VEGF mRNA Research Articles

Effects of maternal HF diet and absence of TRPC1 gene on mouse placental growth and fetal intrauterine growth retardation (IUGR)

Placental tissue intracellular calcium (Ca2+) regulates placental development and growth. Maternal high-fat diet (HFD) results in placental lipid accumulation, increased inflammation, reduced nutrient transport expression, and intrauterine growth restriction (IUGR). Currently, whether maternal HFD differentially affects placental and fetal growth and development under reduced Ca2+ influx is not yet known. We hypothesized that maternal HFD feeding decreases placental growth and development resulting in IUGR and that reduction of Ca2+ influx in the placenta worsens maternal HFD-induced placental dysfunction and IUGR. Three-week-old female B6129SF2/J wild type (WT) and transient receptor potential canonical 1 (TRPC1) protein deficient (KO) mice were fed normal fat (NF, 16 kcal % fat) and high fat (HF, 45 kcal % fat) diets for 12 weeks prior to mating with NF diet fed male mice. Fetuses and placentae were examined at mid- (D12) and late- (D18) gestation. At D12, maternal HFD had no effects on placental or fetal weight changes in WT and TRPC1 KO mice while absence of TRPC1 resulted in decreased placental and fetal weights. At D18, maternal HFD increased placental weights in both TRPC1 KO and WT mice, in part, by moderately increasing placental tissue triacylglyceride (TAG, P=.0632). At D12, mRNA expression of key placental growth factors including IGF1, PLGF, and VEGF were increased in WT compared to TRPC1 KO mice while IGF2 and VEGF mRNA expression were increased at D18. Results presented in our study demonstrated that maternal HFD increased placental weight, in part, due to increased lipid concentration resulting in IUGR and via an additive adverse effect of genotype and maternal HFD. Future studies are needed to determine the signaling mechanism underlying Ca2+ influx reduction-induced placental dysfunction and IUGR.

HIF-2α regulates proliferation, invasion, and metastasis of hepatocellular carcinoma cells via VEGF/Notch1 signaling axis after insufficient radiofrequency ablation.

Background and AimsAlthough insufficient radiofrequency ablation (RFA) promotes the recurrence and metastasis of liver cancer, the underlying mechanism remains unclear. This study aimed to investigate the role and mechanism of HIF-2α in hepatocellular carcinoma cells (HCCs) after Insufficient RFA.MethodsWe established a model of insufficient RFA in MHCC97H hepatoma cells and screened for stable sublines. We inhibited HIF-2α expression in the Insufficient RFA group using PT2385 and assessed the resulting changes in proliferation and biological function of HCCs. Cell viability and proliferation were detected by the MTT method, and scratch and Transwell chamber invasion tests detected migration and invasion abilities of HCCs. The mRNA and protein expression levels of VEGF, HIF-2α, and Notch1 were detected using qPCR, immunofluorescence, and western blotting.ResultsCompared with normal HCCs without RFA treatment, insufficient RFA enhanced the proliferation and invasion abilities of hepatocellular carcinoma subline MHCC97H (P < 0.001), as well as their migration ability (P = 0.046). The HIF-2α-specific inhibitor PT2385 downregulated the migration (P = 0.009) and invasion (P < 0.001) of MHCC97H cells but did not affect cell proliferation (P > 0.05). Insufficient ablation increased the mRNA and protein expression of VEGF, HIF-2α, and Notch1 in HCCs, whereas inhibition of HIF-2α reversed these changes.ConclusionsInsufficient RFA increases the proliferation, migration, and invasion of HCCs via the HIF-2α/VEGF/Notch1 signaling axis; HIF-2α is a potential target for novel treatments of HCC after insufficient RFA.

Exosomal FZD10 derived from non-small cell lung cancer cells promotes angiogenesis of human umbilical venous endothelial cells in vitro

To investigate the effect of exosomal FZD10 derived from non-small cell lung cancer (NSCLC) cells on angiogenesis of human umbilical venous endothelial cells (HUVECs) and explore the possible mechanism. We analyzed the expression of FZD10 in two NSCLC cell lines (95D and H1299 cells), normal human bronchial epithelial cells (BEAS-2B cells) and their exosomes isolated by ultracentrifugation. Cultured HUVECs were treated with the exosomes derived from NSCLC cells or NSCLC cells transfected with FZD10-siRNA, and the changes in tube formation ability of the cells were analyzed using an in vitro angiogenesis assay. ELISA was performed to determine the concentration of VEGFA and Ang-1 in the conditioned media of HUVECs, and RT-qPCR was used to analyze the mRNA levels of VEGFA and Ang-1 in the HUVECs. The effects of exosomal FZD10 on the activation of PI3K, Erk1/2 and YAP/TAZ signaling pathways were evaluated using Western blotting. Compared with BEAS-2B cells and their exosomes, 95D and H1299 cells and their exosomes all expressed high levels of FZD10 (P < 0.01). The exosomes derived from 95D and H1299 cells significantly enhanced tube formation ability and increased the expressions of VEGFA and Ang-1 protein and mRNA in HUVECs (P < 0.01), but FZD10 knockdown in 95D and H1299 cells obviously inhibited these effects of the exosomes. Exosomal FZD10 knockdown suppressed the activation of PI3K and Erk1/2 signaling pathways, but had no obvious effect on the activation of YAP/TAZ signaling pathway. Exosomal FZD10 derived from NSCLC cells promotes HUVEC angiogenesis in vitro, the mechanism of which may involve the activation of PI3K and Erk1/2 signaling pathways.

Identification of AGR2 Gene-Specific Expression Patterns Associated with Epithelial-Mesenchymal Transition.

The TGF-β signaling pathway is involved in numerous cellular processes, and its deregulation may result in cancer development. One of the key processes in tumor progression and metastasis is epithelial to mesenchymal transition (EMT), in which TGF-β signaling plays important roles. Recently, AGR2 was identified as a crucial component of the cellular machinery responsible for maintaining the epithelial phenotype, thereby interfering with the induction of mesenchymal phenotype cells by TGF-β effects in cancer. Here, we performed transcriptomic profiling of A549 lung cancer cells with CRISPR-Cas9 mediated AGR2 knockout with and without TGF-β treatment. We identified significant changes in transcripts associated with focal adhesion and eicosanoid production, in particular arachidonic acid metabolism. Changes in transcripts associated with the focal adhesion pathway were validated by RT-qPCR of COL4A1, COL4A2, FLNA, VAV3, VEGFA, and VINC mRNAs. In addition, immunofluorescence showed the formation of stress fibers and vinculin foci in cells without AGR2 and in response to TGF-β treatment, with synergistic effects observed. These findings imply that both AGR2 downregulation and TGF-β have a role in focal adhesion formation and cancer cell migration and invasion. Transcripts associated with arachidonic acid metabolism were downregulated after both AGR2 knockout and TGF-β treatment and were validated by RT-qPCR of GPX2, PTGS2, and PLA2G4A. Since PGE2 is a product of arachidonic acid metabolism, its lowered concentration in media from AGR2-knockout cells was confirmed by ELISA. Together, our results demonstrate that AGR2 downregulation and TGF-β have an essential role in focal adhesion formation; moreover, we have identified AGR2 as an important component of the arachidonic acid metabolic pathway.

The combination of EGCG with warfarin reduces deep vein thrombosis in rabbits through modulating HIF-1α and VEGF via the PI3K/AKT and ERK1/2 signaling pathways.

Deep venous thrombosis (DVT) poses a major challenge to public health worldwide. Endothelial cell injury evokes inflammatory and oxidative responses that contribute to thrombus formation. Tea polyphenol (TP) in the form of epigallocatechin-3-gallate (EGCG) has anti-inflammatory and oxidative effect that may ameliorate DVT. However, the precise mechanism remains incompletely understood. The current study was designed to investigate the anti-DVT mechanism of EGCG in combination with warfarin (an oral anticoagulant). Rabbits were randomly divided into five groups. A DVT model of rats was established through ligation of the inferior vena cava (IVC) and left common iliac vein, and the animals were orally administered with EGCG, warfarin, or vehicle for seven days. In vitro studies included pretreatment of human umbilical vein endothelial cells (HUVECs) with different concentrations of EGCG for 2 h before exposure to hydrogen peroxide. Thrombus weight and length were examined. Histopathological changes were observed by hematoxylin-eosin staining. Blood samples were collected for detecting coagulation function, including thrombin and prothrombin times, activated partial thromboplastin time, and fibrinogen levels. Protein expression in thrombosed IVCs and HUVECs was evaluated by Western blot, immunohistochemical analysis, and/or immunofluorescence staining. RT-qPCR was used to determine the levels of AGTR-1 and VEGF mRNA in IVCs and HUVECs. The viability of HUVECs was examined by CCK-8 assay. Flow cytometry was performed to detect cell apoptosis and ROS generation was assessed by 2',7'-dichlorofluorescein diacetate reagent. In vitro and invivo studies showed that EGCG combined with warfarin significantly reduced thrombus weight and length, and apoptosis in HUVECs. Our findings indicated that the combination of EGCG and warfarin protects HUVECs from oxidative stress and prevents apoptosis. However, HIF-1α silencing weakened these effects, which indicated that HIF-1α may participate in DVT. Furthermore, HIF-1α silencing significantly up-regulated cell apoptosis and ROS generation, and enhanced VEGF expression and the activation of the PI3K/AKT and ERK1/2 signaling pathways. In conclusion, our results indicate that EGCG combined with warfarin modifies HIF-1α and VEGF to prevent DVT in rabbits through anti-inflammation via the PI3K/AKT and ERK1/2 signaling pathways.

Abstract P103: Apelin And Elabela Attenuate The Pro-inflammatory Response In Hypoxic HTR-8/SVneo Trophoblastic Cells

Preeclampsia is a significant pregnancy complication characterized by de novo hypertension, proteinuria, and further organ dysfunction. We previously demonstrated both cardio- and renoprotective effects by systemic activation of apelin receptor system (APJ) in preeclampsia. Although the APJ ligands apelin and elabela stimulate trophoblast cell proliferation and migration, the cellular mechanisms underlying these actions are not well understood, particularly in response to hypoxia. To examine the response of the APJ system to cellular hypoxia in human trophoblasts, HTR-8/SVneo trophoblastic cells were treated with cobalt chloride (CoCl 2: 200 μM, 24 hours) to induce hypoxic conditions. The cellular mRNA responses for sFlt-1 variants, VEGF, PLGF, IL-6, and IL-1β were determined by RT-qPCR for control and apelin- or elabela-treated cells with or without CoCl 2 . The presence of APJ mRNA was confirmed in HTR-8/SVneo cells. Hypoxia increased apelin content approximately 2-fold in both the cell lysate (723 ± 52 vs. 1216 ± 20 ng/mg protein, n=6, p&lt;0.01) and the conditioned media (49 ± 5 vs. 102 ± 9 ng/ml, n=6, p&lt;0.01). Elabela content also increased in the media of hypoxic cells (0.11 ± 0.01 vs. 0.14 ± 0.01 ng/ml, n=6, p&lt;0.05), although elabela levels were 100-fold lower than apelin. Hypoxia upregulated hsFlt-1-i13 (2.6 fold), hsFlt-1-e15a (2.9 fold), VEGF (3.7 fold), PLGF (1.3 fold), and IL-6 (3.5 fold) mRNA levels. Treatment with (Pyr-1)-apelin-13, a stable APJ ligand (0.1 μM), decreased VEGF (1.2 fold; n=3, p&lt;0.05) and IL-6 (1.4 fold, n=3, p&lt;0.05) mRNA levels in cells exposed to COCl 2 , but had no effect in normoxic cells. Similarly, elabela (0.1 μM) downregulated VEGF (1.1 fold; n=3, p&lt;0.05) and IL-6 (1.2 fold; n=3, p&lt;0.05). We found no difference in sFlt-1 variants, PLGF, or IL-1β mRNAs following treatment of hypoxic cells with either peptide. We conclude that cobalt-induced hypoxia markedly increases various indices of inflammation, as well as stimulates the endogenous apelin and elabela systems in the HTR-8/SVneo cells. Furthermore, local activation of these peptidergic systems may be a compensatory response of the trophoblast cells to hypoxia as exogenous apelin and elabela treatment effectively ameliorate the hypoxia-induced pro-inflammatory milieu.

The Blastocyst Creates a Specialized Microenvironment to Promote Endometrial Receptivity and Implantation

Background: At the time of implantation, the blastocyst produces a significant amount of lactic acid (LA), creating a microenvironment characterized by high lactate and low pH. Historically considered a ‘by-product’ of metabolism, studies have revealed lactate to be a key regulator of cell function, with a lactate-specific receptor, GPR81, recently identified. Further, lactate is an important signalling molecule in cancers promoting ECM breakdown, angiogenesis and immunosuppression, processes vital for successful implantation. Hence, LA could be an early embryonic signal to facilitate endometrial remodelling for receptivity and implantation. Aim: To determine the role of blastocyst-derived lactate on cellular functions/remodelling and transcriptional changes essential for endometrial receptivity and successful implantation. Methods: To assess functional changes to the endometrium, hormonally primed ECC-1 and Ishikawa cells were exposed to LA, LA + neutralized pH (nLA) or acidic pH (pHL), before analysis of tight junction integrity (TER), cellular proliferation and changes to gene expression. LA’s effects on endometrial stromal cell decidualization and migratory capacity, as well as HUVEC endothelial tube formation/angiogenesis, were also determined. Results: Cellular TER and proliferation were significantly downregulated in ECC-1 cells exposed to 2.5, 5 and 7.5mM LA ( P&lt;0.01), while exposure to nLA or pHL alone had no effect. Upregulation of GLUT4, GPR81, VEGF, SNAI1, and RELA mRNA expression was observed following exposure of Ishikawa cells to combined LA + pHL(P&lt;0.05), while MCT-1 expression decreased. Additionally, 5mM LA increased the migratory capacity of decidualized stromal cells (P&lt;0.05) without changing the extent of decidualization. HUVEC tube formation was significantly increased by 5mM LA exposure ( P&lt;0.01). Conclusion: The specialized microenvironment created by the blastocyst, of both high lactate and low pH, enhances endometrial remodelling for receptivity and implantation through functional changes to epithelial, stromal and endothelial cells in the endometrium. Therefore, LA appears to be an important signalling molecule in the maternal-fetal dialogue underpinning implantation.

수영운동이 고환절제 쥐의 대동맥 혈관내피세포 성장인자 발현에 미치는 영향

The purpose of this experiment was to investigate the effect of swimming exercise on vascular endothelial growth factors expression orchiectomized rats. The experimental animals were divided into three groups (n=10 in each group): the normal control group(CON), control group with orchiectomized(ORC+Con) and swimming exercise group with orchiectomized(ORC+SE). Male orchiectomized rats performed five daily bouts of 30 min swimming per week for 16 weeks. After swimming exercise for 16 weeks, serum testosterone concentration of ORC+SE groups were significantly increased compared to ORC+Con groups(p.05). NO concentration were not significantly different between groups. VEGF mRNA expression of ORC+SE groups were significantly increased compared to ORC+Con groups(p.05). In conclusion, 16 weeks swimming exercise training may prevent to atherosclerosis to negative change carotid artery in orchiectomized rats.

TNFAIP3 mediates FGFR1 activation-induced breast cancer angiogenesis by promoting VEGFA expression and secretion.

To investigate the role and mechanism of TNF-inducible protein 3(TNFAIP3) in breast cancer angiogenesis induced by fibroblast growth factor receptor1 (FGFR1) activation. The immunohistochemical assay was used to detect the expression of vascular endothelial cell marker CD31 and CD105 in mice DCIS.COM-iFGFR1 transplanted tumor (previously established by our group). The effects of TNFAIP3 knockout/knockdown breast cancer cell lines on angiogenesis, migration, and invasion of Human Umbilical Vein Endothelial Cells (HUVEC) were detected by the tubulogenesis and Trewells assay. RNA-seq analysis of TNFAIP3 downstreams differential genes after TNFAIP3 knockdown. The expression and secretion of VEGFA after FGFR1 activation in breast cancer cells were detected by qPCR, Western blot, and ELISA. Immunohistochemistry showed that TNFAIP3 knockout inhibited the expression of CD31 and CD105 in DCIS grafted tumors promoted by FGFR1 activation. Tubulogenesis and Trewells experiments showed that TNFAIP3 gene knockout/knockdown inhibited the angiogenesis, migration, and invasion of HUVEC cells promoted by FGFR1 activation. qPCR assay showed that VEGFA mRNA level in the TNFAIP3 knockdown cell line was significantly down-regulated (p < 0.05). qPCR, Western blot and ELISA results showed that TNFAIP3 gene knockout/knockdown could inhibit the expression and secretion of VEGFA in breast cancer cells induced by FGFR1 activation. TNFAIP3 promotes breast cancer angiogenesis induced by FGFR1 activation through the expression and secretion of VEGFA.

Kuoxin Decoction promotes lymphangiogenesis in zebrafish and in vitro based on network analysis.

Background: Inadequate lymphangiogenesis is closely related to the occurrence of many kinds of diseases, and one of the important treatments is to promote lymphangiogenesis. Kuoxin Decoction (KXF) is an herbal formula from traditional Chinese medicine used to treat dilated cardiomyopathy (DCM), which is associated with lymphangiogenesis deficiency. In this study, we comprehensively verified whether KXF promotes lymphangiogenesis in zebrafish and in vitro based on network analysis. Methods: We performed virtual screening of the active compounds of KXF and potential targets regarding DCM based on network analysis. Tg (Flila: EGFP; Gata1: DsRed) transgenic zebrafish embryos were treated with different concentrations of KXF for 48 h with or without the pretreatment of MAZ51 for 6 h, followed by morphological observation of the lymphatic vessels and an assessment of lymphopoiesis. RT-qPCR was employed to identify VEGF-C, VEGF-A, PROX1, and LYVE-1 mRNA expression levels in different groups. After the treatment of lymphatic endothelial cells (LECs) with different concentrations of salvianolic acid B (SAB, the active ingredient of KXF), their proliferation, migration, and protein expression of VEGF-C and VEGFR-3 were compared by CCK-8 assay, wound healing assay, and western blot. Results: A total of 106 active compounds were identified constituting KXF, and 58 target genes of KXF for DCM were identified. There were 132 pathways generated from KEGG enrichment, including 5 signaling pathways related to lymphangiogenesis. Zebrafish experiments confirmed that KXF promoted lymphangiogenesis and increased VEGF-C and VEGF-A mRNA expression levels in zebrafish with or without MAZ51-induced thoracic duct injury. In LECs, SAB promoted proliferation and migration, and it could upregulate the protein expression of VEGF-C and VEGFR-3 in LECs after injury. Conclusion: The results of network analysis showed that KXF could regulate lymphangiogenesis through VEGF-C and VEGF-A, and experiments with zebrafish confirmed that KXF could promote lymphangiogenesis. Cell experiments confirmed that SAB could promote the proliferation and migration of LECs and upregulate the protein expression of VEGF-C and VEGFR-3. These results suggest that KXF promotes lymphangiogenesis by a mechanism related to the upregulation of VEGF-C/VEGFR-3, and the main component exerting this effect may be SAB.

Metallic Scaffold with Micron-Scale Geometrical Cues Promotes Osteogenesis and Angiogenesis via the ROCK/Myosin/YAP Pathway.

The advent of precision manufacturing has enabled the creation of pores in metallic scaffolds with feature size in the range of single microns. In orthopedic implants, pore geometries at the micron scale could regulate bone formation by stimulating osteogenic differentiation and the coupling of osteogenesis and angiogenesis. However, the biological response to pore geometry at the cellular level is not clear. As cells are sensitive to curvature of the pore boundary, this study aimed to investigate osteogenesis in high- vs low-curvature environments by utilizing computer numerical control laser cutting to generate triangular and circular precision manufactured micropores (PMpores). We fabricated PMpores on 100 μm-thick stainless-steel discs. Triangular PMpores had a 30° vertex angle and a 300 μm base, and circular PMpores had a 300 μm diameter. We found triangular PMpores significantly enhanced the elastic modulus, proliferation, migration, and osteogenic differentiation of MC3T3-E1 preosteoblasts through Yes-associated protein (YAP) nuclear translocation. Inhibition of Rho-associated kinase (ROCK) and Myosin II abolished YAP translocation in all pore types and controls. Inhibition of YAP transcriptional activity reduced the proliferation, pore closure, collagen secretion, alkaline phosphatase (ALP), and Alizarin Red staining in MC3T3-E1 cultures. In C166 vascular endothelial cells, PMpores increased the VEGFA mRNA expression even without an angiogenic differentiation medium and induced tubule formation and maintenance. In terms of osteogenesis-angiogenesis coupling, a conditioned medium from MC3T3-E1 cells in PMpores promoted the expression of angiogenic genes in C166 cells. A coculture with MC3T3-E1 induced tubule formation and maintenance in C166 cells and tubule alignment along the edges of pores. Together, curvature cues in micropores are important stimuli to regulate osteogenic differentiation and osteogenesis-angiogenesis coupling. This study uncovered key mechanotransduction signaling components activated by curvature differences in a metallic scaffold and contributed to the understanding of the interaction between orthopedic implants and cells.

Shikonin inhibits the proliferation of cervical cancer cells via FAK/AKT/GSK3β signalling.

Cervical cancer is one of the most lethal malignancies of the female reproductive system. Shikonin, a naphthoquinone pigment extracted from the traditional medicinal herb, Lithospermum erythrorhizon, has been demonstrated to exert significant inhibitory effects on a variety of tumours in vitro and in vivo. In the present study, the effects of shikonin on cervical cancer and the underlying mechanisms were investigated. The effects of shikonin on the viability on HeLa and SiHa cervical cancer cells was examined using cell counting kit (CCK-8) and colony formation assays. Immunofluorescence assay was performed to detect the levels of the proliferation-related protein, Ki67. Western blot analysis was utilized to measure the phosphorylated and total expression levels of proteins, including focal adhesion kinase (FAK), AKT, and glycogen synthase kinase 3β (GSK3β). Cell migration was determined by using wound healing assay. Metastasis-associated 1 (MTA1), TGFβ1 and VEGF mRNA expression levels were determined using reverse transcription-quantitative PCR. It was demonstrated that, shikonin inhibited cervical cancer cell proliferation and migration. The data of the present study revealed that shikonin inhibited the proliferation of HeLa and SiHa cells in a concentration- and time-dependent manner. Mechanistically, shikonin blocked the proliferation of cervical cancer cells by downregulating the phosphorylation of FAK, AKT and GSK3β induced by EGF. In addition, shikonin significantly suppressed cell migration and reduced the expression of migration-related proteins, including MTA1, TGFβ1 and VEGF. On the whole, the present study demonstrates that shikonin may exert an inhibitory effect on the cervical cancer cell proliferation and migration through the FAK/AKT/GSK3β signaling pathway. These findings suggest that shikonin may function as a potential therapeutic drug for the treatment of cervical cancer.

Fetal hyperhomocysteinemia is associated with placental inflammation and early breakdown of maternal-fetal tolerance in pre-term birth.

Hyperhomocysteinemia (hypHcy) due to impaired folate metabolism is shown to be a risk factor for preterm birth (PTB) and low birth weight (LBW) in mothers. However, the relationship of fetal hypHcy with adverse pregnancy outcomes is under-represented. The present study aims to investigate the association of fetal hypHcy with oxidative stress and placental inflammation that can contribute to an early breakdown of maternal-fetal tolerance in pre-term birth (PTB). Cord blood and placenta tissue were collected from PTB and term infant group. Levels of homocysteine, folic acid, vitamin B12 and oxidative stress markers (MDA, T-AOC, 8-OHdG) were measured in cord blood serum using ELISA and respective standard assay kits. Relative expression of candidate genes (TNF-α, IL-6, IL1-β, VEGF-A, MMP2 and MMP9) was also checked using RT-PCR and immunoblotting/immunohistochemistry. PTB infants showed significantly higher levels of homocysteine (P=.02) and lower levels of vitamin B12 (P=.005) as compared to term infants. We also found that PTB infants with hypHcy had lower T-AOC (P=.003) and higher MDA (P=.04) levels as compared to term infants with normal homocysteine levels. The mRNA and protein levels of TNF-α, VEGF-A, MMP2 and MMP9 were significantly higher in hypHcy PTB infants. Our results show that fetal hypHcy is associated with oxidative stress and an increase in inflammatory markers in the placenta. Thus, in conclusion, our study demonstrates that fetal hypHcy during pregnancy is a potential risk factor that may initiate an early breakdown of uterine quiescence due to activation of inflammatory processes leading to PTB.

Anti-Obesity Effect of Rice Bran Extract on High-Fat Diet-Induced Obese Mice.

Obesity involving adipose tissue growth and development are associated with angiogenesis and extracellular matrix remodeling. Rice bran has antioxidant and cardioprotective properties, and can act as a food supplement with potential health benefits, such as lowering blood pressure, hepatic steatosis, and inflammation. Therefore, we hypothesized that rice bran extract (RBE) can regulate adipose tissue growth and obesity. Male Institute of Cancer Research mice were fed with a high-fat diet (HFD) for 8 weeks and then supplemented with 220 and 1,100 mg/kg/d RBE while the low-fat diet group (control) were not. In addition to body weight, adipose tissue mass, and vessel density, we evaluated the mRNA expression of angiogenic factors such as matrix metalloproteinases, Mmp-2, Mmp-9, and the vascular endothelial growth factor (Vegf) in visceral and subcutaneous adipose tissues using real-time polymerase chain reaction. Administration of RBE to HFD-induced obese mice reduced the body weight and adipose tissue mass compared with untreated mice. It also decreased blood vessel density in the adipose tissue. Furthermore, RBE downregulated Vegf and Mmp-2 mRNA levels in visceral fat tissue. These results demonstrate that RBE, at high concentrations, significantly reduces adipose tissue mass and prevents obesity development in HFD-induced obese mice, which might be partly mediated via an anti-angiogenic mechanism.

ALCOHOL-INDUCED EXPRESSION OF VASCULAR ENDOTHELIAL GROWTH FACTOR AND STRUCTURAL CHANGES IN RAT CARDIOMYOCYTES

The aim of the study is to reveal the ethanol effects on the expression of hypoxia-associated factors (HIF-1α, VEGF-A) and structural changes in cardiomyocytes of healthy rats.&#x0D; Materials and methods. Ethanol-induced structural changes in cardiomyocytes were studied in 60 outbred mature rats. Control animals were intragastrically injected with sterile 0.9% saline, experimental animals – with 40 % ethanol. Ethanol exposure on myocardium lasted 4 and 8 days. Morphological study of the heart was carried out after decapitation preceded by ether anesthesia. PCR tests were used to determine mRNA expression of HIF-1α and VEGF-A genes from the left ventricle and serum concentration of vascular endothelial growth factor. Morphological studies of the left ventricle myocardium were carried out on histological sections using light microscopy.&#x0D; Results. A short ethanol exposure was accompanied by fatty and granular cytoplasm degeneration of cardiomyocytes with erythrocyte stasis in capillaries, arterioles and venules. VEGF-A serum concentration in control rats was lower if compared with ethanol-induced rats. Gender differences to ethanol response were revealed, in particular, a more pronounced HIF-1α and VEGF-A mRNA expression in females compared to males on the background of 4-day ethanol exposure, which correlated with VEGF-A level in blood serum. 8-day alcohol exposure was accompanied by an increase in VEGF-A mRNA expression in both female and male rats.&#x0D; Conclusion. Ethanol exposure causes activation of HIF-1α and VEGF-A transcription factors in cardiomyocytes of rats. It can be regarded as a mechanism of urgent adaptation without the development of pronounced structural maladaptive changes of the myocardium.

Mechanism of ischemic brain injury repair by endothelial progenitor cell‑derived exosomes.

Ischemic stroke is a refractory disease that seriously endangers human health and life. The main treatment aim of stroke is to alleviate brain injury. The present study aimed to investigate the effects and mechanisms of endothelial progenitor cell (EPC)‑derived exosomes in repairing ischemic brain injury. Sprague‑Dawley rat models of cerebral ischemia‑reperfusion (IR) injury were established by middle cerebral artery occlusion. The IR model rats were then treated with PBS, EPC or exosomes; untreated and Sham rats were used as control. EPCs were obtained from tibias and femurs, and exosomes were isolated from the EPCs and characterized. To measure brain injury, 2,3,5‑triphenyltetrazolium chloride staining was used to measure the infarct area, neurological deficit was scored, hematoxylin and eosin staining was used to examine pathological changes and TUNEL staining was used to quantify apoptosis. Immunofluorescence staining and reverse transcription‑quantitative PCR were used to determine CD31 and VEGF protein and mRNA expressions, respectively, and western blot analysis was used out to measure the protein expression levels of Wnt3a, GSK‑3β and phosphorylated (p)‑GSK‑3β. Compared with rats in the Control and Sham groups, in IR model rats the nerve fibers were slightly necrotic and swollen and the number of nerve cells was reduced. Following EPC treatment, the brain tissue exhibited mild liquefaction and degeneration in the small focus area with mild edema in the stroma. The numbers of nerve cells decreased, and the distribution of nerve cells was not very uniform; proliferation of glial cells was observed. Following treatment with exosomes, the distribution of nerve cells was more uniform with less degeneration and necrosis; the proliferation of glial cells was remarkable. Compared with the Control group, the infarct size, neurological defect score, percentage of apoptotic cells, expression of CD31, VEGF, Wnt3a, and p‑GSK‑3β were significantly higher in the IR model (P<0.05). After EPC and exosome treatments, the infarct size, neurological defect score, percentage of apoptotic cells, expression of Wnt3a, and p‑GSK‑3β were significantly reduced (P<0.05), whereas the mRNA and protein expression levels of CD31 and VEGF were significantly increased (P<0.05). Results from the present study demonstrated that EPC‑derived exosomes may alleviate ischemic injury by inhibiting apoptosis and promoting angiogenesis. These findings suggested that exosomes may have a protective role for nerve cells and may be a potentially effective option for treating stroke. However, human clinical studies are needed to validate these findings from animals.

VEGF and VEGFR family members are expressed by neoplastic cells of NF1-associated tumors and may play an oncogenic role in malignant peripheral nerve sheath tumor growth through an autocrine loop

Neurofibromatosis type 1 (NF1) is a common autosomal dominant disorder. The role of angiogenesis and VEGF pathway in the pathogenesis of neurofibromas and malignant peripheral nerve sheath tumors (MPNSTs) remains poorly understood.We assessed the expression of VEGF and VEGFR family members in cohorts of plexiform neurofibromas (pNF), MPNSTs and MPNST cell lines at transcript [pNF, n = 49; MPNST, n = 34] and protein levels [pNF, n = 21; MPNST, n = 9].VEGF and VEGFR members were variably expressed in cell lines. VEGFA (p = 3.10−5), VEGFR1 (p = 0.08), and VEGFR2 (p = 2.10−4) mRNAs were overexpressed in MPNSTs in comparison with pNFs. Both VEGFA and VEGFR1 proteins were expressed by spindle tumor cells of pNFs and MPNSTs. VEGFA was expressed more in MPNSTs than in pNFs (p = 9.10−6) and a trend for VEGFR1 overexpression was observed (p = 0.06). VEGFR2 was not found at the protein level. The microvascular density was significantly reduced in MPNSTs as compared to pNFs (p = 0.0025), with no differences regarding the expression of the activated phosphorylated forms of ERK (P-ERK [p = 0.63]) and AKT (P-AKT [p = 0.41]) in endothelial cells, suggesting that VEGF-dependant angiogenesis may not be critical for MPNST oncogenesis.Altogether, these results indicate that the VEGF-VEGFR pathway may play a role in the development of pNFs and MPNSTs, independently of angiogenesis. Whether or not it drives an oncogenic autocrine/paracrine loop in neoplastic cells, participating in an increased activation of signaling pathways downstream of tyrosine kinase receptors, including VEGFRs, is a tempting hypothesis. Nevertheless, the specific targeting of angiogenesis in MPNSTs may not be sufficient to slow down tumor growth.

TGFβ1 Induces Senescence and Attenuated VEGF Production in Retinal Pericytes.

Diabetic retinopathy (DR) is a microvascular disease of the retina and a serious complication of type I and type II diabetes mellitus. DR affects working-age populations and can cause permanent vision loss if left untreated. The standard of care for proliferative DR is inhibiting VEGF. However, the mechanisms that induce excessive VEGF production in the retina remain elusive, although some evidence links elevated VEGF in the diabetic retina with local and systemic TGFβ1 upexpression. Here, we present evidence from animal models of disease suggesting that excessive TGFβ1 production in the early DR is correlated with VEGF mRNA and protein production by senescent pericytes and other retinal cells. Collectively, these results confirm that TGFβ1 is strongly implicated in the vascular complications of DR.

The effect of endostatin on angiogenesis and osteogenesis of steroid-induced osteonecrosis of the femoral head in a rabbit model.

This study aimed to investigate whether endostatin, a crucial anti-angiogenic factor, plays a negative role in angiogenesis and osteogenesis and aggravates the progression of osteonecrosis of the femoral head induced by steroid use in a rabbit model. 66 New Zealand white rabbits were randomly divided into four groups: glucocorticoid model (GC) group (GC group, n = 18), glucocorticoid model and endostatin group (GC;ES group, n = 18), ES group (ES group, n = 18), and blank control group (CON group, n = 12). In the GC group, 10 μg/ kg lipopolysaccharide (LPS) was intravenously injected into the ear margin, and 24h after LPS injection, 20 mg/kg GC methylprednisolone (MPS) was injected into the gluteus muscle three times, each time at an interval of 24h. The animals of the GC;ES group were given as same treatment as the GC group, except for the addition of ES. MPS was not used in the ES group and CON group. ES group was only given ES, while the CON group was only given the same amount of normal saline. All animals successfully established models of femoral head necrosis, and then the difference among the Immunohistochemistry, Quantitative polymerase chain reaction (qPCR) analysis, Enzyme-linked immunosorbent assay, Biomechanical test, etracyclline-calcein double labeling, and Van Gieson staining indices were compared among the four groups. The combination of MPS and LPS was successful in establishing the femoral head necrosis model in New Zealand white rabbits. The incidence of osteonecrosis after MPS and LPS intervention was 70% (7/10), while that plus ES was 100% (10/10). At the same time, after MPS and LPS intervention, while the empty bone lacuna rate of the femoral head was significantly increased, the number of osteo- blasts was decreased. Also, the expressions of CD31 positive cells, Runx2, Osterix, COL1A1, and VEGF mRNA in the femoral head were decreased, and the levels of osteogenesis-related protein b-ALP, OCN, and angiogenic factor VEGF in the femoral head were decreased. The percentage of the trabecular bone area (%Tb.Ar), trabecular thickness (Tb.Th), trabecular number (Tb.N), labeled perimeter percent (%L.Pm), mineral apposition rate (MAR), and bone formation rate (BFR/BS) in the femoral head after MPs and LPS intervention detected by tetracycline calcein double labeling and Van Gieson staining decreased significantly, except trabecular separation (Tb.Sp) increased significantly. The compressive strength (CS), elastic modulus (EM), and strain energy (SE) of the femoral head examed by biomechanical measurement decreased significantly. All the above changes were more obvious after adding ES intervention. ES mRNA in the femoral head was undifferentiated and increased in the GC, ES, and GC;ES group compared with group CON. This study has revealed that ES can inhibit angiogenesis and osteogenesis in the femoral head and aggravate the occurrence and development of femoral head necrosis. Thus, antiangiogenic factors may play an important role in the pathogenesis of ONFH.

Long Noncoding RNA SCIRT Promotes HUVEC Angiogenesis via Stabilizing VEGFA mRNA Induced by Hypoxia

Ischemia-reperfusion injury (IRI) is closely associated the abnormal expression of long noncoding RNAs (lncRNAs), especially for their regulatory roles in IRI-related angiogenesis. This study applied a hypoxia-reoxygenation (HR) cell model to simulate the IRI condition, as well as RNA sequencing and RNA pull-down experiments to reveal roles of the lncRNA and Stem Cell Inhibitory RNA Transcript (SCIRT), in endothelial angiogenesis. We found that SCIRT was increased under the HR condition and exhibited a high expression correlation with angiogenesis marker VEGFA. RNA-seq data analysis further revealed that VEGFA-related angiogenesis was regulated by SCIRT in HUVECs. Gain and loss of function experiments proved that SCIRT posttranscriptionally regulated VEGFA via affecting its mRNA stability. Furthermore, HuR (ELAVL1), an RNA binding protein (RBP), was identified as a SCIRT-binding partner, which bound and stabilized VEGFA. Moreover, SCIRT promoted HuR expression posttranslationally by inhibiting its ubiquitination under the HR condition. These findings reveal that lncRNA SCIRT can mediate endothelial angiogenesis by stabilizing the VEGFA mRNA via modulating RBP HuR stability under the HR condition.