Fibrosis in DMD Federica Montanaro, Ph.D. MVIMG# 7470: Fundamentals of Muscle Biology: Duchenne Muscular Dystrophy April 3, 2014 What is fibrosis? ¤ Basic response of any organ that undergoes repetitive injury and inflammation. ¤ Characterized by the excessive deposition of extracellular matrix proteins (mainly collagens I and III, fibronectin) thus creating a scar. ¤ Leads to a disordered tissue structure, disruption of organ function, and ultimately organ failure. ¤ Major cause of mortality worldwide. ¤ No available FDA- or EMEA- approved anti-fibrotic therapies. Impact on disease progression in DMD NORMAL DMD - EARLY Perimysium Endomysium DMD - LATE DMD - LATE Endomysial fibrosis is the main histopathological parameter that correlates with poor motor outcome in DMD patients Consequences of Endomysial Fibrosis Quadriceps muscle strength DMD - LATE Consequences of Endomysial Fibrosis ¤ Loss of tight association between muscle fibers and capillaries → decreased oxygenation and nutrients CD31 staining (brown) of capillaries Control DMD ¤ Decreased number of satellite cells → impaired regeneration Consequences of Endomysial Fibrosis REVIEWS a Adjacent myofibroblasts attach to collagen network tissues had a similar mode of action to SM cells, their anatomical effects would mean that they would have to produce synchronous, prolonged contraction to oppose the tensile forces from massive skeletal-muscle A blocks (FIG. 6).The implication that accumulations of ¤ Tissue contracture myofibroblasts could achieve such an opposition is untenable on the simplest of energetic grounds. Instead, connective-tissue contracture involves incre- mental, anatomical shortening of the ECM material114. ¤ Increased tissue stiffness On an anatomical basis, it is clear that myofibroblasts B do not need to generate enormous forces to temporar- ily deform their immediate collagen matrix, as skeletal → inhibits the proliferation and differentiation of muscles spend most time under low basal tension (for example, during sleep). Indeed, contractures of only a few tens of µm per day might occur. This argues satellite cells against simultaneous or coordinated cell contraction, b Myofibroblast B contracts, deforming network B but rather supports an average effect of independent, → Enhances production of matrix proteins by local pericellular shortening events. These would be defined through cytomechanics rather than tissue mechanics. A similar misconception underlies the idea fibrotic cells that cell cooperativity is needed to produce shortening in a particular plane. The force that is generated in tis- → Interferes with muscle contraction REVIEWS sues could be non-directional, as the pre-existing matrix shape and properties will largely dictate the resultant vectors. By analogy, the shape of a balloon is dictated by its envelope, but the inflating force is non- remodel and lay down the shortened ECM. The lim- directional. Once achieved, such ‘contracture’ shorten- ing does not require the continuing action of myofi- ited understanding of the relationship between myofi- (3) Contracture broblasts as the shortened ECM restrains the (1) Traction broblast contraction and ECM remodelling makes it surrounding tissues. The visible appearance of contin- (2) Contraction very difficult to define how this process occurs. uous tension in pathological contractures is simply a Some recent insights intoc New the collagen process secretion of stabilizes connec- contracted structure of consequence of this changed anatomical relationship. network B, relative to network A So, how is the contractile force that is generated by tive-tissue contracture might be emerging from the myofibroblasts translated into a shortened collagen collagen lattice model as outlined in FIG. 8.This model matrix that no longer requires active cell contraction to identifies an in vitro form of contracture. This sup- maintain a tissue tension? So far, the mechanism for Force (dynes) ports the importance of the changing material prop- this has been ascribed simply to collagen crosslinking, but the permanency of this covalent bonding is irrec- erties of the collagen matrix on overall cell and tissue oncilable with the dynamic nature of the process. function130,131.Recently,it has been shown that,with Matrix shortening and increased stiffness indicates that time, there is a progressive increase in matrix stiffness the resident cells have locked a tension into the colla- 131 gen structure, but in an interstitial, incremental man- that occurs while the matrix is under tension .As ner115.This represents a ‘slip and ratchet’theory for this occurs, the shortening/compaction of the colla- contracture (FIG. 7),but it can only realistically occur as 10 20 48 Time (h) gen network ceases to depend on cell-generated New collagen a localized, dynamic and incremental process that is forces, as the increased stiffness and ability to carry secretion not consistent with massive breakdown, re-assembly Figure 8 | Contracture in three-dimensional collagen lattices. The schematic force–time plot and crosslinking. Related concepts in which collagen load is built into the matrix material and is present, represents three phases of force generation in the culture force monitor. Uncertainty and overlap Cell re-spreads and process is repeated fibrils slip past one another locally and are then re- Tomasek et al., 2002, Nature Reviews 3: 349 d 115 between the end of one phase and start of the next is indicated by the dotted sections of the even after the loss of cell contraction .In terms of linked whereas adjacent fibrils are, in their turn, Figure 7 | Model of extracellular-matrix-remodelling phase of matrix contracture. unbonded and slipped have been proposed116–119.It is bars. Addition of CYTOCHALASIN D shortly after the force plateau is reached in the contraction cytomechanics,Matrix contracture this is mediated increase by myofibroblasts. in stress occurs How is the a resultcontraction of of myofibroblasts 51,52 noteworthy that in granulation tissue of a normal heal- phase results in a total loss of force . By contrast, addition of cytochalasin D much later in the thetranslated process into functionalof matrix collagen-network contracture shortening? (FIG. The following 7).This working hypothesis ing wound or in fibrocontractive diseases, collagen putative contracture phase (arrow) results in residual tension remaining in the matrix (REF.131; R. serves to define some basic mechanisms. a | Myofibroblasts are embedded in a collagen mechano-remodelling of collagen is a key component type I is replaced to a great extent by collagen type III, A. Brown and M. Marenzana, unpublished observations). This residual tension is due to network. These cells bind to collagen fibrils through fibronexus adhesion complexes that are of thelinked cycle to intracellular that links stress cytomechanical fibres. b | Cytoskeletal orcontrol, signalling alteredevents in one of these which is known to be present in remodelling tissues irreversible remodelling-shortening of the collagen network, as would be expected with loadmyofibroblasts transmission leads to by stress-fibre asymmetric contraction, matrix which results structures in a local matrix contraction, (for example, during development) or in normal tis- contracture. shortening and bundling of the surrounding pericellular collagen network. As this is a local 120 and production/revision of that structure by resident sues that are subject to mechanical stress (for exam- effect, its consequences are incremental, and affect at most a few surrounding cells and ple, arteries). cells,matrix. of c which| New matrix myofibroblasts components are added and to stabilizeassociated the new collagencell organization, relative to its neighbours. The addition of collagen would potentially increase collagen density Any remodelling process inevitably involves the need to be removed to allow slippage, and this is a key typesand are orientation. the central d | The myofibroblastcomponents. that contracted originally, as well as other surrounding removal of matrix molecules, and is largely mediated area of uncertainty. Several candidates have been con- myofibroblasts, can repeat the process so that this small incremental collagen-matrix by matrix METALLOPROTEINASES (MMPs). Which MMPs remodelling can result in tissue contracture. are used will depend on the matrix components that sidered in analogous models, including PROTEOGLYCANS Conclusions and perspectives such as decorin121,122,collagen types V and XIV (REFS Although the model that is summarized in FIG. 9 pro- 123,124), and procollagen N-propeptide (itself dictated vides358 a |framework MAY 2002 | VOLUME for understanding 3 connective-tis- www.nature.com/reviews/molcellbio by levels of procollagen N-protease)125,and non-col- sue contraction and contracture, several important© 2002 Nature Publishing Group lagenous proteins such as lumican and fibromod- aspects of this complex process remain to be deter- ulin126.The identity ofthe MMPs that might be mined. Some of those points are discussed here. involved in the breakdown of linkage that is impor- Although it is well established that mechanical tant in contractures is presently unknown, although stress can promote the assembly of stress fibres26,43,53, combinations of, for example, MMP3, MMP2 and little is known about how this signal is transduced to MMP9 would be expected to degrade proteoglycans induce the early actin polymerization in fibroblasts and non-collagenous proteins. The simplest mecha- after wounding