Abstract
Endothelial dysfunction is the common molecular basis of multiple human diseases, such as atherosclerosis, diabetes, hypertension, and acute lung injury. Therefore, primary isolation of high-purity endothelial cells (ECs) is crucial to study the mechanisms of endothelial function and disease pathogenesis. Mouse lung ECs (MLECs) are widely used in vascular biology and lung cell biology studies such as pulmonary inflammation, angiogenesis, vessel permeability, leukocyte/EC interaction, nitric oxide production, and mechanotransduction. Thus, in this paper, we describe a simple, and reproducible protocol for the isolation and culture of MLECs from adult mice using collagenase I-based enzymatic digestion, followed by sequential sorting with PECAM1 (also known as CD31)- and ICAM2 (also known as CD102)-coated microbeads. The morphology of isolated MLECs were observed with phase contrast microscope. MLECs were authenticated by CD31 immunoblotting, and immunofluorescent staining of established EC markers VE-cadherin and von Willebrand factor (vWF). Cultured MLECs also showed functional characteristics of ECs, evidenced by DiI-oxLDL uptake assay and THP-1 monocyte adhesion assay. Finally, we used MLECs from endothelium-specific enhancer of zeste homolog 2 (EZH2) knockout mice to show the general applicability of our protocol. To conclude, we describe here a simple and reproducible protocol to isolate highly pure and functional ECs from adult mouse lungs. Isolation of ECs from genetically engineered mice is important for downstream phenotypic, genetic, or proteomic studies.