Disrupted Binding of Cystathionine γ-Lyase to p53 Promotes Endothelial Senescence.

BACKGROUND: Advanced age is unequivocally linked to the development of cardiovascular disease; however, the mechanisms resulting in reduced endothelial cell regeneration remain poorly understood. Here, we investigated novel mechanisms involved in endothelial cell senescence that impact endothelial cell transcription and vascular repair after injury. METHODS: Native endothelial cells were isolated from young (20±3.4 years) and aged (80±2.3 years) individuals and subjected to molecular analyses to assess global transcriptional and metabolic changes. In vitro studies were conducted using primary human and murine endothelial cells. A murine aortic re-endothelialization model was used to examine endothelial cell regenerative capacity in vivo. RESULTS: RNA sequencing of native endothelial cells revealed that aging resulted in p53-mediated reprogramming to express senescence-associated genes and suppress glycolysis. Reduced glucose uptake and ATP contributed to attenuated assembly of the telomerase complex, which was required for endothelial cell proliferation. Enhanced p53 activity in aging was linked to its acetylation on K120 due to enhanced activity of the acetyltransferase MOZ (monocytic leukemic zinc finger). Mechanistically, p53 acetylation and translocation were, at least partially, attributed to the loss of the vasoprotective enzyme, CSE (cystathionine γ-lyase). CSE physically anchored p53 in the cytosol to prevent its nuclear translocation and CSE absence inhibited AKT (Protein kinase B)-mediated MOZ phosphorylation, which in turn increased MOZ activity and subsequently p53 acetylation. In mice, the endothelial cell-specific deletion of CSE activated p53, induced premature endothelial senescence, and arrested vascular repair after injury. In contrast, the adeno-associated virus 9-mediated re-expression of an active CSE mutant retained p53 in the cytosol, maintained endothelial glucose metabolism and proliferation, and prevented endothelial cell senescence. Adenoviral overexpression of CSE in native endothelial cells from aged individuals maintained low p53 activity and reactivated telomerase to revert endothelial cell senescence. CONCLUSIONS: Aging-associated impairment of vascular repair is partly determined by the vasoprotective enzyme CSE.

ADPGK-AS1 long noncoding RNA switches macrophage metabolic and phenotypic state to promote lung cancer growth.

Long noncoding RNAs (lncRNAs) influence the transcription of gene networks in many cell types, but their role in tumor-associated macrophages (TAMs) is still largely unknown. We found that the lncRNA ADPGK-AS1 was substantially upregulated in artificially induced M2-like human macrophages, macrophages exposed to lung cancer cells in vitro, and TAMs from human lung cancer tissue. ADPGK-AS1 is partly located within mitochondria and binds to the mitochondrial ribosomal protein MRPL35. Overexpression of ADPGK-AS1 in macrophages upregulates the tricarboxylic acid cycle and promotes mitochondrial fission, suggesting a phenotypic switch toward an M2-like, tumor-promoting cytokine release profile. Macrophage-specific knockdown of ADPGK-AS1 induces a metabolic and phenotypic switch (as judged by cytokine profile and production of reactive oxygen species) to a pro-inflammatory tumor-suppressive M1-like state, inhibiting lung tumor growth in vitro in tumor cell-macrophage cocultures, ex vivo in human tumor precision-cut lung slices, and in vivo in mice. Silencing ADPGK-AS1 in TAMs may thus offer a novel therapeutic strategy for lung cancer.

Optimization of the Lead Compound NVP-BHG712 as a Colorectal Cancer Inhibitor.

The ephrin type-A receptor 2 (EPHA2) kinase belongs to the largest family of receptor tyrosine kinases. There are several indications of an involvement of EPHA2 in the development of infectious diseases and cancer. Despite pharmacological potential, EPHA2 is an under-examined target protein. In this study, we synthesized a series of derivatives of the inhibitor NVP-BHG712 and triazine-based compounds. These compounds were evaluated to determine their potential as kinase inhibitors of EPHA2, including elucidation of their binding mode (X-ray crystallography), affinity (microscale thermophoresis), and selectivity (Kinobeads assay). Eight inhibitors showed affinities in the low-nanomolar regime (KD <10 nM). Testing in up to seven colon cancer cell lines that express EPHA2 reveals that several derivatives feature promising effects for the control of human colon carcinoma. Thus, we have developed a set of powerful tool compounds for fundamental new research on the interplay of EPH receptors in a cellular context.

Long non-coding RNA PCAT19 safeguards DNA in quiescent endothelial cells by preventing uncontrolled phosphorylation of RPA2.

In healthy vessels, endothelial cells maintain a stable, differentiated, and growth-arrested phenotype for years. Upon injury, a rapid phenotypic switch facilitates proliferation to restore tissue perfusion. Here we report the identification of the endothelial cell-enriched long non-coding RNA (lncRNA) PCAT19, which contributes to the proliferative switch and acts as a safeguard for the endothelial genome. PCAT19 is enriched in confluent, quiescent endothelial cells and binds to the full replication protein A (RPA) complex in a DNA damage- and cell-cycle-related manner. Our results suggest that PCAT19 limits the phosphorylation of RPA2, primarily on the serine 33 (S33) residue, and thereby facilitates an appropriate DNA damage response while slowing cell cycle progression. Reduction in PCAT19 levels in response to either loss of cell contacts or knockdown promotes endothelial proliferation and angiogenesis. Collectively, PCAT19 acts as a dynamic guardian of the endothelial genome and facilitates rapid switching from quiescence to proliferation.

HIF1α-AS1 is a DNA:DNA:RNA triplex-forming lncRNA interacting with the HUSH complex.

DNA:DNA:RNA triplexes that are formed through Hoogsteen base-pairing of the RNA in the major groove of the DNA duplex have been observed in vitro, but the extent to which these interactions occur in cells and how they impact cellular functions remains elusive. Using a combination of bioinformatic techniques, RNA/DNA pulldown and biophysical studies, we set out to identify functionally important DNA:DNA:RNA triplex-forming long non-coding RNAs (lncRNA) in human endothelial cells. The lncRNA HIF1α-AS1 was retrieved as a top hit. Endogenous HIF1α-AS1 reduces the expression of numerous genes, including EPH Receptor A2 and Adrenomedullin through DNA:DNA:RNA triplex formation by acting as an adapter for the repressive human silencing hub complex (HUSH). Moreover, the oxygen-sensitive HIF1α-AS1 is down-regulated in pulmonary hypertension and loss-of-function approaches not only result in gene de-repression but also enhance angiogenic capacity. As exemplified here with HIF1α-AS1, DNA:DNA:RNA triplex formation is a functionally important mechanism of trans-acting gene expression control.

DNA topoisomerase inhibition with the HIF inhibitor acriflavine promotes transcription of lncRNAs in endothelial cells.

The transcription factor hypoxia-inducible factor 1 (HIF1) is an important driver of cancer and is therefore an attractive drug target. Acriflavine (ACF) has been suggested to inhibit HIF1, but its mechanism of action is unknown. Here we investigated the interaction of ACF with DNA and long non-coding RNAs (lncRNAs) and its function in human endothelial cells. ACF promoted apoptosis and reduced proliferation, network formation, and angiogenic capacity. It also induced changes in gene expression, as determined by RNA sequencing (RNA-seq), which could not be attributed to specific inhibition of HIF1. A similar response was observed in murine lung endothelial cells. Although ACF increased and decreased a similar number of protein-coding genes, lncRNAs were preferentially upregulated under normoxic and hypoxic conditions. An assay for transposase accessibility with subsequent DNA sequencing (ATAC-seq) demonstrated that ACF induced strong changes in chromatin accessibility at lncRNA promoters. Immunofluorescence showed displacement of DNA:RNA hybrids. Such effects might be due to ACF-mediated topoisomerase inhibition, which was indeed the case, as reflected by DNA unwinding assays. Comparison with other acridine derivatives and topoisomerase inhibitors suggested that the specific function of ACF is an effect of acridinium-class compounds. This study demonstrates that ACF inhibits topoisomerases rather than HIF specifically and that it elicits a unique expression response of lncRNAs.

MIR503HG Loss Promotes Endothelial-to-Mesenchymal Transition in Vascular Disease.

[Figure: see text].

Mapping the Endothelial Cell S-Sulfhydrome Highlights the Crucial Role of Integrin Sulfhydration in Vascular Function.

BACKGROUND: In vascular endothelial cells, cysteine metabolism by the cystathionine γ lyase (CSE), generates hydrogen sulfide-related sulfane sulfur compounds (H2Sn), that exert their biological actions via cysteine S-sulfhydration of target proteins. This study set out to map the "S-sulfhydrome" (ie, the spectrum of proteins targeted by H2Sn) in human endothelial cells. METHODS: Liquid chromatography with tandem mass spectrometry was used to identify S-sulfhydrated cysteines in endothelial cell proteins and ß3 integrin intraprotein disulfide bond rearrangement. Functional studies included endothelial cell adhesion, shear stress-induced cell alignment, blood pressure measurements, and flow-induced vasodilatation in endothelial cell-specific CSE knockout mice and in a small collective of patients with endothelial dysfunction. RESULTS: Three paired sample sets were compared: (1) native human endothelial cells isolated from plaque-free mesenteric arteries (CSE activity high) and plaque-containing carotid arteries (CSE activity low); (2) cultured human endothelial cells kept under static conditions or exposed to fluid shear stress to decrease CSE expression; and (3) cultured endothelial cells exposed to shear stress to decrease CSE expression and treated with solvent or the slow-releasing H2Sn donor, SG1002. The endothelial cell "S-sulfhydrome" consisted of 3446 individual cysteine residues in 1591 proteins. The most altered family of proteins were the integrins and focusing on ß3 integrin in detail we found that S-sulfhydration affected intraprotein disulfide bond formation and was required for the maintenance of an extended-open conformation of the ß leg. ß3 integrin S-sulfhydration was required for endothelial cell mechanotransduction in vitro as well as flow-induced dilatation in murine mesenteric arteries. In cultured cells, the loss of S-sulfhydration impaired interactions between ß3 integrin and Gα13 (guanine nucleotide-binding protein subunit α 13), resulting in the constitutive activation of RhoA (ras homolog family member A) and impaired flow-induced endothelial cell realignment. In humans with atherosclerosis, endothelial function correlated with low H2Sn generation, impaired flow-induced dilatation, and failure to detect ß3 integrin S-sulfhydration, all of which were rescued after the administration of an H2Sn supplement. CONCLUSIONS: Vascular disease is associated with marked changes in the S-sulfhydration of endothelial cell proteins involved in mediating responses to flow. Short-term H2Sn supplementation improved vascular reactivity in humans highlighting the potential of interfering with this pathway to treat vascular disease.

ZNF354C is a transcriptional repressor that inhibits endothelial angiogenic sprouting.

Zinc finger proteins (ZNF) are a large group of transcription factors with diverse functions. We recently discovered that endothelial cells harbour a specific mechanism to limit the action of ZNF354C, whose function in endothelial cells is unknown. Given that ZNF354C has so far only been studied in bone and tumour, its function was determined in endothelial cells. ZNF354C is expressed in vascular cells and localises to the nucleus and cytoplasm. Overexpression of ZNF354C in human endothelial cells results in a marked inhibition of endothelial sprouting. RNA-sequencing of human microvascular endothelial cells with and without overexpression of ZNF354C revealed that the protein is a potent transcriptional repressor. ZNF354C contains an active KRAB domain which mediates this suppression as shown by mutagenesis analysis. ZNF354C interacts with dsDNA, TRIM28 and histones, as observed by proximity ligation and immunoprecipitation. Moreover, chromatin immunoprecipitation revealed that the ZNF binds to specific endothelial-relevant target-gene promoters. ZNF354C suppresses these genes as shown by CRISPR/Cas knockout and RNAi. Inhibition of endothelial sprouting by ZNF354C is dependent on the amino acids DV and MLE of the KRAB domain. These results demonstrate that ZNF354C is a repressive transcription factor which acts through a KRAB domain to inhibit endothelial angiogenic sprouting.

Shear stress regulates cystathionine γ lyase expression to preserve endothelial redox balance and reduce membrane lipid peroxidation.

Cystathionine γ lyase (CSE) is the major source of hydrogen sulfide-derived species (H2Sn) in endothelial cells and plays an important role in protecting against atherosclerosis. Here we investigated the molecular mechanisms underlying the regulation of CSE expression in endothelial cells by fluid shear stress/flow. Fluid shear stress decreased CSE expression in human and murine endothelial cells and was negatively correlated with the transcription factor Krüppel-like factor (KLF) 2. CSE was identified as a direct target of the KLF2-regulated microRNA, miR-27b and high expression of CSE in native human plaque-derived endothelial cells, was also inversely correlated with KLF2 and miR-27b levels. One consequence of decreased CSE expression was the loss of Prx6 sulfhydration (on Cys47), which resulted in Prx6 hyperoxidation, decamerization and inhibition, as well as a concomitant increase in endothelial cell reactive oxygen species and lipid membrane peroxidation. H2Sn supplementation in vitro was able to reverse the redox state of Prx6. Statin therapy, which is known to activate KLF2, also decreased CSE expression but increased CSE activity by preventing its phosphorylation on Ser377. As a result, the sulfhydration of Prx6 was partially restored in samples from plaque containing arteries from statin-treated donors. Taken together, the regulation of CSE expression by shear stress/disturbed flow is dependent on KLF2 and miR-27b. Moreover, in murine and human arteries CSE acts to maintain endothelial redox balance at least partly by targeting Prx6 to prevent its decamerization and inhibition of its peroxidase activity.

Pleiotropic effects of laminar flow and statins depend on the Krüppel-like factor-induced lncRNA MANTIS.

AIMS: To assess the functional relevance and therapeutic potential of the pro-angiogenic long non-coding RNA MANTIS in vascular disease development. METHODS AND RESULTS: RNA sequencing, CRISPR activation, overexpression, and RNAi demonstrated that MANTIS, especially its Alu-element, limits endothelial ICAM-1 expression in different types of endothelial cells. Loss of MANTIS increased endothelial monocyte adhesion in an ICAM-1-dependent manner. MANTIS reduced the binding of the SWI/SNF chromatin remodelling factor BRG1 at the ICAM-1 promoter. The expression of MANTIS was induced by laminar flow and HMG-CoA-reductase inhibitors (statins) through mechanisms involving epigenetic rearrangements and the transcription factors KLF2 and KLF4. Mutation of the KLF binding motifs in the MANTIS promoter blocked the flow-induced MANTIS expression. Importantly, the expression of MANTIS in human carotid artery endarterectomy material was lower compared with healthy vessels and this effect was prevented by statin therapy. Interestingly, the protective effects of statins were mediated in part through MANTIS, which was required to facilitate the atorvastatin-induced changes in endothelial gene expression. Moreover, the beneficial endothelial effects of statins in culture models (spheroid outgrowth, proliferation, telomerase activity, and vascular organ culture) were lost upon knockdown of MANTIS. CONCLUSION: MANTIS is tightly regulated by the transcription factors KLF2 and KLF4 and limits the ICAM-1 mediated monocyte adhesion to endothelial cells and thus potentially atherosclerosis development in humans. The beneficial effects of statin treatment and laminar flow are dependent on MANTIS.

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.

IL-6 augments IL-4-induced polarization of primary human macrophages through synergy of STAT3, STAT6 and BATF transcription factors.

Macrophages in the tumor microenvironment respond to complex cytokine signals. How these responses shape the phenotype of tumor-associated macrophages (TAMs) is incompletely understood. Here we explored how cytokines of the tumor milieu, interleukin (IL)-6 and IL-4, interact to influence target gene expression in primary human monocyte-derived macrophages (hMDMs). We show that dual stimulation with IL-4 and IL-6 synergistically modified gene expression. Among the synergistically induced genes are several targets with known pro-tumorigenic properties, such as CC-chemokine ligand 18 (CCL18), transforming growth factor alpha (TGFA) or CD274 (programmed cell death 1 ligand 1 (PD-L1)). We found that transcription factors of the signal transducer and activator of transcription (STAT) family, STAT3 and STAT6 bind regulatory regions of synergistically induced genes in close vicinity. STAT3 and STAT6 co-binding further induces the basic leucine zipper ATF-like transcription factor (BATF), which participates in synergistic induction of target gene expression. Functional analyses revealed increased MCF-7 and MDA-MB 231 tumor cell motility in response to conditioned media from co-treated hMDMs compared to cells incubated with media from single cytokine-treated hMDMs. Flow cytometric analysis of T cell populations upon co-culture with hMDMs polarized by different cytokines indicated that dual stimulation promoted immunosuppressive properties of hMDMs in a PD-L1-dependent manner. Analysis of clinical data revealed increased expression of BATF together with TAM markers in tumor stroma of breast cancer patients as compared to normal breast tissue stroma. Collectively, our findings suggest that IL-4 and IL-6 cooperate to alter the human macrophage transcriptome, endowing hMDMs with pro-tumorigenic properties.

A positive feedback loop between IL-1ß, LPS and NEU1 may promote atherosclerosis by enhancing a pro-inflammatory state in monocytes and macrophages.

Inflammation plays an important role in atherosclerosis, a notion supported by the beneficial effects of the IL-1ß inhibitor canakinumab in the CANTOS trial. Sialic acids (Sias), components of the surface glycocalyx, regulate intercellular and intermolecular interactions. We investigated the expression of the Sia cleaving enzyme neuraminidase-1 (NEU1) in atherosclerotic plaques and its potential role in inflammatory processes. In isolated mononuclear blood cells from patients with myocardial infarction, NEU1 expression was increased compared to healthy controls. High expression of NEU1 in macrophages located on the intima layer, in calcified regions and the adventitia of the plaque was observed in human carotid arteries' atherectomies. IL-1ß and LPS induced NEU1 expression in THP-1 monocytic cells. Lentiviral NEU1-overexpression in THP-1-cells enhanced expression of CD80, TNF-α, IL-1ß, number of multinuclear cells, phagocytosis and chemotaxis indicative for M1 monocyte/macrophage polarization. CRISPR/Cas9-mediated knock-out of NEU1 in THP-1-cells did not affect differentiation of monocytes to macrophages but attenuated LPS- and IL-1ß -induced TNF-α and IL-1ß expression. SiRNA-mediated knock-down of NEU1 in M1-macrophages differentiated from primary human CD14+ monocytes reduced the expression of TNF-α and IL-1ß. Thus, in monocytes/macrophages, LPS, NEU1 and IL-1ß act in a positive feedback loop as enhancers of inflammation and may therefore promote atherosclerosis and plaque instability.

Long Noncoding RNA MANTIS Facilitates Endothelial Angiogenic Function.

BACKGROUND: The angiogenic function of endothelial cells is regulated by numerous mechanisms, but the impact of long noncoding RNAs (lncRNAs) has hardly been studied. We set out to identify novel and functionally important endothelial lncRNAs. METHODS: Epigenetically controlled lncRNAs in human umbilical vein endothelial cells were searched by exon-array analysis after knockdown of the histone demethylase JARID1B. Molecular mechanisms were investigated by RNA pulldown and immunoprecipitation, mass spectrometry, microarray, several knockdown approaches, CRISPR-Cas9, assay for transposase-accessible chromatin sequencing, and chromatin immunoprecipitation in human umbilical vein endothelial cells. Patient samples from lung and tumors were studied for MANTIS expression. RESULTS: A search for epigenetically controlled endothelial lncRNAs yielded lncRNA n342419, here termed MANTIS, as the most strongly regulated lncRNA. Controlled by the histone demethylase JARID1B, MANTIS was downregulated in patients with idiopathic pulmonary arterial hypertension and in rats treated with monocrotaline, whereas it was upregulated in carotid arteries of Macaca fascicularis subjected to atherosclerosis regression diet, and in endothelial cells isolated from human glioblastoma patients. CRISPR/Cas9-mediated deletion or silencing of MANTIS with small interfering RNAs or GapmeRs inhibited angiogenic sprouting and alignment of endothelial cells in response to shear stress. Mechanistically, the nuclear-localized MANTIS lncRNA interacted with BRG1, the catalytic subunit of the switch/sucrose nonfermentable chromatin-remodeling complex. This interaction was required for nucleosome remodeling by keeping the ATPase function of BRG1 active. Thereby, the transcription of key endothelial genes such as SOX18, SMAD6, and COUP-TFII was regulated by ensuring efficient RNA polymerase II machinery binding. CONCLUSION: MANTIS is a differentially regulated novel lncRNA facilitating endothelial angiogenic function.

CRISPR/Cas9-mediated knockout of p22phox leads to loss of Nox1 and Nox4, but not Nox5 activity.

The NADPH oxidases are important transmembrane proteins producing reactive oxygen species (ROS). Within the Nox family, different modes of activation can be discriminated. Nox1-3 are dependent on different cytosolic subunits, Nox4 seems to be constitutively active and Nox5 is directly activated by calcium. With the exception of Nox5, all Nox family members are thought to depend on the small transmembrane protein p22phox. With the discovery of the CRISPR/Cas9-system, a tool to alter genomic DNA sequences has become available. So far, this method has not been widely used in the redox community. On such basis, we decided to study the requirement of p22phox in the Nox complex using CRISPR/Cas9-mediated knockout. Knockout of the gene of p22phox, CYBA, led to an ablation of activity of Nox4 and Nox1 but not of Nox5. Production of hydrogen peroxide or superoxide after knockout could be rescued with either human or rat p22phox, but not with the DUOX-maturation factors DUOXA1/A2. Furthermore, different mutations of p22phox were studied regarding the influence on Nox4-dependent H2O2 production. P22phox Q130* and Y121H affected maturation and activity of Nox4. Hence, Nox5-dependent O2•- production is independent of p22phox, but native p22phox is needed for maturation of Nox4 and production of H2O2.

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.

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.

Exportin t and Exportin 5: tRNA and miRNA biogenesis - and beyond.

Abstract The biogenesis of most eukaryotic kinds of RNA requires nuclear export, which is mediated by a variety of specific nuclear transport receptors. The nuclear export receptors Exportin-t (Exp-t) and Exportin 5 (Exp5), and their homologues, are involved in the export of transfer RNA to the cytoplasm. Exp5 is further involved in additional nucleocytoplasmic transport pathways, which include nuclear export of microRNA precursors (pre-miRNAs) and pre-60S ribosomal subunits. Inactivation of Exp5 results in nuclear accumulation of pre-miRNAs and perturbation of gene expression, and its mutation was recently found in malignant diseases. Here, we compare the cellular function of Exp5 and Exp-t with focus on Exp5 substrates and its role in diseases.