N-formyl peptide receptor 3 (FPR3) departs from the homologous FPR2/ALX receptor with regard to the major processes governing chemoattractant receptor regulation, expression at the cell surface, and phosphorylation

Among human N-formyl peptide chemoattractant receptors, FPR2/ALX and FPR3 share the highest degree of amino acid identity (83%), and trigger similar cell responses upon ligand binding. Although FPR2/ALX is a promiscuous receptor, FPR3 has only one specific high affinity ligand, F2L, and a more restricted tissue/cell distribution. In this study, we showed that FPR2/ALX behaved as the prototypical receptor FPR1. The agonist-dependent phosphorylation used a hierarchical mechanism with a prominent role of Ser(329), Thr(332), and Thr(335). Phosphorylation of FPR2/ALX was essential for its desensitization but the lack of phosphorylation did not result in enhanced or sustained responses. In contrast, resting FPR3 displayed a marked level of phosphorylation, which was only slightly increased upon agonist stimulation. Another noticeable difference between the two receptors was their subcellular distribution in unstimulated cells. Although FPR2/ALX was evenly distributed at the plasma membrane FPR3 was localized in small intracellular vesicles. By swapping domains between FPR2/ALX and FPR3, we uncovered the determinants involved in the basal phosphorylation of FPR3. Experiments aimed at monitoring receptor-bound antibody uptake showed that the intracellular distribution of FPR3 resulted from a constitutive internalization that was independent of C terminus phosphorylation. Unexpectedly, exchanging residues 1 to 53, which encompass the N-terminal extracellular region and the first transmembrane domain, between FPR2/ALX and FPR3 switched localization of the receptors from the plasma membrane to intracellular vesicles and vice versa. A clathrin-independent, possibly caveolae-dependent, mechanism was involved in FPR3 constitutive internalization. The peculiar behavior of FPR3 most probably serves distinct physiological functions that remain largely unknown.     

High molecular weight kininogen activates B2 receptor signaling pathway in human vascular endothelial cells

The nonenzymatic cofactor high molecular weight kininogen (HK) is a precursor of bradykinin (BK). The production of BK from HK by plasma kallikrein has been implicated in the pathogenesis of inflammation and vascular injury. However, the functional role of HK in the absence of prekallikrein (PK), the proenzyme of plasma kallikrein, on vascular endothelial cells is not fully defined. In addition, no clinical abnormality is seen in PK-deficient patients. Therefore, an investigation into the effect of HK, in the absence of PK, on human pulmonary artery endothelial cell (HPAEC) function was performed. HK caused a marked and dose-dependent increase in the intracellular calcium [Ca(2+)](i) level in HPAEC. Gd(3+) and verapamil potentiated the HK-induced increase in [Ca(2+)](i). HK-induced Ca(2+) increase stimulated endothelial nitric oxide (NO) and prostacyclin (PGI(2)) production. The inhibitors of B(2) receptor-dependent signaling pathway impaired HK-mediated signal transduction in HPAEC. HK had no effect on endothelial permeability at physiological concentration. This study demonstrated that HK regulates endothelial cell function. HK could play an important role in maintaining normal endothelial function and blood flow and serve as a cardioprotective peptide.     

Activated protein C enhances human keratinocyte barrier integrity via sequential activation of epidermal growth factor receptor and Tie2

Keratinocytes play a critical role in maintaining epidermal barrier function. Activated protein C (APC), a natural anticoagulant with anti-inflammatory and endothelial barrier protective properties, significantly increased the barrier impedance of keratinocyte monolayers, measured by electric cell substrate impedance sensing and FITC-dextran flux. In response to APC, Tie2, a tyrosine kinase receptor, was rapidly activated within 30 min, and relocated to cell-cell contacts. APC also increased junction proteins zona occludens, claudin-1 and VE-cadherin. Inhibition of Tie2 by its peptide inhibitor or small interfering RNA abolished the barrier protective effect of APC. Interestingly, APC did not activate Tie2 through its major ligand, angiopoietin-1, but instead acted by binding to endothelial protein C receptor, cleaving protease-activated receptor-1 and transactivating EGF receptor. Furthermore, when activation of Akt, but not ERK, was inhibited, the barrier protective effect of APC on keratinocytes was abolished. Thus, APC activates Tie2, via a mechanism requiring, in sequential order, the receptors, endothelial protein C receptor, protease-activated receptor-1, and EGF receptor, which selectively enhances the PI3K/Akt signaling to enhance junctional complexes and reduce keratinocyte permeability.     

Characterization and development of a peptide substrate-based phosphate transfer assay for the human vascular endothelial growth factor receptor-2 tyrosine kinase

Vascular endothelial growth factor (VEGF), a critical regulator in angiogenesis, exerts its angiogenic effect via binding to its receptor, VEGF receptor-2 tyrosine kinase (VEGFR2) or kinase insert domain-containing receptor (Kdr), on the surface of endothelial cells. Kdr-mediated signaling plays an important role in the proliferation, migration, differentiation, and survival of endothelial cells. Therefore, the inhibition of this signaling pathway represents a promising therapeutic approach for the discovery of novel anticancer agents by destabilizing the progression of solid tumors via abrogating tumor-induced angiogenesis. To explore Kdr as an anticancer target and further characterize the enzyme, we purified a cytoplasmic domain of human Kdr (Kdr-CD) and characterized its autophosphorylation activity. We also designed and synthesized peptides containing amino acid sequences corresponding to the autophosphorylation sites of Kdr and developed a simple, robust, high-throughput assay for measuring the phosphate transfer activity of the enzyme. This assay was validated by the experiments showing that the phosphate transfer activity of the purified Kdr-CD required Mg2+ or Mn2+ and preactivation by adenosine 5'-triphosphate (ATP) and was inhibited by known Kdr inhibitors. Using this assay, we examined effects of Mg2+ and Mn2+ on the enzyme activity; optimized the concentrations of Kdr-CD, peptide and ATP substrates, and metal ions in the assay; and determined the kinetic properties of the enzyme for the peptide and ATP as well as IC50 values of two known Kdr inhibitors. Thus, the results of these studies have validated the utilities of this assay for biochemical characterizations of the enzyme and its inhibitors. This approach of designing peptides corresponding to the autophosphorylation sites of Kdr as substrates for the enzyme has general practical implications to other kinases.     

Regulation of vascular endothelial growth factor receptor-2 expression in pancreatic cancer cells by Sp proteins

Vascular endothelial growth factor receptor-2 (VEGFR2/KDR) is an important mediator of angiogenesis, and VEGFR2 mRNA is expressed in several pancreatic cancer cell lines. Deletion analysis of the VEGFR2 promoter in Panc-1, AsPC-1, and MiaPaCa-2 pancreatic cancer cells shows that the proximal region of the promoter is primarily responsible for VEGFR2 expression, and two GC-rich sites at -58 and -44 are critical elements in all three cell lines. Panc-1, AsPC-1, and MiaPaCa-2 cells also express Sp1, Sp3, and Sp4 proteins which bind to the GC-rich region of the VEGFR2 promoter in electrophoretic mobility shift and chromatin immunoprecipitation assays. RNA interference with small inhibitory RNAs for Sp1, Sp3, and Sp4 decreases VEGFR2 mRNA and reporter gene activity in transfection assays, confirming that VEGFR2 expression in pancreatic cancer cells is regulated by Sp proteins. These results suggest that VEGFR2 cannot only be targeted by receptor tyrosine kinase inhibitors but also by drugs that downregulate Sp proteins or block Sp-dependent transactivation.     

The P2Y(2) nucleotide receptor mediates tissue factor expression in human coronary artery endothelial cells

The discovery of the role of P2Y(12) receptor in platelet aggregation leads to a new anti-thrombotic drug Plavix; however, little is known about non-platelet P2Y receptors in thrombosis. This study tested the hypothesis that endothelial P2Y receptor(s) mediates up-regulation of tissue factor (TF), the initiator of coagulation cascade. Stimulation of human coronary artery endothelial cells (HCAEC) by UTP/ATP increased the mRNA level of TF but not of its counterpart-tissue factor pathway inhibitor, which was accompanied by up-regulation of TF protein and cell surface activity. RT-PCR revealed a selective expression of P2Y(2) and P2Y(11) receptors in HCAEC. Consistent with this, TF up-regulation was inhibited by suramin or by siRNA silencing of P2Y(2) receptor, but not by NF-157, a P2Y(11)-selective antagonist, suggesting a role for the P2Y(2) receptor. In addition, P2Y(2) receptor activated ERK1/2, JNK, and p38 MAPK pathways without affecting the positive NF-κB and negative AKT regulatory pathways of TF expression. Furthermore, TF up-regulation was abolished or partially suppressed by inhibition of p38 or JNK but not ERK1/2. Interestingly, blockade of the PLC/Ca(2+) pathway did not affect P2Y(2) receptor activation of p38, JNK, and TF induction. However, blockade of Src kinase reduced phosphorylation of p38 but not JNK, eliminating TF induction. In contrast, inhibition of Rho kinase reduced phosphorylation of JNK but not p38, decreasing TF expression. These findings demonstrate that P2Y(2) receptor mediates TF expression in HCAEC through new mechanisms involving Src/p38 and Rho/JNK pathways, possibly contributing to a pro-thrombotic status after vascular injury.     

The antifungal drug itraconazole inhibits vascular endothelial growth factor receptor 2 (VEGFR2) glycosylation, trafficking, and signaling in endothelial cells

Itraconazole is a safe and widely used antifungal drug that was recently found to possess potent antiangiogenic activity. Currently, there are four active clinical trials evaluating itraconazole as a cancer therapeutic. Tumor growth is dependent on angiogenesis, which is driven by the secretion of growth factors from the tumor itself. We report here that itraconazole significantly inhibited the binding of vascular endothelial growth factor (VEGF) to VEGF receptor 2 (VEGFR2) and that both VEGFR2 and an immediate downstream substrate, phospholipase C γ1, failed to become activated after VEGF stimulation. These effects were due to a defect in VEGFR2 trafficking, leading to a decrease in cell surface expression, and were associated with the accumulation of immature N-glycans on VEGFR2. Small molecule inducers of lysosomal cholesterol accumulation and mammalian target of rapamycin (mTOR) inhibition, two previously reported itraconazole activities, failed to recapitulate itraconazole's effects on VEGFR2 glycosylation and signaling. Likewise, glycosylation inhibitors did not alter cholesterol trafficking or inhibit mTOR. Repletion of cellular cholesterol levels, which was known to rescue the effects of itraconazole on mTOR and cholesterol trafficking, was also able to restore VEGFR2 glycosylation and signaling. This suggests that the new effects of itraconazole occur in parallel to those previously reported but are downstream of a common target. We also demonstrated that itraconazole globally reduced poly-N-acetyllactosamine and tetra-antennary complex N-glycans in endothelial cells and induced hypoglycosylation of the epidermal growth factor receptor in a renal cell carcinoma line, suggesting that itraconazole's effects extend beyond VEGFR2.     

Transactivation of the epidermal growth factor receptor by heat shock protein 90 via Toll-like receptor 4 contributes to the migration of glioblastoma cells

Extracellular heat shock protein HSP90α was reported to participate in tumor cell growth, invasion, and metastasis formation through poorly understood signaling pathways. Herein, we show that extracellular HSP90α favors cell migration of glioblastoma U87 cells. More specifically, externally applied HSP90α rapidly induced endocytosis of EGFR. This response was accompanied by a transient increase in cytosolic Ca(2+) appearing after 1-3 min of treatment. In the presence of EGF, U87 cells showed HSP90α-induced Ca(2+) oscillations, which were reduced by the ATP/ADPase, apyrase, and inhibited by the purinergic P(2) inhibitor, suramin, suggesting that ATP release is requested. Disruption of lipid rafts with methyl β-cyclodextrin impaired the Ca(2+) rise induced by extracellular HSP90α combined with EGF. Specific inhibition of TLR4 expression by blocking antibodies suppressed extracellular HSP90α-induced Ca(2+) signaling and the associated cell migration. HSPs are known to bind lipopolysaccharides (LPSs). Preincubating cells with Polymyxin B, a potent LPS inhibitor, partially abrogated the effects of HSP90α without affecting Ca(2+) oscillations observed with EGF. Extracellular HSP90α induced EGFR phosphorylation at Tyr-1068, and this event was prevented by both the protein kinase Cδ inhibitor, rottlerin, and the c-Src inhibitor, PP2. Altogether, our results suggest that extracellular HSP90α transactivates EGFR/ErbB1 through TLR4 and a PKCδ/c-Src pathway, which induces ATP release and cytosolic Ca(2+) increase and finally favors cell migration. This mechanism could account for the deleterious effects of HSPs on high grade glioma when released into the tumor cell microenvironment.     

Large putative PEST-like sequence motif at the carboxyl tail of human calcium receptor directs lysosomal degradation and regulates cell surface receptor level

A deletion between amino acid residues Ser(895) and Val(1075) in the carboxyl terminus of the human calcium receptor (hCaR), which causes autosomal dominant hypocalcemia, showed enhanced signaling activity and increased cell surface expression in HEK293 cells (Lienhardt, A., Garabédian, M. G., Bai, M., Sinding, C., Zhang, Z., Lagarde, J. P., Boulesteix, J., Rigaud, M., Brown, E. M., and Kottler, M. L. (2000) J. Clin. Endocrinol. Metab. 85, 1695-1702). To identify the underlying mechanism(s) for these increases, we investigated the effects of carboxyl tail truncation and deletion in hCaR mutants using a combination of biochemical and cell imaging approaches to define motifs that participate in regulating cell surface numbers of this G protein-coupled receptor. Our data indicate a rapid constitutive receptor internalization of the cell surface hCaR, accumulating in early (Rab7 positive) and late endosomal (LAMP1 positive) sorting compartments, before targeting to lysosomes for degradation. Recycling of hCaR back to the cell surface was also evident. Truncation and deletion mapping defined a 51-amino acid sequence between residues 920 and 970 that is required for targeting to lysosomes and degradation but not for internalization or recycling of the receptor. No singular sequence motif was identified, instead the required sequence elements seem to distribute throughout this entire interval. This interval includes a high proportion of acidic and hydroxylated amino acid residues, suggesting a similarity to PEST-like degradation motif (PESTfind score of +10) and several glutamine repeats. The results define a novel large PEST-like sequence that participates in the sorting of internalized hCaR routed to the lysosomal/degradation pathway that regulates cell surface receptor numbers.     

Expression of vascular endothelial growth factor (VEGF) and its cognate receptors in human pheochromocytomas

Pheochromocytomas are well-vascularized tumors, suggesting that a potent angiogenic factor may be involved in the mechanism of their formation. As vascular endothelial growth factor (VEGF) is a potent mitogen for vascular endothelial cells, here we have investigated the mRNA and protein expression of VEGF and the mRNA expression of its two receptors (Flt-1 and Flk-1/KDR) in pheochromocytomas tissue. An increase in VEGF mRNA (mainly isoforms VEGF(121) and VEGF(165)) and in VEGF protein expression were observed by semi-quantitative RT-PCR and Western blot, respectively, compared to normal adrenomedullary tissue. Flk-1/KDR, and Flt-1 levels of mRNA were also increased markedly in tumors and correlated with levels of VEGF mRNA. Therefore, we speculate that upregulation of VEGF expression and its receptors might be important in the pathogenesis of pheochromocytomas.     

Neuropilin-1 forms complexes with vascular endothelial growth factor receptor-2 during megakaryocytic differentiation of UT-7/TPO cells

We investigated whether the gene expression of vascular endothelial growth factor (VEGF) and its receptors (VEGFR and neuropilin-1 [NRP-1]) could be specifically regulated during the megakaryocytic differentiation of human thrombopoietin (TPO)-dependent UT-7/TPO cells. Undifferentiated UT-7/TPO cells expressed a functional VEGFR-2, leading to VEGF binding and VEGF₁₆₅-induced tyrosine phosphorylation, cell proliferation, and apoptosis inhibition. The megakaryocytic differentiation of UT-7/TPO cells on treatment with phorbol myristate acetate (PMA) was accompanied by a marked up-regulation of NRP-1 mRNA and protein expression and by an increase in VEGF-binding activity, which was mainly mediated by VEGFR-2. VEGF₁₆₅ promoted the formation of complexes containing NRP-1 and VEGFR-2 in undifferentiated UT-7/TPO cells in a dose-dependent manner. Unlike human umbilical vein endothelial cells, PMA-differentiated UT-7/TPO cells exhibited complex formation between NRP-1 and VEGFR-2 even in the absence of VEGF₁₆₅. These findings suggest that NRP-1-VEGFR-2-complex formation may contribute to effective cellular functions mediated by VEGF₁₆₅ in megakaryocytic cells.     

Dissecting mannose 6-phosphate-insulin-like growth factor 2 receptor complexes that control activation and uptake of plasminogen in cells

The plasminogen (Plg) activation cascade on the cell surface plays a central role in cell migration and is involved in a plethora of physiological and pathological processes. Its regulation is coordinated by many receptors, in particular the urokinase-type plasminogen activator receptor (uPAR, CD87), receptors that physically interact and functionally cooperate with uPAR, and Plg binding molecules. Here we studied the impact of one of the Plg binding molecules, the mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P-IGF2R, CD222), on cellular Plg activation. By developing both in vitro and in vivo Plg activation assays on size-fractionated lysates of M6P-IGF2R-silenced cells, we identified Plg-associated complexes with M6P-IGF2R as the regulatory factor. Using lipid raft preserving versus dissolving detergents, we found lipid dependence of the Plg regulatory function of these complexes. Furthermore, M6P-IGF2R-silencing in uPAR-positive human cell lines reduced internalization of Plg, resulting in elevated Plg activation. In contrast, the expression of human M6P-IGF2R in mouse embryonic fibroblasts derived from M6P-IGF2R knock-out mice enhanced Plg internalization. Finally, peptide 18-36 derived from the Plg-binding site within M6P-IGF2R enhanced Plg uptake. Thus, by targeting Plg to endocytic pathways, M6P-IGF2R appears to control Plg activation within cells that might be important to restrict plasmin activity to specific sites and substrates.     

Cyclooxygenase-2 and adenylate cyclase/protein kinase A signaling pathway enhances angiogenesis through induction of vascular endothelial growth factor in rat sponge implants

Angiogenesis is reportedly enhanced by prostaglandins (PGs). In the present experiment, we tested whether or not COX-2 and adenylate cyclase/protein kinase A (AC/PKA)-dependent VEGF induction enhanced angiogenesis in this model. Angiogenesis was enhanced by topical injection of human recombinant basic fibroblast growth factor (bFGF). The enhanced angiogenesis by bFGF was inhibited by indomethacin or selective COX-2 inhibitors, NS398, nimesulide, and JTE-522. Topical daily injections of 8-bromo-cAMP enhanced angiogenesis in a dose-dependent manner. Forskolin, an activator of AC, also facilitated angiogenesis in a dose-dependent manner, as did amrinone, an inhibitor of phosphodiesterase. VEGF induction was confirmed by the increased levels in the fluids in the sponge matrix after topical injection of 8-bromo-cAMP. Immunohistochemical investigation further revealed the VEGF-expressed cells in the sponge granulation tissues to be fibroblasts, and the intensity of positive reactions was enhanced by bFGF, 8-bromo-cAMP, forskolin, and amrinone. Angiogenesis was inhibited by indometacin or selective COX-2 inhibitors, NS-398, nimesulide, and JTE-522. In addition, angiogenesis without topical injections of the above compounds was also suppressed by SQ22,536, an inhibitor for AC. or H-89, an inhibitor for PKA, with concomitant reductions in VEGF levels. Daily topical injections of neutralizing antibody or anti-sense oligonucleotide against VEGF significantly suppressed angiogenesis. These results suggested that COX-2 and AC/PKA-dependent induction of VEGF certainly enhanced angiogenesis, and that pharmacological tools for controlling this signaling pathway may be able to facilitate the management of conditions involving angiogenesis.     

Angiogenic functions of voltage-gated Na+ Channels in human endothelial cells: modulation of vascular endothelial growth factor (VEGF) signaling

Voltage-gated sodium channel (VGSC) activity has previously been reported in endothelial cells (ECs). However, the exact isoforms of VGSCs present, their mode(s) of action, and potential role(s) in angiogenesis have not been investigated. The main aims of this study were to determine the role of VGSC activity in angiogenic functions and to elucidate the potentially associated signaling mechanisms using human umbilical vein endothelial cells (HUVECs) as a model system. Real-time PCR showed that the primary functional VGSC α- and β-subunit isoforms in HUVECs were Nav1.5, Nav1.7, VGSCβ1, and VGSCβ3. Western blots verified that VGSCα proteins were expressed in HUVECs, and immunohistochemistry revealed VGSCα expression in mouse aortic ECs in vivo. Electrophysiological recordings showed that the channels were functional and suppressed by tetrodotoxin (TTX). VGSC activity modulated the following angiogenic properties of HUVECs: VEGF-induced proliferation or chemotaxis, tubular differentiation, and substrate adhesion. Interestingly, different aspects of angiogenesis were controlled by the different VGSC isoforms based on TTX sensitivity and effects of siRNA-mediated gene silencing. Additionally, we show for the first time that TTX-resistant (TTX-R) VGSCs (Nav1.5) potentiate VEGF-induced ERK1/2 activation through the PKCα-B-RAF signaling axis. We postulate that this potentiation occurs through modulation of VEGF-induced HUVEC depolarization and [Ca(2+)](i). We conclude that VGSCs regulate multiple angiogenic functions and VEGF signaling in HUVECs. Our results imply that targeting VGSC expression/activity could be a novel strategy for controlling angiogenesis.     

Epidermal growth factor receptor coexpression modulates susceptibility to Herceptin in HER2/neu overexpressing breast cancer cells via specific erbB-receptor interaction and activation

BACKGROUND: Growth factors and Herceptin specifically and differentially modulate cell proliferation of tumor cells. However, the mechanism of action on erbB-receptor level is incompletely understood. We evaluated Herceptin's capacity to modulate erbB-receptor activation and interaction on the cell surface level and thereby potentially impair cell proliferation of HER2/neu (c-erbB2) overexpressing breast cancer cells, both in the presence and absence of relevant growth factors. METHODS: BT474 and SK-BR-3 breast cancer cell lines were treated with Epidermal Growth Factor (EGF), Heregulin, and with Herceptin in different combinations. Kinetics of cell proliferation were evaluated flow cytometrically based on BrdU-labeling. Fluorescence Resonance Energy Transfer, ELISAs and phosphorylation site specific Western Blotting was performed to investigate erbB-receptor interaction and activation. RESULTS: EGF induced EGFR/EGFR and EGFR/c-erbB2 interactions correlate with stimulation of cell proliferation in BT474 cells. Both homo- and heterodimerization are considerably less pronounced in SK-BR-3 cells and heterointeraction is additionally reduced by EGF treatment, causing inhibition of cell proliferation. Heregulin stimulates cell proliferation extensively in both cell lines. Herceptin drives BT474 cells more efficiently into quiescence than it does with SK-BR-3 cells and thereby blocks cell cycle progress. In SK-BR-3 Herceptin treatment causes c-erbB2 phosphorylation of Y877 and Y1248, EGF induces Y877 and Y1112 phosphorylation. The Y1112 phosphorylation site, activated by EGF in SK-BR-3 cell, is bypassed in BT474. In addition the inhibitory capacity of Herceptin on BT474 and SK-BR-3 cell proliferation depends on the presence and absence of growth factors to a various extent. CONCLUSION: The growth inhibitory effect of Herceptin on c-erbB2 overexpressing breast cancer cells is considerably modulated by EGFR coexpression and consequently EGFR/c-erbB2 homo- and heterointeractions, as well as the presence or absence of growth factors. C-erbB2 overexpression alone is insufficient to predict the impact of growth factors and antibodies on cell proliferation. The optimization and specification of therapeutic approaches based on erbB-receptor targeting requires to account for EGFR coexpression as well as the potential presence of erbB-receptor relevant growth factors.     

血管内皮生长因子及其受体与Cyclin E-CDK2在肝癌发生发展过程中的表达变化

目的通过观察血管内皮生长因子(VEGF)及其受体和细胞周期蛋白E(Cyclin E)在肝癌模型大鼠肝脏中表达情况,探讨VEGF与细胞周期相关蛋白在肝癌发生发展过程中的作用。方法建立诱发性肝癌模型,采用酶联免疫吸附试验检测血清中VEGF量的变化,免疫组化技术检测VEGFR1、Cyclin E和细胞周期蛋白依赖性激酶(CDK2)的表达情况。结果血清中的VEGF含量在对照组中最低,在实验组中逐渐增多,以癌变期含量最高。VEGFR1、Cyclin E和CDK2蛋白表达的平均光密度值均随着肝癌的发生发展有增高的趋势,大鼠血清中的VEGF量与肝脏组织中VEGFR1、Cyclin E和CDK2蛋白表达的平均光密度值随着肝癌的发生发展呈正相关(r=0.834,F=42.1274,P<0.05)。结论 VEGF及其受体VEGFR1在肝癌发生发展中异常表达,促进肝癌的发生发展,可能与Cyclin E、CDK2细胞周期蛋白异常表达有关。     

Complex formation between tissue transglutaminase II (tTG) and vascular endothelial growth factor receptor 2 (VEGFR-2): proposed mechanism for modulation of endothelial cell response to VEGF

We have recently demonstrated that thrombin-activated FXIII (FXIIIA-subunit), a plasma transglutaminase, activates VEGFR-2 by crosslinking it with the alpha(v)beta(3) integrin on the surface of endothelial cells (EC), thereby stimulating angiogenesis. Tissue transglutaminase (tTG), which is functionally and structurally related to FXIIIA, is expressed by numerous cell types, among them EC. However, its role in EC function has not been fully characterized. In the present study, we investigated the potential involvement of tTG in angiogenesis. Using co-immunoprecipitation and immunofluorescent staining experiments, we observed that tTG forms a complex with VEGFR-2 on the cell surface and within the cytoplasm of EC. Stimulation of EC with VEGF resulted in translocation of the tTG-VEGFR-2 complex from the cytoplasm to the nucleus. In VEGF-treated cells, tTG-VEGFR-2 interaction resulted in incorporation of VEGFR-2 into high molecular weight crosslinked complex (es), as revealed by an antibody against gamma-glutamyl-epsilon-lysine isopeptide bond. tTG -VEGFR-2 association was inhibited by a specific VEGFR-2 protein tyrosine kinase inhibitor (PTKI ), as well as by cystamine, inhibitor of the transglutaminase activity of tTG, but not by bacitracin which inhibits the protein-disulfide isomerase (PDI) activity of tTG. Furthermore, cystamine completely abolished the VEGF-induced nuclear translocation of the tTG-VEGFR-2 complex. Blockade of the crosslinking activity of tTG by cystamine enhanced VEGF-induced migration of EC in Boyden chamber by 31% (P < 0.02), and prolonged VEGF-induced signaling response, as demonstrated by sustained activation of the MAP kinase ERK. Taken together, our findings suggest that endothelial cell tTG might be involved in modulation of the cellular response to VEGF by forming an intracellular complex with VEGFR-2, and mediating its translocation into the nucleus upon VEGF stimulation.     

Role of vascular endothelial growth factor in the communication between human osteoprogenitors and endothelial cells

Proper bone remodeling requires an active process of angiogenesis which in turn supplies the necessary growth factors and stem cells. This tissue cooperation suggests a cross‐talk between osteoblasts and endothelial cells. This work aims to identify the role of paracrine communication through vascular endothelial growth factor (VEGF) in co‐culture between osteoblastic and endothelial cells. Through a well defined direct contact co‐culture model between human osteoprogenitors (HOPs) and human umbilical vein endothelial cells (HUVECs), we observed that HUVECs were able to migrate along HOPs, inducing the formation of specific tubular‐like structures. VEGF₁₆₅ gene expression was detected in the HOPs, was up‐regulated in the co‐cultured HOPs and both Flt‐1 and KDR gene expression increased in co‐cultured HUVECs. However, the cell rearrangement observed in co‐culture was promoted by a combination of soluble chemoattractive factors and not by VEGF₁₆₅ alone. Despite having no observable effect on endothelial cell tubular‐like formation, VEGF appeared to have a crucial role in osteoblastic differentiation since the inhibition of its receptors reduced the co‐culture‐stimulated osteoblastic phenotype. This co‐culture system appears to enhance both primary angiogenesis events and osteoblastic differentiation, thus allowing for the development of new strategies in vascularized bone tissue engineering. J. Cell. Biochem. 106: 390–398, 2009. © 2009 Wiley‐Liss, Inc.