Inflammation in Nonhealing Diabetic Wounds

American Journal of Pathology, Vol. 159, No. 2, August 2001 Copyright © American Society for Investigative Pathology

Commentary Inflammation in Nonhealing Diabetic Wounds The Space-Time Continuum Does Matter

Glenn F. Pierce From Selective Genetics, San Diego, California

The article by Goova and colleagues in this issue of The American Journal of Pathology extends long-standing work from this laboratory in which the receptor for advanced glycation endproducts (RAGE) was purified, cloned, and shown in multiple in vivo systems to inhibit diabetes-related sequelae when administered as a soluble receptor fragment for advance glycation products (sRAGE).1– 4 In the present studies, the authors methodically show that the well-recognized impaired wound healing in the db/db mouse, a model that approximates many of the features of type 2 diabetes in humans, could be ameliorated by administration of sRAGE. Given the complex molecular and cellular milieu of the chronic wound state, and the well-recognized negative impact of diabetes on wound healing,5 these novel findings open new avenues of investigation and potential therapeutic intervention.

How Does the AGE-RAGE Axis Influence Our Thinking about Nonhealing Diabetic Wounds? Advanced glycation endproducts have been established to interfere with both extracellular matrix through inappropriate cross-linking of matrix proteins,6 and with cellular function via cell surface receptor-mediated interactions.7,8 RAGE, a member of the immunoglobulin superfamily, is present on numerous cell types important for normal wound healing, including endothelial cells, monocytes, fibroblasts, and smooth muscle cells. Consequences of AGEs in diabetic wounds, as shown by Goova and colleagues, include a complex and nonintuitive sequence of events beginning with delayed inflammatory cell influx into the wound, and leading to a sustained state of chronic inflammation once the inflammatory cells do establish residence. This situation prevents the wound from progressing to matrix deposition and remodeling phases, thus inhibiting healing. The Columbia group has shown previously that sRAGE can prevent AGE-induced complications in several experimental

systems, including accelerated diabetic atherosclerosis9 and diabetes-associated periodontitis.10 In the present work, they have demonstrated restoration of the normal healing response when db/db mice were administered sRAGE.1 RAGE also binds non-AGEs: members of the S100/ calgranulin family of polypeptides, termed extracellular newly identified RAGE-binding proteins (EN-RAGEs);11 in fact these may be the natural ligands for this receptor. EN-RAGEs are synthesized by leukocytes, and trigger inflammatory cell activation, leading to synthesis of proinflammatory cytokines such as interleukin-1␤ and tumor necrosis factor (TNF)-␣. This autocrine and paracrine loop propagates and sustains the inflammatory response. At least some intracellular pathways stimulated by EN-RAGEs and AGEs are identical, such as nuclear factor-␬B signaling, likely further contributing to a chronic inflammatory state in wounds that contain both RAGE ligands.

What Has Been Learned about the Biology of Nonhealing Wounds? Chronic wounds are a heterogeneous collection of dermal lesions that do not undergo the normal healing progression: inflammation, proliferation and matrix deposition, and remodeling.12–14 They include not only diabetic neuropathic and vascular insufficiency ulcers on the lower extremities, but also pressure ulcers (decubiti), venous stasis ulcers, severe burns, and a host of rarer lesions associated with skin or autoimmune diseases. These nonhealing wounds have been the subject of intensive investigation throughout the past 15 years, as recombinant growth factors emerged on the scene. Given that the targets of members of the epidermal growth factor, fibroblast growth factor, platelet-derived growth factor (PDGF), and transforming growth factor-␤ families were cells that participated in the dermal wound

Accepted for publication June 4, 2001. Address reprint requests to Glenn Pierce PhD, MD, Selective Genetics, 11035 Roselle St., San Diego, CA 92121. E-mail: [email protected].

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repair process, it was logical to use this model as the first foray into clinical studies with these growth factors. With one notable exception (PDGF-BB homodimer15), this drug development effort may be considered a failure for several reasons.16 First, most growth factors were evaluated in normal animal models or impaired healing models that did not fully replicate the conditions present in human nonhealing wounds. Second, all nonhealing human wounds are not alike, and biology in a neuropathic diabetic ulcer may be quite distinct from the biology in a diabetic ulcer due primarily to vascular insufficiency. Third, patient and caregiver compliance with treatment regimens confounded treatment versus control comparisons. Fourth, wound-care specialists needed to standardize good wound care, and once this occurred, control groups healed at accelerated rates (placebo effect). Fifth, delivery of the protein therapeutic, retaining enough within distinct wound microenvironments for a sufficient period of time, proved very challenging. And sixth, a fundamental lack of understanding of wound-healing processes— both normal and abnormal—that didn’t fully permit science-based selection of therapeutic candidates. Better understanding the pathophysiology of nonhealing diabetic wounds can only help identify better therapeutic modalities and targets. Chronic wounds can arise from recurrent or chronic injuries (ie, intermittent ischemia) and/or low-level bacterial contamination. Once established, positive autocrine feedback loops and ongoing insults maintain the chronic wound state, preventing progression of the healing process, specifically fibroblast and neovessel accumulation and net matrix deposition. The key is to break this cycle. This may be accomplished surgically, through sharp debridement to bleeding margins of the wound. This procedure, standardized for diabetic ulcers by Steed and colleagues,17serves to jump start the wound-healing process, and disrupts the state of chronic inflammation. Growth factors such as PDGF-BB may be the pharmacological equivalent of sharp debridement, because PDGF exaggerates the inflammatory and proliferative/matrix deposition phases of repair. PDGF does not disrupt the normal sequence of events, unlike other growth factors applied pharmacologically to wounds, such as fibroblast growth factors and transforming growth factor-␤, which do alter the normal healing process.18 Together, surgical debridement and daily application of PDGF-BB have resulted in a modest ⬃15% improvement in the rate of fully healed diabetic ulcers in patients.15 Thus, more effective treatment strategies are needed. The delivery of the PDGF-B gene (which encodes PDGF-BB protein) in an adenoviral vector has provided for longer term, more localized delivery of PDGF-BB protein.19,20 Interestingly, adenovirus particles are pro-inflammatory, and when not delivering PDGF-B, will inhibit repair, consistent with prolonging the inflammatory phase.21 However, when the adenoviral vector delivered the PDGF-B gene, a single application augmented the inflammatory phase and led to enhanced net matrix deposition, resulting in accelerated closure of compromised animal wounds.22 Intuitively, it may be of

concern to add inflammation inducers such as the PDGF-B gene and adenovirus to wounds at risk for chronic inflammation. However, the inflammation induced by PDGF-BB protein and adenovirus is self-limiting, and PDGF-BB protein leads to progression to the next phase of repair, rather than inhibiting progression.23,24 This may be due, in part, to the ability of PDGF to more selectively stimulate fibronectin-specific integrin receptors on wound fibroblasts, in contrast to the more generalized integrin up-regulation induced by pro-inflammatory cytokines.25 Fibronectin is a critical cell migration-friendly constituent of the provisional extracellular matrix. Other treatment strategies for nonhealing diabetic wounds include administering proteases and protease inhibitors,26 and covering the wound in an artificial skin, ostensibly to down-regulate the chronic inflammatory state.27 Attempting to change protease/anti-protease levels are challenging within the wound microenvironment, because both activities are essential for normal repair,28,29 but are subject to critical spatial and temporal regulation within the wound.26,30

The Roles of Inflammation and Proteases in Tissue Repair: the Precarious Spatiotemporal Balance Inflammation in normal wound healing is a two-edged sword: it is essential, but like proteases, must be tightly regulated both temporally and spatially. Any pathological process that interferes with this self-limited physiological process can result in a nonhealing wound because of net destruction of soluble growth factors and matrix elements. The importance of the inflammatory phase was shown by Liebovich and Ross,31 who found that monocytes were essential for normal wound healing. Nagaoka and colleagues32 recently demonstrated that ICAM-1 knockout mice had delayed wound healing manifested by decreased wound leukocyte accumulation. PDGF was able to normalize the healing, consistent with its recognized ability to stimulate leukocyte chemotaxis and activation. Alternatively, addition of activated fibroblasts directly to wounds was shown to circumvent the need for an inflammatory process, and led directly to rapid granulation tissue formation.33 Thus, self-resolving inflammation is a normal and necessary prerequisite to fibroblast activation and net matrix synthesis. In contrast, prolonged expression of pharmacological levels of granulocyte-macrophage colony stimulating factor in rodent wounds results in sustained inflammation via prolonged residence of neutrophils and monocytes, abrogating normal healing (unpublished observations). Similarly, in diabetes, a disordered and more self-sustained inflammatory response, induced at least in part by AGEs, may contribute to many of the tissue injury complications, including nephropathy, vasculopathy, retinopathy, and nonhealing wounds. Imbalances in wound proteases and their inhibitors, because of sustained production of inflammatory mediators and influx of inflammatory cells, prevent matrix syn-

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thesis and remodeling, essential for progression to a healed wound.26,30,34,35 Matrix metalloproteinases (MMPs) are members of the zinc-dependent endopeptidase family, contain at least 20 members, and can degrade most extracellular matrix constituents. MMPs have been recognized as normal constituents in the woundhealing process for many years).28,29 Specific expression of MMPs is essential for cellular migration into the wound milieu, but is closely regulated to permit provisional matrix deposition as healing progresses.26,35 In elegant studies by Dumin and colleagues,36 a trimolecular complex consisting of the MMP, a cell surface integrin, and the matrix substrate has been shown to provide enzyme at the point of cellular contact with substrate. Anchoring the MMP to the cell provides proteolytic activity in a highly regulated manner, at the point of cell contact with the matrix. Several groups have associated increased levels of proteases, including MMPs with wound inflammatory cells (leukocytes and monocytes), and elevated levels of pro-inflammatory cytokines, such as TNF-␣, interleukin1␤, and interleukin-6.35,37 Pro-inflammatory cytokines such as TNF-␣ play a role in the normal healing response,38 but when secreted within the wound at high levels for longer periods of time, stimulate excess protease activities. MMPs were detected at elevated concentrations in human nonhealing wounds by Wysocki and colleagues39 (MMP-2 and MMP-9, gelatinases A and B). Subsequently, others found that chronic wounds contained spatially and temporally differentially regulated MMPs,40 and that levels of MMP inhibitors were decreased.41 Trengrove and colleagues42 conducted a well-controlled study in patients with venous stasis ulcers, and showed elevated wound MMP levels decreased after the onset of healing. MMPs such as MMP-1 (collagenase-1) are regulated within wounds via an nuclear factor-␬B pathway, possibly triggered by interleukin-1␣, as well as through the MAP kinase pathways, stimulated by growth factors and cytokines.43,44 Similarly, TNF-␣ indirectly stimulates MMP-2 (type IV collagenase) activation within wounds via an nuclear factor-␬B pathway.45 Further, the provisional matrix proteoglycan dermatan sulfate can activate this pathway, leading to endothelial cell ICAM up-regulation, required for leukocyte influx into the wound.46 Thus, nuclear factor-␬B signaling pathways are triggered by, and mediate, numerous pro-inflammatory activities, which may contribute to a sustained inflammatory state.

Pro-inflammatory Cytokines, AGE-RAGE Axis, and Proteases: a Unifying Hypothesis for Nonhealing Diabetic Wounds? In human nonhealing wounds, including diabetic ulcers, multiple deviations from normal healing have been identified. Few would dispute that most chronic human wounds are characterized by a chronic inflammatory state, manifested by imbalances in 1) proteases and their anti-proteases, and 2) pro-inflammatory cytokines and

their natural inhibitors. These imbalances are central to most chronic wounds, and, in fact, are found in chronic inflammation in other tissues as well. Chronic wounds in diabetics, however, also contain RAGE ligands, which further tip the balance toward a chronic inflammatory state. The db/db mouse model47 has been used extensively to probe the pathophysiology of chronic diabetic wounds in humans. However, caution is warranted in extrapolating db/db mouse findings directly to humans with diabetes. The db/db mouse defect is because of a deficiency of the leptin receptor, and is caused by a point mutation at a splice donor site that reduces expression of the long isoform of the receptor.48,49 However, the human equivalent of leptin receptor deficiency results in obesity and pituitary dysfunction.50 These differences notwithstanding, the db/db mouse model has impaired wound healing that is responsive to intervention. Greenhalgh and colleagues,51 and Tsuboi and Rifkin52 were the first to show that the impaired healing could be reversed by polypeptide growth factors, including fibroblast growth factor-2 and PDGF-BB, setting the stage for the subsequent successful use of PDGF-BB in human diabetic ulcers. Others have shown the decreased early inflammatory cell influx, and excess pro-inflammatory cytokines and metalloproteases observed by Goova and colleagues53–55 as well as decreased fibroblast growth factor, PDGF, and PDGF receptor expression in wounds from db/db mice.56,57 In the related ob/ob mouse, which is deficient in leptin, Goodson and Hunt58 have demonstrated decreased wound collagen accumulation. Thus the mouse model seems to be a reasonable surrogate for studying chronic, nonhealing wounds in humans. EN-RAGEs and AGEs activate inflammatory cells to secrete pro-inflammatory cytokines, and, coupled with the ability of these cytokines to secrete proteases in excess of their inhibitors, a chronic inflammatory state can be established, or maintained if other pathophysiological processes have initiated it. Administration of sRAGE restores normal progression of wound healing in the diabetic mouse. However, sRAGE can also abrogate the normal acute inflammatory response, such as that induced via delayed-type hypersensitivity reactions,11 suggesting it may even be possible to prevent the normal inflammatory response required for wound healing. This would be an intriguing hypothesis to test in normally healing animal wounds, and, if warranted, clinically in nonhealing diabetic ulcers.

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