[Show abstract][Hide abstract] ABSTRACT: Background: Atherosclerosis, the major cause of heart disease and stroke, is a lipid driven chronic inflammatory disease that is characterized by the continuous recruitment of inflammatory cells to the atherosclerotic plaque, a process orchestrated by chemokines. Abundant expression of the chemokine CCL18 (PARC) is observed in human atherosclerotic plaques, and especially in unstable regions. As the exact role of CCL18 in atherogenesis is currently unknown, we aimed to map the effects of focal and systemic CCL18 overexpression on plaque progression and stability. Furthermore, we identify CCR6 as a functional receptor for CCL18 that mediates CCL18 effects and activates intracellular signalling.
Methods: The study included in vivo analyses in patients with carotid atherosclerosis and in vitro experiments in cells involved in atherogenesis. For animals study, animals received CCL18 and were characterized the molecular mechanisms by histological (immunohistochemistry, immunofluorescence for TUNEL) and biochemical methods (Western blots, Real Time PCR, Ca2+ mobilization).
Results: We report the profound up-regulation of the expression of CCL18 in ruptured human atherosclerotic plaque, in particular by macrophages. Systemic gain of function in a mouse model of atherosclerosis indicated pro-atherogenic activity of CCL18; while plaque-targeted CCL18 overexpression was associated with increased Ca2+ mobilization in T cells. Importantly, we identify CCR6 as a functional receptor mediating CCL18 chemotaxis, intracellular Ca2+ mobilization and downstream signaling in human Jurkat T cells while in mouse CD4+ and CD8+ T cells these processes were abolished in the absence of CCR6. Subcutaneous CCL18 administration led to profound skin inflammation in WT but not CCR6-/- mice, characterized by edema, marked infiltration of neutrophils/macrophages and T cells, with a clear Th17 signature, compatible with the role of CCR6 as a migration receptor for Th17 cells.
Conclusions: Our studies identify CCR6 as bona fide receptor for CCL18, mediating chemotaxis of neutrophils, macrophages and Th17 cells, and augmenting inflammation and destabilization of the plaque. Our work sheds light on the mechanisms underlying CCL18-induced cardiovascular risk and may pave the way for targeted treatment and prevention of atherosclerosis.
Cardiovascular Research 07/2014; 103(suppl 1):S15-S16. DOI:10.1093/cvr/cvu082.35 · 5.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
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_v3/Myocd_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.
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.
Circulation Research 08/2013; 113(9). DOI:10.1161/CIRCRESAHA.113.301302 · 11.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Objective:
Despite common disbelief that neutrophils are involved in atherosclerosis, evidence is accumulating for a causal role of neutrophils in atherosclerosis. CC chemokine ligand (CCL)3 is an inflammatory chemokine and its expression is significantly increased during atherosclerotic lesion formation in mice. It has recently been shown that under conditions of inflammation neutrophils can migrate along a CCL3 gradient. In this study, we aimed to elucidate the role of leukocyte-derived CCL3 in atherogenesis.
Methods and results:
Irradiated low density lipoprotein receptor(-/-) mice, reconstituted with CCL3(-/-) or littermate bone marrow showed markedly reduced CCL3 response to lipopolysaccharide treatment, establishing the critical relevance of leukocytes as source of CCL3. Hematopoietic deficiency of CCL3 significantly reduced aortic sinus lesion formation by 31% after 12 weeks of western-type diet. Interestingly, whereas plaque macrophage, collagen, and vascular smooth muscle cell content were unchanged, neutrophil adhesion to and presence in plaques was significantly attenuated in CCL3(-/-) chimeras. These mice had reduced circulating neutrophil numbers, which could be ascribed to an increased neutrophil turnover and CCL3(-/-) neutrophils were shown to be less responsive toward the neutrophil chemoattractant CXC chemokine ligand 1.
Our data indicate that under conditions of acute inflammation leukocyte-derived CCL3 can induce neutrophil chemotaxis toward the atherosclerotic plaque, thereby accelerating lesion formation.
[Show abstract][Hide abstract] ABSTRACT: Cytokines play an important role in ischemic injury and repair. However, little is known about their prognostic value in cardiovascular disease. The aim of this study was to investigate the prognostic importance of chemokines CCL3/MIP-1α, CCL5/RANTES and CCL18/PARC for the risk of future cardiovascular events in patients with acute coronary syndromes (ACS). Baseline levels of CCL3/MIP-1α, CCL5/RANTES and CCL18/PARC were determined in ACS patients from the Bad Nauheim ACS II registry (n = 609). During the following 200 days, patients were monitored for the occurrence of fatal and non-fatal cardiovascular events. Patients with CCL3/MIP1α, CCL5/RANTES and CCL18/PARC concentrations in the highest tertile were associated with an increased risk of a fatal event during follow-up (HR: 2.19, 95%CI: 1.04-4.61 for CCL3/MIP1α, HR: 3.45, 95%CI: 1.54-7.72 for CCL5/RANTES and HR: 3.14, 95%CI: 1.33-7.46 for CCL18/PARC). This risk was highest for patients with all three biomarkers concentrations in the upper tertile (HR: 2.52, 95%CI: 1.11-5.65). Together with known risk predictors of cardiovascular events, CCL3/MIP-1α, CCL5/RANTES and CCL18/PARC combined improved the c-statistics from 0.74 to 0.81 (p = 0.007). In conclusion, CCL3/MIP-1α, CCL5/RANTES and CCL18/PARC are independently associated with the risk of short-term mortality in ACS patients. Combining all three biomarkers further increased their prognostic value.
PLoS ONE 09/2012; 7(9):e45804. DOI:10.1371/journal.pone.0045804 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chemokines are instrumental in the initiation and progression of atherosclerosis. Recent advances in genomic technologies and the recognition of atherosclerosis as an inflammatory disease have given great impetus to studies addressing the relevance of chemokines for the clinically manifest stages of atherosclerosis and acute cardiovascular syndromes. In this paper, we will review the current status of these studies, highlighting those chemokines that have already been associated with plaque destabilization and rupture. We will recapitulate recent epidemiologic, genomic, histopathological and experimental support for the prominent role of particular chemokines in acute cardiovascular syndromes. Collectively, these data underpin the potential of chemokines as biomarkers and/or therapeutic targets, but also expose the lacunae in our understanding of the precise function of chemokines in the atherosclerosis-related disorders and in the efficacy of chemokine-targeted clinical trials.
[Show abstract][Hide abstract] ABSTRACT: A dysbalance of proteases and their inhibitors is instrumental in remodeling of atherosclerotic plaques. One of the proteases implicated in matrix degradation is cathepsin-S (CatS). To address its role in advanced lesion composition, we generated chimeric LDLr(-/-) mice deficient in leukocyte CatS by transplantation with CatS(-/-)xLDLr(-/-) or with LDLr(-/-) bone marrow and administered a high-fat diet.
No difference in aortic root lesion size could be detected between CatS(+/+) and CatS(-/-) chimeras. However, leukocyte CatS deficiency markedly changed plaque morphology and led to a dramatic reduction in necrotic core area by 77% and an abundance of large foam cells. Plaques of CatS(-/-) chimeras contained 17% more macrophages, 62% less SMCs, and 33% less intimal collagen. The latter two could be explained by a reduced number of elastic lamina fractures. Moreover, macrophage apoptosis was reduced by 60% with CatS deficiency. In vitro, CatS was found to be involved in cholesterol metabolism and in macrophage apoptosis in a collagen and fibronectin matrix.
Leukocyte CatS deficiency results in considerably altered plaque morphology, with smaller necrotic cores, reduced apoptosis, and decreased SMC content and collagen deposition and may thus be critical in plaque stability.
[Show abstract][Hide abstract] ABSTRACT: Cholesterol ester transfer protein (CETP) plays an important role in HDL cholesterol metabolism. Leucocytes, including monocyte-derived macrophages in the arterial wall synthesize and secrete CETP, but its role in atherosclerosis is unclear. The aim of the current study was to investigate the effect of acute coronary syndromes (ACS) on leucocyte CETP expression.
Peripheral blood mononuclear cells (PBMCs) were freshly isolated from hospitalized ACS patients displaying Braunwald class IIIB unstable angina pectoris (UAP) on admission (t = 0) and at 180 days post inclusion (t = 180) for analysis of CETP expression. In addition, to prove the potential correlation between leucocyte CETP and ACS the effect of acute myocardial infarction on leucocyte CETP expression was studied in CETP transgenic mice.
Upon admission, UAP patients displayed approximately 3-6 fold (P < 0.01) lower CETP mRNA and nearly absent CETP protein expression in PBMCs, as compared to healthy age-/sex-matched controls. Interestingly, CETP mRNA and protein levels were significantly elevated in PBMCs isolated from UAP patients (both stabilized and refractory) at t = 180 as compared to t = 0 (P < 0.01), which was correlated with a reduced inflammatory status after medical treatment. In agreement with the data obtained in UAP patients, markedly down-regulated leucocyte CETP mRNA expression was observed after coronary artery ligation in CETP transgenic mice, which also correlated with increased serum amyloid A levels.
We are the first to report that episodes of UAP in humans and myocardial infarction in CETP transgenic mice are associated with reduced leucocyte CETP expression. We propose that the impairment in leucocyte CETP production is associated with an enhanced inflammatory status, which could be clinically relevant for the pathogenesis of ACS.
Journal of Internal Medicine 10/2008; 264(6):571-85. DOI:10.1111/j.1365-2796.2008.01997.x · 6.06 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: As chemokines are considered instrumental in thrombotic plaque rupture and erosion as well as in ischemia-reperfusion injury processes, we aimed to identify previously unknown chemokines associated with acute coronary syndromes. Plasma of 44 patients with acute myocardial infarction (AMI) and 22 controls were profiled for a panel of chemokines by multiplex analysis. Levels of CCL3 were prospectively verified in 54 patients with unstable angina pectoris (UAP). An AMI mouse model was used to assess the relationship between differentially expressed chemokines and myocardial ischemia. CCL3 levels were significantly elevated in AMI vs. controls (P=0.02) albeit, that adjustment for confounding factors attenuated this association. In support of a direct association with cardiac ischemia CCL3 levels were also seen to be elevated in patients with UAP at baseline and significantly down-regulated after 180 days (P<0.001). Importantly, baseline upper quartile levels were strongly correlated with future acute coronary syndromes (Likelihood Ratio 11.5; P<0.01). Furthermore circulating levels of CCL3 were significantly enhanced after AMI in mice (P=0.02), while CCR5(+) T-cell numbers were increased as well, suggestive of CCL3 driven T-cell homing towards the ischemic area. CCL3 levels are elevated during ACS and released upon ischemia. Since CCL3 specifically predicts future cardiovascular events, it may serve as a predictive biomarker.
Journal of Molecular and Cellular Cardiology 06/2008; 45(3):446-52. DOI:10.1016/j.yjmcc.2008.06.003 · 4.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chemokines play an important role in atherogenesis and in ischemic injury and repair; however, prospective data on individual chemokines in unstable angina pectoris (UAP) are scarce. Therefore, we assessed chemokine patterns in a prospective cohort of patients with UAP.
Plasma samples of 54 patients with Braunwald class IIIB UAP were examined at baseline for 11 chemokines and 5 inflammatory mediators via multiplex analysis. Levels of CC chemokine ligand (CCL)-5 (also known as RANTES [regulated on activation, normally T-cell expressed, and secreted]; 32.7 versus 23.1 ng/mL, P=0.018) and CCL18 (also known as PARC [pulmonary and activation-regulated chemokine]; 104.4 versus 53.7 ng/mL, P=0.011) were significantly elevated in patients with refractory ischemic symptoms versus stabilized patients. Temporal monitoring by ELISA of CCL5, CCL18, and soluble CD40 ligand (sCD40) levels revealed a drop in CCL5 and sCD40L levels in all UAP patients from day 2 onward (CCL5 12.1 ng/mL, P<0.001; sCD40L 1.35 ng/mL, P<0.05), whereas elevated CCL18 levels were sustained for at least 2 days, then were decreased at 180 days after inclusion (34.5 ng/mL, P<0.001). Peripheral blood mononuclear cells showed increased protein expression of chemokine receptors CCR3 and CCR5 in CD3+ and CD14+ cells at baseline compared with 180 days after inclusion, whereas mRNA levels were downregulated, which was attributable in part to a postischemic release of human neutrophil peptide-3-positive neutrophils and in part to negative feedback. Finally, elevated CCL5 and CCL18 levels predicted future cardiovascular adverse events, whereas C-reactive protein and sCD40L levels did not.
We are the first to report that CCL18 and CCL5 are transiently raised during episodes of UAP, and peak levels of both chemokines are indicative of refractory symptoms. Because levels of both chemokines, as well as of cognate receptor expression by circulating peripheral blood mononuclear cells, are increased during cardiac ischemia, this may point to an involvement of CCL5/CCL18 in the pathophysiology of UAP and/or post-UAP responses.
[Show abstract][Hide abstract] ABSTRACT: Atherosclerosis is currently viewed as an inflammatory disease in which the initiation and progression of the atherosclerotic plaque towards a rupture prone, unstable plaque is driven by leukocyte recruitment mediated by various inflammatory mediators. Recently, interest in chemotactic cytokines or chemokines with regard to atherosclerosis has been growing as chemokines mediate the influx of leukocytes that is typical of atherothrombosis. The activity of the majority of chemokines is overlapping and chemokines are not only produced by the various cellular constituents of the atherosclerotic plaque but also by activated platelets. Consequently, the direct influence of individual chemokines on plaque destabilisation and rupture is widespread and rather unclear. Experimental research has already established the role of a number of chemokines in advanced atherosclerosis. Nevertheless, given the complexity and size of the chemokine family, further screening of cardiovascular disease for chemokine level and genetic polymorphisms for chemokines will be warranted as the search for viable biomarkers of plaque destabilization as well as novel therapeutic targets for specific atheroregressive therapeutic compounds is ongoing. With regard to the latter, clinical trials with specific chemokine inhibitory strategies, like chemokine receptor antagonists, are already underway in other inflammatory disorders. Summarizing, chemokine inhibition likely constitutes an important therapeutic option next to already established drugs in the management of cardiovascular disease.
Current pharmaceutical design 02/2007; 13(10):1039-52. DOI:10.2174/138161207780487584 · 3.45 Impact Factor