RESUMO
The vasculature of the central nervous system is a 3D lattice composed of laminar vascular beds interconnected by penetrating vessels. The mechanisms controlling 3D lattice network formation remain largely unknown. Combining viral labeling, genetic marking, and single-cell profiling in the mouse retina, we discovered a perivascular neuronal subset, annotated as Fam19a4/Nts-positive retinal ganglion cells (Fam19a4/Nts-RGCs), directly contacting the vasculature with perisomatic endfeet. Developmental ablation of Fam19a4/Nts-RGCs led to disoriented growth of penetrating vessels near the ganglion cell layer (GCL), leading to a disorganized 3D vascular lattice. We identified enriched PIEZO2 expression in Fam19a4/Nts-RGCs. Piezo2 loss from all retinal neurons or Fam19a4/Nts-RGCs abolished the direct neurovascular contacts and phenocopied the Fam19a4/Nts-RGC ablation deficits. The defective vascular structure led to reduced capillary perfusion and sensitized the retina to ischemic insults. Furthermore, we uncovered a Piezo2-dependent perivascular granule cell subset for cerebellar vascular patterning, indicating neuronal Piezo2-dependent 3D vascular patterning in the brain.
Assuntos
Cerebelo , Neurônios , Retina , Animais , Feminino , Masculino , Camundongos , Cerebelo/metabolismo , Cerebelo/irrigação sanguínea , Cerebelo/citologia , Canais Iônicos/metabolismo , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Retina/citologia , Retina/metabolismo , Células Ganglionares da Retina/metabolismo , Vasos Retinianos/metabolismoRESUMO
The discovery of vascular endothelial-derived growth factor (VEGF) has revolutionized our understanding of vasculogenesis and angiogenesis during development and physiological homeostasis. Over a short span of two decades, our understanding of the molecular mechanisms by which VEGF coordinates neurovascular homeostasis has become more sophisticated. The central role of VEGF in the pathogenesis of diverse cancers and blinding eye diseases has also become evident. Elucidation of the molecular regulation of VEGF and the transformative development of multiple therapeutic pathways targeting VEGF directly or indirectly is a powerful case study of how fundamental research can guide innovation and translation. It is also an elegant example of how agnostic discovery and can transform our understanding of human disease. This review will highlight critical nodal points in VEGF biology, including recent developments in immunotherapy for cancer and multitarget approaches in neovascular eye disease.
Assuntos
Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/fisiologia , Fatores de Crescimento do Endotélio Vascular/fisiologia , Animais , Humanos , Imunoterapia/métodos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Vasos Retinianos/metabolismo , Vasos Retinianos/fisiologia , Transdução de Sinais/fisiologia , Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
The microvascular system consists of two cell types: endothelial and mural (pericytes and vascular smooth muscle cells; VSMCs) cells. Communication between endothelial and mural cells plays a pivotal role in the maintenance of vascular homeostasis; however, in vivo molecular and cellular mechanisms underlying mural cell development remain unclear. In this study, we found that macrophages played a crucial role in TGFß-dependent pericyte-to-VSMC differentiation during retinal vasculature development. In mice with constitutively active Foxo1 overexpression, substantial accumulation of TGFß1-producing macrophages and pericytes around the angiogenic front region was observed. Additionally, the TGFß-SMAD pathway was activated in pericytes adjacent to macrophages, resulting in excess ectopic α-smooth muscle actin-positive VSMCs. Furthermore, we identified endothelial SEMA3C as an attractant for macrophages. In vivo neutralization of SEMA3C rescued macrophage accumulation and ectopic VSMC phenotypes in the mice, as well as drug-induced macrophage depletion. Therefore, macrophages play an important physiological role in VSMC development via the FOXO1-SEMA3C pathway.
Assuntos
Proteína Forkhead Box O1 , Macrófagos , Músculo Liso Vascular , Miócitos de Músculo Liso , Semaforinas , Animais , Macrófagos/metabolismo , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/citologia , Camundongos , Semaforinas/metabolismo , Semaforinas/genética , Proteína Forkhead Box O1/metabolismo , Proteína Forkhead Box O1/genética , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/citologia , Pericitos/metabolismo , Pericitos/citologia , Diferenciação Celular , Transdução de Sinais , Vasos Retinianos/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Fatores de Transcrição Forkhead/genética , Fator de Crescimento Transformador beta1/metabolismo , Camundongos Endogâmicos C57BLRESUMO
Prolyl hydroxylase domain (PHD) proteins are oxygen sensors that use intracellular oxygen as a substrate to hydroxylate hypoxia-inducible factor (HIF) α proteins, routing them for polyubiquitylation and proteasomal degradation. Typically, HIFα accumulation in hypoxic or PHD-deficient tissues leads to upregulated angiogenesis. Here, we report unexpected retinal phenotypes associated with endothelial cell (EC)-specific gene targeting of Phd2 (Egln1) and Hif2alpha (Epas1). EC-specific Phd2 disruption suppressed retinal angiogenesis, despite HIFα accumulation and VEGFA upregulation. Suppressed retinal angiogenesis was observed both in development and in the oxygen-induced retinopathy (OIR) model. On the other hand, EC-specific deletion of Hif1alpha (Hif1a), Hif2alpha, or both did not affect retinal vascular morphogenesis. Strikingly, retinal angiogenesis appeared normal in mice double-deficient for endothelial PHD2 and HIF2α. In PHD2-deficient retinal vasculature, delta-like 4 (DLL4, a NOTCH ligand) and HEY2 (a NOTCH target) were upregulated by HIF2α-dependent mechanisms. Inhibition of NOTCH signaling by a chemical inhibitor or DLL4 antibody partially rescued retinal angiogenesis. Taken together, our data demonstrate that HIF2α accumulation in retinal ECs inhibits rather than stimulates retinal angiogenesis, in part by upregulating DLL4 expression and NOTCH signaling.
Assuntos
Animais Recém-Nascidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Células Endoteliais , Prolina Dioxigenases do Fator Induzível por Hipóxia , Receptores Notch , Neovascularização Retiniana , Transdução de Sinais , Regulação para Cima , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Camundongos , Receptores Notch/metabolismo , Receptores Notch/genética , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/genética , Neovascularização Retiniana/patologia , Células Endoteliais/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Retina/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Vasos Retinianos/metabolismo , AngiogêneseRESUMO
Ferroptosis is a new form of cell death characterized by iron-dependent lipid peroxidation. Whether ferroptosis is involved in retinal microvascular dysfunction under diabetic condition is not known. Herein, the expression of ferroptosis-related genes in patients with proliferative diabetic retinopathy and in diabetic mice was determined with quantitative RT-PCR. Reactive oxygen species, iron content, lipid peroxidation products, and ferroptosis-associated proteins in the cultured human retinal microvascular endothelial cells (HRMECs) and in the retina of diabetic mice were examined. The association of ferroptosis with the functions of endothelial cells in vitro was evaluated. After administration of ferroptosis-specific inhibitor, Fer-1, the retinal microvasculature in diabetic mice was assessed. Characteristic changes of ferroptosis-associated markers, including glutathione peroxidase 4, ferritin heavy chain 1, long-chain acyl-CoA synthetase 4, transferrin receptor protein 1, and cyclooxygenase-2, were detected in the retinal fibrovascular membrane of patients with proliferative diabetic retinopathy, cultured HRMECs, and the retina of diabetic mice. Elevated levels of reactive oxygen species, lipid peroxidation, and iron content were found in the retina of diabetic mice and in cultured HRMECs. Ferroptosis was found to be associated with HRMEC dysfunction under high-glucose condition. Inhibition of ferroptosis with specific inhibitor Fer-1 in diabetic mice significantly reduced the severity of retinal microvasculopathy. Ferroptosis contributes to microvascular dysfunction in diabetic retinopathy, and inhibition of ferroptosis might be a promising strategy for the therapy of early-stage diabetic retinopathy.
Assuntos
Retinopatia Diabética , Ferroptose , Espécies Reativas de Oxigênio , Retinopatia Diabética/patologia , Retinopatia Diabética/metabolismo , Animais , Humanos , Camundongos , Masculino , Espécies Reativas de Oxigênio/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/metabolismo , Peroxidação de Lipídeos , Camundongos Endogâmicos C57BL , Microvasos/patologia , Microvasos/metabolismo , Ferro/metabolismo , Vasos Retinianos/metabolismo , Vasos Retinianos/patologiaRESUMO
Retinal vascular diseases (RVDs), in particular diabetic retinopathy, retinal vein occlusion, and retinopathy of prematurity, are leading contributors to blindness. The pathogenesis of RVD involves vessel dilatation, leakage, and occlusion; however, the specific underlying mechanisms remain unclear. Recent findings have indicated that pericytes (PCs), as critical members of the vascular mural cells, significantly contribute to the progression of RVDs, including detachment from microvessels, alteration of contractile and secretory properties, and excessive production of the extracellular matrix. Moreover, PCs are believed to have mesenchymal stem properties and, therefore, might contribute to regenerative therapy. Here, we review novel ideas concerning PC characteristics and functions in RVDs and discuss potential therapeutic strategies based on PCs, including the targeting of pathological signals and cell-based regenerative treatments.
Assuntos
Pericitos , Pericitos/metabolismo , Humanos , Animais , Vasos Retinianos/metabolismo , Vasos Retinianos/patologia , Doenças Retinianas/terapia , Doenças Retinianas/metabolismo , Doenças Retinianas/patologia , Retinopatia Diabética/metabolismo , Retinopatia Diabética/terapia , Retinopatia Diabética/patologiaRESUMO
Non-proliferative diabetic retinopathy (NPDR) is the early stage of diabetic retinopathy (DR) and is a chronic oxidative stress-related ocular disease. Few treatments are approved for early DR. This study aimed to investigate the pathogenic mechanisms underlying the retinal micro-vasculopathy induced by diabetes and to explore an early potential for treating early DR in a mouse model. The mouse model of type 1 diabetes was established by intraperitoneal injection of streptozotocin (STZ, 180 mg/kg), which was used as the early DR model. The body weight and blood glucose mice were measured regularly; The retinal vascular leakage in the early DR mice was determined by whole-mount staining; Label-free quantitative proteomic analysis and bioinformatics were used to explore the target proteins and signaling pathways associated with the retinal tissues of early DR mice; To detect the effects of target protein on endothelial cell proliferation, migration, and tube formation, knockdown and overexpression of VEGF-B were performed in human retinal vascular endothelial cells (HRECs); Western blotting was used to detect the expression of target proteins in vitro and in vivo; Meanwhile, the therapeutic effect of VEGF-B on vascular leakage has also been evaluated in vitro and in vivo. The protein expressions of vascular endothelial growth factor (VEGF)-B and the Rho GTPases family member CDC42 were reduced in the retinal tissues of early DR. VEGF-B upregulated the expression of CDC42/ZO1/VE-cadherin and prevented hyperglycemia-induced vascular leakage in HRECs. Standard intravitreal VEGF-B injections improved the retinal vascular leakage and neurovascular response in early DR mice. Our findings demonstrated, for the first time, that in diabetes, the retinal vessels are damaged due to decreased VEGF-B expression through downregulation of CDC42/ZO1/VE-cadherin expression. Therefore, VEGF-B could be used as a novel therapy for early DR.
Assuntos
Antígenos CD , Caderinas , Diabetes Mellitus Experimental , Retinopatia Diabética , Hiperglicemia , Proteína cdc42 de Ligação ao GTP , Animais , Proteína cdc42 de Ligação ao GTP/metabolismo , Camundongos , Caderinas/metabolismo , Retinopatia Diabética/metabolismo , Retinopatia Diabética/prevenção & controle , Retinopatia Diabética/etiologia , Retinopatia Diabética/patologia , Hiperglicemia/metabolismo , Masculino , Antígenos CD/metabolismo , Antígenos CD/genética , Humanos , Diabetes Mellitus Experimental/metabolismo , Proteína da Zônula de Oclusão-1/metabolismo , Proteína da Zônula de Oclusão-1/genética , Transdução de Sinais , Camundongos Endogâmicos C57BL , Vasos Retinianos/metabolismo , Vasos Retinianos/patologia , Células Endoteliais/metabolismo , Retina/metabolismo , Retina/patologia , Permeabilidade CapilarRESUMO
AIMS/HYPOTHESIS: Diabetic retinopathy is characterised by neuroinflammation that drives neuronal and vascular degenerative pathology, which in many individuals can lead to retinal ischaemia and neovascularisation. Infiltrating macrophages and activated retina-resident microglia have been implicated in the progression of diabetic retinopathy, although the distinct roles of these immune cells remain ill-defined. Our aim was to clarify the distinct roles of macrophages/microglia in the pathogenesis of proliferative ischaemic retinopathies. METHODS: Murine oxygen-induced retinopathy is commonly used as a model of ischaemia-induced proliferative diabetic retinopathy (PDR). We evaluated the phenotype macrophages/microglia by immunostaining, quantitative real-time RT-PCR (qRT-PCR), flow cytometry and scRNA-seq analysis. In clinical imaging studies of diabetic retinopathy, we used optical coherence tomography (OCT) and OCT angiography. RESULTS: Immunostaining, qRT-PCR and flow cytometry showed expression levels of M1-like macrophages/microglia markers (CD80, CD68 and nitric oxide synthase 2) and M2-like macrophages/microglia markers (CD206, CD163 and macrophage scavenger receptor 1) were upregulated in areas of retinal ischaemia and around neo-vessels, respectively. scRNA-seq analysis of the ischaemic retina revealed distinct ischaemia-related clusters of macrophages/microglia that express M1 markers as well as C-C chemokine receptor 2. Inhibition of Rho-kinase (ROCK) suppressed CCL2 expression and reduced CCR2-positive M1-like macrophages/microglia in areas of ischaemia. Furthermore, the area of retinal ischaemia was reduced by suppressing blood macrophage infiltration not only by ROCK inhibitor and monocyte chemoattractant protein-1 antibody but also by GdCl3. Clinical imaging studies of diabetic retinopathy using OCT indicated potential involvement of macrophages/microglia represented by hyperreflective foci in areas of reduced perfusion. CONCLUSIONS/INTERPRETATION: These results collectively indicated that heterotypic macrophages/microglia differentially contribute to retinal ischaemia and neovascularisation in retinal vascular diseases including diabetic retinopathy. This adds important new information that could provide a basis for a more targeted, cell-specific therapeutic approach to prevent progression to sight-threatening PDR.
Assuntos
Retinopatia Diabética , Isquemia , Macrófagos , Microglia , Retina , Animais , Macrófagos/metabolismo , Microglia/metabolismo , Camundongos , Retinopatia Diabética/metabolismo , Retinopatia Diabética/patologia , Isquemia/metabolismo , Retina/metabolismo , Retina/patologia , Humanos , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/patologia , Camundongos Endogâmicos C57BL , Tomografia de Coerência Óptica , Masculino , Vasos Retinianos/metabolismo , Vasos Retinianos/patologiaRESUMO
Diabetic retinopathy has a high probability of causing visual impairment or blindness throughout the disease progression and is characterized by the growth of new blood vessels in the retina at an advanced, proliferative stage. Microglia are a resident immune population in the central nervous system, known to play a crucial role in regulating retinal angiogenesis in both physiological and pathological conditions, including diabetic retinopathy. Physiologically, they are located close to blood vessels and are essential for forming new blood vessels (neovascularization). In diabetic retinopathy, microglia become widely activated, showing a distinct polarization phenotype that leads to their accumulation around neovascular tufts. These activated microglia induce pathogenic angiogenesis through the secretion of various angiogenic factors and by regulating the status of endothelial cells. Interestingly, some subtypes of microglia simultaneously promote the regression of neovascularization tufts and normal angiogenesis in neovascularization lesions. Modulating the state of microglial activation to ameliorate neovascularization thus appears as a promising potential therapeutic approach for managing diabetic retinopathy.
Assuntos
Retinopatia Diabética , Microglia , Neovascularização Retiniana , Animais , Humanos , Angiogênese/metabolismo , Angiogênese/patologia , Retinopatia Diabética/patologia , Retinopatia Diabética/metabolismo , Microglia/patologia , Microglia/metabolismo , Retina/patologia , Retina/metabolismo , Neovascularização Retiniana/patologia , Neovascularização Retiniana/metabolismo , Vasos Retinianos/patologia , Vasos Retinianos/metabolismoRESUMO
Pathological retinal angiogenesis profoundly impacts visual function in vascular eye diseases, such as retinopathy of prematurity (ROP) in preterm infants and age-related macular degeneration in the elderly. While the involvement of photoreceptors in these diseases is recognized, the underlying mechanisms remain unclear. This study delved into the pivotal role of photoreceptors in regulating abnormal retinal blood vessel growth using an oxygen-induced retinopathy (OIR) mouse model through the c-Fos/A disintegrin and metalloprotease 17 (Adam17) axis. Our findings revealed a significant induction of c-Fos expression in rod photoreceptors, and c-Fos depletion in these cells inhibited pathological neovascularization and reduced blood vessel leakage in the OIR mouse model. Mechanistically, c-Fos directly regulated the transcription of Adam17 a shedding protease responsible for the production of bioactive molecules involved in inflammation, angiogenesis, and cell adhesion and migration. Furthermore, we demonstrated the therapeutic potential by using an adeno-associated virus carrying a rod photoreceptor-specific short hairpin RNA against c-fos which effectively mitigated abnormal retinal blood vessel overgrowth, restored retinal thickness, and improved electroretinographic (ERG) responses. In conclusion, this study highlights the significance of photoreceptor c-Fos in ROP pathology, offering a novel perspective for the treatment of this disease.
Assuntos
Proteína ADAM17 , Proteínas Proto-Oncogênicas c-fos , Neovascularização Retiniana , Animais , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/patologia , Neovascularização Retiniana/genética , Proteína ADAM17/metabolismo , Proteína ADAM17/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-fos/genética , Camundongos , Humanos , Retinopatia da Prematuridade/metabolismo , Retinopatia da Prematuridade/patologia , Retinopatia da Prematuridade/genética , Camundongos Endogâmicos C57BL , Transcrição Gênica , Regulação da Expressão Gênica , Vasos Retinianos/metabolismo , Vasos Retinianos/patologia , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/patologia , Modelos Animais de Doenças , AngiogêneseRESUMO
Angiogenesis in the developing mammalian retina requires patterning cues from astrocytes. Developmental disorders of retinal vasculature, such as retinopathy of prematurity (ROP), involve arrest or mispatterning of angiogenesis. Whether these vascular pathologies involve astrocyte dysfunction remains untested. Here, we demonstrate that the major risk factor for ROP - transient neonatal exposure to excess oxygen - disrupts formation of the angiogenic astrocyte template. Exposing newborn mice to elevated oxygen (75%) suppressed astrocyte proliferation, whereas return to room air (21% oxygen) at postnatal day 4 triggered extensive proliferation, massively increasing astrocyte numbers and disturbing their spatial patterning prior to the arrival of developing vasculature. Proliferation required astrocytic HIF2α and was also stimulated by direct hypoxia (10% oxygen), suggesting that astrocyte oxygen sensing regulates the number of astrocytes produced during development. Along with astrocyte defects, return to room air also caused vascular defects reminiscent of ROP. Strikingly, these vascular phenotypes were more severe in animals that had larger numbers of excess astrocytes. Together, our findings suggest that fluctuations in environmental oxygen dysregulate molecular pathways controlling astrocyte proliferation, thereby generating excess astrocytes that interfere with retinal angiogenesis.
Assuntos
Astrócitos/metabolismo , Proliferação de Células/fisiologia , Neovascularização Patológica/metabolismo , Neovascularização Fisiológica/fisiologia , Oxigênio/metabolismo , Retina/crescimento & desenvolvimento , Animais , Animais Recém-Nascidos , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Hipóxia/metabolismo , Camundongos , Neovascularização Fisiológica/efeitos dos fármacos , Oxigênio/farmacologia , Retina/anormalidades , Retina/metabolismo , Retina/patologia , Vasos Retinianos/metabolismo , Retinopatia da PrematuridadeRESUMO
BACKGROUND: Diabetic retinopathy (DR), the principal cause of acquired blindness among the working-age population, is the most frequent microvascular complication of diabetes. Although metabolic disorders are hypothesized to play a role in its pathogenesis, the underlying mechanism remains largely elusive. METHODS: To elucidate the mechanism, we initially compared metabolite profiles of vitreous fluid between 23 patients with DR and 12 non-diabetic controls using liquid chromatography/tandem mass spectrometry, identifying the distinct metabolite indoxyl sulfate (IS). Subsequently, streptozotocin (STZ)-induced diabetic and IS-injected rat models were established to examine the effects of IS on retinal microvasculature. RNA sequencing was conducted to identify potential regulatory mechanisms in IS-treated human retinal endothelial cells (HREC). Finally, target gene knockdown in HREC and treatment of IS-injected rats with inhibitors (targeting IS production or downstream regulators) were employed to elucidate the detailed mechanisms and identify therapeutic targets for DR. RESULTS: Metabolomics identified 172 significantly altered metabolites in the vitreous humor of diabetics, including the dysregulated tryptophan metabolite indoxyl sulfate (IS). IS was observed to breach the blood-retinal barrier and accumulate in the intraocular fluid of diabetic rats. Both in vivo and in vitro experiments indicated that elevated levels of IS induced endothelial apoptosis and disrupted cell junctions. RNA sequencing pinpointed prostaglandin E2 (PGE2) synthetase-cyclooxygenase 2 (COX-2) as a potential target of IS. Validation experiments demonstrated that IS enhanced COX-2 expression, which subsequently increased PGE2 secretion by promoting transcription factor EGR1 binding to COX-2 DNA following entry into cells via organic anion transporting polypeptides (OATP2B1). Furthermore, inhibition of COX-2 in vivo or silencing EGR1/OATP2B1 in HREC mitigated IS-induced microcapillary damage and the activation of COX-2/PGE2. CONCLUSION: Our study demonstrated that indoxyl sulfate (IS), a uremic toxin originating from the gut microbiota product indole, increased significantly and contributed to retinal microvascular damage in diabetic retinopathy (DR). Mechanistically, IS impaired retinal microvascular integrity by inducing the expression of COX-2 and the production of PGE2. Consequently, targeting the gut microbiota or the PGE2 pathway may offer effective therapeutic strategies for the treatment of DR.
Assuntos
Ciclo-Oxigenase 2 , Diabetes Mellitus Experimental , Retinopatia Diabética , Dinoprostona , Indicã , Microvasos , Retinopatia Diabética/patologia , Retinopatia Diabética/metabolismo , Animais , Humanos , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/metabolismo , Masculino , Microvasos/patologia , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Ratos Sprague-Dawley , Células Endoteliais/metabolismo , Células Endoteliais/efeitos dos fármacos , Vasos Retinianos/metabolismo , Vasos Retinianos/patologia , Vasos Retinianos/efeitos dos fármacos , Ratos , Pessoa de Meia-Idade , Retina/patologia , Retina/metabolismo , Retina/efeitos dos fármacos , Apoptose/efeitos dos fármacosRESUMO
This study investigated retinal changes in a Western diet (WD)-induced nonhuman primate model of type 2 diabetes. Rhesus nonhuman primates, aged 15 to 17 years, were fed a high-fat diet (n = 7) for >5 years reflective of the traditional WD. Age-matched controls (n = 6) were fed a standard laboratory primate diet. Retinal fundus photography, optical coherence tomography, autofluorescence imaging, and fluorescein angiography were performed before euthanasia. To assess diabetic retinopathy (DR), eyes were examined using trypsin digests, lipofuscin autofluorescence, and multimarker immunofluorescence on cross-sections and whole mounts. Retinal imaging showed venous engorgement and tortuosity, aneurysms, macular exudates, dot and blot hemorrhages, and a marked increase in fundus autofluorescence. Post-mortem changes included the following: decreased CD31 blood vessel density (P < 0.05); increased acellular capillaries (P < 0.05); increased density of ionized calcium-binding adaptor molecule expressing amoeboid microglia/macrophage; loss of regular distribution in stratum and spacing typical of ramified microglia; and increased immunoreactivity of aquaporin 4 and glial fibrillary acidic protein (P < 0.05). However, rhodopsin immunoreactivity (P < 0.05) in rods and neuronal nuclei antibody-positive neuronal density of 50% (P < 0.05) were decreased. This is the first report of a primate model of DR solely induced by a WD that replicates key features of human DR.
Assuntos
Diabetes Mellitus Tipo 2 , Retinopatia Diabética , Animais , Humanos , Retinopatia Diabética/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Diabetes Mellitus Tipo 2/complicações , Dieta Ocidental , Vasos Retinianos/metabolismo , Primatas , Tomografia de Coerência Óptica/métodosRESUMO
Dysfunctional pericytes and disruption of adherens or tight junctions are related to many microvascular diseases, including diabetic retinopathy. In this context, visualizing retinal vascular architecture becomes essential for understanding retinal vascular disease pathophysiology. Although flat mounts provide a demonstration of the retinal blood vasculature, they often lack a clear view of microaneurysms and capillary architecture. Trypsin and elastase digestion are the two techniques for isolating retinal vasculatures in rats, mice, and other animal models. Our observations in the present study reveal that trypsin digestion impacts the association between pericytes and endothelial cells. In contrast, elastase digestion effectively preserves these features in the blood vessels. Furthermore, trypsin digestion disrupts endothelial adherens and tight junctions that elastase digestion does not. Therefore, elastase digestion emerges as a superior technique for isolating retinal vessels, which can be utilized to collect reliable and consistent data to comprehend the pathophysiology of disorders involving microvascular structures.
Assuntos
Camundongos Endogâmicos C57BL , Elastase Pancreática , Pericitos , Vasos Retinianos , Tripsina , Animais , Elastase Pancreática/metabolismo , Tripsina/metabolismo , Vasos Retinianos/metabolismo , Vasos Retinianos/patologia , Pericitos/metabolismo , Pericitos/patologia , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Células Endoteliais/enzimologia , Junções Íntimas/metabolismo , Camundongos , MasculinoRESUMO
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.
Assuntos
Apoptose , Barreira Hematorretiniana , Sobrevivência Celular , Retinopatia Diabética , Células Endoteliais , Células Ependimogliais , Exossomos , Exossomos/metabolismo , Retinopatia Diabética/metabolismo , Retinopatia Diabética/patologia , Retinopatia Diabética/fisiopatologia , Células Ependimogliais/metabolismo , Células Ependimogliais/patologia , Humanos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Barreira Hematorretiniana/metabolismo , Barreira Hematorretiniana/patologia , Células Cultivadas , Proteína da Zônula de Oclusão-1/metabolismo , Permeabilidade Capilar , Sinalização do Cálcio , Linhagem Celular , Vasos Retinianos/metabolismo , Vasos Retinianos/patologia , Vasos Retinianos/fisiopatologiaRESUMO
Pathological retinal angiogenesis is not only the hallmark of retinopathies, but also a major cause of blindness. Guanylate binding protein 2 (GBP2) has been reported to be associated with retinal diseases such as diabetic retinopathy and hypoxic retinopathy. However, GBP2-mediated pathological retinal angiogenesis remains largely unknown. The present study aimed to investigate the role of GBP2 in pathological retinal angiogenesis and its underlying molecular mechanism. In this study, we established oxygen-induced retinopathy (OIR) mice model for in vivo study and hypoxia-induced angiogenesis in ARPE-19 cells for in vitro study. We demonstrated that GBP2 expression was markedly downregulated in the retina of mice with OIR and ARPE-19 cells treated with hypoxia, which was associated with pathological retinal angiogenesis. The regulatory mechanism of GBP2 in ARPE-19 cells was studied by GBP2 silencing and overexpression. The regulatory mechanism of GBP2 in the retina was investigated by overexpressing GBP2 in the retina of OIR mice. Mechanistically, GBP2 downregulated the expression and secretion of vascular endothelial growth factor (VEGFA) in ARPE-19 cells and retina of OIR mice. Interestingly, overexpression of GBP2 significantly inhibited neovascularization in OIR mice, conditioned medium of GBP2 overexpressing ARPE-19 cells inhibited angiogenesis in human umbilical vein endothelial cells (HUVECs). Furthermore, we confirmed that GBP2 downregulated VEGFA expression and angiogenesis by inhibiting the AKT/mTOR signaling pathway. Taken together, we concluded that GBP2 inhibited pathological retinal angiogenesis via the AKT/mTOR/VEGFA axis, thereby suggesting that GBP2 may be a therapeutic target for pathological retinal angiogenesis.
Assuntos
Modelos Animais de Doenças , Proteínas de Ligação ao GTP , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-akt , Neovascularização Retiniana , Vasos Retinianos , Transdução de Sinais , Serina-Treonina Quinases TOR , Fator A de Crescimento do Endotélio Vascular , Animais , Humanos , Camundongos , Hipóxia Celular , Linhagem Celular , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/genética , Oxigênio/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/patologia , Neovascularização Retiniana/genética , Neovascularização Retiniana/prevenção & controle , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Vasos Retinianos/metabolismo , Vasos Retinianos/patologia , Serina-Treonina Quinases TOR/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Diabetic retinopathy (DR) is the leading cause of vision loss and blindness among working-age adults. Pericyte loss is an early pathological feature of DR. Under hyperglycemic conditions, reactive oxygen species (ROS) production increases, leading to oxidative stress and subsequent mitochondrial dysfunction and apoptosis. Dysfunctional pericyte can cause retinal vascular leakage, obliteration, and neovascularization. Glutaredoxin 2 (Grx2) is a mitochondrial glutathione-dependent oxidoreductase which protects cells against oxidative insults by safeguarding mitochondrial function. Whether Grx2 plays a protective role in diabetes-induced microvascular dysfunction remains unclear. Our findings revealed that diabetes-related stress reduced Grx2 expression in pericytes, but not in endothelial cells. Grx2 knock-in ameliorated diabetes-induced microvascular dysfunction in vivo DR models. Decreased Grx2 expression led to significant pericyte apoptosis, and pericyte dysfunction, namely reduced pericyte recruitment towards endothelial cells and increased endothelial cell permeability. Conversely, upregulating Grx2 reversed these effects. Furthermore, Grx2 regulated pericyte apoptosis by modulating complex I activity, which is crucial for pericyte mitochondrial function. Overall, our study uncovered a novel mechanism whereby high glucose inhibited Grx2 expression in vivo and in vitro. Grx2 downregulation exacerbated pericyte apoptosis, pericyte dysfunction, and retinal vascular dysfunction by inactivating complex I and mediating mitochondrial dysfunction in pericytes.
Assuntos
Apoptose , Diabetes Mellitus Experimental , Retinopatia Diabética , Regulação para Baixo , Glutarredoxinas , Pericitos , Vasos Retinianos , Pericitos/metabolismo , Pericitos/patologia , Animais , Glutarredoxinas/metabolismo , Glutarredoxinas/genética , Retinopatia Diabética/metabolismo , Retinopatia Diabética/patologia , Vasos Retinianos/patologia , Vasos Retinianos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Masculino , Células Cultivadas , Permeabilidade Capilar , Western BlottingRESUMO
Diabetic retinopathy (DR), a most common microangiopathy of diabetes, causes vision loss and even blindness. The mechanisms of exosomal lncRNA remain unclear in the development of DR. Here, we first identifed the pro-angiogenic effect of exosomes derived from vitreous humor of proliferative diabetic retinopathy patients, where lncRNA-MIAT was enriched inside. Secondly, lncRNA-MIAT was demonstrated significantly increased in exosomes from high glucose induced human retinal vascular endothelial cell, and can regulate tube formation, migration and proliferation ability to promote angiogenesis in vitro and in vivo. Mechanistically, the pro-angiogenic effect of lncRNA-MIAT was via the lncRNA-MIAT/miR-133a-3p/MMP-X1 axis. The reduced level of lncRNA-MIAT in this axis mitigated the generation of retinal neovascular in mouse model of oxygen-induced retinopathy (OIR), providing crucial evidence for lncRNA-MIAT as a potential clinical target. These findings enhance our understanding of the role of exosomal lncRNA-MIAT in retinal angiogenesis, and propose a promising therapeutic strategy against diabetic retinopathy.
Assuntos
Retinopatia Diabética , Exossomos , MicroRNAs , RNA Longo não Codificante , Neovascularização Retiniana , Animais , Humanos , Masculino , Camundongos , Movimento Celular , Proliferação de Células , Células Cultivadas , Diabetes Mellitus Experimental , Retinopatia Diabética/metabolismo , Retinopatia Diabética/genética , Retinopatia Diabética/patologia , Modelos Animais de Doenças , Exossomos/metabolismo , Exossomos/genética , Regulação da Expressão Gênica , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/genética , Neovascularização Retiniana/patologia , Vasos Retinianos/metabolismo , Vasos Retinianos/patologia , RNA Longo não Codificante/genéticaRESUMO
Retinal vascular leakage is a major event in several retinal diseases, including diabetic retinopathy (DR). In a previous study, we demonstrated that the aqueous humor concentration of Cystatin C (CST3), a physiological inhibitor of cysteine protease, is negatively correlated with the severity of diabetic macular edema. However, its function in the retina has not been clearly elucidated. In this study, we found a significant decrease in the aqueous humor concentration of CST3 with DR progression. Furthermore, we found that CST3 was expressed in retinal endothelial cells and that its expression was significantly downregulated in high glucose-treated human retinal microvascular endothelial cells (HRMECs) and the retinal vessels of oxygen-induced retinopathy (OIR) mice. Silencing CST3 expression resulted in decreased HRMEC migration and tubule formation ability. Exogenous addition of the CST3 protein significantly improved HRMEC migration and tubular formation. In-vivo experiments demonstrated that CST3 silencing induced retinal vascular leakage in WT mice, while its intravitreal injection significantly reduced retinal leakage in OIR mice. Mechanistically, CST3 promoted the expression of the downstream adhesion molecules, claudin5, VE-cadherin, and ZO-1, in retinal vascular cells by regulating the Rap1 signaling pathway. Therefore, this study revealed a novel mechanism by which CST3 improves retinal vascular function and provided evidence that it is a potential therapeutic target for retinal vascular leakage.
Assuntos
Permeabilidade Capilar , Cistatina C , Retinopatia Diabética , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Vasos Retinianos , Transdução de Sinais , Proteínas rap1 de Ligação ao GTP , Animais , Humanos , Camundongos , Humor Aquoso/metabolismo , Barreira Hematorretiniana , Western Blotting , Movimento Celular , Células Cultivadas , Cistatina C/genética , Cistatina C/metabolismo , Retinopatia Diabética/metabolismo , Retinopatia Diabética/genética , Retinopatia Diabética/patologia , Regulação da Expressão Gênica , Injeções Intravítreas , Proteínas rap1 de Ligação ao GTP/metabolismo , Proteínas rap1 de Ligação ao GTP/genética , Vasos Retinianos/metabolismo , Vasos Retinianos/patologia , Complexo Shelterina , Transdução de Sinais/fisiologia , Proteínas de Ligação a Telômeros/metabolismo , Proteínas de Ligação a Telômeros/genéticaRESUMO
Diabetic retinopathy, a leading cause of vision impairment, is marked by microvascular complications in the retina, including pericyte loss, a key indicator of early-stage disease. This study explores the therapeutic potential of exosomes derived from immortalized adipose-mesenchymal stem cells differentiated into pericyte-like cells in restoring the function of mouse retinal microvascular endothelial cells damaged by high glucose conditions, thereby contributing to the understanding of early diabetic retinopathy intervention strategies. To induce immortalized adipose-mesenchymal stem cells differentiation into pericyte-like cells, the study employed pericyte growth supplement. And confirmed the success of cell differentiation through the detection of α-smooth muscle actin and neural/glial antigen 2 expression by Western blot and immunofluorescence. Exosomes were isolated from the culture supernatant of immortalized adipose-mesenchymal stem cells using ultracentrifugation and characterized through Western blot for exosomal markers (CD9, CD81, and TSG101), transmission electron microscopy, and nanoparticle tracking analysis. Their influence on mouse retinal microvascular endothelial cells under high glucose stress was assessed through various functional assays. Findings revealed that exosomes, especially those from pericyte-like immortalized adipose-mesenchymal stem cells, were efficiently internalized by retinal microvascular endothelial cells and effectively counteracted high glucose-induced apoptosis. These exosomes also mitigated the rise in reactive oxygen species levels and suppressed the migratory and angiogenic properties of retinal microvascular endothelial cells, as demonstrated by Transwell and tube formation assays, respectively. Furthermore, they preserved endothelial barrier function, reducing hyperglycemia-induced permeability. At the molecular level, qRT-PCR analysis showed that exosome treatment modulated the expression of critical genes involved in angiogenesis (VEGF-A, ANG2, MMP9), inflammation (IL-1ß, TNF-α), gap junction communication (CX43), and cytoskeletal regulation (ROCK1), with the most prominent effects seen with exosomes from pericyte-like immortalized adipose-mesenchymal stem cells. High glucose increased the expression of pro-angiogenic and pro-inflammatory markers, which were effectively normalized post-exosome treatment. In conclusion, this research highlights the reparative capacity of exosomes secreted by pericyte-like differentiated immortalized adipose-mesenchymal stem cells in reversing the detrimental effects of high glucose on retinal microvascular endothelial cells. By reducing apoptosis, oxidative stress, inflammation, and abnormal angiogenic behavior, these exosomes present a promising avenue for therapeutic intervention in early diabetic retinopathy. Future studies can focus on elucidating the precise molecular mechanisms and exploring their translational potential in vivo.