Abstract P134: Identification of Tissue Inhibitor of Metalloproteinases (TIMP)-4 as a Novel PPARγ Target in Smooth Muscle Cell

Abstract

Patients with PPARγ mutations develop severe early onset hypertension, type 2 diabetes and lipodystrophy. Our recent findings demonstrated that transgenic mice expressing a dominant negative (DN) PPARγ mutant in vascular smooth muscle (S-P467L) exhibited exacerbated DOCA-salt induced hypertension and vascular remodeling in conduit and resistance arteries. Here, we hypothesized that predisposition to vascular injury during hypertension in S-P467L mice is through altered expression of PPARγ target genes in smooth muscle cells (SMC). Gene expression profiling in aorta and mesenteric arteries revealed a significant loss of Tissue Inhibitor of Metalloproteinases (TIMP)-4 in S-P467L compared to non-transgenic (NT) littermates. Interference with PPARγ activity either by treatment with PPARγ inhibitor, GW9662 or expressing P467L PPARγ markedly suppressed TIMP-4 in primary SMC, suggesting that loss of TIMP-4 in S-P467L arteries is a direct result of PPARγ inhibition. Downregulation of TIMP-4 in SMC by GW9662 was correlated with a significant increase in total MMP activity (Fluorescent signal subtracted from negative control; vehicle: 1±0.5, GW9662: 2.2±0.7, p<0.05), consistent with TIMP-4 function as an endogenous inhibitor of MMPs. Overexpressing TIMP-4 in SMC significantly blunted cell migration compared to those with empty plasmid (change in open area after 8 hr; Empty: 1±0.05, TIMP-4: 0.8±0.051, p<0.05), whereas decreased TIMP-4 expression caused by mutant PPARγ resulted in increased migration (change in open area after 10 hr; GFP: 1±0.25, P467L: 1.63±0.26, p<0.05). The significance of TIMP-4 was also underscored during hypertension since the compensatory increase in TIMP-4 during DOCA-salt in NT mesenteric arteries was lost in S-P467L. We identified two highly conserved potential PPAR response elements (PPREs) close to TIMP-4 promoter region using a sequence-based model. Chromatin immunoprecipitation assay showed strong binding of PPARγ at one of the suggested PPREs in SMC (% input±sem; GFP: 37±0.6, P467L: 66±0.7, p<0.05 vs. IgG), suggesting that TIMP-4 is a direct target of PPARγ. Our findings highlight one of protective mechanisms of PPARγ during hypertension and provide a novel link between PPARγ and TIMP-4.