Conversion Of Fibroblasts Research Articles

Targeting fibroblast phenotype switching in cardiac remodelling as a promising antifibrotic strategy.

Myocardial fibrosis, a common feature of heart disease, remains an unsolved clinical challenge. Fibrosis resolution requires activation of cardiac fibroblasts exhibiting context-dependent beneficial and detrimental dichotomy. Here, we explored the hypothesis of fibroblast reversible transition between quiescence and activated myofibroblastic states as a manifestation of cell phenotypic switching in myocardial remodelling. In support, gene regulatory networks executing conversion of cardiac fibroblasts to myofibroblasts and vice versa in fibrosis resolution are reconstructed using TRANSPATH database. In a scenario of fibroblast activation triggered by transforming growth factor β, a cardinal mediator of tissue fibrosis, signalling cascades governing entry into or exit from specific fibroblast statures in cardiac fibrotic remodelling were dissected. It is suggested that fibroblast phenotypic switching constitutes the central gait toward guiding cell state-gating strategies to counteract adverse cardiac fibrosis, a devastating disorder with no approved therapeutic option.

Evidence that CRISPR-Cas9 Y537S-mutant expressing breast cancer cells activate Yes-associated protein 1 to driving the conversion of normal fibroblasts into cancer-associated fibroblasts

BackgroundEndocrine therapy (ET) has improved the clinical outcomes of Estrogen receptor alpha-positive (ERɑ +) breast cancer (BC) patients, even though resistance to ET remains a clinical issue. Mutations in the hormone-binding domain of ERɑ represent an acquired intrinsic mechanism of ET resistance. However, the latter also depends on the multiple functional interactions between BC cells and the tumor microenvironment (TME). Here, we investigated how the most common Y537S-ERɑ mutation may influence the behavior of fibroblasts, the most prominent component of the TME.MethodsWe conducted coculture experiments with normal human foreskin fibroblasts BJ1-hTERT (NFs), cancer-associated fibroblasts (CAFs), isolated from human BC specimens, and Y537S CRISPR-expressing MCF-7 BC cells (MCF-7YS). Mass spectrometry (MS) and Metacore analyses were performed to investigate how the functional interactions between BC cells/fibroblasts may affect their proteomic profile. The impact of fibroblasts on BC tumor growth and metastatic potential was evaluated in nude mice.ResultsMutant BC conditioned medium (CM) affected the morphology/proliferation/migration of both NFs and CAFs. 198 deregulated proteins signed the proteomic similarity profile of NFs exposed to the YS-CM and CAFs. Among the upregulated proteins, Yes-associated protein 1 (YAP1) was the main central hub in the direct interaction network. Increased YAP1 protein expression and activity were confirmed in NFs treated with MCF-7YS-CM. However, YAP1 activation appears to crosstalk with the insulin growth factor-1 receptor (IGF-1R). Higher amount of IGF-1 were noticed in the MCF-7YS-CM cells compared to the MCF-7P, and IGF-1 immunodepletion reversed the enhanced YAP1 expression and activity. Mutant cells upon exposure to the NF- and CAF-CM exhibited an enhanced proliferation/growth/migration/invasion compared to the MCF-7P. MCF-7YS cells when implanted with CAFs showed an early relative increased tumor volume compared to YS alone. No changes were observed when MCF-7P cells were co-implanted with CAFs. Compared with that in MCF-7P cells, the metastatic burden of MCF-7YS cells was intrinsically greater, and this effect was augmented upon treatment with NF-CM and further increased with CAF-CM.ConclusionsYS mutant BC cells induced the conversion of fibroblasts into CAFs, via YAP, which represent a potential therapeutic target which interrupt the functional interactions between mutant cells/TME and to be implemented in the novel therapeutic strategy of a subset of metastatic BC patients carrying the frequent Y537S mutations.

Age-associated metabolic and epigenetic barriers during direct reprogramming of mouse fibroblasts into induced cardiomyocytes.

Heart disease is the leading cause of mortality in developed countries, and novel regenerative procedures are warranted. Direct cardiac conversion (DCC) of adult fibroblasts can create induced cardiomyocytes (iCMs) for gene and cell-based heart therapy, and in addition to holding great promise, still lacks effectiveness as metabolic and age-associated barriers remain elusive. Here, by employing MGT (Mef2c, Gata4, Tbx5) transduction of mouse embryonic fibroblasts (MEFs) and adult (dermal and cardiac) fibroblasts from animals of different ages, we provide evidence that the direct reprogramming of fibroblasts into iCMs decreases with age. Analyses of histone posttranslational modifications and ChIP-qPCR revealed age-dependent alterations in the epigenetic landscape of DCC. Moreover, DCC is accompanied by profound mitochondrial metabolic adaptations, including a lower abundance of anabolic metabolites, network remodeling, and reliance on mitochondrial respiration. Invitro metabolic modulation and dietary manipulation invivo improve DCC efficiency and are accompanied by significant alterations in histone marks and mitochondrial homeostasis. Importantly, adult-derived iCMs exhibit increased accumulation of oxidative stress in the mitochondria and activation of mitophagy or dietary lipids; they improve DCC and revert mitochondrial oxidative damage. Our study provides evidence that metaboloepigenetics plays a direct role in cell fate transitions driving DCC, highlighting the potential use of metabolic modulation to improve cardiac regenerative strategies.

MiR-24-3p secreted as extracellular vesicle cargo by cardiomyocytes inhibits fibrosis in human cardiac microtissues.

Cardiac fibrosis in response to injury leads to myocardial stiffness and heart failure. At the cellular level, fibrosis is triggered by the conversion of cardiac fibroblasts (CF) into extracellular matrix-producing myofibroblasts. miR-24-3p regulates this process in animal models. Here, we investigated whether miR-24-3p plays similar roles in human models. Gain- and loss-of-function experiments were performed using human induced pluripotent stem cell-derived cardiomyocytes (hCM) and primary hCF under normoxic or ischaemia-simulating conditions. hCM-derived extracellular vesicles (EVs) were added to hCF. Similar experiments were performed using three-dimensional human cardiac microtissues and ex vivo-cultured human cardiac slices.hCF transfection with miR-24-3p mimic prevented TGFβ1-mediated induction of FURIN, CCND1 and SMAD4-miR-24-3p target genes participating in TGFβ1-dependent fibrinogenesis -, regulating hCF-to-myofibroblast conversion. hCM secreted miR-24-3p as EV cargo. hCM-derived EVs modulated hCF activation. Ischaemia-simulating conditions induced miR-24-3p depletion in hCM-EVs and microtissues. Similarly, hypoxia downregulated miR-24-3p in cardiac slices. Analyses of clinical samples revealed decreased miR-24-3p levels in circulating EVs in acute myocardial infarction (AMI) patients, compared with healthy subjects. Post-mortem RNAScope analysis showed miR-24-3p downregulation in myocardium from AMI patients, compared with patients who died from noncardiac diseases. Berberin, a plant-derived agent with miR-24-3p-stimulatory activity, increased miR-24-3p contents in hCM-EVs, downregulated FURIN, CCND1 and SMAD4, and inhibited fibrosis in cardiac microtissues. These findings suggest that hCM may control hCF activation through miR-24-3p secreted as EV cargo. Ischaemia impairs this mechanism, favouring fibrosis.

Neural crest precursors from the skin are the primary source of directly reprogrammed neurons

Direct reprogramming involves the conversion of differentiated cell types without returning to an earlier developmental state. Here, we explore how heterogeneity in developmental lineage and maturity of the starting cell population contributes to direct reprogramming using the conversion of murine fibroblasts into neurons. Our hypothesis is that a single lineage of cells contributes to most reprogramming and that a rare elite precursor with intrinsic bias is the source of reprogrammed neurons. We find that nearly all reprogrammed neurons are derived from the neural crest (NC) lineage. Moreover, when rare proliferating NC precursors are selectively ablated, there is a large reduction in the number of reprogrammed neurons. Previous interpretations of this paradigm are that it demonstrates a cell fate conversion across embryonic germ layers (mesoderm to ectoderm). Our interpretation is that this is actually directed differentiation of a neural lineage stem cell in the skin that has intrinsic bias to produce neuronal progeny.

Cardiac Repair after Myocardial Infarction is Controlled by a Complement C5a Receptor 1-Driven Signaling Cascade.

Cardiac repair following myocardial infarction is important in regenerating functionally viable myocardium to prevent cardiac death. Previous studies have linked C5aR1 to cardiac regeneration and inflammation. However, C5a receptor-driven responses during the late phases, up to 4 weeks of the infarction insult - a time window that specifically reflects the outcome of the repair process - and the underlying mechanisms are poorly defined. Here, we show that C5ar1, but not C5ar2, deficiency attenuates infarct size following coronary artery ligation-induced myocardial infarction (MI). C5ar1 deficiency limited the deposition of collagen and mitigated cell death in infarcted areas leading to overall improved cardiac function four weeks after MI. While infiltration of neutrophils was reduced in both C5ar1-/- and C5ar2-/- infarcted myocardium 24 h after MI, influx of monocytes after 1 week of MI was reduced only upon C5ar1 deficiency. Subsequent analysis revealed reduced accumulation of myofibroblast, elevated expression of transforming growth factor-beta1 (Tgf-β1) and vascular endothelial growth factor-A (Vegf-A) in C5ar1-/- infarcted hearts. In vitro, exogenous TGF-β1 triggered conversion of fibroblasts into myofibroblasts, which expressed Vegf-A mRNA - an effect that was enhanced upon C5ar1 deficiency. Incubation of C5ar1+/+ or C5ar1-/- endothelial cells (ECs) with supernatants from C5ar1+/+ or C5ar1-/- myofibroblasts respectively, mimicking the in vivo microenvironment in our model, led to significant increase in matrigel tube formation in C5ar1-/- ECs. This was consistent with enhanced neoangiogenesis in C5ar1-/- infarcted hearts. Collectively, our study demonstrates that inhibition of C5aR1 has the potential to improve cardiac function after myocardial infarction.

Protein kinase N promotes cardiac fibrosis in heart failure by fibroblast-to-myofibroblast conversion

Chronic fibrotic tissue disrupts various organ functions. Despite significant advances in therapies, mortality and morbidity due to heart failure remain high, resulting in poor quality of life. Beyond the cardiomyocyte-centric view of heart failure, it is now accepted that alterations in the interstitial extracellular matrix (ECM) also play a major role in the development of heart failure. Here, we show that protein kinase N (PKN) is expressed in cardiac fibroblasts. Furthermore, PKN mediates the conversion of fibroblasts into myofibroblasts, which plays a central role in secreting large amounts of ECM proteins via p38 phosphorylation signaling. Fibroblast-specific deletion of PKN led to a reduction of myocardial fibrotic changes and cardiac dysfunction in mice models of ischemia-reperfusion or heart failure with preserved ejection fraction. Our results indicate that PKN is a therapeutic target for cardiac fibrosis in heart failure.

Direct Cardiac Reprogramming in the Age of Computational Biology.

Heart disease continues to be one of the most fatal conditions worldwide. This is in part due to the maladaptive remodeling process by which ischemic cardiac tissue is replaced with a fibrotic scar. Direct cardiac reprogramming presents a unique solution for restoring injured cardiac tissue through the direct conversion of fibroblasts into induced cardiomyocytes, bypassing the transition through a pluripotent state. Since its inception in 2010, direct cardiac reprogramming using the transcription factors Gata4, Mef2c, and Tbx5 has revolutionized the field of cardiac regenerative medicine. Just over a decade later, the field has rapidly evolved through the expansion of identified molecular and genetic factors that can be used to optimize reprogramming efficiency. The integration of computational tools into the study of direct cardiac reprogramming has been critical to this progress. Advancements in transcriptomics, epigenetics, proteomics, genome editing, and machine learning have not only enhanced our understanding of the underlying mechanisms driving this cell fate transition, but have also driven innovations that push direct cardiac reprogramming closer to clinical application. This review article explores how these computational advancements have impacted and continue to shape the field of direct cardiac reprogramming.

An Efficient Direct Conversion Strategy to Generate Functional Astrocytes from Human Adult Fibroblasts.

Direct reprogramming approaches offer an attractive alternative to stem-cell-derived models, allowing the retention of epigenetic information and age-associated cellular phenotypes, and providing an expedited method to generate target cell types. Several groups have previously generated multiple neuronal subtypes, neural progenitor cells, oligodendrocytes, and other cell types directly from fibroblasts. However, while some groups have had success at the efficient conversion of embryonic fibroblasts to astrocytes, they have not yet achieved similar conversion efficiency for adult human fibroblasts. To generate astrocytes for the study of adult-stage disorders, we developed an improved direct conversion strategy employing a combination of small molecules to activate specific pathways that induce trans-differentiation of human adult fibroblasts to astrocytes. We demonstrate that this method produces mature GFAP+/S100β+ cells at high efficiency (40-45%), comparable to previous studies utilizing embryonic fibroblasts. Further, Fibroblast-derived induced Astrocytes (FdiAs) are enriched for markers of astrocyte functionality, including ion-channel buffering, gap-junction communication, and glutamate uptake; and exhibit astrocyte-like calcium signaling and neuroinflammatory phenotypes. RNA-Seq analysis indicates a close correlation to human brain astrocytes and iPSC-derived astrocyte models. Fibroblast-derived induced astrocytes provide a useful tool in studying the adult brain and complement existing in vitro models of induced neurons (iNs), providing an additional platform to study adult-stage brain disorders.

Extracellular vesicles derived from melanoma cells induce carcinoma-associated fibroblasts via miR-92b-3p mediated downregulation of PTEN.

In melanoma, carcinoma-associated fibroblasts (CAFs) are important cellular components in the tumour microenvironment due to their potential to promote tumour growth and metastatic spread of malignant cells. Melanoma cells have the ability to affect non-tumour cells in the microenvironment by releasing extracellular vesicles (EVs). The mechanisms responsible for reprogramming normal dermal fibroblasts (NHDFs) into CAFs remain incompletely understood. However, it is likely thought to be mediated by melanoma-specific miRNAs, which are transported by EVs derived from melanoma cells. Therefore, we wondered if one of the most enriched miRNAs in EVs secreted by melanoma cells, miR-92b-3p, is involved in the conversion of normal fibroblasts into CAFs. We observed that melanoma cell-derived EVs indeed delivered miR-92b-3p into NHDFs and that its accumulation correlated with CAF formation, as demonstrated by enhanced expression of CAF marker genes and increased proliferation and migration. Overexpression of miR-92b-3p in NHDFs revealed similar results, while EVs deficient of miR-92b-3p did not induce a CAF phenotype. As a target we identified PTEN, whose repression led to increased expression of CAF markers. We thus provide a novel pathway of intercellular communication by which melanoma cells control the transformation of CAFs by virtue of EV-transported miRNAs.

Abstract We033: Heart-Specific Histone Methyltransferase SMYD1 Promotes Cardiac Maturation in the Direct Conversion of Human Fibroblasts into Cardiomyocytes.

Introduction: Direct cardiac reprogramming emerges as a promising strategy for in situ conversion of fibroblasts into induced cardiomyocyte-like cells (iCMs). While the conversion of mouse fibroblasts into functional, beating iCMs has been achieved efficiently, the reprogramming of human iCMs remains challenging, particularly their maturation with existing transcription factor combinations. Given the pivotal role of epigenetic regulation in this process, our study sought to leverage heart-specific epigenetic factors alongside cardiac-enriched transcription factors to enhance cardiac maturation, thereby yielding more reliable human iCMs. Result: Through analyzing the proteomic data across various human tissues, we found that while many epigenetic factors are broadly expressed, SMYD1 is uniquely heart-specific. To determine its involvement in reprogramming, we overexpressed SMYD1 and evaluated the reprogramming efficiency and iCM quality via flow cytometry, immunofluorescence staining, and transcriptome analysis. Notably, we observed an increase in iCMs expressing sarcomere markers, such as α-myosin heavy chain and α-actinin, as well as the ventricular specific myosin-light-chain 2v in SMYD1-treated group. Gene set enrichment analysis of RNA-sequencing data further indicated that SMYD1-induced iCMs exhibit ventricular gene signatures. Additionally, we discovered moderate activation of SMYD1 in mature iCMs generated with MEF2C, GATA4, TBX5, and TBX20. TBX20 is the key factor that leads to the co-binding of MEF2C, TBX5, and TBX20 on the SMYD1 promoter region and increases active histone marks, H3K27ac and H3K4me1. Rescue assay and transcriptomic comparison confirmed SMYD1 as the primary downstream target of TBX20 in promoting iCM functional maturation. Mechanistically, SMYD1 directly interacts with MEF2C and GATA4 to modify histone marks on cardiac regulatory regions. Conclusion: Our study provides comprehensive phenotypic and molecular evidence of SMYD1’s role as a cardiac-specific regulator during direct cardiac reprogramming. By interacting with key reprogramming factors and rewriting histone marks, SMYD1 significantly advances the maturation of human iCMs towards a ventricular subtype identity.

Abstract Tu061: Pericyte-mediated perivascular fibrosis in the pressure-overloaded heart is dependent on TGF-β signaling and is restrained by ITGB1.

Background: Pericytes exhibit remarkable phenotypic plasticity and have been implicated in the pathogenesis of fibrosis. Cardiac pericytes are involved in repair and fibrotic remodeling of the infarcted heart; however, their role in the cardiomyopathy induced by pressure overload is not known. We hypothesized that cardiac pericytes may contribute to remodeling of the pressure-overloaded heart by converting into fibroblasts and we examined the molecular signals mediating pericyte activation. Methods and Results: Transverse aortic constriction (TAC) protocols were performed to study the role of pericytes in the pressure-overloaded heart. Using fibroblast:pericyte dual reporter mice and lineage tracing experiments with the inducible NG2 CreER mice (to track pericytes) combined with PDGFRa EGFP reporter mice (to reliably label fibroblasts), we found no pericyte to fibroblast conversion in the pressure-overloaded heart. scRNA-seq demonstrated that pericyte lineage cells do not express fibroblast identity genes after TAC but acquire a fibrogenic phenotype expressing high levels of matricellular genes, growth factors, adhesion molecules and integrins. Bioinformatic analysis identified transforming growth factor (TGF)-b and integrin beta-1 (ITGB1) as important upstream regulators of the transcriptional profile of pericytes after TAC. Pericyte-specific loss of transforming growth factor beta receptor 2 (TGFBR2) attenuated dysfunction and perivascular fibrosis after TAC. In contrast, pericyte-specific ITGB1 loss had deleterious effects increasing mortality, promoting dysfunction, inducing microvascular rarefaction and stimulating a pro-inflammatory and fibrogenic pericyte phenotype. Conclusions: In the pressure-overloaded heart, pericytes acquire a fibrogenic phenotype without converting to fibroblasts. Pericyte-specific activation of TGF-β pathways mediates perivascular myocardial fibrosis after TAC. In contrast, pericyte-specific ITGB1 signaling exerts protective actions, preserving microvascular structure, attenuating inflammation and inhibiting fibrosis.

Nrf2 mediates the effects of shionone on silica-induced pulmonary fibrosis

BackgroundExtended contact with silica particles can lead to Silicosis, a chronic lung condition lacking established treatment protocols or clear mechanisms of development. The urgency for innovative treatments arises from the unavailability of effective treatment methodologies. The origin of silica-induced pulmonary fibrosis includes essential processes such as macrophage activation and the conversion of fibroblasts into myofibroblasts, with oxidative stress playing a pivotal role. Shionone (SHI), a triterpenoid extracted from the Aster tataricus plant, is recognized for its extensive health benefits. This study explores the capability of SHI to alleviate the effects of silica-induced lung fibrosis in mice.MethodsThis investigation explored the impact of SHI on lung inflammation and fibrosis at different stages (early and late) triggered by silica in mice, focusing specifically on the initial and more developed phases. It comprised an analysis of isolated peritoneal macrophages and fibroblasts extracted from mice to elucidate SHI's therapeutic potential and its underlying mechanism. The methodology employed encompassed quantitative PCR, immunofluorescence, flow cytometry, and western blotting to examine macrophage activity and their transition into myofibroblasts. The activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway by SHI was confirmed via immunofluorescence and western blot studies. SHI's antioxidative properties were evidenced by the measurement of reactive oxygen species (ROS) and mitochondrial ROS within both macrophages and fibroblasts, using 2′, 7′-dichlorodihydrofluorescein diacetate and MitoSOX, respectively. The relevance of SHI was further underscored by applying ML385 and Nrf2 siRNA to gauge its effectiveness.ResultsStarting SHI treatment early countered the harmful effects of lung inflammation and fibrosis caused by silica, while initiating SHI at a later phase decelerated the advancement of fibrosis. SHI's action was linked to the activation of the Nrf2 signaling pathway, a boost in antioxidant enzyme levels, and a decrease in oxidative stress and inflammation in macrophages affected by silica. Furthermore, SHI prevented the conversion of fibroblasts into myofibroblasts prompted by TGF-β, along with the resultant oxidative stress. The beneficial outcomes of SHI were negated when ML385 and Nrf2 siRNA were applied, highlighting the pivotal role of the Nrf2 pathway in SHI's efficacy.ConclusionSHI plays a significant role in stimulating the Nrf2 pathway, thereby defending against silica-induced oxidative stress and inflammatory reactions in macrophages, and inhibiting the conversion of fibroblasts to myofibroblasts due to TGF-β. This suggests that SHI is a viable option for treating lung inflammation and fibrosis in mice suffering from silicosis.

Fluorescein-Based SynNotch Adaptors for Regulating Gene Expression Responses to Diverse Extracellular Cues.

We introduce an adaptor-based strategy for regulating fluorescein-binding synthetic Notch (SynNotch) receptors using ligands based on conjugates of fluorescein isomers and analogs. To develop a versatile system, we evaluated the surface expression and activities of multiple constructs containing distinct extracellular fluorescein-binding domains. Using an optimized receptor, we devised ways to regulate signaling via fluorescein-based chemical transformations, including an approach based on a bio-orthogonal chemical ligation and a spatially controllable strategy via the photo-patterned uncaging of an o -nitrobenzyl-caged fluorescein conjugate. We further demonstrate that fluorescein-conjugated extracellular matrix (ECM)-binding peptides can regulate SynNotch activity depending on the folding state of collagen-based ECM networks. Treatment with these conjugates enabled cells to distinguish between folded versus denatured collagen proteins and enact dose-dependent gene expression responses depending on the nature of the signaling adaptors presented. To demonstrate the utility of these tools, we applied them to control the myogenic conversion of fibroblasts into myocytes with spatial and temporal precision and in response to denatured collagen-I, a biomarker of multiple pathological states. Overall, we introduce an optimized fluorescein-binding SynNotch as a versatile tool for regulating transcriptional responses to extracellular ligands based on the widely used and clinically-approved fluorescein dye.

Activation of the Macrophage-Associated Inflammasome Exacerbates Myocardial Fibrosis Through the 15-HETE-Mediated Pathway in Acute Myocardial Infarction

This investigation elucidates the spatiotemporal dynamics of NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activation following myocardial infarction (MI), a process that has not been fully characterized. We revealed early activation of the NLRP3 inflammasome in mice with MI and characterized its dynamic temporal expression. Notably, the knockout and inhibition of Nlrp3 expression were found to significantly mitigate infarct size and enhance cardiac function. Furthermore, our analysis of the spatial characteristics of inflammasome activation revealed predominant activation in macrophages and subsequent activation in fibroblasts on the third day post-MI. To elucidate the nexus between macrophage-associated NLRP3 inflammasome activation and myocardial fibrosis, we employed targeted metabolomics analyses of inflammatory oxylipins, small interfering RNA (siRNA) interference experiments, and various molecular assays. These findings revealed that macrophage-associated inflammasome activation facilitates the conversion of fibroblasts into myofibroblasts via the 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE)-mediated small mother against decapentaplegic (Smad) pathway. Additionally, both mass spectrometry imaging (MSI) and targeted metabolomics analyses confirmed the significant increase in 15-HETE levels in mice with MI and in patients with MI and acute coronary syndrome (ACS). Our comprehensive dataset suggests that NLRP3 inflammasome activation in MI is characterized by distinct temporal and spatial patterns. These insights mark a significant advancement toward precise MI prevention and treatment strategies, particularly early myocardial fibrosis intervention.

Direct conversion of cardiac fibroblasts into endothelial-like cells using Sox17 and Erg

Endothelial cells are a heterogeneous population with various organ-specific and conserved functions that are critical to organ development, function, and regeneration. Here we report a Sox17-Erg direct reprogramming approach that uses cardiac fibroblasts to create differentiated endothelial cells that demonstrate endothelial-like molecular and physiological functions in vitro and in vivo. Injection of these induced endothelial cells into myocardial infarct sites after injury results in improved vascular perfusion of the scar region. Furthermore, we use genomic analyses to illustrate that Sox17-Erg reprogramming instructs cardiac fibroblasts toward an arterial-like identity. This results in a more efficient direct conversion of fibroblasts into endothelial-like cells when compared to traditional Etv2-based reprogramming. Overall, this Sox17-Erg direct reprogramming strategy offers a robust tool to generate endothelial cells both in vitro and in vivo, and has the potential to be used in repairing injured tissue.

Abstract 1101: Tnfa-setdb2 Pathway Regulates Fibroblast To Myofibroblast Transition In Wound Repair

Effective healing following tissue injury is predicated on an intimate coordination between immune cells and structural cells, where recruited immune cells induce or repress structural cell proliferation and gene expression. Early after tissue injury, fibroblasts begin to transition to myofibroblasts to contract and close wounds. In pathologic conditions where wounds fail to heal (such as diabetes), fibroblasts do not increase myofibroblast gene expression; however the mechanisms that control this fibroblast transition, in normal or diabetic conditions are not defined. Our group has previously identified that epigenetic alterations can control these phenotypic switches in immune and structural cells. Here, using human wound scSeq and murine transgenic models, we examined the mechanisms that may regulate this transition in the setting of injury. First, we identified that dermal fibroblasts when treated with the pro-inflammatory cytokine TNFa, significantly repressed myofibroblast genes including acta2, tgln, myl9, cald1, fibronectin and periostin. To identify possible the epigenetic mechanisms downstream of TNFa that modulate fibroblast phenotype, we performed an unbiased epigenetic superarray on TNFa-stimulated dermal fibroblasts, where SETDB2 was significantly upregulated and was also confirmed by qPCR. SiRNA-mediated knockdown of Setdb2 in murine fibroblasts prevented TNFa-mediated repression of myofibroblast genes, suggesting regulation of this transition by Setdb2. Human scSeq identified increased Setdb2 associated with increased TNFa signaling in wound fibroblasts, suggesting an interaction of these pathways. Interestingly, we found increased TNFa in macrophages and SETDB2 in fibroblasts from human diabetic wound scSeq and a murine diabetic (DIO) model, suggesting this pathway may contribute to the decreased conversion of fibroblasts to myofibroblasts seen in non-healing diabetic wounds. This work identifies setdb2 as a TNFa-mediated regulator of fibroblast to myofibroblast transition in wound repair and may identify a new therapeutic target for impaired tissue regeneration.

Macrophages suppress cardiac reprogramming of fibroblasts in vivo via IFN-mediated intercellular self-stimulating circuit.

Direct conversion of cardiac fibroblasts (CFs) to cardiomyocytes (CMs) in vivo to regenerate heart tissue is an attractive approach. After myocardial infarction (MI), heart repair proceeds with an inflammation stage initiated by monocytes infiltration of the infarct zone establishing an immune microenvironment. However, whether and how the MI microenvironment influences the reprogramming of CFs remains unclear. Here, we found that in comparison with cardiac fibroblasts (CFs) cultured in vitro, CFs that transplanted into infarct region of MI mouse models resisted to cardiac reprogramming. RNA-seq analysis revealed upregulation of interferon (IFN) response genes in transplanted CFs, and subsequent inhibition of the IFN receptors increased reprogramming efficiency in vivo. Macrophage-secreted IFN-β was identified as the dominant upstream signaling factor after MI. CFs treated with macrophage-conditioned medium containing IFN-β displayed reduced reprogramming efficiency, while macrophage depletion or blocking the IFN signaling pathway after MI increased reprogramming efficiency in vivo. Co-IP, BiFC and Cut-tag assays showed that phosphorylated STAT1 downstream of IFN signaling in CFs could interact with the reprogramming factor GATA4 and inhibit the GATA4 chromatin occupancy in cardiac genes. Furthermore, upregulation of IFN-IFNAR-p-STAT1 signaling could stimulate CFs secretion of CCL2/7/12 chemokines, subsequently recruiting IFN-β-secreting macrophages. Together, these immune cells further activate STAT1 phosphorylation, enhancing CCL2/7/12 secretion and immune cell recruitment, ultimately forming a self-reinforcing positive feedback loop between CFs and macrophages via IFN-IFNAR-p-STAT1 that inhibits cardiac reprogramming in vivo. Cumulatively, our findings uncover an intercellular self-stimulating inflammatory circuit as a microenvironmental molecular barrier of in situ cardiac reprogramming that needs to be overcome for regenerative medicine applications.

HTRA1-driven detachment of type I collagen from endoplasmic reticulum contributes to myocardial fibrosis in dilated cardiomyopathy

BackgroundThe aberrant secretion and excessive deposition of type I collagen (Col1) are important factors in the pathogenesis of myocardial fibrosis in dilated cardiomyopathy (DCM). However, the precise molecular mechanisms underlying the synthesis and secretion of Col1 remain unclear.Methods and resultsRNA-sequencing analysis revealed an increased HtrA serine peptidase 1 (HTRA1) expression in patients with DCM, which is strongly correlated with myocardial fibrosis. Consistent findings were observed in both human and mouse tissues by immunoblotting, quantitative reverse transcription polymerase chain reaction (qRT-PCR), immunohistochemistry, and immunofluorescence analyses. Pearson’s analysis showed a markedly positive correlation between HTRA1 level and myocardial fibrosis indicators, including extracellular volume fraction (ECV), native T1, and late gadolinium enhancement (LGE), in patients with DCM. In vitro experiments showed that the suppression of HTRA1 inhibited the conversion of cardiac fibroblasts into myofibroblasts and decreased Col1 secretion. Further investigations identified the role of HTRA1 in promoting the formation of endoplasmic reticulum (ER) exit sites, which facilitated the transportation of Col1 from the ER to the Golgi apparatus, thereby increasing its secretion. Conversely, HTRA1 knockdown impeded the retention of Col1 in the ER, triggering ER stress and subsequent induction of ER autophagy to degrade misfolded Col1 and maintain ER homeostasis. In vivo experiments using adeno-associated virus-serotype 9-shHTRA1-green fluorescent protein (AAV9-shHTRA1-GFP) showed that HTRA1 knockdown effectively suppressed myocardial fibrosis and improved left ventricular function in mice with DCM.ConclusionsThe findings of this study provide valuable insights regarding the treatment of DCM-associated myocardial fibrosis and highlight the therapeutic potential of targeting HTRA1-mediated collagen secretion.

Abstract 296: BRAFi-induced ROCK-mediated non-canonical nuclear β-catenin shuttling drives a phenotypic switch in cancer-associated fibroblasts

Abstract The discovery of BRAF mutations in around 60% of melanoma patients and the subsequent development of targeted inhibitors to counter the hyperactive BRAF-driven pro-tumorigenic signaling pathway have marked a significant milestone in melanoma therapy. However, despite this progress, the long-term outcomes remain unsatisfactory for many patients due to the occurrence of drug resistance. We previously reported that cancer-associated fibroblasts (CAFs) are stimulated by BRAF inhibitors (BRAFi) to drive matrix remodeling and therapeutic escape pathways in melanoma cells. However, the underlying mechanism by which CAFs are reprogrammed by BRAFi to obtain enhanced phenotypes remains elusive. Here, we present compelling evidence showing that BRAFi (PLX4032), but not CRAF inhibitor (GW5074) or pan-RAF inhibitor (RAF709), induces the accumulation of nuclear β-catenin in CAFs harboring wild-type BRAF, a process mediated by BRAF and CRAF but not ARAF isoform. BRAFi binding to BRAF and CRAF leads to robust RAF kinase dimerization, including BRAF and CRAF homodimer and BRAF: CRAF heterodimer. RAF dimers are subsequently recruited to the plasma membrane, where BRAF and CRAF are phosphorylated on residues Thr599 and Ser338, respectively, and activated in a RAS-GTP-dependent manner. RAF activation stimulates the downstream Rho kinase (ROCK) and MEK/ERK signaling pathways simultaneously. Notably, ablating RAS and RAF isoforms effectively suppressed BRAFi-induced activation of ROCK1/2 signaling in CAFs, further confirming that the ROCK pathway is a downstream effector of BRAFi-driven RAF activation and is intricately linked to increased nuclear β-catenin in CAFs. The pharmacological blockade of ROCK activity, using HA-1077, diminished BRAFi-induced nuclear β-catenin accumulation in CAFs, suggesting BRAFi-driven cytoskeletal remodeling plays a pivotal role in the nuclear shuttling of β-catenin. Using a mouse model that mimics human BRAF-mutant melanoma, we found that elevated level of nuclear β-catenin drives the conversion of fibroblasts into α-SMA-positive CAFs, leading to elevated collagen deposition and contributing to melanoma progression in vivo. In summary, BRAFi reprograms the transcriptional activity in CAFs by driving increased nuclear β-catenin to increase their ability to remodel the tumor ECM and promote melanoma cell proliferation. Collectively, the data offer new insights into the molecular mechanisms that underlie the paradoxical reprogramming of CAFs by BRAFi in melanoma therapy through the non-canonical β-catenin pathway. Citation Format: Bruna da Silva Soley, Radhika Athalye, Ryan Zhang, Thomas Andl, Yuhang Zhang. BRAFi-induced ROCK-mediated non-canonical nuclear β-catenin shuttling drives a phenotypic switch in cancer-associated fibroblasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 296.