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Real-time quantification of endothelial response to shear stress and vascular modulators.

ABSTRACT: Quiescence is commonly used to describe the inactive state of endothelial cells (ECs) in monolayers that have reached homeostasis. Experimentally quiescence is usually described in terms of the relative change in cell activity (e.g. turnover, speed, etc.) in response to a perturbation (e.g. solute, shear stress, etc.). The objective of this study is to provide new insight into EC quiescence by quantitatively defining the morphology and activity of confluent cell monolayers in response to shear stress and vascular modulators. Confluent monolayers of human umbilical vein ECs (HUVECs) were subjected to a range of shear stresses (4-16 dyne cm-2) under steady flow. Using phase contrast, time-lapse microscopy and image analysis, we quantified EC morphology, speed, proliferation, and apoptosis rates over time and detected differences in monolayer responses under various media conditions: basal media supplemented with growth factors, interleukin-8, or cyclic AMP. In all conditions, we observed a transition from cobblestone to spindle-like morphology in a dose-dependent manner due to shear stress. Cyclic AMP enhanced the elongation and alignment of HUVECs due to shear stress and reduced steady state cell speed. We observed the lowest proliferation rates below 8 dyne cm-2 and found that growth factors and cyclic AMP reduced proliferation and apoptosis; interleukin-8 similarly decreased proliferation, but increased apoptosis. We have quantified the response of ECs in confluent monolayers to shear stress and vascular modulators in terms of morphology, speed, proliferation and apoptosis and have established quantifiable metrics of cell activity to define vascular quiescence under shear stress.

SUBMITTER: DeStefano JG

PROVIDER: S-EPMC5490251 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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Real-time quantification of endothelial response to shear stress and vascular modulators.

DeStefano Jackson G JG Williams Ashley A Wnorowski Alexa A Yimam Nahom N Searson Peter C PC Wong Andrew D AD

Integrative biology : quantitative biosciences from nano to macro 20170401 4

Quiescence is commonly used to describe the inactive state of endothelial cells (ECs) in monolayers that have reached homeostasis. Experimentally quiescence is usually described in terms of the relative change in cell activity (e.g. turnover, speed, etc.) in response to a perturbation (e.g. solute, shear stress, etc.). The objective of this study is to provide new insight into EC quiescence by quantitatively defining the morphology and activity of confluent cell monolayers in response to shear s ...[more]

PMID: 28345713

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Project description:In vivo, vascular endothelial cells (VECs) are anchored to the underlying stroma through a specialization of the extracellular matrix, the basement membrane (BM) which provides a variety of substratum associated biophysical cues that have been shown to regulate fundamental VEC behaviors. VEC function and homeostasis are also influenced by hemodynamic cues applied to their apical surface. How the combination of these biophysical cues impacts fundamental VEC behavior remains poorly studied. In the present study, we investigated the impact of providing biophysical cues simultaneously to the basal and apical surfaces of human aortic endothelial cells (HAECs). Anisotropically ordered patterned surfaces of alternating ridges and grooves and isotropic holed surfaces of varying pitch (pitch = ridge or hole width + intervening groove or planar regions) were fabricated and seeded with HAECs. The cells were then subjected to a steady shear stress of 20 dyne/cm(2) applied either parallel or perpendicular to the direction of the ridge/groove topography. HAECs subjected to flow parallel to the ridge/groove topography exhibited protagonistic effects of the two stimuli on cellular orientation and elongation. In contrast, flow perpendicular to the substrate topography resulted in largely antagonistic effects. Interestingly, the behavior depended on the shape and size of the topographic features. HAECs exhibited a response that was less influenced by the substratum and primarily driven by flow on isotropically ordered holed surfaces of identical pitch to the anistropically ordered surfaces of alternating ridges and grooves. Simultaneous presentation of biophysical cues to the basal and apical aspects of cells also influenced nuclear orientation and elongation; however, the extent of nuclear realignment was more modest in comparison to cellular realignment regardless of the surface order of topographic features. Flow-induced HAEC migration was also influenced by the ridge/groove surface topographic features with significantly altered migration direction and increased migration tortuosity when flow was oriented perpendicular to the topography; this effect was also pitch-dependent. The present findings provide valuable insight into the interaction of biologically relevant apical and basal biophysical cues in regulating cellular behavior and promise to inform improved prosthetic design.

Project description:Objective: The vulnerability of atherosclerotic plaques is among the leading cause of ischemic stroke. High wall shear stress (WSS) promotes the instability of atherosclerotic plaques by directly imparting mechanical stimuli, but the specific mechanisms remain unclear. We speculate that modulation of mechanosensitive genes may play a vital role in accelerating the development of plaques. The purpose of this study was to find mechanosensitive genes in vascular endothelial cells (ECs) through combining microarray data with bioinformatics technology and further explore the underlying dynamics-related mechanisms that cause the progression and destabilization of atherosclerotic plaques. Methods: Microarray data sets for human vascular ECs under high and normal WSS were retrieved from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) were identified through the R language. The performance of enrichment analysis and protein-protein interaction (PPI) network presented the biological function and signaling pathways of the DEGs. Hub genes were identified based on the PPI network and validated by GEO data sets. Predicted transcription factor (TF) genes and miRNAs interaction with potential mechanosensitive genes were identified by NetworkAnalyst. Results: A total of 260 DEGs, 121 upregulated and 139 downregulated genes, were screened between high and normal WSS from GSE23289. A total of 10 hub genes and four cluster modules were filtered out based on the PPI network. The enrichment analysis showed that the biological functions of the hub genes were mainly involved in responses to unfolded protein and topologically incorrect protein, and t to endoplasmic reticulum stress. The significant pathways associated with the hub genes were those of protein processing in the endoplasmic reticulum, antigen processing, and presentation. Three out of the 10 hub genes, namely, activated transcription factor 3 (ATF3), heat shock protein family A (Hsp70) member 6 (HSPA6), and dual specificity phosphatase 1 (DUSP1, also known as CL100, HVH1, MKP-1, PTPN10), were verified in GSE13712. The expression of DUSP1 was higher in the senescent cell under high WSS than that of the young cell. The TF-miRNA-mechanosensitive gene coregulatory network was constructed. Conclusion: In this work, we identified three hub genes, ATF3, HSPA6, and DUSP1, as the potential mechanosensitive genes in the human blood vessels. DUSP1 was confirmed to be associated with the senescence of vascular ECs. Therefore, these three mechanosensitive genes may have emerged as potential novel targets for the prediction and prevention of ischemic stroke. Furthermore, the TF-miRNA-mechanosensitive genes coregulatory network reveals an underlying regulatory mechanism and the pathways to control disease progression.

Dataset Information

Real-time quantification of endothelial response to shear stress and vascular modulators.

Publications

Real-time quantification of endothelial response to shear stress and vascular modulators.

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