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INTRODUCTION: Prompt, appropriate coagulation factor replacement according to injury and bleeding severity in persons with haemophilia is required to prevent acute and long-term complications. AIMS: Increase proportion of persons with haemophilia A (HA) and B (HB) treated appropriately for an acute injury and bleeding episode at a tertiary children's emergency department (ED) from 65% to 85% and sustain for one year. Secondary aim: increase time interval between patient ED encounters with out-of-range factor dosing. METHODS: Utilizing quality improvement methodology and plan-do-study-analyze cycles, ED encounters for individuals with HA/HB receiving coagulation factor replacement for injuries were audited for in-range coagulation factor dosing. Goal factor dose defined as 50% correction for minor bleeds and 100% correction for major bleeds. Optimal dosing range defined as 90%-120% of the calculated goal dose to account for vial size variability. Interventions targeted communication via the EMR problem list and optimization of physician education. RESULTS: Our previous publication demonstrated 33.3% of ED encounters with out-of-range factor replacement. Following several interventions, the cumulative rate of encounters with out-of-range dosing decreased to 18%. Overall, there was an increase in the mean percent of encounters receiving optimal factor dosing for both HA/HB compared to baseline (82.2% vs. 71.1%), though this was not a statistically significant difference. CONCLUSION: Despite implementation of multiple interventions, out-of-range factor dosing continues to occur. Our team plans to reinstate simulation center education for ED staff and continue education efforts of pharmacists and hematology trainees with the goal of further reducing out-of-range dosing in our ED.
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Hemofilia A , Melhoria de Qualidade , Criança , Humanos , Fatores de Coagulação Sanguínea/uso terapêutico , Hemofilia A/tratamento farmacológico , Hemorragia/etiologia , Serviço Hospitalar de EmergênciaRESUMO
INTRODUCTION: Haemophilia is a disorder complicated by bleeding episodes that require emergent medical evaluation. Factor replacement dosing can present challenges for emergency department (ED) care. AIMS: We aimed to reduce out-of-range factor dosing in the ED. Specifically, we sought to increase the number of haemophilia ED patient visits between encounters where sub-optimal factor dosing was administered from a baseline of 4-15 encounters. METHODS: A chart review was completed on all patients with haemophilia A (HA) or B (HB) seen in the ED for injuries requiring factor concentrate from September 2015 to August 2016. Injuries were classified as minor-requiring a 50% factor correction or major-requiring a 100% factor correction. Optimal dosing range was defined as 90%-120% of the institutional guideline goal for the degree of injury. The predicted optimal dose range for each patient was compared to the actual dose administered. RESULTS: Baseline data demonstrated optimal dosing range in 70% of encounters. There was no difference between patients with HA or HB in frequency of out-of-range dosing (P = 0.15). There was no difference in frequency of out-of-range dosing between types of clotting factor concentrate used. After initiation of quality improvement (QI) interventions, we achieved 16 encounters between out-of-range dosing, exceeding our goal of 15. However, this success was not sustained. CONCLUSION: Optimal coagulation factor dosing is important for patient care and resource management. QI interventions promoted increased accuracy of factor dosing for patients with haemophilia seen in the ED.
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Fatores de Coagulação Sanguínea/uso terapêutico , Hemofilia A/tratamento farmacológico , Hemofilia B/tratamento farmacológico , Adolescente , Criança , Pré-Escolar , Cálculos da Dosagem de Medicamento , Serviço Hospitalar de Emergência , Humanos , Lactente , Melhoria de Qualidade , Adulto JovemRESUMO
Heme oxygenase (HO)-2 deficiency impairs wound healing and exacerbates inflammation following injury. We examine the impact of HO-2 deficiency on macrophage function and the contribution of macrophage HO-2 to inflammatory and repair responses to injury. Corneal epithelial debridement was performed in control and macrophage-depleted HO-2(-/-) and wild-type (WT) mice and in bone marrow chimeras. Peritoneal macrophages were collected for determination of phagocytic activity and classically activated macrophage (M1)-alternatively activated macrophage (M2) polarization. Depletion of macrophages delayed corneal healing (13.2%) and increased neutrophil infiltration (54.1%) by day 4 in WT mice, whereas in HO-2(-/-) mice, it did not worsen the already impaired wound healing and exacerbated inflammation. HO-2(-/-) macrophages displayed an altered M1 phenotype with no significant expression of M2 or M2-like activated cells and a 31.3% reduction in phagocytic capacity that was restored by inducing HO-1 activity or supplementing biliverdin. Macrophage depletion had no effect, whereas adoptive transfer of WT bone marrow improved wound healing (34% on day 4) but did not resolve the exaggerated inflammatory response in HO-2(-/-) mice. These findings indicate that HO-2-deficient macrophages are dysfunctional and that macrophage HO-2 is required for proper macrophage function but is insufficient to correct the impaired healing of the HO-2(-/-) cornea, suggesting that corneal epithelial expression of HO-2 is a key to resolution and repair in wound healing.
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Lesões da Córnea/fisiopatologia , Heme Oxigenase (Desciclizante)/deficiência , Macrófagos/enzimologia , Macrófagos/fisiologia , Cicatrização/fisiologia , Animais , Transplante de Medula Óssea , Lesões da Córnea/patologia , Citocinas/biossíntese , Epitélio Corneano/patologia , Epitélio Corneano/fisiopatologia , Feminino , Heme Oxigenase (Desciclizante)/genética , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Inflamação/patologia , Inflamação/fisiopatologia , Ativação de Macrófagos , Macrófagos/patologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infiltração de Neutrófilos/fisiologia , Fagocitose , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Quimeras de Transplante/fisiologiaRESUMO
Heme oxygenase (HO) represents an intrinsic cytoprotective system based on its anti-oxidative and anti-inflammatory properties mediated via its products biliverdin/bilirubin and carbon monoxide (CO). We showed that deletion of HO-2 results in impaired corneal wound healing with associated chronic inflammatory complications. This study was undertaken to examine the role of HO activity and the contribution of HO-1 and HO-2 to corneal wound healing in an in vitro epithelial scratch injury model. A scratch wound model was established using human corneal epithelial (HCE) cells. These cells expressed both HO-1 and HO-2 proteins. Injury elicited a rapid and transient increase in HO-1 and HO activity; HO-2 expression was unchanged. Treatment with biliverdin or CORM-A1, a CO donor, accelerated wound closure by 10% at 24 h. Inhibition of HO activity impaired wound closure by more than 50%. However, addition of biliverdin or CORM-A1 reversed the effect of HO inhibition on wound healing. Moreover, knockdown of HO-2 expression, but not HO-1, significantly impaired wound healing. These results indicate that HO activity is required for corneal epithelial cell migration. Inhibition of HO activity impairs wound healing while amplification of its activity restores and accelerates healing. Importantly, HO-2, which is highly expressed in the corneal epithelium, appears to be critical for the wound healing process in the cornea. The mechanisms by which it contributes to cell migration in response to injury may reside in the cytoprotective properties of CO and biliverdin.
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Epitélio Corneano/enzimologia , Heme Oxigenase (Desciclizante)/deficiência , Cicatrização , Biliverdina/metabolismo , Boranos/farmacologia , Monóxido de Carbono/metabolismo , Carbonatos/farmacologia , Movimento Celular , Células Cultivadas , Citoproteção , Relação Dose-Resposta a Droga , Epitélio Corneano/efeitos dos fármacos , Epitélio Corneano/lesões , Epitélio Corneano/patologia , Heme Oxigenase (Desciclizante)/genética , Heme Oxigenase-1/deficiência , Heme Oxigenase-1/genética , Humanos , Interferência de RNA , Fatores de Tempo , Transfecção , Cicatrização/efeitos dos fármacosRESUMO
PURPOSE: Heme oxygenase (HO)-2 is highly expressed in the corneal epithelium and is a component of the heme oxygenase system that represents an intrinsic cytoprotective and anti-inflammatory system based on its ability to modulate leukocyte migration and to inhibit expression of inflammatory cytokines and proteins via its products biliverdin/bilirubin and carbon monoxide (CO). We have shown that in HO-2 null mice epithelial injury leads to unresolved corneal inflammation and chronic inflammatory complications including ulceration, perforation and neovascularization. In this study, we explore whether a localized corneal suppression of HO-2 is sufficient for disrupting the innate anti-inflammatory and repair capability of the cornea. METHODS: Silencing hairpin RNA (shRNA) against HO-2 was administered subconjunctivally (100 ng/eye) as well as topically (100 ng/eye) starting one day before corneal epithelial debridement and once daily, thereafter. The corneal epithelium was removed using an Alger Brush in anesthetized mice. Re-epithelialization was assessed by fluorescein staining using a dissecting microscope and image analysis. Inflammatory response was quantified by myeloperoxidase activity. Levels of mRNA were measured by RT-PCR. RESULTS: Local injection of HO-2-specific shRNA led to a 50% reduction in corneal HO-2 mRNA. Administration of HO-2-specific shRNA delayed corneal re-epithelialization when compared with the control shRNA-treated group by 14%, 20%, and 12% at days 3, 4, and 7 after injury, respectively (n=18-24). The observed delay in the wound repair process in HO-2 shRNA treated mice was accompanied by a threefold and 3.5 fold increase in the neovascular response at days 4 and 7 after injury. Further, local knockdown of HO-2 lead to an aberrant chronic inflammatory response, as shown by presence of high numbers of inflammatory cells still present in the cornea at day 7 after injury; 1.04±0.45×10(6) in HO-2 knockdown mice versus 0.14±0.03×10(6) inflammatory cells in control mice. Matrix metalloproteinase-2 (MMP-2) but not MMP-9 increased following injury and remained elevated in the injured corneas of the HO-2 shRNA-treated eyes. CONCLUSIONS: Corneal knockdown of HO-2 via local administration of HO-2-specific shRNA leads to delayed re-epithelialization, increased neovascularization and an aberrant inflammatory response similar to what is observed in the HO-2 null mouse. The elevated MMP-2 expression may contribute to the increase in neovascularization in corneas in which HO-2 expression is suppressed.
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Córnea/metabolismo , Epitélio Corneano/metabolismo , Expressão Gênica/efeitos dos fármacos , Heme Oxigenase (Desciclizante)/antagonistas & inibidores , Metaloproteinase 2 da Matriz/metabolismo , RNA Interferente Pequeno/farmacologia , Administração Tópica , Animais , Córnea/efeitos dos fármacos , Lesões da Córnea , Epitélio Corneano/efeitos dos fármacos , Epitélio Corneano/lesões , Fluoresceína/análise , Heme Oxigenase (Desciclizante)/genética , Heme Oxigenase (Desciclizante)/metabolismo , Inflamação/genética , Inflamação/metabolismo , Injeções Intraoculares , Metaloproteinase 2 da Matriz/genética , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Peroxidase/análise , Peroxidase/metabolismo , Interferência de RNA , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para Cima , Cicatrização/efeitos dos fármacos , Cicatrização/genéticaRESUMO
BACKGROUND: Pediatric residents must demonstrate competence in several clinical procedures prior to graduation, including simple laceration repair. However, residents may lack opportunities to perform laceration repairs during training, affecting their ability and confidence to perform this procedure. OBJECTIVE: We implemented a quality improvement initiative to increase the number of laceration repairs logged by pediatric residents from a baseline mean of 6.75 per month to more than 30 repairs logged monthly. METHODS: We followed the Institute for Healthcare Improvement's Model for Improvement with rapid plan-do-study-act cycles. From July 2016 to February 2018, we increased the number of procedure shifts and added an education module on performing laceration repairs for residents in a pediatric emergency department at a large tertiary hospital. We used statistical process control charting to document improvement. Our outcome measure was the number of laceration repairs documented in resident procedure logs. We followed the percentage of lacerations repairs completed by residents as a process measure and length of stay as a balancing measure. RESULTS: Following the interventions, logged laceration repairs initially increased from 6.75 to 22.75 per month for the residency program. After the number of procedure shifts decreased, logged repairs decreased to 13.40 per month and the percentage of lacerations repaired by residents also decreased. We noted an increased length of stay for patients whose lacerations were repaired by residents. CONCLUSIONS: While our objective was not met, our quality improvement initiative resulted in more logged laceration repairs. The most effective intervention was dedicated procedure shifts.
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Lacerações/terapia , Pediatria/educação , Serviço Hospitalar de Emergência , Humanos , Internato e Residência , Informática Médica/métodos , Melhoria de Qualidade , Centros de Atenção TerciáriaRESUMO
In previous studies, we have shown that heme oxygenase (HO)-2 null [HO-2(-/-)] mice exhibit a faulty response to injury; chronic inflammation and massive neovascularization replaced resolution of inflammation and tissue repair. Endothelial cells play an active and essential role in the control of inflammation and the process of angiogenesis. We examined whether HO-2 deletion affects endothelial cell function. Under basal conditions, HO-2(-/-) aortic endothelial cells (mAEC) showed a 3-fold higher expression of vascular endothelial growth factor receptor 1 and a marked angiogenic response compared with wild-type (WT) cells. Compared with WT cells, HO-2(-/-) mAEC showed a 2-fold reduction in HO activity and marked increases in levels of gp91(phox)/NADPH oxidase isoform, superoxide, nuclear factor kappaB activation, and expression of inflammatory cytokines, including interleukin (IL)-1alpha and IL-6. HO-2 deletion transforms endothelial cells from a "normal" to an "activated" phenotype characterized by increases in inflammatory, oxidative, and angiogenic factors. This switch may be the result of reduced HO activity and the associated reduction in the cytoprotective HO products, carbon monoxide and biliverdin/bilirubin, because addition of biliverdin to HO-2(-/-) cells attenuated angiogenesis and reduced superoxide production. This transformation underscores the importance of HO-2 in the regulation of endothelial cell homeostasis.
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Células Endoteliais/enzimologia , Endotélio Vascular/enzimologia , Deleção de Genes , Heme Oxigenase (Desciclizante)/deficiência , Inflamação/enzimologia , Neovascularização Patológica/enzimologia , Estresse Oxidativo , Animais , Western Blotting , Células Cultivadas , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Heme Oxigenase (Desciclizante)/genética , Heme Oxigenase (Desciclizante)/metabolismo , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Camundongos , Camundongos Knockout , Microscopia de Fluorescência , NF-kappa B/imunologia , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Estresse Oxidativo/genética , Receptores de Fatores de Crescimento do Endotélio Vascular/biossíntese , Superóxidos/metabolismo , Fator A de Crescimento do Endotélio Vascular/biossínteseRESUMO
Two studies were conducted of students with and without persisting Specific Learning Disabilities (SLDs-WL) in Grades 4 to 9 (M = 11 years, 11 months) that supported the hypotheses that CELF 4 parent ratings for listening (language by ear), speaking (language by mouth), reading (language by eye), and writing (language by hand) were correlated with both (a) normed, standardized behavioral measures of listening, speaking, reading, and writing achievement (Study 1, 94 boys and 61 girls); and (b) fMRI connectivity or DTI white matter integrity involving brain regions for primary motor functions or motor planning and control, or motor timing in a subsample of right handers who did not wear metal (Study 2, 28 boys and 16 girls). Results of these assessment studies, which have implications for planning instruction for three SLDs-WL (dysgraphia, dyslexia, and oral and written language learning disability [OWL LD]), show that more than multisensory instruction is relevant. Language by ear, by mouth, by eye, and by hand, as well as motor planning, control, and output skills and motor timing should also be considered. Research is also reviewed that supports other processes beyond multisensory input alone that should also be considered for students with SLDs-WL.
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Heme oxygenase (HO-1 and HO-2) represents an intrinsic cytoprotective and anti-inflammatory system based on its ability to modulate leukocyte migration and to inhibit expression of inflammatory cytokines and proteins. HO-2 deletion leads to unresolved corneal inflammation and chronic inflammatory complications including ulceration, perforation and neovascularization. We examined the consequences of HO-2 deletion on hemangiogenesis and lymphangiogenesis in the model of suture-induced inflammatory neovascularization. An 8.0 silk suture was placed at the corneal apex of wild type and HO-2 null mice. Neovascularization was assessed by vital microscopy and quantified by image analysis. Hemangiogenesis and lymphangiogenesis were determined by immunofluorescence staining using anti-CD31 and anti-LYVE-1 antibodies, respectively. Inflammation was quantified by histology and myeloperoxidase activity. The levels of HO-1 expression and inflammatory cytokines were determined by real time PCR and ELISA, respectively. Corneal sutures produced a consistent inflammatory response and a time-dependent neovascularization. The response in HO-2 null mice was associated with a greater increase compared to the wild type in the number of leukocytes (827,600+/-129,000 vs. 294,500+/-57,510; p<0.05), neovessels measured by vital microscopy (21.91+/-1.05 vs. 12.77+/-1.55 mm; p<0.001) 4 days after suture placement. Hemangiogenesis but not lymphangiogenesis was more pronounced in HO-2 null mice compared to wild type mice. Induction of HO-1 in sutured corneas was greatly attenuated in HO-2 null corneas and treatment with biliverdin diminished the exaggerated inflammatory and neovascular response in HO-2 null mice. The demonstration that the inflammatory responses, including expression of proinflammatory proteins, inflammatory cell influx and hemangiogenesis are exaggerated in HO-2 knockout mice strongly supports the notion that the HO system is critical for controlling the inflammatory and neovascular response in the cornea. Hence, pharmacological amplification of this system may constitute a novel therapeutic strategy for the treatment of corneal disorders associated with excessive inflammation and neovascularization.
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Biliverdina/uso terapêutico , Neovascularização da Córnea/prevenção & controle , Ceratite/prevenção & controle , Animais , Córnea/enzimologia , Neovascularização da Córnea/enzimologia , Neovascularização da Córnea/patologia , Progressão da Doença , Avaliação Pré-Clínica de Medicamentos , Feminino , Heme Oxigenase (Desciclizante)/deficiência , Heme Oxigenase (Desciclizante)/metabolismo , Heme Oxigenase (Desciclizante)/fisiologia , Heme Oxigenase-1/metabolismo , Ceratite/enzimologia , Ceratite/patologia , Masculino , Camundongos , Camundongos Knockout , Peroxidase/metabolismoRESUMO
Injury to the cornea leads to formation of mediators that initiate and amplify inflammatory responses and neovascularization. Among these are lipid mediators generated by a cytochrome P450 (CYP) enzyme identified as CYP4B1. Increased corneal CYP4B1 expression increases limbal angiogenic activity through the production of 12-hydroxyeicosatrienoic acid (12-HETrE), a potent inflammatory and angiogenic eicosanoid. We used siRNA duplexes targeting CYP4B1 to substantiate the link between CYP4B1 expression, 12-HETrE production and angiogenesis in a model of suture-induced corneal neovascularization. Intrastromal sutures induced a time-dependent neovascular response which was significantly attenuated by CYP4B1-specific siRNAs but not by nonspecific siRNA. CYP4B1 mRNA was reduced by 60% and 12-HETrE's levels were barely detected in corneal homogenates from eyes treated with the CYP4B1-specific siRNA. The decreased neovascular response in CYP4B1 siRNA-treated eyes was associated with a 75% reduction in corneal VEGF mRNA levels. Transfection of rabbit corneal epithelial cells with CYP4B1 cDNA induced VEGF expression. Conversely, treatment with CYP4B1 siRNA or addition of a CYP4B1 inhibitor significantly decreased VEGF mRNA levels; addition of 12-HETrE potently increased them. The results strongly implicate the corneal CYP4B1 as a component of the inflammatory and neovascular cascade initiated by injury and further suggest that CYP4B1-12-HETrE is a proximal regulator of VEGF expression.
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Hidrocarboneto de Aril Hidroxilases/deficiência , Hidrocarboneto de Aril Hidroxilases/genética , Neovascularização da Córnea/enzimologia , Neovascularização da Córnea/genética , Regulação para Baixo , RNA Interferente Pequeno/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Hidrocarboneto de Aril Hidroxilases/metabolismo , Córnea/citologia , Células Epiteliais/enzimologia , Ácidos Hidroxieicosatetraenoicos/biossíntese , Reação em Cadeia da Polimerase , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Coelhos , Fator A de Crescimento do Endotélio Vascular/biossínteseRESUMO
Injury to the ocular surface increases corneal epithelial production of cyclooxygenase (COX)-derived eicosanoids but this increase correlates poorly to the inflammatory sequelae. Moreover, corticosteroids are effective in treatment of this inflammation but NSAIDs are not. The discovery of COX-2 that is differentially affected by common NSAIDs reopened the question of the role of prostaglandins in ocular surface inflammation. We examined the presence and inducibility of COX-2 in the corneal epithelium following hypoxia-induced injury in vivo and in vitro. COX-2, but not COX-1, protein levels markedly increased following hypoxia. Use of the selective COX-2 inhibitor, NS-398, indicated that COX activity in hypoxic corneas or cells is essentially that of COX-2; in control cells, both COX-1 and COX-2 contributed equally to the production of PGE2. COX-2 protein overexpression induced by hypoxia was not associated with a parallel increase in PGE2 accumulation but the enzyme regained full catalytic activity when cells were re-exposed to normoxia in the presence of heme and arachidonic acid. Decreases in the levels of oxygen and heme, essential substrates/cofactors for COX catalytic activity, contributed to a diminished prostanoid production during hypoxia. These results suggest that in hypoxic injury, molecules other than COX-derived prostanoids play a major pro-inflammatory role. Furthermore, this study provides an explanation for the ineffectiveness of classical NSAIDs in the treatment of hypoxia-related ocular surface inflammation.
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Hipóxia Celular/imunologia , Epitélio Corneano/enzimologia , Epitélio Corneano/imunologia , Isoenzimas/imunologia , Isoenzimas/metabolismo , Ceratite/enzimologia , Ceratite/imunologia , Prostaglandina-Endoperóxido Sintases/imunologia , Prostaglandina-Endoperóxido Sintases/metabolismo , Animais , Linhagem Celular , Técnicas de Cultura , Ciclo-Oxigenase 1 , Ciclo-Oxigenase 2 , Dinoprostona/imunologia , Dinoprostona/metabolismo , CoelhosRESUMO
Injury to the ocular surface provokes an inflammatory response that is mediated, at least in part, by corneal epithelial derived 12-hydroxyeicosanoids (HETEs) including 12-HETE and 12-HETrE; both metabolites exhibit potent inflammatory and angiogenic properties and are formed by a cytochrome P450 (CYP) 4B1. Retinoids are known to mediate wound-healing processes in many tissues and, as such, are integral components of the inflammatory response. We studied the effect of various retinoids on corneal synthesis of 12-hydroxyeicosanoids and on activation of CYP4B1 gene expression. Corneal organ cultures were used to assess the effect of retinoic acid on epithelial metabolism of arachidonic acid to 12-hydroxyeicosanoids. Luciferase reporter vectors containing different lengths of the CYP4B1 3.4 kb-5'-untranslated region were used to examine the effect of vitamin D and retinoids (9-cis-retinoic acid and all-trans retinoic acid) on transcriptional activation of CYP4B1 in transient transfection experiments with HepG2 cells. Vitamin D had no effect on CYP4B1 promoter activity, but 9-cis and all-trans retinoic acids increased promoter activity by up to 70% over control. Addition of both 9-cis and all-trans retinoic acids resulted in an additive effect increasing promoter activity by 2-fold. The increased promoter activity correlated with the presence of RAR/RXR binding motifs. Incubation of corneal organ culture for 24 hours in the presence of 9-cis and all-trans retinoic acids increased the synthesis of 12-HETE and 12-HETrE by 2-fold. The finding that retinoic acid increases the expression of the CYP4B1 gene and enhances production of the inflammatory 12-hydroxyeicosanoids in the corneal epithelium may provide a linkage between wound healing and inflammation in the ocular surface.
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Hidrocarboneto de Aril Hidroxilases/genética , Epitélio Corneano/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Ácidos Hidroxieicosatetraenoicos/biossíntese , Tretinoína/farmacologia , Alitretinoína , Animais , Linhagem Celular Tumoral , Epitélio Corneano/enzimologia , Feminino , Humanos , Luciferases/genética , Masculino , Técnicas de Cultura de Órgãos , Oxigênio/metabolismo , Plasmídeos , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/efeitos dos fármacos , Coelhos , Ativação Transcricional/genética , Vitamina D/farmacologiaRESUMO
PURPOSE: Cyclooxygenase (COX)-, lipoxygenase (LOX)-, and cytochrome P450 monooxygenase (CYP)-derived eicosanoids have been implicated in ocular surface inflammation and neovascularization. These eicosanoids are subjected to regulation by enzymes, such as heme oxygenases (HOs) and ferritin. METHODS: Quantitative polymerase chain reaction and lipidomics based on liquid chromatography-tandem mass spectrometry were performed on pterygia from patients undergoing surgical pterygium excision. Control tissues consisted of donor corneas. In addition, lipidomics based on liquid chromatography-tandem mass spectrometry was performed on tears collected from patients before the surgery. RESULTS: Messenger RNA (mRNA) expression of HO-2, the constitutive HO isoform, was upregulated by 40% in pterygia compared with control tissue, whereas the mRNA level of the inducible form, HO-1, was downregulated by more than 50%. Levels of CYP4B1 mRNA showed an approximate 2-fold increase in pterygia compared with control. Lipidomic analysis of tissues indicated a moderate elevation in Prostaglandin E2 and thromboxane B2 levels in pterygia compared with control. Among the LOX-derived metabolites, the antiinflammatory-hydroxyeicosatetraenoic acid (15-HETE) levels were significantly reduced in pterygia (79.3 ± 48.11 pg/mg protein) compared with control (586.2 ± 213.5 pg/mg protein), whereas the proinflammatory LOX- and CYP4B1-derived 12-HETE levels were 10-fold higher in pterygia (2768 ± 832.3 pg/mg protein) compared with control (231.4 ± 87.35 pg/mg protein). Prostaglandin E2 and HETEs were also present in tears from patients with pterygium but were not detected in tears from healthy volunteers. The mRNA expression levels of both light and heavy chain ferritin were 60% and 30% lower, respectively, in pterygia compared with control. CONCLUSIONS: We believe that a dysfunctional HO-ferritin system leads to increased levels of proinflammatory mediators, thus contributing to the inflammation characteristic of pterygia.
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Ferritinas/genética , Regulação Enzimológica da Expressão Gênica/fisiologia , Heme Oxigenase (Desciclizante)/genética , Heme Oxigenase-1/genética , Pterígio/genética , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/metabolismo , Adulto , Hidrocarboneto de Aril Hidroxilases/genética , Cromatografia Líquida de Alta Pressão , Dinoprostona/metabolismo , Feminino , Humanos , Ácidos Hidroxieicosatetraenoicos/metabolismo , Isoenzimas , Masculino , Pessoa de Meia-Idade , Pterígio/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Lágrimas/metabolismo , Tromboxano B2/metabolismoRESUMO
PURPOSE. The heme oxygenase system (HO-1 and HO-2) represents an intrinsic cytoprotective and anti-inflammatory pathway based on its ability to modulate leukocyte migration and to inhibit the expression of inflammatory cytokines and proteins by its products biliverdin/bilirubin and carbon monoxide. Corneal injury in HO-2 null mice leads to impaired healing and chronic inflammatory complications, including ulceration and neovascularization. The authors examined whether topically administered biliverdin can counteract the effects of HO deficiency in a corneal epithelial injury model. METHODS. HO-2 null mice were treated with biliverdin 1 hour before epithelial injury and twice a day thereafter. Reepithelialization and neovascularization were assessed by fluorescein staining and vital microscopy, respectively, and were quantified by image analysis. Inflammation was quantified by histology and Gr-1-specific immunofluorescence, and oxidative stress was assessed by DHE fluorescence. RESULTS. Treatment with biliverdin accelerated wound closure, inhibited neovascularization and reduced epithelial defects. It also reduced inflammation, as evidenced by a reduction in the appearance of inflammatory cells and the expression levels of inflammatory and oxidant proteins, including KC and NOXs. CONCLUSIONS. The results clearly show that biliverdin, directly or through its metabolism to bilirubin by biliverdin reductase-the expression of which is increased after injury-rescues the aberrant inflammatory phenotype, further underscoring the importance of the HO system in the cornea for the execution of an ordered inflammatory and reparative response.
Assuntos
Biliverdina/administração & dosagem , Neovascularização da Córnea/prevenção & controle , Úlcera da Córnea/tratamento farmacológico , Epitélio Corneano/lesões , Heme Oxigenase (Desciclizante)/fisiologia , Cicatrização/efeitos dos fármacos , Administração Tópica , Animais , Quimiocinas/metabolismo , Doença Crônica , Neovascularização da Córnea/enzimologia , Úlcera da Córnea/enzimologia , Ensaio de Imunoadsorção Enzimática , Epitélio Corneano/enzimologia , Técnica Indireta de Fluorescência para Anticorpo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia de Fluorescência , Estresse Oxidativo/efeitos dos fármacos , Fenótipo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Our studies demonstrated that Heme oxygenase (HO), in particular, the constitutive HO-2, is critical for a self-resolving inflammatory and repair response in the cornea. Epithelial injury in HO-2 null mice leads to impaired wound closure and chronic inflammation in the cornea. This study was undertaken to examine the possible relationship between HO-2 and the recruitment of neutrophils following a corneal surface injury in wild type (WT) and HO-2 knockout (HO-2(-/-)) mice treated with Gr-1 monoclonal antibody to deplete peripheral neutrophils. Epithelial injury was performed by removing the entire corneal epithelium. Infiltration of inflammatory cell into the cornea in response to injury was higher in HO-2(-/-) than in WT. However, the rate of corneal wound closure following neutrophil depletion was markedly inhibited in both WT and HO-2(-/-) mice by 60% and 85%, respectively. Neutropenia induced HO-1 expression in WT but not in HO-2(-/-) mice. Moreover, endothelial cells lacking HO-2 expressed higher levels of the Midkine and VE-cadherin and displayed strong adhesion to neutrophils suggesting that perturbation in endothelial cell function caused by HO-2 depletion underlies the increased infiltration of neutrophils into the HO-2(-/-) cornea. Moreover, the fact that neutropenia worsened epithelial healing of the injured cornea in both WT and HO-2(-/-) mice suggest that cells other than neutrophils contribute to the exaggerated inflammation and impaired wound healing seen in the HO-2 null cornea.
Assuntos
Córnea/imunologia , Lesões da Córnea , Deleção de Genes , Heme Oxigenase (Desciclizante)/deficiência , Heme Oxigenase (Desciclizante)/genética , Neutrófilos/imunologia , Cicatrização/imunologia , Animais , Adesão Celular/imunologia , Contagem de Células , Córnea/enzimologia , Córnea/metabolismo , Células Endoteliais/patologia , Regulação Enzimológica da Expressão Gênica/imunologia , Heme Oxigenase-1/genética , Inflamação/imunologia , Inflamação/metabolismo , Camundongos , Infiltração de Neutrófilos/genética , Neutrófilos/citologia , Neutrófilos/enzimologia , Neutrófilos/metabolismo , Cicatrização/genéticaRESUMO
OBJECTIVE: This study was aimed at obtaining a profile of lipids and proteins with a paracrine function in normal and diabetic vitreous and exploring whether the profile correlates with retinal pathology. RESEARCH DESIGN AND METHODS: Vitreous was recovered from 47 individuals undergoing vitreoretinal surgery: 16 had nonproliferative diabetic retinopathy (NPDR), 15 had proliferative diabetic retinopathy, 7 had retinal detachments, and 9 had epiretinal membranes. Protein and lipid autacoid profiles were determined by protein arrays and mass spectrometry-based lipidomics. RESULTS: Vitreous lipids included lipoxygenase (LO)- and cytochrome P450 epoxygenase (CYP)-derived eicosanoids. The most prominent LO-derived eicosanoid was 5-hydroxyeicosate traenoic acid (HETE), which demonstrated a diabetes-specific increase (P = 0.027) with the highest increase in NPDR vitreous. Vitreous also contained CYP-derived epoxyeicosatrienoic acids; their levels were higher in nondiabetic than diabetic vitreous (P < 0.05). Among inflammatory, angiogenic, and angiostatic cytokines and chemokines, only vascular endothelial growth factor (VEGF) showed a significant diabetes-specific profile (P < 0.05), although a similar trend was noted for tumor necrosis factor (TNF)-alpha. Soluble VEGF receptors R1 and R2 were detected in all samples with lowest VEGF-R2 levels (P < 0.05) and higher ratio of VEGF to its receptors in NPDR and PDR vitreous. CONCLUSIONS: This study is the first to demonstrate diabetes-specific changes in vitreous lipid autacoids including arachidonate and docosahexanoate-derived metabolites indicating an increase in inflammatory versus anti-inflammatory lipid mediators that correlated with increased levels of inflammatory and angiogenic proteins, further supporting the notion that inflammation plays a role the pathogenesis of this disease.
Assuntos
Autacoides/análise , Quimiocinas/análise , Citocinas/análise , Retinopatia Diabética/metabolismo , Eicosanoides/análise , Corpo Vítreo/química , Idoso , Autacoides/metabolismo , Quimiocinas/metabolismo , Citocinas/metabolismo , Progressão da Doença , Eicosanoides/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estatísticas não Paramétricas , Corpo Vítreo/metabolismoAssuntos
Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Sistema Enzimático do Citocromo P-450/metabolismo , Eicosanoides/metabolismo , Traumatismos Oculares/metabolismo , Traumatismos Oculares/patologia , Inflamação/metabolismo , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/metabolismo , Ácido 8,11,14-Eicosatrienoico/metabolismo , Animais , Queimaduras/metabolismo , Queimaduras/patologia , Lentes de Contato , Modelos Animais de Doenças , Neovascularização Fisiológica , Coelhos , CicatrizaçãoRESUMO
PURPOSE: Heme oxygenase (HO) is considered a fundamental endogenous immunomodulatory, cytoprotective, and anti-inflammatory system. This protective function is primarily ascribed to the inducible HO-1. The authors examined the effect of HO-1 induction on corneal inflammation and wound healing in mice undergoing epithelial injury. METHODS: C57BL6 mice were treated with SnCl(2) the day before epithelial injury and once daily thereafter. The corneal epithelium was removed with the use of a corneal rust ring remover in anesthetized mice. Reepithelialization was measured by fluorescein staining. The inflammatory response was examined by histology and was quantified by the myeloperoxidase assay. Inflammatory lipid mediators were detected and quantified by LC/MS/MS-based lipidomic analysis. HO-1 expression was assessed by real-time PCR, and HO activity was determined by measuring HO-dependent carbon monoxide production. RESULTS: Epithelial injury caused a time-dependent transient increase in HO-1 expression and HO activity that was significantly amplified by treatment with SnCl(2), resulting in a twofold to threefold increase in mRNA levels and a similar increase in corneal HO activity. Induction of HO-1 was associated with a significant acceleration of wound healing when compared with a vehicle-treated group and with attenuation of the inflammatory response, evidenced by a significant decrease in the number of infiltrating cells and by a significant reduction in the expression and production of proinflammatory lipid mediators and cytokines. CONCLUSIONS: Increased expression of HO-1 provides a mechanism that modulates inflammation and promotes wound closure; pharmacologic amplification of this system may constitute a novel strategy to treat corneal inflammation while accelerating wound repair after injury.
Assuntos
Epitélio Corneano/fisiologia , Heme Oxigenase-1/biossíntese , Ceratite/prevenção & controle , Cicatrização/fisiologia , Animais , Hidrocarboneto de Aril Hidroxilases/genética , Quimiocina CXCL2/metabolismo , Cromatografia Líquida , Indução Enzimática/efeitos dos fármacos , Epitélio Corneano/lesões , Heme Oxigenase-1/genética , Interleucina-1alfa/metabolismo , Ceratite/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Peroxidase/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas em Tandem , Fatores de Tempo , Compostos de Estanho/farmacologiaRESUMO
Heme oxygenase (HO) represents an intrinsic anti-inflammatory system based on its ability to regulate leukocyte function and inhibit expression of proinflammatory cytokines. This anti-inflammatory function is linked to the inducible isoform HO-1; the role of the constitutive isoform HO-2 is unknown. The current study was undertaken to investigate the role of HO-2 in the regulation of the acute inflammatory and reparative response by using HO-2-null mice and well-established animal models of epithelial injury and antigen-induced peritonitis. Here we show that in vivo deletion of HO-2 disables execution of the acute inflammatory and reparative response after epithelial injury and leads to an exaggerated inflammatory response in antigen-induced peritonitis. HO-2 deletion was associated with impaired HO-1 induction, indicating that HO-2 is critical for HO-1 expression and that the subsequent failure to up-regulate the HO system may contribute to unresolved inflammation and the development of chronic inflammatory conditions. Indeed, supplementation with the HO bioactive product, biliverdin, rescued the acute inflammatory and reparative response in HO-2-null mice. Thus, HO-2 sets in place a basal tone of anti-inflammatory signals that may be a prerequisite for the ordered execution of an inflammatory and reparative response.
Assuntos
Heme Oxigenase (Desciclizante)/metabolismo , Inflamação/imunologia , Cicatrização/fisiologia , Animais , Biliverdina/farmacologia , Córnea/irrigação sanguínea , Córnea/citologia , Córnea/efeitos dos fármacos , Córnea/patologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Heme Oxigenase (Desciclizante)/deficiência , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Mediadores da Inflamação/metabolismo , Leucócitos/imunologia , Metabolismo dos Lipídeos , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Patológica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Cicatrização/efeitos dos fármacosRESUMO
Injury to the ocular surface induces the production of the corneal epithelial-derived 12-hydroxyeicosatetrienoic acid (12-HETrE), which exhibits stereospecific potent inflammatory and angiogenic properties and is formed by a cytochrome P450 (P450) enzyme, CYP4B1. We have cloned the rabbit corneal CYP4B1 into the expression plasmid pIRES2-enhanced green fluorescent protein (EGFP) and examined the effect of CYP4B1 overexpression on corneal inflammation in vivo and limbal vessel sprouting ex vivo. Cultured rabbit corneal epithelial cells transfected with pIRES2-EGFP-CYP4B1 metabolized arachidonic acid to 12-HETrE at a rate five times higher than that of pIRES2-EGFP-transfected cells (3.53 +/- 0.08 versus 0.62 +/- 0.10 nmol/h/10(6) cells; mean +/- S.E.M., n = 6, p < 0.05), indicating a functional expression of the CYP4B1. Injection of either plasmid into the rabbit cornea resulted in EGFP fluorescence in the corneal epithelium. However, corneal neovascularization, as measured by the length of penetrating blood vessels, was significantly greater in the corneas of eyes transfected with the pIRES2-CYP4B1 compared with pIRES2-EGFP. Corneal-limbal explants from eyes transfected with pIRES2-CYP4B1 showed a marked angiogenic activity (46 +/- 10 versus 12 +/- 3 mm capillary length, n = 6, p < 0.05), which correlated with increased levels of 12-HETrE, the CYP4B1-derived angiogenic 12-hydroxyeicosanoid (0.93 +/- 0.18 versus 0.15 +/- 0.02 pmol/explant, n = 6, p < 0.05), and was inhibited (76 +/- 5%) by the P450 inhibitor 17-octadecynoic acid. The results further implicate the corneal CYP4B1 as a component of the inflammatory and angiogenic cascade initiated by injury to the ocular surface and raise the possibility of a new therapeutic target for preventing corneal neovascularization, namely, the CYP4B1-12-HETrE system.