Adiponectin, one of the adipose-derived hormone with metabolic activity, has been reported to conversely affect angiogenesis of endothelial cells in vitro. The previous study in animal models has demonstrated that adiponectin has a protective role in retinal vascular injury following pathological stimuli. However, clinical research regarding the relationship between plasma adiponectin level and diabetic retinopathy (DR) are inconclusive. The aim of this study was to investigate the effect of adiponectin on high glucose-induced retinal angiogenesis and its association with autophagy by using rhesus choroid-retinal endothelial (RF-6A) cells as a model. We found that cell vitality decreased and cell migration and tube formation increased in the high-glucose group. Treatment with adiponectin or 3-methyladenine (3-MA, an autophagy inhibitor) increased cell viability and inhibited cell migration and tube formation. In the high-glucose group, the protein expression of Bax and apoptosis rate of cells increased and the expression of Bcl-2 decreased, whereas treatment with adiponectin or 3-MA reversed these results. Autophagy was activated in the highglucose group to present as more LC3B fluorescent dots and higher expressions of LC3B, Atg5 proteins as well as lower expression of p62. Treatment with adiponectin or 3-MA inhibited autophagy by promoting the expression of p-PI3K, p-AKT, and p-mTOR when compared with the high-glucose group. The results of this study suggested that adiponectin inhibits high glucose-induced angiogenesis of RF/6A cells by inhibiting autophagy, and promotion of the PI3K/AKT/mTOR pathway might be involved in the anti-autophagy activities of adiponectin. K E Y W O R D S adiponectin, angiogenesis, apoptosis, autophagy, diabetic retinopathy
Retinal vascularization is arrested at the early (hyperoxia) stage in retinopathy of prematurity (ROP), a leading cause of blindness in children. Estrogen was reported to alleviate ROP by inhibiting reactive oxygen species, the upstream signaling molecules of endoplasmic reticulum stress (ERS). Astrocytes have long been proposed to guide angiogenesis, because they form a reticular network that provides a substrate for migrating endothelial cells. However, the factors that control the vascularization of the immature retina and the therapeutic mechanism of estrogen in early ROP remain poorly understood. This study aimed to investigate the role of G‐protein‐coupled estrogen receptor (GPER), an estrogen receptor distributed in the endoplasmic reticulum (ER), in protecting retinal astrocytes under hyperoxia and the association with ERS. The results showed that GPER was widely expressed in retinal astrocytes. GPER activation increases cell viability, decreases apoptosis, and autophagy of retinal astrocytes, decreases inositol‐1,4,5‐triphosphate receptor activity, and increases Ca2+ concentration in ER of astrocytes under hyperoxia. GPER blockade reversed all of these changes. Together, our findings indicate that GPER can protect the survival of retinal astrocytes by inhibiting ERS under hyperoxia.
Background To investigate the effect of ghrelin, a brain-gut peptide hormone, on high glucose-induced retinal angiogenesis in vitro and explore its association with endoplasmic reticulum (ER) stress. Methods Human retinal microvascular endothelial cells (HRMECs) were first divided into control and high-glucose groups, and the mRNA and protein expression levels of the receptor for ghrelin [growth hormone secretin receptor 1a, (GHSR-1a)] in cells were determined. HRMECs were then treated with high glucose alone or in combination with ghrelin or siGHSR-1a, and cell viability, migration, tube formation and the expression of the ER stress-related proteins PERK, ATF4 and CHOP were detected. Finally, to clarify whether the effects of ghrelin are related to ER stress, tunicamycin, an inducer of ER stress, was used to treat HRMECs, and cell viability, cell migration, and tube formation were evaluated. Results GHSR-1a expression in HRMECs at both the mRNA and protein levels was inhibited by high-glucose treatment. Under high-glucose conditions, ghrelin promoted cell viability and inhibited migration and tube formation, which were blocked by siGHSR-1a treatment. Ghrelin inhibited the increases in the protein levels of p-PERK, ATF4 and CHOP induced by high-glucose treatment, and combination treatment with siGHSR-1a reversed this effect of ghrelin. When tunicamycin was added, the effects of ghrelin on cell viability, migration and tube formation were all weakened. Conclusions This study experimentally revealed that ghrelin can inhibit high glucose-induced retinal angiogenesis in vitro through GHSR-1a, and alleviation of ER stress may be one of the mechanisms underlying this effect.
Abnormal development of immature retinal vascular structure in preterm infants under the condition of hyperoxia is the primary cause of retinopathy of prematurity (ROP), which has become the leading cause of blindness in children. Retinal ganglion cells (RGCs) play a critical role in the normal growth of retinal vessels. Previous studies have indicated that estrogen can alleviate retinal lesions in the ROP animal model by inhibiting reactive oxygen species, which is associated with endoplasmic reticulum (ER) stress. This study aimed to investigate the protecting effect of G‐protein coupled estrogen receptor (GPER), one of the estrogen receptors distributed in ER, on RGCs in the early stage of ROP and its relationship with ER stress. We found that GPER was widely expressed in primary cultured murine RGCs. GPER activation by its agonist G‐1 increased cell vitality and decreased apoptosis and autophagy of RGCs under hyperoxia. GPER activation by G‐1 decreased the expressions of the ER stress proteins, including inositol‐requiring kinase/endonuclease 1α, pancreatic ER stress kinase, and cleaved activating transcription factor 6 in ER of RGCs under hyperoxia. GPER activation decreased IP3R activity and increased Ca2+ concentration in ER of RGCs under hyperoxia. In addition, GPER antagonist (G‐15) reversed all these effects of the GPER agonist mentioned above. This study suggested that GPER activation can protect the survival of RGCs in the early stage of ROP via reducing ER stress in RGCs under the condition of hyperoxia.
Angiogenic factor with G patch and FHA domains 1 (AGGF1) has strong proangiogenic effects on embryonic vascular development and angiogenesis in disease; however, its role in retinopathy has not been elucidated. Retinopathy of prematurity is a serious retinal disorder of premature infants, which is caused by the arrest of immature retinal vascular growth under hyperoxia. This study aims to investigate the effects of AGGF1 on retinal vascular endothelial cells under hyperoxia and the association with autophagy by using rhesus macaque choroid‐retinal endothelial (RF/6A) cells. Western blot analysis and immunofluorescence staining were used to detect the expression of AGGF1 in RF/6A cells. Cell Counting Kit‐8, flow cytometry, and transwell and matrigel assays were applied to detect the vitality, apoptosis, migration, and tube formation of RF/6A cells, respectively. Western blot analysis was then used to detect the expression of autophagy markers LC3 and Beclin‐1, and mCherry‐GFP‐LC3 adenovirus was used to detect autophagy flux in RF/6A cells. Under hyperoxia, the expression of AGGF1 in RF/6A cells decreased compared with the control. Cell vitality, migration, and tube formation decreased, and apoptosis of RF/6A cells increased under hyperoxia, and these effects of hyperoxia were attenuated by AGGF1. The protein expressions of LC3 and Beclin‐1 increased in RF/6A cells and autophagy flux enhanced under hyperoxia. AGGF1 reduced the expression of LC3 and Beclin‐1 as well as the autophagy flux stimulated by hyperoxia. The results clearly showed that exogenous AGGF1 can protect retinal vascular endothelial cells and promote angiogenesis under hyperoxia, in which the expression of AGGF1 was inhibited. Inhibition of autophagy by AGGF1 may be one of the mechanisms involved.
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