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Objective Effect of fluoxetine on the expression of angiogenic factors in hippocampus of depressive rats.Methods 32 male SD rats (3 month) of SPF level were randomly divided into fluoxetine group (n=8),fluoxetine ± LY294002 intervention group (n=8),model group (n=8) and control group (n =8).Control group without any treatment was free to eat and water for 5 weeks.The rats in the other groups were modeled by chronic,mild and unpredictable multiphase stress.The fluoxetine group was treated with fluoxetine for 2 weeks after modeling,and the intervention group was given LY294002 intervention,then the fluoxetine treatment for 2 weeks,the model group was free to eat and drink for 2 weeks.At the end of the 5th week,all the rats were decapitated and the hippocampus was removed on the ice bath.Western blot was performed to detect the expression of angiogenic factors.Results Compared with the model group (vascular endothelial growth factor(VEGF) (0.44 ± 0.08),fetal liver kinase 1 (FLK1) (0.37 ± 0.15),protein kinase B1 (AKT1) (0.40±0.10),p-AKT1 (0.53±0.07)),the expression of VEGF (0.98± 0.13),FLK1 (1.09± 0.21),AKT1 (1.05±0.05) and p-AKT1 (1.01±0.13)in hippocampus of fluoxetine group were significantly increased,and the differences were statistically significant (P＜ 0.01).And the expression of VEGT,FLK1,p-AKT1 in fluoxetine group increased compared with those of fluoxetine ± LY294002 intervention group (VEGF (0.55±0.08),FLK1 (0.55±0.14) and p-AKT1 (0.60±0.05)),and the difference were statistically significant (P＜0.01).Conclusion Fluoxetine can increase the expression of VEGF/FLK1 and p-AKT1 proteins in the hippocampus of depressed rats,which in turn may be involved in the regeneration of hippocampal blood vessels.This effect of fluoxetine may be related to the PI3K/AKT signaling pathway.
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Los estudios en torno al papel patogénico del Schistosoma mansoni se enfocan en el daño hepático y la respuesta inmune en el hospedador definitivo, en contraste a la escasa información en relación a la patología intestinal. A tal fin, se evaluó el efecto del praziquantel en los receptores angiogenicos, citocinas inflamatorias y anti-inflamatorias en la esquitosomiasis intestinal murina. La respuesta inflamatoria granulomatosa intestinal se midió en secciones histológicas teñidas con H&E; la detección de TNF-α, IL-10, TGF-ß, el VEGF y sus receptores (FLK1 y FLT1) mediante inmunohistoquímica y ELISA, en suero e intestino de ratones BALB/c infectados con S. mansoni a las 8 (RI8S) y 20 semanas (RI20S), y ratones con 8 semanas de infección a los quince días de post-tratamiento, con praziquantel 40 µgr/gr (RPT). En RI8S se observaron granulomas pequeños (11304 µm2 en promedio), sin bordes definidos ni zonas marcadas dentro del granuloma, y granulomas de gran tamaño con bordes definidos (70650 µm2) sin zonas definidas dentro del granuloma, ambos con predominio de macrófagos y presencia de plasmocitos. En RI20S, los granulomas presentan una zona interna constituida por abundantes macrófagos y escasos plasmocitos, y una externa constituida solo por macrófagos (11985 µm2). En RPT no se apreciaron granulomas, solo algunos focos inflamatorios cercanos a la muscularis mucosae y las glándulas de Lieberkuhn. La Inmunolocalización en el intestino solo fue positiva en RPT para VEGF y sus receptores, TGP-ß e IL-10; Los resultados muestran una discreta respuesta en cuanto a tamaño del granuloma, celularidad y la expresión de citocinas a nivel intestinal.
The studies on the pathogenic role of the Schistosoma mansoni focus on the hepatic damage and the immune response of the final host, in constrast to the limited information regarding intestinal lesion. This work evaluates. Intestinal granulomatous inflammation was measured in histological sections the effect of praziquantel in the angiogenic receptors (FLK1 and FLT1), inflammation and inflammatory cytokines on the murine intestinal schistosomiasis stained with H&E; the detection of TNF-α, IL-10, TGF-ß, VEGF and its receptors was carried out by immunohistochemistry and ELISA, in serum and intestine sections of BALB/c mice infected with S. mansoni at 8 (RI8S) and 20 (RI20S) weeks post-infection, and mice infected for 8 weeks and evaluated 15 days post-treatment with praziquantel 40 µg/(RPT). In RI8S small granulomas (11.304 µm2 average), without sharp or marked edges within the granuloma; and large granulomas (70.650 µm2) with defined borders without defined zones within the granuloma, In both groups of mice macrophages predominated and plasma cells were present. In RI20S, granulomas have an inner zone composed by abundant macrophages and few plasma cells, and external zone constituted only by macrophages (11.985 µm2). In RPT, granulomas were not observed, only a few inflammatory foci nearby the muscularis mucosae and the Lieberkuhn glands. Localization of immune molecules in the intestine was only positive in RPT for VEGF, its receptors, TGF-ß and IL-10; the results show a discrete response over the granuloma size, cellularity and cytokine expression at intestinal level.
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Objective To explore the effect of acupuncture-rehabilitation therapy on neurological function and expression of Flt-1 and Flk-1, members of vascular endothelial growth factor receptors, after permanent focal cerebral ischemia in rats. Methods Ninety male Sprague-Dawley rats were divided into five groups, namely sham group, model group, acupuncture group, rehabilitation group and acupunc-ture-rehabilitation group, and each group was further divided into 3-day, 7-day and 14-day subgroups, equally. Their middle cerebral arteries were occluded except those of sham group. The sham and model groups accepted no treatment, while the acupuncture group accepted clus-ter needling of scalp acupuncture, the rehabilitation group accepted treadmill training, and the acupuncture-rehabilitation group accepted both acupuncture and treadmill training. They were assessed with modified Neurologic Severity Score (mNSS) 3, 7 and 14 days after model-ing, while the expression of Flt-1 and Flk-1 were determined with Western blotting. Results The mNSS score reduced in all the treatment groups (P<0.05) compared with that of the model group at every time point, and was the least in the acupuncture-rehabilitation group (P<0.05) 7 and 14 days after modeling among the treatment groups. Meanwhile, the expression of Flt-1 and Flk-1 protein increased in all the treatment groups (P<0.05), and was the most in the acupuncture-rehabilitation group (P<0.05). Conclusion Acupuncture-rehabilitation thera-py can promote the neurological function recovery in rat with permanent focal cerebral ischemia, which may be associated with the continu-ous inducement of Flt-1, Flk-1 protein expression in ischemic penumbra cortex.
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Objective To evaluate the influence Qizhu decoction on VEGF and its receptor KDR/flk-1 protein expression in MGC803 cell. Methods Four groups of MGC-803 Cells were established, and intervened with blank control, DMSO control, high dose Qizhu decoction, and low dose Qizhu decoction respectively. VEGF and KDR/flk-1 protein were detected by flow cytometry and western bloting. Results The value of VEGF expression was 120.0±10.8, 116.8±14.7, 95.0±12.5, and 108.4±13.5 respectively in each group. The difference between high dosage Qizhu decoction group and the bland control group was significant. Value of KDR/flk-1 were 10.4±3.5, 9.0±3.4, 6.8±2.3, and 6.8±3.5 in each group respectively. There was no significant difference among these 4 groups. The grayseale values of VEGF expression was 14.45±4.61, 12.32±3.27, 2.58±0.84, and 3.45±1.12 in each group respectively. There was significant difference between the QiZhu groups and the blank control group. Meanwhile grayseale values of KDR/flk-1 expression were 3.87±1.05, 3.55±1.32, 3.62±1.01, and 3.73±0.88 in each group respectively, showing no significant difference among 4 groups. Conclusion Rough extraction of QiZhu decoction down-regulated the protein expression of VEGF, but had no effects on the expression of KDR/flk-1.
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VEGF and its receptors, flk-1 and flt-1 have been characterized as critical factors in angiogenesis and neurogenesis during development. Here we investigated the expression of VEGF and its receptors in postnatal murine cerebellum in terms of time-dependency and regional distribution. Immunofluorescence staining showed that the expression of VEGF was restricted only to Purkinje cells and was increased in their processes on the postnatal development. Flk-1 was expressed in Purkinje cellular bodies on postnatal day (P) 8, 11, 18. Flt-1 was expressed in Purkinje cells on P8 but gradually disappeared in all of cerebellar layers on the postnatal development. These results suggest that VEGF may contribute to postnatal development of cerebellum via its receptors. And they suggest that changes in the expression of VEGF and its receptors related to the difference in maturation and proliferation of Purkinje cells in the cerebellum.
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Cerebellum , Fluorescent Antibody Technique , Neurogenesis , Purkinje Cells , Vascular Endothelial Growth Factor AABSTRACT
AIM: To investigate the expression of vascular endothelial growth factor receptor 2 (VEGFR-2, also known as FLK-1) in laser-induced choroidal neovascularization (CNV) in mouse.METHODS: CNV was induced in C57BL/6 mouse eyes by krypton laser photocoagulation. Choroidal fiuorescein angiography and histopathological examination were used to assess the development of experimental CNV. Cryostat sections from lesions on day 10 after laser treatment and normal eyes were prepared for Immunohistochemistry for FLK-1.RESULTS: Laser-induced CNV developed in all lesions on day 10. The expression of FLK-1 was detected in endothelial cells, retinal pigmented epithelium (RPE)-like cells and fibroblast-like cells in neovascular lesions. In normal adult mouse retinas, FLK-1 expression was mainly observed in RPE cells, inner nuclear and ganglion cell layers.CONCLUSION: Our findings demonstrated that expression of FLK-1 may play a role in the formation of laser-induced CNV in mice, which suggest that FLK-L may be a promising potential target for antiangiogenesis therapy for CNV.
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· AIM: To investigate the possibility of FLK-1 as a therapeutic agent for choroidal neovascularization (CNV) in mouse by FLK1 monoclonal antibody.· METHODS: CNV was induced in C57BL/6 mouse eyes by krypton laser photocoagulation. Preoperatively and on the 3rd, 6th and 9th day after photocoagulation, the animals were intraperitoneally injected with 500μg FLK1 monoclonal antibody. Choroidal fluorescein angiography and histopathological examination were used to assess the development of experimental CNV quantitatively 10 days after laser treatment.· RESULTS: Laser-induced CNV developed in all lesions on the 10th day after the operation. The development of CNV was significantly inhibited in experimental group, indicated by choroidal fluorescein angiography and histopathological examination (P＜0.001).· CONCLUSION: Our findings suggest that FLK-1 may be a promising agent for anti-angiogenesis therapy for CNV.
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· AIM :To investigate the role of pericytes in growth of retinal microvascular endothelial cells with a co-culture system in order to understand some mechanism of angiogenesis in hypoxia induced retinal neovascular disorders.(RMECs) were isolated by a modified protocol using CD31 coated Dynabeads, and identified by immunocytochemical staining with anti-Factor Ⅷ and CD31 antibodies. Rat retinal pericytes were isolated and characterized by immunofluorescent staining with PDGFR-β; and desmin antibodies. Pericytes and RMECs were cultured in a contact co-culture system both under normoxia and hypoxia by Millicell chamber. RMECs proliferation was evaluated by MTT and cell cycle assay with flow cytometry. RT-PCR was used to detect the alteration of KDR/Flk-1 mRNA level in RMECs under normoxia or hypoxia in the co-culture system.harvested with the modified isolating method. The two cell types were identified by positive Factor Ⅷ, CD31 and PDGFR-β, desmin cytochemical staining respectively.RMECs proliferated significantly under hypoxia from 3 to 9d with a maximal rate on day 6 (24.9%, P ＜ 0.01) by MTT. In the co-culture system, the proliferation of RMECs was inhibited by pericytes. After 6d exposure to hypoxia,the fraction of S-phase RMECs number was greatly increased by 43.9% (P ＜ 0.01). In the co-culture system,RMECs proliferation was inhibited by pericytes through decreasing the fraction of S-phase cell number both under normoxia (3.6%, P＜0.05) and under hypoxia (15.1%,P＜0.01). KDR/Flk-1 mRNA level in single cultured RMECs was shown to increase approximately 1.3-fold when exposed to hypoxia. Compared with single cultured RMECs, co-culture with pericytes could decrease KDR/Flk-1 mRNA by 45.1% (P＜0.05) and 27.7% (P ＜ 0.05) under normoxia and hypoxia condition respectively.pericytes could inhibit proliferation of RMECs under both normoxia and hypoxia. The inhibition effects of pericytes maybe, at least in part, due to downregulation of KDR/Flk-1 of RMECs. These findings confirm that pericytes could be a potential inhibitor in the pathogenesis of retinal neovascularization.
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@#ObjectiveTo observe the therapeutic benefit of administration of endothelial cells derived from rat bone marrow cells in ischemic stroke rats and to explore the related mechanism.MethodsPrepared endothelial cells from bone marrow stromal cells (BMSC) of rats, which were multiplied and differentiated in the medium with 400ng/ml rhGM-CSF in vivo. Rats were subjected to permanent cerebral middle artery occlusion (MCAO) models(n=45). Injected intravenously via tongue vein with 3×106 endothelial cells 24 h after stroke for test groups(n=15); injected same amount PBS for control group 1(n=15); control groups without any intervention after stroke (n=15). Neurologic functional behaviour tests (postural reflex test, limb use asymmetrical test and corner test) were performed before transplantation and 1,3,5,7,14 d after stroke. Meanwhile, immunohistochemistry staining was used to identify for vascular endothelial growth factor (VEGF) and its receptor FLK-1 expression in ischemic brain tissue.ResultsSignificant recovery of neurological function was detected in rats treated with endothelial cells on the 7th day and 14th day after stroke, compared with control group 1 and group 2(P<0.05);The number of positive cells of VEGF, FLK-1 were significant more in the peri-ischemic tissue and ipsilateral cortex, compared with non-ischemic hemisphere. The maximum number of positive cells was in the test group which was treated with endothelial cells(P<0.05);VEGF was mainly expressed at neurons, glial cells and part of endothelial cells; FLK-1 was mainly expressed at endothelial cells and part of neurons and glial cells;capillary hyperplasia was demonstrated more at the ischemic hemisphere in the rats treated with endothelial cells, compared with control group 1 or 2.ConclusionEndothelial cells derived from bone marrow cells in rats could improve neurological outcome in rats with ischemic stroke. The effect starts to be significant on the 7th day after transplantation and it shows more significant effect on the 14th day. Endothelial cells transplantation will enhance VEGF, FLK-1 expression at ischemic area and increases capillary hyperplasia formation, which may relate to the potential mechanism of neurological outcome improvement post stroke in rats.
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PURPOSE: The purpose of this study is to clarify the degree of corneal neovascularization and its expression of MMP-2. 9(matrix metalloproteinase) and TIMP-1, 2(tissue inhibitor of metalloproteinases) according to the different concentrations of VEGF(vascular endothelial growth factor). METHODS: After the pellets with different amounts of VEGF(VEGF 125, 250 ng) were inserted into the corneal stroma of rat model, their degree of corneal neovascularization and the expression of MMP-2, 9 and TIMP-1, 2 were compared with those of control group where pellets were filled with phosphate-buffered saline. RESULTS: At the 7th day after the pellet insertion, degree of neovascularization was most highly scored in the group with pellet which contained the largest amount of VEGF, 250 ng, and there was statistically noticeable increase of eovascularization with the increase of VEGF amount(P<0.05). On immunohistochemical staining, as the amount of VEGF increases, not only MMP-2, 9 and flk-1, but also TIMP-1, 2 were expressed more. CONCLUSIONS: In conclusion, when it comes to neovascularization, MMP-2, 9, which induces angiogenesis, as well as its inhibitor TIMP-1, 2 are increased to maintain the homeostasis of the cornea.
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Cornea , Corneal Neovascularization , Corneal Stroma , Homeostasis , Models, Animal , Tissue Inhibitor of Metalloproteinase-1 , Vascular Endothelial Growth Factor AABSTRACT
Objective To study the anti-agiogenesis effect of Taohong Siwu Decoction Ⅱ(THSDⅡ)and to explore its mechanism.Methods Chicken Chorioallantoic Membrane(CAM)assay was used to study the anti-agiogenesis action of THSDⅡin vivo.MTT assay was used to investigate its effect on the proliferation of human umbilical vein vascular endothelial cells ECV304 in vitro.Immunohistochemistry assay was used to observe its effects on the expression of KDR/FLK-1 in endothelial cells and the amount of micro-vessel density(MVD)in mice with B16 melanoma.Results THSDⅡ at the dosages of 1 g / mL and 2 g / mL could obviously inhibit agiogenesis in the CAM(P
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AIM: This study was designed to investigate the secretion of VEGF and its receptor (flt-1 or flk-1/KDR) protein by cultured bovine thoracic aortic endothelial cells treated with various insulin concentrations. METHODS: Endothelial cells was isolated from bovine thoracic aorta, and cultured in serum-free medium, then incubated with different insulin concentrations (30 mU/L, 300 mU/L, 3 000 mU/L). The level of VEGF and its receptor (flt-1 or flk-1/KDR) protein were detected by immunohistochemical staining. RESULTS: As compared with no insulin group, the expression of VEGF protein in low insulin concentration (30 mU/L and 300 mU/L) groups were significantly increased (P0.05) was observed. CONCLUSION: Low concentration insulin up-regulates the VEGF protein expression, while high concentration insulin down-regulates the VEGF protein expression in bovine thoracic aortic endothelial cells, but insulin had no directly effect on the VEGF receptor (flt-1 or flk-1/KDR) protein expression in bovine thoracic aortic endothelial cells. [
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Purpose:To investigate the effect of Flk-1、LR P and MDR1 genes in the carcinogenesis and development of lung cancer. Methods:The expression of Flk-1、LRP and MDR1 geneproteins in primary lung tumors were studied immunohistochemically. Results:of the 70 lung cancers, 29 cases (49.2%) were positive for MDR1 expression in NSCLCs and 2cases (18.2%) in small cell lung carcinoma(S CLC); 41cases (69.5%)had overexpression of LRP in NSCLCs and 3cases (27.3%) in S CLCs, there was a statistically significant correlation (P
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The aim of this study was to evaluate the role of vascular endothelial growth factor (VECF) and its receptor flk-1 system in angiogenesis and progression of human hepatocellular carcinoma (HCC) in vivo, the flk-1 dominant-negative mutant was cloned into the retroviral expression vector pLXSN, and was then packaged by PA317 cells to produce ecotropic retroviruses expressing mutated receptor constructs. 50mm_3 of LCI D-20 intact tissue were subcutaneously or orthotopically implanted in nude mice respectively. At day 1,3,5 after implantation, growing tumor were injected with 0.1 ml retroviral supematants into the site of tumor implantation. Tumor volumes, vascular density and lung metastasis were investigated. The results showed that the transfectants by flk-1 TM formed very small tumors after 21 days, which were less than 10 folds in size compared with control. There were hardly visible vessels in flk-1 TM transfected tumor tissue, whereas rich neovascularization could be found in control. The metastatic nodules in lungs were markedly decreased by dominant-negative flk-l TM (P
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Objective To explore the effect of Buyang Huanwu Decoction (BYHWD) on vascular endothelial growth factor (VEGF) and its receptor, Fetal liver kinase 1 (Flk1) in rats after focal cerebral ischemia. MethodsRats were randomly divided into following groups including Sham group, model group, Nimodipine group, and BYHWD group. The model of focal cerebral ischemia in rats was repro-duced by middle cerebral artery occlusion. Rats were ig administered and killed after operated, then the expression of VEGF and Flk1 and protein level of VEGF in brain tissue of every groups were measured by immunohistochemisty and enzyme linked immunosorbent assay, and the nervous function deficit scores were evaluated. ResultsThere were a few VEGF and Flk1 positive cells in normal brain tissue of rats. After focal cerebral ischemia the VEGF and Flk1 positive cells were increased. Compared to model group, Nimodipine and BYHWD significantly improved the neurological behavior performance, increased the numbers of VEGF and Flk1 positive cells, and enhanced the VEGF protein level (P
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0 05) between the insulin incubation groups both with and without L NAME incubations Conclusion Insulin has no direct effect on the expression of VEGF receptor (flt 1? flk 1/KDR) protein in bovine thoracic aortic endothelial cells The NOS 3 activation of endothelium is not the main cause that could affect the expression of VEGF receptor (flt 1?flk 1/KDR) protein of endothelium
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Objective To construet a novel DNA vaccine pcDNA3.1+/flk-1_((n1-4)) against tumor angiogenesis.Methods flk-1_((n1-4)) cDNA amplified by RT-PCR was inserted into eucaryotic expression vector pcDNA3.1+ and the recombinant plasmids pcDNA3.1+/flk-1_((n14)) was constructed.Confirmed by DNA sequence analysis,the recombinant plasmids was transfected into eucaryotic expression system COS-7 cells by lipofectamine.The protein expression of flk-1_((n1-4)) was detected by Western blot. Results Extracellular loop 1-4 of flk-1 was cloned and the recombinant plasmid was successfully constructed,which has been proved by DNA sequencing and compared with the data in Gene Bank.The protein expression of flk-1_((n1-4)) was detected in eucaryotic expression system COS-7 cells.Conclusions The DNA vaccine against tumor angiogenesis was successfully constructed,which found a basis for the further study on animal experiment and clinical application.
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AIM: We observed the expression of KDR/Flk1 in post-myocardiac ischemia(MI) SD rats in order to explain the effect of captopril and its relationship with myocardium angiogenesis after long-term administration.METHODS: The MI model was made by LAD ligation.Captopril was administered for 6 weeks.Immuohistological method and FQ-PCR were used to test the myocardium KDR/Flk-1 expression.RESULTS: In captopril group,no inhibitory effect was observed in myocardium factor VIII expression,but KDR/Flk-1 decreased.The copies of KDR/Flk-1 mRNA reduced dramatically when compared to control group,false-operation and normal group(P0.05).CONCLUSION: ACEI down-regulates KDR/Flk-1 and its mRNA expression in ischemic rat myocardium after long-term administration of captopril,but does not inhibit angiogenesis.So we suspect that some other pathways exist,which can not affect by ACEI,or that ACEI just reduces abnormal angiogenesis.
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Objective To study the changes of expression levels of vascular endothelial growth factor(VEGF) and its receptors(FLT-1,FLK-1) mRNA in equal area of penumbra in diabetes rats with cerebral infarction(DMCI).Methods Middle cerebral artery occlusion(MACO) model was made in diabetes rats.The expression levels of VEGF and FLT-1,FLK-1 mRNA in equal area of penumbra were measured with situ hybridization in different times after MACO(1 h,3 h,6 h,12 h,24 h).The difference was compared between DMCI group and no diabetes cerebral infarction(NDMCI) group.Results The expression level of VEGF mRNA was lower in DMCI group than that of NDMCI group at every time point(P