Marcella Maddaluno

Plasmacytoid Dendritic Cells Play a Key Role in Promoting Atherosclerosis in Apolipoprotein E–Deficient Mice

Objective—Clinical studies have identified that reduced numbers of circulating plasmacytoid dendritic cells (pDCs) act as a predictor of cardiovascular events in coronary artery disease and that pDCs are detectable in the shoulder region of human atherosclerotic plaques, where rupture is most likely to occur. Results from animal models are controversial, with pDCs seen to inhibit or promote lesion development depending on the experimental settings. Here, we investigated the role of pDCs in atherosclerosis in apolipoprotein E−deficient mice. Methods and Results—We demonstrated that the aorta and spleen of both apolipoprotein E−deficient and C57BL/6 mice displayed similar numbers of pDCs, with similar activation status. In contrast, assessment of antigen uptake/presentation using the E&agr;/Y-Ae system revealed that aortic pDCs in apolipoprotein E−deficient- mice were capable of presenting in vivo systemically administered antigen. Continuous treatment of apolipoprotein E−deficient mice with anti−mouse plasmacytoid dendritic cell antigen 1 (mPDCA-1) antibody caused specific depletion of pDCs in the aorta and spleen and significantly reduced atherosclerosis formation in the aortic sinus (by 46%; P<0.001). Depletion of pDCs also reduced macrophages (by 34%; P<0.05) and increased collagen content (by 41%; P<0.05) in aortic plaques, implying a more stable plaque phenotype. Additionally, pDC depletion reduced splenic T-cell activation and inhibited interleukin-12, chemokine (C-X-C motif) ligand 1, monokine induced by interferon-&ggr;, interferon &ggr;−induced protein 10, and vascular endothelium growth factor serum levels. Conclusion—These results identify a critical role for pDCs in atherosclerosis and suggest a potential role for pDC targeting in the control of the pathology.

The anti-inflammatory agent bindarit inhibits neointima formation in both rats and hyperlipidaemic mice.

Aims Bindarit is an original compound with peculiar anti-inflammatory activity due to a selective inhibition of a subfamily of inflammatory chemokines, including the monocyte chemotactic proteins MCP-1/CCL2, MCP-3/CCL7, and MCP-2/CCL8. In this study, we investigated the effect of bindarit on neointima formation using two animal models of arterial injury: rat carotid artery balloon angioplasty and wire-induced carotid injury in apolipoprotein E-deficient (apoE−/−) mice. Methods and results Treatment of rats with bindarit (200 mg/kg/day) significantly reduced balloon injury-induced neointima formation by 39% at day 14 without affecting re-endothelialization and reduced the number of medial and neointimal proliferating cells at day 7 by 54 and 30%, respectively. These effects were associated with a significant reduction of MCP-1 levels both in sera and in injured carotid arteries of rats treated with bindarit. In addition, in vitro data showed that bindarit (10–300 µM) reduced rat vascular smooth muscle cell (VSMC) proliferation, migration, and invasion, processes contributing to the injury-induced neointima formation in vivo. Similar results were observed in hypercholesterolaemic apoE−/− mice in which bindarit administration resulted in a 42% reduction of the number of proliferating cells at day 7 after carotid injury and in a 47% inhibition of neointima formation at day 28. Analysis of the cellular composition in neointimal lesions of apoE−/− mice treated with bindarit showed that the relative content of macrophages and the number of VSMCs were reduced by 66 and 30%, respectively, compared with the control group. Conclusion This study demonstrates that bindarit is effective in reducing neointima formation in both non-hyperlipidaemic and hyperlipidaemic animal models of vascular injury by a direct effect on VSMC proliferation and migration and by reducing neointimal macrophage content. All of these data were associated with the inhibition of MCP-1 production.

Matrix Metalloproteinase-8 Promotes Vascular Smooth Muscle Cell Proliferation and Neointima Formation

Objective— We investigated the role of matrix metalloproteinase-8 (MMP8) in neointima formation and in vascular smooth muscle cell (VSMC) migration and proliferation. Approach and Results— After carotid artery wire injuring, MMP8–/–/apoE–/– mice had fewer proliferating cells in neointimal lesions and smaller lesion sizes. Ex vivo assays comparing VSMCs isolated from MMP8 knockout and wild-type mice showed that MMP8 knockout decreased proliferation and migration. Proteomics analysis revealed that a disintegrin and metalloproteinase domain–containing protein 10 (ADAM10) had lower concentrations in MMP8 knockout VSMC culture media than in MMP8 wild-type VSMC culture media. Western blot, flow cytometric, and immunocytochemical analyses showed that MMP8 knockout VSMCs contained more pro-ADAM10 but less mature ADAM10, more N-cadherin, and &bgr;-catenin in the plasma membrane but less &bgr;-catenin in the nucleus and less cyclin D1. Treatment of MMP8 wild-type VSMCs with an ADAM10 inhibitor, GI254023X, or siRNA knockdown of ADAM10 in MMP8 wild-type VSMCs inhibited proliferation and migration, increased N-cadherin and &bgr;-catenin in the plasma membrane, reduced &bgr;-catenin in the nucleus, and decreased cyclin D1 expression. Incubation of MMP8 knockout VSMCs with a recombinant ADAM10 rescued the proliferative and migratory ability of MMP8 knockout VSMCs and increased cyclin D1 expression. Furthermore, immunohistochemical analyses showed colocalization of ADAM10 with VSMCs and N-cadherin, and nuclear accumulation of &bgr;-catenin in the neointima in apoE–/–/MMP8+/+ mice. Conclusions— MMP8 enhances VSMC proliferation via an ADAM10, N-cadherin, and &bgr;-catenin–mediated pathway and plays an important role in neointima formation.

Monocyte chemotactic protein-3 induces human coronary smooth muscle cell proliferation.

Monocyte chemotactic protein-3 (MCP-3), also known as CCL7, belongs to the monocyte chemotactic protein (MCP) subfamily of the CC chemokines that includes MCP-1/CCL2, MCP-2/CCL8, MCP-4/CCL13, and MCP-5/CCL12. Few studies have examined the role of MCP-3 in vascular pathologies such as atherosclerosis and restenosis in which smooth muscle cell (SMC) proliferation plays an important role. In this study, we investigated the effect of MCP-3 on human coronary artery smooth muscle cell (CASMC) proliferation. MCP-3 induced concentration-dependent CASMC proliferation with the maximum stimulatory effect at 0.3 ng/mL (about 50% vs unstimulated cells) assessed by bromodeoxyuridine (BrdU) uptake and direct cell counting. Anti-MCP-3 antibody (20 ng/mL) completely inhibited cell proliferation, demonstrating the specificity of the proliferative effect of MCP-3. Moreover, the MCP-3-induced CASMC proliferation was blocked by RS 102895 (0.06-6 μM), a specific antagonist of chemokine receptor 2 (CCR2). The mitogenic effect of MCP-3 appeared to be dependent on ERK1/2 MAPK and PI3K signaling pathway activation, as demonstrated by the reduction of MCP-3-induced CASMC proliferation observed after the treatment of cells with U0126 (1 μM) and LY-294002 (5μM), selective inhibitors of ERK 1/2 and PI3K activation, respectively. We found no relationship between MCP-3-induced CASMC proliferation and nuclear factor-κB activation. Moreover, we found that tumor necrosis factor-α (TNF-α, 30 ng/mL) and interleukin-1β (IL-1β, 1 ng/mL) both induced time-dependent increase of MCP-3 production by CASMCs, which was reduced by the anti-MCP-3 antibody (20 ng/mL), suggesting that the mitogenic effect of these stimuli is due, at least in part, to MCP-3. In conclusion, our results demonstrate that MCP-3 is produced by human CASMCs and directly induces CASMC proliferation in vitro, suggesting a potential role for this chemokine in vascular pathology.

The IκB Kinase Inhibitor Nuclear Factor-κB Essential Modulator–Binding Domain Peptide for Inhibition of Injury-Induced Neointimal Formation

Objective—The activation of nuclear factor-&kgr;B (NF-&kgr;B) is a crucial step in the arterial walls response to injury. The identification and characterization of the NF-&kgr;B essential modulator-binding domain (NBD) peptide, which can block the activation of the I&kgr;B kinase complex, have provided an opportunity to selectively abrogate the inflammation-induced activation of NF-&kgr;B. The aim of the present study was to evaluate the effect of the NBD peptide on neointimal formation. Methods and Results—In the rat carotid artery balloon angioplasty model, local treatment with the NBD peptide (300 &mgr;g/site) significantly reduced the number of proliferating cells at day 7 (by 40%; P<0.01) and reduced injury-induced neointimal formation (by 50%; P<0.01) at day 14. These effects were associated with a significant reduction of NF-&kgr;B activation and monocyte chemotactic protein-1 expression in the carotid arteries of rats treated with the peptide. In addition, the NBD peptide (0.01 to 1 &mgr;mol/L) reduced rat smooth muscle cell proliferation, migration, and invasion in vitro. Similar results were observed in apolipoprotein E−/− mice in which the NBD peptide (150 &mgr;g/site) reduced wire-induced neointimal formation at day 28 (by 47%; P<0.01). Conclusion—The NBD peptide reduces neointimal formation and smooth muscle cell proliferation/migration, both effects associated with the inhibition of NF-&kgr;B activation.

Inhibition of In-Stent Stenosis by Oral Administration of Bindarit in Porcine Coronary Arteries

Objective—We have previously demonstrated that bindarit, a selective inhibitor of monocyte chemotactic proteins (MCPs), is effective in reducing neointimal formation in rodent models of vascular injury by reducing smooth muscle cell proliferation and migration and neointimal macrophage content, effects associated with the inhibition of MCP-1/CCL2 production. The aim of the current study was to evaluate the efficacy of bindarit on in-stent stenosis in the preclinical porcine coronary stent model. Methods and Results—One or 2 bare metal stents (Multi-Link Vision, 3.5 mm) were deployed (1:1.2 oversize ratio) in the coronary arteries of 42 pigs (20 bindarit versus 22 controls). Bindarit (50 mg/kg per day) was administered orally from 2 days before stenting until the time of euthanasia at 7 and 28 days. Bindarit caused a significant reduction in neointimal area (39.4%, P<0.001, n=9 group), neointimal thickness (51%, P<0.001), stenosis area (37%, P<0.001), and inflammatory score (40%, P<0.001) compared with control animals, whereas there was no significant difference in the injury score between the 2 groups. Moreover, treatment with bindarit significantly reduced the number of proliferating cells (by 45%, P<0.05; n=6 group) and monocyte/macrophage content (by 55%, P<0.01; n=5–6 group) in stented arteries at day 7 and 28, respectively. These effects were associated with a significant (P<0.05) reduction of MCP-1 plasma levels at day 28. In vitro data showed that bindarit (10–300 &mgr;mol/L) reduced tumor necrosis factor-&agr; (50 ng/mL)–induced pig coronary artery smooth muscle cell proliferation and inhibited MCP-1 production. Conclusion—Our results show the efficacy of bindarit in the prevention of porcine in-stent stenosis and support further investigation for clinical application of this compound.

Bindarit Inhibits Human Coronary Artery Smooth Muscle Cell Proliferation, Migration and Phenotypic Switching

Bindarit, a selective inhibitor of monocyte chemotactic proteins (MCPs) synthesis, reduces neointimal formation in animal models of vascular injury and recently has been shown to inhibit in-stent late loss in a placebo-controlled phase II clinical trial. However, the mechanisms underlying the efficacy of bindarit in controlling neointimal formation/restenosis have not been fully elucidated. Therefore, we investigated the effect of bindarit on human coronary smooth muscle cells activation, drawing attention to the phenotypic modulation process, focusing on contractile proteins expression as well as proliferation and migration. The expression of contractile proteins was evaluated by western blot analysis on cultured human coronary smooth muscle cells stimulated with TNF-α (30 ng/mL) or fetal bovine serum (5%). Bindarit (100–300 µM) reduced the embryonic form of smooth muscle myosin heavy chain while increased smooth muscle α-actin and calponin in both TNF-α- and fetal bovine serum-stimulated cells. These effects were associated with the inhibition of human coronary smooth muscle cell proliferation/migration and both MCP-1 and MCP-3 production. The effect of bindarit on smooth muscle cells phenotypic switching was confirmed in vivo in the rat balloon angioplasty model. Bindarit (200 mg/Kg/day) significantly reduced the expression of the embryonic form of smooth muscle myosin heavy chain, and increased smooth muscle α-actin and calponin in the rat carodid arteries subjected to endothelial denudation. Our results demonstrate that bindarit induces the differentiated state of human coronary smooth muscle cells, suggesting a novel underlying mechanisms by which this drug inhibits neointimal formation.

Vaccine adjuvant MF59 promotes the intranodal differentiation of antigen-loaded and activated monocyte-derived dendritic cells

MF59 is an oil-in-water emulsion adjuvant approved for human influenza vaccination in European Union. The mode of action of MF59 is not fully elucidated yet, but results from several years of investigation indicate that MF59 establishes an immunocompetent environment at injection site which promotes recruitment of immune cells, including antigen presenting cells (APCs), that are facilitated to engulf antigen and transport it to draining lymph node (dLN) where the antigen is accumulated. In vitro studies showed that MF59 promotes the differentiation of monocytes to dendritic cells (Mo-DCs). Since after immunization with MF59, monocytes are rapidly recruited both at the injection site and in dLN and appear to have a morphological change toward a DC-like phenotype, we asked whether MF59 could play a role in inducing differentiation of Mo-DC in vivo. To address this question we immunized mice with the auto-fluorescent protein Phycoerythrin (PE) as model antigen, in presence or absence of MF59. We measured the APC phenotype and their antigen uptake within dLNs, the antigen distribution within the dLN compartments and the humoral response to PE. In addition, using Ovalbumin as model antigen, we measured the capacity of dLN APCs to induce antigen-specific CD4 T cell proliferation. Here, we show, for the first time, that MF59 promotes differentiation of Mo-DCs within dLNs from intranodal recruited monocytes and we suggest that this differentiation could take place in the medullary compartment of the LN. In addition we show that the Mo-DC subset represents the major source of antigen-loaded and activated APCs within the dLN when immunizing with MF59. Interestingly, this finding correlates with the enhanced triggering of antigen-specific CD4 T cell response induced by LN APCs. This study therefore demonstrates that MF59 is able to promote an immunocompetent environment also directly within the dLN, offering a novel insight on the mechanism of action of vaccine adjuvants based on emulsions.

Murine Aortic Smooth Muscle Cells Acquire, Though Fail to Present Exogenous Protein Antigens on Major Histocompatibility Complex Class II Molecules

In the present study aortic murine smooth muscle cell (SMC) antigen presentation capacity was evaluated using the Eα-GFP/Y-Ae system to visualize antigen uptake through a GFP tag and tracking of Eα peptide/MHCII presentation using the Y-Ae Ab. Stimulation with IFN-γ (100 ng/mL) for 72 h caused a significant (P < 0.01) increase in the percentage of MHC class II positive SMCs, compared with unstimulated cells. Treatment with Eα-GFP (100 μg/mL) for 48 h induced a significant (P < 0.05) increase in the percentage of GFP positive SMCs while it did not affect the percentage of Y-Ae positive cells, being indicative of antigen uptake without its presentation in the context of MHC class II. After IFN-γ-stimulation, ovalbumin- (OVA, 1 mg/mL) or OVA323–339 peptide-(0.5 μg/mL) treated SMCs failed to induce OT-II CD4+ T cell activation/proliferation; this was also accompanied by a lack of expression of key costimulatory molecules (OX40L, CD40, CD70, and CD86) on SMCs. Finally, OVA-treated SMCs failed to induce DO11.10-GFP hybridoma activation, a process independent of costimulation. Our results demonstrate that while murine primary aortic SMCs express MHC class II and can acquire exogenous antigens, they fail to activate T cells through a failure in antigen presentation and a lack of costimulatory molecule expression.

The I kappa b kinase inhibitor, nemo-binding domain peptide, inhibits rat balloon injury-induced neointimal formation

Meeting Abstract from 10th Annual Conference on Arteriosclerosis, Thrombosis and Vascular Biology held in Washington, DC, on April 29 - May 01, 2009. American Heart Association Council Arteriosclerosis, Thrombosis & Vascular Biology.Meeting Abstract from the 10th Annual Conference on Arteriosclerosis, Thrombosis and Vascular Biology held in Washington, DC, on April 29 - May 01, 2009.Objective: Chronic infusion of angiotensin II (AngII) augments the development of atherosclerosis and induces abdominal aortic aneurysms (AAAs) in hypercholesterolemic mice. Smooth muscle cells (SMCs) play a pivotal role in the development of these two pathologies. AngII-induced AAA is associated with the downregulation of peroxisome proliferator-activated receptor gamma (PPARgamma). In addition, activation of PPARgamma by ligands attenuates atherosclerosis in male mice. The purpose of the study was to define whether deficiency of SMC-specific PPARgamma influences AngII-induced vascular pathologies. Methods and Results: Male and female LDL receptor -/- mice were bred with homozygous floxed PPARgamma alleles (PPARgammaf/f). Parental males were also developed that were hemizygous for Cre under the control of the SM22 promoter. Littermates were generated that did not express Cre (Cre0/0, n=20) or were hemizygous for Cre(Cre1/0, n=16) and fed a saturated fat-enriched diet (21% wt/wt milk fat; 0.15% wt/wt cholesterol). Mice were infused with AngII (1,000 ng/kg/min) by osmotic minipump for 4 weeks. AngII increased systolic blood pressure equivalently in both groups. SMC-specific deficiency of PPARgamma had no effect on serum cholesterol concentrations (Cre0/0, 1544±112; Cre1/0, 1548±87 mg/dL) or lipoprotein-cholesterol distributions. AngII infusion led to a similar incidence of AAA formation (84% of male Cre0/0 versus 80% of male Cre1/0 mice). Ex vivo measurement of maximal diameter of abdominal aorta showed a comparable dilation in Cre0/0 and Cre1/0 mice (2.16±0.16 versus 2.17±0.31 mm, P=NS). Male Cre1/0 mice had significantly larger atherosclerotic lesion areas in both arch and thoracic aortic regions by en face measurement compared to male f/fCre0/0 mice (Arch area intimal lesions; Cre0/0 = 4.74±1.13%; Cre1/0 = 10.24±1.67%, Thoracic area intimal lesions; Cre0/0 = 4.12±0.69%; Cre1/0 = 8.99±2.28%, P<0.05, n=12–18 per group). SMC specific PPARgamma deficiency in female LDL receptor -/- mice did not influence either AngII-induced atherosclerosis or AAAs. Conclusion: SMC specific PPARgamma deficiency augments atherosclerosis but does not contribute to the formation of AAAs induced by AngII in male hyperlipidemic mice.Arteriosclerosis, Thrombosis, and Vascular Biology Annual Conference 2009 April 29–May 1, 2009 Omni Shoreham Hotel Washington, DC Sponsored by the American Heart Association Council on Arteriosclerosis, Thrombosis, and Vascular Biology. Conference Chair: Alan Daugherty, PhD, DSc, FAHA Conference Co-Chairs: Robert A. Hegele, MD, FAHA; Murray W. Huff, PhD, FAHA Conference Program Committee Timothy Hla, PhD; Larry Kraiss, MD, FAHA; Steven R. Lentz, MD, PhD, FAHA; Joseph M. Miano, PhD; Kathryn J. Moore, PhD, FAHA; Susan S. Smyth, MD, PhD, FAHA; Mary Sorci-Thomas, PhD, FAHA; Hartmut Weiler, PhD; Cuihua Zhang, MD, PhD, FAHA Conference Program Committee Liaisons Jeanine D’Armiento, MD, PhD; William P. Fay, MD, FAHA; Kathy Griendling, PhD, FAHA; John S. Parks, PhD, FAHA; Muredach P. Reilly, MB, FAHA; Lawrence L. Rudel, PhD, FAHA D ow nladed from http://journals.org by on N ovem er 5, 2019