Treatment of Glucocorticoids Inhibited Early Immune Responses and Impaired Cardiac Repair in Adult Zebrafish

<div><p>Myocardial injury, such as myocardial infarction (MI), can lead to drastic heart damage. Zebrafish have the extraordinary ability to regenerate their heart after a severe injury. Upon ventricle resection, fibrin clots seal the wound and serve as a matrix for recruiting myeloid-derived phagocytes. Accumulated neutrophils and macrophages not only reduce the risk of infection but also secrete cytokines and growth factors to promote tissue repair. However, the underlying cellular and molecular mechanisms for how immune responses are regulated during the early stages of cardiac repair are still unclear. We investigated the role and programming of early immune responses during zebrafish heart regeneration. We found that zebrafish treated with an anti-inflammatory glucocorticoid had significantly reduced heart regenerative capacities, consistent with findings in other higher vertebrates. Moreover, inhibiting the inflammatory response led to excessive collagen deposition. A microarray approach was used to assess the differential expression profiles between zebrafish hearts with normal or impaired healing. Combining cytokine profiling and immune-staining, our data revealed that impaired heart regeneration could be due to reduced phagocyte recruitment, leading to diminished angiogenesis and cell proliferation post-cardiac injury. Despite their robust regenerative ability, our study revealed that glucocorticoid treatment could effectively hinder cardiac repair in adult zebrafish by interfering with the inflammatory response. Our findings may help to clarify the initiation of cardiac repair, which could be used to develop a therapeutic intervention that may enhance cardiac repair in humans to compensate for the loss of cardiomyocytes after an MI.</p></div

Treatment with beclomethasone caused impaired cardiac repair in zebrafish.

<p>Objects were sacrificed at 30 days post ventricular resection; heart sections were stained with aniline blue to distinguish scar tissue from normal tissue. Blue: scar tissue; Orange: normal tissue. (A, A’) In the vehicle control group, the zebrafish regenerated the injured hearts perfectly 1 month after injury. Only a few scar tissue (arrow) was detected in the wound (n = 17). (B, B’) Exposure to beclomethasone (0.25 µM) hindered the cardiac repair process. A large amount of scar tissue (arrow) was found in the wound instead of renewed cardiomyocytes. Meanwhile, obvious blood clots remained, which could be detected (arrow head) (n = 14, scale bar = 100 µm). The dashed lines indicate the approximate amputation plane.</p

<i>wnt16</i> was inhibited by beclomethasone in the early phase of zebrafish cardiac repair.

<p>RT-PCR assays revealed the normal injury response of <i>wnt16</i> expression patterns, and confirmed the inhibitory effect of beclomethasone on <i>wnt16</i> mRNA expression in zebrafish heart. We then checked the mRNA expression of <i>wnt16</i> before ventricular resection and at 1 dpa, 3 dpa and 7 dpa by RT-qPCR. (A) Zebrafish <i>wnt16</i> mRNA was elevated at 1 dpa after injury. In contrast, its expression was repressed by beclomethasone treatment at 1 dpa. (B) During zebrafish cardiac repair, <i>wnt16</i> transcript was highly elevated at 1 dpa. Its level was diminished by 3 dpa, and gradually reduced to the basal level by 7 dpa. (n = 3) The data represent the mean±SEM. *indicates p<0.05. N.S. indicates no significant difference compared to no-injury control.</p

Cell proliferation was hindered in the beclomethasone-treated zebrafish during cardiac repair.

<p>Cell proliferation was examined in early phase of regeneration. (A) At 1 dpa, mRNA expression of proliferation marker gene, <i>pcna,</i> was reduced after beclomethasone treatment (40.3% reduction). In contract, the expression of the pre-cardiac gene, <i>nkx2.5</i>, showed no significant difference between the two groups. The data represent the mean±SEM. *indicates p<0.05. N.S. indicates no significant difference. (B, B’) In the control animals, numerous proliferating BrdU⁺ cells could be detected in the wound (n = 6). The dashed lines indicate the approximate amputation plane. (C, C’) The beclomethasone-treated animals showed little cell proliferation activities (n = 7, scale bar = 50 µm). (D, D’) In control groups, GATA4⁺ regenerating cells appeared near the injury site at 7 dpa. (arrow, n = 5) (E, E’) Beclomethasone treatment significantly reduced the GATA4⁺ regenerating cells during cardiac repair (arrow, n = 5, scale bar = 100 µm). The dashed lines indicate the approximate amputation plane.</p

Angiogenesis was hindered in the beclomethasone-treated zebrafish during cardiac repair.

<p>We checked pro-angiogenic genes of drug-treated objects at early regeneration and then examined neo-vascularization at 7 dpa. (A) At 1 dpa, mRNA expression of pro-angiogenic genes was reduced after beclomethasone treatment with a 43.7% reduction for <i>fgfr1a</i> and 41% for <i>vegfaa</i>. The data represent the mean±SEM. *indicates p<0.05. (B, B’) At 7 dpa, there were blood vessels appearing in the clotted areas of the control animals. (C, C’) The beclomethasone-treated animals showed little formation of new blood vessels. The yellow dashed lines indicate the approximate amputation plane; the white dashed lines indicate the outline of the apex. Scale bar = 100 µm.</p

Expression analyses of PRSS23 in human cell lines.

<p>Expression levels of PRSS23 as well as ERα were analyzed in eight different cell lines: MCF-7, BT-474, Hs.578t, MDA-MB-231, T-47D (all breast cancer), MCF-10A (mammary epithelial), RL95-2 (endometrial cancer), and Ca-SKi (cervical cancer) cell lines. <b>A.</b> Immunoblot analysis showed protein expression level of ERα in these human cell lines. <b>B.</b> qRT-PCR analysis showed relative gene expression of PRSS23 mRNA level. <b>C.</b> Immunoblot analysis showed protein expression level of PRSS23 and GAPDH in these human cell lines. The cell lysate was loaded 20 µg protein for each well in immunoblot anaylsis. qRT-PCR was performed in duplicate.</p

PRSS23 knockdown reduced estrogen-driven MCF-7 cell proliferation.

<p><b>A.</b> The PRSS23 knockdown efficacy in MCF7 cells treated with nonspecific control (NSC) or PRSS23-specific RNAi was validated by immunoblotting. GAPDH was used as the loading control. The bar chart shows the normalized protein level of PRSS23 in NSC and PRSS23 RNAi cells. <b>B.</b> The tumor sphere formation abilities of cells were evaluated in the soft-agar tumor formation assay in the presence or absence of PRSS23 RNAi. The upper panel shows a representative picture of tumor sphere formation in 0.4% soft-agar (scale bar is 200 µm). The bar chart shows that normalized diameter of examined tumors (n≥50). The results are the average of two individual experiments. <b>C.</b> After culturing in phenol-red-free medium containing 0.5% CDS-FBS for 48 h, the cells were stimulated with 20% CDS-FBS and 1 nM E₂ or 25 ppm ethanol for 24 h. The table shows the DNA distribution profile of the examined cells. Each value is the average count of the cells in three individual experiments. * <i>p</i><0.05 and ** <i>p</i><0.01 by the Mann-Whitney U test.</p

E₂-activated ERα enhances PRSS23 expression in MCF-7 cells.

<p><b>A.</b> MCF-7 cells were treated with 1 nM E₂, 25 ppm ethanol, 5 µM Tam, and 0.5% dimethyl sulfoxide (DMSO) in phenol-red-free culture medium containing 10% CDS-FBS for 24 h. The bar plots depicted the results of time-lapse profiling of PRSS23 mRNA levels at 6, 12, and 24 h. All experiments were performed in triplicate. The bars represent relative expression levels of PRSS23 after treatment, which was normalized to the level of 6 h-treated cells (mean ± S.E.M.). <b>B.</b> MDA-MB-231 cells were treated with 1 nM E₂ in phenol-red-free culture medium containing 10% CDS-FBS for 24 h. Expression of PRSS23 (upper panel) and pS2 (lower panel) was evaluated by qRT-PCR at 0, 6, 12, and 24 h. The bars represented the gene expression levels of PRSS23 after treatment, which was normalized to the level of untreated cells (mean ± S.E.M.).</p

Expression of ERα and PRSS23 in human breast carcinoma.

<p>Immunohistochemical analysis revealed expression level of ERα (<b>A</b>, <b>B</b>, <b>C</b>, <b>D</b>, <b>E</b>, <b>F</b>) and the corresponding PRSS23 expression of the same sample (<b>G</b>, <b>H</b>, <b>I</b>, <b>J</b>, <b>K</b>, <b>L</b>) in 6 different breast cancer specimens. The scale bar is 50 µm.</p