Persistence Of Myofibroblasts Research Articles

Cytokine priming enhances the antifibrotic effects of human adipose derived mesenchymal stromal cells conditioned medium

BackgroundFibrosis is a pathological scarring process characterized by persistent myofibroblast activation with excessive accumulation of extracellular matrix (ECM). Fibrotic disorders represent an increasing burden of disease-associated morbidity and mortality worldwide for which there are limited therapeutic options. Reversing fibrosis requires the elimination of myofibroblasts, remodeling of the ECM, and regeneration of functional tissue. Multipotent mesenchymal stromal cells (MSC) have antifibrotic properties mediated by secreted factors present in their conditioned medium (MSC-CM). However, there are no standardized in vitro assays to predict the antifibrotic effects of human MSC. As a result, we lack evidence on the effect of cytokine priming on MSC’s antifibrotic effects. We hypothesize that the MSC-CM promotes fibrosis resolution in vitro and that this effect is enhanced following MSC cytokine priming.MethodsWe compared the antifibrotic effects of resting versus interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) primed MSC-CM in four in vitro assays: prevention of fibroblast activation, myofibroblasts deactivation, ECM degradation and fibrosis resolution in lung explant cultures. Furthermore, we performed transcriptomic analysis of myofibroblasts treated or not with resting or primed MSC-CM and proteomic characterization of resting and primed MSC-CM.ResultsWe isolated MSC from adipose tissue of 8 donors, generated MSC-CM and tested each MSC-CM independently. We report that MSC-CM treatment prevented TGF-β induced fibroblast activation to a similar extent as nintedanib but, in contrast to nintedanib, MSC-CM reduced fibrogenic myofibroblasts (i.e. transcriptomic upregulation of apoptosis, senescence, and inflammatory pathways). These effects were larger when primed rather than resting MSC-CM were used. Priming increased the ability of MSC-CM to remodel the ECM, reducing its content of collagen I and fibronectin, and reduced the fibrotic load in TGF-β treated lung explant cultures. Priming increased the following antifibrotic proteins in MSC-CM: DKK1, MMP-1, MMP-3, follistatin and cathepsin S. Inhibition of DKK1 reduced the antifibrotic effects of MSC-CM.ConclusionsIn vitro, MSC-CM promote fibrosis resolution, an effect enhanced following MSC cytokine priming. Specifically, MSC-CM reduces fibrogenic myofibroblasts through apoptosis, senescence, and by enhancing ECM degradation. Future studies will establish the in vivo relevance of MSC priming to fibrosis resolution.

Carvacrol inhibits the progression of oral submucous fibrosis via downregulation of PVT1/miR-20a-5p-mediated pyroptosis.

Oral submucous fibrosis (OSF) is a precancerous condition in the oral cavity, which is closely related to the myofibroblast conversion of buccal mucosal fibroblasts (BMFs) after chronic consumption of areca nut. Emerging evidence suggests pyroptosis, a form of programmed cell death that is mediated by inflammasome, is implicated in persistent myofibroblast activation and fibrosis. Besides, numerous studies have demonstrated the effects of non-coding RNAs on pyroptosis and myofibroblast activities. Herein, we aimed to target key long non-coding RNA PVT1 with natural compound, carvacrol, to alleviate pyroptosis and myofibroblast activation in OSF. We first identified PVT1 was downregulated in the carvacrol-treated fBMFs and then demonstrated that myofibroblast features and expression of pyroptosis makers were all reduced in response to carvacrol treatment. Subsequently, we analysed the expression of PVT1 and found that PVT1 was aberrantly upregulated in OSF specimens and positively correlated with several fibrosis markers. After revealing the suppressive effects of carvacrol on myofibroblast characterisitcs and pyroptosis were mediated by repression of PVT1, we then explored the potential mechanisms. Our data showed that PVT1 may serve as a sponge of microRNA(miR)-20a to mitigate the myofibroblast activation and pyroptosis. Altogether, these findings indicated that the anti-fibrosis effects of carvacrol merit consideration and may be due to the attenuation of pyroptosis and myofibroblast activation by targeting the PVT1/miR-20a axis.

The Myofibroblast Fate of Therapeutic Mesenchymal Stromal Cells: Regeneration, Repair, or Despair?

Mesenchymal stromal cells (MSCs) can be isolated from various tissues of healthy or patient donors to be retransplanted in cell therapies. Because the number of MSCs obtained from biopsies is typically too low for direct clinical application, MSC expansion in cell culture is required. However, ex vivo amplification often reduces the desired MSC regenerative potential and enhances undesired traits, such as activation into fibrogenic myofibroblasts. Transiently activated myofibroblasts restore tissue integrity after organ injury by producing and contracting extracellular matrix into scar tissue. In contrast, persistent myofibroblasts cause excessive scarring-called fibrosis-that destroys organ function. In this review, we focus on the relevance and molecular mechanisms of myofibroblast activation upon contact with stiff cell culture plastic or recipient scar tissue, such as hypertrophic scars of large skin burns. We discuss cell mechanoperception mechanisms such as integrins and stretch-activated channels, mechanotransduction through the contractile actin cytoskeleton, and conversion of mechanical signals into transcriptional programs via mechanosensitive co-transcription factors, such as YAP, TAZ, and MRTF. We further elaborate how prolonged mechanical stress can create persistent myofibroblast memory by direct mechanotransduction to the nucleus that can evoke lasting epigenetic modifications at the DNA level, such as histone methylation and acetylation. We conclude by projecting how cell culture mechanics can be modulated to generate MSCs, which epigenetically protected against myofibroblast activation and transport desired regeneration potential to the recipient tissue environment in clinical therapies.

How to Keep Myofibroblasts under Control: Culture of Mouse Skin Fibroblasts on Soft Substrates

During the physiological healing of skin wounds, fibroblasts recruited from the uninjured adjacent dermis and deeper subcutaneous fascia layers are transiently activated into myofibroblasts to first secrete and then contract collagen-rich extracellular matrix into a mechanically resistant scar. Scar tissue restores skin integrity after damage but comes at the expense of poor esthetics and loss of tissue function. Stiff scar matrix also mechanically activates various precursor cells into myofibroblasts in a positive feedback loop. Persistent myofibroblast activation results in pathologic accumulation of fibrous collagen and hypertrophic scarring, called fibrosis. Consequently, the mechanisms of fibroblast-to-myofibroblast activation and persistence are studied to develop antifibrotic and prohealing treatments. Mechanistic understanding often starts in a plastic cell culture dish. This can be problematic because contact of fibroblasts with tissue culture plastic or glass surfaces invariably generates myofibroblast phenotypes in standard culture. We describe a straight-forward method to produce soft cell culture surfaces for fibroblast isolation and continued culture and highlight key advantages and limitations of the approach. Adding a layer of elastic silicone polymer tunable to the softness of normal skin and the stiffness of pathologic scars allows to control mechanical fibroblast activation while preserving the simplicity of conventional 2-dimensional cell culture.

Manipulating mitochondrial pyruvate carrier function causes metabolic remodeling in corneal myofibroblasts that ameliorates fibrosis

Myofibroblasts are key cellular effectors of corneal wound healing from trauma, surgery, or infection. However, their persistent deposition of disorganized extracellular matrix can also cause corneal fibrosis and visual impairment. Recent work showed that the PPARγ agonist Troglitazone can mitigate established corneal fibrosis, and parallel in vitro data suggested this occurred through inhibition of the mitochondrial pyruvate carrier (MPC) rather than PPARγ. In addition to oxidative phosphorylation (Ox-Phos), pyruvate and other mitochondrial metabolites provide carbon for the synthesis of biological macromolecules. However, it is currently unclear how these roles selectively impact fibrosis. Here, we performed bioenergetic, metabolomic, and epigenetic analyses of corneal fibroblasts treated with TGF-β1 to stimulate myofibroblast trans-differentiation, with further addition of Troglitazone or the MPC inhibitor UK5099, to identify MPC-dependencies that may facilitate remodeling and loss of the myofibroblast phenotype. Our results show that a shift in energy metabolism is associated with, but not sufficient to drive cellular remodeling. Metabolites whose abundances were sensitive to MPC inhibition suggest that sustained carbon influx into the Krebs’ cycle is prioritized over proline synthesis to fuel collagen deposition. Furthermore, increased abundance of acetyl-CoA and increased histone H3 acetylation suggest that epigenetic mechanisms downstream of metabolic remodeling may reinforce cellular phenotypes. Overall, our results highlight a novel molecular target and metabolic vulnerability that affects myofibroblast persistence in the context of corneal wounding.

A novel senolytic drug for pulmonary fibrosis: BTSA1 targets apoptosis of senescent myofibroblasts by activating BAX.

Idiopathic pulmonary fibrosis is a progressive and age-related disease that results from impaired lung repair following injury. Targeting senescent myofibroblasts with senolytic drugs attenuates pulmonary fibrosis, revealing a detrimental role of these cells in pulmonary fibrosis. The mechanisms underlying the occurrence and persistence of senescent myofibroblasts in fibrotic lung tissue require further clarification. In this study, we demonstrated that senescent myofibroblasts are resistant to apoptosis by upregulating the proapoptotic protein BAX and antiapoptotic protein BCL-2 and BCL-XL, leading to BAX inactivation. We further showed that high levels of inactive BAX-mediated minority mitochondrial outer membrane permeabilization (minority MOMP) promoted DNA damage and myofibroblasts senescence after insult by a sublethal stimulus. Intervention of minority MOMP via the inhibition of caspase activity by quinolyl-valyl-O-methylaspartyl-[2,6-difluorophenoxy]-methyl ketone (QVD-OPH) or BAX knockdown significantly reduced DNA damage and ultimately delayed the progression of senescence. Moreover, the BAX activator BTSA1 selectively promoted the apoptosis of senescent myofibroblasts, as BTSA1-activated BAX converted minority MOMP to complete MOMP while not injuring other cells with low levels of BAX. Furthermore, therapeutic activation of BAX with BTSA1 effectively reduced the number of senescent myofibroblasts in the lung tissue and alleviated both reversible and irreversible pulmonary fibrosis. These findings advance the understanding of apoptosis resistance and cellular senescence mechanisms in senescent myofibroblasts in pulmonary fibrosis and demonstrate a novel senolytic drug for pulmonary fibrosis treatment.

Hydroxypyridone anti-fungals selectively induce myofibroblast apoptosis in an in vitro model of hypertrophic scars

Hypertrophic scars are a common complication of burn injuries, yet there are no medications to prevent their formation. During scar formation, resident fibroblasts are transformed to myofibroblasts which become resistant to apoptosis. Previously, we have shown that hydroxypyridone anti-fungals can inhibit transformation of fibroblasts, isolated from hypertrophic scars, to myofibroblasts. This study aimed to investigate if these drugs can also target myofibroblast persistence.Primary human dermal fibroblasts, derived from burn scar tissue, were exposed to transforming growth factor beta-1 (TGF-β1) for 72 h to induce myofibroblast transformation. The cells were then incubated with three hydroxypyridone anti-fungals (ciclopirox, ciclopirox ethanolamine and piroctone olamine; 0.03–300 μM) for a further 72 h. The In-Cell ELISA method was utilised to quantify myofibroblast transformation by measuring alpha-smooth muscle actin (α-SMA) expression and DRAQ5 staining, to measure cell viability. TUNEL staining was utilised to assess if the drugs could induce apoptosis.When given to established myofibroblasts, the three hydroxypyridones did not reverse myofibroblast transformation, but instead elicited a concentration-dependent decrease in cell viability. TUNEL staining confirmed that the hydroxypyridone anti-fungals induced apoptosis in established myofibroblasts.This is the first study to show that hydroxypyridone anti-fungals are capable of inducing apoptosis in established myofibroblasts. Together with our previous results, we suggest that hydroxypyridone anti-fungals can prevent scar formation by preventing the formation of new myofibroblasts and by reducing the number of existing myofibroblasts.

Identification of Periostin as a critical niche for myofibroblast dynamics and fibrosis during tendon healing

Tendon injuries are a major clinical problem, with poor patient outcomes caused by abundant scar tissue deposition during healing. Myofibroblasts play a critical role in the initial restoration of structural integrity after injury. However, persistent myofibroblast activity drives the transition to fibrotic scar tissue formation. As such, disrupting myofibroblast persistence is a key therapeutic target. While myofibroblasts are typically defined by the presence of αSMA+ stress fibers, αSMA is expressed in other cell types including the vasculature. As such, modulation of myofibroblast dynamics via disruption of αSMA expression is not a translationally tenable approach. Recent work has demonstrated that Periostin-lineage (PostnLin) cells are a precursor for cardiac fibrosis-associated myofibroblasts. In contrast to this, here we show that PostnLin cells contribute to a transient αSMA+ myofibroblast population that is required for functional tendon healing, and that Periostin forms a supportive matrix niche that facilitates myofibroblast differentiation and persistence. Collectively, these data identify the Periostin matrix niche as a critical regulator of myofibroblast fate and persistence that could be targeted for therapeutic manipulation to facilitate regenerative tendon healing.

Modulating mechanobiology as a therapeutic target for synovial fibrosis to restore joint lubrication

Fibroses are disorders linked to persistence of myofibroblasts due to biochemical (e.g., Transforming growth factor-β) and biophysical cues (e.g., a stiff microenvironment). In the context of osteoarthritis, fibrotic changes in the joint-lining synovium have been linked with disease progression. The objective of this study was to probe synovial fibroblast mechanobiology and how essential functions (i.e., lubrication) are altered in fibrotic environments. Both ex vivo and in vitro synovium models were assessed for fibrotic and lubrication biomarkers to better understand the role of mechanobiology and lubrication. Additionally, in vitro, work on small molecules targeting mechanobiology was assessed. Our results indicated that modulating mechanobiology could rescue the fibrotic phenotype instigated by stiffening microenvironment that resulted in altered lubricant expression. A small molecule therapeutic, fasudil, blocked ROCK-mediated contractility and this inhibition of the fibrotic mechano-response of synovial fibroblasts restored proper lubrication function, providing insight into mechanisms of disease progression as well as a new avenue for therapeutic development. This study identifies synovial fibrosis as a condition that potentially has joint-wide deficits through inhibiting lubrication. Additionally, modulating mechanobiology (i.e., ROCK-mediated contractility) may pose a potential target for small molecule therapies that can be delivered to the joint space. Applied Biological Sciences.

Myofibroblast senescence promotes arrhythmogenic remodeling in the aged infarcted rabbit heart.

Progressive tissue remodeling after myocardial infarction (MI) promotes cardiac arrhythmias. This process is well studied in young animals, but little is known about pro-arrhythmic changes in aged animals. Senescent cells accumulate with age and accelerate age-associated diseases. Senescent cells interfere with cardiac function and outcome post-MI with age, but studies have not been performed in larger animals, and the mechanisms are unknown. Specifically, age-associated changes in timecourse of senescence and related changes in inflammation and fibrosis are not well understood. Additionally, the cellular and systemic role of senescence and its inflammatory milieu in influencing arrhythmogenesis with age is not clear, particularly in large animal models with cardiac electrophysiology more similar to humans than previously studied animal models. Here, we investigated the role of senescence in regulating inflammation, fibrosis, and arrhythmogenesis in young and aged infarcted rabbits. Aged rabbits exhibited increased peri-procedural mortality and arrhythmogenic electrophysiological remodeling at the infarct border zone (IBZ) compared to young rabbits. Studies of the aged infarct zone revealed persistent myofibroblast senescence and increased inflammatory signaling over a 12-week timecourse. Senescent IBZ myofibroblasts in aged rabbits appear to be coupled to myocytes, and our computational modeling showed that senescent myofibroblast-cardiomyocyte coupling prolongs action potential duration (APD) and facilitates conduction block permissive of arrhythmias. Aged infarcted human ventricles show levels of senescence consistent with aged rabbits, and senescent myofibroblasts also couple to IBZ myocytes. Our findings suggest that therapeutic interventions targeting senescent cells may mitigate arrhythmias post-MI with age.

Tracing causes of tubule atrophy in vivo

Acute kidney injury (AKI) is a known risk factor for chronic kidney disease (CKD). The mechanisms for AKI-CKD transition may involve failed tubule repair and progressive interstitial fibrosis but are incompletely understood. Which events lead to failed tubule repair? Can injury spread over time? And are these processes steered by myofibroblasts and fibrotic tissue remodeling?Here, we combined serial intravital 2-photon microscopy and a novel model of partial ischemia-reperfusion injury (partial IRI), which allowed us to investigate injured tissue alongside uninjured tissue in the same kidney and over time. Thus, we occluded only one branch of the left renal artery for 21 min, rendering half the kidney ischemic. We then tracked tubule necrosis and remodeling alongside myofibroblast dynamics for up to 3 weeks. Necrotic cell death was detected by in vivo propidium iodide staining. PDGFRβ-CreERT2-Salsa6F mice (N=5) identified main myofibroblast precursor cells, by tdTomato-expression. Myofibroblast-differentiation was confirmed by ex vivo αSMA staining. Proximal tubule (PT) function was assessed by in vivo uptake of fluorescent albumin.Necrotic cell death was solely detectable upon reperfusion. Border areas in between post-ischemic and non-ischemic tissue presented severely necrotic PTs alongside uninjured PTs. From 1 day after partial IRI, we observed formation of granular casts in the lumen of necrotic tubules, which 1-2 days later also appeared in initially non-necrotic PTs downstream. Tubule necrosis and granular cast accumulation preceded thinned epithelium that lacked albumin endocytosis (day 3-7) and recruitment of myofibroblasts (peaking D4-10). Of note, non-necrotic tubules, without granular cast accumulation, didn’t remodel nor recruit myofibroblasts.Among all remodeling tubule segments, 24% turned atrophic. Importantly, preceding necrotic cell death was higher in atrophic than in recovering tubules (55±13% vs. 39±18% necrotic cells/total segment cell number, (mean±SD), p<.0001). Preceding granular casts accounted for 38±15% vs. 12±11% (mean ±SD) of the luminal area in atrophic and recovering tubules, respectively (p<.0001). Ex vivo staining for pro-fibrotic VCAM-1 demonstrated selective positivity in atrophic tubules, which appeared scattered at D8 while at D21, entire atrophic tubules stained positive (n=3, each). Consistently, we detected persistent myofibroblast enclosure of atrophic tubule, while recruitment around recovered PTs resolved by day 14 (50±21% vs. 23±18% of coverage, p<.001).Tracking the same tubules in vivo and over time, we demonstrated early proximal tubule necrosis, which induced luminal granular cast formation and injury propagation. These events preceded myofibroblast accumulation and predicted tubule atrophy. We further documented de novo expression of pro-inflammatory VCAM-1 and persistent myofibroblast recruitment selectively in atrophic tubules, suggesting an active role of this tubule state in AKI-CKD transition. Novo Nordisk Foundation, Augustinus Foundation, Aarhus University Research Foundation (AUFF) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

P53 and Myofibroblast Apoptosis in Organ Fibrosis.

Organ fibrosis represents a dysregulated, maladaptive wound repair response that results in progressive disruption of normal tissue architecture leading to detrimental deterioration in physiological function, and significant morbidity/mortality. Fibrosis is thought to contribute to nearly 50% of all deaths in the Western world with current treatment modalities effective in slowing disease progression but not effective in restoring organ function or reversing fibrotic changes. When physiological wound repair is complete, myofibroblasts are programmed to undergo cell death and self-clearance, however, in fibrosis there is a characteristic absence of myofibroblast apoptosis. It has been shown that in fibrosis, myofibroblasts adopt an apoptotic-resistant, highly proliferative phenotype leading to persistent myofibroblast activation and perpetuation of the fibrotic disease process. Recently, this pathological adaptation has been linked to dysregulated expression of tumour suppressor gene p53. In this review, we discuss p53 dysregulation and apoptotic failure in myofibroblasts and demonstrate its consistent link to fibrotic disease development in all types of organ fibrosis. An enhanced understanding of the role of p53 dysregulation and myofibroblast apoptosis may aid in future novel therapeutic and/or diagnostic strategies in organ fibrosis.

Regulation of Mesenchymal Cell Fate by Transfer of Active Gasdermin-D via Monocyte-Derived Extracellular Vesicles.

Fibrosis is characterized by inappropriately persistent myofibroblast accumulation and excessive extracellular matrix deposition with the disruption of tissue architecture and organ dysfunction. Regulated death of reparative mesenchymal cells is critical for normal wound repair, but profibrotic signaling promotes myofibroblast resistance to apoptotic stimuli. A complex interplay between immune cells and structural cells underlies lung fibrogenesis. However, there is a paucity of knowledge on how these cell populations interact to orchestrate physiologic and pathologic repair of the injured lung. In this context, gasdermin-D (GsdmD) is a cytoplasmic protein that is activated following cleavage by inflammatory caspases and induces regulated cell death by forming pores in cell membranes. This study was undertaken to evaluate the impact of human (Thp-1) monocyte-derived extracellular vesicles and GsdmD on human lung fibroblast death. Our data show that active GsdmD delivered by monocyte-derived extracellular vesicles induces caspase-independent fibroblast and myofibroblast death. This cell death was partly mediated by GsdmD-independent induction of cellular inhibitor of apoptosis 2 (cIAP-2) in the recipient fibroblast population. Our findings, to our knowledge, define a novel paradigm by which inflammatory monocytes may orchestrate the death of mesenchymal cells in physiologic wound healing, illustrating the potential to leverage this mechanism to eliminate mesenchymal cells and facilitate the resolution of fibrotic repair.

Phenotypic screening identifies hydroxypyridone anti-fungals as novel medicines for the prevention of hypertrophic scars

Although hypertrophic scarring affects ∼91% of burn patients annually, there is no drug to prevent this common complication. Hypertrophic scars are a result of dysregulated wound healing, characterised by persistent myofibroblast transformation and the excessive accumulation of extracellular matrix (ECM). Due to the multi-mechanistic nature of the scarring process, target-based approaches for identifying novel drugs have failed.Primary human dermal fibroblasts, derived from burn scar tissue, were exposed to transforming growth factor-beta 1 (TGF-β1) to induce myofibroblast transformation. A phenotypic screening assay, measuring alpha-smooth muscle actin (α-SMA) expression, was developed to screen 1,954 approved drugs. Drugs that elicited >80% inhibition of α-SMA expression, and >80% cell viability were progressed as candidate drugs. Anti-myofibroblast activity of the candidates was confirmed before investigating their effects on extracellular matrix (ECM) production and keratinocyte epithelial-mesenchymal transition (EMT).TGF-β1 induced myofibroblast transformation in primary human dermal fibroblasts (Emax = >3 ng/mL). The assay was optimised and validated (Z’ = 0.59), before screening 1,954 approved drugs. 90 drugs were identified as hits and hydroxypyridone anti-fungals selected for further testing. Concentration-response curves for these drugs confirmed their concentration-dependent anti-myofibroblast activity (IC50 = 1.4 – 16.7 μM). Hydroxypyridone anti-fungals were also found to successfully reduce ECM production and keratinocyte EMT.This is the first study to screen approved drugs in primary human dermal fibroblasts. Hydroxypyridone anti-fungals were found to prevent myofibroblast transformation, ECM production and keratinocyte EMT suggesting they could be repurposed to prevent hypertrophic scarring.

Andrographolide Inhibits Corneal Fibroblast to Myofibroblast Differentiation In Vitro.

Corneal opacification due to fibrosis is a leading cause of blindness worldwide. Fibrosis occurs from many causes including trauma, photorefractive surgery, microbial keratitis (infection of the cornea), and chemical burns, yet there is a paucity of therapeutics to prevent or treat corneal fibrosis. This study aimed to determine if andrographolide, a labdane diterpenoid found in Andrographis paniculate, has anti-fibrotic properties. Furthermore, we evaluated if andrographolide could prevent the differentiation of fibroblasts to myofibroblasts in vitro, given that the transforming growth factor beta-1(TGF-β1) stimulated persistence of myofibroblasts in the cornea is a primary component of fibrosis. We demonstrated that andrographolide inhibited the upregulation of alpha smooth muscle actin (αSMA) mRNA and protein in rabbit corneal fibroblasts (RCFs), thus, demonstrating a reduction in the transdifferentiation of myofibroblasts. Immunofluorescent staining of TGF-β1-stimulated RCFs confirmed a dose-dependent decrease in αSMA expression when treated with andrographolide. Additionally, andrographolide was well tolerated in vivo and had no impact on corneal epithelialization in a rat debridement model. These data support future studies investigating the use of andrographolide as an anti-fibrotic in corneal wound healing.

Senescent Cells Accumulate in Systemic Sclerosis Skin

Systemic sclerosis (SSc) is characterized by intractable fibrosis in the skin and internal organs. A unifying paradigm across all forms of fibrosis is the transformation of quiescent progenitor cells into activated myofibroblasts, but the pathways underlying the transformation and persistence of myofibroblasts in SSc remain poorly understood. SSc might be viewed as an accelerated aging phenotype, and patients with SSc prematurely display hallmarks of aging, including telomere attrition, genomic instability, epigenetic alterations, deregulated nutrient sensing, stem cell exhaustion, and mitochondrial dysfunction ( Luckhardt and Thannickal, 2015 Luckhardt T.R. Thannickal V.J. Systemic sclerosis-associated fibrosis: an accelerated aging phenotype?. Curr Opin Rheumatol. 2015; 27: 571-576 Crossref PubMed Scopus (31) Google Scholar ; Tsou et al., 2022 Tsou P.S. Shi B. Varga J. Role of cellular senescence in the pathogenesis of systemic sclerosis. Curr Opin Rheumatol. 2022; 34 (343‒50) Crossref PubMed Scopus (0) Google Scholar ). Cellular senescence is another hallmark of aging. Sustained tissue accumulation of senescent cells is detrimental to organ function owing in part to the senescence-associated secretory phenotype, characterized by secretion of cytokines/chemokines, growth factors, extracellular matrix modifiers, and other substances that promote chronic, sterile inflammation and fibrosis.

The Role of Myofibroblasts in Physiological and Pathological Tissue Repair.

Myofibroblasts are the construction workers of wound healing and repair damaged tissues by producing and organizing collagen/extracellular matrix (ECM) into scar tissue. Scar tissue effectively and quickly restores the mechanical integrity of lost tissue architecture but comes at the price of lost tissue functionality. Fibrotic diseases caused by excessive or persistent myofibroblast activity can lead to organ failure. This review defines myofibroblast terminology, phenotypic characteristics, and functions. We will focus on the central role of the cell, ECM, and tissue mechanics in regulating tissue repair by controlling myofibroblast action. Additionally, we will discuss how therapies based on mechanical intervention potentially ameliorate wound healing outcomes. Although myofibroblast physiology and pathology affect all organs, we will emphasize cutaneous wound healing and hypertrophic scarring as paradigms for normal tissue repair versus fibrosis. A central message of this review is that myofibroblasts can be activated from multiple cell sources, varying with local environment and type of injury, to either restore tissue integrity and organ function or create an inappropriate mechanical environment.

Comparative Analysis of Co-Cultured Amniotic Cell-Conditioned Media with Cell-Free Amniotic Fluid Reveals Differential Effects on Epithelial-Mesenchymal Transition and Myofibroblast Activation.

Myofibroblast activation is a cellular response elicited by a variety of physiological or pathological insults whereby cells initiate a coordinated response intended to eradicate the insult and then revert back to a basal state. However, an underlying theme in various disease states is persistent myofibroblast activation that fails to resolve. Based on multiple observations, we hypothesized that the secreted factors harvested from co-culturing amniotic stem cells might mimic the anti-inflammatory state that cell-free amniotic fluid (AF) elicits. We optimized an amnion epithelial and amniotic fluid cell co-culture system, and tested this hypothesis in the context of myofibroblast activation. However, we discovered that co-cultured amniotic cell conditioned media (coACCM) and AF have opposing effects on myofibroblast activation: coACCM activates the epithelial–mesenchymal transition (EMT) and stimulates gene expression patterns associated with myofibroblast activation, while AF does the opposite. Intriguingly, extracellular vesicles (EVs) purified from AF are necessary and sufficient to activate EMT and inflammatory gene expression patterns, while the EV-depleted AF potently represses these responses. In summary, these data indicate that coACCM stimulates myofibroblast activation, while AF represses it. We interpret these findings to suggest that coACCM, AF, and fractionated AF represent unique biologics that elicit different cellular responses that are correlated with a wide variety of pathological states, and therefore could have broad utility in the clinic and the lab.

Extracellular matrix stiffness controls cardiac valve myofibroblast activation through epigenetic remodeling.

Aortic valve stenosis (AVS) is a progressive fibrotic disease that is caused by thickening and stiffening of valve leaflets. At the cellular level, quiescent valve interstitial cells (qVICs) activate to myofibroblasts (aVICs) that persist within the valve tissue. Given the persistence of myofibroblasts in AVS, epigenetic mechanisms have been implicated. Here, we studied changes that occur in VICs during myofibroblast activation by using a hydrogel matrix to recapitulate different stiffnesses in the valve leaflet during fibrosis. We first compared the chromatin landscape of qVICs cultured on soft hydrogels and aVICs cultured on stiff hydrogels, representing the native and diseased phenotypes respectively. Using assay for transposase‐accessible chromatin sequencing (ATAC‐Seq), we found that open chromatin regions in aVICs were enriched for transcription factor binding motifs associated with mechanosensing pathways compared to qVICs. Next, we used RNA‐Seq to show that the open chromatin regions in aVICs correlated with pro‐fibrotic gene expression, as aVICs expressed higher levels of contractile fiber genes, including myofibroblast markers such as alpha smooth muscle actin (αSMA), compared to qVICs. In contrast, chromatin remodeling genes were downregulated in aVICs compared to qVICs, indicating qVICs may be protected from myofibroblast activation through epigenetic mechanisms. Small molecule inhibition of one of these remodelers, CREB Binding Protein (CREBBP), prevented qVICs from activating to aVICs. Notably, CREBBP is more abundant in valves from healthy patients compared to fibrotic valves. Our findings reveal the role of mechanical regulation in chromatin remodeling during VIC activation and quiescence and highlight one potential therapeutic target for treating AVS.

Glutamine uptake and catabolism is required for myofibroblast formation and persistence

BackgroundFibrosis and extracellular matrix remodeling are mediated by resident cardiac fibroblasts (CFs). In response to injury, fibroblasts activate, differentiating into specialized synthetic and contractile myofibroblasts producing copious extracellular matrix proteins (e.g., collagens). Myofibroblast persistence in chronic diseases, such as HF, leads to progressive cardiac dysfunction and maladaptive remodeling. We recently reported that an increase in αKG (alpha-ketoglutarate) bioavailability, which contributes to enhanced αKG-dependent lysine demethylase activity and chromatin remodeling, is required for myofibroblast formation. Therefore, we aimed to determine the substrates and metabolic pathways contributing to αKG biosynthesis and their requirement for myofibroblast formation. MethodsStable isotope metabolomics identified glutaminolysis as a key metabolic pathway required for αKG biosynthesis and myofibroblast formation, therefore we tested the effects of pharmacologic inhibition (CB-839) or genetic deletion of glutaminase (Gls1−/−) on myofibroblast formation in both murine and human cardiac fibroblasts. We employed immunofluorescence staining, functional gel contraction, western blotting, and bioenergetic assays to determine the myofibroblast phenotype. ResultsCarbon tracing indicated enhanced glutaminolysis mediating increased αKG abundance. Pharmacological and genetic inhibition of glutaminolysis prevented myofibroblast formation indicated by a reduction in αSMA+ cells, collagen gel contraction, collagen abundance, and the bioenergetic response. Inhibition of glutaminolysis also prevented TGFβ-mediated histone demethylation and supplementation with cell-permeable αKG rescued the myofibroblast phenotype. Importantly, inhibition of glutaminolysis was sufficient to prevent myofibroblast formation in CFs isolated from the human failing heart. ConclusionsThese results define glutaminolysis as necessary for myofibroblast formation and persistence, providing substantial rationale to evaluate several new therapeutic targets to treat cardiac fibrosis.