The Anti-Inflammatory Effect of the β1-Adrenergic Receptor Antagonist Metoprolol on High Glucose Treated Human Microvascular Retinal Endothelial Cells

Hyperglycemia-induced impairment of the blood-retinal barrier represents the main pathological event in diabetic retinopathy that is elicited by a reduced cellular response to an accumulation of reactive oxygen species (ROS) and increased inflammation. The purpose of the study was to evaluate whether the selective β1-adrenoreceptor (β1-AR) antagonist metoprolol could modulate the inflammatory response to hyperglycemic conditions. For this purpose, human retinal endothelial cells (HREC) were treated with normal (5 mM) or high glucose (25 mM, HG) in the presence of metoprolol (10 μM), epinephrine (1 μM), or both compounds. Metoprolol prevented both the HG-induced reduction of cell viability (MTT assays) and the modulation of the angiogenic potential of HREC (tube formation assays) reducing the TNF-α, IL-1β, and VEGF mRNA levels (qRT-PCR). Moreover, metoprolol prevented the increase in phospho-ERK1/2, phospho-cPLA2, COX2, and protein levels (Western blot) as well as counteracting the translocation of ERK1/2 and cPLA2 (high-content screening). Metoprolol reduced ROS accumulation in HG-stimulated HREC by activating the anti-oxidative cellular response mediated by the Keap1/Nrf2/HO-1 pathway. In conclusion, metoprolol exerted a dual effect on HG-stimulated HREC, decreasing the activation of the pro-inflammatory ERK1/2/cPLA2/COX2 axis, and counteracting ROS accumulation by activating the Keap1/Nrf2/HO-1 pathway.

The Effect of STAT3 Signal Pathway Activation on Retinopathy of Prematurity

Objective: To investigate the mechanism of activation of the signal transducer and activator of transcription 3 (STAT3) signal pathway in the process of retinopathy of prematurity (ROP).Methods: Sixty newborn Sprague-Dawley (SD) rats were randomly separated into the hyperoxia and air control groups (n = 30/in each group). The serum hepcidin level on 21 d was measured using the enzyme-linked immunosorbent assay (ELISA). The expression of HAMP and STAT3 protein in the liver was determined using reverse transcription-polymerase chain reaction (RT-PCR) and western blotting. Retinal neovasculature was evaluated by hematoxylin and eosin (HE) stain and fluorescein lectin. The retinal endothelial cells were treated with 250 μmol/L cobalt chloride for 72 h and added S3I-201. The STAT3 level was determined by western blotting.Results: The expression of STAT3 protein increased significantly after hyperoxia stimulation. The expression of HAMP mRNA in the hyperoxia group was significantly higher than that of the control group. The proliferation of retinal cells was inhibited, and the expression of STAT3 was increased. No significant difference was noted in vascular endothelial growth factor (VEGF) mRNA. The expression of STAT3 and VEGF mRNA was significantly reduced.Conclusion: The activation of the STAT3 signal pathway increased hepcidin expression, contributing to the pathogenesis of ROP. S3I-201 inhibited the expression of STAT3 and VEGF mRNA levels. This information provides potential novel therapeutic approach to the prevention and treatment of ROP.

NF90 Interacts with Components of RISC and Modulates Association of Ago2 with mRNA

Nuclear Factor 90 (NF90) is a double-stranded RNA-binding protein involved in a multitude of different cellular mechanisms such as transcription, translation, viral infection and mRNA stability. Recent data suggest that NF90 might influence the abundance of target mRNAs in the cytoplasm through miRNA- and Argonaute 2 (Ago2)-dependent activity. Here, we identified the interactome of NF90 in the cytoplasm, which revealed several components of the RNA-induced silencing complex (RISC) and associated factors. Co-immunoprecipitation analysis confirmed the interaction of NF90 with the RISC-associated RNA helicase, Moloney leukemia virus 10 (MOV10), and other proteins involved in RISC-mediated silencing, including Ago2. Furthermore, NF90 association with MOV10 and Ago2 was found to be RNA-dependent. Glycerol gradient sedimentation of NF90 immune complexes indicates that these proteins occur in the same protein complex. At target RNAs predicted to bind both NF90 and MOV10 in their 3' UTRs, NF90 association was increased upon loss of MOV10 and vice versa, suggesting that the two proteins may compete for the binding of common target mRNAs. Interestingly, loss of NF90 led to an increase in association of Ago2 as well as a decrease in the abundance of the target mRNA. Similarly, during hypoxia, the binding of Ago2 to vascular endothelial growth factor (VEGF) mRNA increased after loss of NF90, while the level of VEGF mRNA decreased. These findings suggest a role for NF90 in the regulation of RISC-mediated silencing which stabilizes target mRNAs, such as VEGF, during cancer-induced hypoxia.

Xihuang Pill Inhibits The Development of DMBA Combined With Oestrogen- And Progesterone-Induced Precancerous Breast lesions In Rats By The PI3K/AKT/mTOR Signaling Pathway

Background:To study the inhibitory effect of Xihuang pill on the development of DMBA combined with oestrogen- and progesterone-induced breast precancerous lesions in rats by the PI3K/AKT/mTOR signaling pathway and to explore the effect of Xihuang pill in preventing and treating breast cancer. Method: Establishment of a rat model of precancerous breast lesions with DMBA combined with oestrogen and progesterone sequential induction for 10 weeks. Xihuang pill was administered continuously by gavage for 4 weeks. Rat breast tissue was stained with haematoxylin-eosin (HE). The pathomorphological changes were observed with a light microscope. TUNEL staining was used to detect cell apoptosis in breast tissue. Western blotting was used to detect the protein expression of P-PI3K, P-AKT (S473), P-AKT (T308), PTEN, P-tuberin/TSC2, P-tuberin (p-S939), p-mTOR, and P-4E-BP1 in breast tissues. qRT-PCR was used to detect the gene expression of PTEN mRNA and VEGF mRNA. Immunohistochemistry was used to detect the protein expression of P-S6, p-p70s6k and VEGF.Result:Compared with the disease model group, the low-, middle- and high-dose Xihuang pill groups could significantly reduce the degree of breast pathology, and the number of apoptotic precancerous breast lesion cells increased with increasing Xihuang pill dose. The expression levels of P-PI3K, P-AKT (S473), P-AKT (T308), p-mTOR, P-4E-BP1, p-S6, p-p70S6K, VEGF protein and VEGF mRNA dropped with increasing Xihuang pill dose. The expression levels of PTEN, P-tuberin/TSC2, P-tuberin (p-S939) protein and PTEN mRNA increased with increasing Xihuang pill dose. Conclusion:Xihuang pill can promote the apoptosis of precancerous breast lesion cells, reduce the proliferation of vascular endothelial cells, and then inhibit the progression of precancerous breast lesions. Its mechanism is probably associated with the regulation of the PI3K/AKT/mTOR pathway related protein expression.

Cottonseed-derived gossypol and ethanol extracts differentially regulate cell viability and VEGF gene expression in mouse macrophages

Vascular endothelial growth factor (VEGF) plays an important role in chronic inflammation associated with several diseases. Many plant extracts have nutritional and healthy benefits by down-regulating VEGF expression, but there was no report on VEGF regulation by cottonseed extracts in any biological system. The objective was to investigate cell viability and VEGF expression regulated by gossypol and ethanol extracts using lipopolysaccharides (LPS) as a control. MTT, qPCR and immunoblotting techniques were used to monitor cell viability, VEGF mRNA and protein levels in mouse RAW264.7 macrophages. Gossypol dramatically reduced macrophage viability but cottonseed extracts and LPS exhibited minor effect on cell viability. VEGFb mRNA levels were approximately 40 fold of VEGFa in the macrophages. Gossypol increased VEGFa and VEGFb mRNA levels up to 27 and 4 fold, respectively, and increased VEGF protein. LPS increased VEGFa mRNA by sixfold but decreased VEGFb mRNA. LPS increased VEGF protein in 2–4 h but decreased in 8–24 h. Glanded seed extracts showed some stimulating effects on VEGF mRNA levels. Glandless seed coat extract showed increased VEGFb mRNA levels but its kernel extract reduced VEGF mRNA levels. This study demonstrated that gossypol and ethanol extracts differentially regulated cell viability and VEGF expression in mouse macrophages.

Ligustrazine Reduces the Apoptosis of Synovial Cells in Knee Arthritis Rats by Inhibiting the Expression of Glucose Regulatory Protein 78 and CCAAT Enhancer Binding Protein Beta (C/EBPb)

To investigate the effect of Ligustrazine on synovial cells and expression of GRP78 and C/EBPb in knee arthritis rats, 52 healthy SD rats, aged 10-15 months, weighing 185–225 g, were selected and fed routinely. According to the principle of random distribution, the experimental rats were assigned into healthy group, arthritis group, ligustrazine 1 and 2 groups, with 13 rats in each group. HE staining was used to detect the pathological morphology. Mankin score was used to measure the severity of the disease. The apoptosis of synovial cells was assessed by flow cytometry. col2α1 and VEGF levels were detected by RT-PCR and GRP78, XBP1 and C/EBP β levels were detected by Western blot. In the healthy group, the cells were scattered orderly, the cartilage surface was smooth, and the synovial tissue was not damaged; in the arthritis group, the joint tissue was damaged, the synovial tissue proliferated and degenerated, the cells were disordered, and synovial pannus was produced; in Ligustrazine group 1 and Ligustrazine 2 groups had certain improvement, and the effect to Ligustrazine group 2 was more obvious. Mankin score of healthy group was lower than arthritis group (P < 0.05) which had higher Mankin score than Ligustrazine 1 and 2 groups, and the Mankin score of Ligustrazine 2 group was lower than that of Ligustrazine 1 group (P < 0.05). Synovial cells apoptosis in healthy group was lower than arthritis group (P < 0.05) which had higher apoptosis than Ligustrazine 1 group and ligustrazine 2 group (P < 0.05) with lower apoptosis for Ligustrazine 2 group (P < 0.05). The expressions of col2α1 mRNA and VEGF mRNA in healthy group were lower than arthritis group (P < 0.05) which had higher levels than Ligustrazine 1 and 2 groups with Ligustrazine 2 group showing more obvious effects (P < 0.05). Arthritis group had significantly elevated levels of GRP78 and XBP1 and decreased C/EBP β levels (P < 0.05). However, GRP78 and XBP1 levels in TMP-1 group and tmp-2 group were decreased and C/EBP β level was increased (P < 0.05). In a word, Ligustrazine can reduce the apoptosis of synovial cells, promote the repair of cartilage tissue and improve the condition of knee arthritis rats by regulating the expression level of GRP78 and XBP1.

Distinct Cellular Profiles of Hif1a and Vegf mRNA Localization in Microglia, Astrocytes and Neurons during a Period of Vascular Maturation in the Auditory Brainstem of Neonate Rats

Defining the relationship between vascular development and the expression of hypoxia-inducible factors (Hifs) and vascular endothelial growth factor (Vegf) in the auditory brainstem is important to understand how tissue hypoxia caused by oxygen shortage contributes to sensory deficits in neonates. In this study, we used histology, molecular labeling, confocal microscopy and 3D image processing methods to test the hypothesis that significant maturation of the vascular bed in the medial nucleus of the trapezoid body (MNTB) occurs during the postnatal period that precedes hearing onset. Isolectin-B4 histochemistry experiments suggested that the MNTB vasculature becomes more elaborate between P5 and P10. When combined with a cell proliferation marker and immunohistochemistry, we found that vascular growth coincides with a switch in the localization of proliferating cells to perivascular locations, and an increase in the density of microglia within the MNTB. Furthermore, microglia were identified as perivascular cells with proliferative activity during the period of vascular maturation. Lastly, combined in situ hybridization and immunohistochemistry experiments showed distinct profiles of Hif1a and Vegf mRNA localization in microglia, astrocytes and MNTB principal neurons. These results suggest that different cells of the neuro-glio-vascular unit are likely targets of hypoxic insult in the auditory brainstem of neonate rats.

Effect of post-exercise muscle cooling on PGC-1α and VEGF mRNA expression in Thoroughbreds

Besides preventing exertional heat illness, muscle cooling can be a potential strategy to enhance exercise-training induced adaptations. This study aimed to examine the effects of post-exercise cooling on the mRNA expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and vascular endothelial growth factor (VEGF) in Thoroughbred skeletal muscle. Five Thoroughbred horses performed treadmill running until their pulmonary artery temperature reached 42 °C, followed by walking on the treadmill with no additional cooling (CONT) or muscle cooling with a shower using the tap water (26 °C, 0.4 l/s; COOL), for 30 min. Muscle biopsies were obtained before (PRE) and 3 h after exercise (3 Hr-REC) from the gluteus medius muscle. PGC-1α mRNA expression was elevated 3 h after exercise in both the CONT (PRE vs 3 Hr-REC: 1.0±0.1 vs 5.0±0.8, P<0.01) and COOL (PRE vs 3 Hr-REC: 1.1±0.3 vs 6.6±0.9, P<0.01) conditions; however, there was no difference between the two conditions at 3 h after exercise (P=0.17). VEGF mRNA expression was elevated 3 h after exercise in COOL (PRE vs 3 Hr-REC: 1.0±0.2 vs 2.2±0.2, P<0.05) but not in CONT (PRE vs 3 Hr-REC: 1.0±0.1 vs 1.8±0.3, P=0.08). VEGF mRNA expression at 3 h after exercise was significantly negatively correlated with rectal temperature at the end of the 30-min cooling period (r = -0.65, P<0.05). Our results suggest that the decline in body temperature after exercise may lead to greater expression of the key angiogenic gene in Thoroughbred horses.

Absence of TRPC1 Ca2+-permeable Channel Causes Placental and Fetal Growth Restriction in Mice

Objectives Placental tissue intracellular calcium (Ca2+) regulates placental development and growth (e.g., blastocyst development through branching morphogenesis). Maternal high-fat (HF) diet results in placental lipid accumulation, increase in inflammation, reduction in nutrient transport expression and intra uterine growth restriction (IUGR). Currently, whether maternal HF diet affects placental and fetal growth and development differentially under reduction in Ca2 + influx is not known. Thus, we hypothesized that maternal HF diet feeding decreases placental growth and development resulting in IUGR. We further hypothesized that reduction of Ca2 + influx in placenta worsens the maternal HF-induced placental dysfunction. Methods Two-month old female B6129SF2/J wild type (WT) and transient receptor potential canonical 1 (TRPC1) protein deficient (KO) mice were fed normal fat (NF, 16% fat) and high fat (HF, 45%) diets for 12 weeks. Fetuses and placentae were examined at mid- (D12) and late- (D19) gestation. Results Placental length, width, and weight as well as fetal weight were decreased in the TRPC1KO mice at D12 and D19 compared to that of WT mice. Expression of placental growth factor (PLGF) mRNA was decreased at D12 in TRPC1 KO mice while vascular endothelial growth factor (VEGF) mRNA levels were increased at D19 compared to WT mice. Conclusions These findings suggest that genotypic differences rather than maternal HF diet alter placental size and weight as well as fetal weight. Decreased in PLGF mRNA may be responsible for the placental and fetal growth restriction while increase in VEGF mRNA indicates compensatory adaptation to decreased PLGF-associated placental and fetal growth restriction. Future studies are needed to determine the signaling mechanism underlying Ca2 + influx reduction- induced placental dysfunction and IUGR. Funding Sources USDA Agricultural Research Service Project #3062–51,000-054–00D.

The Change of Retinal Microvascular, Microstructure and Expression of IL-6, CD18, ICAM, TNF-α and VEGF in The Early Stage of Rat Diabetic Retinopathy

Background: Diabetic retinopathy (DR) is a leading cause of vision loss and blindness. The purpose of this project is to deeply observe the change of retinal microvascular, microstructure and expression of IL-6, CD18, ICAM, TNF-α and VEGF at the early stage of DR in rats with streptozotocin-induced diabetes mellitus (DM). Methods: The fluorescein fundus angiography was used to examine fundus of living organisms, retinas were obtained for hematoxylin and eosin staining, periodic acid-Schiff staining, fluorescence imaging techniques and quantitative real-time PCR, while vitreous humors were isolated for vascular endothelial growth factor (VEGF)-A ELISA in diabetes group (n=25) and normal group (n=25) at 8th day, 4th week, 6th week, 8th week and 10th week after the onset of DM. Results: In this study, we observed not only the decrease of RGCs and the increase of E/P ratio, acellular capillaries and type IV collagen-positive strands began to occur on 8th day after induction, but the vascular permeability and neovascularization buds began to happen in diabetes group in 8th week, while the expression of VEGF-A, VEGF mRNA, IL-6 mRNA, ICAM mRNA and TNF-α mRNA began significantly higher in diabetes group compared with normal group(P＜0.01) on 8th day after induction and remained high expression level throughout the 10-week observation period. However, the expression of CD18 RNA began significantly higher in 4th week after induction and reached peak in 6th week. Conclusions: In conclusion, the retinal microvascular injury, ganglion cell changes and high expression of VEGF-A, VEGF mRNA, IL-6 mRNA, ICAM mRNA, TNF-α mRNA and CD18 mRNA were happened on very early stage. The results offer new insight into the pathogenesis of diabetic retinopathy, and provide novel targets to inhibit the ocular disease.