ABSTRACT
C-reactive protein (CRP) has been linked to the pathogenesis of atherosclerosis. The dissociation of native, pentameric (p)CRP to monomeric (m)CRP on the cell membrane of activated platelets has recently been demonstrated. The dissociation of pCRP to mCRP may explain local pro-inflammatory reactions at the site of developing atherosclerotic plaques. As a biomarker, pCRP predicts cardiovascular adverse events and so do reduced levels and function of circulating endothelial progenitor cells (EPCs). We hypothesised that mCRP and pCRP exert a differential effect on EPC function and differentiation. EPCs were treated with mCRP or pCRP for 72 h, respectively. Phenotypical characterisation was done by flow cytometry and immunofluorescence microscopy, while the effect of mCRP and pCRP on gene expression was examined by whole-genome gene expression analysis. The functional capacity of EPCs was determined by colony forming unit (CFU) assay and endothelial tube formation assay. Double staining for acetylated LDL and ulex lectin significantly decreased in cells treated with pCRP. The length of tubuli in a matrigel assay with HUVECs decreased significantly in response to pCRP, but not to mCRP. The number of CFUs increased after pCRP treatment. RNA expression profiling demonstrated that mCRP and pCRP cause highly contradictory gene regulation. Interferon-responsive genes (IFI44L, IFI44, IFI27, IFI 6, MX1, OAS2) were among the highly up-regulated genes after mCRP, but not after pCRP treatment. In conclusion, EPC phenotype, genotype and function were differentially affected by mCRP and pCRP, strongly arguing for differential roles of these two CRP conformations. The up-regulation of interferon-inducible genes in response to mCRP may constitute a mechanism for the local regulation of EPC function.
Subject(s)
C-Reactive Protein/pharmacology , Cell Differentiation/drug effects , Endothelium, Vascular/drug effects , Stem Cells/drug effects , Antigens, CD34/metabolism , Cell Differentiation/physiology , Cell Survival/drug effects , Cell Survival/physiology , Cells, Cultured , Endothelium, Vascular/cytology , Endothelium, Vascular/metabolism , Humans , In Vitro Techniques , Interferon-alpha/metabolism , Lipoproteins, LDL/metabolism , Phenotype , Plant Lectins/metabolism , Protein Isoforms/pharmacology , Stem Cells/cytology , Stem Cells/metabolismABSTRACT
BACKGROUND: Matrix metalloproteinase-1 (MMP-1) plays an important role in tissue remodelling and in the pathology of inflammatory diseases including periodontitis. The activity of MMP-1 is firmly controlled by the endogenous tissue inhibitor of metalloproteinase-1 (TIMP-1). OBJECTIVE: The aim of the study was to investigate the production and regulation of MMP-1 and TIMP-1 with special regards to the enzyme protein kinase C (PKC) in human gingival fibroblasts. METHODS: Gingival fibroblasts were treated with substances related to PKC such as phorbol 12-myristate 13-acetate (PMA), interleukin-1beta, Ca2+ -ionophore A231817 and inhibitors of PKC, p38 mitogen-activated protein kinase (p38 MAPK) and tyrosine kinase. RESULTS: The PKC activator PMA stimulated the production of MMP-1 and TIMP-1 at both the transcriptional and the translational level. The production of MMP-1 and TIMP-1 stimulated by PMA was abolished by the PKC inhibitor bisindolylmaleimide. Treatment of the cells with interleukin-1beta or A23187 synergistically increased the stimulatory effect of PMA on MMP-1 production. In contrast, TIMP-1 production was unaffected by interleukin-1beta and reduced by A23187. Tyrosine kinase inhibitor herbimycin A reduced MMP-1 production induced by PMA, whereas the p38 MAPK-inhibitor SB 203580 synergistically increased the stimulatory effect of PMA on both MMP-1 and TIMP-1 production. CONCLUSION: The present study shows that MMP-1 and TIMP-1 production is regulated differently by interleukin-1beta and calcium in human gingival fibroblasts and that this difference is markedly amplified in the presence of the PKC-activator PMA. Taken together, the discrepancy in the production of MMP-1 and TIMP-1 in gingival fibroblasts may contribute to tissue destruction in periodontal diseases.
Subject(s)
Gingiva/enzymology , Matrix Metalloproteinase 1/biosynthesis , Protein Kinase C/physiology , Tissue Inhibitor of Metalloproteinase-1/biosynthesis , Adolescent , Calcium/physiology , Cells, Cultured , Child , Child, Preschool , Fibroblasts/enzymology , Gene Expression Regulation, Enzymologic/drug effects , Gingiva/cytology , Humans , Infant , Interleukin-1/physiology , Protein Kinase C/drug effects , RNA, Messenger/biosynthesis , Tetradecanoylphorbol Acetate/pharmacologyABSTRACT
The in vitro effect of phenytoin (PHT) on the production of interleukin-6 (IL-6) and interleukin-8 (IL-8) in human gingival fibroblasts, challenged with or without interleukin-1beta (IL-1beta), was studied. PHT (20 microg/ml) alone increased the mRNA level for both IL-6 and IL-8, as well as synergistically enhancing the production of IL-6 and IL-8, at both transcriptional and translational level in fibroblasts challenged with IL-1beta (30 pg/ml). The stimulatory effect of PHT on IL-1beta-induced IL-6 production was strongly reduced by the specific cyclooxygenase-2 inhibitor NS-398 (1 microM). The anti-inflammatory drug, dexamethasone (1 microM), abolished the production of both IL-6 and IL-8 in gingival fibroblasts challenged with PHT in the presence or absence of IL-1beta. The ability of PHT, alone as well as in combination with IL-1, to upregulate the production of IL-6 and IL-8 in human gingival fibroblasts may contribute to enhanced recruitment and activation of inflammatory cells. This effect of PHT may thereby give a prerequisite for the establishment of an interaction between cytokines and connective tissue cells in the periodontal tissue, which is suggested to lead to gingival overgrowth.
