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

Bindarit, an anti-inflammatory agent, reduces neointimal hyperplasia in animal models of vascular injury

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. It is well known that chemokines have a crucial role in initiating and progressing neointima formation by controlling each step of the vascular remodelling in response to various noxious stimuli. The induction of MCP-1 not only correlates with macrophage accumulation but there is strong evidence for an important role of MCP-1 in vascular smooth muscle cell (SMC) proliferation and migration, processes that contribute substantially to neointima formation after arterial stenting and balloon angioplasty. In this thesis, we investigated the effect of bindarit on neointima formation using three animal models of arterial injury: rat carotid artery balloon angioplasty, wire-induced carotid injury in apolipoprotein E-deficient (apoE-/-) mice, and in stent stenosis in preclinical porcine coronary stent model. 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 (SMC) 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 SMCs were reduced by 66 and 30%, respectively, compared with the control group. 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%), neointimal thickness (51%), stenosis area (37%), and inflammatory score (40%,) 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%) and monocyte/macrophage content (by 55%) 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 μmol/L) reduced tumor necrosis factor-α (TNF-α, 50 ng/mL)–induced pig coronary artery smooth muscle cell proliferation and inhibited MCP-1 production. 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 (FBS, 5%). Bindarit (100-300 μM) reduced the embryonic form of smooth muscle myosin heavy chain (SMemb) while increased smooth muscle α-actin (α-SMA) and calponin in both TNF-α- and FBS-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 SMemb, while increased α-SMA and calponin in rat carotid arteries subjected to endothelial denudation. The results provided in this thesis show that bindarit given systemically significantly reduced neointimal formation in animal models of arterial injury by inhibiting SMC proliferation/migration, and macrophage infiltration; these effects correlated with a reduction in MCP-1 synthesis. Preclinical studies demonstrated that bindarit has a safe toxicological profile and is devoid of immunosuppressive, mutagenic, and carcinogenic effects. Phase I clinical studies demonstrated that bindarit (up to a dose of 1200 mg BID) is well tolerated and confirmed the lack of overt toxicity suggested by preclinical studies. Results of Phase II clinical studies confirmed the good tolerability profile of bindarit and demonstrated, at 600 mg BID, significant effects in kidney disease patients. Importantly, a double-blind, randomized, placebo-controlled phase II clinical trial, with the aim of investigating the effect of bindarit in human coronary restenosis, showed that bindarit induced a significant reduction of in-stent late loss. In conclusion, evidence of bindarit efficacy could provide clinicians with useful complementary or alternative therapeutic tools

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

Effect of bindarit on MCP-3 production by TNF-α-stimulated CASMCs.

<p>Results are expressed as mean ± SEM of three separate experiments run in triplicate.</p><p>°°<i>P</i><0.01, °°°<i>P</i><0.001 <i>vs</i> unstimulated cells; *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 <i>vs</i> TNF-α-stimulated cells.</p

Effect of bindarit on CASMC proliferation, migration and invasion.

<p>CASMC proliferation assessed by MTT assay (<b>A</b>) and by cell counting expressed as number of cells per field (<b>B</b>). Effect of bindarit on CASMC migration (<b>C</b>) and invasion (<b>D</b>). Results are expressed as mean ± SEM of three separate experiments run in triplicate. <b>°°</b><i>P</i><0.01, <b>°°°</b><i>P</i><0.001 <i>vs</i> unstimulated cells; <b>**</b><i>P</i><0.01 <i>vs</i> TNF-α-stimulated cells; <b>^#</b><i>P</i><0.05, <b>^###</b><i>P</i><0.001 <i>vs</i> FBS-stimulated cells.</p

Effect of bindarit on contractile proteins localization in rat carotid arteries.

<p>Immohistochemical localization of α-SMA (<b>A</b>), SMemb (<b>B</b>) and calponin (<b>C</b>) expression in rat carotid arteries 7, 14 and 28 days after angioplasty. Bar = 100 µm.</p

Effect of bindarit on morphological changes induced by FBS in CASMCs.

<p>Phase-contrast photomicrographs of CASMCs cultured in medium with 5% FBS for 48 hours with or without bindarit (300 µM).</p

Effect of bindarit on contractile protein expression in rat carotid arteries. A

<p>and <b>B</b>. Representative Western blots and relative densitometric analysis showing the effect of the oral administration of bindarit (200 mg/Kg/day) on SMemb, calponin, α-SMA and PCNA expression levels in rat carotid arteries at days 7, 14 and 28 days after injury. Results are expressed as mean ± SEM, where n = 4 pools. *<i>P<</i>0.05, **<i>P<</i>0.01 and ***<i>P<</i>0.001 <i>vs</i> control group.</p

Effect of bindarit on contractile proteins expression in CASMCs.

<p>Representative Western blots and relative densitometric analysis showing the effects of bindarit (100 and 300 µM) on contractile proteins expression levels modulated by (<b>A</b>) TNF-α (30 ng/mL) or (<b>B</b>) FBS (5%). Results are expressed as mean ± SEM of three separate experiments run in triplicate. °°<i>P</i><0.01 <i>vs</i> unstimulated cells; *<i>P</i><0.05, **<i>P</i><0.01 <i>vs</i> untreated cells.</p