Linking Oxidative Stress and DNA Damage to Changes in the Expression of Extracellular Matrix Components

1Redox reactions are central to ensure correct cell function, allowing a dynamic transfer of electrons across different molecules. This occurs, for example, during molecular oxygen metabolism in the mitochondria, which are the central metabolic machinery of cells. Molecular oxygen metabolism is the basis of oxidative phosphorylation, and promotes the production of reactive oxygen species (ROS). ROS also play an important role in immune defenses, where innate immune cells produce ROS as part of their pathogen killing strategies. In fact, neutrophils and some macrophages express myeloperoxidase (MPO), which leads to the generation of different ROS, including hypochlorous acid (HOCl), a potent ROS that is involved in pathogen killing mechanisms.

2However, when in excess, ROS may cause serious damage to different molecules promoting DNA damage, protein oxidation, and lipid peroxidation. The deleterious accumulation of ROS may have multiple origins, including the excessive ROS production by the mitochondria, which drives mitochondrial dysfunction, an important trait of a variety of diseases. Mitochondrial dysfunction can be due to a metabolic imbalance, like, for example, in diabetes, where high levels of glucose promote overproduction of ROS, or it may also arise as a consequence of mutations in mitochondrial DNA, defective OXPHOS, and alterations in ROS production, which are common phenomena in cancer. As mentioned above, another example of excess ROS generation is through MPO in neutrophils, which can have a detrimental outcome if it leads to chronic inflammation, a driver of pathology in a variety of diseases.

3DNA damage can be caused by several factors, including by exogenous agents, e.g., UV light, ionizing radiation, as well as by endogenous triggers, such as errors in DNA replication and oxidative stress. DNA replication is a central process during cell division, where accuracy is essential to maintain the genetic information intact. However, DNA replication can also be a source of deleterious events leading to DNA damage, in particular, due to aberrant replication fork structures such as breaks associated with replication fork stalling and collapse. Oxidative stress is also an important source of DNA damage, since increased levels of ROS can result in DNA oxidation, particularly in oxidized DNA bases. Depending on the source of DNA damage, a specific machinery is activated to promote successful repair. When DNA damage cannot be repaired, cells may undergo programmed cell death or accumulate mutations that drive genomic instability, which can be a trigger for tumorigenesis.

4Even though, dedicated machineries exist to cope with both oxidative stress and DNA damage, when these insults occur, they often lead to a variety of cellular and extracellular changes, which are important to ensure that cells return to their normal physiological equilibrium. One of these changes is extracellular matrix (ECM) remodeling. The ECM provides support to tissues and contributes to maintain their appropriate physiological environment. It consists of a network of extracellular glycoproteins (e.g., collagens, fibronectin, and laminins), elastins, proteoglycans, hyaluronan, and a number of other less well characterized ECM components, collectively termed matrisome (;; Figure 1A). ECM components can be directly linked to the cell surface and the underlying cytoskeleton, allowing a rapid and precise communication between the cells and the ECM. The communication between the extracellular and intracellular environments is mediated by transmembrane proteins, such as integrins, syndecans, and dystroglycan. These transmembrane proteins bind directly to specific domains of ECM molecules on the extracellular side, and to cytoskeletal proteins and different kinases on the intracellular side, thus allowing the transmission of signaling cascades. In addition, the ECM can also act as a route or reservoir for different molecules with important autocrine and paracrine functions that include cytokines, chemokines, and growth factors, which modulate a variety of different cellular processes, such as cell adhesion, migration, differentiation, proliferation, survival, and apoptosis.

5Extracellular matrix remodeling is a normal process that occurs during embryonic development, allowing the progressive formation of tissues and organs, and continues throughout adulthood in order to ensure the balance of organismal homeostasis. It is also an important player in tissue healing and repair but, when not properly regulated, it can result in the loss of normal tissue structure and the development of different pathological conditions. Examples of such conditions are (i) fibrosis, where extensive ECM deposition and scar formation hamper normal tissue and organ function, (ii) diabetes, where ECM remodeling is negatively affected in several aspects of the disease, including in the impaired healing of chronic diabetic wounds and in diabetes-associated fibrosis, and (iii) cancer, where several components involved in ECM remodeling have their specific functions hijacked by cancer cells, enabling their proliferation, epithelial-to-mesenchymal transition, and migration. Interestingly, diseases characterized by extensive ECM remodeling often involve oxidative stress and DNA damage, either as the triggers or as a consequence of their pathophysiology. This is the case of the aforementioned pathologies: fibrosis, a pathological condition marked by high levels of oxidative stress and a direct implication of DNA damage; diabetes, which is associated with high levels of blood glucose that in turn lead to increased ROS levels; and cancer in which both DNA damage and oxidative stress play an important role in disease progression. On the other hand, several diseases due to mutations in ECM components or proteins linking the ECM to the cell cytoskeleton have been shown to display increased oxidative stress and DNA damage in the affected tissues. This is particularly striking when considering the muscular dystrophies, but other examples also exist.

6It is essential to point out that ECM remodeling does not only involve the synthesis and deposition of ECM molecules, which transmit signals to cells through cell surface receptors. The action of specific proteins that degrade ECM components, including metalloproteinases (MMPs), and enzymes that shape ECM structure, such as lysyl oxidase (LOX), also play a crucial role (;;; Figure 1A). Indeed, the importance of MMPs and their inhibitors, tissue inhibitors of metalloproteinases (TIMPs), in the response to oxidative stress and DNA damage has been extensively addressed elsewhere and are beyond the scope of this review.

7Considering the body of work showing the deleterious effects of oxidative stress and DNA damage in the context of a variety of diseases on the one hand, and on the other hand, the impact of changes in the ECM for disease onset and progression, this review aims to examine the potential links between oxidative stress/DNA damage and the ECM in the context of disease development. We explore studies showing how oxidative stress and DNA damage affect the synthesis of ECM molecules, focusing on the major, and best studied, glycoproteins of the ECM, namely collagens, fibronectin, and laminins. We also examine the inverse situation, where mutations in genes encoding some of these key ECM glycoproteins or proteins that link the ECM to the cell cytoskeleton trigger an accumulation of oxidative stress and DNA damage. This dual approach aims to address to what extent oxidative stress and DNA damage are part of the molecular processes that control, and are controlled by, the ECM, and through this perspective, we may deepen our understanding of how they contribute to disease development and progression.

8Analysis of NCBI Gene Expression Omnibus database for stress responses in HeLa cells showed that several different forms of stress, including oxidative stress, lead to changes in the expression of genes encoding structural and signaling components of the ECM. Here, we review the literature reporting when oxidative stress affects the synthesis or structure of the best studied ECM glycoproteins, namely collagens, fibronectin, and laminins.

9Several studies have shown that the synthesis and/or the stability of several collagens, including collagen I, the most abundant fibrous protein of the ECM, are regulated by oxidative stress (Figure 1B). However, the exact role played by oxidative stress in the regulation of collagen synthesis is still not fully understood, and apparent contradictory results have been described. showed that oxidative stress can inhibit collagen I and IV mRNA expression, while other studies have shown that oxidative stress can also lead to increased collagen expression. This apparent discrepancy might be explained by the amount of oxidative stress in the tissue, as suggested by. In this work, human uterosacral ligament-derived fibroblasts were treated with a low concentration of H2O2, an important ROS, which led to a decrease in COL1A1 (gene codifying for collagen type I α1 chain) expression. In contrast, when these cells were exposed to a higher concentration of H2O2, their COL1A1 expression increased. This suggests that mild oxidative stress, associated with normal metabolism or small insults, may promote the reduction of collagen levels, whereas high levels of oxidative stress may favor collagen accumulation. Collagen deposition is required for wound healing, but if in excess, can also account for a tissue injury status like fibrosis. In support of this observation, several studies have shown that the presence of elevated levels of oxidative stress lead to increased collagen I and III levels contributing to myocardial fibrosis. In addition, treatment with antioxidants in the context of cardiac fibrosis has been shown to reduce collagen levels, further suggesting a direct effect between increased oxidative stress and collagen deposition. Glucose deregulation is another mechanism known to cause oxidative stress, as observed for diabetes. Kidney glomerular mesangial cells cultured with high levels of glucose show increased collagen IV synthesis. Moreover, the synthesis of collagen I and III is increased in hearts of experimental diabetic rats, a condition that was countered by antioxidant treatment. The regulation of collagen synthesis by ROS may not only occur at the transcriptional level. It is well established that the antioxidant vitamin C is a cofactor of prolyl hydroxylase and lysyl hydroxylase, required for post translational modifications of procollagen and consequently in the correct formation of the mature collagen triple helix. To corroborate the relationship between collagen levels and oxidative stress, it has recently been suggested that vitamin C, due to its role as an antioxidant, could improve healing associated with musculoskeletal diseases, by reducing the levels of ROS associated with the inflammatory response. Even though, pre-clinical studies have shown that vitamin C can reduce ROS and promote tissue healing via collagen I synthesis during bone fracture recovery and in ruptured tendons, more studies in a clinical setting are needed to assess its therapeutic benefit.

10The expression or stability of fibronectin has also been linked to oxidative stress. Fibronectin (FN1) expression has been shown to be positively regulated in the presence of different sources of oxidative stress (;;;; Figure 1B). Oxidative stress, due to high glucose levels, increases the expression of FN1 in vitro, as well as in vivo, in the context of diabetes. ROS reduction by treatment with the ROS scavenging enzyme superoxide dismutase (SOD) led to a reduction in fibronectin levels in diabetic rats and to less kidney damage. Furthermore, HOCl generated through MPO activity in neutrophils has been shown to induce fibronectin modifications, such as tyrosine chlorination and dichlorination and oxidation of different residues, which reduced cell adhesion and increased proliferation of human coronary artery smooth muscle cells in vitro. The HOCl-induced modifications of fibronectin also led to changes in the expression of ECM genes by these smooth muscle cells, including a significant upregulation of FN1 and LAMA1 and downregulation of LAMB2 expression (; Figure 1B). LAMA1 codifies the laminin α1 chain, which is normally not present in the basement membrane of smooth muscle cells, while downregulation of LAMB2 codifying the laminin β2 chain indicates that cells are prevented from maintaining β2 laminins, characteristic of mature smooth muscle basement membranes. These data indicate that cells attempt to remodel their ECM in response to their oxidant-altered fibronectin substrate and associated effects on cell adhesion and proliferation.

11Altogether, these various studies show that oxidative stress causes changes in both ECM gene expression and in ECM structure which correlate with tissue damage.

12It is well established that DNA integrity is essential for gene expression and for the correct transmission of genetic information to daughter cells during mitosis. It is not surprising that DNA damage is one of the most deleterious forms of injury inflicted on cells. Here, we will consider how DNA damage, independently of the source (e.g., ROS, radiation, and mutagens), can directly or indirectly affect the expression of genes encoding either ECM glycoproteins or proteins linking the ECM to the cell cytoskeleton.

13A central protein involved in the DNA damage response is the tumor suppressor p53, which, when activated, either induces cell cycle arrest to allow for DNA repair or, if the damage inflicted is too large, promotes, for example, apoptosis. p53 has been implicated in the regulation of FN1 expression. Depletion of p53 was shown to increase FN1 mRNA expression, leading to increased cell motility and decreased apoptosis (; Figure 1C), while treatment with the p53 activator RITA caused a decrease in fibronectin levels. These studies thus indicate p53 as a negative regulator of FN1 expression. In accordance with this notion, overexpression of wild type p53 in ovarian carcinoma cells was able to repress the activity of the FN1 promoter, while a mutant p53 failed to do so, showing that p53 can act as a transcription factor directly regulating FN1 transcription. Another indication of an effect of DNA damage on the expression of ECM components came from a study on the CDK inhibitor p21, a negative regulator of the cell cycle, which can be induced by p53 and drive cell cycle arrest. This study revealed that in p21 knockout mice, senescent cells were eliminated, and liver fibrosis was alleviated mainly via transcriptional downregulation of collagen type I α1 (COL1A1;; Figure 1C). In fact, p21 knockout reduced Col1a1 expression, prevented collagen deposition and consequently fibrosis, in mice treated with carbon tetrachloride, a fibrogenic agent known to cause DNA damage. These observations suggest that one of the actions of p21 is to increase COL1A1 expression in response to DNA damage. However, further studies are needed to assess whether these described effects of p53 and p21 on FN1 and COL1A1 expression, respectively, are cell type specific and exactly how the pathways involved control cell survival, senescence, and apoptosis.

14Integrins provide yet another link between ECM structure and DNA damage-induced apoptosis. Nevertheless, the connection between adhesion and apoptosis is not linear and integrin binding to the ECM can either promote or prevent cells, in particular cancer cells, from undergoing apoptosis. It is possible that regulation mediated by factors involved in the DNA damage response, such as p53 and p21, control the expression of integrins or ECM components and therefore modulate adhesion. Indeed, it has been shown that p73, a p53 family member that can also be activated in response to DNA damage, directly promotes transcription of ITGB4, encoding integrin β4, therefore acting as a positive regulator of cell adhesion (; Figure 1C).

15Collectively, this suggests that central DNA damage response factors might be able to directly control the expression of ECM components and integrins, hence being able to regulate cell survival and apoptosis.

16As discussed above, the notion that oxidative stress and DNA damage result in alterations in the ECM in the context of different diseases has been gaining ground. However, does the inverse occur? Does a dysfunctional ECM induce oxidative stress and DNA damage in the cells it harbors? Indeed, some observations suggest that this is the case. For example, culture of human dermal fibroblasts in fragmented collagen matrices, resulted in elevated levels of ROS in these cells compared to fibroblasts cultured in intact collagen matrices. Here, we explore the literature on diseases caused by mutations in genes encoding ECM components and proteins that link the ECM to the cytoskeleton, more specifically collagens, laminin 211, and dystrophin, to assess whether they are linked to oxidative stress and/or DNA damage.

17Collagen I is present in a variety of connective tissues. Mutations in COL1A1 and COL1A2 genes, encoding the α1 and α2 chains of collagen I, respectively, cause osteogenesis imperfecta, a disease characterized by brittle bones, short stature, and muscle weakness. Recently, a study using the mouse model of osteogenesis imperfecta, the Col1a2oim mouse, also known as oim/oim mouse, revealed that the muscles of mice homozygous for the mutation display a mitochondrial dysfunction in that they have decreased levels of electron transport chain complex IV, mitochondrial encoded cytochrome oxidase I, energy production, citrate synthase activity and respiration, and elevated levels of PGC1α, all markers of mitochondrial dysfunction (;;; Figure 2). These results suggest that mitochondrial dysfunction is an important feature of the muscles of oim/oim mice, but the exact mechanism that links collagen I deficiency to mitochondrial dysfunction remains to be determined. Moreover, excessive ROS production in the context of collagen I deficiency may also contribute to the exacerbation of osteogenesis imperfecta pathology in bone. Indeed, high ROS generation is associated with inhibition of new bone formation, suggesting that antioxidant treatment may be useful to treat diseases with bone loss.

18Collagen VI is a type of collagen mainly found in muscles, tendons, and skin. Mutations in the COL6A1, COL6A2, and COL6A3 genes, encoding chains of collagen VI, are associated with Ullrich congenital muscular dystrophy and Bethlem myopathy, a severe and mild form of collagen VI deficiency, respectively. Collagen VI deficiency has also been correlated with oxidative stress. Studies using a mouse model of collagen VI deficiency, Col6a1−/− mice, revealed elevated levels of ROS, mitochondrial dysfunction and elevated levels of monoamine oxidase A (MAO-A) (;;; Figure 2), one of the major contributors to ROS production in the mitochondria. After treatment with MAO inhibitor, muscles from Col6a1−/− mice showed a decrease in ROS levels when compared to untreated mice, highlighting the enhanced production of ROS by these enzymes when collagen VI is absent. Consistent with this, treatment with a MAO inhibitor and treatment with a mitochondrial permeability transition pore inhibitor, reduced the apoptotic phenotype and improved mitochondrial function in Col6a1−/− mice and patients with collagen VI deficiency. Altogether, these studies reinforce the idea that the absence of collagen VI drives excess ROS production in the mitochondria, potentially contributing to the observed muscle fiber damage.

19The LAMA2 gene encodes the laminin α2 chain of laminin 211. Mutations in LAMA2 lead to a congenital muscular dystrophy (LAMA2-CMD; also known as merosin-deficient congenital muscular dystrophy type 1A, MDC1A) which is characterized by muscle weakness, fibrosis, and chronic inflammation. Recently, different mouse models of LAMA2-CMD (the dy2J/dy2J and dy3K/dy3K mouse models) and biopsies from LAMA2-CMD patients were used to address the impact of oxidative stress on this condition. These studies indicate that the muscles of Lama2-deficient mice and LAMA2-CMD patients display an increase in oxidative stress both in early and more advanced stages of the disease, pointing to an important role of oxidative stress throughout disease progression. In fact, dy3K/dy3K mice show high levels of oxidative stress in quadriceps at least from post-natal day 7 and increased Gclm and Sqstm1 expression, markers of oxidative stress, at post-natal day 21. Analysis of muscles from 3-week-old dy2J/dy2J mice and muscle biopsies from LAMA2-CMD patients (age ranging between 22 days and 29 years) also confirmed the presence of elevated levels of ROS in both cases. Moreover, the treatment of dy2J/dy2J mouse quadriceps and triceps muscles with the antioxidant N-acetylcysteine (NAC) partially rescued the motor function of dy2J/dy2J mice, reduced the number of apoptotic fibers, lowered the levels of inflammation markers and prevented fibrosis. Vitamin E, another antioxidant, applied to the same mouse model reduced the number of apoptotic fibers and attenuated inflammation in quadriceps muscles, although, it did not improve motor function or reduce fibrosis. Nevertheless, both antioxidants were able to improve dy2J/dy2J skeletal muscle morphology, in particular, by normalizing the proportion of small fibers, decreasing the number of fibers with centrally located nuclei, and decreasing ROS production.

20Mitochondrial dysfunction, closely associated with elevated oxidative stress, also plays a central role in LAMA2-CMD pathology. In fact, it was observed that Lama2-deficient mice present signs of mitochondrial swollenness and downregulation of a variety of mitochondria specific genes encoding components of the electron transport chain/oxidative phosphorylation (;; Figure 2). Likewise, muscles or muscle-derived cells from LAMA2-CMD patients were described as having an abnormal membrane potential, impaired mitochondrial function and bioenergetic status and downregulation of a variety of mitochondrial related genes. Altogether, these results support the hypothesis that oxidative stress and mitochondrial dysfunction are hallmarks of LAMA2-CMD.

21Dystrophin is part of the dystrophin-glycoprotein complex which links cytoplasmic dystrophin to laminin 211 in the muscle fiber basement membrane, a link that is responsible for conferring cellular stability during skeletal muscle contractions. Mutations in the DMD gene, encoding dystrophin, can cause Duchenne muscular dystrophy (DMD), characterized by muscle weakness, inflammation, and fibrosis. Several mechanisms are known to contribute to DMD pathology, including oxidative stress. Early studies of the disease showed that DMD muscles display oxidative stress features, such as increased lipid peroxidation and increased levels of oxidative stress responsive enzymes. Moreover, in vivo and in vitro analyzes of mdx mice, the most widely used DMD model, showed an increased susceptibility to muscle injury caused by free radicals, suggesting a deficiency in oxidative stress responses in mdx mice. During the last decades, the oxidative stress hypothesis has been gathering support with several studies showing high levels of ROS in muscles of mdx mice and DMD patients. The excessive production of ROS in DMD patients and mdx mice may have multiple sources, including activation of inflammatory cells, increased activity of NADPH oxidase, mitochondrial dysfunction, and alterations in antioxidant pathways, including, for example, the elevated levels of oxidative stress sensor nuclear factor erythroid 2-related factor 2 (Nrf2) and overactivation of the nuclear factor κB (NF-κB) pathway (;; Figure 2). Using antioxidizing drugs, such as vitamin C or SOD, and treatment with the antioxidant NAC reduced the levels of ROS in cardiac and skeletal muscles of mdx mice. It also reduced the expression of markers of inflammation and fibrosis, as well as, the number of apoptotic fibers. This observed rescue of the DMD phenotype using antioxidant treatments suggests a possible role of ROS in the inflammation, fibrosis, and apoptosis characteristic of DMD.

22Elevated levels of NADPH oxidase and its regulator caveolin-3 is also a hallmark of mdx muscles. NADPH oxidase produced in skeletal muscles fibers has been shown to be a major source of stretch-induced ROS in mdx mice, since it allows the generation of the free radical superoxide. Furthermore, NAC treatment reduced caveolin-3 expression and NF-κB activation in skeletal muscles from mdx mice. Another important player in DMD pathology is mitochondrial dysfunction. Elevated levels of MAO were reported in mdx mice, as well as, reduced mitochondrial DNA copy number. Inhibition of MAO reduced tropomyosin oxidation (an oxidation marker), normalized fiber size, and reduced tissue inflammation and apoptosis.

23Reactive oxygen species pose various risks for the integrity of different macromolecules including DNA. In keeping with this notion, DMD patients present high levels of DNA damage associated with increased oxidative stress and cells derived from DMD patients are more sensitive to DNA-damaging agents than control cells. More recently, generated dystrophin-deficient human pluripotent stem cell (DMD hPSC) and showed that these cells have high levels of ROS, which leads to increased levels of ROS-induced DNA damage.

24Although, the precise mechanisms and direct or indirect actions and targets are not yet clarified, all these studies sustain the notion that mutations in the genes encoding ECM components, or associated proteins, may trigger oxidative stress and DNA damage. Importantly, some studies, especially those focusing on muscular dystrophies, have raised the question whether oxidative stress constitutes a primary or a secondary event in disease progression. In most cases, studies are conducted during the active phase of the disease, making it impossible to distinguish between these two events. Thus, in the context of mutations in genes encoding ECM components or proteins that link the ECM to cytoskeleton, it is unclear whether oxidative stress acts as a cause, a consequence or both, of disease progression. Hence, more studies are needed to fully understand how oxidative stress contributes to each phase of the disease. Taking into account the link between oxidative stress and DNA damage, it is likely that oxidative stress induced DNA damage may not be limited to DMD, but may also be a feature of the other diseases discussed here. The concomitant presence of oxidative stress and DNA damage can act as an important driving force for disease progression. Therefore, understanding the exact mechanisms behind the accumulation of oxidative stress in each phase of these diseases and determining the cellular consequences of increased ROS in every phase is crucial to design targeted therapies for these devastating conditions.

25The ECM is an intricate network of different molecules that communicate directly to the intracellular space through cell surface receptors and downstream signaling cascades. It is becoming increasingly clear that altered expression of one of these molecules may perturb this fine-tuned communication, triggering a domino effect, which severely compromises tissue integrity. In this review, we examined several examples, where increased oxidative stress and DNA damage alter the expression of genes encoding key ECM components, and conversely, where mutations in genes encoding some of the best studied ECM glycoproteins and proteins linking the ECM to the cell cytoskeleton elicit an increase in oxidative stress and DNA damage. The crosstalk between these two processes, and their major impact on tissue integrity and function, highlights the necessity to study the molecular nature of this relationship further, opening the possibility of identifying new candidate pathways as targets for therapy.

26In response to oxidative stress and DNA damage, regulation of ECM remodeling may occur at different levels, including (i) transcriptional, via modulation of different transcription factors, (ii) posttranslational, by the action of MMPs, TIMPs, and LOX, which control the degradation of ECM components, and (iii) at the network level, where changes in an ECM components or molecules that bridge the ECM to the cell cytoskeleton, may trigger a chain reaction that can compromise tissue integrity. Here, we discuss how oxidative stress and DNA damage could influence or be influenced by the expression of genes encoding ECM components or proteins linking to the cell cytoskeleton and dissect out which pathways may be required for this regulation.

27TGF-β signaling is one of the key regulators of ECM production, via the activation of Smad transcription factors, which regulate the transcription of ECM genes (Figure 3A), for example, collagens and fibronectin, but also ECM proteolytic enzymes, such as MMPs. In particular, induction of TGF-β signaling by oxidative stress has been associated with increased transcription of collagens and fibronectin. Another study, points to the activation of the TNF pathway as important for the transcription of collagen I and III genes, in response to angiotensin II-induced oxidative stress, possibly via the activation of the NF-κβ transcription factor. NF-κβ is also activated in response to DNA damage, leading to the transcription of several targets, including ECM related genes (;; Figure 3A). NRF2 is another important orchestrator of the oxidative stress response and which acts as a transcription factor. This important stress sensor has been shown to be required for the transcriptional regulation of several matrisome genes, including collagen I, III, and laminin α1 chain genes, raising the possibility that in the presence of oxidative stress, NRF2 may control ECM remodeling (Figure 3A). As mentioned above, the transcriptional regulation of ECM components may also be mediated directly or indirectly via the transcription factors p53 and p73, both playing important roles sensing oxidative stress and DNA damage (;;; Figure 3A). Whether these candidate pathways and transcription factors, known to regulate the expression of ECM genes, act in a tissue or disease specific manner, remains to be further explored.

28As detailed earlier, several studies point to oxidative stress and DNA damage being a consequence, rather than a cause, of changes in the ECM. In line with this, mutations in genes encoding ECM components or molecules that bridge the ECM to the cell cytoskeleton have been described to lead to mitochondrial dysfunction, a driving force for ROS accumulation. There are several different mechanisms that can explain why mitochondria may function as an important sensor for ECM changes, and therefore, why mitochondrial dysfunction is a hallmark of muscular dystrophies. ECM glycoproteins, including laminins and fibronectin, have been shown to promote mitochondrial function via integrin-focal adhesion kinase (FAK) signaling, which culminates in the translocation of phosphorylated STAT3 to the mitochondria (; Figure 3B). Other possible mechanisms are related to the loss of adhesion to the ECM, which changes the cellular uptake of nutrients, namely glucose. These effects together with the reduction in the activity of phosphatidyl-inositol-3-kinase (PI3K) and Akt, also associated with altered ECM composition, elicits dramatic metabolic changes including decreased activity of the pentose phosphate pathway, which represents an important source of NADPH, a powerful antioxidant molecule (; Figure 3B). In addition to signaling pathways linking ECM to mitochondria, biomechanical properties of the ECM, such as stiffness (i.e., how rigid the ECM structure is), can impact mitochondrial function (; Figure 3B). High ECM stiffness has been shown to promote mitochondrial fusion and suppress mitochondrial fission downstream of integrin signaling. The importance of ECM biomechanical properties goes beyond maintaining mitochondrial stability. Low ECM stiffness compromises the DNA damage response, making cells more sensitive to DNA damaging agents, while high ECM stiffness promotes nuclear rupture also leading to increased DNA damage (; Figure 3B). Altogether, these studies suggest that countering mitochondrial dysfunction, and possibly preventing DNA damage, may be a promising strategy to revert the pathology caused by some mutations in genes encoding ECM components, their receptors or molecules that bridge ECM receptors to the cell cytoskeleton.

29In conclusion, future studies are needed to characterize all the players and intermediates involved in the link between oxidative stress/DNA damage and ECM remodeling. Only with this increased knowledge can new candidate pathways and targets be identified. This will be instrumental to establish novel lines of therapy, for diseases where ROS and DNA damage drive ECM remodeling, or where mutations in ECM-related genes elicit an over-accumulation of ROS and DNA damage.

30SGM and ARC performed the literature review and wrote the manuscript. RZ and ST contributed with active discussion and revision. All authors contributed to the article and approved the submitted version.

31The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

32All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.