Gianluca Grassia

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.

MHC Class II–Restricted Antigen Presentation by Plasmacytoid Dendritic Cells Drives Proatherogenic T Cell Immunity

Background— Plasmacytoid dendritic cells (pDCs) bridge innate and adaptive immune responses and are important regulators of immuno-inflammatory diseases. However, their role in atherosclerosis remains elusive. Methods and Results— Here, we used genetic approaches to investigate the role of pDCs in atherosclerosis. Selective pDC deficiency in vivo was achieved using CD11c-Cre × Tcf4–/flox bone marrow transplanted into Ldlr–/– mice. Compared with control Ldlr–/– chimeric mice, CD11c-Cre × Tcf4–/flox mice had reduced atherosclerosis levels. To begin to understand the mechanisms by which pDCs regulate atherosclerosis, we studied chimeric Ldlr–/– mice with selective MHCII deficiency on pDCs. Significantly, these mice also developed reduced atherosclerosis compared with controls without reductions in pDC numbers or changes in conventional DCs. MHCII-deficient pDCs showed defective stimulation of apolipoprotein B100–specific CD4+ T cells in response to native low-density lipoprotein, whereas production of interferon-&agr; was not affected. Finally, the atheroprotective effect of selective MHCII deficiency in pDCs was associated with significant reductions of proatherogenic T cell–derived interferon-&ggr; and lesional T cell infiltration, and was abrogated in CD4+ T cell–depleted animals. Conclusions— This study supports a proatherogenic role for pDCs in murine atherosclerosis and identifies a critical role for MHCII-restricted antigen presentation by pDCs in driving proatherogenic T cell immunity.

Macrophage Autophagy in Atherosclerosis

Macrophages play crucial roles in atherosclerotic immune responses. Recent investigation into macrophage autophagy (AP) in atherosclerosis has demonstrated a novel pathway through which these cells contribute to vascular inflammation. AP is a cellular catabolic process involving the delivery of cytoplasmic contents to the lysosomal machinery for ultimate degradation and recycling. Basal levels of macrophage AP play an essential role in atheroprotection during early atherosclerosis. However, AP becomes dysfunctional in the more advanced stages of the pathology and its deficiency promotes vascular inflammation, oxidative stress, and plaque necrosis. In this paper, we will discuss the role of macrophages and AP in atherosclerosis and the emerging evidence demonstrating the contribution of macrophage AP to vascular pathology. Finally, we will discuss how AP could be targeted for therapeutic utility.

Neutralization of Interleukin-18 Inhibits Neointimal Formation in a Rat Model of Vascular Injury

Background— Studies in humans and animal models suggest that interleukin-18 (IL-18) plays a crucial role in vascular pathologies. IL-18 is a predictor of cardiovascular death in angina and is involved in atherotic plaque destabilization. Higher IL-18 plasma levels also are associated with restenosis after coronary artery angioplasty performed in patients with acute myocardial infarction. We investigated the effective role of IL-18 in neointimal formation in a balloon-induced rat model of vascular injury. Methods and Results— Endothelial denudation of the left carotid artery was performed by use of a balloon embolectomy catheter. Increased expression of IL-18 and IL-18Rα/β mRNA was detectable in carotid arteries from days 2 to 14 after angioplasty. The active form of IL-18 was highly expressed in injured arteries. Strong immunoreactivity for IL-18 was detected in the medial smooth muscle cells at days 2 and 7 after balloon injury and in proliferating/migrating smooth muscle cells in neointima at day 14. Moreover, serum concentrations of IL-18 were significantly higher among rats subjected to vascular injury. Treatment with neutralizing rabbit anti-rat IL-18 immunoglobulin G significantly reduced neointimal formation (by 27%; P<0.01), reduced the number of proliferating cells, and inhibited interferon-γ, IL-6, and IL-8 mRNA expression and nuclear factor-&kgr;B activation in injured arteries. In addition, in vitro data show that IL-18 affects smooth muscle cell proliferation. Conclusions— These results identify a critical role for IL-18 in neointimal formation in a rat model of vascular injury and suggest a potential role for IL-18 neutralization in the reduction of neointimal development.

Deletion of the Dual Specific Phosphatase-4 (DUSP-4) Gene Reveals an Essential Non-redundant Role for MAP Kinase Phosphatase-2 (MKP-2) in Proliferation and Cell Survival

Mitogen-activated protein kinase phosphatase-2 (MKP-2) is a type 1 nuclear dual specific phosphatase (DUSP) implicated in a number of cancers. We examined the role of MKP-2 in the regulation of MAP kinase phosphorylation, cell proliferation, and survival responses in mouse embryonic fibroblasts (MEFs) derived from a novel MKP-2 (DUSP-4) deletion mouse. We show that serum and PDGF induced ERK-dependent MKP-2 expression in wild type MEFs but not in MKP-2−/− MEFs. PDGF stimulation of sustained ERK phosphorylation was enhanced in MKP-2−/− MEFs, whereas anisomycin-induced JNK was only marginally increased. However, marked effects upon cell growth parameters were observed. Cellular proliferation rates were significantly reduced in MKP-2−/− MEFs and associated with a significant increase in cell doubling time. Infection with adenoviral MKP-2 reversed the decrease in proliferation. Cell cycle analysis revealed a block in G2/M phase transition associated with cyclin B accumulation and enhanced cdc2 phosphorylation. MEFs from MKP-2−/− mice also showed enhanced apoptosis when stimulated with anisomycin correlated with increased caspase-3 cleavage and γH2AX phosphorylation. Increased apoptosis was reversed by adenoviral MKP-2 infection and correlated with selective inhibition of JNK signaling. Collectively, these data demonstrate for the first time a critical non-redundant role for MKP-2 in regulating cell cycle progression and apoptosis.

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.

A novel lipid A from Halomonas magadiensis inhibits enteric LPS-induced human monocyte activation.

Lipopolysaccharide (LPS) endotoxin is the bacterial product responsible for the clinical syndrome of Gram‐negative septicemia and endotoxic shock. During sepsis, microbial antigens, such as LPS, activate monocytes and macrophages to produce several pro‐inflammatory cytokines, among which tumor necrosis factor‐α (TNF‐α) appears to be very important for the development of endotoxic shock. The endotoxic properties of LPS principally reside in the lipid A (LIP A) component, which is the primary immunostimulatory center of Gram‐negative bacteria. In recent years there has been a continuous effort to identify molecules able to antagonize the deleterious effects of endotoxic shock. In this study we show that a novel LIP A fraction from the LPS of Halomonas magadiensis (Hm), a Gram‐negative extremophilic and alkaliphilic bacterium, significantly inhibits the synthesis of TNF‐α by human monocytes activated by Escherichia coli LPS. LIP A from Hm exerts these effects by interfering with E. coli LPS for activation of Toll‐like receptor 4 expressed in human cells. This result defines Hm LIP A as a novel class of LPS antagonist whose structural features could be utilized for the design of compounds for the treatment of Gram‐negative sepsis.

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.