Isolation, culturing and gene expression profiling of inner mass cells from stable and vulnerable carotid atherosclerotic plaques

Abstract

The connective tissue components that form the atherosclerotic plaque body are produced by the plaque inner mass cells (PIMC), located inside the plaque. We report an approach to isolate and culture cells from the connective tissue of stable and vulnerable human atherosclerotic plaques based on elimination of non-connective tissue cells such as blood and non-plaque intima cells with a lysis buffer. The resulting plaque cells were characterized by growth capacity, morphology, transcriptome profiling and specific protein expression. Plaque cells slowly proliferated for up to three passages unaffected by the use of proliferation stimulants or changes of culture media composition. Stable plaques yielded more cells than vulnerable ones. Plaque cell cultures also contained several morphological cellular types. RNA-seq profiles of plaque cells were different from any of the cell types known to be involved in atherogenesis. The expression of the following proteins was observed in cultured plaque cells: smooth muscle cells marker α-SMA, macrophage marker CD14, extracellular matrix proteins aggrecan, fibronectin, neovascularisation markers VEGF-A, CD105, cellular adhesion receptor CD31 and progenitor/dedifferentiation receptor CD34. Differential expression of several notable transcripts in cells from stable and vulnerable plaques suggests the value of plaque cell culture studies for the search of plaque vulnerability markers.

Human primary plaque cell cultures to study mechanisms of atherosclerosis

“…We tested two culture media - IMDM-FBS (14) and HAM F12K Complete (designed in-house, see Methods), in combination with Matrigel, Vitronectin, and Fibronectin pre-coated plates, and in the presence and absence of Vitamin C. Figure 1 provides an overview of the methodology of the conditioned outgrowth method, which is based on the capability of plaque cells to migrate from 2-3 mm 2 plaque pieces (Fig. S1 A) and proliferate in the dish.…”

Pathways linking aging and atheroprotection in Mif‐deficient atherosclerotic mice

“…MAIT T cells are unconventional T cells with innate‐like antimicrobial activity but have also been assigned various deleterious and protective functions in autoimmune, inflammatory, and metabolic diseases 78 and might thus play a role in the atherogenic vasculature. Cpne7 has not been investigated in the context of atherosclerosis, but the inner mass cells from vulnerable carotid atherosclerotic plaques express higher Cpne7 levels than the ones from stable plaques, inferring that Cpne7 might have a role in the progression of atherosclerosis 79 …”

“…Cpne7 has not been investigated in the context of atherosclerosis, but the inner mass cells from vulnerable carotid atherosclerotic plaques express higher Cpne7 levels than the ones from stable plaques, inferring that Cpne7 might have a role in the progression of atherosclerosis. 79 We did not observe changes in BCA plaque VSMC numbers, but notably, Col4a1 and Tnnt3 (as well as Tnnt2), were among the top-enriched genes in our transcriptomic analysis, indicating effects on VSMC phenotype. Col4a1 was highly enriched in the 24-week Mif-deficient mice as well as in the 42-week paradigm, when comparing Mif-expressing to Mif-deficient mice.…”

The Mechanisms of the Development of Atherosclerosis in Prediabetes

“…In searching for the source of the microparticles expressing CD105 during the development of atherosclerosis, multiple research groups have identified cells with an unstable, vulnerable atherosclerotic plaque expressing CD105 [ 87 , 88 , 89 ]. During the development of atherosclerotic plaques, it was found that vasculature invades into the plaques.…”