Reactive oxygen species produced by macrophage-derived foam cells regulate the activity of vascular matrix metalloproteinases in vitro. Implications for atherosclerotic plaque stability.

Vulnerable areas of atherosclerotic plaques often contain lipid-laden macrophages and display matrix metalloproteinase activity. We hypothesized that reactive oxygen species released by macrophage-derived foam cells could trigger activation of latent proforms of metalloproteinases in the vascular interstitium. We showed that in vivo generated macrophage foam cells produce superoxide, nitric oxide, and hydrogen peroxide after isolation from hypercholesterolemic rabbits. Effects of these reactive oxygens and that of peroxynitrite, likely to result from simultaneous production of nitric oxide and superoxide, were tested in vitro using metalloproteinases secreted by cultured human vascular smooth muscle cells. Enzymes in culture media or affinity-purified (pro-MMP-2 and MMP-9) were examined by SDS-PAGE zymography, Western blotting, and enzymatic assays. Under the conditions used, incubation with xanthine/xanthine oxidase increased the amount of active gelatinases, while nitric oxide donors had no noticeable effect. Incubation with peroxynitrite resulted in nitration of MMP-2 and endowed it with collagenolytic activity. Hydrogen peroxide treatment showed a catalase-reversible biphasic effect (gelatinase activation at concentrations of 4 microM, inhibition at > or = 10-50 microM). Thus, reactive oxygen species can modulate matrix degradation in areas of high oxidant stress and could therefore contribute to instability of atherosclerotic plaques.

Vascular expression of extracellular superoxide dismutase in atherosclerosis.

We characterized a novel form of extracellular superoxide dismutase (ecSOD) in atherosclerotic vessels. Specific activity and protein expression of ecSOD was increased two- to threefold in apo E-deficient compared with control aortas. RNase protection assays demonstrated that the expected ecSOD transcript was not increased in either apo E-deficient mice or cholesterol-fed LDL receptor-deficient mice, but that a second, lower molecular weight transcript was present and became predominant as atherosclerosis progressed. Sequence analysis revealed that this novel ecSOD has a 10-bp deletion in the 3' untranslated region and an asparagine to aspartic acid mutation at amino acid 21. Studies of isolated macrophages and immunohistochemistry suggested that the truncated ecSOD transcript was expressed by lipid-laden but not control macrophages. Recombinant wild-type and novel ecSODs expressed in Sf9 cells exhibited similar SOD activities. These experiments show that ecSOD expression is increased in atherosclerotic vessels and that this is characterized by an alteration in mRNA and protein structure. Further, the source of this altered ecSOD is likely the lipid-laden macrophage. The enzymatic properties of this novel ecSOD may have important implications for the function of the lipid-laden macrophage and the atherosclerotic process.

Increased expression of matrix metalloproteinases and matrix degrading activity in vulnerable regions of human atherosclerotic plaques.

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.

Vascular superoxide production and vasomotor function in hypertension induced by deoxycorticosterone acetate-salt.

BACKGROUND: Angiotensin II-induced hypertension is associated with increased vascular superoxide production, which contributes to hypertension caused by the octapeptide. In cell culture, stretch increases endothelial and vascular smooth muscle production of reactive oxygen species (ROS). In perfused isolated vessels, elevations of pressure can increase vessel angiotensin II production. The effects of low-renin hypertension on vascular ROS production remain unclear. Furthermore, the role of ROS in vascular function and hypertension in low-renin hypertension is undefined. METHODS AND RESULTS: Rats were treated with DOCA and saline drinking water for 3 weeks. Both systolic blood pressure (189+/-4 versus 126+/-2 mm Hg) and aortic superoxide production (3972+/-257 versus 852+/-287, P<0. 05) were increased compared with controls. Relaxations of vascular segments to acetylcholine (ACh, 100+/-2% versus 75+/-2%, P<0.05) and the calcium ionophore A23187 (92+/-2% versus 72+/-3%, P<0.05) were also impaired in DOCA-salt. Heparin-binding superoxide dismutase (1200 U/d IV for 3 days) had no effect on blood pressure but significantly improved relaxations to ACh and A23187. Losartan (25 mg x kg(-1) x d(-1) PO) for 7 days did not correct the hypertension or endothelium-dependent vessel relaxation in DOCA-salt rats, excluding a role of a local renin/angiotensin II system. CONCLUSIONS: These findings indicate that increased vascular superoxide production occurs not only in angiotensin II-induced hypertension but also in hypertension known to be associated with low-renin states. Increased superoxide production alters large-vessel endothelium-dependent vascular relaxation but does not modulate blood pressure in low-renin hypertension.

Remodeling of carotid artery is associated with increased expression of matrix metalloproteinases in mouse blood flow cessation model.

BACKGROUND: The matrix-degrading activity of matrix metalloproteinases (MMPs), required for cell migration and general tissue reshaping, is thought essential for pathological arterial remodeling in atherosclerosis and restenosis. METHODS AND RESULTS: We triggered remodeling of the carotid artery in C57BL/6 mice by blood flow cessation to study the relationship with gelatinases MMP-9 and MMP-2. Ligated and contralateral carotid arteries from ligated and sham-operated mice were harvested fresh, for biochemical analyses, or were perfusion-fixed, for histological studies, at 0, 1, 3, 7, 14, and 28 days after ligation. An early statistically significant (P:<0.01) 4- to 5-fold increase in MMP-9 expression detected by SDS-PAGE zymography and Western blotting in tissue homogenates of ligated carotid arteries 1 day after flow cessation was maintained through day 7, after which expression gradually fell. Maximal MMP-9 levels were higher than MMP-2 levels, which became significantly increased 7 days after ligation. Proliferating cells, identified by bromodeoxyuridine incorporation, were detectable at day 1 in the adventitia and subsequently throughout the wall. Neointima was visible in 3-day specimens of ligated arteries. Suggested by morphology and predicted by theoretical considerations, maximal MMP-9 expression coincided with cell migration into the neointima, supporting its enabling role. Morphological measurements also demonstrated positive lumen remodeling up to 7 days after ligation. CONCLUSIONS: MMP-9 induction is associated with the formation of intimal hyperplasia and does not require frank mechanical injury. Our data also show that a significant increase in MMP-9 expression preceded the positive geometrical remodeling of arteries, suggesting a potentially beneficial role for this matrix-degrading enzyme.

Increased expression of matrix metalloproteinase-2 in the thickened intima of aged rats.

-To characterize remodeling of elastic arteries with aging and to investigate its potential mechanisms, matrix metalloproteinase-2 (MMP-2), intracellular adhesive molecule-1 (ICAM-1), transforming growth factor-beta (TGF-beta), and fibronectin protein levels were measured in the aortas of young adult (6 months) and aged (30 months) Fischer 344XBN rats. At 30 versus 6 months, the thickness of the intima was 5-fold greater and contained marked increases in TGF-beta and ICAM-1, and fibronectin expression was enhanced throughout the aortic wall. Total MMP-2 protein (Western blot) of 30-month-old rats was increased 8-fold over that of 6-month-old rats (0.166+/-0.032 versus 0.020+/-0.006; P<0.01), and staining and activity were regionally localized to the intima, often near breaks in the internal elastic membrane and lamellae. Early passage, explanted smooth muscle cells (SMC) from aged aorta secreted more MMP-2 than those from young aorta; while basal MMP-2 production did not differ with age, after stimulation with cytokines (interleukin-1, tumor necrosis factor-alpha, or TGF-beta, 10 ng/mL each for 24 hours), MMP-2 production in SMC from 30-month-old rats increased to levels greater than those in 6-month-old rats. Thus, enhanced expression of TGF-beta, MMP-2, and ICAM-1 in the thickened vascular intima of aged rats may in part be produced by exaggerated SMC responses to cytokines and may have potential roles in intimal remodeling with aging.

Enhanced incorporation of [14C]glucosamine into glycosaminoglycans of aortic neointima of balloon-injured and cholesterol-fed rabbits in vitro.

Glycosaminoglycans (GAG), which form the elementary constituent of extracellular matrix proteoglycans (PG), are implicated in the pathogenesis of atherosclerosis, mainly due to their lipoprotein binding capability and their abundance in a developing lesion during atherogenesis. However, the reasons for the increment of GAG content are poorly understood. In the present study, the influence of two well known atherogenic factors on arterial GAG synthesis were examined by estimating the incorporation of [14C]glucosamine into aortic GAG in an in vitro incubation system. Radioactivity associated with GAG was taken to represent their synthesis. GAG synthesis by neointimal tissue of rabbit aortas, 12 weeks following balloon catheter deendothelialization was measured and compared in rabbits fed a normal or 0.25% cholesterol supplemented diet for the preceding 6 weeks. In normolipaemic rabbits synthesis was found to be 12,438 +/- 173, 17,884 +/- 1390 and 15,960 +/- 1355 dpm/mg dry defatted tissue from uninjured (control), deendothelialized (DEA) and reendothelialized (REA) areas of rabbit aortas, respectively. This incorporation of radioactivity was significantly greater in hypercholesterolaemic rabbits corresponding to 13,426 +/- 239, 32,670 +/- 3077 and 27,496 +/- 3287 in the control, DEA and REA, respectively. The results demonstrated a synergistic effect of cholesterol feeding and arterial endothelial denudation in stimulating GAG synthesis. Although GAG synthesis was found to be stimulated by either cholesterol feeding or arterial injury, the stimulation by cholesterol feeding alone was only marginal. Further, results show a much higher retention of newly synthesized GAG by the tissue from REA. This study provided a possible explanation for increased GAG content in a developing proliferative lesion.

In situ ultrastructural characterization of chondroitin sulfate proteoglycans in normal rabbit aorta.

We used a monoclonal antibody recognizing chondroitin sulfate (CS) to investigate by immunocytochemistry the characteristics displayed in situ by aortic proteoglycans (PG) containing CS side chains. The antibody specifically precipitated metabolically labeled PG from aortic extracts. Anti-CS specificity was also tested directly on tissue sections and was confirmed by the virtual abolition of immunolabeling on those previously digested with CS-specific enzymes. The overall CS-PG distribution assessed by light microscopy after embedding in Lowicryl KM4 by silver-enhanced immunogold recapitulated that obtained on frozen sections with immunoperoxidase. Extracellular concentrations of CS-PG were very high in the innermost regions of aorta and decreased in the media. The reaction was weak and diffuse in the adventitia. By electron microscopy, the detailed labeling of CS-PG discriminated patterns of organization at both the regional and the molecular level and enabled morphometric estimations. In relation to other components of the extracellular matrix, we found that CS-PG and elastin mutually excluded each other, while two types of CS-PG were differently associated with collagen within media or adventitia. The use of high-resolution immunodetection for the in situ characterization of aortic CS-PG could add specific information relevant to many biological processes in which these molecules have been implicated.

Enhanced expression of vascular matrix metalloproteinases induced in vitro by cytokines and in regions of human atherosclerotic lesions.

Dysregulated extracellular matrix (ECM) metabolism may contribute to vascular remodeling during atherogenesis. The ability of vascular cells to synthesize the components of ECM is well characterized, but less is known about their capacity to degrade ECM and the factors that may regulate this process. We therefore studied the expression of matrix metalloproteinases (MMPs), enzymes that degrade various components of ECM, and of tissue inhibitors of MMPs (TIMPs) by untreated or cytokine-stimulated human smooth muscle cells (SMC). Messenger RNA was studied by Northern blotting, and proteins secreted in culture by SMC were identified by immunoprecipitation. Gelatinolytic and caseinolytic activity of MMPs was detected zymographically. SMC constitutively produced a 72 kDa type IV gelatinase (GL), TIMP-1, and TIMP-2. Upon stimulation with IL1 or TNF alpha, SMC synthesized in addition 92 kDa GL, stromelysin, and interstitial collagenase, MMPs that together can degrade all of the ECM components. IL1 or TNF alpha did not alter the level of TIMP mRNA and protein, suggesting that a net excess of MMP production under these conditions may promote breakdown of the vascular ECM. To test the in vivo relevance of these in vitro findings, we analyzed immunohistochemically normal human arteries and carotid atheromas. Normal tissue and the medial layer underlying lesions stained uniformly for 72 kDa GL and TIMPs 1 and 2. Lesions showed regionally increased MMP expression: the shoulders of atherosclerotic plaques contained stromelysin and 92 kDa GL associated with SMC, and clusters of macrophage-derived foam cells associated with the lipid core stained intensely for all MMPs studied. Endothelial cells covering atheroma or of the plaque microvasculature contained interstitial collagenase. In pathological conditions associated with local release of cytokines in the vessel wall, enhanced regional expression of vascular MMPs may contribute to SMC migration and weakening of matrix that would favor plaque rupture, events associated with the development or complication of the atherosclerotic lesions.

N-acetyl-cysteine decreases the matrix-degrading capacity of macrophage-derived foam cells: new target for antioxidant therapy?

BACKGROUND: Atherosclerotic plaque destabilization triggers clinical cardiovascular disease and thus represents an attractive therapeutic target. Weakening of tissue through the action of matrix-degrading enzymes, called matrix metalloproteinases (MMPs), released by resident macrophages was previously implicated in unstable vascular syndromes. METHODS AND RESULTS: We used a hypercholesterolemic rabbit model of atherosclerosis to investigate the gelatinolytic activity associated with macrophage-derived foam cells (FCs). Gelatinolytic activity and expression of MMP-9 but not of MMP-2 cosegregated with macrophage FCs in aortic lesions. Macrophage-derived gelatinases were further investigated in vitro. MMP-9 was identified as the main macrophage-derived gelatinase in cells isolated from aortic lesions and from granuloma induced in the same rabbits to increase cell yield. Importantly, detection of activated MMP-9 in the FC culture medium supports the notion that these cells can independently initiate processing of secreted MMP zymogens to active enzymes. We further examined whether FC gelatinolytic activity is dependent on the presence of reactive oxygen species (ROS). We found that treatment (1 to 5 days) with 1 to 10 mmol/L N-acetyl-L-cysteine (NAC), an ROS scavenger, decreased not only gelatinolytic activity but also gelatinase expression by FCs. Similarly, NAC treatment of explanted lesions abolished in situ gelatinolytic activity and MMP-9 expression. CONCLUSIONS: Macrophage FCs are an abundant source of gelatinolytic activity that can be inhibited in vitro and in situ by NAC. This newly described action of antioxidant therapy might prove useful to inhibit matrix degradation and to improve vascular stability.

Sulfated proteoglycans of rabbit aorta: selective extraction and alternative method for glycosaminoglycan moiety analysis.

Three different solutions containing urea, guanidine hydrochloride, or a detergent mixture were used to extract proteoglycan molecules (PG) metabolically labeled with 35S from normal rabbit aortic tissue. The size distribution of whole sulfated PG and the glycosaminoglycan (GAG) compositions in the three extracts were compared and found to be characteristically determined by the type of solution used for extraction. The spectrum of sulfated PG isolated by each solution was maintained at consecutive extractions of the tissue, even if this was used after another type of solution. The extracts obtained by using the urea- or guanidine-containing solutions contained similar, rather balanced populations of large and small PG, while the detergent-containing buffer was found to preferentially extract smaller, heparan sulfate-rich aortic PG. The selectivity of various extracting solutions could be exploited to obtain preparations enriched in certain types of sulfated PG. On the other hand, one could obtain a larger variety of 35S-labeled PG from the tissue by consecutively using two solutions with different capacities of extraction. The distribution of GAG moieties among PG populations, separated by size chromatography, was investigated by one of the commonly used methods and by a new method. The standard method is based on comparison of the chromatographic profiles of the extract before and after enzymatic digestions, requiring several chromatographic runs for a sample. In the alternative method proposed, the fractions obtained after a single chromatographic separation are adsorbed onto a support membrane. Processing of the whole membrane by GAG-specific, enzymatic treatments allows simultaneous assessment of GAG types in each fraction.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of matrix metalloproteinase-2 in the remodeling of cell-seeded vascular constructs subjected to cyclic strain.

Tissue engineering offers the opportunity to develop vascular substitutes that mimic the responsive nature of native arteries. A good blood vessel substitute should be able to remodel its matrix in response to mechanical stimulation, as imposed by the hemodynamic environment. We have developed a novel method of studying the influence of mechanical strain on the remodeling of cell-seeded collagen gel blood vessel analogs. We assessed the remodeling capacity by examining the effect of mechanical conditioning upon the expression of enzymes which remodel the extracellular matrix, called matrix metalloproteinases (MMPs), and upon the mechanical properties of the constructs. We found that subjecting collagen constructs to a 10% cyclic radial distention, over a course of 4 days, resulted in an overall increase in the production of MMP-2. Cyclic mechanical strain also stimulated enzymatic activation of latent MMP-2. We found that cyclic strain also significantly increased the mechanical strength and material modulus, as indicated by an increase in circumferential tensile properties of the constructs. These observations suggested that MMP-2-dependent remodeling affects the material properties of vascular tissue analogs. To further investigate this possible connection we examined the effects of dynamic conditioning in the presence of two nonspecific inhibitors of MMP activity. Interestingly, we found that nonspecific inhibition of MMP ablated the benefits of mechanical conditioning upon mechanical properties. Our observations suggest that a better understanding of the complex relation between mechanical stimulation and construct remodeling is key for the proper design of tissue-engineered blood vessel substitutes.

Mechanical stretching of human saphenous vein grafts induces expression and activation of matrix-degrading enzymes associated with vascular tissue injury and repair.

After coronary artery bypass surgery, saphenous vein graft occlusion occurs through tissue remodeling. Although a likely trigger, the role of preparative mechanical injury incurred by the graft is not yet understood. We studied the early effects of simple mechanical injury on human saphenous vein grafts by exposing them to longitudinal stretch, a deformation which potentially occurs during surgery. We then maintained ex vivo for up to 7 days matched pairs of experimentally stretched and nonstretched (control) vein segments and examined the expression and activation of matrix metalloproteinases (MMPs) and integrin alphav, molecules implicated in vascular remodeling. At peak expression on day 3, stretched vein secreted 177 +/- 16% active MMP-2 (P < 0.01), 161 +/- 36% (P < 0.05) pro-MMP-9, and contained 206 +/- 18% (P < 0.01) alphav, a receptor for active MMP-2, compared to control. In situ gelatinase activity was present in the intima and adventitia of stretched veins, but not of control, and correlated spatially with expression of alphav. Stretch also increased severalfold cell proliferation (1.27 +/- 0.4 vs. 0.23 +/- 0.05% in control, P < 0.05), as assessed by bromodeoxyuridine incorporation. Furthermore, we found that cell proliferation colocalized with gelatinase activity and alphav in the adventitia. Our results show that a single longitudinal stretch of vein grafts produces significant changes in the expression and activation of key molecules in vascular remodeling. We also found support for the notion that the adventitial layer contributes to vein graft remodeling.

Microscopic localization of active proteases by in situ zymography: detection of matrix metalloproteinase activity in vascular tissue.

Many physiological and pathological processes involve tissue remodeling due in part to degradation of extracellular matrix. Several factors limit current approaches used for detection of matrix-degrading enzymes in tissues. Matrix metalloproteinases (MMPs), enzymes specialized in catabolism of extracellular matrix constituents, require processing from inactive zymogen precursors to gain enzymatic function. Presently available antibodies do not distinguish between precursor and proteolytically processed forms of MMPs. Also, ubiquitous tissue inhibitors of metalloproteinases (TIMPs) could prevent matrix degradation by MMPs even if the enzymes were in an active form. For these reasons immunocytochemistry does not provide information regarding the functional state of these enzymes. Biochemical studies of tissue extracts preclude localization and entail the possibility of artifactual activation of the enzymes consequent to tissue disruption. To obviate these problems, we have adapted substrate zymography to frozen tissue sections to assess net proteolytic activity in situ. We report here the details and the validation of this methodology. Initial experiments defined casein fluorescently labeled with resorufin as a useful substrate for detecting stromelysin, and fluoresceinated gelatin or autoradiographic emulsion as suitable for detecting gelatinolytic activity by this approach. Either TIMP-1 or the Zn chelator 1,10-phenanthroline reduced the zymographic activity in cryosections of atheroma from humans or rabbits. Inhibitors of serine proteases did not reduce the extent of substrate lysis substantially. In situ zymography preserves the fine morphological details of the tissue and can complement the study of enzyme expression by other microscopic techniques, such as immunocytochemistry. This approach may prove generally applicable for the detection of protease activity in tissue sections permitting exploration of the roles of these enzymes in pathobiology.

Co-localization of aortic apolipoprotein B and chondroitin sulfate in an injury model of atherosclerosis.

In vitro, chondroitin sulfate (CS) proteoglycans (PGs) bind with high-affinity lipoproteins (LPs) containing apolipoprotein B (apo B), and cultured monocytes incubated with LP-PG complexes transform into foam cells (FCs). Consequently, arterial PGs are thought to contribute to the accumulation of LPs in atherosclerotic lesions, but their in vivo interaction has yet to be demonstrated. Balloon catheterization of rabbit aorta modifies the normal aortic distribution of endogenous LPs containing apo B, and determines their accumulation in normocholesterolemic conditions. The distribution of aortic CS-PGs parallels that of apoB within the neointima of injured aortas and might contribute to a favorable environment for LP sequestration within the lesions. To visualize the in situ relationship between apo B and CS-PG, we performed experiments for double detection by immunofluorescence and by post-embedding electron microscope immunogold. The results indicated that the colocalization of endogenous LPs containing apo B and of the large intimal CS-PGs in advanced lesions developed under the regenerated endothelium. At the ultrastructural level, frequent associations were visualized in the extracellular space and in relation to FCs. The close spatial relation between CS-PG and apo B inside the aortic lesions seems to support the hypothesis of their in situ interaction and of a simultaneous cellular uptake, and may be related to the development of both extracellular lipid deposits and the formation of FCs in atherogenesis.

Transmural pressure induces matrix-degrading activity in porcine arteries ex vivo.

Extracellular matrix components must be degraded and resynthesized for vascular remodeling to occur. We hypothesized that the hemodynamic environment regulates activity of matrix metalloproteinases (MMPs), the primary agents for in vivo matrix degradation, during vascular remodeling in response to changes in transmural pressure and shear stress. Pathological hemodynamic conditions were reproduced in an ex vivo system in which we maintained porcine carotid arteries for 24 and 48 h. Total levels of MMP-2 and MMP-9 extracted from tissue homogenates and analyzed by SDS-PAGE zymography were stimulated by transmural pressure and were unaffected by shear stress changes. Degradation of two specific gelatinase substrates, gelatin and elastin, increased with increasing pressure, but the degradation was not affected by shear stress changes in tissue specimens analyzed using in situ zymography (gelatin) and fluorescent measurement of endogenous elastin degradation (elastin). Our results suggest that transmural pressure activates at least two members of the MMP family and that activity of these enzymes is accompanied by degradation of matrix components, effects that may be implicated in hypertensive vascular remodeling.

Thrombin promotes activation of matrix metalloproteinase-2 produced by cultured vascular smooth muscle cells.

Thrombin generated at sites of vascular injury not only participates in the coagulation cascade but can signal other events related to development and complication of atherosclerotic plaques. We investigated here a novel non-thrombotic action of thrombin: the possibility that this protease influences the expression or activation of matrix metalloproteinases (MMPs) produced by vascular smooth muscle cells (SMCs). Matrix-degrading proteinases likely contribute to several aspects of vascular lesion development. Vascular SMCs constitutively elaborate the zymogen form of gelatinase A (MMP-2), found in cell supernatants complexed with its inhibitor, the tissue inhibitor of metalloproteinases (TIMP)-2. When activated, MMP-2 digests collagens and elastin and may thus promote cell migration and vascular remodeling. Analysis of culture supernatants harvested from either human or rabbit vascular SMCs by gelatin zymography revealed that compared with supernatants of unstimulated SMCs, media conditioned by thrombin-stimulated cells contained increased amounts of proteolytically processed MMP-2, suggesting activation of this MMP. Further experiments tested whether thrombin directly activates MMP-2. In cell-free experiments, when added to medium harvested from unstimulated SMCs, alpha-thrombin increased in a dose- and time-dependent manner the amount of proteolytically processed MMP-2, as shown by zymography and by Western blotting with specific antibodies. Thrombin cleaved pro-MMP-2 within 4 hours, even when the gelatinase was bound with its inhibitor, TIMP-2. Thrombin treatment rendered culture media of unstimulated SMCs able to degrade collagen type IV, consistent with generation of active MMP-2. Addition of inhibitors of either thrombin or MMPs decreased this type IV collagenolytic activity, but thrombin in the absence of SMC-conditioned medium containing pro-MMP-2 exhibited only minimal collagenolysis. Our results suggest that at sites of vascular injury, thrombin may activate locally produced MMP-2 and thereby facilitate cell migration and proliferation. In the case of complicated atherosclerotic plaques, episodes of intraplaque hemorrhage or plaque disruption with thrombosis may promote plaque instability by increasing local matrix-degrading activity.

Distribution of endogenous apoprotein B-containing lipoproteins in normal and injured aortas of normocholesterolemic rabbits.

The distribution of endogenous apolipoprotein B (apo B) was studied in both normal and balloon catheter-injured aortas of standard fed rabbits. Using light and electron microscopy, the distribution within entire aortic walls and individual tissue compartments was investigated by immunocytochemistry using an antibody raised against rabbit apo B. The concentration of apo B across the vessel wall dropped sharply from the luminal front towards the media of the normal aortas. The strong superficial reaction was mainly due to a heavy, yet specific, labelling of endothelial cells. Significant concentrations of apo B were also detected within the innermost regions of the extracellular space. The characteristics associated with the labelling of the intimal layer suggested an intense uptake and transcellular transport of apo B by endothelial cells. In contradistinction, normal smooth muscle cells did not appear to be labelled. In the previously injured aortas, the same features of strong superficial apo B labelling were present in the areas covered by regenerated endothelial cells, but not in those persistently deendothelialized. The smooth muscle cells of these regions appeared to show a low uptake of apo B. The increased concentrations of apo B in deeper interstitial areas of injured aortas, indicated the contribution of the extracellular matrix to apo B accumulation. This was especially prominent in the advanced lesions, selectively developed within neointima covered by regenerated endothelium. A rather uniform labelling pattern accompanying small lipid particle deposits, suggested a direct extracellular accumulation of circulating lipoproteins. Intensely labelled foam cells and irregularly distributed apo B within areas of cellular necrosis were detected as well. Injury-mediated responses of the cellular and extracellular aortic components can trigger the development of lipoprotein accumulations characteristic of atherosclerosis within aortas of normocholesterolemic animals.