Abstract-The migration of vascular smooth muscle cells (VSMCs) plays an essential role during the development of atherosclerosis and restenosis. Extensive studies have implicated the importance of extracellular matrix (ECM)-degrading proteinases in VSMC migration. A recently described family of proteinases, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTs), is capable of degrading vascular ECM proteins. Here, we sought to determine whether ADAMTS-7 is involved in VSMC migration and neointima formation in response to vascular injury. ADAMTS-7 protein accumulated preferentially in neointima of the carotid artery wall after balloon injury. In primary VSMCs, ADAMTS-7 level was enhanced by the proinflammatory cytokine tumor necrosis factor ␣ and growth factor platelet-derived growth factor-BB. ADAMTS-7 overexpression greatly accelerated and small interfering RNA knockdown markedly retarded VSMC migration/invasion in vitro. In addition, luminal delivery of ADAMTS-7 adenovirus to carotid arteries exacerbated intimal thickening nearly sixfold 7 days after injury. Conversely, perivascular administration of ADAMTS-7 small interfering RNA but not scramble small interfering RNA to injured arteries attenuated intimal thickening by 50% at 14 days after injury. Furthermore, ADAMTS-7 mediated degradation of the vascular ECM cartilage oligomeric matrix protein (COMP) in injured vessels. Replenishing COMP circumvented the promigratory effect of ADAMTS-7 on VSMCs. Enforced expression of COMP significantly suppressed VSMC migration and neointima formation postinjury, which indicates that ADAMTS-7 facilitated intimal hyperplasia through degradation of inhibitory matrix protein COMP. ADAMTS-7 may therefore serve as a novel therapeutic target for atherosclerosis and postangioplasty restenosis. Key Words: metalloproteinase Ⅲ vascular smooth muscle cell migration Ⅲ neointima formation Ⅲ extracellular matrix M edia-to-intima migration of vascular smooth muscle cells (VSMCs) is pivotal to intimal thickening in atherosclerosis, restenosis after coronary angioplasty, and late failure of vein grafting. 1 Normally VSMCs are quiescent and are surrounded by and embedded in an extracellular matrix (ECM) scaffold that acts as a barrier to VSMC migration. ECM degradation and remodeling require the activation of extracellular proteases, which in turn facilitate VSMC migration. 2 Previous studies have emphasized potential roles for the matrix metalloproteinases MMP-2, MMP-9, and MT1-MMP; the serine proteinases plasminogen activator and plasminogen; and the cysteine proteinases cathepsins K, L, and S during matrix remodeling and VSMC migration. 3 However, the identity of the matrix-degrading proteinases during pathological vascular remodeling in vivo has remained the subject of speculation.The recently identified metalloproteinase family of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) also degrade ECM. First identified in 1997, ADAMTS already showed strong biological relevance. 4 For example, ADAMTS...

A bdominal aortic aneurysm (AAA), characterized by chronic aortic wall inflammation and destructive connective tissue remodeling, is one of the leading causes of sudden death in aging men (>55 years).1 Despite the current progress of surgical invasive repair or medical treatment, we lack a strategy to predict AAA rupture or to delay its progression. Epidemiological studies have identified several risk factors associated with AAA, including aging, male sex, smoking, and hypertension.1-3 However, the cause of AAA remains far from being fully elucidated. In This Issue, see p 1249Homocysteine (Hcy) is a sulfur-containing nonconstitutive amino acid derived from the essential amino acid methionine. The upper limit of the normal range for circulating Hcy is approximately 15 μmol/L, whereas a sex difference has been found, with approximately 10% to 15% higher levels in men versus women. Compelling evidence suggests that hyperhomocysteinemia (HHcy) is a strong independent risk factor of coronary heart disease and stroke in human.

Key Words: smooth muscle cells Ⅲ phenotype Ⅲ COMP Ⅲ integrin Ⅲ neointima U nlike cardiac or skeletal muscle cells, vascular smooth muscle cells (VSMCs) have a unique property of plasticity and can undergo reversible changes in phenotype. 1,2 Normally, they are mainly restricted to the media of adult blood vessels, express a repertoire of contractile proteins such as smooth muscle (SM) myosin heavy chain, SM ␣-actin, SM-22␣ and calponin, and have low rate of replication. However, on various environmental cues, VSMCs can undergo transition from a quiescent, contractile/differentiated phenotype to a synthetic/dedifferentiated phenotype, with a high rate of migration/proliferation and a concomitant reduction in expression of VSMC marker proteins. 1,3 Phenotypic switching of VSMCs plays an essential role in the development of cardiovascular diseases such as atherosclerosis, postangioplastic restenosis and hypertension. Studies have demonstrated the contribution to cell dedifferentiation of growth factors, mitogenic cytokines, reactive oxygen species, stretch or injury. 2,4,5 Nevertheless, how normal VSMCs maintain the differentiated state is much less understood and has been largely ignored. Understanding the mechanisms that conserve a differentiated phenotype is critical to interfere with the development of cardiovascular diseases. MethodsAnimal care and use of carotid-artery injury model in male SpragueDawley rats were in accordance with institutional guidelines. VSMCs were isolated from the thoracic aortic arteries of rats by collagenase digestion. Small interfering (si)RNA against COMP, ␣ 7 integrin, and ␣ 8 integrin were transfected in vitro by use of Oligofectamine (Invitrogen). The adenovirus for COMP was constructed and a single exposure of 5ϫ10 8 plaque forming units was luminally delivered to balloon-injured carotid segments for in vivo studies. The promoter constructs of SM ␣-actin-luc and SM22␣-luc was transfected into VSMCs and the relative promoter activities were detected by use of a luciferase assay kit. For cell adhesion assay, nontreated 48-well plates were coated with purified COMP, fibronectin, or polymerized collagen I and incubated with neutralizing anti-␣ 7 integrin monoclonal antibody or mouse normal IgG. The contractility of VSMCs and vessel tension measurement was examined as described previously.An expanded Methods section is available in the Online Data Supplement at http://circres.ahajournals.org. Results COMP Level Is Decreased in Balloon-Injured Rat Carotid ArteriesUsing a rat balloon-injury model, we first examined the expression of COMP in carotid arteries after injury. COMP protein level was markedly lower in injured vessels than in sham-operated vessels 4 to 14 days after injury and was paralleled by an increase of COMP degradation fragment (Figure 1). Combined with our previous observation that degradation of COMP promoted VSMC migration and neointima formation, 6 COMP may negatively regulate VSMC activation in response to injury. Association of COMP and VSMC Markers In VitroVSMC...

Although adenosine exerts cardio-and vasculoprotective effects, the roles and signaling mechanisms of different adenosine receptors in mediating skeletal muscle protection are not well understood. We used a mouse hindlimb ischemia-reperfusion model to delineate the function of three adenosine receptor subtypes. Adenosine A(3) receptor-selective agonist 2-chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide (Cl-IBMECA; 0.07 mg/kg ip) reduced skeletal muscle injury with a significant decrease in both Evans blue dye staining (5.4 +/- 2.6%, n = 8 mice vs. vehicle-treated 28 +/- 6%, n = 7 mice, P < 0.05) and serum creatine kinase level (1,840 +/- 910 U/l, n = 13 vs. vehicle-treated 12,600 +/- 3,300 U/l, n = 14, P < 0.05), an effect that was selectively blocked by an A(3) receptor antagonist 3-ethyl-5-benzyl-2-methyl-6-phenyl-4-phenylethynyl-1,4-(+/-)-dihydropyridine-3,5-dicarboxylate (MRS-1191; 0.05 mg/kg). The adenosine A(1) receptor agonist 2-chloro-N(6)-cyclopentyladenosine (CCPA; 0.05 mg/kg) also exerted a cytoprotective effect, which was selectively blocked by the A(1) antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 0.2 mg/kg). The adenosine A(2A) receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS-21680; 0.07 mg/kg)-induced decrease in skeletal muscle injury was selectively blocked by the A(2A) antagonist 2-(2-furanyl)-7-[3-(4-methoxyphenyl)propyl]-7H-pyrazolo[4,3-e] [1,2,4]triazolo[1,5-C]pyrimidin-5-amine (SCH-442416; 0.017 mg/kg). The protection induced by the A(3) receptor was abrogated in phospholipase C-beta2/beta3 null mice, but the protection mediated by the A(1) or A(2A) receptor remained unaffected in these animals. The adenosine A(3) receptor is a novel cytoprotective receptor that signals selectively via phospholipase C-beta and represents a new target for ameliorating skeletal muscle injury.

Aging is associated with shifts in autocrine and paracrine pathways in the cardiac vasculature that may contribute to the risk of cardiovascular disease in older persons. To elucidate the molecular basis of these changes in vivo, phage-display biopanning of 3- and 18-mo-old mouse hearts was performed that identified peptide epitopes with homology to brain-derived neurotrophic factor (BDNF) in old but not young phage pools. Quantification of cardiac phage binding by titration and immunostaining after injection with BDNF-like phage identified a twofold increased density of the BDNF receptor, truncated Trk B, in the aging hearts. Studies focused on the receptor ligand using a rat model of transient myocardial ischemia revealed increases in cardiac BDNF associated with local mononuclear infiltrates in 24- but not 4-mo-old rats. To investigate these changes, both 4- and 24-mo-old rat hearts were treated with intramyocardial injections of BDNF (or PBS control), demonstrating significant inflammatory increases with activated macrophage (ED1+) in BDNF-treated aging hearts compared with aging controls and similarly treated young hearts. Additional studies with permanent coronary occlusion following intramyocardial growth factor pretreatment revealed that BDNF significantly increased the extent of myocardial injury in older rat hearts (BDNF 35 +/- 10% vs. PBS 16.2 +/- 7.9% left ventricular injury; P < 0.05) without affecting younger hearts (BDNF 15 +/- 5.1% vs. PBS 14.5 +/- 6.0% left ventricular injury). Overall, these studies suggest that age-associated changes in BDNF-Trk B pathways may predispose the aging heart to increased injury after acute myocardial infarction and potentially contribute to the enhanced severity of cardiovascular disease in older individuals.

Abstract-The directed generation of cardiac myocytes from endogenous stem cells offers the potential for novel therapies for cardiovascular disease. To facilitate the development of such approaches, we sought to identify and exploit the pathways directing the generation of cardiac myocytes from adult rodent bone marrow cells (BMCs). In vitro cultures supporting the spontaneous generation of functional cardiac myocytes from murine BMCs demonstrated induced expression of platelet-derived growth factor (PDGF)-A and -B isoforms with ␣-and ␤-myosin heavy chains as well as connexin43. Supplementation of PDGF-AB speeded the kinetics of myocyte development in culture by 2-fold. In a rat heart, myocardial infarction pretreatment model PDGF-AB also promoted the derivation of cardiac myocytes from BMCs, resulting in a significantly greater number of islands of cardiac myocyte bundles within the myocardial infarction scar compared with other treatment groups. However, gap junctions were detected only between the cardiac myocytes receiving BMCs alone, but not BMCs injected with PDGF-AB. Echocardiography and exercise testing revealed that the functional improvement of hearts treated with the combination of BMCs and PDGF-AB was no greater than with injections of BMCs or PDGF-AB alone. These studies demonstrated that PDGF-AB enhances the generation of BMC-derived cardiac myocytes in rodent hearts, but suggest that alterations in cellular patterning may limit the functional benefit from the combined injection of PDGF-AB and BMCs. Strategies based on the synergistic interactions of PDGF-AB and endogenous stem cells will need to maintain cellular patterning in order to promote the restoration of cardiac function after acute coronary occlusion. The full text of this article is available online at

Age-associated dysfunction in cardiac microvascular endothelial cells with impaired induction of cardioprotective platelet-derived growth factor (PDGF)-dependent pathways suggests that alterations in critical vascular receptor(s) may contribute to the increased severity of cardiovascular pathology in older persons. In vivo murine phage-display peptide library biopanning revealed a senescent decrease in cardiac microvascular binding of phage epitopes homologous to tumor necrosis factor-α (TNF-α), suggesting that its receptor(s) may be downregulated in older cardiac endothelial cells. Immunostaining demonstrated that TNF-receptor 1 (TNF-R1) density was significantly lower in the subendocardial endothelium of the aging murine heart. Functional studies confirmed the senescent dysregulation of TNF-α receptor pathways, demonstrating that TNF-α induced PDGF-B expression in cardiac microvascular endothelial cells of 4-mo-old, but not 24-mo-old, rats. Moreover, TNF-α mediated cardioprotective pathways were impaired in the aging heart. In young rat hearts, injection of TNF-α significantly reduced the extent of myocardial injury after coronary ligation: TNF-α, 7.9 ± 1.9% left ventricular injury ( n = 4) versus PBS, 16.2 ± 7.9% ( n = 10; P < 0.05). The addition of PDGF-AB did not augment the cardioprotective action of TNF-α. In myocardial infarctions of older hearts, however, TNF-α induced significant postcoronary occlusion mortality (TNF-α 80% vs. PBS 0%; n = 10 each, P < 0.05) that was reversed by the coadministration of PDGF-AB. Overall, these studies demonstrate that aging-associated alterations in TNF-α receptor cardiac microvascular pathways may contribute to the increased cardiovasular pathology of the aging heart. Strategies targeted at restoring TNF-α receptor-mediated expression of PDGF-B may improve cardiac microvascular function and provide novel approaches for treatment and possible prevention of cardiovascular disease in older individuals.

Pretreatment of rodent hearts with platelet-derived growth factor (PDGF)–AB decreases myocardial injury after coronary occlusion. However, PDGF-AB cardioprotection is diminished in older animals, suggesting that downstream elements mediating and/or synergizing the actions of PDGF-AB may be limited in aging cardiac vasculature. In vitro PDGF-AB induced vascular endothelial growth factor (VEGF) and angiopoietin (Ang)-2 expression in 4-mo-old rat cardiac endothelial cells, but not in 24-mo-old heart cells. In vivo injection of young hearts with PDGF-AB increased densities of microvessels staining for VEGF and its receptor, Flk-1, and Ang-2 and its receptor, Tie-2, as well as PDGF receptor (PDGFR)–α. In older hearts, PDGF-AB–mediated induction was primarily limited to PDGFR-α. Studies in a murine cardiac transplantation model demonstrated that synergist interactions of PDGF-AB plus VEGF plus Ang-2 (PVA) provided an immediate restoration of senescent cardiac vascular function. Moreover, PVA injection in young rat hearts, but not PDGF-AB alone or other cytokine combinations, at the time of coronary occlusion suppressed acute myocardial cell death by >50%. However, PVA also reduced the extent of myocardial infarction with an age-associated cardioprotective benefit (4-mo-old with 45% reduction vs. 24-mo-old with 24%; P < 0.05). These studies showed that synergistic cytokine pathways augmenting the actions of PDGF-AB are limited in older hearts, suggesting that strategies based on these interactions may provide age-dependent clinical cardiovascular benefit.