Lack of collagen XVIII accelerates cutaneous wound healing, while overexpression of its endostatin domain leads to delayed healing

Endostatin, the C-terminal fragment of collagen XVIII, is known to suppress tumour growth and angiogenesis by inhibiting endothelial cell proliferation and migration. We have previously shown that endostatin and its precursor are important for the structural organization of basement membranes (BM). The aim of this study was to investigate cutaneous wound healing in mice overexpressing endostatin in keratinocytes (ES-tg) and in mice lacking collagen XVIII (Col18a1(-/-)). Excisional wounds were made on the dorsal skin of mice, the wound areas were measured and the wounds were collected for further analyses after 3, 6 or 14 days. The healing of the wounds was delayed in the ES-tg mice and accelerated in the Col18a1(-/-) mice, and the vascularisation rate was accelerated in the Col18a1(-/-) mice, but not affected in the ES-tg mice. Abnormal capillaries with swollen endothelial cells and narrowed lumens were observed in the wounds of the ES-tg mice. In these mice also the formation of the epidermal BM was delayed, and the structure of the epidermal and capillary BMs was more disorganised. Moreover, detachment of the epidermis from the granulation tissue was observed in half (n=10) of the 6-day-old ES-tg wounds, but in none of the controls, suggesting an increased fragility of the epidermal-dermal junction in the presence of an excess of endostatin.     

Increased transglutaminase activity during skin wound healing in rats

Outer, middle and inner layers from wounded or unwounded rat dorsal skin were separated and extracted first with buffer and then with Triton X-100 and dithiothreitol. The extracts and residues were assayed for transglutaminase activity and tissue transglutaminase antigen. Transglutaminase activities in all skin layers are increased in the period 1-5 days after wounding. Most of the increased activity is in the buffer-soluble fraction in the inner skin layer though there is no corresponding increase in antigen in this fraction. This suggests that there is production of activated soluble tissue transglutaminase in the wounded inner layer. In the 3-5 day wounded outer layer the largest fraction of both activity and antigen is associated with the insoluble residue remaining after extraction with Triton X-100. On DEAE-cellulose chromatography Triton X-100 extracts of the inner layer of wounded skin showed a single major peak of activity, corresponding approximately with rabbit liver transglutaminase; the outer layer showed the same peak plus a different one, eluting at lower salt concentration, which is thought to be epidermal transglutaminase.     

Hemidesmosomes and anchoring fibril collagen appear synchronously during development and wound healing

Bullous pemphigoid antisera and monoclonal antibodies to type VII collagen were used to localize hemidesmosomes and anchoring fibrils, respectively, in tissues of developing eyes and healing corneal wounds of New Zealand white rabbits. In the 17-day fetal rabbit eye, both antibodies colocalize to the epithelial-stromal junction of the lid and conjunctival region, but neither binds to the cornea, and electron microscopy demonstrates hemidesmosomes only where the antibodies bind. By 20 days of fetal development, the antibodies colocalize in cornea, and, by electron microscopy, hemidesmosomes are shown to be present as well. In healing 7-mm corneal wounds, both antibodies colocalize at the wound periphery within 66 h. By electron microscopy, hemidesmosomes along small segments of basal lamina are also shown to be present at the wound periphery at this time. These demonstrations of the synchronous assembly of hemidesmosomes and anchoring fibrils support the hypothesis of linkage of hemidesmosomes through the basement membrane to anchoring fibrils.     

Energy metabolism during cutaneous wound healing in immunocompromised and aged rats

Cutaneous cells primarily depend upon carbohydrate metabolism for their energy requirement during healing process. But, it may be greatly hampered during various pathological and altered physiological conditions. The present study was therefore undertaken to investigate the intermediate steps of energy metabolism by measuring enzyme activities in the granulation tissues of immunocompromised and aged rats following excision-type of cutaneous injury. The activities of key regulatory enzymes hexokinase (HK), phosphofructokinase (PFK), lactate dehydrogenase (LDH), citrate synthase (CS) and glucose-6 phosphate dehydrogenase (G6PD) have been monitored in the wound tissues of immunocompromised and aged rats at different time intervals (2, 7, 14 and 21 days) of postwounding. The activities of HK and CS were found significantly decreased both in immunocompromised and aged rats as compared to control subjects. However G6PD exhibited an elevated activity at early stage followed by a decreased activity at later phase of healing both in immunocompromised and aged rats. The PFK and LDH demonstrated an upward trend in immunocompromised rats but a decreasing trend in aged rats. Thus, the results suggest that significant alterations in the activities of energy metabolizing enzymes in the granulation tissues in both immunocompromised as well as in aged rats may overall affect the energy availability for cellular activity needed for repair process. Hence, this may perhaps be one of the factor responsible for impaired healing in these subjects.     

Matrigel increases the rate of split wound healing and promotes keratinocyte `take' in deep wounds in rats

The influence of matrigel, a mixture of the components of thebasement membrane, on the wound healing was studied in a modelof experimental wounds in rats. Matrigel was found to increasethe rate of epithelization of split-thickness wounds. The modelof deep wound was developed in which the host animal could notprovide enough migrating and proliferating keratinocytes tocover the wound area. The model is relevant to severe burns andinjuries in humans. When rat keratinocyte suspension wastransplanted into deep wounds, cell retention in the wound bedwas only observed if matrigel was added together with the cells.Increasing matrigel concentration in the wound was seen toenhance the rate of wound area coverage by the cells. Althoughthe process of healing seemed macroscopically normal, afterhistological screening of the biopsies cell in the wouldappeared as amorphous aggregates and tubules rather thenstratified epidermis.     

Effect of fibroblast implants on wound healing of irradiated skin: assay of wound strength and quantitative immunohistology of collagen

The role of dermal fibroblasts in the expression of radiation-induced damage to the skin was studied. Fibroblasts from neonatal mice were cultured, harvested, and injected into full-depth surgical incisions in the dorsal area of mouse skin, which had been previously locally irradiated by 18 Gy X rays. As a control, cells irradiated with a dose of 20 Gy were also injected. The effect of radiation and fibroblast implants on the gain of skin wound strength was assayed. In an additional experiment freshly isolated cells were implanted. Two weeks following wounding the irradiated skin had reached only about a third of the strength of unirradiated skin. A significant increase of wound strength in irradiated skin was observed when 1.5-2 x 10(6) cultured fibroblasts or freshly isolated fibroblasts were injected into the 20-mm-long wound bed. Irradiated cells had significantly less effect. This suggests that implanting isolated syngeneic cells may "rescue" wounds from the effect of prior irradiation. Semiquantitative immunohistology of types I and III collagen was performed in parallel using a video image digitizing system. Levels of both types I and III collagen were altered in the dermis and the wound tissues in irradiated skin, but the implant of cultured fibroblasts did not affect notably the total levels and the disposition of the two collagen isotypes.     

Role of fibroblast migration in collagen fiber formation during fetal and adult dermal wound healing

Adult dermal wounds, in contrast to fetal wounds, heal with the formation of scar tissue. A crucial factor in determining the degree of scarring is the ratio of types I and III collagen, which regulates the diameter of the combined fibers. We developed a reaction-diffusion model which focuses on the control of collagen synthesis by different isoforms of the polypeptide transforming growth factor-β (TGFβ). We used the model to investigate the current controversy as to whether the fibroblasts migrate into the wound from the surrounding unwounded dermis or from the underlying subcutaneous tissue. Numerical simulations of a spatially independent, temporal model led to a value of the collagen ratio consistent with that of healthy tissue for the fetus, but corresponding to scarring in the adult. We investigated the effect of topical application of TGFβ and show that addition of isoform 3 reduces scar tissue formation, in agreement with the experiment. However, numerical solutions of the reaction-diffusion system do not exhibit this sensitivity to growth factor application. Mathematically, this corresponds to the observation that behind healing wavefront solutions, a particular healed state is always selected independent of transients, even though there is a continuum of possible positive steady states. We explain this phenomenon using a caricature system of equations, which reflects the key qualitative features of the full model but has a much simpler mathematical form. Biologically, our results suggest that the migration into a wound of fibroblasts and TGFβ from the surrounding dermis alone cannot account for the essential features of the healing process, and that fibroblasts entering from the underlying subcutaneous tissue are crucial to the healing process.     

The effect of disodium cromoglycate on the skin wound healing and collagen content in the wounds of rats

The effect of disodium cromoglycate on skin wound healing and collagen formation in the wounds was studied. Disodium cromoglycate (a mast cell stabilizer) administered to the rats in a dose of 2 mg/animal was found to retard wound healing and markedly increased wound surface in all examined days (3rd, 5th, 7th, 10th, 14th day of healing). The mast cell stabilizer injected directly into wounds decreased collagen content, especially on 10th and 14th day of the healing process.     

Energy metabolism in the granulation tissue of diabetic rats during cutaneous wound healing

The skin cells chiefly depend on carbohydrate metabolism for their energy requirement during cutaneous wound healing. Since the glucose metabolism is greatly hampered in diabetes and this might affect wound repair process. This prompted us to investigate the intermediate steps of energy metabolism by measuring enzyme activities in the wound tissues of normal and streptozotocin-induced diabetic rats following excision-type of cutaneous injury. The activities of key regulatory enzymes namely hexokinase (HK), phosphofructokinase (PFK), lactate dehydrogenase (LDH), citrate synthase (CS) and glucose-6 phosphate dehydrogenase (G6PD) have been monitored in the granulation tissues of normal and diabetic rats at different time points (2, 7, 14 and 21 days) of postwounding. Interestingly, a significant alteration in all these enzyme activities was observed in diabetic rats. The activity of PFK was increased but HK, LDH and CS showed a decreased activity in the wound tissue of diabetics as compared to normal rats. However G6PD exhibited an elevated activity only at early stage of healing in diabetic rats. Thus, the results suggest that significant alterations in the activities of energy metabolizing enzymes in the wound tissue of diabetic rats may affect the energy availability for cellular activity needed for repair process and this may perhaps be one of the factor responsible for impaired healing in these subjects. (Mol Cell Biochem 270: 71–77, 2005)     

Changes in skin angiotensin II receptors in rats during wound healing.

Angiotensin (AII) is associated with increased vascular smooth muscle growth and we have found increased levels of tissue AII during healing of wounded skin. Here we have determined changes in skin AII receptors during wound healing in adult male Sprague-Dawley rats. An abdominal surgical incision was made under anesthesia and rats were sacrificed at different times after wounding. Specific binding of 125I-AII was significantly decreased at 12, 18 and 24 hours in the wounded tissue compared to control tissue from the same rat. By 3 days the binding had recovered to baseline levels. Receptors were mostly AT1, with a high and a low affinity site in the skin both in control and healing tissue. The Bmax of the high affinity site was significantly decreased in healing tissue but there was no significant change in Kd. Our results demonstrate that adult rat skin contains predominantly AT1 receptors and also that these receptors are downregulated for 12-24 hours after wounding.     

Organization of collagen bundles during tendon healing in rats treated with L-NAME

The Achilles tendon can support high tension forces and may experience lesions. The damaged tissue does not regenerate completely, with the organization and mechanical properties of the repaired tendon being inferior to those of a healthy tendon. Nitric oxide (NO) plays an important role in wound repair. We have examined the structural reorganization and repair in Achilles tendon after injury in rats treated with the NO synthase inhibitor L-NAME. The right Achilles tendon of male Wistar rats was partially transected. One group of rats was treated with L-NAME (~300 mg/kg per day, given in drinking water) for 4 days prior to tendon sectioning and throughout the post-operative period. Control rats received water without L-NAME. The tendons were excised at 7, 14, and 21 days post-injury and used to quantify hydroxyproline and for mechanical tests. Tendons were also processed for histomorphological analysis by polarized light microscopy, which showed that the collagen fibers were disorganized by day 7 in non-treated and L-NAME-treated rats. In non-treated rats, the organization of the extracellular matrix was more homogeneous by days 14 and 21 compared with day 7, although this homogeneity was less than that in normal tendon. In contrast, in injured tendons from L-NAME-treated rats, the collagen fibers were still disorganized on day 21. Tendons from treated rats had more hydroxyproline but lower mechanical properties compared with those from non-treated rats. Thus, NO modulates tendon healing, with a reduction in NO biosynthesis delaying reorganization of the extracellular matrix, especially collagen. T.C.T. and W.R.N were supported by studentships from CAPES, and S.H. was supported by a research fellowship from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).     

Efficacy of l-proline administration on the early responses during cutaneous wound healing in rats

Proline (Pro) plays a versatile role in cell metabolism and physiology. Pro and hydroxypro are major imino acids present in collagen, an important connective tissue protein, essential for wound healing, which is a primary response to tissue injury. This study explains the role of l-pro on cutaneous wound healing in rats when administered both topically and orally. Open excision wounds were made on the back of rats, and 200 μl (200 mg) of pro was administered topically and orally once daily to the experimental rats until the wounds healed completely. The control wounds were left untreated. Granulation tissues formed were removed after day 4 and 8 of post excision wounding, and biochemical parameters such as total protein, collagen, hexosamine, and uronic acid were estimated. Levels of enzymatic and non-enzymatic antioxidants such as catalase, superoxide dismutase, glutathione peroxidase, ascorbic acid, and reduced glutathione were evaluated along with lipid peroxides in the granulation tissues. Tensile strength and period of epithelialization were also measured. It was observed that the treated wounds healed very fast as evidenced by augmented rates of epithelialization and wound contraction, which was also confirmed by histological examinations. The results strappingly authenticate the beneficial effects of the topical administration of l-proline in the acceleration of wound healing than the oral administration and control.     

Time-dependent expression and distribution of Egr-1 during skeletal muscle wound healing in rats

Recent studies have shown that early growth response factor-1 (Egr-1) plays an important role in regulation of inflammation and tissue repair, but little is known about its expression after trauma to skeletal muscles. A preliminary study on time-dependent expression and distribution of Egr-1 was performed by immunohistochemistry, immunofluorescence and Western blotting during skeletal muscle wound healing in rats. An animal model of skeletal muscle contusion was established in 45 Sprague-Dawley male rats. Samples were taken at 6 h, 12 h, 1 day, 3 days, 5 days, 7 days, 10 days, 14 days and 21 days post-injury, respectively (5 rats in each posttraumatic interval). 5 rats were employed as control. In the uninjured controls, Egr-1 positive staining was observed in the sarcoplasm and nuclei of normal myofibers. In wounded specimens, a small number of polymorphonuclear cells (PMNs), a number of mononuclear cells (MNCs), fibroblastic cells (FBCs) and regenerated multinucleated myotubes showed positive reaction for Egr-1 in contused zones. By morphometric analysis, an increase in Egr-1 expression was verified at inflammatory phase after contusion, which reached a peak in the regenerated phase overlapping with the fibrotic phase during skeletal muscle wound healing. The expression tendency was further confirmed by Western blotting assay. By immunofluorescent staining for co-localization, the Egr-1-positive MNCs and FBCs in wounds were identified as macrophages and myofibroblasts. The results demonstrate that the expression of Egr-1 is up-regulated and temporally distributed in certain cell types after trauma to skeletal muscles, which may be closely involved in inflammatory response, fibrotic repair and muscle regeneration during skeletal muscle wound healing.