Jiong Hu

MicroRNA-223 Antagonizes Angiogenesis by Targeting β1 Integrin and Preventing Growth Factor Signaling in Endothelial Cells

Rationale: Endothelial cells in situ are largely quiescent, and their isolation and culture are associated with the switch to a proliferative phenotype. Objective: To identify antiangiogenic microRNAs expressed by native endothelial cells that are altered after isolation and culture, as well as the protein targets that regulate responses to growth factors. Methods and Results: Profiling studies revealed that miR-223 was highly expressed in freshly isolated human, murine, and porcine endothelial cells, but those levels decreased in culture. In primary cultures of endothelial cells, vascular endothelial cell growth factor and basic fibroblast growth factor further decreased miR-223 expression. The overexpression of precursor-miR-223 did not affect basal endothelial cell proliferation but abrogated vascular endothelial cell growth factor–induced and basic fibroblast growth factor–induced proliferation, as well as migration and sprouting. Inhibition of miR-223 in vivo using specific antagomirs potentiated postnatal retinal angiogenesis in wild-type mice, whereas recovery of perfusion after femoral artery ligation and endothelial sprouting from aortic rings from adult miR-223−/y animals were enhanced. MiR-223 overexpression had no effect on the growth factor–induced activation of ERK1/2 but inhibited the vascular endothelial cell growth factor–induced and basic fibroblast growth factor–induced phosphorylation of their receptors and activation of Akt. &bgr;1 integrin was identified as a target of miR-223 and its downregulation reproduced the defects in growth factor receptor phosphorylation and Akt signaling seen after miR-223 overexpression. Reintroduction of &bgr;1 integrin into miR-223–ovexpressing cells was sufficient to rescue growth factor signaling and angiogenesis. Conclusions: These results indicate that miR-223 is an antiangiogenic microRNA that prevents endothelial cell proliferation at least partly by targeting &bgr;1 integrin.

Macrophages programmed by apoptotic cells promote angiogenesis via prostaglandin E2

Macrophages contribute to tissue homeostasis in the developing as well as the adult organism. They promote tissue regeneration and remodeling after injury, which requires efficient neoangiogenesis. Signaling pathways activating an angiogenic program in macrophages are still poorly defined. We report that apoptotic cells (ACs), which originate from stressed or damaged tissues, can induce angiogenic properties in primary human macrophages. The signal originating from ACs is the lipid mediator sphingosine‐1‐phosphate (S1P), which activates S1P1/3 on macrophages to upregulate cyclooxygenase‐2. The formation and liberation of prostaglandin E2 (PGE2) then stimulates migration of endothelial cells. This is demonstrated by using PGE2 receptor antagonists or a neutralizing PGE2 antibody in vitro, thereby attenuating endothelial cell migration using a Boyden chamber assay. In vivo, neutralization of PGE2 from proangiogenic macrophage supernatants blocked vessel formation into Matrigel plugs. In particular, apoptotic cancer cells shifted prostanoid formation in macrophages selectively toward PGE2 by up‐regulating cyclooxygenase‐2 and microsomal prostaglandin E synthase‐1 (mPGES1), while down‐regulating the PGE2‐degrading enzyme 15‐hydroxyprostaglandin dehydrogenase (15‐PGDH) or prostaglandin‐D synthase (PGDS). Angiogenic programming of macrophages by ACs, therefore, may control responses to tissue stress such as in tumors, where macrophages support cancer progression.—Brecht, K., Weigert, A., Hu, J., Popp, R., Fisslthaler, B., Korff, T., Fleming, I., Geisslinger, G., Brüne, B. Macrophages programmed by apoptotic cells promote angiogenesis via prostaglandin E2. FASEB J. 25, 2408–2417 (2011). www.fasebj.org

Soluble epoxide hydrolase regulates hematopoietic progenitor cell function via generation of fatty acid diols

Fatty acid epoxides are important lipid signaling molecules involved in the regulation of vascular tone and homeostasis. Tissue and plasma levels of these mediators are determined by the activity of cytochrome P450 epoxygenases and the soluble epoxide hydrolase (sEH), and targeting the latter is an effective way of manipulating epoxide levels in vivo. We investigated the role of the sEH in regulating the mobilization and proliferation of progenitor cells with vasculogenic/reparative potential. Our studies revealed that sEH down-regulation/inhibition impaired the development of the caudal vein plexus in zebrafish, and decreased the numbers of lmo2/cmyb-positive progenitor cells therein. In mice sEH inactivation attenuated progenitor cell proliferation (spleen colony formation), but the sEH products 12,13-dihydroxyoctadecenoic acid (12,13-DiHOME) and 11,12- dihydroxyeicosatrienoic acid stimulated canonical Wnt signaling and rescued the effects of sEH inhibition. In murine bone marrow, the epoxide/diol content increased during G-CSF–induced progenitor cell expansion and mobilization, and both mobilization and spleen colony formation were reduced in sEH−/− mice. Similarly, sEH−/− mice showed impaired functional recovery following hindlimb ischemia, which was rescued following either the restoration of bone marrow sEH activity or treatment with 12,13-DiHOME. Thus, sEH activity is required for optimal progenitor cell proliferation, whereas long-term sEH inhibition is detrimental to progenitor cell proliferation, mobilization, and vascular repair.

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.

Dicer Cleavage by Calpain Determines Platelet microRNA Levels and Function in Diabetes

RATIONALE MicroRNAs (miRNAs) are short noncoding RNA species generated by the processing of longer precursors by the ribonucleases Drosha and Dicer. Platelets contain large amounts of miRNA that are altered by disease, in particular diabetes mellitus. OBJECTIVE This study determined why platelet miRNA levels are attenuated in diabetic individuals and how decreased levels of the platelet-enriched miRNA, miR-223, affect platelet function. METHODS AND RESULTS Dicer levels were altered in platelets from diabetic mice and patients, a change that could be attributed to the cleavage of the enzyme by calpain, resulting in loss of function. Diabetes mellitus in human subjects as well as in mice resulted in decreased levels of platelet miR-142, miR-143, miR-155, and miR-223. Focusing on only 1 of these miRNAs, miR-223 deletion in mice resulted in modestly enhanced platelet aggregation, the formation of large thrombi and delayed clot retraction compared with wild-type littermates. A similar dysregulation was detected in platelets from diabetic patients. Proteomic analysis of platelets from miR-223 knockout mice revealed increased levels of several proteins, including kindlin-3 and coagulation factor XIII-A. Whereas, kindlin-3 was indirectly regulated by miR-223, factor XIII was a direct target and both proteins were also altered in diabetic platelets. Treating diabetic mice with a calpain inhibitor prevented loss of platelet dicer as well as the diabetes mellitus-induced decrease in platelet miRNA levels and the upregulation of miR-223 target proteins. CONCLUSIONS Thus, calpain inhibition may be one means of normalizing platelet miRNA processing as well as platelet function in diabetes mellitus.

The F-BAR protein NOSTRIN participates in FGF signal transduction and vascular development

F‐BAR proteins are multivalent adaptors that link plasma membrane and cytoskeleton and coordinate cellular processes such as membrane protrusion and migration. Yet, little is known about the function of F‐BAR proteins in vivo. Here we report, that the F‐BAR protein NOSTRIN is necessary for proper vascular development in zebrafish and postnatal retinal angiogenesis in mice. The loss of NOSTRIN impacts on the migration of endothelial tip cells and leads to a reduction of tip cell filopodia number and length. NOSTRIN forms a complex with the GTPase Rac1 and its exchange factor Sos1 and overexpression of NOSTRIN in cells induces Rac1 activation. Furthermore, NOSTRIN is required for fibroblast growth factor 2 dependent activation of Rac1 in primary endothelial cells and the angiogenic response to fibroblast growth factor 2 in the in vivo matrigel plug assay. We propose a novel regulatory circuit, in which NOSTRIN assembles a signalling complex containing FGFR1, Rac1 and Sos1 thereby facilitating the activation of Rac1 in endothelial cells during developmental angiogenesis.

Müller glia cells regulate Notch signaling and retinal angiogenesis via the generation of 19,20-dihydroxydocosapentaenoic acid

Cytochrome P450 (CYP) epoxygenases generate bioactive lipid epoxides which can be further metabolized to supposedly less active diols by the soluble epoxide hydrolase (sEH). As the role of epoxides and diols in angiogenesis is unclear, we compared retinal vasculature development in wild-type and sEH−/− mice. Deletion of the sEH significantly delayed angiogenesis, tip cell, and filopodia formation, a phenomenon associated with activation of the Notch signaling pathway. In the retina, sEH was localized in Muller glia cells, and Muller cell–specific sEH deletion reproduced the sEH−/− retinal phenotype. Lipid profiling revealed that sEH deletion decreased retinal and Muller cell levels of 19,20–dihydroxydocosapentaenoic acid (DHDP), a diol of docosahexenoic acid (DHA). 19,20-DHDP suppressed endothelial Notch signaling in vitro via inhibition of the γ-secretase and the redistribution of presenilin 1 from lipid rafts. Moreover, 19,20-DHDP, but not the parent epoxide, was able to rescue the defective angiogenesis in sEH−/− mice as well as in animals lacking the Fbxw7 ubiquitin ligase, which demonstrate strong basal activity of the Notch signaling cascade. These studies demonstrate that retinal angiogenesis is regulated by a novel form of neuroretina–vascular interaction involving the sEH-dependent generation of a diol of DHA in Muller cells.

Deleted in Malignant Brain Tumors 1 is Present in the Vascular Extracellular Matrix and Promotes Angiogenesis

Objective—Deleted in malignant brain tumors 1 (DMBT1) belongs to the scavenger receptor cysteine-rich superfamily of proteins and is implicated in innate immunity, cell polarity, and differentiation. Here we studied the role of DMBT1 in endothelial cells. Methods and Results—DMBT1 was secreted into the extracellular matrix (ECM) by endothelial cells in vitro and in situ and the presence of DMBT1 in the ECM increased endothelial cell adherence. Endothelial cell-derived DMBT1 associated with galectin-3 (coprecipitation), and human recombinant DMBT1 bound EGF, vascular endothelial growth factor and Delta-like (Dll) 4 (specific ELISAs). Compared to cells from wild-type mice, endothelial cells from DMBT1−/− mice demonstrated reduced migration, proliferation, and tube formation. In vivo recovery from hindlimb ischemia was attenuated in DMBT1−/− animals as was vascular endothelial growth factor -induced endothelial sprouting from isolated aortic rings; the latter response could be rescued by the addition of recombinant DMBT1. The Notch pathway is involved in multiple aspects of vascular development, including arterial-venous differentiation and we found that endothelial cells from DMBT1−/− mice expressed more EphrinB2 than cells from wild-type mice. Levels of Dll1, Dll4, Hes1, Hey1, and EphB4, on the other hand, were decreased. Conclusion—Taken together, the results of this study indicate that DMBT1 functions as an important endothelium-derived ECM protein that is able to bind angiogenic factors and promote adhesion, migration, proliferation, and angiogenesis as well as vascular repair. Mechanistically, DMBT1 interacts with galectin-3 and modulates the Notch signaling pathway as well as the differential expression of ephrin-B2 and EphB4.

Role of secreted modular calcium-binding protein 1 (SMOC1) in transforming growth factor β signalling and angiogenesis

AIMS Secreted modular calcium-binding protein 1 (SMOC1) is a matricellular protein that potentially interferes with growth factor receptor signalling. The aim of this study was to determine how its expression is regulated in endothelial cells and its role in the regulation of endothelial cell function. METHODS AND RESULTS SMOC1 was expressed by native murine endothelial cells as well as by cultured human, porcine, and murine endothelial cells. SMOC1 expression in cultured cells was increased by hypoxia via the down-regulation of miR-223, and SMOC1 expression was increased in lungs from miR-223-deficient mice. Silencing SMOC1 (small interfering RNA) attenuated endothelial cell proliferation, migration, and sprouting in in vitro angiogenesis assays. Similarly endothelial cell sprouting from aortic rings ex vivo as well as postnatal retinal angiogenesis in vivo was attenuated in SMOC1(+/-) mice. In endothelial cells, transforming growth factor (TGF)-β signalling via activin-like kinase (ALK) 5 leads to quiescence, whereas TGF-β signalling via ALK1 results in endothelial cell activation. SMOC1 acted as a negative regulator of ALK5/SMAD2 signalling, resulting in altered α2 integrin levels. Mechanistically, SMOC1 associated (immunohistochemistry, proximity ligation assay, and co-immunoprecipitation) with endoglin; an endothelium-specific type III auxiliary receptor for the TGF-β super family and the effects of SMOC1 down-regulation on SMAD2 phosphorylation were abolished by the down-regulation of endoglin. CONCLUSION These results indicate that SMOC1 is an ALK5 antagonist produced by endothelial cells that tips TGF-β signalling towards ALK1 activation, thus promoting endothelial cell proliferation and angiogenesis.

Epigenetic Regulation of Angiogenesis by JARID1B-Induced Repression of HOXA5

Objective— Altering endothelial biology through epigenetic modifiers is an attractive novel concept, which is, however, just in its beginnings. We therefore set out to identify chromatin modifiers important for endothelial gene expression and contributing to angiogenesis. Approach and Results— To identify chromatin modifying enzymes in endothelial cells, histone demethylases were screened by microarray and polymerase chain reaction. The histone 3 lysine 4 demethylase JARID1B was identified as a highly expressed enzyme at the mRNA and protein levels. Knockdown of JARID1B by shRNA in human umbilical vein endothelial cells attenuated cell migration, angiogenic sprouting, and tube formation. Similarly, pharmacological inhibition and overexpression of a catalytic inactive JARID1B mutant reduced the angiogenic capacity of human umbilical vein endothelial cells. To identify the in vivo relevance of JARID1B in the vascular system, Jarid1b knockout mice were studied. As global knockout results in increased mortality and developmental defects, tamoxifen-inducible and endothelial-specific knockout mice were generated. Acute knockout of Jarid1b attenuated retinal angiogenesis and endothelial sprout outgrowth from aortic segments. To identify the underlying mechanism, a microarray experiment was performed, which led to the identification of the antiangiogenic transcription factor HOXA5 to be suppressed by JARID1B. Importantly, downregulation or inhibition of JARID1B, but not of JARID1A and JARID1C, induced HOXA5 expression in human umbilical vein endothelial cells. Consistently, chromatin immunoprecipitation revealed that JARID1B occupies and reduces the histone 3 lysine 4 methylation levels at the HOXA5 promoter, demonstrating a direct function of JARID1B in endothelial HOXA5 gene regulation. Conclusions— JARID1B, by suppressing HOXA5, maintains the endothelial angiogenic capacity in a demethylase-dependent manner.