Katharina Schimmel1, Mira Jung1, Ariana Foinquinos1, Gorka San José2,3, Javier Beaumont2,3, Katharina Bock1, Lea Grote-Levi1, Ke Xiao1, Christian Bär1, Angelika Pfanne1, Annette Just1, Karina Zimmer1, Soeun Ngoy4, Begoña López2,3, Susana Ravassa2,3, Sabine Samolovac1, Heike Janssen-Peters1, Janet Remke1, Kristian Scherf1,5, Seema Dangwal1,5, Maria-Teresa Piccoli1, Felix Kleemiss1, Fabian Philipp Kreutzer1, Franziska Kenneweg1, Julia Leonardy1, Lisa Hobuß1, Laura Santer1, Quoc-Tuan Do6, Robert Geffers7, Jan Hinrich Braesen8, Jessica Schmitz8, Christina Brandenberger9, Dominik N Müller10, Nicola Wilck10,11, Volkhard Kaever12, Heike Bähre12, Sandor Batkai1, Jan Fiedler1, Kevin M Alexander5, Bradley M Wertheim13, Sudeshna Fisch4, Ronglih Liao5,4, Javier Diez2,3,14, Arantxa González2,3, Thomas Thum1,15. 1. Institute of Molecular and Translational Therapeutic Strategies (K.S., M.J., A.F., K.B., L.G.-L., K.X., C. Bär, A.P., A.J., K.Z., S.S., H.J.-P., J.R., K.S., S.D., M.-T.P., F.K., F.P.K., F.K., J.L., L.H., L.S., S.B., J.F., T.T.), Hannover Medical School, Germany. 2. Program of Cardiovascular Diseases, CIMA Universidad de Navarra and IdiSNA, Pamplona, Spain (G.S.J., J.B., B.L., S.R., J.D., A.G.). 3. CIBERCV, Institute of Health Carlos III, Madrid, Spain (G.S.J., J.B., B.L., S.R., J.D., A.G.). 4. Department of Medicine, Divisions of Genetics and Cardiology (S.N., S.F., R.L.), Brigham and Women's Hospital, Harvard Medical School, Boston, MA. 5. Cardiovascular Institute, Stanford University School of Medicine, CA (K.S., S.D., K.M.A., R.L.). 6. Greenpharma SAS, Department of Chemoinformatics, Orléans, France (Q.-T.D.). 7. Helmholtz Centre for Infection Research, Research Group Genome Analytics, Braunschweig, Germany (R.G.). 8. Institute for Pathology, Nephropathology Unit (J.H.B., J.S.), Hannover Medical School, Germany. 9. Institute of Functional and Applied Anatomy (C. Brandenberger), Hannover Medical School, Germany. 10. Experimental and Clinical Research Center, a cooperation of Charité-Universitätsmedizin Berlin and Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Germany (D.N.M., N.W.). 11. Division of Nephrology and Internal Intensive Care Medicine, Charité-Universitätsmedizin Berlin, Germany (N.W.). 12. Research Core Unit Metabolomics, Institute of Pharmacology (V.K., H.B.), Hannover Medical School, Germany. 13. Department of Medicine, Division of Pulmonary and Critical Care Medicine (B.M.W.), Brigham and Women's Hospital, Harvard Medical School, Boston, MA. 14. Department of Cardiology and Cardiac Surgery and Department of Nephrology, Clínica Universidad de Navarra, Pamplona, Spain (J.D.). 15. REBIRTH Center of Translational Regenerative Medicine (T.T.), Hannover Medical School, Germany.
Abstract
BACKGROUND: Myocardial fibrosis is a hallmark of cardiac remodeling and functionally involved in heart failure development, a leading cause of deaths worldwide. Clinically, no therapeutic strategy is available that specifically attenuates maladaptive responses of cardiac fibroblasts, the effector cells of fibrosis in the heart. Therefore, our aim was to develop novel antifibrotic therapeutics based on naturally derived substance library screens for the treatment of cardiac fibrosis. METHODS: Antifibrotic drug candidates were identified by functional screening of 480 chemically diverse natural compounds in primary human cardiac fibroblasts, subsequent validation, and mechanistic in vitro and in vivo studies. Hits were analyzed for dose-dependent inhibition of proliferation of human cardiac fibroblasts, modulation of apoptosis, and extracellular matrix expression. In vitro findings were confirmed in vivo with an angiotensin II-mediated murine model of cardiac fibrosis in both preventive and therapeutic settings, as well as in the Dahl salt-sensitive rat model. To investigate the mechanism underlying the antifibrotic potential of the lead compounds, treatment-dependent changes in the noncoding RNAome in primary human cardiac fibroblasts were analyzed by RNA deep sequencing. RESULTS: High-throughput natural compound library screening identified 15 substances with antiproliferative effects in human cardiac fibroblasts. Using multiple in vitro fibrosis assays and stringent selection algorithms, we identified the steroid bufalin (from Chinese toad venom) and the alkaloid lycorine (from Amaryllidaceae species) to be effective antifibrotic molecules both in vitro and in vivo, leading to improvement in diastolic function in 2 hypertension-dependent rodent models of cardiac fibrosis. Administration at effective doses did not change plasma damage markers or the morphology of kidney and liver, providing the first toxicological safety data. Using next-generation sequencing, we identified the conserved microRNA 671-5p and downstream the antifibrotic selenoprotein P1 as common effectors of the antifibrotic compounds. CONCLUSIONS: We identified the molecules bufalin and lycorine as drug candidates for therapeutic applications in cardiac fibrosis and diastolic dysfunction.
BACKGROUND:Myocardial fibrosis is a hallmark of cardiac remodeling and functionally involved in heart failure development, a leading cause of deaths worldwide. Clinically, no therapeutic strategy is available that specifically attenuates maladaptive responses of cardiac fibroblasts, the effector cells of fibrosis in the heart. Therefore, our aim was to develop novel antifibrotic therapeutics based on naturally derived substance library screens for the treatment of cardiac fibrosis. METHODS: Antifibrotic drug candidates were identified by functional screening of 480 chemically diverse natural compounds in primary human cardiac fibroblasts, subsequent validation, and mechanistic in vitro and in vivo studies. Hits were analyzed for dose-dependent inhibition of proliferation of human cardiac fibroblasts, modulation of apoptosis, and extracellular matrix expression. In vitro findings were confirmed in vivo with an angiotensin II-mediated murine model of cardiac fibrosis in both preventive and therapeutic settings, as well as in the Dahl salt-sensitive rat model. To investigate the mechanism underlying the antifibrotic potential of the lead compounds, treatment-dependent changes in the noncoding RNAome in primary human cardiac fibroblasts were analyzed by RNA deep sequencing. RESULTS: High-throughput natural compound library screening identified 15 substances with antiproliferative effects in human cardiac fibroblasts. Using multiple in vitro fibrosis assays and stringent selection algorithms, we identified the steroid bufalin (from Chinese toad venom) and the alkaloid lycorine (from Amaryllidaceae species) to be effective antifibrotic molecules both in vitro and in vivo, leading to improvement in diastolic function in 2 hypertension-dependent rodent models of cardiac fibrosis. Administration at effective doses did not change plasma damage markers or the morphology of kidney and liver, providing the first toxicological safety data. Using next-generation sequencing, we identified the conserved microRNA 671-5p and downstream the antifibrotic selenoprotein P1 as common effectors of the antifibrotic compounds. CONCLUSIONS: We identified the molecules bufalin and lycorine as drug candidates for therapeutic applications in cardiac fibrosis and diastolic dysfunction.
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