Name: Video: hSATMVEC Isolation from Subcutaneous Adipose Tissue of Patients with CIED to Study Endothelial-Adipocyte Dynamics
Uploaded: 2024-04-05T14:35:00
Duration: 3 min 31 s
Description: 133 Views. hSATMVEC Isolation from Subcutaneous Adipose Tissue of Patients with CIED to Study Endothelial-Adipocyte Dynamics

hsatmvec isolation from subcutaneous adipose tissue of patients with cied to study endothelial-adipocyte dynamics

Human SATMVECs: Isolation and Adipocyte Cross-Talk Studies

Endothelial cells (ECs) are squamous cells that line the inner surface of the blood vessel wall as a monolayer. They have many essential roles, including control of vascular tone, regulation of thrombosis, modulating the inflammatory response, and contributing to angiogenesis1. Given the importance of endothelial cells in cardiometabolic physiology, they are frequently used experimentally to further the understanding of pathophysiology and to examine new pharmacological treatments for cardiometabolic disease.
However, there is enormous heterogeneity in endothelial cell morphology, function, gene expression, and antigen composition depending on the origin of their vascular bed2. While endothelial cells from large arteries are best suited to atherosclerosis studies, endothelial cells from small vessels, known as microvascular endothelial cells (MVECs), are more suitable for angiogenesis studies2. Understanding the molecular basis for endothelial heterogeneity may provide valuable insights into vascular bed-specific therapies. Microvascular endothelial function also significantly differs in numerous diseases, including diabetes, cardiovascular disease, and systemic infection3,4. Therefore, the ability to isolate endothelial cells from defined patient groups allows direct comparison of their endothelial cell function and cellular cross-talk5. 
In this paper, we describe a novel method of isolating human MVECs from subcutaneous adipose tissue (hSATMVEC) taken at the time of cardiac implantable electronic device (CIED) insertion. hSATMVEC isolated following enzymatic digestion of subcutaneous adipose tissue (SAT) were grown and passaged on gelatin-coated plates. We then describe a range of phenotyping assays that have been successfully applied to hSATMVECs in order to validate their phenotype and demonstrate use in routine endothelial cell assays. Finally, we describe an application of hSATMVECs in an experimental model of hSATMVECs-adipocyte cross-talk.

Author Spotlight: Insights into Cardiometabolic Diseases with Subcutaneous Adipose Tissue Microvasculature Studies 

Studying Adipose Endothelial Cell/Adipocyte Cross-Talk in Human Subcutaneous Adipose Tissue

hSATMVEC Isolation from Subcutaneous Adipose Tissue of Patients with CIED to Study Endothelial-Adipocyte Dynamics

hsatmvec-isolation-from-subcutaneous-adipose-tissue-patients-with

Research

JoVE Journal

Medicine

133 Views. hSATMVEC Isolation from Subcutaneous Adipose Tissue of Patients with CIED to Study Endothelial-Adipocyte Dynamics

Video: hSATMVEC Isolation from Subcutaneous Adipose Tissue of Patients with CIED to Study Endothelial-Adipocyte Dynamics

Microvascular endothelial cells (MVECs) have many critical roles, including control of vascular tone, regulation of thrombosis, and angiogenesis. Significant heterogeneity in endothelial cell (EC) genotype and phenotype depends on their vascular bed and host disease state. The ability to isolate MVECs from tissue-specific vascular beds and individual patient groups offers the opportunity to directly compare MVEC function in different disease states. Here, using subcutaneous adipose tissue (SAT) taken at the time of insertion of cardiac implantable electronic devices (CIED), we describe a method for the isolation of a pure population of functional human subcutaneous adipose tissue MVEC (hSATMVEC) and an experimental model of hSATMVEC-adipocyte cross-talk.
hSATMVEC were isolated following enzymatic digestion of SAT by incubation with anti-CD31 antibody-coated magnetic beads and passage through magnetic columns. hSATMVEC were grown and passaged on gelatin-coated plates. Experiments used cells at passages 2-4. Cells maintained classic features of EC morphology until at least passage 5. Flow cytometric assessment showed 99.5% purity of isolated hSATMVEC, defined as CD31+/CD144+/CD45-. Isolated hSATMVEC from controls had a population doubling time of approximately 57 h, and active proliferation was confirmed using a cell proliferation imaging kit. Isolated hSATMVEC function was assessed using their response to insulin stimulation and angiogenic tube-forming potential. We then established an hSATMVEC-subcutaneous adipocyte co-culture model to study cellular cross-talk and demonstrated a downstream effect of hSATMVEC on adipocyte function.
hSATMVEC can be isolated from SAT taken at the time of CIED insertion and are of sufficient purity to both experimentally phenotype and study hSATMVEC-adipocyte cross-talk.

Here, we describe a protocol for isolating, culturing, and phenotyping microvascular endothelial cells from human subcutaneous adipose tissue (hSATMVECs). Additionally, we describe an experimental model of hSATMVEC-adipocyte cross-talk.