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. 2019 May 6;100(2):94–101. doi: 10.1111/iep.12310

MT1‐MMP evaluation in neointimal hyperplasia in the late follow‐up after prosthesis implantation

Marta Bruczko 1,✉, Tomasz Gogiel 1, Małgorzata Wolańska 1, Radosław Kowalewski 2, Krzysztof Sobolewski 1, Lech Romanowicz 1
PMCID: PMC6540695  PMID: 31058412

Summary

Vascular surgical interventions are often burdened with late complications, including thrombosis or restenosis. The latter is generally caused by neointimal hyperplasia. Although extracellular matrix (ECM) remodelling is an important part of neointima formation, this process is not clearly understood. The aim of the study was to assess the content and activity of membrane‐type 1 matrix metalloproteinase in human neointima in the late stages of its development. Matrix metalloproteinase‐2 and tissue inhibitor of matrix metalloproteinase‐2 were also evaluated. The research was performed on neointima samples collected during secondary vascular interventions from patients with chronic limb ischaemia who developed vascular occlusion at 6‐18 months after aorto/ilio‐femoral bypass grafting. The control material consisted of segments of femoral arteries collected from organ donors. Western blot and/or ELISA were used for the determination of MT1‐MMP and TIMP‐2 expression. The activity of MT1‐MMP was measured by fluorometric assay and that of MMP‐2 by zymography. We demonstrated significantly increased MT1‐MMP protein content in neointima when compared to normal arteries. However, the activity of MT1‐MMP was significantly lower in neointima than in control samples. The decreased MT1‐MMP activity was concomitant with reduced activity of MMP‐2. The TIMP‐2 protein levels in neointima and normal arteries were not significantly different. The results of our study suggest that the reduced activity of MT1‐MMP and consequently MMP‐2 in human neointima may play a role in decreased degradation of ECM components and thus promote neointimal overgrowth.

Keywords: extracellular matrix, matrix metalloproteinases, neointimal hyperplasia, vascular anastomoses

1. INTRODUCTION

Arterial reconstruction procedures, such as balloon angioplasty, stenting or artery bypass grafting, cause endothelial dysfunction or damage, as well as a metabolic response in the media layer. Excessive healing response results in neointima formation, which leads to artery stenosis or occlusion that requires vascular re‐intervention.1, 2, 3 In general, neointimal hyperplasia starts with migration of vascular smooth muscle cells (VSMCs) from the media layer. Migrating VSMCs secrete and deposit extracellular matrix (ECM) components, ultimately leading to neointimal overgrowth.4, 5, 6 It seems that neointima development is the result of imbalance between synthesis and degradation of ECM components, mainly collagen.7, 8, 9, 10

In normal arterial remodelling, the synthesis of ECM is balanced by matrix metalloproteinases (MMPs) capable of degrading most ECM protein components. The activities of MMPs are tightly regulated by tissue inhibitors of metalloproteinases (TIMPs).11, 12 Recently, a special role in the arterial system has been attributed to membrane‐type matrix metalloproteinases (MT‐MMPs) embedded in the cell membrane of various cells, including endothelial cells, fibroblasts or VSMCs.13, 14, 15, 16 The most studied membrane‐type matrix metalloproteinase is MT1‐MMP. One of the important functions of MT1‐MMP is degradation activity towards collagen being a major ECM component.17 Membrane‐type 1 matrix metalloproteinase controls local environment by direct matrix degradation and pro‐MMP‐2 activation.18, 19 The process of pro‐MMP‐2 activation is also regulated by TIMP‐2.20 Animal studies have demonstrated that MT1‐MMP and MMP‐2 are implicated in early stages of neointima formation through facilitating VSMC migration.13, 19 Although MT1‐MMP is an important mediator of pericellular proteolysis, its role in the second and more prolonged phase of ECM deposition and rebuilding has not been investigated.

The objective of this study was to assess potential contribution of MT1‐MMP in late stages of neointima development. We studied MT1‐MMP content and activity in human arterial neointima and compared the results with normal artery wall. Since MT1‐MMP could regulate matrix remodelling via pro‐MMP‐2 activation, we additionally evaluated MMP‐2 and TIMP‐2.

2. MATERIALS AND METHODS

2.1. Ethical approval

The studies have been performed according to the Declaration of Helsinki, and the investigation protocol was approved by the Committee for Ethics and Supervision on Human Research of the Medical University of Bialystok. All patients signed informed consent to being included in the study.

2.2. Tissue material

The studied material comprised neointima specimens collected during secondary surgical vascular interventions. The indication for the re‐operation was a recurrence of clinical symptoms due to graft occlusion by neointimal hyperplasia. The first clinical symptoms appeared at different periods of time in different patients, but the minimal time of neointima development was six months. Neointimal hyperplasia was evaluated in the anastomosis region at the level of femoral artery and was collected in similar way as described in a previous study.21 Between 2011 and 2014, invasive treatment of aortoiliac lesions was performed in 932 patients with 610 PTA and 306 open procedures. In a 24‐month follow‐up, late vascular graft occlusion developed in 16 patients after open repair. They underwent secondary vascular interventions at 6‐18 months after initial surgery. Neointima specimens were collected from all patients. To avoid the influence on MMP evaluation, the exclusion criterion was statin therapy.22 Ultimately, twelve patients were enrolled in the study group. At primary intervention, all of them were in stage 3 according to the Rutherford classification and presented type D aortoiliac lesions according to TASC II classification. In the postoperative follow‐up, a standard therapy with ASA 75 mg daily and simvastatin 20 mg daily was administered. All the enrolled subjects stopped statin therapy up to six months from the primary procedure, five due to muscle pain and increased CK levels, and others without any plausible reason. Patients qualified to secondary interventions were in stage 4 according to the Rutherford classification. During surgical treatment, vascular anastomosis was incised and graft thrombectomy was performed. After thrombus removal, the glossy white tissue layer covering the inner anastomosis surface was collected and considered as a neointima. Its dissection started 3‐4 mm above the anastomosis and was continued distally below the suture line to a different extent. Distal anastomosis was reconstructed with patch repair of the deep femoral artery in seven patients and femoropopliteal bypass in five patients.

The control material was collected during organ harvesting from twelve age‐matched organ donors. The operations were carried out in the Department of Vascular Surgery and Transplantation at the Medical University of Bialystok. The molecular composition and metabolism of abdominal aorta and iliac arteries differ from peripheral arteries. We decided to compare neointimal hyperplasia to the most closely located artery—common femoral artery. Because atherosclerosis is one of the contraindications to organ procurement, these patients were generally free from exaggerated atherosclerosis lesions. Only fragments of the arterial wall without any visible lipid infiltrations or atherosclerotic plaques were taken as a control material. The blood vessels wall with all three layers were assumed as the control material.

The collected samples were washed with 0.9% NaCl solution and stored at −70°C until further use. Directly before the assays, the harvested tissue samples were homogenized with knife homogenizer (25 000 rpm for 45 seconds at 4°C) in ice‐cold protein extraction buffer (50 mmol/L Tris‐HCl, 150 mmol/L NaCl, 100 mmol/L CaCl2, 0.2% Triton X‐100; pH = 7.6) in a 1:3 (w/v) ratio, and then sonificated (20 kHz, 4 × 15 seconds at 4°C). The homogenates were centrifuged at 10 000 g for 15 minutes at 4°C, and supernatants were collected.

2.3. Protein determination

Protein concentration in supernatants was determined by the Bradford method.23

2.4. Western blot analysis

Aliquots of tissue extracts (normalized to 20 μg of protein) were subjected to 10% SDS‐polyacrylamide gels. After electrophoresis, separated proteins were transferred on to nitrocellulose membranes (Sigma‐Aldrich, USA). Non‐specific binding sites were blocked with 5% non‐fat milk in TBS‐T (20 mmol/L Tris‐HCl buffer (pH 7.4); 150 mmol/L NaCl; 0.05% Tween‐20) for 1 hour. The membranes were incubated with primary anti‐human MT1‐MMP antibody (R&D Systems, USA) solution overnight at 4°C, and subsequently washed in TBS‐T. The membranes were then exposed to the secondary antibody conjugated to alkaline phosphatase (goat anti‐mouse IgG; Sigma‐Aldrich, 1:2000), for 1 hour at room temperature. The bands were visualized using BCIP/NBT reagent (Sigma‐Aldrich). The molecular mass of MT1‐MMP was estimated according to the molecular weight markers (Bio‐Rad, USA). Western blots were scanned, and densitometric analysis of bands was carried out using ImageJ software. The abundance of MT1‐MMP was normalized to the total amount of protein in the sample, and the control group was set as 100%. The amount of protein in the sample was determined using Ponceau S staining.24

2.5. Total content evaluation of MT1‐MMP and TIMP‐2

Commercially available MT1‐MMP (Cloud‐Clone Corp., USA) and TIMP‐2 (Elabscience, China) ELISA kits were used according to manufacturer's protocol.

2.6. Evaluation of MT1‐MMP activity

Assay of MT1‐MMP activity was performed in a black 96‐flat‐bottom‐well microplate (Greiner Bio‐One, Austria), which was precoated with specific anti‐human MT1‐MMP antibody (R&D Systems, USA).25 The extract samples (100 μL) were added to the wells for immobilization of the MT1‐MMP, and the microplate was incubated overnight at 4°C. The unbound proteins were washed out with TBS‐T buffer (50 mmol/L Tris‐HCl [pH 7.4], 0.9% NaCl, 0.05% Tween‐20). To measure MMP activity, 100 μL of 50 mmol/L Tris‐HCl buffer [pH 7.5] containing 10 mmol/L CaCl2, 150 mmol/L NaCl and 0.025% Brij 35 26 with MCA‐Pro‐Leu‐Ala‐Cys(p‐OMeBz)‐Trp‐Ala‐Arg(Dpa)‐H2 (Merck, Germany) (4 μmol/L final concentration) as a fluorogenic substrate was used. The microplate was incubated at 37°C for 60 minutes with gentle shaking. The enzymatic reaction was stopped by the addition of 25 μL of 100 mmol/L EDTANa2. Fluorescence of the released 7‐amido‐4‐methylcoumarin (AMC) was read with a multimode microplate reader (Tecan Infinite® 200 PRO, Tecan, USA) at the excitation and emission wavelengths set at 325 and 393 nm, respectively. Measurements were standardized with AMC. One unit of enzyme activity (U) was defined as the amount of enzyme releasing 1 μmol of product (AMC) per minute at 37°C.

2.7. MMP‐2 gelatin zymography and quantitation

The presence of MMP‐2 in neointima and arterial tissue extracts was detected with gelatin zymography that distinguishes latent and active forms of MMP‐2.27 The tissue extracts containing 20 μg of protein were applied to 1% SDS‐10% polyacrylamide gel, with gelatin at a concentration of 1.5 mg/mL. Electrophoresis was run under non‐reducing conditions at a constant voltage (150 V). After electrophoresis, SDS was removed by incubation in 2% Triton X‐100 at 37°C for 30 minutes. The gel was then transferred to 0.05 mol/L Tris‐HCl buffer (pH 8.0) containing 5 mmol/L CaCl2, incubated at 37°C for 18 hours and stained with 1% Coomassie Brilliant Blue R‐250. The optical densities of lysis bands were analysed using Quantity One Software (Bio‐Rad).

2.8. Statistical analysis

Statistical analysis was performed using Student's t test, accepting P < 0.05 as significant. Mean values and standard deviations (SD) were presented.

3. RESULTS

The Western blot analysis was performed to detect MT1‐MMP protein expression in neointima and normal artery samples. As shown in Figure 1A, MT1‐MMP protein expression was detected both in control arteries (lanes 3 and 5) and in neointima samples (lanes 2 and 4). Two molecular species at approximately 66 and 53 kDa were detected, which corresponded to the pro‐ and active forms of MT1‐MMP, respectively. An additional band at about 45 kDa representing a fragmented MT1‐MMP was also observed. Semi‐quantitative analysis (Figure 1B) demonstrated a slightly higher MT1‐MMP content in neointima (137.94% ± 12.57) compared to the normal artery wall (100% ± 26.4). The percentage of different MT1‐MMP forms in the total MT1‐MMP content was shown in Figure 1C. The percentage of the pro‐form of MT1‐MMP was observed to be the highest in neointima (68.18% ± 8.51), being significantly higher as compared to the normal artery (26.92% ± 9.78). We also found that the fragmented MT1‐MMP was significantly lower in neointima (3.45% ± 1.97) in comparison with the healthy arterial wall (46.56% ± 13.27). The active form of MT1‐MMP was comparable in both tissues.

Figure 1.

Figure 1

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MT1‐MMP Western blot analysis in the normal arterial wall (lanes 3 and 5) and neointima (lanes 2 and 4). The same amount of protein (20 μg) was run in each lane. The analysis was performed for 12 individual control and 12 individual neointimal tissues. The representative blot for two different neointima samples and two different control materials is shown. The molecular mass standards (lane 1) are indicated on the left side. (A), Total MT1‐MMP expression in the normal artery and neointima. Optical density of MT1‐MMP bands was normalized to the total amount of protein in each sample and control group (normal aorta) was set as 100%. The data are expressed as the mean value ± SD (n = 12); **P < 0.05, vs the normal artery wall (B). Percentage content of individual MT1‐MMP form in total MT1‐MMP content. The data are expressed as the mean value ± SD (n = 12); *P < 0.001, vs the normal artery wall (C)

Figure 2 shows the results of the quantitative assay of the MT1‐MMP content in neointima and artery samples. The total MT1‐MMP protein content was significantly increased in neointima (116.21 ± 23.24 ng/mg of protein) when compared to normal arteries (25.26 ± 7.45 ng/mg of protein).

Figure 2.

Figure 2

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The total content of MT1‐MMP in the normal arterial wall and neointima expressed in ng/mg of protein. Mean values ± SD are presented (n = 12); *P < 0.001, vs the normal artery wall

The results of the MT1‐MMP activity calculated per kg of protein are presented in Figure 3. In contrast to the total MT1‐MMP content, the activity of MT1‐MMP expressed as mU/kg of protein was significantly decreased in neointima (110 ± 28.8) in comparison with control samples (223 ± 75.6).

Figure 3.

Figure 3

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The activity of MT1‐MMP in the normal arterial wall and neointima expressed in mU/kg of protein. Mean values ± SD are presented (n = 12); *P < 0.001, vs the normal artery wall

The total TIMP‐2 protein content in the investigated tissues is shown in Figure 4. It was found that TIMP‐2 protein content was slightly increased in neointima, although the differences between neointima samples (948.61 ± 170.75 ng/mg of protein) and normal arteries (701.49 ± 119.25 ng/mg of protein) were not statistically significant.

Figure 4.

Figure 4

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The total content of TIMP‐2 in the normal arterial wall and neointima expressed in ng/mg of protein. Mean values ± SD are presented (n = 12)

The activity of MMP‐2 was assessed by gelatin zymography (Figure 5). Both active (68 kDa) and latent (72 kDa) MMP‐2 forms were detected (Figure 5A). The clear bands were quantified by densitometry (Figure 5B). The activity of MMP‐2 was significantly decreased in neointima samples compared to normal arteries (18.82% vs 100%). However, there was no significant difference (P > 0.05) in the pro‐MMP‐2 content between neointima and normal artery.

Figure 5.

Figure 5

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Zymography in the normal arterial wall (lane 1; 20 μg of protein) and neointima (lane 2; 20 μg of protein) (A). The lytic activity of pro‐MMP‐2 and MMP‐2 was quantified densitometrically. Optical density of pro‐MMP‐2 and MMP‐2 bands in the normal artery was set as 100%. The gelatinolytic activity in neointima was expressed relative to that in normal artery. Mean values ± SD are presented (n = 12); *P < 0.001, vs the normal artery wall (B)

4. DISCUSSION

The present study showed that later stages of human neointima development are characterized by a simultaneous decrease in the catalytic activity of MT1‐MMP and MMP‐2 when compared to the normal artery wall. This may suggest the hypothesis that excessive deposition of ECM may result from reduced activity of proteolytic enzymes.

The studied material consisted of neointima samples taken at least six months after the initial surgery of bypass grafting. It has been reported that peak neointimal growth in humans is observed at least six months following surgical intervention.28 In a later postoperative follow‐up, neointima is subjected to self‐rearrangement in order to adapt to new blood flow conditions. The process in the human body is accompanied by changes in the proteoglycan composition and replacement of collagen type III with type I.29 Animal studies revealed that total collagen content increased between four and twelve weeks while synthesis rates were declining.8 Such discrepancies may be attributable to the reduced protein degradation rate. Membrane‐type 1 matrix metalloproteinase is a major mediator of pericellular proteolysis responsible for the proteolytic cleavage of many substrates, including different types of collagen.14 Therefore, MT1‐MMP examination in terms of neointima ECM rebuilding seems to be reasonable. The understanding of the mechanisms responsible for ECM deposition could propose an alternative or complementary restenosis therapy, which currently is focused on the inhibition of VSMC migration and proliferation.30

The current study showed that catalytic MT1‐MMP activity was significantly reduced in neointima when compared to the normal arterial wall. Quantitative and semi‐quantitative assessments of the total MT1‐MMP content deny the hypothesis that reduced activity of MT1‐MMP may arise from low expression of MT1‐MMP protein. It was observed that the neointimal MT1‐MMP protein content was even several times higher than in the normal artery. Membrane‐type 1 matrix metalloproteinase is synthesized as a proenzyme (about 66 kDa) that requires proteolytic cleavage of the propeptide to produce a catalytically active form of the enzyme (about 53 kDa). Western blot analysis revealed the presence of molecular species of a size that may correspond to the latent and active forms of MT1‐MMP in neointima, as well as in control arteries. A semi‐quantitative assessment based on the Western blot findings demonstrated that the highest percentage of total MT1‐MMP in neointima corresponds to the latent form. This could explain the discrepancies between the content and activity of MT1‐MMP in neointima. Due to an important role of MT1‐MMP in pericellular proteolysis, its catalytic activity must be tightly regulated at the cell surface. This is ensured by the inhibitory effect of TIMPs. The most active inhibitor of MT1‐MMP is TIMP‐2, and on the other hand, it is an important component of ternary complex, necessary for the generation of active MMP‐2 form.20 Moreover, we demonstrated that the amount of active MMP‐2 assessed by zymography is more than fivefold lower in neointima as compared to the control artery, while the content of latent MMP‐2 is similar in both tissues studied. Matrix metalloproteinase 2 is constitutively expressed in the latent form, both in normal and in injured tissues.31 Its activity may be controlled by endogenous inhibitors and also by efficient activation mechanism dependent on MT1‐MMP activity. It may be speculated that the reduced activity of MMP‐2 results rather from the impaired activation process considering the fact that content of TIMP‐2 in both tissues is similar. One may suggest that TIMP‐2 plays a role of a direct inhibitor of MT1‐MMP rather than a part of the pro‐MMP‐2 activation complex. We additionally observed that reduced MMP‐2 activity in neointima is concomitant with a significantly lower amount of the fragmented form of MT1‐MMP. Other authors have observed32 that high level of 40‐45 kDa form of MT1‐MMP coincides with high pro‐MMP‐2 activation. In contrast, if no activation of MMP‐2 was noted, these fragments were not detected despite the presence of a mature form of MT1‐MMP on the cell surface.32 Interestingly, other authors33 have shown that the species of about 45 kDa may regulate the amount of active MT1‐MMP at the cell surface and thus may influence its enzymatic activity. It has been proved that the expression of ~45 kDa fragments correlates with higher levels of surface MT1‐MMP due to delay in the rate of active MT1‐MMP endocytosis.33 In turn, others have demonstrated that phosphorylation of MT1‐MMP mediates its activity through directing cellular localization, shifting its role from MMP‐2 activation to intracellular signalling.34 It cannot be excluded that the MT1‐MMP active form is kept relatively short in the neointimal cell membrane and consequently the proteolytic activity towards the ECM components is decreased. However, MT1‐MMP developed many other regulatory mechanisms that control the activity and amount of MT1‐MMP on the cell surface, and net content of active MT1‐MMP may depend on the balance between them.35 It has been reported that furin‐related proprotein convertases are very important components of the cellular machinery that prolong the presentation of the MT1‐MMP on the cell surface and support its function in extracellular proteolysis.36 Moreover, Sluijter et al have observed that inhibition of furin‐like proprotein convertases after balloon dilation resulted in a reduction in intimal areas. However, they have also observed that active MT1‐MMP was still present in the furin knockout cells which suggested the existence of a furin‐independent activation mechanism of MT1‐MMP. It was concluded that furin‐like proprotein convertases are probably involved in the arterial response to injury via the TGF‐Smad signalling pathway.37 It is unknown whether there is a preferred regulatory mechanism during specific biological conditions. Therefore, future investigations, both in animal and in human models, are needed to clarify the mechanisms involved in the regulation of MT1‐MMP activity in the vascular wall damage conditions.

In conclusion, although the process of neointima formation has been intensively studied in animal models8, 19; it still remains poorly understood in humans. Human studies in terms of neointima development are uncommon, and therefore, the reported changes are potentially useful to increase our understanding of this process. Our results show some interesting changes in the MT1‐MMP content and activity between the normal arterial wall and a neointimal lesion. This may suggest potential contribution of MT1‐MMP to the pathological remodelling of ECM in neointima development. However, unambiguous conclusions cannot be drawn due to lack of more knowledge on the stage and rate of neointima development. Nevertheless, targeting MT1‐MMP activity may have therapeutic relevance as an approach to attenuating neointima development. The hypothesis is worthy of being considered and checked.34

There are some limitations to this study design that could influence the interpretation of the results. First, the number of samples was limited, and therefore, our observations need to be confirmed in a larger cohort. Second, there is a lack of immunohistochemical analysis that would clarify the cellular source and location of the protein of interest within the cell that synthesizes that protein. The insufficient quantity of tissue available for investigation limited the range of our study.

CONFLICT OF INTEREST

The authors declare that they have no conflict of interest.

Bruczko M, Gogiel T, Wolańska M, Kowalewski R, Sobolewski K, Romanowicz L. MT1‐MMP evaluation in neointimal hyperplasia in the late follow‐up after prosthesis implantation. Int. J. Exp. Path.. 2019;100:94–101. 10.1111/iep.12310

Funding information

This research was carried out in relation to statutory activities of research units financed by the Polish Ministry of Science and Higher Education.

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