Oxidative Stress and “Senescent” Fibroblasts in Non-Healing Wounds as Potential Therapeutic Targets
2008; Elsevier BV; Volume: 128; Issue: 10 Linguagem: Inglês
10.1038/jid.2008.257
ISSN1523-1747
Autores Tópico(s)Pressure Ulcer Prevention and Management
ResumoIn chronic wounds, fibroblast dysfunctions, such as increased apoptosis, premature senescence, senescence-like phenotype, or poor growth response in the absence of senescence markers, have been reported. Some of these differential dysfunctions may be secondary to differences in patient age or sex, ulcer size or duration, edge versus base sampling, or culture technique. Nevertheless, the entire spectrum of fibroblast dysfunction may exist and be secondary to, or a response to, different amounts of oxidative stress. In chronic wounds, fibroblast dysfunctions, such as increased apoptosis, premature senescence, senescence-like phenotype, or poor growth response in the absence of senescence markers, have been reported. Some of these differential dysfunctions may be secondary to differences in patient age or sex, ulcer size or duration, edge versus base sampling, or culture technique. Nevertheless, the entire spectrum of fibroblast dysfunction may exist and be secondary to, or a response to, different amounts of oxidative stress. In their article entitled "Fibroblast dysfunction is a key factor in the non-healing of chronic venous leg ulcers," Wall et al. (Wall et al., 2008Wall I.B. Moseley R. Baird D.M. Kipling D. Giles P. Laffafian I. et al.Fibroblast dysfunction is a key factor in the non-healing of chronic venous leg ulcers.J Invest Dermatol. 2008; 128: 2526-2540Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, this issue) demonstrate that a telomere-independent fibroblast abnormality occurs in chronic venous leg ulcers and posit that oxidative stress underlies this dysfunctional behavior. Although it is dysfunctional, the authors have clearly emphasized both here and in a previous publication (Stephens et al., 2003Stephens P. Cook H. Hilton J. Jones C.J. Haughton M.F. Wyllie F.S. et al.An analysis of replicative senescence in dermal fibroblasts derived from chronic leg wounds predicts that telomerase therapy would fail to reverse their disease-specific cellular and proteolytic phenotype.Exp Cell Res. 2003; 283: 22-35Crossref PubMed Scopus (33) Google Scholar) that the chronic wound fibroblast phenotype observed by them does not represent the replicative senescence-like phenotype as has been reported for chronic wound fibroblasts by a number of other investigators (Agren et al., 1999Agren M.S. Steenfos H.H. Dabelsteen S. Hansen J.B. Dabelsteen E. Proliferation and mitogenic response to PDGF-BB of fibroblasts isolated from chronic venous leg ulcers is ulcer-age dependent.J Invest Dermatol. 1999; 112: 463-469Crossref PubMed Scopus (159) Google Scholar; Mendez et al., 1998Mendez M.V. Stanley A. Park H.Y. Shon K. Phillips T. Menzoian J.O. 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Cultured pressure ulcer fibroblasts show replicative senescence with elevated production of plasmin, plasminogen activator inhibitor-1, and transforming growth factor-beta1.Wound Repair Regen. 2005; 13: 76-83Crossref PubMed Scopus (63) Google Scholar). However, Stephens's group isolated their chronic wound fibroblasts from biopsy specimens that come from the ulcer base while all other groups harvested chronic wound fibroblasts from the ulcer edge. Given the heterogeneity in growth response and other functions among human fibroblasts derived from either papillary or reticular dermis (Harper and Grove, 1979Harper R.A. Grove G. Human skin fibroblasts derived from papillary and reticular dermis: differences in growth potential in vitro.Science. 1979; 204: 526-527Crossref PubMed Scopus (223) Google Scholar; Sorrell et al., 2008Sorrell J.M. Baber M.A. Caplan A.I. Human dermal fibroblast subpopulations; differential interactions with vascular endothelial cells in coculture: nonsoluble factors in the extracellular matrix influence interactions.Wound Repair Regen. 2008; 16: 300-309Crossref PubMed Scopus (52) Google Scholar; Sorrell and Caplan, 2004Sorrell J.M. Caplan A.I. Fibroblast heterogeneity: more than skin deep.J Cell Sci. 2004; 117: 667-675Crossref PubMed Scopus (360) Google Scholar), it is not surprising that Stephens's group has identified a different chronic wound fibroblast phenotype from the ulcer bed than the previously described fibroblast phenotype from the ulcer margin. Nevertheless, the fibroblast phenotypes reported by all groups of investigators are consistent with the variety of functional changes that can be induced by oxidative stress on fibroblasts (Wlaschek and Scharffetter-Kochanek, 2005Wlaschek M. Scharffetter-Kochanek K. Oxidative stress in chronic venous leg ulcers.Wound Repair Regen. 2005; 13: 452-461Crossref PubMed Scopus (173) Google Scholar). As pointed out by Wall et al., 2008Wall I.B. Moseley R. Baird D.M. Kipling D. Giles P. Laffafian I. et al.Fibroblast dysfunction is a key factor in the non-healing of chronic venous leg ulcers.J Invest Dermatol. 2008; 128: 2526-2540Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, the oxidative effects on fibroblast phenotype and function depend on the level and length of exposure to reactive oxygen species (ROS). Low-level, chronic exposure to ROS accelerates telomere shortening (von Zglinicki, 2002von Zglinicki T. Oxidative stress shortens telomeres.Trends Biochem Sci. 2002; 27: 339-344Abstract Full Text Full Text PDF PubMed Scopus (1825) Google Scholar), whereas high levels of ROS induce telomere-independent premature senescence (Song et al., 2005Song Y.S. Lee B.Y. Hwang E.S. Dinstinct ROS and biochemical profiles in cells undergoing DNA damage-induced senescence and apoptosis.Mech Ageing Dev. 2005; 126: 580-590Crossref PubMed Scopus (70) Google Scholar). Another important aspect of the cell "senescence" response to oxidative stress is cell age; aged human dermal fibroblasts demonstrate increased entrance into a senescent state compared with their younger counterparts when exposed to oxidative stress (Gurjala et al., 2005Gurjala A.N. Liu W.R. Mogford J.E. Procaccini P.S. Mustoe T.A. Age-dependent response of primary human dermal fibroblasts to oxidative stress: cell survival, pro-survival kinases, and entrance into cellular senescence.Wound Repair Regen. 2005; 13: 565-575Crossref PubMed Scopus (35) Google Scholar). Furthermore, depending on the physiologic state of the cell, oxidative stress can induce apoptosis rather than senescence (Chen et al., 2000Chen Q.M. Liu J. Merrett J.B. Apoptosis or senescence-like growth arrest: influence of cell-cycle position, p53, p21 and bax in H2O2 response of normal human fibroblasts.Biochem J. 2000; 347: 543-551Crossref PubMed Scopus (223) Google Scholar). Because chronic wounds demonstrate findings that indicate the presence of increased ROS (Wlaschek and Scharffetter-Kochanek, 2005Wlaschek M. Scharffetter-Kochanek K. Oxidative stress in chronic venous leg ulcers.Wound Repair Regen. 2005; 13: 452-461Crossref PubMed Scopus (173) Google Scholar), it is certainly possible that the dysfunctional fibroblast phenotypes reported at ulcer margins and the ulcer base are both attributable to oxidative stress. ROS can be generated by a variety of sources, including H2O2 during purine catabolism (xanthine and hypoxanthine oxidase) and superoxide (O2−) during mitochondrial metabolism in all cells or the respiratory burst associated with microbiocidal activity in phagocytes (Wlaschek and Scharffetter-Kochanek, 2005Wlaschek M. Scharffetter-Kochanek K. Oxidative stress in chronic venous leg ulcers.Wound Repair Regen. 2005; 13: 452-461Crossref PubMed Scopus (173) Google Scholar; Wulf, 2002Wulf D. Free radicals in regulation of physiological functions.Physiol Rev. 2002; 82: 47-95Crossref PubMed Scopus (7488) Google Scholar). Importantly, according to Wall et al., 2008Wall I.B. Moseley R. Baird D.M. Kipling D. Giles P. Laffafian I. et al.Fibroblast dysfunction is a key factor in the non-healing of chronic venous leg ulcers.J Invest Dermatol. 2008; 128: 2526-2540Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, chronic wound fibroblasts generate elevated levels of ROS compared with normal dermal fibroblasts, setting up a positive feedback system for wound degeneration. To understand the mechanisms of ROS-induced alteration and damage to cells, it is important to know that hydroxyl (OH•) and peroxy radicals (OOH•) are generated from O2− and H2O2 via superoxide dismutase (SOD) and the Fenton reaction, respectively (Wulf, 2002Wulf D. Free radicals in regulation of physiological functions.Physiol Rev. 2002; 82: 47-95Crossref PubMed Scopus (7488) Google Scholar). SOD generates one oxygen molecule and one hydrogen peroxide molecule (H2O2) from two superoxide molecules (O2−) by the following reactions, where M is a transition metal that oscillates between its oxidized and reduced state: M(n+1)+−SOD+O2−→Mn+−SOD+O2Mn+−SOD+O2−+2H+→M(n+1)+−SOD+H2O2 The Fenton reaction converts H2O2 to hydroxyl and peroxy radicals via the following reactions: Fe2++H2O2→Fe3++OH⋅+OH−Fe3++H2O2→Fe2++OOH⋅+OH+ In the Fenton reaction, the transition metal is iron, which oscillates between its reduced and oxidized states. Hydrogen peroxide (H2O2) is relatively long-lived in the cell until it is reduced by catalase in the perioxisome or by glutathione peroxidase in the cytoplasm by the following reactions, respectively, where GSH is reduced glutathione and GSSG is oxidized glutathione: 2H2O2→2H2O+O2(Catalasemediated)2GSH+H2O2→GSSG+2H2O(glutathioneperoxidasemediated) Because of its relatively long life span, H2O2 can act as an effector in signal transduction pathways (Wulf, 2002Wulf D. Free radicals in regulation of physiological functions.Physiol Rev. 2002; 82: 47-95Crossref PubMed Scopus (7488) Google Scholar). Examples of genes induced by ROS (probably primarily H2O2) include peroxyredoxin I (a thioredoxin peroxidase that is protective against oxidative stress and apoptosis), heme oxygenase-1 (HO-1), and the cystine transporter xc−. The redox control of the HO-1 gene is one of the best-studied models of redox regulation. For example, in dermal fibroblasts HO-1 induction may serve as an inducible defense pathway to remove oxidant-liberated heme, which is extremely toxic when free (Kumar and Bandyopadhyay, 2005Kumar S. Bandyopadhyay U. Free heme toxicity and its detoxification systems.Toxicol Lett. 2005; 175: 175-188Crossref Scopus (603) Google Scholar). Interestingly, heme toxicity probably is attributable in large part to its iron core, which, when released in great abundance, overwhelms the iron-binding capacity in blood and tissue (i.e., ferritin) and therefore can act as a catalyst in the Fenton reaction to generate more ROS. Because HO-1 mRNA is inducible in many tissues and its expression is relatively stable, it is a useful marker for cellular oxidative stress. As previously stated, ROS can induce cell apoptosis, and this appears to be mediated by H2O2 through its activation of the c-Jun N-terminal kinase (JNK) pathway, a subgroup of the mitogen-activated protein kinase family (Shen and Liu, 2006Shen H.-M. Liu Z. JNK signaling pathway is a key modulator in cell death mediated by reactive oxygen and nitrogen species.Free Radical Biol Med. 2006; 40: 928-939Crossref PubMed Scopus (510) Google Scholar). In general, JNK is activated by environmental stresses, including osmotic shock, UV radiation, heat shock, oxidative stress, and chemotherapeutic agents, as well as the cell death ligands FasL and tumor necrosis factor-α and proinflammatory cytokines such as interleukin-1. Among these inducers of JNK, however, oxidative stress seems particularly important. Although activated JNK induces a large family of genes, its pro-apoptotic activity is more direct (Shen and Liu, 2006Shen H.-M. Liu Z. JNK signaling pathway is a key modulator in cell death mediated by reactive oxygen and nitrogen species.Free Radical Biol Med. 2006; 40: 928-939Crossref PubMed Scopus (510) Google Scholar). In stressed cells activated JNK translocates to the mitochondria, where it phosphorylates and inhibits the anti-apoptotic factor Bcl-2 and phosphorylates and activates the pro-apoptotic agents Bax, Bim, and Bmf. The alteration in balance between pro- and anti-apoptotic factor activities results in the release of cytochrome c from the mitochondria into the cytoplasm. Subsequently, free cytoplasmic cytochrome c activates the downstream effectors of the intrinsic (mitochondrial) apoptosis pathway. Some years ago, it was thought that H2O2 was the primary positive effector of transcription factor NF-κB, a central regulator of immunity, inflammation, and cell survival, and that many of the negative effects of ROS were mediated through NF-κB, including expression of inflammatory cytokines such as IL-1, IL-6, IL-8, and tumor necrosis factor-α. However, new evidence demonstrates that NF-κB exerts negative control on ROS and JNK activities and may prevent apoptosis that ROS would otherwise induce through JNK (Bubici et al., 2006Bubici C. Papa S. Dean K. Franzoso G. Mutual cross-talk between reactive oxygen species and nuclear factor-kappa B: molecular basis and biological significance.Oncogene. 2006; 25: 6731-6748Crossref PubMed Scopus (339) Google Scholar).Oxidative stress affects fibroblast phenotype. Oxidative stress affects fibroblast phenotype. Not all signal transduction proteins are positively regulated by H2O2. Interestingly, hypoxia-inducible factor 1 (HIF-1) is negatively regulated by this ROS. HIF-1α and HIF-1β genes are constitutively expressed, but under normoxic conditions HIF-1α is rapidly degraded by proteasomes in an ROS-dependent manner (Wulf, 2002Wulf D. Free radicals in regulation of physiological functions.Physiol Rev. 2002; 82: 47-95Crossref PubMed Scopus (7488) Google Scholar). Hypoxia decreases the ROS-mediated degradation of HIF-1α and thereby enhances the formation of the heterodimeric complex leading to HIF-1-dependent gene regulation. Many of the gene targets of HIF-1 are important positive regulators of wound healing. In particular, HIF-1 increases the transcription of key factors for blood flow, including vascular endothelial growth factor, HO-1, and both endothelial and inducible nitric oxide synthase (Hellwig-Bürgel et al., 2005Hellwig-Bürgel T. Stiehl D.P. Wagner A.E. Metzen E. Jelkmann W. Hypoxia-inducible factor-1 (HIF-1): a novel transcription factor in immune reactions.J Interferon Cytokine Res. 2005; 25: 297-310Crossref PubMed Scopus (223) Google Scholar). Thus, in the presence of oxidative stress these HIF-1-inducible genes will remain repressed. Parenthetically, it should be pointed out that nitric oxide, a reactive nitrogen species, is also a potent mediator of signal transduction systems, but a discussion of this important topic is beyond the scope of this Commentary. Hydroxyl and peroxy radicals, in contrast to H2O2, are extremely short lived and essentially react with the first susceptible molecule with which they collide. Thus, these so-called free radicals damage cells directly by lipid peroxidation (Figure 1), protein oxidation (Figure 2), and scission of DNA single strands (Figure 3), although the figures oversimplify the actual processes. For example, hydroxyl radials can attack purines and pyrimidines, leading to gene mutations as well as the deoxyribosyl sugar in the DNA backbone (Wulf, 2002Wulf D. Free radicals in regulation of physiological functions.Physiol Rev. 2002; 82: 47-95Crossref PubMed Scopus (7488) Google Scholar). It is important to emphasize that telomeres are particularly sensitive to DNA breaks because they have no DNA repair enzyme system (Wulf, 2002Wulf D. Free radicals in regulation of physiological functions.Physiol Rev. 2002; 82: 47-95Crossref PubMed Scopus (7488) Google Scholar). Another important example of the biologic consequences of ROS-generated free radicals is hydroxyl radical attack on endothelial cell membranes. In addition to compromising the membrane itself, hydroxyl radicals appear to directly activate intracellular calcium-dependent adhesion molecule on the membrane, which makes the endothelium "stickier" for leukocytes passing in the circulation (Sellak et al., 1994Sellak H. Franzini E. Hakim J. Pasquier C. Reactive oxygen species rapidly increase endothelial ICAM ability to bind neutrophils without detectable upregulation.Blood. 1994; 83: 2669-2677Crossref PubMed Google Scholar). This both slows blood flow through the wound's microvascular circulation and leads to an influx of inflammatory cells into the wound.Figure 2Protein oxidation by free radicals. (a) Hydrogen atom elimination at the α-carbon of the protein backbone, resulting in backbone fragmentation, or (b) hydrogen atom elimination at side chains, resulting in different products, including peroxides, alcohols, and carbonyls. As with lipid perioxidation, protein peroxides are unstable and propagate further reactions.(Modified from the presentation "Protein Oxidation: Concepts, Mechanisms and New Insights," by M.J. Davies, Heart Research Institute, Sydney, Australia, with permission.)View Large Image Figure ViewerDownload (PPT)Figure 3DNA single-strand breaks are the most common damage inflicted by ROS. The break results from collapse of the sugar that is illustrated in the process at the right.(Modified from the presentation "DNA Oxidation: A Simple Overview," by F.Q. Schafer, University of Iowa, with permission.)View Large Image Figure ViewerDownload (PPT) Given all the adverse consequences of elevated ROS levels on cell machinery, it is no wonder that fibroblasts isolated from ROS-rich chronic wounds have a dysfunctional phenotype. These ongoing pathobiologic events in chronic wounds raise the question of how drug therapy can attenuate the impact of excessive ROS generation. Logically, the main target should be one or more of the pathways that generate ROS rather than dysfunctional chronic wound fibroblasts. If the hypothesis of Wall et al., 2008Wall I.B. Moseley R. Baird D.M. Kipling D. Giles P. Laffafian I. et al.Fibroblast dysfunction is a key factor in the non-healing of chronic venous leg ulcers.J Invest Dermatol. 2008; 128: 2526-2540Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar is correct, relieving oxidative stress in chronic wounds will ameliorate fibroblast dysfunction. Here, clinicians and patients are fortunate to have a readily available drug that appears to attenuate oxidative stress in chronic wounds. Pentoxifylline has been used for patients with venous leg ulcers for two decades, and a recent meta-analysis by the Cochrane Collaborative Group found that pentoxifylline was effective with a high level of certainty (Jull et al., 2007Jull A. Arroll B. Parag V. Waters J. Pentoxifylline for treating venous leg ulcers (review).Cochrane Collaboration. 2007; 2007: 1-30Google Scholar). Although the mechanisms of action of this drug have not been rigorously defined, they include scavenging hydroxyl radicals (Freitas and Filipe, 1995Freitas J.P. Filipe P.M. Pentoxifylline. A hydroxyl radical scavenger.Biol Trace Element Res. 1995; 47: 307-311Crossref PubMed Scopus (48) Google Scholar), inhibiting xanthine oxidase (Hammerman et al., 1999Hammerman C. Goldschmidt D. Caplan M.S. Kaplan M. Schimmel M.S. Eidelman A.I. et al.Amelioration of ischemia–reperfusion injury in rat intestine by pentoxifylline-mediated inhibition of xanthine oxidase.J Pediatr Gastroenterol Nutr. 1999; 29: 69-74Crossref PubMed Scopus (54) Google Scholar), and reducing circulating levels of tumor necrosis factor-α (Fernandes et al., 2008Fernandes J.L. de Oliveira R.T. Mamoni R.L. Coelho O.R. Nicolau J.C. Blotta M.H. et al.Pentoxifylline reduces pro-inflammatory and increases anti-inflammatory activity in patients with coronary artery disease—a randomized placebo-controlled study.Atherosclerosis. 2008; 196: 434-442Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar; Zeni et al., 1996Zeni F. Pain P. Vindimian M. Gay J.P. Gery P. Bertrand M. et al.Effects of pentoxifylline on circulating cytokine concentrations and hemodynamics in patients with septic shock: results from a double-blind, randomized, placebo-controlled study.Crit Care Med. 1996; 24: 207-214Crossref PubMed Scopus (83) Google Scholar). Surprisingly, no other antioxidants have been tested in well-controlled clinical trials for efficacy in venous leg ulcer disease. The author states no conflict of interest.
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