ECM formation and degradation during fibrosis, repair, and regeneration

The mechano-chemical properties of the extracellular matrix (ECM) (Fig. 1 ) are necessary for cells to differentiate, specialize, locate themselves in relation to other cell populations, and build the functional units that characterize multicellular anatomy. There is a reciprocity between function, developmental stage and ECM structure that results in specialized ECMs, where components are connected in organ- and stage-specific patterns 1 . These ECMs are made of different protein combinations 2 with different turnover cycles, dependent on strictly regulated building, dismantling, and remodeling cycles. Fig. 1 A schematic of ECM structure. Although ECM is organ specific, there are characteristics that are common to most tissues, like the presence of a basement membrane and an interstitial matrix. Equally, disease may induce sui generis remodeling, abnormal formation and degradation are shared by several conditions (e.g., IPF, MASLD, COPD, etc.). Pioneering work on Mass Spectrometry (MS) has cataloged ECM components (the matrisome), divided in core-matrisome (~300) and matrisome-associated proteins (~1000) 2 . The core-matrisome, or structural ECM proteins, includes 28 collagens, elastin, fibronectin, and laminin isoforms, as well as proteoglycans (like perlecan) and glycoproteins (like nidogens). Matrisome-associated proteins regulate ECM structure (e.g., proteolytic enzymes, matrix metalloproteinases-MMP, etc) but are also controlled by ECM, e.g., transforming growth factor beta, (TGF-β), vascular endothelial growth factor, (VEGF) superfamilies, other growth factors and cytokines 2 , 3 . The emergence of spatial proteomics and 3D ECM mapping 4 – 6 is revealing the 3D structure of the ECM, showing that healthy and diseased tissue share ECM components, but their amount, distribution, density, and articulation in space differs. It is likely that functional units within an organ (e.g., nerve trunks and terminals, vessels, specialized structures like follicles, glomeruli, alveoli, or acini) have a function-specific ECM 6 . This seems to be the case of capillary follicles and skin 7 , 8 . ECM structure is generally divided in two compartments (Fig. 1 ): Basement Membrane (BM) and Interstitial Matrix (IM). BM is a cloth-like surface, adhesive to epithelia, glandular epithelia, endothelium, myocytes, and adipocytes, among other cells 5 . The BM is based on a Collagen type IV backbone supporting a Laminin surface 9 . Glycoproteins, such as Nidogens and Perlecan, bind the Laminin and Collagen type IV layers. Others, like Netrin-4, regulate BM mechanical properties 10 . IM is structured by fibrillar collagens (type I, II, III, V) and elastin. In turn, collagen fibrils are bridged by Fibril-associated Collagens with Interrupted Triple Helices (reviewed elsewhere 11 ) and linked to the BM by non-fibrillar collagens, like Collagen type VII 12 . Fibronectin (reviewed here 13 , 14 ), regulates ECM assembly, collagen fiber assembly, embryo development, and is also critical during wound healing, ECM maturation and cancer progression. Cell-secreted fibronectin is a mediator of scar tissue formation 15 . Upon fibrosis progression, IM undergoes extensive remodeling, notably, abundant cross-linking mediated by enzyme families, like transglutaminases and lysyl-oxidases 16 , and collagen glycation, a process associated to diabetes and aging 17 . The overgrowth of crosslinked collagen results in a stiff, more viscoelastic 18 , linearized IM with deleterious consequences for disease progression 19 . ECM stores and releases growth factors, cytokines, and bioactive peptides, controlling their location, density, and activity. Fibronectin binds VEGF, hepatocyte growth factor (HGF), platelet-derived growth factors (PDGF), among others, and growth factor-binding domains are abundant in matrisomal proteins 3 . Injury responses can, by increasing binding sites, signaling oligopeptides, and structural domains, enhance fibrogenesis as well as mechanosignaling 20 . Pioneering experiments exposing radiolabeled PDGF isoforms keratinocyte growth factor and HGF to collagen chains demonstrated binding to collagens type I, III, IV, V, and VI as well as preserved growth factor bioactivity 21 – 23 . The affinity of collagen domains extends to inflammatory mediators and cytokines (e.g., interleukin-2, oncostatin) 24 , 25 . Isolating cell-ECM structure interactions in an ECM scaffold-based bioreactor shows that kinase activity (including growth-factor activity) and cell-driven ECM remodeling follow anatomical cues, supporting the notion of positional regulation 26 . Therefore, the ECM acts as a spatial regulator of cellular activity. By-products of ECM protein synthesis add an additional layer of complexity to ECM dynamics. These by-products can be bioactive, paracrine, and endocrine regulators 27 . Collectively, they are called matrikines 28 . Notable examples of this family 29 include endotrophin, a pro-peptide of collagen type VI, linked to visceral

“Core” pro-fibrotic mechanisms 71 are myofibroblast-associated pathways found in different fibroses. A bare bones myofibroblast definition could be that of an “activated” mesenchymal cell characterized by a dense α-smooth muscle actin (αSMA) cytoskeleton, enhanced contractility, and ECM protein overexpression. Myofibroblast progenitors can be traced to be adipocytes 72 , pericytes 73 , smooth muscle cells 74 , immune cells 75 , mesenchymal stem cells 76 , endothelium 77 , epithelia 78 , bone-marrow derived and organ-specific fibroblasts 79 . This heterogeneity suggests that more than a cell type, “myofibroblast” may denote a behavior. Myofibroblast activation largely depends on a signaling network that hovers around the TGF-β superfamily 41 , 79 , beginning with the release of TGF-β1 from the TGF-β latency associated peptide (LAP) in the ECM, prompted by multiple mechanisms, including mechanical stress sensing by the LAP-binding integrins αVβ1 on myofibroblasts and αvβ6 on activated epithelia 80 , 81 , binding to TGF-β receptors 1 and 2, canonical SMAD signaling, translocation of SMAD2, SMAD3 and SMAD4 to the nucleus and promotion of the genes encoding αSMA ( ACTA2 ) and ECM proteins. Other pathways also lead to activation: TGF-β2 82 , 83 and TGF-β3 84 , and non-canonical TGF-β signaling through mitogen-activated protein (MAP) kinase 85 . Signaling and mechanical changes are linked. Injury attracts fibroblasts, and they contract injured tissue, increasing stiffness. An initially soft ECM is then substituted by scar tissue (discussed below) rich in fibronectin and collagen crosslinked by transglutaminases 86 and lysyl oxidases 87 , 88 . Myofibroblasts transmit and perceive force through cell surface receptors, notably integrins 87 , discoid domain receptors (DDR) 89 , vanilloid receptors 90 , G-protein coupled receptors 91 , and hyaluronan receptor CD44 92 Cell-ECM contact induces the synthesis of cytoskeleton proteins and cell adhesion complexes, calling for further ECM contraction. Integrins αvβ1, αvβ3, αvβ5 and αvβ6 activate latent TGF-β by mechanically pulling LAP 79 , thus linking both biochemical and mechanical signaling in one positive activation loop. The ADAMTS (A disintegrin-like and metalloproteinase with thrombospondin motifs) superfamily has 19 members that remodel the ECM, partly by cleaving latent TGF-β complexes, changing cell mechanics, and increasing tension as well as TGF-β release 93 . A subgroup of ADAMTS members bind to fibrillin and fibronectin 94 , belong in the fibrillin microfibril niche, a mechanosensing hub, and regulate elastic fiber assembly through TGF-β 95 . ADAMTS-like 2 variants produce geleophysic dysplasia, a syndrome associated to cardiac and interstitial fibrosis 96 , and is overexpressed in adults with chronic liver disease 97 . Recessive mutations in ADAMTS10 can cause Weill-Marchesani Syndrome, associated with cardiac fibrosis 98 . The TGF-β superfamily also includes a subgroup of cytokines, activins, that bind to membrane receptors (activin receptors type I and II) to phosphorylate the activin-like kinase 4 (ALK4), which in turn phosphorylates Smad proteins 2 and 3 to transduce activin signaling into the nucleus 99 . Activin action is downregulated by follistatins, which bind to the ECM (e.g., to heparan sulfate proteoglycans) 100 and trap activin, so it can be cleaved by proteolysis. Activin upregulation results in follistatin overexpression and activin attenuation. The activin-follistatin system is implicated in scarring and regeneration across several organs. In the liver, activin is overexpressed in models of liver fibrosis, activating hepatic stellate cells (HSCs) 101 , whilst Follistatin blocks activin and inhibits TGF-β signaling as well as collagen production 102 . In the kidneys, activin is overexpressed after injury and during kidney fibrosis, mimicking TGF-β signaling and hampering regeneration 103 , but follistatin blockade promotes epithelial proliferation and repair 104 . In the lungs, activin promotes myofibroblast proliferation, ECM formation, and TGF-β overexpression, which in turn induces activin, creating a persistent fibrotic niche 99 . Follistatin gene therapy has been trialed for Becker muscular dystrophy, resulting in reduced muscle fibrosis and muscle performance improvement 105 . Activin A inhibition with a monoclonal antibody (Garetosmab 106 ) reduces new heterotopic bone lesion formation in fibrodysplasia ossificans progressive. Sotatercept, an activin inhibitor, reduced the risk of death in patients with pulmonary arterial hypertension 107 . The Wnt/ β-catenin pathway can also drive fibrogenesis. Wnt is a homolog of integrase-1 and the wingless gene in Drosophila 108 . There are 19 Wnt proteins, essential for development and homeostasis. The canonical Wnt pathway involves binding to the Frizzled (FZD) transmembrane receptors, the translocation of β-catenin into the cell nucleus, and activation of target genes by transcription factors T-cel

Upon insult, the adult myocardium answers with fibrogenesis, not regeneration 155 , 156 . Nonetheless, adult cardiomyocytes can re-enter the cell cycle, and conclusive evidence emerged from an elegant study that measured carbon-14 incorporation in cardiomyocytes from individuals born around the partial ban in nuclear testing of 1963 157 , when environmental isotope levels decreased exponentially. The key finding established that ≈0.5-1% of adult human cardiomyocytes re-enter the cell cycle per year, not enough to sustain regeneration. The key to heart regeneration may not be in its beating cells, but in those maintaining its structure (Fig. 3 ). Scar-building cells in the heart come from resident fibroblasts 158 , recruited bone marrow progenitors (fibrocytes), and endothelial cells that complete endothelial-mesenchymal transition. They become myofibroblasts under TGF-β signaling 159 . These cells depose ECM after being exposed to hypoxia or inflammatory cytokines like interleukin-2 and tumor necrosis factor 160 , but in another example of the contextual nature of fibrosis, the persistence of myofibroblasts is not necessarily damaging. Mice engineered to produce myofibroblast-rich post-infarction tissue showed reduced scar formation 161 . Still, groundbreaking work 162 in cardiac fibroblasts engineered to express ovoalbumin peptide to mark them as targets for CAR (chimeric antigen receptor) CD8+ T-cells (i.e., anti-fibroblast immunotherapy) led to a reduction of cardiac fibrotic injury. Tantalizingly, fibrillar collagen deposition was reduced in treated hearts that showed histologically normal myocardium, suggesting that a restitution of normal architecture could take place after eliminating the cells producing abnormal ECM. The optimal window for such a therapy needs to be determined. Cardiac biomarkers are extensively reviewed elsewhere 163 , 164 . Benchmark biomarkers include B-type natriuretic peptide (BNP) and N-terminal BNP (NT-proBNP) (diagnostic and prognostic in heart failure patients), cardiac troponins (myocardial necrosis), Suppression of Tumorigenicity (ST2), an interleukin receptor upregulated in response to injury (prognostic for heart failure), Galectin-3, a lectin, that marks cardiac fibroblastic activity and C-terminal type VIa3 pro-collagen (PRO-C6), prognostic in heart failure with preserved ejection fraction 165 .

At homeostasis, the potential plasticity of tubular epithelium translates into a capacity to repair the kidney parenchyma after acute injury, acting in concert with endothelium, fibroblasts, and macrophages, and through the activation of developmental pathways, like Notch, Wnt/B-catenin, SOX9 transcription factor, and the Sonic hedgehog pathway 178 . However, repeated aggression results in a maladaptive response to damage that subverts these pathways and ultimately results in the recruitment of pro-inflammatory cells, and the activation of myofibroblasts (including tubular epithelial cells that undergo mesenchymal-to-epithelial transition, resident mesenchymal cells, and fibroblasts) 179 . Once the bridge to chronic kidney disease (CKD) has been crossed reparative ability diminishes but is not completely erased. In diabetic patients suffering from CKD, pancreas transplantation reverted ECM remodeling (more specifically, BM thickening, it is unclear if this process extends to the IM) after 10 years 180 (Fig. 3 ). In a porcine preclinical model, a nephrectomy followed by the implantation of a segment of decellularized ECM results in the recellularization of the matrix with structures reminiscent of glomeruli, vessels, and tubules 181 . Unfortunately, clinical progress toward the reversal of kidney fibrosis is still partial. Blocking TGF-β signaling, the main driver of ECM deposition, has shown no beneficial effect on kidney damage 182 . In type 2 diabetics with CKD however, GLP-1 inhibitors reduce the risk of death, heart and kidney outcomes 183 . Biomarkers of ECM remodeling and turnover assess kidney fibrosis progression 184 . Collagen type III turnover biomarkers PRO-C3 and C3M (a segment resulting from MMP-9 collagen type III cleavage) correlate with kidney fibrosis degree. C3M/creatinine ratio is highly discriminative for advanced kidney fibrosis 185 . Lysyl oxidase (LOX) crosslinks fibrillar collagen and is increased in patients with kidney fibrosis 186 . Dickkopf-related protein 3 (DKK-3) is a glycoprotein secreted upon tubular injury that promotes scarring that significantly predicts estimated glomerular filtration rate (eGFR) decline and identifies patients at high risk of CKD progression 187 . PRO-C6 is associated with kidney fibrosis and outcomes in acute kidney injury 188 , 189 .

Fat deposits covering viscera and underlying the skin compose an endocrine organ that regulates metabolism, immunity, and homeostasis. Adipose tissue (AT) dysfunction has wide-ranging, systemic consequences, and fibrosis is both one of its sequels and drivers. White adipose tissue (WAT) (Fig. 3 ) stores energy, while brown-beige (BAT) is thermogenic (i.e., it dissipates energy as heat) 227 – 229 . BAT sits in the paravertebral, axillary, supraclavicular, and periadrenal areas but WAT is widespread: subcutaneous fat lies beneath the dermis and represents ~80% of total body fat; visceral fat surrounds intrathoracic (e.g., pericardial, epicardial) and intraperitoneal organs (e.g., omental, mesenteric). AT secretes signaling polypeptides (adipokines) that regulate metabolism 230 , e.g., adiponectin promotes insulin sensitivity 231 , whilst resistin and lipocain promote insulin resistance 230 . It produces leptin, an adipokine that signals to the hypothalamus and other brain regions, promoting satiety and energy expenditure. Leptin resistance is associated to obesity 232 . WAT regulates immunity through pro-inflammatory cytokines TNF-a and interleukins 1B, 6, 8, and 18 230 . WAT deposits have different expansion-contraction patterns 233 and transcriptomic profiles 233 , but expansion by hypertrophy is associated to hypoxia 234 and hypoxia-factor 1α (HIF1α) secretion, which in WAT calls for the transcription of ECM associated genes 235 , fibrosis, and collagen crosslinking. This AT fibrotic response increases the synthesis of collagen type VI and collagen type VI C-terminal pro-collagen, endotrophin 236 . Collagen type VI correlates with insulin resistance in humans (ref), but interestingly, in COL6 −/− animals, AT hypertrophy fails to invoke a fibrotic response, leading to a soft ECM, probably due to a decrease in circulating levels of endotrophin 30 . ECM formation in distant organs, downstream of AT expansion, is associated to ECM formation in distant organs. Approximately one third of systemic angiotensinogen is produced by WAT 237 , activating angiotensin receptors (e.g., angiotensin 1b receptor) in the kidneys that are inflammatory in mice 238 and humans 239 . Overstimulation of leptin receptors in the kidney is associated to progressing renal disease 240 and overexpression of TGF-β1, collagen type IV, and fibronectin 241 , 242 . Another example of AT driving disease is gut creeping fat, which surrounds the exterior of the intestines wrapping the intestines. Creeping fat is linked to the release of pro-inflammatory cytokines and fibrotic mediators which enhance ECM remodeling and collagen deposition of the affected intestinal tissue and is highly associated with the development of intestinal fibrosis and strictures 226 , 243 .

The dysfunction that produces and sustains ECM structural alterations in an organ reverberates in the body, damaging distant tissues and triggering adverse events (Figs. 3 , 4 ). Fibrotic disease in an organ can drive end-stage disease in distant organs, characterized by simultaneous ECM remodeling, albeit at different rates, and with considerable individual variation. Consider how Metabolic dysfunction-associated steatotic liver disease (MASLD, Fig. 4 ), closely linked to metabolic syndrome, and characterized by excess accumulation of lipids in the liver, inflammation/hepatocyte ballooning degeneration, and hepatic fibrosis 272 , 273 impacts multiple systems. Before developing cirrhosis, MASLD patients die of cardiovascular disease and extrahepatic cancer with more frequency than from a liver-related event 274 , 275 (Fig. 4 ). Fig. 4 Outcomes in adults with MASLD. According to Sanyal AJ et al. 160 , patients with chronic steatohepatitis are at higher risk of cardiovascular and renal adverse events than liver-related events. This schematic illustrates the organ death race driven by MASLD progression. (Line thickness represent probability of event per 100 patients). Liver disease is by no means unique. Fibrotic progression in WAT is correlated with higher risks of infection 276 , cancer (including breast, uterus, ovaries, colon, stomach, esophagus, rectum, liver, pancreas, kidney, meninges, and blood), metabolic, kidney, cardiovascular, and psychiatric disease. Detecting, predicting, and tracking ECM formation or degradation is challenging. There are multiple molecular mechanisms and proteins involved, affecting tissues with different shape, function, resilience, and regenerative potential. Conceptually, a pharmacodynamic biomarker should either measure fibrogenic activity, i.e., determine the de novo formation of ECM proteins, or fibrolysis, i.e., ECM degradation and removal. A combination of such biomarkers could mirror the balance between fibrogenesis and fibrolysis. ECM biomarkers measure ECM component synthesis dynamically, reflecting how active fibrosis progression pathways are during measurement. They can discriminate between cumulative damage (which is the parameter assessed by a biopsy) and an actual snapshot of the biological status of the disease. Dynamic biomarkers would reflect distinct patient endotypes, characterized by different formation-degradation balances represented by different ECM parameters (Fig. 2 ). There are different technological paths to build a biomarker strategy. One passes by combining large-scale biological data and data mining. Omics-based biomarker research have been gaining momentum with the establishment of national biobanks (including the UK Biobank 277 , and Biobank Japan 278 ) and disease specific international patient registries (including the European MASLD Registry 279 ). These large databases have increased the depth, quality, and availability of Omics data as the cost of large-scale data generation has decreased, creating a conducive background for biomarker discovery. Mapping the human proteome 280 , 281 was a significant step towards assessing multiple molecular pathways simultaneously, opening a conceptual window into complex biological processes. Recently, leveraging RNA-seq and plasma proteomics resulted in organ-specific protein profiles that reveal tissue aging, thus building a proteomics-based biomarker strategy 282 . Plasma proteomics, used in Alcohol-related Liver Disease (ALD), detected circulating proteins associated to fibrosis and metabolic dysfunction, predictive of future liver-related events and all-cause mortality 283 . Complementary approaches in MASLD utilizing a proteo-transcriptomic strategy to characterize the liver-derived circulating proteome across the full disease spectrum 284 . However, there is evidence to suggest that different technologies (i.e., based on aptamers or antibodies) may affect protein quantification and comparability 285 , 286 . Epigenetics and metabolomics can also perform to a similar level: mapping DNA methylation in whole blood has found associations between disease, age, ancestry and all-cause mortality and specific cytosine-phosphate-guanine sequences, with substantial prognostic improvement for neoplasia-associated death 287 . A metabolomic platform found prognosticators all-cause mortality in a diverse population 288 , identifying a panel of 14 metabolic biomarkers that could perform as well as conventional risk factors of mortality. These panoramic approaches stand in contrast to individual and composite biomarkers supported by mechanistic research. Sensing post translational changes in a fundamental disease pathway is an effective biomarker strategy, with direct clinical impact. This is made evident by the FDA list of approved companion diagnostic devices, where single genetic biomarkers underpin decisions that have reduced disease burden and mortality for millions of cancer patients (e.g., BRCA1, BRCA2, HER1,