Project description:Pulmonary microvascular endothelial cells were cultured on the inner surface of fibers (each 700 µm inner diameter) coated with either with native- or advanced glycation endproduct (AGE) modified fibronectin in hollow-fiber cartridges (FiberCell Systems, Inc.). Duration of shear stress application was 10 days. Shear stress was gradually increased to 17 dyne/cm2 in 2 samples (GSM32266: native fibronectin high shear stress or GSM45608: AGE-fibronectin) or was kept at constant low level of 1 dyne/cm2 in 1 sample (GSM41248: native fibronectin low shear stress). Keywords: parallel sample
Project description:Pulmonary microvascular endothelial cells were cultured on the inner surface of fibers (each 700 µm inner diameter) coated with either with native- or advanced glycation endproduct (AGE) modified fibronectin in hollow-fiber cartridges (FiberCell Systems, Inc.). Duration of shear stress application was 10 days. Shear stress was gradually increased to 17 dyne/cm2 in 2 samples (GSM32266: native fibronectin high shear stress or GSM45608: AGE-fibronectin) or was kept at constant low level of 1 dyne/cm2 in 1 sample (GSM41248: native fibronectin low shear stress). Keywords: parallel sample
Project description:Shear stress is known to regulate endothelial cell orientation along the direction of flow. We asked wither cellular patterning along, in the absence of shear could have similar biological effects as shear. We used DNA microarrays to examine the effect of cellular patterning on their transcriptome. Human microvascular endothelial cells were cultured in parallel micropatterned channels (30um wide channels, 30um apart) composed of polydimethylsiloxane, followed DNA Microarray analysis (Affymetrix 1.0 ST array)
Project description:Physiological shear stress, produced by blood flow, homeostatically regulates the phenotype of pulmonary endothelial cells exerting anti-inflammatory and anti-thrombotic actions and maintaining normal barrier function. In the pulmonary circulation hypoxia, due to high altitude or diseases such as COPD, causes vasoconstriction, increased vascular resistance and pulmonary hypertension. Hypoxia-induced changes in endothelial function play a central role in the development of this pulmonary hypertension. However, the direct interactive effects of hypoxia and shear stress on the pulmonary endothelial phenotype have not been extensively studied. We cultured human pulmonary microvascular endothelial cells (HPMEC) in normoxia or hypoxia while subjected to physiological shear stress or in static conditions. Unbiased proteomics was used to identify hypoxia-induced changes in protein expression. Using publicly available single cell RNA-seq datasets, differences in gene expression between the alveolar endothelial cells from COPD and healthy lungs were identified. 60 proteins were identified in HPMEC lysates whose expression changed in response to hypoxia in sheared but not in static conditions. mRNA for five of these (ERG, MCRIP1, EIF4A2, HSP90AA1 and DNAJA1) showed similar changes in the endothelial cells of COPD compared to healthy lungs. These data show that the proteomic responses of the pulmonary microvascular endothelium to hypoxia are significantly altered by shear stress and suggest that these differences are important in the development of hypoxic pulmonary vascular disease.
Project description:The lymphatic system removes fluid from the interstitial space and returns it to the blood with a tremendous capacity: during inflammation, lymph flow rates can increase dramatically; however, during chronic lymphedema, there is little or no flow. The ability of lymphatic endothelium to sense and actively regulate this function is unknown, and shear stress is likely a key indicator of lymph flow. We profiled gene expression in human dermal microvascular lymphatic endothelial cells exposed to 0, 2 and 20 dyn/cm2 shear stress as representative of chronic lymphedema, normal, and acute inflammatory conditions, respectively. We found important adaptive responses correlated to multiple aspects of lymphatic function. Importantly, shear stress upregulated intracellular water and solute transporters while decreasing cell-cell adhesion and basement membrane components and increasing cell-matrix interactions. This data indicate that during high loading conditions, both passive and active drainage function increases, while conversely when fluid drainage is blocked, transport function is diminished in the lymphatic endothelium. These data demonstrate the first functional-adaptive response of lymphatic endothelium to flow conditions, thus indicating that the lymphatic endothelium plays an active role in regulating their function. Keywords: Shear stress, dose response, cell type comparison Lymphatic endothelial cells were subjected to 0, 2, or 20 dyn/cm2 shear stress; blood endothelial cells were subjected to 0 or 20 dyn/cm2 shear stress. Four samples were used for each cell type/shear level group for a total of 20 samples. Each sample was independently compared to human universal reference RNA via two-color microarray analysis for a total of 20 arrays. In all cases, the experimental samples were labeled with Cy5 dye while the reference RNA was labeled with Cy3.
Project description:Endothelial cells (ECs) are constantly submitted in vivo to hemodynamical forces derived from the blood circulation, including shear stress (SS). EC are able to detect SS and consequently adapt their phenotype, thus affecting many endothelial functions. If a plethora of shear stress-regulated molecular networks have been described in peripheral EC, less is known about the molecular responses of microvascular brain ECs which constitute the blood-brain barrier (BBB). In this work, we investigated the response of human cerebral microvascular ECs to laminar physiological shear stress using the well characterized hCMEC/D3 cell line. Interestingly, we showed that hCMEC/D3 cells responded to shear stress by aligning perpendicularly to the flow direction, contrary to peripheral endothelial cells which aligned in the flow direction. Whole proteomic profiles were compared between hCMEC/D3 cells cultured either in static condition or under 5 or 10 dyn.cm-2 SS for three days. 3592 proteins were identified and expression levels were significantly affected for 3% of them upon both SS conditions. Pathway analyses were performed which revealed that most proteins overexpressed by SS refer to the antioxidant defense, probably mediated by activation of the NRF2 transcriptional factor. Regarding down-regulated proteins, most of them participate to the pro-inflammatory response, cell motility and proliferation. These findings confirm the induction of EC quiescence by laminar physiological SS and reveal a strong neuroprotective effect of SS on hCMEC/D3 cells, suggesting a similar effect on the BBB. Our results also showed that SS did not significantly increase expression levels nor did it affect the localization of junctional proteins or the functional activity of several ABC transporters (P-glycoprotein and MRPs). This work provides new insights on the response of microvascular brain EC to SS and on the importance of SS for optimizing in vitro BBB models.
Project description:Comparison of Human iPSC-derived Brain Microvascular Endothelial-like Cells (iBMECs) grown in poly(dimethylsiloxane) tissue chips. Data contains RNA-seq profiles of iBMECs exposed to various levels of shear stress ranging from 0, 0.01, 0.5, and 2.4 dyn/cm2; as well as RNA-seq profiles of FACS sorted iBMECs cultured alone or with primary human astrocytes and pericytes or with iPSC-derived neural progenitor cells.
Project description:To investigate sex differences at the transcriptome level in human pulmonary microvascular endothelial cells (HPMECs) from healthy male and female donors basally (in normoxia) and in hypoxic conditions. RNA-seq was performed on male (n=3) and female (n=4) HPMECs that were cultured in conditions of physiological shear stress (PMID: 36730645) in normoxia (21% O2) or in hypoxia (1% O2) for either 24 or 48 hours.
Project description:Many studies have characterized the results of shear stress changes on cultured endothelial cells in different bioreactor systems. However it is still unclear how an invasive intervention like stent procedure may influence the transcriptional response of endothelium. To study the simultaneous effects of shear stress changes and stent application on endothelial gene expression, we have used an experimental apparatus of laminar flow bioreactor (LFB) system with human cultured endothelial cells exposed or not exposed to stent procedure with different flow conditions. Microarray analysis was evaluated in each experimental protocol. HUVECs (2nd and 5th passage) covered on Thermanox slides were submitted to static, low and physiological (0, 1, 5 and 10 dyne/cm2) shear stress in absence (AS) or presence (PS) of stent in LFB system for 24h. Affymetryx analysis has been performed in duplicate by Consortium for Genomic Technologies (Cogentech; Milan, Italy)