ABSTRACT
Title
Induction of human coronary smooth muscle cell proliferation by Monocyte chemotactic protein-3
Authors
M. Maddaluno1 , MV. Di Lauro1, A. Di Pascale1, R. Santamaria1, A. Guglielmotti2, G. Grassia1, A. Ialenti1
1 Dept. of Experimental Pharmacology, University of Naples Federico II, Via Domenico Montesano, 49, 80131 Naples, Italy;2 Angelini R&D, Angelini Research Center, S. Palomba-Pomezia, 00040 Rome, Italy.
Abstract
Monocyte chemotactic protein-3 (MCP-3), also known as CCL7, belongs to the monocyte chemotactic proteins (MCPs) subfamily of CC chemokines. Few studies have examined the possibility that smooth muscle cells (SMCs), which activation plays an important role in vascular pathologies,can react to MCP-3. In the present study we investigated the effect of MCP-3 on human coronary artery smooth muscle cell (CASMC) proliferation. Bromodeoxyuridine (BrdU) uptake and direct cell counting, showed a maximum stimulatory effect of MCP-3 at 0.3 ng/mL (about 50% vs unstimulated cells). Treatment of CASMCs with the neutralizing anti-MCP-3 antibody (20 ng/mL), completely inhibited the proliferation induced by MCP-3, demonstrating the specificity of the stimulation.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 pathways 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 (5 μM), selective inhibitors of ERK 1/2 and PI3K activation respectively.
To investigate the pathophysiological role of MCP-3 we also demonstrated that TNF-α (30 ng/mL) or IL-1β (1 ng/mL) induced a time-dependent increase of MCP-3 production by CASMCs. Moreover, the neutralizing anti-MCP-3 antibody (20 ng/mL) reduced TNF-α- or IL-1β-induced cell proliferation, 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 coronary SMCs and directly induces cell proliferation in vitro,suggesting a potential role for this chemokine in vascular pathology.
The mitogenic effect of MCP-3 appeared to be dependent on ERK1/2 MAPK and PI3K signaling pathways 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 (5 μM), selective inhibitors of ERK 1/2 and PI3K activation respectively.
To investigate the pathophysiological role of MCP-3 we also demonstrated that TNF-α (30 ng/mL) or IL-1β (1 ng/mL) induced a time-dependent increase of MCP-3 production by CASMCs. Moreover, the neutralizing anti-MCP-3 antibody (20 ng/mL) reduced TNF-α- or IL-1β-induced cell proliferation, 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 coronary SMCs and directly induces cell proliferation in vitro,suggesting a potential role for this chemokine in vascular pathology.