Guillaume Carmona

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.

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 beta1 and beta2 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 beta2 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.

Wiscott Aldrich Syndrome protein (WASP) deficiency results in defects in calcium ion signaling, cytoskeletal regulation, gene transcription and overall T cell activation. The activation of WASP constitutes a key pathway for actin filament nucleation. Yet, when WASP function is eliminated there is negligible effect on actin polymerization at the immunological synapse, leading to gaps in our understanding of the events connecting WASP and calcium ion signaling. Here, we identify a fraction of total synaptic F-actin selectively generated by WASP in the form of distinct F-actin 'foci'. These foci are polymerized de novo as a result of the T cell receptor (TCR) proximal tyrosine kinase cascade, and facilitate distal signaling events including PLCγ1 activation and subsequent cytoplasmic calcium ion elevation. We conclude that WASP generates a dynamic F-actin architecture in the context of the immunological synapse, which then amplifies the downstream signals required for an optimal immune response.

Ras-associated protein 1 (Rap1), a small GTPase, attracted attention because of its involvement in several aspects of cell adhesion, including integrin- and cadherin-mediated adhesion. Yet, the role of Rap1 genes and of Rap1 effectors for angiogenesis has not been investigated. Human umbilical vein endothelial cells (HUVECs) express Rap1a and Rap1b mRNA. To determine the contribution of Rap1 activity for angiogenesis, we overexpressed Rap1GAP1, a GTPase-activating protein that inhibits Rap1 activity. Overexpression of Rap1GAP1 significantly blocked angiogenic sprouting and tube-forming activity of HUVECs as well as migration and integrin-dependent adhesion. Silencing of Rap1a, Rap1b, or both significantly blocked HUVECs sprouting under basal and basic fibroblast growth factor-stimulated conditions and reduced HUVEC migration and integrin-dependent adhesion. We found that Rap1a and Rap1b are essential for the conformational activation of beta(1)-integrins in endothelial cells. Furthermore, silencing of Rap1a and Rap1b prevented phosphorylation of tyrosine 397 in focal adhesion kinase (FAK) and vascular endothelial growth factor-induced Akt1-activation. Rap1a(-/-)-deficient and Rap1a(+/-) heterozygote mice displayed reduced neovascularization after hind limb ischemia compared with wild-type mice. Silencing of RAPL significantly blocked the Rap1-induced sprouting of HUVECs, suggesting that the angiogenic activity of Rap1 is partly mediated by RAPL. Our data demonstrate a critical role of Rap1 in the regulation of beta(1)-integrin affinity, adhesion, and migration in endothelial cells and in postnatal neovascularization.