Effect of topographical micropatterning on human endothelial cells
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ABSTRACT: 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: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.
Project description:Angiogenesis is tightly regulated by both soluble growth factors and cellular interactions with the extracellular matrix (ECM). While cell adhesion via integrins has been shown to be required for growth factor signaling and downstream angiogenesis, the effects of quantitative changes in cell adhesion and spreading against the ECM remain less clear. Examining changes in global gene expression in limited versus high adhesion contexts in human umbilical vein endothelial cells, we demonstrated a VEGF-induced upregulation of genes associated with vascular invasion and remodeling when cell adhesion was restricted, whereas cells on highly adhesive surfaces upregulated genes associated with a proliferative response. HUVECs were cultured on micropatterned islands of fibronectin or allowed to spread fully for 2 hrs and then stimulated with 50ng/ml VEGF or no growth factor for 18 hrs in starve media. Sample RNA from three biological replicates was extracted and prepared for hybridization on Affymetrix microarrays.
Project description:Lymphatic valves are specialized units regularly distributed along collecting vessels that allow unidirectional forward propulsion of the lymph, and its efficient transport from tissues to the bloodstream. Lymphatic endothelial cells that cover lymphatic valve sinuses are subjected to complex flow patterns, due to recirculation of the lymph during the collecting vessel pumping cycle. They also express high levels of FOXC2 transcription factor. We used microarrays to study the transcriptional networks controlled by FOXC2 in human lymphatic endothelial cells subjected to oscillatory shear stress or cultured under static conditions. Human lymphatic endothelial cells were transfected with control or FOXC2 siRNAs and subjected to 24-hour oscillatory shear stress (1 dyn/cm2; 1/4 Hz) or kept under static conditions as a control. RNA were amplified and hybridized on Affymetrix Human Gene 1.0 ST Arrays. The experiment was run twice independently, using each time a different siRNA to knockdown FOXC2, as previously described (Sabine et al, 2012, Dev Cell).
Project description:The aim of this study was to investigate the effect of endothelial cell (EC) elongation and alignment, independent of fluid shear stress (FSS), on the endothelial transcriptome. Endothelial cells are continuously exposed to a variety of biomechanical stimuli and must adapt their structure, gene expression, and phenotype to maintain vascular homeostasis. FSS-induced changes in EC morphology are highly associated with functional phenotype. However, to fully investigate EC mechanotransduction pathways, it is essential to decouple cell morphology from FSS. We, therefore, investigated the effects of elongated and aligned endothelial monolayers on the endothelial transcriptome. Baboon carotid artery endothelial cells (ECs) were seeded on either fully non-patterned, planar polyurethane culture substrates or micropatterned (3 µm wide anisotropic ridges and grooves, 1 µm depth) polyurethane culture substrates. We found EC elongation and alignment within static culture induced a highly differential gene transcriptome with a multitude of pro-inflammatory genes downregulated by ECs on micropatterned versus planar substrates. Collectively, our data support that endothelial morphology independently holds a critical role in the genetic transcriptome and may contribute to cardiovascular diseases and inflammation.
Project description:This study characterizes the response of primary human endothelial cells (human umbilical vein endothelial cells, HUVECs) to the relative shear stress changes that occur during the initiation of arteriogenesis at the entrance regions to a collateral artery network. HUVECs were preconditioned to a baseline level of unidirectional shear of 15 dynes/cm2 for 24 hours. After 24 hours preconditioning, HUVECs were subjected to an arteriogenic stimulus that mimics the shear stress changes observed in the opposing entrance regions into a collateral artery network. The arteriogenic stimulus consisted of a 100% step wise increase in shear stress magnitude to a unidirectional 30 dynes/cm2 in either the same or opposite direction of the preconditioned shear stress. This simulates either the feeding entrance to the collateral artery circuit or the region that drains into the vasculature downstream of an obstruction in a major artery, respectively. In vivo analysis of collateral growth in the mouse hindlimb showed enhanced outward remodeling in the re-entrant (direction reversing) region that reconnects to the downstream arterial tree, suggesting reversal of shear stress direction as a key enhancer of arteriogenesis. Transcriptional profiling using microarray techniques identified that the reversal of shear stress direction, but not an increase in shear stress alone, yielded a broad-based enhancement of the mechanotransduction pathways necessary for the induction of arteriogenesis. Human umbilical vein endothelial cells (HUVECs) were preconditioned to a unidirectional clockwise shear stress of 15 dynes/cm2 for 24 hours. An acute increase in shear stress magnitude to 30 dynes/cm2 in either a clockwise (non-reversed) or counter-clockwise (reversed) direction was applied for 6 hours. An additional preconditioned control culture was maintained under a unidirectional clockwise shear stress of 15 dynes/cm2 and harvested at the same time point, 6 hours post-conditioning. Each condition of reversed, non-reversed, and control was performed in tandem from the same starting cell culture as one replicate. The total experiment consisted of four replicates. Gene transcription was then assessed using microarray expression analysis.
Project description:The mechanisms by which physical forces regulate cells to determine complexities of vascular structure and function are enigmatic. Here we show the role the ion channel subunit Piezo1 (FAM38A). Disruption of mouse Piezo1 gene disturbed vascular development and was embryonic lethal within days of the heart beating to cause blood flow. Importance of Piezo channels as sensors of blood flow was indicated by Piezo1 dependence of shear stress-evoked ionic current and calcium influx in endothelial cells and the ability of exogenous Piezo1 to confer shear stress sensitivity on cells that otherwise lacked. Downstream of this calcium influx was proteoase activity and spatial organization of endothelial cells to the polarity of the applied force. Without Piezo1, normal endothelial cell organization was lacking. The data suggest Piezo1 channels as pivotal integrators of vascular architacture with physiological mechanical force.
Project description:The arterial endothelium’s response to its flow environment is critical to vascular homeostasis. The endothelial glycocalyx has been shown to play a major role in mechanotransduction, but the extent to which the components of the glycocalyx affect the overall function of the endothelium remains unclear. The objective of this study was to further elucidate the role of heparan sulfate as a mechanosensor on the surface of the arterial endothelium, by (1) expanding the variety of shear waveforms investigated, (2) continuously suppressing heparan sulfate expression rather than using a pre-flow batch treatment, and (3) performing microarray analysis on post-flow samples. Porcine aortic endothelial cells were exposed to non-reversing, reversing, and oscillatory shear waveforms for 24 hours with or without continuous heparan sulfate suppression with heparinase. All shear waveforms significantly increased the amount of heparan sulfate on the surface of the endothelium. Suppression of heparan sulfate to less than 25% of control levels did not inhibit shear-induced cell alignment or nitric oxide production, or alter gene expression, for any of the shear waveforms investigated. We infer that heparan sulfate on the surface of porcine aortic endothelial cells is not the primary mechanosensor for many shear-responsive endothelial cell functions in this species. Porcine aortic endothelial cells were exposed to 3 different shear waveforms for 24 hours with or without the addition of 300 mU/ml heparinase III to the flow media. The shear waveforms inculded Non-reversing (15 ± 15 dyne/cm2, 1 Hz), Steady (15 dyne/cm2), or Oscillatory (0 ± 15 dyne/cm2, 1 Hz) shear. Four replicates of each condition were performed for a total of 24 experiments. Each experimental sample was hybridized to an oligonucleotide array along with a standard reference sample (static cells).
Project description:Precise vascular patterning is critical for normal growth and development. The ERG transcription factor drives Delta like ligand 4 (DLL4)/Notch signalling and is thought to act as pivotal regulators of endothelial cell (EC) dynamics and developmental angiogenesis. However, molecular regulation of ERG activity remains obscure. Using a series of EC specific Focal Adhesion Kinase (FAK)-knockout (KO) and point-mutant FAK-knockin mice, we show that loss of ECFAK, its kinase activity or phosphorylation at FAK-Y397, but not FAK-Y861, reduces ERG and DLL4 expression levels together with concomitant aberrations in vascular patterning. Rapid Immunoprecipitation Mass Spectrometry of Endogenous Proteins identified that endothelial nuclear-FAK interacts with the de-ubiquitinase USP9x and the ubiquitin ligase TRIM25 enzymes. Further in silico analysis corroborates that ERG interacts with USP9x and TRIM25. Moreover, ERG levels are reduced in FAKKO ECs via a ubiquitin-mediated post-translational modification programme involving USP9x and TRIM25. Re-expression of ERG in vivo and in vitro rescues the aberrant vessel sprouting defects observed in the absence of ECFAK. Our findings identify ECFAK as a regulator of retinal vascular patterning by controlling ERG protein degradation via TRIM25/USP9x.
Project description:Endothelial cells (ECs) in the descending aorta are exposed to high laminar shear stress, which supports an anti-inflammatory phenotype. High laminar shear stress also supports flow-aligned cell elongation and front-rear polarity, but whether these are required for the anti-inflammatory phenotype is unclear. Here, we show that Caveolin-1-rich microdomains polarize to the downstream end of ECs exposed to continuous high laminar flow. These microdomains are characterized by high membrane rigidity, filamentous actin (F-actin), and raft-associated lipids. Transient receptor potential vanilloid-type 4 (Trpv4) ion channels are ubiquitously expressed on the plasma membrane but mediate localized Ca2+ entry only at these microdomains where they physically interact with clustered Caveolin-1. The resultant focal Ca2+ bursts activate endothelial nitric oxide synthase (eNOS) within the confines of these domains. Importantly, we find that signaling at these domains requires both cell body elongation and sustained flow. Finally, Trpv4 signaling at these domains is necessary and sufficient to suppress inflammatory gene expression, and ectopic activation of Trpv4 channels ameliorates the inflammatory response to stimuli both in vitro and in vivo. Our work reveals a novel polarized mechanosensitive signaling hub that dampens inflammatory gene expression in arterial ECs.
Project description:In this study, we characterized the adaptive response of arterial endothelial cells to acute increases in shear stress magnitude in well-defined in vitro settings. Porcine endothelial cells were preconditioned by a basal level shear stress of 15 ± 15 dynes/cm2 at 1 Hz for 24 hours, and an acute increase in shear stress magnitude (30 ± 15 dynes/cm2) was then applied. The transcriptomics studies using microarray identified genes that were sensitive to the elevated shear magnitude. A significant number of the identified genes in our study are previously unknown as sensitive to shear stress. Porcine endothelial cells were preconditioned by a basal level shear stress of 15 ± 15 dynes/cm2 at 1 Hz for 24 hours, and an acute increase in shear stress magnitude (30 ± 15 dynes/cm2) was then applied. Gene expression at multiple time points was measured using microarray.