Provisional Extracellular Matrix Research Articles

Proteins Derived From MRL/MpJ Tendon Provisional Extracellular Matrix and Secretome Promote Pro-Regenerative Tenocyte Behavior.

Proteins enriched in a super-healer mouse strain elicit interspecies utility in promoting pro-regenerative tenocyte behavior.

Single nuclei transcriptomics delineates complex immune and kidney cell interactions contributing to kidney allograft fibrosis

Chronic allograft dysfunction (CAD), characterized histologically by interstitial fibrosis and tubular atrophy, is the major cause of kidney allograft loss. Here, using single nuclei RNA sequencing and transcriptome analysis, we identified the origin, functional heterogeneity, and regulation of fibrosis-forming cells in kidney allografts with CAD. A robust technique was used to isolate individual nuclei from kidney allograft biopsies and successfully profiled 23,980 nuclei from five kidney transplant recipients with CAD and 17,913 nuclei from three patients with normal allograft function. Our analysis revealed two distinct states of fibrosis in CAD; low and high extracellular matrix (ECM) with distinct kidney cell subclusters, immune cell types, and transcriptional profiles. Imaging mass cytometry analysis confirmed increased ECM deposition at the protein level. Proximal tubular cells transitioned to an injured mixed tubular (MT1) phenotype comprised of activated fibroblasts and myofibroblast markers, generated provisional ECM which recruited inflammatory cells, and served as the main driver of fibrosis. MT1 cells in the high ECM state achieved replicative repair evidenced by dedifferentiation and nephrogenic transcriptional signatures. MT1 in the low ECM state showed decreased apoptosis, decreased cycling tubular cells, and severe metabolic dysfunction, limiting the potential for repair. Activated B, T and plasma cells were increased in the high ECM state, while macrophage subtypes were increased in the low ECM state. Intercellular communication between kidney parenchymal cells and donor-derived macrophages, detected several years post-transplantation, played a key role in injury propagation. Thus, our study identified novel molecular targets for interventions aimed to ameliorate or prevent allograft fibrogenesis in kidney transplant recipients.

Whole lung proteome of an acute epithelial injury mouse model in comparison to spatially resolved proteomes.

Epithelial injury is one of the major drivers of acute pulmonary diseases. Recurring injury followed by aberrant repair is considered as the primary cause of chronic lung diseases, such as idiopathic pulmonary fibrosis (IPF). Preclinical in vivo models allow studying early disease-driving mechanisms like the recently established adeno-associated virus-diphtheria toxin receptor (AAV-DTR) mouse model of acute epithelial lung injury, which utilises AAV mediated expression of the human DTR. We performed quantitative proteomics of homogenised lung samples from this model and compared the results to spatially resolved proteomics data of epithelial cell regions from the same animals. In whole lung tissue proteins involved in cGAS-STING and interferon pathways, proliferation, DNA replication and the composition of the provisional extracellular matrix were upregulated upon injury. Besides epithelial cell markers SP-A, SP-C and Scgb1a1, proteins involved in cilium assembly, lipid metabolism and redox pathways were among downregulated proteins. Comparison of the bulk to spatially resolved proteomics data revealed a large overlap of protein changes and striking differences. Together our study underpins the broad usability of bulk proteomics and pinpoints to the benefit of sophisticated proteomic analyses of specific tissue regions or single cell types.

Advancing Engineered Heart Muscle Tissue Complexity with Hydrogel Composites.

A heart attack results in the permanent loss of heart muscle and can lead to heart disease, which kills more than 7 million people worldwide each year. To date, outside of heart transplantation, current clinical treatments cannot regenerate lost heart muscle or restore full function to the damaged heart. There is a critical need to create engineered heart tissues with structural complexity and functional capacity needed to replace damaged heart muscle. The inextricable link between structure and function suggests that hydrogel composites hold tremendous promise as a biomaterial-guided strategy to advance heart muscle tissue engineering. Such composites provide biophysical cues and functionality as a provisional extracellular matrix that hydrogels cannot on their own. This review describes the latest advances in the characterization of these biomaterial systems and using them for heart muscle tissue engineering. The review integrates results across the field to provide new insights on critical features within hydrogel composites and perspectives on the next steps to harnessing these promising biomaterials to faithfully reproduce the complex structure and function of native heart muscle.

Fontan-associated liver disease.

Fontan-associated liver disease.

Fibronectin: Molecular Structure, Fibrillar Structure and Mechanochemical Signaling.

The extracellular matrix (ECM) plays a key role as both structural scaffold and regulator of cell signal transduction in tissues. In times of ECM assembly and turnover, cells upregulate assembly of the ECM protein, fibronectin (FN). FN is assembled by cells into viscoelastic fibrils that can bind upward of 40 distinct growth factors and cytokines. These fibrils play a key role in assembling a provisional ECM during embryonic development and wound healing. Fibril assembly is also often upregulated during disease states, including cancer and fibrotic diseases. FN fibrils have unique mechanical properties, which allow them to alter mechanotransduction signals sensed and relayed by cells. Binding of soluble growth factors to FN fibrils alters signal transduction from these proteins, while binding of other ECM proteins, including collagens, elastins, and proteoglycans, to FN fibrils facilitates the maturation and tissue specificity of the ECM. In this review, we will discuss the assembly of FN fibrils from individual FN molecules; the composition, structure, and mechanics of FN fibrils; the interaction of FN fibrils with other ECM proteins and growth factors; the role of FN in transmitting mechanobiology signaling events; and approaches for studying the mechanics of FN fibrils.

Excess Provisional Extracellular Matrix: A Common Factor in Bicuspid Aortic Valve Formation.

A bicuspid aortic valve (BAV) is the most common cardiac malformation, found in 0.5% to 2% of the population. BAVs are present in approximately 50% of patients with severe aortic stenosis and are an independent risk factor for aortic aneurysms. Currently, there are no therapeutics to treat BAV, and the human mutations identified to date represent a relatively small number of BAV patients. However, the discovery of BAV in an increasing number of genetically modified mice is advancing our understanding of molecular pathways that contribute to BAV formation. In this study, we utilized the comparison of BAV phenotypic characteristics between murine models as a tool to advance our understanding of BAV formation. The collation of murine BAV data indicated that excess versican within the provisional extracellular matrix (P-ECM) is a common factor in BAV development. While the percentage of BAVs is low in many of the murine BAV models, the remaining mutant mice exhibit larger and more amorphous tricuspid AoVs, also with excess P-ECM compared to littermates. The identification of common molecular characteristics among murine BAV models may lead to BAV therapeutic targets and biomarkers of disease progression for this highly prevalent and heterogeneous cardiovascular malformation.

Neutrophils Modulate Fibroblast Function and Promote Healing and Scar Formation after Murine Myocardial Infarction.

Aim: Recruitment of neutrophils to the heart following acute myocardial infarction (MI) initiates inflammation and contributes to adverse post-infarct left ventricular (LV) remodeling. However, therapeutic inhibition of neutrophil recruitment into the infarct zone has not been beneficial in MI patients, suggesting a possible dual role for neutrophils in inflammation and repair following MI. Here, we investigate the effect of neutrophils on cardiac fibroblast function following MI. Methods and Results: We found that co-incubating neutrophils with isolated cardiac fibroblasts enhanced the production of provisional extracellular matrix proteins and reduced collagen synthesis when compared to control or co-incubation with mononuclear cells. Furthermore, we showed that neutrophils are required to induce the transient up-regulation of transforming growth factor (TGF)-ß1 expression in fibroblasts, a key requirement for terminating the pro-inflammatory phase and allowing the reparatory phase to form a mature scar after MI. Conclusion: Neutrophils are essential for both initiation and termination of inflammatory events that control and modulate the healing process after MI. Therefore, one should exercise caution when testing therapeutic strategies to inhibit neutrophil recruitment into the infarct zone in MI patients.

Eosinophil accumulation in postnatal lung is specific to the primary septation phase of development

Abstract Type 2 immune cells and eosinophils are transiently present in lung tissue not only in pathology but also during normal postnatal development. However, the lung developmental processes underlying postnatal airway recruitment of eosinophils after birth remain unexplored. We determined that in mice, mature eosinophils are recruited to the lung during P3–14, which corresponds to the primary septation/alveolarization phase of lung development. Developmental eosinophils peaked during P10–14 and exhibited Siglec-Fmed/highCD11c−/low phenotypes, similar to allergic asthma models. By interrogating the lung transcriptome and proteome during peak eosinophil recruitment in postnatal development, we identified markers that functionally capture mesenchymal-epithelial interface establishment (Nes, Smo, Wnt5a, Nog) and provisional extracellular matrix (ECM) deposition (Tnc, Postn, Spon2, Thbs2) as key lung morphogenetic events associating with eosinophils. We identifiedTenascin-C (TNC) as one of the key ECM markers in the lung epithelial-mesenchymal interface at the RNA and protein levels, consistently associating with eosinophils in development and disease. By RNA-seq analysis, naïve murine eosinophils cultured with TNC-enriched ECM significantly induced expression of Siglec-F, CD11c, eosinophil peroxidase, and other markers typical for activated eosinophils in development and allergic inflammatory responses. TNC knockout mice had an altered eosinophil recruitment profile in development. Collectively, our results indicate that lung morphogenetic processes associated with heightened Type 2 immunity are not merely tissue “background” but specifically guide immune cells both in development and pathology.

Innate tissue properties drive improved tendon healing in MRL/MpJ and harness cues that enhance behavior of canonical healing cells.

Development of tendon therapeutics has been hindered by the lack of informative adult mammalian models of regeneration. Murphy Roth's Large (MRL/MpJ) mice exhibit improved healing following acute tendon injuries, but the driver of this regenerative healing response remains unknown. The tissue-specific attributes of this healing response, despite a shared systemic environment within the mouse, support the hypothesis of a tissue-driven mechanism for scarless healing. Our objective was to investigate the potential of MRL/MpJ tendon extracellular matrix (ECM)-derived coatings to regulate scar-mediated healing. We found that deviations in the composition of key structural proteins within MRL/MpJ vs C57Bl/6tendons occur synergistically tomediate the improvements in structure and mechanics following a 1-mm midsubstance injury. Improvement in mechanical properties of healing MRL/MpJ vs C57Bl/6 tendons that were isolated from systemic contributions via organ culture, highlighted the innate tendon environment as the driver of scarless healing. Finally, we established that decellularized coatings derived from early-deposited MRL/MpJ tendon provisional extracellular matrix (provisional-ECM), can modulate canonical healing B6 tendon cell behavior by inducing morphological changes and increasing proliferation in vitro. This study supports that the unique compositional cues in MRL/MpJ provisional-ECM have the therapeutic capability to motivate canonically healing cells toward improved behavior; enhancing our ability to develop effective therapeutics.

Eosinophil accumulation in postnatal lung is specific to the primary septation phase of development

Type 2 immune cells and eosinophils are transiently present in the lung tissue not only in pathology (allergic disease, parasite expulsion) but also during normal postnatal development. However, the lung developmental processes underlying airway recruitment of eosinophils after birth remain unexplored. We determined that in mice, mature eosinophils are transiently recruited to the lung during postnatal days 3–14, which specifically corresponds to the primary septation/alveolarization phase of lung development. Developmental eosinophils peaked during P10-14 and exhibited Siglec-Fmed/highCD11c−/low phenotypes, similar to allergic asthma models. By interrogating the lung transcriptome and proteome during peak eosinophil recruitment in postnatal development, we identified markers that functionally capture the establishment of the mesenchymal-epithelial interface (Nes, Smo, Wnt5a, Nog) and the deposition of the provisional extracellular matrix (ECM) (Tnc, Postn, Spon2, Thbs2) as a key lung morphogenetic event associating with eosinophils. Tenascin-C (TNC) was identified as one of the key ECM markers in the lung epithelial-mesenchymal interface both at the RNA and protein levels, consistently associating with eosinophils in development and disease in mice and humans. As determined by RNA-seq analysis, naïve murine eosinophils cultured with ECM enriched in TNC significantly induced expression of Siglec-F, CD11c, eosinophil peroxidase, and other markers typical for activated eosinophils in development and allergic inflammatory responses. TNC knockout mice had an altered eosinophil recruitment profile in development. Collectively, our results indicate that lung morphogenetic processes associated with heightened Type 2 immunity are not merely a tissue “background” but specifically guide immune cells both in development and pathology.

Does fibrinogen serve the host or the microbe in Staphylococcus infection?

Fibrin(ogen) is a multifunctional clotting protein that not only has critical roles in hemostasis but is also important in inflammatory processes that control bacterial infection. As a provisional extracellular matrix protein, fibrin(ogen) functions as a physical barrier, a scaffold for immune cell migration, or as a spatially-defined cue to drive inflammatory cell activation. These mechanisms contribute to overall host antimicrobial defense against infection. However, numerous bacterial species have evolved mechanisms to manipulate host fibrin(ogen) to promote microbial virulence and survival. Staphylococcal species, in particular, express numerous virulence factors capable of engaging fibrin(ogen), promoting fibrin formation, and driving the dissolution of fibrin matrices. Recent studies have highlighted both new insights into the molecular mechanisms involved in fibrin(ogen)-mediated host defense and pathogen-driven virulence. Of particular interest is the role of fibrin(ogen) in forming host protective biofilms versus pathogen protective barriers and biofilms as well as the role of fibrin(ogen) in mediating direct host antimicrobial responses. Current data suggest that the role of fibrin(ogen) in staphylococcal infection is highly context-dependent and that better defining the precise cellular and molecular pathways activated will provide unique opportunities of therapeutic intervention to better treat Staphylococcal disease.

Macrophage‐Specific Phenotypes Direct Fibroblast Expression of Matrix Metallopeptidases

IntroductionDuring wound repair, fibroblasts secrete abundant extracellular matrix (ECM) components and participate in remodeling of the provisional ECM. Macrophages and other immune cells play a critical role in all stages of wound healing and are primarily responsible for directing ECM‐producing cells into the wound site to replace it with the new scar tissue. Classically activated macrophages, M1M□, contribute to inflammation early on, whereas alternatively activated macrophages, M2M□, secrete ECM‐degrading enzymes, including matrix metallopeptidases (MMPs), during resolution. Macrophages instruct fibroblast activities, largely via secreted factors. However, the differential effects of M1M□vs M2M□ on fibroblasts remains unknown. This study is designed to evaluate the various roles of differentially polarized macrophages through secreted factors on fibroblast activities that relate to wound resolution.MethodNaïve macrophages(M0) were differentiated into either M1M□ using IFN‐gamma and LPS, or M2M□ using IL‐4. To study the effects of secreted factors and cell‐contact dependent factors, we used indirect (condition media) and direct macrophages‐fibroblast co‐culture systems, respectively with and without TGFβ. Morphologic, phenotypic, and transcriptomic characterization, and cytokine analysis was performed. Cultures were also evaluated for relative gene expression of extracellular matrix genes (MMP8, MMP9, TIMP1, and TIMP2).ResultsWe have shown that M1M□ increases gene expression of matrix degrading enzymes, MMP8 and MMP9, while M2M□ enhanced fibroblast expression of matrix signaling proteins ERK, p38 and AKT. We further have shown that M1M□ highly expresses chemokine CXCL10, a known inhibitor of fibroblast matrix production. We use CXCR3−/− fibroblasts, as CXCR3 is the only known receptor of CXCL10, and treated the cells with both M1M□ vs M2M□ condition media. The fibroblasts showed no change in MMP8 gene expression. However, when we knocked out CXCR3 in M1M□ and cultured them with fibroblasts, fibroblast MMP8 and MM9 expression was diminished.ConclusionThese data suggest that macrophage‐induction of fibroblast expression of MMP8 and MM9 is independent of CXCR3 expression on fibroblasts, but requires the expression of CXCR3 on macrophages. These data points to complex macrophage‐fibroblast crosstalk coordination and the dependency in tissue repair.Support or Funding InformationFunding Source: American Society of Investigative Pathology‐ Summer Research Opportunity in Pathology Program (SROPP), and University of Pittsburgh School of Nursing supported by grants from NIAMS and NIGMS (AR68317 CCY), and support in kind from and the School of Nursing.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Matrix crosslinking enhances macrophage adhesion, migration, and inflammatory activation.

Macrophages are versatile cells of the innate immune system that can adopt a variety of functional phenotypes depending on signals in their environment. In previous work, we found that culture of macrophages on fibrin, the provisional extracellular matrix protein, inhibits their inflammatory activation when compared to cells cultured on polystyrene surfaces. Here, we sought to investigate the role of matrix stiffness in the regulation of macrophage activity by manipulating the mechanical properties of fibrin. We utilize a photo-initiated crosslinking method to introduce dityrosine crosslinks to a fibrin gel and confirm an increase in gel stiffness through active microrheology. We observe that matrix crosslinking elicits distinct changes in macrophage morphology, integrin expression, migration, and inflammatory activation. Macrophages cultured on a stiffer substrate exhibit greater cell spreading and expression of αM integrin. Furthermore, macrophages cultured on crosslinked fibrin exhibit increased motility. Finally, culture of macrophages on photo-crosslinked fibrin enhances their inflammatory activation compared to unmodified fibrin, suggesting that matrix crosslinking regulates the functional activation of macrophages. These findings provide insight into how the physical properties of the extracellular matrix might control macrophage behavior during inflammation and wound healing.

αvβ3 Integrin drives fibroblast contraction and strain stiffening of soft provisional matrix during progressive fibrosis

Fibrosis is characterized by persistent deposition of extracellular matrix (ECM) by fibroblasts. Fibroblast mechanosensing of a stiffened ECM is hypothesized to drive the fibrotic program; however, the spatial distribution of ECM mechanics and their derangements in progressive fibrosis are poorly characterized. Importantly, fibrosis presents with significant histopathological heterogeneity at the microscale. Here, we report that fibroblastic foci (FF), the regions of active fibrogenesis in idiopathic pulmonary fibrosis (IPF), are surprisingly of similar modulus as normal lung parenchyma and are nonlinearly elastic. In vitro, provisional ECMs with mechanical properties similar to those of FF activate both normal and IPF patient-derived fibroblasts, whereas type I collagen ECMs with similar mechanical properties do not. This is mediated, in part, by αvβ3 integrin engagement and is augmented by loss of expression of Thy-1, which regulates αvβ3 integrin avidity for ECM. Thy-1 loss potentiates cell contractility-driven strain stiffening of provisional ECM in vitro and causes elevated αvβ3 integrin activation, increased fibrosis, and greater mortality following fibrotic lung injury in vivo. These data suggest a central role for αvβ3 integrin and provisional ECM in overriding mechanical cues that normally impose quiescent phenotypes, driving progressive fibrosis through physical stiffening of the fibrotic niche.

Promoting effective tendon healing and remodeling.

Daily activities subject our tendons to accumulation of sub-rupture fatigue injury which can lead to tendon rupture. Consequently, tendinopathies account for over 30% of musculoskeletal consultations. We adopted a multidisciplinary approach to determine the role of the extracellular matrix (ECM) in the pathogenesis of tendinopathy and impaired healing of ruptured tendons. We have been investigating three main areas: (i) the pathogenesis of tendon degeneration; (ii) approaches to promoting remodeling of sub-rupture fatigue injuries; and the (iii) role of the ECM in promoting scarless tendon healing. In this Kappa Delta Young Investigator award paper, we describe the key discoveries made in each of our three research areas of focus. Briefly, we discovered that sub-rupture fatigue damage can accumulate from just one bout of fatigue loading. Furthermore, any attempt to repair the fatigue damage diminishes as the severity of induced damage increases. We have utilized exercise to develop animal models of exercise-led degeneration and exercise-led repair of sub-rupture fatigue damage injuries, wherein underlying mechanisms can be uncovered, thereby overcoming a major hurdle to development of therapeutics. Since damage accumulation ultimately leads to rupture that is characterized by formation of a mechanically inferior scar, we have used the MRL/MpJ mouse to evaluate the role of the systemic environment and the local tendon environment in driving regeneration to identify new therapeutic pathways to promote scarless healing. Our data suggests that the therapeutic potential of the MRL/MpJ provisional ECM should be further explored as it may harness biological and structural mechanisms to promote scarless healing. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:3115-3124, 2018.

Fibrin network adaptation to cell-generated forces

Fibrin promotes wound healing by serving as provisional extracellular matrix for fibroblasts that realign and degrade fibrin fibers, and sense and respond to surrounding substrate in a mechanical-feedback loop. We aimed to study mechanical adaptation of fibrin networks due to cell-generated forces at the micron-scale. Fibroblasts were elongated-shaped in networks with ≤ 2 mg/ml fibrinogen, or cobblestone-shaped with 3 mg/ml fibrinogen at 24 h. At frequencies f < 102 Hz, G′ of fibroblast-seeded fibrin networks with ≥ 1 mg/ml fibrinogen increased compared to that of fibrin networks. At frequencies f > 103 Hz, G″ of fibrin networks decreased with increasing concentration following the power-law in frequency with exponents ranging from 0.75 ± 0.03 to 0.43 ± 0.03 at 3 h, and of fibroblast-seeded fibrin networks with exponents ranging from 0.56 ± 0.08 to 0.28 ± 0.06. In conclusion, fibroblasts actively contributed to a change in viscoelastic properties of fibrin networks at the micron-scale, suggesting that the cells and fibrin network mechanically interact. This provides better understanding of, e.g., cellular migration in wound healing.Graphical abstract

Matrix protein Tenascin-C expands and reversibly blocks maturation of eosinophil progenitors

Abstract While eosinophil depletion is a sought after strategy in treating allergic disease, little is known about the local tissue factors regulating eosinophils. In disease, eosinophils encounter provisional extracellular matrix (ECM) glycoproteins such as Tenascin-C (TNC), which strongly correlates with eosinophil expansion in tissues. While restricted in healthy adult lungs, TNC is a hematopoietic niche component in bone marrow stroma and is expressed de novo during wound healing or in pathologic epithelial remodeling in asthma. RNA-Seq shows that exposing naïve murine eosinophils to TNC upregulates immaturity markers (CD34, CD117, and Sca-1) and suppresses IL-5Rα. Thus, we hypothesized that TNC represents a factor in the provisional allergic tissue microenvironment that could support in situ eosinophil progenitor expansion. In murine bone marrow-derived cultures, TNC: 1) 3-fold expanded the available Lin−Sca1+ early precursor pool; 2) downregulated IL-5Rα expression on Lin− CD45+ c-kit+ cells during the eosinophil lineage commitment phase; 3) served as a reversible eosinophil maturation block, as TNC withdrawal rescued maturation and increased final eosinophil yield. Moreover, TNC knockout mice lack in situ lung expansion of Lin− c-kit+ CD34+ common myeloid progenitors and Lin− Siglec-F+ Sca-1+ cells in an allergic inflammation model as well as exhibit accelerated lung eosinophil maturation ex vivo. Adding TNC to lung homogenates cultured in IL-5 ex vivo suppressed eosinophil maturation in a manner consistent with TNC block of eosinophil maturation in bone marrow-derived cultures. Together, our results stress the local tissue factor potential to promote in situ expansion and eosinophil persistence in allergic disease.

Abstract 088: Fibronectin Promotes EphA2 Expression through RGD Integrin Signaling in Vascular Smooth Muscle Cells

Vascular smooth muscle cells undergo a phenotypic shift to a “synthetic” phenotype during atherosclerosis characterized by downregulation of contractile markers and augmented proliferation, migration, and deposition of extracellular matrix. While absent in contractile, medial vascular smooth muscle, the receptor tyrosine kinase EphA2 is detectable in vascular smooth muscle within the plaque and in synthetic vascular smooth muscle cells in vitro , and deletion of EphA2 in ApoE knockout mice attenuates plaque size and progression characterized by a loss of smooth muscle and fibrous tissue content. However, the mechanisms driving EphA2 expression in vascular smooth muscle remains unknown. Both serum treatment and plating on provisional extracellular matrix proteins (e.g. fibronectin) are sufficient to induce EphA2 protein expression and transcriptional activation of the EphA2 promoter. While multiple factors in serum could contribute to EphA2 expression, treating vascular smooth muscle cells with individual growth factors (PDGF, FGF, EGF) or insulin failed to recapitulate the increase in EphA2 expression. However, depleting fibronectin from the serum significantly attenuated expression of EphA2, suggesting fibronectin may critically regulate smooth muscle EphA2 expression. This reduction in serum-induced EphA2 expression upon fibronectin depletion was further enhanced by siRNA knockdown of smooth muscle fibronectin expression, suggesting that both serum fibronectin and smooth muscle-derived fibronectin contribute to the enhanced EphA2 expression. Fibronectin binds to a distinct subset of RGD-binding integrins, and blocking fibronectin-integrin interactions or inhibiting specific fibronectin-binding integrins both attenuate EphA2 expression. Together these data identify a novel role for fibronectin-dependent integrin signaling in the induction of smooth muscle EphA2 expression during phenotypic transition.

Bi‐directional Macrophage‐Fibroblast Crosstalk Directs Wound Resolution Factors

IntroductionDuring the wound repair process, fibroblasts secrete abundant extracellular matrix (ECM) proteins and participate in remodeling of the provisional ECM. Macrophages and other immune cells also play an important role in all stages of wound healing. Classically activated macrophages (CAMs/M1) contribute to inflammation early on, while alternatively activated macrophages (AAMs/M2) secrete ECM‐degrading enzymes including matrix metallopeptidases (MMPs) during the resolution phase. It is also understood that macrophages instruct fibroblast activities and that crosstalk is largely coordinated by secreted factors. However, the differential effect of CAMs and AAMs on fibroblasts remains elusive. CXCR3 is a chemokine receptor known to modulate both fibroblast and macrophage activity during wound healing and its activation acts as a critical stop signal to halt continuous ECM remodeling by fibroblasts. Yet, the importance of CXCR3 in macrophage‐fibroblast crosstalk during tissue repair remains explored. Therefore, the goal of this study was to investigate the interplay between macrophages and fibroblasts and elucidate the role of CXCR3 signaling in this interaction.MethodsWe differentiated naïve bone marrow‐derived macrophages (M0) into either CAMs/M1 using IFN‐gamma and LPS, or AAMs/M2 using IL‐4. To study the effects of secreted and cell‐contact dependent factors, we used both indirect and direct co‐culture systems. The role of CXCR3 in macrophages and fibroblasts was examined using cells from wild‐type (WT) and CXCR3−/− knockout mice.ResultsCytokine profiling revealed that fibroblasts treated with media conditioned (CM) by CAMs secreted higher levels of CXCR3 ligands CXCL10 and CXCL11 compared to untreated fibroblasts. Because both fibroblasts and macrophages express CXCR3, it was unclear which cell type might respond to these ligands. Investigation using qRT‐PCR, showed that treatment with WT CAM CM resulted in increased fibroblast expression of MMP8 and MMP9 compared to no treatment. Fibroblast induction of MMPs by WT CAM CM persisted when CXCR3−/− fibroblasts were treated with WT CAM CM, but was greatly diminished when fibroblasts were cultured with CXCR3−/− CAM CM, suggesting that this crosstalk requires CXCR3 expression on CAMs but not on fibroblasts. The influence of macrophage subtypes on MMP8, MMP9, and TGF‐β‐induced α‐SMA and ECM protein production was observed by IF staining.ConclusionsOur data indicate that CAMs induce fibroblast expression of CXCR3 ligands and key matrix degrading enzymes MMP8 and MMP9, with MMP expression requiring CXCR3 expression on CAMs. Thus, our study suggests for the first time that paracrine interaction between fibroblasts and macrophages dictate the effector functions of fibroblasts and the wound healing process.Support or Funding InformationSupported by grants from NIAMS and NIAD (AR68317 CCY and R56A1097679 NI), and support in kind from the Pittsburgh VA Medical Center (AW), the School of Nursing, and McGowan Institute of Regenerative Medicine (BNB, CCY).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.