HE4 Predicts Progressive Fibrosis and Cardiovascular Events in Patients With Dilated Cardiomyopathy

Figures

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  Figure 1. Circulating levels of HE4 (human epididymis protein 4) were associated with the degree of cardiac fibrosis in patients with dilated cardiomyopathy. A, Representative microscopic images of cardiac tissue biopsy stained with hematoxylin and eosin (H&E) and Masson trichrome staining (MTS). B, The collagen area fraction in the high and low HE4 groups was compared using Mann‐Whitney U test. C, The correlation between ratio of collagen area fraction/serum HE4. Bar=100 μm.

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  Figure 2. Elevated levels of serum HE4 (human epididymis protein 4) were associated with a high risk of future cardiovascular events. Kaplan‐Meier analysis for the probability of primary end point (A) and secondary end point (B) in patients with high and low HE4 levels. Primary end point: composite of all‐cause death, left ventricular assist device (LVAD) implantation, and hospitalization for heart failure events. Secondary end point: composite of all‐cause death and LVAD implantation.

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  Figure 3. Cell‐secreted HE4 (human epididymis protein 4) induced fibroblast activation and extracellular matrix deposition in cardiac fibroblasts. A, Experimental scheme for HE4 overexpression and transfer to cardiac fibroblast. B, Western blotting (WB) for type Ⅰ collagen in whole cell lysate of cardiac fibroblast stimulated by culture medium of human embryonic kidney 293T (HEK293T) cells. GAPDH was used as an internal control. C, Quantification of WB analysis. D, Quantitative reverse transcription–polymerase chain reaction analysis in cardiac fibroblast cultured with supernatant of HEK293T cells. The measurements were standardized to expression of the GAPDH. E, Overlay of images of cells stained for 4′,6‐diamidino‐2‐phenylindole (DAPI) (blue), F‐actin (green), and α‐smooth muscle actin (αSMA) (red). F, Quantification of αSMA‐positive cells as percentage of all cells. n=2 for control, n=2 for HE4. Unpaired t tests with Welch correction were used to compare groups. Col1a1 indicates collagen 1a1; col3a1, collagen 3a1; FGF2, fibroblast growth factor 2; PAI‐1, plasminogen activator inhibitor‐1; SM22, smooth muscle protein 22; and TGF‐β1, transforming growth factor‐β1.

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  Figure 4. HE4 (human epididymis protein 4) facilitated cardiac fibroblast activation through extracellular signal‐regulated kinase (ERK) signaling. A, Western blotting (WB) for ERK in cardiac fibroblasts treated with human embryonic kidney 293T (HEK293T) culture medium. B, Quantification of phosphorylated ERK (pERK)/total ERK WB analysis. C, WB for type Ⅰ collagen in whole cell lysate of cardiac fibroblast stimulated by culture medium of HEK293T cells, and U0126 or dimethyl sulfoxide (DMSO). GAPDH was used as an internal control. D, Quantification of WB analysis. E, WB for ERK in whole cell lysate of cardiac fibroblast stimulated by culture medium of HEK293T cells, and anti‐HE4 antibody or IgG. GAPDH was used as an internal control. F, Quantification of WB analysis. G, Overlay of images of fibroblast cultured with supernatant of HEK293T cells, and anti‐HE4 antibody or IgG stained for 4′,6‐diamidino‐2‐phenylindole (DAPI) (blue), F‐actin (green), and α‐smooth muscle actin (αSMA) (red). H, Quantification of αSMA‐positive cells as percentage of all cells. n=3 for control, n=3 for HE4. Unpaired t tests with Welch correction and multiple comparison correction (Bonferroni method) were used to…

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  Figure 5. Vicious cycle of cardiorenal linkage via high HE4 (human epididymis protein 4). In patients with dilated cardiomyopathy (DCM), HE4 expression is elevated in kidney, and HE4 was secreted into the serum. The circulating HE4 introduced fibroblast activation accompanied by phosphorylation of extracellular signal‐regulated kinase (ERK). These were associated with cardiac remodeling and future adverse cardiovascular events.