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Introduction Vascular calcifi cation is a regulated process, which associates with coronary artery disease (CAD) and occurs through an increase in transcription factor expression such as RUNX2, MSX2 and alkaline phosphatase (ALP), then inducing calcium deposition. Bone morphogenetic protein-2 (BMP2) is a potent osteochondrogenic mediator, which is expressed in CAD. Endothelin-1 (ET1) and leptin have a role in regulating infl ammation and CAD. We hypothesized that BMP2, leptin or both increase ROS formation in C57BL/6 vascular smooth muscle cells (SMC), stimulating osteochondrogenic diff erentiation. We also investigated the eff ect of ET1 in SMC osteochondrogenesis. Our objectives were: to investigate ROS production in SMC after BMP2 (50 ng/ml) and/or leptin (10 ng/ml) incubation for 6 hours; and to assess osteochondrogenic gene expression and calcifi cation of SMC stimulated with BMP2, leptin or ET1 (10 nM). Methods We assessed 2-hydroxyethidium, more specifi c for superoxide, and ethidium which refl ects hydrogen peroxide through HPLC analysis in SMC after stimulation. SMC cells were incubated with these stimuli for 48 to 96 hours and RUNX2, MSX2, ALP mRNA and protein expression were assessed using qPCR and western blotting. We quantifi ed SMC calcifi cation after 14 days of stimulation through Alizarin Red staining. Results The results are shown as mean ± SD and were statistically signifi cant when pHydrogen peroxide and superoxide production increased both in BMP2 and in leptin-incubated SMC (3.77 ± 0.32 and 3.26 ± 0.26) versus control (n = 6); pBMP2 and leptin alone increased SMC calcifi cation (1.25 ± 0.08 and 1.28 ± 0.14) versus control after 14 days (n = 6); pET1 alone did not stimulate osteocondrogenic mRNA expression vs. control. Conclusion We showed that BMP2 and leptin increased ROS formation in SMC, which stimulated osteocondrogenic mRNA/protein expression to induce SMC calcifi cation. ET1 alone did not increase osteochondrogenesis in SMC. P2 Eff ects of rapid repetition of a vascular occlusion test on near-infrared spectroscopy-derived variables in healthy subjects and in critically ill patients