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.
Ageing is the predominant risk factor for cardiovascular diseases and contributes to a significantly worse outcome in patients with acute myocardial infarction. MicroRNAs (miRNAs) have emerged as crucial regulators of cardiovascular function and some miRNAs have key roles in ageing. We propose that altered expression of miRNAs in the heart during ageing contributes to the age-dependent decline in cardiac function. Here we show that miR-34a is induced in the ageing heart and that in vivo silencing or genetic deletion of miR-34a reduces age-associated cardiomyocyte cell death. Moreover, miR-34a inhibition reduces cell death and fibrosis following acute myocardial infarction and improves recovery of myocardial function. Mechanistically, we identified PNUTS (also known as PPP1R10) as a novel direct miR-34a target, which reduces telomere shortening, DNA damage responses and cardiomyocyte apoptosis, and improves functional recovery after acute myocardial infarction. Together, these results identify age-induced expression of miR-34a and inhibition of its target PNUTS as a key mechanism that regulates cardiac contractile function during ageing and after acute myocardial infarction, by inducing DNA damage responses and telomere attrition.
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.DOI: http://dx.doi.org/10.7554/eLife.04953.001
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...
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 tubeforming 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  1 -integrins in endothelial cells. IntroductionThe term angiogenesis refers to the formation of new blood capillaries from preexisting ones. 1,2 Angiogenesis is implicated in many physiologic and pathologic conditions, including embryonic development, wound healing, tumor growth, rheumatoid arthritis, and proliferative retinopathy. 1,2 Vascular endothelial growth factor (VEGF) is an essential cytokine for vasculogenesis and angiogenesis. 3 Through its receptors, which include 2 distinct tyrosine kinases, VEGF exerts multiple effects on endothelial cells, including proliferation, rapid induction of endothelial permeability, promotion of endothelial cell survival, stimulation of migration, and induction of gene expression. 3 Besides VEGF, the fibroblast growth factors (FGFs) are also implicated in angiogenesis. 4 However, the downstream signaling pathways mediating the angiogenic effects of angiogenic growth factors, such as VEGF and FGFs, are poorly understood.Integrins are heterodimeric transmembrane proteins consisting of noncovalent bound ␣-and -subunits mediating cell adhesion to extracellular matrix proteins and bidirectional signaling. 5 Beyond angiogenic growth factors, there is evidence that integrins are implicated in angiogenesis. 6 Specifically,  1 -integrins were shown to play an important role in angiogenesis. 6 Moreover, angiogenic factors, such as VEGF, affect integrin activity and function in endothelial cells. 7 Ras-associated proteins (Rap) define a family of highly homologous small guanosine triphosphate (GTP)-binding proteins belonging to the Ras superfamily, which includes 5 members, Rap1a, Rap1b, Rap2a, Rap2b, and the recently discovered Rap2c, which are grouped into 2 subfamilies, Rap1 and Rap2, based on their sequence homology. [8][9][10][11][12] Small GTPases cycle between an inactive guanosine diphosphate (GDP)-bound conformation and an active GTP-bound conformation. In their active conformation, small GTPases interact with effector proteins, which induce downstream signaling. The GDP/GTP cycle is highly regulated by guanine nucleotide exchange factors...
Cancer invasion is a hallmark of metastasis. The mesenchymal mode of cancer cell invasion is mediated by elongated membrane protrusions driven by the assembly of branched F-actin networks. How deregulation of actin regulators promotes cancer cell invasion is still enigmatic. We report that increased expression and membrane localization of the actin regulator Lamellipodin correlates with reduced metastasis-free survival and poor prognosis in breast cancer patients. In agreement we find that Lamellipodin depletion reduced lung metastasis in an orthotopic mouse breast cancer model. Invasive 3D cancer cell migration as well as invadopodia formation, and matrix degradation were impaired upon Lamellipodin depletion. Mechanistically, we show that Lamellipodin promotes invasive 3D cancer cell migration via both actin-elongating Ena/VASP proteins and the Scar/WAVE complex, which stimulates actin branching. In contrast, Lamellipodin interaction with Scar/WAVE but not Ena/VASP is required for random 2D cell migration. We identify a phosphorylation-dependent mechanism that regulates selective recruitment of these effectors to Lamellipodin: Abl-mediated Lamellipodin phosphorylation promotes its association with both Scar/WAVE and Ena/VASP, while Src-dependent phosphorylation enhances binding to Scar/WAVE but not Ena/VASP. Through these selective, regulated interactions Lamellipodin mediates directional sensing of EGF gradients and invasive 3D migration of breast cancer cells. Our findings imply that increased Lamellipodin levels enhance Ena/VASP and Scar/WAVE activities at the plasma membrane to promote 3D invasion and metastasis.
Abstract-Circulating blood-derived vasculogenic cells improve neovascularization of ischemic tissue by a broad repertoire of potential therapeutic actions. Whereas initial studies documented that the cells incorporate and differentiate to cardiovascular cells, other studies suggested that short-time paracrine mechanisms mediate the beneficial effects. The question remains to what extent a physical incorporation is contributing to the beneficial effects of cell therapy. By using the inducible suicide gene thymidine kinase to deplete transplanted cells, we determined the contribution of physical incorporation in 3 animal models. After acute myocardial infarction, depletion of cells 14 days after infusion resulted in a reduction of capillary density and a substantial deterioration of heart function. Likewise, neovascularization of Matrigel plugs and ischemic limbs was significantly suppressed when infused cells were depleted 7 days after infusion. Key Words: progenitor cells Ⅲ neovascularization Ⅲ cell therapy C ell therapy is a promising option for treating ischemic diseases. Adult stem and progenitor cells from various sources have experimentally been shown to augment the functional recovery after ischemia. Clinical trials confirmed that autologous cell therapy using bone marrow-derived or circulating blood-derived progenitor cells is safe and provides beneficial effects 1-3 (see also recent metaanalysis 4 ). Endothelial progenitor cells (EPCs) were initially isolated from bone marrow or peripheral blood and were characterized by the expression of the hematopoietic stem cell marker CD34 or CD133 and endothelial markers such as vascular endothelial growth factor receptor 2 (KDR). 5,6 Isolated CD34 ϩ cells differentiate to endothelial cells in vitro and incorporate into newly formed vessels during angiogenesis in vivo. 5 These pioneering studies suggested that bone marrow-derived cells can participate in vascular repair. Indeed, a single hematopoietic stem cell was shown to give rise to both blood cells and vascular endothelium. [7][8][9] The contribution of circulating cells to the endothelium was further supported by sexmismatched heart transplantation in humans. 10,11 Other investigators used culture assays to enrich for EPC-yielding cells, which coexpress endothelial and myeloid marker ("early EPCs," proangiogenic cells), or to show a more mature endothelial cell phenotype on further culture and expansion ("late or outgrowing EPCs"). 12,13 Although the origin and the phenotype of the cultured EPCs is not entirely clear and may vary depending on the defined specific culture conditions, 14 the therapeutic potential and improved neovascularization mediated by the cultured cells has been documented extensively in ischemic models. 12,15,16 Progenitor cell-mediated improved neovascularization of ischemic tissue might be attributable to various therapeutic actions including a physical incorporation of the infused cells in the endothelium leading to the formation of new capillaries. 16 -18 Transplanted or infused hema...
Cell therapy is a novel promising option for treatment of ischemic diseases. Administered endothelial progenitor cells (EPCs) are recruited to ischemic regions and improve neovascularization. However, the number of cells that home to ischemic tissues is restricted. The GTPase Rap1 plays an important role in the regulation of adhesion and chemotaxis. We investigated whether pharmacologic activation of Epac1, a nucleotide exchange protein for Rap1, which is directly activated by cAMP, can improve the adhesive and migratory capacity of distinct progenitor cell populations. Stimulation of Epac by a cAMPanalog increased Rap1 activity and stimulated the adhesion of human EPCs, CD34 ؉ hematopoietic progenitor cells, and mesenchymal stem cells (MSCs). Specifically, short-term stimulation with a specific Epac activator increased the 2-integrin-dependent adhesion of EPCs to endothelial cell monolayers, and of EPC and CD34 ؉ cells to ICAM-1. Furthermore, the Epac activator enhanced the 1-integrin-dependent adhesion of EPCs and MSCs to the matrix protein fibronectin. In addition, Epac1 activation induced the 1-and 2-integrin-dependent migration of EPCs on fibronectin and fibrinogen. Interestingly, activation of Epac rapidly increased lateral mobility of 1-and 2-integrins, thereby inducing integrin polarization, and stimulated 1-integrin affinity, whereas the 2-integrin affinity was not increased. Furthermore, prestimulation of EPCs with the Epac activator increased homing to ischemic muscles and neovascularization-promoting capacity of intravenously injected EPCs in the model of hind limb ischemia. These data demonstrate that activation of Epac1 increases integrin activity and integrin-dependent homing functions of progenitor cells and enhances their in vivo therapeutic potential. These results may provide a platform for the development of novel therapeutic approaches to improve progenitor cell homing. IntroductionThe term vasculogenesis describes the de novo formation of new vessels from angioblasts during embryonic development. 1 Vasculogenesis, which can be mediated by circulating bone marrowderived endothelial progenitor or hematopoietic stem cells, also contributes to postnatal neovascularization of adult ischemic tissues. [2][3][4][5][6] Therapeutical administration of endothelial progenitor cells (EPCs) increases neovascularization and improves left ventricular function after myocardial infarction in animal models. [7][8][9] Moreover, first clinical studies demonstrated a beneficial effect of intracoronary administration of EPCs or bone marrow cells on the left ventricular function in patients after myocardial infarction. [10][11][12] Progenitor cells 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. 2,4,6,7,13,14 Both the paracrine effects and the differentiation of progenitor cells to endothelial cells depend on the homing of the progenitor c...
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