A chemically defined culture of VEGFR2+ cells derived from embryonic stem cells reveals the role of VEGFR1 in tuning the threshold for VEGF in developing endothelial cells

Vascular endothelial growth factor (VEGF) is a major growth factor for developing endothelial cells (ECs). Embryonic lethality due to haploinsufficiency of VEGF in the mouse highlighted the strict dose dependency of VEGF on embryonic vascular development. Here we investigated the dose-dependent effects of VEGF on the differentiation of ES cell-derived fetal liver kinase 1 (Flk-1)/VEGF receptor 2(+) (VEGFR2(+)) mesodermal cells into ECs on type IV collagen under a chemically defined serum-free condition. These cells could grow even in the absence of VEGF, but differentiated mostly into mural cells positive for alpha-smooth muscle actin. VEGF supported in a dose-dependent manner the differentiation into ECs defined by the expression of VE-cadherin, platelet-endothelial cell adhesion molecule 1 (PECAM-1)/ CD31, CD34, and TIE2/TEK. VEGF requirement was greater at late than at early phase of culture during EC development, whereas response of VEGFR2(+) cells to VEGF-E, which is a virus-derived ligand for VEGFR2 but not for Flt-1/VEGFR1, was not dose sensitive even at late phase of culture. Delayed expression of VEGFR1 correlated with increased dose dependency of VEGF. These results suggested that greater requirement of VEGF in the maintenance than induction of ECs was due to the activity of VEGFR1 sequestering VEGF from VEGFR2 signal. The chemically defined serum-free culture system described here provides a new tool for assessing different factors for the proliferation and differentiation of VEGFR2(+) mesodermal cells.     

Neuropilin-1 regulates vascular endothelial growth factor-mediated endothelial permeability

Neuropilin-1 (Npn-1) is a cell surface receptor that binds vascular endothelial growth factor (VEGF), a potent mediator of endothelial permeability, chemotaxis, and proliferation. In vitro, Npn-1 can complex with VEGF receptor-2 (VEGFR2) to enhance VEGFR2-mediated endothelial cell chemotaxis and proliferation. To determine the role of Npn-1/VEGFR2 complexes in VEGF-induced endothelial barrier dysfunction, endothelial cells were stably transfected with Npn1 or VEGFR2 alone (PAE/Npn and PAE/KDR, respectively), or VEGFR2 and Npn-1 (PAE/KDR/Npn-1). Permeability, estimated by measurement of transendothelial electrical resistance (TER), of PAE/Npn and PAE/KDR cell lines was not altered by VEGF165. In contrast, TER of PAE/KDR/Npn-1 cells decreased in dose-dependent fashion following VEGF165 (10 to 200 ng/mL). Activation of VEGFR2, and 2 downstream signaling intermediates (p38 and ERK1/2 MAPK) involved in VEGF-mediated permeability, also increased in PAE/KDR/Npn-1. Consistent with these data, inhibition of Npn-1, but not VEGFR2, attenuated VEGF165-mediated permeability of human pulmonary artery endothelial cells (HPAE), and VEGF121 (which cannot ligate Npn-1) did not alter TER of HPAE. Npn-1 inhibition also attenuated both VEGF165-mediated pulmonary vascular leak and activation of VEGFR2, p38, and ERK1/2 MAPK, in inducible lung-specific VEGF transgenic mice. These data support a critical role for Npn-1 in regulating endothelial barrier dysfunction in response to VEGF and suggest that activation of distinct receptor complexes may determine specificity of cellular response to VEGF.     

Vascular endothelial growth factor promotes proliferation and function of hepatocyte-like cells in embryoid bodies formed from mouse embryonic stem cells

BACKGROUND/AIMS: Embryoid bodies (EBs) formed from embryonic stem cells (ESCs) differentiate into hepatocyte-like cells (HLCs), and are thus thought to be a useful cell source for drug testing and bioartificial liver. The aim of this study was to induce proliferation and function of ESC-derived HLCs in EBs using HLC-endothelial cell interaction. METHODS: EBs were cultured in the presence of vascular endothelial growth factor (VEGF) and/or VEGF receptor (VEGFR) inhibitors. To reproduce HLC-endothelial cell interaction, we overexpressed VEGF in ESC-derived HLCs under the control of Cyp7a1 gene in EBs. RESULTS: VEGF added to the cultured EBs increased the proliferation of ESC-derived endothelial cells, resulting in the promotion of proliferation and function of ESC-derived HLCs. In EBs, the VEGFR2 inhibitor suppressed expression of albumin and endothelial cell marker genes, whereas the inhibitor for both VEGFR1 and VEGFR2 suppressed expression of Cyp7a1 and hepatocyte growth factor (Hgf) genes. Upon exposure to VEGF, the endothelial cells in EBs increased Hgf mRNA expression. Forced VEGF expression in ESC-derived HLCs in EBs induced angiogenesis around the HLCs and resulted in an increase in the amount of HLCs. CONCLUSIONS: VEGF indirectly induces the proliferation and function of ESC-derived HLCs through VEGFR1 and VEGFR2 signaling in endothelial cells.     

Pituitary tumor transforming gene (PTTG) stimulates thyroid cell proliferation via a vascular endothelial growth factor/kinase insert domain receptor/inhibitor of DNA binding-3 autocrine pathway

CONTEXT: Vascular endothelial growth factor (VEGF) exerts its biological effects by binding to the tyrosine kinase receptors VEGF receptor type 1 (VEGFR1/Flt-1) and VEGFR2 (Flk-1/KDR). Kinase insert domain receptor (KDR) is the critical receptor controlling proliferation and migration of endothelial cells and has been shown to be expressed in some nonendothelial cells. We recently reported that the proangiogenic pituitary tumor transforming gene (PTTG) stimulates VEGF and up-regulates inhibitor of DNA binding-3 (ID3), an important gene in VEGF-dependent angiogenesis. OBJECTIVE: Our objective was to test whether VEGF, ID3, and KDR confer a PTTG-mediated effect on thyroid cell growth. DESIGN: Gene expression, MAPK stimulation, and cell proliferation were assessed in follicular thyroid cancer FTC133 cells. Gene expression and clinical associations were determined in 21 normal and 38 tumorous thyroid specimens (nine follicular and 29 papillary). RESULTS: ID3 correlated with VEGF mRNA expression in our series of thyroid cancers, which also showed up-regulated KDR mRNA. Stimulation of FTC133 cells with exogenous VEGF enhanced ID3 expression, which could be abrogated by the KDR-specific inhibitor ZM323881, suggesting that VEGF regulation of ID3 is KDR dependent. PTTG significantly correlated with KDR mRNA expression in our thyroid cancer cohort and up-regulated KDR and VEGF expression in FTC133 cells. Finally, cells transfected with PTTG demonstrated increased cell proliferation and phosphorylation of MAPK, which was abrogated by ZM323881. CONCLUSIONS: We report the presence of a VEGF/KDR/ID3-dependent autocrine pathway in FTC133 thyroid cells. By up-regulating both VEGF and KDR expression, we propose a novel PTTG-mediated proliferative pathway that may be critical to thyroid cancer growth and progression.     

Role of vascular endothelial growth factor (VEGF) and placenta-derived growth factor (PlGF) in regulating human haemopoietic cell growth

Vascular endothelial growth factor (VEGF) and placental derived growth factor (PlGF) stimulate cell proliferation and differentiation by binding to their specific receptors, Flk-1/KDR and Flt-1 respectively. Flk-1/KDR-deficient murine embryos manifest failure of blood-island formation and vasculogenesis. The aim of this study was to directly evaluate the importance of VEGF, PlGF/Flt-1 and Flk-1/KDR receptor ligand interactions in regulating normal and malignant human haemopoiesis. Addition of VEGF and PlGF failed to enhance survival or cloning efficiency of human haemopoietic progenitors isolated from adult bone marrows, fetal livers or cord blood samples. This finding may be explained by the apparent absence of mRNA encoding Flt-1 and Flk-1/KDR receptors on stem cell rich CD34⁺ c-kit-R⁺ Rh123^low cells. Further studies revealed that Flt-1 R mRNA, but not Flk-1/KDR mRNA was first detectable in the more mature cells isolated from haemopoietic colonies. Accordingly, VEGF receptors are either absent, or expressed at very low level, on human haemopoietic stem/progenitor cells. Of interest, normal and malignant human haemopoietic cells appeared to secrete VEGF protein. However, in contrast to normal haemopoietic progenitors, VEGF co-stimulated HEL cell proliferation as well as CFU-GM colony formation from ∼15% of the chronic myeloid leukaemia (CML) and acute myeloid leukaemia (AML) patients studied. Therefore, although VEGF appeared to have minimal effects on normal haemopoietic cell growth it would appear to drive malignant haemopoietic cell proliferation to some degree. Of more importance, however, we speculate that VEGF may play an very important role in leukaemogenesis by stimulating growth of vascular endothelium, thereby providing a sufficient blood supply to feed the growing haematological tumour.     

Role of fibroblast growth factor receptors in astrocytic stem cells

There are two well-defined neurogenic regions in the adult brain, the subventricular zone (SVZ) lining the lateral wall of the lateral ventricles and, the subgranular zone (SGZ) in the dentate gyrus at the hippocampus. Within these neurogenic regions, there are neural stem cells with astrocytic characteristics, which actively respond to the basic fibroblast growth factor (bFGF, FGF2 or FGF-β) by increasing their proliferation, survival and differentiation, both in vivo and in vitro. FGF2 binds to fibroblast growth factor receptors 1 to 4 (FGFR1, FGFR2, FGFR3, FGFR4). Interestingly, these receptors are differentially expressed in neurogenic progenitors. During development, FGFR-1 and FGFR-2 drive oligodendrocytes and motor neuron specification. In particular, FGFR-1 determines oligodendroglial and neuronal cell fate, whereas FGFR-2 is related to oligodendrocyte specification. In the adult SVZ, FGF-2 promotes oligodendrogliogenesis and myelination. FGF-2 deficient mice show a reduction in the number of new neurons in the SGZ, which suggests that FGFR-1 is important for neuronal cell fate in the adult hippocampus. In human brain, FGF-2 appears to be an important component in the anti-depressive effect of drugs. In summary, FGF2 is an important modulator of the cell fate of neural precursor and, promotes oligodendrogenesis. In this review, we describe the expression pattern of FGFR2 and its role in neural precursors derived from the SVZ and the SGZ.     

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.     

Effects of vascular endothelial growth factors and their receptors on megakaryocytes and platelets and related diseases

It is well known that vascular endothelial growth factors (VEGFs) and their receptors (vascular endothelial growth factor receptors, VEGFRs) are expressed in different tissues, and VEGF‐VEGFR loops regulate a wide range of responses, including metabolic homeostasis, cell proliferation, migration and tubuleogenesis. As ligands, VEGFs act on three structurally related VEGFRs (VEGFR1, VEGFR2 and VEGFR3 [also termed FLT1, KDR and FLT4, respectively]) that deliver downstream signals. Haematopoietic stem cells (HSCs), megakaryocytic cell lines, cultured megakaryocytes (MKs), primary MKs and abnormal MKs express and secrete VEGFs. During the development from HSCs to MKs, VEGFR1, VEGFR2 and VEGFR3 are expressed at different developmental stages, respectively, and re‐expressed, e.g., VEGFR2, and play different roles in commitment, differentiation, proliferation, survival and polyplodization of HSCs/MKs via autocrine, paracrine and/or even intracrine loops. Moreover, VEGFs and their receptors are abnormally expressed in MK‐related diseases, including myeloproliferative neoplasms, myelodysplastic syndromes and acute megakaryocytic leukaemia (a rare subtype of acute myeloid leukaemia), and they lead to the disordered proliferation/differentiation of bone marrow cells and angiogenesis, indicating that they are closely related to these diseases. Thus, targeting VEGF‐VEGFR loops may be of potential therapeutic value.     

TNP-470对肺腺癌细胞增殖和凋亡的影响

目的 探讨TNP－470对肺腺癌细胞增殖和凋亡的影响及其作用机制。方法 以不同浓度的TNP－470信息处理地培养的肺腺癌AGZY－82A细胞株,以免疫组化S－P法检测肺腺癌细胞增殖核抗原（PCNA）、p53、bcl-2、血管内皮生长因子（VEGF）和VEGF受体Flk-1的表达。结果 随着TNP－470浓度的增加,肺腺癌细胞VEGF、Flk-1、PCNA的表达减少,p53、bcl-2的表达逐渐增加;当TNP－470为10^7μg/L时,细胞增殖几乎停止,随着TNP－470（10^4μg/L）作用时间的延长,肺腺癌细胞VEGF、Flk-1、PCNA的表达逐渐减少,而p53、bcl-2的表达逐渐增加。结论 TNP－470通过使肺腺癌细胞自分泌VEGF减少,Flk表达降低,从而抑制肺腺癌细胞增殖,促进其凋亡。     

Novel role of ARF6 in vascular endothelial growth factor-induced signaling and angiogenesis

Vascular endothelial growth factor (VEGF) stimulates endothelial cell (EC) migration and proliferation primarily through the VEGF receptor-2 (VEGFR2). We have shown that VEGF stimulates a Rac1-dependent NAD(P)H oxidase to produce reactive oxygen species (ROS) that are involved in VEGFR2 autophosphorylation and angiogenic-related responses in ECs. The small GTPase ARF6 is involved in membrane trafficking and cell motility; however, its roles in VEGF signaling and physiological responses in ECs are unknown. In this study, we show that overexpression of dominant-negative ARF6 [ARF6(T27N)] almost completely inhibits VEGF-induced Rac1 activation, ROS production, and VEGFR2 autophosphorylation in ECs. Fractionation of caveolae/lipid raft membranes demonstrates that ARF6, Rac1, and VEGFR2 are localized in caveolin-enriched fractions basally. VEGF stimulation results in the release of VEGFR2 from caveolae/lipid rafts and caveolin-1 without affecting localization of ARF6, Rac1, or caveolin-1 in these fractions. The egress of VEGFR2 from caveolae/lipid rafts is contemporaneous with the tyrosine phosphorylation of caveolin-1 (Tyr14) and VEGFR2 and with their association with each other. ARF6(T27N) significantly inhibits both VEGF-induced responses. Immunofluorescence studies show that activated VEGFR2 and phosphocaveolin colocalize at focal complexes/adhesions after VEGF stimulation. Both overexpression of ARF6(T27N) and mutant caveolin-1(Y14F), which cannot be phosphorylated, block VEGF-stimulated EC migration and proliferation. Moreover, ARF6 expression is markedly upregulated in association with an increase in capillary density in a mouse hindlimb ischemia model of angiogenesis. Thus, ARF6 is involved in the temporal-spatial organization of caveolae/lipid rafts- and ROS-dependent VEGF signaling in ECs as well as in angiogenesis in vivo.     

Vascular endothelial growth factor and its receptor, Flk-1/KDR, are cytoprotective in the extravascular compartment of the ovarian follicle

Vascular endothelial growth factor (VEGF) is a potent mitogen and cytoprotective factor for vascular endothelial cells. Although VEGF is ubiquitously expressed, its role in nonvascular tissues is poorly understood. VEGF interacts with various cell surface receptors to mediate its cellular effects. It previously has been thought that the VEGF receptor Flk-1/KDR, its main signaling receptor, was expressed exclusively by endothelial cells. However, in the present study using bovine and rodent models, we demonstrate that VEGF and Flk-1/KDR are coexpressed in ovarian granulosa cells. VEGF and Flk-1/KDR mRNA and protein were both detectable in follicle tissue sections and in vitro cultured granulosa cells. Expression of both ligand and receptor increased in healthy follicles throughout follicular development. VEGF treatment of serum-starved and cytokine-exposed granulosa cells resulted in enhanced survival, and this cytoprotection was ameliorated when Flk-1/KDR signaling was inhibited. Reduced expression of Flk-1/KDR was also associated with the onset and progression of follicle atresia, suggesting involvement in follicular health in vivo. The results of this study demonstrate for the first time expression of Flk-1/KDR in ovarian granulosa cells and identify a novel extravascular role for VEGF and its receptor in ovarian function.     

Gene therapy knockdown of VEGFR2 in retinal endothelial cells to treat retinopathy

Inhibition of vascular endothelial growth factor (VEGF) in retinopathy of prematurity (ROP) raises concerns for premature infants because VEGF is essential for retinovascular development as well as neuronal and glial health. This study tested the hypothesis that endothelial cell-specific knockdown of VEGF receptor 2 (VEGFR2), or downstream STAT3, would inhibit VEGF-induced retinopathy without delaying physiologic retinal vascular development. We developed an endothelial cell-specific lentiviral vector that delivered shRNAs to VEGFR2 or STAT3 and a green fluorescent protein reporter under control of the VE-cadherin promoter. The specificity and efficacy of the lentiviral vector-driven shRNAs were validated in vitro and in vivo. In the rat oxygen-induced retinopathy model highly representative of human ROP, the effects of endothelial cell knockdown of VEGFR2 or STAT3 were determined on intravitreal neovascularization (IVNV), physiologic retinal vascular development [assessed as area of peripheral avascular/total retina (AVA)], retinal structure, and retinal function. Targeted knockdown of VEGFR2 or STAT3 specifically in retinal endothelial cells by subretinal injection of lentiviral vectors into postnatal day 8 rat pup eyes efficiently inhibited IVNV, and knockdown of VEGFR2 also reduced AVA and increased retinal thickness without altering retinal function. Taken together, our results support specific knockdown of VEGFR2 in retinal endothelial cells as a novel therapeutic method to treat retinopathy.     

Inhibition of both paracrine and autocrine VEGF/ VEGFR-2 signaling pathways is essential to induce long-term remission of xenotransplanted human leukemias

Antiangiogenic agents block the effects of tumor-derived angiogenic factors (paracrine factors), such as vascular endothelial growth factor (VEGF), on endothelial cells (EC), inhibiting the growth of solid tumors. However, whether inhibition of angiogenesis also may play a role in liquid tumors is not well established. We recently have shown that certain leukemias not only produce VEGF but also selectively express functional VEGF receptors (VEGFRs), such as VEGFR-2 (Flk-1, KDR) and VEGFR1 (Flt1), resulting in the generation of an autocrine loop. Here, we examined the relative contribution of paracrine (EC-dependent) and autocrine (EC-independent) VEGF/VEGFR signaling pathways, by using a human leukemia model, where autocrine and paracrine VEGF/VEGFR loops could be selectively inhibited by neutralizing mAbs specific for murine EC (paracrine pathway) or human tumor (autocrine) VEGFRs. Blocking either the paracrine or the autocrine VEGF/VEGFR-2 pathway delayed leukemic growth and engraftment in vivo, but failed to cure inoculated mice. Long-term remission with no evidence of disease was achieved only if mice were treated with mAbs against both murine and human VEGFR-2, whereas mAbs against human or murine VEGFR-1 had no effect on mice survival. Therefore, effective antiangiogenic therapies to treat VEGF-producing, VEGFR-expressing leukemias may require blocking both paracrine and autocrine VEGF/VEGFR-2 angiogenic loops to achieve remission and long-term cure.     

Endothelin inhibits cholangiocarcinoma growth by a decrease in the vascular endothelial growth factor expression

Background: Endothelins (ET‐1, ET‐2, ET‐3) are peptides with vasoactive properties interacting with ET_A and ET_B receptors. ET‐1 inhibits secretin‐stimulated ductal secretion (hallmark of cholangiocyte growth) of cholestatic rats by interaction with ET receptors. Aim: The aims of the studies were to evaluate (i) the effect of ET‐1 on cholangiocarcinoma growth in Mz‐ChA‐1 cells and nude mice and (ii) whether ET‐1 regulation of cholangiocarcinoma growth is associated with changes in the expression of vascular endothelial growth factor‐A (VEGF‐A), VEGF‐C, VEGF receptor‐2 (VEGFR‐2) and VEGFR‐3. Methods: We determined the expression of ET_A and ET_B receptors on normal and malignant (Mz‐ChA‐1) cholangiocytes and human cholangiocarcinoma tissue and the effect of ET‐1 on the proliferation and expression of VEGF‐A, VEGF‐C (regulators of tumour angiogenesis) and its receptors, VEGFR‐2 and VEGFR‐3, in Mz‐ChA‐1 cells. In vivo, Mz‐ChA‐1 cells were injected into the flanks of athymic mice and injections of ET‐1 or saline into the tumours were performed daily. The effect of ET‐1 on tumour size, cell proliferation, apoptosis, collagen quantity and the expression of VEGF‐A and VEGF‐C and VEGFR‐2 and VEGFR‐3 were measured after 73 days. Results: Higher expression of ET_A and ET_B was observed in malignant compared with normal cholangiocytes. ET‐1 inhibited proliferation and VEGF‐A, VEGF‐C, VEGFR‐2 and VEGFR‐3 expression of Mz‐ChA‐1 cells. Chronic ET‐1 treatment decreased tumour volume, tumour cell proliferation and VEGF‐A and VEGF‐C expression but increased apoptosis and collagen tissue deposition compared with controls. Conclusions: Modulation of VEGF‐A and VEGF‐C (by ET‐1) may be important for managing cholangiocarcinoma growth.     

GW654652, the pan-inhibitor of VEGF receptors, blocks the growth and migration of multiple myeloma cells in the bone marrow microenvironment

Previous studies have shown that the multiple myeloma (MM) cell line and MM patient cells express high-affinity vascular endothelial growth factor (VEGF) receptor-1 or Fms-like tyrosine kinase-1 (Flt-1) but not VEGF receptor-2 or Flk-1/kinase insert domain-containing receptor (Flk-1/KDR) and that VEGF triggers MM cell proliferation through a mitogen-activated protein kinase (MAPK)-dependent pathway and migration through a protein kinase C (PKC)-dependent pathway. The present study evaluates the efficacy of the small molecule tyrosine-kinase inhibitor GW654652, which inhibits all 3 VEGF receptors with similar potency. We show that GW654652 acts directly on MM cells and in the bone marrow microenvironment. Specifically, GW654652 (1-10 microg/mL) inhibits, in a dose-dependent fashion, VEGF-triggered migrational activity and cell proliferation of MM cell lines that are sensitive and resistant to conventional therapy. As expected from our previous studies of VEGF-induced signaling and sequelae in MM cells, GW654652 blocked VEGF-induced Flt-1 phosphorylation and downstream activation of AKT-1 and MAPK-signaling cascades. Importantly, GW654652 also inhibits interleukin-6 and VEGF secretion and proliferation of MM cells induced by tumor cell binding to bone marrow (BM) stromal cells. The activity of a pan-VEGF receptor inhibitor against MM cells in the BM milieu, coupled with its lack of major toxicity in preclinical mouse models, provides the framework for clinical trials of this drug class to improve patient outcome in MM.     

IQGAP1, a novel vascular endothelial growth factor receptor binding protein, is involved in reactive oxygen species--dependent endothelial migration and proliferation

Endothelial cell (EC) proliferation and migration are important for reendothelialization and angiogenesis. We have demonstrated that reactive oxygen species (ROS) derived from the small GTPase Rac1-dependent NAD(P)H oxidase are involved in vascular endothelial growth factor (VEGF)-mediated endothelial responses mainly through the VEGF type2 receptor (VEGFR2). Little is known about the underlying molecular mechanisms. IQGAP1 is a scaffolding protein that controls cellular motility and morphogenesis by interacting directly with cytoskeletal, cell adhesion, and small G proteins, including Rac1. In this study, we show that IQGAP1 is robustly expressed in ECs and binds to the VEGFR2. A pulldown assay using purified proteins demonstrates that IQGAP1 directly interacts with active VEGFR2. In cultured ECs, VEGF stimulation rapidly promotes recruitment of Rac1 to IQGAP1, which inducibly binds to VEGFR2 and which, in turn, is associated with tyrosine phosphorylation of IQGAP1. Endogenous IQGAP1 knockdown by siRNA shows that IQGAP1 is involved in VEGF-stimulated ROS production, Akt phosphorylation, endothelial migration, and proliferation. Wound assays reveal that IQGAP1 and phosphorylated VEGFR2 accumulate and colocalize at the leading edge in actively migrating ECs. Moreover, we found that IQGAP1 expression is dramatically increased in the VEGFR2-positive regenerating EC layer in balloon-injured rat carotid artery. These results suggest that IQGAP1 functions as a VEGFR2-associated scaffold protein to organize ROS-dependent VEGF signaling, thereby promoting EC migration and proliferation, which may contribute to repair and maintenance of the functional integrity of established blood vessels.     

Role of protein tyrosine phosphatase 1B in vascular endothelial growth factor signaling and cell-cell adhesions in endothelial cells

Vascular endothelial growth factor (VEGF) binding induces phosphorylation of VEGF receptor (VEGFR)2 in tyrosine, which is followed by disruption of VE-cadherin-mediated cell-cell contacts of endothelial cells (ECs), thereby stimulating EC proliferation and migration to promote angiogenesis. Tyrosine phosphorylation events are controlled by the balance of activation of protein tyrosine kinases and protein tyrosine phosphatases (PTPs). Little is known about the role of endogenous PTPs in VEGF signaling in ECs. In this study, we found that PTP1B expression and activity are markedly increased in mice hindlimb ischemia model of angiogenesis. In ECs, overexpression of PTP1B, but not catalytically inactive mutant PTP1B-C/S, inhibits VEGF-induced phosphorylation of VEGFR2 and extracellular signal-regulated kinase 1/2, as well as EC proliferation, whereas knockdown of PTP1B by small interfering RNA enhances these responses, suggesting that PTP1B negatively regulates VEGFR2 signaling in ECs. VEGF-induced p38 mitogen-activated protein kinase phosphorylation and EC migration are not affected by PTP1B overexpression or knockdown. In vivo dephosphorylation and cotransfection assays reveal that PTP1B binds to VEGFR2 cytoplasmic domain in vivo and directly dephosphorylates activated VEGFR2 immunoprecipitates from human umbilical vein endothelial cells. Overexpression of PTP1B stabilizes VE-cadherin-mediated cell-cell adhesions by reducing VE-cadherin tyrosine phosphorylation, whereas PTP1B small interfering RNA causes opposite effects with increasing endothelial permeability, as measured by transendothelial electric resistance. In summary, PTP1B negatively regulates VEGFR2 receptor activation via binding to the VEGFR2, as well as stabilizes cell-cell adhesions through reducing tyrosine phosphorylation of VE-cadherin. Induction of PTP1B by hindlimb ischemia may represent an important counterregulatory mechanism that blunts overactivation of VEGFR2 during angiogenesis in vivo.     

Regulation of the vascular endothelial growth factor (VEGF) receptor Flk-1/KDR by estradiol through VEGF in uterus

The induction of vascular endothelial growth factor (VEGF) expression by 17beta-estradiol (E(2)) in many target cells, including epithelial cells, fibroblasts and smooth muscle cells, suggests a role for this hormone in the modulation of angiogenesis and vascular permeability. We have already described a cyclic increase in Flk-1/KDR-expressing capillaries in the human endometrium during the proliferative and mid-secretory phases, strongly suggestive of an E(2) effect on Flk-1/KDR expression in the endometrial capillaries. However, it is unclear whether these processes are due to a direct effect of E(2) on endothelial cells. Using immunohistochemistry, we report an increase in Flk-1/KDR expression in endometrial capillaries of ovariectomized mice treated with E(2), or both E(2) and progesterone. This process is mediated through estrogen receptor (ER) activation. In vitro experiments using quantitative RT-PCR analysis demonstrate that Flk-1/KDR expression was not regulated by E(2) in human endothelial cells from the microcirculation (HMEC-1) or macrocirculation (HUVEC), even in endothelial cells overexpressing ERalpha or ERbeta after ER-mediated adenovirus infection. In contrast, Flk-1/KDR expression was up-regulated by VEGF itself, in a time- and dose-dependent manner, with the maximal response at 10 ng/ml. Thus, we suggest that E(2) up-regulates Flk-1/KDR expression in vivo in endothelial cells mainly through the modulation of VEGF by a paracrine mechanism. It is currently unknown whether or not the endothelial origin might account for differences in the E(2)-modulation of VEGF receptor expression, particularly in relation to the vascular bed of sex steroid-responsive tissues.     

Neurogenesis in dentate subgranular zone and rostral subventricular zone after focal cerebral ischemia in the rat

Because neurogenesis persists in the adult mammalian brain and can be regulated by physiological and pathological events, we investigated its possible involvement in the brain's response to focal cerebral ischemia. Ischemia was induced by occlusion of the middle cerebral artery in the rat for 90 min, and proliferating cells were labeled with 5-bromo-2'-deoxyuridine-5'-monophosphate (BrdUrd) over 2-day periods before sacrificing animals 1, 2 or 3 weeks after ischemia. Ischemia increased the incorporation of BrdUrd into cells in two neuroproliferative regions-the subgranular zone of the dentate gyrus and the rostral subventricular zone. Both effects were bilateral, but that in the subgranular zone was more prominent on the ischemic side. Cells labeled with BrdUrd coexpressed the immature neuronal markers doublecortin and proliferating cell nuclear antigen but did not express the more mature cell markers NeuN and Hu, suggesting that they were nascent neurons. These results support a role for ischemia-induced neurogenesis in what may be adaptive processes that contribute to recovery after stroke.