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1.
Nat Commun ; 15(1): 4097, 2024 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-38755144

RESUMO

Angiogenesis, the growth of new blood vessels from pre-existing vasculature, is essential for the development of new organ systems, but transcriptional control of angiogenesis remains incompletely understood. Here we show that FOXC1 is essential for retinal angiogenesis. Endothelial cell (EC)-specific loss of Foxc1 impairs retinal vascular growth and expression of Slc3a2 and Slc7a5, which encode the heterodimeric CD98 (LAT1/4F2hc) amino acid transporter and regulate the intracellular transport of essential amino acids and activation of the mammalian target of rapamycin (mTOR). EC-Foxc1 deficiency diminishes mTOR activity, while administration of the mTOR agonist MHY-1485 rescues perturbed retinal angiogenesis. EC-Foxc1 expression is required for retinal revascularization and resolution of neovascular tufts in a model of oxygen-induced retinopathy. Foxc1 is also indispensable for pericytes, a critical component of the blood-retina barrier during retinal angiogenesis. Our findings establish FOXC1 as a crucial regulator of retinal vessels and identify therapeutic targets for treating retinal vascular disease.


Assuntos
Barreira Hematorretiniana , Células Endoteliais , Fatores de Transcrição Forkhead , Neovascularização Retiniana , Animais , Fatores de Transcrição Forkhead/metabolismo , Fatores de Transcrição Forkhead/genética , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/genética , Neovascularização Retiniana/patologia , Camundongos , Células Endoteliais/metabolismo , Barreira Hematorretiniana/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Pericitos/metabolismo , Cadeia Pesada da Proteína-1 Reguladora de Fusão/metabolismo , Cadeia Pesada da Proteína-1 Reguladora de Fusão/genética , Vasos Retinianos/metabolismo , Humanos , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Transportador 1 de Aminoácidos Neutros Grandes/genética , Camundongos Knockout , Camundongos Endogâmicos C57BL , Retina/metabolismo , Masculino , Angiogênese
2.
Development ; 151(11)2024 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-38770916

RESUMO

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 , Angiogênese
3.
Mol Ther ; 32(5): 1425-1444, 2024 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-38504518

RESUMO

Pathological ocular angiogenesis has long been associated with myeloid cell activation. However, the precise cellular and molecular mechanisms governing the intricate crosstalk between the immune system and vascular changes during ocular neovascularization formation remain elusive. In this study, we demonstrated that the absence of the suppressor of cytokine signaling 3 (SOCS3) in myeloid cells led to a substantial accumulation of microglia and macrophage subsets during the neovascularization process. Our single-cell RNA sequencing data analysis revealed a remarkable increase in the expression of the secreted phosphoprotein 1 (Spp1) gene within these microglia and macrophages, identifying subsets of Spp1-expressing microglia and macrophages during neovascularization formation in angiogenesis mouse models. Notably, the number of Spp1-expressing microglia and macrophages exhibited further elevation during neovascularization in mice lacking myeloid SOCS3. Moreover, our investigation unveiled the Spp1 gene as a direct transcriptional target gene of signal transducer and activator of transcription 3. Importantly, pharmaceutical activation of SOCS3 or blocking of SPP1 resulted in a significant reduction in pathological neovascularization. In conclusion, our study highlights the pivotal role of the SOCS3/STAT3/SPP1 axis in the regulation of pathological retinal angiogenesis.


Assuntos
Modelos Animais de Doenças , Macrófagos , Microglia , Osteopontina , Neovascularização Retiniana , Fator de Transcrição STAT3 , Proteína 3 Supressora da Sinalização de Citocinas , Animais , Proteína 3 Supressora da Sinalização de Citocinas/metabolismo , Proteína 3 Supressora da Sinalização de Citocinas/genética , Macrófagos/metabolismo , Camundongos , Microglia/metabolismo , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/patologia , Neovascularização Retiniana/genética , Neovascularização Retiniana/etiologia , Osteopontina/metabolismo , Osteopontina/genética , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT3/genética , Regulação da Expressão Gênica , Transdução de Sinais , Camundongos Knockout , Neovascularização Patológica/metabolismo , Neovascularização Patológica/genética , Angiogênese
4.
Cardiovasc Res ; 120(7): 745-755, 2024 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-38507654

RESUMO

AIMS: In hypoxia, endothelial cells (ECs) proliferate, migrate, and form new vasculature in a process called angiogenesis. Recent studies have suggested that ECs rely on glycolysis to meet metabolic needs for angiogenesis in ischaemic tissues, and several studies have investigated the molecular mechanisms integrating angiogenesis and endothelial metabolism. Here, we investigated the role of stem cell factor (SCF) and its receptor, cKIT, in regulating endothelial glycolysis during hypoxia-driven angiogenesis. METHODS AND RESULTS: SCF and cKIT signalling increased the glucose uptake, lactate production, and glycolysis in human ECs under hypoxia. Mechanistically, SCF and cKIT signalling enhanced the expression of genes encoding glucose transporter 1 (GLUT1) and glycolytic enzymes via Akt- and ERK1/2-dependent increased translation of hypoxia inducible factor 1A (HIF1A). In hypoxic conditions, reduction of glycolysis and HIF-1α expression using chemical inhibitors significantly reduced the SCF-induced in vitro angiogenesis in human ECs. Compared with normal mice, mice with oxygen-induced retinopathy (OIR), characterized by ischaemia-driven pathological retinal neovascularization, displayed increased levels of SCF, cKIT, HIF-1α, GLUT1, and glycolytic enzymes in the retina. Moreover, cKIT-positive neovessels in the retina of mice with OIR showed elevated expression of GLUT1 and glycolytic enzymes. Further, blocking SCF and cKIT signalling using anti-SCF neutralizing IgG and cKIT mutant mice significantly reduced the expression of HIF-1α, GLUT1, and glycolytic enzymes and decreased the pathological neovascularization in the retina of mice with OIR. CONCLUSION: We demonstrated that SCF and cKIT signalling regulate angiogenesis by controlling endothelial glycolysis in hypoxia and elucidated the SCF/cKIT/HIF-1α axis as a novel metabolic regulation pathway during hypoxia-driven pathological angiogenesis.


Assuntos
Hipóxia Celular , Transportador de Glucose Tipo 1 , Glicólise , Subunidade alfa do Fator 1 Induzível por Hipóxia , Proteínas Proto-Oncogênicas c-kit , Transdução de Sinais , Fator de Células-Tronco , Animais , Humanos , Fator de Células-Tronco/metabolismo , Fator de Células-Tronco/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Transportador de Glucose Tipo 1/metabolismo , Transportador de Glucose Tipo 1/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Proteínas Proto-Oncogênicas c-kit/genética , Células Endoteliais da Veia Umbilical Humana/metabolismo , Camundongos Endogâmicos C57BL , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/patologia , Neovascularização Retiniana/genética , Camundongos , Neovascularização Fisiológica , Células Cultivadas , Modelos Animais de Doenças , Glucose/metabolismo
5.
Arterioscler Thromb Vasc Biol ; 44(2): 366-390, 2024 02.
Artigo em Inglês | MEDLINE | ID: mdl-38126170

RESUMO

BACKGROUND: Retinal neovascularization is a major cause of vision impairment. Therefore, the purpose of this study is to investigate the mechanisms by which hypoxia triggers the development of abnormal and leaky blood vessels. METHODS: A variety of cellular and molecular approaches as well as tissue-specific knockout mice were used to investigate the role of Cttn (cortactin) in retinal neovascularization and vascular leakage. RESULTS: We found that VEGFA (vascular endothelial growth factor A) stimulates Cttn phosphorylation at Y421, Y453, and Y470 residues in human retinal microvascular endothelial cells. In addition, we observed that while blockade of Cttn phosphorylation at Y470 inhibited VEGFA-induced human retinal microvascular endothelial cell angiogenic events, suppression of Y421 phosphorylation protected endothelial barrier integrity from disruption by VEGFA. In line with these observations, while blockade of Cttn phosphorylation at Y470 negated oxygen-induced retinopathy-induced retinal neovascularization, interference with Y421 phosphorylation prevented VEGFA/oxygen-induced retinopathy-induced vascular leakage. Mechanistically, while phosphorylation at Y470 was required for its interaction with Arp2/3 and CDC6 facilitating actin polymerization and DNA synthesis, respectively, Cttn phosphorylation at Y421 leads to its dissociation from VE-cadherin, resulting in adherens junction disruption. Furthermore, whereas Cttn phosphorylation at Y470 residue was dependent on Lyn, its phosphorylation at Y421 residue required Syk activation. Accordingly, lentivirus-mediated expression of shRNA targeting Lyn or Syk levels inhibited oxygen-induced retinopathy-induced retinal neovascularization and vascular leakage, respectively. CONCLUSIONS: The above observations show for the first time that phosphorylation of Cttn is involved in a site-specific manner in the regulation of retinal neovascularization and vascular leakage. In view of these findings, Cttn could be a novel target for the development of therapeutics against vascular diseases such as retinal neovascularization and vascular leakage.


Assuntos
Neovascularização Retiniana , Animais , Humanos , Camundongos , Cortactina/genética , Cortactina/metabolismo , Células Endoteliais/metabolismo , Camundongos Knockout , Oxigênio/metabolismo , Fosforilação , Neovascularização Retiniana/genética , Neovascularização Retiniana/metabolismo , Tirosina/efeitos adversos , Tirosina/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
6.
Genes (Basel) ; 14(7)2023 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-37510342

RESUMO

Retinal angiomatous proliferation (RAP) and other types of choroidal neovascularization (CNV) are very rarely reported in patients with retinitis pigmentosa (RP). We present a case report of a 91-year-old patient with an obvious RP phenotype, who presented with a sudden onset of vision worsening and metamorphopsia in the left eye. Genetic testing on the UK inherited retinal disease panel did not identify a pathogenic variant. Multimodal imaging comprising optical coherence tomography (OCT), OCT angiography, and fluorescein and indocyanine green angiography showed a RAP lesion in the left macula. The patient received three treatments of monthly injections of aflibercept, with excellent morphological and functional outcomes. Taking into account the patient's age at presentation of the RAP lesion, it is not clear whether the RAP was associated or coincidental with RP. This case report highlights the importance of possessing an awareness that RAP lesions can occur in RP. Moreover, due to a good response and potential safety concerns with continuous anti-VEGF injections in RP patients, a pro re nata (PRN) regimen might be the safest option.


Assuntos
Degeneração Macular , Neovascularização Retiniana , Retinose Pigmentar , Humanos , Neovascularização Retiniana/tratamento farmacológico , Neovascularização Retiniana/genética , Angiofluoresceinografia/métodos , Degeneração Macular/tratamento farmacológico , Degeneração Macular/genética , Degeneração Macular/patologia , Retina/patologia , Retinose Pigmentar/tratamento farmacológico , Retinose Pigmentar/genética , Retinose Pigmentar/patologia , Proliferação de Células
7.
Commun Biol ; 6(1): 516, 2023 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-37179352

RESUMO

Vascular adhesion molecules play an important role in various immunological disorders, particularly in cancers. However, little is known regarding the role of these adhesion molecules in proliferative retinopathies. We observed that IL-33 regulates VCAM-1 expression in human retinal endothelial cells and that genetic deletion of IL-33 reduces hypoxia-induced VCAM-1 expression and retinal neovascularization in C57BL/6 mice. We found that VCAM-1 via JunB regulates IL-8 promoter activity and expression in human retinal endothelial cells. In addition, our study outlines the regulatory role of VCAM-1-JunB-IL-8 signaling on retinal endothelial cell sprouting and angiogenesis. Our RNA sequencing results show an induced expression of CXCL1 (a murine functional homolog of IL-8) in the hypoxic retina, and intravitreal injection of VCAM-1 siRNA not only decreases hypoxia-induced VCAM-1-JunB-CXCL1 signaling but also reduces OIR-induced sprouting and retinal neovascularization. These findings suggest that VCAM-1-JunB-IL-8 signaling plays a crucial role in retinal neovascularization, and its antagonism might provide an advanced treatment option for proliferative retinopathies.


Assuntos
Neovascularização Retiniana , Animais , Humanos , Camundongos , Neovascularização Retiniana/genética , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/patologia , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/metabolismo , Interleucina-33/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Células Endoteliais/metabolismo , Camundongos Endogâmicos C57BL , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Hipóxia/metabolismo , Quimiocina CXCL1/metabolismo , Fatores de Transcrição/metabolismo
8.
Genome Biol ; 24(1): 87, 2023 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-37085894

RESUMO

BACKGROUND: Ocular neovascularization is a leading cause of blindness. Retinal microglia have been implicated in hypoxia-induced angiogenesis and vasculopathy, but the underlying mechanisms are not entirely clear. Lactylation is a novel lactate-derived posttranslational modification that plays key roles in multiple cellular processes. Since hypoxia in ischemic retinopathy is a precipitating factor for retinal neovascularization, lactylation is very likely to be involved in this process. The present study aimed to explore the role of lactylation in retinal neovascularization and identify new therapeutic targets for retinal neovascular diseases. RESULTS: Microglial depletion by the colony-stimulating factor 1 receptor (CSF1R) inhibitor PLX3397 suppresses retinal neovascularization in oxygen-induced retinopathy. Hypoxia increased lactylation in microglia and accelerates FGF2 expression, promoting retinal neovascularization. We identify 77 sites of 67 proteins with increased lactylation in the context of increased lactate under hypoxia. Our results show that the nonhistone protein Yin Yang-1 (YY1), a transcription factor, is lactylated at lysine 183 (K183), which is regulated by p300. Hyperlactylated YY1 directly enhances FGF2 transcription and promotes angiogenesis. YY1 mutation at K183 eliminates these effects. Overexpression of p300 increases YY1 lactylation and enhances angiogenesis in vitro and administration of the p300 inhibitor A485 greatly suppresses vascularization in vivo and in vitro. CONCLUSIONS: Our results suggest that YY1 lactylation in microglia plays an important role in retinal neovascularization by upregulating FGF2 expression. Targeting the lactate/p300/YY1 lactylation/FGF2 axis may provide new therapeutic targets for proliferative retinopathies.


Assuntos
Fator 2 de Crescimento de Fibroblastos , Microglia , Neovascularização Retiniana , Fator de Transcrição YY1 , Animais , Camundongos , Fator 2 de Crescimento de Fibroblastos/farmacologia , Hipóxia/metabolismo , Lactatos/metabolismo , Lactatos/farmacologia , Microglia/metabolismo , Processamento de Proteína Pós-Traducional , Neovascularização Retiniana/genética , Neovascularização Retiniana/metabolismo , Ativação Transcricional , Regulação para Cima , Fator de Transcrição YY1/genética , Fator de Transcrição YY1/metabolismo
9.
Arterioscler Thromb Vasc Biol ; 43(6): 927-942, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-37078291

RESUMO

BACKGROUND: Endothelial cell activation is tightly controlled by the balance between VEGF (vascular endothelial cell growth factor) and Notch signaling pathway. VEGF destabilizes blood vessels and promotes neovascularization, which are common features of sight-threatening ocular vascular disorders. Here, we show that BCL6B (B-cell CLL/lymphoma 6 member B protein), also known as BAZF, ZBTB28, and ZNF62, plays a pivotal role in the development of retinal edema and neovascularization. METHODS: The pathophysiological physiological role of BCL6B was investigated in cellular and animal models mimicking 2 pathological conditions: retinal vein occlusion and choroidal neovascularization. An in vitro experimental system was used in which human retinal microvascular endothelial cells were supplemented with VEGF. Choroidal neovascularization cynomolgus monkey model was generated to investigate the involvement of BCL6B in the pathogenesis. Mice lacking BCL6B or treated with BCL6B-targeting small-interfering ribose nucleic acid were examined for histological and molecular phenotypes. RESULTS: In retinal endothelial cells, the BCL6B expression level was increased by VEGF. BCL6B-deficient endothelial cells showed Notch signal activation and attenuated cord formation via blockage of the VEGF-VEGFR2 signaling pathway. Optical coherence tomography images showed that choroidal neovascularization lesions were decreased by BCL6B-targeting small-interfering ribose nucleic acid. Although BCL6B mRNA expression was significantly increased in the retina, BCL6B-targeting small-interfering ribose nucleic acid suppressed ocular edema in the neuroretina. The increase in proangiogenic cytokines and breakdown of the inner blood-retinal barrier were abrogated in BCL6B knockout (KO) mice via Notch transcriptional activation by CBF1 (C promotor-binding factor 1) and its activator, the NICD (notch intracellular domain). Immunostaining showed that Müller cell activation, a source of VEGF, was diminished in BCL6B-KO retinas. CONCLUSIONS: These data indicate that BCL6B may be a novel therapeutic target for ocular vascular diseases characterized by ocular neovascularization and edema.


Assuntos
Neovascularização de Coroide , Ácidos Nucleicos , Neovascularização Retiniana , Doenças Vasculares , Animais , Humanos , Camundongos , Neovascularização de Coroide/genética , Neovascularização de Coroide/metabolismo , Células Endoteliais/metabolismo , Macaca fascicularis/metabolismo , Ácidos Nucleicos/metabolismo , Ácidos Nucleicos/uso terapêutico , Neovascularização Retiniana/genética , Neovascularização Retiniana/metabolismo , Ribose/metabolismo , Ribose/uso terapêutico , Doenças Vasculares/patologia , Fator A de Crescimento do Endotélio Vascular/metabolismo
10.
Front Endocrinol (Lausanne) ; 13: 909207, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36120455

RESUMO

Diabetic retinopathy (DR) is an important microvascular complication of type 1 and type 2 diabetes mellitus (DM) and a major cause of blindness. Retinal neovascularization plays a critical role in the proliferative DR. In this study, high glucose-induced connexin 43 (Cx43) expression in human retinal endothelial cells (hRECs) in a dose-dependent manner. Compared with hRECs under normal culture conditions, high-glucose (HG)-stimulated hRECs showed promoted tubule formation, increased ROS release, and elevated levels of tumor necrosis factor-alpha (TNF-α), interleukin-1ß (IL-1ß), vascular endothelial growth factor A (VEGFA), and intercellular adhesion molecule 1 (ICAM-1) in the culture medium. HG-induced alterations were further magnified after Cx43 overexpression, whereas partially eliminated after Cx43 knockdown. Finally, in the DR mouse model, impaired retinal structure, increased CD31 expression, and elevated mRNA levels of TNF-α, IL-1ß, VEGFA, and ICAM-1 were observed; in-vivo Cx43 knockdown partially reversed these phenomena. Conclusively, Cx43 knockdown could inhibit hREC angiogenesis, therefore improving DR in the mouse model.


Assuntos
Diabetes Mellitus Tipo 2 , Retinopatia Diabética , Neovascularização Retiniana , Animais , Conexina 43/genética , Conexina 43/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Retinopatia Diabética/metabolismo , Células Endoteliais/metabolismo , Glucose/efeitos adversos , Glucose/metabolismo , Humanos , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Interleucina-1beta , Camundongos , Neovascularização Patológica/metabolismo , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Neovascularização Retiniana/genética , Neovascularização Retiniana/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
11.
Aging (Albany NY) ; 14(16): 6594-6604, 2022 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-35980290

RESUMO

Aberrant neovascularization in the retina is an important threat to vision and closely related to several retinal diseases, such as wet form of age-related macular degeneration, diabetic retinopathy, and retinopathy of prematurity. However, the pathogenesis remains largely unknown. MicroRNAs (miRNAs) have been demonstrated to play critical regulatory roles in angiogenesis. Therefore, we aimed to identify the key miRNAs that regulate retinal neovascularization and elucidate the potential underlying mechanisms. In the present study, we performed RNA sequencing of microRNAs in the retina and found that miR-375 was significantly downregulated in the retina of oxygen-induced retinopathy mice. In retinal microvascular endothelial cells (RMECs), overexpression of miR-375 inhibited cell proliferation and angiogenesis. Conversely, inhibition of miR-375 had the opposite effects. Moreover, our results showed that miR-375 negatively regulated the protein expression of JAK2 by inhibiting its translation. The promoting effects of anti-miR-375 on cell proliferation and angiogenesis were attenuated by an inhibitor of STAT3. These results indicate that miR-375 mitigates cell proliferation and angiogenesis, at least in part, through the JAK2/STAT3 pathway in RMECs, which implies an important underlying mechanism of retinal angiogenesis and provides potential therapeutic targets for retinal microangiopathy.


Assuntos
MicroRNAs , Neovascularização Retiniana , Animais , Proliferação de Células/genética , Células Endoteliais/metabolismo , Janus Quinase 2/metabolismo , Camundongos , MicroRNAs/metabolismo , Neovascularização Patológica/metabolismo , Retina/patologia , Neovascularização Retiniana/genética , Neovascularização Retiniana/patologia , Fator de Transcrição STAT3/metabolismo
12.
Exp Eye Res ; 223: 109196, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35872179

RESUMO

Heparanase (HPSE) and vascular endothelial growth factor (VEGF) are believed to play a vital role in hypoxia-induced retinal neovascularization (RNV). HPSE is a target gene of miR-429. Our study aimed to investigate the effect of the miR-429-HPSE-VEGF pathway on hypoxia-induced RNV. The gene and protein expression of miR-429, HPSE and VEGF in human retinal endothelial cells and retinas was determined by real-time PCR and Western blot assays. The effects of miR-429 on human retinal endothelial cells and retinal neovascularization under hypoxia condition were verified by in vitro and in vivo experiments. First, we studied the effect of the miR-429-HPSE-VEGF pathway in HRECs under hypoxic conditions. HREC functions such as migration and tube formation were enhanced under hypoxic conditions. Overexpression of miR-429 in HRECs reversed these changes. Then, we investigated the effect of miR-429 on hypoxia-induced RNV in vivo. When miR-429 agomirs were injected into the vitreous cavity of mice with oxygen-induced retinopathy to overexpress miR-429, the mRNA and protein expression of VEGF was significantly reduced. In addition, indicators of retinal neovascularization, such as the retinal avascular area, and morphology of vessels, were reduced significantly in the miR-429 overexpression group. In this study, our data showed that miR-429 plays an important role by inhibiting the HPSE-VEGF pathway in hypoxia-induced retinopathy.


Assuntos
MicroRNAs , Doenças Retinianas , Neovascularização Retiniana , Animais , Células Endoteliais/metabolismo , Glucuronidase , Humanos , Hipóxia/complicações , Hipóxia/genética , Hipóxia/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Oxigênio/metabolismo , RNA Mensageiro/metabolismo , Doenças Retinianas/metabolismo , Neovascularização Retiniana/genética , Neovascularização Retiniana/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
13.
Exp Eye Res ; 220: 109114, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35584758

RESUMO

Retinal neovascular diseases are major causes of blindness worldwide. As a common epitranscriptomic modification of eukaryotic RNAs, N6-methyladenosine (m6A) is associated with the pathogenesis of many diseases, including angiogenesis, through the regulation of RNA metabolism and functions. The aim of this study was to identify m6A modifications of mRNAs and long noncoding RNAs (lncRNAs) and determine their potential roles in retinal neovascularization. The transcriptome-wide m6A profiles of mRNAs and lncRNAs in the retinal tissues of mice with oxygen-induced retinopathy (OIR) and controls were identified by microarray analysis of immunoprecipitated methylated RNAs. The m6A methylation levels of mRNAs and lncRNAs identified in the microarray data were validated by MeRIP-qPCR. A total of 1321 mRNAs (151 hypermethylated and 1170 hypomethylated) and 192 lncRNAs (15 hypermethylated and 177 hypomethylated) were differentially methylated with the m6A modification in OIR and control mice. Gene ontology analysis showed that hypermethylated mRNAs were enriched in the regulation of multicellular organismal process, intracellular organelle, and protein binding, while hypomethylated mRNAs were enriched in cellular metabolic process, intracellular process, and binding. Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated that hypermethylated mRNAs were involved in dopaminergic synapses, glutamatergic synapse, and PI3K-Akt signaling pathway, while hypomethylated mRNAs were involved in autophagy, ubiquitin-mediated proteolysis, and spliceosome. Moreover, the altered levels of m6A methylation of ANGPT2, GNG12, ROBO4, and ENSMUST00000153785 were validated by MeRIP-qPCR. The results revealed an altered m6A epitranscriptome in OIR retinas. These methylated RNAs may act as novel modulators and targets in retinal neovascularization.


Assuntos
RNA Longo não Codificante , Neovascularização Retiniana , Adenosina/análogos & derivados , Animais , Perfilação da Expressão Gênica/métodos , Camundongos , Oxigênio/toxicidade , Fosfatidilinositol 3-Quinases/genética , RNA Longo não Codificante/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/genética , Neovascularização Retiniana/genética
14.
Int J Mol Sci ; 23(7)2022 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-35408803

RESUMO

Macular neovascularization type 3, formerly known as retinal angiomatous proliferation (RAP), is a hallmark of age-related macular degeneration and is associated with an accumulation of myeloid cells, such as microglia (MG) and infiltrating blood-derived macrophages (MAC). However, the contribution of MG and MAC to the myeloid cell pool at RAP sites and their exact functions remain unknown. In this study, we combined a microglia-specific reporter mouse line with a mouse model for RAP to identify the contribution of MG and MAC to myeloid cell accumulation at RAP and determined the transcriptional profile of MG using RNA sequencing. We found that MG are the most abundant myeloid cell population around RAP, whereas MAC are rarely, if ever, associated with late stages of RAP. RNA sequencing of RAP-associated MG showed that differentially expressed genes mainly contribute to immune-associated processes, including chemotaxis and migration in early RAP and proliferative capacity in late RAP, which was confirmed by immunohistochemistry. Interestingly, MG upregulated only a few angiomodulatory factors, suggesting a rather low angiogenic potential. In summary, we showed that MG are the dominant myeloid cell population at RAP sites. Moreover, MG significantly altered their transcriptional profile during RAP formation, activating immune-associated processes and exhibiting enhanced proliferation, however, without showing substantial upregulation of angiomodulatory factors.


Assuntos
Degeneração Macular , Neovascularização Retiniana , Animais , Proliferação de Células/genética , Angiofluoresceinografia , Degeneração Macular/complicações , Camundongos , Microglia , Neovascularização Patológica/complicações , Neovascularização Retiniana/genética , Tomografia de Coerência Óptica
15.
Arterioscler Thromb Vasc Biol ; 42(4): 407-427, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35236105

RESUMO

BACKGROUND: The major aim of this study is to investigate whether CDC6 (cell division cycle 6), a replication origin recognition complex component, plays a role in retinal neovascularization, and if so, to explore the underlying mechanisms. METHODS: In this study, we used a variety of approaches including cellular and moleculer biological methodologies as well as global and tissue-specific knockout mice in combination with an oxygen-induced retinopathy model to study the role of CDC6 in retinal neovascularization. RESULTS: VEGFA (vascular endothelial growth factor A)-induced CDC6 expression in a time-dependent manner in human retinal microvascular endothelial cells. In addition, VEGFA-induced CDC6 expression was dependent on PLCß3 (phospholipase Cß3)-mediated NFATc1 (nuclear factor of activated T cells c1) activation. Furthermore, while siRNA-mediated depletion of PLCß3, NFATc1, or CDC6 levels blunted VEGFA-induced human retinal microvascular endothelial cell angiogenic events such as proliferation, migration, sprouting, and tube formation, CDC6 overexpression rescued these effects in NFATc1-deficient mouse retinal microvascular endothelial cells. In accordance with these observations, global knockdown of PLCß3 or endothelial cell-specific deletion of NFATc1 or siRNA-mediated depletion of CDC6 levels substantially inhibited oxygen-induced retinopathy-induced retinal sprouting and neovascularization. In addition, retroviral-mediated overexpression of CDC6 rescued oxygen-induced retinopathy-induced retinal neovascularization from inhibition in PLCß3 knockout mice and in endothelial cell-specific NFATc1-deficient mice. CONCLUSIONS: The above observations clearly reveal that PLCß3-mediated NFATc1 activation-dependent CDC6 expression plays a crucial role in VEGFA/oxygen-induced retinopathy-induced retinal neovascularization.


Assuntos
Doenças Retinianas , Neovascularização Retiniana , Animais , Ciclo Celular , Proteínas de Ciclo Celular , Células Endoteliais , Camundongos , Camundongos Knockout , Proteínas Nucleares , Oxigênio , RNA Interferente Pequeno , Neovascularização Retiniana/genética , Fatores de Transcrição , Fator A de Crescimento do Endotélio Vascular
16.
Invest Ophthalmol Vis Sci ; 63(1): 27, 2022 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-35060996

RESUMO

Purpose: Erianin has been reported to inhibit tumor activity by suppressing the expression of integrins. It is hypothesized that erianin can inhibit retinal neovascularization in collagen by suppressing the expression of integrins. With an aim to test this hypothesis, the regulation of erianin on collagen-mediated retinal angiogenesis via the Ras homolog gene family member A (RhoA)/Rho-associated coiled-coil containing protein kinase 1 (ROCK1) signaling pathway induced by α2 and ß1 integrin-collagen interactions was investigated. Methods: The effects of erianin on human retinal vascular endothelial cells (HRVECs) were assessed in vitro using a hypoxia model in a three-dimensional cell culture induced by cobalt (II) chloride (CoCl2). A hypoxia-induced retinopathy model in adult zebrafish and zebrafish embryos was established to assess the antiangiogenic effect of erianin with and without vitreous collagen in vivo. The expression of α2 and ß1 integrin and RhoA/ROCK1 pathway in HRVECs and zebrafish retinas were analyzed. Results: In vitro, collagen improved the angiogenic potential of HRVECs, including migration, adhesion, and tube formation, in a three-dimensional cell culture model. Erianin suppressed the angiogenic processes of the CoCl2-induced hypoxia HRVEC model in a concentration-dependent manner. In vivo, erianin reduced retinal angiogenesis in the hypoxia-induced retinopathy model in adult and embryo zebrafish. Erianin inhibited the expression of α2 and ß1 integrin and RhoA/ROCK1 in a hypoxia-induced model in vitro in three-dimensional cell culture and in vivo in adult zebrafish. Conclusions: Collagen-mediated retinal angiogenesis may be regulated by erianin via the RhoA/ROCK1 signaling pathway induced by α2 and ß1 integrin-collagen interactions. These findings suggest that erianin has the therapeutic potential on intraocular collagen-mediated retinal angiogenesis.


Assuntos
Bibenzilas/farmacologia , Regulação da Expressão Gênica , Integrina alfa1/genética , Integrina beta1/genética , Proteínas Monoméricas de Ligação ao GTP/genética , Fenol/farmacologia , Neovascularização Retiniana/genética , Proteínas de Peixe-Zebra/genética , Quinases Associadas a rho/genética , Animais , Movimento Celular , Células Cultivadas , Colágeno/metabolismo , Modelos Animais de Doenças , Integrina alfa1/biossíntese , Integrina beta1/biossíntese , Proteínas Monoméricas de Ligação ao GTP/biossíntese , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/patologia , Transdução de Sinais , Peixe-Zebra , Proteínas de Peixe-Zebra/biossíntese , Quinases Associadas a rho/biossíntese
17.
Cell Mol Life Sci ; 79(1): 63, 2022 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-35006382

RESUMO

Conventional angiogenic factors, such as vascular endothelial growth factor (VEGF), regulate both pathological and physiological angiogenesis indiscriminately, and their inhibitors may elicit adverse side effects. Secretogranin III (Scg3) was recently reported to be a diabetes-restricted VEGF-independent angiogenic factor, but the disease selectivity of Scg3 in retinopathy of prematurity (ROP), a retinal disease in preterm infants with concurrent pathological and physiological angiogenesis, was not defined. Here, using oxygen-induced retinopathy (OIR) mice, a surrogate model of ROP, we quantified an exclusive binding of Scg3 to diseased versus healthy developing neovessels that contrasted sharply with the ubiquitous binding of VEGF. Functional immunohistochemistry visualized Scg3 binding exclusively to disease-related disorganized retinal neovessels and neovascular tufts, whereas VEGF bound to both disorganized and well-organized neovessels. Homozygous deletion of the Scg3 gene showed undetectable effects on physiological retinal neovascularization but markedly reduced the severity of OIR-induced pathological angiogenesis. Furthermore, anti-Scg3 humanized antibody Fab (hFab) inhibited pathological angiogenesis with similar efficacy to anti-VEGF aflibercept. Aflibercept dose-dependently blocked physiological angiogenesis in neonatal retinas, whereas anti-Scg3 hFab was without adverse effects at any dose and supported a therapeutic window at least 10X wider than that of aflibercept. Therefore, Scg3 stringently regulates pathological but not physiological angiogenesis, and anti-Scg3 hFab satisfies essential criteria for development as a safe and effective disease-targeted anti-angiogenic therapy for ROP.


Assuntos
Inibidores da Angiogênese/farmacologia , Cromograninas/imunologia , Cromograninas/metabolismo , Neovascularização Patológica/genética , Neovascularização Retiniana/patologia , Retinopatia da Prematuridade/patologia , Animais , Capilares/metabolismo , Cromograninas/antagonistas & inibidores , Cromograninas/genética , Modelos Animais de Doenças , Fragmentos Fab das Imunoglobulinas/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxigênio/efeitos adversos , Receptores de Fatores de Crescimento do Endotélio Vascular , Proteínas Recombinantes de Fusão/farmacologia , Neovascularização Retiniana/genética , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores
18.
Biofactors ; 48(3): 683-698, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35080047

RESUMO

Retinal neovascularization (RNV) associated diseases typically exhibit pathological neovascularization and neurodegeneration. Wnt inhibitor factor 1 (WIF1) is a secreted Wnt antagonist that regulates angiogenesis. However, the significance of WIF1 in RNV associated disease has not been explicitly tested. In our study, we found that the WIF1 expressions were strongly downregulated in the vitreous of proliferative diabetic retinopathy (PDR) and retinopathy of prematurity (ROP). Similarly, retinal WIF1 expression was significantly downregulated in OIR mice, relative to normal mice at P17. After injection of WIF1 overexpression lentivirus into the vitreous of OIR mice, overexpressing WIF1 in OIR mice vitreous strongly reduced avascular areas and neovascular tufts, increased vessel branches, raised a-, b-waves and oscillatory potentials amplitudes on ERG, increased retinal thickness and the number of synapses in retina, normalized the Golgi, mitochondria, and outer segments of photoreceptors. Furthermore, overexpression WIF1 suppressed expressions of ß-catenin, vascular endothelial growth factor (VEGF), p-AKT and p-ERK, reduced retinal reactive oxygen species (ROS) and 4-HNE levels, improved autophagic flux, and mitigated apoptosis. In summary, WIF1 plays a key role in alleviating angiogenesis and in improving visual function in OIR mice by suppressing the Wnt/ß-catenin-VEGF signaling pathway and ROS levels. WIF1 is an excellent candidate for targeted therapy against RNV associated diseases.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Neovascularização Patológica , Oxigênio , Neovascularização Retiniana , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Patológica/genética , Oxigênio/toxicidade , Espécies Reativas de Oxigênio/metabolismo , Neovascularização Retiniana/genética , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/patologia , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , beta Catenina/genética
19.
Zool Res ; 43(1): 64-74, 2022 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-34845879

RESUMO

Retinal angiogenesis is a critical process for normal retinal function. However, uncontrolled angiogenesis can lead to pathological neovascularization (NV), which is closely related to most irreversible blindness-causing retinal diseases. Understanding the molecular basis behind pathological NV is important for the treatment of related diseases. Twist-related protein 1 (TWIST1) is a well-known transcription factor and principal inducer of epithelial-mesenchymal transition (EMT) in many human cancers. Our previous study showed that Twist1 expression is elevated in pathological retinal NV. To date, however, the role of TWIST1 in retinal pathological angiogenesis remains to be elucidated. To study the role of TWIST1 in pathological retinal NV and identify specific molecular targets for antagonizing pathological NV, we generated an inducible vascular endothelial cell (EC)-specific Twist1 transgenic mouse model ( Tg-Twist1 iEC+ ). Whole-mount retinas from Tg-Twist1 iEC+ mice showed retarded vascular progression and increased vascular density in the front end of the growing retinal vasculature, as well as aneurysm-like pathological retinal NV. Furthermore, overexpression of Twist1 in the ECs promoted cell proliferation but disturbed cell polarity, thus leading to uncontrolled retinal angiogenesis. TWIST1 promoted pathological NV by activating the Wnt/ß-catenin signaling pathway and inducing the expression of NV formation-related genes, thereby acting as a 'valve' in the regulation of pathological angiogenesis. This study identified the critical role of TWIST1 in retinal pathological NV, thus providing a potential therapeutic target for pathological NV.


Assuntos
Neovascularização Patológica , Neovascularização Retiniana , Doenças dos Roedores , Animais , Células Endoteliais , Camundongos , Camundongos Transgênicos , Neovascularização Patológica/genética , Neovascularização Patológica/veterinária , Neovascularização Retiniana/genética , Neovascularização Retiniana/veterinária , Proteína 1 Relacionada a Twist/genética
20.
Pediatr Res ; 91(7): 1677-1685, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-34285351

RESUMO

BACKGROUND: Pathologic ocular neovascularization in retinopathy of prematurity (ROP) and other proliferative retinopathies are characterized by dysregulation of vascular endothelial growth factor-A (VEGF-A). A study of Vegfa isoform expression during oxygen-induced ischemic retinopathy (OIR) may enhance our understanding of Vegf dysregulation. METHODS: Following induction of OIR, immunohistochemistry and polymerase chain reaction (PCR) was performed on room air (RA) and OIR mice. RESULTS: Total Vegfa messenger RNA (mRNA) expression was stable in RA mice, but increased in OIR mice with a peak at postnatal day 17 (P17), before returning to RA levels. Vegfa164a expression was similar in both OIR and RA mice at P10 (Phase 1 OIR), but 2.4-fold higher in OIR mice compared to RA mice at P16 (Phase 2 OIR). At P10, Vegfa164b mRNA was similar in OIR vs RA mice, but was expressed 2.5-fold higher in OIR mice compared to RA mice at P16. At P10 and P16, Vegfr2/Vegfr1 expression was increased in OIR mice compared to RA mice. Increased activation of microglia was seen in OIR mice. CONCLUSIONS: Vegfa164a, Vegfa164b, and Vegfr1 were overexpressed in OIR mice, leading to abnormal signaling and angiogenesis. Further studies of mechanisms of Vegf dysregulation may lead to novel therapies for ROP and other proliferative retinopathies. IMPACT: Vegfa164 has two major isoforms, a proangiogenic, Vegfa164a, and an antiangiogenic, Vegfa164b, with opposing receptors, inhibitory Vegfr1, and stimulatory Vegfr2, but their role in OIR is unclear. In Phase 1 OIR, both isoforms and receptors are expressed similarly. In Phase 2 OIR, both isoforms are overexpressed, with an increased ratio of inhibitory Vegfr1. Modulation of angiogenesis by Vegf regulation enables pruning of excess angiogenesis during physiology, but results in ineffective angiogenesis during OIR. Knowledge of VEGF dysregulation may have novel therapeutic implications in the management of ROP and retinal proliferative diseases.


Assuntos
Neovascularização Retiniana , Retinopatia da Prematuridade , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Animais Recém-Nascidos , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Patológica/metabolismo , Oxigênio/uso terapêutico , Isoformas de Proteínas , RNA Mensageiro/metabolismo , Neovascularização Retiniana/genética , Retinopatia da Prematuridade/genética , Retinopatia da Prematuridade/patologia
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