Person:

Sukhova, Galina

Formation of the atherosclerotic intima must involve altered metabolism of the elastin-rich arterial extracellular matrix. Proteases potentially involved in these processes remain unclear. This study examined the expression of the potent elastases cathepsins S and K in human atheroma. Normal arteries contained little or no cathepsin K or S. In contrast, macrophages in atheroma contained abundant immunoreactive cathepsins K and S. Intimal smooth muscle cells (SMC), especially cells appearing to traverse the internal elastic laminae, also contained these enzymes. Extracts of atheromatous tissues had approximately twofold greater elastase-specific activity than extracts of uninvolved arteries, mostly due to cysteine proteases. Cultured human SMC displayed no immunoreactive cathepsins K and S and exhibited little or no elastolytic activity when incubated with insoluble elastin. SMC stimulated with the atheroma-associated cytokines IL-1beta or IFN-gamma secreted active cathepsin S and degraded substantial insoluble elastin (15-20 microg/10(6) cells/24 h). A selective inhibitor of cathepsin S blocked > 80% of this elastolytic activity. The presence of cathepsins K and S at sites of vascular matrix remodeling and the ability of SMC and macrophages to use these enzymes to degrade elastin supports a role for elastolytic cathepsins in vessel wall remodeling and identifies novel therapeutic targets in regulating plaque stability.

Activated T lymphocytes accumulate early in atheroma formation and persist at sites of lesion growth and rupture, suggesting that they may play an important role in the pathogenesis of atherosclerosis. Moreover, atherosclerotic lesions contain the Th1-type cytokine IFN-gamma, a potentiator of atherosclerosis. The present study demonstrates the differential expression of the 3 IFN-gamma-inducible CXC chemokines--IFN-inducible protein 10 (IP-10), monokine induced by IFN-gamma (Mig), and IFN-inducible T-cell alpha chemoattractant (I-TAC)--by atheroma-associated cells, as well as the expression of their receptor, CXCR3, by all T lymphocytes within human atherosclerotic lesions in situ. Atheroma-associated endothelial cells (ECs), smooth muscle cells (SMCs), and macrophages (MO) all expressed IP-10, whereas Mig and I-TAC were mainly expressed in ECs and MO, as detected by double immunofluorescence staining. ECs of microvessels within lesions also expressed abundant I-TAC. In vitro experiments supported these results and showed that IL-1beta, TNF-alpha, and CD40 ligand potentiated IP-10 expression from IFN-gamma-stimulated ECs. In addition, nitric oxide (NO) treatment decreased IFN-gamma induction of IP-10. Our findings suggest that the differential expression of IP-10, Mig, and I-TAC by atheroma-associated cells plays a role in the recruitment and retention of activated T lymphocytes observed within vascular wall lesions during atherogenesis.

OBJECTIVES

We tested the hypothesis that regional end-systolic left ventricular (ESLV) wall stress is associated with extracellular matrix remodeling activity after myocardial infarction (MI).

BACKGROUND

Increased left ventricular (LV) wall stress is a stimulus for LV enlargement, and echocardiography can be used to estimate regional wall stress. A powerful validation of a noninvasive method of estimating wall stress would be predicting cellular responses after a MI.

METHODS

Echocardiographic images were obtained in rats 1, 7, 14 or 21 days after coronary ligation (n = 11) or sham surgery (n = 5). End-systolic left ventricular wall stress was calculated by finite element analysis in three regions (infarcted, noninfarcted and border) from short-axis images. Matrix metalloproteinase-9 (MMP-9) and macrophage density were determined by immunohistochemistry, and positive cells were counted in high power fields (hpf).

RESULTS

Average ESLV wall stress was higher in rats with MI when compared to shams irrespective of time point (p < 0.01), and ESLV wall stress in the infarcted regions increased with time (25.1 ± 5.9 vs. 69.9 ± 4.4 kdyn/cm2, day 1 vs. 21; p < 0.01). Matrix metalloproteinase-9 expression was higher in infarcted and border regions when compared to noninfarcted regions (22.1 vs. 25.7 vs. 0.10 cells/hpf, respectively; p < 0.01). Over all regions, ESLV wall stress was associated with MMP-9 (r = 0.76; p < 0.001), macrophage density (r = 0.72; p < 0.001) and collagen content (r = 0.67; p < 0.001). End-systolic left ventricular wall stress was significantly higher when MMP-9 positive cell density was greater than 10 cells/hpf (45 ± 20 vs. 14 ± 10 kdyn/cm2; p < 0.001).

CONCLUSIONS

Regional increases in ESLV wall stress determined by echocardiography-based structural analysis are associated with extracellular matrix degradation activity.

The pathogenesis of atherosclerosis and abdominal aortic aneurysm involves breakdown of the elastic laminae. Elastolytic cysteine proteases, including cathepsins S and K, are overexpressed at sites of arterial elastin damage, but whether endogenous local inhibitors counterbalance these proteases is unknown. We show here that, whereas cystatin C is normally expressed in vascular wall smooth muscle cells (SMCs), this cysteine protease inhibitor is severely reduced in both atherosclerotic and aneurysmal aortic lesions. Furthermore, increased abdominal aortic diameter among 122 patients screened by ultrasonography correlated inversely with serum cystatin C levels. In vitro, cytokine-stimulated vascular SMCs secrete cathepsins, whose elastolytic activity could be blocked when cystatin C secretion was induced by treatment with TGF-β1. The findings highlight a potentially important role for imbalance between cysteine proteases and cystatin C in arterial wall remodeling and establish that cystatin C deficiency occurs in vascular disease.

Adenovirus vectors are capable of high efficiency in vivo arterial gene transfer, and are currently in use as therapeutic agents in animal models of vascular disease. However, despite substantial data on the ability of viruses to cause vascular inflammation and proliferation, and the presence in current adenovirus vectors of viral open reading frames that are translated in vivo, no study has examined the effect of adenovirus vectors alone on the arterial phenotype. In a rabbit model of gene transfer into a normal artery, we examined potential vascular cell activation, inflammation, and neointimal proliferation resulting from exposure to replication-defective adenovirus. Exposure of normal arteries to adenovirus vectors resulted in: (a) pronounced infiltration of T cells throughout the artery wall; (b) upregulation of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in arterial smooth muscle cells; (c) neointimal hyperplasia. These findings were present both 10 and 30 d after gene transfer, with no evidence of a decline in severity over time. Adenovirus vectors have pleiotropic effects on the arterial wall and cause significant pathology. Interpretation of experimental protocols that use adenovirus vectors to address either biological or therapeutic issues should take these observations into account. These observations should also prompt the design of more inert gene transfer vectors.

Dysregulated extracellular matrix (ECM) metabolism may contribute to vascular remodeling during the development and complication of human atherosclerotic lesions. We investigated the expression of matrix metalloproteinases (MMPs), a family of enzymes that degrade ECM components in human atherosclerotic plaques (n = 30) and in uninvolved arterial specimens (n = 11). We studied members of all three MMP classes (interstitial collagenase, MMP-1; gelatinases, MMP-2 and MMP-9; and stromelysin, MMP-3) and their endogenous inhibitors (TIMPs 1 and 2) by immunocytochemistry, zymography, and immunoprecipitation. Normal arteries stained uniformly for 72-kD gelatinase and TIMPs. In contrast, plaques' shoulders and regions of foam cell accumulation displayed locally increased expression of 92-kD gelatinase, stromelysin, and interstitial collagenase. However, the mere presence of MMP does not establish their catalytic capacity, as the zymogens lack activity, and TIMPs may block activated MMPs. All plaque extracts contained activated forms of gelatinases determined zymographically and by degradation of 3H-collagen type IV. To test directly whether atheromata actually contain active matrix-degrading enzymes in situ, we devised a method which allows the detection and microscopic localization of MMP enzymatic activity directly in tissue sections. In situ zymography revealed gelatinolytic and caseinolytic activity in frozen sections of atherosclerotic but not of uninvolved arterial tissues. The MMP inhibitors, EDTA and 1,10-phenanthroline, as well as recombinant TIMP-1, reduced these activities which colocalized with regions of increased immunoreactive MMP expression, i.e., the shoulders, core, and microvasculature of the plaques. Focal overexpression of activated MMP may promote destabilization and complication of atherosclerotic plaques and provide novel targets for therapeutic intervention.