in-vitro-model-integrating-substrate-stiffness-flow-to-study

In Vitro Model Integrating Substrate Stiffness and Flow to Study Endothelial Cell Responses

We present an innovative in vitro model aimed at investigating the combined effects of tissue rigidity and shear stress on endothelial cell (EC) function, ...

Northeastern University

Endothelium health is essential to the regulation of physiological vascular functions. Because of the critical capability of endothelial cells (ECs) to sense and transduce chemical and mechanical signals in the local vascular environment, their dysfunction is associated with a vast variety of vascular diseases and injuries, especially atherosclerosis and subsequent cardiovascular diseases. This review describes the mechanotransduction events that are mediated through ECs, the EC subcellular components involved, and the pathways reported to be potentially involved. Up-to-date research efforts involving in vivo animal models and in vitro biomimetic models are also discussed, including their advantages and drawbacks, with recommendations on future modeling approaches to aid the development of novel therapies targeting atherosclerosis and related cardiovascular diseases.

Atherosclerosis and endothelial mechanotransduction: current knowledge and models for future research.

Hypoxia, an important feature of solid tumors, is a major factor shaping the immune landscape, and several cancer models have been developed to emulate hypoxic tumors. However, to date, they still have several limitations, such as the lack of reproducibility, inadequate biophysical cues, limited immune cell infiltration, and poor oxygen (O  ) control, leading to non-pathophysiological tumor responses. As a result, it is essential to develop new and improved cancer models that mimic key features of the tumor extracellular matrix and recreate tumor-associated hypoxia while allowing cell infiltration and cancer-immune cell interactions. Herein, hypoxia-inducing cryogels (HICs) have been engineered using hyaluronic acid (HA) as macroporous scaffolds to fabricate three-dimensional microtissues and model a hypoxic tumor microenvironment. Specifically, tumor cell-laden HICs have been designed to deplete O  locally and induce long-standing hypoxia. This state of low oxygen tension, leading to HIF-1α stabilization in tumor cells, resulted in changes in hypoxia-responsive gene expression and phenotype, a metabolic adaptation to anaerobic glycolysis, and chemotherapy resistance. Additionally, HIC-supported tumor models induced dendritic cell (DC) inhibition, revealing a phenotypic change in plasmacytoid B220  DC (pDC) subset and an impaired conventional B220  DC (cDC) response in hypoxia. Lastly, our HIC-based melanoma model induced CD8+ T cell inhibition, a condition associated with the downregulation of pro-inflammatory cytokine secretion, increased expression of immunomodulatory factors, and decreased degranulation and cytotoxic capacity of T cells. Overall, these data suggest that HICs can be used as a tool to model solid-like tumor microenvironments and identify a phenotypic transition from cDC to pDC in hypoxia and the key contribution of HA in retaining cDC phenotype and inducing their hypoxia-mediated immunosuppression. This technology has great potential to deepen our understanding of the complex relationships between cancer and immune cells in low O  conditions and may pave the way for developing more effective therapies.

Hypoxia-inducing cryogels uncover key cancer-immune cell interactions in an oxygen-deficient tumor microenvironment.

Hypoxia is a major factor shaping the immune landscape, and several cancer models have been developed to emulate hypoxic tumors. However, to date, they still have several limitations, such as the lack of reproducibility, inadequate biophysical cues, limited immune cell infiltration, and poor oxygen (O) control, leading to non-pathophysiological tumor responses. Therefore, it is essential to develop better cancer models that mimic key features of the tumor extracellular matrix and recreate tumor-associated hypoxia while allowing cell infiltration and cancer-immune cell interactions. Herein, hypoxia-inducing cryogels (HICs) have been engineered using hyaluronic acid (HA) to fabricate three-dimensional microtissues and model a hypoxic tumor microenvironment. Specifically, tumor cell-laden HICs have been designed to deplete O locally and induce long-standing hypoxia. HICs promoted changes in hypoxia-responsive gene expression and phenotype, a metabolic adaptation to anaerobic glycolysis, and chemotherapy resistance. Additionally, HIC-supported tumor models induced dendritic cell (DC) inhibition, revealing a phenotypic change in the plasmacytoid DC (pDC) subset and an impaired conventional DC (cDC) response in hypoxia. Lastly, our HIC-based melanoma model induced CD8 T cell inhibition, a condition associated with the downregulation of pro-inflammatory cytokine secretion, increased expression of immunomodulatory factors, and decreased degranulation and cytotoxic capacity of T cells. Overall, these data suggest that HICs can be used as a tool to model solid-like tumor microenvironments and has great potential to deepen our understanding of cancer-immune cell relationship in low O conditions and may pave the way for developing more effective therapies.

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, an  model emulating  vessel 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.

Article	Year
Atherosclerosis and endothelial mechanotransduction: current knowledge and models for future research. American journal of physiology. Cell physiology\| PubMed ID: 36440856	2023
Hypoxia-inducing cryogels uncover key cancer-immune cell interactions in an oxygen-deficient tumor microenvironment. bioRxiv : the preprint server for biology\| PubMed ID: 36711715	2023
Hypoxia-inducing cryogels uncover key cancer-immune cell interactions in an oxygen-deficient tumor microenvironment. Bioactive materials\| PubMed ID: 37600932	2023
Endothelial glycocalyx sensitivity to chemical and mechanical sub-endothelial substrate properties. Frontiers in bioengineering and biotechnology\| PubMed ID: 38026846	2023

Mohammad Hamrangsekachaee

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