Matrix metalloproteinase 7 is associated with symptomatic lesions and adverse events in patients with carotid atherosclerosis - PubMed (original) (raw)

. 2014 Jan 6;9(1):e84935.

doi: 10.1371/journal.pone.0084935. eCollection 2014.

Pål Aukrust 2, David Russell 3, Kirsten Krohg-Sørensen 4, Trine Almås 5, Dorte Bundgaard 6, Vigdis Bjerkeli 7, Ellen Lund Sagen 7, Annika E Michelsen 7, Tuva B Dahl 7, Sverre Holm 8, Thor Ueland 7, Mona Skjelland 1, Bente Halvorsen 7

Affiliations

Matrix metalloproteinase 7 is associated with symptomatic lesions and adverse events in patients with carotid atherosclerosis

Azhar Abbas et al. PLoS One. 2014.

Abstract

Background: Atherosclerosis is a major cause of cerebrovascular disease. Matrix metalloproteinases (MMPs) play an important role in matrix degradation within the atherosclerotic lesion leading to plaque destabilization and ischemic stroke. We hypothesized that MMP-7 could be involved in this process.

Methods: Plasma levels of MMP-7 were measured in 182 consecutive patients with moderate (50-69%) or severe (≥70%) internal carotid artery stenosis, and in 23 healthy controls. The mRNA levels of MMP-7 were measured in atherosclerotic carotid plaques with different symptomatology, and based on its localization to macrophages, the in vitro regulation of MMP-7 in primary monocytes was examined.

Results: Our major findings were (i) Patients with carotid atherosclerosis had markedly increased plasma levels of MMP-7 compared to healthy controls, with particularly high levels in patients with recent symptoms (i.e., within the last 2 months). (ii) A similar pattern was found within carotid plaques with markedly higher mRNA levels of MMP-7 than in non-atherosclerotic vessels. Particularly high protein levels of MMP-7 levels were found in those with the most recent symptoms. (iii) Immunhistochemistry showed that MMP-7 was localized to macrophages, and in vitro studies in primary monocytes showed that the inflammatory cytokine tumor necrosis factor-α in combination with hypoxia and oxidized LDL markedly increased MMP-7 expression. (iv) During the follow-up of patients with carotid atherosclerosis, high plasma levels of MMP-7 were independently associated with total mortality.

Conclusion: Our findings suggest that MMP-7 could contribute to plaque instability in carotid atherosclerosis, potentially involving macrophage-related mechanisms.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Plasma levels of MMP-7 in patients with carotid plaques in relation to symptomatology.

The figure shows plasma levels of MMP-7 in patients with carotid atherosclerosis (n = 182) divided into (i) patients with the most recent symptoms (within the last 2 months, n = 73) and (ii) patients with symptoms >2 months ago and asymptomatic patients (>2 months, n = 109). For comparison, MMP-7 levels were also analyzed in 23 healthy controls (CTR). Data are shown as a box and whisker plot with median (Q1, Q3) in the box and the whiskers representing the 5 and 95 percentiles. Note that both groups of patients have significantly raised MMP-7 levels as compared with controls.

Figure 2

Figure 2. The expression of MMP-7 within atherosclerotic carotid plaques in relation to plaque symptomatology.

mRNA (A) and protein (B) levels of MMP-7 in atherosclerotic carotid plaques were measured in (i) patients with the most recent symptoms (within the last 2 months, n = 35) and (ii) patients with symptoms >2 months ago and asymptomatic patients (>2 months, n = 29). For comparison, mRNA levels of MMP-7 were also analyzed in non-atherosclerotic vessels (common iliac artery) from 10 organ donors (CTR). mRNA levels were quantified by real-time RT-PCR. The expression of β-actin was used as endogenous control. Protein levels of MMP-7 were measured in plaque lysates by EIA. Data are shown as a box and whisker plot with median (Q1, Q3) in the box and the whiskers representing the 5 and 95 percentile in relation to β -actin expression.

Figure 3

Figure 3. Immunostaining of MMP-7 within atherosclerotic and non-atherosclerotic vessels.

Immuhistochemistry of MMP-7 in carotid atherosclerotic plaques (n = 8, symptoms within the recent 2 months) shows strong immunostaining. Representative images obtained with 100× A and 400× magnification (highlighted, with arrows on positive cells). Panel B shows no or weak immunostaining of MMP-7 in non-atherosclerotic carotid artery obtained from autopsies (n = 5). Panel C shows double immunofluorescent staining of MMP-7 (green fluorescence), CD68 (macrophages, red fluorescence) and nucleus (DAPI, blue fluorescence) from carotid atherosclerotic plaques (n = 4). The lower right panel is a merge of the three pictures.

Figure 4

Figure 4. MMP7 staining in areas with thinner and less organized collagen fibers.

Immunohistochemical staining of MMP7 in human atherosclerotic plaques at 100× magnification A and B. Sirius Red staining of collagen in corresponding sections of human atherosclerotic plaques are shown in C and D. The highlighted sections are from corresponding areas of sequential sections of the same plaques stained with MMP7 and Sirius Red, respectively, at 250× magnification. The arrows indicate area with no positive staining for MMP7 corresponding to areas with more organized and tightly packed collagen fibers.

Figure 5

Figure 5. The regulation of MMP-7 expression in primary monocytes.

Panel A shows the effect of oxLDL (20 µg/ml), TNFα (5 ng/ml) or a combination thereof, panel B shows the effect of hypoxia with or without co-stimulation with oxLDL (20 mg/ml), TNFα (5 ng/ml) or a combination thereof. The cells were cultured for 48 hours before experimental starts and cell pellets were harvested 18 hours thereafter. mRNA levels of MMP-7 were quantified by real-time RT-PCR in relation to the expression of the endogenous control gene β-actin. Data are mean±SEM (n = 6) and are given in relation to cells that received vehicle (Ctr) or were cultured in normoxic condition. *p<0.05 versus Ctr (panel A). ***p<0.001 versus all other conditions (panel B).

Figure 6

Figure 6. Association between plasma levels of MMP-7 and adverse outcome in patients with carotid atherosclerosis.

Panel A shows Kaplan–Meier curve with the cumulative incidence of all-cause mortality during the entire study (mean follow-up 3.5 years) according to dichotomized MMP-7 levels (Cut-off median: 1.96 ng/mL). Panel B shows multi-variable analyses of predictors of all-cause mortality (direct entry). CRP and MMP-7 show expressed per SD change.

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