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 homolog of a Hedgehog protein antagonist. Here, we present our investigation of HHIPL1 function and its role in atherosclerosis.
Epitope tagged HHIPL1 protein is present in the media of HHIPLI transfected cells and immunoprecipitates with GFP tagged Sonic Hedgehog (SHH) protein, demonstrating that HHIPL1 is a secreted interactor of SHH. We measured HHIPL1 gene expression in different cardiovascular cell types and found that it is primarily expressed in aortic smooth muscle cells (AoSMCs). We performed siRNA knock-down of HHIPL1 in AoSMCs and observed a reduction of 32% (+/-11%) in proliferation (p = 0.002 at 72 h) and a reduction of 27% (+/-4%) in migration (p = 0.02 at 12 h). We went on to examine the role of Hhipl1 in atherosclerosis in vivo. In atherosclerotic mouse aortas Hhipl1 expression increased with disease progression (~2-fold increase at 6 weeks vs 32 weeks of age, p = 0.001). We crossed Hhipl1-/- mice onto Apoe-/- and Ldlr-/- atherosclerosis prone backgrounds. Hhipl1-/- mice displayed a reduction of 57% (+/-28%) in lesion area compared with controls on an Ldlr -/- background (en face aorta, n = 10 Hhipl1+/+; Ldlr-/- vs n = 19 Hhipl1-/-; Ldlr-/-, t-test p = 0.00007) and 49% (+/-28%) reduction on an Apoe-/- background (en face aorta, n = 9 Hhipl1+/+; Apoe-/- vs n = 17 Hhipl1-/-; Apoe-/- , t-test p = 0.0004).
Our data represent the first experimental investigation of HHIPL1. We find that HHIPL1 is a proatherogenic protein that regulates smooth muscle cell proliferation and migration, presumably through its involvement in Hedgehog signalling. Ongoing analysis will further define the mechanisms through which HHIPL1 contributes to atherosclerosis.
- Coronary artery disease
- hedgehog signalling
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