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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- "Too intense cell culture medium flow compromises metabolic functions of hepatocytes and induces significant morphological changes. Moreover, the viability of hepatocytes cultured under high shear conditions is usually lower than that of static controls (Park et al. 2007; Nakatsuka et al. 2006; Powers et al. 2002b; Tanaka et al. 2006). Therefore, it is important to adapt the culture medium flow rates in such a manner that an optimal balance between oxygen/nutrient supply and shear stress level is achieved. "
ABSTRACT: Continuously increasing understanding of the molecular triggers responsible for the onset of diseases, paralleled by an equally dynamic evolution of chemical synthesis and screening methods, offers an abundance of pharmacological agents with a potential to become new successful drugs. However, before patients can benefit of newly developed pharmaceuticals, stringent safety filters need to be applied to weed out unfavourable drug candidates. Cost effectiveness and the need to identify compound liabilities, without exposing humans to unnecessary risks, has stimulated the shift of the safety studies to the earliest stages of drug discovery and development. In this regard, in vivo relevant organotypic in vitro models have high potential to revolutionize the preclinical safety testing. They can enable automation of the process, to match the requirements of high-throughput screening approaches, while satisfying ethical considerations. Cultures of primary hepatocytes became already an inherent part of the preclinical pharmaco-toxicological testing battery, yet their routine use, particularly for long-term assays, is limited by the progressive deterioration of liver-specific features. The availability of suitable hepatic and other organ-specific in vitro models is, however, of paramount importance in the light of changing European legal regulations in the field of chemical compounds of different origin, which gradually restrict the use of animal studies for safety assessment, as currently witnessed in cosmetic industry. Fortunately, research groups worldwide spare no effort to establish hepatic in vitro systems. In the present review, both classical and innovative methodologies to stabilize the in vivo-like hepatocyte phenotype in culture of primary hepatocytes are presented and discussed.Archives of Toxicology 12/2012; 87(4). DOI:10.1007/s00204-012-0983-3 · 5.08 Impact Factor
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- "Several reports describe the effects of flow and shear stress on hepatocyte cultures (Mufti and Shuler, 1995; Nakatsuka et al., 2006; Powers et al., 2002b; Tanaka et al., 2006). Moreover, many investigators have shown that the viability of hepatocyte cultures under high shear is usually lower than that of static controls, indicating that the cells are under conditions of stress (Park et al., 2007). "
ABSTRACT: A generic "system on a plate" modular multicompartmental bioreactor array which enables microwell protocols to be transferred directly to the bioreactor modules, without redesign of cell culture experiments or protocols is described. The modular bioreactors are simple to assemble and use and can be easily compared with standard controls since cell numbers and medium volumes are quite similar. Starting from fluid dynamic and mass transport considerations, a modular bioreactor chamber was first modeled and then fabricated using "milli-molding," a technique adapted from soft lithography. After confirming that the shear stress was extremely low in the system in the range of useful flow rates, the bioreactor chambers were tested using hepatocytes. The results show that the bioreactor chambers can increase or maintain cell viability and function when the flow rates are below 500 microL/min, corresponding to wall shear stresses of 10(-5) Pa or less at the cell culture surface.Biotechnology and Bioengineering 05/2010; 106(1):127-37. DOI:10.1002/bit.22671 · 4.16 Impact Factor
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- "Our findings indicate a divergent role for Ets-1 in response to shear stress stimulation, as illustrated by the upregulation of protease inhibitors. While there is precedence for the shear stress dependent transcriptional regulation and increased DNA binding activity of Ets-1, which were observed in rheumatic synovial cells (Sun and Yokota, 2001) and cultured hepatocytes (Nakatsuka et al., 2006), the effect of fluid shear stress on Ets-1 expression in endothelial cells has not been reported previously. Previous studies indicate that shear stress downregulates expression of MMPs and uPA in endothelial cells (Yun et al., 2002; Sokabe et al., 2004; Milkiewicz et al., 2006). "
ABSTRACT: Elevated shear stress within the skeletal muscle microvasculature is implicated in the induction of a longitudinal splitting form of angiogenesis, which is characterized by the lack of basement membrane breakage. We investigated whether the transcriptional regulator, Ets-1, is responsive to changes in hemodynamic forces and if so, whether Ets-1 controls microvascular endothelial cell integrity by inducing the expression of inhibitors of matrix degrading proteases. Rats were treated with prazosin for 2, 4, and 7 days to increase in microvascular shear stress in hindlimb skeletal muscles. In complimentary in vitro experiments, rat microvascular skeletal muscle endothelial cells were exposed to laminar shear stress (15 dyne/cm(2)) for 0.5, 2, and 24 h. TaqMan PCR analysis of laser microdissected capillaries isolated from EDL muscles demonstrated transient (after 2 days) induction of Ets-1 gene expression. In cultured cells, a transient up-regulation of Ets-1 mRNA was observed after 2 h shear stimulation, accompanied by increased phosphorylation of Ets-1 and enhanced Ets-1 DNA binding activity. This response was modulated by ERK1/2 and p38 MAP kinases, but was not dependent on NOS or COX-2 activity. PAI-1, TIMP-1 and TIMP-3 mRNA were elevated significantly in prazosin treated EDL, and in response to shear stimulation in vitro. In cultured endothelial cells, Ets-1 RNA interference abolished the shear-induced increases in Ets-1, PAI-1, TIMP-1, and TIMP-3 mRNA expression. These results suggest that enhanced laminar shear stress may act to preserve the integrity of microvascular walls in part through Ets-1-dependent induction of protease inhibitors.Journal of Cellular Physiology 11/2008; 217(2):502-10. DOI:10.1002/jcp.21526 · 3.87 Impact Factor