Masakatsu Takanashi

Endothelin-1–Induced Cardiac Hypertrophy Is Inhibited by Activation of Peroxisome Proliferator–Activated Receptor-α Partly Via Blockade of c-Jun NH2-Terminal Kinase Pathway

Background—Peroxisome proliferator-activated receptor-&agr; (PPAR-&agr;) is a lipid-activated nuclear receptor that negatively regulates the vascular inflammatory gene response by interacting with transcription factors, nuclear factor-&kgr;B, and AP-1. However, the roles of PPAR-&agr; activators in endothelin (ET)-1–induced cardiac hypertrophy are not yet known. Methods and Results—First, in cultured neonatal rat cardiomyocytes, a PPAR-&agr; activator, fenofibrate (10 &mgr;mol/L), and PPAR-&agr; overexpression markedly inhibited the ET-1–induced increase in protein synthesis. Second, fenofibrate markedly inhibited ET-1–induced increase in c-Jun gene expression and phosphorylation of c-Jun and JNK. These results suggest that this PPAR-&agr; activator interferes with the formation and activation of AP-1 protein induced by ET-1 in cardiomyocytes. Third, fenofibrate significantly inhibited the increase of ET-1 mRNA level by ET-1, which was also confirmed by luciferase assay. Electrophoretic mobility shift assay revealed that fenofibrate significantly decreased the ET-1–stimulated or phorbol 12-myristate 13-acetate–stimulated AP-1 DNA binding activity, and the nuclear extract probe complex was supershifted by anti-c-Jun antibody. Fourth, 24 hours after aortic banding (AB) operation, fenofibrate treatment significantly inhibited left ventricular hypertrophy and hypertrophy-related gene expression pattern (ET-1, brain natriuretic peptide, and &bgr;-myosin heavy chain mRNA) in AB rats. Conclusions—These results suggest that PPAR-&agr; activation interferes with the signaling pathway of ET-1–induced cardiac hypertrophy through negative regulation of AP-1 binding activity, partly via inhibition of the JNK pathway in cultured cardiomyocytes. We also revealed that fenofibrate treatment inhibited left ventricle hypertrophy and phenotypic changes in cardiac gene expression in AB rats in vivo.

The peroxisome proliferator-activated receptor alpha activator fenofibrate inhibits endothelin-1-induced cardiac fibroblast proliferation.

Endothelin-1 has been known to promote tissue fibrosis. We previously reported in our animal experiments that a peroxisome proliferator-activated receptor α (PPARα) inhibited cardiac fibrosis with suppression of endothelin-1 production, and it was also reported that PPARα activation suppressed the production of c-jun, which is a component of activator protein-1. The objective of this study is to clarify on the in vitro level that PPARα activators inhibited cardiac fibroblast proliferation via their suppressive action on c-jun expression. We investigated the effects of the PPARα activator fenofibrate (10 μM) on DNA synthesis in neonatal rat cardiac fibroblasts by [3H]thymidine incorporation. The [3H]thymidine incorporation in cardiac fibroblasts showed an increase of 1.1-fold by endothelin-1 (10-8 M) stimulation. Fenofibrate treatment showed significant inhibition of [3H]thymidine incorporation in both endothelin-1-stimulated and non-stimulated fibroblasts. Additionally, we also evaluated mRNA expressions of cjun and c-fos in the fibroblasts by the reverse transcriptionpolymerase chain reaction method. Fenofibrate treatment markedly reduced c-jun mRNA expression, whereas it did not affect c-fos mRNA expression. In conclusion, we demonstrated that the PPARα activator fenofibrate inhibited endothelin-1-induced proliferation of cardiac fibroblasts and also inhibited non-stimulated proliferation. This inhibition of proliferation may be caused by up-regulation of p27Kip1 by suppressing c-jun expression.

Establishment of hypoxia inducible factor-1alpha overexpressing cells that produce endothelin-1.

Cardiomyocytes produce endothelin-1. Upregulation of endothelin-1 is induced under hypoxic conditions. It has been reported that tissue hypoxia induces transcriptional factor hypoxia inducible factor-1α (HIF-1α) expression. It has also been reported that cellular hypoxia leads to HIF-1α activation. Previously, we reported that HIF-1α bound to the site in the ET-1 gene promoter region. HIF-1α is a master transcriptional factor that controls transcriptional activation of a number of genes responsive to cellular hypoxia, including endothelin-1, vascular endothelial growth factor, erythropoietin and glycolytic enzymes. We aimed to establish the HIF-1α-overexpressing cardiomyocytes. Because it has been reported that HIF-1α is destroyed by the ubiquitin proteasome pathway under normoxic conditions, we constructed the pointmutated HIF-1α that was exchanged at 564 proline with alanine. The point-mutated HIF-1α is not destroyed by ubiquitin proteasome, because ubiquitin ligase cannot bind to the pointmutated area. This mutated HIF-1α was transfected to rat cardiomyocytes. Using the protein extraction from the mutated HIF- 1α transfected cells under normoxic conditions, western blot methods with anti-HIF-1α antibody showed that the band was detected at 120 kDa, which is of identical size to HIF-1α. In the normoxic conditions of the control cardiomyocyte cells, the band was undetectable. The endothelin-1 mRNA was significantly altered in HIF-1α-overexpressing cells. These results indicate that we have established the cells overexpressing-HIF-1α, and that these cells produce endothelin-1 mRNA.

Effects of endothelin-1 eyedrops on the retina in rats

Eye disorder accompanied with chronic retinal microvascular obstruction, such as diabetic retinopathy, exists in many diseases. However, it is difficult to produce this model experimentally in the animal eye. Endothelin-1 eyedrops were prepared in order to examine whether the eyedrops affect the rat retina and whether we can produce an obstruction model. Endothelin-1 eyedrops diluted by artificial tears in seven stages from 4 × 10−5 M to 4 × 10−11 M were arranged. We administered this solution three times a day in the left eye of male Sprague-Dawley rats. Artificial tears alone were applied to the right eye as a control vehicle. After 2 weeks, rats were sacrificed under anesthesia and the retinal tissues were isolated. As an index to the action of endothelin- 1 eyedrops to the retina, the expressions of endothelin-A (ETA) and endothelin-B (ETB) receptors in the retina were compared in both eyes. Frozen sections of the retina were immunostained to reveal the distribution of the ETA and ETB receptors. We also examined ETA and ETB mRNA expression by quantitative real-time polymerase chain reaction. As a result, the expressions of ETA and ETB receptors are reduced with both immunostaining and the mRNA levels in the left eye, in which endothelin-1 eyedrops were applied at 4 × 10−5 M. It is suggested that endothelin-1 eyedrops affected the retina and the possibility of producing the experimental model of chronic microvascular obstruction in the rat retina.