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

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 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 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

Pathophisiological mechanisms and possible therapeutic targets of vascular injury

Percutaneous coronary intervention (PCI) is the current procedure that allows the endovascular treatment of occlusive artery disease, without the need of bypass surgery. The most problematic complication of PCI, with or without stent implantation, is the restenosis defined as the re-narrowing of the enlarged artery and characterized by an immune/inflammatory response going with a hyperplastic reaction, involving smooth muscle cell (SMC) migration/proliferation, and remodelling of the arterial wall. In an effort to improve on current therapy for restenosis we are prompted to consider new strategies for prevention and treatment, focusing on understanding of molecular mechanisms and identifying possible therapeutic targets. This thesis aims at four issues described as follows: 1. Effect of NBD peptide on injury-induced neointimal formation The activation of nuclear factor-κB (NF-κB) is a crucial step in the arterial wall's response to injury. NF-κB essential modulator-binding domain (NBD) peptide blocks the activation of the IκB kinase complex, selectively abrogating the inflammation-induced activation of NF-κB. In this study, we investigated the effect of NBD peptide on neointimal formation using two animal models of arterial injury: rat carotid artery balloon angioplasty and wire-induced carotid injury in apolipoproteinE-deficient (apoE-/-) mice. Local treatment with the NBD peptide (300 μg/site) significantly reduced the number of proliferating cells in rat carotid arteries 7 days after angioplasty (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-κB activation and monocyte chemotactic protein-1 (MCP-1) expression in the carotid arteries of rats treated with the peptide. In addition, the NBD peptide (0.01 to 1 μM) reduced rat SMC proliferation, migration, and invasion in vitro, processes contributing to the injury-induced neointimal formation in vivo. Similar results were observed in apoE-/- mice in which the NBD peptide (150 μg/site) reduced wire-induced neointimal formation at day 28 (by 47%; P<0.01). Our results demonstrate that the NBD peptide reduces neointimal formation and SMC proliferation/migration, both effects associated with the inhibition of NF-κB activation. 2. Use of the anti-inflammatory agent bindarit to control neointimal hyperplasia Chemokines are a family of proteins that regulate the migration of circulating leukocytes to sites of arterial injury as well as the activation of SMCs. Many chemokine genes are under the control of NF-κB. Bindarit is an original compound with peculiar anti-inflammatory activity due to a selective inhibition of the chemokines MCP-1, MCP-3, and MCP-2. In the present study we evaluated the effect of bindarit on neointimal formation using both animal models described above. Treatment of rats with bindarit (200 mg/kg/day) significantly reduced balloon injury-induced neointimal formation by 39% at day 14 without affecting re-endothelialisation 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 SMC proliferation, migration, and invasion. Similar results were observed in 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 neointimal 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 SMCs were reduced by 66% and 30%, respectively, compared with the control group. This study demonstrates that bindarit is effective in reducing neointimal formation in both non-hyperlipidaemic and hyperlipidaemic animal models of vascular injury by a direct effect on SMC proliferation and migration and by reducing neointimal macrophage content. All of these data were associated with the inhibition of MCP-1 production. 3. Role of Monocyte Chemotactic Protein-3 in human coronary smooth muscle cell proliferation Few studies have examined the role of MCP-3 in vascular pathologies such as atherosclerosis and restenosis in which SMC proliferation plays an important role. In this study, we investigated the effect of MCP-3 on human coronary artery SMC (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 Ab (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 signalling 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 NF-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 Ab (20 ng/mL), suggesting that the mitogenic effect of these stimuli is due, at least in part, to MCP-3. 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. 4. Antigen presentation and costimulatory molecules expression by murine smooth muscle cells The findings that SMCs express MHC II molecules during arterial response to injury suggested their active role in cellular immunity. Since it is not known if vascular SMCs can function as antigen presenting cells, in the present study we investigated the contribution of SMC in antigen presentation. Firstly, we examined the MHC II and some costimulatory molecules expression in SMCs. The percentage of MHC II, CD54 (ICAM-1), CD44 and OX40L positive unstimulated SMCs was about 2%, 30%, 87% and 5%, respectively. The stimulation with IFN-γ (100 ng/mL)) significantly caused a 7 to 8 fold increase in the percentage of MHC II positive cells (P<0.01), a 2 fold increase in the percentage of ICAM-1 positive cells (P<0.01), while it did not affect the expression of CD44 and OX40L. To assess the antigen presentation by SMCs we employed the Ealpha (Eα)-GFP/Y-Ae system that allows visualisation of antigen uptake, as the Eα is GFP labelled, and tracking of antigen presentation using the Y-Ae Ab to detects Eα when bound to MHC II. Treatment of SMCs with Eα for 24 h induced an increase in the percentage of GFP positive cells, both in presence or absence of IFN-γ-stimulation, without affecting the percentage of Y-Ae positive cells. Treatment with Eα of dendritic cells, used as positive control, significantly caused a 50 to 60 fold increase in the percentage of both GFP and Y-Ae positive cells. Our results show that cultured murine SMCs express MHC II molecules after stimulation with IFN-γ but are not able to present the antigen in the context of MHC II

A multianalytical approach to investigate the effect of nanofiltration on plasma-derived factor IX clinical lots

Plasma-derived proteins are a subset of relevant biotherapeutics also known as "well-characterized biologicals". They are enriched from plasma through several steps of physical and biochemical methodologies, reaching the regulatory accepted standards of safety, levels of impurities, activity and lot-to-lot consistency. Final products accepted for commercialization are submitted to tight analytical, functional and safety controls by a number of different approaches that fulfill the requirements of sensitivity and reliability. We report here the use of a multianalytical approach for the comparative evaluation of different lots of Factor IX isolated from plasma preparations and submitted or not to a step of nanofiltration. The approach include, among the other, proteomic techniques based on both MALDI-TOF and LC-MS Orbitrap mass spectrometry, circular dichroism for structural characterization, chromatographic and electrophoretic techniques, ELISA and functional assays based on clotting activity and binding to known anticoagulants. Comparative data obtained on two sets of nanofiltered and non-nanofiltered lots with different final activity show that the products have substantially overlapping profiles in terms of activity, contaminants, structural properties and protein content, suggesting that the proposed multi analytical approach is robust enough to be used for the routine validation of clinical lots

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

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.&lt;p&gt;&lt;/p&gt; 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 β-catenin in the plasma membrane but less β-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 β-catenin in the plasma membrane, reduced β-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 β-catenin in the neointima in apoE(-/-)/MMP8(+/+) mice.&lt;p&gt;&lt;/p&gt; CONCLUSIONS: MMP8 enhances VSMC proliferation via an ADAM10, N-cadherin, and β-catenin-mediated pathway and plays an important role in neointima formation.&lt;p&gt;&lt;/p&gt

The IkB kinase inhibitor nuclear factor-kB essential modulator-binding domain peptide for inhibition of injury-induced neointimal formation.

Objective—The activation of nuclear factor-B (NF-B) is a crucial step in the arterial wall’s response to injury. The identification and characterization of the NF-B essential modulator– binding domain (NBD) peptide, which can block the activation of the IB kinase complex, have provided an opportunity to selectively abrogate the inflammation-induced activation of NF-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 g/site) significantly reduced the number of proliferating cells at day 7 (by 40%; P0.01) and reduced injury-induced neointimal formation (by 50%; P0.01) at day 14. These effects were associated with a significant reduction of NF-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 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 g/site) reduced wire-induced neointimal formation at day 28 (by 47%; P0.01). Conclusion—The NBD peptide reduces neointimal formation and smooth muscle cell proliferation/migration, both effects associated with the inhibition of NF-B activation