Flt-1 mRNA Research Articles

Expression of FLT4 in hypoxia-induced neovascular models in vitro and in vivo.

Toinvestigatetheexpression of FLT4in retina with oxygen induced retinopathy (OIR) and in brain endothelial cell lines (bEnd3) under hypoxia conditions in mice. Fifty-two one-week-old C57BL/6J mice were divided into control group and hypoxia group. The mice of hypoxia group were exposed to 75% oxygen for 5 days and then returned to the room air to induce retinal neovascularization. Mice in control group were raised in the environment of room air at the same time. The expressions of FLT4 mRNA and protein were checked with RT-PCR and Western Blot analysis at postnatal day 14, 17 and 21 (P14, P17 and P21) respectively. 125mmol/L CoCl(2) were added to the culture medium of bEnd3 cell, proteins were extracted in 12, 24, 48 and 72 hours and FLT4 levels were examined by Western Blot analysis. The mRNAand protein level ofFLT4expressedinP14 and P17 OIR mice retina statistically up-regulated as compared with those in control group, but there was no statistical difference between OIR group and control group at P21. FLT4 levels increased significantly in 12, 24 and 48 hours hypoxia intervened bEnd3 cells, its levels in 72 hours increased mildly but showed no significance. FLT4 levels increase in OIR mice retinas and bEnd3 cells in hypoxia. It may play an important role in endothelial cells proliferation in hypoxia and retinal neovascularization in OIR mice.

Jumonji domain-containing protein 6 (Jmjd6) is required for angiogenic sprouting and regulates splicing of VEGF-receptor 1

JmjC domain-containing proteins play a crucial role in the control of gene expression by acting as protein hydroxylases or demethylases, thereby controlling histone methylation or splicing. Here, we demonstrate that silencing of Jumonji domain-containing protein 6 (Jmjd6) impairs angiogenic functions of endothelial cells by changing the gene expression and modulating the splicing of the VEGF-receptor 1 (Flt1). Reduction of Jmjd6 expression altered splicing of Flt1 and increased the levels of the soluble form of Flt1, which binds to VEGF and placental growth factor (PlGF) and thereby inhibits angiogenesis. Saturating VEGF or PlGF or neutralizing antibodies directed against soluble Flt1 rescued the angiogenic defects induced by Jmjd6 silencing. Jmjd6 interacts with the splicing factors U2AF65 that binds to Flt1 mRNA. In conclusion, Jmjd6 regulates the splicing of Flt1, thereby controlling angiogenic sprouting.

Expression of placental growth factor in regenerating livers after partial hepatectomy in the rat

Placental growth factor (PlGF) is known for its role in pathological conditions to protect parenchymal cells of different organs from injury, whereas its presence in the liver and its potential importance in stimulating liver regeneration has never been described. This was investigated in this study using a rat model of partial hepatectomy (PH). The rat model of 70, 80, and 90% PH was used. Liver samples were taken peroperatively, 1 h, 1, 2, 3, and 7 days after surgery. Liver regeneration was evaluated by liver weight/body weight ratio, liver regeneration rate (%), and proliferating cell marker Ki67. The expression of PlGF, vascular endothelial growth factor (VEGF), VEGF receptor 1 (Flt-1), VEGF receptor 2 (Flk-1), and hypoxia inducible factor-1α mRNA was measured by quantitative real-time PCR and localized by immunohistochemistry. The mRNA expression of PlGF was upregulated immediately after PH. Compared with 70 and 80% PH groups, the 90% PH group had a significantly lower PlGF and hypoxia inducible factor-1α mRNA expression, in parallel to a delayed liver weight/body weight ratio recovery. Only little differences were observed in VEGF, Flt-1, and Flk-1 mRNA expression among the PH groups. This study shows for the first time the PlGF upregulation in regenerating livers, which is related to hypoxia stimulation and liver growth. The swift PlGF upregulation immediately after PH may indicate an important role for the PlGF/Flt-1 pathway in the early stage of liver regeneration.

Effects of vascular endothelial growth factor on porcine preimplantation embryos produced by in vitro fertilization and somatic cell nuclear transfer

This study examined the effects of vascular endothelial growth factor (VEGF) on porcine embryos produced by in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) at different developmental stages. Four sets of experiments were performed. In the first, supplementation of the in vitro culture medium with 5 ng/mL VEGF was suitable for porcine IVF embryo development, and the blastocyst formation rate was significantly higher than the control and other groups (57.73 ± 6.78% (5 ng/mL VEGF) vs. 43.21 ± 10.22% (control), 42.16 ± 10.24% (50 ng/mL VEGF) and 41.91 ± 11.74% (500 ng/mL VEGF); P < 0.05). The total cell number after supplementation with 5 ng/mL VEGF was significantly higher than the control and other groups (151.85 ± 39.77 (5 ng/mL VEGF) vs. 100.00 ± 34.43 (control), 91.2 ± 31.51 (50 ng/mL VEGF), and 112.53 ± 47.66 (500 ng/mL VEGF); P < 0.05). In the second experiment, when VEGF was added at different developmental stages of IVF derived embryos (early stage, days 1–3, late stage, days 4–7), the blastocyst formation rate and total cell number were significantly higher at the late stage (47.71 ± 9.13% and 131.5 ± 20.70, respectively) than in the control (34.32 ± 7.44% and 85.50 ± 20.41, respectively) and at the early stage (33.60 ± 5.78% and 86.75 ± 25.10, respectively; P < 0.05). There was no significant difference in the blastocyst development rate or total cell number between the whole culture period (days 1–7) and the late stage culture period after supplementation with 5 ng/mL VEGF (P > 0.05). In the third experiment, the cleavage rate was significantly higher when SCNT embryos were cultured with VEGF during the whole culture period than in the late stage (63.56 ± 15.52% vs. 39.72 ± 4.94%; P < 0.05), but there was no significant difference between the control and the early stage culture period (P > 0.05). The blastocyst formation rate was significantly higher at the late stage culture period with VEGF than at the early stage culture period (34.40 ± 15.06% vs. (16.07 ± 5.01%; P < 0.05). There was no significant difference in the total cell number between the groups (P > 0.05). In experiment 4, using real-time PCR, VEGF mRNA expression was detected in all the developmental stages of IVF and SCNT embryos, but the expression level varied according to the developmental stage. VEGF receptor, KDR mRNA was detected in all stages IVF and SCNT embryos. However, flt-1 mRNA was not expressed in all embryonic stages of IVF and SCNT embryos. These data suggest that VEGF supplementation at the late embryonic developmental stage might improve the developmental potential of both IVF and SCNT preimplantation porcine embryos through its receptors.

P121 Serum cystatin C for evaluation of glomerular filtration rate in pregnancy

There is recent interest for uniform placental collection protocols so laboratories can share samples. However, concerns have been raised a wash step at collection causes significant loss of sFlt-1 and soluble endoglin, among the most studied proteins in placentology. We measured Flt-1 and endoglin mRNA and protein in 10 preeclamptic placentas that were washed, or left unwashed. Reassuringly, there was no significant change in the Flt-1, sFlt-1-e15a or sFlt-1-i13 mRNA or Flt-1 or sFlt-1 protein expression or localization. There was also no change in endoglin mRNA expression or protein localization. Washing preeclamptic placental samples does not alter anti-angiogenic factor expression.

Vascular endothelial growth factor, fms‐like tyrosine kinase‐1 (Flt‐1) and soluble Flt‐1 gene expressions in Korean pre‐eclamptic placentas

To assess the expressions of vascular endothelial growth factor (VEGF), fms-like tyrosine kinase-1 (Flt-1), and soluble Flt-1 (sFlt-1) genes in healthy normotensive and pre-eclamptic placentas of Korean women. We investigated 12 healthy normotensive pregnant women and 10 pre-eclamptic pregnant women at Eulji University Hospital. The obtained placental tissues were analyzed using reverse transcription polymerase chain reaction and real-time quantitative polymerase chain reaction. The sFlt-1 messenger ribonucleic acid (mRNA) level was elevated 2.6 times more in pre-eclamptic placentas than in normal control placentas. However, the VEGF mRNA level of pre-eclamptic placentas was decreased. There was no difference in the Flt-1 mRNA level between control and pre-eclamptic placentas. Our study showed that expressions of genes relating to angiogenesis were altered in Korean pre-eclamptic placentas. These results suggest that the alteration in expressions of sFlt-1 and VEGF genes might be associated with the pathogenesis of pre-eclampsia.

Early gestational intrauterine infection induces postnatal lung inflammation and arrests lung development in a rat model

Objective. In order to investigate the early gestational inflammation effect on the prenatal and postnatal lung development, identification of the proinflammatory cytokines (IL-1β and TNF-α), genes implicated in angiogenesis (Vascular endothelial growth factor [VEGF], fms-like tyrosine kinase-1 [Flt-1], fetal liver kinase-1 [Flk-1]), and surfactant proteins (SPs) were observed.Methods. Escherichia coli (E. coli) was inoculated into uterine cervix of pregnant rats at embryonic day 15 (E15) during pseudoglandular period of lung development and the control group was inoculated with normal saline. IL-1β, TNF-α, VEGF, Flt-1, Flk-1, SP-A, and SP-B mRNA in pup's lung at E17, 19, 21 and postnatal day (P) 1, 3, 7, 14 were quantified by real-time RT-PCR. Western blot or immunohistochemistry analysis was also performed for the evaluation of VEGF, Flk-1, Flt-1, and SP-A expression in pup's lung.Results. Compared with the control group, the fetal lung of the E. coli-treated group was more immature, the postnatal lung development was impaired marked by less alveoli, fewer secondary septa, and thicker alveolar wall. The lung weight and lung/body weight ratio were lower in the E. coli-treated group pups. IL-1β and TNF-α mRNA were increased significantly in E. coli-treated pup's lung after birth, but no significant difference of IL-1β and TNF-α mRNA levels in fetal lung were found between the two groups. SP-A expression was depressed at E17, E19, and E21 after intrauterine E. coli treated, accompanied with lower SP-B mRNA level at E19 and E21. Furthermore, intrauterine E. coli treated reduced the VEGF mRNA and protein levels in the fetal lung at E17 and E19, while the expression of Flt-1 and Flk-1 were higher at P7, P14 and P1, P7, P14, respectively, compared to the controls.Conclusions. These results suggested early gestational intrauterine E. coli infection could induce a postnatal pulmonary inflammation and might arrest the alveolarization in developing lung which was involved with the VEGF signaling. However, intrauterine E. coli infection could not induce the increase of proinflammatory cytokines in fetal lung and might fail to accelerate the maturation of fetal lung.

Hypoxia Inducible Factor-1α (HIF-1α) and Some HIF-1 Target Genes are Elevated in Experimental Glaucoma

Low levels of hypoxia have been suggested to be a mechanism of retinal damage in glaucoma. To test the hypothesis that the activation of the hypoxia-responsive transcription factor hypoxia inducible factor-1alpha (HIF-1alpha) is involved in the pathophysiology of glaucoma, we used a rat model of glaucoma to study (1) HIF-1alpha retinal protein levels by immunoblot analysis, (2) cellular localization of HIF-1alpha in the retina by immunohistochemistry, and (3) expression of retinal HIF-1 gene targets by quantitative real-time polymerase chain reaction. Glaucoma was unilaterally induced in rats by injecting hypertonic saline in episcleral veins. We find that HIF-1alpha protein was increased in the retina following elevation of intraocular pressure, specifically in Müller glia and astrocytes but not in activated microglia. Eight established HIF-1 target genes were measured in experimental glaucoma. Retinal Epo, Flt-1, Hsp-27, Pai-1, and Vegfa mRNA levels were increased and Et-1, Igf2, and Tgfbeta3 levels were decreased in the glaucomatous retinas. Thus, the increase in HIF-1alpha levels in Müller glia and astrocytes is accompanied by a marked up regulation of some, but not all, HIF-1 transcriptional targets. These data support the hypothesis that HIF-1alpha becomes transcriptionally active in astrocytes and Müller cells but not microglia or neurons in glaucoma, arguing against a global hypoxia stimulus to the retina.

PTEN Regulates VEGF, VEGFR1 Expression and Its Clinical Significance in Myeloid Leukemia.

Abstract 1001Poster Board I-23Phosphatase and tensin homology deleted on chromosome ten (PTEN) as a novel tumor suppressor gene, plays an important role in regulating proliferation, apoptosis, invasion and migration of many cancer cells. PTEN also modulates angiogenesis mediated by vascular endothelial growth factor (VEGF) via down-regulating the activity of PI3K/Akt pathway in many solid tumors. However, in myeloid leukemia, the effects of PTEN on VEGF and VEGFR1 (FLT1) mediated angiogenesis, migration, invasion of leukemia cells and its clinical significance are still unknown. Therefore, in the present study, we investigated the effect of PTEN on the activity of PI3K/Akt and VEGF/FLT1 pathways. Wild type PTEN gene was transfected into K562 cells, a cell line establish from a chronic myelogenous leukemia in blast crisis, to induce high expression of wild-type PTEN gene and protein by the cells. The correlation between the expression levels of PTEN and VEGF/FLT1 and its clinical significance in myeloid leukemia patients were also observed. We found that the expression reconstitution of wild-type PTEN had significance effect on inhibiting proliferation, migration and invasion ability of K562 cells via down-regulation of Akt phosphorylation and inhibition of VEGF/FLT1 expression. In myeloid leukemia patients, a negative correlation was found between the expression level of PTEN mRNA and that of VEGF and FLT1 mRNA. Low expression of PTEN mRNA and high expression of VEGF and FLT1 mRNA indicated a higher tendency of extramedullary disease in acute myeloid leukemia patients. Taken together, our findings indicated that PTEN could modulate the function of VEGF/VEGFR signaling pathway via down regulating Akt phosphorylation and that PTEN would be a candidate target for the treatment of myeloid leukemia. Disclosures:Pan:Nature science foundation of Hebei Province: Research Funding; Research Fund for the Doctoral Program of Higher Education of China: Research Funding; Emphases follow up pregram of Health Bureau of Hebei Province: Research Funding.

Flt-1 expression influences apoptotic susceptibility of vascular smooth muscle cells through the NF-κB/IAP-1 pathway

Flt-1 is an fms-like tyrosine kinase receptor which binds to vascular endothelial growth factor (VEGF) and placental growth factor (PlGF). Ligand activation and blocking of flt-1 influence several vascular smooth muscle cell (SMC) functions, including apoptotic susceptibility. However, downstream signal transduction pathways by which flt-1 regulates SMC apoptosis have still to be investigated. Flt-1 expression and apoptosis in Wistar rat aortic intimal cells 15 days after ballooning were studied by immunohistochemistry, cytometry, cell sorting, western blotting, and PCR. Anti-flt1 blocking antibody effects were compared with those of anti-PlGF and anti-VEGF antibodies. Rat aortic intimal cells 15 days after injury exhibited increased flt-1 protein and mRNA and lower smooth muscle markers compared with normal media SMCs. Immunoreactivity for flt-1 protein was also observed in apoptotic intimal cells. Anti-flt-1 (EC(50) = 16.5 ng/mL) and anti-PlGF (EC(50) = 20.5 ng/mL) antibodies added to intimal cultures reduced serum-deprived apoptosis but not serum- and PDGF-BB-induced proliferation; the anti-VEGF antibody was ineffective. Sorted flt-1(+) cells were more clonogenic than flt-1(-) and whole intimal SMC populations. Increased nuclear factor-kappaB (NF-kappaB) and inhibitor of apoptosis protein-1 (IAP-1) and reduced bax levels associated with the anti-flt-1-induced increase of intimal SMC survival; the latter was prevented by NF-kappaB activity inhibitor and IAP-1 interfering RNA (RNAi). Blocking of NF-kappaB activity reduced IAP-1 expression and prevented IAP-1 RNAi effects. Increased flt-1 immunoreaction was also documented in human atheromatous lesions. Our results show that anti-flt-1 blocking reduces apoptosis through NF-kappaB and the downstream IAP-1 pathway. The close link between flt-1, PlGF, and apoptotic susceptibility of intimal SMCs suggests new potential strategies aimed at influencing post-injury arterial remodelling.

Stem cell marker expression, proliferation and apoptosis of vascular smooth muscle cells

Vascular endothelial Flt-1 and other stem cell markers are variably expressed in vascular smooth muscle cells (SMCs) during normal and pathological conditions, but their biological role remains uncertain. In normal rat aorta, rare flt-1+ and c-kit+ SMCs were detected. Fifteen days after injury, 61.8+3.8, 45.7+3% of the intimal cells resulted flt-1+ and c-kit+ and expressed low level of alpha-smooth muscle actin; CD133+ cells were 5.6+0.7%. BrDU+/flt-1+ largely predominated in the neointima, whereas BrDU+/CD133+ cells were rare. Forty-five and sixty days after injury, intimal proliferation such as BrDU+ cells was greatly reduced. After sixty days, intimal stem marker expression had almost disappeared whereas alpha-smooth muscle actin was restored. Flk-1 and Oct-4 SMC immunodection was consistently negative. In vitro, intimal cells obtained fifteen days after injury exhibited an epithelioid phenotype and increased flt-1 and c-kit protein and mRNA and low smooth muscle markers compared to spindle-shaped medial and intimal SMCs obtained after sixty days. Epithelioid clones, independently from layer of origin, were similar in stem cell marker expression. The anti-flt-1 blocking antibody added to epithelioid SMC cultures reduced serum-deprived apoptosis and migration but not PDGF-BB-induced proliferation, and increased cell-populated collagen lattice contraction. In conclusion, stem marker expression in vascular SMCs was variable, chronologically regulated and prevailed in epithelioid populations and clones; among stem markers, flt-1 expression critically regulates intimal SMC response to microenviromental changes.

Expression and role of vascular endothelial growth factor and its receptors in acute myeloid leukemia with normal cytogenetics

Objective To evaluate the expression and role of vascular endothelial growth factor (VEGF) and its receptors ( KDR and Flt1) in acute myeloid leukemia with normal cytogenetics (NC- AML). Methods The expression and abundance of VEGF and its receptors mRNA were assayed by RT- PCR and fluorescent quantitative RT-PCR (FQ-RT-PCR). Results Among 45 NC-AML patients, the positive rate of VEGF,KDR and FLt1 mRNA in the bone marrow mononuclear cells (BMMNC) were 93.3% ,75.6% and 82.2% ,respectively. However,the expression of VEGF,KDR and FLt1 mRNA in 10 health people was negative. Real time PCR confirmed the significant variation (P<0. 05) of the VEGF expression if the patient received a chemo therapy. In patients with complete remission(CR), the VEGF expression of VEGF was lower than pre-treatment but still remained relatively higher compared with 10 health people (P<0. 05). However, there was no significant difference for VEGF receptor expression, its receptors had no such significant difference. Conclusion VEGF and its receptors (KDR and Flt1) mRNAs were expressed distinctively in NC-AML ,while the VEGF shows higher levels. Thus ,the assay of VEGF expression levels is of great significance to evaluate the progression, the indication of prognosis and the monitor of micro residual disease. Key words: Acute myeloid leukemia; Normal cytogenetics; Vascular endothelial growth factorReceptor; Prognosis

Effect of extracellular signal-regulated kinase pathway on nitric oxide release by human umbilical vein endothelial cell induced by placental growth factor-1

To investigate the effect of extracellular signal-regulated kinase (ERK) pathway on nitric oxide (NO) release by human umbilical vein endothelial cell (HUVEC) induced by placental growth factor-1 (PLGF-1). During January to April 2006, 50 samples of umbilical vein blood were collected from newborns delivered by cesarean section due to intrauterine distress and abnormal fetal position. HUVEC were primarily cultured by trypsin digestion. Then the following procedures were performed: (1) Cells were identified using the morphology and VIII factor immunohistochemistry methods if the culture was satisfactory. (2) Cells were collected, and fms-like tyrosin kinase (Flt-1) protein and its mRNA expression were detected with immunoprints and RT-PCR methods. (3) The protein was extracted after cells were treated with PLGF-1 (cells were collected before the treatment and 2.5, 5, 10, 20 min after the treatment). The protein levels of ERK were determined by immunoprints. (4) The cells were cultured with serum-free culture medium containing PLGF-1 only (culture media were collected 20, 40, 160, 360, 480, 720 and 1440 min after the treatment). The quantity of NO was detected with nitrate reductase method. (5) The cells were cultured with serum-free culture medium containing PD98059, the inhibitor of mitogen-activated protein kinase (MAPK)/MEK for 60 min. Then the cells were cultured continuously with the serum-free culture medium containing PLGF-1 for 60 min. The culture media were collected. The quantity of NO was detected by nitrate reductase method. The samples were divided into treatment group and control group. Control group was exactly the same in treatment time, culture condition, and time to collect the cells as the treatment group, except that it was not treated with PLGF-1 or PD 98059. (1) By morphology and VIII factor immunohistochemistry the cultured cells were identified to be HUVEC. (2) Flt-1 mRNA and protein were expressed in HUVEC. (3) Expression of ERK protein started to increase at 2.5 min after treatment of HUVEC with PLGF-1, reaching the peak at 5 min, and decreased at 10 min. (4) In comparison with the control group, NO started to increase at 20 min after treatment of HUVEC with PLGF-1 (6.96 +/- 0.34) micromol/L, significantly increased at 40 min (9.45 +/- 0.59) micromol/L, and arrived at the peak at 480 min (15.82 +/- 0.69) micromol/L Comparison between the two groups showed a significant difference (P<0.05). (5) Release of NO from the cells treated with PD98059 for 1 hour and PLGF was significantly inhibited, compared with the cells treated with PLGF-1 only. ERK pathways play an important role in NO release by HUVEC induced by PLGF.

Effect of prostaglandin E2 and tumor necrosis factor α on the VEGF‐Receptor system expression in cultured porcine luteal cells

The purpose of the present study was to investigate the effects of prostaglandin (PG) E(2) and tumor necrosis factor (TNF) alpha on expression of vascular endothelial growth factor (VEGF) and its receptors, fms-like tyrosine kinase (Flt-1) and fetal liver kinase-1/kinase insert domain-containing receptor (Flk-1/KDR), in cultured porcine luteal cells. Real-time PCR was used for quantification of VEGF and its receptors mRNA, whereas VEGF release by luteal cells was determined by radioimmunoassay (RIA). Only the highest dose of PGE(2) (1 microM) after 6 hr of incubation stimulated VEGF release by luteal cells collected in the mid-luteal phase (P < 0.05). Moreover, PGE(2) (100 nM, 1 microM) significantly stimulated VEGF secretion by luteal cells in the late phase and during pregnancy on Days 10-12 (P < 0.05). Elevated mRNA expression of both VEGF 120 and VEGF 164 isoforms was found in luteal cells cultured with PGE(2). The lack of an effect of PGE(2) on VEGF receptors mRNA expression was observed. TNFalpha was able to significantly stimulate VEGF release from cells obtained in the mid- and late luteal phase or during early pregnancy. All tested doses enhanced mRNA levels of VEGF 120 isoform, but not VEGF 164. Additionally, TNFalpha was able to decrease Flk-1/KDR mRNA expression, whereas Flt-1 mRNA levels were not affected. These results indicated that PGE(2) and TNFalpha influenced VEGF ligand-receptor system expression in porcine luteal cells and may therefore play an important role in regulation of luteal functions during the estrous cycle and pregnancy in pigs.

Distribution of VEGF and flt-1 in the Normal Dog Tissues

Angiogenesis is essential for tumor progression and is regulated by several angiogenic factors such as vascular endothelial growth factor (VEGF). We investigated the expression and distribution of VEGF and its receptor flt-1 in twelve normal canine tissues in six beagle dogs using immunohistochemistry, RT-PCR and real-time RT-PCR. Immunochemical staining showed that both VEGF and flt-1 were expressed in many tissues and their mRNAs were detected in all organs examined by RT-PCR. Levels of VEGF164 and flt-1 mRNA expression were high in tissues containing many intensely immunopositive cells. The expression levels of VEGF164 and flt-1 mRNA tended to be similar. These results indicated that VEGF and flt-1 are closely associated in canine, as in human tissues, and quantifying their mRNAs might be helpful in evaluating angiogenesis.

Expression of Vascular Endothelial Growth Factor Receptors in Bovine Cystic Follicles

Cystic follicles have excess fluid derived from blood flow in the theca interna of the follicle; therefore, the vasculature network is related to cystic follicle formation. Vascular endothelial growth factor (VEGF) is a potent stimulator of blood vessel permeability and angiogenesis. The aim of this study was to examine the expression of VEGF receptors proteins and mRNA in cystic follicles to elucidate the VEGF system in cystic follicles. The expression of protein for VEGF receptors; fms-like-tyrosine kinase-1 (Flt-1) and foetal liver kinase-1 (Flk-1) was detected by the immunohistochemical method. The mRNA expression of Flt-1 and Flk-1 in cystic follicles was determined by RT-PCR. Concentration of oestradiol-17beta and progesterone in the follicular fluid of cystic follicles was determined using ELISA. Flt-1- and Flk-1 proteins were localized in granulosa and theca interna cells and endothelial cells of theca layers. The intensity of Flt-1 and Flk-1 immunoreaction was similar among cystic follicles with various ratios of oestradiol-17beta/progesterone concentrations. The expression of Flt-1 and Flk-1 mRNA was similar, regardless of the ratio of oestradiol-17beta to progesterone in follicular fluid. These results demonstrate that cystic follicles have both VEGF receptors in the granulosa and theca interna layers, which may be responsible for the increased permeability of microvessels, causing the accumulation of follicular fluid in cystic follicles.

Upregulation of Vascular Endothelial Growth Factor Receptors Flt-1 and Flk-1 Following Acute Spinal Cord Contusion in Rats

To investigate the possible role of vascular endothelial growth factor (VEGF) in the injured spinal cord, we analyzed the distribution and time course of the two tyrosine kinase receptors for VEGF, Flt-1 and Flk-1, in the rat spinal cord following contusion injury using a weight-drop impactor. The semi-quantitative RT-PCR analysis of Flt-1 and Flk-1 in the spinal cord showed slight upregulation of these receptors following spinal cord injury. Although mRNAs for Flt-1 and Flk-1 were constitutively expressed in neurons, vascular endothelial cells, and some astrocytes in laminectomy control rats, their upregulation was induced in association with microglia/macrophages and reactive astrocytes in the vicinity of the lesion within 1 day in rats with a contusion injury and persisted for at least 14 days. The spatiotemporal expression of Flt-1 in the contused spinal cord mirrored that of Flk-1 expression. In the early phase of spinal cord injury, upregulation of Flt-1 and Flk-1 mRNA occurred in microglia/macrophages that infiltrated the lesion. In addition, the expression of both receptors increased progressively in reactive astrocytes within the vicinity of the lesion, predominately in the white matter, and almost all reactive astrocytes coexpressed Flt-1 or Flk-1 and nestin. These results suggest that VEGF may be involved in the inflammatory response and the astroglial reaction to contusion injuries of the spinal cord via specific VEGF receptors.

EXPRESSIONS OF HYPOXIA-INDUCIBLE FACTOR-1 alpha, VASCULAR ENDOTHELIAL GROWTH FACTOR AND ITS RECEPTORS IN BOVINE ENDOMETRIUM THROUGHOUT THE ESTROUS CYCLE

Hypoxia-inducible factor (HIF)-1α is known to be expressed and function specifically under hypoxic conditions. Blood flow to the bovine uterus has been shown to decrease starting at estrus, which might induce hypoxia. In bovine endometrium, HIF-1α protein expression tended to increase at the follicular and estrous stage and decrease thereafter, but the changes were not significant. Vascular endothelial growth factor (VEGF) participates in the regulation of endometrial function and its expression is strongly activated by HIF-1 (α and β). VEGF has four isoforms (121, 165, 189, 201) and two receptors (Flt-1, Flk-1). In the present study, we determined VEGF165 protein content and mRNA expressions of all isoforms and receptors in bovine endometrium throughout the estrous cycle. VEGF165 protein content was greatest at the early luteal stage, and decreased thereafter.VEGF121 and VEGF165 mRNA were highest at estrus, and decreased thereafter. Flt-1 mRNA expression was less at estrus, the early and developing luteal stages than at the other stages, while Flk-1 mRNA expression was higher at the mid and late luteal stages than at the early luteal stage. Furthermore, the ratio of Flt-1: Flk-1 mRNA was highest at estrus, and decreased thereafter. In bovine endometrial epithelial and stromal cells, VEGF mRNA expressions were higher when the cells were cultured under 3% O2 than when they were cultured under 20% O2. The overall findings suggest that the endometrial VEGF is regulated not only by HIF-1α, i.e. by hypoxic condition, but also by other factors, and that this growth factor locally plays a role in bovine endometrium throughout the estrous cycle. (poster)

Vascular endothelial growth factor increases pulmonary vascular permeability in cystic fibrosis patients undergoing lung transplantation☆☆☆

Vascular endothelial growth factor (VEGF) is the prime regulator of angiogenesis and vascular permeability and its serum levels increase in cystic fibrosis (CF). The mechanisms of VEGF overproduction and its impact on CF lung pathology and pulmonary vascular permeability during lung transplantation are not fully understood. The expression of VEGF, its receptors, hypoxia inducible factor (HIF)-1alpha, beta, angiopoietins, and endothelial cell marker CD31 were studied in lung biopsies of CF and COPD patients and controls, using real time reverse transcription (RT)-PCR and Western blotting. DNA binding activity of HIF-1 to VEGF-A promoter was assessed by electrophoretic mobility shift assay (EMSA) and wet-to-dry lung weight ratios as well as microvascular density (MVD) were determined. Serum VEGF-A concentrations in enzyme-linked immunosorbent assay (ELISA) and wet-to-dry weight ratios of donor lungs were monitored during transplantation in CF and COPD patients. Primary graft dysfunction (PGD) was diagnosed and graded according to the guidelines of the International Society for Heart and Lung Transplantation. VEGF-A165 and Flt-1 mRNA expression (P<0.05), VEGF-A (P<0.05), and HIF-1alpha (P<0.05) protein levels, DNA binding activity of HIF-1 to VEGF promoter (P<0.001) and extravascular lung water content (P<0.05) were increased in CF lungs versus controls, whereas MVD was unchanged. Before and during lung transplantation, VEGF-A serum concentrations were higher in CF versus COPD patients (P<0.05) and 60 min following reperfusion donor lungs transplanted to CF patients had higher tissue water contents than in COPD patients (P<0.05). PGD grade 3 occurred more frequently in CF (22.7%) versus COPD patients (4%). PGD grade 3 patients had significantly higher VEGF serum concentrations versus PGD grade 0-2 patients (P<0.001). These data indicate that upregulated VEGF-A levels are most likely induced by enhanced HIF-1 binding to VEGF-A promoter, possibly contributing to elevated serum VEGF-A levels in CF. Furthermore, CF patients undergoing lung transplantation are possibly more susceptible to PGD because of increased VEGF-A expression that mediates increased lung graft vascular permeability.

Upregulation of Vascular Endothelial Growth Factor Receptors Flt-1 And Flk-1 in Rat Hippocampus after Transient Forebrain Ischemia

This study characterizes the distribution of the two tyrosine kinase receptors for vascular endothelial growth factor (VEGF), Flt-1 and Flk-1, in the rat hippocampus following transient forebrain ischemia. The semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of Flt-1 and Flk-1 in hippocampal CA1 showed upregulation of these receptors following ischemic injury. Expression of Flt-1 and Flk-1 mRNA was restricted to neurons in the pyramidal cell and granule cell layers in control animals; however, upregulation was detected in activated glial cells and in the vascular endothelial cells rather than in neurons, in ischemic hippocampi. Most of the activated glial cells expressing Flt-1 and Flk-1 were reactive astrocytes, although some were microglial cells. The spatiotemporal expression of Flt-1 in the ischemic hippocampus mirrored that of Flk-1 expression. Expression of mRNA for both receptors was induced after 12 h, appeared to be increased progressively until 3 days when the highest expression was reached, and was sustained for more than 2 weeks. Flt-1 and Flk-1 immunoreactivity in the ischemic hippocampus matched the mRNA induction patterns except for a somewhat delayed onset. These data suggest that VEGF may be involved in the glial response via specific VEGF receptors in the rat hippocampus following transient forebrain ischemia.