The NADPH Oxidase Nox2 Mediates Vitamin D-Induced Vascular Regeneration in Male Mice.

1α,25-dihydroxy-vitamin D3 (1,25D) exerts protective effects in the vascular system and promotes myeloid cell differentiation, which are important sources of reactive oxygen species. Given that myeloid cell reactive oxygen species derives from Nox-family NADPH oxidases, we hypothesized that this enzyme family contributes to the beneficial effects of 1,25D on vascular regeneration. The function of Nox enzymes in this context was studied in the murine carotid artery electric injury regeneration model. Male mice were treated with daily injections of 1,25D (100 ng/kg · d) for 5 days and carotid injury was induced after 3 days. After injury, 1,25D increased the expression of Nox2 in the carotid artery. As determined by Evans blue staining on day 6, 1,25D improved vascular regeneration in a Nox2-dependent manner. Healing was lost in mice knockout for Nox2, but not in Nox1 or Nox4, knockout mice. Tissue specific knockouts demonstrated that the myeloid, but not the endothelial Nox2, was required for this effect. Mechanistically, the combination of injury and 1,25D induced the mobilization of angiogenic myeloid cells (AMCs) and increased the vascular expression of the cytokine stem cell derived factor (SDF)1, which attracts AMCs to the site of injury. Vitamin D in a Nox2-dependent manner activated MAPKs, and these are known to contribute to SDF1 induction. Accordingly, SDF1 induction was lost after deletion of Nox2. By inducing SDF1 and enhancing the number of AMCs, VitD3 is a novel approach to promote vascular repair.

Oleoyl-lysophosphatidylcholine limits endothelial nitric oxide bioavailability by induction of reactive oxygen species.

Previously we reported modulation of endothelial prostacyclin and interleukin-8 production, cyclooxygenase-2 expression and vasorelaxation by oleoyl- lysophosphatidylcholine (LPC 18:1). In the present study, we examined the impact of this LPC on nitric oxide (NO) bioavailability in vascular endothelial EA.hy926 cells. Basal NO formation in these cells was decreased by LPC 18:1. This was accompanied with a partial disruption of the active endothelial nitric oxide synthase (eNOS)- dimer, leading to eNOS uncoupling and increased formation of reactive oxygen species (ROS). The LPC 18:1-induced ROS formation was attenuated by the superoxide scavenger Tiron, as well as by the pharmacological inhibitors of eNOS, NADPH oxidases, flavin-containing enzymes and superoxide dismutase (SOD). Intracellular ROS-formation was most prominent in mitochondria, less pronounced in cytosol and undetectable in endoplasmic reticulum. Importantly, Tiron completely prevented the LPC 18:1-induced decrease in NO bioavailability in EA.hy926 cells. The importance of the discovered findings for more in vivo like situations was analyzed by organ bath experiments in mouse aortic rings. LPC 18:1 attenuated the acetylcholine-induced, endothelium dependent vasorelaxation and massively decreased NO bioavailability. We conclude that LPC 18:1 induces eNOS uncoupling and unspecific superoxide production. This results in NO scavenging by ROS, a limited endothelial NO bioavailability and impaired vascular function.

Vitamin D promotes vascular regeneration.

BACKGROUND: Vitamin D deficiency in humans is frequent and has been associated with inflammation. The role of the active hormone 1,25-dihydroxycholecalciferol (1,25-dihydroxy-vitamin D3; 1,25-VitD3) in the cardiovascular system is controversial. High doses induce vascular calcification; vitamin D3 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 D3 (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 promoted SDF1 expression, as revealed by reporter assays and knockout and inhibitory strategies of hypoxia-inducible factor 1-α. CONCLUSIONS: By inducing SDF1, vitamin D3 is a novel approach to promote vascular repair.

SYNCRIP-dependent Nox2 mRNA destabilization impairs ROS formation in M2-polarized macrophages.

AIMS: During sepsis, macrophages are alternatively activated toward an M2-like phenotype on contact with apoptotic cells (ACs) or their secretion products. Simultaneously, NADPH oxidase-dependent reactive oxygen species (ROS) formation is attenuated, thus contributing to immune paralysis. However, the exact mechanism remains elusive. Here, we provide mechanistic insights into diminished mRNA stability of the NADPH oxidase Nox2 on macrophage M2 polarization and therefore reduced ROS formation in sepsis. RESULTS: Murine J774A.1 macrophages were stimulated with conditioned medium (CM) of apoptotic T cells, which reduced Nox2 mRNA and protein expression, consequently decreasing ROS production. An mRNA pulldown approach coupled to mass spectrometry analysis identified the RNA-binding protein SYNCRIP attached to the Nox2 mRNA 3' untranslated region (3'UTR). The binding of SYNCRIP to the 3'UTR of Nox2 mRNA is attenuated after treatment with CM of apoptotic T cells, followed by Nox2 mRNA destabilization. In in vivo models of polymicrobial sepsis such as cecal ligation and puncture, SYNCRIP was strongly downregulated, which was associated with a decreased Nox2 expression in peritoneal macrophages. INNOVATION: Downregulation of SYNCRIP in macrophages after contact to material of ACs destabilized Nox2 mRNA and impaired ROS formation, thereby contributing to an M2 phenotype shift of macrophages in sepsis. CONCLUSION: M2 polarization of macrophages in sepsis results in an attenuated SYNCRIP binding to the 3'UTR of Nox2 mRNA, destabilizing Nox2 mRNA abundance and expression. Consequently, ROS formation needed to fight against recurrent infections is impaired. In conclusion, SYNCRIP-regulated Nox2 mRNA degradation mediates the hypoinflammatory phase of sepsis.

COX-mediated endothelium-dependent contractions: from the past to recent discoveries.

Endothelial cells release various substances to control the tone of the underlying vascular smooth muscle. Nitric oxide (NO) is the best defined endothelium-derived relaxing factor (EDRF). Endothelial cells can also increase vascular tone by releasing endothelium-derived contracting factors (EDCF). The over-production of EDCF contributes to the endothelial dysfunctions which accompanies various vascular diseases. The present review summarizes and discusses the mechanisms leading to the release of EDCFs derived from the metabolism of arachidonic acid. This release can be triggered by agonists such as acetylcholine, adenosine nucleotides or by stretch. All these stimuli are able to induce calcium influx into the endothelial cells, an effect which can be mimicked by calcium ionophores. The augmentation in intracellular calcium ion concentration initiates the release of EDCF. Downstream processes include activation of phospholipase A(2) (PLA(2)), cyclooxygenases (COX) and the production of reactive oxygen species (ROS) and vasoconstrictor prostanoids (endoperoxides, prostacyclin, thromboxane A(2) and other prostaglandins) which subsequently diffuse to, and activate thromboxane-prostanoid (TP) receptors on the vascular smooth muscle cells leading to contraction.