Redox-sensitive osteogenic factors as novel mediators of vascular injury in hypertension

Vascular calcification is associated with cardiovascular disease, including hypertension. Pro-osteogenic factors, such as BMP-2 and osteocalcin (OC) and anti-osteogenic factors such as osteoproteregin (OPG) (which is also pro-inflammatory through activation of RANKL and TRAIL), have been implicated in this process. In addition, reactive oxygen species (ROS) play a role in vascular calcification and hypertension-associated vascular remodeling. Here we postulated that in hypertension, vascular calcification leads to increased levels of osteogenic factors which may further promote vascular injury. Aorta and VSMCs from WKY and SHRSP (16-18 weeks) rats were studied. Vascular calcification was assessed by Von Kossa staining. VSMCs were exposed to control and calcification medium (CaM - Ca2+ 1.8 mmol/L, PO4 2.0 mmol/L) for 10 days. Osteocalcin (OC), BMP-2, BMP-7, OPG, TRAIL and fibronectin expression were assessed by immmunoblotting. ROS generation was studied by chemiluminescence. Aorta from SHRSP, but not WKY, exhibited positive Von Kossa staining. At basal levels, expression of osterix (86%), OPG (20%), TRAIL (28%) and fibronectin (51%) were increased in VSMCs from SHRSP rats. CaM, as predicted, induced an increase in OC (Ctl: 5 AU; CaM: 13 AU) and BMP-2 (Ctl: 0.60 AU; CaM: 0.75AU) (p<0.05), followed by a decrease in BMP-7 (Ctl: 1.12 AU; CaM: 0.7 AU) (p<0.05), expression in VSMCs from WKY, an effect that was inhibited by tempol (antioxidant). ROS production (Ctl: 30 AU/ug protein; CaM: 60 AU/ug protein, p<0.05) was increased by the CaM in VSMCs from WKY. Responses to CaM were augmented in VSMCs from SHRSP with respect to BMP-2 expression (p<0.05). Osteogenic regulation in SHRSP-derived VSMCs were sensitive to tempol . OPG stimulation of VSMCs from WKY and SHRSP induced ROS generation in a concentration-dependent manner (OPG 100ng/5 min: WKY - 40% increase; SHRSP - 75% increase, p<0.05 vs vehicle). In conclusion, the OPG system is upregulated in hypertension and may be related to activation of redox-sensitive pathways. Identification of vascular calcification-derived osteogenic factors as positive modulators of VSMC damage provides new insights into molecular mechanisms of vascular injury in hypertension