UNIVERSITA' DEGLI STUDI DI TORINO PhD Programme in Experimental Medicine and Therapy XXVIII cycle GROWTH FACTOR ADMINISTRATION FOR NERVE, SKELETAL MUSCLE AND CARDIAC TISSUE REPAIR: A KEY TOOL IN REGENERATIVE MEDICINE Presented by: Michela Morano Tutor: Prof. Stefano Geuna PhD Coordinator: Prof. Giuseppe Saglio Scientific disciplinary sector: BIO 16 2012-2016 CONTENT ABSTRACT 5 OUTLINE 11 ABBREVIATIONS 15 CHAPTER 1 PERIPHERAL NERVE INJURY AND REPAIR 17 Introduction and Scientific Background Aim of the Research Scientific Publications Discussion and Future Directions CHAPTER 2 SKELETAL MUSCLE DENERVATION 159 Introduction and Scientific Background Aim of the Research Scientific Publication Discussion and Future Directions CHAPTER 3 CARDIAC MUSCLE: ISCHEMIA/REPERFUSION INJURY 205 Introduction and Scientific Background Aim of the Research Scientific Publication Discussion and Future Directions GENERAL CONCLUSIONS 247 ACKNOWLEDGMENTS 255 ABSTRACT INTRODUCTION: The regenerative medicine is a continuously evolving interdisciplinary field aimed to enhance the regenerative capability of the body itself and to guide the regeneration process taking advantage of endogenous repair mechanisms to restore a tissue or organ morphology and function. Two types of approaches can be distinguished in regenerative medicine: cell based- and cell free- therapies. Particular attractive are growth factors-based therapies, which developed, thank to bioengineering and technical science influences, into a more integrated therapeutic approaches based on biomaterial, implant, engineered factors and controlled drug release. Here a multiple analysis is presented: i) the first part examines the in vitro and in vivo investigation of a set of engineered growth factors combined with biomaterials for promoting peripheral nerve regeneration; ii) the second part focus on the study of Neuregulin 1 (NRG1) growth factor and its receptors in two models of muscle injury (denervated skeletal muscle and ischemia/reperfusion cardiac muscle), aimed to better understand the properties of this factors for future in vivo manipulation of this system in muscle recovery after denervation and amelioration of the existing therapies in cardioprotection field. Traumatic injury in peripheral nerves, as for the central nervous system, results in high morbidity with great changes in patient's life and, obviously, elevated socioeconomic cost. Beside the elevated regenerative abilities of peripheral nervous system, the nerve regeneration often fails, with aberrant sprouting and the development of a neuroma at the proximal nerve stump. When regeneration process takes long time, the neurotrophic factors supply decays over the time, failing to sustain neuron regeneration. In most of nerve injury cases is necessary a surgeon intervention, and the standard approach used is the nerve autograft, which entails the suture of a nerve achieved from the patient himself to bridge the gap of another more important nerve. However autograft often gives unsatisfactory functional recovery, therefore increasing efforts have been made to seek new surgical alternative approaches. Among alternative strategies, the tubulization technique, which consist in the insertion of biological or artificial tubular structure between the nerve stumps, obtained good clinical results. Hollow tube are effective specifically for short nerve defect (≤ 3-4 cm). Researcher are now focus on conduits with more complex design in order to increase nerve conduit performance. Some of the solutions adopted are the use of extracellular matrix structural components that increase cell adhesion and invasion, internal framework, conductive polymers, matrix releasing growth factors and supportive cells. The production of neurotrophic growth factors and the expression of their receptors changes considerably after nerve injury in different nerve cell types and is now accepted that neurotrophic factors play an important role in peripheral nerve injury, influencing and controlling several aspect of nerve regeneration. Thus growth factors controlled and prolonged release inside artificial nerve conduit is actually a goal in peripheral nerve tissue 5 engineering. Incorporation of exogenous growth factors in nerve conduit can be done directly (in solution) in the tube or using an hydrogel, as collagen or agarose, which acts as a scaffold releasing the drug in the lumen. Among factors studied for nerve regeneration there is the neuregulin1 (NRG1) a widely express growth factor existing in numerous isoforms as a result of alternative splicing; it can be a soluble or transmembrane protein, that mediates various cellular process through ErbB tyrosine kinase receptors. Nrg1 isoforms have been shown upregulated after injury. They drive the dedifferentiation of Schwann cells and their migration in the site of injury to create the Bands of Büngner, a tubular structure that direct growing axons to their original targets. Moreover a transmembrane NRG1 expressed by the axons guides the deposit of myelin layers by Schwann cells, regulating remyelination process. Therapies for nerve regeneration should monitor also nerve target behaviour, avoiding muscle atrophy and promoting the correct reinnervation of muscle fibres to obtain total functional recovery. After nerve injury the target muscle undergoes to a molecular and morphological changes that result in muscle atrophy, and when denervation persists permanent changes occur, reducing the possibility to recover the complete functionality after reinnervation. The denervation activates the ubiquitin-proteosome machinery and the autophagy-lysosome machinery that are responsible for protein breakdown. At the same time satellite cells, the resident stem cells, became activated, proliferate and then differentiate and fuse in myofibres. Meanwhile the role of NRG1 and ErbB receptors is well defined in nerve, little is known about NRG1/ErbB system in muscle after nerve injury. It is clear that NRG1 has a role in muscle development and controls spindle maintenance, glucose uptake and neuromuscular junction formation. Moreover ErbB2/ErbB3 expression in satellite cells are able to induce pro-survival signalling in activated cells. How NRG1/ErbB system is influenced by nerve acute injury, and if the system could be a good therapeutic target to maintain the muscle receptive for nerve reinnervation remain to be investigated. In another muscular tissue, the cardiac tissue, is becoming more and more clear that NRG1/ErbB system is a potential target for heart failure therapy. NRG1/ErbB system is essential for a correct cardiac development, furthermore, it is now clear that also in adult heart this signalling plays a critical role in the normal function as well as in ischemia or other pathological conditions. In adult heart, cardiac microvascular endothelial cells (EC) express soluble NRG1 isoforms, which stimulate cell survival and growth, glucose uptake, protein synthesis and “hypertrophic” gene expression in cardiomyocytes, expressing ErbB receptors. The deletion of NRG1 from EC increases the infarct area and the number of TUNEL positive cells after ischemia and reperfusion (I/R) injury. For its pro-survival effect Nrg1 has been proposed as a potential drug for heart failure treatment. Several preclinical studies in rat or mouse models of heart failure and several clinical trials demonstrated that intravenous administration of recombinant soluble NRG1 improved 6 cardiac contractility and relaxation, left ventricular remodelling, decreased apoptosis and attenuated mitochondrial dysfunction. However, the molecular bases of this beneficial effect remain unclear, as well as how the downregulation of NRG1/ErbB system detected in cardiac chronic disease is a cause or an effect of the pathological status. MATERIALS AND METHODS: The first part of the study is related to nerve injury. We investigated in vitro and in vivo a set of engineered growth factors with the final goal of in vivo long-time release inside an artificial nerve guide. NGF, FGF and GDNF, together with the extracellular domain of NRG1 beta were covalently conjugated to iron-oxide nanoparticles (10nm±2 diameter). We tested in vitro the retention of factor bioactivity after nanoparticles conjugation, analyzing neurite outgrowth in adult or neonatal dorsal root ganglion (DRG) cultures. Ascertained the bioactivity, we recreated in vitro a possible environment present in artificial scaffolds, culturing neonatal DRGs inside a layer of NVR (a biocompatible hydrogel composed mainly by laminin and hyaluronic acid), and we evaluated neurite outgrowth. Furthermore we tested the stability of the conjugated factors comparing the effects of non-conjugated or conjugated factors left some 2/4 weeks at 37°C in cell medium and then used to stimulate neurite outgrowth or cell migration. In collaboration with our partner in Israel an in vivo pilot study was performed in rat using GDNF factor. A comparison of nerve regeneration was done after sciatic nerve injury (15 mm gap) and nerve repair among the following groups: 1. autograft (gold standard/control); 2. hollow chitosan tube (17 mm length); 3. chitosan tube filled with NVR gel; 4. chitosan tube filled with NVR plus GDNF; 5. chitosan tube filled with NVR gel plus conjugated GDNF. Functional analysis were performed after one, three and five months from surgery. After 5 months animals were sacrificed and morphological parameter (fibre diameter, axon diameter, number of myelinated fibres, myelin thickness) were analyzed on regenerated