MicroRNAs (miRs) are small noncoding RNAs that regulate gene expression by binding to target messenger RNAs (mRNAs), leading to translational repression or degradation. Here, we show that the miR-17approximately92 cluster is highly expressed in human endothelial cells and that miR-92a, a component of this cluster, controls the growth of new blood vessels (angiogenesis). Forced overexpression of miR-92a in endothelial cells blocked angiogenesis in vitro and in vivo. In mouse models of limb ischemia and myocardial infarction, systemic administration of an antagomir designed to inhibit miR-92a led to enhanced blood vessel growth and functional recovery of damaged tissue. MiR-92a appears to target mRNAs corresponding to several proangiogenic proteins, including the integrin subunit alpha5. Thus, miR-92a may serve as a valuable therapeutic target in the setting of ischemic disease.

Leukocyte recruitment to sites of infection or inflammation requires multiple adhesive events. While numerous players promoting leukocyte-endothelial interactions have been characterized, functionally important endogenous inhibitors of leukocyte adhesion have not been identified. Here, we describe the endothelial-derived secreted molecule, developmental endothelial locus-1 (Del-1), as an anti-adhesive factor that interferes with the integrin LFA-1-dependent leukocyte-endothelial adhesion. Endothelial Del-1-deficiency increased LFA-1-dependent leukocyte adhesion in vitro and in vivo. Del-1-/-mice displayed significantly higher neutrophil accumulation in LPS-induced lung inflammation in vivo, which was reversed in Del-1/LFA-1-double deficient mice. Thus, Del-1 is an endogenous inhibitor of inflammatory cell recruitment and could provide a basis for targeting leukocyte-endothelial interactions in disease.Leukocyte extravasation is integral to the response to infection or injury and to inflammation and autoimmunity. Leukocyte recruitment comprises a well coordinated cascade of adhesive events including selectin-mediated rolling, firm adhesion of leukocytes to endothelial cells and & This manuscript has been accepted for publication in Science. This version has not undergone final editing. Please refer to the complete version of record at http://www.sciencemag.org/. The manuscript may not be reproduced or used in any manner that does not fall within the fair use provisions of the Copyright Act without the prior, written permission of AAAS. †To whom correspondence should be addressed chavakist@mail.nih.gov. * EYC and EC contributed equally # MAC and HL contributed equally NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript their subsequent transendothelial migration. The interaction between LFA-1 (αLβ2, CD11a/ CD18) and endothelial ICAM-1 is crucial during firm endothelial adhesion of leukocytes (1-5). Whereas numerous adhesion receptors promoting inflammatory cell recruitment have been identified, very little information exists about endogenous inhibitors of the leukocyte adhesion cascade (1-7). Developmental endothelial locus-1 (Del-1) is a glycoprotein that is secreted by endothelial cells and can associate with the endothelial cell surface and the extracellular matrix (8-10). Del-1 is regulated upon hypoxia or vascular injury and has been implicated in vascular remodelling during angiogenesis (10-12). Here, we sought to determine whether endothelial-derived Del-1 participates in leukocyte-endothelial interactions. RT-PCR analysis revealed Del-1 mRNA predominantly in the brain and lung, with no expression in liver, spleen, or whole blood (Fig. 1A and fig. S1A). Del-1 was expressed in WT but not in Del-1-/-murine lung endothelial cells (Fig. 1B, 9). Immunohistochemistry of lung tissues demonstrated the presence of Del-1 in vessels, as observed by co-staining with the endothelial marker PECAM-1 ( fig. S1B).To determine whether Del-1 participates in leukocyte recruitmen...

Angiopoietin-2 (ANG-2) is a key regulator of angiogenesis that exerts context-dependent effects on ECs. ANG-2 binds the endothelial-specific receptor tyrosine kinase 2 (TIE2) and acts as a negative regulator of ANG-1/TIE2 signaling during angiogenesis, thereby controlling the responsiveness of ECs to exogenous cytokines. Recent data from tumors indicate that under certain conditions ANG-2 can also promote angiogenesis. However, the molecular mechanisms of dual ANG-2 functions are poorly understood. Here, we identify a model for the opposing roles of ANG-2 in angiogenesis. We found that angiogenesis-activated endothelium harbored a subpopulation of TIE2-negative ECs (TIE2 lo ). TIE2 expression was downregulated in angiogenic ECs, which abundantly expressed several integrins. ANG-2 bound to these integrins in TIE2 lo ECs, subsequently inducing, in a TIE2-independent manner, phosphorylation of the integrin adaptor protein FAK, resulting in RAC1 activation, migration, and sprouting angiogenesis. Correspondingly, in vivo ANG-2 blockade interfered with integrin signaling and inhibited FAK phosphorylation and sprouting angiogenesis of TIE2 lo ECs. These data establish a contextual model whereby differential TIE2 and integrin expression, binding, and activation control the role of ANG-2 in angiogenesis. The results of this study have immediate translational implications for the therapeutic exploitation of angiopoietin signaling.

High‐mobility group box 1 (HMGB1) is released extracellularly upon cell necrosis acting as a mediator in tissue injury and inflammation. However, the molecular mechanisms for the proinflammatory effect of HMGB1 are poorly understood. Here, we define a novel function of HMGB1 in promoting Mac‐1‐dependent neutrophil recruitment. HMGB1 administration induced rapid neutrophil recruitment in vivo. HMGB1‐mediated recruitment was prevented in mice deficient in the β2‐integrin Mac‐1 but not in those deficient in LFA‐1. As observed by bone marrow chimera experiments, Mac‐1‐dependent neutrophil recruitment induced by HMGB1 required the presence of receptor for advanced glycation end products (RAGE) on neutrophils but not on endothelial cells. In vitro, HMGB1 enhanced the interaction between Mac‐1 and RAGE. Consistently, HMGB1 activated Mac‐1 as well as Mac‐1‐mediated adhesive and migratory functions of neutrophils in a RAGE‐dependent manner. Moreover, HMGB1‐induced activation of nuclear factor‐κB in neutrophils required both Mac‐1 and RAGE. Together, a novel HMGB1‐dependent pathway for inflammatory cell recruitment and activation that requires the functional interplay between Mac‐1 and RAGE is described here.

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The mechanisms of homing of endothelial progenitor cells (EPCs) to sites of ischemia are unclear. Here, we demonstrate that ex vivo–expanded EPCs as well as murine hematopoietic Sca-1+/Lin− progenitor cells express β2-integrins, which mediate the adhesion of EPCs to endothelial cell monolayers and their chemokine-induced transendothelial migration in vitro. In a murine model of hind limb ischemia, Sca-1+/Lin− hematopoietic progenitor cells from β2-integrin–deficient mice are less capable of homing to sites of ischemia and of improving neovascularization. Preactivation of the β2-integrins expressed on EPCs by activating antibodies augments the EPC-induced neovascularization in vivo. These results provide evidence for a novel function of β2-integrins in postnatal vasculogenesis.

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Abstract-Endothelial progenitor cells (EPCs) are recruited to ischemic regions and improve neovascularization. Integrins contribute to EPC homing. High-mobility group box 1 (HMGB1) is a nuclear protein that is released extracellularly on cell necrosis and tissue damage, eliciting a proinflammatory response and stimulating tissue repair. In the present study, we investigated the effects of HMGB1 on EPC homing. EPCs express the HMGB1 receptors RAGE (receptor for advanced glycation end products) and TLR2 (Toll-like receptor 2). EPC migration was stimulated by HMGB1 in a RAGE-dependent manner. In addition, the HMGB1-induced migration of EPCs on fibronectin and fibrinogen was significantly inhibited by antibodies against ␤ 1 and ␤ 2 integrins, respectively. Short-term prestimulation of EPCs with HMGB1 also increased EPC adhesion to endothelial cell monolayers, and this effect was blocked by antibodies to ␤ 2 integrins or RAGE. HMGB1 increased EPC adhesion to the immobilized integrin ligands intercellular adhesion molecule-1 and fibronectin in a RAGE-dependent manner. Strikingly, HMGB1 rapidly increased integrin affinity and induced integrin polarization. Using intravital microscopy in a tumor model of neovascularization, prestimulation of EPCs with HMGB1 enhanced the initial in vivo adhesion of EPCs to microvessels and the recruitment of EPCs in the tumor tissue. In addition, prestimulation of EPCs with HMGB1 increased the homing of EPCs to ischemic muscles. In conclusion, these data represent a link between HMGB1 and integrin functions of EPCs and demonstrate that HMGB1 stimulates EPC homing to ischemic tissues. These results may provide a platform for the development of novel therapeutic approaches to improve EPC homing. Key Words: high-mobility group box 1 Ⅲ endothelial progenitor cells Ⅲ homing Ⅲ integrins Ⅲ migration T he term vasculogenesis describes the de novo formation of new vessels from angioblasts during embryonic development. 1 Vasculogenesis, which can be mediated by circulating bone marrow (BM)-derived endothelial progenitor or hematopoietic stem cells, is important in postnatal neovascularization of adult ischemic tissues. [2][3][4][5][6] Ischemia or cytokines such as vascular endothelial growth factor (VEGF) lead to mobilization of endothelial progenitor cells (EPCs) from the BM 7 and support the neovascularization of ischemic tissues or tumors. [7][8][9] Therapeutic administration of EPCs increases the neovascularization of ischemic myocardium and limbs and improves left ventricular function after myocardial infarction. 10 -13 EPCs are preferentially recruited to sites of ischemia and improve neovascularization by being directly incorporated into vascular structures and differentiating to endothelial cells and/or by eliciting paracrine effects. 2,4,6,7,10,14 Both the paracrine effects and the differentiation of EPCs to endothelial cells depend on the homing of EPCs to ischemic sites. In an in vivo intravital microscopy study, EPCs embryonic arrested within tumor microvessels, extravasated into the...