[Show abstract][Hide abstract] ABSTRACT: Background:
Experimental studies characterize adaptive immune response as a critical factor in the progression and complications of atherosclerosis. Yet, it is unclear whether these observations translate to the human situation. This study systematically evaluates cellular components of the adaptive immune response in a biobank of human aortas covering the full spectrum of atherosclerotic disease.
Methods and results:
A systematic analysis was performed on 114 well-characterized perirenal aortic specimens with immunostaining for T-cell subsets (CD3/4/8/45RA/45RO/FoxP3) and the Th1/non-Th1/Th17 ratio (CD4(+)T-bet(+)/CD4(+)T-bet(-)/CD4(+)/interleukin-17(+) double staining). CD20 and CD138 were used to identify B cells and plasma cells, while B-cell maturation was evaluated by AID/CD21 staining and expression of lymphoid homeostatic CXCL13. Scattered CD4 and CD8 cells with a T memory subtype were found in normal aorta and early, nonprogressive lesions. The total number of T cells increases in progressive atherosclerotic lesions (≈1:5 CD4/CD8 T-cell ratio). A further increase in medial and adventitial T cells is found upon progression to vulnerable lesions.This critical stage is further hallmarked by de novo formation of adventitial lymphoidlike structures containing B cells and plasma cells, a process accompanied by transient expression of CXCL13. A dramatic reduction of T-cell subsets, disappearance of lymphoid structures, and loss of CXCL13 expression characterize postruptured lesions. FoxP3 and Th17 T cells were minimally present throughout the atherosclerotic process.
Transient CXCL13 expression, restricted presence of B cells in human atherosclerosis, along with formation of nonfunctional extranodal lymphoid structures in the phase preceding plaque rupture, indicates a "critical" change in the inflammatory footprint before and during plaque destabilization.
Journal of the American Heart Association 03/2015; 4(4):1403. DOI:10.1161/JAHA.114.001403 · 4.31 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Modulation of immune responses may form a powerful approach to treat atherosclerosis. It was shown that clearance of apoptotic cells results in tolerance induction to cleared Ags by dendritic cells (DCs); however, this seems impaired in atherosclerosis because Ag-specific tolerance is lacking. This could result, in part, from decreased emigration of DCs from atherosclerotic lesions because of the high-cholesterol environment. Nonetheless, local induction of anti-inflammatory responses by apoptotic cell clearance seems to dampen atherosclerosis, because inhibition of apoptotic cell clearance worsens atherosclerosis. In this study, we assessed whether i.v. administration of oxLDL-induced apoptotic DCs (apop(ox)-DCs) and, as a control, unpulsed apoptotic DCs could modulate atherosclerosis by inducing tolerance. Adoptive transfer of apop(ox)-DCs into low-density lipoprotein receptor knockout mice either before or during feeding of a Western-type diet resulted in increased numbers of CD103(+) tolerogenic splenic DCs, with a concomitant increase in regulatory T cells. Interestingly, both types of apoptotic DCs induced an immediate 40% decrease in Ly-6C(hi) monocyte numbers and a 50% decrease in circulating CCL2 levels, but only apop(ox)-DC treatment resulted in long-term effects on monocytes and CCL2 levels. Although initial lesion development was reduced by 40% in both treatment groups, only apop(ox)-DC treatment prevented lesion progression by 28%. Moreover, progressed lesions of apop(ox)-DC-treated mice showed a robust 45% increase in collagen content, indicating an enhanced stability of lesions. Our findings clearly show that apoptotic DC treatment significantly decreases lesion development, but only apop(ox)-DCs can positively modulate lesion progression and stability. These findings may translate into a safe treatment for patients with established cardiovascular diseases using patient-derived apop(ox)-DCs.
The Journal of Immunology 02/2015; 194(5). DOI:10.4049/jimmunol.1401843 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: The ubiquitously expressed mannose-6-phosphate receptors (MPRs) are a promising class of receptors for targeted compound delivery into the endolysosomal compartments of a variety of cell types. The development of a synthetic, multivalent, mannose-6-phosphate (M6P) glycopeptide-based MPR ligand is described. The conjugation of this ligand to fluorescent DCG-04, an activity-based probe for cysteine cathepsins, enabled fluorescent readout of its receptor-targeting properties. The resulting M6P-cluster–BODIPY–DCG-04 probe was shown to efficiently label cathepsins in cell lysates as well as in live cells. Furthermore, the introduction of the 6-O-phosphates leads to a completely altered uptake profile in COS and dendritic cells compared to a mannose-containing ligand. Competition with mannose-6-phosphate abolished all uptake of the probe in COS cells, and we conclude that the mannose-6-phosphate cluster targets the MPR and ensures the targeted delivery of cargo bound to the cluster into the endolysosomal pathway.
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Cellular and Molecular Medicine 08/2014; 18(10). DOI:10.1111/jcmm.12357 · 4.01 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Rationale: Activated mast cells have been identified in atherosclerotic plaques and have previously been established to promote plaque progression and destabilization. As mast cells have the ability to release chemokines that mediate leucocyte fluxes, we propose that activated mast cells play a pivotal role in leucocyte recruitment during the development of atherosclerosis.
Methods and Results: Western-type diet fed B cell deficient apoE-/-muChain mice, which lack endogenous IgE, were systemically challenged with either IgE or PBS 6 times over a period of 8 weeks to induce mast cell activation during atherosclerotic lesion development. Mast cell activation in the aortic root was indeed significantly enhanced after IgE treatment (control: 35.2 ± 3.9% versus IgE: 48.2 ± 3.4% of activated mast cells, *P<0.05) and we observed a concomitant increase in plaque size (control: 2.0 ± 0.2*10E5 square μm versus IgE: 2.8 ± 0.3*10E5 square μm, P=0.05). Intriguingly, a striking increase in the amount of perivascular neutrophils was observed in the IgE treated mice (control: 57.6 ± 10.6 neutrophils/square mm tissue versus IgE: 183.0 ± 38.7 neutrophils/square mm tissue; *P<0.05). In order to investigate whether activated mast cells can directly attract neutrophils, we injected C57Bl/6 or mast cell deficient Kit (W-sh/W-sh) mice intraperitoneally with the mast cell activator compound 48/80. Mast cell activation led to a massive neutrophil influx into the peritoneal cavity (controls: 1.0 ± 0.6 versus compound 48/80: 5.1 ± 0.7, given in fold change, **P<0.01), while neutrophil numbers in mast cell deficient mice remained unaffected (controls: 1.0 ± 0.3 versus compound 48/80: 1.0 ± 0.2, P=NS). Furthermore, the newly recruited neutrophils were particularly CXCR2+ and/or CXCR4+. Interestingly, mast cells have been shown to secrete the CXCR2 and CXCR4 ligands CXCL1 and CXCL12, respectively. In vitro, supernatant of activated mast cells caused a 3-fold increase in neutrophil migration (32.3 ± 4.7*10E3 of migrated neutrophils versus 11.6 ± 2.5*10E3 neutrophils after stimulation with control supernatant, **P<0.01), which was seen to be inhibited by anti-CXCR2 (18.8 ± 2.2*10E3 neutrophils,*P<0.05), but not by the CXCR4 receptor antagonist AMD3100 (24.8 ± 6.9*10E3 neutrophils, P=NS).
Conclusions: In this study we demonstrate that chemokines, in particular CXCL1, released from activated perivascular mast cells induce neutrophil recruitment to the atherosclerotic plaque, thereby aggravating the inflammatory response which may further enhance atherosclerotic lesion progression and destabilization.
Cardiovascular Research 07/2014; 103(suppl 1):S5. DOI:10.1093/cvr/cvu078.1 · 5.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Toll-like-receptors (TLRs) provide a critical link between innate and adaptive immune responses. It has been shown that TLR5 ligand Flagellin can enhance the suppressive capacity of regulatory T-cells (Treg), but can also functions as an adjuvant. The immune response in atherosclerosis is characterized by an imbalance of pro- and anti-atherogenic T-cells. We aimed to establish if the TLR5/Flagellin axis is involved in the immune response of atherosclerosis. Methods: We first assessed the effect of Flagellin exposure on macrophage maturation and T-cell polarization. Next, we created TLR5-/-/LDLr-/- chimeras to study the TLR5/Flagellin axis in atherosclerosis. Results: Flagellin exposure to primary macrophages did not result in clear polarization differences, but we did observe a less migratory phenotype (decreased MCP-1, CCR2 expression) in TLR5-/- macrophages. Interestingly, expression of the T-cell polarizing cytokine IL-6 was induced by Flagellin exposure, a phenomenon not observed in TLR5-/- macrophages. Next, we assessed potential T cell polarizing properties of Flagellin. Flagellin can induce expansion of regulatory T-cells, however this induction is completely overruled when Flagellin is used as an adjuvant. Hematopoietic absence of TLR5 significantly attenuates atherosclerotic lesion formation by 25% (1.03±0.06×10(6) μm(2) vs 0.79±0.06 ×10(6) μm(2) in TLR5-/-, p = 0.01). This was accompanied by a decrease in macrophage area (-46%, p = 0.01) and necrotic core size (-32%, p<0.05) while collagen content was similar between groups. Interestingly, plasma levels of IL-6 were significantly lower in TLR5-/- chimeras (40.2±6.3 in WT vs. 15.1±2.7 pg/ml in TLR5-/-, p=0.003). Concomitantly, TLR5-/- chimeras displayed defective T-cell responsiveness, as seen by impaired proliferation and decreased splenic T cell content. In conclusion, hematopoietic TLR5 deficiency inhibits atherosclerotic lesion formation by attenuated macrophage accumulation and defective T cell responsiveness.
Cardiovascular Research 07/2014; 103(suppl 1):S5. DOI:10.1093/cvr/cvu078.2 · 5.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Aims
Genetic factors explain a proportion of the inter-individual variation in the risk for atherosclerotic events, but the genetic basis of atherosclerosis and atherothrombosis in families with Mendelian forms of premature atherosclerosis is incompletely understood. We set out to unravel the molecular pathology in a large kindred with an autosomal dominant inherited form of premature atherosclerosis.
Methods and Results
Parametric linkage analysis was performed in a pedigree comprising 4 generations, of which a total of 11 members suffered from premature vascular events. A parametric LOD-score of 3.31 was observed for a 4.4 Mb interval on chromosome 12. Upon sequencing, a non-synonymous variant in KERA (c.920C>G; p.Ser307Cys) was identified. The variant was absent from nearly 28,000 individuals, including 2,571 patients with premature atherosclerosis. KERA, a proteoglycan protein, was expressed in lipid-rich areas of human atherosclerotic lesions, but not in healthy arterial specimens. Moreover, KERA expression in plaques was significantly associated with plaque size in a carotid-collar Apoe−/− mice (r2 = 0.69; p<0.0001).
A rare variant in KERA was identified in a large kindred with premature atherosclerosis. The identification of KERA in atherosclerotic plaque specimen in humans and mice lends support to its potential role in atherosclerosis.
PLoS ONE 05/2014; 9(5):e98289. DOI:10.1371/journal.pone.0098289 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Aim:
Neuropeptide Y is an abundantly expressed neurotransmitter capable of modulating both immune and metabolic responses related to the development of atherosclerosis. NPY receptors are expressed by a number of vascular wall cell types, among which mast cells. However, the direct effects of NPY on atherosclerotic plaque development and progression remain to be investigated. In this study we thus aimed to determine whether NPY is expressed in atherosclerotic plaques and to establish its role in atherosclerotic plaque development.
Methods and results:
NPY expression was seen to be increased up to 2-fold in unstable human endarterectomy plaques, as compared to stable plaques, and to be significantly upregulated during lesion progression in apoE(-/-) mice. In apoE(-/-) mice focal overexpression of NPY in the carotid artery significantly increased atherosclerotic plaque size compared to controls, while plaque composition was unaffected. Interestingly, perivascular mast cell activation was significantly higher in the NPY-overexpressing mice, suggesting that NPY may impact plaque progression in part via mast cell activation. Furthermore, in vitro NPY-induced murine mast cell activation resulted in the release of pro-atherogenic mediators including IL-6 and tryptase.
Our data show that NPY expression is increased during atherogenesis and in particular in unstable plaques. Furthermore, perivascular overexpression of NPY promoted plaque development and perivascular mast cell activation, suggestive of a role for NPY-induced mast cell activation in lesion progression.