HHIPL1, a Gene at the 14q32 Coronary Artery Disease Locus, Positively Regulates Hedgehog Signaling and Promotes Atherosclerosis

Background: Genome-wide association studies have identified chromosome 14q32 as a locus for coronary artery disease. The disease-associated variants fall in a hitherto uncharacterized gene called HHIPL1 (hedgehog interacting protein-like 1), which encodes a sequence homolog of an antagonist of hedgehog signaling. The function of HHIPL1 and its role in atherosclerosis are unknown.

Methods: HHIPL1 cellular localization, interaction with sonic hedgehog (SHH), and influence on hedgehog signaling were tested. HHIPL1 expression was measured in coronary artery disease-relevant human cells, and protein localization was assessed in wild-type and Apoe^-/- (apolipoprotein E deficient) mice. Human aortic smooth muscle cell phenotypes and hedgehog signaling were investigated after gene knockdown. Hhipl1^-/- mice were generated and aortic smooth muscle cells collected for phenotypic analysis and assessment of hedgehog signaling activity. Hhipl1^-/- mice were bred onto both the Apoe^-/- and Ldlr^-/- (low-density lipoprotein receptor deficient) knockout strains, and the extent of atherosclerosis was quantified after 12 weeks of high-fat diet. Cellular composition and collagen content of aortic plaques were assessed by immunohistochemistry.

Results: In vitro analyses revealed that HHIPL1 is a secreted protein that interacts with SHH and increases hedgehog signaling activity. HHIPL1 expression was detected in human smooth muscle cells and in smooth muscle within atherosclerotic plaques of Apoe^-/- mice. The expression of Hhipl1 increased with disease progression in aortic roots of Apoe^-/- mice. Proliferation and migration were reduced in Hhipl1 knockout mouse and HHIPL1 knockdown aortic smooth muscle cells, and hedgehog signaling was decreased in HHIPL1-deficient cells. Hhipl1 knockout caused a reduction of >50% in atherosclerosis burden on both Apoe^-/- and Ldlr^-/- knockout backgrounds, and lesions were characterized by reduced smooth muscle cell content.

Conclusions: HHIPL1 is a secreted proatherogenic protein that enhances hedgehog signaling and regulates smooth muscle cell proliferation and migration. Inhibition of HHIPL1 protein function might offer a novel therapeutic strategy for coronary artery disease.