RESUMO
The current standard of care for moderate to severe ischemic stroke is thrombolytic therapy with tissue plasminogen activator (tPA). Treatment with tPA can significantly improve neurologic outcomes; however, thrombolytic therapy is associated with an increased risk of intracerebral hemorrhage (ICH). The risk of hemorrhage significantly limits the use of thrombolytic therapy, and identifying pathways induced by tPA that increase this risk could provide new therapeutic options to extend thrombolytic therapy to a wider patient population. Here, we investigate the role of protein kinase Cß (PKCß) phosphorylation of the tight junction protein occludin during ischemic stroke and its role in cerebrovascular permeability. We show that activation of this pathway by tPA is associated with an increased risk of ICH. Middle cerebral artery occlusion (MCAO) increased phosphorylation of occludin serine 490 (S490) in the ischemic penumbra in a tPA-dependent manner, as tPA-/- mice were significantly protected from MCAO-induced occludin phosphorylation. Intraventricular injection of tPA in the absence of ischemia was sufficient to induce occludin phosphorylation and vascular permeability in a PKCß-dependent manner. Blocking occludin phosphorylation, either by targeted expression of a non-phosphorylatable form of occludin (S490A) or by pharmacologic inhibition of PKCß, reduced MCAO-induced permeability and improved functional outcome. Furthermore, inhibiting PKCß after MCAO prevented ICH associated with delayed thrombolysis. These results show that PKCß phosphorylation of occludin is a downstream mediator of tPA-induced cerebrovascular permeability and suggest that PKCß inhibitors could improve stroke outcome and prevent ICH associated with delayed thrombolysis, potentially extending the window for thrombolytic therapy in stroke.
Assuntos
AVC Isquêmico , Acidente Vascular Cerebral , Animais , Hemorragia Cerebral/tratamento farmacológico , Hemorragia Cerebral/etiologia , Fibrinolíticos/uso terapêutico , Humanos , Infarto da Artéria Cerebral Média/tratamento farmacológico , Camundongos , Ocludina/genética , Ocludina/metabolismo , Fosforilação , Acidente Vascular Cerebral/complicações , Acidente Vascular Cerebral/etiologia , Terapia Trombolítica/efeitos adversos , Terapia Trombolítica/métodos , Ativador de Plasminogênio Tecidual/metabolismoRESUMO
BACKGROUND: Several retinal pathologies exhibit both inflammation and breakdown of the inner blood-retinal barrier (iBRB) resulting in vascular permeability, suggesting that treatments that trigger resolution of inflammation may also promote iBRB restoration. METHODS: Using the mouse retinal ischemia-reperfusion (IR) injury model, we followed the time course of neurodegeneration, inflammation, and iBRB disruption and repair to examine the relationship between resolution of inflammation and iBRB restoration and to determine if minocycline, a tetracycline derivative shown to reverse microglial activation, can hasten these processes. RESULTS: A 90-min ischemic insult followed by reperfusion in the retina induced cell apoptosis and inner retina thinning that progressed for approximately 2 weeks. IR increased vascular permeability within hours, which resolved between 3 and 4 weeks after injury. Increased vascular permeability coincided with alteration and loss of endothelial cell tight junction (TJ) protein content and disorganization of TJ protein complexes. Shunting of blood flow away from leaky vessels and dropout of leaky capillaries were eliminated as possible mechanisms for restoring the iBRB. Repletion of TJ protein contents occurred within 2 days after injury, long before restoration of the iBRB. In contrast, the eventual re-organization of TJ complexes at the cell border coincided with restoration of the barrier. A robust inflammatory response was evident a 1 day after IR and progressed to resolution over the 4-week time course. The inflammatory response included a rapid and transient infiltration of granulocytes and Ly6C+ classical inflammatory monocytes, a slow accumulation of Ly6Cneg monocyte/macrophages, and activation, proliferation, and mobilization of resident microglia. Extravasation of the majority of CD45+ leukocytes occurred from the superficial plexus. The presence of monocyte/macrophages and increased numbers of microglia were sustained until the iBRB was eventually restored. Intervention with minocycline to reverse microglial activation at 1 week after injury promoted early restoration of the iBRB coinciding with decreased expression of mRNAs for the microglial M1 markers TNF-α, IL-1ß, and Ptgs2 (Cox-2) and increased expression of secreted serine protease inhibitor Serpina3n mRNA. CONCLUSIONS: These results suggest that iBRB restoration occurs as TJ complexes are reorganized and that resolution of inflammation and restoration of the iBRB following retinal IR injury are functionally linked.
Assuntos
Barreira Hematorretiniana/patologia , Inflamação/patologia , Traumatismo por Reperfusão/patologia , Retina/patologia , Vasos Retinianos/patologia , Animais , Apoptose/fisiologia , Permeabilidade Capilar/fisiologia , Fragmentação do DNA , Modelos Animais de Doenças , Camundongos , Microglia/metabolismo , Recuperação de Função Fisiológica/fisiologiaRESUMO
BACKGROUND: Human chorionic gonadotropin (hCG) and luteinizing hormone (LH) are pro-angiogenic gonadotropic hormones, which classically target the reproductive organs. However, hCG, LH, and their shared CG/LH receptor are also present in the human eye. The possibility that a deficiency of these hormones may be involved in the pathogenesis of retinopathy of prematurity (ROP) during its early non-proliferative phase has not been explored. METHODS: We conducted a cross-sectional study of Michigan-born preterm infants utilizing dried blood spots. We analyzed hCG and LH blood levels at 1 week and 4 weeks of age from 113 study participants (60 without ROP; 53 with non-proliferative ROP). We utilized electrochemiluminescence assays on the Mesoscale Discovery platform. RESULTS: Similar levels of hCG are found in preterm infants at both 1 week and 4 weeks after birth. Preterm infants with non-proliferative ROP, after adjusting for sex and gestational age, have 2.42 [95% CI: 1.08-5.40] times the odds of having low hCG at fourth week of age. CONCLUSIONS: We found that hCG is present postnatally in preterm infants and that a deficiency of hCG at 4 weeks of age is potentially associated with non-proliferative ROP. This provides novel evidence to suggest that hCG may participate in human retinal angiogenesis.
Assuntos
Gonadotropina Coriônica/sangue , Recém-Nascido Prematuro/sangue , Retinopatia da Prematuridade/sangue , Biomarcadores/sangue , Gonadotropina Coriônica/deficiência , Estudos Transversais , Teste em Amostras de Sangue Seco , Feminino , Idade Gestacional , Humanos , Recém-Nascido , Hormônio Luteinizante/sangue , Masculino , Michigan , Triagem Neonatal , Estudo de Prova de Conceito , Retinopatia da Prematuridade/diagnóstico , Retinopatia da Prematuridade/etiologia , Medição de Risco , Fatores de Risco , Fatores de TempoRESUMO
INTRODUCTION: Retinopathy of prematurity (ROP) is a potentially serious eye disorder affecting very preterm infants. Non-proliferative ROP (NP-ROP), also known as Early Stage ROP, is characterized by deficient retinal angiogenesis. Proliferative ROP (P-ROP), also known as Late Stage ROP, is characterized by pathologic angiogenesis. The use of neonatal haemoglobin A1C as a biomarker for ROP has not yet been evaluated. MATERIALS AND METHODS: We modified the Haemoglobin A1C assay for use with neonatal dried blood spots (DBS) and then assessed A1C levels via Elisa immunoassay on DBS from 43 preterm infants (with gestational ages 26-28 weeks). We measured A1C on DBS collected at <1 week and 4 weeks of chronological age. RESULTS: Compared to matched counterparts without ROP, there is significantly lower HbA1c in infants who develop NP-ROP, this occurs at Week 4 (p=0.004), but is not seen at Week 1; there is significantly higher HbA1c in infants with P-ROP, this occurs both at Week 1 (p<0.05) and Week 4 (p=0.005). CONCLUSIONS: The A1C test, modified for use with DBS, is a feasible biomarker for ROP; low A1C is a potential biomarker for non-proliferative ROP and high A1C is for proliferative ROP.
Assuntos
Biomarcadores/sangue , Hemoglobinas Glicadas/metabolismo , Recém-Nascido Prematuro/sangue , Retinopatia da Prematuridade/sangue , Feminino , Idade Gestacional , Hemoglobinas Glicadas/isolamento & purificação , Humanos , Lactente , Recém-Nascido , Masculino , Retina/metabolismo , Retina/patologia , Retinopatia da Prematuridade/patologia , Fatores de RiscoRESUMO
Recent evidence suggests an important role for outer retinal cells in the pathogenesis of diabetic retinopathy (DR). Here we investigated the effect of the visual cycle inhibitor retinylamine (Ret-NH2) on the development of early DR lesions. Wild-type (WT) C57BL/6J mice (male, 2 months old when diabetes was induced) were made diabetic with streptozotocin, and some were given Ret-NH2 once per week. Lecithin-retinol acyltransferase (LRAT)-deficient mice and P23H mutant mice were similarly studied. Mice were euthanized after 2 (WT and Lrat(-/-)) and 8 months (WT) of study to assess vascular histopathology, accumulation of albumin, visual function, and biochemical and physiological abnormalities in the retina. Non-retinal effects of Ret-NH2 were examined in leukocytes treated in vivo. Superoxide generation and expression of inflammatory proteins were significantly increased in retinas of mice diabetic for 2 or 8 months, and the number of degenerate retinal capillaries and accumulation of albumin in neural retina were significantly increased in mice diabetic for 8 months compared with nondiabetic controls. Administration of Ret-NH2 once per week inhibited capillary degeneration and accumulation of albumin in the neural retina, significantly reducing diabetes-induced retinal superoxide and expression of inflammatory proteins. Superoxide generation also was suppressed in Lrat(-/-) diabetic mice. Leukocytes isolated from diabetic mice treated with Ret-NH2 caused significantly less cytotoxicity to retinal endothelial cells ex vivo than did leukocytes from control diabetics. Administration of Ret-NH2 once per week significantly inhibited the pathogenesis of lesions characteristic of early DR in diabetic mice. The visual cycle constitutes a novel target for inhibition of DR.
Assuntos
Retinopatia Diabética/tratamento farmacológico , Diterpenos/uso terapêutico , Aciltransferases/deficiência , Aciltransferases/metabolismo , Animais , Separação Celular , Retinopatia Diabética/sangue , Retinopatia Diabética/patologia , Retinopatia Diabética/fisiopatologia , Diterpenos/administração & dosagem , Diterpenos/química , Diterpenos/farmacologia , Relação Dose-Resposta a Droga , Células Endoteliais/metabolismo , Glucose/metabolismo , Inflamação/patologia , Leucócitos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Estresse Oxidativo/efeitos dos fármacos , Permeabilidade , Células Fotorreceptoras de Vertebrados/metabolismo , Retina/efeitos dos fármacos , Retina/patologia , Retina/fisiopatologia , Superóxidos/metabolismoRESUMO
Reactive oxygen species play an important role in the pathogenesis of diabetic retinopathy. We studied the role of adrenergic and serotonin receptors in the generation of superoxide by retina and 661W retinal cells in high glucose and of the α1-adrenergic receptor (AR) on vascular lesions of the retinopathy in experimentally diabetic C57Bl/6J mice (and controls) after 2 and 8 months. Compared with 5 mM glucose, incubating cells or retinal explants in 30 mM glucose induced superoxide generation. This response was reduced or ablated by pharmacologic inhibition of the α1-AR (a Gq-coupled receptor) or Gs-coupled serotonin (5-HT2, 5-HT4, 5-HT6, and 5-HT7) receptors or by activation of the Gi-coupled α2-AR. In elevated glucose, the α1-AR produced superoxide via phospholipase C, inositol triphosphate-induced Ca(2+) release, and NADPH oxidase, and pharmacologic inhibition of these reactions prevented the superoxide increase. Generation of retinal superoxide, expression of proinflammatory proteins, and degeneration of retinal capillaries in diabetes all were significantly inhibited with daily doxazosin or apocynin (inhibitors of α1-AR and NADPH oxidase, respectively), but increased vascular permeability was not significantly affected. Adrenergic receptors, and perhaps other GPCRs, represent novel targets for inhibiting the development of important features of diabetic retinopathy.
Assuntos
Capilares/patologia , Diabetes Mellitus Experimental/complicações , Retinopatia Diabética/patologia , Receptores Adrenérgicos/metabolismo , Receptores de Serotonina/metabolismo , Degeneração Retiniana/patologia , Superóxidos/metabolismo , Animais , Capilares/metabolismo , Células Cultivadas , AMP Cíclico/metabolismo , Diabetes Mellitus Experimental/fisiopatologia , Retinopatia Diabética/etiologia , Retinopatia Diabética/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores Adrenérgicos/genética , Receptores de Serotonina/genética , Retina/citologia , Retina/metabolismo , Degeneração Retiniana/etiologia , Degeneração Retiniana/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family proteins mediate the adherence of infected erythrocytes to microvascular endothelia of various organs, including the placenta, thereby contributing to cerebral, placental, and other severe malaria pathogenesis. Several parasite proteins, including KAHRP and PfEMP3, play important roles in the cytoadherence by mediating the clustering of PfEMP1 in rigid knoblike structures on the infected erythrocyte surface. The lack of a subtelomeric region of chromosome 2 that contains kahrp and pfemp3 causes reduced cytoadherence. In this study, microarray transcriptome analysis showed that the absence of a gene cluster, comprising kahrp, pfemp3, and four other genes, results in the loss of parasitized erythrocytes adhering to chondroitin 4-sulfate (C4S). The role of one of these genes, PF3D7_0201600/PFB0080c, which encodes PHISTb (Plasmodium helical interspersed subtelomeric b) domain-containing RESA-like protein 1 expressed on the infected erythrocyte surface, was investigated. Disruption of PFB0080c resulted in increased var2csa transcription and VAR2CSA surface expression, leading to higher C4S-binding capacity of infected erythrocytes. Further, PFB0080c-knock-out parasites stably maintained the C4S adherence through many generations of growth. Although the majority of PFB0080c-knock-out parasites bound to C4S even after culturing for 6 months, a minor population bound to both C4S and CD36. These results strongly suggest that the loss of PFB0080c markedly compromises the var gene switching process, leading to a marked reduction in the switching rate and additional PfEMP1 expression by a minor population of parasites. PFB0080c interacts with VAR2CSA and modulates knob-associated Hsp40 expression. Thus, PFB0080c may regulate VAR2CSA expression through these processes. Overall, we conclude that PFB0080c regulates PfEMP1 expression and the parasite's cytoadherence.
Assuntos
Antígenos de Protozoários/genética , Sulfatos de Condroitina/química , Eritrócitos/parasitologia , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Antígenos de Protozoários/metabolismo , Antígenos CD36/genética , Antígenos CD36/metabolismo , Adesão Celular , Sulfatos de Condroitina/metabolismo , Cromossomos , Eritrócitos/química , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP40/metabolismo , Humanos , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Família Multigênica , Peptídeos/deficiência , Peptídeos/genética , Plasmodium falciparum/metabolismo , Ligação Proteica , Proteínas de Protozoários/metabolismoRESUMO
BACKGROUND: Many retinal diseases are associated with vascular dysfunction accompanied by neuroinflammation. We examined the ability of minocycline (Mino), a tetracycline derivative with anti-inflammatory and neuroprotective properties, to prevent vascular permeability and inflammation following retinal ischemia-reperfusion (IR) injury, a model of retinal neurodegeneration with breakdown of the blood-retinal barrier (BRB). METHODS: Male Sprague-Dawley rats were subjected to 45 min of pressure-induced retinal ischemia, with the contralateral eye serving as control. Rats were treated with Mino prior to and following IR. At 48 h after reperfusion, retinal gene expression, cellular inflammation, Evan's blue dye leakage, tight junction protein organization, caspase-3 activation, and DNA fragmentation were measured. Cellular inflammation was quantified by flow-cytometric evaluation of retinal tissue using the myeloid marker CD11b and leukocyte common antigen CD45 to differentiate and quantify CD11b+/CD45low microglia, CD11b+/CD45hi myeloid leukocytes and CD11bneg/CD45hi lymphocytes. Major histocompatibility complex class II (MHCII) immunoreactivity was used to determine the inflammatory state of these cells. RESULTS: Mino treatment significantly inhibited IR-induced retinal vascular permeability and disruption of tight junction organization. Retinal IR injury significantly altered mRNA expression for 21 of 25 inflammation- and gliosis-related genes examined. Of these, Mino treatment effectively attenuated IR-induced expression of lipocalin 2 (LCN2), serpin peptidase inhibitor clade A member 3 N (SERPINA3N), TNF receptor superfamily member 12A (TNFRSF12A), monocyte chemoattractant-1 (MCP-1, CCL2) and intercellular adhesion molecule-1 (ICAM-1). A marked increase in leukostasis of both myeloid leukocytes and lymphocytes was observed following IR. Mino treatment significantly reduced retinal leukocyte numbers following IR and was particularly effective in decreasing the appearance of MHCII+ inflammatory leukocytes. Surprisingly, Mino did not significantly inhibit retinal cell death in this model. CONCLUSIONS: IR induces a retinal neuroinflammation within hours of reperfusion characterized by inflammatory gene expression, leukocyte adhesion and invasion, and vascular permeability. Despite Mino significantly inhibiting these responses, it failed to block neurodegeneration.
Assuntos
Barreira Hematorretiniana/efeitos dos fármacos , Inflamação/patologia , Minociclina/farmacologia , Fármacos Neuroprotetores/farmacologia , Traumatismo por Reperfusão/patologia , Retina/patologia , Animais , Barreira Hematorretiniana/patologia , Permeabilidade Capilar/efeitos dos fármacos , Imunofluorescência , Expressão Gênica/efeitos dos fármacos , Masculino , Degeneração Neural/patologia , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Retina/efeitos dos fármacosRESUMO
Mucins and mucin-type glycoproteins, collectively referred to as mucin-type O-glycans, are implicated in many important biological functions and pathological conditions, including malignancy. Presently, there is no reliable method to measure the total mucin-type O-glycans of a sample, which may contain one or more of these macromolecules of unknown structures. We report the development of an improved microassay that is based on the binding of lectins to the unique and constant GalNAc-Ser/Thr structural feature of mucin-type O-glycans. Since the sugar-amino acid linkage in the mucin-type O-glycans is invariably cryptic, we first chemically removed the heterogeneous peripheral and core saccharides of model glycoconjugates before examining for their interactions using an enzyme-linked lectin assay (ELLA). Desialylation of the model glycoconjugates led to maximal binding of the lectins but additional treatments such as Smith degradation did not result in increased binding. Of the lectins tested for their ability to probe the desialylated O-glycans, jacalin showed the highest sensitivity followed by champedak galactose binding (CGB) lectin and Vicia villosa agglutinin. Further improvement in the sensitivity of ELLA was achieved by using microtiter plates that were pre-coated with the CGB lectin, which increased the specificity of the assay to mucin-type O-glycans. Finally, the applicability of the developed sandwich ELLA to crude samples was demonstrated by estimating trace quantities of the mucin-type O-glycans in the human serum.
Assuntos
Análise Química do Sangue/métodos , Mucinas/sangue , Lectinas de Plantas/metabolismo , Animais , Artocarpus/química , Biotinilação , Humanos , Mucinas/química , Mucinas/metabolismoRESUMO
BACKGROUND: Immobilized recombinant perlecan domain I (PlnDI) binds and modulates the activity of heparin-binding growth factors, in vitro. However, activities for PlnDI, in solution, have not been reported. In this study, we assessed the ability of soluble forms to modulate vascular endothelial growth factor-165 (VEGF165) enhanced capillary tube-like formation, and VEGF receptor-2 phosphorylation of human bone marrow endothelial cells, in vitro. RESULTS: In solution, PlnDI binds VEGF165 in a heparan sulfate and pH dependent manner. Capillary tube-like formation is enhanced by exogenous PlnDI; however, PlnDI/VEGF165 mixtures combine to enhance formation beyond that stimulated by either PlnDI or VEGF165 alone. PlnDI also stimulates VEGF receptor-2 phosphorylation, and mixtures of PlnDI/VEGF165 reduce the time required for peak VEGF receptor-2 phosphorylation (Tyr-951), and increase Akt phosphorylation. PlnDI binds both immobilized neuropilin-1 and VEGF receptor-2, but has a greater affinity for neuropilin-1. PlnDI binding to neuropilin-1, but not to VEGF receptor-2 is dependent upon the heparan sulfate chains adorning PlnDI. Interestingly, the presence of VEGF165 but not VEGF121 significantly enhances PlnDI binding to Neuropilin-1 and VEGF receptor-2. CONCLUSIONS: Our observations suggest soluble forms of PlnDI are biologically active. Moreover, PlnDI heparan sulfate chains alone or together with VEGF165 can enhance VEGFR-2 signaling and angiogenic events, in vitro. We propose PlnDI liberated during basement membrane or extracellular matrix turnover may have similar activities, in vivo.
Assuntos
Células da Medula Óssea/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Proteoglicanas de Heparan Sulfato/química , Proteoglicanas de Heparan Sulfato/farmacologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Células Endoteliais/citologia , Proteoglicanas de Heparan Sulfato/isolamento & purificação , Proteoglicanas de Heparan Sulfato/metabolismo , Heparitina Sulfato/metabolismo , Humanos , Neovascularização Fisiológica/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Estrutura Terciária de Proteína , Transdução de Sinais/efeitos dos fármacos , SolubilidadeRESUMO
The anabolic effect of intermittent PTH on bone is variable depending on the species studied, duration/mode of administration, and location of skeletal response investigated. We tested the hypothesis low dose, short term, intermittent PTH 1-34 administration is sufficient to enhance bone formation without altering bone resorption. To test our hypothesis, mice were treated intermittently with one of three concentrations of PTH 1-34 (1 microg/kg; low, 10 microg/kg, or 20 microg/kg; high) for three weeks. The skeletal response was identified by quantifying: serum markers of bone turnover, cancellous bone parameters in distal femur, proximal tibia, and lumbar vertebrae by microCT, and number of osteoblasts and osteoclasts in distal femur. Mice receiving 20 microg/kg of PTH 1-34 demonstrated a 30% increase in serum osteocalcin, but no differences in serum calcium, type I collagen teleopeptides, or TRACP 5b. For all bones, microCT analysis suggested mice receiving 20 microg/kg of PTH 1-34 had increased cancellous bone mineral density, trabecular thickness and spacing, but decreased trabecular number. A 60% increase in the number of alkaline phosphatase positive osteoblasts in the distal femur was also observed in tissue sections; however, the number of TRAP positive osteoclasts was not different between test and control groups. While animals administered 10 microg/kg demonstrated similar trends for all bone turnover indices, such alterations were not observed in animals administered PTH 1-34 at 1 microg/kg per day. Thus, PTH 1-34, administered intermittently for three weeks at 20 microg/kg is sufficient to enhance bone formation without enhancing resorption.
Assuntos
Osteogênese/efeitos dos fármacos , Hormônio Paratireóideo/administração & dosagem , Animais , Peso Corporal/efeitos dos fármacos , Remodelação Óssea/efeitos dos fármacos , Remodelação Óssea/fisiologia , Relação Dose-Resposta a Droga , Esquema de Medicação , Avaliação Pré-Clínica de Medicamentos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Osteoblastos/efeitos dos fármacos , Osteoblastos/fisiologia , Osteoclastos/efeitos dos fármacos , Osteoclastos/fisiologia , Hormônio Paratireóideo/farmacologia , Periodicidade , Pigmentação/genética , Fatores de Tempo , Regulação para Cima/efeitos dos fármacosRESUMO
BACKGROUND/OBJECTIVE: Vascular Endothelial Growth Factor (VEGF) is the main driver of angiogenesis during neurodevelopment (i.e., brain and retina). VEGF165 and VEGF121 are the two most prevalent human VEGF isoforms. Although retinopathy of prematurity (ROP), a neuroretinal disorder, is associated with VEGF dysregulation, little is known about the interaction of VEGF isoforms on neuroretinal angiogenesis. We hypothesized that: (a) A specific VEGF165/VEGF121 correlation, at a given time point, is associated with normal retinal development (no ROP) and (b) An altered correlation, of such, is associated with aberrant retinal development (ROP). Utilizing pre-collected dried blood spots (DBS) from <1-week-old preterm infants, we aimed to determine whether correlations between VEGF165 and VEGF121 precede the diagnosis of early stage, non-proliferative ROP (NP-ROP). METHODOLOGY: We conducted a case/control study, utilizing DBS from 65 preterm infants. We measured DBS levels of VEGF165 on the Mesoscale Discovery Platform and VEGF121 via Cloud Clone Elisa Assay. RESULTS: In infants with NP-ROP, VEGF165 is significantly higher in males (than females). In infants without ROP, there is a significant correlation between VEGF165 and VEGF121 in females (but not males). In infants with NP-ROP, the opposite is so; there is a significant correlation between VEGF165 and VEGF121 in males (but not females). CONCLUSIONS: This pilot study, utilizing de-identified data, suggests the potential importance of examining interactions between VEGF isoforms, at <1 week after birth, to better understand ROP development. Our study also suggests that retinal angiogenesis may not be a sex-neutral process. A prospective study is needed to confirm our novel findings.
Assuntos
Isoformas de Proteínas/metabolismo , Retinopatia da Prematuridade/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Estudos de Casos e Controles , Feminino , Humanos , Lactente , Recém-Nascido , Recém-Nascido Prematuro , MasculinoRESUMO
Vascular endothelial growth factor (VEGF) helps to control angiogenesis and vascular permeability in the kidney. Renal disorders, such as diabetic nephropathy, are associated with VEGF dysregulation in the kidney. The factors that govern VEGF under physiologic conditions in the kidney are not well-understood. Luteinizing hormone (LH), a pro-angiogenic hormone, helps regulate physiologic VEGF expression in reproductive organs. Given that LH receptors are found in the kidney, we, at Zietchick Research Institute, hypothesized here that LH also helps regulate VEGF expression in the kidney as well. To provide evidence, we aimed to show that LH levels are able to predict VEGF levels in the mammalian kidney. Most VEGF-related investigations involving the kidney have used lower order mammals as models (i.e., rodents and rabbits). To translate this work to the human body, it was decided to examine the relationship between VEGF and LH in higher order mammals (i.e., bovine and porcine models). This protocol uses the total protein lysate from the kidney cortex. Keys to this method's success include procurement of kidneys from slaughterhouse animals immediately after death as well as normalization of analyte levels (in the kidney extract) by total protein. This study successfully demonstrates a significant linear relationship between LH and VEGF in both bovine and porcine kidneys. The results are reproducible in two different species. The study provides supporting evidence that the use of kidney extracts from cows and pigs are an excellent, economical, and abundant resource for the study of renal physiology, particularly for examining the correlation between VEGF and other analytes.
Assuntos
Córtex Renal/metabolismo , Hormônio Luteinizante/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , HumanosRESUMO
BACKGROUND: Luteinizing hormone (LH) and human chorionic gonadotropin (hCG), generally considered reproductive hormones, have potent proangiogenic properties. Both of these hormones and their joint receptor (CG/LH receptor) are found in the human eye. We hypothesized that an excess of these hormones is associated with proliferative retinopathy of prematurity (P-ROP). METHODS: Dried blood spots (DBS) were used to perform a cross-sectional study of infants (gestational age of <26 weeks) with and without P-ROP, born in Michigan between August 1, 2012, and March 15, 2015. The DBS were collected at 1 week and 4 weeks of age from 45 preterm infants (27 no-ROP and 18 P-ROP). The DBS were linked to hospital records and then deidentified. ICD-9 codes were used to identify P-ROP cases. Hormones levels were measured via electrochemiluminescence assays on the Meso Scale Discovery platform. Associations between hormone levels at 1 and 4 weeks of age and the presence or absence of P-ROP were assessed. RESULTS: In female infants, we noted a trend toward higher LH levels in ROP cases at week 1 (P = 0.11) and significantly higher LH levels in cases at week 4 (P = 0.03). In male infants, no ROP-related differences in LH levels were found at either time point. For hCG levels, no associations with P-ROP were found in either sex at either time point. CONCLUSIONS: The association of high LH with P-ROP in female but not male infants raises the possibility that there are sex-specific hormonal determinants of aberrant retinal angiogenesis.
Assuntos
Retinopatia da Prematuridade , Estudos Transversais , Feminino , Idade Gestacional , Humanos , Lactente , Recém-Nascido , Recém-Nascido Prematuro , Hormônio Luteinizante , Masculino , Fatores de RiscoRESUMO
The adherence of Plasmodium falciparum-infected red blood cells (IRBCs) in human placenta is mediated by chondroitin 4-sulfate (C4S). The C4S-adherent parasites selected from laboratory strains have been widely used for determining the C4S structural elements involved in IRBC binding and for the identification of parasite adhesive protein(s). However, as far as we know, the relative binding strength of the placental versus laboratory-selected parasites has not been reported. In this study, we show that IRBCs from the infected placentas bind to C4S about 3-fold higher than those selected for C4S adherence from laboratory strains. Although adherent parasites selected from several laboratory strains have comparable binding strengths, the one obtained from 3D7 parasites designated as 3D7N61 used for malaria genome sequencing, exhibits markedly lower binding strength. Furthermore, 3D7N61-CSA parasites lose most of the binding capacity by tenth generation in continuous culture.
Assuntos
Sulfatos de Condroitina/metabolismo , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Placenta/parasitologia , Plasmodium falciparum/metabolismo , Animais , Bovinos , Adesão Celular , Feminino , Interações Hospedeiro-Parasita , Humanos , Laboratórios , Malária Falciparum/parasitologia , Plasmodium falciparum/classificação , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/isolamento & purificação , Gravidez , Complicações Parasitárias na Gravidez/parasitologia , Ligação ProteicaRESUMO
Purpose/Aim: Vascular endothelial growth factor (VEGF) dysregulation is implicated in the pathogenesis of retinopathy of prematurity (ROP). Identifying the factors that contribute to VEGF regulation during normal retinal vascularization is the key to ROP prevention. Currently, physiologic hypoxia is thought to be responsible for retinal VEGF regulation in utero. However, a potential hormonal contribution to VEGF regulation during eye development has not been fully investigated. The placental hormone, human chorionic gonadotropin and the pituitary hormone, and luteinizing hormone (LH) induce VEGF expression in several tissue types. Both of these gonadotropins activate the same LH receptor (LHR) in the human body; LHRs are expressed in the retina. In this study, we aimed to show that LHR signaling participates in VEGF regulation in the developing eye. METHODS: When offspring from breeding pairs of LHR knockout mice (lhrkos) reached 21 days old, eyes and serum were extracted from homozygote lhrkos and wildtype (WT) siblings. VEGF levels were measured using Mouse VEGF Quantikine immunoassay kit. Retinas were incubated with isolectin for endothelial cell staining, flat mounted and imaged by confocal microscopy. Retinal vascular density was quantified using Imaris software. Some eyes were sectioned and stained for histopathologic review. RESULTS: Ocular VEGF and retinal vascular volumes were significantly reduced by ~ 15% in lhrko eyes. Serum VEGF was not changed. The lhrko retinas did not display any anomalies. CONCLUSIONS: We provide evidence that LHR signaling plays a role in VEGF regulation and vascularization in the developing eye. Given that human preterm infants may have altered LHR-activity, the effect of gonadotropins on eye development should be further studied to identify novel strategies for ROP prevention.
Assuntos
Olho/crescimento & desenvolvimento , Receptores do LH/fisiologia , Neovascularização Retiniana/prevenção & controle , Retinopatia da Prematuridade/metabolismo , Transdução de Sinais/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Animais Recém-Nascidos , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia de Fluorescência , Neovascularização Retiniana/metabolismo , Vasos Retinianos/metabolismo , Vasos Retinianos/patologiaRESUMO
Purpose/Aim: Luteinizing hormone (LH) is known to function as a key regulator of vascular endothelial growth factor (VEGF) expression in reproductive organs. In recent years, LH has also been detected in human vitreous and LH receptors have been identified in human retina. This study was aimed to investigate a potential correlation between LH and VEGF levels in healthy mammalian eyes to provide supporting evidence of LH's potential involvement in intraocular VEGF regulation. METHODS: 18 bovine and 30 porcine eyes were procured from an abattoir and VEGF and LH levels were measured in the vitreous extracted from these eyes by commercially available bovine & porcine ELISA assay kits. Total protein of the vitreous was measured by using Micro BSA protein assay kit. RESULTS: After total protein normalization, the Pearson Correlation Coefficients (PCC) showed a strong and significant correlation between LH and VEGF levels. (Bovine LH/VEGF PCC: 0.89, p < 0.001; Porcine LH/VEGF PCC: 0.80, p < 0.001). Linear regression analyses, adjusted for gender, showed significant linear relationships between LH and VEGF levels in both bovine and porcine vitreous. (Bovine: t-value = 7.69, p < 0.0001, adjusted r2 = .79; Porcine: t-value = 6.71, p < 0.001, adjusted r2 = .62) Conclusions: We show that VEGF and LH are strongly correlated in healthy, adult mammalian eyes. The robustness of the correlation is shown both by its strength of association and reproducibility in two species. Given that LH is well known to regulate VEGF levels in several tissue types, the LH/VEGF linear relationship in vitreous potentially implicates LH in homeostatic VEGF regulation of the eye. Because we also found that the correlation between LH and VEGF only became manifest when our targeted analytes were normalized by total amount of protein, preclinical and clinical investigators should consider normalizing analytes in vitreous by total protein when assessing potential correlations among them.
Assuntos
Hormônio Luteinizante/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Corpo Vítreo/metabolismo , Animais , Bovinos , Ensaio de Imunoadsorção Enzimática , Modelos Animais , Valores de Referência , SuínosRESUMO
Human chorionic gonadotropin (hCG) is known to be a powerful vascular endothelial growth factor (VEGF)-regulating hormone. It stimulates vascularization of the gravid uterus by upregulating VEGF expression. In the body, hCG activates the same receptor as luteinizing hormone (LH). Like hCG, LH is also strongly proangiogenic. Recently, it has been shown that LH/hCG receptors are present in the retina and that both LH and hCG are found in the eye. In fact, the human eye can synthesize its own hCG. We have previously shown that LH and VEGF are significantly correlated in mammalian eyes, potentially implicating LH-receptor/hCG-receptor activation in intraocular VEGF regulation. Given that elevated VEGF is associated with progression of two vasoproliferative pediatric retinal disorders, retinopathy of prematurity and retinoblastoma, our objective was to determine whether hCG may potentially affect VEGF production and pathologic retinal vascularization in vasoproliferative disorders affecting the immature retina. In this study, we used (a) oxygen-induced retinopathy mouse model (standard model for retinopathy of prematurity) and (b) Y79 retinoblastoma cells (a human cell line derived from immature retinal cells). In the oxygen-induced retinopathy model, number of preretinal nuclei (representing pathologic retinal neovascularization) significantly increases by 57% (P<0.05) in hCG-treated mice. In Y79 cells, VEGF production significantly increases by 37% (P<0.05) in hCG-treated cells. These findings suggest that hCG is potentially able to influence retinal vascularization and VEGF production and thus, the hCG receptor may potentially represent a therapeutic target for vasoproliferative retinal disorders affecting the young eye.
Assuntos
Gonadotropina Coriônica/farmacologia , Neovascularização Patológica/metabolismo , Retina/efeitos dos fármacos , Neovascularização Retiniana/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Animais Recém-Nascidos , Linhagem Celular Tumoral , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Retina/patologia , Retinoblastoma/metabolismo , Retinoblastoma/patologiaRESUMO
Luteinizing hormone (LH), produced in the anterior pituitary, has been detected in cadaver eyes and LH receptors (LHRs) have been identified in the retina, with the highest density in cone photoreceptors. Our aim was to confirm the presence of LH in the living, human eye as well as to examine the potential impact of a reduction in LHR signaling on visual processing. Vitreous samples were collected from 40 patients (23 diabetics, 17 non-diabetics) who were undergoing vitrectomies for various indications. LH concentration was quantified in each sample via an electro-chemiluminescence immunoassay and Meso Scale Discovery platform and normalized to total protein. In addition, full-field electroretinography (ERG) was performed on 11 adult LHR knockout heterozygous mice (B6;129X1-Lhcgrtm1Zmlei/J) and 11 wild types using the Celeris-Diagnosys system. The median LH values (pg/mg total protein) for non-diabetics, diabetics without proliferative diabetic retinopathy (PDR) and diabetics with PDR were 40.7, 41.9 and 167.8 respectively. LH levels were significantly higher in diabetics with PDR. In our ERG investigation, heterozygous LHRKOs were found to have significantly reduced amplitudes of a-wave and b-waves at high stimulus intensities with no significant change in a-wave or b-wave amplitudes at lower intensities; this is consistent with a selective impairment of cone-mediated responses. Our findings confirm LH is present in the adult human eye. Our findings also suggest that a reduction in LH receptor signaling negatively impacts visual processing of the cone photoreceptors. Overall, our study results support the theory that LH likely plays a physiologic role in the eye.
Assuntos
Hormônio Luteinizante/metabolismo , Receptores do LH/metabolismo , Retina/metabolismo , Corpo Vítreo/metabolismo , Animais , Retinopatia Diabética/metabolismo , Eletrorretinografia , Humanos , Camundongos , Camundongos Knockout , Receptores do LH/genéticaRESUMO
The glycosylphosphatidylinositol (GPI) anchors of Plasmodium falciparum are indispensable for parasite survival since merozoite surface proteins-1, -2, -4, -5, and -10, crucial for erythrocyte invasion, are GPI-anchored. Therefore, the GPI biosynthetic pathway can offer potential targets for novel anti-malarial drugs. Here, we characterized the putative P. falciparum PIG-B gene (PfPIGB) that encodes mannosyltransferase-III of GPI biosynthesis. PfPIGB mRNA is transcribed in a developmental stage specific manner. A protein corresponding to the expected size of PfPIG-B is expressed by the parasite and is localized in the endoplasmic reticulum. Treatment of parasites with PfPIG-B specific siRNA caused reduction in GPI synthesis, affecting the PIG-B specific GPI intermediate. These data demonstrate that PfPIG-B is functional and encodes mannosyltransferase-III of the parasite GPI biosynthesis. The parasite PfPIG-B is novel in that its signature sequence HKEHKI is unique and is only partially conserved as compared to HKEXRF signature motif of mammalian PIG-B enzymes.