Regulatory role of endothelium in the expression of genes affecting arterial calcification q Clarissa Cola, Maria Almeida, Dayuan Li, Francesco Romeo, and Jawahar L. Mehta * Departments of Internal Medicine and Physiology and Biophysics, University of Arkansas for Medical Sciences and Central Arkansas Veterans Healthcare System, Little Rock, AR, USA Department of Internal Medicine, Section of Cardiology, University of Tor Vergata, Rome, Italy Received 18 May 2004 Available online 15 June 2004 Abstract Vascular calcification is a highly regulated process sharing features of bone mineralization. Since endothelium regulates many of the processes during atherogenesis, we monitored the expression of genes involved in calcification upon exposure of human coronary artery endothelial cells (HCAECs) to atherogenic stimuli. Genes studied were: core binding factor a-1 (Cbfa1/Runx2), a pivotal transcriptional regulator of osteogenesis; bone morphogenetic protein-2 (BMP2), an inducer of cartilage and bone; and matrix gla- protein (MGP), a potent inhibitor of calcification, which exerts its action by blocking BMP2. HCAECs were treated with oxidized- low density lipoprotein (ox-LDL, 80 lg/mL) or tumor necrosis factor-a (TNFa, 10 ng/mL), and the expression of Cbfa1, BMP2, and MGP was quantified by real-time PCR. Cbfa1 was expressed at low levels in untreated HCAECs, and its expression did not change with ox-LDL or TNFa treatment. The expression of BMP2 and MGP increased early after exposure to ox-LDL or TNFa (at 2–8 h), and the increase was not evident at 24 h. Ox-LDL exerted a stronger effect on MGP than on BMP2 expression. The effects of ox- LDL, but not TNFa, on MGP and BMP2 expression were inhibited by pretreatment of cells with an antibody directed at LOX-1, a lectin-like receptor for ox-LDL (10 lg/mL). Thus, the endothelium, when exposed to atherogenic stimuli, ox-LDL in particular, regulates the process of calcification by enhancing the expression of the bone inhibitory MGP, while the expression of Cbfa1 remains unchanged. Upregulation of BMP2 may represent a feedback upregulation in response to increase in MGP. The effect of ox-LDL appears to be mediated by LOX-1 activation. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Bone forming genes; Endothelial cells; Oxidized-LDL; Tumor necrosis factor-a Vascular calcification is a key feature of atheroscle- rosis [1]. This ectopic mineralization is a highly regu- lated process, analogous to skeletal bone formation [2]. Vascular smooth muscle cells (SMCs) and pericytes as- sume osteoblastic/chondrocytic phenotype [3], which implies a phenotypic modulation of resident vascular cells in a permissive matrix environment. The factors that regulate this phenotypic transition are unknown; however, a number of potent transcription factors that regulate the expression of many of the osteoblast and chondrocyte functions have been identified in the growing atherosclerotic lesion. These include: the runt- domain transcription factor core binding factor a-1 (Cbfa-1), the master regulator of bone differentiation; this protein serves as the transcription factor triggering the expression of major osteoblast-specific lineage genes [4]. Cbfa1 is co-localized within the calcified athero- sclerotic areas [1]. Bone morphogenetic protein-2 (BMP2), member of the transforming growth factor b- superfamily, induces ectopic bone formation [5], and angiogenesis via induction of VEGF-A expression. It is expressed in small amounts in healthy arterial walls [6]. Matrix gla-protein (MGP) is a vitamin K dependent calcification inhibitor, and its deficiency results in pre- mature calcification in bone and cartilage and ectopic calcification [7]. MGP is expressed in normal as well as in atherosclerotic blood vessels in humans. It is absent in q This work was supported by grants from the American Heart Association and the Department of Veteran Affairs. * Corresponding author. Fax: 1-501-686-6180. E-mail address: mehtajl@uams.edu (J.L. Mehta). 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.05.181 Biochemical and Biophysical Research Communications 320 (2004) 424–427 BBRC www.elsevier.com/locate/ybbrc