Effects of aflibercept and bevacizumab on cell viability, cell metabolism and inflammation in hypoxic human Müller cells.

Anti-VEGF (vascular endothelial growth factor) drugs such as aflibercept (AFL) and bevacizumab (BVZ) inhibit pathological neo-angiogenesis and vascular permeability in retinal vascular diseases. As cytokines and growth factors are produced by Müller glial cells under stressful and pathological conditions, we evaluated the in vitro effect of AFL (Eylea®, 0.5 mg/mL) and BVZ (Avastin®, 0.5 mg/mL) on cell viability/metabolism, and cytokine/growth factor production by Müller cells (MIO-M1) under cobalt chloride (CoCl2)-induced hypoxia after 24h, 48h and 72h. Cell viability/metabolism were analyzed by Trypan Blue and MTT assays and cytokine/growth factors in supernatants by Luminex xMAP-based multiplex bead-based immunoassay. Cell viability increased with AFL at 48h and 72h and decreased with BVZ or hypoxia at 24h. BVZ-treated cells showed lower cell viability than AFL at all exposure times. Cell metabolism increased with AFL but decreased with BVZ (72h) and hypoxia (48h and72h). As expected, AFL and BVZ decreased VEGF levels. AFL increased PDGF-BB, IL-6 and TNF-α (24h) and BVZ increased PDGF-BB (72h). Hypoxia reduced IL-1ß, -6, -8, TNF-α and PDGF-BB at 24h, and its suppressive effect was more prominent than AFL (EGF, PDGF-BB, IL-1ß, IL-6, IL-8, and TNF-α) and BVZ (PDGF-BB and IL-6) effects. Hypoxia increased bFGF levels at 48h and 72h, even when combined with anti-VEGFs. However, the stimulatory effect of BVZ predominated over hypoxia for IL-8 and TNF-α (24h), as well as for IL-1ß (72h). Thus, AFL and BVZ exhibit distinct exposure times effects on MIO-M1 cells viability, metabolism, and cytokines/growth factors. Hypoxia and BVZ decreased MIO-M1 cell viability/metabolism, whereas AFL likely induced gliosis. Hypoxia resulted in immunosuppression, and BVZ stimulated inflammation in hypoxic MIO-M1 cells. These findings highlight the complexity of the cellular response as well as the interplay between anti-VEGF treatments and the hypoxic microenvironment.

Changes in the Concentrations of Proangiogenic Cytokines in Human Brain Glioma and Acute Lymphoblastic Leukemia.

Acute lymphoblastic leukemia (ALL) and glioma are some of the most common malignancies, with ALL most often affecting children and glioma affecting adult men. Proangiogenic cytokines and growth factors play an important role in the development of both of these tumors. Glioma is characterized by an extremely extensive network of blood vessels, which continues to expand mainly in the process of neoangiogenesis, the direct inducers of which are cytokines from the family of vascular endothelial growth factors, i.e., vascular endothelial growth factor (VEGF-A) and its receptor vascular endothelial growth factor receptor 2 (VEGF-R2), as well as a cytokine from the fibroblast growth factor family, fibroblast growth factor 2 (FGF-2 or bFGF). Growth factors are known primarily for their involvement in the progression and development of solid tumors, but there is evidence that local bone marrow angiogenesis and increased blood vessel density are also present in hematological malignancies, including leukemias. The aim of this study was to examine changes in the concentrations of VEGF-A, VEGF-R2, and FGF-2 (with a molecular weight of 17 kDa) in a group of patients divided into specific grades of malignancy (glioma) and a control group; changes of VEGF-A and FGF-2 concentrations in childhood acute lymphoblastic leukemia and a control group; and to determine correlations between the individual proteins as well as the influence of the patient's age, diet, and other conditions that may place the patient in the risk group. During the statistical analysis, significant differences in concentrations were found between the patient and control groups in samples from people with diagnosed glioma and from children with acute lymphoblastic leukemia, but in general, there are no significant differences in the concentrations of VEGF-A, VEGF-R2, and FGF-2 between different grades of glioma malignancy. Among individuals treated for glioma, there was no significant impact from the patient's gender and age, consumption of food from plastic packaging, frequency of eating vegetables and fruit, smoking of tobacco products, the intensity of physical exercise, or the general condition of the body (Karnofsky score) on the concentrations of the determined cytokines and receptor. The listed factors do not bring about an actual increase in the risk of developing brain glioma.

Ononin promotes radiosensitivity in lung cancer by inhibiting HIF-1α/VEGF pathway.

BACKGROUND: In our previous study, we provided evidence that Astragalus mongholicus Bunge(AM) and its extracts possess a protective capability against radiation-induced damage, potentially mediated through the reduction of reactive oxygen species (ROS) and nitric oxide (NO). However, we were pleasantly surprised to discover during our experimentation that AM not only offers protection against radiation damage but also exhibits a radiation sensitization effect. This effect may be attributed to a specific small molecule present in AM known as ononin. Currently, radiation sensitizers are predominantly found in nitrazole drugs and nanomaterials, with no existing reports on the radiation sensitization properties of ononin, nor its underlying mechanism. PURPOSE: This study aims to investigate the sensitization effect of the small molecule ononin derived from AM on lung cancer radiotherapy, elucidating its specific molecular mechanism of action. Additionally, the safety profile of combining astragalus small molecule ononin with radiation therapy will be evaluated. METHODS: The effective concentration of ononin was determined through cell survival experiments, and the impact of ononin combined with varying doses of radiation on lung cancer cells was observed using CCK-8 and cell cloning experiments. The apoptotic effect of ononin combined with radiation on lung cancer cells was assessed using Hochester staining, flow cytometry, and WB assay. Additionally, WB and immunofluorescence analysis were conducted to investigate the influence of ononin on HIF-1α/VEGF pathway. Furthermore, Molecular Dynamics Simulation was employed to validate the targeted binding ability of ononin and HIF-1α. A lung cancer cell line was established to investigate the effects of knockdown and overexpression of HIF-1α. Subsequently, the experiment was repeated using tumor bearing nude mice and C57BL/6 mouse models in an in vivo study. Tumor volume was measured using a vernier caliper, while HE, immunohistochemistry, and immunofluorescence techniques were employed to observe the effects of ononin combined with radiation on tumor morphology, proliferation, and apoptosis. Additionally, Immunofluorescence was employed to examine the impact of ononin on HIF-1α/VEGF pathway in vivo, and its effect on liver function in mice was assessed through biochemistry analysis. RESULTS: At a concentration of 25 µM, ononin did not affect the proliferation of lung epithelial cells but inhibited the survival of lung cancer cells. In vitro experiments demonstrated that the combination of ononin and radiation could effectively inhibit the growth of lung cancer cells, induce apoptosis, and suppress the excessive activation of the Hypoxia inducible factor 1 alpha/Vascular endothelial growth factor pathway. In vivo experiments showed that the combination of ononin and radiation reduced the size and proliferation of lung cancer tumors, promoted cancer cell apoptosis, mitigated abnormal activation of the Hypoxia inducible factor 1 alpha pathway, and protected against liver function damage. CONCLUSION: This study provides evidence that the combination of AM and its small molecule ononin can enhance the sensitivity of lung cancer to radiation. Additionally, it has been observed that this combination can specifically target HIF-1α and exert its effects. Notably, ononin exhibits the unique ability to protect liver function from damage while simultaneously enhancing the tumor-killing effects of radiation, thereby demonstrating a synergistic and detoxifying role in tumor radiotherapy. These findings contribute to the establishment of a solid basis for the development of novel radiation sensitizers derived from traditional Chinese medicine.

A Systematic Review of Tear Vascular Endothelial Growth Factor and External Eye Diseases.

We aim to summarize the current evidence of Vascular endothelial growth factors (VEGF)s in external eye diseases and determine whether serum and plasma VEGF levels are associated with tear and ocular surface tissues. A systematic search of PUBMED and EMBASE was conducted using PRISMA guidelines between October 2022 and November 2023, with no restriction on language or publication date. Search terms included relevant MESH terms. These studies were evaluated for quality, and an assessment of the risk of bias was also carried out. Extracted data were then visually represented through relevant tables or figures. The initial literature search yielded 777 studies from PUBMED, 944 studies from EMBASE, and 10 studies from manual searches. Fourteen eligible studies were identified from 289 articles published from 2000 to 2023 in the English language or with English translations, including rabbit models, murine models, and human-derived samples. Most studies were retrospective in nature and case-control studies. Various common external eye diseases, such as dry eye disease (DED) and allergic eye disease were investigated. Despite limitations and small sample sizes, researchers have found elevated tissue levels of the VEGF in the vascularized cornea, especially in animal models, but there is no evidence of clear changes in the tear concentrations of VEGF in DED and allergic eye disease. Tear VEGF is associated with corneal vascularization. Anti-VEGF therapies may have the potential to manage such conditions.

Heparan Sulfate Modulation Affects Breast Cancer Cell Adhesion and Transmigration across In Vitro Blood-Brain Barrier.

The disruption of endothelial heparan sulfate (HS) is an early event in tumor cell metastasis across vascular barriers, and the reinforcement of endothelial HS reduces tumor cell adhesion to endothelium. Our recent study showed that while vascular endothelial growth factor (VEGF) greatly reduces HS at an in vitro blood-brain barrier (BBB) formed by human cerebral microvascular endothelial cells (hCMECs), it significantly enhances HS on a breast cancer cell, MDA-MB-231 (MB231). Here, we tested that this differential effect of VEGF on the HS favors MB231 adhesion and transmigration. We also tested if agents that enhance endothelial HS may affect the HS of MB231 and reduce its adhesion and transmigration. To test these hypotheses, we generated an in vitro BBB by culturing hCMECs on either a glass-bottom dish or a Transwell filter. We first quantified the HS of the BBB and MB231 after treatment with VEGF and endothelial HS-enhancing agents and then quantified the adhesion and transmigration of MB231 across the BBB after pretreatment with these agents. Our results demonstrated that the reduced/enhanced BBB HS and enhanced/reduced MB231 HS increase/decrease MB231 adhesion to and transmigration across the BBB. Our findings suggest a therapeutic intervention by targeting the HS-mediated breast cancer brain metastasis.

Morphological evaluation of the feline placenta correlates with gene expression of vascular growth factors and receptors†.

Placental angiogenesis is critical for normal development. Angiogenic factors and their receptors are key regulators of this process. Dysregulated placental vascular development is associated with pregnancy complications. Despite their importance, vascular growth factor expression has not been thoroughly correlated with placental morphologic development across gestation in cats. We postulate that changes in placental vessel morphology can be appreciated as consequences of dynamic expression of angiogenic signaling agents. Here, we characterized changes in placental morphology alongside expression analysis of angiogenic factor splice variants and receptors throughout pregnancy in domestic shorthair cats. We observed increased vascular and lamellar density in the lamellar zone during mid-pregnancy. Immunohistochemical analysis localized the vascular endothelial growth factor A (VEGF-A) receptor KDR to endothelial cells of the maternal and fetal microvasculatures. PlGF and its principal receptor Flt-1 were localized to the trophoblasts and fetal vasculature. VEGF-A was found in trophoblast cells and associated with endothelial cells. We detected expression of two Plgf splice variants and four Vegf-a variants. Quantitative real-time polymerase chain reaction analysis showed upregulation of mRNAs encoding pan Vegf-a and all Vegf-a splice forms at gestational days 30-35. Vegf-A showed a marked relative increase in expression during mid-pregnancy, consistent with the pro-angiogenic changes seen in the lamellar zone at days 30-35. Flt-1 was upregulated during late pregnancy. Plgf variants showed stable expression during the first two-thirds of pregnancy, followed by a marked increase toward term. These findings revealed specific spatiotemporal expression patterns of VEGF-A family members consistent with pivotal roles during normal placental development.

Development of high-performance point-of-care aqueous VEGF detection system and proof-of-concept validation in RVO patients.

OBJECTIVES: To develop a sensitive point-of-care testing (POCT) aqueous vascular endothelial growth factor (VEGF) detection system, and assess its role for predicting the response to anti-VEGF treatment in macular edema secondary to retinal vein occlusion (RVO-ME) patients. METHODS: An automatic point-of-care aqueous humor Magnetic Particle Chemiluminescence Enzyme Immuno-Assay (MPCLEIA) VEGF detection system was developed. The predictive values of aqueous cytokine levels, in combination with imaging parameters, on anatomical treatment response (ATR, the relative central macular thickness change [ΔCMT/bl-CMT]) were analyzed. RESULTS: The automatic MPCLEIA system was able to provide results in 45 min with only 20 µL sample. Among the 57 eyes with available pre- and post-treatment evaluation, ATR significantly correlated with levels of interleukin (IL)-6, IL-8, monocyte chemoattractant protein-1 (MCP-1) and VEGF measured by Luminex xMAP platform, and VEGF measured by MPCLEIA. Optimal cut-off values for these biomarkers were 13.26 ng/L, 23.57 ng/L, 1,110.12 ng/L, 105.52 ng/L, and 85.39 ng/L, respectively. Univariate analysis showed significant associations between ATR category (good response if ATR≤-25 % or poor response otherwise) and IL-6, IL-8, MCP-1, VEGF-xMAP, and VEGF-MPCLEIA (p<0.05). Multivariate logistic regression revealed that ATR category was significantly associated with aqueous VEGF-MPCLEIA (p=0.006) and baseline(bl)-CMT (p=0.008). Receiver operating characteristics analysis yielded an AUC of 0.959 for the regression model combining VEGF-MPCLEIA and bl-CMT, for predicting ATR category. CONCLUSIONS: Our novel MPCLEIA-based automatic VEGF detection system enables accurate POCT of aqueous VEGF, which shows promise in predicting the treatment response of RVO-ME to anti-VEGF agents when combined with bl-CMT.

Immunoexpression of vascular endothelial growth factor and vWF-regulating angiogenesis in cyclic corpus luteum of Indian buffalo.

The present study was conducted to localize the immunoexpression of VEGF-A (Vascular Endothelial Growth Factor) and von Willebrand factor (vWF) in corpora lutea of healthy buffaloes (24) collected from local slaughterhouses. CL collected were categorized into early (stage I, 1-5 days, n = 6), mid (stage II, 6-11 days, n = 6), late luteal phase (stage III, 12 to 16 days, n = 6) and regressing phase (stage IV, 17 to 20 days, n = 6). The percent positive immunostaining for VEGF-A was significantly (p < 0.05) higher in mid-luteal phase than the other three stages of CL. However, it was higher in early luteal phase as well indicated intense angiogenesis in both early and mid-luteal phases. The number of capillary endothelium expressing vWF was significantly (p < 0.05) highest in mid-luteal phase among all the phases. However, in late luteal phase, the percent area positive for VEGF-A immunostaining was reduced but it was significantly (p < 0.05) higher than corpus albicans phase. Thus, in regressing phase or corpus albicans, it was lowest and reduced considerably. However, in late luteal phase, the number of capillaries with vWF immunoexpression reduced significantly (p < 0.05) but it was lowest in corpus albicans phase. Therefore, the immunotaining pattern for VEGF-A and vWF concluded that there was a spositive linear correlation between the two, that is, as the VEGF-A expression was increased, the number of vWF positive capillaries also increased and vice versa. The VEGF-A expressed by the luteal parenchyma in different stages of development and regression of corpus luteum was thus observed to be involved in promoting the angiogenesis and luteal cell proliferation as supported by vWF expressed by endothelium of proliferating capillaries in buffalo corpus luteum throughout the estrous cycle.

Vascular endothelial growth factor induces the migration of human airway smooth muscle cells by activating the RhoA/ROCK pathway.

BACKGROUND: Airway remodeling due to increased airway smooth muscle cell (ASMC) mass, likely due to enhanced proliferation, hypertrophy, and migration, has been proven to be highly correlated with decreased lung function in asthma patients. Vascular endothelial growth factor (VEGF) mediates vascular and extravascular remodeling and inflammation and has been proven to be involved in the progression of asthma. Previous studies have focused on the effects of VEGF on ASMC proliferation, but few researchers have focused on the effects of VEGF on human ASMC migration. The purpose of this study was to explore the effect of VEGF on the migration of ASMCs and its related signaling pathway mechanism to provide evidence for the treatment of airway remodeling. METHODS: We examined the effects of VEGF induction on ASMC migration and explored the mechanisms involved in ASMC migration. RESULTS: We found by wound healing and Transwell assays that VEGF promoted ASMC migration. Through the Cell Counting Kit-8 (CCK-8) experiment, we found that VEGF had no significant effect on the proliferation of ASMCs, which excluded the involvement of cell proliferation in the process of wound healing. Moreover, a cellular immunofluorescence assay showed that VEGF promoted F-actin reorganization, and Western blotting showed that VEGF improved RhoA activation and myosin phosphatase targeting subunit-1 (MYPT1) and myosin light chain (MLC) phosphorylation in ASMCs. Treatment with the ROCK inhibitor Y27632 significantly attenuated the effects of VEGF on MYPT1/MLC activation and cell migration. CONCLUSION: In conclusion, the results suggest that the promigratory function of VEGF activates the RhoA/ROCK pathway, induces F-actin reorganization, improves the migration of ASMCs, and provides a better rationale for targeting the RhoA/ROCK pathway for therapeutic approaches in airway remodeling.

Vitreous Levels of Vascular Endothelial Growth Factor and Platelet-Derived Growth Factor in Patients with Proliferative Diabetic Retinopathy: A Clinical Correlation.

Background: The global epidemic status of diabetic retinopathy (DR) and its burden presents an ongoing challenge to health-care systems. It is of great interest to investigate potential prognostic biomarkers of DR. Such markers could aid in detecting early stages of DR, predicting DR progression and its response to therapeutics. Herein, we investigate the prognostic value of intravitreal concentrations of vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) in a DR cohort. Materials and methods: Vitreous sample acquisition was conducted at King Abdullah University Hospital (KAUH) between December 2020 and June 2022. Samples were obtained from any patient scheduled to undergo a pars plana vitrectomy (PPV) for any indication. Included patients were categorized into a DR group or a corresponding non-diabetic (ND) control group. Demographics, clinicopathological variables, standardized laboratory tests results, and optical coherence tomography (OCT) data were obtained for each included individual. Intravitreal concentrations of VEGF and PDGF were assessed using commercial enzyme-linked immunosorbent assay (ELISA). Results: A total of 80 eyes from 80 patients (DR group: n = 42 and ND control group: n = 38) were included in the analysis. The vitreous VEGF levels were significantly higher in the DR group compared to the ND control group (DR group 5744.06 ± 761.5 pg/mL versus ND control group 817.94 ± 403.1 pg/mL, p = 0.0001). In addition, the vitreous PDGF levels were also significantly higher in the DR group than those in the ND control group (DR group 4031.51 ± 410.2 pg/mL versus ND control group 2691.46 ± 821.0 pg/mL, p = 0.001). Bassline differences between test groups and clinical factors impacting VEGF and PDGF concentrations were investigated as well. Multiple regression analysis indicated PDGF as the sole independent risk factor affecting best-corrected visual acuity (BCVA) at the last follow-up visit: the higher the PDGF vitreous levels, the worst the BCVA. Conclusions: Vitreous concentrations of VEGF and PDGF are correlated with DR severity and may exhibit a possible prognostic potential value in DR. Further clinical and experimental data are warranted to confirm the observed findings and to help incorporate them into daily practice.

Vascular endothelial growth factor antagonist peptides inhibit tumor growth and metastasis in breast cancer through repression of c-src and STAT3 genes.

BACKGROUND: Breast cancer is one of the most decisive causes of cancer death in women worldwide. Cancer progression and tumor metastasis depend on angiogenesis. Vascular endothelial growth factor (VEGF) and its receptors (VEGFR1 and VEGFR2) are critically required for tumor angiogenesis. Src is involved in many of the VEGF-mediated pathways. The VEGFRs activate Src via different mechanisms. Given that Src activates STAT3 (signal transducers and activators of transcription) repressing apoptosis and promoting the cell cycle, it may be an important object for cancer treatment. METHODS AND RESULTS: A series of VEGF antagonistic peptides, referred to as VGB 1,3 and 4, were designed to bind and block both VEGFR1 and VEGFR2 inhibiting the proliferation of different tumoral cells. We investigated c-Src and STAT3 gene expression changes in murine 4T1 tumors treated by the VGBs. The treated group received 1 and 10 mg kg-1 of the peptides, while the control mice received PBS, intraperitoneally for two weeks. Both of the groups underwent a resection of breast tissue 14 days after treatment. The results of qRT-PCR showed that the expression levels of c-Src and STAT3 genes were significantly decreased, in a dose-dependent manner, after treatment with the different types of VEGF antagonist peptides, compared to the control groups (P < 0.05). The groups treated with 1 mg kg-1 of all three types of VGB showed decreased expression of c-Src and STAT3 less than the groups receiving 10 mg kg-1 of the anti-angiogenic peptides. CONCLUSIONS: In conclusion, peptides VGB1, 3, and 4, could be effective therapeutic molecules in breast cancer by inhibiting angiogenesis and progression of the disease.

Differential expression of angiogenesis-related genes in goat uterus and corpus luteum during pregnancy.

Vascularization and the control of luteal and endometrial development are regulated by hypoxia-inducible factors (HIFs) and vascular endothelial growth factor (VEGF) during pregnancy. In this study, the mRNA and protein expression levels of HIFs (HIF1A, HIF2A and HIF3A) and VEGF in goat uterine and ovarian tissues during various stages of pregnancy were evaluated. A total of 42 Hair goats were used and were allocated into six groups, namely embryo-positive (G1), early pregnancy (G2), mid-term pregnancy (G3), late pregnancy (G4), oocyte-positive group (G5) and diestrus group (G6). The mRNA expression of the examined genes was evaluated by RT-qPCR, and protein expression was evaluated by immunohistochemistry (IHC). In caruncles, HIF1A mRNA expression was greater in G1, G2 and G4 than in G3 (p < .05). HIF1A and HIF2A expression was greater in G1 than in G5 (p < .05). In cotyledons, HIF1A, HIF2A and HIF3A mRNA expression was greater in G2 and G3 compared to G4 (p < .05). In luteal tissue, HIF1A mRNA expression was greater in G1 and G2 than in G3 and G4 (p < .05). In the immunohistochemical examination, HIF1A, HIF2A, HIF3A and VEGF immunoreactions were detected in uterine and luteal tissues. Findings suggest that HIFs and VEGF are involved in the regulation of ovarian functions as well as the processes of implantation and placentation.

A novel 3D spheroid model of rheumatoid arthritis synovial tissue incorporating fibroblasts, endothelial cells, and macrophages.

Objective: Rheumatoid Arthritis (RA) is a progressive and systemic autoimmune disorder associated with chronic and destructive joint inflammation. The hallmarks of joint synovial inflammation are cellular proliferation, extensive neoangiogenesis and infiltration of immune cells, including macrophages. In vitro approaches simulating RA synovial tissue are crucial in preclinical and translational research to evaluate novel diagnostic and/or therapeutic markers. Two-dimensional (2D) settings present very limited in vivo physiological proximity as they cannot recapitulate cell-cell and cell-matrix interactions occurring in the three-dimensional (3D) tissue compartment. Here, we present the engineering of a spheroid-based model of RA synovial tissue which mimics 3D interactions between cells and pro-inflammatory mediators present in the inflamed synovium. Methods: Spheroids were generated by culturing RA fibroblast-like-synoviocytes (RAFLS), human umbilical vein endothelial cells (ECs) and monocyte-derived macrophages in a collagen-based 3D scaffold. The spheroids were cultured in the presence or absence of vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (bFGF) or RA synovial fluid (SF). Spheroid expansion and cell migration were quantified for all conditions using confocal microscopy and digital image analysis. Results: A novel approach using machine learning was developed to quantify spheroid outgrowth and used to reexamine the existing spheroid-based model of RA synovial angiogenesis consisting of ECs and RAFLS. A 2-fold increase in the spheroid outgrowth ratio was demonstrated upon VEGF/bFGF stimulation (p<0.05). The addition of macrophages within the spheroid structure (3.75x104 RAFLS, 7.5x104 ECs and 3.0x104 macrophages) resulted in good incorporation of the new cell type. The addition of VEGF/bFGF significantly induced spheroid outgrowth (p<0.05) in the new system. SF stimulation enhanced containment of macrophages within the spheroids. Conclusion: We present a novel spheroid based model consisting of RAFLS, ECs and macrophages that reflects the RA synovial tissue microenvironment. This model may be used to dissect the role of specific cell types in inflammatory responses in RA, to study specific signaling pathways involved in the disease pathogenesis and examine the effects of novel diagnostic (molecular imaging) and therapeutic compounds, including small molecule inhibitors and biologics.

Fructose promotes angiogenesis by improving vascular endothelial cell function and upregulating VEGF expression in cancer cells.

BACKGROUND: Fructose is a very common sugar found in natural foods, while current studies demonstrate that high fructose intake is significantly associated with increased risk of multiple cancers and more aggressive tumor behavior, but the relevant mechanisms are not fully understood. METHODS: Tumor-grafting experiments and in vitro angiogenesis assays were conducted to detect the effect of fructose and the conditioned medium of fructose-cultured tumor cells on biological function of vascular endothelial cells (VECs) and angiogenesis. 448 colorectal cancer specimens were utilized to analyze the relationship between Glut5 expression levels in VECs and tumor cells and microvascular density (MVD). RESULTS: We found that fructose can be metabolized by VECs and activate the Akt and Src signaling pathways, thereby enhancing the proliferation, migration, and tube-forming abilities of VECs and thereby promoting angiogenesis. Moreover, fructose can also improve the expression of vascular endothelial growth factor (VEGF) by upregulating the production of reactive oxygen species (ROS) in colorectal cancer cells, thus indirectly enhancing the biological function of VECs. Furthermore, this pro-angiogenic effect of fructose metabolism has also been well validated in clinical colorectal cancer tissues and mouse models. Fructose contributes to angiogenesis in mouse subcutaneous tumor grafts, and MVD is positively correlated with Glut5 expression levels of both endothelial cells and tumor cells of human colorectal cancer specimens. CONCLUSIONS: These findings establish the direct role and mechanism by which fructose promotes tumor progression through increased angiogenesis, and provide reliable evidence for a better understanding of tumor metabolic reprogramming.

Ophthalmic Intervention of Naringenin Decreases Vascular Endothelial Growth Factor by Counteracting Oxidative Stress and Cellular Damage in In Vivo Zebrafish.

Diabetes Mellitus is a metabolic disease that leads to microvascular complications like Diabetic retinopathy (DR), a major cause of blindness worldwide. Current medications for DR are expensive and report multiple side effects; therefore, an alternative medication that alleviates the disease condition is required. An interventional approach targeting the vascular endothelial growth factor (VEGF) remains a treatment strategy for DR. Anti-VEGF medicines are being investigated as the main therapy for managing vision-threatening complications of DR, such as diabetic macular oedema. Therefore, this study investigated the effect of flavonoid naringenin (NG) from citrus fruits on inhibiting early DR in zebrafish. When exposed to 130 mM glucose, the zebrafish larvae developed a hyperglycaemic condition accompanied by oxidative stress, cellular damage, and lipid peroxidation. Similarly, when adult zebrafish were exposed to 4% Glucose, high glucose levels were observed in the ocular region and massive destruction in the retinal membrane. High glucose upregulated the expression of VEGF. In comparison, the co-exposure to NG inhibited oxidative stress and cellular damage and restored the glutathione levels in the ocular region of the zebrafish larvae. NG regressed the glucose levels and cellular damage along with an inhibition of macular degeneration in the retina of adult zebrafish and normalized the overexpression of VEGF as a promising strategy for treating DR. Therefore, intervention of NG could alleviate the domestication of alternative medicine in ophthalmic research.

BMECs Ameliorate High Glucose-Induced Morphological Aberrations and Synaptic Dysfunction via VEGF-Mediated Modulation of Glucose Uptake in Cortical Neurons.

It has been demonstrated that diabetes cause neurite degeneration in the brain and cognitive impairment and neurovascular interactions are crucial for maintaining brain function. However, the role of vascular endothelial cells in neurite outgrowth and synaptic formation in diabetic brain is still unclear. Therefore, present study investigated effects of brain microvascular endothelial cells (BMECs) on high glucose (HG)-induced neuritic dystrophy using a coculture model of BMECs with neurons. Multiple immunofluorescence labelling and western blot analysis were used to detect neurite outgrowth and synapsis formation, and living cell imaging was used to detect uptake function of neuronal glucose transporters. We found cocultured with BMECs significantly reduced HG-induced inhibition of neurites outgrowth (including length and branch formation) and delayed presynaptic and postsynaptic development, as well as reduction of neuronal glucose uptake capacity, which was prevented by pre-treatment with SU1498, a vascular endothelial growth factor (VEGF) receptor antagonist. To analyse the possible mechanism, we collected BMECs cultured condition medium (B-CM) to treat the neurons under HG culture condition. The results showed that B-CM showed the same effects as BMEC on HG-treated neurons. Furthermore, we observed VEGF administration could ameliorate HG-induced neuronal morphology aberrations. Putting together, present results suggest that cerebral microvascular endothelial cells protect against hyperglycaemia-induced neuritic dystrophy and restorate neuronal glucose uptake capacity by activation of VEGF receptors and endothelial VEGF release. This result help us to understand important roles of neurovascular coupling in pathogenesis of diabetic brain, providing a new strategy to study therapy or prevention for diabetic dementia. Hyperglycaemia induced inhibition of neuronal glucose uptake and impaired to neuritic outgrowth and synaptogenesis. Cocultured with BMECs/B-CM and VEGF treatment protected HG-induced inhibition of glucose uptake and neuritic outgrowth and synaptogenesis, which was antagonized by blockade of VEGF receptors. Reduction of glucose uptake may further deteriorate impairment of neurites outgrowth and synaptogenesis.

Real-time measurements of vascular permeability in the mouse eye using vitreous fluorophotometry.

Breakdown of blood-retinal barrier integrity underpins pathological changes in numerous ocular diseases, including neovascular age-related macular degeneration (nAMD) and diabetic macular edema (DME). Whilst anti-vascular endothelial growth factor (VEGF) therapies have revolutionised disease treatment, novel therapies are still required to meet patients' unmet needs. To help develop new treatments, robust methods are needed to measure changes in vascular permeability in ocular tissues in animal models. We present here a method for detecting vascular permeability using fluorophotometry, which enables real-time measurements of fluorescent dye accumulation in different compartments of the mouse eye. We applied this method in several mouse models with different increased vascular leakage, including models of uveitis, diabetic retinopathy and choroidal neovascularization (CNV). Furthermore, in the JR5558 mouse model of CNV, we observed with anti-VEGF post-treatment a longitudinal reduction in permeability, in the same animal eyes. We conclude fluorophotometry is a useful method for measuring vascular permeability in the mouse eye, and can be used over multiple time points, without the need to sacrifice the animal. This method has the potential to be used in both basic research for studying the progression and factors underlying disease, but also for drug discovery and development of novel therapeutics.

Effect of chlorpyrifos on VEGF gene expression.

Chlorpyrifos (CPF; 0,0-diethyl 0-(3,5,6-trichloro-2-pyridinyl)-phosphorothioate), a cholinesterase inhibitor, compromised the integrity of the blood-brain barrier (BBB) when used at low concentrations during our previous experiments in vitro. To determine if BBB leakage would also occur in vivo, we used FITC-dextrans to evaluate BBB permeability in CPF-dosed mice. Results indicated BBB leakages that were evident at 2 h after treatment with 70 mg/kg CPF ip. Since vascular endothelial growth factor (VEGF), a potent vasopermeability factor, is a signaling protein that promotes the growth of new blood vessels, we investigated the possible involvement of VEGF in BBB disruption by CPF. We found that VEGF serum concentration was significantly increased at 24 h after CPF exposure. To further explore VEGF involving BBB disruption by CPF treatment, the receptor antagonist for VEGF (sFlt-1) was used for pretreatment before CPF exposure. After sFlt-1 pretreatment, gene expressions of the tight junction (TJ) proteins claudin5 and occludin were significantly downregulated at 1, 2, and 3 h, but returned to control levels at 24 h after CPF treatment. These results suggest that VEGF is involved in BBB disruption by CPF through BBB-TJs regulation.

Single-Cell RNA-Seq Reveals LRRC75A-Expressing Cell Population Involved in VEGF Secretion of Multipotent Mesenchymal Stromal/Stem Cells Under Ischemia.

Human multipotent mesenchymal stromal/stem cells (MSCs) have been utilized in cell therapy for various diseases and their clinical applications are expected to increase in the future. However, the variation in MSC-based product quality due to the MSC heterogeneity has resulted in significant constraints in the clinical utility of MSCs. Therefore, we hypothesized that it might be important to identify and ensure/enrich suitable cell subpopulations for therapies using MSC-based products. In this study, we aimed to identify functional cell subpopulations to predict the efficacy of angiogenic therapy using bone marrow-derived MSCs (BM-MSCs). To assess its angiogenic potency, we observed various levels of vascular endothelial growth factor (VEGF) secretion among 11 donor-derived BM-MSC lines under in vitro ischemic culture conditions. Next, by clarifying the heterogeneity of BM-MSCs using single-cell RNA-sequencing analysis, we identified a functional cell subpopulation that contributed to the overall VEGF production in BM-MSC lines under ischemic conditions. We also found that leucine-rich repeat-containing 75A (LRRC75A) was more highly expressed in this cell subpopulation than in the others. Importantly, knockdown of LRRC75A using small interfering RNA resulted in significant inhibition of VEGF secretion in ischemic BM-MSCs, indicating that LRRC75A regulates VEGF secretion under ischemic conditions. Therefore, LRRC75A may be a useful biomarker to identify cell subpopulations that contribute to the angiogenic effects of BM-MSCs. Our work provides evidence that a strategy based on single-cell transcriptome profiles is effective for identifying functional cell subpopulations in heterogeneous MSC-based products.

Expression and localization of vascular endothelial growth factor and its receptors in the pig uterus during peri-implantation and determination of the in vitro effect of the angiogenesis inhibitor SU5416 on VEGF system expression.

The aim of this work was to determine endometrial mRNA expression and uterine protein localization of vascular endothelial growth factor (VEGF) and its receptors VEGFR1 and VEGFR2 during the estrous cycle and peri-implantation period in sows. Uterine tissues were collected from pregnant sows on days 12, 14, 16, and 18 after artificial insemination and from non-pregnant animals on days 2 and 12 of the estrous cycle (day 0 = day of estrus). Using immunohistochemistry, a positive signal for VEGF and its receptor VEGFR2 was found in uterine luminal epithelial cells, endometrial glands, stroma, blood vessels, and myometrium. A VEGFR1 signal was only found in endometrial and myometrial blood vessels and stroma. By day 18 of gestation, the mRNA expression levels of VEGF, VEGFR1, and VEGFR2 were higher than those observed on days 2 and 12 of the estrous cycle and on days 12, 14, and 16 of gestation. Then, a primary culture of sow endometrial epithelial cells was established to define the potential of the selective inhibition of VEGFR2 after treatment with inhibitor SU5416 and determine its effects on the expression pattern of the VEGF system. The endometrial epithelial cells treated with SU5416 showed a dose-dependent decrease in VEGFR1 and VEGFR2 mRNA expression. The present study provides additional evidence on the importance of the VEGF system during peri-implantation, as well as on the specific inhibitory activity of SU5416 in epithelial cells, which, as demonstrated, express the protein and mRNA of VEGF and its receptors VEGFR1 and VEGFR2.