Topical photodynamic therapy following excisional wounding of human skin increases production of transforming growth factor-ß3 and matrix metalloproteinases 1 and 9, with associated improvement in dermal matrix organization.

BACKGROUND: Animal studies report photodynamic therapy (PDT) to improve healing of excisional wounds; the mechanism is uncertain and equivalent human studies are lacking. OBJECTIVES: To explore the impact of methyl aminolaevulinate (MAL)-PDT on clinical and microscopic parameters of human cutaneous excisional wound healing, examining potential modulation through production of transforming growth factor (TGF)-ß isoforms. METHODS: In 27 healthy older men (60-77 years), a 4-mm punch biopsy wound was created in skin of the upper inner arm and treated with MAL-PDT three times over 5 days. An identical control wound to the contralateral arm was untreated and both wounds left to heal by secondary intention. Wounds were re-excised during the inflammatory phase (7 days, n = 10), matrix remodelling (3 weeks, n = 8) and cosmetic outcome/dermal structure (9 months, n = 9). Production of TGF-ß1, TGF-ß3 and matrix metalloproteinases (MMPs) was assessed by immunohistochemistry alongside microscopic measurement of wound size/area and clinical assessment of wound appearance. RESULTS: MAL-PDT delayed re-epithelialization at 7 days, associated with increased inflammation. However, 3 weeks postwounding, treated wounds were smaller with higher production of MMP-1 (P = 0·01), MMP-9 (P = 0·04) and TGF-ß3 (P = 0·03). TGF-ß1 was lower than control at 7 days and higher at 3 weeks (both P = 0·03). At 9 months, MAL-PDT-treated wounds showed greater, more ordered deposition of collagen I, collagen III and elastin (all P < 0·05). CONCLUSIONS: MAL-PDT increases MMP-1, MMP-9 and TGF-ß3 production during matrix remodelling, ultimately producing scars with improved dermal matrix architecture.

Clinically relevant doses of lidocaine and bupivacaine do not impair cutaneous wound healing in mice.

BACKGROUND: Lidocaine and bupivacaine are commonly infiltrated into surgical cutaneous wounds to provide local anaesthesia after surgical procedures. However, very little is known about their effects on cutaneous wound healing. If an inhibitory effect is demonstrated, then the balance between the benefits of postoperative local anaesthesia and the negatives of impaired cutaneous wound healing may affect the decision to use local anaesthesia or not. Furthermore, if a difference in the rate of healing of lidocaine- and bupivacaine-treated cutaneous wounds is revealed, or if an inhibitory effect is found to be dose-dependent, then this may well influence the choice of agent and its concentration for clinical use. METHODS: Immediately before incisional wounding, we administered lidocaine and bupivacaine intradermally to adult female mice, some of which had been ovariectomized to act as a model of post-menopausal women (like post-menopausal women, ovariectomized mice heal wounds poorly, with increased proteolysis and inflammation). Day 3 wound tissue was analysed histologically and tested for expression of inflammatory and proteolytic factors. RESULTS: On day 3 post-wounding, wound areas and extent of re-epithelialization were comparable between the control and local anaesthetic-treated animals, in both intact and ovariectomized groups. Both tested drugs significantly increased wound activity of the degradative enzyme matrix metalloproteinase-2 relative to controls, while lidocaine also increased wound neutrophil numbers. CONCLUSIONS: Although lidocaine and bupivacaine influenced local inflammatory and proteolytic factors, they did not impair the rate of healing in either of two well-established models (mimicking normal human wound healing and impaired age-related healing).

5alpha-dihydrotestosterone (DHT) retards wound closure by inhibiting re-epithelialization.

The ongoing search for explanations as to why elderly males heal acute skin wounds more slowly than do their female counterparts (and are more strongly disposed to conditions of chronic ulceration) has identified endogenous oestrogens and androgens as being respectively enhancers and inhibitors of repair. We previously demonstrated that blocking the conversion of testosterone to 5alpha-dihydrotestosterone (DHT) limits its ability to impair healing, suggesting that DHT is a more potent inhibitor of repair than is testosterone. The present study aimed to delineate the central mechanisms by which androgens delay repair. Whilst the contractile properties of neither rat wounds in vivo nor fibroblast-impregnated collagenous discs in vitro appeared to be influenced by androgen manipulations, the global blockade of DHT biosynthesis markedly accelerated re-epithelialization of incisional and excisional wounds and reduced local expression of beta-catenin, a key inhibitor of repair. Moreover, DHT retarded the in vitro migration of epidermal keratinocytes following scratch wounding. By contrast, it failed to influence the migratory and proliferative properties of dermal fibroblasts, suggesting that its primary inhibitory effect is upon re-epithelialization. These novel findings may be of particular significance in the context of chronic ulceration, for which being male is a key risk factor.

Polymorphisms spanning the 0N exon and promoter of the estrogen receptor-beta (ERbeta) gene ESR2 are associated with venous ulceration.

Venous ulcers are characterized by excessive inflammation and raised levels of proinflammatory cytokines. Estrogen has been shown to accelerate the rate of wound healing in elderly subjects by dampening the inflammatory response. The estrogen receptor (ER) proteins, ER-alpha (ERalpha) and ER-beta (ERbeta) mediate the actions of estrogen during wound repair through the activation or repression of target gene transcription. Recent evidence implicates the chromosomal region harboring the ERbeta gene with venous ulceration in a British Caucasian population, highlighting the need to conduct further genetic interrogation. To address this, we conducted a case-control study to investigate whether single nucleotide polymorphisms in the ERbeta gene are associated with venous ulceration in elderly (age >50 years) subjects. We recruited a case group (n = 124, 56 males and 68 females) consisting of patients with an active venous ulcer and a control group consisting of individuals from the general population with no evidence of venous disease or history of venous ulceration (n = 380, 189 males and 191 females). Polymorphisms in close proximity to upstream regulatory regions of the ERbeta gene, including the 0N exon and promoter transcribed in inflammatory cells, were significantly (p < 0.05) associated with venous ulceration. A major susceptibility haplotype carried by 23% (26/112) of cases compared with only 10% (27/276) of controls (odds ratio = 2.8, 95% confidence interval = 1.6-5.0) was significantly (p < 0.01) associated with elevated serum levels of tumor necrosis factor-alpha. In conclusion, common variation in the regulatory regions of the ERbeta gene may pre-dispose to venous ulceration in a British Caucasian population.

Sex steroids and cutaneous wound healing: the contrasting influences of estrogens and androgens.

The increased prevalence in the elderly of chronic wound-healing conditions, such as venous and diabetic ulceration, is firmly established. This same population additionally suffers from impaired healing of acute wounds, which are characterized by delayed closure, increased local inflammation, and excessive proteolytic activity. In females, this decline in the effectiveness of skin repair mechanisms follows the menopause, and a series of clinical studies has identified estrogens as being endogenous enhancers of healing processes. The administration of 17beta-estradiol, either systemically or topically, has been shown to reverse the fundamental repair defects observed in postmenopausal women. By contrast, androgenic species retard repair and interfere with the accumulation of the structural proteins that reconstitute the damaged dermis. Since estrogen-based hormone replacement therapy produces wide-ranging effects, not all of which are considered to be desirable, more recent studies have sought to identify downstream mediators of estrogenic effects in order to formulate better targeted strategies for improving skin repair in the elderly.

Secretory leukocyte protease inhibitor mediates non-redundant functions necessary for normal wound healing.

Secretory leukocyte protease inhibitor (SLPI) is a serine protease inhibitor with anti-microbial properties found in mucosal fluids. It is expressed during cutaneous wound healing. Impaired healing states are characterized by excessive proteolysis and often bacterial infection, leading to the hypothesis that SLPI may have a role in this process. We have generated mice null for the gene encoding SLPI (Slpi), which show impaired cutaneous wound healing with increased inflammation and elastase activity. The altered inflammatory profile involves enhanced activation of local TGF-beta in Slpi-null mice. We propose that SLPI is a pivotal endogenous factor necessary for optimal wound healing.

Incisional wound healing in transforming growth factor-beta1 null mice.

Expression of endogenous transforming growth factor-beta1 is reduced in many animal models of impaired wound healing, and addition of exogenous transforming growth factor-beta has been shown to improve healing. To test the hypothesis that endogenous transforming growth factor-beta1 is essential for normal wound repair, we have studied wound healing in mice in which the transforming growth factor-beta1 gene has been deleted by homologous recombination. No perceptible differences were observed in wounds made in 3-10-day-old neonatal transforming growth factor-beta1 null mice compared to wild-type littermates. To preclude interference from maternally transferred transforming growth factor-beta1, cutaneous wounds were also made on the backs of 30-day-old transforming growth factor-beta1 null and littermate control mice treated with rapamycin, which extends their lifetime and suppresses the inflammatory response characteristic of the transforming growth factor-beta1 null mice. Again, no impairment in healing was seen in transforming growth factor-beta1 null mice. Instead these wounds showed an overall reduction in the amount of granulation tissue and an increased rate of epithelialization compared to littermate controls. Our data suggest that release of transforming growth factor-beta1 from degranulating platelets or secretion by infiltrating macrophages and fibroblasts is not critical to initiation or progression of tissue repair and that endogenous transforming growth factor-beta1 may actually function to increase inflammation and retard wound closure.

Loss of Smad3 modulates wound healing.

TGF-beta plays a central and critical role in tissue repair. The recent identification of TGF-beta signal transduction pathways involving Smad proteins has now made it possible to explore their contribution to the activities of TGF-beta in vivo. Both Smad3 and its closely related homolog Smad2 act as latent nuclear transcriptional activators and mediate intracellular signaling by TGF-betas and activin, each of which regulates cellular functions pivotal to cutaneous wound healing. Mice null for Smad3 (Smad3(ex8/ex8)) survive into adulthood and show accelerated cutaneous wound healing characterized by an increased rate of re-epithelialization and a reduced local inflammatory infiltrate. These data implicate Smad3 in specific pathways of tissue repair and suggest that it could be a target for the development of therapeutic strategies to modulate wound healing.

Bidirectional regulation of macrophage function by TGF-beta.

The dual role of transforming growth factor-beta (TGF-beta) in modulating macrophage function is an important concept gaining increasing recognition. In addition to its role as a 'macrophage-deactivating' agent, TGF-beta functions as a monocyte activator, inducing cytoke production and mediating host defence. These functions are context-dependent, modulated by the differentiation state of the cell, the local cytokine environment, and the local levels of TGF-beta in itself. In general, during the initial stages of inflammation, TGF-beta locally acts as a proinflammatory agent by recruiting and activating resting monocytes. As these cells differentiate specific immunosuppressive actions of TGF-beta predominate, leading to resolution of the inflammatory response. Increasing our understanding of the bidirectional regulation of macrophage function will facilitate prediction of the ultimate outcome of modulating TGF-beta levels in vivo.

Mice lacking Smad3 show accelerated wound healing and an impaired local inflammatory response.

The generation of animals lacking SMAD proteins, which transduce signals from transforming growth factor-beta (TGF-beta), has made it possible to explore the contribution of the SMAD proteins to TGF-beta activity in vivo. Here we report that, in contrast to predictions made on the basis of the ability of exogenous TGF-beta to improve wound healing, Smad3-null (Smad3ex8/ex8) mice paradoxically show accelerated cutaneous wound healing compared with wild-type mice, characterized by an increased rate of re-epithelialization and significantly reduced local infiltration of monocytes. Smad3ex8/ex8 keratinocytes show altered patterns of growth and migration, and Smad3ex8/ex8 monocytes exhibit a selectively blunted chemotactic response to TGF-beta. These data are, to our knowledge, the first to implicate Smad3 in specific pathways of tissue repair and in the modulation of keratinocyte and monocyte function in vivo.

Topical estrogen accelerates cutaneous wound healing in aged humans associated with an altered inflammatory response.

The effects of intrinsic aging on the cutaneous wound healing process are profound, and the resulting acute and chronic wound morbidity imposes a substantial burden on health services. We have investigated the effects of topical estrogen on cutaneous wound healing in healthy elderly men and women, and related these effects to the inflammatory response and local elastase levels, an enzyme known to be up-regulated in impaired wound healing states. Eighteen health status-defined females (mean age, 74.4 years) and eighteen males (mean age, 70.7 years) were randomized in a double-blind study to either active estrogen patch or identical placebo patch attached for 24 hours to the upper inner arm, through which two 4-mm punch biopsies were made. The wounds were excised at either day 7 or day 80 post-wounding. Compared to placebo, estrogen treatment increased the extent of wound healing in both males and females with a decrease in wound size at day 7, increased collagen levels at both days 7 and 80, and increased day 7 fibronectin levels. In addition, estrogen enhanced the strength of day 80 wounds. Estrogen treatment was associated with a decrease in wound elastase levels secondary to reduced neutrophil numbers, and decreased fibronectin degradation. In vitro studies using isolated human neutrophils indicate that one mechanism underlying the altered inflammatory response involves both a direct inhibition of neutrophil chemotaxis by estrogen and an altered expression of neutrophil adhesion molecules. These data demonstrate that delays in wound healing in the elderly can be significantly diminished by topical estrogen in both male and female subjects.

An elastin gene mutation producing abnormal tropoelastin and abnormal elastic fibres in a patient with autosomal dominant cutis laxa.

Elastin is the protein responsible for the characteristic elastic properties of many tissues including the skin, lungs and large blood vessels. Loss-of-function mutations in the elastin gene are known to cause the heart defect supravalvular aortic stenosis (SVAS). We and others have identified deletions, nonsense mutations and splice site mutations in SVAS patients that abolish the function of one elastin gene. We have now identified an elastin mutation in a patient with a completely different phenotype, the rare autosomal dominant condition cutis laxa. A frameshift mutation in exon 32 of the elastin gene is predicted to replace 37 amino acids at the C-terminus of elastin by a novel sequence of 62 amino acids. mRNA and immunoprecipitation studies show that the mutant allele is expressed. Electron microscopy of skin sections shows abnormal branching and fragmentation in the amorphous elastin component, and immunocytochemistry shows reduced elastin deposition in the elastic fibres and fewer microfibrils in the dermis. These findings suggest that the mutant tropoelastin protein is synthesized, secreted and incorporated into the elastic matrix, where it alters the architecture of elastic fibres. Interference with cross-linking would reduce elastic recoil in affected tissues and explain the cutis laxa phenotype.

Aging alters the inflammatory and endothelial cell adhesion molecule profiles during human cutaneous wound healing.

Age-related changes in the human inflammatory response in vivo have been largely ignored, resulting in a lack of understanding of the patho-physiologic processes involving inflammation that become increasingly important with age, of which wound repair is an important example. We have tested the hypothesis that the delay in wound healing resulting from old age is associated with an altered inflammatory response and endothelial cell adhesion molecule (CAM) profile, because CAMs influence the temporal and lineage profiles of extravasated leukocytes within a wound. Cutaneous punch biopsies were taken from 138 healthy subjects, aged 19 to 96 years; the wounds were rebiopsied at fixed time-points from Day 1 up to 3 months postwounding. Quantitative image analysis showed that there was a marked early increase in the neutrophil response in the aged with a less pronounced peak in the wounds of young subjects. Monocyte/macrophage and lymphocyte appearance was delayed in the aged with cell numbers peaking at Day 84, compared to Day 7 for monocytes and Day 21 for lymphocytes in the young, but with increased numbers of mature macrophages in the aged. E-selectin was strongly expressed in a perivascular distribution in the early wounds of the aged; however, only faint staining was seen from Day 3 to 7 in the wounds of the young. Intracellular CAM-1 and vascular CAM-1 expression exhibited an age-related delay in appearance and a reduction in staining intensity. This altered CAM profile may affect the early inflammatory wound healing response in aged humans and suggests a target for future therapeutic manipulations.

Human ageing impairs injury-induced in vivo expression of tissue inhibitor of matrix metalloproteinases (TIMP)-1 and -2 proteins and mRNA.

Proteolysis is an essential component of wound healing but, if uncontrolled, it may lead to degradation of the neo-matrix and a delay in wound repair. Despite numerous reports of impaired wound healing associated with increasing age, the control of proteolysis is completely unknown. Tissue inhibitor of matrix metalloproteinases (TIMP)-1 and -2 inhibit the activity of matrix metalloproteinases and the pattern of regulation of these molecules determines in part the spatial and temporal regulation of proteolytic activity. This study reports on TIMP-1 and -2 protein localization using immunocytochemistry in healing wounds of healthy subjects of different ages from day 1 to 6 months post-wounding, and has quantified the mRNA levels for both inhibitors using reverse transcriptase-polymerase chain reaction (RT-PCR). TIMP-1 and TIMP-2 proteins are up-regulated from 24 h post-wounding, with a decrease in staining intensity by day 7 for TIMP-2 and by day 14 for TIMP-1. Steady-state mRNA levels for both TIMPs were significantly greater in normal young skin than in aged skin. In the young, there was a significant increase in mRNA expression for TIMP-1 and -2 by day 3 post-wounding, which decreased by day 14 and had returned to basal levels at day 21. In the wounds of the aged subjects, basal levels were observed for TIMP-1 and -2 at all time-points. These results suggest that intrinsic cutaneous ageing is associated with reduced levels of TIMP mRNA both in normal skin and during acute wound repair. These levels may be instrumental in dermal tissue breakdown in normal skin, retarded wound healing, and the predisposition of the elderly to chronic wound healing states.

Estrogen accelerates cutaneous wound healing associated with an increase in TGF-beta1 levels.

The cellular and molecular mechanisms underlying the effects of aging on human cutaneous wound healing are poorly understood, and the possible role of reproductive hormones in this process has never been investigated. We report that aging in healthy females was associated with a reduced rate of cutaneous wound healing, but an improved quality of scarring both microscopically and macroscopically, and with reduced levels of transforming growth factor-beta1 (TGF-beta1) immunostaining and steady-state mRNA in the wound. These age-related changes were reversed by the systemic administration of hormone replacement therapy (HRT). Moreover, ovariectomized young female rodents exhibited a marked delay in repair of acute incisional wounds, which was reversed by the topical application of estrogen. The cellular mechanism underlying these changes appears to involve an estrogen-induced increase in latent TGF-beta1 secretion by dermal fibroblasts. These results suggest that both the rate and quality of wound healing depend on reproductive hormone levels.

Age-related changes in the temporal and spatial distributions of fibrillin and elastin mRNAs and proteins in acute cutaneous wounds of healthy humans.

Elasticity and resilience of the skin are determined largely by the elastin framework, whose microfibrillar scaffold is composed of fibrillin. To date, the spatial and temporal patterns of expression of human elastin and fibrillin during would healing have not been described. Ninety healthy human subjects underwent 4 mm cutaneous punch biopsy wounds from the upper inner arm, which were re-excised from day 3 to 3 months post-wounding. There were marked changes in the patterns of distribution and the amounts of elastin and fibrillin in sun-protected skin with ageing. However, there were no major age-related differences in the mRNA levels for elastin, fibrillin-1 and fibrillin-2 using in situ hybridization. Elastin and fibrillin appeared in greatest amounts in the wounds of the elderly, particularly in females. A regenerative pattern of elastin and fibrillin arcades at the dermo-epidermal junction was observed in the wounds of aged subjects. mRNA expression of elastin was greatest in the wounds of the aged (from day 3 to day 14 post-wounding) with a similar spatial and temporal pattern to fibrillin-1 expression; this suggests that fibrillin-1 is the major contributor to dermal elastic fibre construction during wound repair. Fibrillin-2 was expressed only in the wounds of the aged and expression was confined to areas proximal to dermal blood vessels. The clear-cut differences in the localization of the two members of the fibrillin family suggest that these have well-defined roles in normal skin and wound tissue. In summary, these data indicate that ageing is associated with increased expression of fibrillin and elastin during acute wound healing and concomitant restoration of the papillary dermal architecture with an improved quality of scarring.

The effects of ageing on wound healing: immunolocalisation of growth factors and their receptors in a murine incisional model.

A number of reports suggest that the process of ageing impairs wound repair and that strategies to manipulate the age-related wound healing environment are necessary in order to stimulate repair. The process of cutaneous wound repair is controlled by growth factors in an autocrine and paracrine fashion: it is therefore surprising that the localisation of specific growth factors and their receptors has not been documented in wound healing with respect to chronological age. In this study the temporal profile of growth factor and receptor immunostaining was assessed within acute incisional wounds in an ageing mouse colony. A delay in appearance of platelet derived growth factor (PDGF) A and B isoforms, and PDGF-alpha and -beta receptors was evident with increasing animal age, paralleled by a similar finding for epidermal growth factor (EGF) and EGF receptor. Transforming growth factor (TGF)-beta 1 and 2 isoforms were increased at all time points in the wounds of younger animals, but the TGF-beta 3 isoform increased in intensity from d 7 postwounding in the old mice wounds, and basic fibroblast growth factor (bFGF) from d 14. The quantity and distribution patterns of the various growth factors and their receptors may explain the age-related differences in wound healing speed and quality, and possibly suggest new therapeutic targets for manipulating wound healing in the aged.

Aging is associated with reduced deposition of specific extracellular matrix components, an upregulation of angiogenesis, and an altered inflammatory response in a murine incisional wound healing model.

The concept that aging impairs wound healing is largely unsubstantiated, the literature being contradictory because of poor experimental design and a failure to adequately characterize animal models. This study tested the hypothesis that aging retards the rate of wound repair using standardized cutaneous incisional wounds in a well-characterized aging mouse colony. Against the background of age-related changes in normal dermal composition, marked differences in healing were observed. Immunostaining for fibronectin was decreased in the wounds of the old mice, with a delay in the inflammatory response, re-epithelialization, and the appearance of extracellular matrix components. Heparan sulfate and blood vessel staining were both unexpectedly increased in the wounds of the old animals at late time points. Despite an overall decrease in collagen I and III deposition in the wounds of old mice, the dermal organization was surprisingly similar to that of normal dermal basket-weave collagen architecture. By contrast, young animals developed abnormal, dense scars. Intriguingly, some of these age-related changes in scar quality and inflammatory cell profile are similar to those seen in fetal wound healing. The rate of healing in young animals appears to be increased at the expense of the scar quality, perhaps resulting from an altered inflammatory response.

Age-related differences in the temporal and spatial regulation of matrix metalloproteinases (MMPs) in normal skin and acute cutaneous wounds of healthy humans.

Despite the association of increasing age with chronic wound-healing disorders and an impaired rate of healing of acute cutaneous wounds, the role of matrix metalloproteinases (MMPs) is unknown. To determine the spatial and temporal patterns and activities of MMP-1, -2, -3 and -9, 132 healthy humans aged between 19 and 96 years underwent 4-mm punch biopsies followed by wound excision between day 1 and day 180 post-wounding. Wounds showed an age-related increase in MMP-2 and MMP-9 immunostaining from day 3; this was associated with degradation of gelatin as shown by zymograms and with increased proteinase activity as shown by azocoll assays. Distinct spatial localisations for each MMP were observed: MMP-2 was found in epidermal structures; MMP-9 was observed in inflammatory cells up to day 21; MMP-1 was localised to keratinocytes at the wound margin. Normal old skin showed pro-MMP-2 bands on zymography and increased MMP-2 immunostaining. These results indicate that: (1) intrinsic ageing is associated with the up-regulation of MMPs previously associated with chronic wound healing; (2) wound-tissue proteinases are essentially active up to day 21 postwounding; and (3) intrinsic ageing may predispose to tissue breakdown disorders because of MMP-2 up-regulation in normal skin.