Investigating Mechanisms of Age-Related Increase in Fibrosis
Fibrosis is a form of scarring, important in many medical conditions, notably those of the liver, and a process that increases in many internal organ tissues with advancing age. Inappropriate levels of cellular construction of fibrotic structures disrupts the proper function of tissues, leading to dysfunction and disease. Researchers here look into the underlying mechanisms driving that age-related increase in fibrosis, and suggest that the problem lies in a reduced ability to clear out fibrosis rather than an increased tendency to generate these structures in response to damage. The researchers point to the presence of cross-links as one possible contributing cause for that change, which is yet another reason to push for greater support of efforts to produce therapies to clear cross-links.
Liver fibrosis results from a sustained wound healing response due to chronic liver injury and occurs when extracellular matrix (ECM) production exceeds ECM degradation. Activated hepatic stellate cells (aHSCs) are the main cells involved in fibrogenesis as the key source of ECM compounds and a major modulator of hepatic inflammation. Next to aHSCs, the hepatic macrophages also promote fibrosis progression by driving HSCs activation, by releasing pro-inflammatory and pro-fibrogenic factors and by supporting the infiltration of pro-fibrogenic immune cells. Liver fibrosis reversibility has been documented for several years. In animal models, liver damages reverse and fibrotic scar degradation occurs when the hepatotoxic agent is removed or when a normal biliary outflow is restored after common bile duct ligation. Evidences of fibrosis regression come also from clinical practice, especially after the arrival of new anti-viral therapies enabling high rate of hepatitis C virus (HCV) eradication. During fibrosis resolution, aHSCs disappear by senescence, inactivation or apoptosis while inflammatory and pro-fibrogenic macrophages differentiate into pro-resolution cells able to secrete large quantities of fibrolytic matrix metalloproteinases (MMP) and anti-inflammatory cytokines.
The human liver is affected by aging. It manifests by a reduced volume and blood flow as well as by cellular changes such as increased oxidative stress, decreased number and dysfunction of mitochondria, accelerated cellular senescence and decreased regenerative ability. Aging is also a risk factor for several specific hepatic diseases. In non-alcoholic fatty liver disease (NAFLD), evolution from simple steatosis to steatohepatitis and fibrosis occurs more frequently in old patients. In HCV chronic infection, age at time of infection is a strong determinant of fibrosis progression. Although those data emphasize the susceptibility due to aging to develop more severe disease and significant fibrosis, the mechanisms underlying this propensity are not fully understood. In viral hepatitis, an impaired immune response against foreign antigens may explain a different immunopathogenesis in the elderly and more sustained hepatic fibrotic process. In rodents, a more severe fibrosis is also observed in older animals but mechanisms remain debated. Aging-dependent hepatic susceptibility to toxic agents, reduced ECM proteins degradation and variation in inflammatory cells infiltrating the injured liver are discussed as differences in rodent genetic strains may explain at least partially divergent results. Interestingly, evidence points to a quantitatively different ECM turnover according to the age of rat models. Indeed, type I and II collagen turnover was significantly reduced in old compared to young animals, while type IV and V collagen and biglycan degradation biomarkers were significantly upregulated in old rats.
In this work, we reproduced a higher susceptibility to fibrosis in old mice compared to young mice after repetitive administrations of carbon tetrachloride (CCl4). A single dose of CCl4 disrupts hepatocytes integrity that wound healing processes tend to restore. In case of repeated exposures, recurrent profibrotic stimulation occurs prior to the resolution of the previous healing round. In our study, Collagen I and alphaSma mRNA were significantly upregulated in treated groups compared to controls but no difference was observed between age-groups, mitigating the role of variable fibrogenic processes in the severity of ECM deposition. Rather, this is in favor of an equal propensity to initiate profibrotic events in response to a toxic injury.
MMPs are involved both in fibrosis progression and resolution through their ability to degrade virtually all compounds of the ECM. The capacity of the liver to resorb scar or in the contrary to "preserve" the pathologic matrix accumulated after injury will depend on the balance between MMPs and their respective inhibitors. Among all MMPs, MMP-13 is the main interstitial collagenase in rodents and largely involved in fibrosis resolution. We observed a strong induction of Mmp13 gene expression in young mice at peak of fibrosis while old mice expressed significantly less Mmp13 mRNA. No difference was noticed concerning tissue inhibitor metalloproteinases (TIMPs) expression suggesting that the balance MMP/TIMP was overtly tilted in favor of matrix degradation in young mice but less so in old ones. This was confirmed by the nearly complete clearance of scar matrix in young animals 4 days after the last toxic injection while virtually no remodeling occurred in old mice, and by the reduced collagenolytic activity in this last group.
We demonstrated a higher proportion of thick and dense collagen fibers in old mice as well as an enhanced expression of the enzymes involved in collagen maturation changes. There are features that limit fibrosis remodeling: old, pauci-cellular, thick and heavily cross-linked septae resist proteases degradation. More than biochemical impact on matrix fibrils, cross-linking enzymes support also HSCs activation by maintaining a stiff environment and may have immunomodulatory functions in liver fibrosis influencing the changes in balance between fibrogenesis and fibrolysis.
To date, no antifibrotic therapy exists besides the suppression of the causative agent. Our work, demonstrating that liver fibrosis is less prone to reverse in old animals, has several clinical implications. First, impact of aging on reduced ability for fibrosis degradation may partially explain some disappointing results of antifibrotic agents in clinical trials while promising when preclinically tested. Indeed, pre-clinical studies usually use young animals (6-8 weeks old) while patients concerned by treatment classically suffer from fibrosis that has developed over decades rather than weeks in animals. Secondly, our study highlights the importance to target the correct underlying processes in the perspective of an effective therapy. Based on our results, this target may be different according to the age of the patients, and therapies supporting the fibrolysis or opposing the cross-linking of the matrix might be of particular interest in an old population.
...the problem lies in a reduced ability to clear out fibrosis rather than an increased tendency to generate these structures in response to damage.
Meaning that, as with everything else in biology, the process is dynamic and therefore reversible. One of the most difficult hurtles in convincing people that aging is reversible/curable is the notion that biology is static, like a building or a car. Once you realize the inherent dynamism of biology, curing aging seems far more plausible.