Project description:Blood flow within the vasculature is a critical determinant of endothelial cell (EC) identity and functionality, yet the intricate interplay of various hemodynamic forces and their collective impact on endothelial and vascular responses are not fully understood. Specifically, the role of hydrostatic pressure in the context of flow response is understudied, despite its known significance in vascular development and disease. To address this gap, we developed in vitro models to investigate how pressure influences EC responses to flow. Our study demonstrates that elevated pressure conditions significantly modify shear-induced flow alignment and increase endothelial cell density, a phenomenon often observed in vascular diseases. Utilizing both bulk and single-cell RNA sequencing, we found that while flow is the primary driver of transcriptional changes from static conditions, pressure distinctly modulates this flow response by upregulating gene sets linked to arterial cell phenotypes. Conserved pressure-responsive transcriptional signatures identified in human ECs were upregulated during the onset of circulation in early mouse embryonic vascular development, where pressure was notably associated with transcriptional programs essential to arterial and hemogenic EC fates. Our findings emphasize the necessity of an integrative approach to endothelial cell mechanotransduction, one that encompasses the effects induced by pressure alongside other hemodynamic forces.
Project description:The luminal surface of the endothelium is exposed to dynamic blood flow patterns that are known to affect endothelial cell phenotype. In this study, human aortic endothelial cells (HAECs) were exposed to unidirectional laminar flow (20 dynes/cm2) or disturbed flow (±4 dynes/cm2, 2 Hz) for 48 hrs and then subjected to RNA sequencing. The sequencing data was used to analyze changes in endothelial cell gene expression and pathways associated with lipid metabolism.
Project description:Objective The vessel wall is continuously exposed to hemodynamic forces generated by blood flow. Endothelial mechanosensors perceive and translate mechanical signals via cellular signaling pathways into biological processes that control endothelial development, phenotype and function. Here, we aim to unravel the molecular mechanisms underlying endothelial mechanosensing. Approach and results We applied a quantitative mass spectrometry approach combined with cell surface chemical footprinting to assess the hemodynamic effects on the endothelium on a system-wide level. These studies revealed that of the >5000 quantified proteins 104 were altered, which were highly enriched for extracellular matrix proteins and proteins involved in cell-matrix adhesion. Cell surface proteomics furthermore indicated that LAMA4 was proteolytically processed upon flow-exposure, which corresponded to the decreased LAMA4 mass observed on immunoblot. Immunofluorescence microscopy studies highlighted that the endothelial basement membrane was drastically remodeled upon flow exposure. We observed a network-like pattern of LAMA4 and LAMA5, which corresponded to the localization of laminin-adhesion molecules ITGA6 and ITGB4. Furthermore, the adaptation to flow-exposure did not affect the inflammatory response to tumor necrosis factor α, indicating that inflammation and flow trigger fundamentally distinct endothelial signaling pathways with limited reciprocity and synergy. Conclusions Taken together, this study uncovers the blood flow-induced remodeling of the basement membrane and stresses the importance of the subendothelial basement membrane in vascular homeostasis.
Project description:TBI causes the disruption of blood vessels in brain leading to hemorrhage, edema, and changes in cerebral blood flow. How such altered vascular and flow condition affect endothelial cells, the cellular unit of blood vessels, is not known. We used single cell RNA sequencing (scRNAseq) to analyze the heterogenity in endothelial cell clusters in brain, and how key cellular and signaling signatures of these clusters are temporally changed post-TBI at the site of injury.
Project description:BACKGROUND: The deSUMOylase SENP2 exerts athero-protective effects by inhibiting endothelial cell (EC) activation through attenuating ERK5 and p53 SUMOylation. Publicly available datasets show that SENP2 S344 is phosphorylated by Checkpoint Kinase 1 (CHK1), but the functional role remains unknown. METHODS: Mouse SENP2 S343A (human S344A) phosphodeficient knock in (KI) mutant was generated by CRISPR/Cas9, and vascular-specific function was assessed via bone marrow transplantation (BMT). ECs from KI and wild type (WT) mice were exposed to smooth (laminar flow; l-flow) or grooved (disturbed flow; d-flow) cone-and-plate devices and characterized by RNA sequencing (RNA-seq). RESULTS: L-flow increased CHK1 S280 and SENP2 S344 phosphorylation, which inhibited ERK5 and p53 SUMOylation and atherogenesis in vivo. BMT-generated vascular specific SENP2 S344A KI showed more atherogenesis but thinner fibrous cap formation specifically in the aortic arch area (d-flow) compared to that of WT mice. Ionizing radiation (IR) decreased CHK1 expression and SENP2 S344 phosphorylation, which might account for differences between systemic and BMT-generated vascular specific SENP2 S344A KI models. RNA-seq data analysis showed that SENP2 S344 phosphorylation in ECs in response to l-flow inhibited EC activation and fibrotic changes without interfering EC lineage phenotype. Lastly, l-flow-induced expression of genes was regulated by SENP2 S344 phosphorylation through ERK5 activation and inhibited EC apoptosis. CONCLUSIONS: We uncovered a novel mechanism by which l-flow inhibits EC activation, including proliferation, migration, inflammation, and fibrotic changes, via upregulating CHK1-mediated SENP2 S344 phosphorylation to attenuate atherogenesis. We also uncovered a unique expression pattern of fibrotic changes without affecting EC lineage, which is distinct from endothelial-to-mesenchymal transition and therefore should be considered a unique type of EC activation for its potential role in vulnerable plaque formation.
Project description:The objective of this study was to investigate the effect of Direct Current Stimulation (DCS) on the gene expression of human astrocytes (HA). DCS at 0.1 or 1mA was applied to monolayers of HA for 10 minutes. Expression of a set of neuroactive genes was assessed immediately of 1 hour after DCS using RT-qPCR. Because DCS can produce electroosmotic flow and fluid shear stress known to influence cell function, we compared three interventions: pressure-driven flow across the monolayer alone (conP), pressure-driven flow plus DCS (DCS P), and DCS alone with flow blocked (DCS S).
Project description:Biomechanical forces such as wall shear stress induced by flowing blood influence the behavior of vascular cells and have been shown to be crucial in the onset of vascular disease. Primary cilia are increasingly recognized as important biological sensors of low WSS. The goal of the study was to investigate gene expression profile of endothelial cell under low flow (2 dynes/cm2) as compared to physiological flow (30 dynes/cm2) in presence or absence of primary cilia.
Project description:Extracellular vesicles released by tumors (tEVs) disseminate via circulatory networks and promote microenvironmental changes in distant organs favoring metastatic seeding. Despite their abundance in the bloodstream, how hemodynamics affect the function of circulating tEVs remains unsolved. We experimentally tuned flow profiles in vitro (microfluidics) and in vivo (zebrafish) and demonstrated that efficient uptake of tEVs occurs in endothelial cells subjected to capillary-like hemodynamics. Such flow profiles partially reroute internalized tEVs towards non-acidic and non-degradative Rab14-positive endosomes, at the expense of lysosomes, suggesting that endothelial mechanosensing diverts tEVs from degradation. Subsequently, tEVs promote the expression of pro-angiogenic transcription factors in flow-stimulated endothelial cells and favor vessel sprouting in zebrafish. Altogether, we demonstrate that capillary-like flow profiles potentiate the pro-tumoral function of circulating tEVs by promoting their uptake and rerouting their trafficking. We propose that tEVs contribute to pre-metastatic niche formation by exploiting endothelial mechanosensing in specific vascular regions with permissive hemodynamics. This set of experiments represents RNAseq of HUVEC cells subjected to 400 µm/s flow versus no flow (static conditon).