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The eukaryotic initiation factor 4E binding protein (4E-BP) family is involved in translational control of cell proliferation and pro-angiogenic factors. The zebrafish eukaryotic initiation factor 4E binding protein 3 like (eif4ebp3l) is a member of the 4E-BPs and responsible for activity-dependent myofibrillogenesis, but whether it affects cardiomyocyte (CM) proliferation or heart regeneration is unclear. We examined eif4ebp3l during zebrafish vascular development and heart regeneration post cryoinjury in adult zebrafish. Using morpholino injections we induced silencing of eif4ebp3l in zebrafish embryos, which led to increased angiogenesis at 94 h post fertilization (hpf). For investigation of eif4ebp3l in cardiac regeneration, zebrafish hearts were subjected to cryoinjury. Regenerating hearts were analyzed at different time points post-cryoinjury for expression of eif4ebp3l by in situ hybridization and showed strongly decreased eif4ebp3l expression in the injured area. We established a transgenic zebrafish strain, which overexpressed eif4ebp3l under the control of a heat-shock dependent promotor. Overexpression of eif4ebp3l during zebrafish heart regeneration caused only macroscopically a reduced amount of fibrin at the site of injury. Overall, these findings demonstrate that silencing of eif4ebp3l has pro-angiogenic properties in zebrafish vascular development and when eif4ebp3l is overexpressed, fibrin deposition tends to be altered in zebrafish cardiac regeneration after cryoinjury.

Dedifferentiated vascular smooth muscle cells (vSMCs) play an essential role in neointima formation, and we now aim to investigate the role of the bone morphogenetic protein (BMP) modulator BMPER (BMP endothelial cell precursor-derived regulator) in neointima formation. To assess BMPER expression in arterial restenosis, we used a mouse carotid ligation model with perivascular cuff placement. Overall BMPER expression after vessel injury was increased; however, expression in the tunica media was decreased compared to untreated control. Consistently, BMPER expression was decreased in proliferative, dedifferentiated vSMC in vitro. C57BL/6_Bmper+/− mice displayed increased neointima formation 21 days after carotid ligation and enhanced expression of Col3A1, MMP2, and MMP9. Silencing of BMPER increased the proliferation and migration capacity of primary vSMCs, as well as reduced contractibility and expression of contractile markers, whereas stimulation with recombinant BMPER protein had the opposite effect. Mechanistically, we showed that BMPER binds insulin-like growth factor-binding protein 4 (IGFBP4), resulting in the modulation of IGF signaling. Furthermore, perivascular application of recombinant BMPER protein prevented neointima formation and ECM deposition in C57BL/6N mice after carotid ligation. Our data demonstrate that BMPER stimulation causes a contractile vSMC phenotype and suggest that BMPER has the potential for a future therapeutic agent in occlusive cardiovascular diseases.

The purpose of this study is to investigate the role of platelet bone morphogenetic proteins (BMP)-4 during vascular inflammation and remodeling in a mouse model of carotid wire injury. Transgenic mice with a platelet-specific deletion of BMP-4 (BMP4Plt−/−) were generated. Intravital microscopy was performed to evaluate leukocyte adhesion to the vessel wall. Expression of adhesion molecules and chemokines were analyzed. Platelet-leukocyte aggregates (PLAs) were evaluated using flow cytometry. For carotid wire injury, BMP4Plt−/− mice were further crossed with LDLr−/− mice (BMP4Plt−/−/LDLr−/−) and fed with a high cholesterol diet for 2-weeks. Carotid wire injury was performed, and re-endothelialization and neointimal formation were evaluated. In comparison to the control mice, stimulation with TNFα resulted in fewer rolling and adherent leukocytes to the vessel wall in the BMP4Plt−/− mice. mRNA and protein expression of P-selectin and adhesion molecules were reduced in the aorta of the BMP4Plt−/− mice. In platelets from the BMP4Plt−/− mice, the expression of P-selectin was reduced, and fewer PLA formations were measured than in the control mice. Loss of platelet BMP-4 further prevented neointima formation after carotid wire injury. Endothelial regeneration after injury was decelerated in the BMP4Plt−/− mice, and confirmed in-vitro, where the deletion of platelet BMP-4 inhibited endothelial cell proliferation and migration. We demonstrate for the first time that platelet BMP-4 is involved during vascular inflammation and remodeling. This is partially mediated by the inhibition of platelet activation, reduced expression of adhesion molecules and inflammatory responses. Our findings identify platelet BMP-4 as a mediator of vascular inflammation in early atherosclerosis and restenosis.

Leukocyte transmigration through the blood vessel wall is a fundamental step of the inflammatory response and requires expression of adhesion molecule PECAM-1. Accumulating evidence implicates that semaphorin (Sema) 3F and its receptor neuropilin (NRP) 2 are central regulators in vascular biology. Herein, we assess the role of Sema3F in leukocyte migration in vitro and in vivo. To determine the impact of Sema3F on leukocyte recruitment in vivo, we used the thioglycollate-induced peritonitis model. After the induction of peritonitis, C57BL/6 mice were intraperitoneally (i.p.) injected daily with recombinant Sema3F or solvent for 3 days. Compared with solvent-treated controls, leukocyte count was increased in the peritoneal lavage of Sema3F-treated mice indicating that Sema3F promotes leukocyte extravasation into the peritoneal cavity. In line with this observation, stimulation of human endothelial cells with Sema3F enhanced the passage of peripheral blood mononuclear cells (PBMCs) through the endothelial monolayer in the transwell migration assays. Conversely, silencing of endothelial Sema3F by siRNA transfection dampened diapedesis of PBMCs through the endothelium in vitro. xMechanistically, Sema3F induced upregulation of adhesion molecule PECAM-1 in endothelial cells and in murine heart tissue shown by immunofluorescence and western blotting. The inhibition of PECAM-1 by blocking antibody HEC7 blunted Sema3F-induced leukocyte migration in transwell assays. SiRNA-based NRP2 knockdown reduced PECAM-1 expression and migration of PBMCs in Sema3F-treated endothelial cells, indicating that PECAM-1 expression and leukocyte migration in response to Sema3F depend on endothelial NRP2. To assess the regulation of Sema3F in human inflammatory disease, we collected serum samples of patients from day 0 to day 7 after survived out-of-hospital cardiac arrest (OHCA, n = 41). First, we demonstrated enhanced migration of PBMCs through endothelial cells exposed to the serum of patients after OHCA in comparison to the serum of patients with stable coronary artery disease or healthy volunteers. Remarkably, serum samples of OHCA patients contained significantly higher Sema3F protein levels compared with CAD patients (CAD, n = 37) and healthy volunteers (n = 11), suggesting a role of Sema3F in the pathophysiology of the inflammatory response after OHCA. Subgroup analysis revealed that elevated serum Sema3F levels after ROSC are associated with decreased survival, myocardial dysfunction, and prolonged vasopressor therapy, clinical findings that determine the outcome of post-resuscitation period after OHCA. The present study provides novel evidence that endothelial Sema3F controls leukocyte recruitment through a NRP2/PECAM-1-dependent mechanism. Sema3F serum concentrations are elevated following successful resuscitation suggesting that Sema3F might be involved in the inflammatory response after survived OHCA. Targeting the Sema3F/NRP2/PECAM-1 pathway could provide a novel approach to abolish overwhelming inflammation after resuscitation.

Aldosterone is a key factor in adverse cardiovascular remodeling by acting on the mineralocorticoid receptor (MR) in different cell types. Endothelial MR activation mediates hypertrophy, inflammation and fibrosis. Cardiovascular remodeling is often accompanied by impaired angiogenesis, which is a risk factor for the development of heart failure. In this study, we evaluated the impact of MR in endothelial cells on angiogenesis. Deoxycorticosterone acetate (DOCA)-induced hypertension was associated with capillary rarefaction in the heart of WT mice but not of mice with cell type-specific MR deletion in endothelial cells. Consistently, endothelial MR deletion prevented the inhibitory effect of aldosterone on the capillarization of subcutaneously implanted silicon tubes and on capillary sprouting from aortic ring segments. We examined MR-dependent gene expression in cultured endothelial cells by RNA-seq and identified a cluster of differentially regulated genes related to angiogenesis. We found opposing effects on gene expression when comparing activation of the mineralocorticoid receptor in ECs to treatment with vascular endothelial growth factor (VEGF), a potent activator of angiogenesis. In conclusion, we demonstrate here that activation of endothelial cell MR impaired angiogenic capacity and lead to capillary rarefaction in a mouse model of MR-driven hypertension. MR activation opposed VEGF-induced gene expression leading to the dysregulation of angiogenesis-related gene networks in endothelial cells. Our findings underscore the pivotal role of endothelial cell MR in the pathophysiology of hypertension and related heart disease.

The bone morphogenetic protein (BMP) signaling pathway plays a central role during vasculature development. Mutations or dysregulation of the BMP pathway members have been linked to arteriovenous malformations. In the present study, we investigated the effect of the BMP modulators bone morphogenetic protein endothelial precursor-derived regulator (BMPER) and twisted gastrulation protein homolog 1 (TWSG1) on arteriovenous specification during zebrafish development and analyzed downstream Notch signaling pathway in human endothelial cells. Silencing of bmper and twsg1b in zebrafish embryos by morpholinos resulted in a pronounced enhancement of venous ephrinB4a marker expression and concomitant dysregulated arterial ephrinb2a marker expression detected by in situ hybridization. As arteriovenous specification was disturbed, we assessed the impact of BMPER and TWSG1 protein stimulation on the Notch signaling pathway on endothelial cells from different origin. Quantitative real-time PCR (qRT-PCR) and western blot analysis showed increased expression of Notch target gene hairy and enhancer of split, HEY1/2 and EPHRINB2. Consistently, silencing of BMPER in endothelial cells by siRNAs decreased Notch signaling and downstream effectors. BMP receptor antagonist DMH1 abolished BMPER and BMP4 induced Notch signaling pathway activation. In conclusion, we found that in endothelial cells, BMPER and TWSG1 are necessary for regular Notch signaling activity and in zebrafish embryos BMPER and TWSG1 preserve arteriovenous specification to prevent malformations.

Leukocyte recruitment is a fundamental event in the response of the innate immune system to injury. This process is promoted in part by the opening of endothelial cell adherens junctions that allows leukocyte extravasation through gaps between adjacent endothelial cells. VE-cadherin is a key component of endothelial cell adherens junctions and a negative regulator of leukocyte emigration. Accumulating evidence implicates bone morphogenetic protein (BMP) 4 as a critical regulator in vascular biology, but its role in leukocyte extravasation in vitro and in vivo has not been investigated so far. To assess the impact of BMP4 on leukocyte emigration in vivo, we used the thioglycollate-induced peritonitis model. C57BL/6 mice were intraperitoneally (i.p.) injected with recombinant BMP4 in addition to thioglycollate. Compared to solvent-treated controls, we observed higher accumulation of leukocytes in the peritoneal lavage of BMP4-treated mice indicating that BMP4 promotes leukocyte diapedesis into the inflamed peritoneal cavity. Endothelial cell-specific deletion of BMP4 in mice markedly diminished leukocyte diapedesis following thioglycollate administration suggesting that endothelial BMP4 is required for leukocyte recruitment. Consistent with these in vivo results, transwell migration assays with human umbilical vein endothelial cells (HUVECs) in vitro revealed that recombinant BMP4 enhanced leukocyte transmigration through the endothelial monolayer. Conversely, silencing of endothelial BMP4 by siRNA dampened leukocyte diapedesis in vitro. Mechanistic studies showed that loss of BMP4 improved endothelial junction stability by upregulation of VE-cadherin expression in vitro and in vivo. Vice versa, treatment of HUVECs with recombinant BMP4 decreased expression of VE-cadherin and impaired endothelial junction stability shown by Western blotting and immunocytochemistry. Finally, severe endothelial damage in HUVECs in response to serum of patients collected 24 h after survived cardiac arrest was accompanied by increase in leukocyte migration in transwell assays and activation of the BMP pathway most probably by upregulation of endothelial BMP4 RNA and protein expression. Collectively, the present study provides novel evidence that endothelial BMP4 controls leukocyte recruitment through a VE-cadherin-dependent mechanism and that BMP4-induced inflammation might be involved in the pathogenesis of endothelial cell damage following successful resuscitation after cardiac arrest.

The endothelium serves as a selective barrier and controls the exchange of nutrients, hormones, and leukocytes between blood and tissues. Molecular mechanisms contributing to the pathogenesis of endothelial barrier dysfunction remain incompletely understood. Accumulating evidence implicates bone morphogenetic protein (BMP)-modulator BMPER as a key regulator in endothelial biology. Herein, we analyze the impact of BMPER in the control of endothelial barrier function. To assess the role of BMPER in vascular barrier function in mice, we measured the leakage of Evans blue dye from blood into interstitial lung tissue. BMPER+/− mice exhibited a significantly higher degree of vascular leak compared with wild-type siblings. In accordance with our in vivo observation, siRNA-based BMPER knockdown in human umbilical endothelial cells increased endothelial permeability measured by FITC-dextran passage in transwell assays. Mechanistically, BMPER knockdown reduced the expression of VE-cadherin, a pivotal component of endothelial adherens junctions. Conversely, recombinant human BMPER protein upregulated VE-cadherin protein levels and improved endothelial barrier function in transwell assays. The effects of BMPER knockdown on VE-cadherin expression and endothelial permeability were induced by enhanced BMP activity. Supporting this notion, activation of BMP4-Smad-Id1 signaling reduced VE-cadherin levels and impaired endothelial barrier function in vitro. In vivo, Evans blue dye accumulation was higher in the lungs of BMP4-treated C57BL/6 mice compared to controls indicating that BMP4 increased vascular permeability. High levels of BMPER antagonized BMP4-Smad5-Id1 signaling and prevented BMP4-induced downregulation of VE-cadherin and endothelial leakage, suggesting that BMPER exerts anti-BMP effects and restores endothelial barrier function. Taken together, this data demonstrates that BMPER-modulated BMP pathway activity regulates VE-cadherin expression and vascular barrier function.

Aims Angiogenesis is defined as the sprouting of capillaries from pre-existing vasculature. It is a complex process that includes endothelial proliferation, migration, and tube formation. Previous data have demonstrated a high expression level of manganese-superoxide dismutase (MnSOD) in endothelial cells and suggested an important role of MnSOD in several cardiovascular diseases. In addition, manganese (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP) has been shown to mimic some of the effects of MnSOD in various tissues. However, its effect on the vasculature remains unknown. Methods and results HUVECs were treated with MnTBAP. Migration, tube formation, and capillary sprouting assays were performed to evaluate the pro-angiogenic effect in vitro . Matrigel plug assay was performed to assess capillary ingrowth in vivo . Compared to control, treatment with MnTBAP revealed increased cell migration, tube formation and capillary sprouting along with more capillary ingrowth in the Matrigel plug assay. This effect was mediated through a mitofusin (Mfn)-1-dependent pathway. Expression of Tie-2, Ang-2 and VEGF mRNA was increased in muscle tissues after ligation in MnTBAP treated mice. However, revascularization in the hindlimb ischemia model was not statistically significant at day 10 in MnTBAP treated mice. Conclusion In summary, our data demonstrate a strong pro-angiogenic, but less pro-arteriogenic effect of MnTBAP in HUVECs mediated by Mfn-1.

SummaryMicroRNAs are small non-coding RNAs that negatively regulate posttranscriptional gene expression. Several microRNAs have been described to regulate the process of angiogenesis. Previously, we have shown that bone morphogenetic protein 4 (BMP4) increased the proangiogenic activity of endothelial cells. In this project, we now investigated how the pro-angiogenic BMP4 effect is mediated by microRNAs. First, we performed a microRNA array with BMP4-stimulated human umbilical vein endothelial cells (HUVECs). Among the topregulated microRNAs, we detected a decreased expression of miR-494 and increased expression of miR-126–5p. Next, we analysed the canonical Smad and alternative signalling pathways, through which BMP4 would regulate miR-126–5p and miR-494 expression. Furthermore, the functional effect of miR-494 and miR-126–5p on endothelial cells was investigated. MicroRNA-494 overexpression decreased endothelial cell proliferation, migration and sprout formation. Consistently, miR-494 inhibition increased endothelial cell function. As potential miR-494 targets, bFGF and BMP endothelial cell precursorderived regulator (BMPER) were identified and confirmed by western blot. Luciferase assays showed direct miR-494 binding in BMPER 3’UTR. In contrast, miR-126–5p overexpression increased pro-angiogenic endothelial cell behaviour and, accordingly, miR-126–5p inhibition decreased endothelial cell function. As a direct miR-126–5p target we identified the anti-angiogenic thrombospondin-1 which was confirmed by western blot analysis and luciferase assays. In the Matrigel plug assay application of antagomiR-494 increased endothelial cell ingrowth, whereas antagomiR-126–5p treatment decreased cell ingrowth in vivo. Taken together, through differential regulation of the anti-angiomiR-494 and the angiomiR-126–5p by BMP4 both microRNAs contribute to the pro-angiogenic BMP4 effect on endothelial cells.Supplementary Material to this article is available online at www.thrombosis-online.com.

High platelet reactivity (HPR) after P2Y12-inhibition in patients undergoing coronary stenting is associated with an increased risk for thromboembolic events and coronary death. So far it is not known how HPR affects the clinical outcome of different treatment strategies in patients with atrial fibrillation (AF) undergoing coronary stenting. In this single centre, observational study the antiplatelet effect of P2Y12-inhibitors in AF patients undergoing coronary stenting was investigated using impedance aggregometry. Patients received either dual antiplatelet therapy (DAPT) or triple therapy (TT). HPR was defined as the ratio of ADP-to TRAP-induced aggregation (r-ADP-agg) ≥50 %. Thromboembolic and bleeding events were assessed within the first 30 days after stenting. Out of 910 screened patients 167 patients were available for the present analysis. HPR was found in 5 of 43 (12 %) patients treated with DAPT and in 18 of 124 (15 %) patients treated with TT. In patients receiving TT, HPR was not a risk factor for thromboembolic events compared to patients with adequate response to P2Y12-inhibitors (6 vs. 8 %, p = 0.712). There was a trend for less bleeding events in patients with HPR compared to r-ADP-agg

Rationale Regarding branching morphogenesis, neurogenesis and angiogenesis share common principle mechanisms and make use of the same molecules. Therefore, the investigation of neuronal molecules involved in vascular morphogenesis provides new possibilities for pro-angiogenic approaches in cardiovascular diseases. Objective In this study, we investigated the role of the neuronal transcription factor NPAS4 in angiogenesis. Methods and results Here, we demonstrate that the neuronal transcription factor NPAS4 is expressed in endothelial cells of different origin using reverse transcription PCR and western blot analysis. To investigate how NPAS4 affects endothelial cell function, NPAS4 was overexpressed by plasmid transfection or depleted from human umbilical vein endothelial cells (HUVECs) by specific siRNAs. In vitro HUVEC sprouting assays showed that sprouting and branching of endothelial cells was enhanced by NPAS4 overexpression. Consistently, silencing of NPAS4 resulted in reduced HUVEC sprouting and branching. Mechanistically, we identified as target gene vascular endothelial adhesion molecule VE-cadherin to be involved in the pro-angiogenic function of NPAS4. In endothelial cell mosaic spheroid sprouting assays, NPAS4 was involved in tip cell formation. In vivo experiments in mouse and zebrafish confirmed our in vitro findings. NPAS4-deficient mice displayed reduced ingrowth of endothelial cells in the Matrigel plug assay. Consistent with a regulatory role of NPAS4 in endothelial cell function silencing of NPAS4 in zebrafish by specific morpholinos resulted in perturbed intersegmental vessels growth. Conclusions NPAS4 is expressed in endothelial cells, regulates VE-cadherin expression and regulates sprouting angiogenesis.

We thank Dr Welten and colleagues for taking the time to comment on our recently published article.1 In their letter, Welten and colleagues express concern about the microRNA expression profile after the induction of hind-limb ischemia in mice, which differs in part from their own finding. We would like to point out that the techniques for femoral artery occlusion were different between our studies: We used a surgical ligation of the carefully dissected femoral artery to minimize unspecific trauma, whereas Welten et al used electrocoagulation, which could possibly alter microRNA (miR)-155 expression as a result of an increased inflammatory response. …

Simultaneous generation of neural cells and that of the nutrient-supplying vasculature during brain development is called neurovascular coupling. We report on a transgenic mouse with impaired transforming growth factor β (TGFβ)-signalling in forebrain-derived neural cells using a Foxg1-cre knock-in to drive the conditional knock-out of the Tgfbr2. Although the expression of FOXG1 is assigned to neural progenitors and neurons of the telencephalon, Foxg1(cre/+);Tgfbr2(flox/flox) (Tgfbr2-cKO) mutants displayed intracerebral haemorrhage. Blood vessels exhibited an atypical, clustered appearance were less in number and displayed reduced branching. Vascular endothelial growth factor (VEGF) A, insulin-like growth factor (IGF) 1, IGF2, TGFβ, inhibitor of DNA binding (ID) 1, thrombospondin (THBS) 2, and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) 1 were altered in either expression levels or tissue distribution. Accordingly, human umbilical vein endothelial cells (HUVEC) displayed branching defects after stimulation with conditioned medium (CM) that was derived from primary neural cultures of the ventral and dorsal telencephalon of Tgfbr2-cKO. Supplementing CM of Tgfbr2-cKO with VEGFA rescued these defects, but application of TGFβ aggravated them. HUVEC showed reduced migration towards CM of mutants compared with controls. Supplementing the CM with growth factors VEGFA, fibroblast growth factor (FGF) 2 and IGF1 partially restored HUVEC migration. In contrast, TGFβ supplementation further impaired migration of HUVEC. We observed differences along the dorso-ventral axis of the telencephalon with regard to the impact of these factors on the phenotype. Together these data establish a TGFBR2-dependent molecular crosstalk between neural and endothelial cells during brain vessel development. These findings will be useful to further elucidate neurovascular interaction in general and to understand pathologies of the blood vessel system such as intracerebral haemorrhages, hereditary haemorrhagic telangiectasia, Alzheimeŕs disease, cerebral amyloid angiopathy or tumour biology.

Introduction: Bone morphogenetic proteins (BMP) play a key role in vascular development. Previously, we have identified BMP endothelial cell precursor-derived regulator (BMPER), an extracellular BMP modulator, to increase the angiogenic activity of endothelial cells in a concentration-dependent manner. In this project we now investigate how the BMPER effect is mediated by key molecules of angiogenesis. Methods and Results: To assess the effect of BMPER on angiogenesis-related molecules we performed an angiogenesis antibody array with BMPER-stimulated human umbilical venous endothelial cells (HUVECs) and vice versa with BMPER-silenced HUVECs compared to control conditions, respectively. We detected increased protein expression of the anti-angiogenic thrombospondin-1 (TSP-1) 48 hours after siBMPER transfection and, consistently, decreased TSP-1 expression after stimulation with BMPER (60 ng/ml; 39% ± 7.3 N=4). Furthermore, the pro-angiogenic protein bFGF was increased after BMPER stimulation, which was confirmed by realtime-PCR and western blot analysis (288.8% ± 74.8 N=3). Additionally, we detected increased FGF receptor-1 protein expression (137.7% ± 0.4 N=3) as well as FGF signaling pathway activation. Next, we investigated the interaction of BMPER and the FGF signaling pathway in endothelial cell function. BMPER stimulation increased HUVEC angiogenic activity in matrigel, migration and spheroid assays and concomitant inhibition of FGF signaling by an anti-bFGF antibody effectively inhibited the pro-angiogenic BMPER effect. Silencing of BMPER decreased the expression of FGFR1 and, accordingly, stimulation of BMPER-silenced cells with bFGF showed decreased angiogenic endothelial cell activity (65%) compared to control. The angiogenic activity of bFGF was also reduced in C57BL/6_Bmper +/- mice as assessed in the matrigel plug assay. Ex vivo aortic ring assays of C57BL/6_Bmper +/- mice confirmed a specific effect for bFGF but not for VEGF. Conclusion: In summary, BMPER inhibits the expression of the anti-angiogenic TSP-1 and increased the expression as well as activation of the pro-angiogenic FGF signaling pathway, which overall lead to the promotion of angiogenesis.

Aims Proper blood vessel formation is essential in health and is often dysregulated in ischaemic diseases. Therefore, regulatory mechanisms that control angiogenesis and arteriogenesis are required to improve treatment of ischaemic diseases. The aim of this study was to investigate the role of homeobox transcription factor HoxB5 overexpression during revascularization in ischaemic disease. Methods and results To assess the effect of HoxB5 overexpression on blood vessel formation in vivo , we subjected C57BL/6 mice to femoral artery ligation with local intramuscular injection of HoxB5 or green fluorescent protein control adenoviral vectors. Laser Doppler perfusion imaging revealed that HoxB5 enhanced perfusion restoration in mice and immunohistochemistry analysis revealed increased capillary density. To identify a potential mechanism of HoxB5 in blood vessel formation, a ‘proteome-profiler’ array was performed. HoxB5 overexpression in endothelial cells increased the expression of pro-inflammatory molecules such as monocyte chemotactic protein-1 (MCP-1) and interleukin-6 (IL-6) in vitro and in vivo . Functionally, HoxB5 enhanced monocyte as well as endothelial cell migration in vitro and increased leucocyte infiltration into ischaemic tissues. HoxB5-induced migration of monocytes was antagonized by the presence of an MCP-1 blocking antibody. Conclusions Our data suggest that overexpression of HoxB5 enhances blood vessel perfusion in vivo by up-regulation of MCP-1 and IL-6 as well as in enhanced leucocyte infiltration and blood vessel remodelling.

Background— Adaptive neovascularization after arterial occlusion is an important compensatory mechanism in cardiovascular disease and includes both the remodeling of pre-existing vessels to collateral arteries (arteriogenesis) and angiogenic capillary growth. We now aimed to identify regulatory microRNAs involved in the modulation of neovascularization after femoral artery occlusion in mice. Methods and Results— Using microRNA-transcriptome analysis, we identified miR-155 as a downregulated microRNA during hindlimb ischemia. Correspondingly, inhibition of miR-155 in endothelial cells had a stimulatory effect on proliferation and angiogenic tube formation via derepression of its direct target gene angiotensin II type 1 receptor. Surprisingly, miR-155–deficient mice showed an unexpected phenotype in vivo, with a strong reduction of blood flow recovery after femoral artery ligation (arteriogenesis) dependent on the attenuation of leukocyte-endothelial interaction and a reduction of proarteriogenic cytokine expression. Consistently, miR-155–deficient macrophages exhibit a specific alteration of the proarteriogenic cytokine expression profile, which is partly mediated by the direct miR-155 target gene SOCS-1. Conclusions— Our data demonstrate that miR-155 exerts an antiangiogenic but proarteriogenic function in the regulation of neovascularization via the suppression of divergent cell-specific target genes and that its expression in both endothelial and bone marrow–derived cells is essential for arteriogenesis in response to hindlimb ischemia in mice.