Masanori Aikawa

Targeted deletion of matrix metalloproteinase-9 attenuates left ventricular enlargement and collagen accumulation after experimental myocardial infarction

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-diastolic and end-systolic ventricular dimensions at both midpapillary and apical levels, compared with infarcted WT mice; these differences persisted at 15 days after MI. MMP-9 KO mice had less collagen accumulation in the infarcted area than did WT mice, and they showed enhanced expression of MMP-2, MMP-13, and TIMP-1 and a reduced number of macrophages. We conclude that targeted deletion of the MMP9 gene attenuates LV dilation after experimental MI in mice. The decrease in collagen accumulation and the enhanced expression of other MMPs suggest that MMP-9 plays a prominent role in extracellular matrix remodeling after MI.

An HMG-CoA Reductase Inhibitor, Cerivastatin, Suppresses Growth of Macrophages Expressing Matrix Metalloproteinases and Tissue Factor In Vivo and In Vitro

BackgroundUnstable atherosclerotic plaques that cause acute coronary events usually contain abundant macrophages expressing matrix metalloproteinases (MMPs) and tissue factor (TF), molecules that probably contribute to plaque rupture and subsequent thrombus formation. Lipid lowering with HMG-CoA reductase inhibitors reduces acute coronary events. Methods and ResultsTo test whether lipid lowering with an HMG-CoA reductase inhibitor retards macrophage accumulation in rabbit atheroma, we administered cerivastatin to immature Watanabe heritable hyperlipidemic rabbits (cerivastatin group, n=10, cerivastatin 0.6 mg · kg−1 · d−1; control group, n=9, saline 0.6 mL · kg−1 · d−1) for 32 weeks and measured macrophage accumulation and expression of MMPs and TF. Serum cholesterol levels after 32 weeks were 809±40 mg/dL (control group) and 481±24 mg/dL (treated group). Cerivastatin diminished accumulation of macrophages in aortic atheroma. Macrophage expression of MMP-1, MMP-3, MMP-9, and TF also decreased with cerivastatin treatment. Cerivastatin reduced the number of macrophages expressing histone mRNA (a sensitive marker of cell proliferation) detected by in situ hybridization but did not alter macrophages bearing a marker of death (TUNEL staining). Cerivastatin treatment (≥0.01 &mgr;mol/L) also reduced growth, proteolytic activity due to MMP-9, and TF expression in cultured human monocyte/macrophages. ConclusionsThese 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.

Lipid Lowering by Diet Reduces Matrix Metalloproteinase Activity and Increases Collagen Content of Rabbit Atheroma A Potential Mechanism of Lesion Stabilization

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 (n=5) and a lipid-lowering group consumed a purified chow diet with no added cholesterol or fat for 8 (n=3) or 16 months (n=10). 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 lipid-lowering 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.

Stabilization of atherosclerotic plaques: New mechanisms and clinical targets

Research points to pivotal roles for lipids in the development of atherosclerotic plaques. Lipid-lowering statins substantially reduce acute coronary events resulting from plaque development, but only modestly reduce arterial stenosis. This apparent paradox has shifted the goal of therapy towards plaque stabilization rather than enlargement of the lumen. More thorough understanding of the biology of atherosclerosis should enable us to manipulate plaque stability, and reduce further the acute complications of atherosclerosis.

Host bone-marrow cells are a source of donor intimal smooth- muscle–like cells in murine aortic transplant arteriopathy

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 β-galactosidase transgenic recipients showed that intimal SMCs derived almost exclusively from host cells. Bone-marrow transplantation of β-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.

MACROPHAGES AND ATHEROSCLEROTIC PLAQUE STABILITY

Physical disruption of atheroma frequently causes coronary thrombosis. Ruptured plaques usually have thin fibrous caps overlying a large thrombogenic lipid core rich in lipid-laden macrophages. The biology of plaque monocyte-derived macrophages thus assumes critical importance in understanding plaque instability. Monocyte recruitment involves binding to leukocyte adhesion receptors on the endothelial surface such as intercellular adhesion molecule-1 and vascular cell adhesion molecule-1. Once adherent to the endothelial surface, monocytes enter the intima at sites of lesion predilection. This process probably requires directed migration of the mononuclear cells. A number of chemoattractant molecules, such as the monocyte chemoattractant molecule-1, may participate in signaling this entry of adherent monocytes into the artery wall. Once resident in the arterial intima, monocytes accumulate lipid, via increasingly well characterized receptor-mediated uptake, and transform into macrophage foam cells. These lesional macrophages also acquire other functional properties including production of the potent procoagulant, tissue factor, apolipoprotein E, and an increasing list of cytokines (protein mediators of information and immunity) that may participate importantly in autocrine and paracrine signaling among leukocytes and vascular endothelial and smooth muscle cells. Fatty streaks seldom cause clinical events but may evolve into complicated atheromatous plaques characterized by an accumulation of smooth muscle cells and extracellular matrix and formation of a central core containing extracellular lipid. Death of macrophages, including programmed cell death or apoptosis, probably promotes formation of this thrombogenic lipid pool whose size correlates with plaque instability. Lesion complication often culminates in rupture of the fibrous cap overlying this lipid core. The integrity of the fibrous cap, and thus its resistance to rupture, depends critically on the collagenous extracellular matrix of the plaques fibrous cap. This aspect of plaque structure in turn depends upon the balance between synthesis and degradation of the macromolecules that comprise the extracellular matrix of the cap, principally interstitial forms of collagen derived from arterial smooth muscle cells. Collagen breakdown, however, appears to depend critically on macrophages. Plaque macrophages express a variety of matrixdegrading enzymes that can contribute to the weakening of the fibrous cap. In this way, macrophages can critically influence aspects of the biology of human atheroma related to lesion stability. We hypothesize that lipid-lowering reduces clinical events, as shown in recent trials, by stabilizing lesions in part by reversing some of the maladaptive functions of macrophages described above.

Matrix Metalloproteinase Inhibition Attenuates Early Left Ventricular Enlargement After Experimental Myocardial Infarction in Mice

BACKGROUND Extracellular matrix synthesis and degradation contribute to the morphological changes that occur after myocardial infarction (MI). METHODS AND RESULTS We tested the hypothesis that inhibition of matrix metalloproteinases (MMPs) attenuates left ventricular remodeling in experimental MI. Seventy-one male FVB mice that survived ligation of the left anterior coronary artery were randomized to a broad-spectrum MMP inhibitor (CP-471,474) or placebo by gavage. Echocardiographic studies were performed before randomization (within 24 hours of surgery) and 4 days later and included short-axis imaging at the midpapillary and apical levels. Infarction as defined by wall motion abnormality was achieved in 79% of the procedures (n=56), and mortality rate during the 4-day protocol was 23% (9 of 36 on treatment vs 7 of 35 on placebo; P=NS). Baseline end-diastolic and end-systolic dimensions and areas were similar (P=NS) between treated and placebo groups. At follow-up, infarcted mice allocated to MMP inhibitor had significantly smaller increases in end-systolic and end-diastolic dimensions and areas at both midpapillary and apical levels compared with infarcted mice allocated to placebo (all P<0.05). In addition, infarcted animals that received MMP inhibitor had no change in fractional shortening (-3+/-13%), whereas animals that received placebo had a decrease in fractional shortening (-12+/-12%) (P<0.05). In an analysis stratified by baseline end-diastolic area, the effects of MMP inhibition on the changes in end-systolic area and end-diastolic area were most prominent in animals that had more initial left ventricular dilatation (both P<0.05). CONCLUSIONS -Administration of an MMP inhibitor attenuates early left ventricular dilation after experimental MI in mice. Further studies in genetically altered mice and other models will improve understanding of the role of MMPs in left ventricular remodeling.

Osteogenesis Associates With Inflammation in Early-Stage Atherosclerosis Evaluated by Molecular Imaging In Vivo

Background— Arterial calcification is associated with cardiovascular events; however, mechanisms of calcification in atherosclerosis remain obscure. Methods and Results— We tested the hypothesis that inflammation promotes osteogenesis in atherosclerotic plaques using in vivo molecular imaging in apolipoprotein E−/− mice (20 to 30 weeks old, n=35). A bisphosphonate-derivatized near-infrared fluorescent imaging agent (excitation 750 nm) visualized osteogenic activity that was otherwise undetectable by x-ray computed tomography. Flow cytometry validated the target specifically in osteoblast-like cells. A spectrally distinct near-infrared fluorescent nanoparticle (excitation 680 nm) was coinjected to simultaneously image macrophages. Fluorescence reflectance mapping demonstrated an association between osteogenic activity and macrophages in aortas of apolipoprotein E−/− mice (R2=0.93). Intravital dual-channel fluorescence microscopy was used to further monitor osteogenic changes in inflamed carotid arteries at 20 and 30 weeks of age and revealed that macrophage burden and osteogenesis concomitantly increased during plaque progression (P<0.01 and P<0.001, respectively) and decreased after statin treatment (P<0.0001 and P<0.05, respectively). Fluorescence microscopy on cryosections colocalized near-infrared fluorescent osteogenic signals with alkaline phosphatase activity, bone-regulating protein expression, and hydroxyapatite nanocrystals as detected by electron microscopy, whereas von Kossa and alizarin red stains showed no evidence of calcification. Real-time reverse-transcription polymerase chain reaction revealed that macrophage-conditioned media increased alkaline phosphatase mRNA expression in vascular smooth muscle cells. Conclusions— This serial in vivo study demonstrates the real-time association of macrophage burden with osteogenic activity in early-stage atherosclerosis and offers a cellular-resolution tool to identify preclinical microcalcifications.

Statins Alter Smooth Muscle Cell Accumulation and Collagen Content in Established Atheroma of Watanabe Heritable Hyperlipidemic Rabbits

Background —Acute coronary syndromes often result from rupture of vulnerable plaques. The collagen content of plaques probably regulates their stability. This study tested whether HMG-CoA reductase inhibitors (statins) alter interstitial collagen gene expression or matrix metalloproteinase (MMP) levels in rabbit atheroma. Methods and Results —We administered equihypocholesterolemic doses of pravastatin (a hydrophilic statin, 50 mg · kg−1 · d−1, n=9), fluvastatin (a cell-permeant lipophilic statin, 20 mg · kg−1 · d−1, n=10), or placebo (n=10) to mature Watanabe heritable hyperlipidemic rabbits for 52 weeks. The fluvastatin group achieved a much higher peak plasma concentration (23.7 &mgr;mol/L) than did the pravastatin group (1.3 &mgr;mol/L) under these conditions. Immunohistochemistry revealed that MMP-1, MMP-3, and MMP-9 expression by macrophages in the intima was lower in both the pravastatin and fluvastatin groups than in the placebo group, whereas there was no difference in macrophage numbers. Numbers of intimal smooth muscle cells (SMCs) (identified by immunohistochemistry) and expression of type I procollagen mRNA (detected by in situ hybridization), however, were significantly higher in the pravastatin group than in the fluvastatin group. Treatment with pravastatin, but not fluvastatin, preserved interstitial collagen content in vivo (detected by picrosirius red polarization). In vitro, fluvastatin, but not pravastatin, decreased numbers of rabbit and human aortic SMCs without altering procollagen I mRNA expression. Conclusions —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.

Human smooth muscle myosin heavy chain isoforms as molecular markers for vascular development and atherosclerosis.

Smooth muscle myosin heavy chains (MHCs) exist in multiple isoforms. Rabbit smooth muscles contain at least three types of MHC isoforms: SM1 (204 kD), SM2 (200 kD), and SMemb (200 kD). SM1 and SM2 are specific to smooth muscles, but SMemb is a nonmuscle-type MHC abundantly expressed in the embryonic aorta. We recently reported that these three MHC isoforms are differentially expressed in rabbit during normal vascular development and in experimental arteriosclerosis and atherosclerosis. The purpose of this study was to clarify whether expression of human smooth muscle MHC isoforms is regulated in developing arteries and in atherosclerotic lesions. To accomplish this, we have isolated and characterized three cDNA clones from human smooth muscle: SMHC94 (SM1), SMHC93 (SM2), and HSME6 (SMemb). The expression of SM2 mRNA in the fetal aorta was significantly lower as compared with SM1 mRNA, but the ratio of SM2 to SM1 mRNA was increased after birth. SMemb mRNA in the aorta was decreased after birth but appeared to be increased in the aged. To further examine the MHC expression at the histological level, we have developed three antibodies against human SM1, SM2, and SMemb using the isoform-specific sequences of the carboxyl terminal end. Immunohistologically, SM1 was constitutively positive from the fetal stage to adulthood in the apparently normal media of the aorta and coronary arteries, whereas SM2 was negative in fetal arteries of the early gestational stage. In human, unlike rabbit, aorta or coronary arteries, SMemb was detected even in the adult. However, smaller-sized arteries, like the vasa vasorum of the aorta or intramyocardial coronary arterioles, were negative for SMemb. Diffuse intimal thickening in the major coronary arteries was found to be composed of smooth muscles, reacting equally to three antibodies for MHC isoforms, but reactivities with anti-SM2 antibody were reduced with aging. With progression of atherosclerosis, intimal smooth muscles diminished the expression of not only SM2 but also SM1, whereas alpha-smooth muscle actin was well preserved. We conclude from these results that smooth muscle MHC isoforms are important molecular markers for studying human vascular smooth muscle cell differentiation as well as the cellular mechanisms of atherosclerosis.