Objective Growth differentiation factor 15(GDF-15) is a member of Transforming growth factor-β superfamily which is one of the most important profibrotic protein released during Inflammation. It has been suggested that GDF-15 seems to be related to the remodelling processes by anti-hypertrophic in cardiomyocytes. Cardiac fibrosis is one part of the important pathology mechanisms during remodelling. It was still unknown whether GDF-15 could influence remodelling by modulating cardiac fibroblast proliferation and extracellular matrix (ECM) metabolism. Therefore, our study aims to investigate the expression and effects of GDF-15 in the fibroblasts.
Methods Primary myocardial fibroblasts were isolated and cultured from neonatal rats and divided into six groups subjected to different conditions: 10−6mol/l Endothelin-1 (ET-1), 10−5mol/l norepinephrine (NE), 3 ng/ml TGF-β1, 5 pg/ml rhGDF-15, 150 pg/ml rhGDF-15 and no stimuli. The expression of GDF-15 mRNA was detected by Realtime-PCR and the expression of GDF-15 protein was measured by Enzyme-linked Immunosorbent Assay (ELISA). The proliferation of fibroblast induced by rhGDF-15 was measured by 3-(4, 5-dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide (MTT) and Flow cytometry; the migration of fibroblast was shown by Scarification test. The level of phosphor- ERK was determined.
Results GDF-15 is upregulated in ET-1, NE and transforming growth factor (TGF)β1 group; the GDF-15 mRNA is respectively 1.21±0.03, 1.84±0.09, 1.95±0.39 folds of control group (respectively p<0.05, p<0.05, p<0.01); and protein in the supernatant is respectively 3.27±0.81 pg/ml, 3.55±0.20 pg/ml and 3.75±0.70 pg/ml, vs 0.41±0.17 pg/ml of control group (respectively p<0.05, p<0.001, p<0.01). rhGDF-15 (150 pg/ml) increased the OD value as compared to control group (0.56±0.03 vs 0.46±0.02, p<0.05), but 5 pg/ml rhGDF-15 did not (0.43±0.01 vs 0.46±0.02, p>0.05). rhGDF-15 (150 pg/ml) could also raise the cell in S phase nearly three times more than control group (9.62±1.17% vs 3.80±0.78%, p<0.01). The scarification test showed that rhGDF-15 could enhance the migration of fibroblast. The ERK was activated after treatment with rhGDF-15.
Conclusion GDF-15 is upregulated by ET-1, norepinephrine and TGF-β1 in myocardial fibroblasts. rhGDF-15 (150 pg/ml) could enhance the proliferation and migration of fibroblast, which may participate in the progression of myocardial fibrosis and thus was associated with ERK activation. Abbreviations: Growth differentiation factor 15(GDF-15), Extra cellular matrix (ECM), Endothelin-1(ET-1), norepinephrine (NE), Enzyme-linked Immunosorbent Assay (ELISA), 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide (MTT), Transforming growth factor (TGF), Extracellular Signal-Regulated Kinase (ERK).
- Growth differentiation factor 15
- cardiac fibroblasts