Endothelial Growth Factor Receptors Flt-1 Research Articles

SFlt‐1 Splicing Regulation by U2AF65 and JMJD6 in Endothelial Cells

The obstetric disorder preeclampsia (PE) is a condition presenting with new onset hypertension and other co‐morbidities such as proteinuria and endothelial dysfunction. Women with preeclamptic pregnancies have significantly increased circulating levels of the soluble vascular endothelial growth factor (VEGF) receptor Flt‐1 (sFlt‐1). Acting as a VEGF antagonist, which is crucial for the growth and proliferation of endothelial cells, sFlt‐1 causes decreased bioavailability of VEGF. The mechanism regulating the alternative splicing of full length Flt‐1 to the soluble variant are not understood, though U2AF65 and JMJD6 are two proteins which have been implicated in splicing event. Previous research has suggested that U2AF65 promotes the splicing of sFlt‐1, while JMJD6 antagonizes splicing by marking U2AF65 for degradation. Using human umbilical vein endothelial cells (HUVECs), we tested the hypothesis that U2AF65 and JMJD6 were both necessary for alternative splicing in endothelial cells. siRNA was utilized to knock down both U2AF65 (1± 0.012 Fold vs 0.136± 0.017 Fold; p<0.001) and JMJD6 (0.87±0.17 Fold vs 0.077 ± 0.019 Fold; p<0.001) when normalized to β‐actin. Surprisingly, when we measured sFlt‐1 variants 2 and 3, the two most common variants in humans, we found no difference in sFlt‐1 expression. Additionally, we measured expression of the full length Flt‐1, and similarly saw no changes. Next, we overexpressed both U2AF65 and JMJD6 and assessed levels by qRT‐PCR. We observed a trend for ~2‐fold increase in U2AF65 200% (0.85 ± 0.20 Fold vs 1.899 ± 0.39 Fold; p=0.22) though this did not reach significance. Interestingly, in addition to significantly overexpressing JMJD6 (1.27 ± 0.27 Fold vs 2.40 ± 0.31 Fold; p<0.01), cells overexpressing JMJD6 also had significantly higher expression of U2AF65 (0.85 ± 0.20 Fold vs 2.63 ± 0.30 Fold; p<0.05). We measured expression of the FLT1 gene, with no changes seen in the expression of Flt‐1 or sFlt‐1 version 2. The sFlt‐1 variant 3, however, was significantly higher in cells overexpressing JMJD6 (1.008 ± 0.09 Fold vs 1.56 ± 0.14 Fold; p<0.05). Despite the message levels remaining unchanged, sFlt‐1 protein was affected by U2AF65 and JMJD6, with strong trends toward decreased sFlt‐1 in with knock down of U2AF65 (5066 ± 644.3 pg/mL vs 3231 ± 193.5 pg/mL; p=0.067) and JMJD6 (5066 ± 644.3 pg/mL vs 3447 ± 605 pg/mL; p=0.09). Cells which had expression of both U2AF65 and JMJD6 knocked down exhibited an additive effect of sFlt‐1 protein decrease (5066 ± 644.3 pg/mL vs 2166 ± 63.6 pg/mL; p<0.01). Similar, though non‐significant, trends of a positive correlation were seen in cells overexpressing U2AF65 and JMJD6 having slightly increased sFlt‐1 in the media. Because the message levels did not differ greatly among the transformed cells, we looked at heparanase (HPSE) expression as a modulator of sFlt‐1 release into the media. Though there was no change in HPSE expression in knockdown cells, significantly higher HPSE expression was seen in cells overexpressing both U2AF65 and JMJD6 (1.0 ± 0.03 Fold vs 1.97 ± 0.15 Fold; p<0.001). These data suggest that U2AF65 and JMJD6 play an important role in the release of sFlt‐1, but may not be part of the alternative splicing machinery. Future experiments will look at other potential modulators regulating both sFlt‐1 and heparanase, such as MMP‐9.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

RNAi modulation of placental sFLT1 for the treatment of preeclampsia.

Preeclampsia (PE) is a placentally-induced hypertensive disorder of pregnancy that is associated with significant morbidity and mortality to mothers and fetuses. Clinical manifestations of preterm PE result from excess circulating soluble vascular endothelial growth factor receptor FLT1 (sFLT1 or sVEGFR1) of placental origin. Here we identify short interfering RNAs (siRNAs) that selectively silence the three sFLT1 mRNA isoforms primarily responsible for placental overexpression of sFLT1. Full chemical stabilization in the context of hydrophobic modifications enables productive siRNA accumulation in the placenta (up to 7% of injected dose) and reduces circulating sFLT1 in pregnant mice (up to 50%). In a baboon PE model, single dose of siRNAs suppressed sFLT1 overexpression and clinical signs of PE. Our results demonstrate RNAi-based extra-hepatic modulation of gene expression with non-formulated siRNAs in non-human primates and establish a path toward a new treatment paradigm for patients with preterm PE.

Overexpression of Thioredoxin1 enhances functional recovery in a mouse model of hind limb ischemia

BackgroundThere is keen interest in finding nonsurgical treatments for peripheral vascular disease (PVD). Previously, we demonstrated that selective activation of Thioredoxin1 (Trx1), a 12-kDa cytosolic protein, initiates redox-dependent signaling and promotes neovascularization after ischemic heart disease. Therefore, Trx1 might possess immense potential to not only treat murine hind limb ischemia (HLI) through effective angiogenesis but also provide PVD patients with nonsurgical therapy to enhance neovascularization and improve blood perfusion. MethodsTo determine whether activation of Trx1 increases blood perfusion in HLI, two different strategies were used—gene therapy and transgenic model system. In adenoviral-mediated gene therapy, 8- to 12-wk-old mice were divided into two groups: (1) control Adeno-LacZ (Ad-LacZ) and (2) Adeno-Thiroedoxin1 (Ad-Trx1). The mice underwent surgical intervention to induce right HLI followed by injection with Ad-LacZ or Ad-Trx1, respectively. In the second strategy, we used wild-type and transgenic mice overexpressing Trx1 (Trx1Tg/+). All the animals underwent Doppler imaging for the assessment of limb perfusion followed by immunohistochemistry and Western blot analysis. ResultsSignificant increases in perfusion ratio were observed in all the Trx1 overexpressed groups compared with their corresponding controls. Expressions of heme oxygenase-1, vascular endothelial growth factor, and the vascular endothelial growth factor receptors Flk-1 and Flt-1 were increased in Trx1 overexpressed mice compared with their respective controls. Blood perfusion in the ischemic limb gradually improved and significantly recovered in Trx1Tg/+ and Ad-Trx1 groups compared with their corresponding controls. The capillary and arteriolar density in the ischemic zone were found to be higher in Trx1Tg/+ group compared with wild type. ConclusionsThe overall outcomes of our study demonstrate that Trx1 enhances blood perfusion and increases angiogenic protein expression in a rodent HLI model. These results suggest that Trx1 is a potential target for clinical trials and drug therapy for the treatment of PVD.

VEGF, Flt-1, and microvessel density in primary tumors as predictive factors of colorectal cancer prognosis.

Angiogenesis in the primary tumor is known to be necessary for tumor progression in adenocarcinomas of the colon. However, whether angiogenesis in the primary tumors of patients with colorectal cancer affects their prognosis has yet to be fully elucidated. The aim of the present study was to assess the association between selected pathoclinical parameters and overall survival of resectable colorectal cancer patients with the expression of angiogenesis-promoting factors, including vascular endothelial growth factor (VEGF) and Fms-like tyrosine kinase receptor (Flt-1), and microvessel density (MVD) in the primary tumor. VEGF and Flt-1 expression were assessed, as well as MVD (with anti-CD34) by immunohistochemistry in 139 archived primary colorectal cancer tissue samples. These results were compared with the overall survival of the patients and potential prognostic pathoclinical parameters. A higher MVD in the tumors expressing Flt-1 (P=0.04) was identified. However, there was no correlation between the pathoclinical parameters of colon cancer and Flt-1 expression, VEGF expression, or MVD in the tumor. Furthermore, the intensity of VEGF expression, Flt-1 expression and tumor MVD did not correlate with the overall survival of the patients. Therefore, although increased expression of VEGF and Flt-1 was correlated with an increased expression of MVD in the primary tumors of resectable colorectal cancer patients, these factors were not correlated with prognostic pathoclinical factors and overall survival.

Angiogenic Imbalance and Residual Myocardial Injury in Recovered Peripartum Cardiomyopathy Patients.

Recent studies suggest that angiogenic imbalance during pregnancy may lead to acute peripartum cardiomyopathy (PPCM). We propose that angiogenic imbalance and residual cardiac dysfunction may exist even after recovery from PPCM. Twenty-nine women at least 12 months after presentation with PPCM, who exhibited recovery of left ventricular (LV) ejection fraction (≥50%), were included in the study (mean age 35±6 years, LV ejection fraction 61.0±3.9%). The number of circulating endothelial progenitor cells (EPCs) and plasma levels of proangiogenic vascular endothelial growth factor and of soluble vascular endothelial growth factor receptor Flt1 (sFlt1) were measured. All patients underwent comprehensive cardiac function assessment, including tissue Doppler imaging and 2-dimensional (2D) strain echocardiography. All measurements were compared with healthy controls. Patients with a history of PPCM have significantly higher sFlt1 concentrations (median [25th-75th percentile]; 149.57, [63.14-177.89] versus 20.29, [15.00-53.89] pg/mL, P<0.001) and significantly decreased vascular endothelial growth factor/sFlt1 ratio (P=0.012) compared with controls, with a trend toward lower concentration of circulating CD34+/KDR+ levels. In addition, patients with PPCM had lower early velocities E' septal (9.9±2.1 versus 11.0±1.5 cm/s, P=0.02), with a significantly lower systolic velocity S' septal (7.6±1.2 versus 8.5±1.2 cm/s, P=0.003) by tissue Doppler imaging. Significantly lower LV global longitudinal (-19.1±3.3 versus -22.7±2.2%, P<0.001) and apical circumferential 2D strain (-16.6±4.9 versus -21.2±7.9, P=0.02) were present in patients with PPCM compared with controls. Higher concentration of sFlt1 with concomitant decreased circulating endothelial progenitor cell levels along with inappropriate attenuated vascular endothelial growth factor levels may imply an angiogenic imbalance that exists even after recovery and may thus predispose to PPCM. In addition, tissue Doppler imaging and 2D strain were able to identify residual myocardial injury in post-PPCM women with apparent recovery of LV systolic function. Both angiogenic imbalance and residual myocardial injury may play an important role in the recurrence of LV dysfunction during subsequent pregnancies.

Differentiation of nerve-derived adult pluripotent stem cells into osteoblastic and endothelial cells

Background ContextStem cell-involved tissue engineering has gained dramatic attention as a therapeutic strategy for tissue regeneration including bone repair. However, the currently available possibilities to use embryonic stem cells and induced pluripotent stem cells (iPCs) face potential ethical issues, as well as risks of malignant transformation and immune rejection. Recently identified peripheral nerve-derived adult pluripotent stem cells (NEDAPS) that quickly proliferate after exposure to bone morphogenetic protein-2 (BMP-2) or nerve trauma and exhibit many embryonic stem cell characteristics may provide an attractive source cells for a variety of regenerative therapies. PurposeThe study aimed to examine the differentiation potential of the NEDAPS cells into osteoblastic cells and endothelial cells. Study Design/SettingAn in vitro investigation was undertaken to induce mouse NEDAPS cells into the phenotypes of osteoblastic and endothelial cells. MethodsNEDAPS cells were isolated from low-dose BMP-2-exposed mouse sciatic nerves by collagenase and trypsin extraction. The cells were cultured in a stem cell maintenance medium, and the expression of KLF4, Sox2, c-Myc, and Oct4 before differentiation was confirmed. The cells were then subcultured in a complete osteogenic cell induction medium or endothelial cell growth medium, respectively, at 37°C and 5%CO2 atmosphere. Histologic, morphologic, and molecular assessments were performed 7 days later. ResultsThe cells propagated in complete osteogenic medium for 7 days showed strong staining for type I collagen and alkaline phosphatase, suggesting the structural and functional properties of the osteoblastic cells. Further, real-time polymerase chain reaction (RT-PCR) revealed a significant expression of the osteoblast markers osteocalcin, osteopontin, and type I collagen. Similarly, the cells in endothelial growth medium were successfully differentiated into cobblestone-shaped endothelial cells expressing vascular endothelial growth factor (VEGF) receptors Flk-1 and Flt-1 demonstrated by RT-PCR. ConclusionsNEDAPS cells are readily induced to osteoblastic and endothelial cells, suggesting therapeutic potential for bone repair and other regenerative therapies.

Maternal hemoglobin concentration and hematocrit values may affect fetus development by influencing placental angiogenesis

Objective: Vasculogenesis and angiogenesis are crucial for maintaining proper placental perfusion and optimal fetal development. Among other physical and chemical factors, hypoxia is known to stimulate angiogenic processes. Preplacental type of hypoxia is often associated with maternal anemia and is thought to enhance vascularization within the fetoplacental unit. The goal of this study was to establish the correlation between the local expression of vascular endothelial growth factor (VEGF) and placenta growth factor (PlGF) receptors (flt-1, flk-1) with maternal hemoglobin (Hb) concentration, hematocrit (Ht) values and the infant birthweight.Methods: In total, 43 specimens of term placentas obtained from normal course pregnancies delivered at term were included in the study. The expression of flt-1 and flk-1 receptors was analyzed by immunohistochemical staining. Vascular/extravascular tissular index (V/EVTI) was measured by assessing a total vascular area. Nonparametric Mann–Whitney U-test and Spearman’s rank correlation were used to compare the various parameters and their differences between the groups.Results: Among the patients with low Hb concentration, nearly 2-fold greater expression of the flt-1 receptor was positively correlated with infants birthweight (p = 0.028).Conclusions: Increased placental vascular density (increased flt-1 expression), during a physiological course of gestation, may be an adaptive response to lowered maternal Hb concentration and Ht values encountered during pregnancy.

Effect of different enamel matrix derivative proteins on behavior and differentiation of endothelial cells

ObjectivesEnamel matrix derivative (EMD) is an effective biomaterial for periodontal tissue regeneration and might stimulate angiogenesis. In order to clarify mechanisms underlying its biological activity, we separated two EMD fractions with different molecular weight protein components and investigated their effects on human umbilical vein endothelial cells (HUVECs) in vitro. MethodsFraction Low-Molecular Weight (LMW) included proteins with a molecular weight (M.W.)<8kDa. Fraction LMW-depleted included proteins with M.W.>8kDa and lower than approximately 55kDa. The effect of EMD fractions on proliferation/viability, apoptosis, migration and expression of angiopoetin-2 (ang-2), von Willebrand factor (vWF), E-selectin, intracellular adhesion molecules 1 (ICAM-1), vascular endothelial growth factor (VEGF) receptors Flt-1 and KDR was investigated. ResultsThe proliferation/viability of HUVECs was inhibited by both LMW and LMW-depleted at concentrations 100μg/ml, whereas EMD slightly increased cell proliferation/viability. The expression of all investigated proteins was up-regulated by EMD. However, differences in the effect of EMD fractions on the protein expression were observed. The effect of LMW-depleted on the expression of ICAM-1 and E-selectin was markedly higher compared to LMW. In contrast, the expression of vWF and VEGF receptors Flt-1 and KDR was primarily affected LMW than by LMW depleted. The expression of ang-2 was not influenced by LMW and LMW-depleted. HUVECs migration was stimulated more strongly by LMW than by EMD and LMW-depleted. ConclusionOur in vitro study shows that the proteins composing EMD have different and specific biological activities and consequently have the ability to cover different aspects of EMD's biological and clinical effects.

FOXF1 transcription factor is required for formation of embryonic vasculature by regulating VEGF signaling in endothelial cells.

Inactivating mutations in the Forkhead Box transcription factor F1 (FOXF1) gene locus are frequently found in patients with alveolar capillary dysplasia with misalignment of pulmonary veins, a lethal congenital disorder, which is characterized by severe abnormalities in the respiratory, cardiovascular, and gastrointestinal systems. In mice, haploinsufficiency of the Foxf1 gene causes alveolar capillary dysplasia and developmental defects in lung, intestinal, and gall bladder morphogenesis. Although FOXF1 is expressed in multiple mesenchyme-derived cell types, cellular origins and molecular mechanisms of developmental abnormalities in FOXF1-deficient mice and patients with alveolar capillary dysplasia with misalignment of pulmonary veins remain uncharacterized because of lack of mouse models with cell-restricted inactivation of the Foxf1 gene. In the present study, the role of FOXF1 in endothelial cells was examined using a conditional knockout approach. A novel mouse line harboring Foxf1-floxed alleles was generated by homologous recombination. Tie2-Cre and Pdgfb-CreER transgenes were used to delete Foxf1 from endothelial cells. FOXF1-deficient embryos exhibited embryonic lethality, growth retardation, polyhydramnios, cardiac ventricular hypoplasia, and vascular abnormalities in the lung, placenta, yolk sac, and retina. Deletion of FOXF1 from endothelial cells reduced endothelial proliferation, increased apoptosis, inhibited vascular endothelial growth factor signaling, and decreased expression of endothelial genes critical for vascular development, including vascular endothelial growth factor receptors Flt1 and Flk1, Pdgfb, Pecam1, CD34, integrin β3, ephrin B2, Tie2, and the noncoding RNA Fendrr. Chromatin immunoprecipitation assay demonstrated that Flt1, Flk1, Pdgfb, Pecam1, and Tie2 genes are direct transcriptional targets of FOXF1. FOXF1 is required for the formation of embryonic vasculature by regulating endothelial genes critical for vascular development and vascular endothelial growth factor signaling.

The angiogenic behaviors of human umbilical vein endothelial cells (HUVEC) in co-culture with osteoblast-like cells (MG-63) on different titanium surfaces

ObjectivesInteraction between osteogenesis and angiogenesis plays an important role in implant osseointegration. In the present study we investigated the influence of titanium surface properties on the angiogenic behaviors of endothelial cells grown in direct contact co-culture with osteoblasts. MethodsHuman umbilical vein endothelial cells (HUVECs) and osteoblast-like cells (MG-63 cells) were grown in direct co-culture on the following titanium surfaces: acid-etched (A), hydrophilic A (modA), coarse-gritblasted and acid-etched (SLA) and hydrophilic SLA (SLActive). Cell proliferation was evaluated by cell counting combined with flow cytometry. The expression of von Willebrand Factor (vWF), thrombomodulin (TM), endothelial cell protein C receptor (EPCR), E-Selectin, as well as vascular endothelial growth factor (VEGF) receptors Flt-1 and KDR in HUVECs and VEGF in MG-63 were measured by qPCR. The dynamic behavior of endothelial cells was recorded by time-lapse microscopy. ResultsProliferation of HUVECs was highest on A, followed by SLA, modA and SLActive surfaces. The expression of vWF, TM, EPCR, E-Selectin and Flt-1 in HUVECs was significantly higher on A than on all other surfaces. The expression of KDR in HUVECs grown on A surface was below detection limit. VEGF expression in MG-63 cells was significantly higher on SLActive vs SLA and modA vs A surfaces. Time-lapse microscopy revealed that HUVECs moved quickest and formed cell clusters earlier on A surface, followed by SLA, modA and SLActive surface. ConclusionsIn co-culture conditions, proliferation and expression of angiogenesis associated genes in HUVECs are promoted by smooth hydrophobic Ti surface, which is in contrast to previous mono-culture studies.

Immunohistochemical expression of vascular endothelial growth factor receptors Flt1 and KDR in the endometrium during the estrus cycle in albino rats

Background Angiogenesis is an important process in endometrial development and embryonic implantation and is regulated through vascular endothelial growth factor (VEGF); its receptors Flt1 and KDR. Aim This work aimed to study the immunoexpression of VEGF receptors (VEGF-Rs) in the endometrium at different ages and reproductive phases and correlate them with the histological profiles in these phases. Materials and methods Seventy female albino rats were included in this study. They were divided into seven groups of 10 rats each: one group consisted of rats in the prepubertal period at age 4–6 weeks; five groups consisted of rats in the reproductive period at age 6–10 months, which were divided according to estrus cycle phases into proestrus, estrus, metestrus, diestrus, and pregnant groups; and the sixth group consisted of rats in the postmenopausal period at age 15–18 months. The uteri of all rats were removed and processed for staining with H&E and were subjected to immunohistochemical staining for Flt1 and KDR. For morphometric measurements, uterine wall thickness and Flt1 and KDR optical density in the endometrial surface epithelium, glandular epithelium, stromal cells, and endometrial endothelial cells were measured using image analysis. Results were statistically compared. Results The expression of VEGF-Rs was highest in the pubertal age group with marked expression of these receptors in the proestrus phase followed by the estrus phase. This supports the role of sex hormones, especially the estrogen hormone, in regulating VEGF-R expression. The Flt1 receptor was predominantly expressed in endometrial and stromal cells as well as in blastocysts, whereas the KDR receptor was predominantly expressed in endometrial endothelial cells. Comparison among all groups and then between each two groups revealed statistically significant differences in the measured morphometric parameters. Conclusion The upregulation of Flt1 and KDR could be involved in the regulation of endometrial endothelial cell proliferation and in increase in endometrial vascular permeability, especially at implantation sites.

Placental Growth Factor (PlGF) and Soluble fms-Like Tyrosine Kinase-1 (SFLT-1) Predict Mortality in Stable Patients with Heart Failure with Preserved Ejection Fraction

The pro-angiogenic growth factor PlGF and anti-angiogenic soluble vascular endothelial growth factor receptor sFlt-1 have been extensively studied in the pathogenesis of preeclampsia. Recently, elevated levels of PlGF and sFlt-1 in the setting of acute coronary syndrome have been associated with an increased short-term risk of decompensated heart failure, myocardial infarction, and death. The prognostic utility of these angiogenic factors in the setting of heart failure (HF) has not been examined.

Cutting Edge: Vascular Endothelial Growth Factor-Mediated Signaling in Human CD45RO+ CD4+ T Cells Promotes Akt and ERK Activation and Costimulates IFN-γ Production

In this study, we find that CD45RO+ memory populations of CD4+ T lymphocytes express the vascular endothelial growth factor (VEGF) receptors KDR and Flt-1 at both the mRNA and protein levels. Furthermore, by Western blot analysis, we find that VEGF increases the phosphorylation and activation of ERK and Akt within CD4+CD45RO+ T cells. These VEGF-mediated signaling responses were inhibited by a KDR-specific small interfering RNA in a VEGF receptor-expressing Jurkat T cell line and by SU5416, a pharmacological KDR inhibitor, in CD4+CD45RO+ T cells. We also find that VEGF augments mitogen-induced production of IFN-gamma in a dose-dependent manner (p < 0.001) and significantly (p < 0.05) increases directed chemotaxis of this T cell subset. Collectively, our results for the first time define a novel function for VEGF and KDR in CD45RO+ memory T cell responses that are likely of great pathophysiological importance in immunity.

Effects of recombinant human erythropoietin on platelet activation in acute myocardial infarction: Results of a double-blind, placebo-controlled, randomized trial

Erythropoietin mitigates myocardial damage and improves ventricular performance after experimental ischemic injury. This study assessed safety and efficacy markers relevant to the biological activity of recombinant human erythropoietin (rHuEpo) in patients with acute myocardial infarction (MI). We conducted a prospective, placebo-controlled, randomized, double-blind trial to determine the effects of intravenous rHuEpo (200 U/kg daily for 3 consecutive days) on measures of platelet and endothelial cell activation, soluble Fas ligand, and peripheral blood mononuclear cell (PBMC) expression of angiogenesis signaling proteins in 44 subjects with acute MI treated with aspirin and clopidogrel after successful percutaneous coronary intervention. Recombinant human erythropoietin did not alter bleeding time, platelet function assay closure time, von Willebrand factor levels, soluble P-selectin, or soluble Fas ligand levels when compared with placebo. By contrast, rHuEpo significantly increased expression of erythropoietin receptor, vascular endothelial growth factor receptor Flt-1, and phosphorylated phosphatidylinositol 3-kinase in PBMCs when compared with placebo (all Ps < .05). In acute MI patients treated with aspirin and clopidogrel, short-term administration of rHuEpo did not alter markers of platelet and endothelial cell activation associated with thrombosis, yet did increase expression of angiogenesis signaling proteins in PBMCs when compared with placebo. These data provide preliminary evidence of safety and biologic activity of rHuEpo at this dosing and support continued enrollment in ongoing efficacy trials.

Local Guidance of Emerging Vessel Sprouts Requires Soluble Flt-1

Blood vessel networks form via sprouting of endothelial cells from parent vessels. Extrinsic cues guide sprouts after they leave the initiation site, but these cues are likely insufficient to regulate initial outward movement, and many embryonic vessel networks form in the absence of a strong extrinsic gradient. We hypothesized that nascent sprouts are guided by spatial cues produced along their own vessels, and that soluble Flt-1 (sFlt-1) participates in this guidance. Analysis of developing vessels with perturbed flt-1 function revealed misguided emerging sprouts, and transgenic sFlt-1 rescued sprout guidance parameters. sflt-1 activity in endothelial cells immediately adjacent to the emerging sprout significantly improved local sprout guidance. Thus, we propose that a vessel-intrinsic system initially guides emerging sprouts away from the parent vessel, utilizing spatially regulated expression of sFlt-1 in conjunction with exogenous VEGF-A. Local sprout guidance defects are predicted to contribute to vessel dysmorphogenesis during perturbed development and disease.

Expression of vascular endothelial growth factor receptors Flt-1 and Flk-1 in embryonic rat forebrain

To define better the putative targets of vascular endothelial growth factor (VEGF) in the developing brain we have examined the ontogeny of the two VEGF tyrosine kinase receptors, Flt-1 and Flk-1, in embryonic rat forebrain. Semiquantitative reverse transcriptase-polymerase chain reaction and immunoblot analysis showed expression of both receptors in the forebrain at all embryonic ages studied. Messenger RNAs for Flt-1 and Flk-1 appeared along most of the ventricular zone of the lateral ventricle as early as embryonic day (E) 13. Messages gradually became restricted to a limited ventricular zone at E20. Expression of VEGF receptors was also observed in the cerebral cortex, hippocampus and thalamic nuclei. In the cortex, expression of mRNA for both receptors was detected in the cortical plate around E15, and became relatively weak and restricted to the deeper layers of the cortical plate at E20. These data suggest that VEGF may contribute to early developmental processes including the proliferation, differentiation and maturation of specific neuronal populations via specific VEGF receptors in the developing rat forebrain.

Synergistic antiproliferative effects of KIT tyrosine kinase inhibitors on neoplastic canine mast cells

Aggressive mast cell (MC) tumors are hematopoietic neoplasms characterized by uncontrolled growth of MC and resistance to conventional drugs. In most cases, the tyrosine kinase (TK) receptor KIT is involved in malignant cell growth. Therefore, several KIT TK-targeting drugs are currently being tested for their ability to block growth of neoplastic MC. We examined the effects of four TK inhibitors (imatinib, midostaurin, nilotinib, and dasatinib) on C2 canine mastocytoma cells, as well as primary neoplastic canine MC. As assessed by (3)H-thymidine incorporation experiments, all TK inhibitors produced dose-dependent inhibition of proliferation in C2 cells with the following IC(50) values: imatinib: 269 +/- 180 nM, midostaurin: 157 +/- 35 nM, nilotinib: 55 +/- 24 nM, dasatinib: 12 +/- 3 nM. Growth-inhibitory effects of TK inhibitors were also observed in primary neoplastic mast cells, although IC(50) values for each drug varied from patient to patient, with midostaurin being the most potent agent in all samples tested. In consecutive experiments, we were able to show that TK inhibitors cooperate with each other in producing growth inhibition in C2 cells with synergistic effects observed with most drug combinations. In flow cytometry and TUNEL assay experiments, growth-inhibitory effects of TK inhibitors were found to be associated with cell-cycle arrest and apoptosis. Together, these data show that several TK-targeting drugs induce apoptosis and inhibit proliferation in canine mastocytoma cells in vitro, and that synergistic drug interactions can be obtained. Clinical trials are now warranted to explore whether these TK inhibitors also counteract growth of neoplastic cells in vivo in patients with aggressive MC tumors.

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.

Expression and function of vascular endothelial growth factor receptors (Flt-1 and Flk-1) in vascular adventitial fibroblasts

Vascular endothelial growth factor receptors (VEGFRs) are previously considered to exist exclusively in endothelial cells. However, little is known if the receptors are expressed in other non-endothelial cells. In this study, we measured activation of two VEGFRs, Flk-1 and Flt-1, and their biological functions in cultured adventitial fibroblasts and injured rat carotid injury arteries induced by balloon angioplasty. Our results indicated that Flt-1, but not Flk-1, existed in adventitial fibroblasts. Angiotensin II increased Flt-1 protein expression in a time- and concentration-dependent manner. Adventitial fibroblast migration stimulated by vascular endothelial growth factor (VEGF) and placental growth factor (PIGF) required Flt-1 expression. The Flt-1-induced adventitial fibroblast migration was blocked by anti-Flt-1 neutralizing antibody and soluble VEGFR1 protein (sFlt-1). However, Flt-1 activation did not enhance cell proliferation. In addition, Flt-1 expression was significantly increased in the neointima and adventitia in injured rat carotid arteries. We concluded that functional expression of Flt-1 in adventitial fibroblasts might be an important mediator in the pathogenesis of vascular remodeling after arterial injury.

Effect of arsenic trioxide on vascular endothelial cell proliferation and expression of vascular endothelial growth factor receptors Flt-1 and KDR in gastric cancer in nude mice

To investigate the effect of arsenic trioxide (As2O3) on expression of vascular endothelial growth factor receptor-1 (VEGFR-1, Flt-1) and VEGFR-2 (KDR) in human gastric tumor cells and proliferation of vascular endothelial cells. The solid tumor model was formed in nude mice with the gastric cancer cell line SGC-7901. The animals were treated with As2O3. Microvessel density (MVD) and expression of Flt-1 and KDR were detected by immunofluorescence laser confocal microscopy. SGC-7901 cells were treated respectively by exogenous recombinant human VEGF165 or VEGF165 + As2O3. Cell viability was measured by MTT assay. Cell viability of ECV304 cells was measured by MTT assay, and cell cycle and apoptosis were analyzed using flow cytometry. The tumor growth inhibition was 30.33% and 50.85%, respectively, in mice treated with As2O3 2.5 and 5 mg/kg. MVD was significantly lower in arsenic-treated mice than in the control group. The fluorescence intensity levels of Flt-1 and KDR were significantly less in the arsenic-treated mice than in the control group. VEGF165 may accelerate growth of SGC7901 cells, but As2O3 may disturb the stimulating effect of VEGF165. ECV304 cell growth was suppressed by 76.51%, 71.09% and 61.49% after 48 h treatment with As2O3 at 0.5, 2.5 and 5 micromol/L, respectively. Early apoptosis in the As2O3-treated mice was 2.88-5.1 times higher than that in the controls, and late apoptosis was 1.17-1.67 times higher than that in the controls. Our results showed that As2O3 delays tumor growth, inhibits MVD, down-regulates Flt-1 and KDR expression, and disturbs the stimulating effect of VEGF165 on the growth of SGC7901 cells. These results suggest that As2O3 might delay growth of gastric tumors through inhibiting the paracrine and autocrine pathways of VEGF/VEGFRs.