Integrative pathway dissection of molecular mechanisms of moxLDL-induced vascular smooth muscle phenotype...

Atherosclerosis (AT) is a chronic inflammatory disease characterized by the accumulation of inflammatory cells, lipoproteins and fibrous tissue in the walls of arteries. AT is the primary cause of heart attacks and stroke and is the leading cause of death in Western countries. To date, the pathogenesis of AT is not well-defined. Studies have shown that the dedifferentiation of contractile and quiescent vascular smooth muscle cells (SMC) to the proliferative, migratory and synthetic phenotype in the intima is pivotal for the onset and progression of AT. To further delineate the mechanisms underlying the pathogenesis of AT, we analyzed the early molecular pathways and networks involved in the SMC phenotype transformation.

Quiescent human coronary artery SMCs were treated with minimally-oxidized LDL (moxLDL), for 3 hours and 21 hours, respectively. Transcriptomic data was generated for both time-points using microarrays and was subjected to pathway analysis using Gene Set Enrichment Analysis, GeneMANIA and Ingenuity software tools. Gene expression heat maps and pathways enriched in differentially expressed genes were compared to identify functional biological themes to elucidate early and late molecular mechanisms of moxLDL-induced SMC dedifferentiation.

Differentially expressed genes were found to be enriched in cholesterol biosynthesis, inflammatory cytokines, chemokines, growth factors, cell cycle control and myogenic contraction themes. These pathways are consistent with inflammatory responses, cell proliferation, migration and ECM production, which are characteristic of SMC dedifferentiation. Furthermore, up-regulation of cholesterol synthesis and dysregulation of cholesterol metabolism was observed in moxLDL-induced SMC. These observations are consistent with the accumulation of cholesterol and oxidized cholesterol esters, which induce proinflammatory reactions during atherogenesis. Our data implicate for the first time IL12, IFN-α, HGF, CSF3, and VEGF signaling in SMC phenotype transformation. GPCR signaling, HBP1 (repressor of cyclin D1 and CDKN1B), and ID2 and ZEB1 transcriptional regulators were also found to have important roles in SMC dedifferentiation. Several microRNAs were observed to regulate the SMC phenotype transformation via an interaction with IFN-γ pathway. Also, several "nexus" genes in complex networks, including components of the multi-subunit enzyme complex involved in the terminal stages of cholesterol synthesis, microRNAs (miR-203, miR-511, miR-590-3p, miR-346*/miR- 1207-5p/miR-4763-3p), GPCR proteins (GPR1, GPR64, GPRC5A, GPR171, GPR176, GPR32, GPR25, GPR124) and signal transduction pathways, were found to be regulated.

The systems biology analysis of the in vitro model of moxLDL-induced VSMC phenotype transformation was associated with the regulation of several genes not previously implicated in SMC phenotype transformation. The identification of these potential candidate genes enable hypothesis generation and in vivo functional experimentation (such as gain and loss-of-function studies) to establish causality with the process of SMC phenotype transformation and atherogenesis.