RP105-/- chimeras displayed a 57% reduced plaque burden in the proximal .... classified into groups of stable and vulnerable plaques using Trichrome stain-.
Atherosclerosis
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P2389 | BENCH RP105 (cd180) as a TLR-4 regulator ameliorates atherosclerosis via its role on B-cells
P2391 | BENCH Myeloid A Disintegrin And Metalloprotease ADAM10 deficiency promotes atherosclerotic plaque stability by increasing fibrosis
J. Karper 1 , S.C.A. De Jager 2 , M.M. Ewing 1 , M.R. De Vries 1 , R. Arens 3 , I. Bot 1 , J.W. Jukema 4 , J. Kuiper 2 , P. Quax 1 . 1 Leiden University Medical Center, Department of Surgery, Leiden, Netherlands; 2 Leiden University, Div of Biopharmaceutics, LACDR, Leiden, Netherlands; 3 Leiden University Medical Center, Department of Immunohematology and Blood Transfusion, Leiden, Netherlands; 4 Leiden University Medical Center, Department of Cardiology, Leiden, Netherlands
E. Van Der Vorst 1 , I.M.J. Wolfs 1 , S. Weber 2 , M.J. Gijbels 1 , S. Rose-John 2 , A. Ludwig 3 , M.P.J. De Winther 4 , E.A.L. Biessen 1 , P. Saftig 2 , M.M.P.C. Donners 1 . 1 Maastricht University, Cardiovascular Research Institute Maastricht (CARIM), Maastricht, Netherlands; 2 Institute of Biochemistry University Medical Center Kiel, Kiel, Germany; 3 RWTH University Hospital Aachen, Medical Faculty, Aachen, Germany; 4 Academic Medical Center, University of Amsterdam, Department of Medical Biochemistry, Amsterdam, Netherlands
Background: Toll Like Receptors (TLR) are major contributors to cardiovascular disease development and TLR4 is strongly involved in atherosclerosis. RP105, a structural TLR4 homolog, is an important regulator of TLR4 signaling. RP105 dampens the TLR4 response in macrophages and dendritic cells while it enhances B-cells activation. A role for RP105 in atherosclerosis is currently unknown. Methods and results: Irradiated LDLR-/- mice received RP105-/- or wild-type bone marrow. After recovery, mice were fed a hypercholesterolemic diet for 9wks. RP105-/- chimeras displayed a 57% reduced plaque burden in the proximal aorta and lesions contained significantly less macrophages and CD3+T-cells. FACS analysis revealed no differences in activation of DCs, macrophages or Tcells. 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 affects inflammatory B2-B-cells. Moreover BAFF expression was reduced in the spleens of the RP105-/-chimeras. Further in vitro analysis of CD19+ B-cells from RP105-/-mice showed reduced activation, proliferation and cell cycle entry in a TLR4-dependent manner. Anti-oxLDL and anti-MDA-LDL IgG2c antibody levels were significantly lower in RP105-/- chimeras. IgM levels were unaltered, confirming effects on B2-B-cells rather than B1-B-cells. Conclusion: We demonstrate that RP105 deficiency on hematopoietic cells reduced atherosclerotic plaque formation in a B cell dependent manner. RP105 can modulate atherosclerotic lesion development by affecting inflammatory B2 B-cells via modulation of antibody and cytokine production in a TLR4 dependent manner.
Rationale: ADAM10 is a metalloprotease involved in cleavage of various cell surface molecules such as adhesion molecules, chemokines and growth factor receptors. Although we previously showed an association of ADAM10 expression with atherosclerotic plaque progression, a causal role of ADAM10 in atherosclerosis has not been investigated so far. Methods and results: Bone marrow of conditional knockout mice lacking ADAM10 in the myeloid lineage or littermate controls was transplanted into LDLr-/- mice on an atherogenic diet. Myeloid ADAM10 deficiency did not affect plaque size, but increased plaque collagen content. Furthermore, macrophage content in advanced atherosclerotic lesions was decreased while circulating "inflammatory" Ly6Chi monocytes were elevated. ADAM10-deficient macrophages displayed a more anti-inflammatory phenotype by the production of more IL-10, while pro-inflammatory mediators such as TNF, IL-12 and NO were decreased. Furthermore, production of MMP-9 and -13 was significantly reduced in ADAM10deficient macrophages, indicating a detrimental role of ADAM10 in extracellular matrix degradation enhancing the risk of plaque rupture. Conclusion: This is the first study showing a causal role for myeloid ADAM10 in modulating atherosclerotic plaque stability by decreasing plaque inflammation and increasing plaque fibrosis. Clinical Relevance: The observed causal role for myeloid ADAM10 in modulating atherosclerotic plaque stability opens new possibilities for the development of targeted therapeutics for cardiovascular disease.
P2390 | BENCH The dual PPAR-alpha/gamma agonist aleglitazar increases number and function of endothelial progenitor cells: implications for vascular function and atherogenesis C. Werner 1 , C. Gensch 1 , V. Pavlickova 1 , J. Poess 1 , M.B. Wright 2 , M. Boehm 1 , U. Laufs 1 . 1 Universitätsklinikum des Saarlandes - Klinik für Innere Medizin III, Homburg, Germany; 2 F. Hoffmann-La-Roche AG, Basel, Switzerland Background: Endothelial progenitor cells (EPC) improve endothelial function and promote vascular repair. Aleglitazar combines lipid-modifying effects of PPAR-α agonists (fibrates) and insulin-sensitizing effects of PPAR-γ agonists (thiazolidinediones). We studied the effects of the novel dual peroxisome proliferator-activated receptor (PPAR)-α/γ agonist aleglitazar on glucose tolerance, EPC, endothelial function, neoangiogenesis and atherosclerosis in mice. Methods and results: C57Bl/6 wild-type (WT, normal chow), ApoE-/- mice (Western-type diet) and eNOS-/- mice were treated with aleglitazar (10 mg/kg/d, i.p.) or vehicle. Aleglitazar enhanced the expression of both the PPAR-α and PPAR-γ target genes and normalized glucose tolerance in cholesterol-fed ApoE/- mice. In WT mice, aleglitazar treatment upregulated sca-1/VEGFR-2-positive EPC in the blood (153±10%) and bone marrow (197±22%), and upregulated spleen-derived diLDL/lectin-positive EPC (182±8%). Furthermore, aleglitazar augmented EPC migration (186±6% vs. controls) and enhanced neoangiogenesis (vascularized disk area 178±18% vs. controls). The effects of the dual PPARα/γ agonist on EPC number and function were abolished in eNOS knock-out mice. In ApoE-/- mice, aleglitazar upregulated EPC number and function, potently improved endothelium-dependent vasodilation and markedly reduced the formation of atherosclerotic plaques (plaque area/total lumen area 2.3±0.8% vs. 10.1±1.9% after 6 weeks and 22±2.2% vs. 36±2.1% after 8 weeks treatment). OilRed staining of hepatic sections showed a profound reduction of liver steatosis in ApoE-/- mice. In cultured human EPC, aleglitazar increased migration and colony forming units in a concentration-dependent manner. Furthermore, oxidative stress-induced EPC apoptosis and protein expression of p53 were reduced, while telomerase activity and expression of phospho-eNOS (S1177) and phospho-Akt were elevated. Comparative and inhibitor experiments revealed that aleglitazar’s effects on EPC migration and colony forming units (CFU) were mediated by both PPAR-α and -γ signaling. E.g., aleglitazar treatment (10nM/l) induced EPC migration (304±21% vs. controls) and CFU (300±67% vs. controls) comparably to a co-stimulation of EPC with pioglitazone 10μM/l+fenofibric acid (150μM/l) (Migration: 332±28%; CFU: 289±72%). Conclusions: The dual PPAR-α/γ agonist aleglitazar augments number, function and survival of endothelial progenitor cells in an Akt- and eNOS-dependent fashion. The effects on EPC correlate with improved neoangiogenesis, restored endothelial function and prevention of atherosclerosis.
P2392 | BENCH Migration of regulatory T cells into human atherosclerotic lesions is associated with plaque stability and correlates inversely with infiltrated mature dendritic cells B. Dietel, R. Altendorf, I. Cicha, C. Garlichs. University of Erlangen-Nuremberg, Department of Cardiology and Angiology, Erlangen, Germany Purpose: Atherosclerotic lesions are characterized by an extensive inflammation of the vessel wall, which can contribute to plaque instability. Dendritic cells (DC) play a crucial role in the regulation of inflammatory processes within the atherosclerotic lesions, by inducing a T cell-specific immune response. However, DCs are also involved in immunosuppressive processes, by the induction of regulatory T cells (Tregs). We investigated a possible correlation between infiltrated DCs and Tregs in atherosclerotic plaques. Methods: Cross-sections of 40 human carotid endarterectomy specimens were classified into groups of stable and vulnerable plaques using Trichrome staining. Immunohistochemical staining of plaques was used to detect infiltrated total (S100) and mature DCs (Fascin, DC-LAMP, CD83), Tregs (CD3, Foxp3), and to quantify the inflammatory state of the plaques (CD3, COX-2, CD68). In addition, RNA was isolated from plaque specimens and quantitative real-time PCR was performed to analyze transcription rates of DC markers (CD11c, CD209, HLADR), maturation markers (CD80, CD83, CD86), Treg-associated genes (CTLA-4, Foxp3) and of cytokine (TGFβ-family, IL-10, IFN-γ, IL-17α, IL-6). Migration assays and flow experiments were performed, to investigate the effects of Tregs on mature DCs in-vitro. Results: As compared with stable plaques, vulnerable plaques were characterized by increased numbers of COX-2-expressing cells and T lymphocytes, indicating an enhanced inflammatory process in these atherosclerotic lesions. In stable plaques, numbers of total and mature DCs were significantly lower in the inflammatory plaque shoulder. On the contrary, the numbers of Tregs were higher in the plaque shoulder and the fibrous cap of stable as compared to vulnerable plaques. The inverse correlation of mature DCs and Tregs, and the association of Tregs infiltration with plaque stability, were confirmed by PCR analysis showing a decreased transcription of DC- and maturation markers and an increased mRNA expression of Treg-associated genes and anti-inflammatory cytokines in stable atherosclerotic plaques. In-vitro, co-incubation of mature DCs with Tregs resulted in a decreased migration and a suppression of the adhesion to an endothelial cell layer under conditions of non-uniform shear stress. Conclusions: The results of our study suggest the association of a decreased occurrence of Tregs with a vulnerable plaque phenotype.
