Ϫ ) react with nitric oxide (NO) to form peroxynitrite (ONOO Ϫ ), a process that limits NO availability, results in NO synthase (NOS) uncoupling, and, through the action of ONOO Ϫ , leads to protein and thiol oxidation as well as tyrosine nitration. 1 Hydrogen peroxide (H 2 O 2 ), the dismutation product of O 2 Ϫ , also elicits multiple effects, among them smooth muscle cell hypertrophy, activation of metalloproteinases, and, in higher concentrations, NOS inhibition by phosphorylation of tyrosine 657 through the redox-activated tyrosine kinase Pyk2. 2 Interestingly, H 2 O 2 also induces positive endothelial effects because it can activate protein kinase-G I␣ by thiol oxidation and subsequent dimerization. 3 Moreover, H 2 O 2 induces as well as activates endothelial NOS (eNOS). 4

The H2O2-producing NADPH oxidase Nox4 is an endogenous anti-atherosclerotic enzyme. Nox4 inhibitors, currently under clinical evaluation, should be carefully monitored for cardiovascular side-effects.

V itamin D is a misnomer. The body produces this hormone through precursors with final hydroxylation to the most active metabolite 1, 1,. Vitamin D is ingested with food or synthesized from the precursor 7-dehydrocholesterol, which is then photochemically converted in the sunlight-exposed skin into cholecalciferol (vitamin D 3 ). Active 1,25-VitD3 is generated by 2 hydroxylation steps: 25β-hydroxylation in the liver followed by 1α-hydroxylation in the kidney. The latter reaction is particularly rate limiting, and thus the effective concentration of 1,25-VitD3 is much lower than that of vitamin D 3 or 25-hydroxy-vitamin D 3 . By a positive feedback loop, 1,25-VitD3 induces 24-hydroxylase expression. Because 1,24, is water soluble and cleared by the kidney, this pathway limits the effects of the hormone, and 24-hydroxylase expression can be used as a marker for the biological activity of 1,25-VitD3. Unless vitamin D is supplemented, intake with food is usually insufficient, and thus endogenous production contributes significantly to plasma levels. This leads to vitamin D deficiencies in up to 70% in some population groups in which sunlight exposure is low because of modern lifestyle, clothing, and few sunshine hours during winter. 1,2 Clinical Perspective on p 986Background-Vitamin D deficiency in humans is frequent and has been associated with inflammation. The role of the active hormone 1, 1, in the cardiovascular system is controversial. High doses induce vascular calcification; vitamin D 3 deficiency, however, has been linked to cardiovascular disease because the hormone has anti-inflammatory properties. We therefore hypothesized that 1,25-VitD3 promotes regeneration after vascular injury. Methods and Results-In healthy volunteers, supplementation of vitamin D 3 (4000 IU cholecalciferol per day) increased the number of circulating CD45-CD117+Sca1+Flk1+ angiogenic myeloid cells, which are thought to promote vascular regeneration. Similarly, in mice, 1,25-VitD3 (100 ng/kg per day) increased the number of angiogenic myeloid cells and promoted reendothelialization in the carotid artery injury model. In streptozotocin-induced diabetic mice, 1,25-VitD3 also promoted reendothelialization and restored the impaired angiogenesis in the femoral artery ligation model. Angiogenic myeloid cells home through the stromal cell-derived factor 1 (SDF1) receptor CXCR4. Inhibition of CXCR4 blocked 1,25-VitD3-stimulated healing, pointing to a role of SDF1. The combination of injury and 1,25-VitD3 increased SDF1 in vessels. Conditioned medium from injured, 1,25-VitD3-treated arteries elicited a chemotactic effect on angiogenic myeloid cells, which was blocked by SDF1-neutralizing antibodies. Conditional knockout of the vitamin D receptor in myeloid cells but not the endothelium or smooth muscle cells blocked the effects of 1,25-VitD3 on healing and prevented SDF1 formation. Mechanistically, 1,25-VitD3 increased hypoxia-inducible factor 1-α through binding to its promoter. Increased hypoxia-inducible factor signaling subsequently...

Rationale: Polarity proteins are involved in the apico-basal orientation of epithelial cells, but relatively little is known regarding their function in mesenchymal cells. Objective: We hypothesized that polarity proteins also contribute to endothelial processes like angiogenesis. Methods and Results: Screening of endothelial cells revealed high expression of the polarity protein Scribble (Scrib). On fibronectin-coated carriers Scrib siRNA (siScrib) blocked directed but not random migration of human umbilical vein endothelial cells and led to an increased number and disturbed orientation of cellular lamellipodia. Coimmunoprecipitation/mass spectrometry and glutathione S-transferase (GST) pulldown assays identified integrin α5 as a novel Scrib interacting protein. By total internal reflection fluorescence (TIRF) microscopy, Scrib and integrin α5 colocalize at the basal plasma membrane of endothelial cells. Western blot and fluorescence activated cell sorting (FACS) analysis revealed that silencing of Scrib reduced the protein amount and surface expression of integrin α5 whereas surface expression of integrin αV was unaffected. Moreover, in contrast to fibronectin, the ligand of integrin α5, directional migration on collagen mediated by collagen-binding integrins was unaffected by siScrib. Mechanistically, Scrib supported integrin α5 recycling and protein stability by blocking its interaction with Rab7a, its translocation into lysosomes, and its subsequent degradation by pepstatin-sensitive proteases. In siScrib-treated cells, reinduction of the wild-type protein but not of PSD95, Dlg, ZO-1 (PDZ), or leucine rich repeat domain mutants restored integrin α5 abundance and directional cell migration. The downregulation of Scrib function in Tg(kdrl:EGFP) s843 transgenic zebrafish embryos delayed the angiogenesis of intersegmental vessels. Conclusions: Scrib is a novel regulator of integrin α5 turnover and sorting, which is required for oriented cell migration and sprouting angiogenesis.