Matrix metalloproteinase-2-induced epidermal growth factor receptor transactivation impairs redox balance in vascular smooth muscle cells and facilitates vascular contraction

Figures

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  Fig. 1. MMP-2-induced increase in ROS concentrations in vascular smooth muscle cells is prevented by MMP inhibitors. (A) ROS concentrations in vascular smooth muscle cells (A7r5 cells) assessed with dihydroethidium (DHE; 10 µmol/l) by flow cytometry after incubation with MMP-2 (16 nmol/l) for 10 or 30 min. (B) Effects of antioxidant agents including apocynin 100 µmol/l (Apo), diphenyl iodonium (DPI; a flavoprotein inhibitor) 10 µmol/l, or PEG-catalase 3000 U/ml (PG-Cat, which catalyzes the breakdown of H2O2) in both vehicle and in MMP-2-treated cells. Panel C shows the effects of MMP inhibitors doxycycline 100 µmol/l (Doxy) or GM6001 1 µmol/l in both vehicle and in MMP-2-treated cells. Data are shown as the mean ± SEM (n = 4/group). * P < 0.05 vs. Vehicle. ** P < 0.05 vs. Vehicle and 10 min. # P < 0.05 vs. Vehicle in MMP-2-treated cells.

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  Fig. 2. MMP-2 mediates HB-EGF shedding and induces EGFR-dependent increases in ROS concentrations. (A) MMP-2 promotes HB-EGF shedding, as revealed by quantification of HB-EGF shedding measured by alkaline phosphatase (AP) activity in the supernatant of HEK293 cells after incubation of MMP-2 (16 nmol/l) or Vehicle for 10 and 30 min. Shedding activity is presented as the ratio of the AP activity in the supernatant and in the cell lysate as described in Section 2. (B) Incubation with MMP-2 (16 nmol/l) for 10 or 30 min increases ROS concentrations in vascular smooth muscle cells (A7r5 cells) assessed with dihydroethidium (DHE; 10 µmol/l) by flow cytometry, and this effect is prevented by the EGFR kinase inhibitor (Ag1478; 3 or 10 μmol/l). Data are shown as the mean ± SEM (n = 4/group). * P < 0.05 vs. respective Control (Vehicle). *P < 0.05 vs. respective Control (Vehicle) and vs. MMP-2 10 min. # P < 0.05 vs. Vehicle + Vehicle treated cells. ## P < 0.05 vs. Vehicle + Vehicle treated cells and vs. MMP-2 + Vehicle treated cells for 10 min.

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  Fig. 3. MMP-2-induced increases in ROS concentrations in vascular smooth muscle cells is abolished by a phospholipase C inhibitor or by a protein kinase C inhibitor. (A) Quantification of ROS production after 30 min of MMP-2 incubation with VSMC in the absence or presence of the phospholipase C inhibitor (A778 10 μM) or the PKC inhibitor (Chelerythrine, Chel, 3 μM). ROS concentrations were assessed with DHE probe by flow cytometry. (B) Control experiments showing quantification of ROS production stimulated by an EGFR agonist (recombinant HB-EGF) or by a PKC activator (phorbol 12-myristate 13-acetate; PMA) after 30 min of incubation with MMP-2. Data are shown as the mean ± SEM (n = 4/group). *P < 0.05 vs. Vehicle + Vehicle treated cells. # P < 0.05 vs. Vehicle + MMP-2 treated cells.

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  Fig. 4. Intraluminal incubation of rabbit aorta with MMP-2 for 30 min increases gelatinolytic activity in all layers of the vessel. (A) Representative confocal photomicrographs of rabbit aortic sections incubated with DAPI, DQ-Gelatin and anti-MMP-2 antibody (Alexa 647 used as secondary antibody). DAPI´s blue fluorescence indicates the nuclei; green fluorescence reflects the total gelatinolytic activity, and red fluorescence indicates the staining with anti-MMP-2 antibody (this antibody reacts only with the recombinant MMP-2 protein and not with endogenous rabbit MMP-2). (B) Aortic gelatinolytic activity quantified by in situ zymography in aortas exposed to intraluminal incubation with MMP-2 or Vehicle (control).(C) Quantification of the Alexa 647 signal (which reflects aortic MMP-2 concentration) in photomicrographs of rabbit aortas that were intraluminally incubated with recombinant MMP-2. Data are shown as the mean ± SEM (n = 4–5/group). * P < 0.05 vs. Vehicle group.

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  Fig. 5. MMP inhibitors prevent the increases in vascular gelatinolytic activity, in ROS concentrations, and in lucigenin chemiluminescence after intraluminal incubation of rabbit aorta with MMP-2. (A) Representative photomicrographs of aortic in situ zymography experiments showing that MMP-2 gelatinolytic activity was totally inhibited by phenanthroline (Phe, 100 µM) or doxycycline (Doxy, 100 µM), but not by phenylmethylsulfonyl fluoride (PMSF, 100 µM). The arteries were incubated with the inhibitors together with MMP-2 (16 nM) intraluminally for 30 min. (B) Quantification of aortic gelatinolytic activity measured in experiments described in A. (C) Representative photomicrographs of aortic sections incubated with DHE. Red fluorescence reflects ROS production and shows that incubation with MMP-2 (16 nM) intraluminally for 30 min increased ROS production, and this effect was totally inhibited by phenanthroline (Phe, 100 µM) or doxycycline (Doxy, 100 µM), but not by phenylmethylsulfonyl fluoride (PMSF, 100 µM). The experiments were carried out as described in A. (D), Quantification of aortic fluorescence of DHE measured in experiments described in C. (E), Quantification of lucigenin…

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  Fig. 6. Markers of increased aortic oxidative stress increase in proportion to the increases in aortic MMP-2 gelatinolytic activity. (A) Aortic concentrations of reactive oxygen species (ROS) measured by DHE technique increase in proportion to the increases in gelatinolytic activity. This association analysis was carried out using data reported in Fig. 5D. (B), Aortic lucigenin chemiluminescence increase in proportion to the increases in gelatinolytic activity. This association analysis was carried out using data reported in Fig. 5E. The regression line and the 95% confidence interval are plotted.

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  Fig. 7. The antioxidant apocynin or PEG-catalase prevent pro-oxidant effects of MMP-2. (A) Representative photomicrographs of aortic sections incubated with MMP-2 (16 nM) intraluminally for 30 min. ROS production assessed by DHE was totally inhibited by apocynin (Apo 100 µM) or by PEG-catalase (PG-CAT 2500 U/mL). (B) Quantification of aortic fluorescence stimulated by DHE. Data are shown as the mean ± SEM (n = 4–5/group). *P < 0.05 vs. Vehicle+Vehicle group. # P < 0.05 vs. Vehicle+MMP-2 group.

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  Fig. 8. Intraluminal incubation of rabbit aorta with MMP-2 increases the vascular contractility to phenylephrine (PE). (A) Cumulative concentration-effect curves to PE were performed in endothelium denuded (E-) aortic rings obtained from vessels filled with MMP-2 (16 nM) or vehicle. (B) Quantification of maximum effect (Emax) induced by PE in E- aortic rings filled with MMP-2 or vehicle. (C) Cumulative concentration-effect curves to PE in E- aortic rings in presence or absence of the MMP inhibitor GM6001 (10 µM), or apocyanin (Apo 100 µM), or PEG-Catalase (PG-Cat 3000 U/mL). (D) Quantification of maximum effect (Emax) induced by PE in E- aortic rings filled with MMP-2 in presence or absence of GM6001, Apocyanin or PEG-catalase. Data are shown as the mean ± SEM (n = 7–9/group). * P < 0.05 vs. Vehicle group. # P < 0.05 vs. MMP-2 group.

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  Fig. 9. MMP-2 exerts pro-oxidant effects in vascular smooth muscle cell. MMP-2 induces shedding of HB-EGF which activates the EGF receptor (EGFR) activating phospholipase C (PLC-y) leading to diacylglycerol (DAG) formation and protein kinase C (PKC) activation. PKC enhances NADPH oxidase activity and increases ROS production.