Therapeutic targets of fibrosis: Translational advances and current challenges

In a physiological context, the extracellular matrix (ECM) provides an important scaffold for organs. Dysregulation of ECM in disease conditions, characterised by excess deposition of connective tissue and extracellular matrix in response to a pathological insult, is a key driver of disease progression in multiple organs. The resultant fibrosis is predominantly an irreversible process and directly contributes to, and exacerbates, dysfunction of an affected organ. This is particularly paramount in the kidney, liver, heart and lung. A hybrid Joint Meeting of NC‐IUPHAR and British Pharmacological Society was held in Paris and via a webinar in November 2020, when two successive sessions were devoted to translational advances in fibrosis as a therapeutic target. On the upsurge of response to these sessions, the concept of a special themed issue on this topic emerged, and is entitled Translational Advances in Fibrosis as a Therapeutic Target. In this special issue, we seek to provide an up‐to‐date account of the diverse molecular mechanisms and causal role that fibrosis plays in disease progression (contributing to, and exacerbating, dysfunction of affected organs). Recent developments in the understanding of molecular targets involved in fibrosis, and how their actions can be manipulated therapeutically, are included.


| FIBROSIS AS A DRIVER OF ORGAN FAILURE: OVERVIEW OF THERAPEUTIC APPROACHES THAT TARGET REMODELLING
Fibrosis is a hallmark of many diseases.Remodelling is a key characteristic in organs targeted by disease, where deposition of collagen and extracellular matrix can lead to organ failure.As illustrated in our Figure 1 inhibitors potentially offer the promise of halting excess extracellular matrix deposition and allowing for tissue repair in multiple organ systems (Ohm et al., 2023).
An accompanying review by Hao et al. (2023) similarly addresses fibrosis across multiple organs, drilling down on the role of necroptosis involved in fibrosis deposition, and the available small-molecule compounds that can target this form of programmed cell death.
Caspase 8 is described as a key switch between apoptosis and necroptosis, due to the cleaving capacity of caspase 8.As illustrated in their fig.3, when caspase 8 is inhibited, active RIPK1 is available to interact with RIPK3 to cause its phosphorylation, thereby activating MLKL and forming the necrosome complex (Hao et al., 2023).
This action can trigger increased cell membrane permeability, cytoplasm and organelles swelling and overflow of cell components into the microenvironment (Galluzzi et al., 2017), with subsequent necroptotic cell death.This review discusses the core components of necroptosis, the activation of the signalling pathway and the role that necroptosis plays in tissue fibrosis, focussing on stellate cells, macrophages, neutrophils and fibroblasts, and then various disease settings including cardiac fibrosis, liver fibrosis, kidney fibrosis, pulmonary fibrosis and pancreatitis fibrosis.The authors also consider the therapeutic potential of targeting necroptosis in fibrosis of various organs, including the use of inhibitors of RIPK1, RIPK3 and MLKL (Hao et al., 2023).

| THERAPEUTIC TARGETING OF HEPATIC FIBROSIS
The remaining manuscripts in this special themed issue focus on particular organs that are susceptible to dysfunction as a result of fibrosis.
Beginning with the liver, hepatic fibrosis is the consequence of scar tissue replacing healthy liver cells due to chronic and severe injury or inflammation, including viral infections, autoimmune disorders, alcohol abuse or fatty liver disease.Hepatic fibrosis impairs regular liver function and can lead to cirrhosis when left untreated.Borrello and Mann provide an overview of the major causes of chronic liver disease biology and the major processes that lead to, and regulate, hepatic fibrosis.As the most common pathology associated with chronic liver disease, hepatic fibrosis is an attractive therapeutic target (Borrello & Mann, 2023).The major causes of chronic liver disease are described by Borrello and Mann also showcase selected drugs in phase II and phase III development, including agonists for the farnesoid X receptor (FXR) and peroxisome proliferator-activated receptor (PPAR), as well as inhibitors of galectin 3 (e.g., belapectin) and chemokine receptor antagonists, as potential therapeutics for liver fibrosis.The review concludes with the observation that understanding the pathology of liver fibrosis remains a difficult clinical challenge, with an urgent requirement for appropriate preclinical animal models and therapeutic approaches to arrest or reverse its progression (Borrello & Mann, 2023). in women and men, respectively.The authors describe evidence for sex differences in cardiac remodelling (Sharma et al., 2023), which is more likely to develop as eccentric in men, with greater cardiomyocyte size and enhanced left ventricular fibrosis, but more likely to develop concentric cardiac remodelling in women (Kessler et al., 2019;Paulus & Dal Canto, 2018;Yap et al., 2019).Sex differences in cardiac and systemic changes evident in cardiovascular disease and its management preclinical contexts (Sharma et al., 2023).Patients with diabetesinduced heart failure are treated with both heart failure and antidiabetic therapies, to separately target the individual diabetic and cardiovascular complications, although treatments that specifically target both in parallel have been lacking (Ritchie & Abel, 2020).

| THERAPEUTIC TARGETING OF RENAL FIBROSIS
Traditional heart failure treatments, including ACE inhibitors (ACEi), β-adrenoceptor antagonists, mineralocorticoid receptor antagonists (MRAs), angiotensin receptor / neprilysin inhibitors (ARNi) and angiotensin II type I receptor blockers (ARBs), prolong survival and reduce hospitalisations in HFrEF patients, but this has not extended to similar benefits in patients with concomitant HFpEF and diabetes (Ponikowski et al., 2016).Furthermore, some older diabetes treatments (e.g., thiazolidinediones and sulfonylureas) lead to increased risk of heart failure (Seferovi c et al., 2020).As discussed here and illustrated in (particularly with respect to potential sex differences).This is particularly relevant for the management of fibrosis in the diabetic heart, to further enable development of more personalised and efficacious treatment approaches (Sharma et al., 2023).

| CONCLUDING REMARKS
Fibrosis remains a major health problem that affects many organs and will require multidisciplinary approaches for its detection,

| NOMENCLATURE OF TARGETS AND LIGANDS
Key protein targets and ligands in this article are hyperlinked to corresponding entries in https://www.guidetopharmacology.org and are permanently archived in the Concise Guide to PHARMACOLOGY 2021/22 (Alexander et al., 2021).
,Ohm et al. (2023) have reviewed those cells involved in fibrosis development and organ impairment and considered the role of CD26 and its dipeptidyl peptidase-4 (DPP4) inhibition as a potential antifibrotic treatment for multiple organs (in particular lung, heart, liver, kidney and skin)(Alexander et al., 2021).CD26/DPP4 is expressed in endothelial, epithelial and acinar cells in multiple organs.CD26 also is expressed on T-cells, B cells, NK cells and activated lymphocytes.CD26/DPP4 cleaves N-terminal dipeptides from substrates, altering substrate function by receptor modulation and/or substrate degradation.The soluble DPP4 isoform modulates Smad/NF-κΒ signalling (Lee et al., 2020) and, when secreted into the circulation, serves as a marker of chronic inflammatory diseases (e.g., diabetes mellitus, coronary artery disease, and cancer).The review by Ohm and colleagues highlights CD26/DPP4 modulation of profibrotic TGF-β signalling (illustrated in their fig.2), suggesting that inhibition of CD26/DPP4 may regulate TGF-β production in a diseasespecific and cell-type manner by blunting TGF-β expression in fibrotic diseases.CD26/DPP4 inhibition also reduces profibrotic endothelialto-mesenchymal transition (EndoMT) in a drug and organ-specific manner, particularly in kidney and pulmonary fibrosis.DPP4 inhibitors are safe and currently in use for patients with type 2 diabetes.Ohm and colleagues propose here that DPP4 inhibitors may be repurposed as a promising therapeutic approach against fibrosis of multiple organs, considering the potential utility of CD26/ DPP4 inhibition as therapeutics for renal fibrosis/injury, hepatic fibrosis/steatohepatitis, cardiomyopathy, pulmonary fibrosis, scleroderma and cutaneous wound healing and scar formation.With DPP4 inhibitors in current clinical use, the authors set forth that CD26/DPP4 Borrello and Mann, including non-alcoholic fatty liver disease (NAFLD, which is the fastest growing cause of chronic liver disease;Arshad et al., 2020).An in-depth description of the process of fibrosis in the liver is provided, including architectural changes such as net deposition of collagen-rich fibril-forming extracellular matrix (which leads to scarlike tissue deposition), formation of fibrous septa (which can mature to link to vascular structures) and increased deposition of collagens I and III, laminin and fibronectin(Borrello & Mann, 2023).The major cell types F I G U R E 1 Overview of reported antifibrotic mechanisms reported in preclinical models for CD26/dipeptidyl peptidase 4 (DPP4) inhibition, as reviewed byOhm et al. (2023) (figure reproduced from their review in this special themed issue).EMT, epithelialto-mesenchymal transition; EndoMT, endothelial-to-mesenchymal transition.implicated in the progression of hepatic fibrosis are considered here, including hepatocytes, cholangiocytes, sinusoidal endothelial cells, monocyte-derived macrophages, T lymphocytes and stellate cells.Experimental and clinical evidence for the regression of fibrosis are reviewed, and if hepatic remodelling has the potential to be reversible via breakdown of the fibrotic extracellular matrix by fibrolysis and by resolution of inflammation.Unfortunately, effective antifibrotic therapies to reverse chronic liver disease are lacking.Only two clinically approved antifibrotic medicines (pirfenidone and nintedanib) are available, and these are indicated only for idiopathic pulmonary fibrosis(Cameli et al., 2020).Borrello and Mann highlight the clinical need for well-tolerated drugs that specifically target liver fibrosis (without causing further liver injury), drawing attention to current clinical trials that have mainly focussed on drugs that treat non-alcoholic steatohepatitis (NASH, which is the most severe form of NAFLD), and those trials plagued by many confounders (e.g., high rate of placebo effect).The paucity of suitable preclinical models that can resolve NASH poses a further confounder to drug discovery for this disease.A number of mediators implicated in the development and progression of hepatic fibrosis (including MMPs and their endogenous TIMP inhibitors, as well as lysyl oxidases) are amongst the current strategies to treat liver fibrosis.

Fibrosis
in the kidney, leading to chronic kidney disease (CKD), is a common outcome of kidney injury and of multiple diseases including diabetes, high blood pressure and cardiovascular disease.The original research arising from the work of Kaur et al. (2023) seeks to further contribute to drug discovery in the CKD field and search for treatments to reverse renal fibrosis.A range of GPCRs expressed on the surface of multiple cell types have been proposed as key therapeutic F I G U R E 2 Transcription factor 21 (Tcf21) is a potential therapeutic target for treatment of renal fibrosis, as revealed by Kaur et al. (2023).UUO, unilateral ureteral obstruction (murine model of kidney fibrosis).Figure is reproduced from their graphical abstract in this special themed issue.targets for fibrosis in the kidney (and other organs).The work undertaken by Kaur et al. reveals the potential role for transcription factor 21 (Tcf21) expressed in kidney fibroblasts as a potential therapeutic target for renal fibrosis.As illustrated in our Figure 2, the authors utilised kidney RNA sequencing and single-cell GPCR transcriptomic profiling in a murine preclinical model of kidney inflammation and fibrosis.Bulk RNA sequencing revealed >5000 up-regulated and 1200 down-regulated genes in male renal fibrosis mice, of which the top 10 up-regulated genes were expected markers of inflammation and fibrosis (Kaur et al., 2023).Curating a library of cell markers, fibrotic genes and GPCRs, the authors probed single fibroblastenriched cells, identifying which transcriptional markers of activated fibroblasts (including periostin and PDGF receptor A) are increased.Single-cell qRT-PCR of fibroblast-enriched cells from mice with renal fibrosis revealed Tcf21-positive fibroblasts were enriched for differentially up-regulated fibrotic genes, including Col1a2, Timp1, Col1a1, Fn1, Ccn2 and Tgfb1.Subsequent analyses confirmed Tcf21 was an effective marker for activated fibroblasts in fibrotic kidneys, including in both the context of diabetes and patient samples.It was concluded that activation of interstitial fibroblasts was largely responsible for the decline in kidney function.The single-cell GPCR screening undertaken here may identify new treatment targets warranting investigation for chronic kidney disease (Kaur et al., 2023).4| THERAPEUTIC TARGETING OF FIBROSIS IN THE CONTEXT OF DIABETES: FOCUS ON THE HEART Cardiac fibrosis can underlie or further exacerbate cardiomyopathy, a form of cardiac dysfunction that can subsequently lead to overt heart failure.Diabetic cardiomyopathy is commonplace in diabetes mellitus, for which myocardial interstitial and perivascular fibrosis are key features.Sharma et al. (2023) comprehensively review the development, pathophysiology and treatment of cardiac fibrosis and its drivers in this context, and the extent to which the potential for sex-specific differences has been considered to date.The impact of diabetes on susceptibility to heart failure across its different phenotypes is outlined, focussing on heart failure with preserved ejection fraction (HFpEF) and heart failure with reduced ejection fraction (HFrEF), which are common

(
including cardiac fibrosis) are detailed, across both clinical and F I G U R E 3 Mechanism of action of traditional heart failure therapies which inhibit the renin-angiotensin-aldosterone system (RAASi), contrasted to modern glucose-lowering therapies, in the context of diabetic cardiomyopathy, as reviewed by Sharma et al. (2023) (figure reproduced from their review in this special themed issue).

Figure 3 ,
Figure3, modern glucose-lowering therapies (including sodium-glucose co-transporter 2 inhibitors [SGLT2i] and glucagon-like peptide 1 receptor agonists [GLP-1RAs]), as well as renin-angiotensinaldosterone system inhibitors (RAASi), offer varying degrees of promise for limiting fibrosis in the failing heart(Sharma et al., 2023).RAASi reduce markers of fibrosis (such as serum procollagen type I C-terminal pro-peptide) in heart failure patients(Cleland et al., 2021), with greater reductions in cardiac fibrosis markers in females compared with males observed in preclinical studies(Kanashiro-Takeuchi et al., 2009;Walsh- Wilkinson et al., 2019).Excitingly, SGLT2i (dapagliflozin and empagliflozin) and GLP-1RAs (liraglutide, dulaglutide, albiglutide and semaglutide) reduce three-point major adverse cardiovascular events monitoring and treatment.Fibrosis often underlies, and further exacerbates, impaired function of the affected organ, thereby increasing risk of complications in many disease settings.Both new and repurposed pharmacological treatments are now available for fibrosis, depending on the cause, location and severity of the disease being treated.As illustrated in Figure 4, these treatments have been identified through both hypothesis-driven investigation (e.g., targeting pro-inflammatory/profibrotic pathways) in addition to interrogated as a consequence of serendipitous observation (such as those emerging from the cardiovascular outcome studies for modern glucose-lowering therapies).The contributions included in this special themed issue highlight some recent and innovative research approaches for targeting fibrosis.More research is required, however, to find effective and safe therapies that can prevent, halt or even reverse the progression of fibrosis in disease.Future studies focussing on the identification of additional novel druggable targets and treatment intervention strategies are encouraged to modulate the fibrotic response, and their path to translation defined, to improve the quality of life of patients with fibrosis.