Knockdown of IL4I1 Improved High Glucose-evoked Insulin Resistance in HepG2 Cells by Alleviating Inflammation and Lipotoxicity Through AHR Activation.

Insulin resistance (IR) is one of the leading causes of Type 2 diabetes mellitus (T2DM). Inflammation, as a result of the disordered immune response, plays important roles in IR and T2DM. Interleukin-4-induced gene 1 (IL4I1) has been shown to regulate immune response and be involved in inflammation progress. However, there was little known about its roles in T2DM. Here, high glucose (HG)-treated HepG2 cells were used for T2DM investigation in vitro. Our results indicated that the expression of IL4I1 was up-regulated in peripheral blood samples of T2DM-patients and HG-induced HepG2 cells. The silencing of IL4I1 alleviated the HG-evoked IR through elevating the expressions of p-IRS1, p-AKT and GLUT4, and enhancing glucose consumption. Furthermore, IL4I1 knockdown inhibited inflammatory response by reducing the levels of inflammatory mediators, and suppressed the accumulation of lipid metabolites triglyceride (TG) and palmitate (PA) in HG-induced cells. Notably, IL4I1 expression was positively correlated with aryl hydrocarbon receptor (AHR) in peripheral blood samples of T2DM-patients. The silencing of IL4I1 inhibited the AHR signaling by reducing the HG-induced expressions of AHR and CYP1A1. Subsequent experiments confirmed that 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), an agonist of AHR, reversed the suppressive effects of IL4I1 knockdown on HG-caused inflammation, lipid metabolism and IR in cells. In conclusion, we found that the silencing of IL4I1 attenuated inflammation, lipid metabolism and IR in HG-induced cells via inhibiting AHR signaling, suggesting that IL4I1 might be a potential therapy target for T2DM.

Different effects of anti-VEGF drugs (Ranibizumab, Aflibercept, Conbercept) on autophagy and its effect on neovascularization in RF/6A cells.

INTRODUCTION: Choroidal neovascularization (CNV) is the main pathological change of wet age-related macular degeneration. Anti-VEGF drugs are the most commonly used treatment for CNV. The biggest drawback of anti-VEGF drugs is the recurrence of CNV, which requires repeated therapy several times. Autophagy activation may be involved in reducing the therapeutic effect of anti-VEGF drugs. So, this study aims to elucidate the effect and mechanism of anti-VEGF drugs on endothelial autophagy and neovascularization in vitro. METHODS: RF/6A cells were randomly divided into five groups: The control group, hypoxia group (1% O2, 5% CO2, 94% N2), anti-VEGF group (group1: Ranibizumab 100 µg/ml; group2: Aflibercept, 400 µg/ml; group3: Conbercept, 100 µg/ml). Autophagy-related proteins were examined by Western blot. RFP-GFP-LC3 was used to detect autophagy and autophagic flow. Subsequently, we used autophagy inhibitors (3-MA or CQ) to inhibit Conbercept induced autophagy and to observe its effect on angiogenesis in vitro. Proliferation, migration, and tube formation of endothelial cells can be used to study neovascularization in vitro. In this research, the CCK-8 assay was used to detect cell proliferation. Cell migration and tube formation were assessed by wound assay and matrix method, respectively. Flow cytometry and Tunel were used to detect cell apoptosis. Finally, the mechanism of Conbercept activated autophagy was studied. Western blot was used to detect the expression of p53 and DRAM (damage-regulated autophagy modulator), upstream activators of autophagy. RESULTS: The protein levels of Beclin-1 and LC3-2/1 in Ranibizumab and Conbercept groups were significantly higher than in the hypoxia group(P < 0.05). While the expression of P62 was decreased (P < 0.05). The autophagic flux was showed the same results. However, Aflibercept showed the opposite effect on autophagy. Compared with the Conbercept group, autophagy inhibitor 3-MA or CQ can further inhibit cell proliferation and promotes cell apoptosis (P < 0.05). Conbercept significantly inhibited cell migration compared with the hypoxia group (633.08 ± 72.52 vs. 546.33 ± 24.61), while the autophagy inhibitor group (3-MA or CQ) had a more obvious inhibition effect (309.75 ± 86.36 and 263.33 ± 68.67) (P < 0.05). For tube formation, the number of tube formation was decreased significantly in the Conbercept group (32.00 ± 2.00) compared to the hypoxia group (39.00 ± 1.53) and even further reduced in 3-MA or CQ group (24.00 ± 3.61, 20.00 ± 2.65). The length of master segments in the hypoxia group was 15,668.00 ± 894.11. It was decreased in Conbercept (13,885.34 ± 730.03). In 3-MA or CQ group, the length of master segments dropped further (11,997.00 ± 433.66, 10,617.67 ± 543.21). Compare with the hypoxia group, the expression P53 and DRAM were increased in the Conbercept group (P < 0.05). Autophagy-related proteins LC-3, Beclin-1, and DRAM were inhibited by P53 inhibitor Pifithrin-α (PFTα) (P < 0.05). CONCLUSION: Ranibizumab and Conbercept can trigger the autophagy of vascular endothelial cells while Aflibercept can inhibit it. The combination of Conbercept and autophagy inhibitor can significantly inhibit the formation of angiogenesis in vitro. The mechanism of autophagy activation is related to the activation of the p53/DRAM pathway.

Angiogenic factor with G patch and FHA domains 1 protects retinal vascular endothelial cells under hyperoxia by inhibiting autophagy.

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.

Adiponectin inhibits high glucose-induced angiogenesis via inhibiting autophagy in RF/6A cells.

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 high-glucose 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.

Interleukin-6 and Diabetic Retinopathy: A Systematic Review and Meta-Analysis.

BACKGROUND: Interleukin-6 (IL-6) is produced by a variety of cells involved in inflammation and acts as local intensification signals in pathological processes associated with chronic eye inflammation. This meta-analysis was performed to provide a better understanding of the relationship between IL-6 and diabetic retinopathy. METHODS: The study was started with systematic search for literatures by using the PubMed, Web of Science and Embase online databases. The standard mean difference (SMD) and its 95% confidence intervals (CIs) were was included and then pooled with a random effects model. RESULTS: Thirty-one articles, containing1099 DR patients and 1010 controls, were included in this meta-analysis. The level of IL-6 in the DR group was found to be higher than that in the control group (SMD: 2.12, 95% CI: 1.53-2.70, p < 0.00001).Obvious heterogeneity existed between the studies (p < 0.00001, I2 = 96%). So a subgroup analysis and sensitivity analysis were performed. Removing the sensitivity studies, the stability of the overall treatment effect was good. Subgroup analysis showed that the levels of IL-6 in case group were observed to be higher than those in the control group; and the IL-6 levels in the proliferative diabetic retinopathy (PDR) group were also higher than those in the non-proliferative diabetic retinopathy (NPDR) group. (SMD: 0.78, 95% CI: 0.26-1.31, p=  0.003) Conclusion: The results from this current meta-analysis indicated that increased level of IL-6 generally exist in DR patients. And it may associated with the severity of DR. However, large-scale and high-quality studies in future are required to confirm the present findings.

Tumor necrosis factor-α and diabetic retinopathy: Review and meta-analysis.

BACKGROUND: Tumor necrosis factor-alpha (TNF-α) is produced by multinuclear giant cells and acts as local intensification signals in pathological processes associated with chronic eye inflammation. This meta-analysis was performed to provide a better understanding of the relationship between TNF-α and diabetic retinopathy (DR). METHOD: Online electric databases were searched to retrieve all relevant articles published before October 2017. The standard mean difference (SMD) and their 95% confidence intervals (CI) were included and then pooled with a random effects model. RESULTS: A total of 16 articles with 1286 participants were included in this meta-analysis. No difference in the level of TNF-α was found between DR patients and healthy controls (SMD = 0.39, 95% CI = -0.09 to 0.68, P = 0.01). Subgroup analysis showed that with respect to the level of TNF-α, the association was significant for studies conducted in Europe (SMD: 0.57, 95% CI: 0.11-1.02, P = 0.01), patients with type 1 DM (SMD: 1.06, 95% CI: 0.09-2.04, P = 0.03), studies based on serum samples (SMD: 0.57, 95% CI: 0.12-1.02, P = 0.01) and studies with a sample size >50 (SMD: 0.39, 95% CI: 0.03-0.75, P = 0.04). CONCLUSION: The results this meta-analysis indicated that the level of TNF-α in DR patients was significantly different from that in the healthy controls, so TNF-α represents a candidate biomarker for DR.

Manipulation of autophagy: a novelly potential therapeutic strategy for retinal neovascularization.

BACKGROUND: The relationship between the role of VEGF and autophagy in the process of retinal angiogenesis is still unclear. In this study, we explored this issue by using the mouse retinal vascular endothelial cell (RVEC) as a model. METHODS: RVECs were divided into the following groups: control, hypoxia (H), 3-methyladenine (3-MA) + H, VEGF + H, 3-MA + VEGF+H, anti-VEGF antibody + H, 3-MA+ anti-VEGF antibody + H. We then examined activation of autophagy by detecting formation of autophagosomes with transmission electron microscopy (TEM) and by counting the number of green fluorescent protein-positive (GFP+) puncta in RVECs. The turnover of microtubule associated protein 1 light chain 3 B (LC3B) and VEGF were examined by western blot. Cell migratory capacity was measured with wound healing assay and transwell assay. The capillary formation assay was performed to investigate the angiogenic capacity. RESULTS: Hypoxia led to an increased number of autophagosome and of the GFP+ puncta, an increased ratio of LC3B-II/I and enhanced migratory and capillary-formation capacities of RVECs. Pre-treatment with 3-MA attenuated activation of autophagy and abrogated the enhanced cell migration and capillary formation under hypoxia. Exposure to VEGF significantly increased migratory and capillary formation capacities of RVECs under hypoxia and 3-MA decreased VEGF-induced angiogenesis without its expression. Formation of autophagosome, the number of GFP+ puncta of RVECs and expression of LC3B-II/I were both elevated in cells treated with anti-VEGF antibody and these effects were partially inhibited by 3-MA pretreatment. CONCLUSION: Our present data may identify autophagic response as a novel target for enhancing the therapeutic efficacy of angiogenesis inhibitors.

Transient receptor potential vanilloid 4-expressing macrophages and keratinocytes contribute differentially to allergic and nonallergic chronic itch.

BACKGROUND: Chronic itch is a highly debilitating symptom that underlies many medical disorders with no universally effective treatments. Although unique neuronal signaling cascades in the sensory ganglia and spinal cord have been shown to critically promote the pathogenesis of chronic itch, the role of skin-associated cells remains poorly understood. OBJECTIVE: We sought to examine the cutaneous mechanisms underlying transient receptor potential vanilloid 4 (TRPV4)-mediated allergic and nonallergic chronic itch. METHODS: Expression of TRPV4 in chronic itch and healthy control skin preparations was examined by using real-time RT-PCR. Trpv4eGFP mice were used to study the expression and function of TRPV4 in the skin by means of immunofluorescence staining, flow cytometry, calcium imaging, and patch-clamp recordings. Genetic and pharmacologic approaches were used to examine the role and underlying mechanisms of TRPV4 in mouse models of dry skin-associated chronic itch and spontaneous scratching associated with squaric acid dibutylester-induced allergic contact dermatitis. RESULTS: TRPV4 is selectively expressed by dermal macrophages and epidermal keratinocytes in mice. Lineage-specific deletion of TRPV4 in macrophages and keratinocytes reduces allergic and nonallergic chronic itch in mice, respectively. Importantly, TRPV4 expression is significantly increased in skin biopsy specimens from patients with chronic idiopathic pruritus in comparison with skin from healthy control subjects. Moreover, TRPV4-dependent chronic itch requires 5-hydroxytryptamine (5-HT) signaling secondary to activation of distinct 5-HT receptors in mice with allergic and those with nonallergic chronic itch conditions. CONCLUSION: Our study reveals previously unrecognized mechanisms by which TRPV4-expressing epithelial and immune cells in the skin critically and dynamically mediate chronic itch and unravels novel targets for therapeutics in the setting of chronic itch.

Can Fundus Fluorescein Angiography be Performed for Diabetic Patients on Oral Metformin?.

Metformin is a kind of biguanide hypoglycemic agent that has been widely used in patients with diabetes mellitus. In clinical practice, whether metformin should be stopped before Fundus fluorescein angiography (FFA) remains largely unclear. Some endocrinologists suggest stop metformin before FFA. However, ophthalmologists do not always adopt this opinion in their practice. This situation may lead to disputes between physicians and patients. This article analyzed contrast-induced nephropathy(CIN) and the related contrast agent, as well as the adverse reactions of fluorescein angiography. It pointed out that the discrepancy may be caused by misunderstanding of contrast agents used in FFA. For angiography using iodine contrast agent, metformin must be stopped because of the increased possibility of CIN, while for FFA using fluorescein sodium, no CIN has been reported yet. Therefore, the authors believe FFA is safe for diabetic patients with oral metformin and it is unnecessary to stop metformin before the examination.

Role of Autophagy in Hypoxia-Induced Angiogenesis of RF/6A Cells in vitro.

PURPOSE: Autophagy plays a role in the pathogenesis of tumor angiogenesis and cardiovascular diseases. The autophagy level in mammalian cells was found to increase in the state of hypoxia or ischemia reperfusion. However, the role of autophagy in ocular angiogenesis has not yet been elucidated. The aim of this study was to investigate the effects of autophagy on hypoxia-induced choroidal and retinal angiogenesis in vitro using a rhesus macaque choroid-retinal endothelial (RF/6A) cell line. MATERIALS AND METHODS: RF/6A cells were cultured and randomly divided into three groups according to the different culture media: control, hypoxia model[adding 125µMcobalt chloride (CoCl2) to the culture medium], and hypoxia with an autophagy inhibition group [pretreatment with 5 mmol/L 3-methyladenine (3-MA) for 1.5 h and then adding125µmol/LCoCl2 to the culture medium]. The impact of 3-MA's effect on the level of autophagy proteins (Beclin-1 and LC3) was tested by Western blot analysis. The cell proliferation was assessed using the chromogenicmethylthiazol tetrazolium bromide (MTT) dye after 24 and 48 hours. Cell migration was investigated by wound assay. The tube formation was measured on Matrigel. RESULTS: Under chemical hypoxia conditions, Beclin-1 and LC3 levels increased and this change can be inhibited by 3-MA. Cell viability was decreased in cells treated with CoCl2 for 24 and 48 h compared with the control, and pretreatment with 3-MA slightly promoted CoCl2-inhibited cell proliferation. Cell migration and tube formation were increased in cells treated with CoCl2 for 24 and 48 h compared with the control. Pretreatment with 3-MA significantly inhibited CoCl2-induced cell migration and tube formation. CONCLUSIONS: Hypoxia-induced autophagy decreased the cell viability and increased the cell migration and tube formation of RF/6A cells. 3-MA can inhibit hypoxia-induced angiogenesis of RF/6A cells in vitro. The present study suggests that autophagy plays a role in retinal and choroidal angiogenesis and the autophagy inhibitor can be a potential candidate for the treatment of choroidal or retinal neovascularization.

Increased Serum Chemerin Levels in Diabetic Retinopathy of Type 2 Diabetic Patients.

PURPOSE: To compare serum levels of chemerin in type 2 diabetes mellitus (T2DM) with or without retinopathy, and to investigate the relationship between serum chemerin levels and diabetes retinopathy. MATERIALS AND METHODS: A total of 60 T2DM patients and 20 healthy subjects (control group) were enrolled in this study. Of the T2DM patients, 15 had proliferative diabetic retinopathy (PDR group), 20 had non-proliferative retinopathy (NPDR group) and 25 had no retinopathy (T2DM group). Their serum samples were collected for testing the levels of chemerin, vascular endothelial growth factor (VEGF), C-reactive protein (CRP) and so on. The values were analyzed to compare the differences among the groups. Simple linear regression analysis and multiple stepwise linear regression analysis were used to determine the correlations between variables and chemerin. Trend chi-square was used to determine the correlations between chemerin and the severity of diabetic retinopathy (DR). RESULTS: Chemerin levels in group PDR, NPDR and no DR were 147.56 ± 35.98 µg/l, 128.09 ± 16.33 µg/l and 113.19 ± 19.89 µg/l, with the significant difference across the three groups (p < 0.05). But there was no difference between control group (109.55 ± 20.98 µg/l) and T2DM group. Simple linear regression show that serum chemerin was correlated with duration of diabetes, body mass index (BMI), serum triglycerides, total-cholesterol, CRP and VEGF, and not correlated with age, systolic and diastolic blood pressure in T2DM patients. Stepwise regression analysis showed that BMI, CRP and VEGF were significantly associated with serum chemerin (p = 0.006, p = 0.011 and p = 0.036, respectively). In addition, the more severity of DR as the chemerin levels increased (χ(2) = 16.07, p < 0.001). CONCLUSIONS: Serum levels of chemerin were significantly increased in the NPDR and PDR group. Elevated serum level of chemerin and its positive correlation with BMI, CRP and VEGF suggested that chemerin was associated with obesity, inflammation and neovascularization and might be involved in the development of DR.

Retinoids activate the irritant receptor TRPV1 and produce sensory hypersensitivity.

Retinoids are structurally related derivatives of vitamin A and are required for normal vision as well as cell proliferation and differentiation. Clinically, retinoids are effective in treating many skin disorders and cancers. Application of retinoids evokes substantial irritating side effects, including pain and inflammation; however, the precise mechanisms accounting for the sensory hypersensitivity are not understood. Here we show that both naturally occurring and synthetic retinoids activate recombinant or native transient receptor potential channel vanilloid subtype 1 (TRPV1), an irritant receptor for capsaicin, the pungent ingredient of chili peppers. In vivo, retinoids produced pain-related behaviors that were either eliminated or significantly reduced by genetic or pharmacological inhibition of TRPV1 function. These findings identify TRPV1 as an ionotropic receptor for retinoids and provide cellular and molecular insights into retinoid-evoked hypersensitivity. These findings also suggest that selective TRPV1 antagonists are potential therapeutic drugs for treating retinoid-induced sensory hypersensitivity.