Essential role of angiotensin II type 1a receptors in the host vascular wall, but not the bone marrow, in the pathogenesis of angiotensin II-induced atherosclerosis.

The angiotensin II (Ang II) type 1a (AT1a) receptor is expressed on multiple cell types in atherosclerotic lesions, including bone marrow-derived cells and vascular wall cells, and mediates inflammatory and proliferative responses. Indeed, Ang II infusion accelerates atherogenesis in hyperlipidemic mice by recruiting monocytes and by activating vascular wall cells. Here, we investigated the relative roles of AT1a receptors in the bone marrow vs. the vascular wall in Ang II-induced atherogenesis. Apolipoprotein E-knockout (ApoE(-/-)) mice with or without bone marrow AT1a receptor were generated by experimental bone marrow transplantation using AT1a(+/+) or AT1a(-/-) recipients. In these mice, 28-d Ang II infusion induced significant atherosclerosis in the aorta, and the severity of plaque formation was not affected by the absence of bone marrow AT1a receptor. We then generated AT1a(-/-)ApoE(-/-) mice with or without bone marrow AT1a receptor. Ang II-induced plaque formation was blunted irrespective of the presence of bone marrow AT1a receptor. Host AT1a receptor deficiency was found to suppress Ang II-induced reactive oxygen species production. In addition, AT1a receptor deficiency also impaired monocyte chemoattractant protein-1 (MCP-1) and vascular cell adhesion molecule-1 (VCAM-1) expression in the arterial wall 7 d after Ang II initiation. These molecules normally initiate later macrophage-mediated inflammation in the vascular wall. By contrast, AT1a receptor deficiency in the bone marrow did not affect MCP-1-induced monocyte chemotaxis in vitro. In conclusion, AT1a receptors in the host vascular wall, but not in the bone marrow, are essential in Ang II-induced atherogenesis.

Local delivery of anti-monocyte chemoattractant protein-1 by gene-eluting stents attenuates in-stent stenosis in rabbits and monkeys.

OBJECTIVE: We have previously shown that the intramuscular transfer of the anti-monocyte chemoattractant protein-1 (MCP-1) gene (called 7ND) is able to prevent experimental restenosis. The aim of this study was to determine the in vivo efficacy and safety of local delivery of 7ND gene via the gene-eluting stent in reducing in-stent neointima formation in rabbits and in cynomolgus monkeys. METHODS AND RESULTS: We here found that in vitro, 7ND effectively inhibited the chemotaxis of mononuclear leukocytes and also inhibited the proliferation/migration of vascular smooth muscle cells. We then coated stents with a biocompatible polymer containing a plasmid bearing the 7ND gene, and deployed these stents in the iliac arteries of rabbits and monkeys. 7ND gene-eluting stents attenuated stent-associated monocyte infiltration and neointima formation after one month in rabbits, and showed long-term inhibitory effects on neointima formation when assessments were carried out at 1, 3, and 6 months in monkeys. CONCLUSIONS: Strategy of inhibiting the action of MCP-1 with a 7ND gene-eluting stent reduced in-stent neointima formation with no evidence of adverse effects in rabbits and monkeys. The 7ND gene-eluting stent could be a promising therapy for treatment of restenosis in humans.

Upregulation of the ligand-RAGE pathway via the angiotensin II type I receptor is essential in the pathogenesis of diabetic atherosclerosis.

The receptor for advanced glycation end products (RAGE) and the angiotensin II type I receptor (AT1R) have been separately linked to the pathogenesis of diabetic atherosclerosis. However, no prior study has addressed a linkage between RAGE and AT1R in diabetic atherogenesis. Therefore, we tested the hypothesis that upregulation of the ligand-RAGE axis via AT1R is an essential process underlying the disease. Diabetes was induced in apolipoprotein E-deficient (ApoE(-/-)) mice by streptozotocin, and diabetic mice were treated with AT1 receptor blocker (ARB) for 6 weeks. Diabetic ApoE(-/-) mice that were AT1R-deficient (ApoE(-/-)AT1aR(-/-)) were also investigated. In diabetic ApoE(-/-) mice, AT1R was found to increase within 1 week of diabetes induction, before ligand-RAGE pathway activation and other inflammatory changes were observed. Both ARB treatment and AT1aR deficiency suppressed diabetic atherosclerosis, ligand-RAGE expression and inflammatory changes. In contrast, upregulation of the ligand-RAGE pathway was noted in atherosclerotic plaques from non-diabetic ApoE(-/-) mice infused with angiotensin II. In cultured vascular smooth muscle cells, angiotensin II increased RAGE protein levels via AT1R stimulation. Upregulation of the ligand-RAGE pathway via AT1R is an essential mechanism in diabetic atherosclerosis, implying that ARB might decrease diabetic atherogenesis by inhibiting ligand-RAGE signals.

Catheter-based adenovirus-mediated anti-monocyte chemoattractant gene therapy attenuates in-stent neointima formation in cynomolgus monkeys.

We have previously demonstrated great benefit from anti-monocyte chemoattractant protein-1 (MCP-1) gene therapy by "systemic" transfer of an N-terminal deletion mutant of human MCP-1 (called 7ND) gene into skeletal muscle for treatment of restenosis and atherosclerosis. However, recent evidence suggests that "local" gene transfer may be a clinically relevant approach. We therefore tested the hypothesis that catheter-based adenovirus-mediated anti-MCP-1 gene therapy attenuates stent-associated neointima formation. Bare metal stents were implanted in iliac arteries of cynomolgus monkeys fed a high cholesterol diet. Immediately after the stenting procedure, normal saline or recombinant adenoviral vector containing LacZ or the 7ND gene was administered locally into the stenting site through a Remedy channel-delivery catheter. Compared to saline infusion or LacZ gene transfer, 7ND gene transfer markedly reduced inflammatory changes at an early stage and attenuated neointima formation after 4 weeks. This strategy also reduced the increased production of pro-inflammatory and growth-promoting factors such platelet-derived growth factor. No systemic adverse effects of 7ND gene transfer were detected. There were no significant differences in serum cholesterol levels among the three groups. These data suggest that catheter-based adenovirus-mediated anti-MCP-1 gene therapy may be a clinically relevant and feasible strategy for treatment of in-stent restenosis.

Stent-based local delivery of nuclear factor-kappaB decoy attenuates in-stent restenosis in hypercholesterolemic rabbits.

BACKGROUND: Nuclear factor-kappaB (NF-kappaB) plays a critical role in the vascular response to injury. However, the role of NF-kappaB in the mechanism of in-stent restenosis remains unclear. We therefore tested the hypothesis that blockade of NF-kappaB by stent-based delivery of a cis-element "decoy" of NF-kappaB reduces in-stent neointimal formation. METHODS AND RESULTS: Stents were coated with a polymer containing or not containing NF-kappaB decoy, which represented a fast-release formulation (<7 days). Bare, polymer-coated, and NF-kappaB decoy-eluting stents were implanted in iliac arteries of hypercholesterolemic rabbits. Increased NF-kappaB activity was noted at early stages after stenting, which was suppressed by stent-based delivery of NF-kappaB decoy. NF-kappaB decoy-eluting stents also reduced monocyte infiltration and monocyte chemoattractant protein-1 expression and suppressed CD14 activation on circulating leukocytes. Importantly, NF-kappaB decoy-eluting stents attenuated neointimal formation on day 28. There was no evidence of an incomplete healing process (persistent inflammation, hemorrhage, fibrin deposition, impaired endothelial regeneration) at the site of NF-kappaB decoy-eluting stents. Transfection of NF-kappaB decoy suppressed proliferation of human coronary artery smooth muscle cells in vitro. No systemic adverse effects of NF-kappaB decoy were detected. CONCLUSIONS: Stent-based local delivery of NF-kappaB decoy reduced in-stent neointimal formation with no evidence of incomplete healing. These data suggest that this strategy may be a practical and promising means for prevention of in-stent restenosis in humans.

Angiotensin II type 1 receptor blockade attenuates in-stent restenosis by inhibiting inflammation and progenitor cells.

The precise mechanism by which angiotensin II type 1 receptor blocker reduces in-stent restenosis in clinical trials is unclear. We, therefore, investigated the mechanism of in-stent neointima formation. Male cynomolgus monkeys and rabbits were fed a high-cholesterol diet and were allocated to untreated control and type 1 receptor blocker groups. Five days after grouping, multilink stents were implanted in the iliac artery. The type 1 receptor blocker reduced the development of in-stent neointima formation by approximately 30% in rabbits and monkeys. To investigate potential mechanisms, we examined the expression of renin-angiotensin system markers, all of which increased in monocytes and smooth muscle-like cells in the neointima and media within 7 days. The type 1 receptor blocker attenuated increased oxidative stress, the enhanced expression of markers of the rennin-angiotensin system and monocyte chemoattractant protein-1, and macrophage infiltration. The effects of type 1 receptor blocker on the differentiation of peripheral blood mononuclear cells into vascular progenitor cells were also examined. Treatment with type 1 receptor blocker suppressed the enhanced differentiation to smooth muscle progenitor cells induced by stenting. The type 1 receptor blocker attenuated in-stent neointima formation by inhibiting redox-sensitive inflammatory changes and by reducing recruitment of the progenitor cells. These potential actions of type 1 receptor blocker on inflammation and progenitor cells constitute a novel mechanism of suppression of in-stent restenosis by type 1 receptor blocker.

Bone marrow-derived monocyte chemoattractant protein-1 receptor CCR2 is critical in angiotensin II-induced acceleration of atherosclerosis and aneurysm formation in hypercholesterolemic mice.

UNLABELLED: Angiotensin II (Ang II) is implicated in atherogenesis by activating inflammatory responses in arterial wall cells. Ang II accelerates the atherosclerotic process in hyperlipidemic apoE-/- mice by recruiting and activating monocytes. Monocyte chemoattractant protein-1 (MCP-1) controls monocyte-mediated inflammation through its receptor, CCR2. The roles of leukocyte-derived CCR2 in the Ang II-induced acceleration of the atherosclerotic process, however, are not known. We hypothesized that deficiency of leukocyte-derived CCR2 suppresses Ang II-induced atherosclerosis. METHODS AND RESULTS: A bone marrow transplantation technique (BMT) was used to develop apoE-/- mice with and without deficiency of CCR2 in leukocytes (BMT-apoE-/-CCR2+/+ and BMT-apoE-/-CCR2-/- mice). Compared with BMT-apoE-/-CCR2+/+ mice, Ang II-induced increases in atherosclerosis plaque size and abdominal aortic aneurysm formation were suppressed in BMT-apoE-/-CCR2-/- mice. This suppression was associated with a marked decrease in monocyte-mediated inflammation and inflammatory cytokine expression. CONCLUSIONS: Leukocyte-derived CCR2 is critical in Ang II-induced atherosclerosis and abdominal aneurysm formation. The present data suggest that vascular inflammation mediated by CCR2 in leukocytes is a reasonable target of therapy for treatment of atherosclerosis.