Shiro Kitamoto

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.

New Anti–Monocyte Chemoattractant Protein-1 Gene Therapy Attenuates Atherosclerosis in Apolipoprotein E–Knockout Mice

BackgroundMonocyte recruitment into the arterial wall and its activation may be the central event in atherogenesis. Monocyte chemoattractant protein-1 (MCP-1) is an important chemokine for monocyte recruitment, and its receptor (CCR2) may mediate such in vivo response. Although the importance of the MCP-1/CCR2 pathway in atherogenesis has been clarified, it remains unanswered whether postnatal blockade of the MCP-1 signals could be a unique site-specific gene therapy. Methods and ResultsWe devised a new strategy for anti-MCP-1 gene therapy to treat atherosclerosis by transfecting an N-terminal deletion mutant of the human MCP-1 gene into a remote organ (skeletal muscle) in apolipoprotein E-knockout mice. This strategy effectively blocked MCP-1 activity and inhibited the formation of atherosclerotic lesions but had no effect on serum lipid concentrations. Furthermore, this strategy increased the lesional extracellular matrix content. ConclusionsWe conclude that this anti-MCP-1 gene therapy may serve not only to reduce atherogenesis but also to stabilize vulnerable atheromatous plaques. This strategy may be a useful and feasible form of gene therapy against atherosclerosis in humans.

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.

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.

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.

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.

Monocyte Chemoattractant Protein-1 Is an Essential Inflammatory Mediator in Angiotensin II-Induced Progression of Established Atherosclerosis in Hypercholesterolemic Mice

Objective—Chronic inflammatory processes might be involved in the progression and destabilization of atherosclerotic plaques. Therefore, identification of the mechanism underlying arterial inflammatory function might lead to the development of novel therapeutic strategies. Angiotensin II (AngII) is implicated in atherogenesis by activating the vascular inflammation system, mainly through monocyte chemotaxis. Therefore, we hypothesized that AngII increases plaque size and promotes destabilization of established atheromas by activating the monocyte chemoattractant protein-1 (MCP-1) pathway. Methods and Results—We report here that 4-week infusion of AngII not only increased plaque size but also induced a destabilization phenotype (ie, increased macrophages and lipids and decreased collagen and smooth muscle cells) of pre-existing atherosclerotic lesions of hypercholesterolemic mice. AngII also enhanced the gene expression of inflammatory cytokines (TNF&agr;, IL-6, etc.) and chemokines (MCP-1, CCR2, etc). Blockade of MCP-1, by transfecting the deletion mutant of the human MCP-1 gene into the skeletal muscles, limited AngII-induced progression and destabilization of established atherosclerotic lesions and suppressed the induction of proinflammatory genes. Conclusions—These data suggest that MCP-1 functions as a central inflammatory mediator in the AngII-induced progression and changes in plaque composition of established atheroma.

Pathogenic Role of Oxidative Stress in Vascular Angiotensin-Converting Enzyme Activation in Long-Term Blockade of Nitric Oxide Synthesis in Rats

Inhibition of nitric oxide (NO) synthesis with N(omega)-nitro-L-arginine methyl ester (L-NAME) activates vascular angiotensin-converting enzyme (ACE) and causes oxidative stress. We investigated the role of oxidative stress in the pathogenesis of ACE activation in rats. Studies involved aortas of rats receiving no treatment, L-NAME, L-NAME plus L-arginine, or L-NAME plus an antioxidant drug (N-acetylcysteine, allopurinol, or ebselen) for 7 days. L-NAME significantly increased oxidative stress (O(2)(-)) and ACE activity. The increased O(2)(-) production was normalized by removal of endothelium. Immunohistochemistry showed the increased ACE activity in the endothelial layer. Treatment with antioxidant drugs did not affect the L-NAME-induced increase in systolic arterial pressure but did prevent increases in vascular O(2)(-) production and ACE activity. These results implicate oxidative stress in the pathogenesis of vascular ACE activation in rats with long-term inhibition of NO synthesis. The observed effects of antioxidant drugs on ACE activation do not appear to involve the hypertension induced by L-NAME.

Role of Monocyte Chemoattractant Protein-1 in Cardiovascular Remodeling Induced by Chronic Blockade of Nitric Oxide Synthesis

BackgroundChronic inhibition of endothelial nitric oxide (NO) synthesis by the administration of N&ohgr;-nitro-l-arginine methyl ester (L-NAME) to rats induces early vascular inflammatory changes (monocyte infiltration into coronary vessels and monocyte chemoattractant protein-1 [MCP-1] expression) as well as subsequent arteriosclerosis (medial thickening and perivascular fibrosis) and cardiac fibrosis. However, the role of MCP-1 in this process is not known. Methods and ResultsWe investigated the effect of a specific monoclonal anti–MCP-1 neutralizing antibody in rats treated with L-NAME to determine the role of monocytes in the regulation of cardiovascular remodeling. We found increased expression of MCP-1 mRNA in vascular endothelial cells and monocytes in inflammatory lesions. Cotreatment with an anti–MCP-1 antibody, but not with control IgG, prevented the L-NAME–induced early inflammation and reduced late coronary vascular medial thickening. In contrast, the anti–MCP-1 antibody did not decrease the development of perivascular fibrosis, the expression of transforming growth factor (TGF)-&bgr;1 mRNA, or systolic pressure overload induced by L-NAME administration. ConclusionsThese results suggest that MCP-1 is necessary for the development of medial thickening as well as monocyte recruitment. In contrast, the pathogenesis of fibrosis may involve other factors, such as TGF-&bgr;1.

Cathepsin L Deficiency Reduces Diet-Induced Atherosclerosis in Low-Density Lipoprotein Receptor–Knockout Mice

Background— Remodeling of the arterial extracellular matrix participates importantly in atherogenesis and plaque complication. Increased expression of the elastinolytic and collagenolytic enzyme cathepsin L (Cat L) in human atherosclerotic lesions suggests its participation in these processes, a hypothesis tested here in mice. Methods and Results— We generated Cat L and low-density lipoprotein receptor (LDLr) double-deficient (LDLr−/−Cat L−/−) mice by crossbreeding Cat L-null (Cat L−/−) and LDLr-deficient (LDLr−/−) mice. After 12 and 26 weeks of a Western diet, LDLr−/−Cat L−/− mice had significantly smaller atherosclerotic lesions and lipid cores compared with littermate control LDLr−/−Cat L+/− and LDLr−/−Cat L+/+ mice. In addition, lesions from the compound mutant mice showed significantly reduced levels of collagen, medial elastin degradation, CD4+ T cells, macrophages, and smooth muscle cells. Mechanistic studies showed that Cat L contributes to the degradation of extracellular matrix elastin and collagen by aortic smooth muscle cells. Smooth muscle cells from LDLr−/−Cat L−/− mice or those treated with a Cat L-selective inhibitor demonstrated significantly less degradation of elastin and collagen and delayed transmigration through elastin in vitro. Cat L deficiency also significantly impaired monocyte and T-lymphocyte transmigration through a collagen matrix in vitro, suggesting that blood-borne leukocyte penetration through the arterial basement membrane requires Cat L. Cysteine protease active site labeling demonstrated that Cat L deficiency did not affect the activity of other atherosclerosis-associated cathepsins in aortic smooth muscle cells and monocytes. Conclusions— Cat L directly participates in atherosclerosis by degrading elastin and collagen and regulates blood-borne leukocyte transmigration and lesion progression.