The pleiotropic cytokine TGF-β is a multifaceted regulator that affects fundamental biological processes such as cell division, proliferation, apoptosis, and tissue homeostasis. Three isoforms of TGF-β are known so far. TGF-β is stable as a non-covalently bound complex with the latency-associated peptide (LAP). This complex is associated with latent TGF-β binding protein (LTBP) via disulfide bonds and is stored within the ECM post-secretion (91, 92). Various factors and conditions, including bioactivation through integrins, mechanical forces, and acidic pH, can trigger the activation of TGF-β in vitro (92–95).

Integrins, transmembrane proteins consisting of an α-subunit and a β-subunit, mediate communication between the ECM and fibroblasts. They play a crucial role in the dynamics of tissue fibrosis (70, 72). By connecting the inner cytoskeleton to the ECM, integrins contribute to important catabolic reactions for ECM degradation (70). Despite limited data on the interaction between integrins and OA FLS, differences in integrin expression were observed between the lining and sublining layers of the synovium. In particular, increased expression of integrins such as α5, αv and β1 were found in the lining layer compared to the sublining layer (96). Furthermore, there is convincing evidence that lining layer FLS in OA consistently show strong expression of the integrin subunit αv, while being more diverse and heterogeneous among synovial lining FLS in RA (97).

Upon binding to αv integrins on adjacent cells, the latent complex releases the captive TGF-β through mechanisms specific to the types of αv integrins on the given cell. Integrins αvβ6 and αvβ8 on fibroblasts activate TGF-β1 by binding the RGD-motif on LAP and inducing a conformational change (98, 99). By blocking αv integrins with the small molecule CWHM 12, it is possible to inhibit TGF-β1 signaling and achieve the desired anti-fibrotic effect (69). In OA FLS, integrin αvβ6 plays a critical role due to its upregulated expression upon TGF-β1 stimulation and in mediating the activation of TGF-β. Furthermore, the vitronectin fragment VTN_{(381-397 a.a.)}, highly abundant in the serum of OA patients, competes with TGF-β1 for integrin binding and can attenuate TGF-β1 activation (100). Fibronectin can bind to integrins such as α5β1, ανβ3, ανβ5 (101) and ED-A-FN is associated with myofibroblasts and OA (77). Injection of fibronectin fragments into rabbit joints is now an established animal model of OA, characterized by cartilage degradation and osteophyte formation (77, 102). Kragstrup further showed that ED-A-FN is produced in response to TGF-β and self-induces the production of TNF-α in macrophages (77), suggesting ED-A-FN as an interesting target for therapeutic intervention to reduce pro-inflammatory responses in OA.

The initiation of the canonical TGF-β signaling pathways involves β-glycan serving as a co-receptor to present TGF-β1 to transmembrane serine/threonine kinase type I and type II receptors (TGFBR1 and TGFBR2, respectively) (103). The receptor complex formation upon TGF-β1 binding by TGFBR2 leads to the phosphorylation of TGFBR1 (ALK5), which in turn phosphorylates Smad2 and Smad3 forming a complex with Smad4 ( Figure 6 ). The trimeric complex translocates to the nucleus and regulates different target gene expressions in concert with transcriptional co-activators and co-repressors. One of these target genes is the myofibroblast marker α-SMA (104, 105). Smad signaling is attenuated by inhibitory Smads (I-Smads, Smad6 and Smad7), dephosphorylation and dissociation from the trimeric complex, allowing for either Smad recycling or degradation (106).

Apart from the canonical TGF-β1/Smad2/3-signaling TGF-β can signal via non-canonical pathways. Upon TGF-β stimulation, ALK1 another TGFBR1 forms a complex with ALK5, leading to Smad1/5 activation (107). Furthermore, it has been demonstrated that TGF-β activates a number of other non-canonical (non-Smad) signal pathways in different cell types, such as mitogen-activated protein kinases (extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38), phosphatidylinositol-3-kinase, and GTPases that resemble Rho ( Figure 6 ) (108).

RA synovial fluid, rich in TGF-β, has been demonstrated to induce α-SMA expression in adipose-derived mesenchymal stromal cells (MSCs) via Smad2 signaling, indicating TGF-β’s pivotal role in fibrotic phenotype progression (109). Moreover, the deletion of Tgfβr1 prevented TGF-β-mediated differentiation (110) whereas a constitutively active TGFBR1 mutant promoted myofibroblast formation (111) confirming the importance of the canonical signaling in myofibroblast differentiation. Furthermore, inhibition of TGF-β-mediated activation of ALK4/5/7 in osteoarthritic human FLS prevented the induction of the pyridinoline cross-linking enzyme pro-collagen lysine-2-oxoglutarate 5-dioxygenase 2 (PLOD2), which suppresses collagen degradation, as well as CTGF and COL1A1. In contrast, inhibition of ALK1/2/3/6 blocked the induction of CTGF and COL1A1 (112). However, the non-canonical TGF-β-mediated signaling cascades are also implicated in fibrosis. In patients with the fibrotic disease systemic sclerosis, again TGF-β-dependent upregulation of COL1A1 and CTGF is conveyed via ALK1/Smad1/5- and also ERK1/2-signal pathways rather than ALK5/Smad2/3-activation (113). The activation of the aforementioned non-canonical TGF-β-mediated signaling cascades have been implicated in the pathogenesis of fibrosis and fibrotic diseases including renal fibrosis, liver fibrosis, systemic sclerosis and osteoarthritis (107, 108).

In cells from articular cartilage, synovial tissue and fluid from patients with OA, elevated levels of the transcription factor hypoxia inducible factor (HIF)-1α were found, indicating hypoxic conditions, that are associated with progressive joint damage (114, 115). Once hypoxia is established in the tissue microenvironment, cellular HIF-1α is not hydroxylated and therefore forms a heterodimer with HIF-1β initiating a cellular adaptive program by inducing the transcription of genes such as vascular endothelial growth factor A (VEGFA), TGFB, insulin-like growth factor 2 (IGF2), collagen type 2 alpha 1 (COL2A1), and aggrecan (ACAN). This adaptive transcriptional program promotes angiogenesis, shifts the metabolic program towards glycolysis and maintains ECM homeostasis (116, 117). Hypoxia-mediated TGF-β1 expression led to the release of IL-1β and further induces TGF-β1, suggesting a positive self-enhancing feedback loop between inflammation and fibrosis during myofibroblast activation. This emphasizes the importance of studying synovial fibroblasts in the context of OA with a focus on hypoxia. Boer et al. performed a genome-wide association analysis for OA across 21 cohorts (118). They investigated differential gene expression, methylation or protein abundance in osteophytic cartilage compared to low-grade (intact) cartilage. This study identified genes that are important in TGF-β signaling and function and that fibrosis is a major contributor to the degenerative changes in OA (118). Additional fibrosis-associated genes activated by TGF-β in OA include ECM-encoding genes COL1A1, COL3A1, COL5A1, FN1, and key enzymes important for collagen synthesis such PLOD2, prolyl 4-hydroxylase subunit alpha 3 (P4HA3), and lysyl oxidases (LOXs) (91, 106, 119). In FLS from OA patients stimulated with TGF-β, Remst et al. further observed an upregulation of the tissue inhibitor of metalloproteinase-1 (TIMP1), indicating inhibition of MMPs (120). Furthermore, exposure of OA FLS to TGF-β has been shown to upregulate PRG4 (121). This molecule also plays a distinct role in myofibroblast differentiation and in the expression of pro-fibrotic genes (122). For in vitro studies, various groups have utilized a broad range of TGF-β concentrations over different time intervals, as summarized in Table 1 . The pathophysiological concentrations of active TGF-β in the synovial fluid of patients with OA and RA were determined to be 4 ng/mL and 10 ng/mL, respectively (129, 130). However, after acidic activation, increased concentrations of up to 12 ng/mL and 37.5 ng/mL were detected in the synovial fluid of OA and RA patients, respectively, indicating the presence of higher concentrations of latent TGF-β (129).

The TGF-β target CTGF, also known as CCN2, plays a crucial role in physiological and pathological processes such as inflammation, wound healing, tumorigenesis, and fibrosis (131). Several factors such as TGF-β (120), macrophage-colony stimulating factor (M-CSF) (132), VEGF (133), PGE2 (134), and mechanical stressors (135, 136) induce high expression of CTGF. However, CTGF can function independently and promote a positive feedback loop itself by increasing e.g., TGF-β, VEGF, and integrins (137). Along with TGF-β, CTGF is abundantly expressed in the synovial fluid of OA (138, 138) and RA patients (139) positively correlating with disease severity. The transfection of the synovial lining layer of mice with an adenovirus expressing human CTGF exacerbated synovial fibrosis, as evidenced by increased pro-collagen type 1 levels, accumulation of ECM, and depletion of proteoglycans in articular cartilage (140). Cartilage degradation may be triggered by CTGF expression or CTGF-mediated cytokine secretion from fibrotic synovial tissue. Davidson et al. further demonstrated that CTGF-induced fibrosis is transient (140), in contrast to the relatively persistent fibrosis induced by TGF-β (39). Liu and colleagues showed that stimulation of OA FLS with CTGF induced concentration-dependent production of the pro-inflammatory cytokine IL-6 via the CTGF-αvβ5-JNK pathway (141). They further revealed that CTGF can trigger the migration of monocytes in OA by enhancing monocyte chemoattractant protein-1 (MCP-1) expression through interaction with integrin avβ5 (142). The study by Yang et al. reported that CTGF increased the activity of focal adhesion kinase (FAK), mitogen-activated protein kinase kinase (MEK), and ERK proteins (143), which is consistent with the findings of Tan and colleagues that CTGF promotes chondrosarcoma cell migration by increasing the expression of MMP-13 via the αvβ3 integrin, FAK, ERK, and NF-κB signal pathways (144). In summary, CTGF causes synovial fibrosis by activating fibroblast and their transition into myofibroblast, stimulates the production of pro-inflammatory cytokines and thus promotes synovitis and catabolic, destructive processes in the articular cartilage of OA patients.

The binding of cytokines and growth factors such as TGF-β or CTGF to their respective receptors is the predominant way of transmitting a signal and triggering signaling cascades in the responding cell, while electrical and mechanical stimuli also play an important role in signal transmission in a rather non-selective manner (145). In this way, mechanical forces are capable to regulate downstream signaling within the TGF-β pathway at multiple levels and influence both gene and protein expression (146). Additionally, myofibroblast contraction, tissue stiffening and aberrant tensile forces in the ECM activate latent TGF-β from ECM stores, thereby inducing e.g., Smad2/3-signaling and contributing to fibrosis (147). Beyond tensile forces, cells also respond to other forms of mechanical stimuli including compression, shear stress, and hydrostatic pressure ( Figure 7 ) (148).

In joint tissues, shear and compression are the predominant mechanical forces. However, tensile forces notably influence the joint tissues as well. The sinister combination of high levels of pro-fibrotic factors such as TGF-β and CTGF in osteoarthritic joints with short- and long-term pathologically high mechanical forces facilitate the differentiation of FLS into myofibroblasts ─ a hallmark of OA (149–152).

While most research in OA in recent decades has focused on the role of cartilage and subchondral bone, synovial tissue and fibroblasts are gaining increasing attention. They are, in particular, recognized for their role in maintaining cartilage homeostasis and mitigating “wear and tear maintenance” (153). The gathered research demonstrates a nuanced correlation between mechanical stimuli and TGF-β signaling regulating various cellular pathways ( Table 2 ). Upregulation of COL1A1 and FN1 genes in OA FLS and elevated levels of pro-inflammatory markers such as PGE2, IL6, and IL8 in mechanically loaded samples demonstrate the interchangeable influences of mechanical forces and TGF-β signaling (154, 155). Cyclic strain on FLS elevates their TIMP1 levels, while MMP1 and MMP13 appear to be diminished, suggesting regulation of ECM dynamics (157). PRG4 expression, which plays a role in lubrication, is also induced and is further enhanced by TGF-β stimulation (159). Furthermore, mechanical stimulation of FLS increases the expression of TGFB1, with the resulting conditioned medium increasing pro-inflammatory markers in untreated FLS (161). Lysyl oxidases, crucial for collagen and elastin cross-linking, are generally increased after 6 h of physiological stretch (except LOXL-3) and decreased after pathological stretch (except LOXL-2). Here, a distinct temporal profile is evident, with a maximum of expression around 2-3 h and variations noted under differing stretching conditions (162). While some observations, such as IL-1α-mediated mechanosensitivity, are not analogous to TGF-β effects, they may contribute to the complexity of the fibroblast response to mechanical and biochemical stimuli (158). Table 2 summarizes the mechanical stimuli applied to synovial fibroblasts, their intensity and mode of application, their effects, and any analogies to TGF-β signaling.

However, a critical limitation of the studies performed on synovial fibroblasts exposed to mechanical stimuli is the lack of studies on the key molecule α-SMA, which is encoded by the ACTA2 gene and is a well-documented marker for myofibroblast differentiation to underpin the impact of mechanical strain on fibrosis.

Acute sterile inflammation is usually a transient and controlled response aimed at eliminating the cause of the injury and promoting tissue repair. However, if inflammation persists and becomes chronic, this well-controlled process can be pathologically disrupted (163). Inflammatory stimuli and innate immune responses play a central role in the development of OA, leading to chronic inflammation and tissue remodeling closely associated with the development of fibrosis. In OA, synovitis is a feature in the early stages and is characterized by the release of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 (164). Cytokines and other inflammatory mediators initiate cascades that contribute to the perpetuation of inflammation, synovial hyperplasia, hypoxia, subsequent synovial angiogenesis, progressive joint degradation, the activation of fibroblasts and their transition to myofibroblasts. Inflammatory cytokines contribute directly to the activation of synovial fibroblasts and promote the recruitment and activation of immune cells that further enhance inflammatory and fibrotic responses (165). Current studies do not provide evidence to conclude whether fibrosis can occur independently of synovitis, so it is not yet entirely clear which is the hen and which is the egg.

Usually, the early host defense of acute inflammation against invading pathogens implicates the recognition of conserved pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) (165, 166). The latter include calcium phosphate crystals, hydroxyapatite crystals, and uric acid found in the knee and hip joints of patients with OA. PAMPs and DAMPs are recognized by the pattern recognition receptors (PRRs) (167). and activate mechanisms of NLRP3 inflammasome activation leading to the production of inflammatory cytokines such as TNF-α and pro-IL-1β. Three prominent PRRs have been discovered, namely TLRs, nucleotide-binding oligomerization domain–like receptors (NLRs), and intracellular retinoic acid–inducible gene I receptors (RIG-I) (168, 169). They are present in various cell types, including immune cells and structural cells such as fibroblasts. Fibroblasts express a repertoire of PRRs, including TLRs, NLRs and IL-1R. For instance, DAMPS can activate mechanisms of NLRP3 inflammasome activation which trigger an innate immune response profile (167). This includes the production of numerous pro-inflammatory cytokines such as TNF-α and pro-IL-1β and chemokines which induce signaling cascades that promote the differentiation of FLS into myofibroblasts via auto- and paracrine signaling (169). Ospelt et al. compared FLS derived from trauma, RA, or OA patients, demonstrating constitutive gene expression for TLR1-6 but not for TLR7-10 (170). The TLR3 and TLR4 were the most abundant among TLR genes expressed in FLS, followed by members of the TLR2 subfamily. TLR2/4-signaling can be activated by hyaluronan fragments of distinct sizes which usually interact with CD44, a receptor for hyaluronic acid and other ligands, such as collagens, and MMPs (171, 172). As a result, a cellular pro-angiogenic and immunostimulatory response is initiated. Moreover, ED-A of FN, an endogenous TLR4 ligand, triggers the upregulation of MMP-9 supporting fibrosis (173, 174). Abdollahi-Roodsaz et al. showed that overexpression of IL-1β ─ a key cytokine in OA pathogenesis ─ triggers TLR4-mediated synovitis, whereas TLR2 plays a less significant role (175). Moreover, the severity of IL-1β-induced joint degradation in TLR4^-/- mice was reduced, with similar levels of inflammation, suggesting independent processes (175). Therefore, interfering with TLR signaling may be an approach to limit the activation of the FLS-mediated innate immune response, reducing inflammation, and limiting fibrosis.

Besides TLR-mediated FLS stimulation, FLS stimulation with pro-inflammatory cytokines such as TNF-α and IL-17A caused an increase in matrix mineralization, thereby inducing the expression of Wnt5a (176). Canonical Wnt/β-catenin signaling and inflammatory processes are strongly activated in OA synovium in mice and humans (177, 178). Using a PTOA mouse model, Knights and colleagues revealed that the Wnt/β-catenin signaling agonist Rspo2 was strongly upregulated after injury and exclusively secreted by Prg4⁺ lining layer FLS (73). In rats with collagen-induced arthritis, activation of the Wnt/β-catenin signaling pathway was triggered by TNF-α, leading to a concomitant upregulation of α-SMA and cadherin-11 (CAD-11) expression in synovial fibroblasts (179). This subpopulation was found predominantly in the synovial lining layer in RA and was associated with high expression of PDPN (180). The strongest expression of PDPN⁺ FLS is also present in the lining layer in OA (181), suggesting a myofibroblast-like α-SMA⁺ ED-A-FN⁺ CAD-11⁺ PDPN⁺ phenotype. In addition, XAV-939, an inhibitor of the Wnt/β-catenin signaling, decreased the proliferation of human OA FLS and further inhibited the synthesis of collagen type I (178). This underscores the importance of the canonical Wnt/β-catenin signaling pathway in OA progression, which ultimately leads to synovial fibrosis in which the lining layer appears to be predominantly affected.

The onset and progression of OA are closely linked to the cellular aging process in which cells accumulate in a state of senescence (182, 183). These senescent cells (SnCs) are characterized by permanent growth arrest, enhanced ROS synthesis, resistance to apoptosis, and a permanent low-level release of pro-inflammatory molecules. This phenotype is known as senescence-associated secretory phenotype (SASP). Removal of SnCs by administration of UBX0101(a compound inducing apoptosis in SnCs) in transgenic mice with anterior cruciate ligament transection (ACLT)-induced OA, inhibited cartilage erosion, reduced pain, and decreased inflammatory markers such as mmp13 and il1β, and enhanced the number of ki-67-positive synovial fibroblasts (184).

While senescence’s impact on OA has been extensively studied in chondrocytes, its role in synovial fibroblasts and myofibroblasts still remains unclear (182, 185). In pulmonary fibrosis, the senescence of fibroblasts is evident (186), while senescence of other cell lineages such as macrophages, chondrocytes or cardiomyocytes have been related to various aging associated pathologies such as OA or cardiomyopathy (184, 187). Recently, Chen et al. demonstrated that FLS which are associated with impaired autophagy, show a SASP and accumulate in OA (188). These OA FLS express GATA binding protein 4 (GATA4), a regulator of cellular senescence, which is linked to increased expression of p16INK4a, p21 and a SASP. Restoration of autophagy in OA FLS by rapamycin treatment was effective in reducing the expression of GATA4 (188). Impaired autophagy (and mitophagy) leading to an accumulation of dysfunctional mitochondria and excessive ROS production (189). Chronic inflammatory diseases such as OA are characterized by elevated oxidative stress and ROS production (190, 191). Excessive ROS production is a major contributor to mitochondrial damage and dysfunction and to genomic damage as a cause for the onset of cellular senescence (189). In addition, mitochondrial dysfunction has been observed in the development of synovial fibrosis in OA (192). Mitochondrial ROS (mtROS) represent by-products of (patho)physiological processes with profound cellular consequences, ranging from modulation of intracellular signaling cascades to posttranslational modifications. The primary source of superoxide (O₂ ^─) in the cell, which is a product of NAD(P)H oxidase, arises from electron losses at complexes I and III in the electron transport chain (ETC). In various cell types, ROS formation is enhanced in response to TGF-β1 (193–195). These observations are of great importance for fibrogenesis, as fibroblasts are not only responsible for the generation of ROS, but ROS are also directly associated with the transformation of fibroblasts into α-SMA expressing myofibroblasts. A study conducted by Jain and colleagues demonstrated that the elevated mtROS generated at complex III are required for TGF-β-induced gene expression (193). In addition, the transcriptional activation of NADPH oxidase 4 (NOX4) by TGF-β requires the generation of mtROS. Notably, TGF-β-exposed fibroblasts derived from patients with pulmonary fibrosis exhibited higher mtROS production and increased pro-fibrotic gene expression compared to healthy subjects. Noteworthy reductions in TGF-β-induced pro-fibrotic gene expression and NOX4 expression were observed upon the inhibition of mtROS production (193). These findings underscore the crucial role of complex III generated mtROS in TGF-β-mediated fibroblast to myofibroblast transition and suggest potential therapeutic avenues.

A study by Bondi and colleagues showed that rat kidney fibroblasts stimulated in vitro with TGF-β induced the expression of α-Sma, Fn, Nox2, and Nox4 and increased NAD(P)H oxidase activity, leading to mtROS production (195). The siRNA-mediated reduction of Nox4 significantly reduced the expression of α-Sma and Fn. Inhibition of TGF-β receptor 1 blocked Smad3 phosphorylation, decreased TGF-β-induced NADPH oxidase activity, and reduced the expression of Nox4, α-Sma, and Fn. Furthermore, TGF-β stimulated the phosphorylation of ERK1/2, which was inhibited by blocking TGF-β1 receptor 1 or Smad3. ERK1/2 activation also increased α-Sma and Fn (195). These results demonstrate that TGF-β-induced transition from fibroblasts to myofibroblasts involves canonical TGF-β/Smad2/3 signaling, non-canonical TGF-β/ERK1/2 signaling, and ROS production via NAD(P)H oxidase. Guido and colleagues generated fibroblasts overexpressing the mitochondrial fission factor (MFF). This factor regulates the mitochondrial network by continuous fission expanding the interconnected network of mitochondria when expressed at physiological levels. Overexpression of MFF caused mitochondrial dysfunction, mitophagy and autophagy, intracellular ATP depletion, increased ROS production, L-lactate secretion, and increased expression of α-SMA (196). Thus, MFF-induced mitochondrial dysfunction shifts cellular metabolism to a catabolic state in fibroblasts inducing α-SMA a key marker of myofibroblasts.

Metabolic alterations in glycolysis play a central role regarding energy supply, biosynthesis, cell growth, and cell differentiation. Bardon, Ceder, and Kollberg were the first to demonstrate significantly increased activities of glycolytic enzymes such as hexokinase (HK), phosphofructokinase-1 (PFK1), pyruvate kinase (PKM) and lactate dehydrogenase (LDH) in activated fibroblasts from cystic fibrosis patients (204). The upregulation of numerous glycolytic genes that act as metabolic checkpoints has already been verified in various fibroblast populations. Aguilar and colleagues cultured rat cardiac fibroblasts in 5 mM glucose (normal) or 25 mM glucose (high) for 48 h (205). The results show that the O-GlcNAcylation of proteins and the protein content of TGF-β1 in cardiac fibroblasts increases with higher glucose concentrations, thereby activating pro-fibrotic signaling pathways and inducing an increase in collagen synthesis (205).

Assuming that extracellular glucose concentration alone is sufficient to induce fibroblast to myofibroblast transformation and thus fibrosis, Xie et al. inhibited glycolysis in lung fibroblasts by inhibiting 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) (206). In glycolysis, PFK1 is the first glycolysis-specific enzyme, a rate-limiting enzyme, and a key control point for regulating glycolytic flux. PFK1 is allosteric activated via the derivative fructose-2,6-bisphosphate, regulated by PFKFB3 (feedforward regulation). Therefore, inhibiting PFKFB3 prevented the differentiation of lung fibroblasts isolated from patients with IPF into myofibroblasts and attenuated the pro-fibrotic phenotype (206). Furthermore, upregulation of PFKFB3 was shown to be dependent on TGF-β1-activated Smad2/3, as both blocking the TGF-β1 receptor and silencing Smad2/3 abolished the induction of PFKFB3. Moreover, HIF-1α is downstream of increased glycolysis and represents a direct mechanism by which glycolysis is involved in pathologic myofibroblast differentiation (206). Activation of hepatic stellate cells (HSCs) is a key event during liver fibrosis. Since aerobic glycolysis is one of the metabolic hallmarks, Ban and colleagues investigated the effects of targeted inhibition on HSC activation (207). The glycolysis inhibitor 2-desoxy-D-glucose (2-DG) and costunolide showed similar effects in inhibiting the rate-limiting enzyme HK2, leading to decreased expression of HSC activation markers such as α-SMA and collagen I. This could be reversed by overexpression of HK2 induced by plasmid transfection, suggesting that inhibition of HK2 represents a new therapeutic option for (liver) fibrosis (207).

Another glycolytic enzyme that appears to be crucial for the formation of myofibroblasts is pyruvate kinase M2 (PKM2). PKM2 is an important factor in the Warburg effect of cancer cells (208) and can occur in two forms in proliferating cells such as fibroblasts: a catalytically highly active tetrameric and a less active dimeric form. While the tetrameric form is routing glucose metabolism to pyruvate and into the tricarboxylic acid (TCA) cycle for energy metabolism, the PKM2 dimer transfers the metabolic pathway to the pentose phosphate pathway (PPP), the uronic acid pathway (UAP), the polyol pathway (PYP), etc. for the material synthesis such as the synthesis of the subsequent five-carbon ribose and non-essential amino acids (209). Satyanarayana et al. analyzed the expression of PKM2 forms in cultured fibroblasts and myofibroblasts from tissues with fibrotic diseases (210). They showed that the less catalytically active PKM2 dimer is upregulated in myofibroblasts with a concomitant increasing NADPH production. This protects myofibroblasts from apoptosis, thereby feeding the de novo glycine synthesis needed for myofibroblasts’ collagen production and deposition. Moreover, conversion of the dimeric form to the catalytically active, tetrameric PKM2 form inhibited fibrosis progression in mouse models of liver, lung, and pancreatic fibrosis (210).

Smith and Hewitson showed that renal fibroblasts stimulated with TGF-β1 downregulate acetyl-CoA biosynthesis by inhibition of the pyruvate dehydrogenase complex (211). The increase in PDK1 in response to TGF-β1 stimulation corresponds to the increase in inactive PDH-E1α (211). Although lactate has traditionally been considered a by-product of the glycolytic pathway, recent studies suggest a possible direct pathophysiological role of lactate in the development of fibrosis. The study by Nho et al. showed increased lactate formation in fibrotic lung fibroblasts due to increased LDHA and a concomitant decrease in LDHB (212). LDHA converts pyruvate into lactate and NAD⁺, while LDHB is responsible for the conversion of lactate into pyruvate (213). High lactate concentrations lead to the conversion of lactate into pyruvate and the production of NADH, which results in an inhibitory feedback on glycolysis (213). In addition, lactate itself was shown to promote the differentiation of myofibroblasts into normal lung fibroblasts (from deceased individuals with no evidence of lung disease) via the lactate receptor GPR-81 (212). LDH5, one of the five isoenzymes of LDH, is most effective in catalyzing the conversion of pyruvate to lactate. In cancer cells, overexpression of LDH5 results in an increased glycolytic metabolism and a decreased need on oxygen. Consistently, Schruf and colleagues demonstrated that inhibition of LDH5 in primary lung fibroblasts attenuated TGF-β1-mediated metabolic switch to aerobic glycolysis (214). Nevertheless, LDH5 inhibition had no significant effect on TGF-β1-mediated transformation of fibroblasts to myofibroblasts in primary human lung fibroblasts, suggesting that the LDH5-dependent metabolic shift to aerobic glycolysis alone is not the decisive factor (214).

The fatty acid metabolism, encompassing de novo synthesis, uptake, oxidation, and disposal of fatty acids, serves crucial roles at the cellular and organ levels. Fatty acid oxidation (FAO) is the preferred energy source for cells with a high metabolism. FAO generates more ATP than the oxidation of glucose, which is why it is necessary for the activation and proliferation of fibroblasts. In FAO, long-chain fatty acids are more easily absorbed via CD36. Medium-chain and short-chain fatty acids enter the cell without the need for specific transporters. Here, the FAO pathway is promoted by the upregulation of carnitine O-palmitoyltransferase 1 (CPT1) and peroxisome proliferator-activated receptor (PPAR) signaling. Nan and colleagues demonstrated that treatment with rosiglitazone, a ligand activating PPARγ, prevents the development of fibrosing steatohepatitis induced by a methionine-choline deficiency (MCD) diet in C57BL6/J mice (215). Furthermore, targeted overexpression of PPARγ by an adenovirus reduced the extent of liver fibrosis in male C57BL6 mice on MCD diet (216).

Basal autophagy is ubiquitously present in all cell types. It is rapidly upregulated as an adaptive response under cellular stress conditions to obtain intracellular nutrients and energy. In liver injury, it has been shown that autophagy is primarily upregulated in activated stellate cells while inducing fibrogenic markers. In this context, autophagy contributes to the intracellular degradation of lipids and correlates with increased fatty acid β-oxidation and mitochondrial OXPHOS (217). Hernández-Gea et al. demonstrated that HSCs from autophagy-deficient C57BL/6 mice were unable to process cytoplasmic lipid droplets, reducing the availability of free fatty acids (218). As a result, mitochondrial β-oxidation and ATP production were reduced, which attenuated fibrogenesis. Targeted inhibition of β-oxidation with etomoxir, simulating the effect of autophagy blockade, resulted in reduced expression of the fibrogenic genes α-Sma, collagen 1α1, collagen 1α2, Pdgfr-β, and Mmp-2 (218). Obviously, blockade of autophagy is different to impaired autophagy and mitochondrial dysfunction since the later induces mtROS and cellular senescence also capable of inducing α-SMA expression supporting fibrosis (188, 192, 196). Conversely, glycolysis upregulation and FAO downregulation associated with higher lipid accumulation and accompanied by succinate accumulation has been demonstrated in fibroblasts and myofibroblasts from idiopathic pulmonary fibrosis (219). Furthermore, TGF-β1 induced succinate dehydrogenase (SDH) and succinate elevation. Elevated levels of succinate contribute to enhanced glycolysis and reduced FAO by stabilizing HIF-1α. Inhibition of SDH subunit A in fibroblasts prevents fibrosis formation and respiratory dysfunction deeming SDH as a new therapeutic target in fibrosis (219).

Moreover, acetyl-CoA carboxylase (ACC) catalyzes the rate-limiting step of de novo lipogenesis and regulates fatty acid β-oxidation. Using HSCs, Bates and colleagues investigated the role of ACC in liver fibrosis (220). Based on α-SMA expression and collagen production, they showed that inhibition of ACC reduces the activation of TGF-β-stimulated HSCs and thus reduces fibrosis. Inhibition of de novo lipogenesis also blocks glycolysis and induces FAO, demonstrating that de novo lipogenesis is required for fibroblast activation (220). It is known that the fibroproliferative effects of TGF-β are dependent on metabolic adaptation to maintain pathological growth. Herein, fatty acid synthase (FASN) is an essential anabolic enzyme in TGF-β-mediated fibrosis. Jung et al. showed that TGF-β increases FASN expression, which is mediated via the mammalian target of rapamycin complex 1 (mTORC1) (221). FASN expression correlated with the extent of pulmonary fibrosis in bleomycin-treated mice. Inhibition of FASN reduced the expression of pro-fibrotic targets and stabilized lung function, as analyzed by peripheral blood oxygenation (221). Metabolic comparison of synovial fibroblasts obtained from patients with RA, patients with OA, and seronegative controls showed that basal and maximal mitochondrial respiration was significantly lower in RA FLS compared to control FLS. In all donors, basal respiration was largely dependent on FAO, whereas glycolysis was highly utilized in FLS from RA patients (222). However, the evidence for the significance of FAO and FAS in OA and synovial fibrosis remains insufficiently understood and needs further research.

Glutamine is the most abundant amino acid in the human body. It plays an anaplerotic role replenishing the TCA cycle intermediates α-ketoglutarate (α-KG) during increased aerobic glycolysis and reduced OXPHOS (223). Fibroblasts stimulated with TGF-β show increased glutaminolysis, which contributes to increased α-KG content (224). The increased glutaminolysis can be achieved in fibroblasts by upregulation of the rate-limiting enzyme glutaminase 1 (GLS1) (224) and TGF-β (223). The study by Ge et al. found that increased glutaminolysis in myofibroblasts is required for collagen translation and stability (224). The amino acid composition of collagen is unique due to its high glycine content. Targeted inhibition of glutaminolysis with the Gls1 inhibitor CB-839 and BPTES, and genetic silencing by Gls1 short interfering (si)RNA, only reduced the expression of collagens, but not that of FN or α-SMA. This is consistent with the increased α-KG content in myofibroblasts, as α-KG activates mTORC1, which promotes the expression of collagen (224). Importantly, Bernard’s data from 2018 show that suppression of glutaminolysis after myofibroblast differentiation reverses TGF-β induced metabolic reprogramming (223).

Myofibroblasts are synthetic and contractile cells, and these functions are dependent on OXPHOS and ATP production. Glutaminolysis stimulated by TGF-β may therefore support myofibroblast functions by meeting bioenergetic needs by increasing OXPHOS and biosynthetic needs by providing anabolic carbons for e.g., FAS when succinate levels are high (219). Depletion of extracellular glutamine as well as silencing of GLS1 expression in the presence of glutamine prevented TGF-β induced myofibroblast differentiation (223). Studies showed that TGF-β induces the expression of the de novo serine [phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase 1 (PSAT1) and phosphoserine phosphatase (PSPH)] and glycine [serine hydroxymethyltransferase 2 (SHMT2)] synthesis pathways in human fibroblasts (225). Genetic attenuation of PHGDH or SHMT2 and pharmacologic inhibition of PHGDH in human lung fibroblasts have shown that these enzymes are required for collagen synthesis downstream of TGF-β. Thereby, PHGDH is the first and rate-limiting enzyme in this pathway (225). Hamanaka and colleagues investigated whether inhibiting de novo serine and glycine synthesis mitigates lung fibrosis in vivo (226). TGF-β stimulation induces Phgdh expression at both mRNA and protein levels in mouse fibroblasts, and this effect is reflected by an increase in Phgdh expression in mouse lungs after intratracheal administration of bleomycin. However, treatment of murine and human lung fibroblasts with a small molecule Phgdh inhibitor (NCT-503) reduced TGF-β induced collagen protein synthesis. Moreover, mice treated with the Phgdh inhibitor seven days after intratracheal instillation of bleomycin exhibited attenuation of lung fibrosis (226). In summary, these results demonstrate that glucose-derived serine and glycine metabolism plays a pivotal role in the fibrotic response both in vitro and in vivo.

The transition from functional fibroblasts to fibroblast activation and expansion to senescent myofibroblasts involves several steps of metabolic alterations, highlighting glycolysis, FAO and glutaminolysis as key processes. This transition is critical in the development and progression of fibrotic diseases. Interventions targeting glycolysis, such as inhibition of PFKFB3, a key regulator of glycolytic flux, demonstrate potential in attenuating myofibroblast differentiation. Furthermore, Alterations in enzymes such as PKM2 and shifts in metabolic pathways (e.g., from TCA cycle to PPP) play a critical role in myofibroblast functionality and survival, thus influencing fibrosis progression. Similarly, FAO is essential for fibroblast activation. Targeting key metabolic enzymes in FAO such as ACC and FASN could provide promising anti-fibrotic effects. Moreover, TGF-β-induced metabolic reprogramming leads to increased glutaminolysis and serine/glycine synthesis, essential for collagen production and myofibroblast differentiation. Thus, targeting key enzymes such as GLS1 and PHGDH offers promising avenues for therapeutic intervention in fibrotic diseases. These findings emphasize the importance of metabolic pathways in fibrosis and the potential of metabolic interventions as therapeutic strategies.