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Rationale: Acquisition of a procalcific phenotype by resident or circulating cells is important for calcification of atherosclerotic plaques, which is common in diabetes., Objective: We aim to identify and characterize circulating calcifying cells, and to delineate a pathophysiological role for these cells in type 2 diabetes., Methods and Results: We demonstrate for the first time that a distinct subpopulation of circulating cells expressing osteocalcin and bone alkaline phosphatase (OC(+)BAP(+)) has procalcific activity in vitro and in vivo. The study of naïve patients with chronic myeloid leukemia indicated that OC(+)BAP(+) cells have a myeloid origin. Myeloid calcifying OC(+)BAP(+) cells (MCCs) could be differentiated from peripheral blood mononuclear cells, and generation of MCCs was closely associated with expression of the osteogenic transcription factor Runx2. In gender-mismatched bone marrow-transplanted humans, circulating MCCs had a much longer half-life compared with OC(-)BAP(-) cells, suggesting they belong to a stable cell repertoire. The percentage of MCCs was higher in peripheral blood and bone marrow of type 2 diabetic patients compared with controls but was lowered toward normal levels by optimization of glycemic control. Furthermore, diabetic carotid endoarterectomy specimens showed higher degree of calcification and amounts of cells expressing OC and BAP in the α-smooth muscle actin-negative areas surrounding calcified nodules, where CD68(+) macrophages colocalize. High glucose increased calcification by MCCs in vitro, and hypoxia may regulate MCC generation in vitro and in vivo., Conclusions: These data identify a novel type of blood-derived procalcific cells potentially involved in atherosclerotic calcification of diabetic patients.

Objective: Hyperglycemia is the main determinant of long-term diabetic complications, mainly through induction of oxidative stress. NAD(P)H oxidase is a major source of glucose-induced oxidative stress. In this study, we tested the hypothesis that rosiglitazone (RSG) is able to quench oxidative stress initiated by high glucose through prevention of NAD(P)H oxidase activation., Methods and Results: Intracellular ROS were measured using the fluoroprobe TEMPO-9-AC in HUVECs exposed to control (5 mmol/L) and moderately high (10 mmol/L) glucose concentrations. NAD(P)H oxidase and AMPK activities were determined by Western blot. We found that 10 mmol/L glucose increased significantly ROS production in comparison with 5 mmol/L glucose, and that this effect was completely abolished by RSG. Interestingly, inhibition of AMPK, but not PPARgamma, prevented this effect of RSG. AMPK phosphorylation by RSG was necessary for its ability to hamper NAD(P)H oxidase activation, which was indispensable for glucose-induced oxidative stress. Downstream of AMPK activation, RSG exerts antioxidative effects by inhibiting PKC., Conclusions: This study demonstrates that RSG activates AMPK which, in turn, prevents hyperactivity of NAD(P)H oxidase induced by high glucose, possibly through PKC inhibition. Therefore, RSG protects endothelial cells against glucose-induced oxidative stress with an AMPK-dependent and a PPARgamma-independent mechanism.

Endogenous ouabain may play a role in the control of cardiovascular system function. In this study, we investigated the effects of a long-term ouabain treatment on basal and endothelin-1 (ET-1)-induced phosphorylation of cardiac and vascular extracellular signal-regulated kinases 1 and 2 (ERK-1 and ERK-2), which are involved in several cardiac and vascular physiologic and pathologic conditions. Our results show that the hearts from ouabain-treated rats have a higher basal level of ERK-1 and ERK-2 phosphorylation compared with untreated rats. Perfusion of the hearts with ET-1 increased ERK-1 and ERK-2 phosphorylation both in ouabain-treated and in control rats, with a larger stimulatory effect in ouabain-treated animals. On the contrary, exposure of endothelium-free tail artery to ET-1 increased ERKs phosphorylation both in treated and untreated rats, but this effect was blunted in ouabain-treated rats. These findings demonstrate that ouabain treatment has opposite effects on basal and ET-1-induced ERKs phosphorylation in the heart and in the tail artery of the rat.