Abstract-Arterial calcification, common in vascular diseases, involves vascular smooth muscle cell (VSMC) transformation to an osteoblast phenotype. Clinical studies suggest that magnesium may prevent this, but mechanisms are unclear. We assessed whether increasing magnesium levels reduce VSMC calcification and differentiation and questioned the role of the Mg 2ϩ transporter, transient receptor potential melastatin (TRPM)7 cation channels in this process. Rat VSMCs were exposed to calcification medium in the absence and presence of magnesium (2.0 to 3.0 mmol/L) or 2-aminoethoxy-diphenylborate (2-APB) (TRPM7 inhibitor). VSMCs from mice with genetically low (MgL) or high-normal (MgH) [Mg 2ϩ ] i were also studied. Calcification was assessed by von Kossa staining. Expression of osteocalcin, osteopontin, bone morphogenetic protein (BMP)-2, BMP-4, BMP-7, and matrix Gla protein and activity of TRPM7 (cytosol:membrane translocation) were determined by immunoblotting. Calcification medium induced osteogenic differentiation, reduced matrix Gla protein content, and increased expression of the sodium-dependent cotransporter Pit-1. Magnesium prevented calcification and decreased osteocalcin expression and BMP-2 activity and increased expression of calcification inhibitors, osteopontin and matrix Gla protein. TRPM 7 activation was decreased by calcification medium, an effect reversed by magnesium. 2-APB recapitulated the VSMC osteoblastic phenotype in VSMCs. Osteocalcin was increased by calcification medium in VSMCs and intact vessels from MgL but not MgH, whereas osteopontin was increased in MgH, but not in MgL mice. Magnesium negatively regulates vascular calcification and osteogenic differentiation through increased/restored TRPM7 activity and increased expression of anticalcification proteins, including osteopontin, BMP-7, and matrix Gla protein. New molecular insights are provided whereby magnesium could protect against VSMC calcification. (Hypertension. 2010;56:453-462.)

Rationale Although Nox5, (Nox2 homologue), has been identified in the vasculature, its regulation and functional significance remain unclear. Objectives To test if vasoactive agents regulate Nox5 through Ca2+/calmodulin-dependent processes and whether Ca2+ -sensitive Nox5, associated with Rac-1, generates superoxide (•O2−) and activates growth and inflammatory responses via MAP kinases in human endothelial cells (ECs). Methods and results Cultured ECs, exposed to AngII and ET-1 in the absence and presence of diltiazem (Ca2+ channel blocker), calmidazolium (calmodulin inhibitor) and EHT1864 (Rac-1 inhibitor), were studied. Nox5 was downregulated with siRNA. AngII and ET-1 increased Nox5 expression (mRNA and protein). Effects were inhibited by actinomycin D and cycloheximide and blunted by diltiazem, calmidazolium and low extracellular Ca2+ ([Ca2+]e). Ang II and ET-1 activated NADPH oxidase, an effect blocked by low [Ca2+]e, but not by EHT1864. Nox5 knockdown abrogated agonist-stimulated •O2− production and inhibited phosphorylation of ERK1/2, but not p38MAPK or SAPK/JNK. Nox5 siRNA blunted AngII-induced, but not ET-1-induced, upregulation of PCNA and VCAM-1, important in growth and inflammation. Conclusions Human ECs possess functionally active Nox5, regulated by AngII and ET-1 through Ca2+/calmodulin-dependent, Rac-1-independent mechanisms. Nox5 activation by AngII and ET-1 induces ROS generation and ERK 1/2 phosphorylation. Nox5 is involved in ERK1/2-regulated growth and inflammatory signaling by AngII but not by ET-1. We elucidate novel mechanisms whereby vasoactive peptides regulate Nox5 in human ECs and demonstrate differential Nox5-mediated functional responses by AngII and ET-1. Such phenomena link Ca2+/calmodulin to Nox5 signaling, potentially important in the regulation of endothelial function by AngII and ET-1.