Unravel the complexity of the cellular and molecular process of fibrosis
When the body sustains an injury, the formation of an extracellular matrix (ECM) is an important protective mechanism for tissue regeneration and healing. It acts as a glue holding broken tissues together, filling the space left after cellular death, and providing a medium suitable for the growth of new tissues. Fibrosis is a dangerous condition where an excessive accumulation of ECM, in response to chronic injury or illness, leads to organ dysfunction and failure.
There are multiple versions of fibrotic disorders that exist in all organs. In the USA, it has been estimated that 45% of deaths there can be attributed to fibrotic disorders. Some examples of potentially fatal fibrotic diseases include idiopathic pulmonary fibrosis, renal fibrosis (chronic kidney disease), hepatic cirrhosis, and cardiac fibrosis.
When injured, tissues emit inflammatory signals, like cytokines and growth factors. These attract and stimulate fibroblasts, the primary cellular effector of wound healing and fibrosis. Under the influences of these factors, fibroblasts differentiate into myofibroblasts and produce ECM in the wound. They then promote the growth of new blood vessels and cells, which fill the gap left after the injury. Over time, the ECM deposited by fibroblasts is remodeled and replaced by new cells to achieve a regenerated state. In fibrotic disorders, this process is altered by persistent inflammation, which forces the fibroblasts to deposit excessive amounts of ECM into the wound. This results in the scar tissues that characterize such disorders.
PerkinElmer I Cisbio has made available a wide range of documents with educational content (Guide, application notes) and reagents dedicated to fibrosis to help you in your research. We have developed a panel of ready-to-use tests to monitor biomarkers, phosphoproteins, and transcription factors involved in disease development and progression of fibrotic disorders.
Fibroblasts & ECM Management
Fibroblasts are ubiquitous mesenchymal cells that fulfil multiple roles in tissue development, tissue repair, and the overall management of inflammation. They are the main source of collagen and the primary cellular effectors of fibrosis, which makes them one of the most widely investigated aspects of that disease.
In the adult body, fibroblasts remain in a resting state until prompted to activation and differentiation. This initial signal is usually received in the form of mechanical stress resulting from a loss of integrity of nearby tissues. Fibroblasts sense these subtle differences via a cortical network of actin and fibronectin/integrin complexes, and start differentiating themselves into proto-myofibroblasts. Changes include cytoplasmic actin filaments, expression of alternatively spliced ED-A fibronectin, and the growth of focal adhesion points that distort their shape. The full differentiation of fibroblasts requires them to migrate to injury sites, where they receive additional stimuli and TGF-β1. Differentiated myofibroblasts are then characterized by their large amount of ED-A fibronectin, high collagen secretions, and criss-crossing α-SMA filaments that confer them contractility.
The complete differentiation of fibroblasts into myofibroblasts involves a complex pro-fibrotic and inflammatory environment, with multiple cytokines, growth factors, and external stimuli. TGF-β1 signaling is utterly essential, with SMAD2 and SMAD3 being key promoters of collagen expression. However other pathways contribute to the process, such as PDGF and pro-inflammatory cytokine signaling. Along with TGF-β1, mechanical tension is the second most important stimulus without which the myofibroblast phenotype cannot be fully expressed. It is mediated through cells via the transmembrane integrins linked to the ECM. The transduction relies on the resulting fibrillar or globular state of actin, which regulates the hippo pathway. Successful transduction of mechanical stress promotes the expression of Alpha Smooth Muscular Actin (α-SMA), which forms contractile filaments across myofibroblasts and further enhances their sensitivity to mechanical stress.
Signaling pathways involved in fibrosis
The transforming growth factor beta (TGF-β) is a highly-important cytokine acting as a central mediator in several mechanisms related to injury and inflammation, including fibroblast regulation, ECM remodeling, wound healing, immunoregulation, proliferation, survival, and differentiation. In particular, TGF-β1 is known as the principal and necessary mediator of fibroblast activation and the expression of pro-fibrotic phenotype. As such, TGF-β1 is of major interest in fibrosis-related research, but its involvement in multiple mechanisms makes it an unrealistic therapeutic target in itself. The different pathways signaling from TGF-β receptors are therefore generally considered to be better therapeutic approaches.
The TGF-β1 canonical signaling pathway is SMAD-dependent and mediates TGF-β1 binding to its TbRII/ALK5 Ser/Thr kinase transmembrane receptor. It involves the successive phosphorylation and association of SMAD2, 3, & 4. The SMAD2/3/4 complex then acts as a transcription factor promoting the expression of pro-fibrotic phenotypes. The regulation loop is ensured by YB-1 and SMAD7, which respectively promote the latter and inhibit SMAD2 & 3 phosphorylation. Non-canonical SMAD-dependent signaling differs from the canonical pathway by the receptor binding TGF-β1, which incorporates an ALK1 subunit in the place of TbRII. The SMAD complex is also different, and made up of SMAD1, 5, & 4. This pathway results in pro-fibrotic phenotypes as well, and is known to inhibit its canonical equivalent at the SMAD2/3 complex level. SMAD6 acts as a regulator.
Apart from the SMAD-dependent pathways that mediate most of its known pro-fibrotic effects, TGF-β1 also signals through non-canonical SMAD-independent pathways with wide-ranging result, including cell survival, proliferation, cell size increase, inflammation, and sometimes apoptosis.
The P13K-AKT and ERK pathways mediate effects related to stress resistance and survival. They also exhibit interaction with the canonical SMAD-dependent pathway, which they respectively promote and inhibit. In particular, the phosphorylated state of AKT promotes or inhibits SMAD3 phosphorylation via GSK3. The JNK/p38 MAPK and JAK2/STAT3 pathways are related to inflammatory signaling. While the former mediates its effects through the inflammatory mediators NF-kB and JNK, the latter signals via the usual JAK phosphorylation at the membrane and STAT phosphorylation, dimerization, and translocation to the nucleus. Lastly, the ROCK kinase pathway mediates through myosin light chain (MLC) and cofilin, with results in the cytoskeletal architecture of cells. In particular, this signalization may be critical to the actin filament polymerization that is required for the full expression of pro-fibrotic phenotypes.
The role of innate and adaptive immunity in fibrosis
Fibrosis as a process is not a systematically pathological event, and is an essential part of wound healing and tissue repair in response to injuries. The mechanism only becomes pathological when the regulation of fibroblasts is compromised or lost.
In particular, cells of both innate and adaptive immunity are recruited and activated at injury/inflammation sites, and the range of their activities makes them a non-negligible source of signals that can tip the fibrosis process either toward healthy scarring or pathological accumulation of scar tissues.
Innate immunity cells are believed to mostly contribute pro-fibrotic signals to the wound-healing process. Macrophages are resident cells with a dual role. When classically activated (M1), they act as pro-inflammatory pathogen-clearing effectors, while their alternatively activated phenotype (M2) is the main source of TGF-β1 for fibroblast differentiation and the secondary cellular effector of fibrosis. There are three types of Granulocyte pro-fibrotic signals. First, the pro-inflammatory cytokines they secrete and which accumulate at an injury site promotes macrophage and fibroblast activity. Second, the cytotoxic contents of eosinophils and neutrophils are sources of cellular damage and inflammatory DAMPs. Third, basophils and eosinophils promote the M2 macrophage phenotype with secretions of IL-4 and IL-13. Eosinophils are also a direct source of TGF-β1.
The contribution of Adaptive immunity cells is more nuanced. On the one hand, helper T-cells 2 (Th2) are promoters of the pro-fibrotic M2 macrophage phenotype via their secretions of IL-4 and IL-13. Helper T-cells 17 (Th17) are also assimilated to pro-fibrotic actors, as their secretion of IL-17A creates a durable pro-inflammatory environment and promotes neutrophil degranulation of their cytotoxic contents.
On the other hand, helper T-cells 1 (Th1) appear to have anti-fibrotic properties due to their IFN-γ secretion, which inhibits the M2 macrophage phenotype and therefore reduces the availability of TGF- β1. Regulatory T-cells (T-reg) are more controversial. Most studies agree on their anti-fibrotic properties, which result from their anti-inflammatory IL-10 and adenosine secretions, which directly downregulate other T-cells and pro-inflammatory cell activity. However, T-reg cells are also a source of TGF-β1, which has been found to confer an overall pro-fibrotic effect in some experimental contexts.
Fibrogenesis in NASH disease and idiopathic pulmonary fibrosis
Human fibrotic diseases are a significant health problem worldwide, due to the large number of people affected and our incomplete knowledge of the pathogenesis of the fibrotic process. Fibrotic disorders also tend to have poor outcomes as no therapeutic approach has proven entirely effective in reversing and curing them. Two fibrotic disorders are currently considered of particular relevance:
- NASH (Non-Alcoholic Steatohepatitis) is closely linked to the triple epidemics of obesity, pre-diabetes, and diabetes. Symptoms are often silent or non-specific to NASH for years, making this disease difficult to diagnose and therefore prone to escalate to liver fibrosis over time.
- Idiopathic Pulmonary Fibrosis (IPF), the most common form of idiopathic interstitial pneumonia, is a chronic, progressive, irreversible, and generally fatal lung disease of unknown cause. Pulmonary fibrosis in general can be triggered by pulmonary infection, and the COVID-19 pandemic now raises concerns about a potential wave of pulmonary fibrosis in the coming years.
Non-Alcoholic Steatohepatitis, or NASH, is the aggravation of Non-Alcoholic Fatty Liver Disease (NAFLD), where an accumulation of fat in the liver results in metabolic changes and a chronic inflammatory environment. This chronic inflammation is a favorable ground for fibrosis development and potential escalation to liver cancer.
Find more information in the NASH dedicated universe.
Covid-19, caused by the highly pathogenic Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), presents high morbidity and mortality due to the development of SARS associated with extensive pulmonary fibrosis. SARS-CoV-2 is dependent on binding to the ACE2, part of the renin-angiotensin system (RAS). The down-regulation of ACE2 on the binding of the virus disrupts the RAS downstream activities, leading to increased inflammation and the development of fibrosis. The poor prognosis and the risk of developing pulmonary fibrosis are associated with increased expression of ACE2 in at-risk groups (e.g. cases with obesity, heart disease, and/or aging), giving the virus numerous opportunities to bind and internalize ACE2, thereby preventing the enzyme from acting as an anti-inflammatory and antifibrotic agent.
Review on pulmonary fibrosis in COVID-19
Converging pathways in pulmonary fibrosis and Covid-19 – The fibrotic link to disease severity