Department of Cardiovascular Sciences and Center for Metabolic Disease Research, Temple University Lewis Katz School of Medicine, Philadelphia, PA, USA.
Methods Mol Biol. 2024;2782:113-122. doi: 10.1007/978-1-0716-3754-8_8.
Mitochondria-associated membranes (MAMs) are regions where the endoplasmic reticulum (ER) interacts with mitochondria and regulate lipid trafficking, calcium signaling, ER stress, and inflammation activation. Isolation of MAMs from endothelial cells is vital for studying insight into the immune regulation of many inflammatory diseases. Endothelial cells (ECs) are critical innate immune cells due to their paracrine function of secreting interleukins, chemokines, cytokines, and growth factors, as well as expressing levels of pattern recognition receptors including toll-like receptors (TLRs). Furthermore, ECs regulate and recruit monocytes by expressing adhesion molecules including vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), P-selectin, and E-selectin, to facilitate monocyte diapedesis in areas of damage and inflammation. This protocol consists of step-by-step instructions on isolating pure MAMs and other subcellular fractions from endothelial cells, which is critical to understanding ER and mitochondria crosstalks in endothelial functions in health and disease.
线粒体相关膜（MAMs）是内质网（ER）与线粒体相互作用并调节脂质转运、钙信号、ER 应激和炎症激活的区域。从内皮细胞中分离 MAMs 对于深入研究许多炎症性疾病的免疫调节至关重要。内皮细胞（ECs）是重要的先天免疫细胞，因为它们具有旁分泌功能，可分泌白细胞介素、趋化因子、细胞因子和生长因子，并且还表达包括 Toll 样受体（TLRs）在内的模式识别受体。此外，ECs 通过表达黏附分子，如血管细胞黏附分子-1（VCAM-1）、细胞间黏附分子-1（ICAM-1）、P 选择素和 E 选择素，来调节和招募单核细胞，以促进单核细胞在损伤和炎症部位的穿出。本协议包括从内皮细胞中分离纯 MAMs 和其他亚细胞成分的分步说明，这对于理解 ER 和线粒体在健康和疾病中的内皮功能中的相互作用至关重要。
