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Final Report Summary - MACS (The contribution of cellular adhesions to matrix remodeling in health and disease)

1.1 Project objectives

In healthy tissue, cells can positively contribute to tissue functioning by synthesizing additional matrix components, a process initiated at matrix adhesions, the connection between cells and matrix. However, in various fibrous tissue pathologies, a progressive increase in matrix disorganization is observed. In search of the underlying cause of this matrix disorganization, the project focused on the role of mechanoresponsive matrix adhesion proteins under mechanical stimulation and the resulting impact on matrix remodeling in healthy and diseased tissue. Hereby an attempt was made to unravel the underlying cause for the adverse tissue remodeling observed during fibrous tissue pathology, to ultimately provide a treatment strategy.

In a multidisciplinary approach, novel 3D tissue platforms were used to engineer micro-tissues that were subsequently subjected to mechanical stimuli. The aim was to answer the central question of the proposal: “how do mechanosensitive matrix adhesion-associated proteins react to mechanical perturbations and impact matrix remodeling in tissue models that mimic health and disease?”

1.2 Work performed and main results

1.2.1 Impact of mechanical and biochemical stimuli on cell behavior

Insight was gained into the ECM-driven signaling of human fibroblasts, anchored to a fibronectin or to collagen-decorated matrix, in the absence or presence of cyclic mechanical strain. The study revealed that fibroblasts have a high ECM remodeling/repair capacity in contact with fibronectin alone (representative for an early wound healing event), which is reduced in the presence of collagen 1 (representative for a later wound healing event), thereby down-tuning fibroblast activity, a processes which would be required in a tissue repair process to finally reach tissue homeostasis. The pronounced effect of the presence of different ECM components to cellular behavior was further explored using a 3D microtissue platform. Namely, trampoline-shaped collagen microtissues were engineered in a high-throughput platform using not only fibroblasts but also fibroblasts that are unable to synthesize fibronectin (fibronectin-knockout). Fibroblasts were observed to migrate towards the microtissue surfaces in the presence of full-length fibronectin, but not when fibronectin fragments were added. Building order from randomly assembled cells requires cell migration and force generation, and both were shown to be promoted by full-length fibronectin. Next, fibronectin equipped with a stretch-sensitive biosensor was deployed, showing that gradients of fibronectin conformation and ECM composition existed in the microtissues that might provide critical cell migration cues. These data suggest novel mechanisms of how plasma fibronectin, and possibly the amount of stretch present in the fibronectin fibers, contributes to tissue morphogenesis and cellular migration in 3D tissue.

1.2.2 Matrix adhesion protein turnover in 3D tissue, subjected to mechanical stimuli

Human fibroblasts were mixed with collagen type I and seeded in a tissue platform, allowing for the application of cyclic stretch upon formation of microtissues. After 3 days of static constraint, tissues were either left unstrained or were cyclically stretched (unidirectional) for 1, 2 or 3 additional days. Cyclic stretch resulted in a transient change in the orientation of actin stress fibers, i.e. cells reoriented in the microtissues towards the direction where cyclic loading was absent. Since cells are linked to the ECM via adhesion proteins, critical matrix adhesion proteins at various time-points were quantified using western blot and results were compared between statically and cyclically loaded samples. With applied cyclic stretch, a transient decrease in the expression of p130Cas, Paxillin and phosphorylation sites on Paxillin was found and levels of Talin and focal adhesion kinase slightly elevated, which was contrary to statically loaded samples were protein levels remained stable throughout culture. Paxillin and p130Cas are tightly linked and highly involved in cellular motility, which may explain the cyclic stretch-induced reorientation of the cells. In order to further support that the decreased expression of Paxillin and p130Cas may affect cellular motility/reorientation, the downstream effector of p130Cas / Paxillin, i.e. Rac1, and the downstream effector of FAK, i.e. MAPK are currently being evaluated. Rac1 and MAPK / ERK1/2 are proteins that are extensively studied. Potential changes in their presence during cyclic stretch can thus further unravel what the physical response of cells is to cyclic stretch in a 3D microenvironment.

1.2.3 Effect of matrix organization on cellular capacity for functional remodeling

In a collaboration with the Orthopaedic Biomechanics lab of Prof Jess Snedeker, tendon fibroblasts were seeded in reconstituted collagen microtissues. Microtissues anchored around various setups of silicone posts. Namely, a 2 post setup resulting in highly uniaxially aligned tissues with a collagen orientation in the direction of the constraint, where the platform progresses to 2by2, 3by3 and 4by4 posts and to a 12 posts setup in a square configuration. The latter results in formed tissues that are trampoline shaped and have a chaotically organized collagen core. Setups were thus chosen to mimic health (2 posts: healthy tendon tissue is uniaxially aligned), progressively towards disease (12 posts: tendon pathology associates with a disorganized collagen matrix). The central question was whether the cells that reside within the ‘healthy’ vs ‘diseased’ environment would respond directly to this microenvironment and attempt to heal or further progress the diseased matrix. Strikingly, results reveal that cells indeed directly respond to their microenvironment; i.e. when being embedded in a progressively diseased microenvironment, cells also progressively express markers that represent tendinopathy (high expression of genes encoding for MMPs, myofibroblastic smooth muscle actin and collagen type III).

1.3 Impact and implications

Fibrous tissue pathology remains a challenging clinical problem that millions of people suffer from. This is partly related to the many unknowns to how cells respond to mechanical triggers, when being embedded in a 3D environment. This project aimed at unravelling how fibroblastic cells respond to a variety of signals, from direct cyclic mechanical stretch, to structural and compositional stimuli originating from the cellular microenvironment. This study revealed that all these stimuli have a profound effect on cellular behavior. (1) Cyclic stretch alters the turnover of cellular matrix adhesions, proteins that link the intracellular to the extracellular space. Ongoing research focusses on the responsible proteins underlying these changes, to ultimately being able to steer or even prevent such changes. (2) The composition of the extracellular environment highly affects cellular behavior, providing support for the hypothesis that initial changes in the matrix can lead to dramatic effects driven by the cells that reside in the matrix, potentially progressing pathology, for instance during the initiation of tissue fibrosis. Thus, during the progression of fibrous tissue pathology, it appears to be of high importance to aim at reversing these compositional changes. (3) Finally, apart from the composition of the matrix, the structural organization of the matrix also highly affects cell behavior. Mimicking a pathologically organized matrix (in analogy with impact damage induced structural changes in native tissue), appears to directly to steer the cells to contribute to progressing the disease, rather than recovering it. This should therefore be the focus of future studies, i.e. how can cells be tuned to contribute to healing when a diseased environment is presented.

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