Annette B. Wysocki

Interactions between extracellular matrix and growth factors in wound healing

Dynamic interactions between growth factors and extracellular matrix (ECM) are integral to wound healing. These interactions take several forms that may be categorized as direct or indirect. The ECM can directly bind to and release certain growth factors (e.g., heparan sulfate binding to fibroblast growth factor‐2), which may serve to sequester and protect growth factors from degradation, and/or enhance their activity. Indirect interactions include binding of cells to ECM via integrins, which enables cells to respond to growth factors (e.g., integrin binding is necessary for vascular endothelial growth factor‐induced angiogenesis) and can induce growth factor expression (adherence of monocytes to ECM stimulates synthesis of platelet‐derived growth factor). Additionally, matrikines, or subcomponents of ECM molecules, can bind to cell surface receptors in the cytokine, chemokine, or growth factor families and stimulate cellular activities (e.g., tenascin‐C and laminin bind to epidermal growth factor receptors, which enhances fibroblast migration). Growth factors such as transforming growth factor‐β also regulate the ECM by increasing the production of ECM components or enhancing synthesis of matrix degrading enzymes. Thus, the interactions between growth factors and ECM are bidirectional. This review explores these interactions, discusses how they are altered in difficult to heal or chronic wounds, and briefly considers treatment implications.

Temporal expression of urokinase plasminogen activator, plasminogen activator inhibitor and gelatinase-B in chronic wound fluid switches from a chronic to acute wound profile with progression to healing

The plasminogen activator/plasmin system is known to initiate a proteolytic cascade resulting in the activation of matrix metalloproteinases in vitro leading to the degradation of extracellular matrix. To investigate whether or not this cascade is present during delayed wound healing and contributes to the pathophysiological basis of impaired healing we examined the temporal expression of urokinase plasminogen activator, plasminogen activator inhibitor‐1 and gelatinase‐B in fluid collected from chronic venous leg ulcers compared to acute surgical mastectomy wounds. Using a chromogenic substrate assay, levels of active urokinase plasminogen activator in chronic wounds were found to be about five fold higher compared to sera and two fold higher compared to mastectomy wounds. Levels of active plasminogen activator inhibitor‐1 in chronic wounds were four times higher than those found in sera and two times higher than those found in mastectomy wound fluid. Using a fibrin overlay system and reverse zymography, we found that when the wound was not healing, the expression of urokinase plasminogen activator in chronic wound fluid was initially detected in the active forms (50 and 33 kDa), but that as the wound healed and decreased in size, was detected as an inhibitor‐ bound urokinase plasminogen activator–plasminogen activator inhibitor‐1 complex (≅ 80–116 kDa). When the expression of active urokinase plasminogen activator was highest, no plasminogen activator inhibitor‐1 was detectable. In contrast, urokinase plasminogen activator was always detected in the inhibitor bound form as a urokinase plasminogen activator–plasminogen activator inhibitor‐1 complex in blood‐ and plasma‐derived serum and mastectomy wound fluid. Plasminogen activator inhibitor‐1 was detected in blood‐derived serum and mastectomy wound fluid but not in plasma derived serum. Expression of matrix metalloproteinase‐9 in chronic wound fluids, analyzed by gelatin zymography, showed that when urokinase plasminogen activator was detected in the active forms, matrix metalloproteinase‐9 was overexpressed by approximately twice that found in mastectomy wounds and approximately 30 times that detected in blood‐derived sera. When urokinase plasminogen activator appeared almost entirely as an enzyme‐ inhibitor complex, the level of expression of matrix metalloproteinase‐9 was similar to that seen in mastectomy wound fluid. We conclude that the switch in urokinase plasminogen activator expression from an active to inhibitor bound form correlates with the decrease seen in matrix metalloproteinase‐9 expression suggesting the presence of a proteolytic cascade initiated by the plasminogen activator/plasmin system during wound healing leading to the activation of matrix metalloproteinase‐9. In addition, expression of urokinase plasminogen activator and matrix metalloproteinase‐9 appear to be useful biomarkers to determine clinical wound healing status.

Wound fluids and the pathogenesis of chronic wounds.

Purpose To describe two areas of ongoing investigation into analysis of wound fluids that may eventually lead to better understanding of pathophysiology of chronic wounds and to improved care and treatment. Methods Studies used Lowry protein assay, sodium dodecyl sulfate–polyacrylamide gel electrophoresis, Western blotting, and zymography to analyze fluids from acute and chronic wounds and serum samples collected from healthy and affected volunteers. Subjects Thirty-one subjects with ages ranging from 32 to 79 years participated in the research; fluid was collected from chronic wounds in 10 patients (two female, four male, and four unrecorded), fluid was collected from acute mastectomy wounds in 15 patients (all female); blister fluid and blood were collected from two volunteers (one male, one female); and blood for serum preparation was collected from four volunteers (two female, two male). Primary outcome variables (1) Fibronectin degradation and (2) expression of matrix metalloproteinases. Results Fibronectin can be degraded in fluid from chronic wounds but remains intact in blood-derived serum, plasma-derived serum, blister fluid, and mastectomy wound fluid. Matrix metalloproteinases are overexpressed in fluid from chronic wounds compared with mastectomy wound fluid, blood-derived serum, and plasma-derived serum. Matrix metalloproteinases are also expressed at somewhat higher levels in mastectomy fluid than in blood-derived and plasma-derived serum. Conclusions These studies identified two factors that may contribute to delayed healing of chronic wound: fibronectin degradation and overexpression of matrix metalloproteinases.

Microbial diversity in chronic open wounds

Chronic wounds expose the dermal matrix and underlying tissue to a diversity of microbes from the body and surrounding environment. We determined the microbial diversity of 19 chronic wounds using both molecular methods (sequence analysis of rRNA genes) and routine clinical culturing methods using swab samples. We identified 93 phylotypes in 2,653 rRNA clone sequences and found that compared with other environments, the microbial diversity of chronic wounds is relatively well characterized, i.e., 95% of sequences have ≥97% identity with known human commensals. In total, 75% of sequences belonged to four well‐known wound‐associated phylotypes: Staphylococcus (25%), Corynebacterium (20%), Clostridiales (18%), and Pseudomonas (12%). Approximately 0.5% of sequences (seven phylotypes) belonged to potentially new species. Individual wound samples contained four to 22 phylotypes, but in all wounds only a few (one to three) phylotypes were dominant. In more than half the wound specimens, polymerase chain reaction and culturing methods gave different diversity and dominance information about the microbes present. This exploratory study suggests that combining molecular and culturing methods provides a more complete characterization of the microbial diversity of chronic wounds, and can thereby expand our understanding of how microbiology impacts chronic wound pathology and healing.

Adenoviral overexpression and small interfering RNA suppression demonstrate that plasminogen activator inhibitor-1 produces elevated collagen accumulation in normal and keloid fibroblasts.

Keloids are tumor-like skin scars that grow as a result of the aberrant healing of skin injuries, with no effective treatment. We provide new evidence that both overexpression of plasminogen activator inhibitor-1 (PAI-1) and elevated collagen accumulation are intrinsic features of keloid fibroblasts and that these characteristics are causally linked. Using seven strains each of early passage normal and keloid fibroblasts, the keloid strains exhibited inherently elevated collagen accumulation and PAI-1 expression in serum-free, 0.1% ITS+ culture; larger increases in these parameters occurred when cells were cultured in 3% serum. To demonstrate a causal relationship between PAI-1 overexpression and collagen accumulation, normal fibroblasts were infected with PAI-1-expressing adenovirus. Such cells exhibited a two- to fourfold increase in the accumulation of newly synthesized collagen in a viral dose-dependent fashion in both monolayers and fibrin gel, provisional matrix-like cultures. Three different PAI-1-targeted small interfering RNAs, alone or in combination, produced greater than an 80% PAI-1 knockdown and reduced collagen accumulation in PAI-1-overexpressing normal or keloid fibroblasts. A vitronectin-binding mutant of PAI-1 was equipotent with wild-type PAI-1 in inducing collagen accumulation, whereas a complete protease inhibitor mutant retained approximately 50% activity. Thus, PAI-1 may use more than its protease inhibitory activity to control keloid collagen accumulation. PAI-1-targeted interventions, such as small interfering RNA and lentiviral short hairpin RNA-containing microRNA sequence suppression reported here, may have therapeutic utility in the prevention of keloid scarring.

Angiogenic Laminin-Derived Peptides Stimulate Wound Healing

Acceleration of the wound healing process by using angiogenic peptides has been demonstrated previously. Here we used select laminin-111 peptides, A13 and C16, from the laminin alpha1 and gamma1 chain, respectively, to test whether they are able to stimulate wound healing in a rat full thickness wound model. The 12-mer peptides C16 and A13 are highly angiogenic and bind to integrins alphavbeta3 and alpha5beta1. We show that A13 increases wound re-epithelialization as much as 17% over controls by day 4 and C16 increases coverage by 11%. Contraction of the treated wounds was increased as much as 11% for A13 and 8% for C16 at day 4. No differences were observed at day 7 with either peptide. The peptides also stimulated fibroblast migration in Boyden chamber assays. A13 increased cell migration as much as 2.4-fold on uncoated filters and as much as 16-fold on collagen type IV-coated filters over negative controls. Similarly, C16 also stimulated migration 1.8-fold on uncoated filters and as much as 12-fold on collagen-coated filters. A13 and C16 significantly decreased expression of the pro and active forms of matrix metalloproteinase 2 in foreskin fibroblasts indicating their role in collagen accumulation. We conclude that small bioactive angiogenic peptides can promote dermal wound healing and may offer a new class of stable and chemically manipulable therapeutics for wound healing.

Wound Healing in Development

Wound healing is the inherent ability of an organism to protect itself against injuries. Cumulative evidence indicates that the healing process patterns in part embryonic morphogenesis and may result in either organ regeneration or scarring, phenomena that are developmental stage- or age-dependent. Skin is the largest organ. Its morphogenesis and repair mechanisms have been studied extensively due not only to its anatomical location, which allows easy access and observation, but also to its captivating structure and vital function. Thus, this review will focus on using skin as a model organ to illustrate new insights into the mechanisms of wound healing that are developmentally regulated in mammals, with special emphasis on the role of the Wnt signaling pathway and its crosstalk with TGF-β signaling. Relevant information from studies of other organs is discussed where it applies, and the clinical impact from such knowledge and emerging concepts on regenerative medicine are discussed in perspective.

Rat Models of Skin Wound Healing

Rats provide an excellent model for skin wound healing by allowing the standardization of the type, size, shape, and depth of the wound injury, which facilitates comparison of data between studies of healing in all mammalian species. The rat is often selected for skin wound-healing models because of its ready availability, low cost, and small size, which result in a more economical and efficient use of limited laboratory space and housing facilities. Despite species differences, the availability of animals with well-defined health and genetic backgrounds along with a bounty of literature documenting biological responses and parameters for rats allows the rat to serve as a valuable research tool in the search for faster, stronger, and more anatomically correct wound healing with the ultimate goal of exact skin replacement.

Memory Encoded Throughout Our Bodies: Molecular and Cellular Basis of Tissue Regeneration

When a sheep loses its tail, it cannot regenerate it in the manner of lizards. On the other hand, it is possible to clone mammals from somatic cells, showing that a complete developmental program is intact in a wounded sheeps tail the same way it is in a lizard. Thus, there is a requirement for more than only the presence of the entire genetic code in somatic cells for regenerative abilities. Thoughts like this have motivated us to assemble more than just a factographic synopsis on tissue regeneration. As a model, we review skin wound healing in chronological order, and when possible, we use that overview as a framework to point out possible mechanisms of how damaged tissues can restore their original structure. This article postulates the existence of tissue structural memory as a complex distributed homeostatic mechanism. We support such an idea by referring to an extremely fragmented literature base, trying to synthesize a broad picture of important principles of how tissues and organs may store information about their own structure for the purposes of regeneration. Selected developmental, surgical, and tissue engineering aspects are presented and discussed in the light of recent findings in the field.When a sheep loses its tail, it cannot regenerate it in the manner of lizards. On the other hand, it is possible to clone mammals from somatic cells, showing that a complete developmental program is intact in a wounded sheeps tail the same way it is in a lizard. Thus, there is a requirement for more than only the presence of the entire genetic code in somatic cells for regenerative abilities. Thoughts like this have motivated us to assemble more than just a factographic synopsis on tissue regeneration. As a model, we review skin wound healing in chronological order, and when possible, we use that overview as a framework to point out possible mechanisms of how damaged tissues can restore their original structure. This article postulates the existence of tissue structural memory as a complex distributed homeostatic mechanism. We support such an idea by referring to an extremely fragmented literature base, trying to synthesize a broad picture of important principles of how tissues and organs may store information about their own structure for the purposes of regeneration. Selected developmental, surgical, and tissue engineering aspects are presented and discussed in the light of recent findings in the field.

Proteolytic activity by multiple bacterial species isolated from chronic venous leg ulcers degrades matrix substrates.

Background: A major feature of chronic wounds is the loss of tissue, with the exposure of dermal components preventing primary closure and leading to bacterial colonization. Bacterial colonization has been proposed as one of the common underlying pathologies present in chronic wounds. The objective of this exploratory study was to identify bacteria cultured from chronic venous leg ulcers and test for proteolytic activity that degrades matrix substrates. Method: Bacteria were isolated, cultured, and identified from six subjects (average age = 62.8 years) over 2–10 months under an approved protocol using swabs and microbiological culture media. Proteolytic activity against (a) gelatin, (b) an elastin substrate, and (c) a serine/trypsin-sensitive substrate was determined using a colorimetric plate assay with an ELISA plate reader and zymography. Results: We identified 13 bacteria that expressed proteolytic activity against one or more of the tested substrates. Of these, six were Gram-positive (Staphylococcus aureus, Enterococcus faecalis, Staphylococcus epidermidis, Streptococcus agalactiae, Corynebacterium, and Streptococcus bovis) and seven were Gram-negative (Pseudomonas aeruginosa, Escherichia coli, Proteus mirabilis, Morganella morganii, Klebsiella pneumoniae, Bacteroides fragilis, and Serratia marcescens) organisms. Two of these, S. aureus and P. aeruginosa, are recognized wound pathogens. Conclusions: Multiple bacteria species isolated from colonized venous leg ulcers have the capacity to secrete proteases capable of degrading components of the extracellular matrix important for wound healing. Matrix degradation by bacteria may contribute to delays in tissue deposition and repair, suggesting that treatment of chronic wounds should include appropriate management of colonizing bacteria.