Shear stress induces hepatocyte PAI-1 gene expression through cooperative Sp1/Ets-1 activation of transcription.
ABSTRACT Partial hepatectomy causes hemodynamic changes that increase portal blood flow in the remaining lobe, where the expression of immediate-early genes, including plasminogen activator inhibitor-1 (PAI-1), is induced. We hypothesized that a hyperdynamic circulatory state occurring in the remaining lobe induces immediate-early gene expression. In this study, we investigated whether the mechanical force generated by flowing blood, shear stress, induces PAI-1 expression in hepatocytes. When cultured rat hepatocytes were exposed to flow, PAI-1 mRNA levels began to increase within 3 h, peaked at levels significantly higher than the static control levels, and then gradually decreased. The flow-induced PAI-1 expression was shear stress dependent rather than shear rate dependent and accompanied by increased hepatocyte production of PAI-1 protein. Shear stress increased PAI-1 transcription but did not affect PAI-1 mRNA stability. Functional analysis of the 2.1-kb PAI-1 5'-promoter indicated that a 278-bp segment containing transcription factor Sp1 and Ets-1 consensus sequences was critical to the shear stress-dependent increase of PAI-1 transcription. Mutations of both the Sp1 and Ets-1 consensus sequences, but not of either one alone, markedly prevented basal PAI-1 transcription and abolished the response of the PAI-1 promoter to shear stress. EMSA and chromatin immunoprecipitation assays showed binding of Sp1 and Ets-1 to each consensus sequence under static conditions, which increased in response to shear stress. In conclusion, hepatocyte PAI-1 expression is flow sensitive and transcriptionally regulated by shear stress via cooperative interactions between Sp1 and Ets-1.
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ABSTRACT: Laminar shear stress promotes vascular integrity by inhibiting proteolysis of the extracellular matrix (ECM) surrounding the microvasculature. We hypothesized that the matrix metalloproteinase inhibitor TIMP-1 would be upregulated in endothelial cells exposed to shear stress. Microvascular endothelial cells isolated from rat or mouse skeletal muscles were exposed to laminar shear stress for 2, 4, or 24 h. A biphasic increase in TIMP-1 protein was observed at 2 and 24 h of shear stress exposure. Sp-1 siRNA prevented the increase in TIMP-1 after 2, but not 24, hours of shear exposure. TGFβ production and Smad2/3 phosphorylation are increased by shear stress. Inhibition of TGFβ signaling, either by use of the TGFβ receptor 1 inhibitor SB-431542 or with Smad 2/3 siRNA, abrogated the shear stress-induced increase in TIMP-1 mRNA after 24 h of shear stress exposure. These results suggest that both acute and chronic elevated laminar shear stress act to maintain vessel integrity through increasing TIMP-1 production, but that the TGFβ signaling pathway is essential to maintain TIMP-1 expression during chronic shear stress.Biochemistry and Cell Biology 02/2014; 92(1):77-83. · 2.35 Impact Factor
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ABSTRACT: Endothelial progenitor cells (EPCs) are mobilized from bone marrow to peripheral blood, and contribute to angiogenesis in tissues. In the process EPCs are exposed to the shear stress generated by blood flow and tissue fluid flow. Our previous study showed that shear stress promotes differentiation of EPCs into mature endothelial cells. In this study we investigated whether EPCs differentiate into arterial or venous endothelial cells in response to shear stress. When cultured EPCs derived from human peripheral blood were exposed to controlled levels of shear stress in a flow-loading device, the mRNA levels of the arterial endothelial cell markers ephrinB2, Notch1/3, Hey1/2, and ALK1 increased, but the mRNA levels of the venous endothelial cell markers EphB4 and NRP2 decreased. Both the ephrinB2 increase and the EphB4 decrease were shear-stress- rather than shear-rate-dependent. EphrinB2 protein was increased in shear-stressed EPCs, and the increase in ephrinB2 expression was due to activated transcription and not mRNA stabilization. Deletion analysis of the ephrinB2 promoter indicated that the cis-element (shear stress response element) is present within 106 bp 5' upstream from the transcription initiation site. This region contains the Sp1 consensus sequence, and a mutation in its sequence decreased the basal level of transcription and abolished shear stress-induced ephrinB2 transcription. Electrophoretic mobility shift assays and chromatin immunoprecipitation assays showed that shear stress markedly increased binding of Sp1 to its consensus sequence. These results indicate that shear stress induces differentiation of EPCs into arterial endothelial cells by increasing ephrinB2 expression in EPCs through Sp1 activation.01/2009;
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ABSTRACT: In this paper, we discuss the basic design requirements for the development of physiologically meaningful in vitro systems comprising cells, scaffolds and bioreactors, through a bottom up approach. Very simple micro- and milli-fluidic geometries are first used to illustrate the concepts, followed by a real device case-study. At each step, the fluidic and mass transport parameters in biological tissue design are considered, starting from basic questions such as the minimum number of cells and cell density required to represent a physiological system and the conditions necessary to ensure an adequate nutrient supply to tissues. At the next level, we consider the use of three-dimensional scaffolds, which are employed both for regenerative medicine applications and for the study of cells in environments which better recapitulate the physiological milieu. Here, the driving need is the rate of oxygen supply which must be maintained at an appropriate level to ensure cell viability throughout the thickness of a scaffold. Scaffold and bioreactor design are both critical in defining the oxygen profile in a cell construct and are considered together. We also discuss the oxygen-shear stress trade-off by considering the levels of mechanical stress required for hepatocytes, which are the limiting cell type in a multi-organ model. Similar considerations are also made for glucose consumption in cell constructs. Finally, the allometric approach for generating multi-tissue systemic models using bioreactors is described.Processes. 07/2014; 2(3):548-569.