Minako Ishibashi

Gene Transfer of Stromal Cell–Derived Factor-1α Enhances Ischemic Vasculogenesis and Angiogenesis via Vascular Endothelial Growth Factor/Endothelial Nitric Oxide Synthase–Related Pathway Next-Generation Chemokine Therapy for Therapeutic Neovascularization

Background—Stromal cell–derived factor-1&agr; (SDF-1&agr;) is implicated as a chemokine for endothelial progenitor cells (EPCs). We therefore hypothesized that SDF-1&agr; gene transfer would induce therapeutic neovascularization in vivo by functioning as a chemokine of EPC. Methods and Results—To examine SDF-1&agr;–induced mobilization of EPC, we used bone marrow–transplanted mice whose blood cells ubiquitously express β-galactosidase (LacZ). We produced unilateral hindlimb ischemia in the mice and transfected them with plasmid DNA encoding SDF-1&agr; or empty plasmids into the ischemic muscles. SDF-1&agr; gene transfer mobilized EPCs into the peripheral blood, augmented recovery of blood perfusion to the ischemic limb, and increased capillary density associated with partial incorporation of LacZ-positive cells into the capillaries of the ischemic limb, suggesting that SDF-1&agr; induced vasculogenesis and angiogenesis. SDF-1&agr; gene transfer did not affect ischemia-induced expression of vascular endothelial growth factor (VEGF) but did enhance Akt and endothelial nitric oxide synthase (eNOS) activity. Blockade of VEGF or NOS prevented all such SDF-1&agr;–induced effects. Conclusions—SDF-1&agr; gene transfer enhanced ischemia-induced vasculogenesis and angiogenesis in vivo through a VEGF/eNOS-related pathway. This strategy might become a novel chemokine therapy for next generation therapeutic neovascularization.

Increased inflammatory gene expression in ABC transporter deficient macrophages: free cholesterol accumulation, increased signaling via Toll-like receptors and neutrophil infiltration of atherosclerotic lesions

Background— Two macrophage ABC transporters, ABCA1 and ABCG1, have a major role in promoting cholesterol efflux from macrophages. Peritoneal macrophages deficient in ABCA1, ABCG1, or both show enhanced expression of inflammatory and chemokine genes. This study was undertaken to elucidate the mechanisms and consequences of enhanced inflammatory gene expression in ABC transporter–deficient macrophages. Methods and Results— Basal and lipopolysaccharide-stimulated thioglycollate-elicited peritoneal macrophages showed increased inflammatory gene expression in the order Abca1−/−Abcg1−/−>Abcg1−/−>Abca1−/−>wild-type. The increased inflammatory gene expression was abolished in macrophages deficient in Toll-like receptor 4 (TLR4) or MyD88/TRIF. TLR4 cell surface concentration was increased in Abca1−/−Abcg1−/−>Abcg1−/−> Abca1−/−> wild-type macrophages. Treatment of transporter-deficient cells with cyclodextrin reduced and cholesterol-cyclodextrin loading increased inflammatory gene expression. Abca1−/−Abcg1− bone marrow–derived macrophages showed enhanced inflammatory gene responses to TLR2, TLR3, and TLR4 ligands. To assess in vivo relevance, we injected intraperitoneally thioglycollate in Abcg1−/− bone marrow–transplanted, Western diet–fed, Ldlr-deficient mice. This resulted in a profound inflammatory infiltrate in the adventitia and necrotic core region of atherosclerotic lesions, consisting primarily of neutrophils. Conclusions— The results suggest that high-density lipoprotein and apolipoprotein A-1 exert anti-inflammatory effects by promoting cholesterol efflux via ABCG1 and ABCA1 with consequent attenuation of signaling via Toll-like receptors. In response to a peripheral inflammatory stimulus, atherosclerotic lesions containing Abcg1−/− macrophages experience an inflammatory “echo,” suggesting a possible mechanism of plaque destabilization in subjects with low high-density lipoprotein levels.

Pioglitazone, a Peroxisome Proliferator-Activated Receptor-γ Agonist, Attenuates Left Ventricular Remodeling and Failure After Experimental Myocardial Infarction

Background—Peroxisome proliferator–activated receptor-&ggr; activators have recently been implicated as regulators of cellular proliferation and inflammatory response such as cytokine expression. Because proinflammatory cytokines play a critical role in left ventricular (LV) remodeling after myocardial infarction (MI), we examined the effects of pioglitazone treatment in an experimental model of chronic heart failure. Methods and Results—Mice with extensive anterior MI were treated with placebo or pioglitazone (3 mg · kg−1 · d−1) as a dietary supplement for 4 weeks starting 6 hours after surgery. Infarct size and glucose levels were similar among all groups. LV cavity dilatation and dysfunction by echocardiography were significantly attenuated in MI mice given pioglitazone. LV end-diastolic pressure was increased in MI mice and was significantly reduced by pioglitazone treatment. Pioglitazone partially normalized LV dP/dtmax and dP/dtmin, indices of LV contractile function, which were significantly reduced in MI mice. Improvement of LV function by pioglitazone was accompanied by a decrease in myocyte hypertrophy and interstitial fibrosis and a reduced expression of tumor necrosis factor-&agr;, transforming growth factor-&bgr;, and monocyte chemoattractant protein-1 genes in the noninfarcted LV from MI mice. LV inducible nitric oxide synthase and gelatinase B protein levels were increased in MI and were not altered by pioglitazone treatment. Conclusions—Pioglitazone improved LV remodeling and function in mice with post-MI heart failure. This effect was associated with an attenuated LV expression of inflammatory cytokines and chemokines. Peroxisome proliferator–activated receptor-&ggr; ligands have promise as preventive and therapeutic agents against heart failure.

Anti-Monocyte Chemoattractant Protein-1 Gene Therapy Limits Progression and Destabilization of Established Atherosclerosis in Apolipoprotein E–Knockout Mice

Background—Monocyte infiltration into the arterial wall and its activation is the central event in atherogenesis. Thus, monocyte chemoattractant protein-1 (MCP-1) might be a novel therapeutic target against atherogenesis. We and others recently reported that blockade or abrogation of the MCP-1 pathway attenuates the initiation of atheroma formation in hypercholesterolemic mice. It remains unclear, however, whether blockade of MCP-1 can limit progression or destabilization of established lesions. Methods and Results—We report here that blockade of MCP-1 by transfecting an N-terminal deletion mutant of the MCP-1 gene limited progression of preexisting atherosclerotic lesions in the aortic root in hypercholesterolemic mice. In addition, blockade of MCP-1 changed the lesion composition into a more stable phenotype, ie, containing fewer macrophages and lymphocytes, less lipid, and more smooth muscle cells and collagen. This strategy decreased expression of CD40 and the CD40 ligand in the atherosclerotic plaque and normalized the increased chemokine (RANTES and MCP-1) and cytokine (tumor necrosis factor &agr;, interleukin-6, interleukin-1&bgr;, and transforming growth factor &bgr;1) gene expression. These data suggest that MCP-1 is a central mediator in the progression and destabilization of established atheroma. Conclusions—The results of the present study suggest that the inflammatory responses mediated by MCP-1 are important in atherosclerosis and its complications.

The critical role of ocular-infiltrating macrophages in the development of choroidal neovascularization

Choroidal neovascularization (CNV) is directly related to visual loss in some eye diseases, such as age‐related macular degeneration. Although several human histological studies have suggested the participation of macrophages in CNV formation, the precise mechanisms are still not fully understood. In this study, we elucidated the role of ocular‐infiltrating macrophages in experimental CNV using CCR2 knockout (KO) mice, wild‐type mice, and C57BL/6 (B6) mice. CCR2 is the receptor of monocyte chemoattractant protein‐1, and the number of infiltrating macrophage and the area of CNV were significantly reduced in CCR2 KO mice. Enriched ocular‐infiltrating macrophages from B6 mice actually showed angiogenic ability in a dorsal air sac assay. Moreover, their expression of class II, CD40, B7‐1 and B7‐2 molecules, and the mRNA for potential angiogenic factors, such as vascular endothelial growth factor, basic fibroblast growth factor, and tumor necrosis factor α, was also observed. Collectively, we conclude that ocular‐infiltrating macrophages play an important role in CNV generation.

Critical Role of Monocyte Chemoattractant Protein-1 Receptor CCR2 on Monocytes in Hypertension-Induced Vascular Inflammation and Remodeling

Activated monocytes are present in the arterial walls of hypertensive patients and animals. Monocyte chemoattractant protein-1 (MCP-1), which controls monocyte function through its receptor (CCR2), is implicated in hypertensive inflammatory changes in the arterial wall. The role of CCR2 expression on monocytes in hypertension-induced vascular remodeling, however, has not been addressed. We hypothesized that CCR2 on monocytes is critical in hypertension-induced vascular inflammation and remodeling. Hypertension was induced by infusion of angiotensin II (Ang II) into wild-type mice, CCR2-deficient (CCR2−/−) mice, and bone marrow-transferred mice with a leukocyte-selective CCR2 deficiency (BMT-CCR2−/−). In wild-type mice, Ang II increased CCR2 intensity in circulating monocytes, which was prevented by an Ang II type-1 (AT1) receptor blocker or blunted in AT1 receptor–deficient mice. Enhanced CCR2 intensity on monocytes was observed in hypertensive patients and rats, and was reduced by treatment with the Ang II receptor blocker, supporting the clinical relevance of the observation in mice. In CCR2−/− and BMT-CCR2−/− mice, Ang II–induced vascular inflammation and vascular remodeling (aortic wall thickening and fibrosis) were blunted as compared with control mice. In contrast, Ang II–induced left ventricular hypertrophy developed in CCR2−/− and BMT-CCR2−/− mice. The present study suggests that CCR2 expression in monocytes has a critical role in vascular inflammation and remodeling in Ang II–induced hypertension, and possibly in other forms of hypertension.

Essential Role of Vascular Endothelial Growth Factor in Angiotensin II–Induced Vascular Inflammation and Remodeling

Angiotensin II (Ang II) upregulates vascular endothelial growth factor (VEGF) and activates vascular inflammation. However, the decisive role of VEGF in Ang II–induced vascular inflammation and remodeling has not been addressed. Ang II infusion to wild-type mice increased local expression of VEGF and its receptors in cells of aortic wall and plasma VEGF, and caused aortic inflammation (monocyte infiltration) and remodeling (wall thickening and fibrosis). Hypoxia-inducible factor-1&agr; colocalized with VEGF-positive cell types. Blockade of VEGF by the soluble VEGF receptor 1 (sFlt-1) gene transfer attenuated the Ang II–induced inflammation and remodeling. The sFlt-1 gene transfer also inhibited the increased expression of VEGF and inflammatory factors such as monocyte chemoattractant protein-1. In contrast, sFlt-1 gene transfer did not affect Ang II–induced arterial hypertension and cardiac hypertrophy. VEGF is an essential mediator in Ang II–induced vascular inflammation and structural changes through its proinflammatory actions.

Antiinflammatory and Antiarteriosclerotic Effects of Pioglitazone

Abstract—Peroxisome proliferator-activated receptor-&ggr; (PPAR&ggr;) ligands are widely used in patients with insulin resistance and diabetes. Because coronary artery disease is a major complication for such patients, it is important to determine the effects of PPAR&ggr; activation on arteriosclerosis. Long-term inhibition of endothelial NO synthesis by administration of N&ohgr;-nitro-l-arginine methyl ester (L-NAME) to rats induces coronary vascular inflammation (monocyte infiltration, monocyte chemoattractant protein-1 [MCP-1] expression) and subsequent arteriosclerosis. We examined the effects of pioglitazone (a PPAR&ggr; ligand) in this rat model to determine whether PPAR&ggr; activation with pioglitazone inhibits arteriosclerosis by its indirect effects on metabolic conditions or by direct effects on the cells participating to the pathogenesis of arteriosclerosis. We found that pioglitazone did not affect metabolic states, systolic blood pressure, or serum NO levels, but did prevent the L-NAME–induced coronary inflammation and arteriosclerosis. Pioglitazone did not reduce local expression of MCP-1 but markedly attenuated increased expression of the MCP-1 receptor C-C chemokine receptor 2 (CCR2) in lesional and circulating monocytes. PPAR&ggr; activation with pioglitazone prevented coronary arteriosclerosis, possibly by its antiinflammatory effects (downregulation of CCR2 in circulating monocytes). Inhibition of the CCR2-mediated inflammation may represent novel antiinflammatory actions of pioglitazone beyond improvement of metabolic state.

Bone Marrow–Derived Monocyte Chemoattractant Protein-1 Receptor CCR2 Is Critical in Angiotensin II–Induced Acceleration of Atherosclerosis and Aneurysm Formation in Hypercholesterolemic Mice

Abstract—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. Conclusion—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.

Blockade of Vascular Endothelial Growth Factor Suppresses Experimental Restenosis After Intraluminal Injury by Inhibiting Recruitment of Monocyte Lineage Cells

Background—Therapeutic angiogenesis by delivery of vascular endothelial growth factor (VEGF) has attracted attention. However, the role and function of VEGF in experimental restenosis (neointimal formation) after vascular intraluminal injury have not been addressed. Methods and Results—We report herein that blockade of VEGF by soluble VEGF receptor 1 (sFlt-1) gene transfer attenuated neointimal formation after intraluminal injury in rabbits, rats, and mice. sFlt-1 gene transfer markedly attenuated the early vascular inflammation and proliferation and later neointimal formation. sFlt-1 gene transfer also inhibited increased expression of inflammatory factors such as monocyte chemoattractant protein-1 and VEGF. Intravascular VEGF gene transfer enhanced angiogenesis in the adventitia but did not reduce neointimal formation. Conclusions—Increased expression and activity of VEGF are essential in the development of experimental restenosis after intraluminal injury by recruiting monocyte-lineage cells.