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Introduction: Endoglin is a co-receptor of Transforming Growth Factorβ (TGFβ) and is crucial for the formation of new blood vessels. Endoglin-haploinsuffiency is the leading cause of the severe vascular disease Hereditary hemorrhagic telangiectasia 1 (HHT-1). The aim of this study is to investigate whether endoglin haplo-insufficiency impairs macrophage polarization towards a regenerative phenotype to induce angiogenesis and tissue repair in ischemic disease.Materials and Methods: HHT-1 was studied in Eng+/- mice and in HHT-1 patients with endoglin mutation. In Eng+/- and Eng+/+ mice, angiogenesis and tissue repair were studied by the induction of hindlimb ischemia, myocardial infarction or in matrigel plugs. Macrophage phenotypes were screened after a differentiation assay in-vitro after stimulation with GM-CSF or in tissue sections with immuno-histochemistry.Results: Angiogenic capacity was impaired in Eng+/- mice, which was associated by the infiltration of macrophages. Ischemic muscle or heart sections of Eng+/- mice showed prolonged inflammation 7 days postischemia. We can explain this by an extended polarization of bone marrow-derived monocytes (BMM) from Eng+/- mice towards pro-inflammatory macrophage (CD11b+/LY6Chi/CD206-) at the expense of regenerative macrophages. In addition, stimulation of macrophages with TGFβ resulted in the polarization of the more regenerative phenotype only in WT BMM-cultures. This TGFβ response was blunted in macrophages from Eng+/- mice, but we were able to rescue it by the inhibition of the Bone Morphogenetic Protein (BMP)-pathway (by LDN-193189). Subsequently, LDN also improved blood flow recovery and myocardial function in Eng+/- mice after hind limb ischemia and MI, respectively. Interestingly, blood monocytes from HHT-1 patients show increased number of CD14+/CD16+ monocytes, which is associated with pro-inflammatory/non-regenerative phenotype.Discussion and Conclusions: Endoglin haplo-insufficiency results in defective tissue repair due to impaired polarization of regenerative macrophages, directed by TGFβ. The defective responses were rescued by the inhibition of another TGFβ familiy member, BMP, and thereby counterbalance its pathway. These results have major implications for the treatment of HHT-1 patients, as we show the same impaired macrophage polarization. [ABSTRACT FROM AUTHOR]

Restenosis is a complex disease for which the pathophysiological mechanisms have not yet been fully elucidated, but are thought to include inflammation, proliferation, and matrix remodeling. Over the years, many predictive clinical, biological, (epi)genetic, lesion-related, and procedural risk factors for restenosis have been identified. These factors are not only useful in risk stratification of patients, they also contribute to our understanding of this condition. Furthermore, these factors provide evidence on which to base treatment tailored to the individual and aid in the development of novel therapeutic modalities. In this Review, we will evaluate the available evidence on the pathophysiological mechanisms of restenosis and provide an overview of the various risk factors, together with the possible clinical application of this knowledge. [ABSTRACT FROM AUTHOR]

Background: Arterial blood pressure-induced shear stress causes endothelial cell apoptosis and inflammation in vein grafts after coronary artery bypass grafting. As the inflammatory protein type IIA secretory phospholipase A 2 (sPLA 2 -IIA) has been shown to progress atherosclerosis, we hypothesized a role for sPLA 2 -IIA herein., Methods: The effects of PX-18, an inhibitor of both sPLA 2 -IIA and apoptosis, on residual endothelium and the presence of sPLA 2 -IIA were studied in human saphenous vein segments (n = 6) perfused at arterial blood pressure with autologous blood for 6 hrs., Results: The presence of PX-18 in the perfusion blood induced a significant 20% reduction in endothelial cell loss compared to veins perfused without PX18, coinciding with significantly reduced sPLA 2 -IIA levels in the media of the vein graft wall. In addition, PX-18 significantly attenuated caspase-3 activation in human umbilical vein endothelial cells subjected to shear stress via mechanical stretch independent of sPLA 2 -IIA., Conclusions: In conclusion, PX-18 protects saphenous vein endothelial cells from arterial blood pressure-induced death, possibly also independent of sPLA 2 -IIA inhibition., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Birth defects, which are in part caused by exposure to environmental chemicals and pharmaceutical drugs, affect 1 in every 33 babies born in the United States each year. The current standard to screen drugs that affect embryonic development is based on prenatal animal testing; however, this approach yields low-throughput and limited mechanistic information regarding the biological pathways and potential adverse consequences in humans. To develop a screening platform for molecules that affect human embryonic development based on endothelial cells (ECs) derived from human pluripotent stem cells, we differentiated human pluripotent stem cells into embryonic ECs and induced their maturation under arterial flow conditions. These cells were then used to screen compounds that specifically affect embryonic vasculature. Using this platform, we have identified two compounds that have higher inhibitory effect in embryonic than postnatal ECs. One of them was fluphenazine (an antipsychotic), which inhibits calmodulin kinase II. The other compound was pyrrolopyrimidine (an antiinflammatory agent), which inhibits vascular endothelial growth factor receptor 2 (VEGFR2), decreases EC viability, induces an inflammatory response, and disrupts preformed vascular networks. The vascular effect of the pyrrolopyrimidine was further validated in prenatal vs. adult mouse ECs and in embryonic and adult zebrafish. We developed a platform based on human pluripotent stem cell-derived ECs for drug screening, which may open new avenues of research for the study and modulation of embryonic vasculature.

Background: In order to identify factors that stimulate arteriogenesis after ischemia, we followed gene expression profiles in two extreme models for collateral artery formation over 28 days after hindlimb ischemia, namely "good-responding" C57BL/6 mice and "poor-responding" BALB/c mice., Methods and Results: Although BALB/c mice show very poor blood flow recovery after ischemia, most known proarteriogenic genes were upregulated more excessively and for a longer period than in C57BL/6 mice. In clear contrast, chemokine genes Ccl19 , Ccl21a , and Ccl21c and the chemokine receptor CCR7 were upregulated in C57BL/6 mice 1 day after hindlimb ischemia, but not in BALB/C mice. CCL19 and CCL21 regulate migration and homing of T lymphocytes via CCR7. When subjecting CCR7 -/- /LDLR -/- mice to hindlimb ischemia, we observed a 20% reduction in blood flow recovery compared with that in LDLR -/- mice. Equal numbers of α-smooth muscle actin-positive collateral arteries were found in the adductor muscles of both mouse strains, but collateral diameters were smaller in the CCR7 -/- /LDLR -/- . Fluorescence-activated cell sorter analyses showed that numbers of CCR7 + T lymphocytes (both CD4 + and CD8 + ) were decreased in the spleen and increased in the blood at day 1 after hindlimb ischemia in LDLR -/- mice. At day 1 after hindlimb ischemia, however, numbers of activated CD4 + T lymphocytes were decreased in the draining lymph nodes of LDLR -/- mice compared with CCR7 -/- /LDLR -/- mice., Conclusions: These data show that CCR7-CCL19/CCL21 axis facilitates retention CD4 + T lymphocytes at the site of collateral artery remodeling, which is essential for effective arteriogenesis., (© 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.)

Purpose of Review: The review discusses the recent literature on plaque angiogenesis and its relation to inflammation and plaque destabilization. Furthermore, it discusses how plaque angiogenesis can be used to monitor atherosclerosis and serve as a therapeutic target., Recent Findings: Histopathologic studies have shown a clear relationship between plaque angiogenesis, intraplaque hemorrhage (IPH), plaque vulnerability, and cardiovascular events. Hypoxia is a main driver of plaque angiogenesis and the mechanism behind angiogenesis is only partly known. IPH, as the result of immature neovessels, is associated with increased influx of inflammatory cells in the plaques. Experimental models displaying certain features of human atherosclerosis such as plaque angiogenesis or IPH are developed and can contribute to unraveling the mechanism behind plaque vulnerability. New imaging techniques are established, with which plaque angiogenesis and vulnerability can be detected. Furthermore, antiangiogenic therapies in atherosclerosis gain much attention., Summary: Plaque angiogenesis, IPH, and inflammation contribute to plaque vulnerability. Histopathologic and imaging studies together with specific experimental studies have provided insights in plaque angiogenesis and plaque vulnerability. However, more extensive knowledge on the underlying mechanism is required for establishing new therapies for patients at risk.

Occlusive arterial disease is a leading cause of morbidity and mortality worldwide. Aside from balloon angioplasty, bypass graft surgery is the most commonly performed revascularization technique for occlusive arterial disease. Coronary artery bypass graft surgery is performed in patients with left main coronary artery disease and three-vessel coronary disease, whereas peripheral artery bypass graft surgery is used to treat patients with late-stage peripheral artery occlusive disease. The great saphenous veins are commonly used conduits for surgical revascularization; however, they are associated with a high failure rate. Therefore, preservation of vein graft patency is essential for long-term surgical success. With the exception of 'no-touch' techniques and lipid-lowering and antiplatelet (aspirin) therapy, no intervention has hitherto unequivocally proven to be clinically effective in preventing vein graft failure. In this Review, we describe both preclinical and clinical studies evaluating the pathophysiology underlying vein graft failure, and the latest therapeutic options to improve patency for both coronary and peripheral grafts.

Arteriovenous fistulas (AVF) for hemodialysis access have a 1-year primary patency rate of only 60%, mainly as a result of maturation failure that is caused by insufficient outward remodeling and intimal hyperplasia. The exact pathophysiology remains unknown, but the inflammatory vascular response is thought to play an important role. In the present study we demonstrate that targeted liposomal delivery of prednisolone increases outward remodeling of the AVF in a murine model. Liposomes accumulate in the post-anastomotic area of the venous outflow tract in which the vascular pathology is most prominent in failed AVFs. On a histological level, we observed a reduction of lymphocytes and granulocytes in the vascular wall. In addition, a strong anti-inflammatory effect of liposomal prednisolone on macrophages was demonstrated in vitro. Therefore, treatment with liposomal prednisolone might be a valuable strategy to improve AVF maturation.

Background: Staphylococcus aureus cell wall components can induce IL-10 responses by immune cells, which may be atheroprotective. Therefore, in this study, we investigated whether heat-killed S. aureus (HK-SA) could inhibit the development of atherosclerosis., Methods: Atherosclerosis-susceptible LDL receptor-deficient mice were administered intraperitoneal HK-SA twice weekly and fed a Western-type diet for 6 weeks., Results: HK-SA administration resulted in a 1.6-fold increase in IL-10 production by peritoneal macrophages and splenocytes, and a 12-fold increase in serum IL-10 levels. Moreover, aortic plaque ICAM-1, VCAM-1 and CCL2 expression levels were significantly downregulated by on average 40%. HK-SA-treated mice had reduced numbers of inflammatory Ly-6C(hi) monocytes as well as Th1 and Th17 cells in the circulation and spleen, respectively. Attenuated leucocyte recruitment resulted in a significant inhibition of macrophage and T cell infiltration in atherosclerotic plaques, culminating in a significant 34% reduction in the development of atherosclerosis. To determine the effects of intraperitoneal HK-SA treatment, we stimulated macrophages with HK-SA in vitro. This resulted in a significant toll-like receptor 2 (TLR2)-dependent increase in IL-10, arginase-1, iNOS, TNF-α, PD-L1, CCL22 and indoleamine 2,3-dioxygenase expression. It was found that phosphoinositide 3-kinase crucially determined the balance of pro- and anti-inflammatory gene expression. The HK-SA-induced macrophage phenotype resembled M2b-like immunoregulatory macrophages., Conclusions: We have shown that HK-SA treatment induces strong anti-inflammatory IL-10 responses by macrophages, which are largely dependent on TLR2 and PI3K, and protects against the development of atherosclerosis. Commensalism with S. aureus could thus reduce cardiovascular events., (© 2016 The Association for the Publication of the Journal of Internal Medicine.)

Vascular remodelling is a multifactorial process that involves both adaptive and maladaptive changes of the vessel wall through, among others, cell proliferation and migration, but also apoptosis and necrosis of the various cell types in the vessel wall. Vascular remodelling can be beneficial, e.g. during neovascularization after ischaemia, as well as pathological, e.g. during atherosclerosis and aneurysm formation. In recent years, it has become clear that microRNAs are able to target many genes that are involved in vascular remodelling processes and either can promote or inhibit structural changes of the vessel wall. Since many different processes of vascular remodelling are regulated by similar mechanisms and factors, both positive and negative vascular remodelling can be affected by the same microRNAs. A large number of microRNAs has been linked to various aspects of vascular remodelling and indeed, several of these microRNAs regulate multiple vascular remodelling processes, including both the adaptive processes angiogenesis and arteriogenesis as well as maladaptive processes of atherosclerosis, restenosis and aneurysm formation. Here, we discuss the multifactorial role of microRNAs and microRNA clusters that were reported to play a role in multiple forms of vascular remodelling and are clearly linked to cardiovascular disease (CVD). The microRNAs reviewed are miR-126, miR-155 and the microRNA gene clusters 17-92, 23/24/27, 143/145 and 14q32. Understanding the contribution of these microRNAs to the entire spectrum of vascular remodelling processes is important, especially as these microRNAs may have great potential as therapeutic targets for treatment of various CVDs., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2016. For permissions please email: journals.permissions@oup.com.)

The arteriovenous fistula (AVF) still suffers from a high number of failures caused by insufficient remodeling and intimal hyperplasia from which the exact pathophysiology remains unknown. In order to unravel the pathophysiology a murine model of AVF-failure was developed in which the configuration of the anastomosis resembles the preferred situation in the clinical setting. A model was described in which an AVF is created by connecting the venous end of the branch of the external jugular vein to the side of the common carotid artery using interrupted sutures. At a histological level, we observed progressive stenotic intimal lesions in the venous outflow tract that is also seen in failed human AVFs. Although this procedure can be technically challenging due to the small dimensions of the animal, we were able to achieve a surgical success rate of 97% after sufficient training. The key advantage of a murine model is the availability of transgenic animals. In view of the different proposed mechanisms that are responsible for AVF failure, disabling genes that might play a role in vascular remodeling can help us to unravel the complex pathophysiology of AVF failure.

Objectives: Unstable atherosclerotic lesions in carotid arteries require surgical endarterectomy to reduce the risk of ischemic stroke. We aimed to identify microRNAs that exert a broad effect on atherosclerotic plaque formation and stability in the carotid artery., Background: We made a selection of 164 genes involved in atherosclerosis. Using www.targetscan.org, we determined which microRNAs potentially regulate expression of these genes. We identified multiple microRNAs from the 14q32 microRNA cluster, which is highly involved in vascular remodeling. In human plaques, collected during carotid endarterectomy surgery, we found that 14q32 microRNA (miR-494) was abundantly expressed in unstable lesions., Methods: We induced atherosclerotic plaque formation in hypercholesterolemic ApoE mice by placing semiconstrictive collars around both carotid arteries. We injected "Gene Silencing Oligonucleotides" against miR-494 (GSO-494) or negative control (GSO-control). Using fluorescently labeled GSOs, we confirmed uptake of GSOs in affected areas of the carotids, but not elsewhere in the vasculature., Results: After injection of GSO-494, we observed significant downregulation of miR-494 expression in the carotid arteries, although miR-494 target genes were upregulated. Further analyses revealed a 65% decrease in plaque size after GSO-494 treatment. Plaque stability was increased in GSO-494-treated mice, determined by an 80% decrease in necrotic core size and a 50% increase in plaque collagen content. Inhibition of miR-494 also resulted in decreased cholesterol levels and decreased very low-density lipoprotein (VLDL) fractions., Conclusions: Treatment with GSO-494 results in smaller atherosclerotic lesions with increased plaque stability. Inhibition of miR-494 may decrease the risk of surgical complications or even avert endarterectomy surgery in some cases.

Background: Coronary collateral arteries function as natural bypasses in the event of coronary obstruction. The degree of collateral network development significantly impacts the outcome of patients after an acute myocardial infarction (AMI). MicroRNAs (miRNAs, miRs) have arisen as biomarkers to identify heterogeneous patients, as well as new therapeutic targets in cardiovascular disease. We sought to identify miRNAs that are differentially expressed in chronic total occlusion (CTO) patients with well or poorly developed collateral arteries., Methods and Results: Forty-one CTO patients undergoing coronary angiography and invasive assessment of their coronary collateralization were dichotomized based on their collateral flow index (CFI). After miRNA profiling was conducted on aortic plasma, four miRNAs were selected for validation by real-time quantitative reverse transcription polymerase chain reaction in patients with low (CFI<0.39) and high (CFI>0.39) collateral artery capacity. We confirmed significantly elevated levels of miR423-5p (p<0.05), miR10b (p<0.05), miR30d (p<0.05) and miR126 (p<0.001) in patients with insufficient collateral network development. We further demonstrated that each of these miRNAs could serve as circulating biomarkers to discriminate patients with low collateral capacity (p<0.01 for each miRNA). We also determined significantly greater expression of miR30d (p<0.05) and miR126 (p<0.001) in CTO patients relative to healthy controls., Conclusion: The present study identifies differentially expressed miRNAs in patients with high versus low coronary collateral capacity. We have shown that these miRNAs can function as circulating biomarkers to discriminate between patients with insufficient or sufficient collateralization. This is the first study to identify miRNAs linked to coronary collateral vessel function in humans.

Fibrinogen as precursor of fibrin plays an essential role in clot formation. There are three main mechanisms associated with a reduction in fibrinogen concentration during cardiac surgery: hemodilution, consumption, and degradation. Moreover, early fibrinogen degradation products (FgDPs) can interfere with normal fibrin formation of intact fibrinogen. The aim of this study was to determine the relative contributions of hemodilution, consumption, and degradation to fibrinogen loss in cardiac surgery and to evaluate the effects fibrinogen degradation products on blood clot formation in vitro. First, fibrin and fibrinogen concentrations, their degradation products, hematocrit, and albumin concentrations were compared in 10 patients before and after isolated coronary artery bypass graft (CABG) surgery with cardiopulmonary bypass. Second, ex-vivo fibrinogen supplementation experiments were performed. Finally, the effects of purified FgDPs on clotting time and clot firmness were established in vitro in whole blood by ROTEM. Fibrinogen plasma concentration decreased 30% during surgery. This drop appears to be mainly caused by hemodilution, as both hematocrit and albumin levels decreased and no relevant increase in D-dimer levels and FgDPs was observed. Furthermore, the coagulation profile normalized after addition of purified fibrinogen. Early FgDPs demonstrated a significant impact on in-vitro whole blood clotting. Although early FgDPs have a pronounced effect on blood clot formation in vitro and therefore may induce or enhance in vivo coagulopathy, the drop of fibrinogen concentration seen after CABG surgery (using tranexamic acid) is primarily caused by hemodilution.

Aims: Activated mast cells have been identified in the intima and perivascular tissue of human atherosclerotic plaques. As mast cells have been described to release a number of chemokines that mediate leukocyte fluxes, we propose that activated mast cells may play a pivotal role in leukocyte recruitment during atherosclerotic plaque progression., Methods and Results: Systemic IgE-mediated mast cell activation in apoE(-/-)μMT mice resulted in an increase in atherosclerotic lesion size as compared to control mice, and interestingly, the number of neutrophils was highly increased in these lesions. In addition, peritoneal mast cell activation led to a massive neutrophil influx into the peritoneal cavity in C57Bl6 mice, whereas neutrophil numbers in mast cell deficient Kit(W(-sh)/W(-sh)) mice were not affected. Within the newly recruited neutrophil population, increased levels of CXCR2(+) and CXCR4(+) neutrophils were observed after mast cell activation. Indeed, mast cells were seen to contain and release CXCL1 and CXCL12, the ligands for CXCR2 and CXCR4. Intriguingly, peritoneal mast cell activation in combination with anti-CXCR2 receptor antagonist resulted in decreased neutrophil recruitment, thus establishing a prominent role for the CXCL1/CXCR2 axis in mast cell-mediated neutrophil recruitment., Conclusions: Our data suggest that chemokines, and in particular CXCL1, released from activated mast cells induce neutrophil recruitment to the site of inflammation, thereby aggravating the ongoing inflammatory response and thus affecting plaque progression and destabilization., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Galectin-2 is a monocyte-expressed carbohydrate-binding lectin, for which increased expression is genetically determined and associated with decreased collateral arteriogenesis in obstructive coronary artery disease patients. The inhibiting effect of galectin-2 on arteriogenesis was confirmed in vivo, but the mechanism is largely unknown. In this study we aimed to explore the effects of galectin-2 on monocyte/macrophage phenotype in vitro and vivo, and to identify the receptor by which galectin-2 exerts these effects. We now show that the binding of galectin-2 to different circulating human monocyte subsets is dependent on monocyte surface expression levels of CD14. The high affinity binding is blocked by an anti-CD14 antibody but not by carbohydrates, indicating a specific protein-protein interaction. Galectin-2 binding to human monocytes modulated their transcriptome by inducing proinflammatory cytokines and inhibiting pro-arteriogenic factors, while attenuating monocyte migration. Using specific knock-out mice, we show that galectin-2 acts through the CD14/toll-like receptor (TLR)-4 pathway. Furthermore, galectin-2 skews human macrophages to a M1-like proinflammatory phenotype, characterized by a reduced motility and expression of an anti-arteriogenic cytokine/growth factor repertoire. This is accompanied by a switch in surface protein expression to CD40-high and CD206-low (M1). In a murine model we show that galectin-2 administration, known to attenuate arteriogenesis, leads to increased numbers of CD40-positive (M1) and reduced numbers of CD206-positive (M2) macrophages surrounding actively remodeling collateral arteries. In conclusion galectin-2 is the first endogenous CD14/TLR4 ligand that induces a proinflammatory, non-arteriogenic phenotype in monocytes/macrophages. Interference with CD14-Galectin-2 interaction may provide a new intervention strategy to stimulate growth of collateral arteries in genetically compromised cardiovascular patients.

Objectives: Maturation failure is the major limitation of arteriovenous fistulas (AVFs) as hemodialysis access conduits. Indeed, 30-50% of AVFs fail to mature due to intimal hyperplasia and insufficient outward remodeling. Elastin has emerged as an important determinant of vascular remodeling. Here the role of elastin in AVF remodeling in elastin haplodeficient (eln(+/-)) mice undergoing AVF surgery has been studied., Methods: Unilateral AVFs between the branch of the jugular vein and carotid artery in an end to side manner were created in wild-type (WT) C57BL/6 (n = 11) and in eln(+/-) mice (n = 9). Animals were killed at day 21 and the AVFs were analyzed histologically and at an mRNA level using real-time quantitative polymerase chain reaction., Results: Before AVF surgery, a marked reduction in elastin density in the internal elastic lamina (IEL) of eln(+/-) mice was observed. AVF surgery resulted in fragmentation of the venous internal elastic lamina in both groups while the expression of the tropoelastin mRNA was 53% lower in the eln(+/-) mice than in WT mice (p < .001). At 21 days after AVF surgery, the circumference of the venous outflow tract of the AVF was 21% larger in the eln(+/-) mice than in the WT mice (p = .037), indicating enhanced outward remodeling in the eln(+/-) mice. No significant difference in intimal hyperplasia was observed. The venous lumen of the AVF in the eln(+/-) mice was 53% larger than in the WT mice, although this difference was not statistically significant (eln(+/-), 350,116 ± 45,073 μm(2); WT, 229,405 ± 40,453 μm(2); p = .064)., Conclusions: In a murine model, elastin has an important role in vascular remodeling following AVF creation, in which a lower amount of elastin results in enhanced outward remodeling. Interventions targeting elastin degradation might be a viable option in order to improve AVF maturation., (Copyright © 2015 European Society for Vascular Surgery. Published by Elsevier Ltd. All rights reserved.)

Objective: Toll like receptor 4 (TLR4) plays a key role in inflammation and previously it was established that TLR4 deficiency attenuates atherosclerosis. RadioProtective 105 (RP105) is a structural homolog of TLR4 and an important regulator of TLR4 signaling, suggesting that RP105 may also be an important effector in atherosclerosis. We thus aimed to determine the role of RP105 in atherosclerotic lesion development using RP105 deficient mice on an atherosclerotic background., Methods and Results: Atherosclerosis was induced in Western-type diet fed low density lipoprotein receptor deficient (LDLr(-/-)) and LDLr/RP105 double knockout (LDLr(-/-)/RP105(-/-)) mice by means of perivascular carotid artery collar placement. Lesion size was significantly reduced by 58% in LDLr(-/-)/RP105(-/-) mice, and moreover, plaque macrophage content was markedly reduced by 40%. In a model of acute peritonitis, monocyte influx was almost 3-fold reduced in LDLr(-/-)/RP105(-/-) mice (P = 0.001), while neutrophil influx remained unaltered, suggestive of an altered migratory capacity of monocytes upon deletion of RP105. Interestingly, in vitro stimulation of monocytes with LPS induced a downregulation of CCR2, a chemokine receptor crucially involved in monocyte influx to atherosclerotic lesions, which was more pronounced in LDLr(-/-)/RP105(-/-) monocytes as compared to LDLr(-/-) monocytes., Conclusion: We here show that RP105 deficiency results in reduced early atherosclerotic plaque development with a marked decrease in lesional macrophage content, which may be due to disturbed migration of RP105 deficient monocytes resulting from CCR2 downregulation., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Rupture of an atherosclerotic plaque is the major underlying cause of adverse cardiovascular events such as myocardial infarction or stroke. Therapeutic interventions should therefore be directed towards inhibiting growth of atherosclerotic lesions as well as towards prevention of lesion destabilization. Interestingly, the presence of mast cells has been demonstrated in both murine and human plaques, and multiple interventional murine studies have pointed out a direct role for mast cells in early and late stages of atherosclerosis. Moreover, it has recently been described that activated lesional mast cells correlate with major cardiovascular events in patients suffering from cardiovascular disease. This review focuses on the effect of different mast cell derived mediators in atherogenesis and in late stage plaque destabilization. Also, possible ligands for mast cell activation in the context of atherosclerosis are discussed. Finally, we will elaborate on the predictive value of mast cells, together with therapeutic implications, in cardiovascular disease.

Critical limb ischemia (CLI) is often poorly treatable by conventional management and alternatives such as autologous cell therapy are increasingly investigated. Whereas previous studies showed a substantial impairment of neovascularization capacity in primary bone-marrow (BM) isolates from patients, little is known about dysfunction in patient-derived BM mesenchymal stromal cells (MSCs). In this study, we have compared CLI-MSCs to healthy controls using gene expression profiling and functional assays for differentiation, senescence and in vitro and in vivo pro-angiogenic ability. Whereas no differentially expressed genes were found and adipogenic and osteogenic differentiation did not significantly differ between groups, chondrogenic differentiation was impaired in CLI-MSCs, potentially as a consequence of increased senescence. Migration experiments showed no differences in growth factor sensitivity and secretion between CLI- and control MSCs. In a murine hind-limb ischemia model, recovery of perfusion was enhanced in MSC-treated mice compared to vehicle controls (71 ± 24% versus 44 ± 11%; P < 1 × 10(-6)). CLI-MSC- and control-MSC-treated animals showed nearly identical amounts of reperfusion (ratio CLI:Control = 0.98, 95% CI = 0.82-1.14), meeting our criteria for statistical equivalence. The neovascularization capacity of MSCs derived from CLI-patients is not compromised and equivalent to that of control MSCs, suggesting that autologous MSCs are suitable for cell therapy in CLI patients.

Background: Toll-like receptor-4 (TLR4), a receptor of the innate immune system, is suggested to have detrimental effects on cardiac function after myocardial infarction (MI). RP105 (CD180) is a TLR4 homolog lacking the intracellular signaling domain that competitively inhibits TLR4-signaling. Thus, we hypothesized that RP105 deficiency, by amplifying TLR4 signaling, would lead to aggravated cardiac dysfunction after MI., Methods and Results: First, whole blood from RP105-/- and wild-type (WT) male C57Bl/6N mice was stimulated with LPS, which induced a strong inflammatory TNFα response in RP105-/- mice. Then, baseline heart function was assessed by left ventricular pressure-volume relationships which were not different between RP105-/- and WT mice. Permanent ligation of the left anterior descending coronary artery was performed to induce MI. Infarct sizes were analyzed by (immuno)histology and did not differ. Fifteen days post MI heart function was assessed and RP105-/- mice had significantly higher heart rate (+21%, P<0.01), end systolic volume index (+57%, P<0.05), end systolic pressure (+22%, P<0.05) and lower relaxation time constant tau (-12%, P<0.05), and a tendency for increased end diastolic volume index (+42%, P<0.06), compared to WT mice. In the area adjacent to the infarct zone, compared to the healthy myocardium, levels of RP105, TLR4 and the endogenous TLR4 ligand fibronectin-EDA were increased as well as the number of macrophages, however this was not different between both groups., Conclusion: Deficiency of the endogenous TLR4 inhibitor RP105 leads to an enhanced inflammatory status and more pronounced cardiac dilatation after induction of MI, underscoring the role of the TLR4 pathway in post-infarction remodeling., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Complement factor C5a and its receptor C5aR are expressed in vulnerable atherosclerotic plaques; however, a causal relation between C5a and plaque rupture has not been established yet. Accelerated atherosclerosis was induced by placing vein grafts in male apoE(-/-) mice. After 24 days, when advanced plaques had developed, C5a or PBS was applied locally at the lesion site in a pluronic gel. Three days later mice were killed to examine the acute effect of C5a on late stage atherosclerosis. A significant increase in C5aR in the plaque was detectable in mice treated with C5a. Lesion size and plaque morphology did not differ between treatment groups, but interestingly, local treatment with C5a resulted in a striking increase in the amount of plaque disruptions with concomitant intraplaque haemorrhage. To identify the potential underlying mechanisms, smooth muscle cells and endothelial cells were treated in vitro with C5a. Both cell types revealed a marked increase in apoptosis after stimulation with C5a, which may contribute to lesion instability in vivo. Indeed, apoptosis within the plaque was seen to be significantly increased after C5a treatment. We here demonstrate a causal role for C5a in atherosclerotic plaque disruptions, probably by inducing apoptosis. Therefore, intervention in complement factor C5a signalling may be a promising target in the prevention of acute atherosclerotic complications., (© 2014 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Rationale: Effective neovascularization is crucial for recovery after cardiovascular events., Objective: Because microRNAs regulate expression of up to several hundred target genes, we set out to identify microRNAs that target genes in all pathways of the multifactorial neovascularization process. Using www.targetscan.org, we performed a reverse target prediction analysis on a set of 197 genes involved in neovascularization. We found enrichment of binding sites for 27 microRNAs in a single microRNA gene cluster. Microarray analyses showed upregulation of 14q32 microRNAs during neovascularization in mice after single femoral artery ligation., Methods and Results: Gene silencing oligonucleotides (GSOs) were used to inhibit 4 14q32 microRNAs, miR-329, miR-487b, miR-494, and miR-495, 1 day before double femoral artery ligation. Blood flow recovery was followed by laser Doppler perfusion imaging. All 4 GSOs clearly improved blood flow recovery after ischemia. Mice treated with GSO-495 or GSO-329 showed increased perfusion already after 3 days (30% perfusion versus 15% in control), and those treated with GSO-329 showed a full recovery of perfusion after 7 days (versus 60% in control). Increased collateral artery diameters (arteriogenesis) were observed in adductor muscles of GSO-treated mice, as well as increased capillary densities (angiogenesis) in the ischemic soleus muscle. In vitro, treatment with GSOs led to increased sprout formation and increased arterial endothelial cell proliferation, as well as to increased arterial myofibroblast proliferation., Conclusions: The 14q32 microRNA gene cluster is highly involved in neovascularization. Inhibition of 14q32 microRNAs miR-329, miR-487b, miR-494, and miR-495 provides a promising tool for future therapeutic neovascularization., (© 2014 American Heart Association, Inc.)

Aims: We investigated the role of the TLR4-accessory molecule RP105 (CD180) in post-ischemic neovascularization, i.e. arteriogenesis and angiogenesis. TLR4-mediated activation of pro-inflammatory Ly6Chi monocytes is crucial for effective neovascularization. Immunohistochemical analyses revealed that RP105+ monocytes are present in the perivascular space of remodeling collateral arterioles. As RP105 inhibits TLR4 signaling, we hypothesized that RP105 deficiency would lead to an unrestrained TLR4-mediated inflammatory response and hence to enhanced blood flow recovery after ischemia., Methods and Results: RP105-/- and wild type (WT) mice were subjected to hind limb ischemia and blood flow recovery was followed by Laser Doppler Perfusion Imaging. Surprisingly, we found that blood flow recovery was severely impaired in RP105-/- mice. Immunohistochemistry showed that arteriogenesis was reduced in these mice compared to the WT. However, both in vivo and ex vivo analyses showed that circulatory pro-arteriogenic Ly6Chi monocytes were more readily activated in RP105-/- mice. FACS analyses showed that Ly6Chi monocytes became activated and migrated to the affected muscle tissues in WT mice following induction of hind limb ischemia. Although Ly6Chi monocytes were readily activated in RP105-/- mice, migration into the ischemic tissues was hampered and instead, Ly6Chi monocytes accumulated in their storage compartments, bone marrow and spleen, in RP105-/- mice., Conclusions: RP105 deficiency results in an unrestrained inflammatory response and monocyte over-activation, most likely due to the lack of TLR4 regulation. Inappropriate, premature systemic activation of pro-inflammatory Ly6Chi monocytes results in reduced infiltration of Ly6Chi monocytes in ischemic tissues and in impaired blood flow recovery.

The role of the CD200 ligand-CD200 receptor (CD200-CD200R) inhibitory axis is highly important in controlling myeloid cell function. Since the activation of myeloid cells is crucial in arteriogenesis, we hypothesized that disruption of the CD200-CD200R axis promotes arteriogenesis in a murine hindlimb ischemia model. Female Cd200-/- and wildtype (C57Bl/6J) mice underwent unilateral femoral artery ligation. Perfusion recovery was monitored over 7 days using Laser-Doppler analysis and was increased in Cd200-/- mice at day 3 and 7 after femoral artery ligation, compared to wildtype. Histology was performed on hindlimb muscles at baseline, day 3 and 7 to assess vessel geometry and number and inflammatory cell influx. Vessel geometry in non-ischemic muscles was larger, and vessel numbers in ischemic muscles were increased in Cd200-/- mice compared to wildtype. Furthermore, T lymphocyte influx was increased in Cd200-/- compared to wildtype. CD200R agonist treatment was performed in male C57Bl/6J mice to validate the role of the CD200-CD200R axis in arteriogenesis. CD200R agonist treatment after unilateral femoral artery ligation resulted in a significant decrease in vessel geometry, perfusion recovery and T lymphocyte influx at day 7 compared to isotype treatment. In this study, we show a causal role for the CD200-CD200R inhibitory axis in arteriogenesis in a murine hindlimb ischemia model. Lack of CD200R signaling is accompanied by increased T lymphocyte recruitment to the collateral vasculature and results in enlargement of preexisting collateral arteries.

Objective: In arteriogenesis, pre-existing anastomoses undergo enlargement to restore blood flow in ischemic tissues. Chemokine (C-X-C motif) ligand 10 (CXCL10) is secreted after Toll-like receptor activation. Toll-like receptors are involved in arteriogenesis; however, the role of CXCL10 is still unclear. In this study, we investigated the role for CXCL10 in a murine hindlimb ischemia model., Approach and Results: Unilateral femoral artery ligation was performed in wild-type (WT) and CXCL10(-/-) knockout (KO) mice and perfusion recovery was measured using laser-Doppler perfusion analysis. Perfusion recovery was significantly lower in KO mice compared with WT at days 4 and 7 after surgery (KO versus WT: 28±5% versus 81±13% at day 4; P=0.003 and 57±12% versus 107±8% at day 7; P=0.003). Vessel measurements of α-smooth muscle actin-positive vessels revealed increasing numbers in time after surgery, which was significantly higher in WT when compared with that in KO. Furthermore, α-smooth muscle actin-positive vessels were significantly larger in WT when compared with those in KO at day 7 (wall thickness, P<0.001; lumen area, P=0.003). Local inflammation was assessed in hindlimb muscles, but this did not differ between WT and KO. Chimerization experiments analyzing perfusion recovery and histology revealed an equal contribution for bone marrow-derived and circulating CXCL10. Migration assays showed a stimulating role for both intrinsic and extrinsic CXCL10 in vascular smooth muscle cell migration., Conclusions: CXCL10 plays a causal role in arteriogenesis. Bone marrow-derived CXCL10 and tissue-derived CXCL10 play a critical role in accelerating perfusion recovery after arterial occlusion in mice probably by promoting vascular smooth muscle cell recruitment and maturation of pre-existing anastomoses.

Background: Recently, we have shown that intraplaque mast cell numbers are associated with atherosclerotic plaque vulnerability and with future cardiovascular events, which renders inhibition of mast cell activation of interest for future therapeutic interventions. However, the endogenous triggers that activate mast cells during the progression and destabilization of atherosclerotic lesions remain unidentified. Mast cells can be activated by immunoglobulins and in the present study, we aimed to establish whether specific immunoglobulins in plasma of patients scheduled for carotid endarterectomy were related to (activated) intraplaque mast cell numbers and plasma tryptase levels. In addition, the levels were related to other vulnerable plaque characteristics and baseline clinical data., Methods and Results: OxLDL-IgG, total IgG and total IgE levels were measured in 135 patients who underwent carotid endarterectomy. No associations were observed between the tested plasma immunoglobulin levels and total mast cell numbers in atherosclerotic plaques. Furthermore, no associations were found between IgG levels and the following plaque characteristics: lipid core size, degree of calcification, number of macrophages or smooth muscle cells, amount of collagen and number of microvessels. Interestingly, statin use was negatively associated with plasma IgE and oxLDL-IgG levels., Conclusions: In patients suffering from carotid artery disease, total IgE, total IgG and oxLDL-IgG levels do not associate with plaque mast cell numbers or other vulnerable plaque histopathological characteristics. This study thus does not provide evidence that the immunoglobulins tested in our cohort play a role in intraplaque mast cell activation or grade of atherosclerosis.

Background: Leukotriene B4 (LTB4) has been associated with the initiation and progression of atherosclerosis and abdominal aortic aneurysm (AAA) formation. However, associations of LTB4 levels with tissue characteristics and adverse clinical outcome of advanced atherosclerosis and AAA are scarcely studied. We hypothesized that LTB4 levels are associated with a vulnerable plaque phenotype and adverse clinical outcome. Furthermore, that LTB4 levels are associated with inflammatory AAA and adverse clinical outcome., Methods: Atherosclerotic plaques and AAA specimens were selected from two independent databases for LTB4 measurements. Plaques were isolated during carotid endarterectomy from asymptomatic (n = 58) or symptomatic (n = 317) patients, classified prior to surgery. LTB4 levels were measured without prior lipid extraction and levels were corrected for protein content. LTB4 levels were related to plaque phenotype, baseline patient characteristics and clinical outcome within three years following surgery. Seven non-diseased mammary artery specimens served as controls. AAA specimens were isolated during open repair, classified as elective (n = 189), symptomatic (n = 29) or ruptured (n = 23). LTB4 levels were measured similar to the plaque measurements and were related to tissue characteristics, baseline patient characteristics and clinical outcome. Twenty-six non-diseased aortic specimens served as controls., Results: LTB4 levels corrected for protein content were not significantly associated with histological characteristics specific for vulnerable plaques or inflammatory AAA as well as clinical presentation. Moreover, it could not predict secondary manifestations independently investigated in both databases. However, LTB4 levels were significantly lower in controls compared to plaque (p = 0.025) or AAA (p = 0.017)., Conclusions: LTB4 levels were not associated with a vulnerable plaque phenotype or inflammatory AAA or clinical presentation. This study does not provide supportive evidence for a role of LTB4 in atherosclerotic plaque destabilization or AAA expansion. However, these data should be interpreted with care, since LTB4 measurements were performed without prior lipid extractions.

C-X-C motif ligand 10 (CXCL10), or interferon-inducible protein-10, is a small chemokine belonging to the CXC chemokine family. Its members are responsible for leukocyte trafficking and act on tissue cells, like endothelial and vascular smooth muscle cells. CXCL10 is secreted by leukocytes and tissue cells and functions as a chemoattractant, mainly for lymphocytes. After binding to its receptor CXCR3, CXCL10 evokes a range of inflammatory responses: key features in cardiovascular disease (CVD). The role of CXCL10 in CVD has been extensively described, for example for atherosclerosis, aneurysm formation, and myocardial infarction. However, there seems to be a discrepancy between experimental and clinical settings. This discrepancy occurs from differences in biological actions between species (e.g. mice and human), which is dependent on CXCL10 signaling via different CXCR3 isoforms or CXCR3-independent signaling. This makes translation from experimental to clinical settings challenging. Furthermore, the overall consensus on the actions of CXCL10 in specific CVD models is not yet reached. The purpose of this review is to describe the functions of CXCL10 in different CVDs in both experimental and clinical settings and to highlight and discuss the possible discrepancies and translational difficulties. Furthermore, CXCL10 as a possible biomarker in CVD will be discussed.

Toll-like receptors (TLRs) are important in innate immune responses, which are crucial in collateral artery formation (arteriogenesis). TLR4⁻/⁻ mice undergoing hind limb ischemia show decreased perfusion recovery accompanied by an impaired infiltration of inflammatory cells. TLR antagonists are currently developed and tested with the objective to inhibit acute exacerbation of organ damaging immune responses. However, systemic inhibition of innate immune responses may negatively influence arteriogenesis. In this study, we evaluated if TLR4 inhibition by a potent TLR4 inhibitor (TAK-242) would negatively influence perfusion recovery in a mouse model for arteriogenesis. Whole blood from human and mouse origin was stimulated with the TLR4 ligand lipopolysaccharide following TAK-242 incubation. After stimulation, cellular TLR4 activation was measured using fluorescence-activated cell sorting and tumor necrosis factor alpha release was measured using enzyme-linked immunosorbent assay. Next, the effect of TAK-242 was tested in a mouse model for arteriogenesis on perfusion recovery. TLR4 responses measured by tumor necrosis factor alpha levels were inhibited by TAK-242 in human and mouse blood after long-term stimulation. TAK-242 attenuated TLR4 responses in vivo but did not inhibit perfusion recovery in mice. In conclusion, TAK-242 does not negatively influence perfusion recovery following hind limb ischemia despite its TLR4 inhibiting properties.

Objective: The arteriovenous fistula (AVF) still suffers from a high number of failures caused by insufficient outward remodeling and intimal hyperplasia (IH) formation from which the exact mechanism is largely unknown. A suitable animal model is of vital importance in the unraveling of the underlying pathophysiology. However, current murine models of AVF failure do not incorporate the surgical configuration that is commonly used in humans. Because the hemodynamic profile is one of the key determinants that play a role in vascular remodeling in the AVF, it is preferable to use this same configuration in an animal model. Here we describe a novel murine model of AVF failure in which the configuration (end-to-side) is similar to what is most frequently performed in humans., Methods: An AVF was created in 45 C57BL/6 mice by anastomosing the end of a branch of the external jugular vein to the side of the common carotid artery with interrupted sutures. The AVFs were harvested and analyzed histologically at days 7, 14, and 28. Identical veins of unoperated-on mice served as controls. Intravenous near-infrared fluorescent fluorophores were used to assess the patency of the fistula., Results: The patency rates at days 7, 14, and 28 days were 88%, 90%, and 50%, respectively. The mean circumference increased up to day 14, with a maximum 1.4-fold increase at day 7 compared with the control group (1.82 ± 0.7 vs 1.33 ± 0.3 mm; P = .443). Between days 14 and 28, the circumference remained constant (2.36 ± 0.2 vs 2.45 ± 0.2 mm; P = .996). At 7 days after surgery, the intimal area consisted mainly of an acellular layer that was structurally analogous to a focal adherent thrombus. Starting at 14 days after surgery, venous IH increased significantly compared with the unoperated-on group (14 days: 115,090 ± 22,594 μm(2), 28 days: 234,619 ± 47,828 μm(2), unoperated group: 2368 ± 1056 μm(2); P = .001 and P < .001, respectively) and was mainly composed of cells positive for α-smooth muscle actin. We observed leukocytes in the adventitial side of the vein at all time points., Conclusions: Our novel murine AVF model, which incorporates a clinically relevant configuration of the anastomosis, displays similar features that are characteristic of failing human AVFs. Moreover, our findings suggest that coagulation and inflammation could both potentially play an important role in the formation of IH and subsequent AVF failure. Near-infrared fluoroscopy was a suitable alternative for conventional imaging techniques. This murine AVF-model is a valuable addition to the AVF animal model arsenal., (Copyright © 2014 Society for Vascular Surgery. All rights reserved.)

In this study, we target the hypoxia inducible factor-1 alpha (HIF-1-alpha) pathway by short hairpin RNA interference therapy targeting prolyl hydroxylase-2 (shPHD2). We use the minicircle (MC) vector technology as an alternative for conventional nonviral plasmid (PL) vectors in order to improve neovascularization after unilateral hindlimb ischemia in a murine model. Gene expression and transfection efficiency of MC and PL, both in vitro and in vivo, were assessed using bioluminescence imaging (BLI) and firefly luciferase (Luc) reporter gene. C57Bl6 mice underwent unilateral electrocoagulation of the femoral artery and gastrocnemic muscle injection with MC-shPHD2, PL-shPHD2, or phosphate-buffered saline (PBS) as control. Blood flow recovery was monitored using laser Doppler perfusion imaging, and collaterals were visualized by immunohistochemistry and angiography. MC-Luc showed a 4.6-fold higher in vitro BLI signal compared with PL-Luc. BLI signals in vivo were 4.3×10(5)±3.3×10(5) (MC-Luc) versus 0.4×10(5)±0.3×10(5) (PL-Luc) at day 28 (p=0.016). Compared with PL-shPHD2 or PBS, MC-shPHD2 significantly improved blood flow recovery, up to 50% from day 3 until day 14 after ischemia induction. MC-shPHD2 significantly increased collateral density and capillary density, as monitored by alpha-smooth muscle actin expression and CD31(+) expression, respectively. Angiography data confirmed the histological findings. Significant downregulation of PHD2 mRNA levels by MC-shPHD2 was confirmed by quantitative polymerase chain reaction. Finally, Western blot analysis confirmed significantly higher levels of HIF-1-alpha protein by MC-shPHD2, compared with PL-shPHD2 and PBS. This study provides initial evidence of a new potential therapeutic approach for peripheral artery disease. The combination of HIF-1-alpha pathway targeting by shPHD2 with the robust nonviral MC plasmid improved postischemic neovascularization, making this approach a promising potential treatment option for critical limb ischemia.

Objective: In atherosclerosis, Toll-like receptors (TLRs) are traditionally linked to effects on tissue macrophages or foam cells. RP105, a structural TLR4 homolog, is an important regulator of TLR signaling. The effects of RP105 on TLR signaling vary for different leukocyte subsets known to be involved in atherosclerosis, making it unique in its role of either suppressing (in myeloid cells) or enhancing (in B cells) TLR-regulated inflammation in different cell types. We aimed to identify a role of TLR accessory molecule RP105 on circulating cells in atherosclerotic plaque formation., Approach and Results: Irradiated low density lipoprotein receptor deficient mice received RP105(-/-) or wild-type bone marrow. RP105(-/-) chimeras displayed a 57% reduced plaque burden. Interestingly, total and activated B-cell numbers were significantly reduced in RP105(-/-) chimeras. Activation of B1 B cells was unaltered, suggesting that RP105 deficiency only affected inflammatory B2 B cells. IgM levels were unaltered, but anti-oxidized low-density lipoprotein and anti-malondialdehyde-modified low-density lipoprotein IgG2c antibody levels were significantly lower in RP105(-/-) chimeras, confirming effects on B2 B cells rather than B1 B cells. Moreover, B-cell activating factor expression was reduced in spleens of RP105(-/-) chimeras., Conclusions: RP105 deficiency on circulating cells results in an intriguing unexpected TLR-associated mechanisms that decrease atherosclerotic lesion formation with alterations on proinflammatory B2 B cells.

Aims: Human autopsy, animal, and cell culture studies together have merged in a concept suggesting participation of mast cells (MCs) in the generation of atherosclerotic plaques. More specifically, these studies have suggested MC-induced intraplaque neovascularization as one mechanism by which MCs may render the plaques vulnerable. The present study was designed to assess the association between MC numbers and neovascularization in human atherosclerotic plaques, and to relate the abundance of plaque MCs to the occurrence of adverse cardiovascular events during the follow-up., Methods and Results: Atherosclerotic plaques of 270 patients suffering from carotid artery stenosis were stained for the presence of MCs (MC tryptase). Furthermore, during a follow-up of 3 years, cardiovascular-related endpoints were assessed in 253 patients. On average a high number of MCs were observed per plaque cross-section [median 108 (55-233) cells per section]. Plaques with high MC numbers revealed an unstable lipid-rich inflammatory phenotype and were associated with symptomatic patients. In addition, MC numbers were positively associated with microvessel density (r = 0.416, P < 0.001). Patients with high intraplaque MC numbers showed significantly more cardiovascular events during the follow-up (58/142 vs. 31/111 events, P = 0.029). In a multivariate analysis with correction for the main risk factors of cardiovascular diseases, MCs remained independently associated with adverse cardiovascular events (P = 0.025)., Conclusion: Mast cells are highly prevalent in human carotid atherosclerotic lesions and associated with plaque microvessel density. Furthermore, intraplaque MC numbers associate with future cardiovascular events.

Objective: Soluble ST2 (sST2), a novel biomarker predictive for heart disease, has recently been shown associated with the progression of atherosclerotic disease in a mouse model. The present study was designed to assess sST2 plasma levels in patients scheduled for carotid endarterectomy and relate it with the occurrence of adverse cardiovascular events during follow-up. In addition, sST2 levels were associated to patient clinical data and atherosclerotic plaque characteristics., Methods and Results: Plasma sST2 levels were measured in 391 patients who underwent carotid endarterectomy and were subsequently followed for 3 years. Primary composite endpoint was the occurrence of an adverse cardiovascular event. At baseline, no differences were observed in sST2 levels between asymptomatic (n = 75) and symptomatic (n = 316) patients (85 [49-122] versus 90 [58-137] pg/ml, p = 0.263). Soluble ST2 plasma levels did not differ between patients who experienced a secondary manifestation of cardiovascular disease and patients who remained free of symptoms (90 [60-129] versus 88 [46-140] pg/ml, p = 0.519). There was no association between sST2 levels and any of the following plaque characteristics: size of a lipid core, degree of calcification, number of macrophages or smooth muscle cells, amount of collagen and number of microvessels., Conclusions: Soluble ST2 plasma levels have no predictive value for future cardiovascular events in patients with significant carotid artery stenosis. In addition, we did not observe an association between plasma sST2 levels and the histopathological features of a rupture prone plaque. This study does not provide supportive evidence that sST2 reflects a progressive state of advanced atherosclerotic disease., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Objectives: To study the role of microRNAs in hypertension-induced vascular pathology before the onset of symptoms of severe cardiovascular disease., Background: MicroRNAs play a crucial role in cardiovascular disease. However, microRNAs are often studied in full-blown cardiovascular disease models, not during development of cardiovascular pathology., Methods: Angiotensin II was infused into healthy adult rats, inducing chronic hypertension, and microRNA expression profiles were obtained. The most prominently regulated microRNA, miR-487b, was further investigated, using primary cultures of rat aortic and human umbilical cord arterial cells., Results: MiR-487b is predicted to target insulin receptor substrate 1 (IRS1). IRS1 plays an important role in both insulin signaling and cell proliferation and survival. IRS1 mRNA and protein levels were downregulated in aortae of hypertensive rats. MiR-487b binds directly to both rat and human IRS1 3'UTR and inhibits reporter gene expression in vitro. In primary rat and human arterial adventitial fibroblasts, inhibition of miR-487b leads to upregulation of IRS1 expression. Upregulation of miR-487b had the opposite effect, confirming direct targeting of IRS1 by miR-487b.Immunohistochemistry of aortic cross sections and rt/qPCR analyses of the separate aortic wall layers showed that both IRS1 and miR-487b were present mainly in the adventitia and less or not at all in the intima and tunica media. IRS1 expression in adventitial fibroblasts was predominantly nuclear and nuclear IRS1 is known to have antiapoptotic effects. Indeed, inhibition of miR-487b protected adventitial fibroblasts, and also medial smooth muscle cells, against serum starvation-induced apoptosis and increased cell survival., Conclusions: Angiotensin II-induced hypertension leads to upregulation of miR-487b, which targets IRS1. Via downregulation of IRS1, miR-487b can contribute to cell death and loss of adventitial and medial integrity during hypertension-induced vascular pathology.

Objective: NK cells are known to be involved in cardiovascular disease processes. One of these processes, vascular remodeling, may strongly differ between individuals and mouse strains such as the C57BL/6 and BALB/c. Moreover, C57BL/6 and BALB/c mice vary in immune responses and in the composition of their Natural Killer gene Complex (NKC). Here we study the role of NK cells, and in particular the C57BL/6 NKC in vascular remodeling and intimal hyperplasia formation., Methods and Results: C57BL/6, BALB/c and CMV1(r) mice, a BALB/c strain congenic for the C57BL/6 NKC, were used in an injury induced cuff model and a vein graft model. NK cell depleted C57BL/6 mice demonstrated a 43% reduction in intimal hyperplasia after femoral artery cuff placement compared to control C57BL/6 mice (p<0.05). Cuff placement and vein grafting resulted in profound intimal hyperplasia in C57BL/6 mice, but also in CMV1(r) mice, whereas this was significantly less in BALB/c mice. Significant more leukocyte infiltrations and IFN-γ staining were seen in both C57BL/6 and CMV1(r) vein grafts compared to BALB/c vein grafts., Conclusions: These data demonstrate an important role for NK cells in intimal hyperplasia and vascular remodeling. Furthermore, the C57BL/6 NKC in CMV1(r) mice stimulates vascular remodeling most likely through the activation of (IFN-γ-secreting) NK-cells that modulate the outcome of vascular remodeling., (© 2013.)

Rationale: RNA-binding proteins are critical post-transcriptional regulators of RNA and can influence pre-mRNA splicing, RNA localization, and stability. The RNA-binding protein Quaking (QKI) is essential for embryonic blood vessel development. However, the role of QKI in the adult vasculature, and in particular in vascular smooth muscle cells (VSMCs), is currently unknown., Objective: We sought to determine the role of QKI in regulating adult VSMC function and plasticity., Methods and Results: We identified that QKI is highly expressed by neointimal VSMCs of human coronary restenotic lesions, but not in healthy vessels. In a mouse model of vascular injury, we observed reduced neointima hyperplasia in Quaking viable mice, which have decreased QKI expression. Concordantly, abrogation of QKI attenuated fibroproliferative properties of VSMCs, while potently inducing contractile apparatus protein expression, rendering noncontractile VSMCs with the capacity to contract. We identified that QKI localizes to the spliceosome, where it interacts with the myocardin pre-mRNA and regulates the splicing of alternative exon 2a. This post-transcriptional event impacts the Myocd&#95;v3/Myocd&#95;v1 mRNA balance and can be modulated by mutating the quaking response element in exon 2a of myocardin. Furthermore, we identified that arterial damage triggers myocardin alternative splicing and is tightly coupled with changes in the expression levels of distinct QKI isoforms., Conclusions: We propose that QKI is a central regulator of VSMC phenotypic plasticity and that intervention in QKI activity can ameliorate pathogenic, fibroproliferative responses to vascular injury.

Objective: T-cells are central to the immune response responsible for native atherosclerosis. The objective of this study is to investigate T-cell contribution to post-interventional accelerated atherosclerosis development, as well as the role of the CD28-CD80/86 co-stimulatory and Cytotoxic T-Lymphocyte Antigen (CTLA)-4 co-inhibitory pathways controlling T-cell activation status in this process., Methods and Results: The role of T-cells and the CD28-CD80/86 co-stimulatory and CTLA-4 co-inhibitory pathways were investigated in a femoral artery cuff mouse model for post-interventional remodeling, with notable intravascular CTLA-4+ T-cell infiltration. Reduced intimal lesions developed in CD4(-/-) and CD80(-/-)CD86(-/-) mice compared to normal C57Bl/6J controls. Systemic abatacept-treatment, a soluble CTLA-4Ig fusion protein that prevents CD28-CD80/86 co-stimulatory T-cell activation, prevented intimal thickening by 58.5% (p=0.029). Next, hypercholesterolemic ApoE3*Leiden mice received abatacept-treatment which reduced accelerated atherosclerosis development by 78.1% (p=0.040) and prevented CD4 T-cell activation, indicated by reduced splenic fractions of activated KLRG1+, PD1+, CD69+ and CTLA-4+ T-cells. This correlated with reduced plasma interferon-γ and elevated interleukin-10 levels. The role of CTLA-4 was confirmed using CTLA-4 blocking antibodies, which strongly increased vascular lesion size by 66.7% (p=0.008), compared to isotype-treated controls., Conclusions: T-cell CD28-CD80/86 co-stimulation is vital for post-interventional accelerated atherosclerosis development and is regulated by CTLA-4 co-inhibition, indicating promising clinical potential for prevention of post-interventional remodeling by abatacept., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Background: Coronary restenosis after percutaneous coronary intervention (PCI) still remains a significant limitation of the procedure. The causative mechanisms of restenosis have not yet been fully identified. The goal of the current study was to perform gene-set analysis of biological pathways related to inflammation, proliferation, vascular function and transcriptional regulation on coronary restenosis to identify novel genes and pathways related to this condition., Methods: The GENetic DEterminants of Restenosis (GENDER) databank contains genotypic data of 556,099SNPs of 295 cases with restenosis and 571 matched controls. Fifty-four pathways, related to known restenosis-related processes, were selected. Gene-set analysis was performed using PLINK, GRASS and ALIGATOR software. Pathways with a p<0.01 were fine-mapped and significantly associated SNPs were analyzed in an independent replication cohort., Results: Six pathways (cell-extracellular matrix (ECM) interactions pathway, IL2 signaling pathway, IL6 signaling pathway, platelet derived growth factor pathway, vitamin D receptor pathway and the mitochondria pathway) were significantly associated in one or two of the software packages. Two SNPs in the cell-ECM interactions pathway were replicated in an independent restenosis cohort. No replication was obtained for the other pathways., Conclusion: With these results we demonstrate a potential role of the cell-ECM interactions pathway in the development of coronary restenosis. These findings contribute to the increasing knowledge of the genetic etiology of restenosis formation and could serve as a hypothesis-generating effort for further functional studies.

Acute cardiovascular syndromes such as myocardial infarction and stroke are a major cause of death in the Western society and are generally caused by rupture of an atherosclerotic plaque. Treatment of atherosclerosis, the main underlying cause of acute cardiovascular syndromes, is still inadequate for most of the patients. Therefore, there is a need for new therapeutic strategies in addition to the existing lipid-lowering drugs such as statins. Lipid accumulation, inflammation and matrix degradation are generally considered key processes in the pathogenesis of atherosclerosis and that of plaque rupture. Furthermore, apoptosis or programmed cell death of plaque cells, depending on the disease stage, is thought to be of importance in the development and progression of atherosclerosis and the incidence of acute cardiovascular syndromes. Serine protease inhibitors or so-called serpins have been demonstrated to be involved in both the induction and inhibition of apoptosis and may thus be of interest as therapeutics in cardiovascular diseases such as atherosclerosis. In this review, we will discuss the current knowledge on the role of serpins in cardiovascular diseases with particular emphasis on apoptotic cell death and the potential therapeutic applications.

Objective: Therapeutic arteriogenesis, that is, expansive remodeling of preexisting collaterals, using single-action factor therapies has not been as successful as anticipated. Modulation of factors that act as a master switch for relevant gene programs may prove more effective. Transcriptional coactivator p300-CBP-associated factor (PCAF) has histone acetylating activity and promotes transcription of multiple inflammatory genes. Because arteriogenesis is an inflammation-driven process, we hypothesized that PCAF acts as multifactorial regulator of arteriogenesis., Approach and Results: After induction of hindlimb ischemia, blood flow recovery was impaired in both PCAF(-/-) mice and healthy wild-type mice treated with the pharmacological PCAF inhibitor Garcinol, demonstrating an important role for PCAF in arteriogenesis. PCAF deficiency reduced the in vitro inflammatory response in leukocytes and vascular cells involved in arteriogenesis. In vivo gene expression profiling revealed that PCAF deficiency results in differential expression of 3505 genes during arteriogenesis and, more specifically, in impaired induction of multiple proinflammatory genes. Additionally, recruitment from the bone marrow of inflammatory cells, in particular proinflammatory Ly6C(hi) monocytes, was severely impaired in PCAF(-/-) mice., Conclusions: These findings indicate that PCAF acts as master switch in the inflammatory processes required for effective arteriogenesis.

Background: RP105 (CD180) is TLR4 homologue lacking the intracellular TLR4 signaling domain and acts a TLR accessory molecule and physiological inhibitor of TLR4-signaling. The role of RP105 in vascular remodeling, in particular post-interventional remodeling is unknown., Methods and Results: TLR4 and RP105 are expressed on vascular smooth muscle cells (VSMC) as well as in the media of murine femoral artery segments as detected by qPCR and immunohistochemistry. Furthermore, the response to the TLR4 ligand LPS was stronger in VSMC from RP105(-/-) mice resulting in a higher proliferation rate. In RP105(-/-) mice femoral artery cuff placement resulted in an increase in neointima formation as compared to WT mice (4982 ± 974 µm(2) vs.1947 ± 278 µm(2),p = 0.0014). Local LPS application augmented neointima formation in both groups, but in RP105(-/-) mice this effect was more pronounced (10316±1243 µm(2) vs.4208 ± 555 µm(2),p = 0.0002), suggesting a functional role for RP105. For additional functional studies, the extracellular domain of murine RP105 was expressed with or without its adaptor protein MD1 and purified. SEC-MALSanalysis showed a functional 2∶2 homodimer formation of the RP105-MD1 complex. This protein complex was able to block the TLR4 response in whole blood ex-vivo. In vivo gene transfer of plasmid vectors encoding the extracellular part of RP105 and its adaptor protein MD1 were performed to initiate a stable endogenous soluble protein production. Expression of soluble RP105-MD1 resulted in a significant reduction in neointima formation in hypercholesterolemic mice (2500 ± 573 vs.6581 ± 1894 µm(2),p<0.05), whereas expression of the single factors RP105 or MD1 had no effect., Conclusion: RP105 is a potent inhibitor of post-interventional neointima formation.