Regulation Of Neovascularization Research Articles

Efficacy of tacrolimus capsules in patients undergoing high-risk keratoplasty

Purpose: To evaluate the efficacy of tacrolimus capsules in patients undergoing high-risk keratoplasty.
 Methods: 40 high-risk patients who underwent penetrating keratoplasty at The Third Hospital of Hebei Medical University, China between January 2016 and December 2021 were included in this study. These patients were divided into two groups based on the specific immunosuppressant administered post-surgery. Twenty patients were administered oral tacrolimus capsules in conjunction with 0.1 % tacrolimus eye drops, constituting the combination with systemic treatment group (group 1), while another twenty patients were solely administered 0.1 % tacrolimus eye drops, forming the topical treatment group (group 2). The occurrence of rejection, corneal neovascularization, corneal graft edema and visual acuity were documented in both groups.
 Results: In comparison to patients in group 2, patients in group 1 exhibited a significant reduction in rejection rate (p < 0.05). Additionally, the average time of neovascularization in group 1 was delayed and the number of cases was lower (p < 0.05). Furthermore, a smaller proportion of patients in group 1 experienced corneal graft edema (25 vs 60 %, p < 0.05), while a higher percentage of patients in group 1 demonstrated improved visual acuity (90 vs 60 %, p < 0.05).
 Conclusion: Concurrent administration of tacrolimus eye drops and capsules orally effectively mitigates anti-rejection reactions, regulation of neovascularization, management of graft edema and enhancement of visual acuity in high-risk keratoplasty patients when compared to the use of tacrolimus eye drops as a standalone treatment. These results have the potential to stimulate novel avenues of investigation in future clinical research.

Role of ATG7-dependent non-autophagic pathway in angiogenesis.

ATG7, one of the core proteins of autophagy, plays an important role in various biological processes, including the regulation of autophagy. While clear that autophagy drives angiogenesis, the role of ATG7 in angiogenesis remains less defined. Several studies have linked ATG7 with angiogenesis, which has long been underappreciated. The knockdown of ATG7 gene in cerebrovascular development leads to angiogenesis defects. In addition, specific knockout of ATG7 in endothelial cells results in abnormal development of neovascularization. Notably, the autophagy pathway is not necessary for ATG7 regulation of angiogenesis, while the ATG7-dependent non-autophagic pathway plays a critical role in the regulation of neovascularization. In order to gain a better understanding of the non-autophagic pathway-mediated biological functions of the autophagy-associated protein ATG7 and to bring attention to this expanding but understudied research area, this article reviews recent developments in the ATG7-dependent non-autophagic pathways regulating angiogenesis.

Transcription factor Foxp1 stimulates angiogenesis in adult rats after myocardial infarction

Forkhead box protein P1 (FoxP1) is essential for cardiac development and the regulation of neovascularization, but its potential for cardiac angiogenesis has not been explored. This study aims to investigate the angiogenic role of FoxP1 in a rat model of myocardial infarction (MI). Adult male rats were subjected to MI, and Foxp1 was knocked down with lentivirus FoxP1 siRNA. Endothelial cell proliferation, angiogenesis, and cardiac function were also assessed. Cell scratch assay and tubule formation analysis were used to detect the migration ability and tube formation ability of human umbilical vein endothelial cells (HUVECs). Compared with that in the sham group, results showed that the expression of FoxP1 was significantly increased in the MI group. Foxp1 knockdown decreases FoxP1 expression, reduces angiogenesis, and increases collagen deposition. When Foxp1 was knocked down in HUVECs using FoxP1 siRNA lentivirus, cell proliferation, migration, and tube formation abilities decreased significantly. Our study showed that FoxP1 elicits pleiotropic beneficial actions on angiogenesis in the post-MI heart by promoting the proliferation of endothelial cells. FoxP1 should be considered a candidate for therapeutic cardiac angiogenesis.

Decreased endostatin in db/db retinas is associated with optic disc intravitreal vascularization

Endostatin, a naturally cleaved fragment of type XVIII collagen with antiangiogenic activity, has been involved in the regulation of neovascularization during diabetic retinopathy. Here, the intracellular distribution of endostatin in healthy mouse and human neuroretinas has been analyzed. In addition, to study the effect of experimental hyperglycemia on retinal endostatin, the db/db mouse model has been used. Endostatin protein expression in mouse and human retinas was studied by immunofluorescence and Western blot, and compared with db/db mice. Eye fundus angiography, histology, and immunofluorescence were used to visualize mouse retinal and intravitreal vessels.For the first time, our results revealed the presence of endostatin in neurons of mouse and human retinas. Endostatin was mainly expressed in bipolar cells and photoreceptors, in contrast to the optic disc, where endostatin expression was undetectable. Diabetic mice showed a reduction of endostatin in their retinas associated with the appearance of intravitreal vessels at the optic disc in 50% of db/db mice. Intravitreal vessels showed GFAP positive neuroglia sheath, basement membrane thickening by collagen IV deposition, and presence of MMP-2 and MMP-9 in the vascular wall. All together, these results point that decreased retinal endostatin during experimental diabetes is associated with optic disc intravitreal vascularization. Based on their phenotype, these intravitreal vessels could be neovessels. However, it cannot be ruled out the possibility that they may also represent persistent hyaloid vessels.

MicroRNA-146a regulates angiogenesis and functional regeneration after myocardial infarction

Abstract Background The growth or regeneration of new blood vessels is critical for physiological angiogenesis and for tissue repair in response to injury, such as myocardial infarction. However, the mechanisms underlying the angiogenic processes and, specifically, the regulation of neo-vascularization by microRNAs are not well understood. Purpose To study the potential beneficial role of miRNA-146a in vessel regeneration after myocardial infarction. Methods Mice were subjected to myocardial infarction or hind limb ischemia and vascular development was followed in zebra fish. microRNA-146a was blocked or overexpressed in endothelial cells in vitro or in mice or zebra fish in vivo. Furthermore, Blood samples from patients with ST-elevation myocardial infarction (STEMI) were studied for miR-146a expression. Results MicroRNA-146a was upregulated in the ischemic hind limb following ligation of the femoral artery and in the ischemic myocardium in male C57BL/6 mice following ligation of the left anterior descending (LAD) artery, as well as in patients with STEMI. In vitro, overexpression of microRNA-146a significantly attenuated endothelial cell proliferation and migration, abolished endothelial capillary network formation, and inhibited cell sprouting from endothelial spheroids. In contrast, microRNA-146a silencing had opposite effects. Mechanistically, Nox4, Notch1, and nRas were identified by in silico prediction and microarray mRNA expression analysis, and validated as direct targets and effector molecules of microRNA-146a in endothelial cells according to mRNA and protein expression profiles, as well as luciferase gene reporter assays and respective rescue experiments. In zebrafish, overexpression of microRNA-146a inhibited the development of the trunk vasculature. Blocking the upregulation of endothelial microRNA-146a in mice,using specific inhibitors, resulted in significantly enhanced 1 angiogenesis and revascularization both in an ischemic hind limb model and in the infarcted myocardium. This was accompanied by a significantly reduced myocardial infarct size and preserved cardiac function post MI. Conclusions Our findings identify microRNA-146a as a critical regulator of angiogenesis and vascular regeneration. Inhibition of microRNA-146a may represent an attractive strategy for the angiogenic therapy of ischemic tissue diseases. Funding Acknowledgement Type of funding source: None

Myeloid-derived suppressor cells improve corneal graft survival through suppressing angiogenesis and lymphangiogenesis.

Myeloid-derived suppressor cells (MDSC) are one of the major negative regulators of immune responses during many pathological conditions such as cancer and transplantation. Emerging evidence indicates that MDSC also contribute to tumor progression through their pro-angiogenic activity in addition to immunosuppressive function. However, virtually nothing is known about the role of MDSC in the regulation of neovascularization after transplantation. Here we showed that antibody-mediated depletion of MDSC in mice led to robust growth of blood and lymphatic neovessels and rapid allograft rejection after corneal penetrating keratoplasty. In contrast, adoptive transfer of ex vivo generated MDSC from cytokine-treated bone marrow cells (evMDSC) suppressed neovascularization and prolonged corneal allograft survival in an inducible nitric oxide synthase (iNOS)-dependent manner. Mechanistically, compared to naïve MDSC control, evMDSC have increased expression of an anti-angiogenic factor thrombospondin 1 (Tsp-1) and decreased expression of two critical pro-angiogenic factors, vascular endothelial growth factor A (VEGF-A), and VEGF-C. These findings demonstrate MDSC as a critical anti-angiogenic regulator during transplantation. Our study also indicates that evMDSC are a valuable candidate agent for development of novel cell therapy to improve allograft survival after transplantation.

MiR-96 and miR-183 differentially regulate neonatal and adult postinfarct neovascularization.

Following myocardial infarction (MI), the adult heart has minimal regenerative potential. Conversely, the neonatal heart can undergo extensive regeneration, and neovascularization capacity was hypothesized to contribute to this difference. Here, we demonstrate the higher angiogenic potential of neonatal compared with adult mouse cardiac endothelial cells (MCECs) in vitro and use this difference to identify candidate microRNAs (miRs) regulating cardiac angiogenesis after MI. miR expression profiling revealed miR-96 and miR-183 upregulation in adult compared with neonatal MCECs. Their overexpression decreased the angiogenic potential of neonatal MCECs in vitro and prevented scar resolution and neovascularization in neonatal mice after MI. Inversely, their inhibition improved the angiogenic potential of adult MCECs, and miR-96/miR-183–KO mice had increased peri-infarct neovascularization. In silico analyses identified anillin (ANLN) as a direct target of miR-96 and miR-183. In agreement, Anln expression declined following their overexpression and increased after their inhibition in vitro. Moreover, ANLN expression inversely correlated with miR-96 expression and age in cardiac ECs of cardiovascular patients. In vivo, ANLN+ vessels were enriched in the peri-infarct area of miR-96/miR-183–KO mice. These findings identify miR-96 and miR-183 as regulators of neovascularization following MI and miR-regulated genes, such as anillin, as potential therapeutic targets for cardiovascular disease.

Crosstalk Between Long Non-coding RNAs, Micro-RNAs and mRNAs: Deciphering Molecular Mechanisms of Master Regulators in Cancer

Cancer is a complex disease, and its study requires deep understanding of several biological processes and their regulation. It is an accepted fact that non-coding RNAs are vital components of the regulation and cross-talk among cancer-related signaling pathways that favor tumor aggressiveness and metastasis, such as neovascularization, angiogenesis, and vasculogenic mimicry. Both long non-coding RNAs (lncRNAs) and micro-RNAs (miRNAs) have been described as master regulators of cancer on their own; yet there is accumulating evidence that, besides regulating mRNA expression through independent mechanisms, these classes of non-coding RNAs interact with each other directly, fine-tuning the effects of their regulation. While still relatively scant, research on the lncRNA-miRNA-mRNA axis regulation is growing at a fast rate, it is only in the last 5 years, that lncRNA-miRNA interactions have been identified in tumor-related vascular processes. In this review, we summarize the current progress of research on the cross-talk between lncRNAs and miRNAs in the regulation of neovascularization, angiogenesis and vasculogenic mimicry.

The function of αA-crytstallin and effects in ocular diseases

αA-crystallin is an important protein in lens, with molecular chaperone founction, apoptosis regulation effect, neovascularization regulation, oxidation-reduction reaction process regulation and inflammation reaction regulation.Now, more and more studies have verified that αA-crystallin exists not only in lens, but also in other intraocular structures, such as retina.αA-crystallin can not only maintain the internal environment homeostasis, but also participate in many other eye diseases, such as optic nerve injury diseases, retinal degeneration, ocular neoplasmas, inflammatory diseases and retinal choroidal vascular diseases.This article reviewed the distribution, structure and founction of αA-crystallin and its effects in ocular diseases. Key words: αA-crystallin; Ocular diseases; Function; Distribution; Structure

RGD Conjugated Hyperbranched Cationic Amylopectin Nanoparticle as Hypoxia Inducible Factor-1α Targeting Carrier Promotes Neovascularization in Cerebral Infarction

Promoting the regeneration of new blood vessels is a crucial treatment in cerebral infarction and hypoxia-inducible factor 1-α (HIF-1α) plays a pivotal role in the regulation of neovascularization. Nevertheless, the degradation of HIF-1α and poor homing into ischemic sites greatly limit its clinical application. In this study, we encapsulated mutant HIF-1α in RGD modified hyperbranched cationic amylopectin derivative conjugated with 3-(dimethylamino)-1-propylamine (RGD-DMAPA-Amyp) nanoparticles. RGD-DMAPA-Amyp nanoparticles exhibited good blood compatibility and low cytotoxicity in hemolysis and MTT assay, and in vivo study showed low concentrations of RGD-DMAPA-Amyp and DMAPA-Amyp had no significant toxicity to zebrafish embryos. Further to investigate the targeting ability of RGD-DMAPA-Amyp in human umbilical vein endothelial cells and rats model of transient middle cerebral artery occlusion, the in vitro and in vivo results showed RGD-DMAPA-Amyp could specifically bind to vascular endothelial cells. Moreover, the RGD-DMAPA-Amyp/HIF-1α administration significantly reduced the incidence of cerebral ischemia in zebrafish. The pro-angiogenic effect of RGD-DMAPA-Amyp/HIF-1α was evaluated on zebra fish model of vascular loss, RGD-DMAPA-Amyp/HIF-1α were found could improve the growth of subintestinal vessels (SIVs). Our results imply that RGD-DMAPA-Amyp/HIF-1α might alleviate cerebral infarction in vivo via promoting neovascularization and could potentially be a very promising approach for cerebral infarction or ischemic diseases.

Effects of miR-153 on angiogenesis in MCAO rats through Shh signaling pathway.

Cerebrovascular disease is a disease which has the highest mortality in China. Angiogenesis in the ischemic region after cerebral infarction is closely related to its prognosis. Recent studies found that microRNAs (miRNAs) are involved in the regulation of neovascularization. MicroRNA-153 (MiR-153) has protective effects on the ischemic injury, but its relationship with the Sonic Hedgehog (Shh) signaling pathway is still unclear. This work aimed to investigate the role of miR-153 in angiogenesis of middle cerebral artery occlusion (MCAO) rats through the Shh signaling pathway. The rat cerebral ischemic injury (MCAO) model was established by thread embolism and treated by Agomir-153 and 5-EI. MiR-153 expression was detected using Real Time-Polymerase Chain Reaction (RT-PCR). The neurological function was assessed. The infarct area of the brain and the capillary density were determined using 2,3,5-triphenyl tetrazolium chloride (TTC) method. The Shh signaling pathway and angiogenesis-related factors were tested by Western blot assay. Agomir-153 or Agomir-153 combined with 5-EI significantly increased miR-153 expression, reduced the infarct area, and promoted the generation of cerebral capillaries in the MCAO model. 5-EI partially blocked the protective effects of Agomir-153 and angiogenesis. The up-regulation of miR-153 markedly inhibited patched (PTC) expression and activated the Shh signaling pathway. The up-regulation of miR-153 rats activated the Shh signaling pathway to promote angiogenesis and improve prognosis through lipid-coated Patch (PTC) in MCAO. MiR-153 was considered to be a new therapeutic target for promoting angiogenesis after MCAO.

Remodeling Tumor-Associated Macrophages and Neovascularization Overcomes EGFRT790M -Associated Drug Resistance by PD-L1 Nanobody-Mediated Codelivery.

Precision medicine has made a significant breakthrough in the past decade. The most representative success is the molecular targeting therapy of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) in non-small-cell lung cancer (NSCLC) with oncogenic drivers, approved by the US Food and Drug Administration (FDA) as first-line therapeutics for substituting chemotherapy. However, the rapidly developed TKI resistance invariably leads to unsustainable treatment. For example, gefitinib is the first choice for advanced NSCLC with EGFR mutation, but most patients would soon develop secondary EGFRT790M mutation and acquire gefitinib resistance. TKI resistance is a severe emergency issue to be solved in NSCLC, but there are a few investigations of nanomedicine reported to address this pressing problem. To overcome EGFRT790M -associated drug resistance, a novel delivery and therapeutic strategy is developed. A PD-L1 nanobody is identified, and first used as a targeting ligand for liposomal codelivery. It is found that simvastatin/gefitinib combination nanomedicine can remodel the tumor microenvironment (e.g., neovascularization regulation, M2-macrophage repolarization, and innate immunity), and display the effectiveness of reversing the gefitinib resistance and enhancing the EGFRT790M -mutated NSCLC treatment outcomes. The novel simvastatin-based nanomedicine provides a clinically translatable strategy for tackling the major problem in NSCLC treatment and demonstrates the promise of an old drug for new application.

Research advances of cancer therapy targeting HIF-1α-VEGF-VEGFR-2 signaling pathway for malignant tumors

Hypoxia inducible factor-1α (HIF-1α)-vascular endothelial growth factor (VEGF)-vascular endothelial growth factor receptor-2 (VEGFR-2) signaling pathway plays a vital role in the regulation of neovascularization, and the activation of this pathway is closely related with tumor invasion and metastasis. Key words: Neoplasms; Neovascularization, pathologic; Vascular endothelial growth factors; Receptors, vascular endothelial growth factor; Signal transduction

Abstract 221: Anti-angiogenic Therapy Rescues the Defect in Ischemia-Induced Neovasculature in Type II Diabetic Mice

Objective: Type II diabetes mellitus (DM) leads to the development of microvascular diseases and is associated with impaired angiogenesis. The presence of VEGF can block PDGF-dependent regulation of neovascularization and vessel normalization. We tested the hypothesis that the inhibition of VEGF rescues the defect in Ischemia-Induced neovasculature by inducing PDGF-BB activation. Methods and Results: Hindlimb ischemia was generated by femoral artery occlusion in diet-induced diabetic mice and age-matched normal chow (NC)-fed mice. Bevacizumab, a humanized monoclonal antibody against VEGF-A, was intraperitoneally administered to mice (200 μg/mouse; once weekly for 2 weeks) to determine if bevacizumab restores neovasculature formation in DM mice. Color Laser Doppler showed that the blood flow perfusion in ischemic hindlimb was significantly increased in the DM treated by bevacizumab compared to vehicles at 7, 12, and 14 days (n=6/group, p<0.05). Real-time PCR revealed that the expression of PDGF-BB was significantly enhanced in the DM treated by bevacizumab compared to vehicles. Furthermore, anti-PDGF drug significantly inhibit the blood flow perfusion in the DM mice treated by bevacizumab. Conclusions: These results indicate that inhibition of VEGF could restore PDGF-BB expression, and possibly imply the molecular target of diabetic foot.

MicroRNA-155 Exerts Cell-Specific Antiangiogenic but Proarteriogenic Effects During Adaptive Neovascularization

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. 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. 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.

Vascular endothelial growth factor B coordinates metastasis of non-small cell lung cancer.

Neovascularization is critical for the invasion and metastasis of non-small cell lung cancer (NSCLC). However, the molecular mechanism underlying the control of neovascularization of NSCLC is not completely understood. Both vascular endothelial growth factor B (VEGF-B) and matrix metalloproteinases 9 (MMP9) play essential roles in neovascularization of NSCLC. Here, we examined whether VEGF-B and MMP9 may affect each other to coordinate the neovascularization process in NSCLC. We found strong positive correlation of VEGF-B and MMP9 levels in the NSCLC from the patients. Moreover, patients that had NSCLC with metastasis had significantly higher levels of VEGF-B and MMP9 in the primary cancer. Using a human NSCLC line A549, we found that overexpression of VEGF-B increased expression of MMP9, while inhibition of VEGF-B decreased expression of MMP9. On the other hand, overexpression of MMP9 increased expression of VEGF-B, while inhibition of MMP9 decreased expression of VEGF-B. These data suggest that expression of VEGF-B and MMP9 may activate each other to enhance neovascularization. We then analyzed the underlying mechanism. Application of a specific ERK/MAPK inhibitor but not a PI3K/Akt inhibitor to VEGF-B-overexpressing A549 cells substantially abolished the effect of VEGF-B on MMP9 activation, while application of a specific PI3K/Akt inhibitor but not an ERK/MAPK inhibitor to MMP9-overexpressing A549 cells substantially abolished the effect of MMP9 on VEGF-B activation, suggesting that VEGF-B may activate MMP9 via ERK/MAPK signaling pathway, while MMP9 may activate VEGF-B via PI3K/Akt signaling pathway. Thus, our data highlight a coordinating relationship between VEGF-B and MMP9 in the regulation of neovascularization in NSCLC.

Regulation of neovascularization by S-glutathionylation via the Wnt5a/sFlt-1 pathway.

S-glutathionylation occurs when reactive oxygen or nitrogen species react with protein-cysteine thiols. Glutaredoxin-1 (Glrx) is a cytosolic enzyme which enzymatically catalyses the reduction in S-glutathionylation, conferring reversible signalling function to proteins with redox-sensitive thiols. Glrx can regulate vascular hypertrophy and inflammation by regulating the activity of nuclear factor κB (NF-κB) and actin polymerization. Vascular endothelial growth factor (VEGF)-induced endothelial cell (EC) migration is inhibited by Glrx overexpression. In mice overexpressing Glrx, blood flow recovery, exercise function and capillary density were significantly attenuated after hindlimb ischaemia (HLI). Wnt5a and soluble Fms-like tyrosine kinase-1 (sFlt-1) were enhanced in the ischaemic-limb muscle and plasma respectively from Glrx transgenic (TG) mice. A Wnt5a/sFlt-1 pathway had been described in myeloid cells controlling retinal blood vessel development. Interestingly, a Wnt5a/sFlt-1 pathway was found also to play a role in EC to inhibit network formation. S-glutathionylation of NF-κB components inhibits its activation. Up-regulated Glrx stimulated the Wnt5a/sFlt-1 pathway through enhancing NF-κB signalling. These studies show a novel role for Glrx in post-ischaemic neovascularization, which could define a potential target for therapy of impaired angiogenesis in pathological conditions including diabetes.

CXCL4L1 and CXCL4 signaling in human lymphatic and microvascular endothelial cells and activated lymphocytes: involvement of mitogen-activated protein (MAP) kinases, Src and p70S6 kinase

CXC chemokines influence a variety of biological processes, such as angiogenesis, both in a physiological and pathological context. Platelet factor-4 (PF-4)/CXCL4 and its variant PF-4var/CXCL4L1 are known to favor angiostasis by inhibiting endothelial cell proliferation and chemotaxis. CXCL4L1 in particular is a potent inhibitor of angiogenesis with anti-tumoral characteristics, both through regulation of neovascularization and through attraction of activated lymphocytes. However, its underlying signaling pathways remain to be elucidated. Here, we have identified various intracellular pathways activated by CXCL4L1 in comparison with other CXCR3 ligands, including CXCL4 and interferon-γ-induced protein 10/CXCL10. Signaling experiments show involvement of the mitogen-activated protein kinase (MAPK) family in CXCR3A-transfected cells, activated lymphocytes and human microvascular endothelial cells (HMVEC). In CXCR3A transfectants, CXCL4 and CXCL4L1 activated p38 MAPK, as well as Src kinase within 30 and 5 min, respectively. Extracellular signal-regulated kinase (ERK) phosphorylation occurred in activated lymphocytes, yet was inhibited in microvascular and lymphatic endothelial cells. CXCL4L1 and CXCL4 counterbalanced the angiogenic chemokine stromal cell-derived factor-1/CXCL12 in both endothelial cell types. Notably, inhibition of ERK signaling by CXCL4L1 and CXCL4 in lymphatic endothelial cells implies that these chemokines might also regulate lymphangiogenesis. Furthermore, CXCL4, CXCL4L1 and CXCL10 slightly enhanced forskolin-stimulated cAMP production in HMVEC. Finally, CXCL4, but not CXCL4L1, induced activation of p70S6 kinase within 5 min in HMVEC. Our findings confirm that the angiostatic chemokines CXCL4L1 and CXCL4 activate both CXCR3A and CXCR3B and bring new insights into the complexity of their signaling cascades.

FoxP1 stimulates angiogenesis by repressing the inhibitory guidance protein semaphorin 5B in endothelial cells.

Forkhead box (Fox) transcription factors are important regulators of cardiovascular development and several Fox-proteins have recently been shown to modulate embryonic and post-natal angiogenesis. However, the role of the FoxP subfamily, which is highly expressed in cardiovascular tissue, has not been investigated so far. Here, we show that the transcription factor FoxP1 is the highest expressed FoxP-protein in endothelial cells and that it is upregulated at the site of neovascularization during hindlimb ischemia in mice. Silencing of FoxP1 results in a strong inhibition of proliferation, tube formation and migration of cultured endothelial cells. Accordingly, knockdown of FoxP1 in zebrafish was followed by a disruption of intersomitic vascular formation. Using gene expression profiling, we show that FoxP1 induces a specific change of the endothelial transcriptome and functions as a suppressor of semaphorin 5B, which has previously been described as a neuronal inhibitory factor. Our findings now demonstrate that semaphorin 5B also acts as a FoxP1- dependent suppressor of endothelial cell proliferation, migration and sprouting, mediating the effects of FoxP1. In summary, our data indicate that the transcription factor FoxP1 is essential for the angiogenic function of endothelial cells and functions as a suppressor of the inhibitory guidance cue semaphorin 5B, suggesting an important function of FoxP1 in the regulation of neovascularization.

MicroRNA-155 exerts anti-angiogenic but pro-arteriogenic effects in the regulation of adaptive neovascularization in mice by cell specific regulation of divergent target genes

Background: MicroRNAs (miRNAs) are small non-coding RNAs, which regulate mRNAs by inhibition of translation or degradation. The adaptive growth of blood vessels is known to be modulated by miRNAs and includes both the sprouting of new endothelial capillaries (angiogenesis) as well as the enlargement of pre-existing arteriolar and arterial anastomoses to functional collateral arteries (arteriogenesis) after occlusion or narrowing of a major artery. We now aimed to identify new regulatory miRNAs involved in regulation of neovascularization in a murine model of femoral artery occlusion. Methods and results: We explored the expression profile of miRNAs after occlusion of the femoral artery in mice using miRNA-transcriptome analysis and identified miR-155 as one of the top down-regulated miRNAs. Following this, we examined the angiogenic properties of miR-155 in human umbilical endothelial cells (HUVECS) and found an inhibitory function of miR-155 on proliferation and angiogenic tube formation by suppression of the direct miR-155 target angiotensin II receptor type one (AGTR1). Surprisingly, miR-155 deficient mice showed an unexpected phenotype in vivo, with an impaired blood flow recovery after femoral artery ligation, due to a reduction of pro-arteriogenic cytokine expression, a reduction of infiltrating monocytes/macrophages and an attenuation of collateral artery growth (arteriogenesis). Based on this, we examined leukocyte-endothelial interaction and migration capacity of circulating cells in this mouse strain. Intravital imaging of mesenteric venules after systemic TNF-alpha treatment revealed a lower number of rolling and adherent cells on the endothelium compared to wildtype mice. In addition, we observed a significantly reduced migration capacity of miR-155 deficient bone marrow derived macrophages (BMDM) compared to wildtype cells and in a model of sterile induced peritonitis miR-155 deficiency led to a significant reduction of leukocytes in peritoneal lavage. Subsequently, we examined the cytokine expression profile in BMDM and found a decreased expression of pro-arteriogenic cytokines in cells lacking miR-155, which was partly mediated by the direct miR-155 target SOCS-1. Conclusion: Our data demonstrate that miR-155 exerts a cell-specific anti-angiogenic but pro-arteriogenic function in the regulation of neovascularization by targeting divergent genes and that its expression in bone marrow derived cells is essential for arteriogenesis in response to hindlimb ischemia in mice. MiRNAs could represent attractive target molecules for modulation of blood vessel growth.