IntroductionThe extent of ventricular dilation after a myocardial infarction (MI) depends on the magnitude of the initial ischemic damage as well as the tissue healing process (1, 2). This dynamic pathological process includes the expression and activation of the matrix metalloproteinases (MMPs), which may mediate many of the morphological changes that occur after MI in both infarcted and noninfarcted regions (3-5). Members of the MMP family of enzymes degrade specific extracellular matrix components; clinical and experimental studies have shown that MMP expression and activity increase in both MI (5) and dilated cardiomyopathy (6, 7).Because extracellular matrix deposition and organization play a major role in left ventricular (LV) remodeling, MMP inhibition has emerged as a potential therapeutic strategy for patients at risk for the development of congestive heart failure. Administration of a broad-spectrum MMP inhibitor attenuates LV enlargement in pacing-induced congestive heart failure (8) and in the early period after MI occurs (9). Whether this effect depends on the inhibition of many MMPs, or selective inhibition of MMPs can prevent ventricular dilation remains unexplored. Recently, Heymans et al. reported a decreased incidence of rupture at 4 days after MI in MMP-9-deficient animals, suggesting that MMP-9 may have a specific role in early myocardial healing (10). In this study we evaluated the influence of targeted deletion of the MMP-9 gene on LV remodeling after experimental MI in mice. We performed this study with sibling wild-type (WT) controls after at least six backcrosses to minimize genetic variability. All analyses were blinded to genotype; the animals were studied for 15 days. MethodsAnimals and surgery. We used the progeny of heterozygous breeding pairs of mice with targeted disruption of MMP-9, as described by Vu et al. (11). MMP-9-deficient mice have delayed long-bone growth and development due to delayed angiogenesis and ossification; however, by adulthood, these changes result in only a 10% shortening in the long bones. Animals with an FVB background were backcrossed; our studies used the homozygous MMP-9-deficient and sibling WT offspring of generation six or higher. Offspring were eartagged and coded, with tail DNA samples harvested for genotyping using PCR. For MMP-9, we used a sense oligonucleotide primer (5′-GCA TAC TTG TAC CGC TAT GG -3′) and an antisense primer (5′-TAA CCG GAG GTC CAA ACT GG-3′). For the neomycin cassette, we also used a sense oligonucleotide primer (5′-GAA Matrix metalloproteinase-9 (MMP-9) is prominently overexpressed after myocardial infarction (MI). We tested the hypothesis that mice with targeted deletion of MMP9 have less left ventricular (LV) dilation after experimental MI than do sibling wild-type (WT) mice. Animals that survived ligation of the left coronary artery underwent echocardiographic studies after MI; all analyses were performed without knowledge of mouse genotype. By day 8, MMP9 knockout (KO) mice had significantly smaller increases in end-diasto...

Arterial conduits are increasingly preferred for surgical bypass because of inherent functional properties conferred by arterial endothelial cells, especially nitric oxide production in response to physiologic stimuli. Here we tested whether endothelial progenitor cells (EPCs) can replace arterial endothelial cells and promote patency in tissue-engineered small-diameter blood vessels (4 mm). We isolated EPCs from peripheral blood of sheep, expanded them ex vivo and then seeded them on decellularized porcine iliac vessels. EPC-seeded grafts remained patent for 130 days as a carotid interposition graft in sheep, whereas non-seeded grafts occluded within 15 days. The EPC-explanted grafts exhibited contractile activity and nitric-oxide-mediated vascular relaxation that were similar to native carotid arteries. These results indicate that EPCs can function similarly to arterial endothelial cells and thereby confer longer vascular-graft survival. Due to their unique properties, EPCs might have other general applications for tissue-engineered structures and in treating vascular diseases.

Background-The mechanisms of extracellular matrix changes accompanying myxomatous valvular degeneration are uncertain. Methods and Results-To test the hypothesis that valvular interstitial cells mediate extracellular matrix degradation in myxomatous degeneration by excessive secretion of catabolic enzymes, we examined the functional characteristics of valvular interstitial cells in 14 mitral valves removed for myxomatous degeneration from patients with mitral regurgitation and in 11 normal mitral valves obtained at autopsy. Immunohistochemical staining assessed (1) cell phenotype using antibodies to ␣-actin (microfilaments), vimentin and desmin (intermediate filaments), smooth muscle myosin (SM1), and SMemb (a nonmuscle myosin produced by activated mesenchymal cells) and (2) the expression of proteolytic activity using antibodies to collagenases (matrix metalloproteinase [MMP]-1, MMP-13), gelatinases (MMP-2, MMP-9), cysteine endoproteases (cathepsin S and K), and interleukin-1␤, a cytokine that can induce secretion of proteolytic enzymes. Although interstitial cells in normal valves stained positively for vimentin, but not ␣-actin or desmin, cells in myxomatous valves contained both vimentin and ␣-actin or desmin (characteristics of myofibroblasts). Moreover, cells in myxomatous valves strongly expressed SMemb, MMPs, cathepsins, and interleukin-1␤, which were weakly stained in controls. Nevertheless, interstitial cells in both groups strongly expressed procollagen-I mRNA (in situ hybridization), suggesting preserved ability to synthesize collagen in myxomatous valves. Conclusions-Interstitial cells in myxomatous valves have features of activated myofibroblasts and express excessive levels of catabolic enzymes, without altered levels of interstitial collagen mRNA. We conclude that valvular interstitial cells regulate matrix degradation and remodeling in myxomatous mitral valve degeneration.

This report demonstrates that atheromatous rather than fibrous plaques might be prone to rupture due to increased collagenolysis associated with macrophages, probably mediated by the interstitial collagenases MMP-1 and MMP-13.

These results suggest that lipid lowering with HMG-CoA reductase inhibitors alters plaque biology by reducing proliferation and activation of macrophages, prominent sources of molecules responsible for plaque instability and thrombogenicity.

Background-Proteolytic enzyme activity in lipid-rich atheroma may promote plaque rupture and precipitate acute coronary syndromes. This study tested the hypothesis that lipid lowering stabilizes plaques by reducing proteolytic activity. Methods and Results-We produced experimental atheroma in 33 rabbits by balloon injury and an atherogenic diet (0.3% cholesterol and 4.7% coconut oil) for 4 months. At that time, 15 rabbits were killed (baseline group). The remaining animals were divided into two groups: a hyperlipemic group continued to consume a cholesterol-enriched diet (0.05% to 0.2%) for 16 more months (nϭ5) and a lipid-lowering group consumed a purified chow diet with no added cholesterol or fat for 8 (nϭ3) or 16 months (nϭ10). Macrophage accumulation and interstitial collagenase (matrix metalloproteinase-1, MMP-1) expression in the lesion were measured by quantitative image analysis of standardized sections of immunostained aortas. Baseline lesions expressed high levels of MMP-1 and contained many macrophages. These features of plaque instability persisted in the hyperlipemic group. However, the lipid-lowering group showed progressive reduction in both macrophage content and MMP-1 immunoreactivity with time. Aortic rings of the baseline and hyperlipemic groups elaborated gelatinolytic, caseinolytic, and elastinolytic activity attributable to MMP-2, MMP-3, or MMP-9, monitored by SDS-PAGE zymography. Proteolytic activity decreased markedly in the lipidlowering group. Aortic content of interstitial collagen, determined by sirius red staining, increased in the lipid-lowering group compared with the baseline or continued hyperlipemic groups, indicating that lipid lowering reinforced the fibrous skeleton of the atheroma. Conclusions-These results establish a mechanism by which lipid lowering may stabilize vulnerable plaques by reduced expression and activity of enzymes that degrade the arterial extracellular matrix and render atheroma less susceptible to disruption and thrombosis by favoring collagen accumulation in the fibrous cap. (Circulation. 1998;97:2433-2444.)

Long-term solid-organ allografts typically develop diffuse arterial intimal lesions (graft arterial disease; GAD), consisting of smooth-muscle cells (SMC), extracellular matrix and admixed mononuclear leukocytes. GAD eventually culminates in vascular stenosis and ischemic graft failure. Although the exact mechanisms are unknown, chronic low-level alloresponses likely induce inflammatory cells and/or dysfunctional vascular wall cells to secrete growth factors that promote SMC intimal recruitment, proliferation and matrix synthesis. Although prior work demonstrated that the endothelium and medial SMCs lining GAD lesions in cardiac allografts are donor-derived, the intimal SMC origin could not be determined. They are generally presumed to originate from the donor media, leading to interventions that target donor medial SMC proliferation, with limited efficacy. However, other reports indicate that allograft vessels may contain host-derived endothelium and SMCs (refs. 8,9). Moreover, subpopulations of bone-marrow and circulating cells can differentiate into endothelium, and implanted synthetic vascular grafts are seeded by host SMCs and endothelium. Here we used murine aortic transplants to formally identify the source of SMCs in GAD lesions. Allografts in beta-galactosidase transgenic recipients showed that intimal SMCs derived almost exclusively from host cells. Bone-marrow transplantation of beta-galactosidase--expressing cells into aortic allograft recipients demonstrated that intimal cells included those of marrow origin. Thus, smooth-muscle--like cells in GAD lesions can originate from circulating bone--marrow-derived precursors.

This study showed that statins can reduce MMP expression in atheroma and that cell-permeant statins can decrease SMC number and collagen gene expression in vivo.