442Fibroblast-activation-protein alpha is a cell surface dipeptidyl peptidase and gelatinase expressed by fibroblasts at the tissue remodelling interface after myocardial infarction

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Fibroblast-activation-protein a (FAP) is a membrane-bound dipeptidyl peptidase with gelatinolytic activity and is expressed in thin-cap fibroatheromas and activated fibroblasts during wound healing. Whether FAP is expressed in the heart after myocardial infarction (MI) and whether it exerts effects on matrix remodelling is unknown. Therefore, MI was induced by LAD ligation in rats and FAP expression was analyzed by western blot (WB) and immunohistochemistry (IHC). Human cardiac fibroblasts (HCF) were analyzed for TGFβ1-dependent expression of MMP-2 and FAP (WB, ELISA), cell migration and gelatinolytic activity. TGFβ1-signaling was blocked using the TGFbR1-inhibitor SB431542. FAP, SMAD2 and SMAD3 were silenced by siRNA. FAP was upregulated after MI, as evidenced by large numbers of FAP-expressing cells in the peri-infarct area at 3d and 7d post-MI (1339 ± 249* and 2072 ± 185* cells/mm2; * p < 0.05 vs. sham). FAP + cells were rarely found in hearts of sham animals. Co-localization analysis with prolyl-4-hydroxylase beta (P4H) identified the majority of FAP + cells as collagen-producing myofibroblasts. FAP was also expressed by fibroblasts in fibrotic scar tissue of human hearts with ischemic heart disease, while it was not detected in control hearts. In vitro, inhibition of FAP-expression reduced migratory capacity by 31% (40147 ± 2048* vs. 58737 ± 2199 AU; p < 0.05 FAP-knockdown vs. control). Stimulation with TGFβ1 at 10ng/ml resulted in increased FAP protein expression (1.5 ± 0.5-fold, p < 0.05 vs. unstimulated). Newly produced FAP was primarily bound to the cell surface and not secreted. Similar to FAP, MMP-2 increased after stimulation with 10ng/ml TGFβ1 (1.8 ± 0.3-fold, p < 0.05 vs. unstimulated). Gelatin zymography of fibroblast lysates revealed significant gelatinase activity from FAP and MMP-2. Induction of FAP by TGFβ1 was blocked after preincubation with SB431542. In control HCF, activation of TGFbR1 induced phosphorylation of SMAD2 and SMAD3. In SMAD2- or SMAD3-deficient HCF, TGFβ1 stimulation resulted in markedly reduced levels of p-SMAD2/p-SMAD3, together with a blunted increase of FAP in both cases. These results demonstrated that induction of FAP by TGFβ1 is dependent on phosphorylation of SMAD2 and SMAD3. Similar to FAP, induction of MMP-2 was blunted by the TGFbR1-blocker SB431542. In conclusion, this study demonstrates for the first time expression of FAP after MI in activated fibroblasts at the tissue remodelling interface after myocardial infarction and indicates a functional role of FAP, together with MMP2, in TGFβ1-mediated matrix remodelling.

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