Dr. Matthew Regulski, DPM
Chronic wounds are associated with very unique intracellular and extracellular/ ECM interactions both in cellular senescence and in growth factor deficiency but also with receptor dysfunction.
Integrins are the main cellular receptors that mediate cell-ECM interaction and cell-cell mediated interactions. They anchor cells to their surrounding environment and transduce a variety of signals that influence cell behavior. In chronic wounds integrins can be up regulated or down regulated by chronic inflammation in the form of excessive ROS that leads to oxidative stress. There is a deficiency of α5-β1 integrin which is necessary to adhere keratinocytes to fibronectin to stimulate and enhance their migration and proliferation. Also,we see the up-regulation of αV-β6 integrin, which leads to premature and excessive secretion of TGFbeta causing premature senescence of fibroblasts and myofibroblasts. Significant production CCN1 binding to α6-β1 integrin leads to the production of excessive ROS, DNA damage and activation of the cyclin dependent kinase inhibitor p53, the master regulator of senescence. This causes irreversible growth arrest and the senescent phenotype ensues. This receptor dysfunction results in increased senescent cells including fibroblasts, myofibroblasts, and keratinocytes- this will produce an ECM that is corrupt and a wound bed that is inflamed, corrosive and un-responsive. By understanding this process of cellular senescence and thus growth arrest, we should intervene earlier, when the biofilm has been reduced and thus chronic inflammation extinguished, with cell based therapies that will re-capitulate the proliferative phase in order to repair and regenerate before chronicity becomes terminal.
What Factors Can Affect Wound Healing?
In hostile environments that are rife with microbial invaders, human respond to wounding and tissue injury with a vigorous inflammatory response coupled to the rapid synthesis and deposition of the extra cellular matrix, thereby maintaining tissue integrity and providing defense against microbes while the wounded tissue is being repaired and remodeled.
In virtually all organ systems, wound healing occurs similarly in three overlapping but distinct phases; inflammation, ECM deposition and tissue formation, and tissue remodeling (2,3,4). Each of these steps must be tightly regulated for optimal wound healing.
However, excessive ECM deposition may occur in wound repair, particularly in association with chronic injury and inflammation (4,5,6). When excessive non-functional ECM replaces functioning parenchyma, the resulting fibrosis, scarring, and loss of tissue function may lead to severe consequences. Examples being fibrotic scarring in the liver due to viral infections, in the lung from obstructive pulmonary disease, and in the heart following myocardial infarction which can lead to organ failure and death (2).
These types of exaggerated, dysregulated wound healing adversely affect a large number of people worldwide and this can be a tremendous burden on public health.
The vast majority of chronic wounds that fail to heal in a timely fashion fall into one of three categories: pressure ulcers, diabetic ulcers, and venous ulcers (2,7). Although these wounds all have different etiologies, chronic wound development is most often associated with a background pathophysiologic sequence of an ischemia-reperfusion injury with resultant prothrombotic and procoagulant phenotype, biofilm formation which results in a prolonged inflammatory response (8,9,10).
In many cases, healing does not occur despite adequate standard of care (7,10). Failure to re-epithelialize can be a consequence of a number of factors, including prolonged inflammation, an imbalance of regulatory growth factors and cytokines, defective extracellular matrix that fails to support keratinocyte migration, modified fibroblast function, and defective capillary function leading to inadequate tissue oxygenation (11). The ECM of these wounds has been referred to as corrupt or corrosive (11,12), characterized by chronic wound fibroblasts that are unresponsive to growth factors, cytokines, and other signals (13), containing high levels of matrix metalloproteinases (MMPs), neutrophil elastase, serine proteases, cathepsins and lacking of receptors such as integrins which are quite necessary for fibronectin binding and keratinocyte migration (14).
There is vast evidence that has shown chronic wound healing may be related to increased or reduced integrin receptors, production of senescent cells that are unresponsive to growth factors and cytokines, and with changes relating to cell signal or receptor dysfunction (7,11,13-21). Integrins coordinate and translate these effector signals that control cell signaling, proliferation, ECM maturation, and cellular spreading. This is quite germane to the chronic wound state where specific changes in integrin type and receptor sensitivity may trigger the changes in cellular phenotype that contribute to this chronicity (2).
Integrins provide a mechanical connection between matrix components and the cytoskeleton and transduce an astonishing variety of signals, either alone or in collaboration with growth factor receptors or other protein signals (2, 22-26).
They were originally discovered as receptors that anchor cells to their surroundings by concomitantly binding to the cytoskeleton and the ECM. Also, integrins were shown to activate cellular signalling pathways and, in this way, to contribute to the decisions of both cell behavior and fate (22,27). Direct binding to growth factors and regulation of endocytosis and trafficking of growth factors makes integrins even more multifunctional than previously appreciated (17,22).
How Does Senescence Corrode Wound Healing
Traditionally senescent in culture is associated with telomere shorting during repeated cell divisions eventually leading to cell cycle arrest, which is known as replicative senescence (28). In the chronic wound, cellular senescence to a degree is associated with telomere length, but rather this phenotypic cell change is initiated by oxidative stress, ER stress, carbonyl and nitrosative stress, activated oncogenes, repressor proteins, and cyclin-dependent kinase inhibitors (20). In the chronic wound environment, ROS attack DNA, (causing an accumulation of lipofuscin), which is un-degradable by the cell, thus stimulating the DNA Damage Response (DDR) and then DNA damage-induced cell cycle arrest (29).
Fibroblast senescence in chronic wounds is also related to chronic inflammation and molecular dysfunctions(20). When exposed to chronic wound fluid, normal fibroblasts in culture appear to switch to a senescent mode, showing changes in morphology and increase in pro-inflammatory protein synthesis, which is consistent with senescent associated secretory phenotype (SASP) (20). Chronic wound fluid rapidly degrades integrin receptors, exogenous growth factors, decreases the production of cyclin, D1, phosphorylated retinoblastoma protein RB, and increases p21 (30).
The main cell type that contributes to the synthesis and deposition of ECM in healing wounds is the myofibroblast, which expresses alpha smooth muscle actin and promotes wound contraction (6). Myofibroblast can be sourced from a variety of cell lines including differentiation of activated resident fibroblast and recruited fibrocytes, and epithelial and endothelial-mesenchymal transitions of epithelial and endothelial cells respectively (6,7).
Activated myofibroblast will proliferate and initially promote wound repair by producing ECM components, fibrosis may result when wound healing becomes chronic or if the ECM producing activity of myofibroblast continues unchecked.The conversion of fibroblast to myofibroblast plays a significant role in the stimulation of wound contraction and healing by producing ECM constituents to form granulation tissue. Therefore, this system is kept in balance by myofibroblast being driven into senescence at later stages of wound healing, thereby converting these ECM-producing cells into ECM-degrading cells, thus balancing and limiting fibrosis of the wound (28).
Triggering of Myofibroblast Senescence is a Necessary Step in the Genesis of Chronicity
In skin wound healing, myofibroblast senescence is triggered by the dynamically expressed matricellular protein CCN1 (also known as CYR61) through integrin signalling (28). CCN1 is an angiogenic matrix cellular protein of the CCN family. CCN is the acronym of the first three members: CYR61, CTGF, and Nov. CCN1 is normally expressed at a low level in most tissues but becomes highly expressed because of bacterial, viral infections (28,31) or in tissue repair (28,32,33).
The physiology of CCN1 will induce fibroblast senescence through its direct binding to the integrin alpha 6 beta1 and cell-surface heparin sulfate proteoglycans (HSPG) (28). This triggers formation and accumulation of ROS, DNA damage, and p53 activation resulting in these cells being driven into senescence and thus the production of inflammatory proteins that will stimulate the degradation of the ECM.
Through integrin signaling, myofibroblast senescence brings about increased expression of a multitude of pro-inflammatory cytokines/chemokines (e.g. IL-1, IL-6, IL-8, MCP-2, MCP-4, MIP-1a, MIP-3a,) and ECM-degrading enzymes MMPs, which will down regulate expression of certain ECM components (e.g. collagen) (28). Therefore, senescent myofibroblast will accumulate as part of this normal process of tissue repair and function to limit the extent of fibrogenesis associated with wound healing (28, 34).
Also, premature cellular senescence is stimulated by events often present in the evolution of a chronic wound (infection particularly biofilm, corrosive CWF). This results in CCN1 binding to integrin alpha 6- beta 1, ROS stimulation, fibroblast, and then myofibroblast senescence, increased inflammation, and degradation of the ECM.
Therefore, during skin wound healing, recruited fibroblast and differentiated myofibroblasts proliferate and deposit ECM to form the granulation tissue. Myofibroblast will then be driven into senescence at later stages of wound healing, where they cease to proliferate and up regulate the expression of matrix degrading enzymes (MMP2, MMP3, MMP9), concomitant with down regulation of collagen and TGF-beta, thereby exerting anti-fibrotic effects (35).
At this point you can see that the control of fibrogenesis during wound healing is efficient and balanced – the very cells that synthesize ECM in wound healing, the myofibroblasts, are themselves converted to matrix-degrading senescent cells to produce a self-limiting effect. These senescent cells may also promote tissue remodeling and clearance of the myofibroblast during wound maturation (35, 36).
In a clinical presentation, chronic wound bed changes have direct implications on healing. The length of venous leg ulcerations can have a direct correlation with ultimate healing (11). It is apparent that the longer a wound remains open and in the inflammatory phase, the more cellular defects arise and accumulate, therefore becoming less responsive to treatment. Premature senescence with the production of the SASP leads to ongoing ECM degradation. This has been shown to be a contributing factor to chronic wound prevalence (11, 20, 28). Accumulation of greater than 15 percent of senescent fibroblasts has been described as a threshold beyond which wounds become very hard to heal (11). Therefore evidence for this declares that there is a positive relationship between higher levels of fibroblasts with a senescent phenotype that have bee identified in VLU and DFU, will portend for a poor prognosis (11, 37).
While much attention has focused on fibroblast senescence in the chronic wound, other cells have been shown to be affected by cellular senescence formation. Many of the same reasons that cause abnormal changes in the fibroblast also affect keratinocytes and endothelial cells (20). Increases in proteolytic enzymes and chronic inflammation can lead to a gradual loss of endothelial cell function, mimicking replicative senescence in fibroblasts and keratinocytes (20).
Therefore, induction of fibroblast senescence may have a direct effect on the induction of senescence in endothelial cells and keratinocytes. This has been termed “the bystander effect”. This is due to the formation of the SASP that occurs in senescent cells. This phenomena is also found within the cells of our immune system . Macrophages also have been shown to produce significantly less vascular endothelial growth factor (VEGF) when isolated from chronic wound tissue with increased senescence compared to young controls (20).
CCN1, Contributor to Senescence and Chronic Wound Healing
CCN1 protein can directly induce fibroblast senescence, both as a cellular factor and as an immobilized cell adhesion substrate (35).
CCN1 induces fibroblast senescence through its direct binding to integrin alpha 6-beta 1, and cell surface heparin sulfate proteoglycans (HSPG), thereby activating RAC1 and the RAC1-dependent NADPH oxidase 1 to trigger a robust and sustained accumulation of reactive oxygen species (ROS).
Consequently, CCN1 induces a DNA damage response and p53 activation, and triggers the ROS-dependent activation of p38 MAPK, and ERK, which in turn activate p16 INK4A/pRb pathway to induce senescence (35).
Both p53 and p16 INK4A/pRb pathway also contributes to CCN1 induced senescence (38, 39). Cell adhesion to CCN1 induces a much higher and more sustained level of ROS than cell adhesion to other ECM proteins, such as collagen, fibronectin, and laminin, which do not induce senescence. The accumulation of substantial level of ROS sustained for at least 10 hours appears necessary for efficient induction of senescence in fibroblasts (35).
Apart from cellular senescence induction, CCN1 can up regulate plasminogen activator inhibitor-1 (PAI-1) possibly through the activation of p53 (20, 28, 40). Plasminogen activators activate plasminogen to produce plasmin. Plasmin participates in the breakdown of other glycoproteins in the ECM and the activation of MMP’s (28). Chronic wounds are associated with elevated levels of plasmin, collagenases, and other degradative enzymes. Overexpression of PAI-1 is sufficient to drive fibroblast into senescence (20, 28).
As you can see it would appear that there is a dichotomy occurring in the ECM of a chronic wound. Fibroblasts secrete factors that will stimulate matrix degradation, yet they also produce matrix-promoting factors (TGF-beta 1 and PAI-1). PAI-1 and CCN1 may then stimulate the senescent myofibroblast phenotype but will result in ratios of PAI-1 and TGF-beta1 to plasmin that will lead to a degraded and defective ECM (41).
The association between fibroblast senescence and ECM degradation appears to be supported by these changing ratios that will shift the balance towards degradation of the ECM as senescence is propagated (41).
The secretory function of fibroblasts may decrease with senescence, although Herrick et al (42) have shown that chronic wound fibroblast show no decrease in matrix secretion – thus the observed degradation of ECM appears to be possibly related to increases in matrix remodeling enzymes and their proteolytic activity.
Fibroblast Unresponsivness Can Lead to ECM Degradation, The How and Why?
Another reason for the continuance of ECM degradation is its unresponsiveness to the stimulatory action of a multitude of growth factors and cytokines, including TGF-beta, PDGF, and EGF, as well as bFGF (42). Research has shown that VLU fibroblasts have decreased TGF-beta type II receptor expression (13). This was accompanied by failure of ulcer fibroblasts to phosphorylate Smad2, Smad3, and p42/44 mitogen-activated protein kinase, and was associated with a slower proliferative rate in response to TGF-beta (13).
This continues to highlight the possibility that non-healing chronic wounds are associated and related to a decrease in receptor expression and a failure in specific signal transduction pathways (2). Much of the corrosive ECM that is associated with cellular senescence relates to the increased presence of MMPs. In acute wounds, there is a balance between protease activity and ECM deposition encouraged by tissue inhibitors of metalloproteases (TIMPs). The ratio of MMP-9/TIMP-1 in chronic wounds is an important healing measurement parameter (20, 43).
Chronic wound fibroblasts will have decreased capacity to react to a multitude of growth factors and other signaling proteins. This decreased response to bFGF, EGF and PDGF does not appear to be related to a decrease in receptor quantity, but rather a dysfunction in intracellular signalling (42).
The more reasonable approach to changing the degradative ECM wound milieu is wound bed preparation, including sharp debridement to stimulate vascularization and decrease bacterial burden thru reduction of the wound biofilm (9,10,11). Also by removing the senescent cell burden and their SASP this will contribute greatly to this change. Only then can exogenously applied growth factors, cytokines, various stimulatory proteins and biologics can then be expected to exert their stimulatory effect on the surrounding healthy tissue and promote proper and organized wound healing.
Proteolysis by MMPs exposes specific amino acid binding sites on ECM proteins, such as fibronectin, fibrogen, osteopontin, tenascin, and vitronectin. These specific sequences are referred to as the RGD sequence (arginine, glycine, aspartic acid). This is a very well studied and elucidated binding motif that is quite necessary and incredibly critical for facilitating cell surface integrin recognition and attachment of the cell to the ECM protein (44). This then will initiate growth factor and paracrine signaling (44). Chronic wounds are associated with increased inflammation, and copious corrosive wound exudate, with increased proteolysis and degradation of the ECM, which will result in an abnormal wound matrix that induces less growth factor action because of the diminished integrin binding (9, 10, 11, 15, 16, 17, 24, 25, 26, 45, 27, 46, 47).
Aside from increased protease activity, (particularly MMP’s, gelatinases, collagenases, stromelysins), chronic wound edge keratocytes also display growth factor – ECM receptor dysfunction, which fails to convert them into a migratory phenotype because their intregrin receptors which are necessary for their locomotion and therefore their movement, are degraded by the chronic inflammation (2, 48). In chronic wounds, VEGF wound fluid levels were also significantly higher in non-healing VLU ‘s than in normal wounds despite excessive degradation of VGEF by plasmin (48, 49).
However, the VGEF inhibitor, soluble VEGFR-1, was increased fourfold in wound fluids of chronic VLU’s compared with wound fluids from acute excisional wounds on the lower leg (48).
Thus, it is more than likely that abnormal ECM composition which fails to activate adhesion receptors on fibroblasts, thus preventing their binding to the ECM, which is a prerequisite for maximal growth factor stimulation, responsiveness, migration, and proliferation (48, 15, 18).
Wound chronicity is a timely process. The typical biochemical abnormalities described in this review occur over time and the resulting wound milieu often renders the wound non-responsive to most treatments. We must find a way to have early focus and target the Biofilm mass causing subsequent chronic inflammation that leads to cellular senescence and receptor dysfunction in the wound milieu so that we can change the course of this chronicity. This will be very relevant for patients that have chronic wounds with multiple co-morbidities that slow the healing process. Patients with diabetic, venous, or pressure ulcers often do not respond adequately to several weeks of standard care due to multiple pathogens, biochemical and molecular dysfunctions.
Because of the consequences of the biofilm mass, defective degraded ECM, cellular senescence with growth factor/cytokine/chemokine dysfunctional signalling, we must find strategies that aim to repair and regenerate the entire wound healing paradigm so that it will be beneficial to these high risk groups to reduce their morbidity and mortality.
As it has been noted previously, topically applied growth factors have been tested extensively in chronic wounds with very mixed results (50, 51, 52, 53). It has been postulated that topical application of these agents in the corrosive environment of the chronic wound bed are rapidly degraded by these destructive proteases (9, 54).
Therefore, a plausible explanation for the relative lack of success of growth factors in the treatment of chronic wounds may reside in the phenotypic abnormalities of wound cells and their relative unresponsiveness to stimulatory signals.
The keys to understanding the pathogenesis of chronic wounds in all age-related wound defects, as well as age-related diseases and tissue dysfunction, is identifying cellular receptors that mediate the responses of the epidermis to provisional wound matrix and determining how these changes in the receptors contribute to impaired wound healing through cellular senescence. Integrins are the major cell surface receptors for cell adhesion and migration (15, 16, 17), and many cell lines, including epidermal keratinocytes, fibroblasts, and endothelial cells express several integrins that bind ECM proteins in this provisional wound matrix (28, 29, 50).
As we can see, chronic wound healing is a complicated and fascinating series of molecular pertubations, which occur at the most basic of structural, biochemical, and molecular levels. Cellular senescence is a state of irreversible growth arrest but with metabolically active cells that secrete pro-inflammatory cytokines, proteases, chemokines growth factors, and many other proteins that can have a negative effect upon wound healing. This inflammatory repertoire is termed the senescence associated secretory phenotype (SASP). In this review, I have shown that cellular senescence can be triggered by receptor dysfunction, oxidative stress, chronic inflammation, DNA damage, as well as oncogene activation and telomere shortening. Cellular senescence underlies a vast majority of age-related chronic diseases including diabetes, chronic wound healing, and atherosclerosis. We encounter these pathologies every day in our wound patients.
Therefore, would it not make sense that in order to repair dysfunctional and degraded ECM, paracrine protein dysfunction, and non –proliferative cellular dysfunction, should we be looking at adding robust, young, healthy cell lines into this wound milieu to extinguish the chronic inflammation, and replace the senescent and thus non-proliferative cells? Applying young cellular constructs that are not fettered or molested by age, senescence or chronic inflammatory disease processes, might provide a better platform or scaffolding for enhanced proliferation and epithelialization. As physicians, clinicians, scientists, we must learn and understand these dysfunctions in order to avail ourselves of the proper therapies and paradigms to correct these abnormalities. I believe by adding robust cellular constructs that will coordinate the proper cell signalling pathways, secretory pathways, proper scaffolding arrangements and proper protein profiles, we will be able to change the chronic wound environment in order to re-establish a proliferative and proper wound healing trajectory.
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Dr. Matthew Regulski, DPM
Dr. Regulski is a Graduate of Temple University School of Medicine, where he graduated Summa Cum Laude. He performed his residency at the Main Line Health System in Philadelphia, PA, and is currently the Medical Director of the Wound Care Institute of Ocean County, LLC. Dr. Regulski is also Co-Director of The Center for Wound Healing & Hyperbaric Medicine at Community Medical Center and is involved at the Wound Care Center at the Kimball Institute. He has been the principal investigator for multiple clinical trials for wound healing, diabetic foot, and venous leg ulcers, and has authored several peer-reviewed articles for the treatment of chronic wound healing and limb-salvage surgery.
Dr. Regulski is currently on the Board of Directors of the Federation of International Podiatry and is the Scientific Chairman for the Federation of International Podiatry. He is also the Communications Director for the Foot Working Group of the American Diabetes Association. Dr. Regulski is a wound care certified physician and fellow of both the Academy of Physicians in Wound Healing and the American Professional Wound Care Association. He is also a National Delegate for the Association for the Advancement of Wound Care and is a member of the Wound Healing Society. An International and National lecturer of wound healing and limb-salvage surgery, Dr. Regulski has multiple certifications in wound healing, diabetic wounds, and limb-salvage surgery.
Dr. Matthew Regulski is a fellow faculty member at the Royal College of Physicians and Surgeons of Glasgow, Scotland.