Proteomic profiling of concurrently isolated primary microvascular endothelial cells, pericytes, and vascular smooth muscle cells from adult mouse heart

Abstract

The microcirculation serves crucial functions in adult heart, distinct from those carried out by epicardial vessels. Microvessels are governed by unique regulatory mechanisms, impairment of which leads to microvessel-specific pathology. There are few treatment options for patients with microvascular heart disease, primarily due to limited understanding of underlying pathology. High throughput mRNA sequencing and protein expression profiling in specific cells can improve our understanding of microvessel biology and disease at the molecular level. Understanding responses of individual microvascular cells to the same physiological or pathophysiological stimuli requires the ability to isolate the specific cell types that comprise the functional units of the microcirculation in the heart, preferably from the same heart, to ensure that different cells have been exposed to the same in-vivo conditions. We developed an integrated process for simultaneous isolation and culture of the main cell types comprising the microcirculation in adult mouse heart: endothelial cells, pericytes, and vascular smooth muscle cells. These cell types were characterized with isobaric labeling quantitative proteomics and mRNA sequencing. We defined microvascular cell proteomes, identified novel protein markers, and confirmed established cell-specific markers. Our results allow identification of unique markers and regulatory proteins that govern microvascular physiology and pathology.

A possible role for proinflammatory activation via cGAS-STING pathway in atherosclerosis induced by accumulation of DNA double-strand breaks

“…Based on recent evidence, VSMCs seem to drive the inflammatory process of atherosclerosis 30 – 32 . We confirmed that the isolated cells were VSMCs by immunocytochemistry, using antibodies against the VSMC specific isoform of alpha-actin (Supplemental Figure 2A ) 33 – 35 . We performed a time course analysis to determine the effect of Ku80 deficiency on DSB formation and DDR activation in response to hydrogen peroxide exposure.…”

Approaches for the isolation and long-term expansion of pericytes from human and animal tissues

“…Pre-warmed media is then added to cover the fragments completely. Explanted cultures are kept undisturbed for a few days until pericytes become visible as migrated cells from the tissue pieces ( 31 , 41 , 94 ). The advantages of this isolation technique are the increased yield of collected cells, low cost, and avoidance of the destructive stress caused by enzymes ( 95 ).…”

“…The labeled cells are retained in the tube walls, and the unlabeled cells are discarded. MACS columns and Dynabeads are widely used to isolate pericytes due to the simplicity and rapidity of the procedure ( 32 – 35 , 94 , 103 ). The harsh magnetic field may cause damage or rupture of the cell membranes of fragile cells.…”