Hypoxia increases reepithelialization via an αvβ6-dependent pathway

Reepithelialization is an essential step in successful cutaneous wound healing. Human keratinocytes, integral in this process, have been shown to have increased motility in the hypoxic healing edge of wounds correlating with the clinical success of semiocclusive hypoxic dressings, although the underlying mechanisms remain poorly understood. Subconfluent human keratinocyte cell monolayers were exposed to 1% hypoxia for up to 24 hours or control conditions. Re-oxygenation studies were performed up to 72 hours. Cellular alphav subunit and alphavbeta6 integrin expression was measured by flow cytometry. Migration scratch assays on fibronectin following hypoxic exposure were performed over 24 hours. Relative matrix metallo-proteinase (MMP)-2, 9 activity was determined by gelatin zymography with TIMP-1 levels assayed by enzyme-linked immunoassay. Sustained increases in alphav and alphavbeta6 expression were shown up to 48 hours in re-oxygenation studies (P < 0.001). Standardized scratch assays confirmed increased migration in the hypoxic group (P < 0.05). This effect was attenuated by the addition of a specific inhibitor of the alphavbeta6 integrin. MMP-2 and -9 activity was up-regulated following hypoxic exposure (P < 0.001; P < 0.05, respectively), whereas increased MMP expression was significantly retarded by addition of an alphavbeta6 inhibitor (P < 0.05). Migration on fibronectin was attenuated by a specific gelatinase inhibitor. We conclude that integrin alphavbeta6-dependent MMP-2 and -9 up-regulation is an important feature of increased migration in hypoxic human keratinocytes.     

Mice lacking β6 integrin in skin show accelerated wound repair in dexamethasone impaired wound healing model

Integrin αvβ6 is an epithelial-specific receptor that is absent from the healthy epidermis but synthesized de novo during wound repair. However, its function in wound repair is unknown. Integrin-mediated transforming growth factor-β1 (TGF-β1) activation is the main activation mechanism of this key cytokine in vivo. Impaired wound healing caused by glucocorticoids is a major clinical problem and is associated with a disturbed balance of TGF-β1 activity. Therefore, αvβ6 integrin-mediated regulation of TGF-β1 activity may be involved in this process. To determine the function of αvβ6 integrin in glucocorticoid-induced impaired wound healing, both β6 integrin-deficient (β6−/−) and wild-type mice were exposed to dexamethasone treatment. Multiple wound parameters, keratinocyte proliferation, inflammation, and TGF-β1 activation were assessed. Wound healing was significantly accelerated in the dexamethasone-treated β6−/− mice compared with the corresponding wild-type mice. The dexamethasone-treated β6−/− mice showed enhanced keratinocyte proliferation in both wound epithelium and hair follicles while the production of proinflammatory cytokines and TGF-β1 activation were reduced. Accelerated wound repair in the dexamethasone-treated β6−/− mice might be associated with the reduced antiproliferative and proinflammatory effects of TGF-β1. Inhibition of αvβ6 integrin may provide a future target for treatment of impaired wound healing.     

AlphaV beta3 (αvβ3) integrin inhibition reduces leukocyte–endothelium interaction in a pressure-induced reperfusion model

The leukocyte-endothelium interaction is known to contribute to reperfusion injury, which is considered to participate in the pathophysiology of pressure ulcers, and integrin alphaV beta3 (alphavbeta3) has been shown to mediate the processes of cellular adhesion in various types of cells. This study aims to clarify leukocyte behavior in our original microcirculatory pressure-induced reperfusion model, which can visualize the microcirculation in vivo. We also estimated the effect of alphavbeta3 integrin inhibition on the reduction of the leukocyte-endothelium interaction. Mice with dorsal skinfold chambers were divided into three groups: the baseline group (n=6), in which animals received no compression; the compression-reperfusion group (n=6), in which animals underwent 2-hour compression of the dorsal skin, followed by release, and the inhibitor-treated group (n=7), in which an alphavbeta3 inhibitor, CP4715, was administered in addition to the compression-release procedure. Staining with rhodamine 6G quantitatively visualized leukocyte behavior under the intravital fluorescent microscope. Compression-reperfusion induced a significant increase in rolling, sticking, and extravasation of the leukocytes. Treatment with the inhibitor strikingly reduced leukocyte sticking and extravasation. The present experiment has provided evidence that alphavbeta3 inhibition reduces leukocyte-endothelium interaction in our original pressure-induced reperfusion model.     

β-1 and β-2, but not α-1 and α-2, adrenoceptor blockade delays rat cutaneous wound healing

The sympathetic nervous system plays an important role in wound healing, but its mechanism of action is poorly understood. The aim of this study was to investigate the effects of β- and α-adrenoceptor blockade on cutaneous wound healing. Male rats were treated with propranolol (β1- and β2-antagonist), atenolol (β1-antagonist), or phentolamine (α1- and α2-antagonist) dissolved in drinking water. A full-thickness excisional lesion was created and the wound area was measured. Fourteen days after wounding, lesions and adjacent skin were removed, formalin-fixed, and paraffin-embedded. Sections were stained with hematoxylin–eosin and toluidine blue, and immunostained for α-smooth muscle actin and proliferating cell nuclear antigen. Wound contraction was delayed in propranolol- and atenolol-treated animals but not in phentolamine-treated animals. Reepithelialization was decreased only in propranolol-treated animals. β1- and β2-adrenoceptor blockade delayed leukocyte migration, epidermal and connective tissue cell proliferation, myofibroblastic differentiation, and mast cell migration. The volume density of blood vessels was increased in the propranolol- and atenolol-treated animals compared with controls. The levels of matrix metalloproteases (MMP-2 and MMP-9) decreased in the propranolol- and atenolol-treated animals. α1- and α2-adrenoceptor blockade only affected leukocyte migration, epithelial and connective tissue cell proliferation, and pro-MMP-9 levels. In conclusion, β-1 and β-2, but not α-1 and α-2, adrenoceptor blockade delays cutaneous wound healing.     

Localization of integrin β1, α1, α5 and α9 subunits in the rat testis

Very late activation ( VLA, β₁; α₁; α₅, α₉) integrins were studied by immunoblotting and immunohistochemistry in the testes of sexually mature rats. All integrin subunits were present in membrane fractions of homogenized testes. Immunohistochemistry revealed that the anti β₁ antibody recognized peritubular cells and the basement membrane of blood vessels. Immunoreactivity was also demonstrated in the lamina propria, basement membrane, and the basal cytoplasm of Sertoli cells. In elongating spermatids, β₁ integrin was localized to the acrosome. The α₁ subunit was expressed in peritubular cells and in the lamina propria. In the adluminal compartment, round spermatids were stained diffusely for the α₁ subunit. Immunoreactivity for α₁ integrin was found additionally in the acrosomes of elongating spermatids shortly before their release into the seminiferous tubule lumen. The α₅ subunit was expressed in the acrosomes of elongating spermatids as well as in their distal cytoplasm during stages III–VI; the cytoplasmic lobes of elongate spermatids and/or residual bodies also appeared to be immunostained in seminiferous tubules at stages VII–VIII. The α9 subunit was immunolocalized only in the basement membrane and in peritubular cells. These data suggest that integrins are involved in spermatogenesis, in particular in the process of spermatid maturation.     

Aminated β-1,3-d-glucan improves wound healing in diabetic db/db mice

Delayed wound healing in diabetes is caused by neuropathy, vascular changes, and impaired cellular response to the injury. Macrophages are crucial in normal wound healing, and impaired functions of these cells have been shown in diabetes. beta-1,3-D-glucans stimulate macrophage function. This open-label study was performed to see if aminated beta-1,3-D-glucan (AG) stimulates wound healing in diabetes. Four groups (1-4) of diabetic db/db mice and one nondiabetic control group, db/+(5) were studied: group 1 (n=11): topical AG; group 2 (n=10): topical AG and subcutaneous insulin; group 3 (n=14): topical placebo and subcutaneous insulin; group 4 (n=10): diabetic control (placebo); group 5 (n=12): normal control (placebo). At the end of the experiments fasting blood glucose and A1C were (mean +/- SE) as follows: Group 1: 30.5 +/- 1.9 mmol/L and 11.3 +/- 0.6%; group 2: 12.0 +/- 1.7 mmol/L and 8.0 +/- 0.6%; group 3: 15.4 +/- 2.4 mmol/L and 7.4 +/- 0.3%; group 4: 32.6 +/- 2.6 mmol/L and 12.3 +/- 0.6%; group 5: 7.2 +/- 0.4 mmol/L and 3.9 +/- 0.04%, respectively. The closed wound area was the same in group 1 (AG alone) and group 2 (AG plus insulin) after 17 days, 57.3 +/- 4.7 vs. 50.1 +/- 4.9% (p=0.7).The results of these two groups were superior to group 3 (insulin treatment alone, 32.0 +/- 4.3%, p<0.001) and diabetic controls (38.2 +/- 5.1%, p=0.001). The macrophage-stimulant AG improves wound healing in db/db mice.     

Acute Cellular Rejection Following Human Heart Transplantation is Associated with Increased Expression of Vitronectin Receptor (Integrin αvβ3)

The vitronectin receptor (integrin alphavbeta3), a cell-surface adhesion receptor, has been shown to play a significant role in endothelial cell migration, apoptosis, atherosclerosis, and T-lymphocyte activation. This study was undertaken to test the hypothesis that cardiac allograft rejection is associated with increased expression of alphavbeta3. We also determined whether fibronectin receptor (alpha5beta1) and tissue factor are up-regulated in the presence of acute cellular rejection. We evaluated endomyocardial biopsy specimens with histologic evidence of different degrees of acute cellular rejection (grade 0, n = 10; grade 1A, n = 10; grade 2, n = 10; grade 3A, n = 10). Biopsies were obtained 2-4weeks after cardiac transplantation. Immunoperoxidase staining was performed for alphavbeta3, tissue factor, and alpha5beta1, and protein levels were further determined by Western blot analysis. Specimens with grade 2 and grade 3A rejection showed positive staining of alphavbeta3 in lymphocytic aggregates and vascular endothelial cells. By immunoblotting, we identified significantly increased expression of alphavbeta3 in the presence of acute rejection, grade 2 (3-fold, p = 0.01) and grade 3A (3.6-fold, p = 0.005) compared to grade 0 and 1 A specimens. There was no evidence of increased expression of alpha5beta1 or tissue factor. Acute cellular rejection, a process characterized by T-lymphocyte activation and release of inflammatory cytokines, is associated with increased expression of alphavbeta3.     

On Some Glycosidases (β-D-Mannosidase, α-L-Fucosidase, N-Acetyl- β-D-Glucosaminidase) of Human Seminal Plasma/Über einige Glykosidase-Aktivitäten (β-D-Mannosidase, α-L-Fukosidase und N-acetyl-β-D-Glukosaminidase) des menschlichen Spermaplasmas

The glycosidase activities (beta-D-mannosidase, alpha-L-fucosidase and N-acetyl-beta-D-glucosaminidase) have been compared in whole and split ejaculate samples from men whose couple suffers from infertility. The site of secretion of enzymatic activities is either prostatic or epididymal. The three enzymatic activities have possibly different origin and should constitute new biochemical markers of male genital tract.     

Tissue expression of transforming growth factor-β1 and transforming growth factor-α during wound healing in human skin explants

In the dynamic and complex process of wound healing, locally produced growth factors are important mediators, although their actual roles have not been fully established. In the present study, the presence of transforming growth factor-beta1 and -alpha during the re-epithelialization of full-thickness wounds was investigated in an in vitro model of wound healing in human skin. The amounts of transforming growth factor-beta1 and -alpha secreted from the wound area were measured with enzyme immunoassays, and immunohistochemistry was used to study the localization of these two growth factors in the healing wound. The wounds were followed until they were completely re-epithelialized. The results showed a continuous increase in secreted transforming growth factor-beta1 throughout the re-epithelialization phase of healing followed by a decrease after its completion. The keratinocytes migrating out from the wound edges showed intense staining for transforming growth factor-beta1 which declined to the level of the surrounding epidermis after the wound was covered by a new epidermis. After the skin was wounded, a decrease both in secreted transforming growth factor-alpha and in immunostaining for this growth factor was apparent. Even though a minor increase in the immunoreactivity for transforming growth factor-alpha occurred after the completion of re-epithelialization, no increase in secreted transforming growth factor-alpha could be detected by enzyme immunoassay. These data suggest that keratinocytes modulate their expression of transforming growth factor-beta1 and -alpha during the wound healing process in human skin and that these changes may be controlled in part by autocrine pathways.     

MMP-3 plays a major role in calcium pantothenate-promoted wound healing after fractional ablative laser treatment

Lasers in Medical Science - Ablative fractional laser treatment leads to a loss of matrix metalloproteinase-3 (MMP-3) expression; therefore, in the present in vitro study, we addressed the role of...     

Incisional wound healing in transforming growth factor-β1 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.     

The role of lymphocytes in wound healing

In the past, lymphocytes have usually been associated with chronic inflammatory conditions and only recently have animal experiments indicated a possible role in wound healing. The present paper describes a study of lymphocytes in human wounds and scars using monoclonal antibody stains. The results suggest that T lymphocytes may play an important regulatory role in wound healing and scar formation.     

The role of iNOS in wound healing

BACKGROUND: We have previously shown that the blockade of nitric oxide (NO) synthesis impairs wound healing, in particular collagen synthesis. Conversely, impaired wound healing is accompanied by decreased wound NO synthesis. Fibroblast collagen synthesis, proliferation, and fibroblast-mediated matrix contraction are critical to wound healing. We examined the wound healing-related phenotypic changes that are induced by the loss of inducible nitric oxide synthase (iNOS) gene function in fibroblasts. METHODS: Dermal fibroblasts were obtained from 8- to 12-week-old iNOS--knock out (KO; C57BL/Ai-[KO] Nos2 N5) and wild type mice by an explant technique and used after 1 to 3 passages. Proliferation ([(3)H]-thymidine incorporation) and collagen synthesis ([(3)H]-proline incorporation into collagenase-sensitive protein) were studied after stimulation with 10% fetal bovine serum. Matrix remodeling was assessed by the measurement of the contraction of fibroblast-populated collagen lattices. RESULTS: iNOS-KO fibroblasts proliferated more slowly, synthesized less collagen, and contracted fibroblast-populated collagen lattices more slowly than wild-type fibroblast. Collagen synthesis was restored to normal in KO fibroblasts in response to NO donors (s-nitroso-N-acetylpenicillamine). CONCLUSIONS: iNOS deficiency causes significant impairment in wound healing-related properties of fibroblasts, which suggests that NO plays an important role in wound healing.     

The role of hyaluronan in wound healing

The polysaccharide hyaluronan (HA) (synonyms – hyaluronic acid, hyaluronate) is a versatile, polymorphic, glycosoaminoglycan with vast biological functions. HA is found throughout the body, primarily residing in skin, thus playing an important role in wound healing. Research regarding HA's function has changed over the years, primarily focussing on a particular aspect or function. The contribution of HA in each stage of normal wound healing as well as its clinical wound dressing applications will be examined.     

The role of growth factors in wound healing

Growth factors are mediators with essential importance for undisturbed repair process after wounding. The well coordinated concert of these substances is necessary for healing with complete restoration of function and morphology. These complex mechanisms are disturbed during secondary and delayed repair. The result is protracted healing course and inferior scar quality--either hypo- or hypertrophic. Local and systemic application of these growth factors seems to add important instruments for therapeutic use in the treatment of chronic wounds. Knowledge from experimental research is encouraging, although the exact mechanisms of synergistic action are not completely understood. However, the results from clinical use in controlled studies do not meet these expectations by far. The main reasons for this dilemma are thought to be little understanding in the complex interactions of these substances. In fact, different wound entities seem to reveal different cytokine profiles during the course of repair. Further intensive research therefore is required for the rational use of growth factors in the clinical setting.     

NO在创伤愈合过程中的作用

创伤修复是损伤组织恢复其完好结构的必需过程，大致分为三个阶段：①局部炎症反应阶段：②细胞增殖分化及肉芽组织形成阶段：③组织重建阶段。有研究发现包括一氧化氮（nitric oxide，NO）在内的小分子自由基对伤口的良好愈合起到了关键作用。现就NO在创伤修复过程中的作用作以阐述。     

The prognostic value of E-cadherin, α-, β- and γ-catenin in bladder cancer patients who underwent radical cystectomy

OBJECTIVES: To determine the value of the loss of expression of E-cadherin and cadherin associated molecules as useful markers for both prognosis and chemosensitivity in bladder cancer patients who have undergone radical cystectomy. PATIENTS AND METHODS: In 55 paraffin embedded specimens of radical cystectomy at our hospital from 1982 to 2000, the expression of E-cadherin, alpha-, beta- and gamma-catenin was examined by immunohistochemical staining. To evaluate the prognostic significance of these molecules, Kaplan-Meier survival curves were constructed and a statistical analysis was calculated by a log-rank test. A multivariate test (tumor stage, tumor grade, lymph node metastasis, configuration, the expression of E-cadherin, alpha-, beta- and gamma-catenin) was performed to detect prognostic markers. RESULTS: Normal expression was found in 33 cases (60.0%) for E-cadherin, 29 (52.7%) for alpha-catenin, 31 cases (56.4%) for beta-catenin, and 31 cases (56.4%) for gamma-catenin. The expression patterns for E-cadherin, alpha-, beta- and gamma-catenin were significantly correlated with each other (P < 0.01). Survival analysis showed a significant difference between normal and aberrant expression in each staining. A multivariate analysis revealed that the expression of alpha- catenin was an independent prognostic factor (P = 0.0191). In 23 patients that received adjuvant chemotherapy, there was a significant difference in survival between the normal and aberrant expression of alpha-catenin, but not other molecules. CONCLUSION: Alpha-catenin may not only be a good prognostic marker, but also one of key molecules that determine the chemosensitivities in patients with invasive bladder cancer.     

Biochemical modulation of 5-fluorouracil with interferon α/β and γ on murine renal cell carcinoma

BACKGROUND: Conventional therapy for renal cell carcinoma (RCC) using interferon (IFN) has shown limited antitumor action. The purpose of the present study was to investigate the synergistic antitumor effects of IFN and 5-fluorouracil (5-FU) and to elucidate the mechanism underlying antitumor effects. METHODS: The antitumor effects and biochemical modulation of murine IFN and 5-FU were determined using murine renal cell carcinoma (RENCA). The activity of thymidylate synthase, thymidine kinase and the concentration of 5-FU incorporated into RNA was measured using cytosolic extracts of tumors. RESULTS: Triple combination therapy (5-FU, IFN alpha/beta and IFN gamma) showed a synergistic antitumor effect on RENCA tumors, because triple combination therapy suppressed growth significantly compared to combination therapy (IFN alpha/beta and IFN gamma, P = 0.0258) and 5-FU (P < 0.0001). Total thymidylate synthase was decreased by triple combination therapy (5-FU, IFN alpha/beta and IFN gamma, P = 0.0019) and combination therapy (5-FU and IFN gamma, P = 0.0018) compared to 5-FU alone. Thymidine kinase activity was decreased by triple combination therapy (5-FU, IFN alpha/beta and IFN gamma, P < 0.0001) and combination therapy (5-FU and IFN alpha/beta, P < 0.0001) compared to 5-FU alone. The concentration of 5-FU incorporated into RENCA tumors was increased by triple combination therapy (P = 0.0132) and combination therapy (5-FU and IFN alpha/beta, P = 0.0124) compared to 5-FU alone. CONCLUSIONS: Interferons alpha/beta and gamma showed different biochemical modulation for 5-FU. Therefore, combination therapy using 5-FU and IFN showed synergistic antitumor effects on murine RCC.     

The role of mast cells in wound healing

Mast cells are predominantly found in the vicinity of connective tissue vessels of skin and mucosa. The main immunological functions of mast cells are in IgE‐mediated reactions and in helminth infestations. Mast cells respond to tissue injury by releasing inflammatory mediators and have been implicated in diseases of excessive fibrosis of the dermis such as scleroderma. Current evidence suggests that mast cells exert its role during inflammation and cellular proliferation. Animal models have shown that by stabilising mast cells at the early stages of wound healing, wound contraction is reduced. Mast cells are an ideal candidate to play a pivotal role in wound healing due to its location, substances released and clinical associations.