VEGF Protein Levels Research Articles

MiR-320 Overexpression Affects Lung Cancer Macrophage M2 Polarization Through Targeting Vascular Endothelial Growth Factor A (VEGFA)

miR-320 overexpression’s effect on macrophages polarization in lung cancer was assessed to seek the related therapeutic target for providing theoretical foundation on drug development. THP-1 and A549 cell was induced into macrophage M2 type and then divided into no-polarization group (MΦ) and M2 type of macrophage’s polarization group (M2). Cells were transfected with miR-320mimic and assigned into BC group, NC group and miR-320mimic group followed by analysis of TGF-β level by ELISA, TNF-α, IL-1β, CCL22, CCL17 and VEGFA level by real-time PCR, VEGFA, CCL22 and IL-1β protein level by western blot, as well as CD206 level by flow cytometry. The proportion of CD206+ cell was significantly increased after induction with the elevated CCL22 and CCL17 mRNA level. There was no significance of TNF-α, IL-1β RNA level between two groups (P > 0.05). TGF-β level was significantly increased in macrophage of M2 type. CCL2 and VEGFA level was lower and IL-1β was higher in miR-320 overexpression group than other two groups (P < 0.05). In conclusion, macrophage polarization in lung cancer influenced by VEGFA could be regulated by miR-320 overexpression.

Protective effect of royal jelly on fluoride-induced nephrotoxicity in rats via the some protein biomarkers signalling pathways: a new approach for kidney damage

Introduction Protective effect of royal jelly (RJ) on fluoride-induced nephrotoxicity was investigated in this study. Methods 42 healthy male Wistar rats (n = 42, 8 weeks of age) were divided equally into 6 groups with 7 rats in each; (1) Group-1: Controls fed with standard diet; (2) Group-2: RJ [100 mg/kg] bw (body weight), by oral gavage; (3) Group-3: Fluoride [50 mg/kg] bw, in drinking water; (4) Group-4: Fluoride [100 mg/kg] bw, in drinking water; (5) Group-5: RJ [100 mg/kg] bw, by oral gavage + Fluoride [50 mg/kg] bw, in drinking water; (6) Group-6: RJ [100 mg/kg] bw, by oral gavage + Fluoride [100 mg/kg] bw, in drinking water. After 8 weeks, all rats were decapitated and their kidney tissues were removed for further analysis. The protein expression levels of caspase-3, caspase-6, caspase-9, Bcl-2, Bax, VEGF, GSK-3, BDNF, COX-2 and TNF-α proteins in kidney tissue were analysed by western blotting technique Results RJ increased Bcl-2, COX-2, GSK-3, TNF-α and VEGF protein levels and a decreased caspase-3, caspase −6, caspase-9, Bax and BDNF protein levels in fluoride-treated rats. Conclusion RJ application may have a promising therapeutical potential in the treatment of many diseases in the future by reducing kidney damage.

The traditional Chinese medicine (TCM), Sheng-xue-xiaoban, inhibits the angiogenesis in ovarian cancer in vitro

Purpose: The traditional Chinese medicine (TCM), Sheng-xue-xiao-van (SXXB) is reported to be effective in treatment of thrombocytopenic purpura and chemotherapy-induced thrombocytopenia in clinical practices. The purpose of this study was to explore the functions of the SXXB Capsule in ovarian cancer in vitro.Methods: The toxicity of the medicine was measured using CCK8 method in ovarian cancer cell lines A2780 and OC-3-VGH and also the human ovarian epithelial cell line IOSE80. The cells were treated using the DMSO-diluted SXXB solution with the concentration levels at0, 3.125 and 6.25 μg/ml. Western blot was applied to measure the VEGFA proteins in each group. Later, the cells treated with 0 and 25μg/ml SXXB solution for 48h were collected for supernatant. Then HUVECs were co-cultured with the supernatant. Migration and tube formation assays were performed thereafter.Results: SXXB solution showed higher toxicity in ovarian cancer cells than in IOSE80. The protein levels of VEGFA were reduced as the SXXB concentrations increased in ovarian cancer cells but not in IOSE80. Migration and formed tubes were inhibited in HUVECs co-cultured with the supernatant collected from SXXB-treated ovarian cancer cells.Conclusion: The SXXB Capsule might inhibit the angiogenesis in ovarian cancer in vitro.

Abstract 3209: Everolimus inhibits angiogenesis and lymphangiogenesis to affect tumor growth in TP53 mutant HNSCC

Abstract Head and neck squamous cell carcinoma (HNSCC) constitutes the eighth most common cancer globally, with the eighth highest mortality rate amongst all cancer types. TP53 is the most frequently mutated gene in HPV -negative HNSCC. HPV-negative HNSCC harboring p53 mutations have the worst clinical outcomes with 50-60% local regional and distant recurrences causing increased morbidity and mortality. TP53 mutations are associated with shorter recurrence-free and overall survival. Angiogenesis and lymphangiogenesis play a causal role in tumor recurrence and lymph node metastasis. Therefore, inhibiting tumor angiogenesis and lymphangiogenesis is important in to preventing tumor recurrence and metastasis of TP53 mutant HNSCC. Previous studies demonstrated that mTOR activates STAT3 to upregulate HIF-1α and its target proteins, VEGF-A and VEGF-C, key molecules involved in angiogenesis and lymphangiogenesis, respectively. Further, mTOR inhibitors suppress autocrine and paracrine growth stimulation of tumor and lymphatic endothelial cells by impairing the VEGF-C/VEGF-3 axis and releasing soluble VEGFR-2. Accordingly, our objective was to investigate the effect of an mTOR inhibitor, everolimus, on angiogenesis and lymphangiogenesis of HPV-negative TP53 mutant HNSCC. Everolimus treatment of HPV-negative HNSCC cell lines harboring a variety of TP53 mutations, significantly downregulated protein and mRNA levels of VEGF-A and VEGF-C. Moreover, everolimus downregulated HIF-1α protein levels suggesting a HIF-1alpha-dependent VEGF modulation. Interestingly, treatment of human microvascular and lymphatic endothelial cells (HMEC-1 and HMEC-1A, respectively) with everolimus was also associated with a significant reduction in cell proliferation, in vitro tube formation, and migration. Protein and mRNA levels of HIF-1α, VEGF-A, and VEGF-C were downregulated by everolimus treatment in both HMEC-1 and HMEC-1A cell lines. Taken together, our data suggest that everolimus prevents angiogenesis and lymphangiogenesis through the inhibition of HIF-1α, indicating a promising role for mTOR inhibitors in treating HPV-negative TP53 mutant HNSCC patients at high risk for recurrence. Citation Format: Md Maksudul Alam, Janmaris Marin Fermin, Mark Knackstedt, Mackenzie J. Noonan, Emily K. Daniel, Tara Moore-Medlin, Xiaohua Rong, Alok R. Khandelwal, Cherie-Ann O. Nathan. Everolimus inhibits angiogenesis and lymphangiogenesis to affect tumor growth in TP53 mutant HNSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3209.

Berberis dictyophylla F. inhibits angiogenesis and apoptosis of diabetic retinopathy via suppressing HIF-1α/VEGF/DLL-4/Notch-1 pathway

Ethnopharmacological relevanceXiao Bopi (XBP, སྐྱེར་བའི་བར་ཤུན།), as a classical Tibetan medicinal plant in China, which derived from the stem bark of Berberis dictyophylla F., has the function of “clearing heat and decreasing mKhris-pa”. And it traditionally is utilized to treat the diabetes mellitus and its complications, such as diabetic retinopathy (DR). However, its underlying mechanisms remain unclear. Aim of the studyThe purpose of this study aimed to explore the microvascular protection of water extract of XBP against the spontaneous retinal damage of db/db mice. Meanwhile, the underlying mechanisms of XBP on angiogenesis and apoptosis were further interpreted. Materials and methodsWe firstly used high-performance liquid chromatography to detected the representative chemical ingredients in the water extract of XBP. The DR model of db/db mice was then randomly divided into five groups: model group, calcium dobesilate (0.23 g/kg) group, and the water extract of XBP (0.375, 0.75 and 1.5 g/kg, respectively) groups. After 8 weeks of continuous administration, the parameters including body weight, fasting blood glucose, oral glucose tolerance test and insulin tolerance test were measured. The pathological changes and abnormal angiogenesis of the retina were detected by optical coherence tomography, HE, periodic acid–Schiff staining and transmission electron microscopy. Simultaneously, molecular docking was used to predict the potential connections between representative ingredients in XBP and angiogenesis/apoptosis-related proteins. The level of angiogenesis-related proteins and gene hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth (VEGF), delta-like ligand 4 (DLL-4) and Notch-1 were estimated by immunofluorescence analyses and real time-PCR. Further, TUNEL staining and immunofluorescence analyses were performed to investigate the apoptotic phenomenon and the expression of Bax, Bcl-2, Apaf-1, Cyto-c and cleaved caspase-3 and cleaved caspase-9 in the retina. ResultsPhytochemical analysis revealed that magnoflorine, jatrorrhizine, palmatine and berberine were principally representative ingredients in XBP. The results demonstrated that XBP effectively increased glucose tolerance and insulin sensitivity, whereas no effect on body weight of DR mice. Moreover, retinal thickening, pathological and retinal ultrastructure changes in DR mice were evidently ameliorated by XBP. The molecular docking results demonstrated that the main components of XBP and the protein of angiogenesis and apoptosis had a potential bind. XBP restrained the gene and protein levels of HIF-1α, VEGF, DLL-4 and Notch-1 in retina. Additionally, the TUNEL-positive cell rate and the down-regulated proteins of Bax, Apaf-1, Cyto-c, cleaved Caspase-3 and cleaved Caspase 9 and increased Bcl-2 level were revised by XBP. ConclusionsTo sum up, the results suggested that XBP against DR could attribute to alleviating angiogenesis and apoptosis by suppressing the HIF-1α/VEGF/DLL-4/Notch-1 pathway. This evidence sheds a new light on the potential mechanisms of XBP in the treatment of DR.

PTP1B inhibition ameliorates inflammatory injury and dysfunction in ox-LDL-induced HUVECs by activating the AMPK/SIRT1 signaling pathway via negative regulation of KLF2.

Atherosclerosis is a key pathogenic factor of cardiovascular diseases. However, the role of protein tyrosine phosphatase 1B (PTP1B) in oxidized low-density lipoprotein (ox-LDL)-treated vascular endothelial cells remains unclear. The aim of the present study was to explore the possible physiological roles and mechanism of PTP1B in atherosclerosis using HUVECs as an in vitro model. PTP1B expression was assessed by reverse transcription-quantitative PCR. Cell viability was measured using the Cell Counting Kit-8 and lactate dehydrogenase activity assays. Levels of inflammatory factors, including IL-1β, IL-6 and TNF-α, and oxidative stress factors, including malondialdehyde, superoxide dismutase and glutathione peroxidase, were assessed using ELISA and commercially available kits, respectively. Furthermore, TUNEL assay and western blotting were performed to assess the extent of apoptosis-related factors, including Bcl-2, Bax, Cleaved caspase-3 and Caspase-3. Tube formation assay was used to assess tubule formation ability and western blotting was to analyze VEGFA protein level. Binding sites for the transcription factor Kruppel-like factor 2 (KLF2) on the PTP1B promoter were predicted using the JASPAR database and verified using luciferase reporter assays and chromatin immunoprecipitation. The protein levels of phosphorylated 5'AMP-activated protein kinase (p-AMPK), AMPK and SIRT1 were measured using western blotting. The results demonstrated that the PTP1B mRNA and protein expression levels were significantly upregulated in oxidized low-density lipoprotein (ox-LDL)-induced HUVECs. In addition, ox-LDL-induced HUVECs transfected with short hairpin RNA against PTP1B exhibited a significant increase in cell viability, reduced inflammatory factor levels, apoptosis and oxidative stress, as well as increased tubule formation ability. KLF2 was found to negatively regulate the transcriptional activity of PTP1B. KLF2 knockdown reversed the protective effects of PTP1B knockdown on ox-LDL-induced HUVECs. KLF2 knockdown also abolished PTP1B knockdown-triggered AMPK/SIRT1 signaling pathway activation in ox-LDL-induced HUVECs. To conclude, the results of the present study suggested that PTP1B knockdown can prevent ox-LDL-induced inflammatory injury and dysfunction in HUVECs, which is regulated at least in part by the AMPK/SIRT1 signaling pathway through KLF2.

Construction of Tissue-engineered Cartilage In Vivo from Microtia Chondrocytes After Transfection with Human VEGF165 Genes Mediated by a Recombinant Adeno-Associated Viral Vector.

To evaluate the transfection efficiency of cultured chondrocytes from individuals with microtia (microtia chondrocytes) with the recombinant adeno-associated virus vector rAAV2-hVEGF165-IRES-EGFP and hVEGF165 in vitro. To test whether VEGF165 gene-modified microtia chondrocytes can enhance the survival and quality of tissue-engineered cartilage. The recombinant plasmid rAAV2-hVEGF165-IRES-EGFP was inserted into rAAV2 virus vectors to construct rAAV2-hVEGF165-IRES-EGFP using the AATMaxTM system. The second-passage microtia chondrocytes were divided into 3 groups in vitro: the Ctr group (without transfection), Exp1 group (transfected with rAAV2-IRES-EGFP), and Exp2 group (transfected with rAAV2-hVEGF165-IRES-EGFP). At 24 h, 48 h, 72 h and 7 d after transfection, cell viability was measured by MTT staining. Transfection efficiency was determined by the rate of fluorescence-positive cells. The mRNA expression of hVEG165 was detected by RT-PCR (reverse transcription PCR) and agarose gel electrophoresis, and the VEGF165 protein levels in the supernatant fluids were measured by ELISAs. The second passage microtia chondrocytes with (Exp) and without (Ctr) transfection of VEGF165 genes were mixed with 0.5 ml 30% Pluronic F-127 at 4°C and then injected subcutaneously into the opposing side of the back of nude mice. Eight weeks after injection, the cartilage-like tissues of nude mice were harvested for morphological and histologic examination. Chondrocyte viability increased in a time-dependent manner but did not differ among the 3 groups at the same time point. The mRNA and protein levels of VEGF increased in a time-dependent manner in the 3 groups. The mRNA and protein levels of VEGF165 were much higher in the Exp 2 group than in the Ctr and Exp 1 groups at the same time point, but the levels were not significantly different between the Exp 1 and Exp 2 groups. Both the Ctr group and the Exp1 group formed mature cartilage with mature lacunar structures, metachromatic matrices, collagen, and elastic fibers, and the structure of neonatal cartilage was not significantly different between the 2 groups. However, the wet weight of the neonatal cartilage was much larger in the Exp group (127.4±12.4 mg) than in the Ctr group (58.5±12.2 mg, p < 0.05). VEGF protein staining also showed a higher level in the Exp group. The HVEGF165 gene was transfected efficiently into microtia chondrocytes using the recombinant adeno-associated virus vector rAAV2-hVEGF165-IRES-EGFP. After transfection, the mRNA and protein levels of hVEGF165 increased in a time-dependent manner. VEGF165 gene-modified microtia chondrocytes showed enhanced survival in vivo but did not improve the texture of tissue-engineered cartilage. This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description ofthese Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

CCDC88A Post-Transcriptionally Regulates VEGF via miR-101 and Subsequently Regulates Hepatocellular Carcinoma.

BackgroundmiR-101 is one of the most abundantly expressed microRNA (miRNA) and exerst a critical role in hepatocellular carcinoma (HCC) by targeting to 3’ -untranslated region (UTR) of Girders of actin filaments (CCDC88A) and Vascular endothelial growth factor (VEGF) mRNA, but the underlying molecular mechanism remains to be elucidated. This study aimed to investigate the potential role of CCDC88A on malignancies and stemness by regulating VEGF via miR-101 in HCC.MethodsGene Expression Profiling Interactive Analysis (GEPIA) was employed to analyze the relevance of CCDC88A expression with prognosis in HCC. Tissue slides were performed to confirm the protein level of CCDC88A in HCC. Correlation between CCDC88A and VEGF was transcriptionally and post-transcriptionally detected, followed by evaluation of malignancies.ResultsBy employing Immunohistochemistry, we found CCDC88A protein was upregulated in HCC tissues, which is closely correlated to poor prognosis and survival rate. Employment of GEPIA revealed the positive correlation between CCDC88A and VEGF in HCC, but not in liver tissue. Silencing of CCDC88A in Huh-7 and SK-HEP-1 cells significantly decreased proliferation, cell cycle phases, migration, invasion, colony formation, and tumor formation. Introduction of miR-101 mimics specifically targeting CCDC88A and VEGF decreased protein levels of both CCDC88A and VEGFA. Notably, inhibition of miR-101 reversed the correlation between CCDC88A and VEGFA protein levels, indicating that CCDC88A and VEGF may exert as a miR-101 sponge. The addition of SKLB1002, a VEGFR2 inhibitor inhibited malignant behaviors, which was further inhibited by the introduction of miR-101 mimics, indicating that CCDC88A regulates malignant behaviors partially via regulating VEGF. Moreover, CCDC88A also promotes the stemness of cancer stem-like cells derived from HCC cells depending on VEGF modification.ConclusionTaken together, our findings suggested that the miR-101/CCDC88A/VEGF axis could be a potential therapeutic target of HCC treatment.

Effects of miR-363 on the Biological Activities of Eutopic Endometrial Stromal Cells in Endometriosis.

EMs is a kind of benign disease with certain malignant behaviors. The adhesion, invasive growth, and angiogenesis of ectopic endometrial cells are the pathological basis of EMs occurrence, but its etiology and pathogenesis have not been completely illustrated yet. In our research, we aim to investigate the role of miR-363 in the pathogenesis of endometriosis. Real-time quantitative PCR was used to detect the expression of miR-363 before and after ESC/NSC transfection. CCK-8, flow cytometry, and transwell assay were used to detect the effect of the miR-363 expression on cell proliferation, apoptosis, and invasion. The effects of the miR-363 expression on the contents of Fas/APO-1 and ICAM-1 in cell culture supernatant were detected by ELISA. qRT-PCR and WB assay were used to detect the effects of the miR-363 expression on the mRNA and protein expression levels of ICAM-1, MMP-7, and VEGF in ESC. The increased expression of miR-363 could inhibit the proliferation and invasion of ESC, promote apoptosis, and inhibit the secretion of FAS/APO-1 and ICAM-1. The knockdown expression of miR-363 promoted proliferation and invasion of NSC, inhibited apoptosis, and promoted secretion of FAS/APO-1 and ICAM-1. VCAM-1, VEGF, and MMP-7 were detected in ESCs before transfection. The protein expression level was higher than that of NSCs. Compared with pretransfection, the protein levels of VCAM-1, VEGF, and MMP-7 in the M-363 group were significantly downregulated. The downregulated expression of miR-363 was associated with a stronger cell proliferation ability, a lower cell apoptosis rate, and a stronger ESC. Migration is associated with invasiveness, proliferation, angiogenesis, and immune escape. The low expression of miR-363 promotes endogenesis through posttranscriptional regulation of target genes VCAM-1, MMP-7, and VEGF. The differential expression of miR-363 between ESC and NSC may be an important factor in the many biological differences between ESC and NSC.

Ascorbic acid 2-glucoside preconditioning enhances the ability of bone marrow mesenchymal stem cells in promoting wound healing

BackgroundNowadays, wound is associated with a complicated repairing process and still represents a significant biomedical burden worldwide. Bone marrow mesenchymal stem cells (BMSCs) possess multidirectional differentiation potential and secretory function, emerging as potential cellular candidates in treating wounds. Ascorbic acid 2-glucoside (AA2G) is a well-known antioxidant and its function in BMSC-promoting wound healing is worth exploring.MethodsThe in vitro cell proliferation, migration, and angiogenesis of BMSCs and AA2G-treated BMSCs were detected by flow cytometry, EDU staining, scratch assay, transwell assay, and immunofluorescence (IF). Besides, the collagen formation effect of AA2G-treated BMSCs conditioned medium (CM) on NIH-3T3 cells was evaluated by hydroxyproline, qRT-PCR and IF staining detection. Next, in the wound healing mouse model, the histological evaluation of wound tissue in PBS, BMSCs, and AA2G-treated BMSCs group were further investigated. Lastly, western blot and ELISA were used to detect the expression levels of 5-hmc, TET2 and VEGF protein, and PI3K/AKT pathway activation in BMSCs treated with or without AA2G.ResultsThe in vitro results indicated that AA2G-treated BMSCs exhibited stronger proliferation and improved the angiogenesis ability of vascular endothelial cells. In addition, the AA2G-treated BMSCs CM enhanced migration and collagen formation of NIH-3T3 cells. In vivo, the AA2G-treated BMSCs group had a faster wound healing rate and a higher degree of vascularization in the new wound, compared with the PBS and BMSCs group. Moreover, AA2G preconditioning might enhance the demethylation process of BMSCs by regulating TET2 and up-regulating VEGF expression by activating the PI3K/AKT pathway.ConclusionsAA2G-treated BMSCs promoted wound healing by promoting angiogenesis and collagen deposition, thereby providing a feasible strategy to reinforce the biofunctionability of BMSCs in treating wounds.

Investigation of the Effect of High-Intensity Interval Training (HIIT) on the Expression of the Genes miR-126, miR-296, HGS and VEGF-A Protein Levels in Tumor Tissue in Female Mice

Background &amp; Objective: The studies of the last two decades have shown that regular training is associated with the reduction of mortality among patients with breast cancer, and has an important role in inhibiting breast cancer progression as well as treatment of the disease. However, the micro-molecular mechanisms involved are not fully understood yet. However, the aim of the present study was to investigate the effect of a high-intensity interval training (HIIT) on the expression of miR-126, miR-296, HGS gene, and VEGF-A protein level in tumor tissue of the mice with breast cancer. Materials &amp; Methods: For this purpose, 12 BALB/c mice (6-8 weeks, weight 19 ± 1.05 g), after induction of cancer (MC4-L2 subcutaneous injection into the right side of the mice), were randomly divided into two groups with 6 in each group: HIIT and control. Each session of HIIT group program includes six intervals of three minutes and 20 seconds with an intensity of 85 to 90 percent of VO2max and 1-minute recovery with 30 to 35 percent VO2max intensity between each interval; and the animals were sacrificed 24 hours after the last training session and the expression of miR-126, miR-296, HBS gene and VEGF protein levels in tumor tissues were examined. Results: The statistical results showed that the implementation of HIIT significantly reduced the expression of miR-296 and subsequently increased the HGS gene expression and led to increased expression levels of miR-126 and decreased levels of VEGF-A protein in the training group compared to the control group (p&lt;0.05). Conclusion: It seems that HIIT, by suppressing tumor angiogenesis signaling pathways, could be an effective intervention to inhibit the growth of breast tumors.

Effects of different types of exercise training on angiogenic responses in the left ventricular muscle of aged rats.

We evaluated angiogenic responses in the left ventricular muscle and aerobic capacity according to exercise type (aerobic, resistance, combined) in aged rats. In total, 24 male Sprague-Dawley rats (100weeks old) were used. To investigate the effect of regular training, the rats were divided into non-exercise (NE), aerobic exercise (AE), resistance exercise (RE), and combined exercise (CE) groups (six rats per group). Regular training tailored to each exercise type was performed for 8weeks (five times a week, 1h per day). After 8weeks of training, aerobic capacity was evaluated by a treadmill running test. Left ventricular muscle tissue was collected and the protein levels of angiogenesis indicators (eNOS, HIF-1α, PGC-1α, VEGF, FLK-1, Ang-1, Ang-2) were analyzed by Western blotting. Capillaries were observed by immunohistochemical staining for CD31. Body weight, heart weight, and heart/body weight ratio showed no difference among the groups. The AE and CE groups showed higher treadmill running capacity than the NE and RE groups. The eNOS, VEGF, HIF-1α, PGC-1α, and Ang-2 protein levels were significantly higher in the AE than NE group. The PGC-1α and FLK-1 protein levels were significantly higher in the RE than NE group. In addition, in the CE group, the eNOS, FLK-1, and PGC-1α protein levels were significantly higher than in the NE group. Expression of CD31 in cardiac tissue was higher in the AE and CE groups than in the other groups. Taken together, the results suggest that regular exercise training, irrespective of exercise type, might improve cardiovascular function by inducing angiogenic responses in the aged myocardium; however, AE may be the most effective.

VEGF alleviates lower limb ischemia in diabetic mice by altering muscle fiber types.

Lower limb ischemia caused by diabetic foot (DF) is one of the most serious complications of diabetes. The therapeutic role of VEGF in DF is well documented. However, the mechanism for action of VEGF is still not clear. The present study aimed to explore the effects of VEGF-mediated skeletal muscle fiber type switch in angiogenesis and the treatment of DF. C57BL/6 mice housed in cages equipped with a voluntary running wheel were used to access VEGF protein level and citrate synthase activity (by ELISA) as well as muscle fiber type changes (by immunofluorescence) in the gastrocnemius muscle. C57BL/6 mice were fed on a high-fat diet for 6 weeks and then injected with streptozocin to induce diabetic lower limb ischemia model. Control adenovirus (Ad-GFP) or Ad-VEGF-GFP were then injected into the left gastrocnemius of the ischemic diabetic mice. Blood flow perfusion was examined by laser Doppler imaging at 1, 3, 7 and 14 days after adenovirus transduction. On day 14, all mice were anesthetized and sacrificed. VEGF expression levels, citrate synthase activity and muscle fiber type changes in the gastrocnemius muscle were assayed by ELISA and immunofluorescence analysis of myosin heavy chain IIa (MHCIIa) expression, respectively. Transwell assays were performed to determine whether VEGF-treated C2C12 myotubes played a role on tubule formation and migration of HUVECs. It was found that VEGF levels and citrate synthase activity were upregulated after voluntary exercise, along with the increased frequency of oxidized muscle fibers. Notably, adenovirus-mediated VEGF overexpression in the muscle also increased the frequency of oxidized (MHCIIa-positive) muscle fibers, enhanced citrate synthase activity and ameliorated lower limb ischemia in diabetic mice. VEGF treatment enhanced the phosphorylation of PI3K, Akt and AMPK (assayed by western blotting), as well as glucose consumption and metabolism (assayed by western blotting and glucose uptake assay), in the C2C12 myotubes. Interestingly, VEGF-treated C2C12 myotubes promoted the migration and tubule formation of HUVEC cells. The present findings suggest that skeletal muscle fiber conversion might be a potential approach for VEGF-mediated angiogenesis and disease treatment, which may provide new options for the prevention and treatment of DF.

The effects of combined training and activity in an enriched environment on the brain BDNF and VEGF protein levels in the pre-pubertal male rats

Background. Neurotrophic factors play an important role in the brain plasticity. The brain developmental processes remarkably occur in the pre-pubertal period, and training interventions during this sensitive period may lead to specific effects. The purpose of this study was to investigate the effects of combined training and activity in an enriched environment on the brain BDNF and VEGF protein levels in the pre-pubertal male rats. Methods. The pre-pubertal male Wistar rats (n=18) were randomly divided into three groups of combined training (CT), enriched environment (EE), and control (C) at postnatal day 22. The combined training included endurance and resistance physical exercises, which performed six days a week. A large cage with various rich stimuli (wheel running, ladder, toys, tunnels) was provided for voluntary physical activity in an enriched environment. The interventions lasted for three weeks, and the brain tissue (right hemisphere) was removed for biochemical evaluations. The BDNF and VEGF protein levels of the brain were measured by the western blotting method. Results. Significant differences found in the BDNF and VEGF levels in this study (P&lt;0.01). The neurotrophic factors were significantly increased in the CT and EE groups. Although VEGF protein level increased in both groups, only enriched rats showed a significant difference. Conclusion. It can be concluded that combined training and voluntary physical activity in an enriched environment in the pre-pubertal period can effective in brain plasticity by increasing the neurotrophic factors.

Blocking Ocular Sympathetic Activity Inhibits Choroidal Neovascularization.

Purpose: To investigate how modulating ocular sympathetic activity affects progression of choroidal neovascularization (CNV), a hallmark feature of wet age-related macular degeneration (AMD).Methods: In the first of two studies, Brown Norway rats underwent laser-induced CNV and were assigned to one of the following groups: daily eye drops of artificial tears (n = 10; control group); daily eye drops of the β-adrenoreceptor agonist isoproterenol (n = 10); daily eye drops of the β-adrenoreceptor antagonist propranolol (n = 10); sympathetic internal carotid nerve (ICN) transection 6 weeks prior to laser-induced CNV (n = 10). In the second study, rats underwent laser-induced CNV followed by ICN transection at different time points: immediately after the laser injury (n = 6), 7 days after the laser injury (n = 6), and sham surgery 7 days after the laser injury (n = 6; control group). All animals were euthanized 14 days after laser application. CNV development was quantified with fluorescein angiography and optical coherence tomography (in vivo), as well as lesion volume analysis using 3D confocal reconstruction (postmortem). Angiogenic growth factor protein levels in the choroid were measured with ELISA.Results: In the first study, blocking ocular sympathetic activity through pharmacological or surgical manipulation led to a 75% or 70% reduction in CNV lesion volume versus the control group, respectively (P < 0.001). Stimulating ocular sympathetic activity with isoproterenol also led to a reduction in lesion volume, but only by 27% versus controls (P < 0.05). VEGF protein levels in the choroid were elevated in the three treatment groups (P < 0.01). In the second study, fluorescein angiography and CNV lesion volume analysis indicated that surgically removing the ocular sympathetic supply inhibited progression of laser-induced CNV, regardless of whether ICN transection was performed on the same day or 7 days after the laser injury.Conclusion: Surgical and pharmacological block of ocular sympathetic activity can inhibit progression of CNV in a rat model. Therefore, electrical block of ICN activity could be a potential bioelectronic medicine strategy for treating wet AMD.

Doxycycline inhibits the progression of metastases in early-stage osteosarcoma by downregulating the expression of MMPs, VEGF and ezrin at primary sites

IntroductionOsteosarcoma (OS) is the most common primary osseous malignant tumour, with high propensity to metastasise in lungs. Pulmonary micro-metastases are present in up to 80% of patients at initial diagnosis and they are associated with significantly worse prognosis. Doxycycline (Dox) is a synthetic tetracycline that has been shown to have anti-cancer properties in vitro and in vivo, and inhibit angiogenesis - effects that may prove beneficial for several types of cancer. The aim of the present work was to study how Dox affects OS cell growth in vitro and in vivo and OS-driven pulmonary metastasis in vivo. MethodsIn vitro, the effect of Dox was measured in MG-63 and 143B human OS cell viability, apoptosis, invasion and migration. In vivo, highly metastatic 143B cells were orthotopically implanted into the tibia of SCID mice. The tumour growth and pulmonary metastases between Dox treated and untreated, non-amputated and early amputated xenografts were examined. ResultsIn vitro, Dox decreased viability, inhibited invasion, migration, and induced the apoptosis of OS cells. In vivo, Dox significantly enhanced tumour necrosis at primary OS sites, similarly to its in vitro effect, and downregulated the expression of Ki67, MMP2, MMP9, VEGFA and ezrin. It also decreased circulating VEGFA and MMP9 protein levels, in line with the decreased metastatic burden in Dox-treated mice (non-amputated and early-amputated). ConclusionsReprofiling of Dox can prevent the evolvement of pulmonary micro-metastases to clinically detectable macro-metastases and suppress the lethal progress of OS by inhibiting the expression of MMPs, VEGFA and ezrin at primary sites.

Oridonin represses epithelial-mesenchymal transition and angiogenesis of thyroid cancer via downregulating JAK2/STAT3 signaling

Oridonin, a bioactive diterpenoid isolated from Rabdosia rubescens, has been reported to exert anticancer activity in various cancers. However, the molecular mechanism of oridonin in thyroid cancer has not yet been elucidated. In the present study, oridonin was found to significantly inhibit migration and invasion of thyroid cancer TPC-1 and BCPAP cells, as evidenced by wound healing assay, transwell migration assay and Matrigel invasion assay. In addition, oridonin could partially impede epithelial-mesenchymal transition by upregulating E-Cadherin expression and downregulating N-Cadherin and vimentin expressions in a concentration-dependent manner. Accumulating evidence indicated that JAK2 (Janus kinase-2)/STAT3 (Signal Transducer and Activator of Transcription 3) signaling pathway was associated with epithelial-mesenchymal transition. As expected, the protein levels of phosphorylated-JAK2 and phosphorylated-STAT3 were dramatically reduced upon oridonin treatment in thyroid cancer TPC-1 and BCPAP cells. Subsequently, the findings revealed that JAK2 overexpression could weaken the anti-metastatic effect and partially attenuate MET (mesenchymal-to-epithelial transition) by oridonin, while AG490, a JAK2 antagonist, enhanced the above process in thyroid cancer cells. The subsequent results showed that oridonin inhibited angiogenesis and VEGFA expression in thyroid cancer cells by tube formation assay, western blot and ELISA assay. Meanwhile, AG490 could further attenuate oridonin-treated VEGFA protein level. In addition, the in vivo results further confirmed that oridonin inhibited tumorigenicity in thyroid cancer xenograft. In conclusion, the results demonstrated that oridonin repressed metastatic phenotype, angiogenesis and modulated EMT (epithelial-mesenchymal transition) of thyroid cancer cells via the inactivation of JAK2/STAT3 signaling pathway, suggesting that JAK2 may be a novel therapeutic target of oridonin against thyroid cancer.

IGF-I combined with exercise improve diabetes-induced vascular dysfunction in heart of male Wistar rats.

Introduction: This research investigates the impact of insulin-like growth factor-I (IGF -I)and exercise on mediators associated with angiogenesis (VEGF-A, TSP-1, and NF-кβ) and capillarization status of the diabetic rats’ hearts. Methods: Splitting of forty Wistar male rats into five groups occurred as following: control,diabetes, diabetes+IGF-I, diabetes+exercise, and diabetes+exercise+IGF-I.Through intraperitoneal administration of 60 mg/kg streptozotocin, the condition of Type 1diabetes was escalated. After four weeks of treatment with IGF-I (2 mg/kg/day) or treadmill exercise (17 m/min, zero degrees slope, 30 min/day), in the heart, microvascular density and protein levels of VEGF-A, TSP-1, and NF-кβ were determined by H&E staining and ELISA,respectively. Results: Within the diabetic group, observations present a significant decrease in VEGF-A and MVD levels, whereas an increase in the TSP-1 and NF-Κb levels. While these impacts were reversed by either IGF-I or exercise treatments, simultaneous treatment had synergistic effects. Moreover, among diabetic rats, undesirable histologic alterations of the heart were demonstrated, including myonecrosis, interstitial edema, hemorrhage, and mononuclear immune cell infiltration, whereas treatments improved these changes. Conclusion: These data manifest that IGF-I and exercise can increase the cardiac angiogenesis of diabetic rats through increasing expression of VEGF-A, and decreasing TSP-1 and NF-кβproteins level, also can improve myocardial tissue damages.

Exposure to widespread drinking water chemicals, blood inflammation markers, and colorectal cancer.

Trihalomethanes (THMs) and nitrate are widespread chemicals in drinking water associated with colorectal cancer risk but mechanisms are not well understood. We explored the association between exposure to THMs and nitrate in drinking water and inflammation markers, and the link with colorectal cancer risk. A subset of 198 colorectal cancer cases and 205 controls from the multicase-control study MCC-Spain were included. Average concentration of THMs (chloroform, bromodichloromethane, dibromochloromethane, bromoform) and nitrate in tap water at the residence was estimated from age 18 until 2years before the interview ("long term") and for a recent period (3years before diagnosis). Serum levels of EGF, eotaxin, G-CSF, IL-17E, IL-1rA, IL-8, IP-10, MDC, MPO, periostin, VEGF, and C-reactive protein (CRP) were measured. We estimated the linear association between inflammation markers and exposure among controls, and the odds ratio of colorectal cancer associated with THM and nitrate exposure, and inflammation markers. A mediation analysis was conducted to identify inflammation markers in the pathway between THM/nitrate exposure and colorectal cancer. Serum concentrations of EGF, IL-8, IL-17E and eotaxin increased with recent residential levels of brominated THMs, chloroforom and/or total THM. No associations were observed for nitrate and for long-term residential THM levels. All residential exposures except chloroform were positively associated with colorectal cancer. Serum concentrations of VEGF and periostin were positively associated with colorectal cancer, while EGF was inversely associated. One protein-exposure combination (periostin-recent ingested brominated THMs) slightly mediated the association with colorectal cancer risk. Results suggest that estimated THM exposure is involved in inflammation processes. However, the study design was limited to stablish etiologically relevant associations between the protein levels and colorectal cancer risk. The lack of association between nitrate exposure and inflammation markers suggests other biological mechanisms are involved in the link with colorectal cancer.

MiR-376a inhibits glioma proliferation and angiogenesis by regulating YAP1/VEGF signalling via targeting of SIRT1

BackgroundGlioma is the most common cancer in the central nervous system. Previous studies have revealed that the miR-376 family is crucial in tumour development; however, its detailed mechanism in glioma is not clear. MethodsCellular mRNA or protein levels of miR-376a, SIRT1, VEGF and YAP1 were detected via qRT–PCR or Western blotting. We analysed the proliferation, angiogenesis and migration abilities of glioma cell lines using colony formation, tube formation and Transwell assays. A luciferase assay was performed to determine whether miR-376a could recognize SIRT1 mRNA. Xenograft experiments were performed to analyse the tumorigenesis capacity of glioma cell lines in nude mice. The angiogenesis marker CD31 in xenograft tumours was detected via immunohistochemistry (IHC). ResultsmiR-376a expression was lower in glioma cells than in normal astrocytes. miR-376a mimic inhibited SIRT1, YAP1, and VEGF expression and suppressed the proliferation, migration and angiogenesis abilities of the glioma cell lines LN229 and A172, whereas miR-376a inhibitor exerted the opposite functions. In a luciferase assay, miR-376a inhibited the luciferase activity of WT-SIRT1. SIRT1 overexpression upregulated YAP1 and VEGF in glioma cells and promoted proliferation, migration and angiogenesis. Xenografts with ectopic miR-376a expression exhibited lower volumes and weights and a slower growth curve. Overexpression of miR-376a inhibited YAP1/VEGF signalling and angiogenesis by inhibiting SIRT1 in xenograft tissues. ConclusionmiR-376a directly targets and inhibits SIRT1 in glioma cells. Downregulation of SIRT1 resulted in decreased YAP1 and VEGF signalling, which led to suppression of glioma cell proliferation, migration and angiogenesis.