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

Abstract

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.