INTRODUCTION:Epithelial Ovarian Cancer (EOC) is a highly aggressive gynecological malignancy with a high mortality rate primarily due to late-stage diagnosis and metastatic dissemination. Regulatory T cells (Tregs) have emerged as critical mediators of immune evasion, yet the role of Foxp3+ Tregs in modulating Tumor-Associated Macrophage (TAM) polarization and the underlying molecular mechanisms in EOC remains unclear. METHODS:An orthotopic EOC mouse model and in vitro co-culture systems were employed to investigate the effects of Foxp3+ Tregs on TAM polarization. Quantitative Real-Time PCR (qRTPCR), flow cytometry, Western blotting, wound healing, and transwell assays were performed to assess gene expression, immune cell infiltration, and tumor cell migration/invasion. Foxp3 knockdown was achieved using Adeno-Associated Virus (AAV)-mediated delivery to evaluate its effects in vivo. RESULTS:Foxp3+ Tregs induced macrophage polarization toward the M2 phenotype, characterized by downregulation of M1 markers (IL-1β, iNOS) and upregulation of M2 markers (IL-10, Arg-1). Mechanistically, Foxp3+ Tregs activated the Sirt1-ERK1/2-STAT3 signaling pathway while suppressing NF-κB activity. In vitro, Foxp3+ Tregs enhanced the migratory and invasive capacities of ovarian cancer cells, whereas in vivo Foxp3 knockdown significantly reduced tumor growth and M2 macrophage infiltration. DISCUSSION:These findings suggest that Foxp3+ Tregs play a pivotal role in shaping the immunosuppressive tumor microenvironment in EOC by promoting M2 macrophage polarization through Sirt1-ERK1/2-STAT3 signaling and NF-κB suppression, ultimately facilitating tumor progression. CONCLUSION:Foxp3+ Tregs drive immunosuppressive macrophage polarization and ovarian cancer progression, highlighting Foxp3 as a potential therapeutic target for EOC treatment.

Glucose metabolic reprogramming describes the alterations in intracellular metabolic pathways in response to variations in the body’s internal environment. This metabolic reprogramming has been the subject of extensive research. The primary function is to enhance glycolysis for rapid ATP production, even with sufficient oxygen, leading to a significant accumulation of lactic acid, which subsequently affects the functions of tumor cells and immune cells within TME. Lactylation represents a newly identified post-translational modification (PTM) that occurs due to lactate accumulation and is observed in various proteins, encompassing both histone and non-histone types. Lactylation alters the spatial configuration of proteins, influences gene transcription, and thereby regulates gene expression. This modification serves as a significant epigenetic regulatory factor in numerous diseases. Glucose metabolic reprogramming and lactylation are intricately linked in the process of tumorigenesis. Glucose reprogramming activates essential enzymes, including hexokinase 2 (HK2), pyruvate kinase M2 (PKM2), and lactate dehydrogenase A (LDHA), through transcription factors such as HIF-1α and c-Myc, thereby enhancing glycolysis and lactate accumulation. Lactate functions as a metabolite and signaling molecule, acting as a substrate for lactylation facilitated by histone acetyltransferases such as CBP/p300. This epigenetic modification inhibits antitumor immunity through the upregulation of oncogenic signaling pathways, the induction of M2-type macrophage polarization, and the dysfunction of T-cells. Glucose metabolic reprogramming not only influences lactate synthesis but also provides sufficient substrates for lactate modification. The two factors jointly affect gene expression and protein function, acidify the tumor microenvironment, regulate immune evasion, and promote carcinogenesis. This review systematically details the mechanisms of lactylation and glucose metabolic reprogramming, their impacts on immune cells within the tumor microenvironment, and their interrelations in tumor progression, immunity, and inflammation.

Our previous research identified pathogenic variants in RetNet genes in 23.4% of individuals with early-onset high myopia. This study aims to analyze the genetic defects in patients with high myopia complicated by rhegmatogenous retinal detachment. Whole-exome sequencing was performed on 40 patients with high myopia accompanied by retinal detachment. Variants were filtered from 281 RetNet genes, 178 genes related to syndromic high myopia, 23 non-syndromic high myopia-associated genes, and 29 rhegmatogenous retinal detachment-related genes using a multistep bioinformatics approach. Clinical data were collected for genotype-phenotype correlation analysis. Pathogenic variants were detected in 47.5% (19/40) in patients with high myopia accompanied by retinal detachment, specifically in RetNet genes (18/40), rhegmatogenous retinal detachment-related genes (11/40), and syndromic high myopia associated genes (10/40). No variants were found in non-syndromic genes. The most prevalent pathogenic genes for high myopia with retinal detachment were Stickler-related genes, including COL2A1 (10.0%, 4/40) and COL11A1 (5.0%, 2/40). Patients with Stickler-related gene variants presented the youngest average age of retinal detachment onset (35.17 ± 18.03 years) and shortest axial length (27.63 ± 1.01 mm). RetNet genes are the predominant causative genes (18/40, 45.0%) in patients with high myopia and retinal detachment. The findings affirm that Stickler syndrome (15%) is a significant etiological factor for high myopia accompanied by retinal detachment. We recommend enhanced comprehensive systemic and ophthalmic examinations for patients with high myopia to enable early detection and prevention of retinal detachment.

BACKGROUND The relationship between Alzheimer's disease biomarkers and postoperative complications, such as postoperative delirium (POD) and postoperative cognitive dysfunction (POCD), remains a subject of ongoing debate. OBJECTIVE This meta-analysis aimed to determine whether there is an association between perioperative Alzheimer's disease biomarkers and postoperative complications. DESIGN We conducted a meta-analysis of observational clinical studies that explored the correlation between Alzheimer's disease biomarkers and POD or POCD in patients who have undergone surgery, following PRISMA guidelines. The protocol was previously published (INPLASY: INPLASY202350001). DATA SOURCES A comprehensive search was conducted across PubMed, Embase, Web of Science, and Cochrane databases until March 2023. ELIGIBILITY CRITERIA Surgical patients aged at least 18 years, studies focusing on POD or POCD, research involving Alzheimer's disease biomarkers, including Aβ or tau in blood or cerebrospinal fluid (CSF), and availability of the full text. RESULTS Our meta-analysis included 15 studies: six focusing on POD and nine on POCD. The findings revealed a negative correlation between preoperative CSF β-amyloid 42 (Aβ42) levels and the onset of POD [mean difference −86.1, 95% confidence interval (CI), −114.15 to −58.05, I 2 : 47%]; this association was strongly supported by trial sequential analysis (TSA). A similar negative correlation was discerned between preoperative CSF Aβ42 levels and the incidence of POCD (−165.01, 95% CI, −261.48 to −68.53, I 2 : 95%). The TSA also provided robust evidence for this finding; however, the evidence remains insufficient to confirm a relationship between other Alzheimer's disease biomarkers [β-amyloid 40 (Aβ40), total tau (T-tau), phosphorylated tau (P-tau), and Aβ42/T-tau ratio] and POD or POCD. CONCLUSION The study results indicate a negative correlation between preoperative CSF Aβ42 levels and the occurrence of both POD and POCD. Future investigations are warranted to identify the predictive cutoff value of preoperative CSF Aβ42 for POD and POCD.

Diabetic retinopathy (DR) is characterized by chronic, low-grade inflammation. This state may be related to the heightened production of neutrophil extracellular traps (NETs) induced by high glucose (HG). Human cathelicidin antimicrobial peptide (LL37) is an endogenous ligand of G protein-coupled chemoattractant receptor formyl peptide receptor 2 (FPR2), expressed on neutrophils and facilitating the formation and stabilization of the structure of NETs. In this study, we detected neutrophils cultured under different conditions, the retinal tissue of diabetic mice, and fibrovascular epiretinal membranes (FVM) samples of patients with proliferative diabetic retinopathy (PDR) to explore the regulating effect of LL37/FPR2 on neutrophil in the development of NETs during the process of DR. Specifically, HG or NG with LL37 upregulates the expression of FPR2 in neutrophils, induces the opening of mitochondrial permeability transition pore (mPTP), promotes the increase of reactive oxygen species and mitochondrial ROS, and then leads to the rise of NET production, which is mainly manifested by the release of DNA reticular structure and the increased expression of NETs-related markers. The PI3K/AKT signaling pathway was activated in neutrophils, and the phosphorylation level was enhanced by FPR2 agonists in vitro. In vivo, increased expression of NETs markers was detected in the retina of diabetic mice and in FVM, vitreous fluid, and serum of PDR patients. Transgenic FPR2 deletion led to decreased NETs in the retina of diabetic mice. Furthermore, in vitro, inhibition of the LL37/FPR2/mPTP axis and PI3K/AKT signaling pathway decreased NET production induced by high glucose. These results suggested that FPR2 plays an essential role in regulating the production of NETs induced by HG, thus may be considered as one of the potential therapeutic targets.

Inrtoduction In proliferative diabetic retinopathy (PDR), retinal neovascularization is the essential pathogenic process that is linked to endothelial-to-mesenchymal transition (EndoMT) induced by high glucose (HG). This pathophysiological process may be regulated by a G-protein-coupled chemoattractant receptor FPR2 (mouse Fpr2), involved in inflammatory cell migration and proliferation. In the current study, we investigated the role of Fpr2 in regulating EndoMT and the underlying mechanisms during diabetic retinopathy progression.Methods FPR2 agonist or inhibitor was added to human microvascular endothelial cells (HMECs) exposed to normal glucose (NG) or HG. Morphologic, phenotypic, and functional changes of HMECs as well as the formation of microvasculature related to EndoMT were assessed. EndoMT biomarkers were detected in the retinal tissues of diabetic mice and fibrovascular epiretinal membranes (FVM) from patients with PDR.Results HG upregulated FPR2 in HMECs, which triggered morphological changes and the cells acquired mesenchymal phenotype, with enhanced cell migration, viability and angiogenic process shown by tube formation and aortic ring sprouting. Inhibition of FPR2 attenuated HG-induced EndoMT and endothelial cell migration to form vessel-like tube structures. RNA sequence and protein analysis further revealed that inhibition of FPR2 decreased the expression of genes associated with EndoMT. ERK 1/2 and P38 signaling pathway was activated in HMECs, promoting neovascularization in HG-induced EndoMT of HMECs. In vivo, increased expression of mesenchymal markers was detected in the retina of diabetic mice and FVMs from patients with PDR. FPR2 deficiency was associated with diminished EndoMT-related phenotypic changes in the retina of diabetic mice.Conclusions FPR2 is actively involved in the progression of EndoMT that may contribute to the pathogenesis of PDR. Thus, FPR2 may be a potential therapeutic target for PDR.

To find out the incidence and risk factors of opaque bubble layer (OBL) in eyes with myopia and myopic astigmatism following femtosecond laser–assisted in situ keratomileusis (FS-LASIK) and small incision lenticule extraction (SMILE). A total of 1076 eyes from 569 patients who had FS-LASIK or SMILE were included in the retrospective research. For each kind of surgery, eyes were separated into two groups: "OBL" groups and "no OBL" groups. In the FS-LASIK group, eyes that developed OBL were split into "hard OBL" and "soft OBL" groups. The incidence and size of OBL were analyzed after watching the surgical procedure videotaped during the operation and taking screenshots. Surgical parameters, including sphere, cylinder, keratometry, corneal thickness, flap thickness, cap thickness, lenticule thickness, and visual acuity, were compared. In the FS-LASIK surgery, the incidence of OBL was 63.2

Introduction: In proliferative diabetic retinopathy (PDR), retinal neovascularization is the essential pathogenic process that is linked to endothelial-to-mesenchymal transition (EndoMT) induced by high glucose (HG). This pathophysiological process may be regulated by a G-protein-coupled chemoattractant receptor FPR2 (mouse Fpr2), involved in inflammatory cell migration and proliferation. In the current study, we investigated the role of Fpr2 in regulating EndoMT and the underlying mechanisms during diabetic retinopathy progression. Methods: FPR2 agonist or inhibitor was added to human microvascular endothelial cells (HMECs) exposed to normal glucose or HG. Morphologic, phenotypic, and functional changes of HMECs as well as the formation of microvasculature related to EndoMT were assessed. EndoMT biomarkers were detected in the retinal tissues of diabetic mice and fibrovascular epiretinal membranes (FVMs) from patients with PDR. Results: HG upregulated FPR2 in HMECs, which triggered morphological changes, and the cells acquired mesenchymal phenotype, with enhanced cell migration, viability, and angiogenic process shown by tube formation and aortic ring sprouting. Inhibition of FPR2 attenuated HG-induced EndoMT and endothelial cell migration to form vessel-like tube structures. RNA sequence and protein analysis further revealed that inhibition of FPR2 decreased the expression of genes associated with EndoMT. ERK1/2 and P38 signaling pathway was activated in HMECs, promoting neovascularization in HG-induced EndoMT of HMECs. In vivo, increased expression of mesenchymal markers was detected in the retina of diabetic mice and FVMs from patients with PDR. FPR2 deficiency was associated with diminished EndoMT-related phenotypic changes in the retina of diabetic mice. Conclusions: FPR2 is actively involved in the progression of EndoMT that may contribute to the pathogenesis of PDR. Thus, FPR2 may be a potential therapeutic target for PDR.

To evaluate the incidence of an opaque bubble layer (OBL) in femtosecond laser–assisted in situ keratomileusis (FS-LASIK) flaps created with VisuMax Flap 2.0 as a result of a modification in the parameters of the flap programming. This retrospective study was comprised of 1400 eyes of 715 patients who received FS-LASIK surgery. OBLs were measured and reported as a percentage of the flap area to identify the incidence and extent. Flap creation, which is a modification technique, was performed with 8.1-mm flap diameters plus 0.3-mm enlarged interlamellar photodisruption (group Flap 2.0). The same flap diameters without extra photodisruption as the previous standard setting were also implemented (group Flap 1.0). The preoperative measurements, including sphere, cylinder, keratometry, and intraoperative characteristics such as flap size and thickness, were documented. Possible risk factors for the occurrence of OBLs were investigated in this study. The incidence of an OBL was reduced when using the Flap 2.0 program (31.4

目的 探讨甲酰肽受体Fpr2在氧诱导视网膜病变(OIR)小鼠中对胶质细胞改变的影响,并探讨其在新生血管形成中的作用及机制.方法 将新生C57BL/6J小鼠、Fpr2-/-小鼠分别诱导建立OIR模型后分为氧诱导组和Fpr2-/-氧诱导组.正常对照组和Fpr2-/-组小鼠正常环境饲养.收集各组小鼠视网膜组织并制作成冰冻切片,免疫荧光染色观察小鼠视网膜中波形蛋白(Vimentin)、神经胶质纤维酸性蛋白(GFAP)和离子化钙结合适配分子1(IBA1)变化;实时荧光定量PCR(RT-PCR)检测小鼠视网膜中炎症因子mRNA的表达;Western blot检测小鼠视网膜中细胞外调节蛋白激酶(ERK)和P38的蛋白磷酸化表达.免疫荧光染色法观察小鼠视网膜中新生血管和无灌注区情况.结果 免疫荧光染色结果显示,与正常对照组相比,氧诱导组和Fpr2-/-氧诱导组小鼠视网膜中Vimentin、GFAP、IBA1表达均明显增加,而与氧诱导组相比,Fpr2-/-氧诱导组小鼠视网膜中Vimentin、GFAP、IBA1表达均降低(均为P<0.05).RT-PCR检测结果显示,与Fpr2-/-氧诱导组相比,氧诱导组小鼠视网膜中白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)mRNA相对表达量均增加(均为P<0.05).Western blot检测结果显示,与正常对照组相比,氧诱导组和Fpr2-/-氧诱导组小鼠视网膜中p-E RK 1/2和p-P38蛋白相对表达量均增加,而与氧诱导组相比,Fpr2-/-氧诱导组小鼠视网膜中p-ERK 1/2和p-P38蛋白相对表达量均减少(均为P<0.05).与氧诱导组相比,Fpr2-/-氧诱导组小鼠视网膜中新生血管形成和无灌注区面积均减少(均为P<0.05).结论 抑制Fpr2能够抑制OIR模型小鼠视网膜胶质细胞活化,下调炎症因子表达,减少视网膜新生血管形成和无灌注区面积,此过程中涉及E RK、P38信号通路的作用.

Diabetic retinopathy (DR) as a retinal neovascularization-related disease is one of the leading causes of irreversible blindness in patients. The goal of this study is to determine the role of a G-protein-coupled chemoattractant receptor (GPCR) FPR2 (mouse Fpr2) in the progression of DR, in order to identify novel therapeutic targets. We report that Fpr2 was markedly upregulated in mouse diabetic retinas, especially in retinal vascular endothelial cells, in associated with increased number of activated microglia and Muller glial cells. In contrast, in the retina of diabetic Fpr2(-/-) mice, the activation of vascular endothelial cells and glia was attenuated with reduced production of proinflammatory cytokines. Fpr2 deficiency also prevented the formation of acellular capillary during diabetic progression. Furthermore, in oxygen-induced retinopathy (OIR) mice, the absence of Fpr2 was associated with diminished neovasculature formation and pathological vaso-obliteration region in the retina. These results highlight the importance of Fpr2 in exacerbating the progression of neuroglial degeneration and angiogenesis in DR and its potential as a therapeutic target.

Of the seven P2X receptor subtypes, P2X4 receptor (P2X4R) is widely distributed in the central nervous system, including in neurons, astrocytes, and microglia. Accumulating evidence supports roles for P2X4R in the central nervous system, including regulating cell excitability, synaptic transmission, and neuropathic pain. However, little information is available about the distribution and function of P2X4R in the peripheral nervous system. In this study, we find that P2X4R is mainly localized in the lysosomes of Schwann cells in the peripheral nervous system. In cultured Schwann cells, TNF-a not only enhances the synthesis of P2X4R protein but also promotes P2X4R trafficking to the surface of Schwann cells. TNF-a-induced BDNF secretion in Schwann cells is P2X4R dependent. in vivo experiments reveal that expression of P2X4R in Schwann cells of injured nerves is strikingly upregulated following nerve crush injury. Moreover, overexpression of P2X4R in Schwann cells by genetic manipulation promotes motor and sensory functional recovery and accelerates nerve remyelination via BDNF release following nerve injury. Our results suggest that enhancement of P2X4R expression in Schwann cells after nerve injury may be an effective approach to facilitate the regrowth and remyelination of injured nerves.

Purpose:Age-related macular degeneration (AMD) is the leading cause of central visual loss among patients over the age of 55 years worldwide. Neovascular-type AMD (nAMD) accounts for approximately 10% of patients with AMD and is characterized by choroidal neovascularization (CNV). The proliferation of choroidal endothelial cells (CECs) is one important step in the formation of new vessels. Transcriptional coactivator Yes-associated protein (YAP) can promote the proliferation of multiple cancer cells, corneal endothelial cells, and vascular smooth muscle cells, which participate in angiogenesis. This study intends to reveal the expression and functions of YAP during the CNV process.Methods:In the study, a mouse CNV model was generated by laser photocoagulation. YAP expression was detected with western blotting and immunohistochemistry. YAP siRNA and ranibizumab, a VEGF monoclonal antibody, were injected intravitreally in CNV mice. The YAP and VEGF expression levels after injection were detected with western blotting. The incidence and leakage area of CNV were measured with fundus fluorescein angiography, choroidal flat mounting, and hematoxylin and eosin (HE) staining. Immunofluorescent double staining was used to detect YAP cellular localization with CD31 (an endothelial cell marker) antibody. Proliferating cell nuclear antigen (PCNA) expression in CNV mice without or with YAP siRNA intravitreal injection and the colocalization of PCNA and CD31 were measured with western blotting and immunofluorescent double staining, respectively.Results:YAP expression increased following laser exposure, in accordance with vascular endothelial growth factor (VEGF) expression. YAP siRNA and ranibizumab decreased VEGF expression and the incidence and leakage area of CNV. YAP was localized in the vascular endothelium within the CNV site. Additionally, after laser exposure, YAP siRNA inhibited the increased expression of PCNA, which was colocalized with endothelial cells.Conclusions:This study showed that YAP upregulation promoted CNV formation by upregulating the proliferation of endothelial cells, providing evidence for the molecular mechanisms of CNV and suggesting a novel molecular target for nAMD treatment.

In proliferative diabetic retinopathy (PDR), activated Müller glial cells (MGCs) exhibit increased motility and a fibroblast-like proliferation phenotype that contribute to the formation of fibrovascular membrane. In this study, we investigated the capacity of high glucose (HG) to regulate the expression of cell surface receptors that may participate in the proinflammatory responses of MGCs. We found that MGCs express a G-protein coupled chemoattractant receptor formyl peptide receptor 2 (Fpr2) and fibroblast growth factor receptor 1 (FGFR1), which mediated MGC migration and proliferation in response to corresponding ligands. HG upregulated Fpr2 through an NF-κB pathway in MGCs, increased the activation of MAPKs coupled to Fpr2 and FGFR1, which also further enhanced the production of vascular endothelial growth factor by MGCs in the presence of HG. In vivo, Fpr2 was more highly expressed by retina MGCs of diabetic mice and the human counterpart FPR2 was detected in the retina MGCs in fibrovascular membrane of PDR patients. To support the potential pathological relevance of Fpr2, an endogenous Fpr2 agonist cathelin-related antimicrobial peptide was detected in mouse MGCs and the retina, which was upregulated by HG. These results suggest that Fpr2, together with FGFR1, may actively participate in the pathogenesis of PDR thus may be considered as one of the potential therapeutic targets.

Purpose. To evaluate decentration following femtosecond laser small incision lenticule extraction (SMILE) and sub-Bowman keratomileusis (SBK) and its impact on higher-order aberrations (HOAs). Methods. Prospective, nonrandom, and comparison study. There were 96 eyes of 52 patients who received SMILE and 96 eyes of 49 patients who received SBK in this study. Decentration was calculated 6 months after surgery with Pentacam. HOAs and visual acuity after the surgery were examined for patients in both groups before and 6 months after surgery. Results. The mean decentration displacement in SMILE group was significantly less than SBK group (P = 0.020). 89 eyes were decentered within 0.50 mm after SMILE and SBK. The association between vertical decentration and the induced spherical aberration was insignificant in SMILE group (P = 0.035). There was an association between decentration and safety index, efficacy index, vertical coma, spherical aberration, and HOAs in root mean square (RMS, μm) after SBK (all P < 0.05). No difference was found in uncorrected and corrected distance visual acuity, safety index, efficacy index, and wavefront aberrations between the two subgroups at any delimited value after SMILE (all P > 0.05). Decentration exceeding 0.37 mm affected vertical coma and RMSh of SBK eyes (P = 0.002, 0.005). Conclusion. SMILE surgery achieved more accurate centration than SBK surgery. Vertical decentration is associated with the induced spherical aberration in SMILE.

Cell cycle re-entry, in which Fra-1 (transcription factor FOS-related antigen 1) plays an important role, is a key process in neuronal apoptosis. However, the expression and function of Fra-1 in retinal ganglion cell (RGC) apoptosis are unknown. To investigate whether Fra-1 was involved in RGC apoptosis, we performed a light-induced retinal damage model in adult rats. Western blot revealed that up-regulation of Fra-1 expression appeared in retina after light exposure (LE). Immunostaining indicated that increased Fra-1 was mainly expressed in RGCs in retinal ganglion cell layer (GCL) after LE. Co-localization of Fra-1 with active caspase-3 or TUNEL-positive cells in GCL after LE was also detected. In addition, Fra-1 expression increased in parallel with cyclin D1 and phosphorylated mitogen-activated protein kinase p38 (p-p38) expression in retina after LE. Furthermore, Fra-1, cyclin D1, and active caspase-3 protein expression decreased by intravitreal injection of SB203580, a highly selective inhibitor of p38 MAP kinase (p38 MAPK). All these results suggested that Fra-1 may be associated with RGC apoptosis after LE regulated by p38 MAPK through cell cycle re-entry mechanism.

Immune responses play a key role in the pathogenesis and progression of polypoidal choroidal vasculopath (PCV) and age-related macular degeneration (AMD). In this study, we determined the Th cell-associated immune responses by measuring the cytokine expression of peripheral blood mononuclear cells (PBMC) in both PCV and neovascular AMD (nAMD) patients.

Purpose The purpose of the present study was to investigate the potential signal mechanism of tissue factor (TF) in the regulation of the expression of vascular endothelial growth factor (VEGF) in human retinal pigment epithelial (ARPE-19) cells. Methods An in vitro RPE cell chemical hypoxia model was established by adding cobalt chloride (CoCl2) in the culture medium. The irritative concentration of CoCl2 was determined with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay kit. VEGF production in ARPE-19 cells was measured with enzyme-linked immunosorbent assay (ELISA) and western blotting. The Wnt signaling pathway–associated molecules, including phospho-glycogen synthase kinase 3β (p-GSK3β), GSK3β, p-β-catenin and β-catenin, were detected with western blotting. pEGFP-N3-hTF was constructed and verified with digestion of the restriction enzyme and sequencing analysis. Human TF overexpression and silencing plasmids were transfected into the ARPE-19 cells to clarify the causal relationship between TF and VEGF expression. The Transwell coculture system of ARPE-19 cells and RF/6A rhesus macaque choroid–retinal endothelial cells was performed to evaluate cell invasion and tube formation ability. Results Our anoxic model of ARPE-19 cells showed that TF expression was upregulated in accordance with variations in hypoxia-inducible factor 1-alpha (HIF-1α) and VEGF levels. Silencing and overexpression of TF decreased and increased VEGF expression, respectively. The Wnt/β-catenin signaling pathway played an important role in this effect. Results from the ARPE-19 cell and RF/6A cell coculture system showed that the enhancement of TF expression in the ARPE-19 cells led to significantly faster invasion and stronger tube-forming ability of the RF/6A cells, while siRNA-mediated TF silencing caused the opposite effects. Pharmacological disruption of Wnt signaling IWR-1-endo inhibited the effects compared to the TF-overexpressing group, indicating the importance of the Wnt/β-catenin signaling pathway in the process of TF-induced VEGF expression and angiogenesis. Conclusions Involvement of the activation of the Wnt/β-catenin signaling pathway is an important mediator for TF-induced VEGF production during the process of angiogenesis. Thus, our findings may ascertain the molecular regulation of TF in neovessel formation and show significant therapeutic implications.

Background: Diabetic retinopathy (DR) is a severe ocular complication of diabetes. Kallistatin has multiple biological functions including anti-inflammation and antiangiogenesis. Our aim was to detect the level of kallistatin in the vitreous of proliferative DR (PDR) and its effect on proliferation, migration, and tube formation of human retinal endothelial cells (HRECs) under high glucose in an in vitro model. Methods: Vitreous humor samples were obtained through pars plana vitrectomy from 7 nondiabetic patients with idiopathic macular holes or idiopathic preretinal membranes and 10 PDR patients. The vitreous levels of kallistatin were measured by ELISA. HRECs were cultured with different concentrations of glucose and 1,000 nM kallistatin. The proliferation of HRECs was evaluated by a Cell Counting Kit-8 assay. Cell migration was assessed by using Transwell chambers. Cell sprouting was detected by tube formation assay. The RNA interference technique was used to create the knockdown of the kallistatin gene in HRECs for evaluating its effect on the proliferation, migration, and tube formation of HRECs. Results: The vitreous levels of kallistatin were significantly lower in PDR patients in comparison with nondiabetic control patients (p < 0.05). Compared with 5 mM of normal glucose treatment, high glucose (30 mM) in culture significantly increased the proliferation and migration of HRECs, which was attenuated by 1,000 nM kallistatin. In addition, 1,000 nM kallistatin was shown to suppress high-glucose-induced tube formation and the expression of vascular endothelial growth factor of HRECs. Furthermore, the knockdown of kallistatin enhanced the proliferation, migration, and tube formation of HRECs. Conclusions: Our data indicated that kallistatin might be a potent inhibitory factor for PDR. The molecule plays its role by inhibiting high-glucose-induced proliferation of HRECs. The findings suggest that the upregulation of kallistatin might be an effective strategy for PDR prevention.