A Neutrophil Timer Coordinates Immune Defense and Vascular Protection.

Neutrophils eliminate pathogens efficiently but can inflict severe damage to the host if they over-activate within blood vessels. It is unclear how immunity solves the dilemma of mounting an efficient anti-microbial defense while preserving vascular health. Here, we identify a neutrophil-intrinsic program that enabled both. The gene Bmal1 regulated expression of the chemokine CXCL2 to induce chemokine receptor CXCR2-dependent diurnal changes in the transcriptional and migratory properties of circulating neutrophils. These diurnal alterations, referred to as neutrophil aging, were antagonized by CXCR4 (C-X-C chemokine receptor type 4) and regulated the outer topology of neutrophils to favor homeostatic egress from blood vessels at night, resulting in boosted anti-microbial activity in tissues. Mice engineered for constitutive neutrophil aging became resistant to infection, but the persistence of intravascular aged neutrophils predisposed them to thrombo-inflammation and death. Thus, diurnal compartmentalization of neutrophils, driven by an internal timer, coordinates immune defense and vascular protection.

Chemokines and galectins form heterodimers to modulate inflammation.

Chemokines and galectins are simultaneously upregulated and mediate leukocyte recruitment during inflammation. Until now, these effector molecules have been considered to function independently. Here, we tested the hypothesis that they form molecular hybrids. By systematically screening chemokines for their ability to bind galectin-1 and galectin-3, we identified several interacting pairs, such as CXCL12 and galectin-3. Based on NMR and MD studies of the CXCL12/galectin-3 heterodimer, we identified contact sites between CXCL12 ß-strand 1 and Gal-3 F-face residues. Mutagenesis of galectin-3 residues involved in heterodimer formation resulted in reduced binding to CXCL12, enabling testing of functional activity comparatively. Galectin-3, but not its mutants, inhibited CXCL12-induced chemotaxis of leukocytes and their recruitment into the mouse peritoneum. Moreover, galectin-3 attenuated CXCL12-stimulated signaling via its receptor CXCR4 in a ternary complex with the chemokine and receptor, consistent with our structural model. This first report of heterodimerization between chemokines and galectins reveals a new type of interaction between inflammatory mediators that can underlie a novel immunoregulatory mechanism in inflammation. Thus, further exploration of the chemokine/galectin interactome is warranted.

Vascular CXCR4 Limits Atherosclerosis by Maintaining Arterial Integrity: Evidence From Mouse and Human Studies.

BACKGROUND: The CXCL12/CXCR4 chemokine ligand/receptor axis controls (progenitor) cell homeostasis and trafficking. So far, an atheroprotective role of CXCL12/CXCR4 has only been implied through pharmacological intervention, in particular, because the somatic deletion of the CXCR4 gene in mice is embryonically lethal. Moreover, cell-specific effects of CXCR4 in the arterial wall and underlying mechanisms remain elusive, prompting us to investigate the relevance of CXCR4 in vascular cell types for atheroprotection. METHODS: We examined the role of vascular CXCR4 in atherosclerosis and plaque composition by inducing an endothelial cell (BmxCreERT2-driven)-specific or smooth muscle cell (SMC, SmmhcCreERT2- or TaglnCre-driven)-specific deficiency of CXCR4 in an apolipoprotein E-deficient mouse model. To identify underlying mechanisms for effects of CXCR4, we studied endothelial permeability, intravital leukocyte adhesion, involvement of the Akt/WNT/ß-catenin signaling pathway and relevant phosphatases in VE-cadherin expression and function, vascular tone in aortic rings, cholesterol efflux from macrophages, and expression of SMC phenotypic markers. Finally, we analyzed associations of common genetic variants at the CXCR4 locus with the risk for coronary heart disease, along with CXCR4 transcript expression in human atherosclerotic plaques. RESULTS: The cell-specific deletion of CXCR4 in arterial endothelial cells (n=12-15) or SMCs (n=13-24) markedly increased atherosclerotic lesion formation in hyperlipidemic mice. Endothelial barrier function was promoted by CXCL12/CXCR4, which triggered Akt/WNT/ß-catenin signaling to drive VE-cadherin expression and stabilized junctional VE-cadherin complexes through associated phosphatases. Conversely, endothelial CXCR4 deficiency caused arterial leakage and inflammatory leukocyte recruitment during atherogenesis. In arterial SMCs, CXCR4 sustained normal vascular reactivity and contractile responses, whereas CXCR4 deficiency favored a synthetic phenotype, the occurrence of macrophage-like SMCs in the lesions, and impaired cholesterol efflux. Regression analyses in humans (n=259 796) identified the C-allele at rs2322864 within the CXCR4 locus to be associated with increased risk for coronary heart disease. In line, C/C risk genotype carriers showed reduced CXCR4 expression in carotid artery plaques (n=188), which was furthermore associated with symptomatic disease. CONCLUSIONS: Our data clearly establish that vascular CXCR4 limits atherosclerosis by maintaining arterial integrity, preserving endothelial barrier function, and a normal contractile SMC phenotype. Enhancing these beneficial functions of arterial CXCR4 by selective modulators might open novel therapeutic options in atherosclerosis.

Hyperreactivity of junctional adhesion molecule A-deficient platelets accelerates atherosclerosis in hyperlipidemic mice.

RATIONALE: Besides their essential role in hemostasis, platelets also have functions in inflammation. In platelets, junctional adhesion molecule (JAM)-A was previously identified as an inhibitor of integrin αIIbß3-mediated outside-in signaling and its genetic knockdown resulted in hyperreactivity. OBJECTIVE: This gain-of-function was specifically exploited to investigate the role of platelet hyperreactivity in plaque development. METHODS AND RESULTS: JAM-A-deficient platelets showed increased aggregation and cellular and sarcoma tyrosine-protein kinase activation. On αIIbß3 ligation, JAM-A was shown to be dephosphorylated, which could be prevented by protein tyrosine phosphatase nonreceptor type 1 inhibition. Mice with or without platelet-specific (tr)JAM-A-deficiency in an apolipoprotein e (apoe(-/-)) background were fed a high-fat diet. After ≤12 weeks of diet, trJAM-A(-/-)apoe-/- mice showed increased aortic plaque formation when compared with trJAM-A(+/+) apoe(-/-) controls, and these differences were most evident at early time points. At 2 weeks, the plaques of the trJAM-A(-/-) apoe(-/-) animals revealed increased macrophage, T cell, and smooth muscle cell content. Interestingly, plasma levels of chemokines CC chemokine ligand 5 and CXC-chemokine ligand 4 were increased in the trJAM-A(-/-) apoe(-/-)mice, and JAM-A-deficient platelets showed increased binding to monocytes and neutrophils. Whole-blood perfusion experiments and intravital microscopy revealed increased recruitment of platelets and monocytes to the inflamed endothelium in blood of trJAM-A(-/-) apoe(-/-)mice. Notably, these proinflammatory effects of JAM-A-deficient platelets could be abolished by the inhibition of αIIbß3 signaling in vitro. CONCLUSIONS: Deletion of JAM-A causes a gain-of-function in platelets, with lower activation thresholds and increased inflammatory activities. This leads to an increase of plaque formation, particularly in early stages of the disease.

Endothelial junctional adhesion molecule-a guides monocytes into flow-dependent predilection sites of atherosclerosis.

BACKGROUND: Junctional adhesion molecule (JAM)-A expressed in endothelial, epithelial, and blood cells can regulate permeability and leukocyte extravasation. Atherosclerosis develops at sites of disturbed flow in large arteries, but the mechanisms guiding inflammatory cells into these predilection sites remain unknown. METHODS AND RESULTS: To characterize cell-specific functions of JAM-A in atherosclerosis, we used apolipoprotein E-deficient mice with a somatic or endothelium-specific deficiency in JAM-A and bone marrow chimeras with JAM-A-deficient leukocytes. We show that impaired JAM-A expression in endothelial cells reduced mononuclear cell recruitment into the arterial wall and limited atherosclerotic lesion formation in hyperlipidemic mice. In contrast, JAM-A deficiency in bone marrow cells impeded monocyte de-adhesion, thereby increasing vascular permeability and lesion formation, whereas somatic JAM-A deletion revealed no significant effects. Regions with disturbed flow displayed a focal enrichment and luminal redistribution of endothelial JAM-A and were preferentially protected by its deficiency. The functional expression and redistribution of endothelial JAM-A was increased by oxidized low-density lipoprotein, but confined by atheroprotective laminar flow through an upregulation of microRNA (miR)-145, which repressed JAM-A. CONCLUSIONS: Our data identify endothelial JAM-A as an important effector molecule integrating atherogenic conditions to direct inflammatory cell entry at predilection sites of atherosclerosis.

MicroRNA-26b Attenuates Platelet Adhesion and Aggregation in Mice.

Platelets are key regulators of haemostasis, making platelet dysfunction a major driver of thrombosis. Numerous processes that determine platelet function are influenced by microRNAs (miRs). MiR-26b is one of the highest-expressed miRs in healthy platelets, and its expression in platelets is changed in a diseased state. However, the exact effect of this miR on platelet function has not been studied yet. In this study, we made use of a whole-body knockout of miR-26b in ApoE-deficient mice in order to determine its impact on platelet function, thrombus formation and platelet signalling both ex vivo and in vivo. We show that a whole-body deficiency of miR-26b exacerbated platelet adhesion and aggregation ex vivo. Additionally, in vivo, platelets adhered faster, and larger thrombi were formed in mice lacking miR-26b. Moreover, isolated platelets from miR-26b-deficient mice showed a hyperactivated Src and EGFR signalling. Taken together, we show here for the first time that miR-26b attenuates platelet adhesion and aggregation, possibly through Src and EGFR signalling.

C5a receptor targeting in neointima formation after arterial injury in atherosclerosis-prone mice.

BACKGROUND: Receptor binding of complement C5a leads to proinflammatory activation of many cell types, but the role of receptor-mediated action during arterial remodeling after injury has not been studied. In the present study, we examined the contribution of the C5a receptor (C5aR) to neointima formation in apolipoprotein E-deficient mice employing a C5aR antagonist (C5aRA) and a C5aR-blocking monoclonal antibody. METHODS AND RESULTS: Mice fed an atherogenic diet were subjected to wire-induced endothelial denudation of the carotid artery and treated with C5aRA and anti-C5aR-blocking monoclonal antibody or vehicle control. Compared with controls, neointima formation was significantly reduced in mice receiving C5aRA or anti-C5aR-blocking monoclonal antibody for 1 week but not for 3 weeks, attributable to an increased content of vascular smooth muscle cells, whereas a marked decrease in monocyte and neutrophil content was associated with reduced vascular cell adhesion molecule-1. As assessed by immunohistochemistry, reverse transcription polymerase chain reaction, and flow cytometry, C5aR was expressed in lesional and cultured vascular smooth muscle cells, upregulated by injury or tumor necrosis factor-alpha, and reduced by C5aRA. Plasma levels and neointimal plasminogen activator inhibitor-1 peaked 1 week after injury and were downregulated in C5aRA-treated mice. In vitro, C5a induced plasminogen activator inhibitor-1 expression in endothelial cells and vascular smooth muscle cells in a C5aRA-dependent manner, possibly accounting for higher vascular smooth muscle cell immigration. CONCLUSIONS: One-week treatment with C5aRA or anti-C5aR-blocking monoclonal antibody limited neointimal hyperplasia and inflammatory cell content and was associated with reduced vascular cell adhesion molecule-1 expression. However, treatment for 3 weeks failed to reduce but rather stabilized plaques, likely by reducing vascular plasminogen activator inhibitor-1 and increasing vascular smooth muscle cell migration.

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-1alpha. 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.

Transcriptome signature of miRNA-26b KO mouse model suggests novel targets.

BACKGROUND: MicroRNAs (miRNAs) are short (20-24 nt) non-coding RNAs that are involved in post-transcriptional regulation of gene expression in multicellular organisms by affecting both the stability and translation of mRNAs. One of the miRNAs that has been shown to play a role in various pathologies like cancer, neurological disorders and cardiovascular diseases is miRNA-26b. However, these studies only demonstrated rather ambiguous associations without revealing a causal relationship. Therefore, the aim of this study is to establish and validate a mouse model which enables the elucidation of the exact role of miRNA-26b in various pathologies. RESULTS: A miRNA-26b-deficient mouse model was established using homologous recombination and validated using PCR. miRNA-26b-deficient mice did not show any physiological abnormalities and no effects on systemic lipid levels, blood parameters or tissue leukocytes. Using next generation sequencing, the gene expression patterns in miRNA-26b-deficient mice were analyzed and compared to wild type controls. This supported the already suggested role of miRNA-26b in cancer and neurological processes, but also revealed novel associations of miRNA-26b with thermogenesis and allergic reactions. In addition, detailed analysis identified several genes that seem to be highly regulated by miRNA-26b, which are linked to the same pathological conditions, further confirming the role of miRNA-26b in these pathologies and providing a strong validation of our mouse model. CONCLUSIONS: miRNA-26b plays an important role in various pathologies, although causal relationships still have to be established. The described mouse model of miRNA-26b deficiency is a crucial first step towards the identification of the exact role of miRNA-26b in various diseases that could identify miRNA-26b as a promising novel diagnostic or even therapeutic target in a broad range of pathologies.

Neutrophil secretion products pave the way for inflammatory monocytes.

The leukocyte response in inflammation is characterized by an initial recruitment of polymorphonuclear leukocytes (PMN) preceding a second wave of monocytes to the site of injury or infection. In the mouse, 2 populations of monocytes have been identified, Gr1(-)CCR2(-)CX3CR1(hi) resident monocytes and Gr1(+)CCR2(+)CX3CR1(lo) inflammatory monocytes. Here, intravital microscopy of the musculus cremaster and a subcutaneous air pouch model were used to investigate a possible link between PMN extravasation and the subsequent emigration of inflammatory monocytes in response to local stimulation with PAF. In mice that were made neutropenic by injection of a PMN-depleting antibody, the extravasation of inflammatory monocytes, but not resident monocytes, was markedly reduced compared with mice with intact white blood cell count but was restored by local treatment with secretion of activated PMN. Components of the PMN secretion were found to directly activate inflammatory monocytes and further examination revealed PMN-derived LL-37 and heparin-binding protein (HBP/CAP37/azurocidin) as primary mediators of the recruitment of inflammatory monocytes via activation of formyl-peptide receptors. These data show that LL-37 and HBP specifically stimulate mobilization of inflammatory monocytes. This cellular cross-talk functionally results in enhanced cytokine levels and increased bacterial clearance, thus boosting the early immune response.

Protective role of CXC receptor 4/CXC ligand 12 unveils the importance of neutrophils in atherosclerosis.

The CXC ligand (CXCL)12/CXC receptor (CXCR)4 chemokine-receptor axis controls hematopoiesis, organ development, and angiogenesis, but its role in the inflammatory pathogenesis of atherosclerosis is unknown. Here we show that interference with Cxcl12/Cxcr4 by a small-molecule antagonist, genetic Cxcr4 deficiency, or lentiviral transduction with Cxcr4 degrakine in bone marrow chimeras aggravated diet-induced atherosclerosis in apolipoprotein E-deficient (Apoe-/-) or LDL receptor-deficient (Ldlr-/-) mice. Chronic blockade of Cxcr4 caused leukocytosis and an expansion of neutrophils and increased neutrophil content in plaques, associated with apoptosis and a proinflammatory phenotype. Whereas circulating neutrophils were recruited to atherosclerotic lesions, depletion of neutrophils reduced plaque formation and prevented its exacerbation after blocking Cxcr4. Disrupting Cxcl12/Cxcr4 thus promotes lesion formation through deranged neutrophil homeostasis, indicating that Cxcl12/Cxcr4 controls the important contribution of neutrophils to atherogenesis in mice.

Importance of junctional adhesion molecule-C for neointimal hyperplasia and monocyte recruitment in atherosclerosis-prone mice-brief report.

OBJECTIVE: Although junctional adhesion molecule (JAM)-C has been implicated in the control of inflammatory leukocyte recruitment, its role in neointima formation after arterial injury has not been elucidated. METHODS AND RESULTS: In apolipoprotein E-deficient (Apoe(-/-)) mice fed an atherogenic diet, antibody blockade of JAM-C significantly reduced neointimal hyperplasia after wire injury of carotid arteries without altering medial area and decreased neointimal macrophage but not smooth muscle cell (SMC) content. An increased expression of JAM-C was detected in colocalization with luminal SMCs 1 day after injury and neointimal SMCs after 3 weeks. Blocking JAM-C inhibited monocytic cell arrest and leukocyte adhesion to carotid arteries perfused ex vivo and in vivo. Furthermore, monocyte adhesion to activated coronary artery SMCs under flow conditions in vitro was diminished by blocking JAM-C. CONCLUSIONS: Our data provide the first evidence for a crucial role of JAM-C in accelerated lesion formation and leukocyte recruitment in atherosclerosis-prone mice.

Autophagy unleashes noncanonical microRNA functions.

MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression which act by guiding AGO (argonaute) proteins to target RNA transcripts in the RNA-induced silencing complex (RISC). This macromolecular complex includes multiple additional components (e.g., TNRC6A) that allow for interaction with enzymes mediating inhibition of translation or RNA decay. However, miRNAs also reside in low-molecular weight complexes without being engaged in target repression, and their function in this context is largely unknown. Our recent findings show that endothelial cells exposed to protective high-shear stress or MTORC inhibition activate the macroautophagy/autophagy machinery to sustain viability by promoting differential trafficking of MIR126 strands and by enabling unconventional features of MIR126-5p. Whereas MIR126-3p is degraded upon autophagy activation, MIR126-5p interacts with the RNA-binding protein MEX3A to form a ternary complex with AGO2. This complex forms on the autophagosomal surface and facilitates its nuclear localization. Once in the nucleus, MIR126-5p dissociates from AGO2 and establishes aptamer-like interactions with the effector CASP3 (caspase 3). The binding to MIR126-5p prevents dimerization and proper active site formation of CASP3, thus inhibiting proteolytic activity and limiting apoptosis. Disrupting this pathway in vivo by genetic deletion of Mex3a or by specific deficiency of endothelial autophagy aggravates endothelial apoptosis and exacerbates the progression of atherosclerosis. The direct inhibition of CASP3 by MIR126-5p reveals a non-canonical mechanism by which miRNAs can modulate protein function and mediate the autophagy-apoptosis crosstalk.

Noncanonical inhibition of caspase-3 by a nuclear microRNA confers endothelial protection by autophagy in atherosclerosis.

MicroRNAs (miRNAs) are versatile regulators of gene expression with profound implications for human disease including atherosclerosis, but whether they can exert posttranslational functions to control cell adaptation and whether such noncanonical features harbor pathophysiological relevance is unknown. Here, we show that miR-126-5p sustains endothelial integrity in the context of high shear stress and autophagy. Bound to argonaute-2 (Ago2), miR-126-5p forms a complex with Mex3a, which occurs on the surface of autophagic vesicles and guides its transport into the nucleus. Mutational studies and biophysical measurements demonstrate that Mex3a binds to the central U- and G-rich regions of miR-126-5p with nanomolar affinity via its two K homology domains. In the nucleus, miR-126-5p dissociates from Ago2 and binds to caspase-3 in an aptamer-like fashion with its seed sequence, preventing dimerization of the caspase and inhibiting its activity to limit apoptosis. The antiapoptotic effect of miR-126-5p outside of the RNA-induced silencing complex is important for endothelial integrity under conditions of high shear stress promoting autophagy: ablation of Mex3a or ATG5 in vivo attenuates nuclear import of miR-126-5p, aggravates endothelial apoptosis, and exacerbates atherosclerosis. In human plaques, we found reduced nuclear miR-126-5p and active caspase-3 in areas of disturbed flow. The direct inhibition of caspase-3 by nuclear miR-126-5p reveals a noncanonical mechanism by which miRNAs can modulate protein function.

C1-esterase inhibitor protects against neointima formation after arterial injury in atherosclerosis-prone mice.

BACKGROUND: Although activation of the complement system has been implicated in the progression of human atherosclerosis, its function during arterial remodeling after injury has not been investigated. Here, we examined the contribution of the complement cascade to neointima formation in apolipoprotein E-deficient mice using a C1-esterase inhibitor (C1-inhibitor). METHODS AND RESULTS: Apolipoprotein E-deficient mice fed an atherogenic diet were subjected to wire-induced endothelial denudation of the carotid artery and treated with C1-inhibitor (Berinert; 15 IU i.v.) or vehicle perioperatively and subsequently every 2 days. The effectiveness of C1-inhibitor treatment was confirmed by measurement of plasma C1-inhibitor activity. A significant reduction in serum triglyceride levels was observed in C1-inhibitor-treated mice, whereas cholesterol levels did not differ. After 3 weeks, neointimal area was significantly reduced in C1-inhibitor-treated mice versus controls, whereas medial area was unaltered. This was associated with a significant decrease in neointimal and medial macrophage and CD3+ T-cell content. Expression of C3 mRNA was significantly reduced in plaques of C1-inhibitor-treated mice 10 days after injury, as assessed by reverse-transcription polymerase chain reaction. The peak in serum C3 levels after injury was markedly downregulated by C1-inhibitor, as evidenced by ELISA. Immunohistochemistry revealed strong expression of C3 and C3c, which colocalized to plaque macrophages and was reduced in C1-inhibitor-treated mice. C1-inhibitor impaired monocyte arrest on activated endothelium and platelets under flow conditions in vitro and leukocyte recruitment to carotid arteries 1 day after injury in vivo. CONCLUSIONS: C1-inhibitor limits neointimal plaque formation and inflammation. This may involve blockade of complement activation, inhibition of leukocyte recruitment, and reduced triglyceride levels, thus providing a multimodal approach to treat arterial disease.

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 beta(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 beta-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.

Ccr5 but not Ccr1 deficiency reduces development of diet-induced atherosclerosis in mice.

OBJECTIVE: Chemokines and their receptors are crucially involved in the development of atherosclerotic lesions by directing monocyte and T cell recruitment. The CC-chemokine receptors 1 (CCR1) and 5 (CCR5) expressed on these cells bind chemokines implicated in atherosclerosis, namely CCL5/RANTES. Although general blockade of CCL5 receptors reduces atherosclerosis, specific roles of CCR1 and CCR5 have not been unequivocally determined. METHODS AND RESULTS: We provide two independent lines of investigation to dissect the effects of Ccr1 and Ccr5 deletion in apolipoprotein E-deficient (ApoE-/-) mice in a collaboration between Aachen/Germany and Geneva/Switzerland. Different strains of ApoE-/- Ccr5-/- mice, ApoE-/- Ccr1-/- mice or respective littermates, were fed a high-fat diet for 10 to 12 weeks. Plaque areas were quantified in the aortic roots and thoracoabdominal aortas. Concordantly, both laboratories found that lesion formation was reduced in ApoE-/- Ccr5-/- mice. Plaque quality and immune cells were assessed by immunohistochemistry or mRNA analysis. Whereas lesional macrophage content, aortic CD4, and Th1-related Tim3 expression were reduced, smooth muscle cell (SMC) content and expression of interleukin-10 in plaques, lesional SMCs, and splenocytes were elevated. Protection against lesion formation by Ccr5 deficiency was sustained over 22 weeks of high-fat diet or over 26 weeks of chow diet. Conversely, plaque area, T cell, and interferon-gamma content were increased in ApoE-/- Ccr1-/- mice. CONCLUSIONS: Genetic deletion of Ccr5 but not Ccr1 in ApoE-/- mice protects from diet-induced atherosclerosis, associated with a more stable plaque phenotype, reduced mononuclear cell infiltration, Th1-type immune responses, and increased interleukin-10 expression. This corroborates CCR5 as a promising therapeutic target.

CD73/ecto-5'-nucleotidase protects against vascular inflammation and neointima formation.

BACKGROUND: Although CD73/ecto-5'-nucleotidase has been implicated in maintaining vasoprotection, its role in regulating endothelial adhesion molecule or inflammatory monocyte recruitment (eg, in the context of vascular injury) remains to be defined. METHODS AND RESULTS: Compared with wild-type mice, CD73-deficient (CD73(-/-)) mice exhibit increased luminal staining and protein and transcript expression for vascular cell adhesion molecule (VCAM)-1 in carotid arteries. In vitro, aortic endothelial cells (ECs) from CD73(-/-) mice display an upregulation of mRNA and protein expression of VCAM-1, associated with increased nuclear factor (NF)-kappaB activity, as determined by chromatin cross-linking and immunoprecipitation or quantitative p65 binding assays. CD73(-/-) ECs and carotid arteries perfused ex vivo supported increased monocyte arrest under flow conditions, which was mediated by alpha(4beta1) integrin. After wire injury of carotid arteries, CD73 expression and activity were upregulated in wild-type mice, whereas neointimal plaque formation and macrophage content were increased in CD73(-/-) mice versus wild-type mice, concomitant with elevated NF-kappaB activation, luminal VCAM-1 expression, and soluble VCAM-1 concentrations. In contrast, reconstitution of wild-type mice with CD73(-/-) versus CD73(+/+) BM did not significantly exacerbate neointima formation. Treatment with the specific A2A receptor agonist ATL-146e reversed the increased VCAM-1 transcript and protein expression in CD73(-/-) ECs and inhibited monocyte arrest on CD73(-/-) ECs. Continuous infusion of ATL-146e prevented neointima formation in CD73(-/-) mice. CONCLUSIONS: Our data epitomize the importance of vascular CD73 in limiting endothelial activation and monocyte recruitment via generation of adenosine acting through the A2A receptor, providing a molecular basis for therapeutic protection against vascular inflammation and neointimal hyperplasia.