Expression Of Fetal Liver Kinase-1 Research Articles

Polydopamine-Coated Poly(l-lactide) Nanofibers with Controlled Release of VEGF and BMP-2 as a Regenerative Periosteum.

The periosteum plays an important role in vascularization and ossification during bone repair. However, in most studies, an artificial periosteum cannot restore both functions of the periosteum concurrently. In this study, a novel nanofiber that can sustain the release of vascular endothelial growth factor (VEGF) and bone morphogenetic protein-2 (BMP-2) was fabricated to enhance the durability of angiogenesis and osteogenesis during bone regeneration. A cell-free tissue engineered periosteum based on an electrospinning poly-l-lactic acid (PLLA) nanofiber was fabricated, on which VEGF and BMP-2 were immobilized through a polydopamine (PDA) coating conveniently and safely (BVP@PLLA membrane). The results indicated a significantly improved loading rate as well as a slow and sustained release of VEGF and BMP-2 with the help of the PDA coating. BMP-2 immobilized on nanofibers successfully induced the osteogenic differentiation of human bone marrow mesenchymal stem cells (BMSCs) in vitro with high expression of runt-related transcription factor 2 (Runx2), osteopontin (OPN), and alkaline phosphatase (ALP). Similarly, angiogenic differentiation of BMSCs with the expression of fetal liver kinase-1 (Flk-1) and vascular endothelial cadherin (VE-cadherin) was observed under the environment of VEGF sustained release. Moreover, an in vivo study revealed that the BVP@PLLA membrane could enhance vascular formation and new bone formation, which accelerates bone regeneration in rat femoral defects along with a massive periosteum defect. Therefore, our study suggests that the novel artificial periosteum with dual growth factor controlled release is a promising system to improve bone regeneration in bone defects along with a massive periosteum defect.

MiR-92a regulates endothelial progenitor cells (EPCs) by targeting GDF11 via activate SMAD2/3/FAK/Akt/eNOS pathway.

The effects of miR-92a on EPCs are still poorly elucidated. This study aimed to investigate the effects of miR-92a on EPCs (Endothelial progenitor cells) in a model of hypoxia (HO) or high glucose (HG)-induced EPCs injury by targeting GDF11 (Differentiation growth factor 11). The effects of miR-92a on EPCs subjected to HO or HG were investigated firstly. Subsequently, the action mechanism of miR-92a on EPCs by targeting GDF11 was elucidated. Proliferation, apoptosis, migration, angiogenesis was measured with MTT, flow cytometry, transwell, tube formation respectively. After 24 h, levels of reactive oxygen species (ROS) were measured by fluorescence intensity. LDH and NO (nitric oxide) levels were determined by ELISA. The expression of FLK-1 (fetal liver kinase 1) and vWF (von Willebrand factor) was detected by immunofluorescence. mRNA and protein expression levels were examined using PCR and western blotting respectively. The interaction between miR-92a and GDF11 was evaluated by dual-luciferase reporter assay. Our results showed that HO or HG increased apoptosis, production of LDH and generation of ROS, but decreased the ability of migration and tube formation and generation of NO in EPCs; inhibiting of miR-92a decreased HO or HG-induced injury of EPCs, whereas miR-92a over-expression had the opposite effect; the protective effects induced by inhibiting of miR-92a on EPCs could be reversed by GDF11 siRNA and the harmful effects induced by over-expression of miR-92a could be rescued by over-expression of GDF11, which showed that the harmful effects of miR-92a be related to its inhibition of GDF11 and subsequent inactivation of the SMAD2/3/FAK/Akt/eNOS signaling pathway. Inhibiting miR-92a can protect EPCs from HO or HG-induced injury. The effect of miR-92a on EPCs are mediated by regulating of GDF11 and downstream SMAD2/3/FAK/Akt/eNOS signaling pathway.

Effect of fluoxetine on the expression of angiogenic factors in hippocampus of depressive rats

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. Key words: Depression; Hippocampus; Angiogenic factors; Rat; PI3K/AKT signaling pathway; VEGF /FLK1; AKT1/p-AKT1

EXPERIMENTAL RESEARCH ABOUT EFFECT OF CELL–CELL CONTACT TO MESENCHYMAL STEM CELLS DIFFERENTIATE INTO VASCULAR ENDOTHELIAL CELLS

ObjectivesUse the method of HUVECs were cultured with human mesenchymal stem cells by cell–cell contact to simulate intravascular environment after angioplasty and stent implantation, and research the role of cell–cell...

The expressions of periostin and the related factors during healing process of full-thickness cutaneous wound in rat

To observe the expression of osteoblast-specific factor 2 (periostin, PN), angiopoietin-1 (Ang-1), vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor-2 [VEGFR-2/fetal liver kinase-1 (FLK-1)] in wound surface and its peripheral skin, and their effects on wound healing in rats. Forty-eight Sprague-Dawley (SD) rats were randomly divided into six groups, with 8 rats in each group. An area of 2 cm×2 cm full-thickness skin was excised on both sides of the back of rats. Specimens from wounds were obtained on 1, 4, 7, 10, 14, 21 days after operation, and histological evaluation and immunohistochemical staining of PN, Ang-1, VEGF and FLK-1 were made to determine their expression levels. Normal skin specimens were obtained as normal controls. The expressions of PN, Ang-1, VEGF and FLK-1 were significantly increased in wound surface after operation. Compared with the skin of normal controls, the expression of PN in the tissues of wound increased by 234.4% on the 1st day, and then increased continuously up to 597.9% on the 7th day (reaching the peak) after operation, followed by a decrease, the increase rate was 280.9% on the 21st day, and still remained at a high level (all P < 0.05). The expression of Ang-1 in the tissue of wound increased by 128.1% on the 1st day and 327.5% on the 4th day (reaching the peak), and then, it was gradually decreased. The increase rate was only 80.5% on the 14th day and it rose slightly later (all P < 0.05). The expression of VEGF in the tissues of wound reached the peak (165.8%) on the 7th day. Then it decreased with a slight fluctuation (all P < 0.05). The expression of FLK-1 in the tissues of wound was increased by 56.1% on the 1st day, and the level remained. It reached the peak by an increase of 70.1% on the 7th day (both P < 0.05). Then, it was lowered after the 10th day (all P > 0.05). The expressions of PN, Ang-1, VEGF, FLK-1 were obviously increased during healing of skin wound, with different peaking time and expressing rates. The increase in expression of PN showed the longest duration and highest peak value. The PN, Ang-1, VEGF, FLK-1 all play a role in the wound healing process, and PN might play an important role during the healing process of a full-thickness cutaneous wound.

NANOG induction of fetal liver kinase-1 (FLK1) transcription regulates endothelial cell proliferation and angiogenesis

AbstractNANOG is a master transcription factor associated with the maintenance of stem cell pluripotency. Here, we demonstrate that transcription factor NANOG is expressed in cultured endothelial cells (ECs) and in a subset of tumor cell lines. Importantly, we provide evidence that WNT3A stimulation of ECs induces the transcription of NANOG which mediates the expression of vascular endothelial growth factor receptor-2, also known as fetal liver kinase-1 (FLK1). We defined ATTA as a minimal binding site for NANOG. Accordingly, a luciferase reporter assay showed that NANOG binds to and activates 4 ATTA binding sites identified in the FLK1 promoter after WNT3A stimulation. Consistent with this data, we found that, under basal conditions and in response to WNT3A stimulation, NANOG binding to these ATTA sequences markedly induced the expression of FLK1. Thus, our data indicate an essential role in angiogenesis for NANOG binding to these 4 ATTA sites. Surprisingly, NANOG depletion not only decreased FLK1 expression but also reduced cell proliferation and angiogenesis. These findings show the necessary and sufficient role of NANOG in inducing the transcription of FLK1 to regulate the angiogenic phenotypes of ECs.

Blocking effects of siRNA on VEGF expression in human colorectal cancer cells

To investigate the expression of vascular endothelial cell growth factor (VEGF) and its receptors Fms-like tyrosine kinase 1 (FLT-1) and fetal liver kinase 1 (FLK-1) in colorectal carcinoma (CRC), and the blocking effects of small interfering RNAs (siRNAs) on VEGF expression in human colorectal cancer HCT116 cells. Immunohistochemical staining for VEGF, FLT-1 and FLK-1 proteins was performed in 82 cases of CRC and 14 normal colorectal mucosae. A siRNA targeting VEGF was synthesized and transfected into HCT116 cells using lipofectamine 2000. Immunocytochemical staining and Western blotting analyses were performed to detect the expression of VEGF protein. The suppressive effect of the siRNA on cell proliferation was detected using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltertrazolium bromide (MTT) assay. Cellular apoptosis was detected using flow cytometry (FCM). The expression of VEGF, FLT-1 and FLK-1 in tumor tissues was significantly higher than that in normal tissues (P = 0.008, P = 0.000, P = 0.000). The expression of VEGF was positively correlated with both lymph node metastasis and clinical stage (P = 0.009 and P = 0.025, respectively). Immunocytochemistry showed that the expression of VEGF was weakly positive and Western blotting indicated a significant reduction in VEGF-siRNA cell protein levels. VEGF-siRNA cell growth inhibition was assessed by the MTT assay, and the tumor cell proliferation rate was significantly different at 24, 48, and 72 h after transfection. FCM results showed that the VEGF-siRNA group had an apparent aneuploid peak. VEGF, FLT-1 and FLK-1 are associated with colorectal carcinogenesis. siRNA silencing of the VEGF gene suppresses proliferation, and induces apoptosis in HCT116 cells. The results suggest that VEGF may be a new gene therapy target for colorectal cancer.

Mechanisms underlying TGF-β1-induced expression of VEGF and Flk-1 in mouse macrophages and their implications for angiogenesis

TGF-beta induces vascular endothelial growth factor (VEGF), a potent angiogenic factor, at the transcriptional and protein levels in mouse macrophages. VEGF secretion in response to TGF-beta1 is enhanced by hypoxia and by overexpression of Smad3/4 and hypoxia-inducible factor-1alpha/beta (HIF-1alpha/beta). To examine the transcriptional regulation of VEGF by TGF-beta1, we constructed mouse reporters driven by the VEGF promoter. Overexpression of HIF-1alpha/beta or Smad3/4 caused a slight increase of VEGF promoter activity in the presence of TGF-beta1, whereas cotransfection of HIF-1alpha/beta and Smad3/4 had a marked effect. Smad2 was without effect on this promoter activity, whereas Smad7 markedly reduced it. Analysis of mutant promoters revealed that the one putative HIF-1 and two Smad-binding elements were critical for TGF-beta1-induced VEGF promoter activity. The relevance of these elements was confirmed by chromatin immunoprecipitation assay. p300, which has histone acetyltransferase activity, augmented transcriptional activity in response to HIF-1alpha/beta and Smad3/4, and E1A, an inhibitor of p300, inhibited it. TGF-beta1 also increased the expression of fetal liver kinase-1 (Flk-1), a major VEGF receptor, and TGF-beta1 and VEGF stimulated pro-matrix metalloproteinase 9 (MMP-9) and active-MMP-9 expression, respectively. The results from the present study indicate that TGF-beta1 can activate mouse macrophages to express angiogenic mediators such as VEGF, MMP-9, and Flk-1.

Evaluation of vascular endothelial growth factor addition in vitro on mouse inner ear progenitor cell cultures

We analyzed progenitor cell cultures of inner ear tissue from newborn mice, and found proliferating cells, morphologically differentiating cells and subpopulations of cells expressing either neuronal or glial markers. In addition, we observed the expression of fetal liver kinase-1, a receptor for the vascular endothelial growth factor in a subpopulation of the cultured cells. Consistent with the expression of fetal liver kinase-1, addition of vascular endothelial growth factor at a dose of 10 ng/ml increased the expansion rate of inner ear-derived progenitor cells. Together with other published data, these results suggest that the vascular endothelial growth factor might be involved in inducing or supporting cochlear repair processes.

Participation of the PI-3K/Akt-NF-κB signaling pathways in hypoxia-induced mitogenic factor-stimulated Flk-1 expression in endothelial cells

BackgroundHypoxia-induced mitogenic factor (HIMF), a lung-specific growth factor, promotes vascular tubule formation in a matrigel plug model. We initially found that HIMF enhances vascular endothelial growth factor (VEGF) expression in lung epithelial cells. In present work, we tested whether HIMF modulates expression of fetal liver kinase-1 (Flk-1) in endothelial cells, and dissected the possible signaling pathways that link HIMF to Flk-1 upregulation.MethodsRecombinant HIMF protein was intratracheally instilled into adult mouse lungs, Flk-1 expression was examined by immunohistochemistry and Western blot. The promoter-luciferase reporter assay and real-time RT-PCR were performed to examine the effects of HIMF on Flk-1 expression in mouse endothelial cell line SVEC 4–10. The activation of NF-kappa B (NF-κB) and phosphorylation of Akt, IKK, and IκBα were examined by luciferase assay and Western blot, respectively.ResultsIntratracheal instillation of HIMF protein resulted in a significant increase of Flk-1 production in lung tissues. Stimulation of SVEC 4–10 cells by HIMF resulted in increased phosphorylation of IKK and IκBα, leading to activation of NF-κB. Blocking NF-κB signaling pathway by dominant-negative mutants of IKK and IκBα suppressed HIMF-induced Flk-1 upregulation. Mutation or deletion of NF-κB binding site within Flk-1 promoter also abolished HIMF-induced Flk-1 expression in SVEC 4–10 cells. Furthermore, HIMF strongly induced phosphorylation of Akt. A dominant-negative mutant of PI-3K, Δp85, as well as PI-3K inhibitor LY294002, blocked HIMF-induced NF-κB activation and attenuated Flk-1 production.ConclusionThese results suggest that HIMF upregulates Flk-1 expression in endothelial cells in a PI-3K/Akt-NF-κB signaling pathway-dependent manner, and may play critical roles in pulmonary angiogenesis.

Shear Stress Induces Expression of Vascular Endothelial Growth Factor Receptor Flk-1/KDR Through the CT-Rich Sp1 Binding Site

Fluid shear stress is 1 of the major factors that control gene expression in vascular endothelial cells. We investigated the role of shear stress in the regulation of the expression of fetal liver kinase-1/kinase domain region (Flk-1/KDR), a vascular endothelial growth factor receptor, by using human umbilical vein endothelial cells. Laminar shear stress (15 dyne/cm2) elevated Flk-1/KDR mRNA levels by approximately 3-fold for 8 hours, and the expression was upregulated within the range of 5 to 40 dyne/cm2. Deletion analysis of the 5'-flanking region of the Flk-1/KDR gene promoter by use of a luciferase reporter vector revealed that a shear stress-responsive element resided in the sequence between -94 and -31 bp, which contained putative nuclear factor-kappaB, activator protein-2, and GC-rich Sp1 and CT-rich Sp1 binding sites. Electrophoretic mobility shift assay demonstrated that nuclear extract was bound to the GC-rich Sp1 sites and the CT-rich Sp1 site with a similar pattern. However, shear stress enhanced the DNA-protein interactions only on the CT-rich Sp1 site but not on the GC-rich Sp1 sites. A 3-bp mutation in the CT-rich Sp1 site eliminated the response to shear stress in electrophoretic mobility shift assay and luciferase reporter assay. These results suggest that shear stress induces Flk-1/KDR expression through the CT-rich Sp1 binding site.

Expression of vascular endothelial growth factor receptors in human prostate cancer

Objectives. We recently reported the expression and cytokine regulation of vascular endothelial growth factor (VEGF) in human prostate cancer (PCa). VEGF exerts its angiogenic and pro-tumorigenic properties by way of two high affinity receptors, fms-like tyrosine kinase 1 (FLT-1) and fetal liver kinase 1 (FLK-1). We hypothesized that these receptors are expressed and control VEGF functions in the PCa microenvironment. Herein, we evaluate the expression of these receptors in ex vivo PCa tissue, benign prostatic hypertrophy (BPH) tissue, and cultured PCa cell lines. Methods. Ex vivo PCa specimens were obtained from patients undergoing radical retropubic prostatectomy. Specimens were selected to contain both PCa and BPH tissue (n = 15). Immunohistochemical analysis using antihuman FLT-1 and FLK-1 was performed and specimens were analyzed to characterize the expression and distribution of both receptors. Immunocytochemical analysis for FLT-1 and FLK-1 was also performed on cultured PCa cell lines (DU-145 and LNCaP). Results. PCa cells expressed the VEGF receptor FLT-1 in 100% of specimens evaluated. Expression of FLK-1 was variable and related to tumor grade; high-grade tumors displayed little or no FLK-1 expression. Vascular endothelial cells (VECs) within areas of PCa consistently expressed both FLT-1 and FLK-1 receptors. FLT-1 and FLK-1 were both expressed in BPH tissue. FLT-1 was expressed in the glandular epithelial cells in BPH, but in most cases FLK-1 was localized specifically to the basal cell layer of hypertrophic glands. FLT-1, but not FLK-1, was expressed by the DU-145 and LNCaP cell lines. Conclusions. Although they are differentially expressed, both FLT-1 and FLK-1 are present in PCa and BPH. Expression of receptors on VECs of tumor vessels supports the well-established role of VEGF in paracrine stimulation of VECs in the tumor microenvironment. The expression of FLT-1 and FLK-1 on tumor cells themselves suggests a potential autocrine function for VEGF (such as regulating tumor cell proliferation). These findings imply that a novel dual role may exist for VEGF, such that it is involved in tumor cell activation (autocrine), in addition to paracrine actions whereby it regulates endothelial cell functions and subsequent neovascular development.