Abstract
Risk of hypertension, peripheral artery disease, myocardial infarction and development of human atherosclerosis has been linked to vitamin D deficiency. Atheromatous cytokines, including IL-6, TNF-α and epidermal growth factor receptor (EGFR) family growth factors are released at the site of atherosclerosis and boost the activity of proteolytic enzymes such as ADAMs (a disintegrin and metalloproteinases). ADAM-12 cleaves proHB-EGF (Heparin-binding EGF-like growth factor) activating EGFR, resulting in increased proliferation of smooth muscle cells (VSMCs). Our lab has reported that vitamin D supplementation reduces the intimal hyperplasia in hypercholesterolemic swine, but the underlying mechanisms are not well understood. The aim of this study was to examine the effect of vitamin D on IL-6 and TNF-α-induced ADAM-12 mediated growth factor HB-EGF release, EGFR phosphorylation and SMC proliferation due to activation of EGFR.|In this study, animal model i.e. micro-swine (Sus scrofa domesticus) were fed with vitamin D-deficient high cholesterol diet, high cholesterol diet containing 900 IU of vitamin D, and high cholesterol diet containing 3000 IU of vitamin D for total of 12 months. Serum levels of 25(OH)D and cholesterol were measured each month until euthanasia. After six months, serum cholesterol levels of 500-600 mg/dL were achieved in all the three groups. The protein levels of ADAM-12 & pEGFR, and HB-EGF release, in presence or absence of IL-6, TNF-α and Calcitriol, in VSMCs was quantified by western blot. HB-EGF release was measured by ELISA. Cell proliferation was assayed by [3H]-Thymidine incorporation and cell counting.|Vitamin D-deficient swine carotid artery VSMCs expressed more ADAM-12, released more HB-EGF, and had higher levels of EGFR phosphorylation was increased compared to D-sufficient and D- supplemented swine VSMCs. Treatment of VSMCs with ADAM-12 siRNA inhibited ADAM-12 expression, HB-EGF release and EGFR phosphorylation. HB-EGF treatment induced proliferation in carotid artery VSMCs of vitamin D-deficient, D-sufficient and D-supplemented swine VSMCs. Treatment of VSMCs with both HB-EGF and ADAM-12 siRNA together inhibited proliferation. IL-6 and TNF-α treatment increased the protein expression of ADAM-12, EGFR phosphorylation and HB-EGF release in carotid artery VSMCs of vitamin D-deficient, -sufficient and - supplemented swine VSMCs. Proliferation was higher in VSMCs isolated from vitamin D-deficient swine carotid artery and potentiated by IL-6 and TNF-α. Calcitriol inhibited ADAM-12 and EGFR expression and HB-EGF release in VSMCs of hypercholesterolemic swine. Calcitriol also inhibited the proliferation of carotid artery VSMCs isolated from vitamin D-deficient, -sufficient and -supplemented swine. Calcitriol mediated inhibition of proliferation of VSMCs also occurred in the presence or absence of IL-6 or TNF- α.|Together, these results suggest that vitamin D deficiency enhances proliferation of VSMCs, which is potentiated by atheromatous cytokines. In contrast, vitamin D supplementation reduces ADAM-12-mediated cleavage of proHB-EGF and activation of EGFR inhibiting SMC proliferation. This study provides a new approach for researching therapeutic effect of vitamin D. Vitamin D as therapeutic agent is inexpensive and safe for patients and could be useful in developing better therapeutic modalities applicable to carotid artery disease.