1089
Cardiovascular Biology and Cell Signalling
Matrix metalloproteinase-2 of human carotid atherosclerotic plaques promotes platelet activation Correlation with ischaemic events Massimo Lenti1; Emanuela Falcinelli2; Marcella Pompili2; Paola De Rango1; Valentina Conti2; Giuseppe Guglielmini2; Stefania Momi2; Teresa Corazzi2; Giuseppe Giordano1; Paolo Gresele2 of Vascular and Endovascular Surgery, Hospital S. M. Misericordia, Perugia, Italy; 2Department of Internal Medicine, Section of Internal and Cardiovascular Medicine, University of Perugia, Perugia, Italy
Summary Purified active matrix metalloproteinase-2 (MMP-2) is able to promote platelet aggregation. We aimed to assess the role of MMP-2 expressed in atherosclerotic plaques in the platelet-activating potential of human carotid plaques and its correlation with ischaemic events. Carotid plaques from 81 patients undergoing endarterectomy were tested for pro-MMP-2 and TIMP-2 content by zymography and ELISA. Plaque extracts were incubated with gel-filtered platelets from healthy volunteers for 2 minutes before the addition of a subthreshold concentration of thrombin receptor activating peptide-6 (TRAP-6) and aggregation was assessed. Moreover, platelet deposition on plaque extracts immobilised on plastic coverslips under high shear-rate flow conditions was measured. Forty-three plaque extracts (53%) potentiated platelet aggregation (+233 ± 26.8%), an effect prevented by three different specific MMP-2 inhibitors (inhibitor II, TIMP-2, moAb
Correspondence to: Paolo Gresele, MD, PhD Dept. of Internal Medicine Section of Internal and Cardiovascular Medicine University of Perugia Via E. Dal Pozzo, 06126 Perugia, Italy Tel.: +39/075 5783989, Fax: +39/075 5716083 E-mail: [email protected]
anti-MMP-2). The pro-MMP-2/TIMP-2 ratio of plaques potentiating platelet aggregation was significantly higher than that of plaques not potentiating it (3.67 ± 1.21 vs 1.01 ± 0.43, p24 h and associated with new ischaemic lesions on a CT scan. Patients were defined as symptomatic if had experienced TIA or stroke compatible with the affected carotid artery within the six months prior to CEA.
Pro-MMP-2 in plaque extracts was measured by SDS-PAGE zymography, as previously described (20). Gelatinolytic activities were detected as transparent bands against the background of Coomassie Brilliant Blu-stained gelatine. A standard curve for each gel was generated using increasing concentrations (0.25 to 5 ng) of recombinant MMP-2 (kindly provided by Dr. Rafael Fridman, Wayne State University, Detroit, MI, USA). Minimal detectable amount (2 SD from 0) was 18 ± 7 pg. Intraand inter-assay coefficients of variation of MMP-2 measurements were 2.3% and 3.9%, respectively (n=8). Recoveries ranged from 85 to 112%, with a mean recovery of 96 ± 6.5%. Gel images were acquired with a Camedia 4040 digital camera (Olympus, Center Valley, PA, USA) and gelatinase activity areas were analysed using the Image-J 1.38x (NIH, Bethesda, MD, USA) software. TIMP-2 and active MMP-2 (GE Healthcare, Buckinghamshire, UK) were measured by ELISA, as described (15, 21).
Follow-up and clinical outcome A blood sample was taken on the day of hospitalisation for CEA. Clinical events on follow-up were evaluated by periodic clinical visits or, when not feasible, by phone interviews, at six months intervals. The primary outcome was defined as any adverse cardiovascular event, including any cardiovascular-related death, non fatal myocardial infarction (MI) or non-fatal stroke.
Carotid endarterectomy (CEA) and plaque extraction CEA was performed by standard techniques. Carotid plaques were defined as "lipidic" or "calcific" according to the preoperative duplex ultrasound scan appearance as hypoechoic or hyperechoic, respectively. Plaques were defined as complicated when highly hypoechoic, compatible with the presence of haemorrhage, thrombus, or fatty tisThrombosis and Haemostasis 111.6/2014
Platelet aggregation Venous blood was collected in the morning under fasting conditions, from twenty-five unrelated healthy subjects who had not taken any medication for at least 10 days before blood sampling, using a 19-gauge needle without tourniquet. Gel-filtered platelets (GFP) were prepared as described (14, 20) and adjusted to 1x108/ml in Hepes-Tyrode’s buffer containing 0.1% bovine serum albumin (BSA) and physiological magnesium concentration, pH 7.4. CaCl2 (2 mmol/l) was added to gel filtered platelets before incubation with plaques. Platelet aggregation was measured by light transmission aggregometry (LTA) using an APACT 4 aggregometer (Alfa Wasserman, Milan, Italy). Plaque extracts (72 ng/ml) or their solvent were incubated with 250 μl of GFP for 2 min before the addition of thrombin receptor © Schattauer 2014
Downloaded by: University of the Sciences in Philadelphia. Copyrighted material.
1090
Lenti et al. Plaque MMP-2 potentiates platelet aggregation
Platelet deposition on a plaque extract-covered surface under flow-conditions Platelet deposition on plaque extract-covered surfaces under highshear stress conditions (3,000 s-1) was carried out as described (24, 25). Briefly, plastic coverslips were covered with 100 μl of a suspension of plaque extract dissolved in PBS (1 mg of protein/ml), incubated for 1 h at 37°C and then rinsed with BSA/saline buffer (0.35%) for 1h at room temperature (18). Plaque extracts dissolved in PBS were preincubated, or not, with the MMP-2 inhibitor II (10 μM) or, in selected experiments, with a blocking anti-MMP-2 monoclonal antibody (5 μg/ml) for 20 min at room temperature prior to coverslip coating. Then extract-covered plastic coverslips were mounted in a rectangular parallel plate perfusion chamber and then citrated whole blood (0.5 ml) was passed through it at a wall shear rate of 3,000 s-1 for 5 min at 37°C, as described (26). Moreover, in order to assess the role of platelet GPVI in the platelet activating activity of plaques, we tested the effect of an antibody against GPVI (Fab 9O12.2, 50 µg/ml, kindly given by Prof. L. De Marco, CRO, Italy), for its effect on platelet deposition. To this end whole blood was incubated with the antibody for 15 min and then perfused over plaque extract-covered slides under high shear conditions. Moreover, to further explore the effect of MMP-2 on platelet thrombus formation in flowing blood, active MMP-2 (100 ng/ml) was either added to the plaque extract and the resulting mixture was used for coverlsips coating (1 h), or plaque extract was stratified over the coverslip (1 h) and then MMP-2 (100 ng/ml) was added to the stratified extract and incubated for further 1 h, prior to perfusion experiments. After whole blood perfusion, 0.1% bovine serum albumin in physiologic solution, pH 7.3, was passed to remove all residual blood and the coverslips were then harvested, gently washed with 10 mM HEPES, pH 7.3, and fixed with 0.25% glutaric-dialdehyde in PBS, pH 7.4. Coverslips were stained with May-Grünwald/ Giemsa and observed under an optical microscope. Microscopic © Schattauer 2014
images were captured from at least 10 different fields (50x), randomly chosen, and analysed off-line (NHI-ImageJ). The surface covered by deposited platelets was expressed as % of total surface area (24, 25).
Immunohistochemistry Serial atherosclerotic plaque sections were stained for CD68, a monocyte antigen, and for MMP-2, according to standard procedures (27). Formalin-fixed, paraffin-embedded, 3.5-μm sections of tissue plaques were layered onto polylysine-coated slides, deparaffinised and then treated with proteinase K (DAKO Corp, Glostrup, Denmark) for 10 min at 37°C for antigen retrieval. After endogenous peroxidase activity was blocked with 1% H2O2 in PBS, sections were stained with monoclonal mouse anti-human CD68 (clone KP1, 1: 50, DAKO Cytomation, Glostrup, Denmark), or monoclonal mouse anti-human MMP-2 (1: 50, cod. MAB3308, Millipore Temecula, CA, USA), or monoclonal mouse anti-human IgG1 class (1: 50, DAKO Cytomation). After rinsing with PBS, slides were developed with biotinylated rabbit anti-mouse IgG (1: 200, DAKO Cytomation). Antibody reactivity was detected using horse radish peroxidase-conjugated biotin-avidin complexes (Vector Laboratories, Burlingame, CA, USA) and developed with diaminobenzidine tetrahydrochloride substrate (Dako, Glostrup, Denmark). The sections were photographed with a Leica Microscope and the presence of macrophages and MMP-2 were evaluated. Immunohistologic images were acquired at the resolution 2592x1944 and the analysis of plaques stained for CD68 or MMP-2 was performed by an investigator blinded as to the platelet aggregation-potentiating activity or MMP-2 content of plaques using the NIH ImageJ Software (v 1.47h, http: //imagej.nih.gov/ij) and expressed as mean pixel intensity (28). Collagen content in plaque sections was evaluated by picrosirius red staining and visualised under a polarised light.
hsCRP analysis Venous blood samples were collected into a tube without additives, left to clot for 60 min at 37°C and then centrifuged at 2,000 xg for 10 min, serum was removed and stored at −80°C for later assay. High-sensitivity C-reactive protein (hs-CRP) was measured using a commercially available kit based on nephelometry (N-Latex CRP II, Dade Behring Marburg Gmbh, Marburg, Germany).
Statistical analysis Platelet aggregation, deposition on plaque extracts, MMPs measurements and histologic analyses were carried out by different investigators blinded as to the patient characteristics, plaque type and results of the other assays. Conformity to normal distribution of the various parameters was assessed with the D’Agostino and Pearson omnibus normality test. Dichotomous variables were analysed with the Chi-square test, while the Mann-Whitney test was used to compare pro MMP-2, Thrombosis and Haemostasis 111.6/2014
Downloaded by: University of the Sciences in Philadelphia. Copyrighted material.
activating peptide-6 (TRAP-6, Bachem AG, Bubendorf, Switzerland) at a subthreshold concentration (12, 22). Based on the results of LTA, plaque extracts were defined as potentiating platelet aggregation when the ratio of the maximal amplitude of plaque+TRAP6/solvent+TRAP-6 was ≥ 2 (indicating that maximal amplitude of platelet aggregation with the plaque extract was twice or more, i.e. +100% or more, that obtained without the plaque extract). Subsequently, the same experiment was repeated after preincubation for 2 min with three different selective inhibitors of MMP-2, i.e. the synthetic MMP-2 inhibitor II (10 μM) (23), the naturally occurring inhibitor TIMP-2 (3 ng/ml) (both from Calbiochem, La Jolla, CA, USA), and a blocking anti-MMP-2 monoclonal antibody (50 ng/ml) (clone CA-4001, Millipore, Temecula, CA, USA). In selected experiments, aspirin (100 μM) or the P2Y12 receptor antagonist AR-C66096 (100 μM) were also tested. Each plaque extract was tested in triplicate with gel-filtered platelets from three different donors.
1091
Lenti et al. Plaque MMP-2 potentiates platelet aggregation
active MMP-2 and TIMP-2 levels in plaque extracts from potentiating and not potentiating plaque extracts. Multiple comparisons were performed by the one-way ANOVA followed by the Bonferroni’s multiple comparison test or the Dunn’s post test, where appropriate. The relation of the platelet potentiating effect of plaque extracts with the occurrence of ischaemic events at follow-up was evaluated by the Chi-square test. All data were analysed with NCSS 2007 (Version 7.1.21) (Hintze J (2007) NCSS and GESS. NCSS, LLc. Kaysville, UT, USA; www.ncss.com) and are presented as means and 95% confidence intervals (CIs).
Clinical characteristics, demographics, risk factors, and treatments of the patients enrolled are reported in ▶ Table 1.
Forty-three out of 81 plaque extracts (53%) potentiated TRAP-6 induced platelet aggregation (+233%, 95% CIs 135 to 281%, p
Cardiovascular Biology and Cell Signalling
Matrix metalloproteinase-2 of human carotid atherosclerotic plaques promotes platelet activation Correlation with ischaemic events Massimo Lenti1; Emanuela Falcinelli2; Marcella Pompili2; Paola De Rango1; Valentina Conti2; Giuseppe Guglielmini2; Stefania Momi2; Teresa Corazzi2; Giuseppe Giordano1; Paolo Gresele2 of Vascular and Endovascular Surgery, Hospital S. M. Misericordia, Perugia, Italy; 2Department of Internal Medicine, Section of Internal and Cardiovascular Medicine, University of Perugia, Perugia, Italy
Summary Purified active matrix metalloproteinase-2 (MMP-2) is able to promote platelet aggregation. We aimed to assess the role of MMP-2 expressed in atherosclerotic plaques in the platelet-activating potential of human carotid plaques and its correlation with ischaemic events. Carotid plaques from 81 patients undergoing endarterectomy were tested for pro-MMP-2 and TIMP-2 content by zymography and ELISA. Plaque extracts were incubated with gel-filtered platelets from healthy volunteers for 2 minutes before the addition of a subthreshold concentration of thrombin receptor activating peptide-6 (TRAP-6) and aggregation was assessed. Moreover, platelet deposition on plaque extracts immobilised on plastic coverslips under high shear-rate flow conditions was measured. Forty-three plaque extracts (53%) potentiated platelet aggregation (+233 ± 26.8%), an effect prevented by three different specific MMP-2 inhibitors (inhibitor II, TIMP-2, moAb
Correspondence to: Paolo Gresele, MD, PhD Dept. of Internal Medicine Section of Internal and Cardiovascular Medicine University of Perugia Via E. Dal Pozzo, 06126 Perugia, Italy Tel.: +39/075 5783989, Fax: +39/075 5716083 E-mail: [email protected]
anti-MMP-2). The pro-MMP-2/TIMP-2 ratio of plaques potentiating platelet aggregation was significantly higher than that of plaques not potentiating it (3.67 ± 1.21 vs 1.01 ± 0.43, p24 h and associated with new ischaemic lesions on a CT scan. Patients were defined as symptomatic if had experienced TIA or stroke compatible with the affected carotid artery within the six months prior to CEA.
Pro-MMP-2 in plaque extracts was measured by SDS-PAGE zymography, as previously described (20). Gelatinolytic activities were detected as transparent bands against the background of Coomassie Brilliant Blu-stained gelatine. A standard curve for each gel was generated using increasing concentrations (0.25 to 5 ng) of recombinant MMP-2 (kindly provided by Dr. Rafael Fridman, Wayne State University, Detroit, MI, USA). Minimal detectable amount (2 SD from 0) was 18 ± 7 pg. Intraand inter-assay coefficients of variation of MMP-2 measurements were 2.3% and 3.9%, respectively (n=8). Recoveries ranged from 85 to 112%, with a mean recovery of 96 ± 6.5%. Gel images were acquired with a Camedia 4040 digital camera (Olympus, Center Valley, PA, USA) and gelatinase activity areas were analysed using the Image-J 1.38x (NIH, Bethesda, MD, USA) software. TIMP-2 and active MMP-2 (GE Healthcare, Buckinghamshire, UK) were measured by ELISA, as described (15, 21).
Follow-up and clinical outcome A blood sample was taken on the day of hospitalisation for CEA. Clinical events on follow-up were evaluated by periodic clinical visits or, when not feasible, by phone interviews, at six months intervals. The primary outcome was defined as any adverse cardiovascular event, including any cardiovascular-related death, non fatal myocardial infarction (MI) or non-fatal stroke.
Carotid endarterectomy (CEA) and plaque extraction CEA was performed by standard techniques. Carotid plaques were defined as "lipidic" or "calcific" according to the preoperative duplex ultrasound scan appearance as hypoechoic or hyperechoic, respectively. Plaques were defined as complicated when highly hypoechoic, compatible with the presence of haemorrhage, thrombus, or fatty tisThrombosis and Haemostasis 111.6/2014
Platelet aggregation Venous blood was collected in the morning under fasting conditions, from twenty-five unrelated healthy subjects who had not taken any medication for at least 10 days before blood sampling, using a 19-gauge needle without tourniquet. Gel-filtered platelets (GFP) were prepared as described (14, 20) and adjusted to 1x108/ml in Hepes-Tyrode’s buffer containing 0.1% bovine serum albumin (BSA) and physiological magnesium concentration, pH 7.4. CaCl2 (2 mmol/l) was added to gel filtered platelets before incubation with plaques. Platelet aggregation was measured by light transmission aggregometry (LTA) using an APACT 4 aggregometer (Alfa Wasserman, Milan, Italy). Plaque extracts (72 ng/ml) or their solvent were incubated with 250 μl of GFP for 2 min before the addition of thrombin receptor © Schattauer 2014
Downloaded by: University of the Sciences in Philadelphia. Copyrighted material.
1090
Lenti et al. Plaque MMP-2 potentiates platelet aggregation
Platelet deposition on a plaque extract-covered surface under flow-conditions Platelet deposition on plaque extract-covered surfaces under highshear stress conditions (3,000 s-1) was carried out as described (24, 25). Briefly, plastic coverslips were covered with 100 μl of a suspension of plaque extract dissolved in PBS (1 mg of protein/ml), incubated for 1 h at 37°C and then rinsed with BSA/saline buffer (0.35%) for 1h at room temperature (18). Plaque extracts dissolved in PBS were preincubated, or not, with the MMP-2 inhibitor II (10 μM) or, in selected experiments, with a blocking anti-MMP-2 monoclonal antibody (5 μg/ml) for 20 min at room temperature prior to coverslip coating. Then extract-covered plastic coverslips were mounted in a rectangular parallel plate perfusion chamber and then citrated whole blood (0.5 ml) was passed through it at a wall shear rate of 3,000 s-1 for 5 min at 37°C, as described (26). Moreover, in order to assess the role of platelet GPVI in the platelet activating activity of plaques, we tested the effect of an antibody against GPVI (Fab 9O12.2, 50 µg/ml, kindly given by Prof. L. De Marco, CRO, Italy), for its effect on platelet deposition. To this end whole blood was incubated with the antibody for 15 min and then perfused over plaque extract-covered slides under high shear conditions. Moreover, to further explore the effect of MMP-2 on platelet thrombus formation in flowing blood, active MMP-2 (100 ng/ml) was either added to the plaque extract and the resulting mixture was used for coverlsips coating (1 h), or plaque extract was stratified over the coverslip (1 h) and then MMP-2 (100 ng/ml) was added to the stratified extract and incubated for further 1 h, prior to perfusion experiments. After whole blood perfusion, 0.1% bovine serum albumin in physiologic solution, pH 7.3, was passed to remove all residual blood and the coverslips were then harvested, gently washed with 10 mM HEPES, pH 7.3, and fixed with 0.25% glutaric-dialdehyde in PBS, pH 7.4. Coverslips were stained with May-Grünwald/ Giemsa and observed under an optical microscope. Microscopic © Schattauer 2014
images were captured from at least 10 different fields (50x), randomly chosen, and analysed off-line (NHI-ImageJ). The surface covered by deposited platelets was expressed as % of total surface area (24, 25).
Immunohistochemistry Serial atherosclerotic plaque sections were stained for CD68, a monocyte antigen, and for MMP-2, according to standard procedures (27). Formalin-fixed, paraffin-embedded, 3.5-μm sections of tissue plaques were layered onto polylysine-coated slides, deparaffinised and then treated with proteinase K (DAKO Corp, Glostrup, Denmark) for 10 min at 37°C for antigen retrieval. After endogenous peroxidase activity was blocked with 1% H2O2 in PBS, sections were stained with monoclonal mouse anti-human CD68 (clone KP1, 1: 50, DAKO Cytomation, Glostrup, Denmark), or monoclonal mouse anti-human MMP-2 (1: 50, cod. MAB3308, Millipore Temecula, CA, USA), or monoclonal mouse anti-human IgG1 class (1: 50, DAKO Cytomation). After rinsing with PBS, slides were developed with biotinylated rabbit anti-mouse IgG (1: 200, DAKO Cytomation). Antibody reactivity was detected using horse radish peroxidase-conjugated biotin-avidin complexes (Vector Laboratories, Burlingame, CA, USA) and developed with diaminobenzidine tetrahydrochloride substrate (Dako, Glostrup, Denmark). The sections were photographed with a Leica Microscope and the presence of macrophages and MMP-2 were evaluated. Immunohistologic images were acquired at the resolution 2592x1944 and the analysis of plaques stained for CD68 or MMP-2 was performed by an investigator blinded as to the platelet aggregation-potentiating activity or MMP-2 content of plaques using the NIH ImageJ Software (v 1.47h, http: //imagej.nih.gov/ij) and expressed as mean pixel intensity (28). Collagen content in plaque sections was evaluated by picrosirius red staining and visualised under a polarised light.
hsCRP analysis Venous blood samples were collected into a tube without additives, left to clot for 60 min at 37°C and then centrifuged at 2,000 xg for 10 min, serum was removed and stored at −80°C for later assay. High-sensitivity C-reactive protein (hs-CRP) was measured using a commercially available kit based on nephelometry (N-Latex CRP II, Dade Behring Marburg Gmbh, Marburg, Germany).
Statistical analysis Platelet aggregation, deposition on plaque extracts, MMPs measurements and histologic analyses were carried out by different investigators blinded as to the patient characteristics, plaque type and results of the other assays. Conformity to normal distribution of the various parameters was assessed with the D’Agostino and Pearson omnibus normality test. Dichotomous variables were analysed with the Chi-square test, while the Mann-Whitney test was used to compare pro MMP-2, Thrombosis and Haemostasis 111.6/2014
Downloaded by: University of the Sciences in Philadelphia. Copyrighted material.
activating peptide-6 (TRAP-6, Bachem AG, Bubendorf, Switzerland) at a subthreshold concentration (12, 22). Based on the results of LTA, plaque extracts were defined as potentiating platelet aggregation when the ratio of the maximal amplitude of plaque+TRAP6/solvent+TRAP-6 was ≥ 2 (indicating that maximal amplitude of platelet aggregation with the plaque extract was twice or more, i.e. +100% or more, that obtained without the plaque extract). Subsequently, the same experiment was repeated after preincubation for 2 min with three different selective inhibitors of MMP-2, i.e. the synthetic MMP-2 inhibitor II (10 μM) (23), the naturally occurring inhibitor TIMP-2 (3 ng/ml) (both from Calbiochem, La Jolla, CA, USA), and a blocking anti-MMP-2 monoclonal antibody (50 ng/ml) (clone CA-4001, Millipore, Temecula, CA, USA). In selected experiments, aspirin (100 μM) or the P2Y12 receptor antagonist AR-C66096 (100 μM) were also tested. Each plaque extract was tested in triplicate with gel-filtered platelets from three different donors.
1091
Lenti et al. Plaque MMP-2 potentiates platelet aggregation
active MMP-2 and TIMP-2 levels in plaque extracts from potentiating and not potentiating plaque extracts. Multiple comparisons were performed by the one-way ANOVA followed by the Bonferroni’s multiple comparison test or the Dunn’s post test, where appropriate. The relation of the platelet potentiating effect of plaque extracts with the occurrence of ischaemic events at follow-up was evaluated by the Chi-square test. All data were analysed with NCSS 2007 (Version 7.1.21) (Hintze J (2007) NCSS and GESS. NCSS, LLc. Kaysville, UT, USA; www.ncss.com) and are presented as means and 95% confidence intervals (CIs).
Clinical characteristics, demographics, risk factors, and treatments of the patients enrolled are reported in ▶ Table 1.
Forty-three out of 81 plaque extracts (53%) potentiated TRAP-6 induced platelet aggregation (+233%, 95% CIs 135 to 281%, p
