C-Reactive Protein Induces Matrix Metalloproteinase-1 and -10 in Human Endothelial Cells
Implications for Clinical and Subclinical Atherosclerosis
Ines Montero, BS*, ,
Josune Orbe, PhD*, ,
Nerea Varo, PhD,,
Oscar Beloqui, MD, PhD ,
José I. Monreal, MD, PhD,
José A. Rodríguez, PhD*,,
Javier Díez, MD, PhD,
Peter Libby, MD|| and
José A. Páramo, MD, PhD*,,*
* Laboratory of Atherosclerosis, School of Medicine/University Clinic, Center for Applied Medical Research, University of Navarra, Pamplona, Spain
Division of Cardiovascular Sciences, School of Medicine/University Clinic, Center for Applied Medical Research, University of Navarra, Pamplona, Spain
Internal Medicine, School of Medicine/University Clinic, Center for Applied Medical Research, University of Navarra, Pamplona, Spain
Biochemistry Laboratory, School of Medicine/University Clinic, Center for Applied Medical Research, University of Navarra, Pamplona, Spain
|| D.W. Reynolds Foundation Cardiovascular Research Center of Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts.
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Figure 1 C-reactive protein (CRP) increases matrix metalloproteinase (MMP)-1 and MMP-10 messenger ribonucleic acid (mRNA) expression in human endothelial cell. Reverse transcription-polymerase chain reaction for MMP-1, -9, -10, and tissue inhibitor of metalloproteinases (TIMP)-1 mRNA was performed in CRP-stimulated human umbilical vein endothelial cells (HUVEC) (12 h, 50 µg/ml) and mRNA normalized values (target gene/ß-actin mRNA copies) obtained. C-reactive protein-induced MMP-1 and -10 expression in human aortic endothelial cell (HAEC) is also shown. Data are presented (mean ± SEM, n = 4) as percentage from controls set as 100%. *p < 0.05 compared with control.
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Figure 2 C-reactive protein (CRP) increases endothelial matrix metalloproteinase (MMP)-1 and -10 protein in a concentration and time-dependent manner. (A) Matrix metalloproteinase-1 and -10 dose response after incubation with CRP (10 to 100 µg/ml) for 12 h. (B) Time course of MMP-1 and -10 secretion in conditioned medium after incubation with CRP (50 µg/ml) for 24 h. (C) C-reactive protein-induced (50 µg/ml) global MMP activity assessed by fluorogenic substrates. (D) Representative Western blot of MMP-1 and -10 and densitometric analysis corresponding to increased active form/zymogen ratio of MMP-1 and -10 after CRP (50 µg/ml) stimulation. Data are presented as mean ± SEM (n = 4). *p < 0.05 compared with control. RFU = relative fluorescent unit.
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Figure 3 Mitogen-activated protein kinase-mediated C-reactive protein (CRP)-induced matrix metalloproteinase (MMP)-1 (A) and MMP-10 (B) up-regulation. (A) An MEK inhibitor (PD98059) abolished the CRP-induced MMP-1 expression, whereas a p38 inhibitor (SB203580) partially reduced it. (B) Both SB203580 and SP600125 (a JNK inhibitor) blocked the CRP-induced MMP-10 up-regulation. *p < 0.05 vs. control; p < 0.05 vs. CRP.
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Figure 4 (A to C) Representative immunohistochemical analysis of C-reactive protein (CRP), matrix metalloproteinases (MMP)-1, and -10 in adjacent sections of human atherosclerotic plaques. Area squared in red corresponds to magnified details. Higher magnification revealed intense positive signal for CRP, MMP-1, and MMP-10 within macrophage-rich areas (CD-68 positive) (D). Magnification detail of endothelial layer (x100) showed that endothelial cell, positive for von Willebrand factor (vWf), also expressed MMP-10 (E and F). (G to I) Inmmunolocalization of CRP, MMP-1, and MMP-10 in control mammary arteries.
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Figure 5 Confocal microscopy in advanced human atherosclerotic plaques (n = 5). Matrix metalloproteinases (MMP)-10 (red) and C-reactive protein (CRP) (green) colocalized (yellow to orange) in macrophage-rich areas (A) and endothelial layer (B). No signal was detected in the absence of the primary antibodies (control). Nuclei were counterstained with TOPRO-3 (blue).
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