RESUMO
Inner blood-retina barrier (iBRB) is primarily formed of retinal microvascular endothelial cells (ECs) with tight junctions, which are surrounded and supported by retinal microvascular pericytes (RMPs) and basement membrane. Pericytes are believed to be critically involved in the physiology and pathology of iBRB. However, the underlying mechanism remains to be fully elucidated. We developed a novel in vitro iBRB model which was composed of primary cultures of rat retinal ECs and RMPs based on Transwell system. We tested the involvement of pericytes in the migration and invasion of ECs, examined the expression and activity of matrix metalloproteinase- (MMP-) 2/MMP-9 in the culture, evaluated the TEER and permeability of iBRB, and assessed the expression of ZO-1, occludin, claudin-5, and VE-cadherin of endothelial junctions. We found that RMPs with indirect contact of ECs can increase the expression of MMP-2 and upgrade the activity of MMP-2/9 in the coculture, which subsequently decreased TJ protein abundance of ZO-1 and occludin in ECs, promoted the migration of ECs, and finally reduced the integrity of iBRB. Taken together, our data show that RMP relative location with ECs is involved in the integrity of iBRB via MMP-2/9 and has important implications for treating diabetic retinopathy and other retinal disorders involving iBRB dysfunction.
Assuntos
Barreira Hematorretiniana/citologia , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Pericitos/citologia , Retina/citologia , Animais , Barreira Hematorretiniana/metabolismo , Movimento Celular , Células Cultivadas , Técnicas de Cocultura , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Masculino , Modelos Biológicos , Pericitos/metabolismo , Cultura Primária de Células , Ratos , Retina/metabolismo , Junções Íntimas/metabolismoRESUMO
Activation of P2X7 signaling, due to high glucose levels, leads to blood retinal barrier (BRB) breakdown, which is a hallmark of diabetic retinopathy (DR). Furthermore, several studies report that high glucose (HG) conditions and the related activation of the P2X7 receptor (P2X7R) lead to the over-expression of pro-inflammatory markers. In order to identify novel P2X7R antagonists, we carried out virtual screening on a focused compound dataset, including indole derivatives and natural compounds such as caffeic acid phenethyl ester derivatives, flavonoids, and diterpenoids. Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) rescoring and structural fingerprint clustering of docking poses from virtual screening highlighted that the diterpenoid dihydrotanshinone (DHTS) clustered with the well-known P2X7R antagonist JNJ47965567. A human-based in vitro BRB model made of retinal pericytes, astrocytes, and endothelial cells was used to assess the potential protective effect of DHTS against HG and 2'(3')-O-(4-Benzoylbenzoyl)adenosine-5'-triphosphate (BzATP), a P2X7R agonist, insult. We found that HG/BzATP exposure generated BRB breakdown by enhancing barrier permeability (trans-endothelial electrical resistance (TEER)) and reducing the levels of ZO-1 and VE-cadherin junction proteins as well as of the Cx-43 mRNA expression levels. Furthermore, HG levels and P2X7R agonist treatment led to increased expression of pro-inflammatory mediators (TLR-4, IL-1ß, IL-6, TNF-α, and IL-8) and other molecular markers (P2X7R, VEGF-A, and ICAM-1), along with enhanced production of reactive oxygen species. Treatment with DHTS preserved the BRB integrity from HG/BzATP damage. The protective effects of DHTS were also compared to the validated P2X7R antagonist, JNJ47965567. In conclusion, we provided new findings pointing out the therapeutic potential of DHTS, which is an inhibitor of P2X7R, in terms of preventing and/or counteracting the BRB dysfunctions elicited by HG conditions.
Assuntos
Barreira Hematorretiniana/efeitos dos fármacos , Furanos/farmacologia , Fenantrenos/farmacologia , Antagonistas do Receptor Purinérgico P2X/farmacologia , Receptores Purinérgicos P2X7/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/toxicidade , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Sítios de Ligação , Barreira Hematorretiniana/citologia , Barreira Hematorretiniana/metabolismo , Permeabilidade Capilar , Linhagem Celular , Conexina 43/metabolismo , Citocinas/metabolismo , Citoproteção , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Furanos/química , Humanos , Pericitos/efeitos dos fármacos , Pericitos/metabolismo , Fenantrenos/química , Ligação Proteica , Agonistas do Receptor Purinérgico P2X/toxicidade , Antagonistas do Receptor Purinérgico P2X/química , Quinonas , Espécies Reativas de Oxigênio/metabolismo , Receptores Purinérgicos P2X7/químicaRESUMO
The nervous system demands an adequate oxygen and metabolite exchange, making pericytes (PCs), the only vasoactive cells on the capillaries, essential to neural function. Loss of PCs is a hallmark of multiple diseases, including diabetes, Alzheimer's, amyotrophic lateral sclerosis (ALS) and Parkinson's. Platelet-derived growth factor receptors (PDGFRs) have been shown to be critical to PC function and survival. However, how PDGFR-mediated PC activity affects vascular homeostasis is not fully understood. Here, we tested the hypothesis that imatinib, a chemotherapeutic agent and a potent PDGFR inhibitor, alters PC distribution and thus induces vascular atrophy. We performed a morphometric analysis of the vascular elements in sham control and imatinib-treated NG2-DsRed mice. Vascular morphology and the integrity of the blood-retina barrier (BRB) were evaluated using blood albumin labeling. We found that imatinib decreased the number of PCs and blood vessel (BV) coverage in all retinal vascular layers; this was accompanied by a shrinkage of BV diameters. Surprisingly, the total length of capillaries was not altered, suggesting a preferential effect of imatinib on PCs. Furthermore, blood-retina barrier disruption was not evident. In conclusion, our data suggest that imatinib could help in treating neurovascular diseases and serve as a model for PC loss, without BRB disruption.
Assuntos
Barreira Hematorretiniana/efeitos dos fármacos , Mesilato de Imatinib/farmacologia , Pericitos/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Animais , Barreira Hematorretiniana/citologia , Camundongos , Camundongos Endogâmicos C57BL , Pericitos/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismoRESUMO
Retinal disorders are leading causes of blindness. Still, treatment strategies are limited and the challenging anatomical barriers of the eye limit the evaluation and development of new therapeutics. Among these layers of barriers is the blood-retinal barrier, which separates the retina from the choroid by the Bruch's membrane. This work aimed to establish a 3D cellular model that recapitulates barrier properties of the BRB and diffusion through the vitreous, the main barriers encountered upon intravitreal injection. Several parameters were evaluated namely co-culture time of ARPE-19 and HUVECs and different biomaterial compositions of hydrogels to better mimic the human vitreous. The developed vitreous mimic has viscoelastic properties similar to human vitreous. Co-culture of human retinal and endothelial cells showed increased transepithelial resistance with longer co-culture times concomitant with reduced permeability to FITC-dextran 40â¯kDa. The proposed models lay the foundation of a platform for faster assessment of a large number of samples and without the use of animals.
Assuntos
Barreira Hematorretiniana/citologia , Células Endoteliais/citologia , Modelos Biológicos , Alternativas aos Testes com Animais , Animais , Barreira Hematorretiniana/metabolismo , Técnicas de Cocultura , Dextranos/administração & dosagem , Dextranos/farmacocinética , Células Endoteliais/metabolismo , Fluoresceína-5-Isotiocianato/administração & dosagem , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/farmacocinética , Humanos , Injeções Intravítreas , Permeabilidade , Corpo Vítreo/metabolismoRESUMO
Increased O-GlcNAcylation, a well-known post-translational modification of proteins causally linked to various detrimental cellular functions in pathological conditions including diabetic retinopathy (DR). Previously we have shown that endothelial activation induced by inflammation and hyperglycemia results in the endoplasmic reticulum (ER) stress-mediated intercellular junction alterations accompanied by visual deficits in a tie2-TNF-α transgenic mouse model. In this study, we tested the hypothesis that increased ER stress via O-GlcNAcylation of VE-Cadherin likely contribute to endothelial permeability. We show that ER stress leads to GRP78 translocation to the plasma membrane, increased O-GlcNAcylation of proteins, particularly VE-Cadherin resulting in a defective complex partnering leading to the loss of retinal endothelial barrier integrity and increased transendothelial migration of monocytes. We further show an association of GRP78 with the VE-Cadherin under these conditions. Interestingly, cells exposed to ER stress inhibitor, tauroursodeoxycholic acid partially mitigated all these effects. Our findings suggest an essential role for ER stress and O-GlcNAcylation in altering the endothelial barrier function and reveal a potential therapeutic target in the treatment of DR.
Assuntos
Antígenos CD/metabolismo , Barreira Hematorretiniana/metabolismo , Caderinas/metabolismo , Permeabilidade Capilar , Estresse do Retículo Endoplasmático , Células Endoteliais/metabolismo , Proteínas de Choque Térmico/metabolismo , Barreira Hematorretiniana/citologia , Membrana Celular/metabolismo , Movimento Celular , Células Cultivadas , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Chaperona BiP do Retículo Endoplasmático , Glicosilação , Humanos , Monócitos/fisiologia , Transporte Proteico , Ácido Tauroquenodesoxicólico/farmacologiaRESUMO
This study investigated the mechanism of transporting imperatorin across the inner blood-retinal barrier (iBRB). The carotid artery single injection method was used to calculate the retinal uptake index (RUI) of [3H]imperatorin in vivo, whereas the retinal capillary endothelial cell lines were used for the in vitro uptake and mRNA expression assays. RUI value of [3H]imperatorin was greater than that of the reference compound ([14C]n-butanol). [3H]Imperatorin significantly reduced the RUI in the presence of neuroprotective organic cationic drugs at 10 mM. However, tetraethylammonium and p-aminohippuric acid showed no significant effects. [3H]Imperatorin uptake by TR-iBRB2 cells was time-, pH-, energy-, and concentration-dependent with a Km value of 679 ± 130 µM. In addition, the uptake study showed insensitivity to sodium and membrane potential. Various organic cations including pyrilamine, nicotine, and clonidine significantly reduced the uptake of [3H]imperatorin, whereas organic anions and monocarboxylic acids did not. Furthermore, the mRNA expression level dropped markedly with rOCTN1, rOCTN2, rPMAT, and rMATE1 small interfering RNAs in the transfection study. Moreover, [3H]imperatorin uptake remained neutral with small interfering RNA transfections. Our results indicate that imperatorin transport across the iBRB involves carrier-mediated transporter system.
Assuntos
Barreira Hematorretiniana/metabolismo , Furocumarinas/farmacocinética , Proteínas Carreadoras de Solutos/metabolismo , Animais , Barreira Hematorretiniana/citologia , Linhagem Celular , Células Endoteliais , Endotélio Vascular/citologia , Furocumarinas/administração & dosagem , Perfilação da Expressão Gênica , Humanos , Injeções Intra-Arteriais , Masculino , Permeabilidade , RNA Interferente Pequeno/metabolismo , Ratos , Doenças Retinianas/tratamento farmacológico , Vasos Retinianos/citologia , Proteínas Carreadoras de Solutos/genéticaRESUMO
ß-Catenin signaling controls the development and maintenance of the blood-brain barrier (BBB) and the blood-retina barrier (BRB), but the division of labor and degree of redundancy between the two principal ligand-receptor systems-the Norrin and Wnt7a/Wnt7b systems-are incompletely defined. Here, we present a loss-of-function genetic analysis of postnatal BBB and BRB maintenance in mice that shows striking threshold and partial redundancy effects. In particular, the combined loss of Wnt7a and Norrin or Wnt7a and Frizzled4 (Fz4) leads to anatomically localized BBB defects that are far more severe than observed with loss of Wnt7a, Norrin, or Fz4 alone. In the cerebellum, selective loss of Wnt7a in glia combined with ubiquitous loss of Norrin recapitulates the phenotype observed with ubiquitous loss of both Wnt7a and Norrin, implying that glia are the source of Wnt7a in the cerebellum. Tspan12, a coactivator of Norrin signaling in the retina, is also active in BBB maintenance but is less potent than Norrin, consistent with a model in which Tspan12 enhances the amplitude of the Norrin signal in vascular endothelial cells. Finally, in the context of a partially impaired Norrin system, the retina reveals a small contribution to BRB development from the Wnt7a/Wnt7b system. Taken together, these experiments define the extent of CNS region-specific cooperation for several components of the Norrin and Wnt7a/Wnt7b systems, and they reveal substantial regional heterogeneity in the extent to which partially redundant ligands, receptors, and coactivators maintain the BBB and BRB.
Assuntos
Barreira Hematoencefálica/crescimento & desenvolvimento , Barreira Hematoencefálica/fisiologia , Barreira Hematorretiniana/crescimento & desenvolvimento , Barreira Hematorretiniana/fisiologia , Proteínas do Olho/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Wnt/fisiologia , Animais , Barreira Hematoencefálica/citologia , Barreira Hematorretiniana/citologia , Técnicas de Cultura de Células , Proteínas do Olho/genética , Receptores Frizzled/deficiência , Receptores Frizzled/genética , Receptores Frizzled/fisiologia , Camundongos , Camundongos Knockout , Modelos Biológicos , Modelos Neurológicos , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Transdução de Sinais , Tetraspaninas/deficiência , Tetraspaninas/genética , Tetraspaninas/fisiologia , Proteínas Wnt/deficiência , Proteínas Wnt/genética , beta Catenina/fisiologiaRESUMO
BACKGROUND: Aster koraiensis extract (AKE) is a standard dietary herbal supplement. The aim of this study is to investigate the inhibitory effects of AKE on diabetes-induced retinal vascular dysfunction in Spontaneously Diabetic Torii (SDT) rats. METHODS: AKE (50 and 100 mg/kg body weight/day) was administered for 16 weeks. The effects of orally administered AKE on blood glucose levels, retinal vascular leakage, apoptosis, and accumulation of advanced glycation end products (AGEs) in the retina were evaluated. RESULTS: SDT rats exhibited hyperglycemia and retinal vascular leakage, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was clearly detected apoptosis in the retinal microvasculature. Immunofluorescence staining revealed the accumulation of AGEs in the retinal vasculature of the SDT rats. However, oral administration of AKE for 16 weeks blocked diabetes-induced blood-retinal barrier (BRB) breakdown and the loss of occludin, which is an important tight junction protein. Apoptosis of retinal vascular cells and AGE accumulation were significantly inhibited after AKE treatment. CONCLUSION: These results indicate that, as a dietary herbal supplement, AKE may have beneficial effects on patients with diabetic retinopathy.
Assuntos
Aster/química , Barreira Hematorretiniana/efeitos dos fármacos , Retinopatia Diabética/metabolismo , Extratos Vegetais/farmacologia , Animais , Apoptose/efeitos dos fármacos , Glicemia/efeitos dos fármacos , Barreira Hematorretiniana/citologia , Barreira Hematorretiniana/patologia , Diabetes Mellitus Experimental , Produtos Finais de Glicação Avançada/análise , Produtos Finais de Glicação Avançada/metabolismo , Masculino , Extratos Vegetais/química , Ratos , Ratos Sprague-Dawley , Proteínas de Junções Íntimas/análise , Proteínas de Junções Íntimas/metabolismoRESUMO
Immune privilege is a complex process that protects organs from immune-mediated attack and damage. It is accomplished by a series of cellular barriers that both control immune cell entry and promote the development of tolerogenic immune cells. In this Review, we describe the vascular endothelial and epithelial barriers in organs that are commonly considered to be immune privileged, such as the brain and the eye. We compare these classical barriers with barriers in the intestine, which share features with barriers of immune-privileged organs, such as the capacity to induce tolerance and to protect from external insults. We suggest that when intestinal barriers break down, disruption of other barriers at distant sites can ensue, and this may underlie the development of various neurological, metabolic and intestinal disorders.
Assuntos
Endotélio Vascular/imunologia , Endotélio Vascular/metabolismo , Epitélio/imunologia , Epitélio/metabolismo , Homeostase , Mucosa/imunologia , Mucosa/metabolismo , Animais , Barreira Hematoencefálica/citologia , Barreira Hematoencefálica/imunologia , Barreira Hematoencefálica/metabolismo , Barreira Hematorretiniana/citologia , Barreira Hematorretiniana/imunologia , Barreira Hematorretiniana/metabolismo , Suscetibilidade a Doenças , Endotélio Vascular/citologia , Microbioma Gastrointestinal/imunologia , Humanos , Privilégio Imunológico , Imunidade , Imunomodulação , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Mucosa/citologia , Especificidade de Órgãos/imunologiaRESUMO
More than 1 billion world's population actively smokes tobacco containing the bioactive component nicotine (NT). The biological role of this molecule is mediated through the activation of nicotinic cholinergic receptors, widely distributed in various human tissues including retinal pigmented epithelium. The long-term assumption of NT contributes to several diseases development such as diabetic retinopathy. The major complication of this pathology is the diabetic macular edema (DME), characterized by macular area thinning and blood-retinal barrier (BRB) breakdown. Retinal hyperglycemic/hypoxic microenvironment represents one of the main factors favoring DME progression by eliciting the hypoxia-inducible factors (HIFs) expression. The latter induce new vessels formation by stimulating cellular secretion of vascular endothelial growth factor (VEGF). The etiology of DME is multifactorial, but little is known about the risk factors linked to cigarette smoking, in particular to nicotine's contribution. In the present study, we have investigated the NT role in a model, in vitro, of DME, by evaluating its effect on outer BRB permeability and HIFs/VEGF expression following exposure to hyperglycemic/hypoxic insult. Our results have demonstrated that this compound alters outer BRB integrity exposed to high glucose and low oxygen pressure microenvironment by upregulating HIF-1α/HIF-2α, VEGF expression and ERK1/2 phosphorylation. These data have suggested that NT may play a negative role in active smokers affected by DME.
Assuntos
Barreira Hematorretiniana/efeitos dos fármacos , Complicações do Diabetes/metabolismo , Células Epiteliais/efeitos dos fármacos , Edema Macular/metabolismo , Nicotina/toxicidade , Proteínas Reguladoras de Apoptose , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Barreira Hematorretiniana/citologia , Barreira Hematorretiniana/metabolismo , Linhagem Celular , Células Epiteliais/metabolismo , Humanos , Hiperglicemia/metabolismo , Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Permeabilidade/efeitos dos fármacos , Proteínas Repressoras , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Retinal diseases leading to impaired vision and ultimately blindness are mainly characterized by ischemic and hypoxic stress. Targeting the retinal ρ-containing γ-aminobutyric acid type A receptors (ρ GABAARs) and thereby decreasing the retinal neuronal activity has been proposed as a novel therapeutic approach. The taurine transporter (TAUT) plays a key role in the retinal transport of GABA and has been previously suggested to display a higher functional activity in the retina compared to the brain. TAUT would therefore stand as a suitable target for the selective delivery of ρ GABAAR ligands into the retina. Consequently, an in vitro model of TAUT at the outer blood-retinal barrier (BRB) was developed and characterized using the ARPE-19 cell line. Furthermore, the structural requirements of GABAAR ligands for interacting with TAUT at the BRB were investigated for a series of standard GABAAR ligands by testing their ability to inhibit the TAUT-mediated influx of taurine in ARPE-19 cells. Results showed that taurine influx was seven-fold higher when the ARPE-19 cells were cultured under hyperosmotic conditions and was demonstrated to display saturable kinetics (Km=27.7±2.2µM and Jmax=24.2±0.6pmol/cm2·min). Furthermore, the taurine influx was significantly inhibited in a concentration-dependent manner by GABA and imidazole-4-acetic acid (IAA), which is a naturally occurring metabolite of histamine. These compounds display similar Ki values of 644.2µM and 658.6µM, respectively. Moreover, IAA demonstrated higher inhibitory properties than the other tested GABA analogs: 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), 4,5,6,7-tetrahydropyrazolo[5,4-c]pyridin-3-ol (Aza-THIP), muscimol, and thiomuscimol. These studies demonstrated that IAA interacts with TAUT, which makes IAA a new lead structure in the development of new compounds, which are not only interacting with TAUT but also potent ρ GABAAR ligands.
Assuntos
Barreira Hematorretiniana/metabolismo , Imidazóis/farmacologia , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Receptores de GABA-A/metabolismo , Receptores de GABA/metabolismo , Barreira Hematorretiniana/citologia , Linhagem Celular , Humanos , Imidazóis/química , Pressão Osmótica , Taurina/metabolismoRESUMO
Zika virus (ZIKV) is an important pathogen that causes not only neurologic, but also ocular, abnormalities. Thus, it is imperative that models to study ZIKV pathogenesis in the eye are developed to identify potential targets for interventions. Here, we studied ZIKV interactions with human retinal cells and evaluated ZIKV's pathobiology in mouse eyes. We showed that cells lining the blood-retinal barrier (BRB), the retinal endothelium, and retinal pigment epithelium (RPE) were highly permissive and susceptible to ZIKV-induced cell death. Direct inoculation of ZIKV in eyes of adult C57BL/6 and IFN-stimulated gene 15 (ISG15) KO mice caused chorioretinal atrophy with RPE mottling, a common ocular manifestation of congenital ZIKV infection in humans. This response was associated with induced expression of multiple inflammatory and antiviral (IFNs) response genes in the infected mouse retina. Interestingly, ISG15 KO eyes exhibited severe chorioretinitis, which coincided with increased retinal cell death and higher ZIKV replication. Collectively, our study provides the first evidence to our knowledge that ZIKV causes retinal lesions and infects the cells lining the BRB and that ISG15 plays a role in retinal innate defense against ZIKV infection. Our mouse model can be used to study mechanisms underlying ZIKV-induced chorioretinitis and to gauge ocular antiviral therapies.
Assuntos
Barreira Hematorretiniana/virologia , Coriorretinite/virologia , Corioide/virologia , Endotélio/virologia , Epitélio Pigmentado da Retina/virologia , Infecção por Zika virus/patologia , Zika virus , Animais , Atrofia , Barreira Hematorretiniana/citologia , Morte Celular , Linhagem Celular , Coriorretinite/patologia , Corioide/patologia , Citocinas/genética , Modelos Animais de Doenças , Endotélio/citologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Retina/patologia , Retina/virologia , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/patologia , Ubiquitinas/genética , Replicação ViralRESUMO
Neural vascular barrier is essential for the life of multicellular organisms, and its impairment by tissue hypoxia is known to be a central of pathophysiology accelerating the progression of various intractable neural diseases. Therefore, the molecules involved in hypoxia-induced impairment of vascular barrier can be the targets to establish new therapies for intractable diseases. Here, we demonstrate that a disintegrin and metalloproteinases (ADAMs) 12 and 17 expressed in endothelial cells are the molecules responsible for the impairment of neural vascular barrier by hypoxia. Brain microvascular endothelial cells in vitro lost their barrier properties immediately after hypoxic stimulation through diminished localization of claudin-5, a tight junction molecule, on cell membranes. Hypoxic disappearance of claudin-5 from cell membranes and the consequent loss of barrier properties were completely suppressed by inhibition of the metalloproteinase activity which was found to be attributed to ADAM12 and ADAM17. Inhibition of either ADAM12 or ADAM17 was sufficient to rescue the in vivo neural vasculature under hypoxia from the loss of barrier function. This is the first report to specify the molecules which are responsible for hypoxia-induced impairment of neural vascular barrier and furthermore can be the targets of new therapeutic strategies for intractable neural diseases.
Assuntos
Proteínas ADAM/fisiologia , Células Endoteliais/enzimologia , Proteína ADAM12 , Proteína ADAM17 , Animais , Barreira Hematorretiniana/citologia , Barreira Hematorretiniana/enzimologia , Hipóxia Celular , Linhagem Celular , Membrana Celular/metabolismo , Claudina-5/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Retina/enzimologia , Vasos Retinianos/citologia , Vasos Retinianos/enzimologiaRESUMO
Inflammation in the diabetic retina is mediated by leukocyte adhesion to the retinal vasculature and alteration of the blood-retinal barrier (BRB). We investigated the role of chemokines in the alteration of the BRB in diabetes. Animals were made diabetic by streptozotocin injection and analyzed for gene expression and monocyte/macrophage infiltration. The expression of CCL2 (chemokine ligand 2) was significantly up-regulated in the retinas of rats with 4 and 8 weeks of diabetes and also in human retinal endothelial cells treated with high glucose and glucose flux. Additionally, diabetes or intraocular injection of recombinant CCL2 resulted in increased expression of the macrophage marker, F4/80. Cell culture impedance sensing studies showed that purified CCL2 was unable to alter the integrity of the human retinal endothelial cell barrier, whereas monocyte conditioned medium resulted in significant reduction in cell resistance, suggesting the relevance of CCL2 in early immune cell recruitment for subsequent barrier alterations. Further, using Cx3cr1-GFP mice, we found that intraocular injection of CCL2 increased retinal GFP+ monocyte/macrophage infiltration. When these mice were made diabetic, increased infiltration of monocytes/macrophages was also present in retinal tissues. Diabetes and CCL2 injection also induced activation of retinal microglia in these animals. Quantification by flow cytometry demonstrated a two-fold increase of CX3CR1+/CD11b+ (monocyte/macrophage and microglia) cells in retinas of wildtype diabetic animals in comparison to control non-diabetic ones. Using CCL2 knockout (Ccl2-/-) mice, we show a significant reduction in retinal vascular leakage and monocyte infiltration following induction of diabetes indicating the importance of this chemokine in alteration of the BRB. Thus, CCL2 may be an important therapeutic target for the treatment of diabetic macular edema.
Assuntos
Barreira Hematorretiniana/citologia , Movimento Celular , Quimiocina CCL2/metabolismo , Retinopatia Diabética/metabolismo , Monócitos/citologia , Animais , Barreira Hematorretiniana/efeitos dos fármacos , Barreira Hematorretiniana/imunologia , Permeabilidade Capilar/efeitos dos fármacos , Contagem de Células , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Quimiocina CCL2/deficiência , Quimiocina CCL2/genética , Retinopatia Diabética/genética , Retinopatia Diabética/imunologia , Relação Dose-Resposta a Droga , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Técnicas de Inativação de Genes , Glucose/farmacologia , Humanos , Inflamação/metabolismo , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Ratos , Ratos Sprague-Dawley , Regulação para Cima/efeitos dos fármacosRESUMO
HIV-1-associated ocular complications, such as microvasculopathies, can lead to the loss of vision in HIV-1-infected patients. Even in patients under highly active antiretroviral therapy, ocular lesions are unavoidable. Ocular complications have been demonstrated to be closely related to the breakdown of the blood-retinal-barrier (BRB); however, the underlying mechanism is not clear. The data from this study indicated that the HIV-1 Tat protein induced the apoptosis of human retinal microvascular endothelial cells (HRMECs) and retinal pigmen epithelium (RPE) cells, which compose the inner BRB and the outer BRB, respectively. In addition, this study found that the activation of N-methyl-D-aspartate receptors (NMDARs) was involved in the apoptosis of RPE cells, but it caused no changes in HRMECs. Furthermore, both cell types exhibited enhanced expression of Bak, Bax and Cytochrome c. The inhibition of Tat activity protected against the apoptosis induced by NMDAR activation and prevented the dysregulation of Bak, Bax and Cytochrome c, revealing an important role for the mitochondrial pathway in HIV-1 Tat-induced apoptosis. Together, these findings suggest a possible mechanism and may identify a potential therapeutic strategy for HIV-1-associated ocular complications.
Assuntos
Apoptose/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , HIV-1/química , Epitélio Pigmentado da Retina/efeitos dos fármacos , Produtos do Gene tat do Vírus da Imunodeficiência Humana/farmacologia , Anticorpos Neutralizantes/farmacologia , Barreira Hematorretiniana/citologia , Barreira Hematorretiniana/efeitos dos fármacos , Barreira Hematorretiniana/metabolismo , Linhagem Celular , Citocromos c/genética , Citocromos c/metabolismo , Relação Dose-Resposta a Droga , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Regulação da Expressão Gênica , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Modelos Biológicos , Especificidade de Órgãos , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/metabolismo , Transdução de Sinais , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/antagonistas & inibidoresRESUMO
Retinal degenerations are a major cause of impaired vision in the elderly. Degenerations originate in either photoreceptors or the retinal pigment epithelium (RPE). RPE forms the outer blood-retinal barrier and functions intimately with photoreceptors. Animal models and cultures of RPE are commonly used to screen potential pharmaceuticals or explore RPE replacement therapy, but human RPE differs from that of other species. Human RPE forms a barrier using tight junctions composed of a unique set of claudins, proteins that determine the permeability and selectivity of tight junctions. Human adult RPE fails to replicate these properties in vitro. To develop a culture model for drug development and tissue-engineering human retina, RPE were derived from human embryonic stem cells (hESCs). Barrier properties of RPE derived from the H1 and H9 hESC lines were compared with a well-regarded model of RPE function, human fetal RPE isolated from 16-week-gestation fetuses (hfRPE). A serum-free medium (SFM-1) that enhanced the redifferentiation of hfRPE in culture also furthered the maturation of hESC-derived RPE. In SFM-1, the composition, selectivity, and permeability of tight junctions were similar to those of hfRPE. Comparison of the transcriptomes by RNA sequencing and quantitative reverse transcription-polymerase chain reaction revealed a high correlation between the hESCs and hfRPE, but there were notable differences in the expression of adhesion junction and membrane transport genes. These data indicated that hESC-derived RPE is highly differentiated but may be less mature than RPE isolated from 16-week fetuses. The study identified a panel of genes to monitor the maturation of RPE.
Assuntos
Barreira Hematorretiniana/citologia , Células-Tronco Embrionárias/citologia , Epitélio Pigmentado da Retina/citologia , Engenharia Tecidual/métodos , Transcriptoma , Transporte Biológico/genética , Barreira Hematorretiniana/fisiologia , Linhagem Celular , Células Cultivadas , Claudina-3/genética , Claudina-3/metabolismo , Células-Tronco Embrionárias/fisiologia , Feto/citologia , Humanos , RNA Mensageiro/genética , Epitélio Pigmentado da Retina/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Junções Íntimas/genética , Junções Íntimas/metabolismoRESUMO
Formation and maintenance of the blood-retinal barrier is required for proper vision and loss of this barrier contributes to the pathology of a wide number of retinal diseases. The retina is responsible for converting visible light into the electrochemical signal interpreted by the brain as vision. Multiple cell types are required for this function, which are organized into eight distinct cell layers. These neural and glial cells gain metabolic support from a unique vascular structure that provides the necessary nutrients while minimizing interference with light sensing. In addition to the vascular contribution, the retina also possesses an epithelial barrier, the retinal pigment epithelium, which is located at the posterior of the eye and controls exchange of nutrients with the choroidal vessels. Together the vascular and epithelial components of the blood-retinal barrier maintain the specialized environment of the neural retina. Both the vascular endothelium and pigment epithelium possess a well-developed junctional complex that includes both adherens and tight junctions conferring a high degree of control of solute and fluid permeability. Understanding induction and regulation of the blood-retinal barrier will allow the development of therapies aimed at restoring the barrier when compromised in disease or allowing the specific transport of therapies across this barrier when needed. This chapter will highlight the anatomical structure of the blood-retinal barrier and explore the molecular structure of the tight junctions that provide the unique barrier properties of the blood--retinal barrier.
Assuntos
Barreira Hematorretiniana/citologia , Barreira Hematorretiniana/fisiologia , Animais , Barreira Hematorretiniana/ultraestrutura , Humanos , Retina/citologia , Retina/metabolismo , Retina/ultraestrutura , Junções Íntimas/metabolismo , Junções Íntimas/ultraestruturaRESUMO
The outer blood-retinal barrier is composed of a monolayer of retinal pigment epithelium (RPE), Bruch's membrane, and the choriocapillaris, which is fenestrated. An in vitro model that includes all these layers within a 3-D architecture confers a clear advantage over traditional monolayer cultures. Cells here, whether endothelial or epithelial, reside in conditions resembling that in vivo and can participate in cell-cell and cell-matrix cross talk. This chapter describes how a human trilayer culture model was generated with RPE (ARPE-19) cells cultured on the epithelial surface of amniotic membrane and with human umbilical vein derived endothelial cells (HUVEC) on the interstitial surface. This model resembles the outer retinal barrier both in restricting transport of small molecules (<4 kDa), possession of occludin-rich tight junctions in the RPE and fenestrated endothelial cells. Techniques used to test the generated trilayer properties are also described and these include imaging of structure and molecular occupancy of tight and adherens junctions, estimation of the barrier efficiency of trilayer by measurement of fluorescein and fluorescein-conjugated tracers under flow, measurement of secreted vascular endothelial growth factor-A and ultrastructural studies, which allow analyses of the fine structure of the tight junctions in the RPE, and the endothelial fenestra.
Assuntos
Barreira Hematorretiniana/fisiologia , Modelos Biológicos , Barreira Hematorretiniana/citologia , Técnicas de Cultura de Células , Células Cultivadas , Células Endoteliais/metabolismo , Humanos , Epitélio Pigmentado da Retina/irrigação sanguínea , Epitélio Pigmentado da Retina/metabolismo , Junções Íntimas/metabolismoRESUMO
The retina maintains homeostasis through the blood-retinal barrier (BRB). Although it is ideal to deliver the drug to the retina via systemic administration, it is still challenging due to the BRB strictly regulating permeation from blood to the retina. Herein, we demonstrated that intravenously administered gold nanoparticles could pass through the BRB and are distributed in all retinal layers without cytotoxicity. After intravenous injection of gold nanoparticles into C57BL/6 mice, 100 nm nanoparticles were not detected in the retina whereas 20 nm nanoparticles passed through the BRB and were distributed in all retinal layers. 20 nm nanoparticles in the retina were observed in neurons (75 +/- 5%), endothelial cells (17 +/- 6%) and peri-endothelial glial cells (8 +/- 3%), where nanoparticles were bound on the membrane. In the retina, cells containing nanoparticles did not show any structural abnormality and increase of cell death compared to cells without nanoparticles. Gold nanoparticles never affected the viability of retinal endothelial cells, astrocytes and retinoblastoma cells. Furthermore, gold nanoparticles never led to any change in expression of representative biological molecules including zonula occludens-1 and glut-1 in retinal endothelial cells, neurofilaments in differentiated retinoblastoma cells and glial fibrillary acidic protein in astrocytes. Therefore, our data suggests that small gold nanoparticles (20 nm) could be an alternative for drug delivery across the BRB, which could be safely applied in vivo.
Assuntos
Barreira Hematorretiniana/efeitos dos fármacos , Barreira Hematorretiniana/metabolismo , Ouro/administração & dosagem , Ouro/toxicidade , Nanopartículas Metálicas/administração & dosagem , Nanopartículas Metálicas/toxicidade , Tamanho da Partícula , Animais , Apoptose/efeitos dos fármacos , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Barreira Hematorretiniana/citologia , Barreira Hematorretiniana/ultraestrutura , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Transportador de Glucose Tipo 1/metabolismo , Ouro/farmacocinética , Humanos , Injeções Intravenosas , Proteínas de Membrana/metabolismo , Nanopartículas Metálicas/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Fosfoproteínas/metabolismo , Retinoblastoma/metabolismo , Retinoblastoma/patologia , Distribuição Tecidual/efeitos dos fármacos , Proteína da Zônula de Oclusão-1RESUMO
The purpose of this study was to identify the uptake mechanism of gamma-aminobutyric acid (GABA) via taurine transporter (Slc6a6/TauT) and its relationship with GABA transport at the inner BRB. Rat Slc6a6/TauT-transfected HeLa cells exhibited Na(+)-, Cl(-)-, and concentration-dependent [3H]GABA uptake with a Km of 1.5 mM. Taurine, beta-alanine, and GABA markedly inhibited Slc6a6/TauT-mediated uptake of [3H]GABA. The uptake of [3H]GABA by a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2) was Na(+)-, Cl(-)-, and concentration-dependent with a Km of 2.0 mM. This process was more potently inhibited by substrates of Slc6a6/TauT, taurine and beta-alanine, than those of GABA transporters, GABA and betaine. In the presence of taurine, there was competitive inhibition with a Ki of 74 microM. [3H]Taurine also exhibited competitive inhibition with a Ki of 1.8 mM in the presence of GABA. In conclusion, rat Slc6a6/TauT has the ability to use GABA as a substrate and Slc6a6/TauT-mediated GABA transport appears to be present at the inner BRB.