Myocardin-Related Transcription Factor Mediates Epithelial Fibrogenesis in Polycystic Kidney Disease.

Polycystic kidney disease (PKD) is characterized by extensive cyst formation and progressive fibrosis. However, the molecular mechanisms whereby the loss/loss-of-function of Polycystin 1 or 2 (PC1/2) provokes fibrosis are largely unknown. The small GTPase RhoA has been recently implicated in cystogenesis, and we identified the RhoA/cytoskeleton/myocardin-related transcription factor (MRTF) pathway as an emerging mediator of epithelium-induced fibrogenesis. Therefore, we hypothesized that MRTF is activated by PC1/2 loss and plays a critical role in the fibrogenic reprogramming of the epithelium. The loss of PC1 or PC2, induced by siRNA in vitro, activated RhoA and caused cytoskeletal remodeling and robust nuclear MRTF translocation and overexpression. These phenomena were also manifested in PKD1 (RC/RC) and PKD2 (WS25/-) mice, with MRTF translocation and overexpression occurring predominantly in dilated tubules and the cyst-lining epithelium, respectively. In epithelial cells, a large cohort of PC1/PC2 downregulation-induced genes was MRTF-dependent, including cytoskeletal, integrin-related, and matricellular/fibrogenic proteins. Epithelial MRTF was necessary for the paracrine priming of the fibroblast-myofibroblast transition. Thus, MRTF acts as a prime inducer of epithelial fibrogenesis in PKD. We propose that RhoA is a common upstream inducer of both histological hallmarks of PKD: cystogenesis and fibrosis.

Modeling mechanical activation of macrophages during pulmonary fibrogenesis for targeted anti-fibrosis therapy.

Pulmonary fibrosis is an often fatal lung disease. Immune cells such as macrophages were shown to accumulate in the fibrotic lung, but their contribution to the fibrosis development is unclear. To recapitulate the involvement of macrophages in the development of pulmonary fibrosis, we developed a fibrotic microtissue model with cocultured human macrophages and fibroblasts. We show that profibrotic macrophages seeded on topographically controlled stromal tissues became mechanically activated. The resulting co-alignment of macrophages, collagen fibers, and fibroblasts promoted widespread fibrogenesis in micro-engineered lung tissues. Anti-fibrosis treatment using pirfenidone disrupts the polarization and mechanical activation of profibrotic macrophages, leading to fibrosis inhibition. Pirfenidone inhibits the mechanical activation of macrophages by suppressing integrin αMß2 and Rho-associated kinase 2. These results demonstrate a potential pulmonary fibrogenesis mechanism at the tissue level contributed by macrophages. The cocultured microtissue model is a powerful tool to study the immune-stromal cell interactions and the anti-fibrosis drug mechanism.

Cardioprotection by the adiponectin receptor agonist ALY688 in a preclinical mouse model of heart failure with reduced ejection fraction (HFrEF).

AIMS: Adiponectin has been shown to mediate cardioprotective effects and levels are typically reduced in patients with cardiometabolic disease. Hence, there has been intense interest in developing adiponectin-based therapeutics. The aim of this translational research study was to examine the functional significance of targeting adiponectin signaling with the adiponectin receptor agonist ALY688 in a mouse model of heart failure with reduced ejection fraction (HFrEF), and the mechanisms of cardiac remodeling leading to cardioprotection. METHODS AND RESULTS: Wild-type mice were subjected to transverse aortic constriction (TAC) to induce left ventricular pressure overload (PO), or sham surgery, with or without daily subcutaneous ALY688-SR administration. Temporal analysis of cardiac function was conducted via weekly echocardiography for 5 weeks and we observed that ALY688 attenuated the PO-induced dysfunction. ALY688 also reduced cardiac hypertrophic remodeling, assessed via LV mass, heart weight to body weight ratio, cardiomyocyte cross sectional area, ANP and BNP levels. ALY688 also attenuated PO-induced changes in myosin light and heavy chain expression. Collagen content and myofibroblast profile indicated that fibrosis was attenuated by ALY688 with TIMP1 and scleraxis/periostin identified as potential mechanistic contributors. ALY688 reduced PO-induced elevation in circulating cytokines including IL-5, IL-13 and IL-17, and the chemoattractants MCP-1, MIP-1ß, MIP-1alpha and MIP-3α. Assessment of myocardial transcript levels indicated that ALY688 suppressed PO-induced elevations in IL-6, TLR-4 and IL-1ß, collectively indicating anti-inflammatory effects. Targeted metabolomic profiling indicated that ALY688 increased fatty acid mobilization and oxidation, increased betaine and putrescine plus decreased sphingomyelin and lysophospholipids, a profile indicative of improved insulin sensitivity. CONCLUSION: These results indicate that the adiponectin mimetic peptide ALY688 reduced PO-induced fibrosis, hypertrophy, inflammation and metabolic dysfunction and represents a promising therapeutic approach for treating HFrEF in a clinical setting.

Discovery of phosphorylated lantibiotics with proimmune activity that regulate the oral microbiome.

Lantibiotics are ribosomally synthesized and posttranslationally modified peptides (RiPPs) that are produced by bacteria. Interest in this group of natural products is increasing rapidly as alternatives to conventional antibiotics. Some human microbiome-derived commensals produce lantibiotics to impair pathogens' colonization and promote healthy microbiomes. Streptococcus salivarius is one of the first commensal microbes to colonize the human oral cavity and gastrointestinal tract, and its biosynthesis of RiPPs, called salivaricins, has been shown to inhibit the growth of oral pathogens. Herein, we report on a phosphorylated class of three related RiPPs, collectively referred to as salivaricin 10, that exhibit proimmune activity and targeted antimicrobial properties against known oral pathogens and multispecies biofilms. Strikingly, the immunomodulatory activities observed include upregulation of neutrophil-mediated phagocytosis, promotion of antiinflammatory M2 macrophage polarization, and stimulation of neutrophil chemotaxis-these activities have been attributed to the phosphorylation site identified on the N-terminal region of the peptides. Salivaricin 10 peptides were determined to be produced by S. salivarius strains found in healthy human subjects, and their dual bactericidal/antibiofilm and immunoregulatory activity may provide new means to effectively target infectious pathogens while maintaining important oral microbiota.

Controlled release of low-molecular weight, polymer-free corticosteroid coatings suppresses fibrotic encapsulation of implanted medical devices.

Inflammation-driven foreign body reactions, and the frequently associated encapsulation by fibrogenic fibroblasts, reduce the functionality and longevity of implanted medical devices and materials. Anti-inflammatory drugs, such as dexamethasone, can suppress the foreign body reaction for a few days post-surgery, but lasting drug delivery strategies for long-term implanted materials remain an unmet need. We here establish a thin-coating strategy with novel low molecular weight corticosteroid dimers to suppress foreign body reactions and fibrotic encapsulation of subcutaneous silicone implants. The dimer coatings are >75% dexamethasone by mass and directly processable into conformal coatings using conventional solvent-based techniques, such as casting or spray coating without added polymers or binding agents. In vitro, surface erosion of the coating, and subsequent hydrolysis, provide controlled release of free dexamethasone. In a rat subcutaneous implantation model, the resulting slow and sustained release profile of dexamethasone is effective at reducing the number and activation of pro-fibrotic macrophages both acutely and at chronic time points. Consequently, fibroblast activation, collagen deposition and fibrotic encapsulation are suppressed at least 45 days post-implantation. Thus, our approach to protect implants from host rejection is advantageous over polymeric drug delivery systems, which typically have low drug loading capacity (<30%), initial burst release profiles, and unpredictable release kinetics.

Physics and Physiology of Cell Spreading in Two and Three Dimensions.

Cells spread on surfaces and within three-dimensional (3-D) matrixes as they grow, divide, and move. Both chemical and physical signals orchestrate spreading during normal development, wound healing, and pathological states such as fibrosis and tumor growth. Diverse molecular mechanisms drive different forms of cell spreading. This article discusses mechanisms by which cells spread in 2-D and 3-D and illustrates new directions in studies of this aspect of cell function.

Multipotent stromal cells: One name, multiple identities.

Multipotent stromal cells (MSCs) are vital for development, maintenance, function, and regeneration of most tissues. They can differentiate along multiple connective lineages, but unlike most other stem/progenitor cells, they carry out various other functions while maintaining their developmental potential. MSCs function as damage sensors, respond to injury by fostering regeneration through secretion of trophic factors as well as extracellular matrix (ECM) molecules, and contribute to fibrotic reparative processes when regeneration fails. Tissue-specific MSC identity, fate(s), and function(s) are being resolved through fate mapping coupled with single cell "omics," providing unparalleled insights into the secret lives of tissue-resident MSCs.

Implant Fibrosis and the Underappreciated Role of Myofibroblasts in the Foreign Body Reaction.

Body implants and implantable medical devices have dramatically improved and prolonged the life of countless patients. However, our body repair mechanisms have evolved to isolate, reject, or destroy any object that is recognized as foreign to the organism and inevitably mounts a foreign body reaction (FBR). Depending on its severity and chronicity, the FBR can impair implant performance or create severe clinical complications that will require surgical removal and/or replacement of the faulty device. The number of review articles discussing the FBR seems to be proportional to the number of different implant materials and clinical applications and one wonders, what else is there to tell? We will here take the position of a fibrosis researcher (which, coincidentally, we are) to elaborate similarities and differences between the FBR, normal wound healing, and chronic healing conditions that result in the development of peri-implant fibrosis. After giving credit to macrophages in the inflammatory phase of the FBR, we will mainly focus on the activation of fibroblastic cells into matrix-producing and highly contractile myofibroblasts. While fibrosis has been discussed to be a consequence of the disturbed and chronic inflammatory milieu in the FBR, direct activation of myofibroblasts at the implant surface is less commonly considered. Thus, we will provide a perspective how physical properties of the implant surface control myofibroblast actions and accumulation of stiff scar tissue. Because formation of scar tissue at the surface and around implant materials is a major reason for device failure and extraction surgeries, providing implant surfaces with myofibroblast-suppressing features is a first step to enhance implant acceptance and functional lifetime. Alternative therapeutic targets are elements of the myofibroblast mechanotransduction and contractile machinery and we will end with a brief overview on such targets that are considered for the treatment of other organ fibroses.

The inflammatory speech of fibroblasts.

Activation of fibroblasts is a key event during normal tissue repair after injury and the dysregulated repair processes that result in organ fibrosis. To most researchers, fibroblasts are rather unremarkable spindle-shaped cells embedded in the fibrous collagen matrix of connective tissues and/or deemed useful to perform mechanistic studies with adherent cells in culture. For more than a century, fibroblasts escaped thorough classification due to the lack of specific markers and were treated as the leftovers after all other cells have been identified from a tissue sample. With novel cell lineage tracing and single cell transcriptomics tools, bona fide fibroblasts emerge as only one heterogeneous sub-population of a much larger group of partly overlapping cell types, including mesenchymal stromal cells, fibro-adipogenic progenitor cells, pericytes, and/or perivascular cells. All these cells are activated to contribute to tissue repair after injury and/or chronic inflammation. "Activation" can entail various functions, such as enhanced proliferation, migration, instruction of inflammatory cells, secretion of extracellular matrix proteins and organizing enzymes, and acquisition of a contractile myofibroblast phenotype. We provide our view on the fibroblastic cell types and activation states playing a role during physiological and pathological repair and their crosstalk with inflammatory macrophages. Inflammation and fibrosis of the articular synovium during rheumatoid arthritis and osteoarthritis are used as specific examples to discuss inflammatory fibroblast phenotypes. Ultimately, delineating the precursors and functional roles of activated fibroblastic cells will contribute to better and more specific intervention strategies to treat fibroproliferative and fibrocontractive disorders.

Kindlin-2 Mediates Mechanical Activation of Cardiac Myofibroblasts.

We identify the focal adhesion protein kindlin-2 as player in a novel mechanotransduction pathway that controls profibrotic cardiac fibroblast to myofibroblast activation. Kindlin-2 is co-upregulated with the myofibroblast marker α-smooth muscle actin (α-SMA) in fibrotic rat hearts and in human cardiac fibroblasts exposed to fibrosis-stiff culture substrates and pro-fibrotic TGF-ß1. Stressing fibroblasts using ferromagnetic microbeads, stretchable silicone membranes, and cell contraction agonists all result in kindlin-2 translocation to the nucleus. Overexpression of full-length kindlin-2 but not of kindlin-2 missing a putative nuclear localization sequence (∆NLS kindlin-2) results in increased α-SMA promoter activity. Downregulating kindlin-2 with siRNA leads to decreased myofibroblast contraction and reduced α-SMA expression, which is dependent on CC(A/T)-rich GG(CArG) box elements in the α-SMA promoter. Lost myofibroblast features under kindlin-2 knockdown are rescued with wild-type but not ∆NLS kindlin-2, indicating that myofibroblast control by kindlin-2 requires its nuclear translocation. Because kindlin-2 can act as a mechanotransducer regulating the transcription of α-SMA, it is a potential target to interfere with myofibroblast activation in tissue fibrosis.

CXCR3A promotes the secretion of the antifibrotic decoy receptor sIL-13Rα2 by pulmonary fibroblasts.

CXC chemokine receptor 3 (CXCR3) A and its IFN-inducible ligands CXCL9 and CXCL10 regulate vascular remodeling and fibroblast motility. IL-13 is a profibrotic cytokine implicated in the pathogenesis of inflammatory and fibroproliferative conditions. Previous work from our laboratory has shown that CXCR3A is negatively regulated by IL-13 and is necessary for the basal regulation of the IL-13 receptor subunit IL-13Rα2. This study investigates the regulation of fibroblast phenotype, function, and downstream IL-13 signaling by CXCR3A in vitro. CXCR3A was overexpressed via transient transfection. CXCR3A-/- lung fibroblasts were isolated for functional analysis. Additionally, the contribution of CXCR3A to tissue remodeling following acute lung injury was assessed in vivo with wild-type (WT) and CXCR3-/- mice challenged with IL-13. CXCR3 and IL-13Rα2 displayed a reciprocal relationship after stimulation with either IL-13 or CXCR3 ligands. CXCR3A reduced expression of fibroblast activation makers, soluble collagen production, and proliferation. CXCR3A enhanced the basal expression of pERK1/2 while inducing IL-13-mediated downregulation of NF-κB-p65. CXCR3A-/- pulmonary fibroblasts were increasingly proliferative and displayed reduced contractility and α-smooth muscle actin expression. IL-13 challenge regulated expression of the CXCR3 ligands and soluble IL-13Rα2 levels in lungs and bronchoalveolar lavage fluid (BALF) of WT mice; this response was absent in CXCR3-/- mice. Alveolar macrophage accumulation and expression of genes involved in lung remodeling was increased in CXCR3-/- mice. We conclude that CXCR3A is a central antifibrotic factor in pulmonary fibroblasts, limiting fibroblast activation and reducing extracellular matrix (ECM) production. Therefore, targeting of CXCR3A may be a novel approach to regulating fibroblast activity in lung fibrosis and remodeling.

Dancing with the Cells: Acoustic Microflows Generated by Oscillating Cells.

The interaction of a sound or ultrasound wave with an elastic object, such as a microbubble, can give rise to a steady-state microstreaming flow in its surrounding liquid. Many microfluidic strategies for cell and particle manipulation, and analyte mixing, are based on this type of flow. In addition, there are reports that acoustic streaming can be generated in biological systems, for instance, in a mammalian inner ear. Here, new observations are reported that individual cells are able to induce microstreaming flow, when they are excited by controlled acoustic waves in vitro. Single adherent cells are exposed to an acoustic field inside a microfluidic device. The cell-induced microstreaming is then investigated by monitoring flow tracers around the cell, while the structure and extracellular environment of the cell are altered using different chemicals. The observations suggest that the maximum streaming flow induced by an MDA-MB-231 breast cancer cell can reach velocities on the order of mm s-1 , and this maximum velocity is primarily governed by the overall cell stiffness. Therefore, such cell-induced microstreaming measurements, including flow pattern and velocity magnitude, may be used as label-free proxies of cellular mechanical properties, such as stiffness.

TGF-ß1 - A truly transforming growth factor in fibrosis and immunity.

'Jack of all trades, master of everything' is a fair label for transforming growth factor ß1 (TGF-ß) - a cytokine that controls our life at many levels. In the adult organism, TGF-ß1 is critical for the development and maturation of immune cells, maintains immune tolerance and homeostasis, and regulates various aspects of immune responses. Following acute tissue damages, TGF-ß1 becomes a master regulator of the healing process with impacts on about every cell type involved. Divergence from the tight control of TGF-ß1 actions, for instance caused by chronic injury, severe trauma, or infection can tip the balance from regulated physiological to excessive pathological repair. This condition of fibrosis is characterized by accumulation and stiffening of collagenous scar tissue which impairs organ functions to the point of failure. Fibrosis and dysregulated immune responses are also a feature of cancer, in which tumor cells escape immune control partly by manipulating TGF-ß1 regulation and where immune cells are excluded from the tumor by fibrotic matrix created during the stroma 'healing' response. Despite the obvious potential of TGF-ß-signalling therapies, globally targeting TGF-ß1 receptor, downstream pathways, or the active growth factor have proven to be extremely difficult if not impossible in systemic treatment regimes. However, TGF-ß1 binding to cell receptors requires prior activation from latent complexes that are extracellularly presented on the surface of immune cells or within the extracellular matrix. These different locations have led to some divergence in the field which is often either seen from the perspective of an immunologists or a fibrosis/matrix researcher. Despite these human boundaries, there is considerable overlap between immune and tissue repair cells with respect to latent TGF-ß1 presentation and activation. Moreover, the mechanisms and proteins employed by different cells and spatiotemporal control of latent TGF-ß1 activation provide specificity that is amenable to drug development. This review aims at synthesizing the knowledge on TGF-ß1 extracellular activation in the immune system and in fibrosis to further stimulate cross talk between the two research communities in solving the TGF-ß conundrum.

The circadian clock protein REVERBα inhibits pulmonary fibrosis development.

Pulmonary inflammatory responses lie under circadian control; however, the importance of circadian mechanisms in the underlying fibrotic phenotype is not understood. Here, we identify a striking change to these mechanisms resulting in a gain of amplitude and lack of synchrony within pulmonary fibrotic tissue. These changes result from an infiltration of mesenchymal cells, an important cell type in the pathogenesis of pulmonary fibrosis. Mutation of the core clock protein REVERBα in these cells exacerbated the development of bleomycin-induced fibrosis, whereas mutation of REVERBα in club or myeloid cells had no effect on the bleomycin phenotype. Knockdown of REVERBα revealed regulation of the little-understood transcription factor TBPL1. Both REVERBα and TBPL1 altered integrinß1 focal-adhesion formation, resulting in increased myofibroblast activation. The translational importance of our findings was established through analysis of 2 human cohorts. In the UK Biobank, circadian strain markers (sleep length, chronotype, and shift work) are associated with pulmonary fibrosis, making them risk factors. In a separate cohort, REVERBα expression was increased in human idiopathic pulmonary fibrosis (IPF) lung tissue. Pharmacological targeting of REVERBα inhibited myofibroblast activation in IPF fibroblasts and collagen secretion in organotypic cultures from IPF patients, thus suggesting that targeting of REVERBα could be a viable therapeutic approach.

Tracking adiponectin biodistribution via fluorescence molecular tomography indicates increased vascular permeability after streptozotocin-induced diabetes.

Adiponectin, a highly abundant polypeptide hormone in plasma, plays an important role in the regulation of energy metabolism in a wide variety of tissues, as well as providing important beneficial effects in diabetes, inflammation, and cardiovascular disease. To act on target tissues, adiponectin must move from the circulation to the interstitial space, suggesting that vascular permeability plays an important role in regulating adiponectin action. To test this hypothesis, fluorescently labeled adiponectin was used to monitor its biodistribution in mice with streptozotocin-induced diabetes (STZD). Adiponectin was, indeed, found to have increased sequestration in the highly fenestrated liver and other tissues within 90 min in STZD mice. In addition, increased myocardial adiponectin was detected and confirmed using computed tomography (CT) coregistration. This provided support of adiponectin delivery to affected cardiac tissue as a cardioprotective mechanism. Higher adiponectin content in the STZD heart tissues was further examined by ex vivo fluorescence molecular tomography (FMT) imaging, immunohistochemistry, and Western blot analysis. In vitro mechanistic studies using an endothelial monolayer on inserts and three-dimensional microvascular networks on microfluidic chips further confirmed that adiponectin flux was increased by high glucose. However, in the in vitro model and mouse heart tissue, high glucose levels did not change adiponectin receptor levels. An examination of the tight junction (TJ) complex revealed a decrease in the TJ protein claudin (CLDN)-7 in high glucose-treated endothelial cells, and the functional significance of this change was underscored by increased endothelium permeability upon siRNA-mediated knockdown of CLDN-7. Our data support the idea that glucose-induced effects on permeability of the vascular endothelium contribute to the actions of adiponectin by regulating its transendothelial movement from blood to the interstitial space. These observations are physiologically significant and critical when considering ways to harness the therapeutic potential of adiponectin for diabetes.

Dynamic fibroblast contractions attract remote macrophages in fibrillar collagen matrix.

Macrophage (Mϕ)-fibroblast interactions coordinate tissue repair after injury whereas miscommunications can result in pathological healing and fibrosis. We show that contracting fibroblasts generate deformation fields in fibrillar collagen matrix that provide far-reaching physical cues for Mϕ. Within collagen deformation fields created by fibroblasts or actuated microneedles, Mϕ migrate towards the force source from several hundreds of micrometers away. The presence of a dynamic force source in the matrix is critical to initiate and direct Mϕ migration. In contrast, collagen condensation and fiber alignment resulting from fibroblast remodelling activities or chemotactic signals are neither required nor sufficient to guide Mϕ migration. Binding of α2ß1 integrin and stretch-activated channels mediate Mϕ migration and mechanosensing in fibrillar collagen ECM. We propose that Mϕ mechanosense the velocity of local displacements of their substrate, allowing contractile fibroblasts to attract Mϕ over distances that exceed the range of chemotactic gradients.

Cadherin-11-mediated adhesion of macrophages to myofibroblasts establishes a profibrotic niche of active TGF-ß.

Macrophages contribute to the activation of fibroblastic cells into myofibroblasts, which secrete collagen and contract the collagen matrix to acutely repair injured tissue. Persistent myofibroblast activation leads to the accumulation of fibrotic scar tissue that impairs organ function. We investigated the key processes that turn acute beneficial repair into destructive progressive fibrosis. We showed that homotypic cadherin-11 interactions promoted the specific binding of macrophages to and persistent activation of profibrotic myofibroblasts. Cadherin-11 was highly abundant at contacts between macrophages and myofibroblasts in mouse and human fibrotic lung tissues. In attachment assays, cadherin-11 junctions mediated specific recognition and strong adhesion between macrophages and myofibroblasts. One functional outcome of cadherin-11-mediated adhesion was locally restricted activation of latent transforming growth factor-ß (TGF-ß) between macrophage-myofibroblast pairs that was not observed in cocultures of macrophages and myofibroblasts that were not in contact with one another. Our data suggest that cadherin-11 junctions maintain latent TGF-ß-producing macrophages and TGF-ß-activating myofibroblasts in close proximity to one another. Inhibition of homotypic cadherin-11 interactions could be used to cause macrophage-myofibroblast separation, thereby destabilizing the profibrotic niche.

Novel differences in gene expression and functional capabilities of myofibroblast populations in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF), a chronic progressive interstitial pneumonia, is characterized by excessive fibroproliferation. Key effector cells in IPF are myofibroblasts that are recruited from three potential sources: resident fibroblasts, fibrocytes, and epithelial cells. We hypothesized that IPF myofibroblasts from different sources display unique gene expression differences and distinct functional characteristics. Primary human pulmonary fibroblasts (normal and IPF), fibrocytes, and epithelial cells were activated using the profibrotic factors TGF-ß and TNF-α. The resulting myofibroblasts were characterized using cell proliferation, soluble collagen, and contractility assays, ELISA, and human fibrosis PCR arrays. Genes of significance in human whole lung were validated by immunohistochemistry on human lung sections. Fibroblast-derived myofibroblasts exhibited the greatest increase in expression of profibrotic genes and genes involved in extracellular matrix remodeling and signal transduction. Functional studies demonstrated that myofibroblasts derived from fibrocytes expressed mostly soluble collagen and chemokine (C-C) motif ligand (CCL) 18 but were the least proliferative of the myofibroblast progeny. Activated IPF fibroblasts displayed the highest levels of contractility and CCL2 production. This study identified novel differences in gene expression and functional characteristics of different myofibroblast populations. Further investigation into the myofibroblast phenotype may lead to potential therapeutic targets in future IPF research.

The big five in fibrosis: Macrophages, myofibroblasts, matrix, mechanics, and miscommunication.

Scarring is part of the normal healing response to tissue injury in all organs and required to rapidly repair acute damages, mostly with extracellular matrix. A variety of different cells are activated into myofibroblasts to produce and remodel the scar matrix. Temporal and spatial coordination of myofibroblast activities with inflammatory macrophages is crucial for the controlled healing process. Miscommunication can result in either insufficient (chronic) or exacerbated (fibrotic) repair. In addition to soluble biochemical signals and intercellular contacts, cell-to-cell communication is mediated by biophysical and chemical signals transmitted through the extracellular matrix. Over the course of healing, the matrix takes over the role of a master coordinator; failure to do so produces poor healing outcomes that reduce organ function. Understanding the mechanical and chemical state of the matrix and its effects on cellular processes will be essential to address diseases that are characterized by dysfunctional matrix, such as fibrosis.

Collagen scaffold enhances the regenerative properties of mesenchymal stromal cells.

MSCs are widely applied to regenerate heart tissue in myocardial diseases but when grown in standard two-dimensional (2D) cultures exhibit limited potential for cardiac repair and develop fibrogenic features with increasing culture time. MSCs can undergo partial cardiomyogenic differentiation, which improves their cardiac repair capacity. When applied to collagen patches they may improve cardiac tissue regeneration but the mechanisms remain elusive. Here, we investigated the regenerative properties of MSCs grown in a collagen scaffold as a three-dimensional (3D) culture system, and performed functional analysis using an engineered heart tissue (EHT) model. We showed that the expression of cardiomyocyte-specific proteins by MSCs co-cultured with rat neonatal cardiomyocytes was increased in collagen patches versus conventional cultures. MSCs in 3D collagen patches were less fibrogenic, secreted more cardiotrophic factors, retained anti-apoptotic and immunomodulatory function, and responded less to TLR4 ligand lipopolysaccharide (LPS) stimulation. EHT analysis showed no effects by MSCs on cardiomyocyte function, whereas control dermal fibroblasts abrogated the beating of cardiac tissue constructs. We conclude that 3D collagen scaffold improves the cardioprotective effects of MSCs by enhancing the production of trophic factors and modifying their immune modulatory and fibrogenic phenotype. The improvement in myocardial function by MSCs after acquisition of a partial cardiac cell-like phenotype is not due to enhanced MSC contractility. A better understanding of the mechanisms of MSC-mediated tissue repair will help to further enhance the therapeutic potency of MSCs.