VEGF mRNA Levels Research Articles

Protective role of estrogen against excessive erythrocytosis in Monge\u2019s disease

Monge’s disease (chronic mountain sickness (CMS)) is a maladaptive condition caused by chronic (years) exposure to high-altitude hypoxia. One of the defining features of CMS is excessive erythrocytosis with extremely high hematocrit levels. In the Andean population, CMS prevalence is vastly different between males and females, being rare in females. Furthermore, there is a sharp increase in CMS incidence in females after menopause. In this study, we assessed the role of sex hormones (testosterone, progesterone, and estrogen) in CMS and non-CMS cells using a well-characterized in vitro erythroid platform. While we found that there was a mild (nonsignificant) increase in RBC production with testosterone, we observed that estrogen, in physiologic concentrations, reduced sharply CD235a+ cells (glycophorin A; a marker of RBC), from 56% in the untreated CMS cells to 10% in the treated CMS cells, in a stage-specific and dose-responsive manner. At the molecular level, we determined that estrogen has a direct effect on GATA1, remarkably decreasing the messenger RNA (mRNA) and protein levels of GATA1 (p < 0.01) and its target genes (Alas2, BclxL, and Epor, p < 0.001). These changes result in a significant increase in apoptosis of erythroid cells. We also demonstrate that estrogen regulates erythropoiesis in CMS patients through estrogen beta signaling and that its inhibition can diminish the effects of estrogen by significantly increasing HIF1, VEGF, and GATA1 mRNA levels. Taken altogether, our results indicate that estrogen has a major impact on the regulation of erythropoiesis, particularly under chronic hypoxic conditions, and has the potential to treat blood diseases, such as high altitude severe erythrocytosis.

Resveratrol Suppresses Severe Acute Pancreatitis-Induced Microcirculation Disturbance through Targeting SIRT1-FOXO1 Axis.

Background Resveratrol (RSV), one of the SIRT1 agonists, has the ability of alleviating severe acute pancreatitis (SAP); however, the concrete protective mechanism remains unknown. It is noteworthy that microcirculation disturbance plays a vital role in SAP, and the SIRT1/FOX1 axis can regulate microcirculation. Therefore, this study is aimed at ascertaining what is the underlying mechanism of the protective effect of RSV on SAP, and whether it is associated with alleviating microcirculation disturbance by regulating the SIRT1/FOX1 axis. Method The model of SAP was induced by retrograde injection of sodium taurodeoxycholate into the bile duct of the rats. The pancreatic wet/dry weight, ET/NO, and TXB2/6-keto-PGF1α ratios; microcirculatory function; and SIRT1 activity were examined. ELISA was used to examine the serum level of lipase, amylase, hemorheology, ET, NO, TXB2, and 6-keto-PGF1α and the content of SIRT1, VEGF, Ang I, and Ang II in the pancreas. RT-PCR was used to examine the mRNA level of VEGF, Ang I, and Ang II. Western blotting was used to detect SIRT1, FOXO1, and acetyl-FOXO1. Immunoprecipitation was used to examine the interaction of SIRT1 and FOXO1. Results Resveratrol can significantly decrease the expression of lipase, amylase, acetyl-FOXO1, VEGF, Ang II, ET, NO, TXB2, and 6-keto-PGF1α and the ratio of wet/dry weight, ET/NO, and TXB2/6-keto-PGF1α by improving microcirculatory dysfunction and blood viscosity in SAP. Moreover, resveratrol can also promote the interaction of SIRT1 and FOXO1 and increase SIRT1 activity and the expression of SIRT1 and Ang I. The SIRT1 inhibitor, Sirtinol (EX527), obliviously reversed the effects of RSV on SAP. Conclusion Resveratrol can protect rats against SAP, and its protective mechanism is associated with suppressing microcirculation disturbance through activating SIRT1-FOXO1 axis.

Accelerative effect of topical Zataria multiflora essential oil against infected wound model by modulating inflammation, angiogenesis, and collagen biosynthesis

Context Zataria multiflora Boiss (Lamiaceae) essential oil (ZME) is believed to be a bactericide herbal medicine and might alleviate negative effects of infection. Objective This study evaluates the effects of an ointment prepared from ZME (ZMEO) on infected wounds. Materials and methods A full-thickness excisional skin wound was surgically created in each mouse and inoculated with 5 × 107 suspension containing Pseudomonas aeruginosa and Staphylococcus aureus. The BALB/c mice (n = 72) were divided into four groups: (1) negative control that received base ointment (NCG), (2) positive control that daily received Mupirocin® (MG), (3) therapeutic ointment containing 2% ZMEO and (4) therapeutic ointment containing 4% ZMEO, for 21 days. Wound contraction, total bacterial count, histopathological parameters, antioxidant activity, qRT-PCR analysis for expression of IL-1β, TNF-α, VEGF, IGF-1, TGF-β, IL-10, and FGF-2 mRNA levels were assessed on days 3, 7, and 14 following the wounding. Results Topical administration of ZMEO significantly decreased the total bacterial count and wound area and also expression of IL-1β and TNF-α compared to the control groups (p < 0.05) in all days. This could also increase significantly the expression of TGF-β, IL-10 IGF-1, FGF-2, and VEGF, and also angiogenesis, fibroblasts, fibrocytes, epithelialization ratio, and collagen deposition and improve antioxidant status compared to the control group (p < 0.05). Discussion and conclusion ZMEO accelerated the healing process of infected wounds by shortening the inflammatory factors and increasing proliferative phase. Applying ZMEO only and/or in combination with chemical agents for the treatment of wound healing could be suggested.

Studies on the therapeutic effects of three TCM methods on steroid-induced osteonecrosis of femoral head

Purpose: To determine the therapeutic effects of kidney yang-tonifying, blood-activating and phlegmeliminating methods on steroid-induced osteonecrosis of femoral head (SONFH), and their mechanisms of action.Methods: A rabbit model of SONFH was established. Six rabbit groups were used: control, SONFH (given normal saline); tongluo shenggu group, kidney-tonifying group, blood-activating group, and phlegm--eliminating group, which received tongluo shenggu capsule (0.28 g/kg/day), yougui yin (YGY, 2.52 g/kg/day), taohong siwu decoction (TSD, 2.38 g/kg/day) and linggui zhugan decoction (LZD,1.54 g/kg/day), respectively, for 8 weeks. The following serum indices were assayed on the 2nd, 4th and 8th weeks: total cholesterol, triglycerides, thromboxane B2 (TXB2), 6-keto-PGF1α, osteoprotegerin (OPG), and receptor activator of nuclear factor kappa-Β ligand (RANKL). At the end of the 8th week, mRNA levels of OPG, BMP-2 and VEGF were assayed using real-time fluorescent quantitative polymerase chain reaction (RTF-qPCR).Results: Serum OPG in the kidney-tonifying group was increased, while its RANKL was decreased on the 2nd week, but these parameters, along with mRNA of OPG and BMP-2, reached normal levels at the 8th week (p &lt;0.05). The TXB2 of blood-activating group was decreased, while its 6-keto-PGF1 level was increased at the 2nd week, but both of them attained normal levels at the 8th week, along with VEGF mRNA. Serum total cholesterol, triglycerides and TXB2 in the phlegm-eliminating group were decreased, while its 6-keto-PGF1α was increased at the 2nd week, but the levels of serum total cholesterol and triglycerides were normalized at the 8th week (p &lt;0.05). Histological examination showed increases in bone trabeculae and bone marrow cells, while the proliferation of fat cells was decreased in kidney-tonifying, blood-activating and phlegm-eliminating groups.Conclusion: These findings suggest that the therapeutic effects of the three different TCM methods on SONFH involve different mechanisms.&#x0D; Keywords: Steroid-induced osteonecrosis, Femoral head, Yougui yin, Taohong siwu, Linggui zhugan decoction, Kidney-tonifying yang, Phlegm

Downregulation of astrocyte elevated gene-1 expression inhibits the development of vasculogenic mimicry in gliomas.

Vasculogenic mimicry (VM) contributes to the resistance of anti-angiogenic therapies in glioma. Certain genes, including MMP-2 and VEGF may be associated with the development of VM. Astrocyte elevated gene-1 (AEG-1) is considered to be an oncogene that promotes autophagy, invasion, metastasis, angiogenesis and drug resistance; however, the association between AEG-1 and VM formation is still unknown. The present study investigated the effects of AEG-1 downregulation on VM formation in the U87 glioma cell line in vitro and in xenograft models of glioma, and the potential underlying mechanisms of action. In the present study, U87 glioma cells were infected with the AEG-1 short hairpin RNA lentivirus. A Matrigel-based tube formation assay was performed to evaluate VM formation in vitro. Reverse transcription-quantitative PCR and western blot analysis were conducted to investigate the mRNA and protein expression levels of MMP-2 and VEGF. Glioma xenograft models were generated through the intracerebral implantation of U87 glioma cells into nude rats; CD34/Periodic Acid-Schiff double-staining was performed to detect VM channels in vivo. Following AEG-1 downregulation in U87 cells, the development of VM was significantly decreased in vitro and in vivo. In addition, the expression levels of MMP-2 and VEGF in glioma cells were decreased compared with the control group. These results suggested that downregulation of AEG-1 expression could significantly inhibit the development of VM in gliomas, both in vitro and in vivo, and may be partially related to the regulation of VEGF and MMP-2 expression.

YB-1 transferred by gastric cancer exosomes promotes angiogenesis via enhancing the expression of angiogenic factors in vascular endothelial cells

BackgroundAngiogenesis is important for the progression of gastric cancer (GC). Y-box binding protein 1 (YB-1) predicts advanced disease and indicates neovasculature formation in GC tissues, while the related mechanisms remain elusive. Exosomes mediate intercellular communications via transferring various molecules including proteins, lipids, mRNAs, and microRNAs, while the cargos of GC exosomes and the related mechanisms in GC angiogenesis were rarely reported except for several microRNAs.MethodsIn this study, human umbilical vein endothelial cells (HUVECs) were, respectively, treated by the exosomes isolated from the YB-1 transfected and the control SGC-7901 cells (SGC-7901-OE-Exo and SGC-7901-NC-Exo), and their apoptosis, proliferation, migration, invasion, and angiogenesis were, sequentially, compared. The levels of angiogenic factors including VEGF, Ang-1, MMP-9 and IL-8 in the exosome-treated HUVECs and the GC-derived exosomes were, separately, detected using PCR and Western blotting as well as RNA sequencing assays.ResultsWe observed the consistent level of YB-1 in the exosomes and their originated GC cells, and the internalization of exosomes into HUVECs. Comparing with SGC-7901-NC-Exo, SGC-7901-OE-Exo significantly inhibited the apoptosis but promoted the proliferation, migration, invasion, and angiogenesis of HUVECs, within which the increased mRNA and protein levels of VEGF, Ang-1, MMP-9 and IL-8 were demonstrated. Meanwhile, mRNA levels of VEGF, Ang-1, MMP-9 and IL-8 showed no significant difference between SGC-7901-NC-Exo and SGC-7901-OE-Exo, although statistically higher mRNA of YB-1 was detected in the SGC-7901-OE-Exo.ConclusionsOur findings illustrate YB-1 as the key component of exosome to promote GC angiogenesis by upregulating specific angiogenic factors in the exosome-treated endothelial cells but not in the exosomes themselves.

Interaction of sperm with endometrium can regulate genes involved in endometrial receptivity pathway in mice: An experimental study.

BackgroundMany researchers consider implantation and endometrial receptivity as pertinent issues in reproductive science. Although, several experiments have been performed and their results evaluated, yet there is no confirmed evidence about the related factors and the role of sperm in endometrial receptivity.ObjectiveTo investigate the effect of the sperm-endometrium interaction in regulating genes involved in the endometrial receptivity pathway.Materials and Methods In this experimental study, 10 male and 30 female NMRI mice were included, and half of the male cases were vasectomized. The subjects were divided into two groups as follows; group 1 (case) comprised of 15 females mated with 5 non-vasectomized male mice, while group 2 (control) consisted of 15 females mated with 5 vasectomized males. Cases were sacrificed and assessed after 36 hr and the endometrial tissue was extracted and kept at -80°C until the next use. The expression of the endometrial receptivity pathway genes, including VEGF, HBEGF, FGF2, EGF, LIF, LIFR, HOXA10, MUC1, PGR, and CSF, was examined in both groups. For statistical analysis, an independent samples test (Mean SD) was used.ResultsThe mRNA levels of LIF (p = 0.045), LIFR (p = 0.040), MUC1 (p = 0.032), VEGF (p = 0.022), EFG (p = 0.035), and FGF2 (p = 0.040) were significantly upregulated in the case group compared with the control group.ConclusionFinally, seminal plasma was observed to be effective in expressing the involved genes in the successful implantation pathway, including LIF, LIFR, MUC1, VEGF, EGF, and FGF2.

Expression of Notch-Hif-1α signaling pathway in liver regeneration of rats.

ObjectiveTo investigate whether the Notch–Hif-1α signaling pathway is involved in liver regeneration.MethodsRats were divided into two groups and treated with daily intraperitoneal injections of saline (control) or the gamma-secretase inhibitor, Fli-06, for 2 days. Two-thirds of the rat livers were resected and rats were later euthanized at specific time points post-resection to analyze the remnant livers. Each group's liver/body weight ratio was calculated, and immunostaining and western blotting were used to determine the cell proliferation marker, PCNA and Ki-67 expression. Real-time PCR and western blotting were used to compare the mRNA expression of Notch homolog-1 (Notch1), hairy and enhancer of split-1 (Hes1), and vascular endothelial growth factor (Vegf), and the protein expression of NICD and HIF-1α, respectively.ResultsThe liver/body weight ratios and number of Ki-67- and PCNA-positive cells were significantly lower in the experimental group than the control group, indicating lower levels of liver regeneration following the disruption of Notch signaling by Fli-06. The Hes1 and Vegf mRNA levels and NICD and HIF-1α protein expression levels were all down-regulated by Fli-06 treatment.ConclusionNotch–Hif-α signaling pathway activation plays an important role in liver regeneration, where it may contribute toward liver cell proliferation.

A NOVEL PRE-VASCULARIZED TISSUE ENGINEERING CHAMBER SERVES AS A BIO-ARTIFICIAL PANCREAS FOR AN ATTRACTIVE SITE OF ALLOGENIC AND XENOGENEIC ISLET TRANSPLANTATION

Background: Although the sites for clinical or experimental islet transplantation have been well established, the pancreatic islet survival and function in homing locations still remain unsatisfied[1]. One of possible reasons that account for this is local hypoxia due to the shortage of blood supply[2]. Here we modified a pre-vascularized tissue engineering chamber (TEC) in vivo which facilitated the vitality and function of seeded islets by providing a microvascular network prior to transplantation. Method: A TEC filled with MatrigelTM and bFGF-2 was created and embedded in the groin of streptozotocin-induced diabetic mice. The micro-vascularization in the TEC was analyzed by histology, real-time PCR and western blotting. Three hundreds of syngeneic islets were seeded into the chamber on day 0, 14, and 28 post-chamber implantation to research the degree of pre-vascularized in the TEC. In addition, different numbers of syngenic islets (300, 200, and 100) were seeded into the chamber on day 28 post implantation to study the TEC function influenced by islets quantities. Furthermore, we explore the feasibility of TEC as a transplantation site for islet allograft and xenograft which was treated by anti-CD45RB and/or anti-CD40L (MR-1). Results and Discussion: A highly developed micro-vascularized network was established in the TEC on day 28 post implantation. The normalization of blood glucose levels of diabetic mice was negatively correlated with the time required for pre-vascularization and the number of seeded syngenic islets. The combined treatment of anti-CD45RB and MR-1 guaranteed the grafts long-term survival after allotransplantation (5/5, 100%) and xenotransplantation (16/20, 80%). Flow cytometry showed the frequency of CD4+Foxp3 Tregs and CD4+IL-4+Th2 cells significantly increased in tolegenic xenograft transplantation, compared with the decrease of CD4+IFN-γTh1 cells. Conclusion: These findings highlight that the highly developed micro-vascularized construct facilitates islet transplantation in the TEC, implicating the possibility of application as an artificial pancreas in the future.Fig. 1. (A) HE images (1st row; scale bars: 10 μm) and histology analysis (2nd and 3rd rows; scale bars: 40 μm) of pre-vascularization TECs on day 7, 14, 28, and 35. Blood vessels were marked by black arrows in HE (1st row; scale bars: 10 μm). The micro-vascularize were stained by anti-VEGF and anti-CD31, and marked by brown and black arrows seperately (2nd and 3rd rows; scale bars: 40 μm). (B)The level of VEGF mRNA was quantified. The extent of pre-vascularization confirmed a significant increase in the 28d group compared with VEGF levels in the 7d and 14d groups (p**<0.001, p***<0.001), and no significant difference between the 28d group and the 35day group (P > 0.05). Sham of inguinal adipose tissue set as control. Data was shown as the mean ± SD and representative of three separate experiments. (C) Western blot analysis showed the protein levels of VEGF at different time points in TEC.Fig. 2. The time required for the blood glucose returning to normal level (11.1mol/dl) in diabetic mice was negatively correlated with the pre-vascularization time (A) and the seeding quantity of syngeneic islets (B). (C) Non-fasting blood glucose maintained normoglycemia after syngeneic islets transplanted in the TEC of diabetic mice, and became hyperglycaemia when the TEC removed (black arrow). (D) Insulin stained donor islet in recipient TEC at 90d posttransplant (shown brown), the islet was pointed by the red arrow (Scale bars:40 μm).Txpl: Transplantation post-implantation.Fig. 3. (A) Islet allografts (Balb/c to C57BL/6) were rejected in the untreated group within 22±2.2 d. Islet allografts treated with anti-CD45RB or anti-CD40L alone were also rejected in recipients within 24±3.12 d, 26±1.74 d. Islet allografts with the dual antibody treatment of anti-CD45RB and anti-CD40L presented long-term graft survival in all recipients. The Kaplan–Meier curves were compared with the log-rank test. (B) Non-fasting blood glucose maintained normoglycemia (5/5, 80%) in the diabetic mice after allogeneic islets transplanted, and became hyperglycaemia when the TEC removed (black arrow) (C) Islet xenograft (SD rat to C57BL/6) rejected in the untreated group, treated-CD45RB group and treated-CD40L group within 13±3.1d, 16±3.12d, 18±1.74d, respectively. Islet xenografts with the dual antibody treatment of anti-CD45RB and anti-CD40L resulted in long-term graft survival in all recipients. The Kaplan–Meier curves were compared with the log-rank test. (D)Non-fasting blood glucose maintained normoglycemia (16/20, 80%) after xenogeneic islets transplanted in the diabetic mice, and showed hyperglycaemia when the TEC removed (black arrow).(E) Another 300 xenogeneic islets were re-transplanted into diabetic mice again, which were failures for the acceptance of the xenograft. Three of four diabetic mice showing that hyperglycemia was ameliorated until the TEC was removed (black arrow). (F)Insulin stained donor allograft and xenograft in recipients TEC at 90d of posttransplant (shown brown), the islets were pointed by the red arrow (Scale bars:40 μm). National Natural Science Foundation of China (nos. 81172832 and 81771723). Special Program for Sichuan Youth Science and Technology Innovation (no. 2014TD0010). Sichuan Youth Science and Technology Foundation (no. 2013JQ0020). The Health and Family Planning Commission of Sichuan Province (no. 110190).

Newly crocin-coated magnetite nanoparticles induce apoptosis and decrease VEGF expression in breast carcinoma cells

Nanoparticles can act on tumor cells at the nanoscale, causing biological therapeutic responses. In the current work, magnetic nanoparticles (MNPs) were obtained by means of a co-precipitation method and functionalized with dextran (D) and crocin, a saffron metabolite with potential antiproliferative properties. These MNPs were characterized using X-ray diffraction (XRD), transmission electron microscopy (TEM), field emission-scanning electron microscopy (FESEM), and Fourier transform infrared (FTIR) techniques. The crystalline structure of the functionalized MNPs was confirmed by XRD with a crystallite size of ca. 12 nm. The successful coating of MNPs surface with dextran and crocin was confirmed by FTIR spectroscopy. The functionalized MNPs were then used in order to elucidate their antiproliferative and apoptosis-inducing activities in the MCF-7 breast tumor cell line using the tetrazolium-based colorimetric (MTT) and Annexin V/PI flow cytometric assays, respectively. Real time-PCR analysis was used to determine the mRNA levels of VEGF as a marker of tumor angiogenesis. The antitumoral effects of crocin-coated D-MNPs were significantly higher than those of free crocin after 24 (P = 0.01) and 48 h (P < 0.0001) of incubation. Results of Annexin V/PI staining indicated that the rate of early apoptosis in MCF-7 cells treated with crocin-coated D-MNPs was higher than the cell fraction undergoing late apoptosis after 6 (P = 0.002), 12 (P < 0.0001), 24 (P < 0.0001), and 48 h (P < 0.0001) of incubation when compared to administration of free crocin. These results indicate that crocin-coated D-MNPs are a much successful drug nanocarrier with enhanced therapeutic effects than the free administration of the bioactive compound, crocin, becoming an attractive alternative option for the therapeutic use of this class of bioactive principles.

Inhibition of retinal neovascularization by VEGF siRNA delivered via bioreducible lipid-like nanoparticles.

Previously, we have demonstrated the use of lipidoid (lipid-like) nanoparticles (e.g., "1-O16B") for gene delivery to live cells, as an alternative to viral vectors. Here, we encapsulate VEGF siRNA (siVEGF) in bioreducible lipidoid nanoparticles and examine whether these nanocomplexes can reduce intravitreal neovascularization in a rodent model of oxygen-induced retinopathy (OIR). Firstly, we constructed siVEGF-nanoparticles (NPs) and transfected human umbilical vein endothelial cells, which caused significantly reduced expression of VEGF, compared to exposure to siVEGF in solution. Secondly, we compared the effect of intravitreal siVEGF-NPs and an anti-VEGF drug (ranibizumab) on retinal vascular development and VEGF mRNA/protein expression in the retinas of a rat model of OIR. Compared to a non-functional lipid vehicle control group, the level of VEGF mRNA and protein was significantly lower in the siVEGF-NP group (p < 0.01), but the level of VEGF mRNA was not significantly lower in the ranibizumab group. Anatomically, the number of retinal neovascular endothelial nuclei that had protruded through the internal limiting membrane and the number of areas of non-perfusion of the retina were both significantly lower in the siVEGF-NP group and the ranibizumab group than in the OIR group (p < 0.01). Our results demonstrate that bioreducible lipidoid nanoparticles conveying VEGF siRNA can effectively inhibit retinal neovascularization in a rodent model of OIR, and reduce the expression of VEGF mRNA and protein. This novel treatment modality could have profound implications for treating retinal angiogenic diseases.

Reciprocal Interplay Between Astrocytes and CD4+ Cells Affects Blood-Brain Barrier and Neuronal Function in Response to β Amyloid.

Background: In Alzheimer’s disease (AD) neuronal degeneration is associated with gliosis and infiltration of peripheral blood mononuclear cells (PBMCs), which participate in neuroinflammation. Defects at the blood-brain barrier (BBB) facilitate PBMCs migration towards the central nervous system (CNS) and in particular CD4+ T cells have been found in areas severely affected in AD. However, the role of T cells, once they migrate into the CNS, is not well defined. CD4+ cells interact with astrocytes able to release several factors and cytokines that can modulate T cell polarization; similarly, astrocytic properties are modulated after interaction with T cells.Methods: In in vitro models, astrocytes were primed with β-amyloid (Aβ; 2.5 μM, 5 h) and then co-cultured with magnetically isolated CD4+ cells. Cytokines expression was evaluated both in co-cultured CD4+ cells and astrocytes. The effects of this crosstalk were further evaluated by co-culturing CD4+ cells with the neuronal-like SH-SY5Y cell line and astrocytes with endothelial cells.Results: The pattern of cytokines and trophic factors expressed by CD4+ cells were strongly modulated in the presence of Aβ-primed astrocytes. Specifically, the percentage of IL-4+ and IFNγ+ CD4+ cells was significantly increased and reduced, respectively. Further, increased BDNF mRNA levels were observed in CD4+ cells. When SH-SY5Y cells were co-cultured with astrocyte-conditioned CD4+ cells and exposed to Aβ, the reduction of the presynaptic protein synaptophysin was prevented with a BDNF-dependent mechanism. In astrocytes co-cultured with CD4+ cells, reduced mRNA levels of inflammatory cytokines and VEGF were observed. This was paralleled by the prevention of the reduction of claudin-5 when astrocytes were co-cultured with endothelial cells.Conclusion: Following Aβ exposure, there exists reciprocal crosstalk between infiltrating peripheral cells and astrocytes that in turn affects not only endothelial function and thus BBB properties, but also neuronal behavior. Since astrocytes are the first cells that lymphocytes interact with and are among the principal players in neuroinflammation occurring in AD, understanding this crosstalk may disclose new potential targets of intervention in the treatment of neurodegeneration.

Transcriptional Activation Of Hypoxia Sensitive Genes Following Repeated Sprint Exercise In Hypoxia

Repeated sprint training in hypoxia (RSTH) utilizes short supramaximal efforts followed by incomplete rest intervals. RSTH has been shown to improve repeated sprint performance compared to repeated sprint training in normoxia. Because of the relatively short exposure to hypoxia compared to live high, train low approaches, the benefits of RSTH have been attributed to local muscular adaptations rather than hypoxia-induced erythropoiesis. Repeated activation of the hypoxia sensitive transcription factors HIF-1α and PGC-1α, and regulation of their gene targets related to angiogenesis (VEGF), mitophagy (BNIP3, PINK1), and glucose metabolism (PDK-M, GLUT4) may underlie these muscular adaptations. PURPOSE: To investigate the transcriptional activation of HIF-1α, PGC-1α and several HIF-1α-target genes following repeated sprint exercise in normoxia and hypoxia. METHODS: Eight recreationally active males (n=8) and one female (n=1) performed 20, 10s all-out sprints in normoxia (1600m) and hypobaric hypoxia (4600m) on a cycle ergometer on two days separated by 2 weeks. Skeletal muscle samples from the vastus lateralis were analyzed for mRNA levels of HIF-1α, PGC-1α, BNIP3, PINK1, VEGF, PDK-M, and GLUT4 pre, post and 3h post exercise in hypoxia and normoxia. Comparisons between condition and time were made using two-way repeated measures ANOVAs. RESULTS: There was a significant increase in mRNA levels for HIF-1α (fold change: 2.6±1.8) and VEGF (fold change: 3.0±1.6) 3h post-exercise in hypoxia (p<.05) but not normoxia. PGC-1α, was higher 3h post-exercise in both hypoxia (fold change: 9.2±4.6) and normoxia (fold change: 6.2±3.8; p<.05). No significant effect of time or group was observed for BNIP3, PINK1, PDK-M, or GLUT4. CONCLUSIONS: Acute sprint exercise in both hypoxia and normoxia induces an increase in the transcription of PGC-1α. However, hypoxia sensitive genes HIF-1α and VEGF were only greater following sprint exercise in hypoxia. Collectively, these findings suggest that sprint exercise stimulates mitochondrial biogenesis, but an additional hypoxic stress is required to induce changes in hypoxia sensitive genes. The downstream effect of these transcriptional activations may increase angiogenesis and mitochondrial signaling in response to RSTH, which may in part explain the benefits of RSTH.

Pigment epithelium-derived factor attenuates angiogenesis and collagen deposition in hypertrophic scars.

Scar forming wounds are often characterized by higher levels of vascularity than non-scarring wounds and normal skin, and inhibition of angiogenesis has been shown to inhibit scar formation in some model systems. The rabbit ear hypertrophic scar (HS) model has been widely used to study the mechanisms that underlie the development of HS as well as the effectiveness of various treatments. Although the rabbit ear HS model is well characterized in terms of scar formation, the rate and level of angiogenesis has not been investigated in this model, and the cause-effect relationship between angiogenesis and rabbit HSs has not been examined. In the current study, full-thickness excisional wounds were created on the ventral side of New Zealand White rabbit ears to induce HS formation, and the dynamic pattern of angiogenesis and the expression of angiogenic regulatory factors were examined over time. Blood vessel density was found to peak at 2.7% on day 14 post-wounding, decreasing to 1.7% by day 28. mRNA levels of the proangiogenic factor VEGF-A peaked at day 14, while the expression of the antiangiogenic factors pigment epithelium-derived factor (PEDF) and thrombospondin 1 (TSP1) peaked at day 28 post-wounding. To examine whether inhibition of angiogenesis influences HS formation in this model, wounds were treated with exogenous soluble antiangiogenic agents including recombinant PEDF (rPEDF) and a functional PEDF peptide (PEDF-335). rPEDF and PEDF-335 were administered intradermally from day 4 post-wounding every 3 days until day 19. Intradermal injection of rPEDF or PEDF-335 both led to decreased angiogenesis and decreased collagen deposition at the wound site. The results support the utility of antiangiogenic therapies, including rPEDF/PEDF-335, as a potential new strategy for the prevention or treatment of HSs.

Pigment epithelium-derived factor (PEDF) negates hyperandrogenic PCOS features.

Polycystic ovary syndrome (PCOS), one of the most common female endocrine disorder, is a prevalent cause of infertility. Hyperandrogenism is a key feature in PCOS and is correlated with increased expression of VEGF and cytokines in the ovaries. We have previously shown that pigment epithelium-derived factor (PEDF), an endogenous protein, presents potent anti-angiogenic and anti-inflammatory activities in the ovary and negates the effects of cytokines and VEGF. Additionally, PEDF plays a role in both pathophysiology and treatment of ovarian-hyperstimulation syndrome (OHSS), frequently seen in PCOS patients. We established hyperandrogenic-PCOS models, both in vivo, using mice exposed prenatally to dihydrotestosterone (DHT) and, in vitro, using human primary granulosa cells (hpGCs) and human granulosa cell line (KGN). In PCOS-induced mice, the mRNA levels of I l-6, V egf and Amh were higher than those of control; yet, treatment with rPEDF decreased these levels. Moreover, treating OHSS-induced PCOS-mice with rPEDF alleviated all OHSS symptoms. Stimulation of hpGCs with DHT resulted in downregulation of PEDF mRNA expression, concomitantly with a significant increase in IL-6 and IL-8 mRNAs expression. However, co-stimulation of DHT with rPEDF attenuated the increase in cytokines expression. The anti-inflammatory effect of PEDF was found to be mediated via PPARγ pathway. Our findings suggest that rPEDF treatment may normalize the ovarian angiogenic-inflammatory imbalance, induced by PCOS-associated hyperandrogenism. Moreover, the therapeutic potency of PEDF in preventing OHSS symptomes offers a rationale for using PEDF as novel physiological treatment for PCOS sequels.

Differential Effects of Ketamine on Peripheral Blood VEGFA and PEDF(SERPINF1) mRNA in Patients With Depression

Through interacting with BDNF, vascular endothelial growth factor (VEGF) has been implicated in the antidepressant action of ketamine. VEGF is also linked to pigment epithelial-derived factor (PEDF), which inhibits VEGF and is neurotrophic. Plasma PEDF is increased in depressed patients and further increased post-ECT. We therefore explored the effect of single ketamine infusions on peripheral blood mRNA levels of VEGFA and PEDF (SERPINF1) and their relationship in patients with depression.

Effect of Jiajian-Zhujing Decoction on the AKT/mTOR signaling pathway in ARPE-19 cells after AKT transfection

Objective To study the mechanism of the effect of Jiajian-Zhujing Decoction on the expression of VEGF on ARPE-19 cells after AKT transfection. Methods To prepare the serum and blank serum of Jiajian-Zhujing Decoction and divide ARPE-19 cells into the normal group, model group, blank serum group, medicated serum group, Conbercept group and combined group. Except normal group, this research established AKT transfected cell model. Then cultured the normal group and model group with conventional method, and the blank serum group was cultured with 10% blank serum, the medicated serum group was cultured with 10% medicated serum, the Conbercept group was cultured with 20 μg/ml Conbercept, the combined group was cultured with 10% medicated serum and 20 μg/m Conbercept. The proliferation of ARPE-19 cells in each group was detect by the CCK-8 method. The levels of AKT, mTOR and VEGF mRNA were detected by real-time quantitative PCR. Western blot was used to detect the expression of AKT, mTOR and VEGF. Results After being cultured for 24, 48 and 72 hours, compared with the model group, the cell proliferation rate in blank serum group, medicated serum group, Conbercept group and combined group significantly decreased (P<0.05). Compared with the model group, the expression of AKT mRNA (24 h: 3.10 ± 0.48, 1.97 ± 0.14, 1.26 ± 0.24 vs. 4.77 ± 0.68; 48 h: 3.52 ± 0.82, 2.62 ± 0.77, 1.10 ± 0.19 vs. 6.12 ± 1.21), mTOR mRNA (24 h: 3.02 ± 0.26, 2.45 ± 0.75, 1.13 ± 0.15 vs. 4.48 ± 0.80; 48 h: 1.29 ± 0.30, 1.30 ± 0.57, 0.65 ± 0.19 vs. 2.54 ± 0.62), VEGF mRNA (24 h: 3.33 ± 0.62, 2.18 ± 0.20, 1.55 ± 0.28 vs. 5.53 ± 1.02; 48 h: 2.35 ± 0.54, 1.23 ± 0.28, 0.93 ± 0.25 vs. 3.59 ± 0.40), AKT protion (24 h: 0.45 ± 0.09, 0.25 ± 0.05, 0.14 ± 0.04 vs. 0.62 ± 0.04; 48 h: 0.36 ± 0.06, 0.23 ± 0.04, 0.14 ± 0.03 vs. 0.54 ± 0.08), mTOR protion (24 h: 0.35 ± 0.05, 0.24 ± 0.02, 0.18 ± 0.02 vs. 0.52 ± 0.09; 48 h: 0.23 ± 0.04, 0.29 ± 0.04, 0.14 ± 0.03 vs. 0.40 ± 0.10), VEGF protion (24 h: 0.14 ± 0.03, 0.33 ± 0.04, 0.24 ± 0.03 vs. 0.54 ± 0.10; 48 h: 0.24 ± 0.03, 0.17 ± 0.02, 0.11 ± 0.02 vs. 0.42 ± 0.10) significantly decreased (P<0.05), and the combined group was significantly lower than that of the Conbercept group (P<0.05). Conclusions AKT transfection can promote the proliferation of ARPE-19 cells, and Jiajian-Zhujing Decoction can significantly inhibit this proliferation. Jiajian-Zhujing Decoction may inhibit the activity of AKT/mTOR signaling pathway to reduce the expression of VEGF. Key words: Serum pharmacology (TCD); Retinal pigment epithelium; Transfection; Vascular endothelial growth factors; Jiajian-Zhujing Decoction

Regulation of eIF2α by RNF4 Promotes Melanoma Tumorigenesis and Therapy Resistance

Among the hallmarks of melanoma are impaired proteostasis and rapid development of resistance to targeted therapy that represent a major clinical challenge. However, the molecular machinery that links these processes is unknown. Here we describe that by stabilizing key melanoma oncoproteins, the ubiquitin ligase RNF4 promotes tumorigenesis and confers resistance to targeted therapy in melanoma cells, xenograft mouse models, and patient samples. In patients, RNF4 protein and mRNA levels correlate with poor prognosis and with resistance to MAPK inhibitors. Remarkably, RNF4 tumorigenic properties, including therapy resistance, require the translation initiation factor initiation elongation factor alpha (eIF2α). RNF4 binds, ubiquitinates, and stabilizes the phosphorylated eIF2α (p-eIF2α) but not activating transcription factor 4 or C/EBP homologous protein that mediates the eIF2α-dependent integrated stress response. In accordance, p-eIF2α levels were significantly elevated in high-RNF4 patient-derived melanomas. Thus, RNF4 and p-eIF2α establish a positive feed-forward loop connecting oncogenic translation and ubiquitin-dependent protein stabilization in melanoma.

The epigallocatechin gallate derivative Y6 inhibits human hepatocellular carcinoma by inhibiting angiogenesis in MAPK/ERK1/2 and PI3K/AKT/ HIF-1α/VEGF dependent pathways

Ethnopharmacological relevanceHypervascularity has been considered as one of the major features of many solid tumors. Green tea is one of the commonly drink resources in China, and its active component, Epigallocatechin gallate (EGCG), exhibits antiangiogenic activities in various experimental tumor models. However, EGCG has many shortages, e.g., relatively unstable, low lipid solubility, poor bioavailability, and short duration of action. Aim of the studyTo overcome the shortages of EGCG for antiangiogenic antitumor usage, our study developed a novel EGCG derivate, Y6(5,3′,4′,3″,4″,5″-6-0-ethyl-EGCG). The underlying mechanism was also elucidated. Material and methodswe evaluated the effects of EGCG, Y6 on HCC and angiogenesis in vivo and in vitro. Moreover, to understand their antitumor mechanisms, key factors within angiogenesis-related signaling pathways (MAPK/ERK1/2, PI3K/AKT, HIF-1 VEGF) were analyzed by using western blot, immunohistochemistry (IHC), quantitative real-time quantitative PCR (RT-PCR). HepG2 xenograft model and the chorioallantoic membrane (CAM) were used to investigate the effects of Y6 and EGCG on tumors and anti-angiogenesis in vivo. Micro-vessel density (MVD) was analyzed by IHC of CD34 staining. IHC, qRT-PCR and Western blot were used to detect the expression of HIF-1α and VEGF protein in tumor tissues. The protein levels of MAPK/ERK1/2, PI3K/AKT, HIF-1α, and VEGF in tumor tissues were detected by western blot. ResultsOur results demonstrated that both EGCG and Y6 displayed antiangiogenetic and antitumor effects against HCC cells in vitro and in vivo. We found that rather than equal amount of EGCG, Y6 displayed better abilities in inhibiting the growth of HCC tumor cells, as well as inhibiting the growth of neovascularization in the chick embryos and HepG2 xenograft tumors bearing-mice, based on the data obtained from MTT assay, immunohistochemistry (IHC), chick chorioallantoic membrane (CAM) assays. In the comparison of equivalent dose of EGCG, qRT-PCR data showed that Y6 induced more significant decrease of the mRNA levels of HIF-1α and VEGF in supernatant-treated SMMC-7721 cells under hypoxic condition, as well as in the in xenograft tumor tissues; whereas Y6 also significantly reduced the protein levels of MAPK/ERK1/2, PI3K/AKT, HIF-1α, and VEGF to a greater extent than EGCG, determined by western blotting assay. Conclusionsour work suggests that the new EGCG derivate Y6 could significantly inhibit tumor growth and angiogenesis which is possibly involved with the signaling intervention of MAPK/ERK1/2 and PI3K/AKT/HIF-1α/VEGF pathways, and is supposed to be a potential therapeutic reagent for anti-angiogenesis treatment of solid tumors.

MiR-134, Mediated by IRF1, Suppresses Tumorigenesis and Progression by Targeting VEGFA and MYCN in Osteosarcoma.

Osteosarcoma (OS) is a prevalent primary bone malignancy and its distal metastasis remains the main cause of mortality in OS patients. MicroRNAs (miRNAs) play critical roles during cancer metastasis. Thus, elucidating the role of miRNA dysregulation in OS metastasis may provide novel therapeutic targets. The previous study found a low miR-134 expression level in the OS specimens compared with paracancer tissues. Overexpression of miR-134 stable cell lines was established. Cell viability assay, cell invasion and migration assay and apoptosis assay were performed to evaluate the role of miR-134 in OS in vitro. We found that miR-134 overexpression inhibits cell proliferation, migration and invasion, and induces cell apoptosis in both MG63 and Saos-2 cell lines. Mechanistically, miR-134 targets the 3'-UTR of VEGFA and MYCN mRNA to silence its translation, which was confirmed by luciferase-reporter assay. The real-time PCR analysis illustrated that miR-134 overexpression decreases VEGFA and MYCN mRNA levels. Additionally, the overexpression of VEGFA or MYCN can partly attenuate the effects of miR-134 on OS cell migration and viability. Furthermore, the overexpression of miR-134 dramatically inhibits tumor growth in the human OS cell line xenograft mouse model in vivo. Moreover, bioinformatic and luciferase assays indicate that the expression of miR-134 is regulated by Interferon Regulatory Factor (IRF1), which binds to its promoter and activates miR-134 expression. Our study demonstrates that IRF1 is a key player in the transcriptional control of miR-134, and it inhibits cell proliferation, invasion and migration in vitro and in vivo via targeting VEGFA and MYCN.