Functional analysis of the coronary artery disease associated gene HHIPL1

Genome-wide association studies have identified chromosome 14q32 as a locus for coronary artery disease in humans. The disease associated variants fall in a gene called Hedgehog interacting protein-like 1 (HHIPL1), which encodes an uncharacterised sequence homologue of an antagonist of Hedgehog signalling. Here, is presented the investigation of HHIPL1 function and its role in atherosclerosis. In vitro analysis was undertaken in order to determine the molecular and cellular function of the protein. Epitope tagged HHIPL1 protein was present in the media of transfected cells and immunoprecipitated with GFP tagged Sonic Hedgehog (SHH) protein, demonstrating that HHIPL1 is a secreted interactor of SHH. HHIPL1 gene expression was also measured in cardiovascular cell types and found that it is expressed in aortic smooth muscle cells (HASMC), but not in other disease relevant cell types. During atherogenesis smooth muscle cells migrate and proliferate into the tunica intima. Therefore, the effect of HHIPL1 on HASMC phenotype was examined following HHIPL1 knockdown. Down regulation of HHIPL1 through siRNA resulted in a significant reduction in both HASMC proliferation and migration, suggesting a regulatory role for HHIPL1 in smooth muscle cell phenotype. Next, the role of HHIPL1 in atherosclerosis in vivo was examined. In atherosclerotic mouse aortas (Apoe-/-) Hhipl1 expression increased with disease progression. In order to further investigate the effect of Hhipl1 on atherosclerosis Hhipl1 knockout mice, which are phenotypically normal, were crossed onto two hyperlipidemic atherosclerosis prone backgrounds. Double knockout mice (Hhipl1-/-; Apoe-/-, Hhipl1-/-; Ldlr-/-) displayed a sunstantial reduction of over 60% in atherosclerotic lesion size compared with controls. Moreover, Hhipl1-/- lesions were characterised by reduced smooth muscle cell content, but unaltered lipid and macrophage profile. These data represent the first experimental investigation of HHIPL1 and demonstrate that HHIPL1 is a proatherogenic protein that regulates smooth muscle cell proliferation and migration, presumably through its involvement in Hedgehog signalling.