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We present an innovative in vitro model aimed at investigating the combined effects of tissue rigidity and shear stress on endothelial cell (EC) function, which are crucial for understanding vascular health and the onset of diseases such as atherosclerosis. Traditionally, studies have explored the impacts of shear stress and substrate stiffness on ECs, independently. However, this integrated system combines these factors to provide a more precise simulation of the mechanical environment of the vasculature. The objective is to examine EC mechanotransduction across various tissue stiffness levels and flow conditions using human ECs. We detail the protocol for synthesizing gelatin methacrylate (GelMA) hydrogels with tunable stiffness and seeding them with ECs to achieve confluency. Additionally, we describe the design and assembly of a cost-effective flow chamber, supplemented by computational fluid dynamics simulations, to generate physiological flow conditions characterized by laminar flow and appropriate shear stress levels. The protocol also incorporates fluorescence labeling for confocal microscopy, enabling the assessment of EC responses to both tissue compliance and flow conditions. By subjecting cultured ECs to multiple integrated mechanical stimuli, this model enables comprehensive investigations into how factors such as hypertension and aging may affect EC function and EC-mediated vascular diseases. The insights gained from these investigations will be instrumental in elucidating the mechanisms underlying vascular diseases and in developing effective treatment strategies.
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Endothelial glycocalyx sensitivity to chemical and mechanical sub-endothelial substrate properties
Glycocalyx (GCX) is a carbohydrate-rich structure that coats the surface of endothelial cells (ECs) and lines the blood vessel lumen. Mechanical perturbations in the vascular environment, such as blood vessel stiffness, can be transduced and sent to ECs through mechanosensors such as GCX. Adverse stiffness alters GCX-mediated mechanotransduction and leads to EC dysfunction and eventually atherosclerotic cardiovascular diseases. To understand GCX-regulated mechanotransduction events, anin vitromodel emulatingin vivovessel conditions is needed. To this end, we investigated the impact of matrix chemical and mechanical properties on GCX expression via fabricating a tunable non-swelling matrix based on the collagen-derived polypeptide, gelatin. To study the effect of matrix composition, we conducted a comparative analysis of GCX expression using different concentrations (60–25,000 μg/mL) of gelatin and gelatin methacrylate (GelMA) in comparison to fibronectin (60 μg/mL), a standard coating material for GCX-related studies. Using immunocytochemistry analysis, we showed for the first time that different substrate compositions and concentrations altered the overall GCX expression on human umbilical vein ECs (HUVECs). Subsequently, GelMA hydrogels were fabricated with stiffnesses of 2.5 and 5 kPa, representing healthy vessel tissues, and 10 kPa, corresponding to diseased vessel tissues. Immunocytochemistry analysis showed that on hydrogels with different levels of stiffness, the GCX expression in HUVECs remained unchanged, while its major polysaccharide components exhibited dysregulation in distinct patterns. For example, there was a significant decrease in heparan sulfate expression on pathological substrates (10 kPa), while sialic acid expression increased with increased matrix stiffness. This study suggests the specific mechanisms through which GCX may influence ECs in modulating barrier function, immune cell adhesion, and mechanotransduction function under distinct chemical and mechanical conditions of both healthy and diseased substrates.
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- Award ID(s):
- 1846962
- PAR ID:
- 10512975
- Publisher / Repository:
- Frontiers in Bioengineering and Biotechnology
- Date Published:
- Journal Name:
- Frontiers in Bioengineering and Biotechnology
- Volume:
- 11
- ISSN:
- 2296-4185
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3389/fbioe.2023.1250348
