Novel anti-inflammatory actions of amlodipine in a rat model of arteriosclerosis induced by long-term inhibition of nitric oxide synthesis.

Amlodipine (a new class of calcium channel antagonist) has been shown to limit the progression of arteriosclerosis and decrease the incidence of cardiovascular events. The mechanisms underlying the beneficial effects of amlodipine, however, remain unclear. Therefore, we hypothesized that amlodipine attenuates the development of arteriosclerosis through the inhibition of inflammation in vivo. Long-term inhibition of nitric oxide (NO) by administration of a NO synthase inhibitor, N(omega)-nitro-L-arginine methyl ester (L-NAME), to rats induces coronary vascular inflammation [monocyte infiltration, monocyte chemoattractant protein-1 (MCP-1) expression, increased activity of angiotensin-converting enzyme (ACE)], and arteriosclerosis. Here, we used the rat model to investigate the anti-inflammatory effects of amlodipine in vivo. Treatment with amlodipine markedly inhibited the L-NAME-induced increase in vascular inflammation, oxidative stress, and local ACE and Rho activity and prevented arteriosclerosis. Interestingly, amlodipine prevented the L-NAME-induced increase in MCP-1 receptor CCR2 expression in circulating monocytes. Amlodipine markedly attenuated the high mortality rate at 8 wk of treatment. These data suggest that amlodipine attenuated arteriosclerosis through inhibiting inflammatory disorders in the rat model of long-term inhibition of NO synthesis. The anti-inflammatory effects of amlodipine seem to be mediated not only by the inhibition of local factors such as MCP-1 but also by the decrease in CCR2 in circulating monocytes. Inhibition of the MCP-1 to CCR2 pathway may represent novel anti-inflammatory actions of amlodipine beyond blood pressure lowering.

Anti-monocyte chemoattractant protein-1 gene therapy attenuates pulmonary hypertension in rats.

Monocyte/macrophage chemoattractant protein-1 (MCP-1), a potent chemoattractant chemokine and an activator for mononuclear cells, may play a role in the initiation and/or progression of pulmonary hypertension (PH). To determine whether blockade of a systemic MCP-1 signal pathway in vivo may prevent PH, we intramuscularly transduced a naked plasmid encoding a 7-NH(2) terminus-deleted dominant negative inhibitor of the MCP-1 (7ND MCP-1) gene in monocrotaline-induced PH. We also simultaneously gave a duplicate transfection at 2-wk intervals or skeletal muscle-directed in vivo electroporation (EP) to evaluate whether a longer or higher expression might be more effective. The intramuscular reporter gene expression was enhanced 10 times over that by EP than by simple injection, and a significant 7ND MCP-1 protein in plasma was detected only in the EP group. 7ND MCP-1 gene transfer significantly inhibited the progression of MCT-induced PH as evaluated by right ventricular systolic pressure, right ventricular hypertrophy, medial hypertrophy of pulmonary arterioles, and mononuclear cell infiltration into the lung. Differential effects of longer or higher transgene expression were not apparent. Although the in vivo kinetics of 7ND MCP-1 gene therapy should be studied further, these encouraging results suggest that an anti-inflammatory strategy via blockade of the MCP-1 signal pathway may be an alternative approach to treat subjects with PH.

Anti-monocyte chemoattractant protein-1 gene therapy inhibits restenotic changes (neointimal hyperplasia) after balloon injury in rats and monkeys.

Prevention of restenosis after coronary intervention is a major clinical challenge, which highlights the need of new therapeutic options. Vascular injury may involve inflammatory responses that accelerate the recruitment and activation of monocytes through the activation of chemotactic factors, including monocyte chemoattractant protein-1 (MCP-1). However, there is no definitive evidence supporting the role of MCP-1 in restenosis. We recently devised a new strategy for anti-MCP-1 gene therapy by transfecting an N-terminal deletion mutant of the MCP-1 gene into skeletal muscles. We demonstrate here that this strategy suppressed monocyte infiltration/activation in the injured site and markedly inhibited restenotic changes (neointimal hyperplasia) after balloon injury of the carotid artery in rats and monkeys. This strategy also suppressed the local production of MCP-1 and inflammatory cytokines. Therefore, monocyte infiltration and activation mediated by MCP-1 are essential in the development of restenotic changes after balloon injury. This strategy may be a useful form of gene therapy against human restenosis.

Essential role of monocyte chemoattractant protein-1 in development of restenotic changes (neointimal hyperplasia and constrictive remodeling) after balloon angioplasty in hypercholesterolemic rabbits.

BACKGROUND: Renarrowing of dilated arterial sites (restenosis) hampers the clinical benefits of coronary angioplasty. Infiltration and activation of monocytes in the arterial wall mediated by monocyte chemoattractant protein-1 (MCP-1) might be a major cause of restenosis after angioplasty. However, there is no direct evidence to support a definite role of MCP-1 in the development of restenosis. Methods and Results- We recently devised a new strategy for anti-MCP-1 gene therapy by transfecting an N-terminal deletion mutant of the MCP-1 gene into skeletal muscles. We used this strategy to investigate the role of MCP-1 in the development of restenotic changes after balloon injury in the carotid artery in hypercholesterolemic rabbits. Intramuscular transfection of the mutant MCP-1 gene suppressed monocyte infiltration/activation in the injured arterial wall and thus attenuated the development of neointimal hyperplasia and negative remodeling. CONCLUSIONS: MCP-1-mediated monocyte infiltration is necessary in the development of restenotic changes to balloon injury in hypercholesterolemic rabbits. This strategy may be a useful and practical form of gene therapy against human restenosis.

Importance of monocyte chemoattractant protein-1 pathway in neointimal hyperplasia after periarterial injury in mice and monkeys.

Neointimal hyperplasia is a major cause of restenosis after coronary intervention. Because vascular injury is now recognized to involve an inflammatory response, monocyte chemoattractant protein-1 (MCP-1) might be involved in underlying mechanisms of restenosis. In the present study, we demonstrate the important role of MCP-1 in neointimal hyperplasia after cuff-induced arterial injury. In the first set of experiments, placement of a nonconstricting cuff around the femoral artery of intact mice and monkeys resulted in inflammation in the early stages and subsequent neointimal hyperplasia at the late stages. We transfected with an N-terminal deletion mutant of the human MCP-1 gene into skeletal muscles to block MCP-1 activity in vivo. This mutant MCP-1 works as a dominant-negative inhibitor of MCP-1. This strategy inhibited early vascular inflammation (monocyte infiltration, increased expression of MCP-1, and inflammatory cytokines) and late neointimal hyperplasia. In the second set of experiments, the cuff-induced neointimal hyperplasia was found to be less in CCR2-deficient mice than in control CCR2(+/+) mice. The MCP-1/CCR2 pathway plays a central role in the pathogenesis of neointimal hyperplasia in cuffed femoral artery of mice and monkeys. Therefore, the MCP-1/CCR2 pathway can be a therapeutic target for human restenosis after coronary intervention.

Important role of Rho-kinase in the pathogenesis of cardiovascular inflammation and remodeling induced by long-term blockade of nitric oxide synthesis in rats.

Chronic inhibition of endothelial NO synthesis by the administration of N(G)-nitro-L-arginine methyl ester (L-NAME) to rats induces early vascular inflammation (monocyte infiltration into coronary vessels and monocyte chemoattractant protein-1 expression) as well as subsequent arteriosclerosis. The small GTPase Rho controls cell adhesion, motility, and proliferation and is activated by several growth factors such as angiotensin II. We investigated the effect of a specific inhibitor of Rho-kinase, Y-27632, in rats treated with L-NAME to determine the role of the Rho/Rho-kinase pathway in the development of arteriosclerosis. We found here increased activity of Rho/Rho-kinase after L-NAME administration and its prevention by angiotensin II type 1 receptor blockade. Hydralazine or lecithinized superoxide dismutase (l-SOD) did not affect Rho/Rho-kinase activity. Co-treatment with Y-27632 did not affect the L-NAME-induced increase in cardiovascular tissue ACE activity or L-NAME-induced decrease in plasma NO concentrations, but did prevent the L-NAME-induced early inflammation and late coronary arteriosclerosis. In addition, Y-27632 prevented the increased gene expression of monocyte chemoattractant protein-1 and transforming growth factor-beta1 as well as cardiac fibrosis and glomerulosclerosis. These findings suggest that increased activity of Rho/Rho-kinase pathway mediated via the angiotensin II type 1 receptor may thus be important in the pathogenesis of early vascular inflammation and late remodeling induced by chronic inhibition of NO synthesis. The beneficial effects of Rho-kinase inhibition are not mediated by restoration of NO production. The Rho-kinase pathway could be a new therapeutic target for treatment of vascular diseases.