The polarity protein Scrib limits atherosclerosis development in mice.

AIMS: The protein Scrib (Scribble 1) is known to control apico-basal polarity in epithelial cells. The role of polarity proteins in the vascular system remains poorly characterized; however, we previously reported that Scrib maintains the endothelial phenotype and directed migration. On this basis, we hypothesized that Scrib has anti-atherosclerotic functions. METHODS AND RESULTS: Tamoxifen-induced Scrib-knockout mice were crossed with ApoE-/- knockout mice and spontaneous atherosclerosis under high-fat diet (HFD), as well as accelerated atherosclerosis in response to partial carotid artery ligation and HFD, was induced. Deletion of Scrib resulted in increased atherosclerosis development in both models. Mechanistically, flow- as well as acetylcholine-induced endothelium-dependent relaxation and AKT phosphorylation was reduced by deletion of Scrib, whereas vascular permeability and leucocyte extravasation were increased after Scrib knockout. Scrib immune pull down in primary carotid endothelial cells and mass spectrometry identified Arhgef7 (Rho Guanine Nucleotide Exchange Factor 7, ßPix) as interaction partner. Scrib or Arhgef7 down-regulation by siRNA reduced the endothelial barrier function in human umbilical vein endothelial cells. Gene expression analysis from murine samples and from human biobank material of carotid endarterectomies indicated that loss of Scrib resulted in endothelial dedifferentiation with a decreased expression of endothelial signature genes. CONCLUSIONS: By maintaining a quiescent endothelial phenotype, the polarity protein Scrib elicits anti-atherosclerotic functions.

The Histone Demethylase PHF8 Is Essential for Endothelial Cell Migration.

Epigenetic marks critically control gene expression and thus the cellular activity state. The functions of many epigenetic modifiers in the vascular system have not yet been studied. We screened for histone modifiers in endothelial cells and observed a fairly high expression of the histone plant homeodomain finger protein 8 (PHF8). Given its high expression, we hypothesize that this histone demethylase is important for endothelial cell function. Overexpression of PHF8 catalyzed the removal of methyl-groups from histone 3 lysine 9 (H3K9) and H4K20, whereas knockdown of the enzyme increased H3K9 methylation. Knockdown of PHF8 by RNAi also attenuated endothelial proliferation and survival. As a functional readout endothelial migration and tube formation was studied. PHF8 siRNA attenuated the capacity for migration and developing of capillary-like structures. Given the impact of PHF8 on cell cycle genes, endothelial E2F transcription factors were screened, which led to the identification of the gene repressor E2F4 to be controlled by PHF8. Importantly, PHF8 maintains E2F4 but not E2F1 expression in endothelial cells. Consistently, chromatin immunoprecipitation revealed that PHF8 reduces the H3K9me2 level at the E2F4 transcriptional start site, demonstrating a direct function of PHF8 in endothelial E2F4 gene regulation. Conclusion: PHF8 by controlling E2F4 expression maintains endothelial function.

The NADPH oxidase Nox4 has anti-atherosclerotic functions.

AIMS: Oxidative stress is thought to be a risk for cardiovascular disease and NADPH oxidases of the Nox family are important producers of reactive oxygen species. Within the Nox family, the NADPH oxidase Nox4 has a unique position as it is constitutively active and produces H2O2 rather than [Formula: see text] . Nox4 is therefore incapable of scavenging NO and its low constitutive H2O2 production might even be beneficial. We hypothesized that Nox4 acts as an endogenous anti-atherosclerotic enzyme. METHODS AND RESULTS: Tamoxifen-induced Nox4-knockout mice were crossed with ApoE⁻/⁻ mice and spontaneous atherosclerosis under regular chow as well as accelerated atherosclerosis in response to partial carotid artery ligation under high-fat diet were determined. Deletion of Nox4 resulted in increased atherosclerosis formation in both models. Mechanistically, pro-atherosclerotic and pro-inflammatory changes in gene expression were observed prior to plaque development. Moreover, inhibition of Nox4 or deletion of the enzyme in the endothelium but not in macrophages resulted in increased adhesion of macrophages to the endothelial surface. CONCLUSIONS: 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.

Polarity Protein Scrib Facilitates Endothelial Inflammatory Signaling.

OBJECTIVE: The polarity protein Scrib is highly expressed in endothelial cells and is required for planar cell polarity. Scrib also facilitates recycling of integrin α5 to the plasma membrane. Because integrin α5 signals the presence of the inflammatory matrix protein fibronectin, we hypothesized that Scrib contributes to endothelial inflammatory signaling. APPROACH AND RESULTS: Cytokine treatment of human umbilical vein endothelial cells induced an inflammatory response as evident by the induction of vascular cell adhesion molecule-1 (VCAM-1). Downregulation of Scrib greatly attenuated this effect. In endothelial-specific conditional Scrib knockout mice, in vivo lipopolysaccharide treatment resulted in an impaired VCAM-1 induction. These effects were functionally relevant because Scrib small interfering RNAs in human umbilical vein endothelial cells attenuated the VCAM-1-mediated leukocyte adhesion in response to tumor necrosis factor-α. In vivo, tamoxifen-induced endothelial-specific deletion of Scrib resulted in a reduced VCAM-1-mediated leukocyte adhesion in response to tumor necrosis factor-α in the mouse cremaster model. This effect was specific for Scrib and not mediated by other polarity proteins. Moreover, it did not involve integrin α5 or classic pathways supporting inflammatory signaling, such as nuclear factor κ light chain enhancer of activated B-cells or MAP kinases. Co-immunoprecipitation/mass spectrometry identified the zinc finger transcription factor GATA-like protein-1 as a novel Scrib interacting protein. Small interfering RNA depletion of GATA-like protein-1 decreased the tumor necrosis factor-α-stimulated VCAM-1 induction to a similar extent as loss of Scrib did. Silencing of Scrib reduced GATA-like protein-1 protein, but not mRNA abundance. CONCLUSIONS: Scrib is a novel proinflammatory regulator in endothelial cells, which maintains the protein expression of GATA-like protein-1.

Nox4 supports proper capillary growth in exercise and retina neo-vascularization.

KEY POINTS: We provide evidence for two distinct functions of the NADPH oxidase Nox4 in angiogenesis using Nox4 knockout mice. First, Nox4 maintains vascular endothelial growth factor expression and prevents an increase in angiopoietin 1 expression, thereby contributing to angiogenesis in exercise. Second, deletion of Nox4, via an enhanced angiopoietin 1 expression, contributes to stabilization of new formed vessels and prevents an exacerbated neo-angiogenesis in oxygen-induced retinopathy. By contrast, Nox4 does not influence developmental angiogenesis. ABSTRACT: By producing H2 O2 , the NADPH oxidase Nox4 is involved in hypoxia-induced angiogenesis, as present in vascular remodelling of the hypertrophic heart or blood flow recovery after hind limb ischaemia. In the present study, we hypothesized that Nox4 contributes to proper capillary growth in the retina and in exercised muscles and investigated this in wild-type and Nox4(-/-) mice. Exercise, as induced by voluntary running in a running wheel or forced running on a treadmill, stimulated capillary growth in wild-type but not Nox4(-/-) mice. As an underlying mechanism, we identified both vascular endothelial growth factor (VEGF) expression to be reduced and angiopoietin 1 (Ang1) expression to be increased in response to Nox4 knockout. To differentiate the two factors, oxygen-induced retinopathy was investigated. In this model, deletion of Nox4 protected from neo-angiogenesis and stabilized the network of regrown vessels, which is a typical feature of Ang1. However the angiogenesis in the developing retina was similar between Nox4(-/-) and wild-type mice. Thus, Nox4 contributes to exercise- and hypoxia-induced angiogenesis through a dual mechanism of maintaining VEGF and preventing Ang-1 expression, whereas the developmental angiogenesis is Nox4 independent.

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.

The polarity protein Scrib is essential for directed endothelial cell migration.

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.

Nox4 is a protective reactive oxygen species generating vascular NADPH oxidase.

RATIONALE: The function of Nox4, a source of vascular H(2)O(2), is unknown. Other Nox proteins were identified as mediators of endothelial dysfunction. OBJECTIVE: We determined the function of Nox4 in situations of increased stress induced by ischemia or angiotensin II with global and tamoxifen-inducible Nox4(-/-) mice. METHODS AND RESULTS: Nox4 was highly expressed in the endothelium and contributed to H(2)O(2) formation. Nox4(-/-) mice exhibited attenuated angiogenesis (femoral artery ligation) and PEG-catalase treatment in control mice had a similar effect. Tube formation in cultured Nox4(-/-) lung endothelial cells (LECs) was attenuated and restored by low concentrations of H(2)O(2,) whereas PEG-catalase attenuated tube formation in control LECs. Angiotensin II infusion was used as a model of oxidative stress. Compared to wild-type, aortas from inducible Nox4-deficient animals had development of increased inflammation, media hypertrophy, and endothelial dysfunction. Mechanistically, loss of Nox4 resulted in reduction of endothelial nitric oxide synthase expression, nitric oxide production, and heme oxygenase-1 (HO-1) expression, which was associated with apoptosis and inflammatory activation. HO-1 expression is controlled by Nrf-2. Accordingly, Nox4-deficient LECs exhibited reduced Nrf-2 protein level and deletion of Nox4 reduced Nrf-2 reporter gene activity. In vivo treatment with hemin, an inducer of HO-1, blocked the vascular hypertrophy induced by Nox4 deletion in the angiotensin II infusion model and carbon monoxide, the product of HO-1, blocked the Nox4-deletion-induced apoptosis in LECs. CONCLUSION: Endogenous Nox4 protects the vasculature during ischemic or inflammatory stress. Different from Nox1 and Nox2, this particular NADPH oxidase therefore may have a protective vascular function.