Expression of heat shock proteins in mouse skin during wound healing

Wound healing conditions generate a stressful environment for the cells involved in the regeneration process and are therefore postulated to influence the expression of heat shock proteins (Hsps). We have examined the expression of four Hsps (Hsp27, Hsp60, Hsp70 and Hsp90) and a keratin (keratin 6) by immunohistochemistry during cutaneous wound repair from Day 1 to Day 21 after wounding in the mouse. Hsps were constitutively expressed in normal mouse epidermis and their patterns of expression were modified during the healing process. The changes were not directly linked to the time course of the healing process but rather were dependent on the location of cells in the regenerating epidermis. In the thickened epidermis, Hsp60 was induced in basal and low suprabasal cells, Hsp70 showed a reduced expression, and Hsp90 and Hsp27 preserved a suprabasal pattern with an induction in basal and low suprabasal cells. All Hsps had a uniform pattern of expression in the migrating epithelial tongue. These observations suggest that the expression of Hsps in the neoepidermis is related to the proliferation, the migration, and the differentiation states of keratinocytes within the wound.     

Keratinocyte growth factor-2 accelerates wound healing in incisional wounds

BACKGROUND: Keratinocyte growth factor-2 (KGF-2) also described as fibroblast growth factor-10 (FGF-10) is a newly identified member of the fibroblast growth factor family. KGF-2 is 96% identical to the recently identified rat FGF-10 and specifically stimulates growth of normal human epidermal keratinocytes. The present study was undertaken to examine the effects of topically applied KGF-2 in an incisional wound healing model. KGF-2 treatment resulted in an improvement in incisional wound healing as characterized by an increase in breaking strength, collagen content, and epidermal thickness. METHODS: KGF-2 was topically applied to linear incisions made in the dorsal skin of Sprague-Dawley rats. Biomechanical testing was done using an Instron tensiometer for breaking and tensile strength determinations. Wound collagen content was determined using the Sircol collagen assay. Epidermal thickness measurements were conducted using Masson's trichrome-stained sections of the wound. RESULTS: A single topical application of KGF-2 at the time of wounding resulted in an increase in wound breaking and tensile strength at Day 5 after wounding. Breaking strength of KGF-2-treated wounds was significantly higher compared with the buffer control (1 microgram, 222.1 +/- 13.5 g, P = 0.0007; 4 microgram, 248.7 +/- 15.4 g, P = 0.0001; 10 microgram, 247.2 +/- 21.9 g, P = 0.001; buffer, 141.0 +/- 9.7 g). Epidermal thickness and wound collagen content were significantly increased following treatment with KGF-2. CONCLUSIONS: Based on our findings, KGF-2 is a potent stimulator of wound healing as demonstrated by increased mechanical strength accompanied by an increase in wound collagen content. KGF-2 could be an important cellular mediator responsible for the initiation and acceleration of wound healing and may enhance the healing of surgical wounds.     

Migration of human keratinocytes in electric fields requires growth factors and extracellular calcium

Currents that leak out of wounds generate electric fields lateral to the wound. These fields induce directional locomotion of human keratinocytes in vitro and may promote wound healing in vivo. We have examined the effects of growth factors and calcium, normally present in culture medium and the wound fluid, on the directional migration of human keratinocytes in culture. In electric fields of physiologic strength (100 mV per mm), keratinocytes migrated directionally towards the cathode at a rate of about 1 microm per min. This directional migration requires several growth factors. In the absence of these growth factors, the cell migration rate decreased but directionality was maintained. Epidermal growth factor alone restored cell migration rates at concentrations as low as 0.2 ng per ml. Insulin at 5-100 microg per ml or bovine pituitary extract at 0.2%-2% vol/vol also stimulated keratinocyte motility but was not sufficient to fully restore the migration rate. Keratinocyte migration in electric fields requires extracellular calcium. Changes in calcium concentrations from 3 microM to 3.3 mM did not significantly change keratinocyte migration rate nor directionality in electric fields; however, addition of the chelator ethyleneglycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid to migration medium reduced, and eventually abolished, keratinocyte motility. Our results show that (i) growth factors and extracellular calcium are required for electric field-induced directional migration of human keratinocytes, and (ii) keratinocytes migrate equally well in low and high calcium media.     

Diabetic periodontitis: possible lipid-induced defect in tissue repair through alteration of macrophage phenotype and function

BACKGROUND: Diabetes mellitus is a major health problem in the United States affecting approximately 13 million people. The five 'classic' complications which have historically been associated with the condition are microangiopathy, neuropathy, nephropathy, microvascular disease, and delayed wound healing. Recently, periodontal disease (PD) has been declared the 'sixth' major complication of diabetes as diabetics demonstrate an increased incidence and severity of PD. The cellular and molecular basis for diabetic PD is unknown. HYPOTHESIS: Recent evidence suggests that PD and delayed dermal wound healing may be manifestations of the same general systemic deficit in diabetes involving impairment of the cellular and molecular signal of wounding via alterations in macrophage phenotype. Diabetes-induced hyperlipidemia may interfere with the normal cellular and molecular signal of wounding by alteration of macrophage function and subsequent dysregulation of cytokines at the wound site. RESULTS: Preliminary data in both animal models and humans suggests that hyperglycemia, in combination with elevations of serum low density lipoproteins and triglycerides, leads to formation of advanced glycation end products (AGEs) which may alter macrophage phenotype. This may be responsible for dysregulation of macrophage cytokine production and increased inflammatory tissue destruction and alveolar bone loss. IMPLICATIONS: Future investigations will consider diabetic PD in the context of a generalized systemic wound healing deficit that manifests as PD in the face of constant pathologic wounding of the gingiva (bacterial plaque) or delayed dermal wound healing in instances of periodic traumatic wounding to other parts of the body. These types of studies will provide information concerning defective tissue repair in diabetics that will have clinical relevance for the understanding of PD and delayed dermal healing as well as applications of appropriate and specific therapies.     

Protein kinase C mediates up-regulation of urokinase and its receptor in the migrating keratinocytes of wounded cultures, but urokinase is not required for movement across a substratum in vitro

Both in cell culture and in vivo, keratinocytes that are migrating in response to a wound express enhanced levels of both urokinase-type plasminogen activator (uPA) and the uPA cell surface receptor (uPA-R). To explore the mechanism of this up-regulation, keratinocyte cultures were treated proir to wounding with a variety of metabolic and growth factor inhibitors in order to evaluate their effect on uPA and uPA-R expression. Actinomycin D and cycloheximide inhibited the up-regulation of both uPA and uPA-R, as determined by immunohistochemistry, indicating that RNA and protein syntheses are required for their induction in migrating keratinocytes. Neither removal of protein growth factors from the medium nor addition of inhibitory antibodies to a number of growth factors depressed uPA or uPA-R induction; these findings suggest that a variety of exogenous or endogenous growth factors [i.e., basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), amphiregulin, and tumor necrosis factor-alpha (TNF-alpha) do not have a critical role in the induction of uPA or uPA-R. In contrast, when protein kinase C (PKC) was either down-regulated with bryostatin 5 or inhibited with Ro31-8220 or staurosporine, the expression of both uPA and uPA-R was greatly decreased in migrating keratinocytes. Furthermore, pharmacologic activation of PKC enhanced uPA levels in non-wounded cultures. These data suggest that the enhanced expression of uPA and uPA-R in migrating keratinocytes is mediated by selective activation of PKC in these cells, perhaps secondary to alterations in the cytoskeleton induced by wounding. To test the requirement for uPA during keratinocyte migration in vitro, the extent of migration was quantified in the presence and absence of a variety of inhibitors in the wounded culture model. Migration was not altered by actinomycin D, cycloheximide, any of the above growth factor inhibitors, anti-uPA antibodies, a variety of inhibitors of uPA or plasmin enzymatic activity, or exogenous uPA. The independence of keratinocyte migration in vitro from uPA was further suggested by experiments which combined the phagokinetic assay of migration and the zymographic assay for pericellular uPA activity; no relationship was observed between pericellular uPA activity and the motility of individual cells.     

Heterogeneity in endothelium-derived nitric oxide-mediated relaxation of different sized pulmonary arteries of newborn lambs

Extensive documentation has validated the role of UV irradiation as a tumor initiator and promoter, inducing both squamous and basal cell carcinomas. Human epidermis is a tissue which undergoes active metabolism of arachidonic acid to prostaglandins which is regulated by the action of prostaglandin H synthase (also known as cyclooxygenase). One mechanism for the promotional activity of UV light may involve its ability to induce prostaglandin formation. Work in our laboratory has demonstrated that acute exposure of human keratinocytes to UVB irradiation results in increased production of prostaglandin E2 (PGE2). When cultured human keratinocytes were examined after irradiation with 30 mJ/cm2 UVB in vitro, Western blot analysis showed a 6-fold increase in COX-2 protein which was evident at 6 h and peaked 24 h after irradiation. Furthermore, when human subjects were irradiated on sun-protected skin with up to four times their minimal erythema dosage (MED) and biopsied 24 h later, upregulation of COX-2 protein expression was observed via immunofluorescence microscopy. RNAase protection assays supported this observation, showing induction of COX-2 message which peaked at approximately 12 h following irradiation in vitro. Furthermore, human squamous cell carcinoma biopsies exhibited strongly enhanced staining for COX-2 protein via immunohistochemistry and Western analysis when compared to normal non-sun-exposed control skin. Together, these data demonstrate acute upregulation of COX-2 via UVB irradiation and suggest the need for further studies of COX-2 expression as a potential pharmacological target mediating human skin tumor development.     

Influence of methylprednisolone and transforming growth factor-beta on wound healing

This study was aimed at evaluating the influence of transforming growth factor-beta (TGF-beta) on methylprednisolone induced inhibition of wound healing. C57BL/6 mice underwent a standardized dorsal incision. At regular intervals after wounding the mice were sacrificed and their pelts were excised. The fresh breaking strength (FBS) of the pelts was then measured with a constant-speed tension meter. 1) In the first experiment, designed to determine if methylprednisolone did in fact have any inhibiting effect on wound healing, mice received methylprednisolone and a control group received saline. Both methylprednisolone and saline were administered for a four day period. In this experiment the FBS of methylprednisolone treated mice was weaker than that of the control group on the 14th and the 21st day. 2) In the second experiment, designed to determine when the administration of methylprednisolone most noticeably inhibited wound healing, mice were divided into three groups which received methylprednisolone in the following manner: for three days prior to wounding, on the day of wounding, and for three days immediately following wounding. The fourth group received no methylprednisolone at all. The FBS of mice treated with methylprednisolone for three days prior to wounding was weaker than that of the control group on the 14th day after wounding, but showed no significant difference on the 21st day after wounding. The FBS of mice treated on the operative day was weaker on both the 14th and the 21st day after wounding. The FBS of mice treated three days after wounding showed no significant difference on the 14th day after wounding, but was weaker than the control group on the 21st day after wounding. 3) In the third experiment, designed to determine at what time the administration of TGF-beta most accelerated wound healing, mice were divided into three groups which received TGF-beta at different intervals. The first group received TGF-beta on the day of wounding, the second group received TGF-beta on the third day after wounding, and the third group received TGF-beta on the 7th day after wounding. A control group received no treatment. In this experiment the FBS of mice treated with TGF-beta on the third day after wounding was stronger than that of the control group when measured on the 7th and 11th day after wounding, but there was no significant difference on the 14th day. The FBS of mice treated on the day of wounding and mice treated on the 7th day after wounding was not significantly different from that of the control group. 4) In the fourth experiment, designed to determine if TGF-beta can prevent methylprednisolone-induced inhibition of wound healing, mice were divided into three groups. The first group received methylprednisolone for four days prior to wounding. On the third day after wounding they were given saline. The second group also received methylprednisolone for four days prior to wounding, but was treated with TGF-beta on the third day after wounding. The third group received no methylprednisolone, and was given saline three days after wounding. In this experiment the FBS of mice which received only methylprednisolone and saline was weaker than that of the control group on both the 14th and the 21st day after wounding. However, there was no significant difference between the FBS of methylprednisolone treated mice which received TGF-beta and the control group on both the 14th and the 21st day after wounding. From these results the following conclusions were drawn: 1) Methylprednisolone does inhibit wound healing. 2) The influence of methylprednisolone on wound healing is stronger if it is received on operative day. 3) TGF-beta can accelerate wound healing. 4) TGF-beta can prevent methylprednisolone induced inhibition of wound healing.     

Hexokinase, glucose-6-phosphate dehydrogenase and antioxidant enzymes in diabetic reticulocytes: effects of insulin and vanadate

Growth factors of the transforming growth factor-beta superfamily are involved in cutaneous wound healing. In this study we analyze the expression of the bone morphogenetic protein-6 (BMP-6) gene, a transforming growth factor-beta related gene, in skin wounds. In normal mouse skin high levels of BMP-6 mRNA and protein are expressed by postmitotic keratinocytes of stratified epidermis until day 6 after birth. BMP-6 expression is strongly reduced in adult epidermis with diminished mitotic activity. After skin injury we found large induction of BMP-6-specific RNA and protein in keratinocytes at the wound edge and keratinocytes of the newly formed epithelium as well as in fibroblast shaped cells in the wound bed. BMP-6-specific RNA was induced within 24 h after injury, whereas significant upregulation of BMP-6 on the protein level was detected only 2-3 d after injury. Protein was confined to outermost suprabasal epidermal layers, whereas BMP-6-specific RNA was distributed throughout all epidermal layers including basal keratinocytes and the leading edge of the migrating keratinocytes. We also detected high levels of BMP-6-specific RNA and protein in chronic human wounds of different etiology. In contrast to the overall distribution pattern of BMP-6-specific RNA, the protein was not detected in keratinocytes directly bordering the wound. In order to test the influence of BMP-6 abundance on the progress of wound healing, we analyzed the wound response of transgenic mice overexpressing BMP-6 in the epidermis. In these mice, reepitheliazation of skin wounds was significantly delayed, suggesting that strict spatial and temporal regulation of BMP-6 expression is necessary not only for formation but also for reestablishment of a fully differentiated epidermis.     

Augmented inflammatory responses and altered wound healing in cathepsin G-deficient mice

BACKGROUND: Cathepsin G is a neutral serine proteinase that exists primarily in azurophilic granules of neutrophils, but also as a proteolytically active membrane-bound form. While the specificity and many in vitro biological activities have been described for cathepsin G, little is known about the role of this enzyme in neutrophil function in vivo, particularly as it applies to the wound-healing process. OBJECTIVE: To determine the role of cathepsin G in cutaneous tissue repair by examination of full-thickness incisional wound healing in mice with a null mutation for cathepsin G. METHODS: Paired, full-thickness linear incisions were made on the backs of cathepsin G +/+ and cathepsin G -/- mice, and wound tissue was harvested at days 1, 2, 3, 5, 7, 10, and 14 after wounding. Neutrophil influx, myeloperoxidase activity, and migration were examined using light microscopy, the myeloperoxidase assay, and modified Boyden chamber technique, respectively. Wound-breaking strength was measured using tensiometry. RESULTS: The absence of cathepsin G led to a 42% decrease in wound-breaking strength at day 7 after wounding (n=28; P<.002), which returned to the level of control mice by day 10 after wounding. Wound tissue sections in mice lacking cathepsin G also showed a 26% increase in neutrophil myeloperoxidase activity (n=12; P=.001) and an 18% increase in neutrophil influx (n=14; P=.002) at day 3 after wounding. Wound fluid collected on day 5 after wounding from cathepsin G-deficient mice attracted 58% more neutrophils than wound fluid collected from control mice (n=4; P<.05). CONCLUSIONS: Neutrophil cathepsin G is important during the early inflammatory stage of wound healing. Cathepsin G may be involved in processing 1 (or more) soluble mediator(s) in the wound milieu that is responsible for neutrophil chemotaxis. Our findings suggest that tight regulation of inflammation is necessary to prevent impaired healing during early tissue repair.     

Sequencing of double-stranded PCR products

Glutathione is a low molecular weight tripeptide that is a major intracellular antioxidant, modulates DNA synthesis, and may regulate signal transduction mechanisms. Our previous studies in rats suggested that intracellular stores of glutathione were sensitive to skin ischemia and, therefore, may regulate the early temporal course of wound healing. A 4-cm incision was placed on a rat's back and in vivo wound strength was measured over time. Animals were depleted of glutathione using L-buthionine-(S,R)-sulfoximine (BSO), an inhibitor of the enzyme gamma-glutamylcysteine synthetase. Some animals were treated in combination with allopurinol/BSO or with allopurinol alone. The data demonstrated at 4 days that BSO treatment produced a fourfold reduction in glutathione (3.51 +/- 1.78) over baseline (16.15 +/- 2.18) levels and twofold reduction (5.0 +/- 1.1) over untreated sham controls (11.1 +/- 2.3) (P < 0.05). Allopurinol provided no protection to glutathione levels. BSO treatment alone reduced wound burst strength compared to the other groups (P < 0.05). Allopurinol treatment enhanced wound strength over sham controls and BSO groups at 9 days after wounding (P < 0.05). Hydroxyproline content in wounds accumulated faster by Day 4 in the BSO-treatment groups compared to sham controls (P < 0.05), whereas the BSO-treatment groups had lower hydroxyproline levels measured at Day 6 (P < 0.05). These data provide the first evidence that wound healing is related to the temporal course of glutathione metabolism. The effect may not be related to oxidant stress since allopurinol provided enhanced wound burst strength without protecting wound glutathione levels.     

Cultured keratinocyte allografts and wound healing in severe recessive dystrophic epidermolysis bullosa

BACKGROUND: Patients with recessive dystrophic epidermolysis bullosa (RDEB) frequently have painful erosions that are slow to heal. There is no definitive treatment; therefore any therapy that improves wound healing would be beneficial to these patients. OBJECTIVE: Our purpose was to assess the effects of cultured allogeneic keratinocytes on wound healing in RDEB. METHODS: Ten patients with RDEB and dermatome-induced superficial dermal wounds were studied. Cultured keratinocyte grafts were applied to part of the wound, with another part left ungrafted. Both sites were assessed clinically and microscopically, particularly with regard to basement membrane zone reconstitution. RESULTS: Apart from minor differences in keratinocyte differentiation and a moderate analgesic effect induced by the graft, there were no other distinguishing findings in wound healing in the grafted and nongrafted sites. CONCLUSION: There was little clinical benefit from cultured keratinocyte allografts in wound healing in RDEB. However, this study showed that RDEB keratinocytes have an inherent capacity to express some type VII collagen epitopes transiently during wound healing, although this was not associated with the detection of anchoring fibrils.     

Enhancement of cell interactions with collagen/glycosaminoglycan matrices by RGD derivatization

The interaction of three cell types important to the wound repair process with collagen/glycosaminoglycan (GAG) dermal regeneration matrices covalently modified with an Arg-Gly-Asp (RGD)-containing peptide was characterized. Function-blocking monoclonal antibodies directed against various integrin subunits were used to demonstrate that human fibroblasts attached to the unmodified matrix through the integrin, alpha2beta1. Human endothelial cells and human keratinocytes, however, attached minimally to the unmodified matrix. After modification of the collagen/GAG matrix with RGD-containing peptide, endothelial cells and keratinocytes attached and spread well on the matrix. This attachment was RGD dependent as evidenced by essentially complete inhibition with competing soluble peptide. In terms of overall cell number, fibroblast cell attachment remained unchanged on the RGD peptide-modified matrix compared to the unmodified material. Antibody and peptide inhibition studies demonstrate, however, that attachment to the modified matrix was mediated by both alpha2beta1 and RGD-binding integrins. We have successfully introduced a specific RGD receptor-mediated attachment site on collagen/GAG dermal regeneration matrices, resulting in enhanced cell interaction of important wound healing cell types. This modification could have important implications for the performance of these matrices in promoting dermal regeneration.     

Basal membrane heparan sulphate proteoglycan expression during wound healing in human skin

Heparan sulphate proteoglycans (HSPGs) are integral components of the basement membrane (BM) in various tissues. HSPGs are important in the assembly and structure of the BM, and their putative functions include regulation of basement membrane permeability, binding of growth factors, and a role in cellular adhesion. In this study the expression of HSPG was examined during wound healing in human skin, using monoclonal antibodies (MAbs) that recognize the HSPG core protein and two different heparan sulphate (HS) epitopes, and the dynamics of HSPG expression were investigated in relation to epidermal cellular proliferation and permeability of the BM. Healing of excisional wounds in healthy volunteers was studied from day 0 up to 1 year. Intact human skin showed strong continuous staining of the dermo-epidermal BM and the vascular BM with all MAbs. Up to day 4 after wounding, staining for HSPG was absent under the ingrowing epidermis, with any of the MAbs, indicating that no complete BM was present. From day 7 onwards, the BM of the neo-epidermis showed positive staining for the HSPG core protein and a low sulphated HS epitope, and after day 14, the staining intensity was similar to normal skin. The staining patterns of these HSPG epitopes were similar to that of laminin. The staining pattern with a MAb against an epitope in the highly sulphated part of HS was found to be distinct from the other BM markers studied. This epitope was absent under the neo-epidermis up to 2 months after wounding. One year after wounding, the epitope was found to be present again. We observed that only in the time period between 2 months and 1 year had the epidermis normalized with respect to the number of cycling cells and the absence of high molecular weight plasma proteins. These findings suggest a correlation between normalization of epidermal proliferation, BM permeability, and regeneration of BM HS. It is proposed that complete BM maturation following skin wounding is a slow process and may account for the epidermal abnormalities that persist for a considerable period of time after wound healing.