T. Lin1, X. Wang1, R. Guzman1 1Beth Israel Deaconess Medical Center,Surgery,Boston, MA, USA
Arterial calcification is associated with increased cardiovascular morbidity and mortality. It is also associated with increased rates of restenosis after angioplasty. During arterial calcification, medial smooth muscle cells (SMCs) undergo phenotypic transformation into a more osteogenic cell type and it is thought that Bone Morphogenetic Proteins (BMPs) act as regulators of this process. We sought to evaluate the effects of the synthetic, non-peptide, BMP inhibitor DMH1 on migration and proliferation of calcifying SMCs.
Methods: Human aortic SMCs were induced to calcify by stimulation with 3mM inorganic phosphate (Pi) in DMEM containing 10%FBS or control medium without Pi. Medium was changed every other day with or without increasing doses of DMH1. Calcium content was measured using the o-Cresolphthalein Complexone method normalized to protein content after 7 days. Total RNA from SMCs was isolated at 4 and 7 days using the RNeasy mini kit (Qiagen, USA) and qRT-PCR was performed with respective gene-specific primers. Changes in alpha-SM-actin, alkaline phosphatase (ALPL) and BMP signaling proteins p-Smad1/5/8 were assessed by western blot. Proliferation was assayed using the BrdU cell proliferation assay kit (EMDMillipore, USA). Migration was assayed by using transwell migration assay (Costar, USA).
Results: Cells grown in Pi-containing medium had increased amounts of calcification compared with controls. DMH1 decreased calcium accumulation in Pi-stimulated SMCs in a dose dependent manner. Addition of DMH1 decreased the expression of smooth muscle cell markers (alpha-SM actin and SM22-alpha) while increasing the expression of the bone markers ALPL, RANK ligand and Runx2. In calcifying SMCs, DMH1 inhibited proliferation more significantly than in non-calcifying SMCs suggesting that calcifying SMCs are more sensitive to BMP receptor activation. In a transwell assay, addition of DMH1 increased migration in calcifying but not uncalcified cells.
Conclusions: We find that the synthetic BMP inhibitor DMH1 inhibits Pi-induced calcification and reduces proliferation of calcifying SMCs more strongly than untransformed cells. This suggests that cells from calcifying arteries are different, and that they may respond to BMP stimulation more like osteocytes than smooth muscle cells. DMH1 or similar BMP inhibiting compounds may have clinical utility in decreasing SMC proliferation after endovascular interventions in patients with calcified arteries due to diabetes and renal disease.