Abstract 527: The BMP Inhibitor DMH1 Inhibits Aortic Smooth Muscle Cell Calcification

Abstract

Arterial calcification is associated with increased morbidity and mortality in patients with lower extremity occlusive disease. In calcifying arteries, vascular smooth muscle cells (SMC) undergo phenotypic transformation to a more bone-like cell. It is thought that Bone Morphogenetic Proteins (BMPs) may regulate this process. We sought to evaluate the effects of the novel, synthetic, non-peptide, BMP inhibitor DMH1 on SMC calcification. Methods: Confluent human aortic SMCs were incubated in calcification medium containing 3 mM Phosphate (Pi). Cells in calcification medium were treated with increasing concentrations of DMH1. After 7 days, cells were incubated in 0.6N HCl overnight and the amount of soluble calcium was measured in the media. Calcium levels were normalized to protein content as measured using the BCA assay. Total RNA from SMCs was isolated with RNeasy mini kit and qRT-PCR was performed with respective gene-specific primers. Rat aorta rings were induced to calcify by stimulation with phosphate in DMEM with or without DMH1 for 7days. Calcium was measured as described above. Changes in BMP signaling proteins p-smad1/5/8 and total smad1/5/8 were assessed by western blot at 24 hours. Findings: Cells grown in calcification medium had increased amounts of calcification compared with controls; however, this was significantly reduced by addition of DMH1 in a dose dependent manner. Similar findings were seen in aortic rings. Interestingly, DMH1 increased the expression of the two bone-related proteins alkaline phosphatase and RANK ligand while decreasing the expression levels of the SMC-specific genes α-SM-actin and SM22-α. Western blot analysis revealed decreased p-smad1/5/8 levels in DMH1-treated cells. Conclusions: The synthetic BMP inhibitor DMH1 inhibits Pi-induced calcification of human vascular SMCs and rat aorta rings. BMP-inhibiting compounds may have clinical utility in inhibiting arterial calcification and reducing amputation in our patients with critical limb ischemia.