Targeting cluster of differentiation 26 / dipeptidyl peptidase 4 (CD26/DPP4) in organ fibrosis

Cluster of differentiation 26 (CD26)/dipeptidyl peptidase 4 (DPP4) is an exopeptidase that is expressed as a transmembrane protein in many organs but also present in a circulating soluble form. Beyond its enzymatic and costimulatory activity, CD26/DPP4 is involved in the pathogenesis of chronic fibrotic diseases across many organ types, such as liver cirrhosis, kidney fibrosis and lung fibrosis. Organ fibrosis is associated with a high morbidity and mortality, and there are no causative therapies that can effectively attenuate the progress of the disease. Growing evidence suggests that inhibiting CD26/DPP4 can modulate the profibrotic tissue microenvironment and thus reduce fibrotic changes within affected organs. This review summarizes the role of CD26/DPP4 in fibroproliferative disorders and highlights new opportunities for an antifibrotic treatment by CD26/DPP4 inhibition. As a major advantage, CD26/DPP4 inhibitors have been in safe and routine clinical use in type 2 diabetes for many years and thus qualify for repurposing to repurpose as a promising therapeutic against fibrosis.


| CLUSTER OF DIFFERENTIATION 26 /DIPEPTIDYL PEPTIDASE 4 (CD26/DPP4) AS A REGULATOR IN EXTRACELLULAR MATRIX AND FIBROSIS
Fibrosis is the pathomorphological hallmark of many chronic inflammatory diseases, such as systemic sclerosis, graft-versus-host disease, liver cirrhosis, myocardial infarction, arteriosclerosis, interstitial renal fibrosis and pulmonary fibrosis.Fibrotic remodelling can occur in nearly every tissue eventually leading to organ failure and thus contributing to approximately a third of all natural deaths in the industrialized world (Henderson et al., 2020).Despite this pressing disease burden, causative treatment options that prevent or reverse the fibrotic process are not yet available for any organ.However, the common histomorphological features pointing towards common profibrotic pathways provide hope in the search for potential therapeutic drug targets across different organ systems.
Fibrosis is the final pathological outcome of a dysregulated repair response upon tissue injury and is characterized by excess deposition of ECM components.The ECM forms a complex three-dimensional network consisting of fibrillar macromolecules, such as collagens, fibronectin and elastin, as well as non-fibrillar components, such as Abbreviations: COPD, chronic obstructive pulmonary disease; CXCL12, CXC-motif chemokine ligand 12; ECM, extracellular matrix; EMT, epithelial-to-mesenchymal transition; EndoMT, endothelial-to-mesenchymal transition; IPF, idiopathic pulmonary fibrosis; miRNA, microRNA; NAFLD, non-alcoholic fatty liver disease; NASH, non-alcoholic steatohepatitis; proteoglycans, hyaluronan and glycoproteins (Theocharis et al., 2016).
It does not only act as a passive scaffold embedding cellular components in tissues but also actively communicates with cells by binding cell surface receptors or via mechanical cues (Dong et al., 2017).Even under normal conditions, the ECM undergoes continuous remodelling during which matrix degradation coexists simultaneously with the production of new matrix components.Consequently, the ECM is a critical player in maintaining tissue homeostasis, and abnormal ECM remodelling can drive disease progression by modulating cell-matrix interactions and intracellular signalling pathways (Herrera et al., 2018).
Fibrotic diseases originate from dysregulation of the repair response during wound healing.Upon tissue injury, an inflammatory response is initiated and ultimately results in the activation of myofibroblasts, mainly via transforming growth factor-β1 (TGF-β1).Myofibroblasts are the key cellular drivers of fibrotic remodelling by ECM deposition and are characterized by the expression of α-smooth muscle actin (α-SMA).They can arise from different progenitors, such as tissue-resident mesenchymal cells, inflammatory monocytes and possibly epithelial and endothelial cells (Wynn, 2008).The newly formed ECM then provides mechanical stabilization of the injured tissue, the immobilization of growth factors, and aids in cell migration to the area of repair.During physiological wound healing, the fibrotic process ends with the timely clearing of myofibroblasts.However, under pathological conditions such as a sustained inflammatory stimulus, failure to resolve the injury results in a continuous cycle of fibroblast activation and ECM deposition resulting in tissue fibrosis and functional impairment.
CD26/DPP4 is widely expressed as a type II transmembrane protein.It can be found in all organs, mainly on endothelial, epithelial and acinar cells (Heike et al., 1988).CD26 is also expressed on a variety of cells of the adaptive immune system, such as T-cells, B cells and natural killer cells, and is strongly up-regulated in activated lymphocytes.In T-cells, it has been characterized as a costimulatory molecule (Ishii et al., 2001).Cells of the myeloid lineage also show CD26 expression, and growing evidence suggests a proinflammatory role of CD26 in innate immunity (Yazbeck et al., 2021).As an exopeptidase, CD26/ DPP4 exerts enzymatic activity cleaving N-terminal dipeptides from substrates carrying a proline or alanine residue at their penultimate position (Kenny et al., 1976).To date, a wide variety of substrates or potential substrates have been identified including chemokines, cytokines and growth factors.Their cleavage alters their functional activity via modulation of receptor selectivity or by facilitating their degradation.
DPP4 is also present systemically in a soluble form (sDPP4) as it is shed from the cell surface by matrix metalloproteinases (MMPs) (Röhrborn et al., 2014).During this process, DPP4 retains its enzymatic region as the catalytic centre remains intact.Accordingly, 95% of all dipeptidyl peptidase activity in human serum can be attributed to sDPP4 (Durinx et al., 2000).However, sDPP4 can also exert biological functions targeting cellular effectors.To date, knowledge of sDPP4 signalling is still limited.Protease-activated receptor 2 (PAR2) has been identified as a functional sDPP4 receptor in human vascular smooth muscle cells (Wronkowitz et al., 2014).PAR2 is a seven-transmembrane domain G protein-coupled receptor that is widely expressed in human tissues and immune cells and is activated by several systemically available proteases, and its signalling is implicated in a wide variety of inflammatory diseases (Heuberger & Schuepbach, 2019).
Recently, CD26/DPP4 has been identified as a marker of a highly activated subset of myofibroblasts, and its inhibition resulted in reduced ECM deposition in skin fibroblast cultures (Rinkevich et al., 2015;Soare et al., 2020).Moreover, CD26 has been shown to bind to the ECM components collagen 1 and fibronectin, thereby promoting cell migration and forming the architecture of the fibrotic microenvironment (Ghersi et al., 2002(Ghersi et al., , 2006;;Hanski et al., 1985; F I G U R E 1 Impact of cluster of differentiation 26 (CD26)/dipeptidyl peptidase 4 (DPP4) on fibroblasts.CD26/DPP4 is expressed in a highly active subset of fibroblasts and facilitates the production of extracellular matrix (ECM) components and profibrotic mediators by enhancing intracellular transforming growth factor-β (TGF-β) signalling.On the cell surface, CD26/DPP4 forms a complex with seprase/fibroblast activation protein-α (FAP-α), which aids in cell migration and fibroblast assembly due to its collagen-binding and ECM-degrading capabilities.Piazza et al., 1989).Forming complexes with seprase (or fibroblast activation protein-α [FAP-α]), CD26 also assists in the degradation of ECM proteins, which further facilitates cell invasion to the site of injury (Ghersi et al., 2002(Ghersi et al., , 2006)).The specific role for CD26/DPP4 in fibroblasts is detailed in Figure 1.CD26/DPP4 recently gained increased attention as a potential target for antifibrotic therapy: Indeed, CD26/DPP4 inhibition markedly reduces fibrotic changes in various animal models of organ fibrosis as discussed below.To date, different CD26-dependent signalling pathways have been identified in fibrotic disorders, providing mechanistic evidence for the antifibrotic effects of CD26/DPP4 inhibition (Chang et al., 2017;Lee et al., 2020;Shi et al., 2015;Suzuki et al., 2017).

| CD26/DPP4 MODULATES TGF-β SIGNALLING IN FIBROGENESIS
The profibrotic role of TGF-β signalling has been intensively described in a wide range of fibrotic diseases, and it is known as the key activator of fibroblasts in both human and murine systems (Henderson et al., 2020).However, therapeutic approaches directly targeting TGFβ systemically have only shown limited success due to the pleiotropic effects of the cytokine resulting in serious side effects such as autoimmune reactions and an increased incidence of malignancies (Lacouture et al., 2015).Therefore, local modulation of downstream effects of TGF-β signalling seems to be a more promising strategy in antifibrotic drug development (Lachapelle et al., 2018).Nintedanib and pirfenidone, two antifibrotic drugs in clinical use against idiopathic pulmonary fibrosis (IPF), modulate the downstream effects of TGF-β even though their exact mechanisms of action remain poorly understood (Lachapelle et al., 2018).Several studies have shown that the inhibition of CD26/DPP4 significantly reduces TGF-β-induced fibroblast activation and collagen release (Kaji et al., 2014;Li, Zhang, et al., 2019;Liu et al., 2020;Liu & Qi, 2020;Soare et al., 2020).
Under proinflammatory conditions, TGF-β is produced by immune cells, epithelial cells (Juban et al., 2018) or platelets (Meyer et al., 2012) in a latent form.Latent TGF-β is then stored extracellularly inside the ECM (Falcone et al., 1993), where biologically active TGF-β can be released from its latent complexes.Local activation of TGF-β is proposed as a key mechanism in establishing and controlling a profibrotic microenvironment (Lodyga et al., 2019).Several different proteases, such as cathepsins, serine proteases and MMPs, have been demonstrated to activate TGF-β (Frangogiannis, 2020;Okuno et al., 2001).
Direct effects of CD26/DPP4 on TGF-β activation or production have not been demonstrated conclusively.TGF-β lacks an appropriate DPP4 truncation site and therefore is not subject to the enzymatic activity of CD26 (Ou et al., 2013).Interestingly, up-regulation of TGFβ production upon CD26/DPP4 inhibition has been reported in activated lymphocytes (Reinhold, Bank, Bühling, Lendeckel, et al., 1997;Reinhold, Bank, Bühling, Täger, et al., 1997).More recent studies in a model of autoimmune encephalitis support these observations as they show increased TGF-β release from Th1 cells upon CD26/DPP4 inhibition (Preller et al., 2007;Steinbrecher et al., 2001).Most likely, these effects should be attributed to specific effects of CD26/DPP4 in Th1 autoimmunity.In contrast, local reduction of TGF-β expression upon CD26/DPP4 inhibition has been described in fibrotic disease models, which do not rely on Th1 immune responses (Li, Lian, et al., 2019).In a randomized control trial in healthy individuals in which TGF-β production was evaluated as the primary outcome measure, sitagliptin treatment failed to influence systemic levels of the cytokine and did not alter TGF-β release from peripheral blood cells (Price et al., 2013).
Generally, the inhibition of CD26/DPP4 may regulate TGF-β production in a disease-specific manner mainly depending on the cell type involved.Although CD26/DPP4 inhibition has no major effects on systemic TGF-β levels, it generally results in a local reduction of TGF-β expression in fibrotic disease.
Recently, Lee et al. (2020) discovered a role for soluble DPP4 (sDPP4) in modulating the Smad pathway via PAR2 as they detected an increase of phosphorylated Smad2/3 in murine fibroblasts upon stimulation with sDPP4 alone.Apart from modulating the classical Smad pathway, sDPP4 was also shown to activate the NF-κB pathway (Lee et al., 2020), which is non-canonically involved in TGF-β signalling.Treatment with the CD26/DPP4 inhibitor linagliptin prevented fibrotic gene expression patterns, Smad phosphorylation and NF-κB activation in murine fibroblasts (Lee et al., 2020).
These findings point out a profibrotic role for sDPP4 via the PAR2 receptor by further promoting the intracellular signalling pathways classically activated by TGF-β, and pharmacological DPP4 inhibition successfully reduces these effects.
In addition, CD26/DPP4 is essential for TGF-β receptor expression and assembly.Shi et al. (2015) reported a reduction of the expression of TGF-β receptor type I and II and their heterodimerization upon CD26/DPP4 inhibition by siRNA transfection in human dermal microvascular endothelial cells.They also reported reduced receptor dimerization in a murine model of diabetic kidney fibrosis upon linagliptin treatment and propose that CD26-integrin-β1 interaction is required for TGF-β receptor formation (Shi et al., 2015).In human dermal fibroblasts, enhanced expression of CD26/DPP4 has been demonstrated upon TGF-β stimulation (Soare et al., 2020) further supporting a lively crosstalk between CD26 and the TGF-β receptors.
Taken together, CD26/DPP4 interacts with TGF-β signalling on multiple levels as depicted in Figure 2a.CD26 aids in functional TGF-β receptor expression and enhances its downstream signalling via canonical and non-canonical pathways.In fibroblasts, TGF-β signalling promotes the release of collagen and proinflammatory mediators by myofibroblast differentiation, which is reduced by CD26/DPP4 inhibition.It is therefore highly suggestive that CD26/DPP4 qualifies as an antifibrotic drug target modulating TGF-β signalling.(Huang et al., 2013).Most studies delineating the role of EMT in tissue repair have been conducted in the context of cutaneous wound healing where keratinocytes acquire a migratory phenotype during re-epithelialization (Haensel & Dai, 2018).However, EMT programming also occurs in extracutaneous tissues upon injury, and the up-regulation of EMT markers is a hallmark of fibrotic disorders in models of renal, pulmonary, hepatic, cardiac and systemic fibrosis (Stone et al., 2016).The exact pathophysiological contribution of EMT programming in fibrogenesis remains subject to debate.Although initial lineage-tracing studies identified EMT as a mechanism contributing to the myofibroblast pool (Iwano et al., 2002), these results could not be consistently corroborated in subsequent studies (LeBleu et al., 2013).Therefore, the contribution of EMT to the myofibroblast pool may be organ specific or EMT may occur in damaged tissueresident cells and thus promote them to contribute to a profibrotic microenvironment (Lovisa et al., 2015).
There is ample evidence for CD26/DPP4 modulating EMT induction in a variety of different disease models, and DPP4 inhibition is often associated with a reduction of EMT: A role for sDPP4 in TGFβ-induced EMT has been described in human bronchial epithelial cells, and addition of a CD26/DPP4 inhibitor diminished EMT marker expression suggesting a function for CD26/DPP4 in airway remodelling (Sun et al., 2020).In a murine model of diabetic kidney injury, CD26/DPP4 inhibition effectively blocked the up-regulation of EMT transcription factors and restored epithelial markers while reducing interstitial fibrotic changes (Takagaki et al., 2019).Chang et al. (2017) presented similar results with saxagliptin reducing EMT in diabetic nephropathy, confirming these results in vitro in podocytes.Other authors suggest that EMT reduction upon DPP4 inhibition is mediated by the sustained presence of stromal-cell derived factor 1 (SDF-1/ CXC-motif chemokine ligand 12 [CXCL12]) modulating TGF-β signalling via inhibition of reactive oxygen species (ROS) generation and ERK phosphorylation (Chang et al., 2017).Furthermore, Hu et al. 3.2 | CD26/DPP4 inhibition reduces profibrotic endothelial-to-mesenchymal transition (EndoMT) in a drug-and organ-specific manner Similar to the mechanism of EMT, endothelial cells can acquire a migratory phenotype and detach from the endothelial layer in EndoMT.During this process, the cells lose endothelial markers while gaining mesenchymal markers (Pardali et al., 2017).EndoMT is initiated by TGF-β via Smad-dependent and non-canonical pathways (Pérez et al., 2017).
A reduction of EndoMT markers upon CD26/DPP4 inhibition has so far been demonstrated in pulmonary (Suzuki et al., 2017) and renal fibrosis (Kanasaki et al., 2014;Shi et al., 2015).The influence of CD26/DPP4 on EndoMT has primarily been studied in diabetic kidney remodelling.Shi et al. (2015) demonstrated a reduction of EndoMT in streptozotocin-induced diabetic kidney fibrosis upon treatment with the CD26/DPP4 inhibitor linagliptin.Mechanistically, they proposed that the impairment of the TGF-β signalling pathway via CD26-integrin-β1 crosstalk leads to differential expression of endothelial vascular endothelial growth factor (VEGF)-A receptors, tipping the balance towards VEGF-R2 and consequently reducing EndoMT (Shi et al., 2015).However, they were not able to replicate these results using sitagliptin, pointing towards a drug-rather than target-specific effect (Shi et al., 2016).In addition, the influence of CD26/DPP4 inhibitors seems to be organ specific as EndoMT reduction in pulmonary endothelium was observed upon both sitagliptin and vildagliptin treatment (Suzuki et al., 2017;Xu et al., 2018).Kanasaki et al. (2014) presented micro RNA (miRNA) regulation as an antifibrotic target for linagliptin in diabetic kidneys.miRNAs are small non-coding RNAs post-transcriptionally regulating the expression of other protein-coding messenger RNAs (mRNAs) by translational repression and induction of mRNA degradation (Srivastava et al., 2019).Generally, miRNA expression is highly organ specific and depends on the metabolic status.In diabetic kidneys, a decrease in the antifibrotic miRNA-29 family is observed.Linagliptin treatment restored miRNA-29 levels, reduced EndoMT and ameliorated experimentally induced murine diabetic kidney fibrosis (Kanasaki et al., 2014).In addition, a crosstalk between miRNA-29 and CD26/DPP4 in endothelial cells was demonstrated as miRNA-29 suppression resulted in enhanced CD26/ DPP4 expression in human vascular endothelial cells in vitro (Kanasaki et al., 2014).miRNAs have since been discussed as potential antifibrotic therapeutic agents, and miR-448-3p treatment has recently been demonstrated to reduce EndoMT by CD26/DPP4 modulation in experimental diabetic kidney fibrosis (Guan et al., 2020).
Taken together, CD26/DPP4 seems to be involved in profibrotic EndoMT processes in both pulmonary and renal endothelia.However, the effects of CD26/DPP4 inhibition are highly drug and organ specific, and their mechanism of action in modulating EndoMT largely remains unclear.

| EVIDENCE TO REPURPOSE PHARMACOLOGICAL CD26/DPPINHIBITION IN FIBROTIC DISORDERS
Pharmacological inhibition of CD26/DPP4 was first proposed in 1995 as a therapeutic approach for type 2 diabetes as it hinders the degradation of the incretin hormones glucagon-like peptide 1 (GLP-1) and gastric inhibitory polypeptide (GIP), which in turn potentiate glucoseinduced insulin release and suppress glucagon secretion (Deacon et al., 1995).This therapeutic concept was then supported by clinical trials demonstrating improved glycaemic control in diabetic patients upon treatment with a small molecule DPP4 inhibitor (Ahrén et al., 2002).In 2006, the Food and Drug Administration (FDA) approved sitagliptin as the first selective inhibitor of CD26/DPP4 for treating type 2 diabetes.DPP4 inhibitors are now in wide clinical use without significant side effects.Importantly, they lack hypoglycaemic risk and have a very low potential for drug interactions.Initial concerns about a risk of pancreatitis or pancreatic cancer are not supported by a recent meta-analysis showing no evidence for a causal association with DPP4 inhibitor intake (Dicembrini et al., 2020).More recently, post-marketing studies demonstrated that joint pain may rarely be caused by DPP4 inhibitor treatment (Rai et al., 2019).To date, more than 10 different DPP4 inhibitors of the gliptin class have received marketing approval from regulatory authorities worldwide (Deacon, 2020).
However, the clinical use of DPP4 inhibitors is still limited to treating diabetes, despite our growing understanding of the diverse roles of CD26/DPP4 in health and disease.As detailed above, evidence suggest multiple functions for CD26/DPP4 in fibrotic tissue remodelling.Thus, it seems apt to propose drug repurposing of DPP4 inhibitors to attempt the treatment or prevention of fibrotic disorders.
Therefore, we will now provide an organ-and disease-specific overview of the current evidence for the antifibrotic properties of approved CD26/DPP4 inhibitors.Figure 3  Out of all mammalian organs, kidneys show the highest expression density of CD26/DPP4.Its expression has been demonstrated in various renal cell types, such as preglomerular microvascular smooth muscle cells, glomerular podocytes and mesangial cells, and proximal tubular cells, and in the epithelium of the descending portion of Henle's loop (Hartel et al., 1988;Jackson et al., 2012).The level and pattern of CD26/DPP4 expression are further dependent on metabolic and inflammatory status as cell surface expression in glomerular podocytes can only be detected in diabetic nephropathy whereas it is absent in healthy individuals (Sharkovska et al., 2014).Mechanistically, these preclinical studies observed changes in EMT and EndoMT programming, modulation of miRNA signalling and reduced integrin-β1 activation (Chang et al., 2017;Kanasaki et al., 2014;Shi et al., 2015;Takagaki et al., 2019).
In the non-diabetic murine model of unilateral ureteral obstruction, treatment with gemigliptin, alogliptin or evogliptin ameliorates renal fibrosis.Mechanistically, a reduction in Smad signalling has been observed in all of these studies (Kim et al., 2020;Min et al., 2014;Uchida et al., 2017).Furthermore, anti-inflammatory effects of CD26/ DPP4 inhibition were observed in this model: Both gemigliptin and alogliptin treatment caused decreased infiltration of macrophages (Min et al., 2014;Uchida et al., 2017), and polarization of the infiltrating macrophage pool towards the anti-inflammatory, alternatively activated phenotype was observed (Uchida et al., 2017).
Taken together, preclinical studies suggest an antifibrotic effect for a wide range of CD26/DPP4 inhibitors based on pleiotropic mechanisms under both diabetic and non-diabetic conditions.(ClinicalTrials.govidentifier: NCT01792518) evaluated the urinary albumin-to-creatinine ratio as a surrogate parameter for acute glomerular damage after a 24-week treatment period with linagliptin.Overall, CD26/DPP4 inhibition did not significantly ameliorate acute glomerular damage in this trial.However, a meaningful improvement in albuminuria was observed in more patients receiving linagliptin than in the placebo group (Groop et al., 2017).
The 'Cardiovascular and Renal Microvascular Outcome Study with Linagliptin in Patients with Type 2 Diabetes Mellitus' (CARMELINA) trial (ClinicalTrials.govidentifier: NCT01897532) reported a reduction in albuminuria progression during long-term linagliptin treatment.Notably, this observation did not depend on changes in HbA1c or baseline renal function (Perkovic et al., 2020).
These clinical trials demonstrate that CD26/DPP4 treatment in diabetic patients shows mild nephroprotective effects during longterm use.These effects might be unrelated to glycaemic control.As preclinical studies further suggest protective effects in non-diabetic renal fibrosis, a clinical evaluation of CD26/DPP4 inhibition in nondiabetic kidney injury may be warranted.

| Hepatic fibrosis and steatohepatitis
Hepatic fibrosis results from hepatotoxic and cholestatic forms of chronic or iterative liver injury.Advanced fibrotic remodelling causes cirrhosis, portal hypertension and end-stage organ failure and is a major risk factor for the development of hepatocellular carcinoma.
However, both clinical and experimental hepatic fibrosis has the capacity to regress significantly upon removal of the causative agent.
It is controversial whether a complete regression to a normal hepatic architecture can be obtained (Kisseleva & Brenner, 2021).
Myofibroblasts in fibrotic liver tissue primarily originate from resident portal fibroblasts and hepatic stellate cells.Bone marrow-derived mesenchymal progenitors or circulatory fibrocytes contribute to the myofibroblast pool to a lesser extent (Kisseleva & Brenner, 2021).
Mesenchymal transition of hepatocytes or cholangiocytes has also been implicated in hepatic fibrosis.However, fate-mapping studies in a murine experimental setting were unable to support any contribution of EMT (Chu et al., 2011;Taura et al., 2010).
In the liver, CD26/DPP4 expression was demonstrated along the entire biliary tree.In addition, sinusoidal endothelial cells in murine hepatic tissue also stain positive for CD26/DPP4 (Iwanaga & Nio-Kobayashi, 2021).In human cirrhotic liver specimens, only a mild alteration of the zonal distribution of CD26/DPP4 expression was observed (Matsumoto et al., 1992).Furthermore, elevated levels of soluble CD26/DPP4 in serum were described as a potential biomarker for hepatic fibrotic remodelling in patients suffering from primary biliary cirrhosis (PBC), chronic cholestasis, chronic hepatitis C, alcoholic liver disease or non-alcoholic fatty liver disease (NAFLD) (Perner et al., 1999;Wang et al., 2017;Williams et al., 2015).CD26/DPP4 inhibition has been employed in several different preclinical models of fibrotic liver injury.All of these studies reported a histological reduction in collagen deposition upon gliptin treatment.
In a murine model of carbon tetrachloride (CCl 4 )-induced liver fibrosis, vildagliptin treatment significantly improved histological changes and prevented the elevation of liver enzymes.Mechanistically, a decrease in TGF-β1 expression and subsequent Smad signalling as well as a modulation of the ERK and NF-κB pathways was described (Khalil et al., 2020).Similarly, sitagliptin treatment reduced fibrotic changes and TGF-β1 expression in a rat model of porcine serum-induced fibrosis (Kaji et al., 2014).In this study, the authors suggested a modulation of the ERK and Smad pathways in activated hepatic stellate cells (Kaji et al., 2014).Apart from down-regulation of profibrotic signalling, immunomodulatory properties have been demonstrated in different preclinical models as CD26/DPP4 inhibitor treatment reduces hepatic leucocyte infiltration and intrahepatic lymphocyte composition (Wang et al., 2017) or achieves a polarization of the infiltrating macrophage pool towards an alternatively activated phenotype (Wang et al., 2018).
In addition, CD26/DPP4 inhibition achieved beneficial vascular effects reducing portal hypertension in a rat model of CCl 4 -induced cirrhosis (Wang et al., 2021).Furthermore, CD26/DPP4 inhibition may have carcinopreventive effects in the context of hepatic fibrosis.In a murine model of nonalcoholic steatohepatitis (NASH), melanocortin 4 receptor-deficient mice on Western diet spontaneously develop hepatic tumours reminiscent of human hepatocellular carcinoma, and long-term anagliptin treatment significantly reduced the size and number of hepatic tumours (Kawakubo et al., 2020).
Preclinical studies in mouse and rat models of different fibrotic liver injuries revealed beneficial effects of CD26/DPP4 inhibition on histological changes, hepatic function, portal hypertension and hepatic tumour development.Mechanistically, CD26/DPP4 inhibition modulates the inflammatory milieu and profibrotic signalling pathways in experimental hepatic fibrosis.
Despite these convincing preclinical observations, clinical evidence of antifibrotic properties of CD26/DPP4 inhibition in hepatic fibrosis is scarce.In a randomized control trial, sitagliptin treatment did not significantly contribute to the alleviation of fibrotic changes in patients suffering from NASH while undergoing lifestyle change.However, steatotic changes and hepatic function were further improved by sitagliptin treatment in this study (Alam et al., 2018).Thus, CD26/DPP4 inhibition may preferentially prevent early hepatic injury, but may not be suitable in advanced fibrotic remodelling.In support of this, a retrospective study in diabetic patients with established hepatic cirrhosis described an elevated risk of decompensation and hepatic failure during intake of CD26/DPP4 inhibitors (Yen et al., 2021).

| Cardiomyopathy, myocardial infarction and heart failure
Fibrotic remodelling occurs in almost all forms of heart disease, such as myocardial infarction, and hypertrophic or dilated cardiomyopathy.
Pathological remodelling of the cardiac tissue results in increased matrix stiffness impairing cardiac function and filling capacity, ultimately resulting in systolic and diastolic heart failure (Liu, de Juan, et al., 2021).In the myocardium, fibrotic changes show a characteristic distribution depending on the type of cardiac injury.Interstitial fibrosis, as seen in pressure-overload cardiomyopathy, results in increased ECM deposition without numerical loss of cardiomyocytes.In contrast, myocardial infarction causes damage to cardiomyocytes, which are in turn replaced by scar tissue (Liu, de Juan, et al., 2021).ECMproducing myofibroblasts in the myocardium partly originate from resident cardiac fibroblasts.However, EndoMT has been demonstrated to generate up to 70% of the cardiac myofibroblast pool in pressureoverload cardiomyopathy (Zeisberg et al., 2007).Additional myofibroblast progenitors can be recruited from the periphery, as bone marrow-derived fibrocytes contribute to scar formation after myocardial infarction (van Amerongen et al., 2008).CD26/DPP4 in cardiac tissue is mainly expressed on the surface of vascular endothelial cells of the coronary capillaries as well as in infiltrating immune cells (Cuijpers et al., 2021;Iwanaga & Nio-Kobayashi, 2021).Cytosolic expression of CD26/DPP4 was further observed in some cardiomyocytes (Cuijpers et al., 2021).
The effects of CD26/DPP4 inhibition on cardiac remodelling have been studied in preclinical models of different types of cardiac injury.
In a rat model of experimental myocardial infarction, vildagliptin treatment attenuated oxidative stress, histological fibrosis and improved left ventricular function (Inthachai et al., 2015).Similarly, sitagliptin treatment improved left ventricular compliance in streptozotocininduced diabetic rats undergoing experimental myocardial infarction (Connelly et al., 2013).This study further demonstrated a reduction of cardiac deposition of collagen 1 upon CD26/DPP4 inhibition (Connelly et al., 2013).However, systolic function and infarct size remained unchanged.Mechanistically, the authors proposed that preserved SDF-1 signalling upon sitagliptin treatment aids in recruitment of bone marrow-derived early proangiogenic cells and thus achieves an increased density of cardiac microvasculature (Connelly et al., 2013).
The absence of CD26 showed cardioprotective effects in a murine model of pressure overload-induced cardiac hypertrophy by aortic constriction: CD26/DPP4-deficient animals demonstrate a significant reduction of histological interstitial and perivascular fibrosis as well as improved left ventricular function, and treatment with the sitagliptin analogue MK-0626 achieved similar results (Hirose et al., 2017).Mechanistically, the preservation of GLP-1 levels by CD26/DPP4 inhibition may increase left ventricular contractility (Shigeta et al., 2012).In a similar experimental setting, Mulvihill et al. (2016) demonstrated preservation of cardiac function and lower cardiac collagen deposition in CD26/DPP4-deficient mice compared to littermate controls.However, they were unable to confirm a cardioprotective effect of MK-0626 treatment in streptozotocin-induced diabetic and high-fat diet-fed animals undergoing transverse aortic constriction.Surprisingly, MK-0626-treated mice showed elevated markers of fibrotic remodelling, increased left ventricular mass, a significant reduction of ejection fraction upon treatment and lower exercise tolerance (Mulvihill et al., 2016).Even though the mechanistic base of these observations remains unclear, the authors hypothesized that the combination of hyperglycaemia, a high-fat diet, pressure overload and prolonged treatment with the CD26/DPP4 inhibitor in older mice promotes a profibrotic switch in the milieu of growth factors, cytokines and neurohumoral agents (Mulvihill et al., 2016).
Several preclinical studies in rats, mice and rabbits have evaluated the effect of pharmacological CD26/DPP4 inhibition in cardiac injury caused by experimental diabetes or hypertension without additional surgically induced cardiac impairment.These studies uniformly describe a cardioprotective effect of CD26/DPP4 inhibition resulting in a reduction of histological perivascular and interstitial fibrosis, prevention of diastolic dysfunction and preserved left ventricular compliance (Cuijpers et al., 2021;Esposito et al., 2017;Shigeta et al., 2012;Yamamoto et al., 2018;Zhang et al., 2018).
The effect of CD26/DPP4 inhibition on histological cardiac fibrosis has not been evaluated in clinical trials.However, several largescale randomized controlled trials have evaluated the clinical cardiovascular outcome of saxagliptin, alogliptin and sitagliptin treatment (SAVOR-TIMI 53, EXAMINE and TECOS trials) (Green et al., 2015;Scirica et al., 2013;White et al., 2013).Generally, these studies found that CD26/DPP4 inhibition has no effect on the combined major adverse cardiovascular event outcome, including cardiovascular death, myocardial infarction or stroke.However, the SAVOR-TIMI 53 trial reported an absolute increase of 0.7% in risk of hospitalization for heart failure in saxagliptin-treated patients (3.5% vs. 2.8%, hazard ratio [HR] 1.27, 95% confidence interval [CI] 1.07-1.51)(Scirica et al., 2013).Both the TECOS and EXAMINE trials could not confirm a statistically significant increase in risk for hospitalization for heart failure in the CD26/DPP4 inhibitor-treated group (Green et al., 2015;White et al., 2013).Furthermore, several meta-analyses describe only a non-significantly elevated risk for heart failure upon CD26/DPP4 treatment (Elgendy et al., 2017;McGuire et al., 2016).
Importantly, these results may be heavily influenced by the large sample size of the SAVOR-TIMI 53 trial being included in the analysis.
Thus, it remains unclear if the SAVOR-TIMI 53 results reflect a specific side effect of saxagliptin treatment or merely a chance phenomenon.Generally, there is only weak evidence for an increased risk of heart failure during antidiabetic treatment with drugs of the gliptin class.However, none of the aforementioned large-scale clinical trials demonstrated cardioprotective effects that may have been expected from preclinical work upon CD26/DPP4 inhibition.Interestingly, GLP-1 receptor agonists exert solid clinical cardioprotective effects (Nauck et al., 2017).To date, it remains unclear why DPP4 inhibition fails to mimic these effects by the upkeep of GLP-1 signalling.

| Pulmonary fibrosis
Fibrotic tissue remodelling occurs in many pulmonary diseases, including the progressively worsening parenchymal fibrosis in IPF, but also airway remodelling in asthma or chronic obstructive pulmonary disease (COPD) (Liu, Philp, et al., 2021).As described in most other organs, the main ECM-producing cells in the lung are myofibroblasts believed to arise from resident mesenchymal cells.Immunohistopathological findings from human fibrotic lung tissue as well as in vitro studies further suggest a contribution of bone marrow-derived precursors as well as EMT and EndoMT (Borthwick et al., 2009;Dolgachev et al., 2009).
In the lung, CD26/DPP4 is expressed in alveolar pneumocytes, leucocytes, endothelia and bronchial submucosal glands (Vliegen et al., 2017).Furthermore, CD26/DPP4 has been demonstrated to have an impact on the profibrotic programming of activated pulmonary fibroblasts, as the addition of a CD26/DPP4 inhibitor reduces proliferation, α-SMA expression and ECM production in vitro (Jungraithmayr et al., 2021;Liu et al., 2020;Liu & Qi, 2020).In this context, a pathological overexpression of CD26 has been demonstrated in loosely fibrotic regions in lung tissue of patients suffering from fibrotic remodelling after lung transplantation due to chronic allograft rejection (Jungraithmayr et al., 2021).Similarly, CD26 expression is elevated in IPF lung tissue, and sitagliptin treatment shows an antifibrotic effect in primary fibroblasts isolated from IPF lungs (Montero Magall o et al., 2020).
The impact of CD26/DPP4 inhibition on fibrotic remodelling has been investigated in different models of murine lung injury and pulmonary fibrosis.In a model of pulmonary fibrosis induced by repetitive intratracheal bleomycin injection, vildagliptin treatment achieves a reduction of histological fibrosis and ECM deposition, accompanied by reduced expression levels of α-SMA.Furthermore, a reduction in different inflammatory cytokines including TGF-β as well as inflammatory cellular infiltrates was observed in the bronchioalveolar lavage fluid in vildagliptin-treated animals (Liu & Qi, 2020).
Similarly, linagliptin treatment ameliorates pulmonary fibrotic changes in a murine model of systemic sclerosis induced by subcutaneous application of bleomycin (Pei et al., 2022).Mechanistically, the authors demonstrate a reduction of markers of EndoMT and reduced Akt/mTOR signalling in treated animals (Pei et al., 2022).Likewise, a reduction in histological changes as well as reduced EndoMT have been demonstrated in a murine model of septic lung injury by intraperitoneal lipopolysaccharide (LPS) injection upon vildagliptin treatment (Suzuki et al., 2017).
Preclinical studies strongly suggest a beneficial effect of CD26/ DPP4 by reducing not only pulmonary inflammation but also the generation of myofibroblasts from EndoMT and their subsequent ECM production.However, chemically induced murine models of pulmonary fibrosis are based on repetitive acute lung injuries and thus do not reflect the slowly progressive nature of human fibrotic lung disease.To date, clinical investigations towards antifibrotic pulmonary effects of CD26/DPP4 inhibition are lacking.

| Scleroderma, cutaneous wound healing and scar formation
Formation of ECM in the skin is a precondition to the physiological process of cutaneous wound healing.Excess ECM deposition during this process leads to hypertrophic scarring or keloid formation.In addition, fibrotic changes of the skin occur in pathological conditions such as scleroderma or graft-versus-host disease.
CD26/DPP4 has recently gained attention as a marker of skin fibroblasts with enhanced profibrotic potential (Rinkevich et al., 2015).CD26 + fibroblasts can be found in human skin in physiological conditions (Mah et al., 2017), and increased numbers of these cells are found in keloid (Xin et al., 2017), hypertrophic scars (Vorstandlechner et al., 2021) or in patients suffering from systemic scleroderma (Soare et al., 2020).In vitro, CD26 + cutaneous fibroblasts show a higher proliferation rate, enhanced expression of TGF-β and increased production of ECM proteins and display an invasively growing phenotype (Xin et al., 2017).Furthermore, CD26/DPP4 seems to mediate the profibrotic effect of TGF-β stimulation, as skin fibroblasts isolated from CD26-deficient mice demonstrate impaired collagen deposition and α-SMA expression compared to those isolated from littermate controls.
The effects of pharmacological CD26/DPP4 inhibition have been evaluated in murine models of scleroderma as well as in diabetic and non-diabetic wound healing.The inhibition of DPP4-like enzymatic activity has been found to reduce dermal thickening, collagen synthesis and α-SMA expression in experimental cutaneous fibrosis induced by repetitive intracutaneous injection of TGF-β (Thielitz et al., 2008).
Similarly, treatment with the CD26/DPP4 inhibitor sitagliptin led to a reduction of dermal thickness and a lower degree of myofibroblast differentiation in murine skin fibrosis induced by subcutaneous bleomycin injections and in a murine model of sclerodermatous graft-vshost disease (Soare et al., 2020).In these scleroderma models, the main effect of CD26/DPP4 inhibition seems to be mediated by tissue-resident rather than bone marrow-derived cells, as chimeric mice receiving CD26-deficient bone marrow transplants show a similar extent of fibrotic remodelling compared to animals receiving wildtype bone marrow (Soare et al., 2020).However, these toxin-induced models do not fully mirror human scleroderma as they are unable to reflect the autoimmune nature of the disease.CD26/DPP4 inhibition further shows beneficial effects in preclinical models of cutaneous wound healing.Jiang et al. (2021) demonstrated that CD26 + cells accumulate in the wound area in both murine excisional and burn wounds.The subcutaneous application of sitagliptin in burn wounds achieved a reduction of CD26/DPP4 expression and decreased collagen deposition in the wound area (Jiang et al., 2021).In excisional wounds, oral administration of sitagliptin improved wound healing, accelerated re-epithelialization and increased blood flow in the wound area (Shih et al., 2018).Presumably, the upkeep of SDF-1 signalling upon CD26/DPP4 inhibition facilitates angiogenesis in wounded skin due to enhanced migration of bone marrow-derived precursor cells (Whittam et al., 2019).Also, SDF-1 has been demonstrated to enhance mesenchymal transition in keratinocytes and promote their migratory capacities, thus facilitating re-epithelialization (Long et al., 2018).
In both human hypertrophic and murine developing scars, Vorstandlechner et al. (2021) found an up-regulation of different genes encoding members of the serine protease superfamily, including DPP4 and urokinase.Both were also found to be up-regulated at the protein level.In primary human skin fibroblasts, siRNA-mediated knockdown and pharmacological inhibition of these serine proteases reduced the TGF-β-dependent acquisition of a myofibroblast phenotype and ECM production (Vorstandlechner et al., 2021).In a murine wound healing assay, pharmacological inhibition achieved a reduction of ECM deposition and the structural thickness of deposited collagen bundles for both DPP4 and urokinase (Vorstandlechner et al., 2021).
In general, functional wound healing assays conducted in mice do not allow for a direct translation to human skin.Murine skin is characterized by a subcutaneous muscle layer that aids in early wound contraction (Zhou et al., 2000).In contrast, human wound contraction only occurs in the late healing stages well after the establishment of granulation tissue.
Clinical trials confirmed the beneficial effects of CD26/DPP4 inhibition on wound closure in terms of the healing process of diabetic ulcers: In a randomized, open-label trial, the addition of vildagliptin to the antidiabetic treatment regimen in patients suffering from chronic non-healing foot ulcers achieved a significantly higher rate of wound closure, enhanced granulation tissue and a higher capillary density within the wounds (Marfella et al., 2012).Another randomized controlled trial (ClinicalTrials.govidentifier: NCT02742233) employing saxagliptin could confirm the finding of a significantly improved rate of healing and wound closure in diabetic foot ulcers (Long et al., 2018).Mechanistically, wounds in saxagliptin-treated patients demonstrated higher levels of SDF-1 and higher numbers of cells undergoing mesenchymal transition (Long et al., 2018).
Regarding the excessive ECM deposition during the formation of hypertrophic scars and keloid, a large retrospective Japanese cohort study revealed a reduced risk of hypertrophic scar formation after median sternotomy in patients receiving antidiabetic DPP4 inhibitor treatment within a year prior to the surgical procedure (Suwanai et al., 2020).

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| CD26/DPP4 REGULATES TGFβ-MEDIATED MESENCHYMAL TRANSITION 3.1 | CD26/DPP4 inhibition reduces profibrotic epithelial-to-mesenchymal transition (EMT) One of the profibrotic programmes initiated by TGF-β is EMT.EMT describes the constitutional change of stationary epithelial cells towards a migratory mesenchymal phenotype associated with the loss of cell-cell adhesion and basal-apical polarity.However, these changes are neither necessarily complete nor permanent, as EMT often results in cells showing both epithelial and mesenchymal characteristics
(2021) recently described the effects of siRNA-mediated knockdown of DPP4 in papillary thyroid carcinoma cells modulating MAPK pathways and demonstrated subsequent reduction of EMT markers.In contrast, some studies describe an aggravation of EMT upon CD26/ DPP4 inhibition:Long et al. (2018) demonstrated an enhanced presence of EMT in the context of saxagliptin treatment in diabetic wound healing while contrastingly describing a reduction in scar tissue formation.Similarly, siRNA-mediated knockdown of DPP4 aggravated EMT in breast cancer cells independent of TGF-β signalling(Yang et al., 2019).These observations point towards a context-specific role of EMT in disease progression and an accordingly differential relevance for CD26/DPP4 in EMT regulation.In the context of fibrotic disease, a recent study byLi et al. (2021) convincingly demonstrated the reduction of peritoneal fibrosis by CD26/DPP4 inhibition in a rat model as well as in a clinical application in peritoneal dialysis patients.They demonstrated diminished EMT programming and Smad3 phosphorylation in peritoneal mesothelial cells(Li et al., 2021) favouring a TGF-β-mediated role for CD26/DPP4 in profibrotic EMT induction under inflammatory conditions.Overall, these studies suggest a disease-specific role for CD26/DPP4 in EMT.In fibrotic processes, CD26/DPP4 promotes EMT by furthering TGF-β signalling as detailed in Figure2a,b.Different mechanisms of action have been proposed in this context such as SDF-1-mediated antiinflammatory effects or interaction with canonical or non-canonical TGF-β pathways.Reduced EMT programming upon CD26/DPP4 inhibitor treatment has been demonstrated in epithelial cells of different tissue origin.
gives an organ-specific mechanistic overview of CD26/DPP4 inhibition in experimental fibrotic conditions.4.1 | Renal fibrosis and kidney injury Interstitial fibrosis with tubule atrophy is the common histopathological endpoint in any form of sustained kidney injury and chronic kidney disease.It is characterized by excess deposition of ECM molecules between the tubular basement membrane and peritubular capillaries.Resident stromal cells, such as kidney pericytes, mesenchymal stem cell-like cells or perivascular fibroblasts, appear to constitute the primary source of myofibroblasts in renal fibrotic remodelling.Mesenchymal transition processes of epithelial and endothelial cells have been observed, but the extent of their contribution to the renal myofibroblast pool remains subject to debate.
To our knowledge, at least six gliptins have been evaluated for their antifibrotic properties in preclinical models of diabetic and nondiabetic renal fibrosis.These studies uniformly demonstrated significant reductions in fibrotic tissue remodelling upon CD26/DPP4 inhibition.In streptozotocin-induced diabetic mice, linagliptin, F I G U R E 3 Organ-specific overview of the antifibrotic mechanisms investigated upon cluster of differentiation 26 (CD26)/dipeptidyl peptidase 4 (DPP4) inhibition in preclinical models.CD26/DPP4 inhibition results in reduced extracellular matrix (ECM) deposition in preclinical models of organ fibrosis.However, the beneficial effects of CD26/DPP4 inhibition in different models appear to be based on different mechanisms of action.EMT, epithelial-to-mesenchymal transition; EndoMT, endothelial-to-mesenchymal transition.teneligliptin and saxagliptin successfully ameliorated renal fibrosis.
With regard to clinical translation, streptozotocin-induced renal fibrosis is accepted as a model of diabetic kidney disease as it reflects the development of interstitial fibrosis upon diabetic metabolic changes.Unilateral ureteral obstruction is the most commonly used model for non-immune and non-diabetic interstitial renal fibrosis.However, it does not adequately reflect the human condition, as the model is based on a full ligation of the ureter resulting in acute parenchymal injury.Even though obstructive uropathy is a major cause of end-stage renal failure in children, an acute and absolute obstruction does not occur in humans.Therefore, the model represents an accelerated course of the fibrotic process.Another major drawback of the model of unilateral ureteral obstruction is that renal function cannot be monitored due to compensation by the unobstructed contralateral kidney.Generally, the clinical relevance of fibrotic organ remodelling can only be defined by organ function.To date, clinical trials regarding the renal effects of CD26/DPP4 inhibitors are limited to diabetic patients.The randomized, controlled 'Efficacy, Safety and Modification of Albuminuria in Type 2 Diabetes Subjects with Renal Disease with LINAgliptin' (MARLINA-T2D) study Taken together, evidence gathered from this experimental and clinical work strongly suggests beneficial effects of CD26/DPP4 inhibition in pathological cutaneous fibrosis and during physiological wound healing.Mainly, these effects are mediated by local inhibitory effects on fibroblasts resulting in reduced amounts of ECM deposition.During wound healing, CD26/DPP4 inhibition enhances the local expression of SDF-1, promoting angiogenesis in the wound area and wound closure by keratinocyte mesenchymal transition.In clinical application, the use of CD26/DPP4 inhibitors can thus be especially beneficial in diabetic patients suffering from foot ulcers.Furthermore, a functional relevance in cutaneous scarring has been demonstrated for other members of the serine protease superfamily, such as urokinase.These proteases could likewise qualify as antifibrotic therapeutic targets in the future.5 | CONCLUDING REMARKS Fibrotic tissue remodelling is the predominant pathological feature of multiple chronic diseases eventually resulting in fatal organ failure.Due to the excruciating disease burden and limited treatment options, there is an urgent need for novel therapeutic targets in fibrotic diseases.Pharmacological inhibition of CD26/DPP4 has demonstrated substantial beneficial effects in multiple preclinical fibrosis models.Mechanistically, CD26/DPP4 inhibition exerts pleiotropic effects at different levels, such as immunomodulation, cell migration and fibroblast activation.Modulating profibrotic intracellular signalling cascades, CD26/DPP4 inhibition shows potential to impede excess ECM deposition while allowing for physiological tissue repair.CD26/DPP4 inhibitors have been in routine clinical use in type 2 diabetes and are characterized by an excellent safety profile.Clinical evidence supporting the antifibrotic properties of CD26/DPP4 inhibitors is scarce: To the best of our knowledge, the antifibrotic effect of CD26/DPP4 inhibition has not yet been evaluated as a primary outcome measure in any clinical trial.Moreover, current clinical evidence is limited to studies enrolling diabetic patients.However, ample preclinical evidence suggests that the antifibrotic properties of CD26/DPP4 inhibition are not attributable to its antidiabetic action.Therefore, clinical trials