RESUMO
Reclaimed Asphalt Pavement (RAP) as recycled aggregates is a relatively new construction process of rigid pavements due to the scarcity and degradation of natural aggregates. This study aims at the sequential characterization of RAP aggregate to obtain optimized proportions for strength. For this purpose, RAP aggregates were used for the replacement of natural aggregates (NA) in the concrete mix which was achieved by varying from 0-100%. Furthermore, zirconia silica fume (ZSF) was used as a partial replacement of the cement in the concrete mix, replacing Ordinary Portland Cement (OPC). Experimental studies have shown that the incorporation of washed RAP (WRAP) slightly reduces the compressive strength of concrete by 2.7-37.35% as compared to the reference control concrete mix. Although the 7-days, 28-days and 56-day compressive strength of WRAP recycled aggregate-based concrete is slightly better than the 7-days, 28-days and 56-day compressive strength of dirty RAP (DRAP) recycled aggregate-based concrete. A similar trend was observed in the flexural strength and split tensile strength of WRAP recycled aggregate-based. Overall, the results show that 40% WRAP recycled aggregates with 10% ZSF as a replacement for cement outperform DRAP aggregates in concrete mixes. According to the ANOVA results, the combination of ZSF and WRAP aggregates met the cement concrete pavement strength standard, thereby contributing to sustainable development. Reclaimed Asphalt Concrete Pavements (RACP) are now seen as a potential and long-term answer to the present environmental and economic crisis.
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The objective of this study was to provide optimized processing for examination of rat incisors in nonclinical toxicity studies that enables analysis using immunohistochemistry (IHC). Rat maxillas and mandibles were decalcified in Immunocal, a formic acid decalcifier, and Decal Stat, a hydrochloric acid decalcifier, to evaluate tissue quality when with hematoxylin and eosin (H&E) stain and an IHC. Following necropsy of 10 to 13-week-old male Sprague Dawley rats, tissues were collected, trimmed, fixed in neutral buffered formalin (NBF), and placed into the corresponding decalcifying solution. After a pilot study with multiple timepoints for both decalcifying solutions, times were selected for the definitive study. Incisors in the definitive study were decalcified for 72, 96 or 120 hours in Immunocal and 24 hours in Decal Stat, trimmed, processed, embedded in paraffin, and sectioned. The microtomy process and sections were evaluated by histotechnologists. Sections were stained withH&E or an IHC to detect vimentin. Veterinary pathologists used blinded assessment to evaluate staining and tissue quality. The H&E sections from Immunocal timepoints scored higher based on criteria such as cellular morphology. However, tissue quality decreased at 120 hours with Immunocal but was adequate after 24 hours with Decal Stat. For IHC, moderate to excellent expression of vimentin was observed at timepoints for both decalcifiers. Optimal tissue sectioning and histological quality were achieved on incisor sections decalcified for 96 hours with Immunocal and 24 hours with Decal Stat.
Assuntos
Incisivo , Maxila , Animais , Técnica de Descalcificação , Imuno-Histoquímica , Masculino , Projetos Piloto , Ratos , Ratos Sprague-Dawley , VimentinaRESUMO
Glomerular filtration rate is the gold-standard method for assessment of renal function but is rarely performed in routine toxicity studies. Standard serum biomarkers of renal function are insensitive and become elevated only with significant loss of organ function. Symmetric dimethylarginine (SDMA) is a ubiquitous analyte that is freely filtered by the glomerulus and can be detected in serum. It has shown utility for the detection of renal injury in dogs and cats in clinical veterinary practice, but the potential utility of SDMA to detect renal injury in preclinical species or toxicity studies has not been thoroughly investigated. We utilized a well-characterized glomerular toxicant, puromycin aminonucleoside, to induce podocyte injury and subsequent proteinuria in young male Sprague-Dawley rats. At the end of 1 or 2 weeks, blood, urine, and kidney tissue were collected for analysis. One week following a single 50 mg/kg dose, urea nitrogen, creatinine, and albumin mean values were within historical control ranges, while SDMA was increased. Glomerular changes in these animals included periodic acid-Schiff positive globules within podocytes, podocyte hypertrophy by light microscopy, and podocyte degeneration with effacement of foot processes by electron microscopy (EM). Taken together, our data indicate that SDMA may be a useful biomarker for early detection of glomerular toxicities in rats.
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Doenças do Gato , Doenças do Cão , Insuficiência Renal Crônica , Animais , Arginina/análogos & derivados , Biomarcadores , Gatos , Cães , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: There is microscopic spatial and temporal heterogeneity of pathological changes in idiopathic pulmonary fibrosis (IPF) lung tissue, which may relate to heterogeneity in pathophysiological mediators of disease and clinical progression. We assessed relationships between gene expression patterns, pathological features, and systemic biomarkers to identify biomarkers that reflect the aggregate disease burden in patients with IPF. METHODS: Gene expression microarrays (N=40 IPF; 8 controls) and immunohistochemical analyses (N=22 IPF; 8 controls) of lung biopsies. Clinical characterisation and blood biomarker levels of MMP3 and CXCL13 in a separate cohort of patients with IPF (N=80). RESULTS: 2940 genes were significantly differentially expressed between IPF and control samples (|fold change| >1.5, p<0.05). Two clusters of co-regulated genes related to bronchiolar epithelium or lymphoid aggregates exhibited substantial heterogeneity within the IPF population. Gene expression in bronchiolar and lymphoid clusters corresponded to the extent of bronchiolisation and lymphoid aggregates determined by immunohistochemistry in adjacent tissue sections. Elevated serum levels of MMP3, encoded in the bronchiolar cluster, and CXCL13, encoded in the lymphoid cluster, corresponded to disease severity and shortened survival time (p<10(-7) for MMP3 and p<10(-5) for CXCL13; Cox proportional hazards model). CONCLUSIONS: Microscopic pathological heterogeneity in IPF lung tissue corresponds to specific gene expression patterns related to bronchiolisation and lymphoid aggregates. MMP3 and CXCL13 are systemic biomarkers that reflect the aggregate burden of these pathological features across total lung tissue. These biomarkers may have clinical utility as prognostic and/or surrogate biomarkers of disease activity in interventional studies in IPF.
Assuntos
Quimiocina CXCL13/genética , Regulação da Expressão Gênica , Fibrose Pulmonar Idiopática/genética , Pulmão/patologia , Metaloproteinase 3 da Matriz/genética , Idoso , Idoso de 80 Anos ou mais , Linfócitos B , Quimiocina CXCL13/biossíntese , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Humanos , Fibrose Pulmonar Idiopática/metabolismo , Fibrose Pulmonar Idiopática/patologia , Imuno-Histoquímica , Pulmão/metabolismo , Masculino , Metaloproteinase 3 da Matriz/biossíntese , Pessoa de Meia-Idade , Prognóstico , Índice de Gravidade de DoençaRESUMO
This paper describes a simple, highly efficient and robust proteomic workflow for routine liquid-chromatography tandem mass spectrometry analysis of Laser Microdissection Pressure Catapulting (LMPC) isolates. Highly efficient protein recovery was achieved by optimization of a "one-pot" protein extraction and digestion allowing for reproducible proteomic analysis on as few as 500 LMPC isolated cells. The method was combined with label-free spectral count quantitation to characterize proteomic differences from 3000-10,000 LMPC isolated cells. Significance analysis of spectral count data was accomplished using the edgeR tag-count R package combined with hierarchical cluster analysis. To illustrate the capability of this robust workflow, two examples are presented: 1) analysis of keratinocytes from human punch biopsies of normal skin and a chronic diabetic wound and 2) comparison of glomeruli from needle biopsies of patients with kidney disease. Differentially expressed proteins were validated by use of immunohistochemistry. These examples illustrate that tissue proteomics carried out on limited clinical material can obtain informative proteomic signatures for disease pathogenesis and demonstrate the suitability of this approach for biomarker discovery.
Assuntos
Criopreservação/métodos , Microdissecção e Captura a Laser/métodos , Espectrometria de Massas/métodos , Proteômica/métodos , Biomarcadores/análise , Biomarcadores/metabolismo , Biópsia , Feminino , Humanos , MasculinoRESUMO
OBJECTIVE: The primary objective was to ascertain the therapeutic usefulness of homeopathic medicine in the management of chronic sinusitis (CS). MATERIALS AND METHODS: Multicentre observational study at Institutes and Units of the Central Council for Research in Homoeopathy, India. Symptoms were assessed using the chronic sinusitis assessment score (CSAS). 17 pre-defined homeopathic medicines were shortlisted for prescription on the basis of repertorisation for the pathological symptoms of CS. Regimes and adjustment of regimes in the event of a change of symptoms were pre-defined. The follow-up period was for 6 months. Statistical analysis was done using SPSS version 16. RESULTS: 628 patients suffering from CS confirmed on X-ray were enrolled from eight Institutes and Units of the Central Council for Research in Homoeopathy. All 550 patients with at least one follow-up assessment were analyzed. There was a statistically significant reduction in CSAS (P = 0.0001, Friedman test) after 3 and 6 months of treatment. Radiological appearances also improved. A total of 13 out of 17 pre-defined medicines were prescribed in 550 patients, Sil. (55.2% of 210), Calc. (62.5% of 98), Lyc. (69% of 55), Phos. (66.7% of 45) and Kali iod. (65% of 40) were found to be most useful having marked improvement. 4/17 medicines were never prescribed. No complications were observed during treatment. CONCLUSION: Homeopathic treatment may be effective for CS patients. Controlled trials are required for further validation.
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Homeopatia/métodos , Sinusite/tratamento farmacológico , Adolescente , Adulto , Criança , Doença Crônica , Feminino , Humanos , Índia , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Radiografia , Sinusite/diagnóstico por imagem , Resultado do Tratamento , Adulto JovemRESUMO
The economic, social, and public health burden of chronic ulcers and other compromised wounds is enormous and rapidly increasing with the aging population. The growth factors derived from platelets play an important role in tissue remodeling including neovascularization. Platelet-rich plasma (PRP) has been utilized and studied for the last four decades. Platelet gel and fibrin sealant, derived from PRP mixed with thrombin and calcium chloride, have been exogenously applied to tissues to promote wound healing, bone growth, hemostasis, and tissue sealing. In this study, we first characterized recovery and viability of as well as growth factor release from platelets in a novel preparation of platelet gel and fibrin matrix, namely platelet-rich fibrin matrix (PRFM). Next, the effect of PRFM application in a delayed model of ischemic wound angiogenesis was investigated. The study, for the first time, shows the kinetics of the viability of platelet-embedded fibrin matrix. A slow and steady release of growth factors from PRFM was observed. The vascular endothelial growth factor released from PRFM was primarily responsible for endothelial mitogenic response via extracellular signal-regulated protein kinase activation pathway. Finally, this preparation of PRFM effectively induced endothelial cell proliferation and improved wound angiogenesis in chronic wounds, providing evidence of probable mechanisms of action of PRFM in healing of chronic ulcers.
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Proliferação de Células , Endotélio Vascular/citologia , Fibrina/fisiologia , Neovascularização Fisiológica/fisiologia , Plasma Rico em Plaquetas/fisiologia , Cicatrização/fisiologia , Adulto , Animais , Plaquetas/metabolismo , Plaquetas/fisiologia , Sobrevivência Celular , Células Cultivadas , Meios de Cultivo Condicionados , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Pessoa de Meia-Idade , Sus scrofa , Adulto JovemRESUMO
BACKGROUND: Chronic inflammation is a characteristic feature of diabetic cutaneous wounds. We sought to delineate novel mechanisms involved in the impairment of resolution of inflammation in diabetic cutaneous wounds. At the wound-site, efficient dead cell clearance (efferocytosis) is a pre-requisite for the timely resolution of inflammation and successful healing. METHODOLOGY/PRINCIPAL FINDINGS: Macrophages isolated from wounds of diabetic mice showed significant impairment in efferocytosis. Impaired efferocytosis was associated with significantly higher burden of apoptotic cells in wound tissue as well as higher expression of pro-inflammatory and lower expression of anti-inflammatory cytokines. Observations related to apoptotic cell load at the wound site in mice were validated in the wound tissue of diabetic and non-diabetic patients. Forced Fas ligand driven elevation of apoptotic cell burden at the wound site augmented pro-inflammatory and attenuated anti-inflammatory cytokine response. Furthermore, successful efferocytosis switched wound macrophages from pro-inflammatory to an anti-inflammatory mode. CONCLUSIONS/SIGNIFICANCE: Taken together, this study presents first evidence demonstrating that diabetic wounds suffer from dysfunctional macrophage efferocytosis resulting in increased apoptotic cell burden at the wound site. This burden, in turn, prolongs the inflammatory phase and complicates wound healing.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Inflamação , Macrófagos/metabolismo , Adulto , Animais , Apoptose , Citocinas/metabolismo , Proteína Ligante Fas/metabolismo , Feminino , Homozigoto , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Estresse Oxidativo , Pele/patologia , CicatrizaçãoRESUMO
Ischemia complicates wound closure. Here, we are unique in presenting a murine ischemic wound model that is based on bipedicle flap approach. Using this model of ischemic wounds we have sought to elucidate how microRNAs may be implicated in limiting wound re-epithelialization under hypoxia, a major component of ischemia. Ischemia, evaluated by laser Doppler as well as hyperspectral imaging, limited blood flow and lowered tissue oxygen saturation. EPR oximetry demonstrated that the ischemic wound tissue had pO(2) <10 mm Hg. Ischemic wounds suffered from compromised macrophage recruitment and delayed wound epithelialization. Specifically, epithelial proliferation, as determined by Ki67 staining, was compromised. In vivo imaging showed massive hypoxia inducible factor-1alpha (HIF-1alpha) stabilization in ischemic wounds, where HIF-1alpha induced miR-210 expression that, in turn, silenced its target E2F3, which was markedly down-regulated in the wound-edge tissue of ischemic wounds. E2F3 was recognized as a key facilitator of cell proliferation. In keratinocytes, knock-down of E2F3 limited cell proliferation. Forced stabilization of HIF-1alpha using Ad-VP16- HIF-1alpha under normoxic conditions up-regulated miR-210 expression, down-regulated E2F3, and limited cell proliferation. Studies using cellular delivery of miR-210 antagomir and mimic demonstrated a key role of miR-210 in limiting keratinocyte proliferation. In summary, these results are unique in presenting evidence demonstrating that the hypoxia component of ischemia may limit wound re-epithelialization by stabilizing HIF-1alpha, which induces miR-210 expression, resulting in the down-regulation of the cell-cycle regulatory protein E2F3.
Assuntos
Regulação da Expressão Gênica , Isquemia/patologia , Queratinócitos/citologia , MicroRNAs/química , MicroRNAs/genética , Animais , Proliferação de Células , Modelos Animais de Doenças , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Isquemia/metabolismo , Antígeno Ki-67/biossíntese , Fluxometria por Laser-Doppler/métodos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , CicatrizaçãoRESUMO
The compromised antioxidant defense system in chronic lymphocytic leukemia (CLL) suggested a potential use for reactive oxygen species (ROS) generating arsenic trioxide (ATO) and ascorbic acid. While both ATO and ascorbic acid mediate cytotoxicity in CLL B cells as single agents, the efficacy of ATO is enhanced by ascorbic acid. This effect is dependent on increased ROS accumulation, as pretreatment of B-CLL cells with a glutathione reducing buthionine sulfoximine or catalase inhibiting aminotriazole, enhanced ATO/ascorbic acid-mediated cytotoxicity. Pretreatment with reducing agents such as catalase, or thiol antioxidant, N-acetyl cysteine or GSH also abrogated ATO/ascorbic acid-mediated cytotoxicity. Furthermore, Hu1D10-mediated cell death was enhanced with ATO and ascorbic acid, thus justifying potential combination of ATO/arsenic trioxide therapy with antibodies such as Hu1D10 that also cause accumulation of ROS.
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Arsenicais/farmacologia , Ácido Ascórbico/farmacologia , Leucemia Linfocítica Crônica de Células B/patologia , Óxidos/farmacologia , Clorometilcetonas de Aminoácidos/farmacologia , Amitrol (Herbicida)/farmacologia , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados , Trióxido de Arsênio , Linfócitos B/efeitos dos fármacos , Linfócitos B/patologia , Butionina Sulfoximina/farmacologia , Catalase/antagonistas & inibidores , Catalase/farmacologia , Linhagem Celular Tumoral/efeitos dos fármacos , Cisteína Proteases/fisiologia , Inibidores de Cisteína Proteinase/farmacologia , Avaliação Pré-Clínica de Medicamentos , Sinergismo Farmacológico , Ativação Enzimática/efeitos dos fármacos , Glutationa/metabolismo , Humanos , Proteínas de Neoplasias/fisiologia , Oxidantes/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismoRESUMO
Our laboratory has published the first evidence obtained from fast low-angle-shot cine magnetic resonance imaging (11.7 T) studies demonstrating secondary myocyte death after ischemia/reperfusion (IR) of the murine heart. This work provides the first evidence from 11.7-T magnet-assisted pixel-level analysis of the post-IR murine myocardial infarct patches. Changes in function of the remodeling heart were examined in tandem. IR compromised cardiac function and induced LV hypertrophy. During recovery, the IR-induced increase in LV mass was partly offset. IR-induced wall thinning was noted in the anterior aspect of LV and at the diametrically opposite end. Infarct size was observed to be largest on post-IR days 3 and 7. With time (day 28), however, the infarct size was significantly reduced. IR-induced absolute signal-intensity enhancement was highest on post-IR days 3 and 7. As a function of post-IR time, signal-intensity enhancement was attenuated. The threshold of hyperenhanced tissue resulted in delineation of contours that identified necrotic (bona fide infarct) and reversibly injured infarct patches. The study of infarct transmurality indicated that whereas the permanently injured tissue volume remained unchanged, part of the reversibly injured infarct patch recovered in 4 weeks after IR. The approach validated in the current study is powerful in noninvasively monitoring remodeling of the post-IR beating murine myocardium.
Assuntos
Meios de Contraste , Imageamento por Ressonância Magnética/métodos , Isquemia Miocárdica/patologia , Animais , Masculino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Chronic ischemic wounds presenting at wound clinics are heterogeneous with respect to etiology, age of the wound, and other factors complicating wound healing. In addition, there are ethical challenges associated with collecting repeated biopsies from a patient to develop an understanding of the temporal dynamics of the mechanisms underlying chronic wounds. The need for a preclinical model of ischemic wound is therefore compelling. The porcine model is widely accepted as an excellent preclinical model for human wounds. A full-thickness bipedicle flap approach was adopted to cause skin ischemia. Closure of excisional wounds placed on ischemic tissue was severely impaired resulting in chronic wounds. Histologically, ischemic wounds suffered from impaired re-epithelialization, delayed macrophage recruitment and poorer endothelial cell abundance and organization. Compared with the pair-matched nonischemic wound, unique aspects of the ischemic wound biology were examined on days 3, 7, 14, and 28 by systematic screening of the wound tissue transcriptome using high-density porcine GeneChips. Ischemia markedly potentiated the expression of arginase-1, a cytosolic enzyme that metabolizes the precursor of nitric oxide l-arginine. Ischemia also induced the SOD2 in the wound tissue perhaps as survival response of the challenged tissue. Human chronic wounds also demonstrated elevated expression of SOD2 and arginase-1. This study provides a thorough database that may serve as a valuable reference tool to develop novel hypotheses aiming to elucidate the biology of ischemic chronic wounds in a preclinical setting.
Assuntos
Modelos Animais de Doenças , Isquemia/complicações , Pele/metabolismo , Ferimentos e Lesões/genética , Animais , Arginase/genética , Arginase/metabolismo , Doença Crônica , Análise por Conglomerados , Procedimentos Cirúrgicos Dermatológicos , Perfilação da Expressão Gênica , Humanos , Hipóxia/fisiopatologia , Imuno-Histoquímica , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Pele/patologia , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Retalhos Cirúrgicos , Sus scrofa , Ferimentos e Lesões/etiologia , Ferimentos e Lesões/metabolismoRESUMO
AIMS: MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at the post-transcriptional level by either degradation or translational repression of a target mRNA. Encoded in the genome of most eukaryotes, miRNAs have been proposed to regulate specifically up to 90% of human genes through a process known as miRNA-guided RNA silencing. For the first time, we sought to test how myocardial ischaemia-reperfusion (IR) changes miR expression. METHODS AND RESULTS: Following 2 and 7 h of IR or sham operation, myocardial tissue was collected and subjected to miRNA expression profiling and quantification using a Bioarray system that screens for human-, mice-, rat-, and Ambi-miR. Data mining and differential analyses resulted in 13 miRs that were up-regulated on day 2, 9 miRs that were up-regulated on day 7, and 6 miRs that were down-regulated on day 7 post-IR. Results randomly selected from expression profiling were validated using real-time PCR. Tissue elements laser-captured from the infarct site showed marked induction of miR-21. In situ hybridization studies using locked nucleic acid miR-21-specific probe identified that IR-inducible miR-21 was specifically localized in the infarct region of the IR heart. Immunohistochemistry data show that cardiac fibroblasts (CFs) are the major cell type in the infarct zone. Studies with isolated CFs demonstrated that phosphatase and tensin homologue (PTEN) is a direct target of miR-21. Modulation of miR-21 regulated expression of matrix metalloprotease-2 (MMP-2) via a PTEN pathway. Finally, we noted a marked decrease in PTEN expression in the infarct zone. This decrease was associated with increased MMP-2 expression in the infarct area. CONCLUSION: This work constitutes the first report describing changes in miR expression in response to IR in the mouse heart, showing that miR-21 regulates MMP-2 expression in CFs of the infarct zone via a PTEN pathway.
Assuntos
Fibroblastos/enzimologia , Regulação Enzimológica da Expressão Gênica , Metaloproteinase 2 da Matriz/genética , MicroRNAs/metabolismo , Infarto do Miocárdio/genética , Traumatismo por Reperfusão Miocárdica/genética , PTEN Fosfo-Hidrolase/genética , Animais , Células Cultivadas , Análise por Conglomerados , Modelos Animais de Doenças , Fibroblastos/patologia , Perfilação da Expressão Gênica/métodos , Imuno-Histoquímica , Hibridização In Situ , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/enzimologia , Infarto do Miocárdio/patologia , Traumatismo por Reperfusão Miocárdica/enzimologia , Traumatismo por Reperfusão Miocárdica/patologia , Análise de Sequência com Séries de Oligonucleotídeos , PTEN Fosfo-Hidrolase/metabolismo , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/metabolismo , Fatores de TempoRESUMO
This work represents a maiden effort to systematically screen the transcriptome of the healing wound-edge tissue temporally using high-density GeneChips. Changes during the acute inflammatory phase of murine excisional wounds were characterized histologically. Sets of genes that significantly changed in expression during healing could be segregated into the following five sets: up-early (6-24 h; cytokine-cytokine receptor interaction pathway), up-intermediary (12-96 h; leukocyte-endothelial interaction pathway), up-late (48-96 h; cell-cycle pathway), down-early (6-12 h; purine metabolism) and down-intermediary (12-96 h; oxidative phosphorylation pathway). Results from microarray and real-time PCR analyses were consistent. Results listing all genes that were significantly changed at any specific time point were further mined for cell-type (neutrophils, macrophages, endothelial, fibroblasts, and pluripotent stem cells) specificity. Candidate genes were also clustered on the basis of their functional annotation, linking them to inflammation, angiogenesis, reactive oxygen species (ROS), or extracellular matrix (ECM) categories. Rapid induction of genes encoding NADPH oxidase subunits and downregulation of catalase in response to wounding is consistent with the fact that low levels of endogenous H2O2 is required for wound healing. Angiogenic genes, previously not connected to cutaneous wound healing, that were induced in the healing wound-edge included adiponectin, epiregulin, angiomotin, Nogo, and VEGF-B. This study provides a digested database that may serve as a valuable reference tool to develop novel hypotheses aiming to elucidate the biology of cutaneous wound healing comprehensively.
Assuntos
Perfilação da Expressão Gênica , Inflamação/genética , Pele/patologia , Cicatrização/genética , Animais , Análise por Conglomerados , Regulação da Expressão Gênica , Cinética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TempoRESUMO
We have previously reported that H(2)O(2) is actively generated by cells at the wound site and that H(2)O(2)-driven redox signaling supports wound angiogenesis and healing. In this study, we have standardized a novel and effective electron paramagnetic resonance spectroscopy-based approach to assess the redox environment of the dermal wound site in vivo. Rac2 regulates inducible NADPH oxidase activation and other functional responses in neutrophils. Using Rac2-deficient mice we sought to investigate the significance of Rac2 in the wound-site redox environment and healing responses. Noninvasive measurements of metabolism of topically applied nitroxide (15)N-perdeuterated tempone in murine excisional dermal wounds demonstrated that the wound site is rich in oxidants, the levels of which peak 2 days postwounding in the inflammatory phase. Rac2-deficient mice had threefold lower production of superoxide compared to controls with similar wounds. In these mice, a lower wound-site superoxide level was associated with compromised wound closure. Immunostaining of wound edges harvested during the inflammatory phase showed that the numbers of phagocytic cells recruited to the wound site in Rac2-deficient and control mice were similar, but the amount of lipid peroxidation was significantly lower in Rac2-deficient mice, indicating compromised NADPH oxidase activity. Taken together, the findings of this study support that the wound site is rich in oxidants. Rac2 significantly contributes to oxidant production at the wound site and supports the healing process.
Assuntos
Cicatrização , Proteínas rac de Ligação ao GTP/fisiologia , Animais , Espectroscopia de Ressonância de Spin Eletrônica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH Oxidases/fisiologia , Neovascularização Fisiológica , Óxidos de Nitrogênio/metabolismo , Oxidantes/análise , Oxirredução , Superóxidos/metabolismo , Proteína RAC2 de Ligação ao GTPRESUMO
The phenotypic switch of cardiac fibroblasts (CFs) to myofibroblasts is essential for normal and pathological wound healing. Relative hyperoxic challenge during reoxygenation causes myocardial remodeling. Here, we sought to characterize the novel O(2)-sensitive molecular mechanisms responsible for triggering the differentiation of CFs to myofibroblasts. Exposure of CFs to hyperoxic challenge-induced transcription of smooth muscle actin (SMA) and enhanced the stability of both Acta2 transcript as well as of SMA protein. Both p21 deficiency as well as knockdown blunted hyperoxia-induced Acta2 and SMA response. Strikingly, overexpression of p21 alone markedly induced differentiation of CFs under normoxia. Overexpression of p21 alone induced SMA transcription by down-regulating YB1 and independent of TGFbeta1. In vivo, hyperoxic challenge induced p21-dependent differentiation of CFs to myofibroblasts in the infarct boundary region of ischemia-reperfused heart. Tissue elements were laser-captured from infarct boundary and from a noninfarct region 0.5 mm away. Reperfusion caused marked p21 induction in the infarct region. Acta2 as well as SMA expression were markedly up-regulated in CF-rich infarct boundary region. Of note, ischemia-reperfusion-induced up-regulation of Acta2 in the infarct region was completely abrogated in p21-deficient mice. This observation establishes p21 as a central regulator of reperfusion-induced phenotypic switch of CFs to myofibroblasts.
Assuntos
Actinas/metabolismo , Diferenciação Celular , Músculo Liso/metabolismo , Miocárdio/citologia , Miocárdio/metabolismo , Animais , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21 , Fibroblastos , Regulação da Expressão Gênica/efeitos dos fármacos , Coração/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Oxigênio/farmacologia , RNA Mensageiro/genéticaRESUMO
Chronic wounds represent a substantial public health problem. The development of tools that would enable sophisticated scrutiny of clinical wound tissue material is highly desirable. This work presents evidence enabling rapid specific identification and laser capture of blood vessels from human tissue in a manner which lends itself to successful high-density (U133A) microarray analysis. Such screening of transcriptome followed by real-time PCR and immunohistochemical verification of candidate genes and their corresponding products were performed by using 3 mm biopsies. Of the 18,400 transcripts and variants screened, a focused set of 53 up-regulated and 24 down-regulated genes were noted in wound-derived blood vessels compared with blood vessels from intact human skin. The mean abundance of periostin in wound-site blood vessels was 96-fold higher. Periostin is known to be induced in response to vascular injury and its expression is associated with smooth muscle cell differentiation in vitro and promotes cell migration. Forty-fold higher expression of heparan sulfate 6-O-endosulfatase1 (Sulf1) was noted in wound-site vessels. Sulf1 has been recently recognized to be anti-angiogenic. During embryonic vasculogenesis, CD24 expression is down-regulated in human embryonic stem cells. Wound-site vessels had lower CD24 expression. The findings of this work provide a unique opportunity to appreciate the striking contrast in the transcriptome composition in blood vessels collected from the intact skin and from the wound-edge tissue. Sets of genes with known vascular functions but never connected to wound healing were identified to be differentially expressed in wound-derived blood vessels paving the way for innovative clinically relevant hypotheses.
Assuntos
Membranas Intracelulares/metabolismo , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Dermatopatias/genética , Dermatopatias/patologia , Transcrição Gênica/genética , Cicatrização , Doença Crônica , Regulação para Baixo , Humanos , Lasers , Análise de Sequência com Séries de Oligonucleotídeos , Regulação para Cima , Cicatrização/genéticaRESUMO
Oxidant and free radical-generating system were used to promote oxidative damage in erythrocytes. Among the oxidants used, phenylhydrazine represents one of the most investigated intracellular free radical-generating probes, which in the presence of haemoglobin auto-oxidises and give rise to hydroxyl radical, a marker for cellular damage. Erythrocyte, as a single cell, is a good model to be used for studying the haemolytic mechanism of anaemia. Our present investigations reveal increased lipid peroxidation of erythrocyte using phenylhydrazine as well as other oxygen-generating systems (hydrogen peroxide, iron with hydrogen peroxide). It has further been observed that not only lipid peroxidation, phenylhydrazine causes significant elevation in methemoglobin formation, catalase activity and turbidity, in the above system, which are the typical characteristics of haemolytic anaemia. However, exogenous administration of green tea leaf extract and ascorbic acid as natural antioxidants and free radical scavengers were shown to protect separately increased lipid peroxidation caused by phenylhydrazine, though the degree of protection is more in case of green tea leaf extract than ascorbic acid. Results suggest that oxidative damage in vivo due to haemolytic disease may be checked to some extent by using natural antioxidants.
Assuntos
Ácido Ascórbico/farmacologia , Eritrócitos/efeitos dos fármacos , Eritrócitos/patologia , Oxidantes/farmacologia , Extratos Vegetais/farmacologia , Folhas de Planta/química , Chá/química , Animais , Cabras/sangue , Cabras/metabolismo , Hemólise/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/químicaRESUMO
The pathogenetic sequence of reactions mediated by endotoxin (LPS) leading to the production of sepsis involves the oxygen radicals or reactive oxygen species, which has been evaluated in the present review. Among reactive oxygen species hydroxyl radical either singly or in combination with peroxynitrite, produces tissue damage often observed during septic injury. Inactivation of these damaging radicals by antioxidants or nitric oxide inhibitor(s) may be helpful for protecting sepsis mediated derangements but the application of these agents as drugs in humans has not been fully successful. Transcription factor NF-kappa B is reported to be the oxygen sensor in LPS induced endotoxemia. Polyphenols, especially the catechin group of compounds, are important therapeutic agents, which may be used for the treatment of endotoxin mediated sepsis.
Assuntos
Antioxidantes/farmacologia , Endotoxemia/etiologia , Endotoxinas/fisiologia , Radicais Livres/metabolismo , Sepse/etiologia , Animais , Antioxidantes/uso terapêutico , Catequina/uso terapêutico , Endotoxemia/tratamento farmacológico , Endotoxemia/prevenção & controle , Endotoxinas/química , Endotoxinas/imunologia , Radicais Livres/química , Humanos , Lipopolissacarídeos , NF-kappa B/fisiologia , Óxido Nítrico/metabolismo , Ácido Peroxinitroso/metabolismo , Espécies Reativas de Oxigênio/química , Espécies Reativas de Oxigênio/metabolismo , Sepse/tratamento farmacológico , Sepse/prevenção & controleRESUMO
Administration of isoproterenol to mice at a dose of 30 mg/100 g body weight for 3 consecutive days at an interval of 24 h induced lipid peroxidation in cardiac tissue and exhibited a significantly elevated serum glutamate oxaloacetate transaminase (SGOT) level. Increased superoxide dismutase (SOD) activity with a concomitant decrease in catalase activity has also been observed in cardiac tissue with isoproterenol treatment. Quinidine, a class I antiarrhythmic agent has been found to exhibit a protective role in isoproterenol induced myocardial ischaemia. Cardiac tissue of quinidine treated mice showed reduction of lipid peroxidation reaction. In addition, quinidine treatment is found to influence the cardiac antioxidant enzymes - catalase and SOD. The decrease of SOD activity and increase of catalase activity suggests that quinidine also exerts an 'indirect antioxidant' effect in protecting the myocardial tissue from reactive oxygen species. Furthermore, our current in vitro studies with quinidine have clearly shown in this work that it possesses a very convincing hydroxyl radical scavenging potential with almost no ability to scavenge superoxide anion and hydrogen peroxide (H2O2) in vitro. Thus, our present investigation suggests that quinidine, when administered to mice, strengthens the antioxidant defense system to resist the free radical induced damage brought about by isoproterenol induced ischaemic condition.