470 Hypoxia-induced pulmonary hypertension in mice modifies the expression pattern of von willebrand factor gene in lung endothelial cells

Abstract

BackgroundVon Willebrand Factor (VWF) gene expression is restricted to endothelial cells and is commonly used as a marker to distinguish endothelial cells from other cell type. However, consistent with heterogeneity of endothelial cells, VWF is not uniformly expressed by endothelial cells of all vascular beds. In lung, while VWF expression is highly abundant and uniformly expressed in vessels of diameters 40 um or above, expression in capillaries and microvessels is low and displays a mosaic pattern. Increased VWF levels in lung is associated with diseases such as pulmonary hypertension (PH), contributing to the thrombogenicity, and increased mortality. However, the mechanism leading to increased VWF levels in PH has not been determined, and is the aim of our present studies. Analyses of the mechanism of endothelial-specific regulation of VWF gene in vivo has demonstrated that VWF gene contains regions that dictate its activation only in subpopulations of endothelial cells. We previously described a distinct region of the VWF promoter (sequences −487 to +247) that targets expression in vivo only to brain endothelium. We have also demonstrated that another specific regulatory region in intron 51 targets activation in lung endothelium.MethodsOur characterization of the region that targets the VWF promoter activity to lung endothelial cells and generation of transgenic mice that express LacZ gene under the regulation of these VWF regulatory sequences (HSS-VWF-LacZ and VWF-LacZ-HSS) provided the opportunity to explore whether pulmonary hypertension results in modulation of the VWF promoter activity in lung endothelial cells.ResultsOur analyses demonstrated that PH induced by hypoxia exposure results in upregulation of the exogenous VWF promoter activity in brain and lung (demonstrated by RNA and protein analyses of the LacZ transgene), and specifically activates expression in lung microvascular endothelial cells (demonstrated by immunoflourescence and confocal microscopy). This pattern of exogenous VWF promoter activity was concomitant with increased expression of endogenous VWF and a change in its expression pattern from primarily larger vessels endothelium to include those of microvasculature. Additionally promoter activation in hypoxic mice was detected in heart endothelial cells that in control mice do not support the activity of the VWF promoter-HSS (intron 51) sequences.ConclusionThese results suggests that hypoxia/PH leads to a phenotypic switch of microvascular endothelial cells from an anti-coagulant to a procoagulant activity, which is potentially regulated by changes in the activity of transacting factors that participate in regulation of VWF gene expression.Canadian Institutes of Health Research (CIHR) BackgroundVon Willebrand Factor (VWF) gene expression is restricted to endothelial cells and is commonly used as a marker to distinguish endothelial cells from other cell type. However, consistent with heterogeneity of endothelial cells, VWF is not uniformly expressed by endothelial cells of all vascular beds. In lung, while VWF expression is highly abundant and uniformly expressed in vessels of diameters 40 um or above, expression in capillaries and microvessels is low and displays a mosaic pattern. Increased VWF levels in lung is associated with diseases such as pulmonary hypertension (PH), contributing to the thrombogenicity, and increased mortality. However, the mechanism leading to increased VWF levels in PH has not been determined, and is the aim of our present studies. Analyses of the mechanism of endothelial-specific regulation of VWF gene in vivo has demonstrated that VWF gene contains regions that dictate its activation only in subpopulations of endothelial cells. We previously described a distinct region of the VWF promoter (sequences −487 to +247) that targets expression in vivo only to brain endothelium. We have also demonstrated that another specific regulatory region in intron 51 targets activation in lung endothelium. Von Willebrand Factor (VWF) gene expression is restricted to endothelial cells and is commonly used as a marker to distinguish endothelial cells from other cell type. However, consistent with heterogeneity of endothelial cells, VWF is not uniformly expressed by endothelial cells of all vascular beds. In lung, while VWF expression is highly abundant and uniformly expressed in vessels of diameters 40 um or above, expression in capillaries and microvessels is low and displays a mosaic pattern. Increased VWF levels in lung is associated with diseases such as pulmonary hypertension (PH), contributing to the thrombogenicity, and increased mortality. However, the mechanism leading to increased VWF levels in PH has not been determined, and is the aim of our present studies. Analyses of the mechanism of endothelial-specific regulation of VWF gene in vivo has demonstrated that VWF gene contains regions that dictate its activation only in subpopulations of endothelial cells. We previously described a distinct region of the VWF promoter (sequences −487 to +247) that targets expression in vivo only to brain endothelium. We have also demonstrated that another specific regulatory region in intron 51 targets activation in lung endothelium. MethodsOur characterization of the region that targets the VWF promoter activity to lung endothelial cells and generation of transgenic mice that express LacZ gene under the regulation of these VWF regulatory sequences (HSS-VWF-LacZ and VWF-LacZ-HSS) provided the opportunity to explore whether pulmonary hypertension results in modulation of the VWF promoter activity in lung endothelial cells. Our characterization of the region that targets the VWF promoter activity to lung endothelial cells and generation of transgenic mice that express LacZ gene under the regulation of these VWF regulatory sequences (HSS-VWF-LacZ and VWF-LacZ-HSS) provided the opportunity to explore whether pulmonary hypertension results in modulation of the VWF promoter activity in lung endothelial cells. ResultsOur analyses demonstrated that PH induced by hypoxia exposure results in upregulation of the exogenous VWF promoter activity in brain and lung (demonstrated by RNA and protein analyses of the LacZ transgene), and specifically activates expression in lung microvascular endothelial cells (demonstrated by immunoflourescence and confocal microscopy). This pattern of exogenous VWF promoter activity was concomitant with increased expression of endogenous VWF and a change in its expression pattern from primarily larger vessels endothelium to include those of microvasculature. Additionally promoter activation in hypoxic mice was detected in heart endothelial cells that in control mice do not support the activity of the VWF promoter-HSS (intron 51) sequences. Our analyses demonstrated that PH induced by hypoxia exposure results in upregulation of the exogenous VWF promoter activity in brain and lung (demonstrated by RNA and protein analyses of the LacZ transgene), and specifically activates expression in lung microvascular endothelial cells (demonstrated by immunoflourescence and confocal microscopy). This pattern of exogenous VWF promoter activity was concomitant with increased expression of endogenous VWF and a change in its expression pattern from primarily larger vessels endothelium to include those of microvasculature. Additionally promoter activation in hypoxic mice was detected in heart endothelial cells that in control mice do not support the activity of the VWF promoter-HSS (intron 51) sequences. ConclusionThese results suggests that hypoxia/PH leads to a phenotypic switch of microvascular endothelial cells from an anti-coagulant to a procoagulant activity, which is potentially regulated by changes in the activity of transacting factors that participate in regulation of VWF gene expression.Canadian Institutes of Health Research (CIHR) These results suggests that hypoxia/PH leads to a phenotypic switch of microvascular endothelial cells from an anti-coagulant to a procoagulant activity, which is potentially regulated by changes in the activity of transacting factors that participate in regulation of VWF gene expression.