Mihail Hristov

Delivery of MicroRNA-126 by Apoptotic Bodies Induces CXCL12-Dependent Vascular Protection

Apoptotic endothelial cells release microRNA-containing microvesicles to modulate the responses of neighboring cells and reduce atherosclerosis in mice. Sounding the Alarm In addition to its importance in development and homeostasis, apoptotic cell death is implicated in a number of diseases, including atherosclerosis. Apoptotic endothelial cells at atherosclerotic plaques release microvesicles known as apoptotic bodies into the circulation, and their abundance correlates with negative indicators of disease. Zernecke et al. showed that apoptotic bodies from endothelial cells contained microRNA-126 (miR-126). Neighboring vascular cells took up the microvesicles, which allowed miR-126 to reduce the abundance of an inhibitor of the signaling of the chemokine receptor CXCR4, resulting in the increased production of CXCL12, the ligand for CXCR4. CXCL12 mediated the recruitment to atherosclerotic plaques of progenitor cells from the bone marrow, which limited plaque size. Apoptotic bodies isolated from human patients with atherosclerosis reduced the size of plaques in different mouse models of atherosclerosis. Thus, dying endothelial cells send alarm signals in the form of packaged microRNA to neighboring cells to trigger a healing response that reduces atherosclerosis. Apoptosis is a pivotal process in embryogenesis and postnatal cell homeostasis and involves the shedding of membranous microvesicles termed apoptotic bodies. In response to tissue damage, the CXC chemokine CXCL12 and its receptor CXCR4 counteract apoptosis and recruit progenitor cells. Here, we show that endothelial cell–derived apoptotic bodies are generated during atherosclerosis and convey paracrine alarm signals to recipient vascular cells that trigger the production of CXCL12. CXCL12 production was mediated by microRNA-126 (miR-126), which was enriched in apoptotic bodies and repressed the function of regulator of G protein (heterotrimeric guanosine triphosphate–binding protein) signaling 16, an inhibitor of G protein–coupled receptor (GPCR) signaling. This enabled CXCR4, a GPCR, to trigger an autoregulatory feedback loop that increased the production of CXCL12. Administration of apoptotic bodies or miR-126 limited atherosclerosis, promoted the incorporation of Sca-1+ progenitor cells, and conferred features of plaque stability on different mouse models of atherosclerosis. This study highlights functions of microRNAs in health and disease that may extend to the recruitment of progenitor cells during other forms of tissue repair or homeostasis.

Endothelial progenitor cells: mobilization, differentiation, and homing.

Postnatal bone marrow contains a subtype of progenitor cells that have the capacity to migrate to the peripheral circulation and to differentiate into mature endothelial cells. Therefore, these cells have been termed endothelial progenitor cells (EPCs). The isolation of EPCs by adherence culture or magnetic microbeads has been described. In general, EPCs are characterized by the expression of 3 markers, CD133, CD34, and the vascular endothelial growth factor receptor-2. During differentiation, EPCs obviously lose CD133 and start to express CD31, vascular endothelial cadherin, and von Willebrand factor. EPCs seem to participate in endothelial repair and neovascularization of ischemic organs. Clinical studies using EPCs for neovascularization have just been started; however, the mechanisms stimulating or inhibiting the differentiation of EPC in vivo and the signals causing their migration and homing to sites of injured endothelium or extravascular tissue are largely unknown at present. Thus, future studies will help to explore areas of potential basic research and clinical application of EPCs.

Endothelial progenitor cells: characterization, pathophysiology, and possible clinical relevance

Bone marrow and peripheral blood of adults contain a special sub‐type of progenitor cells which are able to differentiate into mature endothelial cells, thus contributing to re‐endothelialization and neo‐vascularization. These angiogenic cells have properties of embryonal angioblasts and were termed endothelial progenitor cells (EPCs). In general, three surface markers (CD133, CD34 and the vascular endothelial growth factor receptor‐2) characterize the early functional angioblast, located predominantly in the bone marrow. Later, when migrating to the systemic circulation EPCs gradually lose their progenitor properties and start to express endothelial marker like VE‐cadherin, endothelial nitric oxide synthase and von Willebrand factor. The number of circulating EPCs in healthy subjects is rather low and a variety of conditions or factors may further influence this number. In the context of possible therapeutic application of EPCs recent clinical studies employing these cells for neo‐vascularization of ischemic organs have just been published. However, the specificity of the observed positive clinical effects, the mechanisms regulating the differentiation of EPCs and their homing to sites of injured tissue remain partially unknown at present.

Endothelial Progenitor Cells Isolation and Characterization

Bone marrow of adults contains a subtype of progenitor cells that have the capacity to differentiate into mature endothelial cells and have therefore been termed endothelial progenitor cells (EPCs). Of the three cell markers (CD133, CD34, and the vascular endothelial growth factor receptor 2) that characterize the early functional EPCs, located predominantly in the bone marrow, EPCs obviously lose CD133/CD34 and start to express CD31, vascular endothelial cadherin, and von Willebrand factor when migrating to the circulation. Various isolation procedures of EPCs from different sources by using adherence culture or magnetic microbeads have been described, but published findings with regard to the number of EPCs in the peripheral circulation of healthy adults are scanty and no data regarding the lifetime of EPCs in vivo exist. Clinical studies employing EPCs for neovascularization of ischemic organs have just been started; however, the mechanisms stimulating or inhibiting the differentiation of bone marrow-derived EPCs in vivo and the signals causing their adhesion, migration, and homing to sites of injured tissue are largely unknown at present.

Importance of CXC Chemokine Receptor 2 in the Homing of Human Peripheral Blood Endothelial Progenitor Cells to Sites of Arterial Injury

Circulating endothelial progenitor cells (EPCs) may contribute to endothelial regeneration; however, the exact mechanisms of their arterial homing remain elusive. We examined the role of the angiogenic chemokine receptor CXCR2 in the homing of human EPCs. Isolated EPCs expressed CXCR2 together with kinase insert domain–containing receptor, CD31, vascular endothelial cadherin, and CXCR4. Adhesion assays under flow conditions showed that EPCs preferentially adhered to &bgr;2-integrin ligands, that firm arrest on fibronectin or fibrinogen was enhanced by the CXCR2 ligands CXCL1 or CXCL7, and that blockade of CXCR2 significantly reduced EPC adhesion on platelet-coated endothelial matrix. This was corroborated by the involvement of CXCR2 in EPC recruitment to denuded areas of murine carotid arteries ex vivo and in vivo. Notably, blocking CXCR2 inhibited the incorporation of human EPCs expressing CXCR2 at sites of arterial injury in athymic nude mice. Immunoreactivity for the &bgr;-thromboglobulin isoform CXCL7 was observed in murine platelets and denuded smooth muscle cells (SMCs) early after wire injury, and transcripts for CXCL7 and CXCL1 were detected in isolated human arterial SMCs. Human KDR+CXCR2+ cells showed better in situ adhesion to injured murine carotid arteries than KDR+CXCR2− cells, were predominantly CD14+, and improved CXCR2-dependent endothelial recovery after injury in nude mice. In conclusion, our data clearly demonstrate the importance of CXCR2 for the homing of circulating EPCs to sites of arterial injury and for endothelial recovery in vivo.

CCL17-expressing dendritic cells drive atherosclerosis by restraining regulatory T cell homeostasis in mice

Immune mechanisms are known to control the pathogenesis of atherosclerosis. However, the exact role of DCs, which are essential for priming of immune responses, remains elusive. We have shown here that the DC-derived chemokine CCL17 is present in advanced human and mouse atherosclerosis and that CCL17+ DCs accumulate in atherosclerotic lesions. In atherosclerosis-prone mice, Ccl17 deficiency entailed a reduction of atherosclerosis, which was dependent on Tregs. Expression of CCL17 by DCs limited the expansion of Tregs by restricting their maintenance and precipitated atherosclerosis in a mechanism conferred by T cells. Conversely, a blocking antibody specific for CCL17 expanded Tregs and reduced atheroprogression. Our data identify DC-derived CCL17 as a central regulator of Treg homeostasis, implicate DCs and their effector functions in atherogenesis, and suggest that CCL17 might be a target for vascular therapy.

Platelet Microparticles Enhance the Vasoregenerative Potential of Angiogenic Early Outgrowth Cells After Vascular Injury

Background— Angiogenic early outgrowth cells (EOCs) have been reported to contribute to endothelial regeneration and to limit neointima formation after vascular injury. Vascular pathologies comprise platelet activation and concomitant generation of platelet microparticles (PMPs). We hypothesized that PMPs may interact with EOCs in the context of vascular injury and modulate their regenerative potential. Methods and Results— Using flow cytometry, confocal microscopy, and scanning electron microscopy, we demonstrated the binding of thrombin/collagen-induced PMPs to EOCs with subsequent membrane assimilation and incorporation. This interaction promoted phenotypic alterations of EOCs with increased expression of endothelial cell markers and transfer of the chemokine receptor CXCR4 to EOCs with enhanced responsiveness to its ligand CXCL12/SDF-1&agr;. In addition, PMPs augmented the adhesion of EOCs to extracellular matrix components and to the injured vessel wall and accelerated cytoskeletal reorganization and migration of EOCs. PMPs induced changes in the EOC secretome toward a more proangiogenic profile and amplified the EOC-mediated induction of proliferation, migration, and capillary tube formation by mature endothelial cells. Compared with untreated EOCs, the injection of PMP-treated EOCs resulted in accelerated reendothelialization after arterial denudation injury in athymic nude mice, whereas the EOC-mediated reduction of neointima formation remained unchanged. Conclusions— Our data provide evidence that PMPs can boost the potential of EOCs to restore endothelial integrity after vascular injury. Major mechanisms involve the enhancement of EOC recruitment, migration, differentiation, and release of proangiogenic factors.

Distinct functions of chemokine receptor axes in the atherogenic mobilization and recruitment of classical monocytes

We used a novel approach of cytostatically induced leucocyte depletion and subsequent reconstitution with leucocytes deprived of classical (inflammatory/Gr1hi) or non‐classical (resident/Gr1lo) monocytes to dissect their differential role in atheroprogression under high‐fat diet (HFD). Apolipoprotein E‐deficient (Apoe−/−) mice lacking classical but not non‐classical monocytes displayed reduced lesion size and macrophage and apoptotic cell content. Conversely, HFD induced a selective expansion of classical monocytes in blood and bone marrow. Increased CXCL1 levels accompanied by higher expression of its receptor CXCR2 on classical monocytes and inhibition of monocytosis by CXCL1‐neutralization indicated a preferential role for the CXCL1/CXCR2 axis in mobilizing classical monocytes during hypercholesterolemia. Studies correlating circulating and lesional classical monocytes in gene‐deficient Apoe−/− mice, adoptive transfer of gene‐deficient cells and pharmacological modulation during intravital microscopy of the carotid artery revealed a crucial function of CCR1 and CCR5 but not CCR2 or CX3CR1 in classical monocyte recruitment to atherosclerotic vessels. Collectively, these data establish the impact of classical monocytes on atheroprogression, identify a sequential role of CXCL1 in their mobilization and CCR1/CCR5 in their recruitment.

Intracoronary infusion of autologous bone marrow cells and left ventricular function after acute myocardial infarction: a meta-analysis

Recent clinical studies have demonstrated that intracoronary infusion of autologous bone marrow cells (BMC) in conjunction with standard treatment may improve left ventricular function after an acute myocardial infarction (AMI). However, the results of these studies remain controversial, as the studies were relatively small in size and partially differed in design. We reviewed primary controlled randomized clinical studies comparing intracoronary transfer of autologous non‐mobilized BMC combined with standard therapy versus standard therapy alone in patients with AMI. We identified five randomized controlled clinical trials, three of which were also placebo‐ and bone marrow aspiration‐controlled. Non‐mobilized BMC were infused into the revascularized coronary target artery 6.6 ± 6.1 days after AMI. The mean follow‐up period of 5.2 ± 1.1 months was completed by 482 patients, 241 of which received infusion of BMC. The effect of BMC on left ventricular ejection fraction (LVEF) as a major functional parameter was evaluated. Analyzing the overall effect on the change in LVEF between baseline and follow‐up value revealed a significant improvement in the BMCtreated group as compared to the control group (P = 0.04). Thus, considering the increase in LVEF during follow‐up, transplantation of BMC may be a safe and beneficial procedure to support treatment of AMI. However, the functional improvement observed with this form of therapy was altogether relatively moderate and the studies were heterogeneous in design. Hence, further efforts aiming at large‐scale, double‐blind, randomized and placebo‐controlled multi‐center trials in conjunction with better definition of patients, which benefit from BMC infusion, appear to be warranted.

Hypoxia-induced endothelial secretion of macrophage migration inhibitory factor and role in endothelial progenitor cell recruitment

Macrophage migration inhibitory factor (MIF) is a pleiotropic inflammatory cytokine that was recently identified as a non‐cognate ligand of the CXC‐family chemokine receptors 2 and 4 (CXCR2 and CXCR4). MIF is expressed and secreted from endothelial cells (ECs) following atherogenic stimulation, exhibits chemokine‐like properties and promotes the recruitment of leucocytes to atherogenic endothelium. CXCR4 expressed on endothelial progenitor cells (EPCs) and EC‐derived CXCL12, the cognate ligand of CXCR4, have been demonstrated to be critical when EPCs are recruited to ischemic tissues. Here we studied whether hypoxic stimulation triggers MIF secretion from ECs and whether the MIF/CXCR4 axis contributes to EPC recruitment. Exposure of human umbilical vein endothelial cells (HUVECs) and human aortic endothelial cells (HAoECs) to 1% hypoxia led to the specific release of substantial amounts of MIF. Hypoxia‐induced MIF release followed a biphasic behaviour. MIF secretion in the first phase peaked at 60 min. and was inhibited by glyburide, indicating that this MIF pool was secreted by a non‐classical mechanism and originated from pre‐formed MIF stores. Early hypoxia‐triggered MIF secretion was not inhibited by cycloheximide and echinomycin, inhibitors of general and hypoxia‐inducible factor (HIF)‐1α‐induced protein synthesis, respectively. A second phase of MIF secretion peaked around 8 hrs and was likely due to HIF‐1α‐induced de novo synthesis of MIF. To functionally investigate the role of hypoxia‐inducible secreted MIF on the recruitment of EPCs, we subjected human AcLDL+ KDR+ CD31+ EPCs to a chemotactic MIF gradient. MIF potently promoted EPC chemotaxis in a dose‐dependent bell‐shaped manner (peak: 10 ng/ml MIF). Importantly, EPC migration was induced by supernatants of hypoxia‐conditioned HUVECs, an effect that was completely abrogated by anti‐MIF‐ or anti‐CXCR4‐antibodies. Thus, hypoxia‐induced MIF secretion from ECs might play an important role in the recruitment and migration of EPCs to hypoxic tissues such as after ischemia‐induced myocardial damage.