Increased expression of bFGF is associated with carotid atherosclerotic plaques instability engaging the NF-κB pathway

Abstract

Unstable atherosclerotic plaques of the carotid arteries are at great risk for the development of ischemic cerebrovascular events. The degradation of the extracellular matrix by matrix metalloproteinases (MMPs) and NO-induced apoptosis of vascular smooth muscle cells (VSMCs) contribute to the vulnerability of the atherosclerotic plaques. Basic fibroblast growth factor (bFGF) through its mitogenic and angiogenic properties has already been implicated in the pathogenesis of atherosclerosis. However, its role in plaque stability remains elusive. To address this issue, a panel of human carotid atherosclerotic plaques was analyzed for bFGF, FGF-receptors-1 and -2 (FGFR-1/-2), inducible nitric oxide synthase (iNOS) and MMP-9 expression. Our data revealed increased expression of bFGF and FGFR-1 in VSMCs of unstable plaques, implying the existence of an autocrine loop, which significantly correlated with high iNOS and MMP-9 levels. These results were recapitulated in vitro by treatment of VSMCs with bFGF. bFGF administration led to up-regulation of both iNOS and MMP-9 that was specifically mediated by nuclear factor-kappaB (NF-kappaB) activation. Collectively, our data demonstrate a novel NF-kappaB-mediated pathway linking bFGF with iNOS and MMP-9 expression that is associated with carotid plaque vulnerability.

Fig 1

bFGF expression is increased in unstable carotid atherosclerotic plaques and correlates with FGFR-1, MMP-9 and iNOS expression. (A) Representative pictures of bFGF immunohistochemical expression in stable and unstable plaques. Insets represent higher magnification of the selected areas. (B) Boxplots depict differences in bFGF expression according to plaque stability and patients’ symptoms. (C) Representative pictures of FGFR-1/2 expression in stable and unstable carotid atherosclerotic plaques. (D) Boxplots showing differences in FGFR-1 and FGFR-2 expression between stable and unstable carotid plaques. (E) Representative pictures from two cases with low and high expression levels of iNOS and MMP-9, respectively. (F) Representative immunoblotting analysis for iNOS and MMP-9 in carotid specimens. (G) Scatterplots showing the correlation among bFGF and iNOS and MMP-9 expression. (H) Immunoblotting analysis of human VSMCs for iNOS and MMP-9 expression, before and after the addition of recombinant bFGF in the culture medium.

Fig 2

NF-κB specifically mediates the up-regulation of MMP-9 and iNOS by bFGF treatment. (A) NF-κB luciferase reporter assay of VSMCs transfected either with empty vector or firefly luciferase reporter plasmid driven by five consecutive artificial NF-κB-binding sites [NF-κB–luc). (B) Immunoblotting analysis of p65-pSer536 and pERK in VSMCs at 30, 60 and 180 min. after addition of bFGF in the culture medium. Actin serves as loading control. The histogram depicts the quantitative estimation of p65-pSer536 expression levels after densitometric analysis. (C) iNOS and MMP-9 immunoblotting analysis in VSMCs treated with bFGF along with either MAPK inhibitor PD98059 or siRelA. The histogram depicts the quantitative estimation, after densitometric analysis, of iNOS and MMP-9 expression levels with several treatments. (D) Representative cases depicting sole cytoplasmic (stable plaque) and cytoplasmic/nuclear (unstable plaque) NF-κB IHC staining in carotid atherosclerotic plaques. (E) IF analysis of case 1 depicting cytoplasmic/nuclear NF-κB localization. (F) Immunoblotting analysis for total p65-pSer536 levels in two representative cases expressing only cytoplasmic (stable plaque) and both cytoplasmic and nuclear (unstable plaque) IHC staining of p65, respectively. (G) Boxplots depicting differences in bFGF, iNOS, MMP-9, FGFR-1 and FGFR-2 expression, respectively, according to NF-κB localization. NS, non-significant.