Human cardiac fibroblasts express B-type natriuretic peptide: fluvastatin ameliorates its up-regulation by interleukin-1alpha, tumour necrosis factor-alpha and transforming growth factor-beta

Abstract

B-type natriuretic peptide (BNP) is a cardiac hormone, which plays a major role in body fluid and cardiovascular homeostasis. Produced by cardiac ventricles, its expression is highly regulated by various mediators. Canine cardiac fibroblasts have been identified as a source of BNP. Cardiac fibroblasts are key regulators of myocardial structure and function. We treated cultured human adult cardiac fibroblasts (HACF) with 2000 U/ml tumour necrosis factor-alpha (TNF-alpha), 200 U/ml interleukin-1alpha (IL-1alpha) or 50 ng/ml transforming growth factor-beta (TGF-beta) in the presence or absence of 500 nM fluvastatin. N-terminal pro-BNP (Nt-proBNP) concentration was determined by a competitive enzyme immunoassay. RealTime polymerase chain reaction (real-time PCR) was performed to investigate changes in BNP mRNA expression. Nt-proBNP peptide was present in the conditioned media of HACF and incubation with fluvastatin significantly reduced Nt-proBNP peptide levels. Treatment of HACF with TNF-alpha, IL-1alpha or TGF-beta significantly increased Nt-proBNP levels compared with untreated cells. This effect was completely abolished in the presence of fluvastatin. Real-time PCR analysis confirmed these changes at the level of mRNA expression. Our data suggest that cardiac fibroblasts are a potential source of BNP in the human heart. Pro-inflammatory cytokines, associated with ventricular dysfunction and cardiac fibrosis, seem to be major inducers of BNP production in cardiac fibroblasts. This effect can be reverted by a statin. Based on our data, we speculate that elevated plasma BNP levels might not only reflect increased myocardial stretch but also inflammatory and remodelling processes. A possible benefit of statin-induced reduction in BNP production requires further studies.

Figure 1

Effect of IL-1α, TNF-α, TGF-β, IL-6, IL-11, OSM and LIF on proBNP production in HACF and HACM. Confluent monolayers of HACF (open bars) were incubated for 24 hrs with or without 2, 20 and 200 U/ml IL-1α; 20, 200 and 2000 U/ml TNF-α; or 0.5, 5 and 50 ng/ml TGF-β, respectively (panel A). Confluent monolayers of HACM (hatched bars) were incubated for 24 hrs with or without 200 U/ml IL-1α; or 2000 U/ml TNF-α (panel A). Confluent monolayers of HACF were incubated for 24 hrs with or without 100 ng/ml IL-6; 100 ng/ml IL-11; 10,000 U/ml LIF; or 100 ng/ml OSM, respectively (panel B). Nt-proBNP was determined in conditioned media using a specific ELISA and values are given in fmol/ml and represent mean values ± S.D. of three independent determinations. Experiments were performed three times with cells isolated from three different donors. A representative experiment is shown. ***P < 0.001, **P < 0.005, *P < 0.05 as compared with untreated control cells.

Figure 1

Effect of IL-1α, TNF-α, TGF-β, IL-6, IL-11, OSM and LIF on proBNP production in HACF and HACM. Confluent monolayers of HACF (open bars) were incubated for 24 hrs with or without 2, 20 and 200 U/ml IL-1α; 20, 200 and 2000 U/ml TNF-α; or 0.5, 5 and 50 ng/ml TGF-β, respectively (panel A). Confluent monolayers of HACM (hatched bars) were incubated for 24 hrs with or without 200 U/ml IL-1α; or 2000 U/ml TNF-α (panel A). Confluent monolayers of HACF were incubated for 24 hrs with or without 100 ng/ml IL-6; 100 ng/ml IL-11; 10,000 U/ml LIF; or 100 ng/ml OSM, respectively (panel B). Nt-proBNP was determined in conditioned media using a specific ELISA and values are given in fmol/ml and represent mean values ± S.D. of three independent determinations. Experiments were performed three times with cells isolated from three different donors. A representative experiment is shown. ***P < 0.001, **P < 0.005, *P < 0.05 as compared with untreated control cells.

Figure 2

Effect of TNF-α, IL-1α, TGF-β and fluvastatin on proBNP production in HACF. Confluent monolayers of HACF were incubated for 24 hrs without (panel A) or with 200 U/ml IL-1α (panel B), 2000 U/ml TNF-α (panel C) or 50 ng/ml TGF-β (panel D), respectively, in the absence (open bars) or presence of 500 nM, 100 nM, 10 nM or 1 nM fluvastatin without or with 100 μM mevalonate or 10 μM GGPP (hatched bars). In panel E, confluent monolayers of HACF were pre-treated with medium (open bars) or 5 μM Y27632 (hatched bars) for 1 hr and then incubated for 24 hrs without or with 200 U/ml IL-1α, 2000 U/ml TNF-α or 50 ng/ml TGF-β, respectively. Nt-proBNP was determined in conditioned media using a specific ELISA and values are given in fmol/ml and represent mean values ± S.D. of three independent determinations. Experiments were performed 3 times with cells isolated from three different donors. A representative experiment is shown. **P < 0.001, *P < 0.05 as compared with untreated control cells; P¤ < 0.001 as compared with cells incubated without fluvastatin or Y27632.

Figure 2

Effect of TNF-α, IL-1α, TGF-β and fluvastatin on proBNP production in HACF. Confluent monolayers of HACF were incubated for 24 hrs without (panel A) or with 200 U/ml IL-1α (panel B), 2000 U/ml TNF-α (panel C) or 50 ng/ml TGF-β (panel D), respectively, in the absence (open bars) or presence of 500 nM, 100 nM, 10 nM or 1 nM fluvastatin without or with 100 μM mevalonate or 10 μM GGPP (hatched bars). In panel E, confluent monolayers of HACF were pre-treated with medium (open bars) or 5 μM Y27632 (hatched bars) for 1 hr and then incubated for 24 hrs without or with 200 U/ml IL-1α, 2000 U/ml TNF-α or 50 ng/ml TGF-β, respectively. Nt-proBNP was determined in conditioned media using a specific ELISA and values are given in fmol/ml and represent mean values ± S.D. of three independent determinations. Experiments were performed 3 times with cells isolated from three different donors. A representative experiment is shown. **P < 0.001, *P < 0.05 as compared with untreated control cells; P¤ < 0.001 as compared with cells incubated without fluvastatin or Y27632.

Figure 2

Effect of TNF-α, IL-1α, TGF-β and fluvastatin on proBNP production in HACF. Confluent monolayers of HACF were incubated for 24 hrs without (panel A) or with 200 U/ml IL-1α (panel B), 2000 U/ml TNF-α (panel C) or 50 ng/ml TGF-β (panel D), respectively, in the absence (open bars) or presence of 500 nM, 100 nM, 10 nM or 1 nM fluvastatin without or with 100 μM mevalonate or 10 μM GGPP (hatched bars). In panel E, confluent monolayers of HACF were pre-treated with medium (open bars) or 5 μM Y27632 (hatched bars) for 1 hr and then incubated for 24 hrs without or with 200 U/ml IL-1α, 2000 U/ml TNF-α or 50 ng/ml TGF-β, respectively. Nt-proBNP was determined in conditioned media using a specific ELISA and values are given in fmol/ml and represent mean values ± S.D. of three independent determinations. Experiments were performed 3 times with cells isolated from three different donors. A representative experiment is shown. **P < 0.001, *P < 0.05 as compared with untreated control cells; P¤ < 0.001 as compared with cells incubated without fluvastatin or Y27632.

Figure 2

Effect of TNF-α, IL-1α, TGF-β and fluvastatin on proBNP production in HACF. Confluent monolayers of HACF were incubated for 24 hrs without (panel A) or with 200 U/ml IL-1α (panel B), 2000 U/ml TNF-α (panel C) or 50 ng/ml TGF-β (panel D), respectively, in the absence (open bars) or presence of 500 nM, 100 nM, 10 nM or 1 nM fluvastatin without or with 100 μM mevalonate or 10 μM GGPP (hatched bars). In panel E, confluent monolayers of HACF were pre-treated with medium (open bars) or 5 μM Y27632 (hatched bars) for 1 hr and then incubated for 24 hrs without or with 200 U/ml IL-1α, 2000 U/ml TNF-α or 50 ng/ml TGF-β, respectively. Nt-proBNP was determined in conditioned media using a specific ELISA and values are given in fmol/ml and represent mean values ± S.D. of three independent determinations. Experiments were performed 3 times with cells isolated from three different donors. A representative experiment is shown. **P < 0.001, *P < 0.05 as compared with untreated control cells; P¤ < 0.001 as compared with cells incubated without fluvastatin or Y27632.

Figure 2

Effect of TNF-α, IL-1α, TGF-β and fluvastatin on proBNP production in HACF. Confluent monolayers of HACF were incubated for 24 hrs without (panel A) or with 200 U/ml IL-1α (panel B), 2000 U/ml TNF-α (panel C) or 50 ng/ml TGF-β (panel D), respectively, in the absence (open bars) or presence of 500 nM, 100 nM, 10 nM or 1 nM fluvastatin without or with 100 μM mevalonate or 10 μM GGPP (hatched bars). In panel E, confluent monolayers of HACF were pre-treated with medium (open bars) or 5 μM Y27632 (hatched bars) for 1 hr and then incubated for 24 hrs without or with 200 U/ml IL-1α, 2000 U/ml TNF-α or 50 ng/ml TGF-β, respectively. Nt-proBNP was determined in conditioned media using a specific ELISA and values are given in fmol/ml and represent mean values ± S.D. of three independent determinations. Experiments were performed 3 times with cells isolated from three different donors. A representative experiment is shown. **P < 0.001, *P < 0.05 as compared with untreated control cells; P¤ < 0.001 as compared with cells incubated without fluvastatin or Y27632.