The pro-fibrotic properties of transforming growth factor on human fibroblasts are counteracted by caffeic acid by inhibiting myofibroblast formation and collagen synthesis

Abstract

Fibrosis is a chronic disorder affecting many organs. A universal process in fibrosis is the formation of myofibroblasts and the subsequent collagen deposition by these cells. Transforming growth factor beta1 (TGFβ1) plays a major role in the formation of myofibroblasts, e.g. by activating fibroblasts. Currently, no treatments are available to circumvent fibrosis. Caffeic acid phenethyl ester (CAPE) shows a broad spectrum of biological activities, including anti-fibrotic properties in vivo in mice and rats. However, little is known about the direct effects of CAPE on fibroblasts. We have tested whether CAPE is able to suppress myofibroblast formation and collagen formation of human dermal and lung fibroblasts exposed to TGFβ1, and found that this was indeed the case. In fact, the formation of myofibroblasts by TGFβ1 and subsequent collagen formation was completely abolished by CAPE. The same was observed for fibronectin and tenascin C. The lack of myofibroblast formation is likely due to the suppression of GLI1 and GLI2 expression by CAPE because of diminished nuclear SMAD2/3 levels. Post-treatment with CAPE after myofibroblast formation even resulted in a partial reversal of myofibroblasts into fibroblasts and/or reduction in collagen formation. Major discrepancies were seen between mRNA levels of collagen type I and cells stained positive for collagen, underlining the need for protein data in fibrosis studies to make reliable conclusions.

Keywords: Caffeic acid; Collagen; Fibrosis; Lysyl hydroxylase; Myofibroblasts.

Fig. 1

Characterization of mRNA levels and protein synthesis as observed in non-activated HDFa and HLFa. Fibroblasts were cultured for 48 h in basal medium supplemented with 0.17 mM L-ascorbic acid 2-phosphate magnesium salt and 0.5 % FBS in the absence of TGFβ1 and CAPE. a–g mRNA levels of ACTA2, TAGLN, GLI1, GLI2, COL1A1, COL1A2 and PLOD2 relative to the reference gene YWHAZ. h–o Representative immunofluorescence stainings (upper panel) and p–s quantification of the % of cells (lower panel) positive for αSMA, SM22α, collagen type I and LH2. § Statistically significant towards HDFa. Scale bar 100 μm

Fig. 2

Effects of TGFβ1 and CAPE on nuclear localization of SMAD2/3. a–c HDFa and d–f HLFa were cultured for 45 min in the presence of TGFβ1 alone, or TGFβ1 in combination with CAPE (co-treatment). Fibroblasts cultured in the presence of TGFβ1 showed a strong nuclear localization of SMAD2/3 (b, e), whereas CAPE prevented the TGFβ1-induced nuclear translocation of SMAD2/3 (c, f). Scale bar 25 μm

Fig. 3

Effects of CAPE, TGFβ1 and TGFβ1 + CAPE on ACTA2 mRNA levels and % αSMA-positive cells of HDFa and HLFa. Fibroblasts were cultured for 48 h in the presence of CAPE alone, TGFβ1 alone, or TGFβ1 in combination with CAPE (co-treatment). a, b mRNA levels of ACTA2 relative to the reference gene YWHAZ and expressed as fold-change compared to untreated control (i.e. the baseline level as provided in Fig. 1). c–j Representative immunofluorescence stainings (upper panel) and k, l quantification of the % of cells (lower panel) positive for αSMA. * Statistically significant towards untreated control, and # statistically significant for cells co-treated with TGFβ1 + CAPE towards TGFβ1-treated cells. Scale bar 100 μm

Fig. 4

Effects of post-treatment of CAPE on cells treated with TGFβ1 regarding the % αSMA-positive cells of HDFa and HLFa. Fibroblasts were cultured for 48 h in the presence of TGFβ1, followed by a post-treatment with CAPE for 24 h. a–d Representative immunofluorescence stainings (upper panel) and e, f quantification of the % of cells (lower panel) positive for αSMA. # Statistically significant for cells post-treated with CAPE towards cells stimulated with TGFβ1 only. Scale bar 100 μm

Fig. 5

Effects of CAPE, TGFβ1 and TGFβ1 + CAPE on TAGLN mRNA levels and % SM22α-positive cells of HDFa and HLFa. Fibroblasts were cultured for 48 h in the presence of CAPE alone, TGFβ1 alone, or TGFβ1 in combination with CAPE (co-treatment). a, b mRNA levels of TAGLN relative to the reference gene YWHAZ and expressed as fold-change compared to untreated control (i.e. the baseline level as provided in Fig. 1). c–j Representative immunofluorescence stainings (upper panel) and k, l quantification of the % of cells (lower panel) positive for SM22α. * Statistically significant towards untreated control, and # statistically significant for cells co-treated with TGFβ1 + CAPE towards TGFβ1-treated cells. Scale bar 100 μm

Fig. 6

Effects of CAPE, TGFβ1 and TGFβ1 + CAPE on COL1A1 and COL1A2 mRNA levels and % collagen type I-positive cells of HDFa and HLFa. Fibroblasts were cultured for 48 h in the presence of CAPE alone, TGFβ1 alone, or TGFβ1 in combination with CAPE (co-treatment). a–d mRNA levels of COL1A1 and COL1A2 relative to the reference gene YWHAZ and expressed as fold-change compared to untreated control (i.e. the baseline level as provided in Fig. 1). e–l Representative immunofluorescence stainings (upper panel) and m, n quantification of the % of cells (lower panel) positive for collagen type I. * Statistically significant towards untreated control, and # statistically significant for cells co-treated with TGFβ1 + CAPE towards TGFβ1-treated cells. Scale bar 100 μm

Fig. 7

Effects of post-treatment of CAPE on cells treated with TGFβ1 regarding the % collagen type I-positive cells of HDFa and HLFa. Fibroblasts were cultured for 48 h in the presence of TGFβ1, followed by a post-treatment with CAPE for 24 h. a–d Representative immunofluorescence stainings (upper panel) and e, f quantification of the % of cells (lower panel) positive for collagen type I. # Statistically significant for cells post-treated with CAPE towards cells stimulated with TGFβ1 only. Scale bar 100 μm

Fig. 8

Effects of CAPE, TGFβ1 and TGFβ1 + CAPE on PLOD2 mRNA levels and % LH2-positive cells of HDFa and HLFa. Fibroblasts were cultured for 48 h in the presence of CAPE alone, TGFβ1 alone, or TGFβ1 in combination with CAPE (co-treatment). a, b mRNA levels of PLOD2 relative to the reference gene YWHAZ and expressed as fold-change compared to untreated control (i.e. the baseline level as provided in Fig. 1). c–j Representative immunofluorescence stainings (upper panel) and k, l quantification of the % of cells (lower panel) positive for LH2. * Statistically significant towards untreated control, and # statistically significant for cells co-treated with TGFβ1 + CAPE towards TGFβ1-treated cells. Scale bar 100 μm

Fig. 9

Effects of TGFβ1 and CAPE on fibronectin (FN1) and tenascin C (TNC) gene expression. a–d Fibroblasts were cultured for 48 h in the presence of CAPE alone, TGFβ1 alone, or TGFβ1 in combination with CAPE (co-treatment) on mRNA levels of FN1 and TNC relative to the reference gene YWHAZ and expressed as fold-change compared to untreated control. * Statistically significant towards untreated control, # statistically significant for cells co-treated with TGFβ1 + CAPE towards TGFβ1-treated cells

Fig. 10

Effects of CAPE, TGFβ1 and TGFβ1 + CAPE on GLI1, GLI2 and SNAIL1 mRNA levels of HDFa and HLFa. a–f Fibroblasts were cultured for 48 h in the presence of CAPE alone, TGFβ1 alone, or TGFβ1 in combination with CAPE (co-treatment) on mRNA levels of GLI1, GLI2 and SNAIL1 relative to the reference gene YWHAZ and expressed as fold-change compared to untreated control (i.e. the baseline level as provided in Fig. 1). * Statistically significant towards untreated control, # statistically significant for cells co-treated with TGFβ1 + CAPE towards TGFβ1-treated cells