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Diabetic nephropathy (DN) is reported as one of the most serious microvascular diabetic complications and the trigger of end-stage renal disease (ESRD), underscoring the concern of any therapeutic intervention directed at ameliorating the development and progression of DN. The current study explored the renoprotective impact of montelukast (Mon) against streptozotocin (STZ)-induced DN in rats compared to a standard anti-hyperglycemic insulin (Ins) treatment. Diabetes was induced by a single dose of STZ (55 mg/kg). Diabetic rats were treated with Mon (10 and 20 mg/kg, oral gavage) for eight weeks. Mon administration for 8 weeks after induction of diabetes conferred significant dose-dependent renoprotection, independent of blood glucose levels (unlike Ins), as evidenced by the improvement in serum creatinine, and blood urea nitrogen (BUN), and ameliorated STZ-induced renal necrotic, inflammatory alterations, and renal fibrosis. Additionally, Mon treatment in diabetic rats significantly restored redox hemostasis as evidenced by malondialdehyde (MDA) and total antioxidant capacity (TAC) levels; significantly reduced the renal expression of high mobility group box (HMGB) 1, toll-like receptor (TLR) 4, nuclear factor kappa B (NF-κB) (in the nucleus), NOD-like receptor family pyrin domain containing (NLRP) 3, and interleukin (IL)-1ß. Moreover, Mon administration ameliorated the dysregulation in autophagy as evidenced by p62 and microtubule-associated protein 1A/1B-light chain 3 (LC3)-II levels. In conclusion, the renoprotective effect of Mon is potentially associated with its modulatory effect on inflammatory cytokines, antioxidant properties, and autophagy.
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Acetatos , Ciclopropanos , Diabetes Mellitus Experimental , Nefropatias Diabéticas , Proteína HMGB1 , Quinolinas , Sulfetos , Animais , Ratos , Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Nefropatias Diabéticas/tratamento farmacológico , NF-kappa B , Estreptozocina/farmacologia , Antioxidantes/farmacologia , Diabetes Mellitus Experimental/tratamento farmacológico , Receptor 4 Toll-Like , InsulinaRESUMO
AIMS: Montelukast, a cysteinyl leukotriene receptor (CysLTR)1 antagonist, is emerging as an attractive strategy to curtail diabetic complications; however, its role in aortic and testicular tissues is unknown. This study investigated the effect of CysLTR1 antagonism by montelukast on toll-like receptor (TLR)4 and nuclear factor kappa B (NF-κB) pathways in diabetes-induced aortic and testicular injury. METHODS: Adult male Sprague-Dawley rats were made diabetic with Streptozotocin (STZ, 55 mg/kg). Following this, Streptozotocin-induced diabetic (SD) rats were administered montelukast (10 and 20 mg/kg, orally) for 8 weeks. Blood glucose, serum malondialdehyde (MDA), inflammatory markers, and histopathology were evaluated. RESULTS: Montelukast showed protection against diabetic complications, as evidenced by the ameliorative effect against STZ-induced alterations in oxidative stress as indicated by serum MDA levels. Moreover, montelukast conferred a significant decrease in the aortic and testicular levels of CysLTR1, TLR4, and NF-κB with a subsequent decrease in the levels of NOD-like receptor family pyrin domain containing (NLRP)3, caspase 1, interleukin (IL)-1ß, IL-6, monocyte chemoattractant protein (MCP)-1, and tumor necrosis factor (TNF)-α. Additionally, administration of montelukast resulted in autophagy stimulation, as shown by decreased p62/Sequestosome (SQSTM)1 levels. Finally, montelukast protection resulted in normal thickness of whole aortic wall, regular tunica (t.) intima, mild vacuolation of smooth muscle fibers in aorta, increased size of seminiferous tubules, and increased spermatogenesis in testis as demonstrated by histopathology. CONCLUSIONS: The protective effect of montelukast against diabetes-induced aortic and testicular injury is due to its antioxidant, anti-inflammatory, and autophagy stimulation characteristics.
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Complicações do Diabetes , Diabetes Mellitus , Ratos , Masculino , Animais , NF-kappa B/metabolismo , Ratos Sprague-Dawley , Estreptozocina , Fator de Necrose Tumoral alfa/metabolismo , Inflamação/tratamento farmacológico , Complicações do Diabetes/tratamento farmacológico , Aorta/metabolismoRESUMO
Type-2 diabetes (T2DM) is known to be highly associated with increased risk for vascular complications including peripheral arterial diseases (PAD). Critical limb ischemia (CLI) is the most advanced stage of PAD. Current therapeutic options for diabetic patients experiencing vascular complications are limited to surgical revascularization with no effective pharmacotherapy available for clinical settings. This study is dedicated to evaluate the angiogenic potential of candesartan an angiotensin-II receptor blocker in an experimental model of vascular complications associating T2DM. T2DM was induced in rats through feeding with high fat diet for 6â¯weeks, followed by injection with streptozotocin (STZ, 30â¯mg/kg; i.p). After establishment of T2DM, unilateral CLI was induced through the ligation and excision of superficial femoral artery. Candesartan treatment (10 or 30â¯mg/kg; orally) was initiated one day post CLI and thereafter once daily for up to 14â¯days. T2DM rats that underwent CLI demonstrated impaired angiogenic signaling, increased inflammation and apoptosis in gastrocnemius muscle (GC). Candesartan reversed ischemic insult in T2DM rats subjected to unilateral CLI and induced reparative angiogenesis that was evident by increase in p-PI3K/PI3K, p-Akt/Akt, p-eNOS/eNOS, p-VEGFR2/VEGFR2 ratios, and VEGF levels. Candesartan treatment also increased levels of HO-1; while decreased caspase-3 apoptotic marker and levels of inflammatory markers; NF-κB and TNF-α, all of which were accompanied by preserved histological manifestations of GC muscles. Candesartan was able to combat limb ischemia under diabetic conditions which could pave the way for its therapeutic utility for diabetic patients experiencing vascular complications in clinical setting.
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Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , Ratos , Animais , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Diabetes Mellitus Experimental/metabolismo , Isquemia/tratamento farmacológico , Isquemia/metabolismo , Transdução de Sinais , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/tratamento farmacológico , Neovascularização FisiológicaRESUMO
Peripheral arterial diseases (PAD) had a great attention owing to devastating consequences of disability and cardiovascular morbidity and mortality. Yet, current therapeutic options are limited to surgical revascularization with no effective pharmacotherapy available. Excessive activity of Rho-associated coiled-coil protein kinase (ROCK) is implicated with several vascular diseases, rendering ROCK inhibition as a potential therapeutic strategy for patients suffering vascular disorders. AIM: The current study was dedicated to investigating the vascular protective potential of Fasudil, a ROCK inhibitor, on an experimentally induced unilateral critical limb ischemia (CLI) model in mice and demonstrated the possible underlying mechanisms. METHODS: Unilateral CLI was induced by ligation and excision of femoral artery followed by daily i.p. injection of Fasudil (10 mg/kg or 25 mg/kg) up to two weeks post-surgery. KEY FINDINGS: Mice underwent CLI showed decreased antioxidant capacity and increased inflammatory signal, evident by elevation of ERK1/2 in both serum and GC muscles that coincided with increases in VEGFA, HIF-1α and CD34+ cells of GC muscles. CLI resulted in structural damage of GC muscle fibers, with marked apoptosis, declined proliferation and deteriorated peripheral limb function. Treatment with Fasudil restored antioxidant capacity and attenuated VEGFA, HIF-1α, CD34+ cells and inflammatory markers in ischemic limbs. Furthermore, Fasudil preserved histological integrity of ischemic GC muscles, with amelioration of apoptosis, preserved proliferation rate and improvement in peripheral limb function. SIGNIFICANCE: Fasudil could protect against experimentally induced unilateral CLI, in a dose-dependent manner, which could pave the way for future clinical application of Fasudil in patients suffering PAD.
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Isquemia Crônica Crítica de Membro , Quinases Associadas a rho , Animais , Camundongos , Quinases Associadas a rho/metabolismo , Antioxidantes/farmacologia , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/uso terapêutico , Transdução de Sinais , Isquemia/tratamento farmacológico , Modelos Animais de Doenças , Extremidade InferiorRESUMO
Mesenchymal stem cells (MSCs) are a promising therapy to improve vascular repair, yet their role in ischemic retinopathy is not fully understood. The aim of this study is to investigate the impact of modulating the neurotrophin receptor; p75NTR on the vascular protection of MSCs in an acute model of retinal ischemia/reperfusion (I/R). Wild type (WT) and p75NTR-/- mice were subjected to I/R injury by increasing intra-ocular pressure to 120 mmHg for 45 min, followed by perfusion. Murine GFP-labeled MSCs (100,000 cells/eye) were injected intravitreally 2 days post-I/R and vascular homing was assessed 1 week later. Acellular capillaries were counted using trypsin digest 10-days post-I/R. In vitro, MSC-p75NTR was modulated either genetically using siRNA or pharmacologically using the p75NTR modulator; LM11A-31, and conditioned media were co-cultured with human retinal endothelial cells (HREs) to examine the angiogenic response. Finally, visual function in mice undergoing retinal I/R and receiving LM11A-31 was assessed by visual-clue water-maze test. I/R significantly increased the number of acellular capillaries (3.2-Fold) in WT retinas, which was partially ameliorated in p75NTR-/- retinas. GFP-MSCs were successfully incorporated and engrafted into retinal vasculature 1 week post injection and normalized the number of acellular capillaries in p75NTR-/- retinas, yet ischemic WT retinas maintained a 2-Fold increase. Silencing p75NTR on GFP-MSCs coincided with a higher number of cells homing to the ischemic WT retinal vasculature and normalized the number of acellular capillaries when compared to ischemic WT retinas receiving scrambled-GFP-MSCs. In vitro, silencing p75NTR-MSCs enhanced their secretome, as evidenced by significant increases in SDF-1, VEGF and NGF release in MSCs conditioned medium; improved paracrine angiogenic response in HREs, where HREs showed enhanced migration (1.4-Fold) and tube formation (2-Fold) compared to controls. In parallel, modulating MSCs-p75NTR using LM11A-31 resulted in a similar improvement in MSCs secretome and the enhanced paracrine angiogenic potential of HREs. Further, intervention with LM11A-31 significantly mitigated the decline in visual acuity post retinal I/R injury. In conclusion, p75NTR modulation can potentiate the therapeutic potential of MSCs to harness vascular repair in ischemic retinopathy diseases.
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Células-Tronco Mesenquimais/citologia , Receptores de Fator de Crescimento Neural/genética , Traumatismo por Reperfusão/metabolismo , Vasos Retinianos/metabolismo , Animais , Capilares/metabolismo , Movimento Celular , Proliferação de Células , Técnicas de Cocultura , Meios de Cultivo Condicionados/química , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Endotélio/metabolismo , Deleção de Genes , Inativação Gênica , Proteínas de Fluorescência Verde/metabolismo , Humanos , Injeções Intravítreas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Patológica , Fator de Crescimento Neural/metabolismo , Proteínas do Tecido Nervoso/genética , Receptor de Fator de Crescimento Neural/metabolismo , Traumatismo por Reperfusão/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Mesenchymal stem cells are multipotent stem cells isolated from various tissue sources, including but not limited to bone marrow, adipose, umbilical cord, and Wharton Jelly. Although cell-mediated mechanisms have been reported, the therapeutic effect of MSCs is now recognized to be primarily mediated via paracrine effects through the secretion of bioactive molecules, known as the "secretome". The regenerative benefit of the secretome has been attributed to trophic factors and cytokines that play neuroprotective, anti-angiogenic/pro-angiogenic, anti-inflammatory, and immune-modulatory roles. The advancement of autologous MSCs therapy can be hindered when introduced back into a hostile/disease environment. Barriers include impaired endogenous MSCs function, limited post-transplantation cell viability, and altered immune-modulatory efficiency. Although secretome-based therapeutics have gained popularity, many translational hurdles, including the heterogeneity of MSCs, limited proliferation potential, and the complex nature of the secretome, have impeded the progress. This review will discuss the experimental and clinical impact of restoring the functional capabilities of MSCs prior to transplantation and the progress in secretome therapies involving extracellular vesicles. Modulation and utilization of MSCs-secretome are most likely to serve as an effective strategy for promoting their ultimate success as therapeutic modulators.
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Isquemia/terapia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Doenças Vasculares/terapia , Animais , Ensaios Clínicos como Assunto , Humanos , Isquemia/complicações , Doenças Vasculares/complicações , CicatrizaçãoRESUMO
AIMS/HYPOTHESIS: Breakdown of the inner blood-retinal barrier (BRB) is an early event in the pathogenesis of diabetic macular oedema, that eventually leads to vision loss. We have previously shown that diabetes causes an imbalance of nerve growth factor (NGF) isoforms resulting in accumulation of its precursor proNGF and upregulation of the p75 neurotrophin receptor (p75NTR), with consequent increases in the activation of Ras homologue gene family, member A (RhoA). We also showed that genetic deletion of p75NTR in diabetes preserved the BRB and prevented inflammatory mediators in retinas. This study aims to examine the therapeutic potential of LM11A-31, a small-molecule p75NTR modulator and proNGF antagonist, in preventing diabetes-induced BRB breakdown. The study also examined the role of p75NTR/RhoA downstream signalling in mediating cell permeability. METHODS: Male C57BL/6 J mice were rendered diabetic using streptozotocin injection. After 2 weeks of diabetes, mice received oral gavage of LM11A-31 (50 mg kg-1 day-1) or saline (NaCl 154 mmol/l) for an additional 4 weeks. BRB breakdown was assessed by extravasation of BSA-AlexaFluor-488. Direct effects of proNGF were examined in human retinal endothelial (HRE) cells in the presence or absence of LM11A-31 or the Rho kinase inhibitor Y-27632. RESULTS: Diabetes triggered BRB breakdown and caused significant increases in circulatory and retinal TNF-α and IL-1ß levels. These effects coincided with significant decreases in retinal NGF and increases in vascular endothelial growth factor and proNGF expression, as well as activation of RhoA. Interventional modulation of p75NTR activity through treatment of mouse models of diabetes with LM11A-31 significantly mitigated proNGF accumulation and preserved BRB integrity. In HRE cells, treatment with mutant proNGF (10 ng/ml) triggered increased cell permeability with marked reduction of expression of tight junction proteins, zona occludens-1 (ZO-1) and claudin-5, compared with control, independent of inflammatory mediators or cell death. Modulating p75NTR significantly inhibited proNGF-mediated RhoA activation, occludin phosphorylation (at serine 490) and cell permeability. ProNGF induced redistribution of ZO-1 in the cell wall and formation of F-actin stress fibres; these effects were mitigated by LM11A-31. CONCLUSIONS/INTERPRETATION: Targeting p75NTR signalling using LM11A-31, an orally bioavailable receptor modulator, may offer an effective, safe and non-invasive therapeutic strategy for treating macular oedema, a major cause of blindness in diabetes.
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Permeabilidade Capilar , Complicações do Diabetes/prevenção & controle , Retinopatia Diabética/metabolismo , Isoleucina/análogos & derivados , Morfolinas/uso terapêutico , Proteínas do Tecido Nervoso/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Glicemia/análise , Barreira Hematorretiniana , Peso Corporal , Células Endoteliais/metabolismo , Deleção de Genes , Humanos , Inflamação , Interleucina-1beta/metabolismo , Isoleucina/uso terapêutico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microcirculação , Receptor de Fator de Crescimento Neural/metabolismo , Retina/metabolismo , Retina/patologia , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo , Regulação para CimaRESUMO
Adipose-derived stem cells (ASCs) are multipotent mesenchymal progenitor cells that have functional and phenotypic overlap with pericytes lining microvessels in adipose tissue. The role of CD140b [platelet-derived growth factor receptor- ß (PDGFR-ß)], a constitutive marker expressed by ASCs, in the angiogenic behavior of human retinal endothelial cells (HREs) is not known. CD140b was knocked down in ASCs using targeted siRNA and lipofectamine transfection protocol. Both CD140b+ and CD140b- ASCs were tested for their proliferation (WST-1 reagent), adhesion (laminin-1 coated plates), and migration (wound-scratch assay). Angiogenic effect of CD140b+ and CD140b- ASCs on HREs was examined by co-culturing ASCs:HREs in 12:1 ratio for 6 days followed by visualization of vascular network by Isolectin B4 staining. The RayBio® Membrane-Based Antibody Array was used to assess differences in human cytokines released by CD140b+ or CD140b- ASCs. Knockdown of CD140b in ASCs resulted in a significant 50% decrease in proliferation rate, 25% decrease in adhesion ability to Laminin-1, and 50% decrease in migration rate, as compared to CD140b+ ASCs. Direct contact of ASCs expressing CD140b+ with HREs resulted in robust vascular network formation that was significantly reduced with using CD140b- ASCs. Of the 80 proteins tested, 45 proteins remained unchanged (>0.5-<1.5 fold), 6 proteins including IL-10 downregulated (<0.5 fold) and 29 proteins including IL-16 & TNF-ß were upregulated (>1.5 fold) in CD140b- ASCs compared to CD140b+ ASCs. Our data demonstrate a substantial role for CD140b in the intrinsic abilities of ASCs and their angiogenic influence on HREs. Future studies are needed to fully explore the signaling of CD140b in ASCs in vivo for retinal regeneration.
RESUMO
BACKGROUND: Early-stage diabetic retinopathy (DR) is characterized by neurovascular defects. In this study, we hypothesized that human adipose-derived stem cells (ASCs) positive for the pericyte marker CD140b, or their secreted paracrine factors, therapeutically rescue early-stage DR features in an Ins2Akita mouse model. METHODS: Ins2Akita mice at 24 weeks of age received intravitreal injections of CD140b-positive ASCs (1000 cells/1 µL) or 20× conditioned media from cytokine-primed ASCs (ASC-CM, 1 µL). Age-matched wildtype mice that received saline served as controls. Visual function experiments and histological analyses were performed 3 weeks post intravitreal injection. Biochemical and molecular analyses assessed the ASC-CM composition and its biological effects. RESULTS: Three weeks post-injection, Ins2Akita mice that received ASCs had ameliorated decreased b-wave amplitudes and vascular leakage but failed to improve visual acuity, whereas Ins2Akita mice that received ASC-CM demonstrated amelioration of all aforementioned visual deficits. The ASC-CM group demonstrated partial amelioration of retinal GFAP immunoreactivity and DR-related gene expression but the ASC group did not. While Ins2Akita mice that received ASCs exhibited occasional (1 in 8) hemorrhagic retinas, mice that received ASC-CM had no adverse complications. In vitro, ASC-CM protected against TNFα-induced retinal endothelial permeability as measured by transendothelial electrical resistance. Biochemical and molecular analyses demonstrated several anti-inflammatory proteins including TSG-6 being highly expressed in cytokine-primed ASC-CM. CONCLUSIONS: ASCs or their secreted factors mitigate retinal complications of diabetes in the Ins2Akita model. Further investigation is warranted to determine whether ASCs or their secreted factors are safe and effective therapeutic modalities long-term as current locally delivered therapies fail to effectively mitigate the progression of early-stage DR. Nonetheless, our study sheds new light on the therapeutic mechanisms of adult stem cells, with implications for assessing relative risks/benefits of experimental regenerative therapies for vision loss.
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Tecido Adiposo/citologia , Diabetes Mellitus Experimental/complicações , Retinopatia Diabética/terapia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Transplante de Células-Tronco Mesenquimais , Tecido Adiposo/metabolismo , Animais , Antígenos de Superfície/química , Antígenos de Superfície/uso terapêutico , Meios de Cultura Livres de Soro/química , Meios de Cultura Livres de Soro/farmacologia , Retinopatia Diabética/etiologia , Retinopatia Diabética/patologia , Humanos , Injeções Intravítreas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , TrombomodulinaRESUMO
Ischemic retinopathy is characterized by ischemia followed by retinal neovascularization (RNV) resulting in visual impairment. Given the role of neuron-secreted growth factors in regulating angiogenesis, we examined how genetic deletion of the neurotrophin receptor; p75NTR can overcome retinal ischemia using oxygen-induced retinopathy (OIR) mouse model. Wildtype (WT) or p75NTR-/- mice pups were subjected to hyperoxia (70% O2, p7-p12) then returned to normal air (relative hypoxia, p12-p17). Vascular alterations were assessed at p12 and p17 time-points. Deletion of p75NTR prevented hyperoxia-associated central vascular cell death (p12) and hypoxia-associated RNV and enhanced central vascular repair (p17). Decreased expression of apoptotic markers; preserved Akt survival signal decreased proNGF were also observed at p12. During hypoxia, deletion of p75NTR maintained VEGF and VEGFR2 activation and restored NGF/proNGF and BDNF/proBDNF levels. Deletion of p75NTR coincided with significant increases in expression and activation of NGF survival receptor, TrkA at basal and hyperoxic condition. Pharmacological inhibition of TrkA using compound K-252a (0.5 µg 1 µl-1/eye) resulted in 2-fold increase in pathological RNV and 1.34-fold increase in central vascular cell death in p75NTR-/- pups. In conclusion, deletion of p75NTR protected against retinal ischemia and prevented RNV, in part, through restoring neurotrophic support and activating TrkA receptor.
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Deleção de Genes , Hiperóxia/terapia , Oxigênio/efeitos adversos , Receptor trkA/genética , Receptores de Fator de Crescimento Neural/metabolismo , Neovascularização Retiniana/prevenção & controle , Animais , Carbazóis/farmacologia , Modelos Animais de Doenças , Humanos , Hiperóxia/induzido quimicamente , Hiperóxia/genética , Hiperóxia/metabolismo , Alcaloides Indólicos/farmacologia , Camundongos , Fator de Crescimento Neural/metabolismo , Precursores de Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor trkA/metabolismo , Receptores de Fator de Crescimento Neural/genética , Neovascularização Retiniana/genética , Neovascularização Retiniana/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Blast concussions are a common injury sustained in military combat today. Inflammation due to microglial polarization can drive the development of visual defects following blast injuries. In this study, we assessed whether anti-inflammatory factors released by the mesenchymal stem cells derived from adipose tissue (adipose stem cells, ASC) can limit retinal tissue damage and improve visual function in a mouse model of visual deficits following mild traumatic brain injury. We show that intravitreal injection of 1 µL of ASC concentrated conditioned medium from cells pre-stimulated with inflammatory cytokines (ASC-CCM) mitigates loss of visual acuity and contrast sensitivity four weeks post blast injury. Moreover, blast mice showed increased retinal expression of genes associated with microglial activation and inflammation by molecular analyses, retinal glial fibrillary acidic protein (GFAP) immunoreactivity, and increased loss of ganglion cells. Interestingly, blast mice that received ASC-CCM improved in all parameters above. In vitro, ASC-CCM not only suppressed microglial activation but also protected against Tumor necrosis alpha (TNFα) induced endothelial permeability as measured by transendothelial electrical resistance. Biochemical and molecular analyses demonstrate TSG-6 is highly expressed in ASC-CCM from cells pre-stimulated with TNFα and IFNγ but not from unstimulated cells. Our findings suggest that ASC-CCM mitigates visual deficits of the blast injury through their anti-inflammatory properties on activated pro-inflammatory microglia and endothelial cells. A regenerative therapy for immediate delivery at the time of injury may provide a practical and cost-effective solution against the traumatic effects of blast injuries to the retina.
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Anti-Inflamatórios/administração & dosagem , Traumatismos por Explosões/complicações , Concussão Encefálica/etiologia , Meios de Cultivo Condicionados/química , Células-Tronco Mesenquimais/metabolismo , Retinite/tratamento farmacológico , Transtornos da Visão/tratamento farmacológico , Tecido Adiposo/citologia , Tecido Adiposo/metabolismo , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Concussão Encefálica/complicações , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Injeções Intravítreas , Masculino , Células-Tronco Mesenquimais/citologia , Camundongos , Microglia/efeitos dos fármacos , Microglia/metabolismo , Retinite/etiologia , Transtornos da Visão/etiologiaRESUMO
Diabetes and hyperglycemia are associated with increased retinal oxidative and nitrative stress and vascular cell death. Paradoxically, high glucose stimulates expression of survival and angiogenic growth factors. Therefore, we examined the hypothesis that high glucose-mediated tyrosine nitration causes inhibition of the survival protein PI3-kinase, and in particular, its regulatory p85 subunit in retinal endothelial cell (EC) cultures. Retinal EC were cultured in high glucose (HG, 25 mM) for 3 days or peroxynitrite (PN, 100 µM) overnight in the presence or absence of a peroxynitrite decomposition catalyst (FeTPPs, 2.5 µM), or the selective nitration inhibitor epicatechin (100 µM). Apoptosis of ECs was assessed using TUNEL assay and caspase-3 activity. Immunoprecipitation and Western blot were used to assess protein expression and tyrosine nitration of p85 subunit and its interaction with the p110 subunit. HG or PN accelerated apoptosis of retinal ECs compared to normal glucose (NG, 5 mM) controls. HG- or PN-treated cells also showed significant increases in tyrosine nitration on the p85 subunit of PI3-kinase that inhibited its association with the catalytic p110 subunit and impaired PI3-kinase/Akt kinase activity. Decomposing peroxynitrite or blocking tyrosine nitration of p85 restored the activity of PI3-kinase, and prevented apoptosis and activation of p38 MAPK. Inhibiting p38 MAPK or overexpression of the constitutively activated Myr-Akt construct prevented HG- or peroxynitrite-mediated apoptosis. In conclusion, HG impairs pro-survival signals and causes accelerated EC apoptosis, at least in part via tyrosine nitration and inhibition of PI3-kinase. Inhibitors of nitration can be used in adjuvant therapy to delay diabetic retinopathy and microvascular complication.
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Impaired maturation of nerve growth factor precursor (proNGF) and its accumulation has been reported in several neurodegenerative diseases, myocardial infarction and diabetes. To elucidate the direct impact of proNGF accumulation identified the need to create a transgenic model that can express fully mutated cleavage-resistant proNGF. Using Cre-Lox technology, we developed an inducible endothelial-specific proNGF transgenic mouse (proNGFLoxp) that overexpresses GFP-conjugated cleavage-resistant proNGF123 when crossed with VE-cadherin-CreERT2 (Cre). Expression of proNGF, inflammatory mediators, NGF and VEGF was evaluated by PCR, Western blot and immunohistochemistry. EC-proNGF overexpression was confirmed using colocalization of anti-proNGF within retinal vasculature. EC-proNGF did not cause retinal neurotoxicity or marked glial activation at 4-weeks. Microvascular preparation from Cre-proNGF mice showed significant imbalance of proNGF/NGF ratio, enhanced expression of TNF-α and p75NTR, and tendency to impair TrkA phosphorylation compared to controls. EC-proNGF overexpression triggered mRNA expression of p75NTR and inflammatory mediators in both retina and renal cortex compared to controls. EC-proNGF expression induced vascular permeability including breakdown of BRB and albuminuria in the kidney without affecting VEGF level at 4-weeks. Histopathological changes were assessed after 8-weeks and the results showed that EC-proNGF triggered formation of occluded (acellular) capillaries, hall mark of retinal ischemia. EC-proNGF resulted in glomerular enlargement and kidney fibrosis, hall mark of renal dysfunction. We have successfully created an inducible mouse model that can dissect the contribution of autocrine direct action of cleavage-resistant proNGF on systemic microvascular abnormalities in both retina and kidney, major targets for microvascular complication.
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Endotélio Vascular/metabolismo , Endotélio Vascular/fisiopatologia , Microvasos/fisiopatologia , Fator de Crescimento Neural/genética , Animais , Modelos Animais de Doenças , Endotélio Vascular/patologia , Regulação da Expressão Gênica , Nefropatias/genética , Nefropatias/metabolismo , Nefropatias/patologia , Nefropatias/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fator de Crescimento Neural/metabolismo , Precursores de Proteínas/genética , Processamento de Proteína Pós-Traducional , Retina/metabolismo , Retina/patologia , Retina/fisiopatologia , Doenças Retinianas/genética , Doenças Retinianas/metabolismo , Doenças Retinianas/patologia , Doenças Retinianas/fisiopatologia , Vasos Retinianos/metabolismo , Vasos Retinianos/patologia , Vasos Retinianos/fisiopatologiaRESUMO
INTRODUCTION: The p75 neurotrophin receptor (p75NTR) is a member of TNF-α receptor superfamily that bind all neurotrophins, mainly regulating their pro-apoptotic actions. Ischemia is a common pathology in different cardiovascular diseases affecting multiple organs, however the contribution of p75NTR remains not fully addressed. The aim of this work is to review the current evidence through published literature studying the impact of p75NTR receptor in ischemic vascular diseases. AREAS COVERED: In the eye, several ischemic ocular diseases are associated with enhanced p75NTR expression. Ischemic retinopathy including diabetic retinopathy, retinopathy of prematurity and retinal vein occlusion are characterized initially by ischemia followed by excessive neovascularization. Beyond the eye, cerebral ischemia, myocardial infarction and critical limb ischemia are ischemic cardiovascular diseases that are characterized by altered expression of neurotrophins and p75NTR expression. We surveyed both clinical and experimental studies that examined the impact of p75NTR receptor in ischemic diseases of eye, heart, brain and peripheral limbs. EXPERT COMMENTARY: p75NTR receptor is a major player in multiple ischemic vascular diseases affecting the eye, brain, heart and peripheral limbs with significant increases in its expression accompanying neuro-vascular injury. This has been addressed in the current review along with the beneficial vascular outcomes of p75NTR inhibition.
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BACKGROUND: Previous work demonstrated that high-fat diet (HFD) triggered thioredoxin-interacting protein (TXNIP) and that silencing TXNIP prevents diabetes-impaired vascular recovery. Here, we examine the impact of genetic deletion of TXNIP on HFD-impaired vascular recovery using hind limb ischemia model. METHODS: Wild type mice (WT, C57Bl/6) and TXNIP knockout mice (TKO) were fed either normal chow diet (WT-ND and TKO-ND) or 60% high-fat diet (WT-HFD and TKO-HFD). After four weeks of HFD, unilateral hind limb ischemia was performed and blood flow was measured using Laser doppler scanner at baseline and then weekly for an additional three weeks. Vascular density, nitrative stress, infiltration of CD68+ macrophages, and expression of inflammasome, vascular endothelial growth factor (VEGF), VEGF receptor-2 were examined by slot blot, Western blot and immunohistochemistry. RESULTS: By week 8, HFD caused similar increases in weight, cholesterol and triglycerides in both WT and TKO. At week 4 and week 8, HFD significantly impaired glucose tolerance in WT and to a lesser extent in TKO. HFD significantly impaired blood flow and vascular density (CD31 labeled) in skeletal muscle of WT mice compared to ND but not in TKO. HFD and ischemia significantly induced tyrosine nitration, and systemic IL-1ß and infiltration of CD68+ cells in skeletal muscle from WT but not from TKO. HFD significantly increased cleaved-caspase-1 and IL-1 ß compared to ND. Under both ND, ischemia tended to increase VEGF expression and increased VEGFR2 activation in WT only but not TKO. CONCLUSION: Similar to prior observation in diabetes, HFD-induced obesity can compromise vascular recovery in response to ischemic insult. The mechanism involves increased TXNIP-NLRP3 (nucleotide-binding oligomerization domain-like receptor protein 3) inflammasome activation, nitrative stress and impaired VEGFR2 activation. Deletion of TXNIP restored blood flow, reduced nitrative stress and blunted inflammasome-mediated inflammation; however, it did not impact VEGF/VEGFR2 in HFD. Targeting TXNIP-NLRP3 inflammasome can provide potential therapeutic target in obesity-induced vascular complication.
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AIM: To elucidate how high diet-induced endoplasmic reticulum-stress upregulates thioredoxin interacting protein expression in Müller cells leading to retinal inflammation. METHODS: Male C57Bl/J mice were fed either normal diet or 60% high fat diet for 4-8 wk. During the 4 wk study, mice received phenyl-butyric acid (PBA); endoplasmic reticulum-stress inhibitor; for 2 wk. Insulin resistance was assessed by oral glucose tolerance. Effects of palmitate-bovine serum albumin (BSA) (400 µmol/L) were examined in retinal Müller glial cell line and primary Müller cells isolated from wild type and thioredoxin interacting protein knock-out mice. Expression of thioredoxin interacting protein, endoplasmic reticulum-stress markers, miR-17-5p mRNA, as well as nucleotide-binding oligomerization domain-like receptor protein (NLRP3) and IL1ß protein was determined. RESULTS: High fat diet for 8 wk induced obesity and insulin resistance evident by increases in body weight and impaired glucose tolerance. By performing quantitative real-time polymerase chain reaction, we found that high fat diet triggered the expression of retinal endoplasmic reticulum-stress markers (P < 0.05). These effects were associated with increased thioredoxin interacting protein and decreased miR-17-5p expression, which were restored by inhibiting endoplasmic reticulum-stress with PBA (P < 0.05). In vitro, palmitate-BSA triggered endoplasmic reticulum-stress markers, which was accompanied with reduced miR-17-5p and induced thioredoxin interacting protein mRNA in retinal Müller glial cell line (P < 0.05). Palmitate upregulated NLRP3 and IL1ß expression in primary Müller cells isolated from wild type. However, using primary Müller cells isolated from thioredoxin interacting protein knock-out mice abolished palmitate-mediated increase in NLRP3 and IL1ß. CONCLUSION: Our work suggests that targeting endoplasmic reticulum-stress or thioredoxin interacting protein are potential therapeutic strategies for early intervention of obesity-induced retinal inflammation.
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Diabetic retinopathy (DR) is the leading cause of blindness in working-age adults in United States. Research indicates an association between oxidative stress and the development of diabetes complications. However, clinical trials with general antioxidants have failed to prove effective in diabetic patients. Mounting evidence from experimental studies that continue to elucidate the damaging effects of oxidative stress and inflammation in both vascular and neural retina suggest its critical role in the pathogenesis of DR. This review will outline the current management of DR as well as present potential experimental therapeutic interventions, focusing on molecules that link oxidative stress to inflammation to provide potential therapeutic targets for treatment or prevention of DR. Understanding the biochemical changes and the molecular events under diabetic conditions could provide new effective therapeutic tools to combat the disease.
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Retinopatia Diabética/tratamento farmacológico , Retinopatia Diabética/etiologia , Inibidores da Angiogênese/uso terapêutico , Complicações do Diabetes , Retinopatia Diabética/metabolismo , Humanos , NADPH Oxidases/efeitos adversos , NADPH Oxidases/metabolismo , Estresse Oxidativo , Ácido Peroxinitroso/efeitos adversos , Ácido Peroxinitroso/metabolismo , Estados UnidosRESUMO
Ischemic diseases such as stroke and proliferative retinopathy are characterized by hypoxia-driven release of angiogenic factors such as vascular endothelial growth factor (VEGF). However, revascularization of the ischemic areas is inadequate, resulting in impaired neuro-vascular function. We aim to examine the vascular protective effects of candesartan, an angiotensin receptor blocker, in an ischemic retinopathy mouse model. Vascular density, number of tip cells, and perfusions of capillaries were assessed. Activation of Muller glial cells and levels of peroxynitrite, VEGF, VEGFR2, inducible nitric oxide synthase, hemeoxygenase-1 (HO-1) were assessed. Proangiogenic effects of candesartan were examined in human endothelial cells (EC) that were cultured in normoxia or hypoxia and transduced with siRNA against HO-1. Candesartan (1 mg/kg) and (10 mg/kg) decreased hypoxia-induced neovascularization by 67 and 70%, respectively. Candesartan (10 mg/kg) significantly stimulated the number of tip cells and physiological revascularization of the central retina (45%) compared with untreated pups. The effects of candesartan coincided with reduction of hypoxia-induced Muller glial activation, iNOS expression and restoration of HO-1 expression with no significant change in VEGF levels. In vitro, silencing HO-1 expression blunted the ability of candesartan to induce VEGF expression under normoxia and VEGFR2 activation and angiogenic response under both normoxia and hypoxia. These findings suggest that candesartan improved reparative angiogenesis and hence prevented pathological angiogenesis by modulating HO-1 and iNOS levels in ischemic retinopathy. HO-1 is required for VEGFR2 activation and proangiogenic action of candesartan in EC. Candesartan, an FDA-approved drug, could be repurposed as a potential therapeutic agent for the treatment of ischemic diseases.
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Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Benzimidazóis/farmacologia , Heme Oxigenase-1/metabolismo , Isquemia/fisiopatologia , Neovascularização Patológica/tratamento farmacológico , Doenças Retinianas/fisiopatologia , Tetrazóis/farmacologia , Animais , Compostos de Bifenilo , Inativação Gênica , Heme Oxigenase-1/genética , Isquemia/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo II/metabolismo , Estresse Oxidativo , Doenças Retinianas/enzimologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Diabetic retinopathy (DR), a major ocular complication of diabetes, is a leading cause of blindness in US working age adults with limited treatments. Neurotrophins (NTs), a family of proteins essential for growth, differentiation and survival of retinal neurons, have emerged as potential players in the pathogenesis of DR. NTs can signal through their corresponding tropomyosin kinase related receptor to mediate cell survival or through the p75 neurotrophin receptor with the co-receptor, sortilin, to mediate cell death. This review focuses on the role of NGF, the first discovered NT, in the development of DR. Impaired processing of proNGF has been found in ocular fluids from diabetic patients as well as experimental models. Evidence from literature and our studies support the notion that NTs appear to play multiple potential roles in DR, hence, understanding their contribution to DR may lead to promising therapeutic approaches for this devastating disease.