VEGF189 stimulates endothelial cells proliferation and migration in vitro and up-regulates the expression of Flk-1/KDR mRNA

The vascular endothelial growth factor (VEGF) is a critical factor for development of the vascular system in physiological and pathological angiogenesis. This growth factor exists under at least three isoforms, VEGF120/121, VEGF164/165 and VEGF188/189 which are generated by alternative splicing. VEGF isoforms have different affinities for heparan sulphate as well as for VEGF receptors, and may play distinct roles in vascular development. The role of VEGF189 as an endothelial mitogen, however, remains controversial. VEGF189 is almost entirely bound to the cell surface or extracellular matrix, and is considered active after its cleavage and release from its extracellular binding site. In the present study, we demonstrate that VEGF189 induces endothelial cell proliferation and migration in vitro. The 30-60% increase observed with VEGF189 (10 ng/ml) in HUVEC proliferation was similar to that observed with VEGF165. However, the proliferative effect observed with VEGF189 appeared dependent on the origin of the endothelial cell, since the proliferation was clearly observed with HUVEC but not with BAEC or capillary endothelial cells from dermis (HMEC). The effect of VEGF189 on endothelial cell migration was also analyzed using the wound healing and the Boyden chamber assays. The migration effect was observed with BAEC which do not proliferate with VEGF189, suggesting that different mechanisms are involved in proliferation and migration. In addition, VEGF189 as well as VEGF165 induced a 2-fold increase of Flk-1/KDR expression in HUVEC, the receptor involved in proliferation and migration of endothelial cells. In the Matrigel plug assay in vivo, both VEGF189 and 165 (100 ng/ml) increased the infiltration of endothelial cells. These data suggest that VEGF189 induced endothelial cell migration and proliferation under certain circumstances.     

C-reactive protein decreases expression of VEGF receptors and neuropilins and inhibits VEGF165-induced cell proliferation in human endothelial cells

C-reactive protein (CRP) is associated with cardiovascular disease. However, its biological functions for the vascular system are largely unknown. The objective of this study was to determine whether CRP could affect endothelial cell proliferation and expression of VEGF receptors (VEGFRs) and/or neuropilins. Human coronary artery endothelial cells (HCAECs) treated with CRP showed a significant reduction of mRNA levels of VEGFR-2, VEGFR-3, NRP-1, and NRP-2 by 34%, 63%, 41%, and 43%, respectively, as compared to untreated control cells (p < 0.05) by real-time PCR analysis. In addition, VEGF165-induced cell proliferation was determined by [3H]thymidine incorporation and MTS assay as well as capillary-like tube formation on Matrigel. HCAECs pretreated with CRP significantly decreased VEGF165-induced [3H]thymidine incorporation by 73%, MTS absorbance by 44%, and capillary-like tube formation by 54% as compared to CRP-untreated cells (p < 0.05). These data demonstrate that CRP significantly attenuates VEGF165-induced HCAEC proliferation and capillary-like tube formation through downregulation of expression of VEGFRs and NRPs. This study suggests a new molecular mechanism underlying the adverse effect of CRP on the vascular system.     

PAI-1 deficiency reduces liver fibrosis after bile duct ligation in mice through activation of tPA

Plasminogen activator inhibitor-1 (PAI-1) increases injury in several liver, lung and kidney disease models. The objective of this investigation was to assess the effect of PAI-1 deficiency on cholestatic liver fibrosis and determine PAI-1 influenced fibrogenic mechanisms. We found that PAI-1(-/-) mice had less fibrosis than wild type (WT) mice after bile duct ligation. This change correlated with increased tissue-type plasminogen activator (tPA) activity, and increased matrix metalloproteinase-9 (MMP-9), but not MMP-2 activity. Furthermore, there was increased activation of the tPA substrate hepatocyte growth factor (HGF), a known anti-fibrogenic protein. In contrast, there was no difference in hepatic urokinase plasminogen activator (uPA) or plasmin activities between PAI-1(-/-) and WT mice. There was also no difference in the level of transforming growth factor beta 1 (TGF-beta1), stellate cell activation or collagen production between WT and PAI-1(-/-) animals. In conclusion, PAI-1 deficiency reduces hepatic fibrosis after bile duct obstruction mainly through the activation of tPA and HGF.     

Hyperbaric oxygen induces VEGF expression through ERK, JNK and c-Jun/AP-1 activation in human umbilical vein endothelial cells

Summary Hyperbaric oxygen (HBO) is increasingly used in a number of areas of medical practice, such as selected problem infections and wounds. The beneficial effects of HBO in treating ischemia-related wounds may be mediated by stimulating angiogenesis. We sought to investigate VEGF, the main angiogenic regulator, regulated by HBO in human umbilical vein endothelial cells (HUVECs). In this study, we found that VEGF was up regulated both at mRNA and protein levels in HUVECs treated with HBO dose- and time-dependently. Since there are several AP-1 sites in the VEGF promoter, and the c-Jun/AP-1 is activated through stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and extracellular signal regulated kinase (ERK), we further examined the c-Jun, JNK and ERK that might be involved in the VEGF induced by HBO. The VEGF mRNA induced by HBO was blocked by both PD98059 and SP600125, the ERK and JNK inhibitors respectively. HBO induced phospho-ERK and phospho-JNK expressions within 15 min. We further demonstrated that c-Jun phosphorylation was induced within 60 min of HBO treatment. HBO also induced the nuclear AP-1 binding ability within 30–60 min, but the AP-1 induction was blocked by treatment with either the ERK or JNK inhibitor. To verify that the VEGF expression induced by HBO is through the AP-1 trans-activation and VEGF promoter, both the VEGF promoter and AP-1 driving luciferase activity were found increased by the cells treated with HBO. The c-Jun mRNA, which is also driven by AP-1, was also induced by HBO, and the induction of c-Jun was blocked by ERK and JNK inhibitors. We suggest that VEGF induced by HBO is through c-Jun/AP-1 activation, and through simultaneous activation of ERK and JNK pathways.     

Differential involvement of TGF-beta1 in mediating the motogenic effects of TSP-1 on endothelial cells, fibroblasts and oral tumour cells

The role of TSP-1 in tumour growth and angiogenesis remains controversial, with both stimulatory and inhibitory roles proposed. The effects of TSP-1 on the migration of endothelial cells, fibroblast and oral tumour cell lines were examined using the transmembrane assay. TSP-1 induced a bi-phasic effect on human and bovine endothelial cells: stimulation at low concentrations (0.1–10 µg/ml) and inhibition at high concentrations (25–100 µg/ml). FGF-2-stimulated endothelial cell migration was either further stimulated or inhibited by TSP-1, following the same bi-phasic dose response as in the absence of FGF-2. In contrast, TSP-1 stimulated the migration of human fibroblast and oral tumour cells in a dose dependent manner; a plateau was reached with 5–25 µg/ml and no inhibitory effect was observed. These effects were partly neutralised by antibodies to αvβ3 integrin. TGF-β1 (0.1–200ng/ml tested) mimicked the effects of TSP-1 on cell migration. Function-neutralising antibodies to TGF-β1 completely abolished both the stimulatory and inhibitory effects of TSP-1 on endothelial migration, but had no effect on TSP-1-stimulated migration of fibroblast and oral tumour cells. The effects of TGF-β1 were not affected by antibodies to TSP-1. These results indicate that the effects of TSP-1 on endothelial cell migration are mediated by TGF-β1, whereas the effects on fibroblast and tumour cell migration are TGF-β1-independent.     

uPA、PAI-1 的表达和MVD 计数与浸润性乳腺癌侵袭性的关系

目的:探讨尿激酶型纤溶酶原激活剂(uPA)、纤溶酶原激活物抑制剂(PAI-1)的表达和血管形成与浸润性乳腺癌侵袭性的关系。方法:应用免疫组化SP法检测80例浸润性乳腺癌、20例良性乳腺肿瘤组织中uPA、PAI-1的表达情况并进行微血管计数。结果:uPA和PAI-1在浸润性乳腺癌组的阳性表达显著高于良性肿瘤组,且其高表达率与乳腺癌的临床病理参数密切相关(P<0.05);微血管计数在乳腺癌组和良性肿瘤组分别为30.87±7.64、20.28±8.72,两组比较有显著性差异(P<0.01)。uPA与PAI-1在浸润性乳腺癌中的表达呈正相关(P<0.05)。结论:uPA、PAI-1的高表达与浸润性乳腺癌的浸润、转移相关,uPA、PAI-1的高表达和MVD计数可作为评价浸润性乳腺癌侵袭性、评估预后和确定治疗方案的生物学指标。     

uPA、PAI-1的表达和MVD计数与浸润性乳腺癌侵袭性的关系

目的：探讨尿激酶型纤溶酶原激活剂（uPA）、纤溶酶原激活物抑制剂（PAI-1）的表达和血管形成与浸润性乳腺癌侵袭性的关系。方法：应用免疫组化SP法检测80例浸润性乳腺癌、20例良性乳腺肿瘤组织中uPA、PAI-1的表达情况并进行微血管计数。结果：uPA和PAI-1在浸润性乳腺癌组的阳性表达显著高于良性肿瘤组,且其高表达率与乳腺癌的临床病理参数密切相关（P〈0.05）;微血管计数在乳腺癌组和良性肿瘤组分别为30.87±7.64、20.28±8.72,两组比较有显著性差异（P〈0.01）。uPA与PAI-1在浸润性乳腺癌中的表达呈正相关（P〈0.05）。结论：uPA、PAI-1的高表达与浸润性乳腺癌的浸润、转移相关,uPA、PAI-1的高表达和MVD计数可作为评价浸润性乳腺癌侵袭性、评估预后和确定治疗方案的生物学指标。     

Effect of FGF-1 and FGF-2 on VEGF binding to human umbilical vein endothelial cells

When FGF-1 or FGF-2 and VEGF were added together, the mitogenic effect of FGF-1 or FGF-2 and VEGF on HUVEC was additive. However, when HUVECs were preincubated for 2 days with 10 ng/ml FGF-1 in the absence of VEGF, the Scatchard plot of [125I]VEGF binding sites was shifted to the right: both affinity classes of VEGF binding sites were equally affected, such that the total number of sites increased twofold. It is suggested that this type of interaction may be related to tumor angiogenesis and wound repair.     

26S Proteasome regulation of Ankrd1/CARP in adult rat ventricular myocytes and human microvascular endothelial cells

Mutations of the cyclic nucleotide binding domain (CNBD) may disrupt human ether-a-go-go-related gene (hERG) K(+) channel function and lead to hereditary long QT syndrome (LQTS). We identified a novel missense mutation located in close proximity to the CNBD, hERG R744P, in a patient presenting with recurrent syncope and aborted cardiac death triggered by sudden auditory stimuli. Functional properties of wild type (WT) and mutant hERG R744P subunits were studied in Xenopus laevis oocytes using two-electrode voltage clamp electrophysiology and Western blot analysis. HERG R744P channels exhibited reduced activating currents compared to hERG WT (1.48±0.26 versus 3.40±0.29μA; n=40). These findings were confirmed by tail current analysis (hERG R744P, 0.53±0.07μA; hERG WT, 0.97±0.06μA; n=40). Cell surface trafficking of hERG R744P protein subunits was not impaired. To simulate the autosomal-dominant inheritance associated with LQTS, WT and R744P subunits were co-expressed in equimolar ratio. Mean activating and tail currents were reduced by 32% and 25% compared to hERG WT (n=40), indicating that R744P protein did not exert dominant-negative effects on WT channels. The half-maximal activation voltage was not significantly affected by the R744P mutation. This study highlights the significance of in vitro testing to provide mechanistic evidence for pathogenicity of mutations identified in LQTS. The functional defect associated with hERG R744P serves as molecular basis for LQTS in the index patient.     

Ganoderma lucidum suppresses angiogenesis through the inhibition of secretion of VEGF and TGF-beta1 from prostate cancer cells

Ganoderma lucidum (G. lucidum) is a popular medicinal mushroom that has been used as a home remedy for the general promotion of health and longevity in East Asia. The dried powder of G. lucidum, which was recommended as a cancer chemotherapy agent in traditional Chinese medicine, is currently popularly used worldwide in the form of dietary supplements. We have previously demonstrated that G. lucidum induces apoptosis, inhibits cell proliferation, and suppresses cell migration of highly invasive human prostate cancer cells PC-3. However, the molecular mechanism(s) responsible for the inhibitory effects of G. lucidum on the prostate cancer cells has not been fully elucidated. In the present study, we examined the effect of G. lucidum on angiogenesis related to prostate cancer. We found that G. lucidum inhibits the early event in angiogenesis, capillary morphogenesis of the human aortic endothelial cells. These effects are caused by the inhibition of constitutively active AP-1 in prostate cancer cells, resulting in the down-regulation of secretion of VEGF and TGF-beta1 from PC-3 cells. Thus, G. lucidum modulates the phosphorylation of Erk1/2 and Akt kinases in PC-3 cells, which in turn inhibits the activity of AP-1. In summary, our results suggest that G. lucidum inhibits prostate cancer-dependent angiogenesis by modulating MAPK and Akt signaling and could have potential therapeutic use for the treatment of prostate cancer.     

Effect of thalidomide affecting VEGF secretion, cell migration, adhesion and capillary tube formation of human endothelial EA.hy 926 cells

Angiogenesis, new blood vessel formation, is a multistep process, precisely regulated by pro-angiogenic cytokines, which stimulate endothelial cells to migrate, proliferate and differentiate to form new capillary microvessels. Excessive vascular development and blood vessel remodeling appears in psoriasis, rheumatoid arthritis, diabetic retinopathy and solid tumors formation. Thalidomide [alpha-(N-phthalimido)-glutarimide] is known to be a potent inhibitor of angiogenesis, but the mechanism of its inhibitory action remains unclear. The aim of the study was to investigate the potential influence of thalidomide on the several steps of angiogenesis, using in vitro models. We have evaluated the effect of thalidomide on VEGF secretion, cell migration, adhesion as well as in capillary formation of human endothelial cell line EA.hy 926. Thalidomide at the concentrations of 0.01 microM and 10 microM inhibited VEGF secretion into supernatants, decreased the number of formed capillary tubes and increased cell adhesion to collagen. Administration of thalidomide at the concentration of 0.01 microM increased cell migration, while at 10 microM, it decreased cell migration. Thalidomide in concentrations from 0.1 microM to 10 microM did not change cell proliferation of 72-h cell cultures. We conclude that anti-angiogenic action of thalidomide is due to direct inhibitory action on VEGF secretion and capillary microvessel formation as well as immunomodulatory influence on EA.hy 926 cells migration and adhesion.     

Cloning and characterization of angiocidin, a tumor cell binding protein for thrombospondin-1

Thrombospondin-1 (TSP-1) is a matrix protein that has been implicated in mechanisms of tumor progression. Our laboratory previously showed that the CSVTCG (cys-ser-val-thr-cys-gly) sequence of TSP-1 functioned as a tumor cell adhesion domain and CSVTCG peptides as well as an anti-peptide antibody possessed anti-metastatic activity in a murine model of lung metastasis. In a subsequent study, a putative TSP-1 binding protein from lung carcinoma was isolated by CSVTCG-peptide affinity chromatography. In this study, we present the full-length cDNA of this binding protein isolated from a prostate cancer cell (PC3-NI) cDNA library. The purified recombinant protein, termed angiocidin, is a potent inhibitor of tumor growth of Lewis Lung carcinoma in vivo and tumor invasion and angiogenesis in vitro. In addition, the recombinant protein inhibits tumor and endothelial cell proliferation and induces apoptosis. The activity of angiocidin both in vivo and in vitro is partially dependent on its TSP-1 binding activity, since an angiocidin deletion mutant missing a high affinity-binding site for TSP-1 failed to inhibit tumor growth in vivo and was less active in its anti-tumor and anti-angiogenic activities in vitro. These results suggest that the anti-tumor activity of TSP-1 reported in many studies may be mediated in part by binding proteins such as angiocidin. Such proteins may function as tumor-suppressor proteins, which limit the growth of tumors by inhibiting angiogenesis and cell matrix interaction.     

Driving vascular endothelial cell fate of human multipotent Isl1+ heart progenitors with VEGF modified mRNA

Distinct families of multipotent heart progenitors play a central role in the generation of diverse cardiac, smooth muscle and endothelial cell lineages during mammalian cardiogenesis. The identification of precise paracrine signals that drive the cell-fate decision of these multipotent progenitors, and the development of novel approaches to deliver these signals in vivo, are critical steps towards unlocking their regenerative therapeutic potential. Herein, we have identified a family of human cardiac endothelial intermediates located in outflow tract of the early human fetal hearts (OFT-ECs), characterized by coexpression of Isl1 and CD144/vWF. By comparing angiocrine factors expressed by the human OFT-ECs and non-cardiac ECs, vascular endothelial growth factor (VEGF)-A was identified as the most abundantly expressed factor, and clonal assays documented its ability to drive endothelial specification of human embryonic stem cell (ESC)-derived Isl1⁺ progenitors in a VEGF receptor-dependent manner. Human Isl1-ECs (endothelial cells differentiated from hESC-derived ISL1⁺ progenitors) resemble OFT-ECs in terms of expression of the cardiac endothelial progenitor- and endocardial cell-specific genes, confirming their organ specificity. To determine whether VEGF-A might serve as an in vivo cell-fate switch for human ESC-derived Isl1-ECs, we established a novel approach using chemically modified mRNA as a platform for transient, yet highly efficient expression of paracrine factors in cardiovascular progenitors. Overexpression of VEGF-A promotes not only the endothelial specification but also engraftment, proliferation and survival (reduced apoptosis) of the human Isl1⁺ progenitors in vivo. The large-scale derivation of cardiac-specific human Isl1-ECs from human pluripotent stem cells, coupled with the ability to drive endothelial specification, engraftment, and survival following transplantation, suggest a novel strategy for vascular regeneration in the heart.     

VEGF induces proliferation, migration, and TGF-beta1 expression in mouse glomerular endothelial cells via mitogen-activated protein kinase and phosphatidylinositol 3-kinase

The role of glomerular endothelial cells in kidney fibrosis remains incompletely understood. While endothelia are indispensable for repair of acute damage, they can produce extracellular matrix proteins and profibrogenic cytokines that promote fibrogenesis. We used a murine cell line with all features of glomerular endothelial cells (glEND.2), which dissected the effects of vascular endothelial growth factor (VEGF) on cell migration, proliferation, and profibrogenic cytokine production. VEGF dose-dependently induced glEND.2 cell migration and proliferation, accompanied by up-regulation of VEGFR-2 phosphorylation and mRNA expression. VEGF induced a profibrogenic gene expression profile, including up-regulation of TGF-beta1 mRNA, enhanced TGF-beta1 secretion, and bioactivity. VEGF-induced endothelial cell migration and TGF-beta1 induction were mediated by the phosphatidyl-inositol-3 kinase pathway, while proliferation was dependent on the Erk1/2 MAP kinase pathway. This suggests that differential modulation of glomerular angiogenesis by selective inhibition of the two identified VEGF-induced signaling pathways could be a therapeutic approach to treat kidney fibrosis.     

The matrix protein CCN1 (CYR61) promotes proliferation, migration and tube formation of endothelial progenitor cells

Neovascularization and re-endothelialization relies on circulating endothelial progenitor cells (EPCs), but their recruitment and angiogenic roles are subjected to regulation by the vascular microenvironment, which remains largely unknown. The present study was designed to investigate the effects of mature ECs and matrix protein CCN1 on the properties of EPCs. In a coculture system, effects of ECs on proliferation, migration and participation in tube-like formation of EPCs were evaluated, and functional assays were employed to identify the exact role of CCN1 in EPCs vitality and function. We demonstrated that ECs, as an indispensable part of the cellular milieu, significantly promoted the proliferation, migration and tube formation activities of EPCs, and more importantly, CCN1 was potentially involved in such effects of ECs. Expression of CCN1 in EPCs was significantly increased by serum, VEGF, ECs-cocultivation and ECs conditioned medium. Moreover, Ad-CCN1-mediated overexpression of CCN1 directly enhanced migration and tube formation of EPCs, whereas silencing of endogenous CCN1 in EPCs inhibits cell functions. Furthermore, CCN1 induced the expressions of chemokines and growth factors, such as MCP-1 and VEGF, suggesting a complex interaction between those proangiogenic factors. Our data suggest that matrix protein CCN1 may play an important role in microenvironment-mediated biological properties of EPCs.     

Genomics of TGF-beta1 signaling in stem cell commitment and dendritic cell development

Dendritic cells are professional antigen presenting cells and central for establishing and maintaining immunity and immunological tolerance. They develop from hematopoietic stem cells through successive steps of lineage commitment and differentiation. Dendritic cell development and function are regulated by specific cytokines, including transforming growth factor type beta1 (TGF-beta1). Our previous work demonstrated the importance of TGF-beta1 signaling for dendritic cell development and subset specification. Here, we used genome-wide gene expression profiling with DNA microarrays to investigate the activity of TGF-beta1 on gene expression in dendritic cell development. This study identified specific gene categories induced by TGF-beta1 with an impact on dendritic cell biology.