Hydrostatic pressure suppresses fibrotic changes via Akt/GSK-3 signaling in human cardiac fibroblasts

Figures

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  Figure 1. (A–B) Pressure loading apparatus and pressurizing method. Ventilation time of 30 min every 60 min of pressurization minimized the pH and dissolved O2 and CO 2 in the culture medium.

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  Figure 2. Phosphorylated antibody array showed that hydrostatic pressure resulted in phosphorylation of several proteins. (A) Phosphorylated antibody map with atmospheric pressure (AP) and hydrostatic pressure (HP). The left map shows phosphorylated proteins in the AP group. The right map shows phosphorylated proteins in the HP group (200 mmHg, 60 min). (B) List of target proteins and phosphorylation sites in phosphorylated antibody array. (C) Histograms of protein phosphorylation under AP conditions (white) and HP conditions (200 mmHg; gray) for 60 min. HP resulted in significant phosphorylation of several proteins including Akt (Ser473); unpaired t‐test, n = 4, * P < 0.05, ** P < 0.01, *** P < 0.001, ns: no significant difference).

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  Figure 3. HP resulted in phosphorylation of Akt (Ser473) in a time‐ and pressure‐dependent manner and enhanced or attenuated the phosphorylation of several kinases and transcription factors. (A) HP for 60 min resulted in the phosphorylation of Akt in a pressure‐dependent manner (one‐way ANOVA with Tukey post hoc test, n = 4, * P < 0.05). (B) HP resulted in the phosphorylation of Akt in a time‐dependent manner when the pressure was fixed at 200 mmHg (one‐way ANOVA with Tukey post hoc test, n = 4, * P < 0.05, ** P < 0.01, ns: no significant difference). (C–E) HP (200 mmHg, 60 min) resulted in phosphorylation of glycogen synthase kinase (GSK)‐3α (Ser21) and in dephosphorylation of p38 (Thr180/Tyr182); unpaired t‐test, n = 4, * P < 0.05, ** P < 0.01, *** P < 0.001).

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  Figure 4. HP resulted in suppression of mRNA expression related to tissue healing factors, such as extracellular matrix, matrix metalloproteinases, and inflammatory cytokines in human cardiac fibroblasts. HP (200 mmHg, 8 h) inhibited mRNA expressions of COL1A1, CO3A1, matrix metalloproteinase (MMP)‐1, MMP‐9, interleukin (IL)‐6, and tumor necrosis factor alpha (TNF‐α) in human cardiac fibroblasts (HCFs); unpaired t‐test, n = 4, * P < 0.05, ** P < 0.01, *** P < 0.001).

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  Figure 5. HP inhibited differentiation of fibroblasts into myofibroblasts and inhibited contractile function in HCFs. (A) Quantitative mRNA expression of alpha‐actin‐2 (ACTA2) after 3 h of HP (white) and 8 h of HP (gray); unpaired t‐test, n = 4, *** P < 0.001, ns: no significant difference). HP inhibited mRNA expression of ACTA2 after 8 h of HP, but not after 3 h. (B) Quantitative mRNA expression of ACTA2 after 8 h of AP (white) or HP (gray); unpaired t‐test, n = 4, *** P < 0.001, ns: no significant difference). HP of 200 mmHg inhibited mRNA expression of ACTA2, but not HP of 100 mmHg. (C) Representative images of western blot (upper) and normalized protein expression of α‐smooth muscle actin (α‐SMA) at 16 h determined by densitometry (lower); unpaired t‐test, n = 4, ** P < 0.01). (D) Representative immunocytochemistry of α‐SMA in HCFs (DAPI, 4′,6‐diamidino‐2‐phenylindole); Magnification = × 40; bar = 50 μm. (E) Effects of AP and HP on the ability of HCF cells to contract floating collagen gels. HP for 16 h inhibited contraction of the collagen matrix in three‐dimensional HCF culture. Representative photographs of floating gels (E) and relative area of collagen matrix (AP and HP; (F));…

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  Figure 6. Apelin receptor antagonist suppressed HP effects. (A) Representative images of western blot analysis of pAkt, GSK‐3α and p38 in the presence of LY294002 or ML221. (B–G) Both ML221 (50 μmol/L), an apelin receptor antagonist, and LY294002 (10 μmol/L), a PI3K‐Akt inhibitor, suppressed HP‐induced phosphorylation of Akt (B) and GSK‐3α (C). (one‐way ANOVA with Tukey post hoc test, n = 4, * P < 0.05, ** P < 0.01, *** P < 0.001). ML221 did not suppress HP‐induced dephosphorylation of p38 (D). ML221 (50 μmol/L) suppressed HP‐induced mRNA transcription of ACTA2 (E), COL1A1 (F), and IL‐6 (G); unpaired t‐test, n = 4, ns: no significant difference).

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  Figure 7. Proposed mechanism of effects of HP on HCFs. HP acts on apelin receptor to suppress the tissue healing response via the PI3K/Akt pathway.