Seppo T. Nikkari

Interstitial Collagenase (MMP-1) Expression in Human Carotid Atherosclerosis

BACKGROUND In human atherosclerosis, most clinical events occur when plaque integrity is compromised and hemorrhage and thrombosis result. One mechanism for this might be the release by plaque cells of matrix-degrading proteases, such as interstitial collagenase (matrix metalloproteinase-1, MMP-1), which degrades two major plaque structural proteins, types I and III collagen. This study was undertaken to determine whether MMP-1 is expressed in human atherosclerotic plaques. METHODS AND RESULTS To determine the cellular source and location of MMP-1 in human carotid atherosclerotic lesions, in situ hybridization and immunohistochemistry were performed on 20 endarterectomy specimens. Six nonatherosclerotic carotid arteries also were studied. Intense MMP-1 expression (mRNA and protein) was detected in a subset of plaque macrophages located at the borders of the lipid cores adjacent to fibrous caps and shoulder regions. Subsets of plaque smooth muscle cells and endothelial cells also expressed MMP-1. There was a strong correlation between the percentage of the lipid core occupied by hemorrhage and the percentage of the lipid core perimeter positive for MMP-1 (r = .823, P = .0001). MMP-1 was not detected in any cell type in nonatherosclerotic carotid arteries. CONCLUSIONS This study demonstrates that MMP-1 is expressed by several cell types in human carotid atherosclerosis and that there is a correlation between the expression of the protease and histopathological evidence of plaque instability. Since MMP-1 may degrade the major structural collagens of the plaque, expression of the protease by macrophages in regions critical to plaque integrity could contribute to plaque expansion, rupture, and hemorrhage.

Overexpression of Tissue Inhibitor of Matrix Metalloproteinase-1 Inhibits Vascular Smooth Muscle Cell Functions In Vitro and In Vivo

Arterial smooth muscle cells (SMCs) are in a quiescent growth state under normal physiological conditions, but they can be stimulated to proliferate and migrate from one tissue compartment to another if the vessel is injured. This response might require a selective and focal increase in tissue degradation, which might be mediated through the increased production of matrix metalloproteinases (MMPs). Blockade of MMP activity might therefore inhibit the SMC response to injury. To test this hypothesis, we developed clones of rat SMCs that overexpress baboon tissue inhibitor of matrix metalloproteinase-I (TIMP-1), using retrovirally mediated gene transfer, and characterized the functional capacity of these cells in vitro and in vivo. SMCs transduced with the TIMP-1 vector (LTSN) grew more slowly and also migrated through a gel matrix in a Boyden chamber assay more slowly than the vector alone (LXSN) cells. The conditioned medium from LTSN cells completely inhibited the platelet-derived growth factor-BB-induced migration of normal SMCs across a matrix-coated filter, while the LXSN cell conditioned medium had no effect. The inhibitor activity in the LTSN conditioned medium could be neutralized with an antibody to TIMP-1. In vivo, local overexpression of TIMP-1 using LTSN cells implanted onto balloon-injured rat carotid artery inhibited intimal hyperplasia. Neutralizing antibodies against TIMP-1 suppressed the effect of LTSN cell seeding on intimal thickening. These data support the conclusion that the process of SMC activation leading to a thickened intima is dependent on MMP activity and that TIMP-1 could be utilized to inhibit this process.

Effect of consumption of red wine, spirits, and beer on serum homocysteine

Serum homocysteine increases after moderate consumption of red wine and spirits, not after moderate consumption of beer. Vitamin B6 in beer seems to prevent the alcohol-induced rise in serum homocysteine.

Heparin inhibits the induction of three matrix metalloproteinases (stromelysin, 92-kD gelatinase, and collagenase) in primate arterial smooth muscle cells.

Heparin inhibits the migration and proliferation of arterial smooth muscle cells and modifies the extracellular matrix. These effects may be the result of heparins effects on proteinases that degrade the matrix. We have previously reported that heparin inhibits the induction of tissue-type plasminogen activator and interstitial collagenase mRNA. We have investigated the possibility that heparin affects other members of the matrix metalloproteinase family. Phorbol ester increased the levels of mRNA of collagenase, 92-kD gelatinase and stromelysin as well as the synthesis of these proteins. These effects were inhibited by heparin, but not by other glycosaminoglycans, in a dose-dependent manner. The induction of these matrix metalloproteinases was also inhibited by staurosporine and pretreatment with phorbol ester indicating the involvement of the protein kinase C pathway. In contrast, the 72-kD gelatinase was expressed constitutively and was not affected by phorbol ester or heparin. Tissue inhibitor of metalloproteinase-1 was expressed constitutively and was slightly increased by phorbol ester. It was not affected by heparin. Thus, heparin inhibits the production of four proteinases (tissue plasminogen activator, collagenase, stromelysin and 92-kD gelatinase) that form an interdependent system capable of degrading all the major components of the extracellular matrix.

Wound healing: A paradigm for lumen narrowing after arterial reconstruction

PURPOSE The intimal hyperplasia hypothesis that equates lumen narrowing after arterial injury with intimal mass has recently been challenged. Evidence has emerged to suggest that lumen narrowing is caused in large part by changes in artery wall geometry rather than intimal mass per se. We have begun to explore this hypothesis in a unique nonhuman primate model of atherosclerosis. METHODS Monkeys who were fed an atherogenic diet for 3 to 5 years underwent experimental angioplasty of the left iliac artery. The contralateral iliac artery served as an intraanimal control. Arteries were removed 2, 4, 7, 14, 28, or 112 days later for analysis (6 or 13 per time point). Angioplasty dilated arteries by fracturing atheroma and stretching or tearing the media. Cross-sections of injured arteries were analyzed for expression of extracellular matrix components and cell surface integrins that are important in wound healing. Antibodies, riboprobes, or histochemical stains specific for fibrin, hyaluronan, versican (chondroitin sulfate-containing proteoglycan), procollagen-I, elastin, and the alpha 2 beta 1 and alpha V beta 3 integrins were used. RESULTS A thin mural thrombus was seen at sites of denudation and plaque fracture (days 2 to 7). This provisional matrix was invaded by leukocytes (days 2 to 4) and alpha-actin-positive smooth muscle cells (SMCs; days 4 to 7). Thrombus was replaced by SMCs expressing hyaluronan and the associated versican proteoglycans (day 14). Versican was expressed throughout the neointima as it enlarged (day 28), but expression later subsided (day 112). Procollagen-I expression initially increased in the adventitia (day 4) and then in the forming neointima (day 14). Procollagen-I expression was found to persist within the adventitia and in the neointima in SMCs nearest the lumen (days 28 to 112). Elastin staining was prominent within the mature neointima (day 112) but not at earlier time points. Integrin expression also increased within the injured artery wall. alpha v beta 3 staining (fibrin[ogen] receptor) increased in the injured media (days 2 to 7) and was then seen throughout the early neointima (day 7). Low level expression of alpha V beta 3 subsequently persisted within the forming neointima (day 28). alpha 2 beta 1 (collagen receptor) expression increased in the neointima in SMCs nearest the lumen (day 28). CONCLUSIONS Lumen narrowing after angioplasty in this model of atherosclerosis is caused largely by decreased artery wall diameter. The pattern of matrix and integrin expression within the injured artery wall is in many ways analogous to that of healing wounds. These observations suggest that tissue contraction may play a role in lumen narrowing at sites of arterial reconstruction. Strategies to inhibit wound contraction may prove effective in preventing restenosis.

Serum matrix metalloproteinase-9 concentration in angiographically assessed coronary artery disease.

Expression of several matrix metalloproteinases (MMPs) in atherosclerotic plaques has been well documented, and there are findings to indicate that arterial inflammation is reflected in increased serum concentration of matrix metalloproteinase-9 (MMP-9). In coronary atherosclerosis, there is enhanced expression of this MMP, which may be predictive of the severity of the disease. We determined the concentrations of serum MMP-9 in 61 patients (47 males, 14 females) who had >50% obstruction in one or more coronary arteries as assessed by coronary angiography before bypass surgery. In a control group of 19 patients (9 males, 10 females) there were no pathological findings in coronary angiography. ANOVA showed that serum MMP-9 concentrations were highest in patients with 3-vessel coronary artery disease (CAD) (57.3 - 39.1 µg/L, p= 0.011). The difference remained statistically significant after adjustment for age, diabetes and sex (p= 0.025, ANCOVA). When the groups were compared with each other, serum MMP-9 concentration was higher in the patients with 3-vessel CAD than in those with 1- or 2-vessel CAD (40.4 - 25.1 µg/L, p= 0.044) or in the controls (32.2 - 16.1 µg/L, p= 0.007). These results show that serum MMP-9 is elevated in patients with severe coronary stenosis compared with controls. Since MMP-9 has been suggested to reflect inflammation in atherosclerotic plaques, it may be useful in the evaluation of the severity of cardiovascular disease.

Decorin is produced by capillary endothelial cells in inflammation-associated angiogenesis.

Decorin is a small extracellular chondroitin/dermatan sulfate proteoglycan that has previously been shown to be involved in the angiogenesis-like behavior of endothelial cells (ECs) in vitro. There is also evidence that decorin plays a role in angiogenesis in vivo. In this study we sought to further explore the involvement of decorin in angiogenesis in vivo, especially in that associated with inflammation. We found by CD31 immunostaining of ECs that in giant cell arteritis there are capillary blood vessels not only in the adventitia as in uninvolved temporal artery wall, but also in the media and the external zone of the thickened intima. Localization of decorin by antiserum LF-30 in adjacent sections showed that in normal temporal artery wall decorin resides mainly in the media and the adventitia, whereas in inflamed temporal artery wall decorin is distributed throughout the vessel wall including the intima. Furthermore, the most intense reaction for decorin was evident in ECs of capillary neovessels within the media and the thickened intima of inflamed temporal artery wall. Decorin was also found in capillary ECs in certain pathological and physiological conditions in which the pivotal role of angiogenesis is more generally accepted. Pyogenic granulomas, granulation tissue of healing dermal wounds, and ovaries at different phases of follicle and corpus luteum formation all contained widely distributed CD31-positive capillaries. Decorin, on the other hand, was found in capillary ECs in pyogenic granulomas and granulation tissue, but not in those in the ovaries. The assessment of the degree of inflammation in the specimens with the presence of CD68-positive macrophages showed that the pyogenic granuloma, granulation tissue, and giant cell arteritis specimens were rich in macrophages around the decorin-positive capillaries. In contrast, the ovarian specimens were populated with fewer macrophages and even they were not located in close vicinity of capillaries negative for decorin. Our results confirm that decorin is involved in angiogenesis in vivo and, particularly, in conditions in which the inflammatory component is dominant.

Identification of different bacterial DNAs in human coronary arteries

Background  Various studies have suggested a link between infection, atherosclerosis and coronary artery disease. We studied whether bacterial DNA is present in coronary specimens obtained from left anterior descending coronary arteries of subjects having sudden deaths of cardiovascular and other causes, as verified by an autopsy.

Differences in the distribution of versican, decorin, and biglycan in atherosclerotic human coronary arteries.

The distributions of versican, biglycan, and decorin have been examined in segments of normal and atherosclerotic human coronary arteries using antibodies directed against the core proteins of these macromolecules. Versican immunostaining was prominent throughout the extracellular matrix (ECM) in regions of the vessels that contained abundant smooth-muscle cells, such as in diffuse intimal thickenings, fibrous caps, and in zones of loose, myxoid connective tissue. Versican also was present in smooth-muscle-rich thrombi and at borders of the lipid-rich cores of advanced atherosclerotic lesions. Biglycan immunostaining was observed in diffuse intimal thickenings, fibrous caps, and myxoid areas, but, unlike versican, it was abundant in the lipid-rich core of advanced plaques. However, biglycan immunostaining was absent in smooth-muscle cell-enriched thrombi. Decorin immunostaining paralleled biglycan immunostaining except that it was conspicuously absent in the myxoid areas of the plaque and markedly reduced in diffuse intimal thickenings. Both biglycan and decorin immunostaining were consistently associated with some of the microvessels in the thrombi and in advanced atherosclerotic plaques. Taken together, these results indicate that specific proteoglycans distribute to topographically defined regions of normal and atherosclerotic human coronary arteries and that these different distributions may indicate a diversity of functions in normal and pathologic processes of the arterial wall.

Restenosis after Vascular Reconstruction

About 20-50% of vascular reconstructions used for restoration of circulation in atherosclerotic vessels fail because of restenosis. Despite progress in both experimental and clinical studies, the underlying mechanism of restenosis remains unclear. This has presented a problem for the targeting of pharmacological therapies, and so far the only effective cure for restenosis remains repetition of the operative treatment. However, the subsequent reconstructions are also subject to luminal narrowing. New approaches in preventing restenosis may involve identifying the patients most likely to be at risk, and treating them selectively with novel suppressive agents, or with combinations of already tested agents.