VEGFR2 mRNA Expression Research Articles

Unravelling the oxygen factor - An investigation of transcriptional activation of hypoxia associated placental angiogenesis in recurrent pregnancy loss (RPL) patients from Assam, India

IntroductionRecurrent pregnancy loss (RPL) is defined as the spontaneous loss of two or more consecutive pregnancies before 20 weeks of gestation, and affects 7.46 % of the Indian population. About 40–50 % of RPL cases are idiopathic making it a therapeutic challenge for clinicians. This study focuses on elucidating the role of hypoxia-associated placental angiogenesis in these idiopathic RPL cases. MethodsWhole blood and product of conception (POCs) were collected from RPL patients (N = 87) and cases of voluntary abortions (medically terminated pregnancy, MTP; n = 110) as controls with informed consent. Serum separated from whole blood was used to study the ROS-antioxidant status in the cases and controls through colorimetric assays and ELISA. The mRNA extracted from placental tissue samples were used to determine the hypoxic and angiogenic status in cases and controls through real time PCR. Statistical analysis was also carried out to correlate the differential hypoxic status between RPL and MTP cohorts with the expression of angiogenic factors (VEGFA, VEGFR1 and VEGFR2). ResultsHIF1α mRNA expression was found to be upregulated in the RPL cases. While the serum levels of H2O2 (p = 0.012), guanine oxides and lipid hydroperoxides (LPO) were increased in the RPL cases, reduced glutathione (GSH) was found to be significantly decreased (p = 0.012). Additionally, AUROC analysis also shows an excellent discriminatory ability of 0.850 for serum H2O2 levels. VEGF-A and VEGF-R1 mRNA expression was also found to be downregulated in the RPL cases compared to MTP. DiscussionThis study indicates that increased oxidative stress may lead to aberrations in the VEGF pathway resulting in improper placentation in RPL cases, and subsequently, pregnancy loss.

The placenta in fetal death: molecular evidence of dysregulation of inflammatory, proliferative, and fetal protective pathways

BackgroundIt is estimated that over 2 million cases of fetal death occur worldwide every year, but, despite the high incidence, several basic and clinical characteristics of this disorder are still unclear. Placenta is suggested to play a central role in fetal death. Placenta produces hormones, cytokines and growth factors that modulate functions of the placental-maternal unit. Fetal death has been correlated with impaired secretion of some of these regulatory factors. Objective(s)The aim of the present study was to evaluate, in placentas collected from fetal death, the gene expression of inflammatory, proliferative and protective factors. Study designCases of fetal death in singleton pregnancy were retrospectively selected, excluding pregnancies complicated by fetal anomalies, gestational diabetes, intrauterine growth restriction and moderate to severe maternal diseases. A group of placentas collected from healthy singleton term pregnancies were used as controls. Groups were compared regarding maternal and gestational age, fetal sex and birth weight. Placental mRNA expression of inflammatory (IL-6), proliferative (Activin A, TGF-β1) and regulatory (VEGF, VEGFR2, ATP-binding cassette (ABC) transporters ABCB1 and ABCG2, sphingosine 1-phosphate (S1P) signaling pathway) markers was conducted using real-time PCR. Statistical analysis and graphical representation of the data were performed using the GraphPad Prism 5 software. For the statistical analysis, Student’s t-test was used, and P values < 0.05 were considered significant. ResultsPlacental mRNA expression of IL-6 and VEGFR2 resulted significantly higher in the fetal death group compared to controls (P<0.01), while activin A, ABCB1 and ABCG2 expression resulted significantly lower (P<0.01). A significant alteration in the S1P signaling pathway was found in the fetal death group, with an increased expression of the specific receptor isoforms sphingosine 1-phosphate receptor 1, 3 and 4 (S1P1, S1P3, S1P4) and of sphingosine kinase 2 (SK2), one of the enzyme isoforms responsible for S1P synthesis (P<0.01). Conclusion(s): The present study confirmed a significantly increased expression of placental IL-6 and VEGFR2 mRNA, and for the first time showed an increased expression of S1P receptors and SK2 as well as a decreased expression of activin A and of selected ATP-binding cassette transporters, suggesting that multiple inflammatory and protective factors are deranged in placenta of fetal death.

Abstract TP310: Prnd in Angiogenic Brain Endothelial Cells is Involved in Neurovascular Repair After Ischemic Stroke

Introduction: The prion protein family member doppel (encoded by the Prnd gene) is essential for the control of angiogenesis during CNS development and has been implicated in pathological angiogenesis. Various data demonstrate that Prnd expression is linked to physiological angiogenesis by regulating VEGFR2 signaling. However, the roled for Prnd in pathological angiogenesis after ischemic stroke is not known. Therefore, we hypothesized that Prnd contributes to stroke-induced brain injury repair by regulating angiogenesis. Methods: We induced ischemic injury in Prnd+/+ and Prnd-/- mice by middle cerebral artery occlusion for 30 minutes. Infarct volume was measured three days and 28 days after stroke and survival rates were analyzed. We also measured the expression of Prnd and angiogenesis-related factors (VEGF, VEGFR2, and VE-Cadherin) in control and Prnd-/- brains. We also determined the activation and localization of pericytes, oligodendrocytes, and astrocytes in brain sections after ischemic stroke. Results: Prnd-/- mice showed worse survival rate than Prnd+/+ mice after ischemic stroke. At three days post-stroke, infarct volume in Prnd-/- and Prnd+/+ mice was similar, but brain infarct volume of Prnd-/- mice was increased at 28 days. Interestingly, Prnd mRNA levels were significantly increased in the ipsilateral region of the brain of Prnd+/+ mice at three days post ischemic stroke. VEGFR2 mRNA expression increased in the Prnd+/+ brain, but this increase was observed to a higher extent in the Prnd-/- brain. Also, the levels of VE-Cadherin which is an essential component of adherens junctions and modulates the VEGFR function in Prnd+/+ was increased in the ipsilateral region compared to contralateral, but not in Prnd-/-mice. Conclusion: Our results indicate that Prnd deficiency is associated with poor stroke outcome and that increased Prnd expression after ischemic stroke is involved in brain injury repair by regulating angiogenesis-related signaling pathways. The data also suggest that Prnd-controlled angiogenesis may be a potential mechanism to improve outcomes after ischemic stroke.

Studying the possibilities of pharmacological correction of hypoxia-induced pulmonary hypertension using a phenolic compound with a laboratory cypher KUD975

The aim of our work was to study a pharmacological activity of a selective arginase-2 and thrombin inhibitor from a phenolic compounds group with a laboratory cypher KUD975 on a model of arterial pulmonary hypertension induced by hypoxia.Materials and methods. To simulate pulmonary hypertension (РН), animals were placed in a normobaric hypoxic chamber and subjected to 5 weeks of hypoxia with an oxygen content of 10% in the air. After 3 weeks of hypoxia, the animals were administered with the test compound KUD975 (intragastrically, at a dose of 2 mg/kg once a day for 2 weeks). L-norvaline (intragastrically, 20 mg/kg) was used as a reference drug. To assess the development and correction of РН, measurements of cardiohemodynamics, analysis of blood gas composition, study of the number of circulating endothelial precursor cells (EPCs), quantitative PCR assessing the expression of mRNA VEGF-R2, SGF-1 (stromal growth factor-1) and MCP-1 (monocyte chemoattractant protein-1). Next, a histological examination of the lungs and heart was performed, the degree of pulmonary edema and the concentration of cardiotrophin-1 and atrial natriuretic peptide were assessed.Results. The administration of the studied phenolic compound with laboratory cypher KUD975, as well as the reference drug L-norvaline, led to a decrease in the right ventricular systolic pressure against the background of modeling РН. The present study shows a more than twice-fold decrease in the number of circulating (EPCs) in the animals group with modeling a hypoxia-induced circulatory РН (171.3±12.1) in comparison with the group of intact animals (296.1±31.7; p=0.0018). The recovery of EPCs was noted in the animals group administered with KUD-975 and L-norvaline, up to 247.5±34.2 (p=0.0009 compared with a pulmonary arterial hypertension (PAH) and 235.6±36.4 (p=0.008 compared to PAH), respectively. The studied compounds had a protective effect by statistically significantly increasing the expression of VEGF-R2 mRNA and decreasing the expression of SGF-1 mRNA, reducing the lung moisture coefficient and the concentrations of cardiotrophin-1 and atrial natriuretic peptide and preventing vascular remodeling caused by hypoxia.Conclusion. When studying the pharmacological activity, it was shown that the phenolic compound with the laboratory cypher KUD975 normalizes hemodynamic parameters, reduces the signs of remodeling of the heart and pulmonary vessels and has a pronounced endothelial protective effect on the model of hypoxia-induced РН, and is superior to the activity of the reference drug L-norvaline.

The EPOR/CD131 heteroreceptoragonist has an endothelioprotective effect against the background of pulmonary hypertension caused by monocrotalin

Introduction: The abnormal increase in pulmonary pressure observed in pulmonary arterial hypertension (PAH) is a consequence of increased pulmonary vascular resistance due to progressive loss and obliteration of pulmonary arteries. The initial trigger is a combination of factors that lead to endothelial damage and impaired vascular regeneration. Aim: research the possibilities of pharmacological correction of pulmonary arterial hypertension induced by monocrotalin using the EPOR/CD131 heteroreceptor agonist with the laboratory code EP-11-3.&#x0D; Materials and Methods: The study of pharmacological activity on a model of monocrotaline induced PAH was carried out on male Sprague-Dawley rats weighing 180-220 grams. Monocrotaline (MCT) pulmonary hypertension was simulated in 30 animals using subcutaneous injection of MCT at a dose of 60 mg/kg. Seven days after the injection of MCT, the administration of the studied compounds began. The erythropoietin derivative with the laboratory code EP-11-3 and pHBSP administered subcutaneously at a dose of 25 mcg/kg once every 3 days for 21 days.&#x0D; Results: On the model of monocrotalin-induced PAH, it was shown that the erythropoietin derivative with the laboratory code EP-11-3 has a pronounced endothelioprotective effect, reducing the coefficient of endothelial dysfunction, statistically significantly increasing the expression of VEGF-R2 mRNA and reducing the expression of SDF-1 mRNA, reducing the concentrations of CT-1 and PNP, and reducing the signs of remodeling of the heart and pulmonary vessels.&#x0D; Conclusion: Erythropoietin derivative with laboratory code EP-11-3 has an endothelioprotective effect and reduces the manifestations of vascular remodeling in pulmonary hypertension caused by monocrotalin.

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

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

Angiogenic genes and proteins are differentially expressed in left and right ventricular tissue in perinatal sheep hearts

Birth places a great stress on the fetal heart due to increased cardiac workload, following a disconnection from the low-resistance placenta and closure of fetal shunts. The mechanisms that regulate myocardial microvascular growth and development during the perinatal period are largely unknown. We hypothesized that birth stimulates an increase in angiogenic gene and protein expression in a ventricular dependent manner to reflect the changing roles for the left and right ventricles (LV, RV). Fetal (135 d gestation, dGA; term=147 dGA) and neonatal (1 and 5 postnatal days, PD) sheep LV and RV were snap frozen. Gene expression of key angiogenic regulators ( VEGFA, VEGFR1, VEGFR2, ANGPT1, ANGPT2, PGC1A), endothelial cell growth ( DDL4, NOTCH4, SPP1, PECAM1, NCL), nitric oxide synthesis ( NOS2, NOS3), and extracellular matrix (ECM) remodeling ( MMP2, MMP9, THBS1) were quantified using real-time qPCR (n=12/age group). Protein expression of VEGFA and VEGF-R2 were measured by Western blot (n=5/age group). Data were analyzed by two-way ANOVA with Tukey’s post-hoc test to determine effects of age, ventricle, and interaction. Significance was set at P&lt;0.05. At 135 dGA, all angiogenic, nitric oxide synthesis, ECM, DDL4, SPP1, and PECAM1 mRNA expression levels were higher in the RV vs. LV. At PD1, there were no differences between LV and RV. At PD5, VEGFA, VEGFR1, ANGPT1, ANGPT2, PGC1A, DDL4, SPP1, PECAM1, NCL, MMP2, and THBS1 mRNA expression levels were again higher in the RV vs. LV. At 135 dGA, VEGFA and VEGF-R2 protein expression levels were 76% and 72% lower, respectively, in the RV vs. LV. Within each ventricle, age related changes to mRNA expression patterns were unique. In the LV, mRNA expression of NOTCH4 and NCL increased stepwise from 135 dGA to PD5; however, in the RV, both were upregulated at PD1 but returned to fetal levels by PD5. In contrast, all angiogenic, ECM, DDL4, SPP1, PECAM1, and NOS2 genes were downregulated in the RV at PD1 but all returned to fetal levels by PD5. In the LV, VEGF-R2 protein expression was 56% and 73% lower at PD 1 and 5, respectively, vs. 135 dGA. In summary, gene and protein expression patterns involved in angiogenesis and vascular remodeling change over the perinatal period and are differentially expressed in the ventricles. Greater angiogenic gene expression levels in the RV vs. the LV at 135 dGA might be due to the RV serving as the main systemic pump in utero, thus requiring relatively increased microcirculation. However, reduced VEGFA and VEGF-R2 protein levels at 135 dGA contradict this hypothesis; histological studies to determine vascularity are ongoing. Likewise, the subsequent downregulation of angiogenic genes in the RV at PD1 might be due to the RV’s sudden decrease in cardiac output as the LV becomes the main pump. The underlying reasons for why LV angiogenic genes are not significantly upregulated in the early postnatal period, given its new role as the main systemic pump, are yet to be elucidated. NIH R21-HL152112 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Discovery of novel VEGFR-2-PROTAC degraders based on the localization of lysine residues via recruiting VHL for the treatment of gastric cancer

VEGFR-2 is an attractive therapeutic target for antitumor drug research by blocking tumor angiogenesis and PROTAC provides a new technology for targeted protein knockout. Herein, a library of novel VEGFR-2-PROTAC degraders were rationally designed and synthesized based on the Lys residue region on the surface of VEGFR-2 protein using protein structure-based drug design strategy. Among them, P7 exhibited preferable antitumor activity against HGC-27 cells and less toxic to human normal HUVEC, HEK293T and GES-1 cells in vitro, as well as the potent degradation activity of VEGFR-2 protein in HGC-27 cells (DC50: 0.084 ± 0.04 μM, Dmax: 73.7%) and HUVEC cells (DC50: 0.51 ± 0.10 μM, Dmax: 76.6%). Additionally, P7 degraded VEGFR-2 protein by the formation of ternary complex and the ubiquitin proteasome pathway in HGC-27 cells. Furthermore, P7 shortened the half-life of VEGFR-2 protein synthesis and had no inhibitory effect on the expression of VEGFR-2 mRNA in HGC-27 cells. Moreover, P7 inhibited the colony formation, migration and invasion of HGC-27 cells in a time- and dose-dependent manner, and meanwhile induced G2/M phase cycle arrest and apoptosis. All the results demonstrated that P7 could be as a promising VEGFR-2-PROTAC degrader for gastric cancer therapy.

Protective role of VEGF/VEGFR2 signaling against high fatality associated with hepatic encephalopathy via sustaining mitochondrial bioenergetics functions

BackgroundThe lack of better understanding of the pathophysiology and cellular mechanisms associated with high mortality seen in hepatic encephalopathy (HE), a neurological complication arising from acute hepatic failure, remains a challenging medical issue. Clinical reports showed that the degree of baroreflex dysregulation is related to the severity of HE. Furthermore, mitochondrial dysfunction in the rostral ventrolateral medulla (RVLM), a key component of the baroreflex loop that maintains blood pressure and sympathetic vasomotor tone, is known to underpin impairment of baroreflex. Realizing that in addition to angiogenic and vasculogenic effects, by acting on its key receptor (VEGFR2), vascular endothelial growth factor (VEGF) elicits neuroprotection via maintenance of mitochondrial function, the guiding hypothesis of the present study is that the VEGF/VEGFR2 signaling plays a protective role against mitochondrial dysfunction in the RVLM to ameliorate baroreflex dysregulation that underpins the high fatality associated with HE.MethodsPhysiological, pharmacological and biochemical investigations were carried out in proof-of-concept experiments using an in vitro model of HE that involved incubation of cultured mouse hippocampal neurons with ammonium chloride. This was followed by corroboratory experiments employing a mouse model of HE, in which adult male C57BL/6 mice and VEGFR2 wild-type and heterozygous mice received an intraperitoneal injection of azoxymethane, a toxin used to induce acute hepatic failure.ResultsWe demonstrated that VEGFR2 is present in cultured neurons, and observed that whereas recombinant VEGF protein maintained cell viability, gene-knockdown of vegfr2 enhanced the reduction of cell viability in our in vitro model of HE. In our in vivo model of HE, we found that VEGFR2 heterozygous mice exhibited shorter survival rate and time when compared to wild-type mice. In C57BL/6 mice, there was a progressive reduction in VEGFR2 mRNA and protein expression, mitochondrial membrane potential and ATP levels, alongside augmentation of apoptotic cell death in the RVLM, accompanied by a decrease in baroreflex-mediated sympathetic vasomotor tone and hypotension. Immunoneutralization of VEGF exacerbated all those biochemical and physiological events.ConclusionsOur results suggest that, acting via VEGFR2, the endogenous VEGF plays a protective role against high fatality associated with HE by amelioration of the dysregulated baroreflex-mediated sympathetic vasomotor tone through sustaining mitochondrial bioenergetics functions and eliciting antiapoptotic action in the RVLM.

The efficiency of anlotinib in osteosarcoma with chemoresistance: Exploratory therapy based on PDX models and next-generation sequencing.

11527 Background: It’s urgent to find a new approach for the treatment of progressed osteosarcoma in neoadjuvant chemotherapy. Using patient-derived xenograft (PDX) models and next-generation sequencing (NGS), we have explored the efficiency of anlotinib in these patients. Methods: Frozen osteosarcoma tissues were used to establish PDX models. Ten samples of each PDX model were randomized to treatment or control group. The treatment group were gavaged at a volume of 0.1 ml/10g body weight for 4 weeks, while the control group were administered with vehicle at the same dose orally. Tumor volume and body weight were measured every 3 days, and the tumour were removed and weighed after 28 days. The number of mitotic cells and tumor necrosis rate were detected by Hematoxylin-Eosin (HE) staining. Immunohistochemistry (IHC) was used to evaluate the number of apoptotic cells and the expression of VEGFR-2, PDGFR-β, FGFR-1, c-Kit and their phosphorylated proteins, CD 31. Additionally, patients who had progressed during neoadjuvant chemotherapy (NAC) were treated with combination of anlotinib. After four cycles NAC, we performed salvage surgery and maintained with anlotinib. The change of tumor size was evaluated for tumor response. We used IHC and NGS to analyze the expression of drug targets. Results: 21 PDX models were established successfully from 43 samples. Tumor specimens from patients, who had pulmonary metastasis, local recurrence or chemoresistance, were easier to establish PDX models (P &lt; 0.001, P = 0.046, P = 0.013). Six models were selected randomly for anlotinib. After anlotinib administration, the tumor inhibition rates were 18.82%, 45.98%, 85.86%, 83.38%, 84.57% and 86.42%. Anlotinib could not only inhibit the mitosis, induce tumor cell apoptosis and necrosis, but also decrease the expression of drug targets. The expression of VEGFR-2, PDGFR-β and CD31 were positively associated with tumor response. Five patients had received anlotinib during NAC. Four patients had tumor regression (69.4%, 28%, 19%, 8.7%), including two with high expression of VEGFR-2 mRNA or/and PDGFR-β and one with medium expression. Conclusions: Based on the results of PDX models and NGS, we suggested that osteosarcoma with high expression of VEGFR-2 or/and PDGFR-β was sensitive to anlotinib, which was alternative for patients progressed during NAC.

Effects of root-end filling materials on vascular endothelial cell proliferation and tube formation

Background/purposeRegarding root-end filling materials in apical surgery, sealing ability and biocompatibility are useful for treatment. Angiogenesis, which occurs in the process of periapical wound healing, is closely related to bone formation. In this study, we investigated the effects of root-end filling materials on vascular endothelial cell proliferation and angiogenesis. Materials and methodsMineral trioxide aggregate (MTA), 4-methacryloxyethyl trimellitate anhydride/methyl methacrylate-tri-n-butyl borane (4-META/MMA-TBB) resin, Super EBA, and CS-BG-multi, bioactive glass-related materials, were used. After curing, each material was soaked in a medium for 1 or 7 days, and then cultured for 1–7 days to investigate the effects on human umbilical vein endothelial cell (HUVEC) proliferation, angiogenesis, and vascular endothelial growth factor receptors (VEGFRs) mRNA expression. ResultsIn the 1-day soaked sample, there was significantly less proliferation in MTA and Super EBA on day 7 of culture. In the 7-day soaked sample, there was significantly less proliferation in Super EBA and CS-BG-multi on day 7 of culture. Tube formation was significantly high in MTA in both the 1-day and 7-day soaked samples, significantly high in SB in the 1-day soaked sample, and significantly low in Super EBA in both the 1-day and 7-day soaked samples. CS-BG-multi was comparable to the control. VEGFR-1 and VEGFR-2 mRNA expressions showed an upward trend in MTA, and a trend similar to the control in SB. ConclusionMTA and 4-META/MMA-TBB resin had a higher pro-angiogenic effect while Super EBA had a less pro-angiogenic effect. CS-BG-multi had low toxicity on tube formation of HUVEC.

Substrate stiffness modulates endothelial cell function via the YAP-Dll4-Notch1 pathway

Endothelial cells adapt their functions as a consequence of sensing extracellular substrate stiffness; these alterations allow them to maintain their vascular structure and function. Substrate stiffness-mediated yes-associated protein 1 (YAP) activation plays an important role in mechano-transduction and pro-angiogenic phenotype of endothelial cells, and Delta-like ligand 4 (Dll4)-Notch1 signaling is closely related to angiogenesis; however, the impact of substrate stiffness-mediated interrelation of these pathways on endothelial cell functions remains elusive. We confirmed that endothelial cells on softer substrates not only elongate cellular aspects but also attenuate YAP activation compared to cells on stiffer substrates. Endothelial cells on softer substrates also upregulate the vascular endothelial growth factor receptor 1 (VEGFR1) and VEGFR2 mRNA expression that is enhanced by VEGF stimulation. We determined that endothelial cells on softer substrates increased Dll4 expression, but not Notch1 expression, via YAP signaling. Moreover, endothelial cells on soft substrates induced not only VEGFRs upregulation but also suppression of pro-inflammatory interleukin-6 and plasminogen activator inhibitor-1 mRNA expression and the facilitation of anti-coagulant thrombomodulin and pro-coagulant tissue factor mRNA expression. Our results suggest that endothelial cells activate the YAP-Dll4-Notch signaling pathway in response to substrate stiffness and dictate cellular function.

Anti-Fibrotic and Anti-Angiogenic Activities of Osbeckia octandra Leaf Extracts in Thioacetamide-Induced Experimental Liver Cirrhosis.

Chronic liver inflammation has become a major global health concern. In the absence of clinical surrogate markers to diagnose inflammatory liver disease, the intervention with effective drugs in modern medicine tends to be late. In Sri Lanka, traditional medical practitioners prescribe herbal preparations from Osbeckia octandra for the prevention and treatment of liver disorders. To test the efficacy of such treatments, we have administered thioacetamide (TAA) to male Wistar rats to induce chronic liver damage (disease control; DC) and examined how various leaf extracts: crude leaf suspension (CLS), boiled leaf extract (BLE), sonicated leaf extract (SLE), methanol leaf extract (MLE) and hexane leaf extract (HLE) of O. octandra ameliorate TAA-induced liver disease. The CLS, BLE and SLE treatments in cirrhotic rats significantly attenuated disease-related changes, such as liver weight and hepato-enzymes. The mRNA levels of Tnf-α were significantly decreased by 3.6, 10 and 3.9 times in CLS, BLE and SLE compared to DC. The same treatments resulted in significantly lower (19.5, 4.2 and 2.4 times) α-Sma levels compared to DC. In addition, Tgf-β1 and Vegf-R2 mRNA expressions were significantly lower with the treatments. Moreover, BLE expressed a strong anti-angiogenic effect. We conclude that CLS, BLE and SLE from O. octandra have potent hepatic anti-fibrotic effects in TAA-induced liver cirrhosis.

Inhibitory Effects of Euphorbia ebracteolata Hayata Extract ECB on Melanoma-Induced Hyperplasia of Blood Vessels in Zebrafish Embryos.

Melanoma is a serious malignant form of skin cancer. Euphorbiaceae compound B (ECB, 2,4-dihydroxy-6-methoxy-3-methylacetophenone) is an acetophenone compound that is isolated from Euphorbia ebracteolata Hayata (EEH), an herbaceous perennial, and has antitumor activity. Here, we transplanted human melanoma cells into zebrafish embryos to establish a zebrafish/melanoma model. We showed that this model can be used to evaluate the therapeutic effect of EEH and ECB and discussed its potential mechanism of action. The results showed that ECB was an active ingredient of EEH in inhibiting melanoma-induced hyperplasia of blood vessels in zebrafish embryos, similar to the angiogenic inhibitor vatalanib. ECB inhibited the number and length of subintestinal veins (p < 0.05), as well as the distribution of melanoma in zebrafish embryos (p < 0.05). More importantly, unlike vatalanib, ECB only inhibited melanoma-induced abnormal and excessive growth of blood vessels in xenografts. In addition, ECB inhibited the mRNA expression of vegfr2 and vegfr3 in zebrafish. Both vegfr2 and vegfr3 are essential genes that regulate blood vessel formation and upregulate the expression of p53 and casp3a genes in zebrafish. Together, the above-mentioned results indicate that ECB has a potential antimelanoma effect in vivo, which may be mediated by inhibiting vascular endothelial growth factor receptors.

The expression of VEGF and VEGFR in endotoxin induced otitis media with effusion in rats

ObjectiveTo investigate the expression and correlation of vascular endothelial growth factor (VEGF) and its receptor with hypoxia-inducible factor-1 α (HIF-1 α) in otitis media with effusion (OME). MethodsA rat model of OME was induced by injection of lipopolysaccharide (LPS) into the middle ear. Hematoxylin and eosin (HE) staining was used to observe the pathomorphological changes of the tympanic cavity in the middle ear of rats. Immunohistochemistry (IHC), western blotting and RT-qPCR were used to determine the mRNA and protein expression of VEGF, VEGFR-1, VEGFR-2 and HIF-1α in mucosa of tympanic cavity mucosa, respectively. ResultsIn the OME group, the epithelial space of the middle ear mucosa was significantly thickened and infiltration of a large number of inflammatory cells was found on postoperative day (POD), and the otitis media basically subsided 2 weeks after operation. VEGF mRNA expression was significantly increased on POD 1, and its protein expression peaked on POD 3. HIF-1α mRNA expression was significantly increased and peaked on POD 1, while its protein expression began to increase on POD 3 and was significantly expressed in the middle ear mucosal epithelium. HIF-1α mRNA showed a positive correlation with VEGF mRNA and VEGFR-1 mRNA expression. ConclusionVEGF mainly plays a role in the acute phase of OME, and it is abundantly expressed mediated by HIF-1α. And then it play a role in vasodilatation and increase of vascular permeability, thus promoting the generation of middle ear effusion.

ONC201 Shows Potent Anticancer Activity Against Medullary Thyroid Cancer via Transcriptional Inhibition of RET, VEGFR2, and IGFBP2

Gain-of-function point mutations in the receptor tyrosine kinase RET, a driver oncogene in medullary thyroid carcinoma (MTC), prevent apoptosis through inhibition of ATF4, a critical transcriptional regulator of endoplasmic reticulum stress. However, the critical regulatory mechanisms driving RET-dependent oncogenesis remain elusive, and there is a clinical need to identify a transcriptional RET inhibitor. Here, we found that RET depletion decreased IGFBP2 and VEGFR2 mRNA and protein expression in MTC cells. IGFBP2 knockdown decreased cell survival and migration of MTC cells. In patients, IGFBP2 expression increased in metastatic MTC, and high IGFBP2 associated with poor overall survival. VEGFR2 protein levels were positively associated with RET expression in primary tumors, and VEGF-mediated increased cell viability was RET dependent. The small-molecule ONC201 treatment of MTC cells caused apoptotic cell death, decreased transcription of RET, VEGFR2, IGFBP2, increased mRNA levels of ATF4, and ATF4 target genes including DDIT3, BBC3, DUSP8, MKNK2, KLF9, LZTFL1, and SESN2 Moreover, IGFBP2 depletion increased ONC201-induced cell death. ONC201 inhibited tumor growth at a well-tolerated dose of 120 mg/kg/week administered by oral gavage and decreased MTC xenograft cell proliferation and angiogenesis. The protein levels of RET, IGFBP2, and VEGFR2 were decreased in ONC201-treated xenografts. Our study uncovered a novel ONC201 mechanism of action through regulation of RET and its targets, VEGFR2 and IGFBP2; this mechanism could be translated into the clinic and represent a promising strategy for the treatment of all patients with MTC, including those with TKI-refractory disease and other cancer with RET abnormalities.

Abstract 387: Role of Aldehyde Dehydrogenase (aldh) 2 in Angiotensin II-induced Decrease in Angiogenesis of Coronary Endothelial Cells

Diabetes-induced coronary endothelial cell (CEC) dysfunction following defective angiogenesis is reported in cardiovascular diseases (CVD). Angiotensin II (Ang II), a vasoactive molecule, is upregulated in diabetes. However, the underlying molecular mechanisms of Ang II-induced CEC dysfunction are not fully understood. Aldehyde dehydrogenase (ALDH) 2 is cytoprotective in diabetic CVD. Thus, we hypothesize that ALDH2 improves Ang II-mediated defective CEC angiogenesis. To test our hypothesis, we treated the cultured mouse CECs with Ang II (0.1, 1 and 10 μM) for 2 and 4 hours. Next, we treated CEC with Alda-1 (10 μM), an ALDH2 activator or disulfiram (2.5 μM), an ALDH2 inhibitor, before challenging MCECs with Ang II. We found that Ang II attenuated tube formation (P&lt;0.05 vs control) which indicates in vitro angiogenesis. Next, we found that Ang II have downregulated the mRNA expressions of vascular endothelial growth factor receptor VEGFR1 (p&lt;0.05) and upregulated angiotensin II type-2 receptor (AT2R) (P&lt;0.05) in cultured CECs compared to controls. ALDH2 inhibition with disulfiram potentiated Ang II-induced decrease in angiogenesis (P&lt;0.005) by decreasing the expressions of VEGFR1 (P&lt;0.0005) and increasing the expression of AT2R (p&lt;0.05) relative to Ang II alone. Additionally, activation of ALDH2 activity with Alda-1 rescued Ang II-induced decrease in angiogenesis (P&lt;0.05) by increasing the expression of VEGFR1 (P&lt;0.05) and decreasing the expression of AT2R (P&lt;0.05) relative to Ang II alone. Finally, we conclude that ALDH2 can be an important therapeutic target to improve coronary angiogenesis in diabetic CVD.

Dual drug-delivering polycaprolactone-collagen scaffold to induce early osteogenic differentiation and coupled angiogenesis

Bone regeneration is a multi-step, overlapping process, in which angiogenesis and osteogenesis are the key players. Several attempts have been made to promote angiogenesis-coupled osteogenesis using scaffolding technology. However, the recreation of functional vasculature during bone regeneration is an unparalleled challenge. In this study, a dual drug-delivering polycaprolactone-collagen fibrous scaffold is reported to promote early osteogenesis and angiogenesis. Simvastatin as a pro-angiogenic and dexamethasone as an osteoinductive drug were encapsulated to functionalize the electrospun fibers. The optically transparent fibrous mat represented the sustained and sequential release of drugs for 28 days. The fibrous mesh increased cell proliferation and enhanced the osteogenic differentiation up to 21 days. The alkaline phosphatase activity and mineral deposition were comparatively higher on dual drug-releasing fibers when compared to control fibers. The dual drug-releasing osteoconductive fibers demonstrated osteogenesis as early as 7 days with a 3.7 and 1.5 fold increase in the expression of osteogenic differentiation markers (RUNX2 and osteocalcin), respectively. In vitro angiogenesis using primary human umbilical vein endothelial cells (pHUVECs) showed no significant difference in cell proliferation among control fibers and dual drug-releasing fibers. However, the angioinductive nature of simvastatin released from the fibers demonstrated tube formation and 2 fold higher angiogenic score. The mRNA and protein expression study of angiogenic markers (VEGFR2 and eNOS) by polymerase chain reaction and western blotting depicted the angioinducing potential of dual drug-releasing fibers. VEGFR2 and eNOS mRNA expressions increased by 1.1 and 1.6 fold, respectively, whereas their protein expression increased by 3.2 and 1.7 fold, respectively. The overall results demonstrate the synergistic effect of osteoconductive substrate and osteoinductive dual drugs to promote early osteogenesis, and release of the pro-angiogenic drug promotes angiogenesis.

The in vitro effect of VEGF receptor inhibition on primary alveolar osteoblast nodule formation.

Vascular endothelial growth factor (VEGF) is a master regulator and is required for the effective coupling of angiogenesis and osteogenesis supporting both skeletal development and postnatal bone repair. A direct role for VEGF in intramembranous-derived osteoblast growth and differentiation is not clear. We investigated the expression of primary alveolar osteoblast VEGF receptors and the subsequent effects on mineralization and nodule formation in vitro following VEGFR inhibition. Primary human alveolar osteoblasts (HAOBs) were cultured in the presence of VEGF receptor inhibitors, exogenous VEGF or the bisphosphonate, zoledronic acid. VEGF, VEGFR1 and VEGFR2 mRNA expression and nodule formation following 21days of culture. VEGFR1 protein expression was examined using immunofluorescence after 48h. The HAOBs expressed high levels of VEGF and VEGFR1 protein but VEGFR2 was not detected. The VEGFR1/2 inhibitors, ZM306416 and KRN633, lead to a dose-dependent decrease in mineralization. Treatment with zoledronic acid showed no difference in HAOB VEGF receptor expression. VEGF/VEGFR1 pathway appears to be important for intramembranous-derived osteoblast differentiation and maturation in vitro.

The efficacy and safety of Apatinib mesylate in the treatment of metastatic osteosarcoma patients who progressed after standard therapy and the VEGFR2 gene polymorphism analysis.

The aim of this study was to investigate the efficacy and safety of Apatinib mesylate in the treatment of metastatic osteosarcoma patients who progressed after standard therapy and the VEGFR2 gene polymorphism analysis. Designed as a retrospective study, a total of 105 metastatic osteosarcoma patients who progressed after standard therapy were included in this study. The metastatic osteosarcoma patients received 500-750mg Apatinib mesylate according to body surface area until disease progression or unacceptable toxicity with 28days one cycle. Overall response was evaluated after two cycles Apatinib treatment, then progression-free survival (PFS) and overall survival (OS) were evaluated, and safety data were recorded. Additionally. peripheral blood and peripheral blood mononuclear cell (PBMC) specimens in the osteosarcoma patients were collected for the genotyping of VEGFR2 genetic variation and mRNA expression, respectively. Analysis on the association between genotype and baseline characteristics and VEGFR2 gene mRNA expression was analyzed. The univariate analysis of genotypes and prognosis was carried out by Kaplan-Meier survival analysis, and multivariate analysis was adjusted by Cox regression analysis. The objective response rate (ORR) of the 105 metastatic osteosarcoma patients was 37.14%, disease control rate (DCR) was 77.14%, median PFS was 4.1months, and median OS was 9.0months. Regarding the VEGFR2 gene polymorphisms analysis, only - 906T > C was of clinical significance. The prevalence of - 906T > C in VEGFR2 among the study population was as follows: TT genotype 62 cases (59.05%), TC genotype 36 cases (34.29%) and CC genotype 7 cases (6.66%), minor allele frequency of - 906T > C was 0.24. Compared with patients with TC/CC genotype, patients with TT genotype showed longer median PFS (5.0 versus 3.1months, P = 0.011) and median OS (9.8 versus 7.6months, P = 0.032). There was no correlation between the polymorphism and adverse reactions. Additionally, the mRNA expression in 69 randomly selected sample indicated that the mRNA expression of VEGFR2 of the patients with CC/TC genotypes were significantly higher than those of the TT genotype patients (P < 0.001). Apatinib was safe and effective in the treatment of metastatic osteosarcoma patients who progressed after standard therapy. The clinical outcomes of Apatinib may be influenced by the polymorphism - 906T > C of VEGFR2 through mediating the mRNA expression of VEGFR2.