Figure 5

CM of M1 polarized, M2 polarized or unstimulated macrophages do not induce myofibroblast differentiation of HDFs. HDFs stimulated with CM of unstimulated macrophages showed, compared to fibroblasts stimulated with CM of M1 polarized macrophages, an upregulated gene expression of ACTA2 after 48 h, 72 h and 144 h. HDFs stimulated with CM of M2 polarized macrophages showed a higher gene expression of ACTA2 compared to HDFs stimulated with CM of M1 macrophages after 144 h (A). No differences were observed in TAGLN gene expression between the three conditions in all time points (A). No differences in ACTA2 protein expression were seen between the three conditions after 144 h. TGFB1 stimulated fibroblasts were used as positive control (B). Fibroblasts stimulated with CM of M1 macrophages contract the collagen 10% more than fibroblasts stimulated with CM of M2 and unstimulated fibroblasts. Fibroblasts stimulated with TGFB1 contract the collagen 50% more compared to the other stimulations (C). * p < 0.05, Difference between HDFs stimulated with CM of M1 polarized and CM of M2 polarized macrophages, *** p < 0.001. # p < 0.05, Difference between HDFs stimulated with CM of M1 polarized and CM of unstimulated macrophages, ## p < 0.01, ### p < 0.001. ^ p < 0.05, Difference between HDFs stimulated with CM of M2 polarized and CM of unstimulated macrophages. ≅≅≅ p < 0.001, Difference between HDFs stimulated with TGFB1 and CM of M1 polarized macrophages. §§§ p < 0.001, Difference between HDFs stimulated with TGFB1 and CM of M2 polarized macrophages. ❖❖❖ p < 0.001, Difference between HDFs stimulated with TGFB1 and CM of unstimulated polarized macrophages. Gene expression analysis data were analyzed using two-way ANOVA followed by Bonferroni’s post-test, n = 4. ACTA2 protein expression was shown in red and nuclei in blue (DAPI), original magnification 200×. Collagen gel contraction analysis data were analyzed using one-way ANOVA followed by Tukey’s post-test, n = 3.