Augmented sphingosine 1 phosphate receptor-1 signaling in cardiac fibroblasts induces cardiac hypertrophy and fibrosis through angiotensin II and interleukin-6

<div><p>Background: Cardiac fibroblasts, together with cardiomyocytes, occupy the majority of cells in the myocardium and are involved in myocardial remodeling. The lysophospholipid mediator sphigosine-1-phosphate (S1P) regulates functions of cardiovascular cells through multiple receptors including S1PR1–S1PR3. S1PR1 but not other S1P receptors was upregulated in angiotensin II-induced hypertrophic hearts. Therefore, we investigated a role of S1PR1 in fibroblasts for cardiac remodeling by employing transgenic mice that overexpressed S1PR1 under the control of α-smooth muscle actin promoter. In S1PR1-transgenic mouse heart, fibroblasts and/or myofibroblasts were hyperplastic, and those cells as well as vascular smooth muscle cells overexpressed S1PR1. Transgenic mice developed bi-ventricular hypertrophy by 12-week-old and diffuse interstitial fibrosis by 24-week-old without hemodynamic stress. Cardiac remodeling in transgenic mice was associated with greater ERK phosphorylation, upregulation of fetal genes, and systolic dysfunction. Transgenic mouse heart showed increased mRNA expression of angiotensin-converting enzyme and interleukin-6 (IL-6). Isolated fibroblasts from transgenic mice exhibited enhanced generation of angiotensin II, which in turn stimulated IL-6 release. Either an AT1 blocker or angiotensin-converting enzyme inhibitor prevented development of cardiac hypertrophy and fibrosis, systolic dysfunction and increased IL-6 expression in transgenic mice. Finally, administration of anti-IL-6 antibody abolished an increase in tyrosine phosphorylation of STAT3, a major signaling molecule downstream of IL-6, in the transgenic mouse heart and prevented development of cardiac hypertrophy in transgenic mice. These results demonstrate a promoting role of S1PR1 in cardiac fibroblasts for cardiac remodeling, in which angiotensin II—AT1 and IL-6 are involved.</p></div

S1PR1 is expressed in fibroblasts in heart of TG mice.

<p>Double immunofluorescence staining of ventricular sections from 8- (A, B), 15- (C, D), and 24-week-old (E, F) WT and TG mice. (A, C and E) Anti-αSMA and anti-S1PR1 staining. In both WT and TG mice, S1PR1 is diffusely expressed in αSMA-negative cells. However, some scattered and solitary S1PR1- and αSMA-double positive apparently non-vascular wall cells (arrowheads) are observed in TG mice but not WT mice, and these double positive non-vascular wall cells increase with aging in TG mice. The arrows in C denote S1PR1- and αSMA-double positive vascular wall cells in WT and TG mice. (B, D and F) Anti-S1PR1 and anti-CD31 staining. In both WT and TG mice, S1PR1 is diffusely expressed in CD31-positive vascular endothelial cells. However, some S1PR1-positive cells are CD31-negative (arrowheads) and these S1PR1-positive and CD31-negative cells increase with aging in TG mice. (G) Quantified data of scattered and solitary αSMA- and S1PR1-positive cells. HF, high power field. **, p<0.01. (H) Anti-EGFP and anti-S1PR1 double immunofluorescence staining of ventricular sections from 24-week-old WT and S1PR1-TG mice with the ColI α2-EGFP background. S1PR1 is overexpressed in EGFP-positive cells (arrowheads) in TG mice but not WT mice.</p

Cardiac fibroblasts overexpressing S1PR1 release a greater amount of Ang II.

<p>(A) Double immunofluorescence staining of fibroblasts isolated from 24-week-old WT and TG mice, using anti-αSMA and anti-S1PR1 antibodies. S1PR1 is more abundantly expressed in fibroblasts from TG mice compared with WT mice. (B) Ang II peptide levels in the conditioned media of WT and TG fibroblasts cultured in the presence and absence of S1P (100 nmol/L) without serum for 24 h. n = 6 per group. (C) Real-time PCR analysis of mRNAs of ACE, angiotensinogen, AT₁ and AT₂ in WT and TG fibroblasts. In B and C, *, p<0.05 and **, p<0.01.</p

Transgenic overexpression of S1PR1 induces pathological cardiac hypertrophy.

<p>(A) Gross views and HE-stained cross-sections of the hearts from WT and TG mice. (B) The HW/BW ratio in 8- to 32-week-old WT and TG mice. n = 7 mice per group. *, p<0.05 and **, p<0.01. (C) Silver-staining of myocardial sections of 12-week-old WT and TG mice (right). Quantified data of mean cross sectional area of cardiomyocytes (left). (D) Images of representative isolated cardiomyocytes from WT and TG mice at 12 weeks (left). Quantification of cell areas of isolated cardiomyocytes (right). (E) Azan staining of myocardial sections of 24-week-old WT and TG mice (left). Quantified data of fibrotic areas in Azan stained sections of the hearts (right). (F) Northern blot analysis of mRNAs of natriuretic peptides ANP and BNP, fetal contractile proteins βMHC and αSKA and type I collagen α1 chain.</p

An AT1 blocker inhibits ventricular hypertrophy and fibrosis in TG mice.

<p>Administration of the AT1 blocker candesartan (CDS) was started at 6-weeks and continued for 18 weeks. (A) Gross views and HE-stained sections of the hearts from WT, non-treated TG, and CDS-treated TG mice at 24-week-old (left panel), and the HW/BW ratio in each mouse group (right panel). n = 6 mice per group. (B) Azan staining of myocardial sections of WT, non-treated TG, and CDS-treated TG mice at 24-weeks (upper panel) and quantified data (lower panel). n = 6 per group. (C) Silver-staining of myocardial sections of WT, non-treated TG, and CDS-treated TG mice at 24-weeks (upper panel) and quantified data (lower panel). n = 6 per group. (D) %FS in WT, non-treated TG, and CDS-treated TG mice at 24-weeks as evaluated with echocardiography. N = 6 per group. (E) Effects of CDS on increased mRNA expression of ANP, BNP and βMHC in the heart of TG mice at 24-weeks. N = 5 mice per group. (F) Phosphorylation of ERK in heart. The extracts of the hearts from WT, non-treated TG, and CDS-treated TG mice at 24-weeks was analyzed by Western blotting using anti-phospho ERK (p-ERK) antibody. n = 5 mice per group. (G) Effects of CDS on mRNA expression of IL-6 in the heart of TG mice at 24-weeks. n = 5 mice per group. In A-G, *, p<0.05, and **, p<0.01.</p

S1P3-mediated cardiac fibrosis in sphingosine kinase 1 transgenic mice involves reactive oxygen species

金沢大学医薬保健研究域医学系Aims Sphingosine kinase 1 (SPHK1), its product sphingosine-1-phosphate (S1P), and S1P receptor subtypes have been suggested to play protective roles for cardiomyocytes in animal models of ischaemic preconditioning and cardiac ischaemia/reperfusion injury. To get more insight into roles for SPHK1 in vivo, we have generated SPHK1-transgenic (TG) mice and analysed the cardiac phenotype.Methods and results SPHK1-TG mice overexpressed SPHK1 in diverse tissues, with a nearly 20-fold increase in enzymatic activity. The TG mice grew normally with normal blood chemistry, cell counts, heart rate, and blood pressure. Unexpectedly, TG mice with high but not low expression levels of SPHK1 developed progressive myocardial degeneration and fibrosis, with upregulation of embryonic genes, elevated RhoA and Rac1 activity, stimulation of Smad3 phosphorylation, and increased levels of oxidative stress markers. Treatment of juvenile TG mice with pitavastatin, an established inhibitor of the Rho family G proteins, or deletion of S1P3, a major myocardial S1P receptor subtype that couples to Rho GTPases and transactivates Smad signalling, both inhibited cardiac fibrosis with concomitant inhibition of SPHK1-dependent Smad-3 phosphorylation. In addition, the anti-oxidant N-2-mercaptopropyonylglycine, which reduces reactive oxygen species (ROS), also inhibited cardiac fibrosis. In in vivo ischaemia/reperfusion injury, the size of myocardial infarct was 30 decreased in SPHK1-TG mice compared with wild-type mice.Conclusion These results suggest that chronic activation of SPHK1-S1P signalling results in both pathological cardiac remodelling through ROS mediated by S1P3 and favourable cardioprotective effects