RESUMEN
Purpose: To examine whether increased ephrin type-B receptor 1 (EphB1) leads to inflammatory mediators in retinal Müller cells. Methods: Diabetic human and mouse retinal samples were examined for EphB1 protein levels. Rat Müller cells (rMC-1) were grown in culture and treated with EphB1 siRNA or ephrin B1-Fc to explore inflammatory mediators in cells grown in high glucose. An EphB1 overexpression adeno-associated virus (AAV) was used to increase EphB1 in Müller cells in vivo. Ischemia/reperfusion (I/R) was performed on mice treated with the EphB1 overexpression AAV to explore the actions of EphB1 on retinal neuronal changes in vivo. Results: EphB1 protein levels were increased in diabetic human and mouse retinal samples. Knockdown of EphB1 reduced inflammatory mediator levels in Müller cells grown in high glucose. Ephrin B1-Fc increased inflammatory proteins in rMC-1 cells grown in normal and high glucose. Treatment of mice with I/R caused retinal thinning and loss of cell numbers in the ganglion cell layer. This was increased in mice exposed to I/R and treated with the EphB1 overexpressing AAVs. Conclusions: EphB1 is increased in the retinas of diabetic humans and mice and in high glucose-treated Müller cells. This increase leads to inflammatory proteins. EphB1 also enhanced retinal damage in response to I/R. Taken together, inhibition of EphB1 may offer a new therapeutic option for diabetic retinopathy.
Asunto(s)
Retinopatía Diabética , Efrina-B1 , Enfermedades de la Retina , Animales , Humanos , Ratones , Ratas , Retinopatía Diabética/genética , Retinopatía Diabética/metabolismo , Células Ependimogliales/metabolismo , Efrina-B1/genética , Efrina-B1/metabolismo , Glucosa/metabolismo , Mediadores de Inflamación/metabolismo , Retina/metabolismo , Enfermedades de la Retina/metabolismoRESUMEN
Exosomes are nanosized vesicles that have been reported as cargo-delivering vehicles between cells. Müller cells play a crucial role in the pathogenesis of diabetic retinopathy (DR). Activated Müller cells in the diabetic retina mediate disruption of barrier integrity and neovascularization. Endothelial cells constitute the inner blood-retinal barrier (BRB). Herein, we aim to evaluate the effect of Müller cell-derived exosomes on endothelial cell viability and barrier function under normal and hyperglycemic conditions. Müller cell-derived exosomes were isolated and characterized using Western blotting, nanoparticle tracking, and electron microscopy. The uptake of Müller cells-derived exosomes by the human retinal endothelial cells (HRECs) was monitored by labeling exosomes with PKH67. Endothelial cell vitality after treatment by exosomes under normo- and hypoglycemic conditions was checked by MTT assay and Western blot for apoptotic proteins. The barrier function of HRECs was evaluated by analysis of ZO-1 and transcellular electrical resistance (TER) using ECIS. Additionally, intracellular Ca+2 in HRECs was assessed by spectrofluorimetry. Analysis of the isolated exosomes showed a non-significant change in the number of exosomes isolated from both normal and hyperglycemic condition media, however, the average size of exosomes isolated from the hyperglycemic group showed a significant rise when compared to that of the normoglycemic group. Müller cells derived exosomes from hyperglycemic condition media markedly reduced HRECs cell count, increased caspase-3 and Annexin V, decreased ZO-1 levels and TER, and increased intracellular Ca+ when compared to other groups. However, treatment of HRECs under hyperglycemia with normo-glycemic Müller cells-derived exosomes significantly decreased cell death, preserved cellular integrity and barrier function, and reduced intracellular Ca+2. Collectively, Müller cell-derived exosomes play a remarkable role in the pathological changes associated with hyperglycemia-induced inner barrier dysfunction in DR. Further in vivo research will help in understanding the role of exosomes as therapeutic targets and/or delivery systems for DR.
Asunto(s)
Apoptosis , Barrera Hematorretinal , Supervivencia Celular , Retinopatía Diabética , Células Endoteliales , Células Ependimogliales , Exosomas , Exosomas/metabolismo , Retinopatía Diabética/metabolismo , Retinopatía Diabética/patología , Retinopatía Diabética/fisiopatología , Células Ependimogliales/metabolismo , Células Ependimogliales/patología , Humanos , Células Endoteliales/metabolismo , Células Endoteliales/patología , Barrera Hematorretinal/metabolismo , Barrera Hematorretinal/patología , Células Cultivadas , Proteína de la Zonula Occludens-1/metabolismo , Permeabilidad Capilar , Señalización del Calcio , Línea Celular , Vasos Retinianos/metabolismo , Vasos Retinianos/patología , Vasos Retinianos/fisiopatologíaRESUMEN
Diabetic retinopathy (DR) is a neurovascular complication of diabetes, driven by an intricate network of cellular and molecular mechanisms. This study sought to explore the mechanisms by investigating the role of 12-hydroxyeicosatetraenoic acid (12-HETE), its receptor GPR31, and microRNA (miR-29) in the context of DR, specifically focusing on their impact on Müller glial cells. We found that 12-HETE activates Müller cells (MCs), elevates glutamate production, and induces inflammatory and oxidative responses, all of which are instrumental in DR progression. The expression of GPR31, the receptor for 12-HETE, was prominently found in the retina, especially in MCs and retinal ganglion cells, and was upregulated in diabetes. Interestingly, miR29 showed potential as a protective agent, mitigating the harmful effects of 12-HETE by attenuating inflammation and oxidative stress, and restoring the expression of pigment epithelium-derived factor (PEDF). Our results underline the central role of 12-HETE in DR progression through activation of a neurovascular toxic pathway in MCs and illuminate the protective capabilities of miR-29, highlighting both as promising therapeutic targets for the management of DR.
Asunto(s)
Retinopatía Diabética , MicroARNs , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/metabolismo , Retinopatía Diabética/genética , Retinopatía Diabética/metabolismo , Células Ependimogliales , MicroARNs/genética , MicroARNs/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Retina/metabolismoRESUMEN
Preservation of retinal barrier function is critical to maintenance of retinal health. Therefore, it is not surprising that loss of barrier integrity is a pathologic feature common to degenerative retinal diseases such as diabetic retinopathy. Our prior studies demonstrate the importance of hydroxycarboxylic acid receptor 2/GPR109A (HCAR2/GPR109A) expression in the retinal pigment epithelium (RPE) to outer retinal barrier integrity. However, whether HCAR2/GPR109A is expressed in retinal endothelial cells and has a similar relationship to inner blood retinal barrier regulation is not known. In the current study, we examined relevance of receptor expression to endothelial cell dependent-blood retinal barrier integrity. siRNA technology was used to modulate HCAR2/GPR109A expression in human retinal endothelial cells (HRECs). Cells were cultured in the presence or absence of VEGF, a pro-inflammatory stimulus, and/or various concentrations of the HCAR2/GPR109A-specific agonist beta-hydyroxybutyrate (BHB). HCAR2/GPR109A expression was monitored by qPCR and electrical cell impedance sensing (ECIS) was used to evaluate barrier function. Complementary in vivo studies were conducted in wildtype and HCAR2/GPR109A knockout mice treated intraperitoneally with lipopolysaccharide and/or BHB. Vascular leakage was monitored using fluorescein angiography and Western blot analyses of albumin extravasation. Additionally, retinal function was evaluated by OptoMotry. Decreased (siRNA knockdown) or absent (gene knockout) HCAR2/GPR109A expression was associated with impaired barrier function both in vitro and in vivo. BHB treatment provided some protection, limiting disruptions in retinal barrier integrity and function; an effect that was found to be receptor (HCAR2/GPR109A)-dependent. Collectively, the present studies support a key role for HCAR2/GPR109A in regulating blood-retinal barrier integrity and highlight the therapeutic potential of the receptor toward preventing and treating retinal diseases such as diabetic retinopathy in which compromised barrier function is paramount.
Asunto(s)
Retinopatía Diabética , Receptores Acoplados a Proteínas G , Enfermedades de la Retina , Animales , Barrera Hematorretinal/metabolismo , Proteínas Portadoras/metabolismo , Retinopatía Diabética/metabolismo , Células Endoteliales/metabolismo , Cetonas/metabolismo , Cetonas/uso terapéutico , Ratones , ARN Interferente Pequeño/uso terapéutico , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Enfermedades de la Retina/metabolismoRESUMEN
Age-related macular degeneration (AMD) is a leading cause of vision loss. Elevated homocysteine (Hcy) (Hyperhomocysteinemia) (HHcy) has been reported in AMD. We previously reported that HHcy induces AMD-like features. This study suggests that N-Methyl-d-aspartate receptor (NMDAR) activation in the retinal pigment epithelium (RPE) is a mechanism for HHcy-induced AMD. Serum Hcy and cystathionine-ß-synthase (CBS) were assessed by ELISA. The involvement of NMDAR in Hcy-induced AMD features was evaluated (1) in vitro using ARPE-19 cells, primary RPE isolated from HHcy mice (CBS), and mouse choroidal endothelial cells (MCEC); (2) in vivo using wild-type mice and mice deficient in RPE NMDAR (NMDARR-/-) with/without Hcy injection. Isolectin-B4, Ki67, HIF-1α, VEGF, NMDAR1, and albumin were assessed by immunofluorescence (IF), Western blot (WB), Optical coherence tomography (OCT), and fluorescein angiography (FA) to evaluate retinal structure, fluorescein leakage, and choroidal neovascularization (CNV). A neovascular AMD patient's serum showed a significant increase in Hcy and a decrease in CBS. Hcy significantly increased HIF-1α, VEGF, and NMDAR in RPE cells, and Ki67 in MCEC. Hcy-injected WT mice showed disrupted retina and CNV. Knocking down RPE NMDAR improved retinal structure and CNV. Our findings underscore the role of RPE NMDAR in Hcy-induced AMD features; thus, NMDAR inhibition could serve as a promising therapeutic target for AMD.
Asunto(s)
Homocisteína/efectos adversos , Homocisteína/sangre , Degeneración Macular/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Anciano , Anciano de 80 o más Años , Animales , Línea Celular , Neovascularización Coroidal/etiología , Cistationina betasintasa/sangre , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Femenino , Humanos , Hiperhomocisteinemia/complicaciones , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Degeneración Macular/inducido químicamente , Degeneración Macular/diagnóstico por imagen , Degeneración Macular/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Neovascularización Patológica/etiología , Cultivo Primario de Células , Epitelio Pigmentado de la Retina/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Clinical studies have suggested that the renin-angiotensin system (RAS) may be a promising therapeutic target in treating diabetic retinopathy (DR). While AT1 receptor blockade decreased the incidence of DR in the DIRECT trial, it did not reduce the DR progression. Lack of understanding of the molecular mechanism of retinal microvascular damage induced by RAS is a critical barrier to the use of RAS blockade in preventing or treating DR. The purpose of this study is to investigate the interaction between soluble epoxide hydrolase (sEH) and the AT1 receptor in Angiotensin II (Ang II)- and diabetes-induced retinal microvascular damage. We demonstrate that Ang II increases retinal sEH levels, which is blunted by an AT1 blocker; administration of 11,12-epoxyeicosatrienoic acid (EET) exacerbates intravitreal Ang II-induced retinal albumin leakage; while sEH knockout (KO) and blockade reduce Ang II-induced retinal vascular remodeling, sEH KO causes retinal vascular leakage in Ang II-sEH KO mice; and sEH KO potentiates diabetes-induced retinal damage via promoting retinal vascular endothelial growth factor (VEGF) but reducing expression of tight junction proteins (ZO-1 and occludin). Our studies hold the promise of providing a new strategy, the use of combined EETs blockade with AT1 blocker, to prevent or reduce DR.
Asunto(s)
Angiotensina II/metabolismo , Diabetes Mellitus Experimental/patología , Epóxido Hidrolasas/metabolismo , Microvasos/patología , Receptor de Angiotensina Tipo 1/metabolismo , Sistema Renina-Angiotensina , Retina/patología , Animales , Diabetes Mellitus Experimental/metabolismo , Modelos Animales de Enfermedad , Epóxido Hidrolasas/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microvasos/metabolismo , Retina/metabolismoRESUMEN
Vascular inflammation plays a critical role in the pathogenesis of diabetic retinopathy. Recently, Interleukin-6 (IL-6) trans-signaling via soluble IL-6 receptor (sIL-6R) has emerged as a prominent regulator of inflammation in endothelial cells. This study was designed to test the hypothesis that selective inhibition of the IL-6 trans-signaling pathway will attenuate inflammation and subsequent barrier disruption in retinal endothelial cells. Human retinal endothelial cells (HRECs) were exposed to IL-6 and sIL-6R to induce IL-6 trans-signaling and the commercially available compound sgp130Fc (soluble gp-130 fused chimera) was used to selectively inhibit IL-6 trans-signaling. IL-6 trans-signaling activation caused a significant increase in STAT3 phosphorylation, expression of adhesion molecules, ROS production and apoptosis in HRECs whereas a significant decrease in mitochondrial membrane potential and NO production was observed in IL-6 trans-signaling activated cells. These changes were not observed in cells pre-treated with sgp130Fc. IL-6 trans-signaling activation was sufficient to cause barrier disruption in endothelial monolayers and pre-treatment of HRECs with sgp130Fc, maintained endothelial barrier function similar to that of untreated cells. Thus, in conclusion, these results indicate that IL-6 trans-signaling is an important mediator of inflammation, apoptosis and barrier disruptive effects in the retinal endothelial cells and inhibition of the IL-6 trans-signaling pathway using sgp130-Fc attenuates vascular inflammation and endothelial barrier disruption.
Asunto(s)
Barrera Hematorretinal/efectos de los fármacos , Células Endoteliales/metabolismo , Inflamación/prevención & control , Interleucina-6/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/farmacología , Transducción de Señal/efectos de los fármacos , Apoptosis/efectos de los fármacos , Western Blotting , Células Cultivadas , Células Endoteliales/patología , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Peróxido de Hidrógeno/metabolismo , Interleucina-6/metabolismo , Potencial de la Membrana Mitocondrial , Microscopía Confocal , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Óxido Nítrico/metabolismo , Fosforilación , Especies Reactivas de Oxígeno/metabolismo , Receptores de Interleucina-6/metabolismo , Retinitis/prevención & control , Factor de Transcripción STAT3/metabolismoRESUMEN
AIMS/HYPOTHESIS: Our earlier studies have established the role of 12/15-lipoxygenase (LO) in mediating the inflammatory reaction in diabetic retinopathy. However, the exact mechanism is still unclear. The goal of the current study was to identify the potential role of endoplasmic reticulum (ER) stress as a major cellular stress response in the 12/15-LO-induced retinal changes in diabetic retinopathy. METHODS: We used in vivo and in vitro approaches. For in vivo studies, experimental diabetes was induced in wild-type (WT) mice and 12/15-Lo (also known as Alox15) knockout mice (12/15-Lo-/-); ER stress was then evaluated after 12-14 weeks of diabetes. We also tested the effect of intravitreal injection of 12-hydroxyeicosatetraenoic acid (HETE) on retinal ER stress in WT mice and in mice lacking the catalytic subunit of NADPH oxidase, encoded by Nox2 (also known as Cybb) (Nox2-/- mice). In vitro studies were performed using human retinal endothelial cells (HRECs) treated with 15-HETE (0.1 µmol/l) or vehicle, with or without ER stress or NADPH oxidase inhibitors. This was followed by evaluation of ER stress response, NADPH oxidase expression/activity and the levels of phosphorylated vascular endothelial growth factor receptor-2 (p-VEGFR2) by western blotting and immunoprecipitation assays. Moreover, real-time imaging of intracellular calcium (Ca2+) release in HRECs treated with or without 15-HETE was performed using confocal microscopy. RESULTS: Deletion of 12/15-Lo significantly attenuated diabetes-induced ER stress in mouse retina. In vitro, 15-HETE upregulated ER stress markers such as phosphorylated RNA-dependent protein kinase-like ER-regulated kinase (p-PERK), activating transcription factor 6 (ATF6) and protein disulfide isomerase (PDI) in HRECs. Inhibition of ER stress reduced 15-HETE-induced-leucocyte adhesion, VEGFR2 phosphorylation and NADPH oxidase expression/activity. However, inhibition of NADPH oxidase or deletion of Nox2 had no effect on ER stress induced by the 12/15-LO-derived metabolites both in vitro and in vivo. We also found that 15-HETE increases the intracellular calcium in HRECs. CONCLUSIONS/INTERPRETATION: ER stress contributes to 12/15-LO-induced retinal inflammation in diabetic retinopathy via activation of NADPH oxidase and VEGFR2. Perturbation of calcium homeostasis in the retina might also play a role in linking 12/15-LO to retinal ER stress and subsequent microvascular dysfunction in diabetic retinopathy.
Asunto(s)
Araquidonato 15-Lipooxigenasa/metabolismo , Retinopatía Diabética/metabolismo , Estrés del Retículo Endoplásmico , Retículo Endoplásmico/metabolismo , Vasos Retinianos/metabolismo , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/química , Animales , Apoptosis , Calcio/metabolismo , Dominio Catalítico , Línea Celular , Células Endoteliales/metabolismo , Humanos , Inflamación , Leucocitos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , NADPH Oxidasas/metabolismo , Fosforilación , Transducción de Señal , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
AIMS: Our previous studies have established a role for 12/15-lipoxygenase (LO) in mediating the inflammatory response in diabetic retinopathy (DR). However, the extent at which the local or systemic induction of 12/15-LO activity involved is unclear. Thus, the current study aimed to characterize the relative contribution of retinal endothelial versus monocytic/macrophagic 12/15-LO to inflammatory responses in DR. MATERIALS & METHODS: We first generated a clustered heat map for circulating bioactive lipid metabolites in the plasma of streptozotocin (STZ)-induced diabetic mice using liquid chromatography coupled with mass-spectrometry (LC-MS) to evaluate changes in circulating 12/15-LO activity. This was followed by comparing the in vitro mouse endothelium-leukocytes interaction between leukocytes isolated from 12/15-LO knockout (KO) versus those isolated from wild type (WT) mice using the myeloperoxidase (MPO) assay. Finally, we examined the effects of knocking down or inhibiting endothelial 12/15-LO on diabetes-induced endothelial cell activation and ICAM-1 expression. RESULTS: Analysis of plasma bioactive lipids' heat map revealed that the activity of circulating 12/15-LO was not altered by diabetes as evident by no significant changes in the plasma levels of major metabolites derived from 12/15-lipoxygenation of different PUFAs, including linoleic acid (13-HODE), arachidonic acid (12- and 15- HETEs), eicosapentaenoic acid (12- and 15- HEPEs), or docosahexaenoic acid (17-HDoHE). Moreover, leukocytes from 12/15-LO KO mice displayed a similar increase in adhesion to high glucose (HG)-activated endothelial cells as do leukocytes from WT mice. Furthermore, abundant proteins of 12-LO and 15-LO were detected in human retinal endothelial cells (HRECs), while it was undetected (15-LO) or hardly detectable (12-LO) in human monocyte-like U937 cells. Inhibition or knock down of endothelial 12/15-LO in HRECs blocked HG-induced expression of ICAM-1, a well-known identified important molecule for leukocyte adhesion in DR. CONCLUSION: Our data support that endothelial, rather than monocytic/macrophagic, 12/15-LO has a critical role in hyperglycemia-induced ICAM-1 expression, leukocyte adhesion, and subsequent local retinal barrier dysfunction. This may facilitate the development of more precisely targeted treatment strategies for DR.
Asunto(s)
Araquidonato 12-Lipooxigenasa/metabolismo , Araquidonato 15-Lipooxigenasa/metabolismo , Retinopatía Diabética/enzimología , Células Endoteliales/enzimología , Leucostasis/enzimología , Macrófagos/enzimología , Monocitos/enzimología , Retina/enzimología , Animales , Araquidonato 12-Lipooxigenasa/genética , Araquidonato 15-Lipooxigenasa/genética , Adhesión Celular/genética , Retinopatía Diabética/genética , Retinopatía Diabética/patología , Células Endoteliales/patología , Humanos , Molécula 1 de Adhesión Intercelular/biosíntesis , Molécula 1 de Adhesión Intercelular/genética , Leucostasis/genética , Leucostasis/patología , Macrófagos/patología , Ratones , Ratones Noqueados , Monocitos/patología , Retina/patología , Células U937RESUMEN
We recently demonstrated that 12/15-lipoxygenase (LOX) derived metabolites, hydroxyeicosatetraenoic acids (HETEs), contribute to diabetic retinopathy (DR) via NADPH oxidase (NOX) and disruption of the balance in retinal levels of the vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF). Here, we test whether PEDF ameliorates retinal vascular injury induced by HETEs and the underlying mechanisms. Furthermore, we pursue the causal relationship between LOX-NOX system and regulation of PEDF expression during DR. For these purposes, we used an experimental eye model in which normal mice were injected intravitreally with 12-HETE with/without PEDF. Thereafter, fluorescein angiography (FA) was used to evaluate the vascular leakage, followed by optical coherence tomography (OCT) to assess the presence of angiogenesis. FA and OCT reported an increased vascular leakage and pre-retinal neovascularization, respectively, in response to 12-HETE that were not observed in the PEDF-treated group. Moreover, PEDF significantly attenuated the increased levels of vascular cell and intercellular adhesion molecules, VCAM-1 and ICAM-1, elicited by 12-HETE injection. Accordingly, the direct relationship between HETEs and PEDF has been explored through in-vitro studies using Müller cells (rMCs) and human retinal endothelial cells (HRECs). The results showed that 12- and 15-HETEs triggered the secretion of TNF-α and IL-6, as well as activation of NFκB in rMCs and significantly increased permeability and reduced zonula occludens protein-1 (ZO-1) immunoreactivity in HRECs. All these effects were prevented in PEDF-treated cells. Furthermore, interest in PEDF regulation during DR has been expanded to include NOX system. Retinal PEDF was significantly restored in diabetic mice treated with NOX inhibitor, apocynin, or lacking NOX2 up to 80% of the control level. Collectively, our findings suggest that interfering with LOX-NOX signaling opens up a new direction for treating DR by restoring endogenous PEDF that carries out multilevel vascular protective functions.
Asunto(s)
Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/antagonistas & inhibidores , Retinopatía Diabética/tratamiento farmacológico , Proteínas del Ojo/farmacología , Ácidos Hidroxieicosatetraenoicos/antagonistas & inhibidores , Factores de Crecimiento Nervioso/farmacología , Retina/efectos de los fármacos , Neovascularización Retiniana/tratamiento farmacológico , Serpinas/farmacología , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/farmacología , Acetofenonas/farmacología , Animales , Araquidonato 12-Lipooxigenasa/genética , Araquidonato 12-Lipooxigenasa/metabolismo , Araquidonato 15-Lipooxigenasa/genética , Araquidonato 15-Lipooxigenasa/metabolismo , Células Cultivadas , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Retinopatía Diabética/genética , Retinopatía Diabética/metabolismo , Retinopatía Diabética/patología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Células Endoteliales/patología , Células Ependimogliales/efectos de los fármacos , Células Ependimogliales/metabolismo , Células Ependimogliales/patología , Regulación de la Expresión Génica , Humanos , Ácidos Hidroxieicosatetraenoicos/farmacología , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Inyecciones Intravítreas , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Noqueados , NADPH Oxidasa 2 , NADPH Oxidasas/antagonistas & inhibidores , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , Retina/metabolismo , Retina/patología , Neovascularización Retiniana/genética , Neovascularización Retiniana/metabolismo , Neovascularización Retiniana/patología , Transducción de Señal , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Molécula 1 de Adhesión Celular Vascular/genética , Molécula 1 de Adhesión Celular Vascular/metabolismo , Proteína de la Zonula Occludens-1/genéticaRESUMEN
Arachidonic acid (AA) is metabolized by cyclooxygenase (COX) and cytochrome P450 (CYP) enzymes into eicosanoids, which are involved in cardiovascular diseases and stroke. Evidence has demonstrated the important functions of these eicosanoids in regulating cerebral vascular tone, cerebral blood flow, and autoregulation of cerebral circulation. Although COX-2 inhibitors have been suggested as potential treatments for stroke, adverse events, including an increased risk of stroke, occur following long-term use of coxibs. It is important to note that prolonged treatment with rofecoxib increased circulating levels of 20-hydroxyeicosatetraenoic acid (20-HETE), and 20-HETE blockade is a possible strategy to prevent coxib-induced stroke events. It appears that 20-HETE has detrimental effects in the brain, and that its blockade exerts cerebroprotection against ischemic stroke and subarachnoid hemorrhage (SAH). There is clear evidence that activation of EP2 and EP4 receptors exerts cerebroprotection against ischemic stroke. Several elegant studies have contributed to defining the importance of stabilizing the levels of epoxyeicosatrienoic acids (EETs), by inhibiting or deleting soluble epoxide hydrolase (sEH), in stroke research. These reports support the notion that sEH blockade is cerebroprotective against ischemic stroke and SAH. Here, we summarize recent findings implicating these eicosanoid pathways in cerebral vascular function and stroke. We also discuss the development of animal models with targeted gene deletion and specific enzymatic inhibitors in each pathway to identify potential targets for the treatment of ischemic stroke and SAH.
Asunto(s)
Ácido Araquidónico/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Eicosanoides/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Accidente Cerebrovascular/metabolismo , Humanos , Ácidos Hidroxieicosatetraenoicos/sangre , Ácidos Hidroxieicosatetraenoicos/metabolismo , Modelos Biológicos , Receptores de Prostaglandina E/metabolismo , Accidente Cerebrovascular/sangre , Hemorragia Subaracnoidea/sangre , Hemorragia Subaracnoidea/metabolismoRESUMEN
Retinal hyperpermeability and subsequent macular edema is a cardinal feature of early diabetic retinopathy (DR). Here, we investigated the role of bioactive lipid metabolites, in particular 12/15-lipoxygenase (LOX)-derived metabolites, in this process. LC/MS lipidomic screen of human retinal endothelial cells (HRECs) demonstrated that 15-HETE was the only significantly increased metabolite (2.4 ± 0.4-fold, P = 0.0004) by high glucose (30 mM) treatment. In the presence of arachidonic acid, additional eicosanoids generated by 12/15-LOX, including 12- and 11-HETEs, were significantly increased. Fluorescein angiography and retinal albumin leakage showed a significant decrease in retinal hyperpermeability in streptozotocin-induced diabetic mice lacking 12/15-LOX compared with diabetic WT mice. Our previous studies demonstrated the potential role of NADPH oxidase in mediating the permeability effect of 12- and 15-HETEs, therefore we tested the impact of intraocular injection of 12-HETE in mice lacking the catalytic subunit of NADPH oxidase (NOX2). The permeability effect of 12-HETE was significantly reduced in NOX2(-/-) mice compared with the WT mice. In vitro experiments also showed that 15-HETE induced HREC migration and tube formation in a NOX-dependent manner. Taken together our data suggest that 12/15-LOX is implicated in DR via a NOX-dependent mechanism.
Asunto(s)
Araquidonato 12-Lipooxigenasa/metabolismo , Araquidonato 15-Lipooxigenasa/metabolismo , Retinopatía Diabética/tratamiento farmacológico , Ácidos Hidroxieicosatetraenoicos/farmacología , Hiperglucemia/tratamiento farmacológico , Glicoproteínas de Membrana/metabolismo , NADPH Oxidasas/metabolismo , Animales , Araquidonato 12-Lipooxigenasa/genética , Araquidonato 15-Lipooxigenasa/genética , Retinopatía Diabética/enzimología , Retinopatía Diabética/genética , Retinopatía Diabética/patología , Humanos , Hiperglucemia/enzimología , Hiperglucemia/genética , Hiperglucemia/patología , Glicoproteínas de Membrana/genética , Ratones , Ratones Noqueados , NADPH Oxidasa 2 , NADPH Oxidasas/genéticaRESUMEN
Mild to moderate hyperhomocysteinemia is prevalent in humans and is implicated in neurovascular diseases, including recently in certain retinal diseases. Herein, we used hyperhomocysteinemic mice deficient in the Cbs gene encoding cystathionine-ß-synthase (Cbs(+/-)) to evaluate retinal vascular integrity. The Cbs(+/+) (wild type) and Cbs(+/-) (heterozygous) mice (aged 16 to 52 weeks) were subjected to fluorescein angiography and optical coherence tomography to assess vasculature in vivo. Retinas harvested for cryosectioning or flat mount preparations were subjected to immunofluorescence microscopy to detect blood vessels (isolectin-B4), angiogenesis [anti-vascular endothelial growth factor (VEGF) and anti-CD105], gliosis [anti-glial fibrillary acidic protein (GFAP)], pericytes (anti-neural/glial antigen 2), blood-retinal barrier [anti-zonula occludens protein 1 (ZO-1) and anti-occludin], and hypoxia [anti-pimonidazole hydrochloride (Hypoxyprobe-1)]. Levels of VEGF, GFAP, ZO-1, and occludin were determined by immunoblotting. Results of these analyses showed a mild vascular phenotype in young mice, which progressed with age. Fluorescein angiography revealed progressive neovascularization and vascular leakage in Cbs(+/-) mice; optical coherence tomography confirmed new vessels in the vitreous by 1 year. Immunofluorescence microscopy demonstrated vascular patterns consistent with ischemia, including a capillary-free zone centrally and new vessels with capillary tufts midperipherally in older mice. This was associated with increased VEGF, CD105, and GFAP and decreased ZO-1/occludin levels in the Cbs(+/-) retinas. Retinal vein occlusion was observed in some Cbs(+/-) mouse retinas. We conclude that mild to moderate elevation of homocysteine in Cbs(+/-) mice is accompanied by progressive alterations in retinal vasculature characterized by ischemia, neovascularization, incompetent blood-retinal barrier, and vascular occlusion.
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Cistationina betasintasa/genética , Modelos Animales de Enfermedad , Hiperhomocisteinemia/patología , Vasos Retinianos/patología , Animales , Heterocigoto , Hiperhomocisteinemia/genética , Ratones , Ratones Mutantes , Microscopía Fluorescente , Reacción en Cadena en Tiempo Real de la Polimerasa , Neovascularización Retiniana/genética , Neovascularización Retiniana/patologíaRESUMEN
Diabetic retinopathy (DR) is one of the most common complications of diabetes mellitus. Vision loss in DR principally occurs due to breakdown of the blood-retinal barrier (BRB), leading to macular edema, retinal detachment and inner retinal and vitreous hemorrhage. Several growth factors have been shown to play crucial role in the development of these vascular changes; however, the cellular and molecular mechanisms of DR are not yet fully revealed. In the current study we investigated the role of bone morphogenetic protein-2 (BMP2) in DR. We examined the changes in the protein levels of BMP2 in human vitreous and retina in addition to the mouse retina of streptozotocin-induced diabetes. To detect the source of BMP2 during diabetes, human retinal endothelial cells (hRECs) were subjected to high glucose (HG) for 5 days and levels of BMP2 protein were analyzed in conditioned media of these cells relative to control. We also evaluated the effect of BMP2 on the levels of VEGF in cultured rat Müller cells (rMC1). In addition, we tested the pro-inflammatory effects of BMP2 by examining its effect on leukocyte adhesion to cultured hRECs, and levels of adhesion molecules and cytokines production. Finally, the effect of different concentrations of BMP2 on permeability of confluent monolayer of hRECs was evaluated using FITC-Dextran flux permeability assay and by measuring Transcellular Electrical Resistance (TER) using Electric Cell-substrate Impedance Sensing (ECIS). Our results show, for the first time, the up-regulation of BMP2 in diabetic human and mouse retinas in addition to its detection in vitreous of patients with proliferative DR (72 ± 7 pg/ml). In vitro, hRECs showed upregulation of BMP2 in HG conditions suggesting that these cells are a potential source of BMP2 in diabetic conditions. Furthermore, BMP2 induced VEGF secretion by Müller cells in-vitro; and showed a dose response in increasing permeability of cultured hRECs. Meanwhile, BMP2 pro-inflammatory effects were recognized by its ability to induce leukocyte adhesion to the hRECs, intercellular adhesion molecule-1 (ICAM-1) and upregulation of interleukin-6 and 8 (IL-6 and IL-8). These results show that BMP2 could be a contributing growth factor to the development of microvascular dysfunction during DR via enhancing both pro-angiogenic and inflammatory pathways. Our findings suggest BMP2 as a potential therapeutic target to prevent/treat DR.
Asunto(s)
Proteína Morfogenética Ósea 2/metabolismo , Diabetes Mellitus Experimental/metabolismo , Retinopatía Diabética/metabolismo , Células Ependimogliales/metabolismo , Análisis de Varianza , Animales , Proteína Morfogenética Ósea 2/fisiología , Adhesión Celular/fisiología , Células Cultivadas , Citocinas/metabolismo , Diabetes Mellitus Experimental/etiología , Impedancia Eléctrica , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Células Ependimogliales/efectos de los fármacos , Humanos , Ratones , Ratas , Retina/citología , Retina/metabolismo , Regulación hacia Arriba , Factor A de Crecimiento Endotelial Vascular/metabolismo , Cuerpo Vítreo/metabolismo , Receptor de Retrovirus Xenotrópico y PolitrópicoRESUMEN
Adenosine provides anti-inflammatory effects in cardiovascular disease via the activation of adenosine A2A receptors; however, the physiological effect of adenosine could be limited due to its phosphorylation by adenosine kinase. We hypothesized that inhibition of adenosine kinase exacerbates extracellular adenosine levels to reduce renal inflammation and injury in streptozotocin-induced diabetes. Diabetes was induced in male C57BL/6 mice by daily injection of streptozotocin (50mg/kg/day, i.p. for 5 days). Control and diabetic mice were then treated with the adenosine kinase inhibitor ABT702 (1.5mg/kg, i.p. two times a week for 8 weeks, n=7-8/group) or the vehicle (5% DMSO). ABT702 treatment reduced blood glucose level in diabetic mice (â¼20%; P<0.05). ABT702 also reduced albuminuria and markers of glomerular injury, nephrinuria and podocalyxin excretion levels, in diabetic mice. Renal NADPH oxidase activity and urinary thiobarbituric acid reactive substances (TBARS) excretion, indices of oxidative stress, were also elevated in diabetic mice and ABT702 significantly reduced these changes. ABT702 increased renal endothelial nitric oxide synthase expression (eNOS) and nitrate/nitrite excretion levels in diabetic mice. In addition, the diabetic mice displayed an increase in renal macrophage infiltration, in association with increased renal NFκB activation. Importantly, treatment with ABT702 significantly reduced all these inflammatory parameters (P<0.05). Furthermore, ABT702 decreased glomerular permeability and inflammation and restored the decrease in glomerular occludin expression in vitro in high glucose treated human glomerular endothelial cells. Collectively, the results suggest that the reno-protective effects of ABT702 could be attributed to the reduction in renal inflammation and oxidative stress in diabetic mice.
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Adenosina Quinasa/antagonistas & inhibidores , Antiinflamatorios/farmacología , Antioxidantes/farmacología , Diabetes Mellitus Experimental/metabolismo , Riñón/efectos de los fármacos , Morfolinas/farmacología , Pirimidinas/farmacología , Adenosina Quinasa/metabolismo , Animales , Antiinflamatorios/uso terapéutico , Antioxidantes/uso terapéutico , Glucemia/análisis , Línea Celular , Dextranos/metabolismo , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/patología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/metabolismo , Humanos , Riñón/metabolismo , Riñón/patología , Masculino , Ratones Endogámicos C57BL , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Morfolinas/uso terapéutico , NADPH Oxidasas/metabolismo , Nitratos/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Nitritos/metabolismo , Proteinuria/tratamiento farmacológico , Proteinuria/metabolismo , Proteinuria/patología , Pirimidinas/uso terapéutico , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismo , Factor de Transcripción ReIA/metabolismoRESUMEN
The role of inflammation has been accepted as a factor in the complications of diabetic retinopathy. Discovery of the upstream regulation of these inflammatory factors has remained a challenge. In this study, we explored the actions of ephrin B1 in retinal Müller cells and their actions on inflammatory proteins. We used diabetic human and mouse samples, as well as Müller cells in culture to measure ephrin B1 in Müller cells. We then generated Müller cell specific ephrin B1 knockout mice. We measure levels of key inflammatory proteins, including high mobility group box 1 (HMGB1) and NOD-like receptor protein 3 (NLRP3) pathway proteins in retinal lysates from the ephrin B1 floxed and ephrin B1 Müller cell specific knockout mice. Data show that ephrin B1 is significantly increased in the retina of diabetic humans and mice, as well as in Müller cells grown in high glucose. Elimination of ephrin B1 in mouse Müller cells led to a significant decline in all inflammatory proteins studied. In conclusion, a reduction in ephrin B1 in the diabetic retina may offer a new therapeutic modality for diabetic retinopathy.
RESUMEN
Extracellular high-mobility group box-1 (HMGB-1) functions as a pro-inflammatory cytokine and exhibits angiogenic effects. The purpose of this study was to investigate the expression of HMGB-1 signaling pathway components in the retinas of diabetic rats and to examine the effect of intravitreal administration of HMGB-1 on the retinas of rats. The retinas of diabetic and intravitreally injected HMGB-1 rats were studied using immunohistochemistry, Western blotting, co-immunoprecipitation and enzyme-linked immunosorbent assay. The effect of HMGB-1 on retinal endothelial cell barrier function was evaluated using electrical cell-substrate impedance sensing system (ECIS). Diabetes induced significant upregulation of the expression of HMGB-1, receptor for advanced glycation end products (RAGE), ERK(1/2) and nuclear transcription factor Kappa B (NF-κB), whereas the expression of toll-like receptor 2 (TLR2) and occludin was significantly downregulated. Co-immunoprecipitation studies revealed significant increase in interaction between HMGB-1 and RAGE. HMGB-1 reduced transendothelial electrical resistance of bovine retinal endothelial cells. Intravitreal administration of HMGB-1 to normal rats induced significant upregulation of intercellular adhesion molecule-1 (ICAM-1), soluble intercellular adhesion molecule-1 (sICAM-1), HMGB-1, RAGE, ERK(1/2), and NF-κB, and significantly increased retinal vascular permeability, whereas the expression of TLR2 and occludin was downregulated. Oral administration of glycyrrhizin, a specific inhibitor of HMGB-1, attenuated diabetes-induced upregulation of HMGB-1 expression, NF-κB activation and downregulation of occludin expression. Our findings provide evidence that in the diabetic retina, HMGB-1 possibly interacts with RAGE and activates ERK(1/2) and NF-κB to generate an inflammatory response and disrupt retinal vascular barrier.
Asunto(s)
Barrera Hematorretinal/metabolismo , Diabetes Mellitus Experimental/metabolismo , Retinopatía Diabética/metabolismo , Proteína HMGB1/fisiología , Transducción de Señal/fisiología , Animales , Antiinflamatorios/farmacología , Western Blotting , Diabetes Mellitus Experimental/patología , Retinopatía Diabética/patología , Impedancia Eléctrica , Endotelio Vascular/metabolismo , Ensayo de Inmunoadsorción Enzimática , Ácido Glicirrínico/farmacología , Proteína HMGB1/antagonistas & inhibidores , Inmunohistoquímica , Inmunoprecipitación , Molécula 1 de Adhesión Intercelular/metabolismo , Inyecciones Intravítreas , Sistema de Señalización de MAP Quinasas/fisiología , Masculino , FN-kappa B/metabolismo , Ocludina/metabolismo , Ratas , Ratas Sprague-Dawley , Receptor para Productos Finales de Glicación Avanzada , Receptores Inmunológicos/metabolismo , Receptor Toll-Like 2/metabolismoRESUMEN
ARTICLES DISCUSSED: Damsgaard, C., Lauridsen, H. Deep vascular imaging in the eye with flow-enhanced ultrasound. Journal of Visualized Experiments. (176), e62986 (2021). Macouzet-Romero, F. J., Ochoa-Máynez, G. A., Pérez-García, O., Pérez-Aragón, B. J., Lima-Gómez, V. Evaluation of capillary and other vessel contribution to macular perfusion density measured with optical coherence tomography angiography. Journal of Visualized Experiments. (180), e63033 (2022). Subirada, P. V. et al. Quantification of vascular parameters in whole mount retinas of mice with non-proliferative and proliferative retinopathies. Journal of Visualized Experiments. (181), e63126 (2022). Vaglienti, M. V., Subirada, P. V., Barcelona, P. F., Bonacci, G., Sanchez, M. C. Quantification of reactive oxygen species using 2',7'-dichlorofluorescein diacetate probe and flow-cytometry in Müller glial cells. Journal of Visualized Experiments. (183), e63337 (2022). Tomaszewsk, R., Rajpurohit, P., Cheng, M., Tawfik, A. Isolation of primary mouse retinal pigmented epithelium cells. Journal of Visualized Experiments. (189), e63543 (2022). Elmasry, K., Moustafa, M., Al-Shabrawey, M. Retinal explant of the adult mouse retina as an ex vivo model for studying retinal neurovascular diseases. Journal of Visualized Experiments. (190), e63966 (2022).
Asunto(s)
Enfermedades de la Retina , Vasos Retinianos , Animales , Ratones , Vasos Retinianos/diagnóstico por imagen , Retina/diagnóstico por imagen , Venas , Angiografía , Células EpendimoglialesRESUMEN
Diabetic retinopathy (DR) is a result of neurovacular insults from hyperglycemia in diabetes mellitus (DM), and it is one of the top causes of vision loss throughout the modern world. This review article explores the role endothelial to mesenchymal transition (EndMT) has on the pathogenesis of DR. EndMT contributes to the disruption of the blood-retinal barrier, vascular leakage, neovascularization, and fibrosis observed in DR. Risk factors and biomarkers associated with DR severity are discussed, highlighting the importance of early detection and targeted therapies. Current treatments primarily focus on anti-vascular endothelial growth factor (anti-VEGF) agents, corticosteroids, and laser photocoagulation. However, emerging therapeutic strategies aimed at inhibiting EndMT and its downstream effects show promise in preventing the development and progression of DR. Understanding the molecular and cellular mechanisms underlying EndMT in DR provides valuable insights into the disease process and offers potential options for the development of potential treatments.
RESUMEN
Bone Morphogenetic Protein 4 (BMP4) is a secreted growth factor of the Transforming Growth Factor beta (TGFß) superfamily. The goal of this study was to test whether BMP4 contributes to the pathogenesis of diabetic retinopathy (DR). Immunofluorescence of BMP4 and the vascular marker isolectin-B4 was conducted on retinal sections of diabetic and non-diabetic human and experimental mice. We used Akita mice as a model for type-1 diabetes. Proteins were extracted from the retina of postmortem human eyes and 6-month diabetic Akita mice and age-matched control. BMP4 levels were measured by Western blot (WB). Human retinal endothelial cells (HRECs) were used as an in vitro model. HRECs were treated with BMP4 (50 ng/mL) for 48 h. The levels of phospho-smad 1/5/9 and phospho-p38 were measured by WB. BMP4-treated and control HRECs were also immunostained with anti-Zo-1. We also used electric cell-substrate impedance sensing (ECIS) to calculate the transcellular electrical resistance (TER) under BMP4 treatment in the presence and absence of noggin (200 ng/mL), LDN193189 (200 nM), LDN212854 (200 nM) or inhibitors of vascular endothelial growth factor receptor 2 (VEGFR2; SU5416, 10 µM), p38 (SB202190, 10 µM), ERK (U0126, 10 µM) and ER stress (Phenylbutyric acid or PBA, 30 µmol/L). The impact of BMP4 on matrix metalloproteinases (MMP2 and MMP9) was also evaluated using specific ELISA kits. Immunofluorescence of human and mouse eyes showed increased BMP4 immunoreactivity, mainly localized in the retinal vessels of diabetic humans and mice compared to the control. Western blots of retinal proteins showed a significant increase in BMP4 expression in diabetic humans and mice compared to the control groups (p < 0.05). HRECs treated with BMP4 showed a marked increase in phospho-smad 1/5/9 (p = 0.039) and phospho-p38 (p = 0.013). Immunofluorescence of Zo-1 showed that BMP4-treated cells exhibited significant barrier disruption. ECIS also showed a marked decrease in TER of HRECs by BMP4 treatment compared to vehicle-treated HRECs (p < 0.001). Noggin, LDN193189, LDN212854, and inhibitors of p38 and VEGFR2 significantly mitigated the effects of BMP4 on the TER of HRECs. Our finding provides important insights regarding the role of BMP4 as a potential player in retinal endothelial cell dysfunction in diabetic retinopathy and could be a novel target to preserve the blood-retinal barrier during diabetes.