Abstract Idiopathic usual interstitial pneumonia (UIP) and bronchiolitis obliterans organizing pneumonia (BOOP) are fibrous pulmonary disorders in both of which there is newly formed connective tissue in distal air spaces. UIP is a progressive and usually fatal lung disease without any efficient treatment, while the prognosis of BOOP is good. In both diseases, an injury of the alveolar epithelium and its basement membrane (BM) leads to migration of fibroblasts and myofibroblasts into air spaces and production of extracellular matrix by these cells. In UIP, the newly formed intraluminal connective tissue lesions cause fusion of alveolar structures and interstitial remodeling, while in BOOP the newly formed connective tissue may resolve completely. One of the major aims of the research on pulmonary fibrosis is to define the mechanisms that lead to persistence of the newly formed connective tissue and thus to irreversible fibrosis in UIP. The aim of the present study was to compare the extent of capillarization, apoptotic activity and re-epithelialization of the newly formed connective tissue in BOOP and UIP. The number of capillaries per tissue surface area was measured. Furthermore, the expression of angiogenic growth factors vascular endothelial growth factor-A (VEGF-A) and basic fibroblast growth factor (bFGF) was evaluated in the same areas, in addition to the expression of Flt-1 and Flk-1, which serve as receptors for VEGF. Apoptotic activity was analyzed using TUNEL-method, and the immunohistochemical expression of apoptosis regulating proteins bcl-2, mcl-1, and bax was studied. Finally, the extent of re-epithelialization was studied with the immunohistochemical and ultrastructural localization of laminin-5 γ2 chain, and the sites of synthesis of laminin-5 γ2 chain mRNA. In BOOP, an efficient repair process with good capillarization along with high expression of VEGF and bFGF, and orderly re-epithelialization of the newly formed connective tissue takes place after lung injury. The apoptotic activity of the newly formed connective tissue is also high, presumably leading to resolution of the intraluminal connective tissue in BOOP. In UIP, the newly formed connective tissue showed poor capillarization, inadequate re-epithelialization and low apoptotic activity. The results suggest disturbed or delayed repair process in UIP, contributing to irreversible interstitial fibrosis and remodeling.

Large doses of recombinant growth factors formulated in solution form directly injected into the body is usual clinical practice in treating second-degree scald injuries, with promising results, but this approach creates side effects; furthermore, it may not allow appropriate levels of the factor to be sensed by the target injured tissue/organ in the specific time frame, owing to complications arising from regeneration. In this research, two delivery methods (infusion pumping and local topical application) were applied to deliver recombinant human erythropoietin (rHuEPO) for skin regeneration. First, rHuEPO was given in deep second-degree scald injury sites in mice by infusion pump. Vascularization was remarkably higher in the rHuEPO pumping group than in controls. Second, local topical application of rHuEPO gel was given in deep second-degree scald injury sites in rats. Histological analysis showed that epithelialization rate was significantly higher in the rHuEPO gel-treated group than in controls. Immunohistochemical studies showed that the rHuEPO gel-treated group showed remarkably higher expression of skin regeneration makers than the control group. An accurate method for visualization and quantification of blood vessel networks in target areas has still not been developed up to this point, because of technical difficulties in detecting such thin blood vessels. A method which utilizes a series of steps to enhance the image, removes noise from image background, and tracks the vessels edges for vessel segmentation and quantification has been used in this study. Using image analysis methods, we were able to detect the microvascular networks of newly formed blood vessels (less than 500 μm thickness), which participate in the healing process, providing not only nutrition and oxygen to grow tissues but also necessary growth factors to grow tissue cells for complete skin regeneration. The rHuEPO-treated group showed higher expression of stem cell markers (CD 31, CD 90, CD 71, and nestin), which actively contribute to in-wound-healing processes for new hair follicle generation as well as skin regeneration. Collectively, both rHuEPO group pumping into the systemic circulation system, and injection into the local injury area, prompted mice and rats to form new blood vessel networks in scald injury sites, which significantly participate in the scald healing process. These results may lead to the development of novel treatments for scald wounds.

Cutaneous wound healing is a complex process involving the migration of inflammatory cells to the wound site, deposition of extracellular matrix, and the reestablishment of an intact epithelial barrier. Re-epithelialization depends on the proliferation and directional migration of keratinocytes from the wound edges. Initially, keratinocytes migrate over a provisional wound matrix that is rich in fibronectin, and as the wound heals the provisional matrix becomes replaced by one consisting of collagen and proteoglycans. Re-epithelialization is tightly regulated by a variety of peptides such as growth factors, cytokines and proteases, and abnormalities may result in chronic non-healing wounds or hypertrophic scars. CCN2 (Connective Tissue Growth Factor) is a multifunctional protein with effects on cells and their interactions with the connective tissue. CCN2 is expressed in a variety of cell types and regulates numerous cell functions including proliferation, differentiation, adhesion, migration and stimulation of collagen production. While the importance of CCN2 for the fibrotic response has been well studied, its involvement in keratinocyte function has not yet been fully explored. Using an in vivo wound model, the expression of CCN2 was captured at the leading keratinocyte edge during re-epithelialization. In vitro, exogenous addition of CCN2 to human keratinocyte cultures promoted keratinocyte migration. Subsequently, integrin a5b1 was identified as an important mediator of CCN2 enhancement of keratinocyte adhesion to fibronectin. CCN2 activated the FAK-MAPK signaling pathway, and pretreatment with MEK1 specific inhibitor PD98059 markedly reduced CCN2-promoted keratinocyte migration. In vitro, CCN2 expression was induced by TGF-β1. Compared with inhibiting the SMAD pathway, blocking MAPK was more effective in reducing TGF-β1-induced CCN2 mRNA and protein expression. In addition, CCN2-induced keratinocyte spreading required FAK. Treatment with CCN2 led to actin disassembly and altered the activity of the Rho proteins and p190RhoGAP in keratinocytes. Furthermore, Cdc42 mediated CCN2-induced cell polarity. In conclusion, using in vivo and in vitro models, CCN2 was shown to regulate keratinocyte function by promoting keratinocyte adhesion, spreading and migration. A complete understanding of CCN2 expression in keratinocytes is crucial in order to develop novel therapies for wound healing.

Made available in DSpace on 2014-06-11T19:27:48Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-02-03Bitstream added on 2014-06-13T18:31:58Z : No. of bitstreams: 1 conceicao_lf_me_jabo.pdf: 2281445 bytes, checksum: c6ad72b6b6c930106b1b1fa9f6e7ea2a (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Estudou-se os efeitos do cetorolaco de trometamina 0,5%, sem conservante, sobre a inflamação ocular, avaliando-se a reparação epitelial e a sensibilidade corneal, em coelhos submetidos à ulceração corneal química com hidróxido de sódio (NaOH) 1 mol/L. Constituíram-se dois grupos (n=12). O primeiro grupo recebeu 30 μl de cetorolaco de tometamina 0,5%, sem conservante (OT), a cada seis horas, em uma das córneas ulceradas e 30 μl de solução salina fisiológica 0.9% (OC) na córnea adelfa, totalizando 4 aplicações diárias, por um período de 24 horas. O limiar de sensibilidade corneal foi avaliado por estesiometria antes e posteriormente a lesão ser realizada, a intervalos regulares de quatro horas. Avaliou-se ainda o edema corneal, a hiperemia conjuntival, o blefarospasmo e o diâmetro das úlceras, empregando-se, biomicroscópio com lâmpada em fenda, teste do tingimento pela fluoresceína, imagem digitalizada para posterior avaliação em programa Image J, até a reepitelização completa. Decorridas 24 horas da abrasão nos animais do primeiro grupo (n=6) e 55 horas nos animais do segundo grupo (n=6), estes foram submetidos à eutanásia. Ambas as córneas (OT e OC) foram avaliadas histologicamente e à microscopia eletrônica de varredura. Utilizou-se Análise de Variância para Medidas Repetidas, considerando significativos os valores de p ≤ 0,05. Conclui-se que mesmo sem a adição de conservantes e a despeito do bom efeito analgésico, o cetorolaco de trometamina a 0,5%, sem conservante, retarda a reparação cicatricial do epitélio corneal em coelhos submetidos a abrasões com hidróxido de sódio 1 mol/L. / Studied the effects of ketorolac tromethamine 0.5% without preservatives on ocular inflammation, evaluating the sensitivity and corneal epithelial repair in rabbits subjected to chemical corneal ulceration with sodium hydroxide (NaOH) 1 mol / L. Constituted two groups (n = 12). The first group received 30 μl of tometamine ketorolac 0.5%, without preservative (OT) every six hours in one of ulcerated corneas and 30 μl of saline solution 0.9% (OC) in the other cornea a total of four applications daily for a period of 24 hours. The threshold of corneal sensitivity was assessed by esthesiometry before and after the injury to be held at regular intervals of four hours. It was also evaluated corneal edema, conjunctival hyperemia, blepharospasm and the diameter of the ulcers, using, biomicroscopy with slit lamp, the fluorescein dye test, the scanned image for further evaluation in program Image J, until complete reepithelialization. After 24 hours of abrasion in the animals of first group (n = 6) and 55 hours in animals of the second group (n = 6), they were euthanized. Both corneas (OT and OC) were evaluated histologically and scanning electron microscopy. We used ANOVA for repeated measures, considering significant p values ≤ 0.05. We conclude that even without the added preservatives and despite the good analgesic effect of ketorolac tromethamine 0.5%, without preservative, slowing the repair of corneal epithelium wound healing in rabbits subjected to abrasion with sodium hydroxide 1 mol/L.

Being the longest-lived rodent, naked mole-rats (NMR; Heterocephalus glaber) are an exceptional model for biogerontological research. However, unlike other rodents, not much is known about their wound healing process. To investigate that, full-thickness wounds were created in the back skin of naked mole rats. Our initial data confirmed that wound closure in NMR skin was achieved primarily by reepithelialization and granulation tissue formation, with only ~26% wound contraction, making them an excellent model to study human cutaneous wound healing. Similar to mice and human skin, changes in wound epithelial tongue included progressive enlargement of wound epithelium, increased proliferation and changes in the expression pattern of epidermal markers including K14, K17, integrin α6 and E-cadherin. Further analysis revealed characteristics of reduced scarring in NMR wounds including low collagen I to III ratio, increased HA expression (HMW) and increased fibronectin expression. Transcriptional profiling of TGFβ isoforms and different pro/anti-inflammatory cytokines revealed a balance in the expression and repression of different cytokines, potentially contributing into reduced scarring. Comparison of RNA-seq data from NMR and human fullthickness wounds revealed a delay in the activation of important biological processes and pathways in NMR skin in response to injury. Further analysis based on cultured human and NMR cells revealed differential regulation of TGFβ signalling pathway between both species. 3-D collagen gel contraction assay revealed that NMR fibroblast showed noticeable contraction but independently of TGFβ treatment, while human fibroblast showed marked increased in gel contraction in the presence of TGFβ. In conclusion, NMR can serve as a very useful model to study human cutaneous wound healing. The reduced scarring in NMR could be a result of multiple factors including HMW-HA, balanced cytokine expression and differential regulation of different TGFβ cytokines as observed in the in vitro studies. / The full text will be available at the end of the embargo: 3rd May 2024

Yes / Ex vivo wounded human skin organ culture is an invaluable tool for translationally relevant preclinical wound healing research. However, studies incorporating this system are still underutilized within the field because of the low throughput of histological analysis required for downstream assessment. In this study, we use intravital fluorescent dye to lineage trace epidermal cells, demonstrating that wound re‐epithelialization of human ex vivo wounds occurs consistent with an extending shield mechanism of collective migration. Moreover, we also report a relatively simple method to investigate global epithelial closure of explants in culture using daily fluorescent dye treatment and en face imaging. This study is the first to quantify healing of ex vivo wounds in a longitudinal manner, providing global assessments for re‐epithelialization and tissue contraction. We show that this approach can identify alterations to healing with a known healing promoter. This methodological study highlights the utility of human ex vivo wounds in enhancing our understanding of mechanisms of human skin repair and in evaluating novel therapies to improve healing outcome. / University of Manchester Strategic Fund; Wellcome Trust; BBSRC; Ministry of Higher Education, Malaysia Universiti; Sains Malaysia

Orientador: José Luiz Laus / Banca: Paola Castro Moraes / Banca: Cristiane dos Santos Honsho / Resumo: Estudou-se os efeitos do cetorolaco de trometamina 0,5%, sem conservante, sobre a inflamação ocular, avaliando-se a reparação epitelial e a sensibilidade corneal, em coelhos submetidos à ulceração corneal química com hidróxido de sódio (NaOH) 1 mol/L. Constituíram-se dois grupos (n=12). O primeiro grupo recebeu 30 μl de cetorolaco de tometamina 0,5%, sem conservante (OT), a cada seis horas, em uma das córneas ulceradas e 30 μl de solução salina fisiológica 0.9% (OC) na córnea adelfa, totalizando 4 aplicações diárias, por um período de 24 horas. O limiar de sensibilidade corneal foi avaliado por estesiometria antes e posteriormente a lesão ser realizada, a intervalos regulares de quatro horas. Avaliou-se ainda o edema corneal, a hiperemia conjuntival, o blefarospasmo e o diâmetro das úlceras, empregando-se, biomicroscópio com lâmpada em fenda, teste do tingimento pela fluoresceína, imagem digitalizada para posterior avaliação em programa Image J, até a reepitelização completa. Decorridas 24 horas da abrasão nos animais do primeiro grupo (n=6) e 55 horas nos animais do segundo grupo (n=6), estes foram submetidos à eutanásia. Ambas as córneas (OT e OC) foram avaliadas histologicamente e à microscopia eletrônica de varredura. Utilizou-se Análise de Variância para Medidas Repetidas, considerando significativos os valores de p ≤ 0,05. Conclui-se que mesmo sem a adição de conservantes e a despeito do bom efeito analgésico, o cetorolaco de trometamina a 0,5%, sem conservante, retarda a reparação cicatricial do epitélio corneal em coelhos submetidos a abrasões com hidróxido de sódio 1 mol/L. / Abstract: Studied the effects of ketorolac tromethamine 0.5% without preservatives on ocular inflammation, evaluating the sensitivity and corneal epithelial repair in rabbits subjected to chemical corneal ulceration with sodium hydroxide (NaOH) 1 mol / L. Constituted two groups (n = 12). The first group received 30 μl of tometamine ketorolac 0.5%, without preservative (OT) every six hours in one of ulcerated corneas and 30 μl of saline solution 0.9% (OC) in the other cornea a total of four applications daily for a period of 24 hours. The threshold of corneal sensitivity was assessed by esthesiometry before and after the injury to be held at regular intervals of four hours. It was also evaluated corneal edema, conjunctival hyperemia, blepharospasm and the diameter of the ulcers, using, biomicroscopy with slit lamp, the fluorescein dye test, the scanned image for further evaluation in program Image J, until complete reepithelialization. After 24 hours of abrasion in the animals of first group (n = 6) and 55 hours in animals of the second group (n = 6), they were euthanized. Both corneas (OT and OC) were evaluated histologically and scanning electron microscopy. We used ANOVA for repeated measures, considering significant p values ≤ 0.05. We conclude that even without the added preservatives and despite the good analgesic effect of ketorolac tromethamine 0.5%, without preservative, slowing the repair of corneal epithelium wound healing in rabbits subjected to abrasion with sodium hydroxide 1 mol/L. / Mestre

Abstract Collagen XVII (BP180) is a transmembrane component of hemidesmosomes, which connect basal keratinocytes to the basement membrane. The extracellular domain of collagen XVII is proteolytically shed from the cell surface and released to the extracellular matrix. Apart from its function in epithelial cell adhesion, collagen XVII has been suggested to participate in keratinocyte motility. The collagen XVII expression pattern was studied in wounds of oral mucosa and in epithelial tumors. During re-epithelialization, collagen XVII was expressed in the keratinocytes distal to the wound edge, but not in the leading cells of the epithelial tip. Collagen XVII upregulation was observed in moderate/severe dysplasias of oral mucosa. In follicular ameloblastomas and basal cell carcinomas, collagen XVII expression was reduced in peripheral cells, whereas cytoplasmic staining was detected in central tumor cells. Tongue squamous cell carcinomas showed increased collagen XVII expression in grade II/III tumors, particularly in areas of invasive growth. The results suggest a correlation between overexpression of collagen XVII and the invasive potential of the tumor. For the first time, the role of collagen XVII in the regulation of malignant migration was explored. The presence of COL15, the cell adhesion domain of collagen XVII, induced migration of tongue squamous cell carcinoma cells in transmigration assays. Experiments with specific function-blocking integrin antibodies revealed that the promigratory function of COL15 is mediated by αv and α5 integrins. The role of the matrix metalloproteinase (MMP) family of proteolytic enzymes in wound re-epithelialization was studied in a transgenic mouse model. In these mice, a specific inhibitor of MMPs, TIMP-1, was overexpressed in cells that normally produce MMP-9. The healing of cutaneous wounds was found to be significantly delayed, but not prevented, due to the impaired ability of keratinocytes to migrate to the wound area. These results suggest that collagen XVII may participate in epithelial tumor progression and invasion by promoting migration of tumor cells. Based on the present study, epithelial cell-derived MMPs play a significant role in the migration of wound keratinocytes during re-epithelialization.

Large doses of recombinant growth factors formulated in solution form directly injected into the body is usual clinical practice in treating second-degree scald injuries, with promising results, but this approach creates side effects; furthermore, it may not allow appropriate levels of the factor to be sensed by the target injured tissue/organ in the specific time frame, owing to complications arising from regeneration. In this research, two delivery methods (infusion pumping and local topical application) were applied to deliver recombinant human erythropoietin (rHuEPO) for skin regeneration. First, rHuEPO was given in deep second-degree scald injury sites in mice by infusion pump. Vascularization was remarkably higher in the rHuEPO pumping group than in controls. Second, local topical application of rHuEPO gel was given in deep second-degree scald injury sites in rats. Histological analysis showed that epithelialization rate was significantly higher in the rHuEPO gel-treated group than in controls. Immunohistochemical studies showed that the rHuEPO gel-treated group showed remarkably higher expression of skin regeneration makers than the control group. An accurate method for visualization and quantification of blood vessel networks in target areas has still not been developed up to this point, because of technical difficulties in detecting such thin blood vessels. A method which utilizes a series of steps to enhance the image, removes noise from image background, and tracks the vessels edges for vessel segmentation and quantification has been used in this study. Using image analysis methods, we were able to detect the microvascular networks of newly formed blood vessels (less than 500 μm thickness), which participate in the healing process, providing not only nutrition and oxygen to grow tissues but also necessary growth factors to grow tissue cells for complete skin regeneration. The rHuEPO-treated group showed higher expression of stem cell markers (CD 31, CD 90, CD 71, and nestin), which actively contribute to in-wound-healing processes for new hair follicle generation as well as skin regeneration. Collectively, both rHuEPO group pumping into the systemic circulation system, and injection into the local injury area, prompted mice and rats to form new blood vessel networks in scald injury sites, which significantly participate in the scald healing process. These results may lead to the development of novel treatments for scald wounds.