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BACKGROUND: Diabetic retinopathy (DR) is a leading cause of vision loss, with complex mechanisms. The study aimed to comprehensively explore vitreous humor of diabetic and non-diabetic individuals, paving the way for identifying the potential molecular mechanisms underlying DR. METHODS: Vitreous samples from type 2 diabetic and non-diabetic subjects, collected post-mortem, were analyzed using liquid chromatography-mass spectrometry. Pathway enrichment and gene ontology analyses were conducted to identify dysregulated pathways and characterize protein functions. RESULTS: Pathway analysis revealed dysregulation in multiple metabolic and signaling pathways associated with diabetes, including glycerolipid metabolism, histidine metabolism, and Wnt signaling. Gene ontology analysis identified proteins involved in inflammation, immune response dysregulation, and calcium signaling. Notably, proteins such as Inositol 1,4,5-trisphosphate receptor type 2 (ITPR2), Calcium homeostasis endoplasmic reticulum protein (CHERP), and Coronin-1A (CORO1A) were markedly upregulated in diabetic vitreous, implicating aberrant calcium signaling, inflammatory responses, and cytoskeletal reorganization in DR. CONCLUSIONS: Our study provides valuable insights into the intricate mechanisms underlying DR and highlights the significance of inflammation, immune dysregulation, and metabolic disturbances in disease progression. Identification of specific proteins as potential biomarkers underscores the multifactorial nature of DR. Future research in this area is vital for advancing therapeutic interventions and translating findings into clinical practice.
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BACKGROUND: Multiple Sclerosis (MS) is a debilitating neurological disease affecting the central nervous system and significantly impacting patients' quality of life. MS is known as an autoimmune disease affecting the white matter. The disease involves inflammation, demyelination, and neurodegeneration, causing irreversible disabilities. Current treatments for MS target the inflammatory phase, with limited effects on long-term disability. While neuronal damage significantly contributes to MS pathology, mechanisms of neurodegeneration are not well studied. METHODS: This study evaluated neurodegenerative changes in the retina during disease progression, using data collected from an experimental MS model (Experimental Autoimmune Encephalomyelitis, EAE). Utilizing Hematoxylin and Eosin-stained retinal sections and assessment using Optical Coherence Tomography (OCT), the study investigated the neurodegenerative changes, such as loss of cells in the retinal ganglion cell layer (GCL) and retinal thinning in the retina of the EAE model and the control groups. RESULTS: Our results showed a significant reduction in the number of cells in the GCL of the EAE retina at two different time points studied, suggesting loss of neurons compared to the control group. Thickness measurements showed a reduction in the total retina and inner retinal layer thicknesses in the EAE retina compared to the controls. Our results indicate evidence of neurodegenerative changes in the retina of the experimental model of MS. No significant differences were observed between the percent losses of cells between the two time points studied. The pattern of cell loss suggests that neurodegeneration occurs at an earlier stage of disease progression. CONCLUSIONS: Overall, the retina is an excellent model to investigate neurodegeneration in MS, and possibly, loss of cells in the GCL could be used as an early indicator of neurodegeneration in MS and to identify novel therapeutic agents to treat the disease.
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Encefalomielitis Autoinmune Experimental , Esclerosis Múltiple , Células Ganglionares de la Retina , Tomografía de Coherencia Óptica , Células Ganglionares de la Retina/patología , Encefalomielitis Autoinmune Experimental/patología , Animales , Femenino , Esclerosis Múltiple/patología , Esclerosis Múltiple/diagnóstico por imagen , Modelos Animales de Enfermedad , Degeneración Nerviosa/patología , Progresión de la Enfermedad , Retina/patología , Retina/diagnóstico por imagenRESUMEN
Diabetes Mellitus presents a formidable challenge as one of the most prevalent and complex chronic diseases, exerting significant strain on both patients and the world economy. It is recognized as a common comorbidity among severely ill individuals, often leading to a myriad of micro- and macro-vascular complications. Despite extensive research dissecting the pathophysiology and molecular mechanisms underlying vascular complications of diabetes, relatively little attention has been paid to potential lung-related complications. This review aims to illuminate the impact of diabetes on prevalent respiratory diseases, including chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome (ARDS), idiopathic pulmonary fibrosis (IPF), tuberculosis (TB), pneumonia infections, and asthma, and compare the vascular complications with other vascular beds. Additionally, we explore the primary mechanistic pathways contributing to these complications, such as the expression modulation of blood-tissue-barrier proteins, resulting in increased paracellular and transcellular permeability, and compromised immune responses rendering diabetes patients more susceptible to infections. The activation of inflammatory pathways leading to cellular injury and hastening the onset of these respiratory complications is also discussed.
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Disruptions in polyamine metabolism have been identified as contributing factors to various central nervous system disorders. Our laboratory has previously highlighted the crucial role of polyamine oxidation in retinal disease models, specifically noting elevated levels of spermine oxidase (SMOX) in inner retinal neurons. Our prior research demonstrated that inhibiting SMOX with MDL 72527 protected against vascular injury and microglial activation induced by hyperoxia in the retina. However, the effects of SMOX inhibition on retinal neovascularization and vascular permeability, along with the underlying molecular mechanisms of vascular protection, remain incompletely understood. In this study, we utilized the oxygen-induced retinopathy (OIR) model to explore the impact of SMOX inhibition on retinal neovascularization, vascular permeability, and the molecular mechanisms underlying MDL 72527-mediated vasoprotection in the OIR retina. Our findings indicate that inhibiting SMOX with MDL 72527 mitigated vaso-obliteration and neovascularization in the OIR retina. Additionally, it reduced OIR-induced vascular permeability and Claudin-5 expression, suppressed acrolein-conjugated protein levels, and downregulated P38/ERK1/2/STAT3 signaling. Furthermore, our results revealed that treatment with BSA-Acrolein conjugates significantly decreased the viability of human retinal endothelial cells (HRECs) and activated P38 signaling. These observations contribute valuable insights into the potential therapeutic benefits of SMOX inhibition by MDL 72527 in ischemic retinopathy.
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Cryptococcus neoformans, an opportunistic fungal pathogen, primarily infects immunodeficient patients frequently causing cryptococcal meningoencephalitis (CM). Increased intracranial pressure (ICP) is a serious complication responsible for increased morbidity and mortality in CM patients. Non-invasive pharmacological agents that mitigate ICP could be beneficial in treating CM patients. The objective of the study was to investigate the efficacy of acetazolamide (AZA), candesartan (CAN), and triciribine (TCBN), in combination with the antifungal fluconazole, on C. neoformans-induced endothelial, brain, and lung injury in an experimental mouse model of CM. Our study shows that C. neoformans increases the expression of brain endothelial cell (BEC) junction proteins Claudin-5 (Cldn5) and VE-Cadherin to induce pathological cell-barrier remodeling and gap formation associated with increased Akt and p38 MAPK activation. All three agents inhibited C. neoformans-induced endothelial gap formation, only CAN and TCBN significantly reduced C. neoformans-induced Cldn5 expression, and only TCBN was effective in inhibiting Akt and p38MAPK. Interestingly, although C. neoformans did not cause brain or lung edema in mice, it induced lung and brain injuries, which were significantly reversed by AZA, CAN, or TCBN. Our study provides novel insights into the direct effects of C. neoformans on BECs in vitro, and the potential benefits of using AZA, CAN, or TCBN in the management of CM patients.
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Cryptococcus neoformans , Meningitis Criptocócica , Meningoencefalitis , Humanos , Animales , Ratones , Fluconazol/farmacología , Fluconazol/uso terapéutico , Meningitis Criptocócica/tratamiento farmacológico , Meningitis Criptocócica/microbiología , Acetazolamida/uso terapéutico , Proteínas Proto-Oncogénicas c-akt , Antifúngicos/farmacología , Antifúngicos/uso terapéutico , Meningoencefalitis/tratamiento farmacológico , Meningoencefalitis/microbiología , Meningoencefalitis/patologíaRESUMEN
Compromised blood-retinal barrier (BRB) integrity is a significant factor in ocular diseases like uveitis and retinopathies, leading to pathological vascular permeability and retinal edema. Adherens and tight junction (AJ and TJ) dysregulation due to retinal inflammation plays a pivotal role in BRB disruption. We investigated the potential of ICG001, which inhibits ß-catenin-mediated transcription, in stabilizing cell junctions and preventing BRB leakage. In vitro studies using human retinal endothelial cells (HRECs) showed that ICG001 treatment improved ß-Catenin distribution within AJs post lipopolysaccharide (LPS) treatment and enhanced monolayer barrier resistance. The in vivo experiments involved a mouse model of LPS-induced ocular inflammation. LPS treatment resulted in increased albumin leakage from retinal vessels, elevated vascular endothelial growth factor (VEGF) and Plasmalemmal Vesicle-Associated Protein (PLVAP) expression, as well as microglia and macroglia activation. ICG001 treatment (i.p.) effectively mitigated albumin leakage, reduced VEGF and PLVAP expression, and reduced the number of activated microglia/macrophages. Furthermore, ICG001 treatment suppressed the surge in inflammatory cytokine synthesis induced by LPS. These findings highlight the potential of interventions targeting ß-Catenin to enhance cell junction stability and improve compromised barrier integrity in various ocular inflammatory diseases, offering hope for better management and treatment options.
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We previously found that global deletion of the mitochondrial enzyme arginase 2 (A2) limits optic nerve crush (ONC)-induced neuronal death. Herein, we examined the cell-specific role of A2 in this pathology by studies using wild type (WT), neuronal-specific calbindin 2 A2 KO (Calb2cre/+ A2 f/f), myeloid-specific A2 KO (LysMcre/+ A2f/f), endothelial-specific A2 KO (Cdh5cre/+ A2f/f), and floxed controls. We also examined the impact of A2 overexpression on mitochondrial function in retinal neuronal R28 cells. Immunolabeling showed increased A2 expression in ganglion cell layer (GCL) neurons of WT mice within 6 h-post injury and inner retinal neurons after 7 days. Calb2 A2 KO mice showed improved neuronal survival, decreased TUNEL-positive neurons, and improved retinal function compared to floxed littermates. Neuronal loss was unchanged by A2 deletion in myeloid or endothelial cells. We also found increased expression of neurotrophins (BDNF, FGF2) and improved survival signaling (pAKT, pERK1/2) in Calb2 A2 KO retinas within 24-hour post-ONC along with suppression of inflammatory mediators (IL1ß, TNFα, IL6, and iNOS) and apoptotic markers (cleavage of caspase3 and PARP). ONC increased GFAP and Iba1 immunostaining in floxed controls, and Calb2 A2 KO dampened this effect. Overexpression of A2 in R28 cells increased Drp1 expression, and decreased mitochondrial respiration, whereas ABH-induced inhibition of A2 decreased Drp1 expression and improved mitochondrial respiration. Finally, A2 overexpression or excitotoxic treatment with glutamate significantly impaired mitochondrial function in R28 cells as shown by significant reductions in basal respiration, maximal respiration, and ATP production. Further, glutamate treatment of A2 overexpressing cells did not induce further deterioration in their mitochondrial function, indicating that A2 overexpression or glutamate insult induce comparable alterations in mitochondrial function. Our data indicate that neuronal A2 expression is neurotoxic after injury, and A2 deletion in Calb2 expressing neurons limits ONC-induced retinal neurodegeneration and improves visual function.
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Arginasa , Traumatismos del Nervio Óptico , Animales , Ratones , Apoptosis , Arginasa/genética , Arginasa/metabolismo , Calbindina 2 , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Glutamatos , Compresión Nerviosa , Nervio Óptico/metabolismo , Traumatismos del Nervio Óptico/metabolismoRESUMEN
Proliferative retinopathies are the leading cause of irreversible blindness in all ages, and there is a critical need to identify novel therapies. We investigated the impact of triciribine (TCBN), a tricyclic nucleoside analog and a weak Akt inhibitor, on retinal neurovascular injury, vascular permeability, and inflammation in oxygen-induced retinopathy (OIR). Post-natal day 7 (P7) mouse pups were subjected to OIR, and treated (i.p.) with TCBN or vehicle from P14-P16 and compared with age-matched, normoxic, vehicle or TCBN-treated controls. P17 retinas were processed for flat mounts, immunostaining, Western blotting, and qRT-PCR studies. Fluorescein angiography, electroretinography, and spectral domain optical coherence tomography were performed on days P21, P26, and P30, respectively. TCBN treatment significantly reduced pathological neovascularization, vaso-obliteration, and inflammation marked by reduced TNFα, IL6, MCP-1, Iba1, and F4/80 (macrophage/microglia markers) expression compared to the vehicle-treated OIR mouse retinas. Pathological expression of VEGF (vascular endothelial growth factor), and claudin-5 compromised the blood-retinal barrier integrity in the OIR retinas correlating with increased vascular permeability and neovascular tuft formation, which were blunted by TCBN treatment. Of note, there were no changes in the retinal architecture or retinal cell function in response to TCBN in the normoxia or OIR mice. We conclude that TCBN protects against pathological neovascularization, restores blood-retinal barrier homeostasis, and reduces retinal inflammation without adversely affecting the retinal structure and neuronal function in a mouse model of OIR. Our data suggest that TCBN may provide a novel therapeutic option for proliferative retinopathy.
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Enfermedades de la Retina , Neovascularización Retiniana , Vitreorretinopatía Proliferativa , Animales , Ratones , Neovascularización Retiniana/patología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Permeabilidad Capilar , Animales Recién Nacidos , Neovascularización Patológica , Oxígeno/efectos adversos , Inflamación/complicaciones , Modelos Animales de Enfermedad , Ratones Endogámicos C57BLRESUMEN
Multiple Sclerosis (MS) is a highly disabling neurological disease characterized by inflammation, neuronal damage, and demyelination. Vision impairment is one of the major clinical features of MS. Previous studies from our lab have shown that MDL 72527, a pharmacological inhibitor of spermine oxidase (SMOX), is protective against neurodegeneration and inflammation in the models of diabetic retinopathy and excitotoxicity. In the present study, utilizing the experimental autoimmune encephalomyelitis (EAE) model of MS, we determined the impact of SMOX blockade on retinal neurodegeneration and optic nerve inflammation. The increased expression of SMOX observed in EAE retinas was associated with a significant loss of retinal ganglion cells, degeneration of synaptic contacts, and reduced visual acuity. MDL 72527-treated mice exhibited markedly reduced motor deficits, improved neuronal survival, the preservation of synapses, and improved visual acuity compared to the vehicle-treated group. The EAE-induced increase in macrophage/microglia was markedly reduced by SMOX inhibition. Upregulated acrolein conjugates in the EAE retina were decreased through MDL 72527 treatment. Mechanistically, the EAE-induced ERK-STAT3 signaling was blunted by SMOX inhibition. In conclusion, our studies demonstrate the potential benefits of targeting SMOX to treat MS-mediated neuroinflammation and vision loss.
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Encefalomielitis Autoinmune Experimental , Esclerosis Múltiple , Neuritis Óptica , Animales , Ratones , Células Ganglionares de la Retina , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/complicaciones , Neuritis Óptica/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/inducido químicamente , Inflamación/tratamiento farmacológico , Inflamación/complicaciones , Nervio Óptico , Agudeza Visual , Modelos TeóricosRESUMEN
Current therapies for treatment of proliferative retinopathy focus on retinal neovascularization (RNV) during advanced disease and can trigger adverse side-effects. Here, we have tested a new strategy for limiting neurovascular injury and promoting repair during early-stage disease. We have recently shown that treatment with a stable, pegylated drug form of the ureohydrolase enzyme arginase 1 (A1) provides neuroprotection in acute models of ischemia/reperfusion injury, optic nerve crush, and ischemic stroke. Now, we have determined the effects of this treatment on RNV, vascular repair, and retinal function in the mouse oxygen-induced retinopathy (OIR) model of retinopathy of prematurity (ROP). Our studies in the OIR model show that treatment with pegylated A1 (PEG-A1), inhibits pathological RNV, promotes angiogenic repair, and improves retinal function by a mechanism involving decreased expression of TNF, iNOS, and VEGF and increased expression of FGF2 and A1. We further show that A1 is expressed in myeloid cells and areas of RNV in retinal sections from mice with OIR and human diabetic retinopathy (DR) patients and in blood samples from ROP patients. Moreover, studies using knockout mice with hemizygous deletion of A1 show worsened RNV and retinal injury, supporting the protective role of A1 in limiting the OIR-induced pathology. Collectively, A1 is critically involved in reparative angiogenesis and neuroprotection in OIR. Pegylated A1 may offer a novel therapy for limiting retinal injury and promoting repair during proliferative retinopathy.
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Neovascularización Retiniana , Retinopatía de la Prematuridad , Animales , Arginasa/genética , Arginasa/metabolismo , Modelos Animales de Enfermedad , Humanos , Recién Nacido , Ratones , Ratones Endogámicos C57BL , Neovascularización Patológica , Oxígeno , Polietilenglicoles/uso terapéutico , Neovascularización Retiniana/patología , Retinopatía de la Prematuridad/tratamiento farmacológico , Retinopatía de la Prematuridad/metabolismo , Retinopatía de la Prematuridad/patologíaRESUMEN
The multi-gene claudin (CLDN) family of tight junction proteins have isoform-specific roles in blood-tissue barrier regulation. CLDN17, a putative anion pore-forming CLDN based on its structural characterization, is assumed to regulate anion balance across the blood-tissue barriers. However, our knowledge about CLDN17 in physiology and pathology is limited. The current study investigated how Cldn17 deficiency in mice affects blood electrolytes and kidney structure. Cldn17-/- mice revealed no breeding abnormalities, but the newborn pups exhibited delayed growth. Adult Cldn17-/- mice displayed electrolyte imbalance, oxidative stress, and injury to the kidneys. Ingenuity pathway analysis followed by RNA-sequencing revealed hyperactivation of signaling pathways and downregulation of SOD1 expression in kidneys associated with inflammation and reactive oxygen species generation, demonstrating the importance of Cldn17 in the maintenance of electrolytes and reactive oxygen species across the blood-tissue barrier.
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Claudinas , Riñón , Estrés Oxidativo , Equilibrio Hidroelectrolítico , Animales , Aniones/metabolismo , Claudinas/genética , Claudinas/metabolismo , Riñón/fisiopatología , Ratones , Ratones Noqueados , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Polyamine oxidation plays a major role in neurodegenerative diseases. Previous studies from our laboratory demonstrated that spermine oxidase (SMOX, a member of the polyamine oxidase family) inhibition using MDL 72527 reduced neurodegeneration in models of retinal excitotoxicity and diabetic retinopathy. However, the mechanisms behind the neuroprotection offered by SMOX inhibition are not completely studied. Utilizing the experimental model of retinal excitotoxicity, the present study determined the impact of SMOX blockade in retinal neuroinflammation. Our results demonstrated upregulation in the number of cells positive for Iba-1 (ionized calcium-binding adaptor molecule 1), CD (Cluster Differentiation) 68, and CD16/32 in excitotoxicity-induced retinas, while MDL 72527 treatment reduced these changes, along with increases in the number of cells positive for Arginase1 and CD206. When retinal excitotoxicity upregulated several pro-inflammatory genes, MDL 72527 treatment reduced many of them and increased anti-inflammatory genes. Furthermore, SMOX inhibition upregulated antioxidant signaling (indicated by elevated Nrf2 and HO-1 levels) and reduced protein-conjugated acrolein in excitotoxic retinas. In vitro studies using C8-B4 cells showed changes in cellular morphology and increased reactive oxygen species formation in response to acrolein (a product of SMOX activity) treatment. Overall, our findings indicate that the inhibition SMOX pathway reduced neuroinflammation and upregulated antioxidant signaling in the retina.
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Enfermedades Neuroinflamatorias/tratamiento farmacológico , Enfermedades Neuroinflamatorias/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/antagonistas & inhibidores , Retina/diagnóstico por imagen , Retina/metabolismo , Animales , Antioxidantes/metabolismo , Retinopatía Diabética/tratamiento farmacológico , Retinopatía Diabética/metabolismo , Modelos Animales de Enfermedad , Masculino , Ratones , Ratones Endogámicos C57BL , Enfermedades Neurodegenerativas/tratamiento farmacológico , Enfermedades Neurodegenerativas/metabolismo , Oxidación-Reducción/efectos de los fármacos , Putrescina/análogos & derivados , Putrescina/farmacología , Transducción de Señal/efectos de los fármacos , Poliamino OxidasaRESUMEN
Arginase 1 (A1) is the enzyme that hydrolyzes the amino acid, L-arginine, to ornithine and urea. We have previously shown that A1 deletion worsens retinal ischemic injury, suggesting a protective role of A1. In this translational study, we aimed to study the utility of systemic pegylated A1 (PEG-A1, recombinant human arginase linked to polyethylene glycol) treatment in mouse models of acute retinal and brain injury. Cohorts of WT mice were subjected to retinal ischemia-reperfusion (IR) injury, traumatic optic neuropathy (TON) or brain cerebral ischemia via middle cerebral artery occlusion (MCAO) and treated with intraperitoneal injections of PEG-A1 or vehicle (PEG only). Drug penetration into retina and brain tissues was measured by western blotting and immunolabeling for PEG. Neuroprotection was measured in a blinded fashion by quantitation of NeuN (neuronal marker) immunolabeling of retina flat-mounts and brain infarct area using triphenyl tetrazolium chloride (TTC) staining. Furthermore, ex vivo retina explants and in vitro retina neuron cultures were subjected to oxygen-glucose deprivation (OGD) followed by reoxygenation (R) and treated with PEG-A1. PEG-A1 given systemically did not cross the intact blood-retina/brain barriers in sham controls but reached the retina and brain after injury. PEG-A1 provided neuroprotection after retinal IR injury, TON and cerebral ischemia. PEG-A1 treatment was also neuroprotective in retina explants subjected to OGD/R but did not improve survival in retinal neuronal cultures exposed to OGD/R. In summary, systemic PEG-A1 administration is neuroprotective and provides an excellent route to deliver the drug to the retina and the brain after acute injury.
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Arginasa/uso terapéutico , Lesiones Encefálicas/tratamiento farmacológico , Fármacos Neuroprotectores/uso terapéutico , Retina/lesiones , Animales , Arginasa/farmacocinética , Barrera Hematoencefálica , Barrera Hematorretinal , Encéfalo/metabolismo , Isquemia Encefálica/tratamiento farmacológico , Supervivencia Celular/efectos de los fármacos , Humanos , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fármacos Neuroprotectores/farmacocinética , Traumatismos del Nervio Óptico/tratamiento farmacológico , Polietilenglicoles , Proteínas Recombinantes/uso terapéutico , Daño por Reperfusión/prevención & control , Retina/metabolismoRESUMEN
Cell junctions maintain the blood-tissue barriers to preserve vascular and tissue integrity. Viral infections reportedly modulate cell-cell junctions to facilitate their invasion. However, information on the effect of COVID-19 infection on the gene expression of cell junction and cytoskeletal proteins is limited. Using the Gene Expression Omnibus and Reactome databases, we analyzed the data on human lung A549, NHBE, and Calu-3 cells for the expression changes in cell junction and cytoskeletal proteins by SARS-CoV-2 (CoV-2) infection. The analysis revealed changes in 3,660 genes in A549, 100 genes in NHBE, and 592 genes in Calu-3 cells with CoV-2 infection. Interestingly, EGOT (9.8-, 3- and 8.3-fold; p < .05) and CSF3 (4.3-, 33- and 56.3-fold; p < .05) were the only two genes significantly elevated in all three cell lines (A549, NHBE and Calu-3, respectively). On the other hand, 39 genes related to cell junctions and cytoskeleton were modulated in lung cells, with DLL1 demonstrating alterations in all cells. Alterations were also seen in several miRNAs associated with the cell junction and cytoskeleton genes modulated in the analysis. Further, matrix metalloproteinases involved in disease pathologies, including MMP-3, -9, and -12 demonstrated elevated expression on CoV-2 infection (p < .05). The study findings emphasize the integral role of cell junction and cytoskeletal genes in COVID-19, suggesting their therapeutic potential. Our analysis also identified a distinct EGOT gene that has not been previously implicated in COVID-19. Further studies on these newly identified genes and miRNAs could lead to advances in the pathogenesis and therapeutics of COVID-19.
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COVID-19 , MicroARNs , Biología Computacional , Proteínas del Citoesqueleto/metabolismo , Células Epiteliales/metabolismo , Humanos , Uniones Intercelulares , Pulmón/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , SARS-CoV-2RESUMEN
Visual dysfunction resulting from optic neuritis (ON) is one of the most common clinical manifestations of multiple sclerosis (MS), characterized by loss of retinal ganglion cells, thinning of the nerve fiber layer, and inflammation to the optic nerve. Current treatments available for ON or MS are only partially effective, specifically target the inflammatory phase, and have limited effects on long-term disability. Fingolimod (FTY) is an FDA-approved immunomodulatory agent for MS therapy. The objective of the current study was to evaluate the neuroprotective properties of FTY in the cellular model of ON-associated neuronal damage. R28 retinal neuronal cell damage was induced through treatment with tumor necrosis factor-α (TNFα). In our cell viability analysis, FTY treatment showed significantly reduced TNFα-induced neuronal death. Treatment with FTY attenuated the TNFα-induced changes in cell survival and cell stress signaling molecules. Furthermore, immunofluorescence studies performed using various markers indicated that FTY treatment protects the R28 cells against the TNFα-induced neurodegenerative changes by suppressing reactive oxygen species generation and promoting the expression of neuronal markers. In conclusion, our study suggests neuroprotective effects of FTY in an in vitro model of optic neuritis.
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Clorhidrato de Fingolimod/uso terapéutico , Fármacos Neuroprotectores/uso terapéutico , Neuritis Óptica/tratamiento farmacológico , Animales , Caspasa 3/metabolismo , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Clorhidrato de Fingolimod/farmacología , Dinámicas Mitocondriales , Proteínas Mitocondriales/metabolismo , Modelos Biológicos , Neuronas/efectos de los fármacos , Neuronas/patología , Fármacos Neuroprotectores/farmacología , Neuritis Óptica/metabolismo , Neuritis Óptica/patología , Ratas , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Estrés Fisiológico/efectos de los fármacos , Factor de Necrosis Tumoral alfa/toxicidad , Proteína bcl-X/metabolismoRESUMEN
The enormous library of pharmaceutical compounds presents endless research avenues. However, several factors limit the therapeutic potential of these drugs, such as drug resistance, stability, off-target toxicity, and inadequate delivery to the site of action. Extracellular vesicles (EVs) are lipid bilayer-delimited particles and are naturally released from cells. Growing evidence shows that EVs have great potential to serve as effective drug carriers. Since EVs can not only transfer biological information, but also effectively deliver hydrophobic drugs into cells, the application of EVs as a novel drug delivery system has attracted considerable scientific interest. Recently, EVs loaded with siRNA, miRNA, mRNA, CRISPR/Cas9, proteins, or therapeutic drugs show improved delivery efficiency and drug effect. In this review, we summarize the methods used for the cargo loading into EVs, including siRNA, miRNA, mRNA, CRISPR/Cas9, proteins, and therapeutic drugs. Furthermore, we also include the recent advance in engineered EVs for drug delivery. Finally, both advantages and challenges of EVs as a new drug delivery system are discussed. Here, we encourage researchers to further develop convenient and reliable loading methods for the potential clinical applications of EVs as drug carriers in the future.
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Vision impairment due to optic neuritis (ON) is one of the major clinical presentations in Multiple Sclerosis (MS) and is characterized by inflammation and degeneration of the optic nerve and retina. Currently available treatments are only partially effective and have a limited impact on the neuroinflammatory pathology of the disease. A recent study from our laboratory highlighted the beneficial effect of arginase 2 (A2) deletion in suppressing retinal neurodegeneration and inflammation in an experimental model of MS. Utilizing the same model, the present study investigated the impact of A2 deficiency on MS-induced optic neuritis. Experimental autoimmune encephalomyelitis (EAE) was induced in wild-type (WT) and A2 knockout (A2-/-) mice. EAE-induced cellular infiltration, as well as activation of microglia and macrophages, were reduced in A2-/- optic nerves. Axonal degeneration and demyelination seen in EAE optic nerves were observed to be reduced with A2 deletion. Further, the lack of A2 significantly ameliorated astrogliosis induced by EAE. In conclusion, our findings demonstrate a critical involvement of arginase 2 in mediating neuroinflammation in optic neuritis and suggest the potential of A2 blockade as a targeted therapy for MS-induced optic neuritis.
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Arginasa/genética , Encefalomielitis Autoinmune Experimental/patología , Inflamación/patología , Neuritis Óptica/patología , Animales , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/genética , Inflamación/genética , Macrófagos/patología , Ratones , Ratones Noqueados , Microglía/patología , Nervio Óptico/patología , Neuritis Óptica/genéticaRESUMEN
The severe acute respiratory syndrome coronavirus 2 that causes coronavirus disease 2019 (COVID-19) binds to the angiotensin-converting enzyme 2 (ACE2) to gain cellular entry. Akt inhibitor triciribine (TCBN) has demonstrated promising results in promoting recovery from advanced-stage acute lung injury in preclinical studies. In the current study, we tested the direct effect of TCBN on ACE2 expression in human bronchial (H441) and lung alveolar (A549) epithelial cells. Treatment with TCBN resulted in the downregulation of both messenger RNA and protein levels of ACE2 in A549 cells. Since HMGB1 plays a vital role in the inflammatory response in COVID-19, and because hyperglycemia has been linked to increased COVID-19 infections, we determined if HMGB1 and hyperglycemia have any effect on ACE2 expression in lung epithelial cells and whether TCBN has any effect on reversing HMGB1- and hyperglycemia-induced ACE2 expression. We observed increased ACE2 expression with both HMGB1 and hyperglycemia treatment in A549 as well as H441 cells, which were blunted by TCBN treatment. Our findings from this study, combined with our previous reports on the potential benefits of TCBN in the treatment of acute lung injury, generate reasonable optimism on the potential utility of TCBN in the therapeutic management of patients with COVID-19.
Asunto(s)
Enzima Convertidora de Angiotensina 2/genética , Tratamiento Farmacológico de COVID-19 , Proteína HMGB1/genética , Proteínas Proto-Oncogénicas c-akt/genética , Células A549 , Bronquios/metabolismo , Bronquios/patología , Bronquios/virología , COVID-19/genética , COVID-19/patología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/virología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Pulmón/efectos de los fármacos , Pulmón/patología , ARN Viral/genética , Ribonucleósidos/administración & dosificación , SARS-CoV-2/genética , SARS-CoV-2/patogenicidadRESUMEN
Ocular diseases such as diabetic retinopathy (DR) and uveitis are associated with injury to the blood-retinal barrier (BRB). Whereas high glucose (HG) and advanced glycation end products (AGE) contribute to DR, bacterial infections causing uveitis are triggered by endotoxins such as lipopolysaccharide (LPS). It is unclear how HG, AGE, and LPS affect human retinal endothelial cell (HREC) junctions. Moreover, tumor necrosis factor-α (TNFα) is elevated in both DR and ocular infections. In the current study, we determined the direct effects of HG, AGE, TNFα, and LPS on the expression and intracellular distribution of claudin-5, VE-cadherin, and ß-catenin in HRECs and how these mediators affect Akt and P38 MAP kinase that have been implicated in ocular pathologies. In our results, whereas HG, AGE, and TNFα activated both Akt and P38 MAPK, LPS treatment suppressed Akt but increased P38 MAPK phosphorylation. Furthermore, while treatment with AGE and HG increased cell-junction protein expression in HRECs, LPS elicited a paradoxical effect. By contrast, when HG treatment increased HREC-barrier resistance, AGE and LPS stimulation compromised it, and TNFα had no effect. Together, our results demonstrated the differential effects of the mediators of diabetes and infection on HREC-barrier modulation leading to BRB injury.
RESUMEN
Epithelial and endothelial cell-cell contacts are established and maintained by several intercellular junctional complexes. These structurally and biochemically differentiated regions on the plasma membrane primarily include tight junctions (TJs), and anchoring junctions. While the adherens junctions (AJs) provide essential adhesive and mechanical properties, TJs hold the cells together and form a near leak-proof intercellular seal by the fusion of adjacent cell membranes. AJs and TJs play essential roles in vascular permeability. Considering their involvement in several key cellular functions such as barrier formation, proliferation, migration, survival, and differentiation, further research is warranted on the composition and signaling pathways regulating cell-cell junctions to develop novel therapeutics for diseases such as organ injuries. The current review article presents our current state of knowledge on various cell-cell junctions, their molecular composition, and mechanisms regulating their expression and function in endothelial and epithelial cells.