensitive calcium channels are present in bovine aortic endothelial cell plasmalemma and if they contribute to the rise in cytosolic calcium produced by bradykinin. After bradykinin (100 nM) exposure, endothelial cell associated fura-2 fluorescence peaked within 10-20 seconds and then declined to a steady level 2- to 3-fold above resting values. Pretreatment with lanthanum (20 μM) abolished the steady level produced by bradykinin but had little effect on the initial, transient rise in cytosolic calcium. Chelation of extracellular calcium with EGTA before addition of bradykinin resulted in a substantial decrease in the fura-2 transient and elimination of the long-lasting component. Nimodipine (3 μM) and nitrendipine (1 μM) were without effect on either phase of the bradykinin-induced response. Moreover, elevation of extracellular potassium failed to produce a rise in intracellular calcium. With the use of the tight seal technique to voltage clamp the cells, inwardly rectifying and calcium-activated potassium currents were found to exist in the endothelial cells. Addition of bradykinin (100 nM) elicited a calcium-activated potassium current that was eliminated in the absence of intracellular potassium. No voltage-sensitive calcium currents were activated when the cells were exposed to 10 mM or 110 mM calcium chloride in the presence or absence of bradykinin. The binding of [3H]( + )PN200-110 to endothelial cell membrane preparations was 1-3 orders of magnitude lower than that observed in PC-12, GH3, or BC3H1 cell membranes. Together, these results suggest that cloned bovine aortic endothelial cells lack voltage-sensitive calcium channels. Therefore, the changes in cytosolic calcium stimulated by bradykinin that are dependent on extracellular calcium must occur via some other calcium influx pathway. (Circulation Research 1987;61:632-640)

Mechanical effects on endothelial cell morphology: In vitro assessment

SummaryEndothelial cells are subjected to fluid mechanical forces which accompany blood flow. These cells become elongated and orient their long axes parallel to the direction of shear stress when the cultured cells are subjected to flow in an in vitro circulatory system. When the substrate is compliant and cyclically deformed, to simulate effects of pressure in the vasculature, the cells elongate an orient perpendicular to the axis of deformation. Cell shape changes are reflected in the alignment of microtubule networks. The systems described provide tools for assessing the individual roles of shear stress, pressure, and mechanical strain on vascular cell structure and function.

Shear stress increases inositol trisphosphate levels in human endothelial cells

To elucidate some of the early mechanisms underlying the response of primary human endothelial cells to the initiation of flow, we investigated the changes in inositol lipid metabolism in cells exposed to arterial and venous levels of shear stress. We used a radioimmunoassay specific for inositol-1,4,5-trisphosphate (Ins1,4,5P3) to demonstrate that initiation of an arterial shear stress caused a rapid rise in Ins1,4,5P3 levels which peaked after approximately 30 seconds of flow (2.1 +/- 0.2 fold stimulation) and remained elevated for at least 6 minutes after the initiation of flow. This increased Ins1,4,5P3 concentration is similar in magnitude to the increase caused by 10 microM histamine (2.8 +/- 0.3 fold stimulation). Thus these cells may detect the presence of mechanical stress by a signal transduction pathway involving inositol lipid metabolism.

Fluid flow decreases preproendothelin mRNA levels and suppresses endothelin-1 peptide release in cultured human endothelial cells

Endothelin-1, a 21-amino acid peptide secreted by endothelial cells, has constrictor and mitogenic activity for vascular smooth muscle cells, and its mitogenic activity is synergistic with that of platelet-derived growth factor. Endothelial cell-derived endothelin-1 might therefore contribute to intimal hyperplasia in reendothelialized segments of vascular grafts or of endarterectomy and angioplasty sites. Because intimal hyperplasia occurs most often at sites with disordered flow patterns and lower fluid shear stress, we tested the effects of static culture versus high laminar shear stress (25 dyne/cm2) on endothelin-1 precursor (preproendothelin) gene mRNA transcript levels and endothelin-1 peptide release in cultured human endothelial cells. Primary cultures of human umbilical vein endothelial cells were subjected to controlled levels of shear stress in parallel plate flow chambers for 24 hours. To detect preproendothelin mRNA we applied a linked reverse transcriptase-polymerase chain reaction (RT/PCR) to RNA extracted from cultures. Southern blots of RT/PCR reaction products were hybridized with radioactive phosphorous (32P) labeled probes for the amplified preproendothelin complementary deoxyribonucleic acid (cDNA). Detection by RT/PCR of mRNA for glyceraldehyde 3-phosphate dehydrogenase was used to measure a constitutively expressed control signal. Endothelin-1 release into culture medium was measured by radioimmunoassay. Application of 25 dyne/cm2 of shear stress for 24 hours sharply reduced endothelial cell levels of precursor preproendothelin mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Response of cultured endothelial cells to steady flow

A system has been developed for subjecting endothelial cell monolayers to prolonged steady flow while maintaining normal culture conditions. Cloned bovine endothelial cells were grown to confluence on one wall of a square glass tube which was then incorporated in the flow circuit. Flow rates of 19-21 ml/min were sustained for periods of 6-45 hr, subjecting the cells along the center line of the wall of the tube to a maximum shear stress of 34 dyn/cm2. The cells in all the experiments remained attached and viable when subjected to this shear stress. Photographic data from experimental runs were qualitatively assessed for changes in cell morphology, confluence, and orientation and were compared to data from matched stationary controls. Five experiments were chosen for quantitative morphometric analysis. In three experiments, the cells showed elongation with their long axes aligned with the direction of flow in 6.5, 21, and 22 hr. In the other experiments, either the cells formed a swirling pattern or no change in morphology was apparent. Although cell shape (form) changed in response to shear stress, cell area remained unaffected by exposure to flow.

Effects of Fluid Shear Stress on Gene Regulation of Vascular Cells

Hemodynamic forces such as fluid shear stress play an active role in many physiological and pathophysiological processes of the cardiovascular system. Shear stress resulting from blood flow and transmural plasma flux alters the function of vascular cell (primarily endothelial cells), leading to both rapid and slower adaptive tissue responses. Transmission of the shear stress signal throughout the vascular cell involves a complex interplay between cytoskeletal and biochemical elements and results in changes in structure, metabolism, and gene expression. Herein we review current knowledge on flow‐induced mechanotransduction in the vascular endothelial cell and the molecular mechanisms believed responsible for shear‐induced endothelial and smooth muscle cell gene regulation with an emphasis on signal transduction.

Endothelial cell responses to atheroprone flow are driven by two separate flow components: low time-average shear stress and fluid flow reversal

To simulate the effects of shear stress in regions of the vasculature prone to developing atherosclerosis, we subjected human umbilical vein endothelial cells to reversing shear stress to mimic the hemodynamic conditions at the wall of the carotid sinus, a site of complex, reversing blood flow and commonly observed atherosclerosis. We compared the effects of reversing shear stress (time-average: 1 dyn/cm(2), maximum: +11 dyn/cm(2), minimum: -11 dyn/cm(2), 1 Hz), arterial steady shear stress (15 dyn/cm(2)), and low steady shear stress (1 dyn/cm(2)) on gene expression, cell proliferation, and monocyte adhesiveness. Microarray analysis revealed that most differentially expressed genes were similarly regulated by all three shear stress regimens compared with static culture. Comparisons of the three shear stress regimens to each other identified 138 genes regulated by low average shear stress and 22 genes regulated by fluid reversal. Low average shear stress induced increased cell proliferation compared with high shear stress. Only reversing shear stress exposure induced monocyte adhesion. The adhesion of monocytes was partially inhibited by the incubation of endothelial cells with ICAM-1 blocking antibody. Increased heparan sulfate proteoglycan expression was observed on the surface of cells exposed to reversing shear stress. Heparinase III treatment significantly reduced monocyte adhesion. Our results suggest that low steady shear stress is the major impetus for differential gene expression and cell proliferation, whereas reversing flow regulates monocyte adhesion.

Expression of CYP1A1 and CYP1B1 in human endothelial cells: regulation by fluid shear stress

AIMS CYP1A1 and CYP1B1, members of the cytochrome P450 protein family, are regulated by fluid shear stress. This study describes the effects of duration, magnitude and pattern of shear stress on CYP1A1 and CYP1B1 expressions in human endothelial cells, towards the goal of understanding the role(s) of these genes in pro-atherogenic or anti-atherogenic endothelial cell functions. METHODS AND RESULTS We investigated CYP1A1 and CYP1B1 expressions under different durations, levels, and patterns of shear stress. CYP1A1 and CYP1B1 mRNA, protein, and enzymatic activity were maximally up-regulated at > or =24 h of arterial levels of shear stress (15-25 dynes/cm2). Expression of both genes was significantly attenuated by reversing shear stress when compared with 15 dynes/cm2 steady shear stress. Small interfering RNA knockdown of CYP1A1 resulted in significantly reduced CYP1B1 and thrombospondin-1 expression, genes regulated by the aryl hydrocarbon receptor (AhR). Immunostaining of human coronary arteries showed constitutive CYP1A1 and CYP1B1 protein expressions in endothelial cells. Immunostaining of mouse aorta showed nuclear localization of AhR and increased expression of CYP1A1 in the descending thoracic aorta, whereas reduced nuclear localization of AhR and attenuated CYP1A1 expression were observed in the lesser curvature of the aortic arch. CONCLUSION CYP1A1 and CYP1B1 gene and protein expressions vary with time, magnitude, and pattern of shear stress. Increased CYP1A1 gene expression modulates AhR-regulated genes. Based on our in vitro reversing flow data and in vivo immunostained mouse aorta, we suggest that increased expression of both genes reflects an anti-atherogenic endothelial cell phenotype.

Differential Regulation of Protease Activated Receptor-1 and Tissue Plasminogen Activator Expression by Shear Stress in Vascular Smooth Muscle Cells

Recent studies have demonstrated that vascular smooth muscle cells are responsive to changes in their local hemodynamic environment. The effects of shear stress on the expression of human protease activated receptor-1 (PAR-1) and tissue plasminogen activator (tPA) mRNA and protein were investigated in human aortic smooth muscle cells (HASMCs). Under conditions of low shear stress (5 dyn/cm2), PAR-1 mRNA expression was increased transiently at 2 hours compared with stationary control values, whereas at high shear stress (25 dyn/cm2), mRNA expression was decreased (to 29% of stationary control; P<0.05) at all examined time points (2 to 24 hours). mRNA half-life studies showed that this response was not due to increased mRNA instability. tPA mRNA expression was decreased (to 10% of stationary control; P<0.05) by low shear stress after 12 hours of exposure and was increased (to 250% of stationary control; P<0.05) after 24 hours at high shear stress. The same trends in PAR-1 mRNA levels were observed in rat smooth muscle cells, indicating that the effects of shear stress on human PAR-1 were not species-specific. Flow cytometry and ELISA techniques using rat smooth muscle cells and HASMCs, respectively, provided evidence that shear stress exerted similar effects on cell surface-associated PAR-1 and tPA protein released into the conditioned media. The decrease in PAR-1 mRNA and protein had functional consequences for HASMCs, such as inhibition of [Ca2+] mobilization in response to thrombin stimulation. These data indicate that human PAR-1 and tPA gene expression are regulated differentially by shear stress, in a pattern consistent with their putative roles in several arterial vascular pathologies.