Doctor of Philosophy (PHD)
Molecular and Cellular Pharmacology (Medicine)
Date of Defense
First Committee Member
Roberto I. Vazquez-Padron
Second Committee Member
Third Committee Member
Fourth Committee Member
Fifth Committee Member
Rationale: c-Kit, as a receptor tyrosine kinase (RTK), is present in multiple cell types including vascular smooth muscle cells (SMCs). However, so far, limited progress has been made to understand how c-Kit mediates SMC biology, and whether the presence of this receptor is critical for vascular pathogenesis such as atherosclerosis. Objectives: In this dissertation, I investigated the roles of c-Kit in atherosclerosis and underlying mechanisms. Methods: c-Kit distribution was determined in both murine and human aortae using a combination of KitBac-eGFP reporter mice, immunofluorescence (IF), and Western blot. c-Kit deficiency studies were performed in c-Kit mutant (KitW/W-v) mice on an ApoE-/- background, which were fed for 16 weeks on a high fat diet (HFD) to induce hyperlipidemia. Cell specific c-Kit roles were investigated using: 1) chimeric mice with global KitW/W-v ApoE-/- cells but Kit+/+ ApoE-/- bone marrow cells, and 2) c-Kit SMC conditional knockout (Kitlox66-71/lox66-71 Myh11CreERT2 ApoE-/-) mice. Mechanistic studies were performed using primary aortic SMCs isolated from c-Kit mutant (KitW/W-v) or wild type control (Kit+/+) mice. Rescued c-Kit SMCs were created by transfecting a murine Kit gene expressing plasmid into the c-Kit deficient (KitW/W-v) SMCs. All these SMCs were treated with various atherosclerotic stimuli (e.g., cholesterol or phospholipid) to determine the roles of c-Kit in the context of hyperlipidemia. Results: IF analysis of the transgenic KitBac-eGFP reporter mice revealed that c-Kit was present in arteries but not veins. Further confocal microscopy analysis of murine aortic tissue demonstrated that the expression of c-Kit was co-localized with SMC marker smooth muscle actin (SMA). Similarly, c-Kit expression was also found in human arterial samples, as determined by IF and Western blot. In c-Kit loss of function studies, both atherosclerotic burden and the lesion site macrophage-like SMC (SMA+, CD68+) ratio were found significantly elevated in hyperlipidemic c-Kit mutant (KitW/W-v ApoE-/-) mice compared to their control littermates (Kit+/+ ApoE-/-). c-Kit positive bone marrow transplantation (BMT) to hyperlipidemic c-Kit deficient (KitW/W-v ApoE-/-) mice failed to alleviate the atherosclerotic phenotype. However, cell specific deletion of c-Kit in SMCs led to exacerbated atherosclerosis in hyperlipidemic mice. These findings pointed out that the observed c-Kit protective role in atherosclerosis is SMC specific. In mechanistic studies, primary aortic SMCs from c-Kit-deficient (KitW/W-v) mice exhibited a “synthetic” phenotype with decreased contractile marker expression, abnormally accelerated growth, and unexpected migration compared to the aortic SMCs isolated from c-Kit wild type (Kit+/+) mice. Furthermore, when treated with cholesterol, these synthetic SMCs were prone to acquire a “foam cell-like (or macrophage-like)” phenotype with enhanced expression and activity of Kruppel-like factor 4 (KLF4), a transcriptional factor that was previously reported to favor the SMC phenotype transformation. Restoration of c-Kit in mutant (KitW/W-v) SMCs decreased the expression and activity of KLF4, subsequently abolished the “foam cell-like” phenotype. In addition, loss of c-Kit led to an inflammatory state in SMCs. By using a high-throughput microarray assay and in silico pathway analysis, NF-κB signaling related gene expression was found upregulated in mutant (KitW/W-v) SMCs, but not in wild type (Kit+/+). Upon the stimulation with oxidized phospholipid, these c-Kit mutant (KitW/W-v) SMCs presented enhanced NF-κB activity with increased NF-κB signaling regulated pro-inflammatory mediator expressions compared to wild type (Kit+/+) SMCs. Further, c-Kit suppression of NF-κB activity in SMCs was in a TGFβ-activated kinase 1 (TAK1) and a Nemo-like kinase (NLK) dependent manner. Conclusions: This work demonstrates for the first time that vascular expression of c-Kit protects against atherosclerosis, and provides mechanistic evidence suggesting a fundamental role of c-Kit in preserving SMCs in a contractile state, therefore preventing their synthetic, foam- cell-like, and pro-inflammatory phenotype switch under various atherosclerotic stimuli.
c-Kit; Smooth Muscle Cell; Atherosclerosis
Song, Lei, "Role of c-Kit in Atherosclerosis: A Mechanistic Study" (2017). Open Access Dissertations. 1972.