Generation and characterization of human smooth muscle cell lines derived from atherosclerotic plaque

The study of atherogenesis in humans has been restricted by the limited ava ilability and brief in vitro life span of plaque smooth muscle cells (SMCs) . We describe plaque SMC lines with extended life spans generated by the ex pression of the human papillomavirus (HPV)-16 E6 and E7 genes, which has be en shown to extend the life span of normal adult human aortic SMCs. Resulti ng cell lines (pdSMC1A and 2) demonstrated at least 10-fold increases in li fe span; pdSMC1A became immortal. The SMC identity of both pdSMC lines was confirmed by SM22 mRNA expression. pdSMC2 were generally diploid but with v arious structural and numerical alterations; pdSMC1A demonstrated several c hromosomal abnormalities, most commonly -Y, +7, -13, anomalies previously r eported in both primary pdSMCs and atherosclerotic tissue. Confluent pdSMC2 appeared grossly similar to HPV-16 E6/E7-expressing normal adult aortic SM Cs (AASMCs), exhibiting typical SMC morphology/growth patterns; pdSMC1A dis played irregular cell shape/organization with numerous mitotic figures. Ded ifferentiation to a synthetic/proliferative phenotype has been hypothesized as a critical step in atherogenesis, because rat neonatal SMCs and adult i ntimal SMCs exhibit similar gene expression patterns. To confirm that our p dSMC lines likewise express this apparent plaque phenotype, osteopontin, pl atelet-derived growth factor B, and elastin mRNA levels were determined in pdSMC1A, pdSMC2, and AASMCs. However, no significant increases in osteopont in or platelet-derived growth factor B expression levels were observed in e ither pdSMC compared with AASMCs. pdSMC2 alone expressed high levels of ela stin mRNA. Lower levels of SM22 mRNA in pdSMC1A suggested greater dediffere ntiation and/or additional population doublings in pdSMC1A relative to pdSM C2. Both pdSMC lines (particularly 1A) demonstrated high message levels for matrix Gla protein, previously reported to be highly expressed by human ne ointimal SMCs in vitro. These results describe 2 novel plaque cell lines ex hibiting Various features of plaque SMC biology; pdSMC2 may represent an ea rlier plaque SMC phenotype, whereas pdSMC1A may be representative of cells comprising an advanced atherosclerotic lesion.