An Ensemble Docking Approach for Analyzing and Designing Aptamer Heterodimers Targeting VEGF165.

Vascular endothelial growth factor 165 (VEGF165) is a prominent isoform of the VEGF-A protein that plays a crucial role in various angiogenesis-related diseases. It is homodimeric, and each of its monomers is composed of two domains connected by a flexible linker. DNA aptamers, which have emerged as potent therapeutic molecules for many proteins with high specificity and affinity, can also work for VEGF165. A DNA aptamer heterodimer composed of monomers of V7t1 and del5-1 connected by a flexible linker (V7t1:del5-1) exhibits a greater binding affinity with VEGF165 compared to either of the two monomers alone. Although the structure of the complex formed between the aptamer heterodimer and VEGF165 is unknown due to the highly flexible linkers, gaining structural information will still be valuable for future developments. Toward this end of accessing structural information, we adopt an ensemble docking approach here. We first obtain an ensemble of structures for both VEGF165 and the aptamer heterodimer by considering both small- and large-scale motions. We then proceed through an extraction process based on ensemble docking, molecular dynamics simulations, and binding free energy calculations to predict the structures of the VEGF165/V7t1:del5-1 complex. Through the same procedures, we reach a new aptamer heterodimer that bears a locked nucleic acid-modified counterpart of V7t1, namely RNV66:del5-1, which also binds well with VEGF165. We apply the same protocol to the monomeric units V7t1, RNV66, and del5-1 to target VEGF165. We observe that V7t1:del5-1 and RNV66:del5-1 show higher binding affinities with VEGF165 than any of the monomers, consistent with experiments that support the notion that aptamer heterodimers are more effective anti-VEGF165 aptamers than monomeric aptamers. Among the five different aptamers studied here, the newly designed RNV66:del5-1 shows the highest binding affinity with VEGF165. We expect that our ensemble docking approach can help in de novo designs of homo/heterodimeric anti-angiogenic drugs to target the homodimeric VEGF165.

Eriocitrin Inhibits Angiogenesis by Targeting VEGFR2-Mediated PI3K/AKT/mTOR Signaling Pathways.

Eriocitrin, a flavanone found in peppermint and citrus fruits, is known to possess many physiological activities. However, the anti-angiogenic effects of eriocitrin are yet to be fully elucidated. Therefore, the objective of this research was to explore the anti-angiogenic effects of eriocitrin both in vitro and in vivo as well as its underlying mechanism. Anti-angiogenic effects of eriocitrin were evaluated utilizing in vitro models of angiogenesis, including inhibition of tube formation, and induction of apoptosis in human umbilical vein endothelial cells (HUVECs). A chorioallantoic membrane (CAM) assay in chick embryos was also performed to evaluate the in vivo effects of eriocitrin on angiogenesis. Results showed significant eriocitrin effects on proliferation, tube formation, migration, and apoptosis in HUVECs. Furthermore, in vivo analysis revealed that eriocitrin significantly suppressed the formation of new blood vessels. In particular, it regulated MAPK/ERK signaling pathway and VEGFR2, inhibited the downstream PI3K/AKT/mTOR signaling pathway, and activated apoptosis signals such as caspase cascades. In HUVECs, the expression of matrix metalloproteinases (MMP-2 and MMP-9) exhibited an inhibitory effect on angiogenesis through the suppression of the signaling pathway. Therefore, eriocitrin presents potential for development into an antiangiogenic therapeutic agent.

Clinical effects of atorvastatin combined with conbercept in the treatment of patients with macular edema secondary to retinal vein occlusion and carotid plaque: study protocol for a prospective randomized controlled trial.

INTRODUCTION: Intravitreal injections of anti-vascular endothelial growth factor (anti-VEGF) drugs have been widely used in patients with macular edema (ME) secondary to retinal vein occlusion (RVO); however, recurrence is a major concern. This study aims to observe the clinical effects of atorvastatin and intravitreal therapy in the treatment of patients with branch or central RVO-ME and coexistent carotid plaques (CP). METHODS AND ANALYSIS: A prospective randomized controlled clinical trial will be conducted. Sixty-four patients diagnosed with branch or central RVO-ME and coexistent CP will be enrolled and randomly allocated in a 1:1 ratio to the control and experimental groups. The control group will be treated with intravitreal conbercept monthly for 3 months, followed by monthly evaluation and injection of pro re nata (PRN) for 12 months, while the experimental group will be treated with oral atorvastatin 20 mg daily combined with the control group treatment. If a drop of best-corrected visual acuity (BCVA) is more than five Early Treatment Diabetic Retinopathy Study (ETDRS) letters (one line) or an increment in central subfield thickness (CSFT) of 100 µm (or a 10% increment from the previous visit), intravitreal re-treatment will be performed. Outcome measurements include CSFT, BCVA, number of injections, and incidence of adverse events during the 12-month follow-up period. Differences between groups will be evaluated using Student's t-test, and comparisons between groups will be evaluated using repeated-measures analysis of variance. ETHICS AND DISSEMINATION: The study has been approved by the Institutional Review Board of Nanjing Lishui People's Hospital, Nanjing, China (approval number 2023KY0418-12, dated 18 April 2023), and has been registered on chictr.org.cn. Written informed consent will be collected from each patient and the results of this trial will be submitted to a peer-reviewed journal. TRIAL REGISTRATION: Chinese Clinical Trial Registry ChiCTR2300071359. Registered on 12 May 2023.

Central subfield thickness of diabetic macular edema: Correlation with the aqueous humor proteome.

Purpose: Diabetic macular edema (DME) is a sight-threatening complication of diabetes. Consequently, studying the proteome of DME may provide novel insights into underlying molecular mechanisms. Methods: In this study, aqueous humor samples from eyes with treatment-naïve clinically significant DME (n = 13) and age-matched controls (n = 11) were compared with label-free liquid chromatography-tandem mass spectrometry. Additional aqueous humor samples from eyes with treatment-naïve DME (n = 15) and controls (n = 8) were obtained for validation by enzyme-linked immunosorbent assay (ELISA). Best-corrected visual acuity (BCVA) was evaluated, and the severity of DME was measured as central subfield thickness (CST) employing optical coherence tomography. Control samples were obtained before cataract surgery. Significantly changed proteins were identified using a permutation-based calculation, with a false discovery rate of 0.05. A human donor eye with DME and a control eye were used for immunofluorescence. Results: A total of 101 proteins were differentially expressed in the DME. Regulated proteins were involved in complement activation, glycolysis, extracellular matrix interaction, and cholesterol metabolism. The highest-fold change was observed for the fibrinogen alpha chain (fold change = 17.8). Complement components C2, C5, and C8, fibronectin, and hepatocyte growth factor-like protein were increased in DME and correlated with best-corrected visual acuity (BCVA). Ceruloplasmin and complement component C8 correlated with central subfield thickness (CST). Hemopexin, plasma kallikrein, monocyte differentiation antigen CD14 (CD14), and lipopolysaccharide-binding protein (LBP) were upregulated in the DME. LBP was correlated with vascular endothelial growth factor. The increased level of LBP in DME was confirmed using ELISA. The proteins involved in desmosomal integrity, including desmocollin-1 and desmoglein-1, were downregulated in DME and correlated negatively with CST. Immunofluorescence confirmed the extravasation of fibrinogen at the retinal level in the DME. Conclusion: Elevated levels of pro-inflammatory proteins, including the complement components LBP and CD14, were observed in DME. DME was associated with the loss of basal membrane proteins, compromised desmosomal integrity, and perturbation of glycolysis.

Brolucizumab in recalcitrant neovascular age-related macular degeneration-real-world data in Chinese population.

This retrospective study aimed to determine the short-term efficacy and safety of brolucizumab treatment for recalcitrant neovascular age-related macular degeneration (nAMD) in a real-world setting in Taiwan. Recalcitrant nAMD patients who were treated with brolucizumab from November 2021 to August 2022 at Taipei Veterans General Hospital were included. Patients were followed for 3 months after switching to brolucizumab. The primary outcomes were changes in mean best-corrected visual acuity (BCVA) and central retinal thickness (CRT) from baseline to the third month. The secondary outcomes included the incidence of intraocular inflammation (IOI), proportion of patients with subretinal and intraretinal fluid (SRF and IRF), and change in pigment epithelial detachment (PED) height from baseline to the third month. The significance level was considered as p < .05 in all tests. A total of 38 patients (40 eyes) with a mean (±SD) age of 76.3 (±10.84) years were included. The baseline BCVA was 0.92±0.64 logMAR, and the CRT and PED height were 329.0±171.18 and 189.8±114.94 um, respectively. The patients had a significant reduction in CRT and resolution of IRF and SRF from baseline to the third month. There were numerical improvements in mean BCVA and PED height, but they were not significant. The percentages of achieving at least 0.1, 0.2, and 0.3 logMAR (equivalent to 5, 10, 15 ETDRS letters) visual gain were 50%, 37.5%, and 30%, respectively, during the first 3 months of follow-up. No IOI occurred in these patients. This study demonstrated that brolucizumab had good short-term structural and functional efficacy in recalcitrant nAMD patients.

Immune checkpoint inhibitors in the treatment of hepatocellular carcinoma.

Despite advances in cancer treatment, hepatocellular carcinoma (HCC), the most common form of liver cancer, remains a major public health problem worldwide. The immune microenvironment plays a critical role in regulating tumor progression and resistance to therapy, and in HCC, the tumor microenvironment (TME) is characterized by an abundance of immunosuppressive cells and signals that facilitate immune evasion and metastasis. Recently, anti-cancer immunotherapies, therapeutic interventions designed to modulate the immune system to recognize and eliminate cancer, have become an important cornerstone of cancer therapy. Immunotherapy has demonstrated the ability to improve survival and provide durable cancer control in certain groups of HCC patients, while reducing adverse side effects. These findings represent a significant step toward improving cancer treatment outcomes. As demonstrated in clinical trials, the administration of immune checkpoint inhibitors (ICIs), particularly in combination with anti-angiogenic agents and tyrosine kinase inhibitors, has prolonged survival in a subset of patients with HCC, providing an alternative for patients who progress on first-line therapy. In this review, we aimed to provide an overview of HCC and the role of the immune system in its development, and to summarize the findings of clinical trials involving ICIs, either as monotherapies or in combination with other agents in the treatment of the disease. Challenges and considerations regarding the administration of ICIs in the treatment of HCC are also outlined.

Switching to the aflibercept (3 mg) therapy for treatment-resistant wet age-related macular degeneration: 1-year outcomes.

This study aimed to elucidate 1-year outcomes following switching to the aflibercept (3 mg) therapy for treatment-resistant wet age-related macular degeneration (wAMD). In this prospective, open-label, non-controlled clinical trial, 18 patients with wAMD who had multiple recurrences or persistent exudation despite intravitreal injections of anti-vascular endothelial growth factor agents (except aflibercept) received a 3-mg intravitreal aflibercept injection every 4 weeks. Each patient received 3 to 8 injections. The central retinal thickness and fibrovascular pigment epithelial detachment height decreased significantly at 1 month after initiation of the aflibercept injection, and the values were 146 and 163.2 µm, respectively, at the final visit. The morphological improvement was sustained. The intraretinal and subretinal fluid was completely absorbed at the end of the follow-up. The logMAR vision increased from baseline 0.68 to 0.59 (P < .05). No ocular or systemic adverse events occurred. The intravitreal injection of 3-mg aflibercept seems to be feasible in the treatment of wAMD unresponsive to other anti-vascular endothelial growth factor agents.

Non-surgical treatment of stage 4A retinopathy of prematurity.

BACKGROUND: Retinopathy of prematurity (ROP) is a major cause of visual impairment in premature infants, often requiring surgical interventions in advanced stages. This retrospective case series study investigates non-surgical management for Stage 4A ROP, specifically the use of combined laser therapy and intravitreal anti-vascular endothelial growth factor (VEGF) injections. METHODS: Ten eyes from five infants with Stage 4A ROP were treated with a combined laser and anti-VEGF approach. Comprehensive follow-up examinations were conducted to evaluate the treatment outcomes. RESULTS: The study demonstrated successful retinal attachment without complications, showcasing the efficacy and safety of this non-surgical method. A comparison with surgical interventions highlighted the potential benefits in terms of reduced adverse effects. DISCUSSION: This combined treatment emerges as a promising first-choice option for Stage 4A ROP, offering rapid regression without surgical intervention, particularly in early stages. However, larger randomized clinical trials are necessary to validate these findings and establish definitive guidelines for managing this complex condition. CONCLUSION: Combined laser and anti-VEGF therapy proved to be an effective and safe non-surgical approach for Stage 4A ROP, with the potential to reduce the need for surgery, especially in its early presentation. Further research is required to confirm these findings and provide comprehensive recommendations for clinical practice.

Machine learning and optical coherence tomography-derived radiomics analysis to predict persistent diabetic macular edema in patients undergoing anti-VEGF intravitreal therapy.

BACKGROUND: Diabetic macular edema (DME) is a leading cause of vision loss in patients with diabetes. This study aimed to develop and evaluate an OCT-omics prediction model for assessing anti-vascular endothelial growth factor (VEGF) treatment response in patients with DME. METHODS: A retrospective analysis of 113 eyes from 82 patients with DME was conducted. Comprehensive feature engineering was applied to clinical and optical coherence tomography (OCT) data. Logistic regression, support vector machine (SVM), and backpropagation neural network (BPNN) classifiers were trained using a training set of 79 eyes, and evaluated on a test set of 34 eyes. Clinical implications of the OCT-omics prediction model were assessed by decision curve analysis. Performance metrics (sensitivity, specificity, F1 score, and AUC) were calculated. RESULTS: The logistic, SVM, and BPNN classifiers demonstrated robust discriminative abilities in both the training and test sets. In the training set, the logistic classifier achieved a sensitivity of 0.904, specificity of 0.741, F1 score of 0.887, and AUC of 0.910. The SVM classifier showed a sensitivity of 0.923, specificity of 0.667, F1 score of 0.881, and AUC of 0.897. The BPNN classifier exhibited a sensitivity of 0.962, specificity of 0.926, F1 score of 0.962, and AUC of 0.982. Similar discriminative capabilities were maintained in the test set. The OCT-omics scores were significantly higher in the non-persistent DME group than in the persistent DME group (p < 0.001). OCT-omics scores were also positively correlated with the rate of decline in central subfield thickness after treatment (Pearson's R = 0.44, p < 0.001). CONCLUSION: The developed OCT-omics model accurately assesses anti-VEGF treatment response in DME patients. The model's robust performance and clinical implications highlight its utility as a non-invasive tool for personalized treatment prediction and retinal pathology assessment.

Flavonoids with Anti-Angiogenesis Function in Cancer.

The formation of new blood vessels, known as angiogenesis, significantly impacts the development of multiple types of cancer. Consequently, researchers have focused on targeting this process to prevent and treat numerous disorders. However, most existing anti-angiogenic treatments rely on synthetic compounds and humanized monoclonal antibodies, often expensive or toxic, restricting patient access to these therapies. Hence, the pursuit of discovering new, affordable, less toxic, and efficient anti-angiogenic compounds is imperative. Numerous studies propose that natural plant-derived products exhibit these sought-after characteristics. The objective of this review is to delve into the anti-angiogenic properties exhibited by naturally derived flavonoids from plants, along with their underlying molecular mechanisms of action. Additionally, we summarize the structure, classification, and the relationship between flavonoids with their signaling pathways in plants as anti-angiogenic agents, including main HIF-1α/VEGF/VEGFR2/PI3K/AKT, Wnt/ß-catenin, JNK1/STAT3, and MAPK/AP-1 pathways. Nonetheless, further research and innovative approaches are required to enhance their bioavailability for clinical application.

Visual outcomes of intraocular inflammation after brolucizumab injection in Japanese patients with neovascular age-related macular degeneration.

PURPOSE: This study investigates the visual outcomes of neovascular age-related macular degeneration (nAMD) patients who developed intraocular inflammation (IOI) after intravitreal brolucizumab injection (IVBr). METHODS: We studied 285 eyes of 279 cases diagnosed with nAMD and focused on 18 eyes (6.3%) of 17 cases which developed IOI after IVBr. IVBr was performed either on the initial treatment or for switching of other anti-vascular endothelial growth factor agents during January 2020 to December 2021. We evaluated clinical features and the course of treatment of a 6-month follow-up after IOI occurred. RESULTS: Of 17 cases, 9 cases were male, 8 cases were female. Baseline logarithm of the minimum angle of resolution(logMAR) best-corrected visual acuity (BCVA) was 0.36, BCVA before IOI occurred was 0.30, and BCVA when IOI occurred was 0.43. 16 eyes (88.9%) had symptoms such as visual loss or floaters when IOI occurred. On the other hand, the remaining 2 eyes (11.1%) had no symptoms. 11 eyes (61.1%) had only IOI, while the remaining 7 eyes (38.9%) had IOI and perivascular sheathing. Steroid sub-tenon injection was performed on 1 eye (5.6%), steroid eye drops were used in 11 eyes (61.1%), and 6 eyes (33.3%) were followed-up without treatment. Neovascular AMD recurred in 16 eyes (88.9%) after IOI occurred and were treated with aflibercept. VA at 3 and 6 months after IOI occurred were significantly improved to 0.34 and 0.30, respectively (P = 0.09 at 3 months and P = 0.02 at 6 months). The symptoms of patients were improved in all cases. We were able to stop steroid treatment in all cases. CONCLUSIONS: IOI occurred in 6.3% of nAMD patients after IVBr treatment. All of which showed significant improvement from logMAR of 0.43 to 0.30 with steroid treatment or without any treatment. We should consider the possibility of IOI after IVBr as a complication, however, they have a relatively good prognosis if treated at an early stage.

One-year visual and anatomical outcomes of intravitreal faricimab injection for neovascular age-related macular degeneration after prior brolucizumab treatment.

This single-center retrospective cohort study analyzed the 1-year real-world treatment outcomes of 63 consecutive eyes (of 60 patients) with neovascular age-related macular degeneration (nAMD) that were switched from intravitreal brolucizumab (IVBr) to intravitreal faricimab (IVF) and managed on a treat-and-extend regimen with discontinuation criteria. After the switch, patients opted to continue IVF, to switch back to IVBr, or receive photodynamic therapy (PDT). Thirty-eight patients continued IVF, 16 patients were switched back to IVBr, 2 patients received PDT, and 4 patients paused treatment. Best-corrected visual acuity (BCVA), central subfield thickness (CST), subfoveal choroidal thickness (sf-CT), and injection intervals were compared immediately before and 1 year after the initial IVF. Whereas there was no change in BCVA and CST; 0 [- 0.0969 to 0.125, P = 0.58], - 1.5 [- 27.8 to 13.5, P = 0.11] µm, respectively, sf-CT decreased significantly; - 19.5 [- 45.5 to 7.75, P = 0.015] µm. The patients switched back showed no significant change in sf-CT. The injection interval extended significantly in the IVF continuation and the switch-back group (2.0 and 3.0 weeks, respectively; [P = 0.0007 and 0.0078]) in eyes with a pre-switching interval of less than 12 weeks. Faricimab shows promise as a safe and effective alternative to brolucizumab for treating nAMD.

Exploring the Impact of Saccharin on Neovascular Age-Related Macular Degeneration: A Comprehensive Study in Patients and Mice.

Purpose: We aimed to determine the impact of artificial sweeteners (AS), especially saccharin, on the progression and treatment efficacy of patients with neovascular age-related macular degeneration (nAMD) under anti-vascular endothelial growth factor (anti-VEGF-A) treatment. Methods: In a cross-sectional study involving 46 patients with nAMD undergoing intravitreal anti-VEGF therapy, 6 AS metabolites were detected in peripheral blood using liquid chromatography - tandem mass spectrometry (LC-MS/MS). Disease features were statistically tested against these metabolite levels. Additionally, a murine choroidal neovascularization (CNV) model, induced by laser, was used to evaluate the effects of orally administered saccharin, assessing both imaging outcomes and gene expression patterns. Polymerase chain reaction (PCR) methods were used to evaluate functional expression of sweet taste receptors in a retinal pigment epithelium (RPE) cell line. Results: Saccharin levels in blood were significantly higher in patients with well-controlled CNV activity (P = 0.004) and those without subretinal hyper-reflective material (P = 0.015). In the murine model, saccharin-treated mice exhibited fewer leaking laser scars, lesser occurrence of bleeding, smaller fibrotic areas (P < 0.05), and a 40% decrease in mononuclear phagocyte accumulation (P = 0.06). Gene analysis indicated downregulation of inflammatory and VEGFR-1 response genes in the treated animals. Human RPE cells expressed taste receptor type 1 member 3 (TAS1R3) mRNA and reacted to saccharin stimulation with changes in mRNA expression. Conclusions: Saccharin appears to play a protective role in patients with nAMD undergoing intravitreal anti-VEGF treatment, aiding in better pathological lesion control and scar reduction. The murine study supports this observation, proposing saccharin's potential in mitigating pathological VEGFR-1-induced immune responses potentially via the RPE sensing saccharin in the blood stream.

Selectivity Studies and Free Energy Calculations of AKT Inhibitors.

Protein kinase B (PKB) or AKT protein is an important target for cancer treatment. Significant advances have been made in developing ATP-competitive inhibitors and allosteric binders targeting AKT1. However, adverse effects or toxicities have been found, and the cutaneous toxicity was found to be linked to the inhibition of AKT2. Thus, selective inhibition of AKT inhibitors is of significance. Our work, using the Schrödinger Covalent Dock (CovDock) program and the Movable Type (MT)-based free energy calculation (ΔG), yielded small mean errors for the experimentally derived binding free energy (ΔG). The docking data suggested that AKT1 binding may require residues Asn54, Trp80, Tyr272, Asp274, and Asp292, whereas AKT2 binding would expect residues Phe163 and Glu279, and AKT3 binding would favor residues Glu17, Trp79, Phe306, and Glu295. These findings may help guide AKT1-selective or AKT3-selective molecular design while sparing the inhibition of AKT2 to minimize the cutaneous toxicity.

PQR309, a dual PI3K/mTOR inhibitor, synergizes with gemcitabine by impairing the GSK-3ß and STAT3/HSP60 signaling pathways to treat nasopharyngeal carcinoma.

End-stage nasopharyngeal carcinoma (NPC) has unsatisfactory survival. The limited benefit of chemotherapy and the scarcity of targeted drugs are major challenges in NPC. New approaches to treat late-stage NPC are urgently required. In this study, we explored whether the dual PI3K/mTOR inhibitor, PQR309, exerted a favorable antineoplastic effect and sensitized the response to gemcitabine in NPC. We observed that PI3K expression was positive and elevated in 14 NPC cell lines compared with that in normal nasopharygeal cell lines. Patients with NPC with higher PI3K levels displayed poorer prognosis. We subsequently showed that PQR309 alone effectively decreased the viability, invasiveness, and migratory capability of NPC cells and neoplasm development in mice xenograft models, and dose-dependently induced apoptosis. More importantly, PQR309 remarkably strengthened the anti-NPC function of gemcitabine both in vivo and in vitro. Mechanistically, PQR309 sensitized NPC to gemcitabine by increasing caspase pathway-dependent apoptosis, blocking GSK-3ß and STAT3/HSP60 signaling, and ablating epithelial-mesenchyme transition. Thus, targeting PI3K/mTOR using PQR309 might represent a treatment option to promote the response to gemcitabine in NPC, and provides a theoretical foundation for the study of targeted drugs combined with chemotherapy for NPC.

Treatment Strategies for Anti-VEGF Resistance in Neovascular Age-Related Macular Degeneration by Targeting Arteriolar Choroidal Neovascularization.

Despite extensive use of intravitreal anti-vascular endothelial growth factor (anti-VEGF) biologics for over a decade, neovascular age-related macular degeneration (nAMD) or choroidal neovascularization (CNV) continues to be a major cause of irreversible vision loss in developed countries. Many nAMD patients demonstrate persistent disease activity or experience declining responses over time despite anti-VEGF treatment. The underlying mechanisms of anti-VEGF resistance are poorly understood, and no effective treatment strategies are available to date. Here we review evidence from animal models and clinical studies that supports the roles of neovascular remodeling and arteriolar CNV formation in anti-VEGF resistance. Cholesterol dysregulation, inflammation, and ensuing macrophage activation are critically involved in arteriolar CNV formation and anti-VEGF resistance. Combination therapy by neutralizing VEGF and enhancing cholesterol removal from macrophages is a promising strategy to combat anti-VEGF resistance in CNV.

Zeaxanthin impairs angiogenesis and tumor growth of glioblastoma: An in vitro and in vivo study.

OBJECTIVES: To investigate the therapeutic effects of Zeaxanthin (Zea), one of the oxidized xanthophyll carotenoids belonging to the isoprenoids, on inhibiting the angiogenesis and tumor growth of glioblastoma (GBM) via an in vitro and in vivo study. METHODS: The effects of Zea on the proliferation, adhesion, migration and invasion of human GBM cell lines were detected by cell proliferation assay, cell adhesion assay and Transwell assay. The effect of Zea on angiogenesis was detected by rat aortic ring assay and human umbilical vein endothelial cells (HUVEC) in vitro tube formation assay. The effects of Zea on PARP, Caspase 3 and VEGFR2 phosphorylation as well as VEGFR2's downstream signaling pathway were detected by Western blot. The in vivo human GBM xenograft mouse model was employed to study the therapeutic efficacy of Zea. RESULTS: Zea impaired the proliferation, adhesion, migration and invasion of U87 and U251 cells as well as HUVECs. Rat aortic ring experiments displayed Zea significantly inhibited angiogenesis during VEGF-induced microvascular germination. In vitro and in vivo vascular experiments verified that Zea inhibited VEGF-induced HUVEC proliferation and capillary-like tube formation. Additionally, Zea induced GBM cells apoptosis via increasing the expression of cleaved PARP and Caspase 3. In HUVECs and U251 GBM cells, Zea down-regulated VEGF-induced activation of the VEGFR2 kinase pathway. Meanwhile the expression of p-AKT, p-ERK, p-STAT3 and FAK were all attenuated in U251 cells. Moreover, the effects of Zea on GBM cells proliferation could be blocked by VEGFR2 kinase inhibitor SU5408. These results suggest that Zea may hinder GBM angiogenesis and tumor growth through down-regulating a cascade of oncogenic signaling pathways, both through the inhibition of angiogenesis and the anti-tumor mechanism of a direct cytotoxic effect. Besides, Zea inhibits GBM angiogenesis and tumor growth exemplified through a xenograft mouse model in vivo. CONCLUSION: Zea impairs angiogenesis and tumor growth of GBM both in vitro and in vivo. It can be declared that Zea is a potential valuable anticancer candidate for the future treatment strategy of GBM.

Intravitreal aflibercept 8 mg in neovascular age-related macular degeneration (PULSAR): 48-week results from a randomised, double-masked, non-inferiority, phase 3 trial.

BACKGROUND: Intravitreal aflibercept 8 mg could improve treatment outcomes and provide sustained disease control in patients with neovascular age-related macular degeneration (nAMD), with extended dosing compared with aflibercept 2 mg. METHODS: PULSAR is a phase 3, randomised, three-group, double-masked, non-inferiority, 96-week trial conducted across 223 sites worldwide. Adults with nAMD were randomised 1:1:1 to aflibercept 8 mg every 12 weeks (8q12), aflibercept 8 mg every 16 weeks (8q16), or aflibercept 2 mg every 8 weeks (2q8), following three initial monthly doses in all groups. From week 16, patients in the aflibercept 8 mg groups had their dosing interval shortened if pre-specified dose regimen modification criteria denoting disease activity were met. The primary endpoint was change from baseline in best-corrected visual acuity (BCVA) at week 48. All patients with at least one dose of study treatment were included in the efficacy and safety analyses. This trial is registered with ClinicalTrials.gov (NCT04423718) and is ongoing. FINDINGS: Of 1011 patients randomised to aflibercept 8q12 (n=336), 8q16 (n=338), or 2q8 (n=337) between Aug 11, 2020, and July 30, 2021, 1009 patients received study treatment (aflibercept 8q12 n=335; aflibercept 8q16 n=338; and aflibercept 2q8 n=336). Aflibercept 8q12 and 8q16 showed non-inferior BCVA gains versus aflibercept 2q8 (mean BCVA change from baseline +6·7 [SD 12·6] and +6·2 [11·7] vs +7·6 [12·2] letters). The least squares mean differences between aflibercept 8q12 versus 2q8 and 8q16 versus 2q8, respectively, were -0·97 (95% CI -2·87 to 0·92) and -1·14 (-2·97 to 0·69) letters (non-inferiority margin at 4 letters). The incidence of ocular adverse events in the study eye was similar across groups (aflibercept 8q12 n=129 [39%]; aflibercept 8q16 n=127 [38%]; and aflibercept 2q8 n=130 [39%]). INTERPRETATION: Aflibercept 8 mg showed efficacy and safety with extended dosing intervals, which has the potential to improve the management of patients with nAMD. FUNDING: Bayer AG and Regeneron Pharmaceuticals.