Interleukin-6 increases vascular endothelial growth factor and angiogenesis in gastric carcinoma

Interleukin-6 (IL-6) is a proinflammatory cytokine associated with the disease status of gastric carcinoma (GC). Vascular endothelial growth factor (VEGF) is a potent tumor angiogenic factor in GC. In this study, we attempted to clarify whether IL-6 can regulate VEGF and angiogenesis in GC. GC samples from 54 surgical specimens were subjected to immunohistochemical examination of IL-6, VEGF, and tumor microvessels, and results showed that IL-6 was positively correlated with VEGF expression and tumor vasculature. We determined VEGF expression in four GC cell lines by ELISA, revealing that GC cells can produce significant amount of VEGF with increasing dose and duration of IL-6 stimulation. Next, a luciferase reporter gene assay was employed to determine the signaling pathway driving the VEGF promoter by IL-6, which showed that the JAK/STAT pathway is involved in the stimulation of VEGF gene expression. The effects of IL-6 on angiogenesis in vitro and in vivo were evaluated by HUVEC studies and the Matrigel plug assay, respectively. Results showed that IL-6 effectively promoted HUVEC proliferation and tube formation in vitro and Matrigel plug vascularization in vivo, primarily by inducing VEGF in GC. This study provides evidence that the multifunctional cytokine, IL-6, may induce VEGF expression which increases angiogenesis in gastric carcinogenesis.     

Cyclic strain-induced endothelial MMP-2: role in vascular smooth muscle cell migration

Matrix metalloproteinases (MMPs) play a vital role in vasculature response to hemodynamic stimuli via the degradation of extracellular matrix substrates. In this study, we investigated the putative role of cyclic strain-induced endothelial MMP-2 (and MMP-9) expression and release in modulating bovine aortic smooth muscle cell (BASMC) migration in vitro. Equibiaxial cyclic strain of bovine aortic endothelial cells (BAECs) leads to elevation in cellular MMP-2 (and MMP-9) expression, activity, and secretion into conditioned media, events which were time- and force-dependent. Subsequent incubation of BASMCs with conditioned media from chronically strained BAECs (5%, 24 h) significantly reduces BASMC migration (38+/-6%), an inhibitory effect which could be completely reversed by targeted siRNA 'knock-down' of MMP-2 (but not MMP-9) expression and activity in BAECs. Moreover, inhibition of strain-mediated MMP-2 expression in BAECs by protein tyrosine kinase (PTK) blockade with genistein (50 microM) was also found to completely reverse this inhibitory effect on BASMC migration. Finally, direct supplementation of recombinant MMP-2 into the BASMC migration assay was found to have no significant effect on migration. However, the effect on BASMC migration of MMP-2 siRNA transfection in BAECs could be reversed by supplementation of recombinant MMP-2 into BAEC media prior to (and for the duration of) strain. These findings reveal a potentially novel role for strain-induced endothelial MMP-2 in regulating vascular SMC migration.     

UVA-mediated down-regulation of MMP-2 and MT1-MMP coincides with impaired angiogenic phenotype of human dermal endothelial cells

UVA irradiation, dose-dependently (5-20 J/cm2), was shown to impair the morphogenic differentiation of human microvascular endothelial cells (HMECs) on Matrigel. Parallely, UVA down-regulated the expression of MMP-2 and MT1-MMP, both at the protein and the mRNA levels. On the contrary, the production of MMP-1 and TIMP-1 by HMECs increased following UVA treatment. The inhibitory effect of UVA on MMP expression and pseudotubes formation was mediated by UVA-generated singlet oxygen (1O2). The contribution of MT1-MMP, but not TIMP-1, to the regulation of HMECs' angiogenic phenotype following UVA irradiation was suggested using elastin-derived peptides and TIMP-1 blocking antibody, respectively.     

Paracrine interleukin-8 affects mesenchymal stem cells through the Akt pathway and enhances human umbilical vein endothelial cell proliferation and migration

Interleukin-8 (IL-8) promotes cell homing and angiogenesis, but its effects on activating human bone marrow mesenchymal stem cells (BMSCs) and promoting angiogenesis are unclear. We used bioinformatics to predict these processes. In vitro, BMSCs were stimulated in a high-glucose (HG) environment with 50 or 100 μg/ml IL-8 was used as the IL-8 group. A total of 5 μmol/l Triciribine was added to the two IL-8 groups as the Akt inhibitor group. Cultured human umbilical vein endothelial cells (HUVECs) were cultured in BMSCs conditioned medium (CM). The changes in proliferation, apoptosis, migration ability and levels of VEGF and IL-6 in HUVECs were observed in each group. Seventy processes and 26 pathways were involved in vascular development, through which IL-8 affected BMSCs. Compared with the HG control group, HUVEC proliferation absorbance value (A value), Gap closure rate, and Transwell cell migration rate in the IL-8 50 and IL-8 100 CM groups were significantly increased (P<0.01, n=30). However, HUVEC apoptosis was significantly decreased (P<0.01, n=30). Akt and phospho-Akt (P-Akt) protein contents in lysates of BMSCs treated with IL-8, as well as VEGF and IL-6 protein contents in the supernatant of BMSCs treated with IL-8, were all highly expressed (P<0.01, n=15). These analyses confirmed that IL-8 promoted the expression of 41 core proteins in BMSCs through the PI3K Akt pathway, which could promote the proliferation and migration of vascular endothelial cells. Therefore, in an HG environment, IL-8 activated the Akt signaling pathway, promoted paracrine mechanisms of BMSCs, and improved the proliferation and migration of HUVECs.     

细胞生长因子对冠状动脉内皮细胞增殖与迁移的影响

目的 :观察肝细胞生长因子 (HGF)和血管内皮细胞生长因子 (VEGF)对体外培养牛冠状动脉内皮细胞(BCAEC)增殖、迁移的影响。方法 :分离和培养BCAEC ,设对照组、VEGF组、HGF组。采用四甲基偶氮唑蓝法(MTT)观察细胞增殖 ;倒置显微镜观察培养的血管内皮细胞的迁移。结果 :对照组、VEGF组、HGF组的OD值分别为 0 .2 2± 0 .0 1、0 .40± 0 .1 4、0 .44± 0 .1 5 ;VEGF组、HGF组BCAEC的增殖率分别为 81 .8%± 1 6 .9%、1 0 0 %±2 1 .1 % ;对照组BCAEC迁移不明显 ,而VEGF组和HGF组BCAEC迁移明显。结论 :VEGF、HGF能促进BCAEC增殖、迁移 ,HGF作用强度不亚于VEGF。     

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.     

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.     

Heat shock proteins and other components of cellular machinery for protein synthesis are up-regulated in vascular endothelial cell growth factor-activated human endothelial cells

Proteome analysis of human umbilical endothelial cells was performed to identify proteins that are modified during vascular endothelial cell growth factor (VEGF)-induced transition from the quiescent into the proliferating-migrative phenotype. Subtractive analysis of two-dimensional gel patterns of human endothelial cells, before and after stimulation with VEGF(165), revealed differences in 85 protein spots. All proteins were identified by peptide sequencing and peptide mass fingerprinting using an electrospray spectrometer. The proteins identified were members of specific families including Ca(2+)-binding proteins, fatty-acid binding proteins, structural proteins, and chaperones. Remarkably, there was a massive activation of cellular machinery for both protein synthesis and protein degradation. Thus, among up-regulated proteins there were members of all groups of heat shock proteins (HSPs; HSP 27, HSP 60, HSP 70p5, HSP 70p8, HSP 90, and HSP 96) and some other proteins showing either chaperone activity or which participate in assembly of multimolecular structures (TCP-1, desmoplakins, junction plakoglobin, GRP 94, thioredoxin related protein, and peptidylprolyl isomerase). The increased expression of HSPs was confirmed at the mRNA level at different stages of treatment with VEGF. Similarly, components of the proteolytic machinery for the degradation of misfolded proteins (ER-60, cathepsin D, proteasome subunits, and protease inhibitor 6) were also up-regulated. On the other hand, changes in the expression of structural proteins (T-plastin, vimentin, alpha tubulin, actin, and myosin) could account, at least in part, for the different morphologies displayed by migrating endothelial cells. In summary, our data show that VEGF levels similar to those during physiological stresses induce a number of genes and multiple endogenous pathways seem to be engaged in restoring cellular homeostasis. To ensure cell survival, the molecular chaperones (the heat shock family of stress proteins) are highly up-regulated providing protein-folding machinery to repair or degrade misfolded proteins.     

VEGF-mediated angiogenesis stimulates neural stem cell proliferation and differentiation in the premature brain

This study investigated the effects of angiogenesis on the proliferation and differentiation of neural stem cells in the premature brain. We observed the changes in neurogenesis that followed the stimulation and inhibition of angiogenesis by altering vascular endothelial growth factor (VEGF) expression in a 3-day-old rat model. VEGF expression was overexpressed by adenovirus transfection and down-regulated by siRNA interference. Using immunofluorescence assays, Western blot analysis, and real-time PCR methods, we observed angiogenesis and the proliferation and differentiation of neural stem cells. Immunofluorescence assays showed that the number of vWF-positive areas peaked at day 7, and they were highest in the VEGF up-regulation group and lowest in the VEGF down-regulation group at every time point. The number of neural stem cells, neurons, astrocytes, and oligodendrocytes in the subventricular zone gradually increased over time in the VEGF up-regulation group. Among the three groups, the number of these cells was highest in the VEGF up-regulation group and lowest in the VEGF down-regulation group at the same time point. Western blot analysis and real-time PCR confirmed these results. These data suggest that angiogenesis may stimulate the proliferation of neural stem cells and differentiation into neurons, astrocytes, and oligodendrocytes in the premature brain.     

Opposite effects of high glucose on MMP-2 and TIMP-2 in human endothelial cells

Diabetes mellitus (DM) is a major risk factor for atherosclerosis and causes multiple cardiovascular complications. Although high glucose can induce matrix metalloproteinases (MMPs), its inhibitors and cell apoptosis, little is known about the roles of MMPs in regulating cell apoptosis in response to high glucose. To address this issue, we elucidated the relationship between MMPs, its inhibitors and cell apoptosis in human umbilical vein endothelial cells (HUVECs). HUVECs were treated with medium containing 5.5 mM or 33 mM of glucose in the presence or the absence of ascorbic acid and MMP inhibitors (GM6001 and endogenous tissue inhibitors of MMPs, TIMP-1, and TIMP-2). For detection of cell apoptosis, the cell death detection ELISA assay was used. The results revealed that high glucose-induced apoptosis could be suppressed by ascorbic acid, GM6001 and TIMP-2, but not by TIMP-1. The activities of MMP-2, MMP-9 and its inhibitors, TIMP-1, TIMP-2 after high glucose treatment, were also detected by ELISA method. We found that the activated form of MMP-2, but not MMP-9, was increased, while the level of TIMP-2, but not TIMP-1, was decreased. In Western blot and RT-PCR analysis, the expression of TIMP-2, but not TIMP-1, after high glucose treatment was downregulated, whereas the levels of MMP-2 and -9 proteins and mRNA were not changed. The present study indicated that oxidative stress induced by high glucose might be involved in the opposite effects on MMP-2 activation and TIMP-2 downregulation. This reactive oxygen species (ROS)-dependent MMP-2 activation in turn mediates high glucose-induced cell apoptosis in HUVECs.     

IGF2: epigenetic regulation and role in development and disease

Insulin-like growth factor II (IGF2) is perhaps the most intricately regulated of all growth factors characterized to date. Its gene is imprinted – only one allele is active, depending on parental origin – and this pattern of expression is maintained epigenetically in almost all tissues. IGF2 activity is further controlled through differential expression of receptors and IGF-binding proteins (IGFBPs) that determine protein availability. This complex and multifaceted regulation emphasizes the importance of accurate IGF2 expression and activity. This review will examine the regulation of the IGF2 gene and what it has revealed about the phenomenon of imprinting, which is frequently disrupted in cancer. IGF2 protein function will be discussed, along with diseases that involve IGF2 overexpression. Roles for IGF2 in sonic hedgehog (Shh) signaling and angiogenesis will also be explored.     

Inhibition of angiogenic differentiation of human umbilical vein endothelial cells by diallyl disulfide (DADS)

Angiogenesis is a crucial step in the growth and metastasis of cancers. The activation of endothelial cells and their further behaviour are very critical during angiogenesis. We analyzed the effect of diallyl disulfide (DADS) on angiogenesis in in vitro models using human umbilical vein endothelial cells (HUVECs). DADS significantly inhibited endothelial cell migration, invasion and tube formation. (3)H-thymidine proliferation assay clearly showed the inhibitory effect of DADS on the proliferation of HUVECs in vitro. The role of metalloproteinases has been shown to be important in angiogenesis; therefore, zymography was performed to determine whether DADS affected protease activity. Gelatin zymographic analysis showed the inhibitory effect of DADS on the activation of matrix metalloproteinases-MMP-2 and MMP-9. These findings suggest that DADS acts as an angiogenesis inhibitor by inhibiting the activation of matrix metalloproteinases during endothelial morphogenesis.     

Knockdown of CD146 reduces the migration and proliferation of human endothelial cells

Our previous study has demonstrated that CD 146 molecule is a biomarker on vascular endothelium,which is involvedin angiogenesis and tumor growth.However the mechanism behind is not clear.Here we have for the first time devel-oped a novel CD146 blockade system using CD146 siRNA to study its function on endothelial cells.Our data showedthat CD146 siRNA specifically blocked the expression of CD146 on both mRNA and protein levels,leading to thesignificant suppression of HUVEC proliferation,adhesion and migration.These results demonstrate that CD146 playsa key role in vascular endothelial cell activity and angiogenesis,and CD146 siRNA can be used as a new inhibitor foranti-angiogenesis therapy.     

Vascular endothelial growth factor: Therapeutic possibilities and challenges for the treatment of ischemia

Vascular endothelial growth factor (VEGF) is a notable chemokine that plays critical roles in angiogenesis and vasculogenesis. The contemporary body of literature contains a substantial amount of information regarding its chemical properties as well as its fundamental role in vascular development. Studies strongly indicate its potential use as a therapeutic agent, especially in the vascular restoration of injured and ischemic tissues. VEGF therapy could be most beneficial for diseases whose pathologies revolve around tissue inflammation and necrosis, such as myocardial infarction and stroke, as well as ischemic bowel diseases such as acute mesenteric ischemia and necrotizing enterocolitis. However, a delicate balance exists between the therapeutic benefits of VEGF and the hazards of tumor growth and neo-angiogenesis. Effective future research surrounding VEGF may allow for the development of effective therapies for ischemia which simultaneously limit its more deleterious side effects. This review will: (1) summarize the current understanding of the molecular aspects and function of VEGF, (2) review potential benefits of its use in medical therapy, (3) denote its role in tumorigenesis and inflammation when overexpressed, and (4) elucidate the qualities which make it a viable compound of study for diagnostic and therapeutic applications.     

Activation of chemokine receptor CXCR4 in malignant glioma cells promotes the production of vascular endothelial growth factor

Numerous studies have showed that chemokine receptors, such as CXCR4, contribute to the growth and metastasis of a variety of malignant tumors. In this study, we investigated the role of CXCR4 in the production of angiogenic factor, vascular endothelial growth factor (VEGF), in various human glioma cells from astrocytic origin. The expression of CXCR4 mRNA and protein in three glioma cell lines, U87-MG, SHG-44, and CHG-5, was determined by RT-PCR and immunocytochemistry, respectively. The malignancies of three gliomas were evaluated by expression of glial fibrillary acidic protein and vimentin, the differentiation markers of astrocytic cells. The role of functional CXCR4 in tumor cell migration was studied with chemotaxis assay. Ca2+ mobilization and VEGF production were measured in the cells after stimulation with CXCR4 ligand, SDF1beta. The results showed that the levels of functional CXCR4 expression at both mRNA and protein levels by several human glioma cell lines were correlated with the degree of differentiation of the tumor cells. Activation of CXCR4 induced glioma cell chemotaxis and could trigger the increase of intracellular [Ca2+]i. Such an activation could result in the increased production of VEGF by the stimulated tumor cells. Our results suggest that CXCR4 may contribute to the high level of VEGF produced by malignant glioma cells and thus constitute a therapeutic target for antiangiogenesis strategy.     

胆管癌组织白介素-6、环氧合酶-2和血管内皮生长因子的表达及临床意义

目的:探讨胆管癌组织白介素-6(IL-6)、环氧合酶-2(COX-2)和血管内皮生长因子(VEGF)的表达及临床意义。方法:将手术切除并经病理诊断确诊的胆管癌石蜡包埋标本80例纳为胆管癌组,另取癌旁正常胆管组织作为对照组,采用免疫组织化学SP法检测两组组织中IL-6、COX-2、VEGF的表达情况并做比较,分析胆管癌组织中VEGF、COX-2、IL-6阳性表达与临床病理特征关系,采用Spearman等级相关分析胆管癌组织中VEGF、COX-2、IL-6表达的相关性。结果:胆管癌组的VEGF、COX-2、IL-6阳性表达率均显著高于对照组,组间比较差异有统计学意义(P0.05)。胆管癌组织中VEGF、COX-2、IL-6阳性表达率与有无淋巴结转移、TNM分期、分化程度有关(P0.05),而与性别、年龄、肿瘤直径无关(P0.05),其中有淋巴结转移、TNM分期Ⅲ～Ⅳ期、低分化程度的胆管癌患者的VEGF、COX-2、IL-6阳性表达率高于无淋巴结转移、TNM分期Ⅰ～Ⅱ期、中高分化程度的胆管癌患者(P0.05)。Spearman等级相关分析显示,胆管癌组织中VEGF与COX-2、IL-6呈正相关(P0.05),COX-2与IL-6也呈正相关(P0.05)。结论:胆管癌组织IL-6、COX-2、VEGF均呈现高表达,并与胆管癌的生长、转移密切相关,检测IL-6、COX-2和VEGF有助于判断胆管癌疾病进展。