Abstract
Objectives: Microvascular dysfunction in skeletal muscle is involved in metabolic and vascular diseases. Microvascular endothelial cells (MEC) are poorly characterized in the progression of associated diseases in part due to lack of availability of MEC from various animal models. The objective was to provide a fast, simple, and efficient method to isolate murine MEC derived from skeletal muscle. Methods: Dissected abdominal skeletal muscles from C57BL/6J mice at 8 - 12 weeks of age were enzymatically dissociated. MEC were isolated using a modified two-step Dynabeads™-based purification method. With a combination of Dynabeads™ - Griffonia simplicifolia lectin-I and Dynabeads™ - monoclonal antibody against CD31/PECAM-1, MEC were isolated and purified twice followed by cultivation. Results: Isolated and purified cells were viable and cultured. MEC were characterized by using immunofluorescence to identify CD31/PECAM-1, an EC marker, and two specific functional assays, which include a capillary-like tube formation and the uptake of Dil-Ac-LDL. The purity of isolated cell populations from skeletal muscle microvessels, which was assessed by flow cytometry, was 88.02% ± 2.99% (n = 6). Conclusions: This method is simple, fast, and highly reproducible for isolating MEC from murine skeletal muscle. The method will enable us to obtain primary cultured MEC from various genetic or diseased murine models, contributing to insightful knowledge of diseases associated with the dysfunction of microvessels.
Highlights
IntroductionGiven the heterogenic properties of vascular endothelial cells, physiological and pathophysiological studies using species- and origin-specific cultured Microvascular endothelial cells (MEC) are critical for gaining novel insights into molecular mechanisms underlying diseases associated with dysfunction of MEC
CD31/PECAM-1 has been widely used as an endothelial cell marker
The immunofluorescence assay with monoclonal antibody against CD31/PECAM-1 showed positive staining of CD31/PECAM-1 in cells isolated by the two-step method (n = 3)
Summary
Given the heterogenic properties of vascular endothelial cells, physiological and pathophysiological studies using species- and origin-specific cultured MEC are critical for gaining novel insights into molecular mechanisms underlying diseases associated with dysfunction of MEC. A large degree of functional heterogeneity was found among MEC derived from different tissues [8]. It would be ideal for insightful molecular and functional studies to utilize MEC derived from the same tissue in disease models or genetically engineered mice, and with sex- and age-matched control groups. The challenge is to obtain large numbers of pure primary cultured MEC
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