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Introduction: Calcification of atherosclerotic plaques is associated with different histopathological and clinical manifestations. Microcalcification promotes inflammation and increases plaque instability, whereas macrocalcification is associated with stable fibroatheroma. The mechanisms of initiation and progression of calcification in atherosclerosis are not completely understood. Hypothesis: Upregulation of tissue-nonspecific alkaline phosphatase (TNAP) in monocytes/macrophages can lead to calcification of atherosclerotic plaques in a mouse model. Methods: TNAP overexpression was induced from a targeted transgene in Hprt locus utilizing Cre recombinase driven by monocyte-specific LyzM gene promoter. Resultant mice overexpressing TNAP in cells of monocyte/macrophage lineage (TNAP-Tg) were bred with animals bearing low density lipoprotein receptor mutation. Hypercholesterolemia was induced by feeding Paigen diet for ten weeks. Average plaque area and calcification (area %) were determined from histological sections of the aortic root (5-10 sections/sample). Blood flow velocity in the ascending aorta and left ventricular function were assessed by echocardiography. Results: In the absence of atherosclerosis, no appreciable soft tissue calcification was observed in TNAP-Tg animals (n=2). During progression of atherosclerosis, calcification was detected in the aortic root lesions of TNAP-Tg mice (4.85% of total plaque area) in significant excess compared to controls (0.02%, p Conclusions: We developed an animal model in which the interaction between early calcium deposition inside atherosclerotic lesions and the stability of such lesions can be studied experimentally.

Objective— Peripheral blood contains primitive (stem cell-like) and monocytic-like endothelial cell progenitors. Diabetes apparently converts these primitive progenitors, from a pro-angiogenic to anti-angiogenic phenotype. Monocytic progenitors seem to be less affected by diabetes, but potential pro-angiogenic activities of freshly isolated monocytic progenitors remain unexplored. We compared the ability of primitive and monocytic endothelial cell progenitors to stimulate vascular growth and healing in diabetes and investigated potential molecular mechanisms through which the cells mediate their in vivo effects. Methods and Results— Human CD34 + primitive progenitors and CD14 + monocytic progenitors were injected locally into the ischemic limbs of diabetic mice. CD14 + cell therapy improved healing and vessel growth, although not as rapidly or effectively as CD34 + cell treatment. Western blot analysis revealed that cell therapy modulated expression of molecules in the VEGF, MCP-1, and angiopoietin pathways. Conclusions— Injection of freshly isolated circulating CD14 + cells improves healing and vascular growth indicating their potential for use in acute clinical settings. Importantly, CD14 + cells could provide a therapeutic option for people with diabetes, the function of whose CD34 + cells may be compromised. At least some progenitor-induced healing probably is mediated through increased sensitivity to VEGF and increases in MCP-1, and possibly modulation of angiopoietins.