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Augmented glycolysis due to metabolic reprogramming in lung myofibroblasts is critical to their profibrotic phenotype. The primary glycolysis byproduct, lactate, is also secreted into the extracellular milieu, together with which myofibroblasts and macrophages form a spatially restricted site usually described as fibrotic niche. Therefore, we hypothesized that myofibroblast glycolysis might have a non-cell autonomous effect through lactate regulating the pathogenic phenotype of alveolar macrophages. Here, we demonstrated that there was a markedly increased lactate in the conditioned media of TGF-β1 (transforming growth factor-β1)-induced lung myofibroblasts and in the BAL fluids (BALFs) from mice with TGF-β1- or bleomycin-induced lung fibrosis. Importantly, the media and BALFs promoted profibrotic mediator expression in macrophages. Mechanistically, lactate induced histone lactylation in the promoters of the profibrotic genes in macrophages, consistent with the upregulation of this epigenetic modification in these cells in the fibrotic lungs. The lactate inductions of the histone lactylation and profibrotic gene expression were mediated by p300, as evidenced by their diminished concentrations in p300-knockdown macrophages. Collectively, our study establishes that in addition to protein, lipid, and nucleic acid molecules, a metabolite can also mediate intercellular regulations in the setting of lung fibrosis. Our findings shed new light on the mechanism underlying the key contribution of myofibroblast glycolysis to the pathogenesis of lung fibrosis.

Altered microarchitecture of collagen type I is a hallmark of wound healing and cancer that is commonly attributed to myofibroblasts. However, it remains unknown which effect collagen microarchitecture has on myofibroblast differentiation. Here, we combined experimental and computational approaches to investigate the hypothesis that the microarchitecture of fibrillar collagen networks mechanically regulates myofibroblast differentiation of adipose stromal cells (ASCs) independent of bulk stiffness. Collagen gels with controlled fiber thickness and pore size were microfabricated by adjusting the gelation temperature while keeping their concentration constant. Rheological characterization and simulation data indicated that networks with thicker fibers and larger pores exhibited increased strain-stiffening relative to networks with thinner fibers and smaller pores. Accordingly, ASCs cultured in scaffolds with thicker fibers were more contractile, expressed myofibroblast markers, and deposited more extended fibronectin fibers. Consistent with elevated myofibroblast differentiation, ASCs in scaffolds with thicker fibers exhibited a more proangiogenic phenotype that promoted endothelial sprouting in a contractility-dependent manner. Our findings suggest that changes of collagen microarchitecture regulate myofibroblast differentiation and fibrosis independent of collagen quantity and bulk stiffness by locally modulating cellular mechanosignaling. These findings have implications for regenerative medicine and anticancer treatments.

During long-term peritoneal dialysis, peritoneal fibrosis (PF) often happens and results in ultrafiltration failure, which directly leads to the termination of dialysis. The accumulation of extracellular matrix produced from an increasing number of myofibroblasts was a hallmark characteristic of PF. To date, glucose degradation products (GDPs, i.e., methylglyoxal (MGO)) that appeared during the heating and storage of the dialysate are considered to be key components to initiating PF, but how GDPs lead to the activation of myofibroblast in fibrotic peritoneum has not yet been fully elucidated. In this study, mesothelial cell line (MeT-5A) and fibroblast cell line (MRC-5) were used to investigate the transcriptomic and proteomic changes to unveil the underlying mechanism of MGO-induced PF. Our transcriptomic data from the MGO-stimulated mesothelial cells showed upregulation of genes involved in pro-inflammatory, apoptotic, and fibrotic pathways. While no phenotypic changes were noted on fibroblasts after direct MGO, supernatant from MGO-stimulated mesothelial cells promoted fibroblasts to change into proto-myofibroblasts, activated fibroblasts in the first stage toward myofibroblasts. In conclusion, this study showed that MGO-stimulated mesothelial cells promoted fibroblast-to-proto-myofibroblast transition; however, additional involvement of other factors or cells (e.g., macrophages) may be needed to complete the transformation into myofibroblasts.

Macrophage-myofibroblast transformation (MMT) transforms macrophages into myofibroblasts in a specific inflammation or injury microenvironment. MMT is an essential biological process in fibrosis-related diseases involving the lung, heart, kidney, liver, skeletal muscle, and other organs and tissues. This process consists of interacting with various cells and molecules and activating different signal transduction pathways. This review deeply discussed the molecular mechanism of MMT, clarified crucial signal pathways, multiple cytokines, and growth factors, and formed a complex regulatory network. Significantly, the critical role of transforming growth factor-β (TGF-β) and its downstream signaling pathways in this process were clarified. Furthermore, we discussed the significance of MMT in physiological and pathological conditions, such as pulmonary fibrosis and cardiac fibrosis. This review provides a new perspective for understanding the interaction between macrophages and myofibroblasts and new strategies and targets for the prevention and treatment of MMT in fibrotic diseases.

Myofibroblasts produce the fibrous scar in hepatic fibrosis. In the carbon tetrachloride (CCl ₄) model of liver fibrosis, quiescent hepatic stellate cells (HSC) are activated to become myofibroblasts. When the underlying etiological agent is removed, clinical and experimental fibrosis undergoes a remarkable regression with complete disappearance of these myofibroblasts. Although some myofibroblasts apoptose, it is unknown whether other myofibroblasts may revert to an inactive phenotype during regression of fibrosis. We elucidated the fate of HSCs/myofibroblasts during recovery from CCl ₄- and alcohol-induced liver fibrosis using Cre-LoxP–based genetic labeling of myofibroblasts. Here we demonstrate that half of the myofibroblasts escape apoptosis during regression of liver fibrosis, down-regulate fibrogenic genes, and acquire a phenotype similar to, but distinct from, quiescent HSCs in their ability to more rapidly reactivate into myofibroblasts in response to fibrogenic stimuli and strongly contribute to liver fibrosis. Inactivation of HSCs was associated with up-regulation of the anti-apoptotic genes Hspa1a/b, which participate in the survival of HSCs in culture and in vivo.

Macrophage-myofibroblast transformation (MMT) has emerged as a discovery in the field of fibrotic disease research. MMT is the process by which macrophages differentiate into myofibroblasts, leading to organ fibrosis following organ damage and playing an important role in fibrosis formation and progression. Recently, many new advances have been made in studying the mechanisms of MMT occurrence in fibrotic diseases. This article reviews some critical recent findings on MMT, including the origin of MMT in myofibroblasts, the specific mechanisms by which MMT develops, and the mechanisms and effects of MMT in the kidneys, lungs, heart, retina, and other fibrosis. By summarizing the latest research related to MMT, this paper provides a theoretical basis for elucidating the mechanisms of fibrosis in various organs and developing effective therapeutic targets for fibrotic diseases.

Background Macular fibrosis causes irreparable vision loss in neovascular age-related macular degeneration (nAMD) even with anti-vascular endothelial growth factor (VEGF) therapy. Inflammation is known to play an important role in macular fibrosis although the underlying mechanism remains poorly defined. The aim of this study was to understand how infiltrating macrophages and complement proteins may contribute to macular fibrosis. Methods Subretinal fibrosis was induced in C57BL/6J mice using the two-stage laser protocol developed by our group. The eyes were collected at 10, 20, 30 and 40 days after the second laser and processed for immunohistochemistry for infiltrating macrophages (F4/80 and Iba-1), complement components (C3a and C3aR) and fibrovascular lesions (collagen-1, Isolectin B4 and α-SMA). Human retinal sections with macular fibrosis were also used in the study. Bone marrow-derived macrophages (BMDMs) from C57BL/6J mice were treated with recombinant C3a, C5a or TGF-β for 48 and 96 h. qPCR, Western blot and immunohistochemistry were used to examine the expression of myofibroblast markers. The involvement of C3a-C3aR pathway in macrophage to myofibroblast transition (MMT) and subretinal fibrosis was further investigated using a C3aR antagonist (C3aRA) and a C3a blocking antibody in vitro and in vivo. Results Approximately 20~30% of F4/80 + (or Iba-1 + ) infiltrating macrophages co-expressed α-SMA in subretinal fibrotic lesions both in human nAMD eyes and in the mouse model. TGF-β and C3a, but not C5a treatment, significantly upregulated expression of α-SMA, fibronectin and collagen-1 in BMDMs. C3a-induced upregulation of α-SMA, fibronectin and collagen-1 in BMDMs was prevented by C3aRA treatment. In the two-stage laser model of induced subretinal fibrosis, treatment with C3a blocking antibody but not C3aRA significantly reduced vascular leakage and Isolectin B4 + lesions. The treatment did not significantly alter collagen-1 + fibrotic lesions. Conclusions MMT plays a role in macular fibrosis secondary to nAMD. MMT can be induced by TGF-β and C3a but not C5a. Further research is required to fully understand the role of MMT in macular fibrosis. Graphical abstract Macrophage to myofibroblast transition (MMT) contributes to subretinal fibrosis. Subretinal fibrosis lesions contain various cell types, including macrophages and myofibroblasts, and are fibrovascular. Myofibroblasts are key cells driving pathogenic fibrosis, and they do so by producing excessive amount of extracellular matrix proteins. We have found that infiltrating macrophages can transdifferentiate into myofibroblasts, a phenomenon termed macrophage to myofibroblast transition (MMT) in macular fibrosis. In addition to TGF-β1, C3a generated during complement activation in CNV can also induce MMT contributing to macular fibrosis. RPE = retinal pigment epithelium. BM = Bruch’s membrane. MMT = macrophage to myofibroblast transition. TGFB = transforming growth factor β. a-SMA = alpha smooth muscle actin. C3a = complement C3a.

Cardiac fibrosis is characterized by net accumulation of extracellular matrix proteins in the cardiac interstitium, and contributes to both systolic and diastolic dysfunction in many cardiac pathophysiologic conditions. This review discusses the cellular effectors and molecular pathways implicated in the pathogenesis of cardiac fibrosis. Although activated myofibroblasts are the main effector cells in the fibrotic heart, monocytes/macrophages, lymphocytes, mast cells, vascular cells and cardiomyocytes may also contribute to the fibrotic response by secreting key fibrogenic mediators. Inflammatory cytokines and chemokines, reactive oxygen species, mast cell-derived proteases, endothelin-1, the renin/angiotensin/aldosterone system, matricellular proteins, and growth factors (such as TGF-β and PDGF) are some of the best-studied mediators implicated in cardiac fibrosis. Both experimental and clinical evidence suggests that cardiac fibrotic alterations may be reversible. Understanding the mechanisms responsible for initiation, progression, and resolution of cardiac fibrosis is crucial to design anti-fibrotic treatment strategies for patients with heart disease.

Hepatic myofibroblasts are activated in response to chronic liver injury of any etiology to produce a fibrous scar. Despite extensive studies, the origin of myofibroblasts in different types of fibrotic liver diseases is unresolved. To identify distinct populations of myofibroblasts and quantify their contribution to hepatic fibrosis of two different etiologies, collagen-α1(I)-GFP mice were subjected to hepatotoxic (carbon tetrachloride; CCl ₄) or cholestatic (bile duct ligation; BDL) liver injury. All myofibroblasts were purified by flow cytometry of GFP ⁺ cells and then different subsets identified by phenotyping. Liver resident activated hepatic stellate cells (aHSCs) and activated portal fibroblasts (aPFs) are the major source (>95%) of fibrogenic myofibroblasts in these models of liver fibrosis in mice. As previously reported using other methodologies, hepatic stellate cells (HSCs) are the major source of myofibroblasts (>87%) in CCl ₄ liver injury. However, aPFs are a major source of myofibroblasts in cholestatic liver injury, contributing >70% of myofibroblasts at the onset of injury (5 d BDL). The relative contribution of aPFs decreases with progressive injury, as HSCs become activated and contribute to the myofibroblast population (14 and 20 d BDL). Unlike aHSCs, aPFs respond to stimulation with taurocholic acid and IL-25 by induction of collagen-α1(I) and IL-13, respectively. Furthermore, BDL-activated PFs express high levels of collagen type I and provide stimulatory signals to HSCs. Gene expression analysis identified several novel markers of aPFs, including a mesothelial-specific marker mesothelin. PFs may play a critical role in the pathogenesis of cholestatic liver fibrosis and, therefore, serve as an attractive target for antifibrotic therapy.

Telocytes (TCs) are distinctive cells widely localized in the stromal compartment of several human organs, including the skin. By means of their peculiar prolongations named telopodes, skin TCs are organized in networks interconnected with a variety of adjacent cells, being thus supposed to take part in skin homeostasis through both cell-to-cell contacts and the release of extracellular vesicles. A disarrangement/loss of the TC network was shown in human fibrotic skin as well as in the murine model of bleomycin-induced cutaneous fibrosis, but whether such TC alterations may represent just a consequence or a trigger of the fibrotic process still remains to be clarified. Thus, we investigated the effects of skin TC secretome as conditioned medium (TC-CM) on the transition of skin fibroblasts into myofibroblasts promoted by the master profibrotic cytokine transforming growth factor β1 (TGFβ1). Primary cultures of both adult human skin TCs and fibroblasts were obtained by means of immunomagnetic cell separation. Nanoparticle tracking analysis was carried out to measure extracellular vesicles in TC-CM. The combination of multiple morphological, gene/protein expression, and functional assessments demonstrated that TC-CM was able to significantly prevent TGFβ1-induced fibroblast-to-myofibroblast transition. TC-CM did not influence cell viability, while it effectively inhibited TGFβ1-induced fibroblast proliferation, migration, and morphological changes. Indeed, TC-CM was able to reduce TGFβ1-mediated skin fibroblast phenotypic and functional differentiation into myofibroblasts, as shown by a significant decrease in FAP, ACTA2, COL1A1, COL1A2, FN1, and CTGF gene expression, α-smooth muscle actin, N-cadherin, COL1A1, and FN-EDA protein levels, and collagen gel matrix contraction. Furthermore, TC-CM significantly lowered TGFβ1-mediated ERK1/2 signaling pathway activation. This in vitro study proves for the first time that TCs may play an important role in skin homeostasis through the prevention of fibroblast-to-myofibroblast transition via paracrine mechanisms and affords the necessary basis to investigate in the future the feasibility of TC secretome as an innovative antifibrotic therapeutic tool.