Dedifferentiation of Human Cardiac Myofibroblasts Is Independent of Activation of COX-2/PGE2 Pathway

Figures

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  Figure 1. Protocol of studies on dedifferentiation of de novo human cardiac myofibroblasts and activation of COX-2/PGE2. TGF-β1 at 2 ng/mL was added into medium for 48 h to differentiate human cardiac fibroblasts to myofibroblasts. After changing medium withoutTGF-β1, cells were treated with PMA, PGE2, or DMSO as vehicle control. To investigate the involvement of COX-2/PGE2 activation in dedifferentiation of myofibroblasts, NS-398 (a selective COX-2 inhibitor) or PF-04418948 (a selective EP2 receptor antagonist) was added 1 hour before the treatment. The protein expression of α-SMA was detected at the end of 48 hours after treatment of the above agents. HCF: Human cardiac fibroblast.

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  Figure 2. PMA but not PGE2 dedifferentiates de novo human cardiac myofibroblasts induced by TGF-β1 from human cardiac fibroblasts (HCFs). (A). Phase-contract images of HCFs or human cardiac myofibroblasts treated with PMA or PGE2 were shown. Magnification: 100×. (B). Double immunostaining of α-SMA (green) and DAPI (blue) in HCFs or human cardiac myofibroblasts was performed. Increased expression of α-SMA was demonstrated in cardiac myofibroblasts after exposure to TGF-β1 at 2 ng/mL for 48 h. PMA at 50 ng/mL reduced expression of α-SMA. PGE2 at 500 nM did not affect the increased α-SMA expression in cardiac myofibroblasts. Scale bar = 20 µm. (C). Representative images of protein expression of α-SMA in cardiac myofibroblasts were shown. Beta-actin was used as a loading control. (D). Immunoblot analysis showed increased protein levels in cardiac myofibroblasts treated with TGF-β1. PMA reduced expression of α-SMA. PGE2 did not affect increased α-SMA expression in cardiac myofibroblasts. n: independent samples per group. Bar: mean ± SEM. ** p < 0.01, *** p < 0.001.

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  Figure 3. PGE2 content in human cardiac myofibroblasts treated with PMA or other indicated agents. Levels of PGE2 in the culture medium of cardiac myofibroblasts were measured using PGE2 ELISA Monoclonal Kit. Samples and standards were assayed in parallel. All assays were performed in duplicate plates. The results were calculated according to the standard curve of PGE2 and expressed as pg/mL. PMA: phorbol 12-myristate 13-acetate; NS: NS-398 (a selective COX-2 inhibitor); PF: PF-04418948 (a selective EP2 receptor antagonist). Data were expressed as mean ± S.E.M. n = 4/per group. * p < 0.05, ** p < 0.01, *** p < 0.001.

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  Figure 4. PMA-induced dedifferentiation of cardiac myofibroblasts does not depend on activation of COX-2/PGE2 pathway. (A). A representative image of Western blotting results of protein expression of α-SMA was shown. Beta-actin (42 kDa) was used as a load control. (B). Immunoblot analysis showed a marked increase in α-SMA protein levels in human cardiac myofibroblasts treated with TGF-β1. PMA at 50 ng/mL reduced protein expression of α-SMA. Pretreatment with NS-398 or PF-04418948 had no effect on reduced expression of α-SMA induced by PMA. PMA: phorbol 12-myristate 13-acetate; NS: NS-398; PF: PF-04418948. n: independent samples per group. Bar: mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001.

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  Figure 5. PMA-induced dedifferentiation of human cardiac myofibroblasts does not depend on activation of COX-2/PGE2 pathway. A schematic outlines dedifferentiation of cardiac myofibroblasts induced by PMA and the role of COX2/PGE2 activation in dedifferentiation of myofibroblasts. TGF-β1 and/or other factors, such as increased mechanical tension, inflammation, or oxidative stress promote differentiation of human cardiac fibroblasts to myofibroblasts though active fibroblasts. PMA dedifferentiates cardiac myofibroblasts to fibroblasts and/or other unidentified cell type. PMA increases activity of COX-2 and formation of PGE2. Deactivation of COX-2 and PGE2 produces no effects on PMA-induced dedifferentiation of cardiac myofibroblasts. (+) denotes increase.