Systemic inhibition of BMP1-3 decreases progression of CCl₄-induced liver fibrosis in rats

Abstract

Liver fibrosis is a progressive pathological process resulting in an accumulation of excess extracellular matrix proteins. We discovered that bone morphogenetic protein 1-3 (BMP1-3), an isoform of the metalloproteinase Bmp1 gene, circulates in the plasma of healthy volunteers and its neutralization decreases the progression of chronic kidney disease in 5/6 nephrectomized rats. Here, we investigated the potential role of BMP1-3 in a chronic liver disease. Rats with carbon tetrachloride (CCl₄)-induced liver fibrosis were treated with monoclonal anti-BMP1-3 antibodies. Treatment with anti-BMP1-3 antibodies dose-dependently lowered the amount of collagen type I, downregulated the expression of Tgfb1, Itgb6, Col1a1, and Acta2 and upregulated the expression of Ctgf, Itgb1, and Dcn. Mehanistically, BMP1-3 inhibition decreased the plasma levels of transforming growth factor beta 1(TGFβ1) by prevention of its activation and lowered the prodecorin production further suppressing the TGFβ1 profibrotic effect. Our results suggest that BMP1-3 inhibitors have significant potential for decreasing the progression of fibrosis in liver cirrhosis.

Keywords:

• Bone morphogenetic protein 1–3
• liver fibrosis
• integrin
• BMP7
• TGFβ1

Acknowledgements

We thank Durdica Car and Mirjana Marija Renic for providing animal care and assisting in rat experiments and Ivancica Bastalic and Lucija Kuc ko for administrative support. We are grateful to Vladimir Trkulja for the data analyses and the graphical presentation of results.

Disclosure statement

The authors declare that they have no conflict of interest.

Notes on contibutors

Lovorka Grgurevic conducts research in the Center for Translational and Clinical Research (CETRI), University of Zagreb School of Medicine, on formulation development and toxicology testing. She explores the structure and function of bone morphogenetic proteins (BMPs) in biological fluids, has discovered circulating osteogenic proteins and associated molecules in the plasma, and then investigated their effectiveness in bone healing and in models of acute and chronic renal failure. She contributed significantly to the discovery of a new carrier for BMPs and tested its efficacy in animal models of bone defects. Dr Grgurevic discovered novel biomarkers for bone repair, breast, and prostate cancer prognosis. She received a new laboratory installation grant from the Croatian Science Foundation and was awarded by the Croatian Academy of Sciences and Arts for scientific achievements.

Igor Erjavec is a postdoctoral fellow at the Laboratory for mineralized tissues and Proteomic center at the Center for Translational and Clinical Research (CETRI), University of Zagreb School of Medicine. Scientific field of his research are animal models, bone remodeling, and regeneration with emphasis on bone morphogenetic proteins and the study of kidney and liver regeneration. Member of Croatian and European Calcified Tissue Society. Co-authored 15 publications in Medline database.

Ivica Grgurevic is an Assistant Professor of Medicine and Board certified Consultant in Internal Medicine, Gastroenterology and Hepatology. He graduated at University of Zagreb School of Medicine and attended postgraduate clinical fellowship in Hepatology at Institute for Liver and Digestive Health, University College London, Royal Free Hospital, UK. Currently works at Department of Gastroenterology, Hepatology and Clinical Nutrition, University hospital Dubrava Zagreb. He was the first person in Croatia to introduce new methods in Hepatology: Quantitative ultrasound elastography (in 2009); Contrast-enchanced ultrasound (2014); and invasive measurement of portal pressure (HVPG) (2015). His principal scientific interests are in the field of Hepatology and Ultrasound. He was awarded the best poster in the category of Liver imaging at United European Gastroenterology Week in Vienna 2016. Member of EASL, EFSUMB, IASGO, and former Secretary general of the Croatian Society of Gastroenterology 2013–2016, currently serves as the Vice-president of the Croatian Society for the Ultrasound in Medicine and Biology.

Ivo Dumic-Cule is now a resident in Radiology at Clinical Hospital Dubrava, Zagreb. For five years he has been working as assistant in Laboratory for mineralized tissues at University of Zagreb School of Medicine, investigating the role of bone morphogenetic proteins in various biological systems.

Jelena Brkljacic is a cell biologist, now working at Xellia R&D. For eight years she has been working in Laboratory for mineralized tissues at University of Zagreb School of Medicine, investigating the activity of bone morphogenetic activity in vitro, focusing on the effect of bone morphogenetic protein 6 (BMP6) on the prevention of heparin induced secondary osteoporosis. She contributed to the production of rhBMP by developing the in vitro assay for BMP activity testing.

Donatella Verbanac works as an Assistant Professor at the School of Medicine University of Zagreb since 2009, leading Department for Intracellular Communication within the Centre for Translational and Clinical Research. In parallel, she participates in several scientific projects, as well as in education activities for the graduate and post-graduate students. She is the Coordinator of translational project ‘Assessment of Microbiota, Inflammatory Markers, Nutritional, and Endocrinological Status in IBD Patients’ funded by Croatian Science Foundation (2014–2018). Her previous experience was within pharmaceutical industies where she worked as Program Leader and Project Manager. For the achievements in the drug development and research projects she has been awarded with PLIVA’s Pharmaceutical Industry and GlaxoSmithKline awards. She has published more than 40 scientific and professional papers. Co-inventor of one patent and coauthor of several chapters in the books, author of few professional books on nutrition and one text-book. She acts also as a reviewer, monitor, and vice-chair for the projects funded by EC, as well as for many distinguished journals in the field of medicinal chemistry and life sciences. She is member of Croatian Biochemical and Molecular Biology Society, Croatian Society of Enteral, and Parenteral Nutrition and British Biochemical Society.

Mario Matijasic is a researcher in the Center for Translational and Clinical Research (CETRI), University of Zagreb School of Medicine, with a decade of experience in anti-inflammatory drug discovery in pharmaceutical industry. Current interests include bone morphogenetic proteins (BMPs), biomarkers, and microbiota research.

Hana Cipcic Paljetak is a researcher in the Center for Translational and Clinical Research (CETRI), University of Zagreb School of Medicine, with almost a decade of experience in drug discovery in pharmaceutical industry in the anti-infective and anti-inflammatory therapeutic areas. Current interests include bone morphogenetic proteins (BMPs), biomarkers, and microbiota research.

Rudjer Novak is a research associate at the Department for Proteomics in the Center for Translational and Clinical Research (CETRI), University of Zagreb School of Medicine. He specializes in the area of mass spectrometry and participates in scientific projects in the area of regenerative medicine and cancer proteomics. His main scientific interest is exploring the function of bone morphogenetic proteins.

Mihovil Plecko is a student at University of Zagreb School of Medicine. He is on his last year of study. He is a volunteer at Laboratory for mineralized tissues since 2012, and his topics of interest are bone morphogenetic proteins and bone metabolism.

Jadranka Bubic Spoljar, PhD, Senior advisor for laboratory animals, Head of animal and breeding facility, designated veterinarian of Animal facilities University of Zagreb, School of Medicine, manage person responsible for the monitoring of preclinical studies, coordination with the Croatian Ministry, and responsible person for animal welfare, quarantine, and health status of animals. Member of the Ethics Committee at the School of Medicine.

Dunja Rogic is a medical biochemistry specialist, professor at Zagreb University School of Pharmacy and Biochemistry, and head of Department of Laboratory Diagnostics, University Hospital Center Zagreb. She is a member of the EFLM Working Group for Postanalytical Phase, of the National Health Council, Croatian Ministry of Health, and of the Ethics Committee, University Hospital Center Zagreb. Her research interests encompass evidence-based medicine, point-of-care testing, critical care, laboratory parameters of renal diseases, and organization and management of a clinical laboratory. She is the author/coauthor of about 50 manuscripts, six book chapters, an editor of several textbooks, a lecturer and organizer of continuous education courses for physicians and medical biochemists, and a member of scientific and organizing committees and an invited speaker at national and international conferences.

Vera Kufner has seventeen years of working experience in biomedical research. She is actively involved in number of scientific projects developing recombinant proteins and anti-osteoporotic therapies. She has extensive experience and expertise in biochemistry, cellular biology, and chemistry, with broad know-how in affinity and ion-exchange chromatography techniques and in training students and young researchers in laboratory practice. Besides scientific work, she is involved in teaching one course of postgraduate program within School of Medicine. She is a team member of on-going HRZZ Research project Assessment of Microbiota, Inflammatory Markers, Nutritional, and Endocrinological Status in IBD Patients and of FP7 project OSTEOGROW.

Martina Pauk works as a senior research assistant in the Laboratory for mineralized tissues, Center for Translational and Clinical Research (CETRI), University of Zagreb School of Medicine. Her research is focused on mechanisms of hepcidin regulation and involvement of other BMPs in the prevention of hemochromatosis and subsequent osteopenia in Bmp6−/− mice. She participated in research projects funded by Croatian Science Foundation named ‘Novel anabolic targeted therapy for osteoporosis: BONE6-BIS Consortium’ (2012-2015) and ‘Newly Discovered Circulating Biomarkers and Therapeutic Goals for Human Diseases’ (2014–2017). Also, she is actively involved in the Framework Program-7 (FP-7) HEALTH program entitled ‘OSTEOGROW‘– Novel Bone Morphogenetic Protein 6 Biocompatible Carrier Device for Bone Regeneration in bioanalytical method validations, writing Standard Operating Procedures (SOP) for work in conditions of GLP (Good laboratory practice), pharmacokinetics of therapeutic proteins, toxicology studies, analysis of clinical trial samples, and stability studies by optimized C2C12-BRE-Luc cell bioassay. She is also involved in teaching activities for students at postgraduate studies in the field of Bone morphogenetic proteins in bone and cartilage regeneration at University of Zagreb School of Medicine.

Tatjana Bordukalo Niksic works as a research associate on several projects in the Laboratory for mineralized tissues in the Center for Translational and Clinical Research, University of Zagreb School of Medicine. She performs molecular biology and biochemical analyses related to the role of bone morphogenetic proteins (BMPs) in various biological systems and actively participates in writing of scientific papers.

Slobodan Vukicevic is a full professor and head of Laboratory of Mineralized Tissues and Proteomic Center at the Center for Translational and Clinical Research, University of Zagreb School of Medicine. Scientific interests comprise bone and cartilage morphogenetic proteins and development of drugs for regeneration of bone, kidney, pancreas, and heart muscle. Is an invited speaker at international conferences and universities. Received international awards for achievements in science. Chairman and organizer of several international conferences on calcified tissues. Member of EMBO, World Academy of Arts and Sciences (WAAS), and Croatian Academy of Sciences and Arts (CASA). Author of more than 190 manuscripts which is cited more than 6600 times, (h-index 44). Editor of four books on BMPs and inventor on 35 patents. Founder of Genera Research, innovation-based biotechnology company developing a novel regenerative therapy for bone defects via coordinating the collaborative FP7 program OSTEOGROW.

Additional information

Funding

This work was in part supported by the Hrvatska Zaklada za Znanost (Croatian Science Foundation), under Grant BMP1-IsoFor [UIP-09-2014-3509] and Scientific Center of Excellence for Reproductive and Regenerative Medicine (project ‘Reproductive and regenerative medicine - exploration of new platforms and potentials‘, Grant Agreement [KK01.1.1.01.0008] which is funded by the European Union through the European Regional Development Fund).