Characterization and expression of a novel alternatively spliced human angiopoietin-2

Angiopoietin-2 (Ang2) is a naturally occurring antagonist of angiopoietin-1 (Ang1) that competes for binding to the Tie2 receptor and blocks Ang1-induced Tie2 autophosphorylation during vasculogenesis. Using the polymerase chain reaction, we isolated a cDNA encoding a novel shorter form of Ang2 from human umbilical vein endothelial cell cDNA and have designated it angiopoietin-2(443) (Ang2(443)), because it contains 443 amino acids. Part of the coiled-coil domain (amino acids 96-148) is absent in Ang2(443) because of alternative splicing of the gene. Like Ang2, recombinant Ang2(443) expressed in COS-7 cells is secreted as a glycosylated homodimeric protein. Recombinant Ang2(443) binds to the Tie2 receptor but does not induce Tie2 phosphorylation. Pre-occupation of Ang2(443) on Tie2 inhibits Ang1 or Ang2 binding and inhibits Ang1-induced phosphorylation. Expression of Ang2(443) mRNA is detectable in primary endothelial cells, several nonendothelial tumor cell lines, and primary tumor tissues. Interestingly, two cervical carcinoma cell lines express relatively moderate levels of Ang2(443) mRNA and protein. Macrophages express mainly Ang2 mRNA, but the expression of Ang2(443) mRNA is temporarily up-regulated during macrophage differentiation. These results suggest that Ang2(443) is a functional antagonist of Ang1 and could be an important regulator of angiogenesis during some tumorigenic and inflammatory processes.     

Molecular cloning and characterization of a novel angiopoietin family protein, angiopoietin-3

Using homology-based PCR, we have isolated cDNA encoding a novel member (491 amino acids) of the angiopoietin (Ang) family from human adult heart cDNA and have designated it angiopoietin-3 (Ang3). The NH2-terminal and COOH-terminal portions of Ang-3 contain the characteristic coiled-coil domain and fibrinogen-like domain that are conserved in other known Angs. Ang3 has a highly hydrophobic region at the N-terminus (approximately 21 amino acids) that is typical of a signal sequence for protein secretion. Ang3 mRNA is most abundant in adrenal gland, placenta, thyroid gland, heart and small intestine in human adult tissues. Additionally, Ang3 is a secretory protein, but is not a mitogen in endothelial cells.     

Role of Src in angiopoietin 1-induced capillary morphogenesis of endothelial cells: Effect of chronic hypoxia on Src inhibition by PP2

Signal transduction pathways leading to angiopoietin 1 (Ang1)-induced capillary morphogenesis by endothelial cells remain poorly defined. Angiogenic cellular responses by endothelial cells may be modulated in vivo by chronic hypoxia, such as that induced by tumors. Here, we studied Ang1-induced capillary morphogenesis in human umbilical-vein endothelial cells (HUVECs) cultured chronically under normoxic (21% oxygen) or hypoxic (1.5% oxygen) conditions. Downregulation of Src using a small interfering RNA (siRNA) inhibited Ang1-induced capillary morphogenesis of HUVECs cultured under both conditions by blocking cell spreading and protrusion. Ang1 upregulated the Src-dependent secretion of vascular endothelial growth factor-A (VEGF-A). Blockade of endogenous VEGF-A also inhibited Ang1-induced capillary morphogenesis. Addition of exogenous VEGF-A restored cell spreading and protrusion, leading to Ang1-induced capillary morphogenesis of Src siRNA-treated HUVECs, suggesting that Ang1-induced VEGF-A secretion through Src was required for capillary morphogenesis. PP2 inhibited both Ang1-induced capillary morphogenesis and Src activation in HUVECs cultured under normoxic conditions, but the PP2 activity was significantly impaired in HUVECs cultured under hypoxic conditions. Expression of multidrug resistance-associated protein 1 (MRP 1) was upregulated in hypoxic HUVECs, and treatment with MRP 1 siRNA restored the inhibitory action of PP2. Taken together, our results suggest that Ang1 induces capillary morphogenesis in HUVECs through Src-dependent upregulation of endogenous VEGF-A. Conditions of chronic hypoxia impaired the effect of PP2, possibly via MRP 1.     

Single chain Fv antibody against angiopoietin-2 inhibits VEGF-induced endothelial cell proliferation and migration in vitro

Angiopoietin-2 (Ang2) promotes tumor growth and metastasis by specifically priming endothelial cells for angiogenesis. Multiple angiogenic factors up-regulate expression of Ang2, suggesting that Ang2 may be the common pathway in growth factor initiated-angiogenesis. Using phage display technology, we generated single chain Fv molecule against human Ang2 (scFv-Ang2) with high affinity (K(d)=0.01 microM) from a mouse phage antibody library. Compared with control scFv, the mouse scFv-Ang2 completely inhibited the proliferation of human umbilical vein endothelial cells (HUVECs) treated with vascular endothelial growth factor (VEGF, 10 ng/ml), but not that of the cells treated with either basic fibroblast growth factor, or angiotensin II, or Ang2. Chemotaxis assay showed that scFv-Ang2 could block completely Ang2-induced (100%) and partially VEGF-induced (49%) migration of HUVECs. The results indicate that Ang2 takes part in the VEGF-induced angiogenesis and scFv-Ang2 might be a promising compound in blocking both VEGF and Ang2 induced angiogenesis.     

Tie1 regulates the Tie2 agonistic role of angiopoietin-2 in human lymphatic endothelial cells

Although Angiopoietin (Ang) 2 has been shown to function as a Tie2 antagonist in vascular endothelial cells, several recent studies on Ang2-deficient mice have reported that, like Ang1, Ang2 acts as a Tie2 agonist during in vivo lymphangiogenesis. However, the mechanism governing the Tie2 agonistic activity of Ang2 in lymphatic endothelial cells has not been investigated. We found that both Ang1 and Ang2 enhanced the in vitro angiogenic and anti-apoptotic activities of human lymphatic endothelial cells (HLECs) through the Tie2/Akt signaling pathway, while only Ang1 elicited such effects in human umbilical vein vascular endothelial cells (HUVECs). This Tie2-agonistic effect of Ang2 in HLECs resulted from low levels of physical association between Tie2 and Tie1 receptors due to a reduced level of Tie1 expression in HLECs compared to HUVECs. Overexpression of Tie1 and the resulting increase in formation of Tie1/Tie2 heterocomplexes in HLECs completely abolished Ang2-mediated Tie2 activation and the subsequent cellular responses, but did not alter the Ang1 function. This inhibitory role of Tie1 in Ang2-induced Tie2 activation was also confirmed in non-endothelial cells with adenovirus-mediated ectopic expression of Tie1 and/or Tie2. To our knowledge, this study is the first to describe how Ang2 acts as a Tie2 agonist in HLECs. Our results suggest that the expression level of Tie1 and its physical interaction with Tie2 defines whether Ang2 functions as a Tie2 agonist or antagonist, thereby determining the context-dependent differential endothelial sensitivity to Ang2.     

Ang2、Tie2基因的RNA干扰及其在体外抑制血管生成的作用

目的：探讨应用RNA干扰（RNA interference,RNAi）技术沉默Ang2、Tie2基因及其在体外抑制血管生成的研究,为将来进一步进行抑制肿瘤血管生成的动物实验研究奠定基础,为肿瘤的基因治疗提供实验依据。方法：用pSilencer 1.0-U6-Ang2/Tie2-siRNA重组质粒转染人脐静脉内皮细胞(HUVECs),采用RT PCR检测各组HUVECs Ang2、Tie2的mRNA表达状况。应用体外血管生成的三维培养模型研究转染后的HUVECs在体外形成血管样结构的情况。结果：pSilencer 1.0-U6-Ang2/Tie2-siRNA重组质粒转染HUVECs后,RT-PCR检测结果显示: Ang2、Tie2基因mRNA的表达水平均受到明显抑制（P<0.05）,并且siRNA的2条重组质粒之间均无明显差别（P>0.05）。转染后的HUVECs在体外三维培养模型中血管样结构形成的数量和长度均明显减少（P<0.05）,表明血管生成受到明显抑制。结论：Ang2-siRNA、Tie2-siRNA能够抑制HUVECs中Ang2和Tie2的mRNA表达,从而在体外抑制血管生成。     

Molecular cloning, sequence analysis and expression of a cDNA encoding human type-1 angiotensin II receptor.

We isolated a cDNA encoding type-1 angiotensin II receptor from a human liver cDNA library. The cDNA had an open reading frame encoding a protein of 359 amino acid residues with a relative Mr of 41,060. The deduced amino acid sequence of the human angiotensin II (Ang II) receptor was 95.3% and 94.2% identical to those of bovine and rat type-1 Ang II receptors, respectively, and had a significant similarity with the G protein-coupled receptor. The rank order of the binding to the receptor expressed in COS-7 cells was Ang II greater than Ang III greater than Ang I. The expression of the Ang II receptor mRNA was detected in human liver, lung, adrenal and adrenocortical adenomas but not in adrenomedullary tumor, pheochromocytoma, by Northern blot analysis.     

Transcription factor Ap-1 mediates proangiogenic MIF expression in human endothelial cells exposed to Angiotensin II

Macrophage migration inhibitory factor (MIF) is an inflammatory cytokine associated with the atherosclerotic process and atherosclerotic plaque stability. MIF was shown to be highly expressed in advanced atherosclerotic lesions. Neutralizing MIF with a blocking antibody induced a regression of established atherosclerotic lesions. In this study, we investigated the mechanism underlying the proangiogenic effect of MIF in human umbilical vein endothelial cells (HUVECs). We showed that MIF induced the expression of angiogenesis-related genes in HUVECs. We also showed that MIF induced tube formation of HUVECs in vitro and in vivo. Angiotensin II (Ang II) could specifically up-regulate MIF expression in HUVECs. Using a luciferase reporter assay, we demonstrated that the AP-1 response element in the 5'-UTR of the MIF gene played a role in Ang II-induced MIF expression. Small hairpin RNA (shRNA) targeting c-Jun, a component of AP-1, and the AP-1 inhibitor CHX both efficiently inhibited MIF expression. The consistent result of electrophoretic mobility shift assay (EMSA) showed that Ang II specifically increased AP-1 activation in HUVECs. Our results suggest that AP-1 mediates Ang II-induced MIF expression which contributes to atherosclerotic plaque destabilization in human endothelial cells.     

AT2 receptor interacting protein 1 (ATIP1) mediates COX-2 induction by an AT2 receptor agonist in endothelial cells

Angiotensin II (Ang II) type 2 receptor (AT2R) is one of the major components of the renin-angiotensin-aldosterone system. Nevertheless, the physiological role is not well defined compared to the understanding of the Ang II type 1 receptor (AT1R), which is a well characterized G-protein coupled receptor in the cardiovascular system. While the AT2R signaling pathway remains unclear, AT2 receptor interacting protein 1 (ATIP1) has been identified as a candidate molecule for interacting with the C-terminal region of AT2R. In this study, we investigated the ATIP1 dependent AT2R inducible genes in human umbilical vein endothelial cells (HUVECs). CGP42112A, an AT2R specific agonist, resulted in an upregulation of inflammatory genes in HUVECs, which were inhibited by knocking down ATIP1 with siRNA (siATIP1). Among them, we confirmed by quantitative PCR that the induction of COX-2 mRNA expression was significantly downregulated by siATIP1. COX-2 was also upregulated by Ang II stimulation. This upregulation was suppressed by treatment with the AT2R specific antagonist PD123319, which was not replicated by the AT1R antagonist telmisartan.These findings suggest that ATIP1 plays an important role in AT2R dependent inflammatory responses. This may provide a new approach to the development of cardio-protective drugs.     

晚期糖基化终产物对人脐静脉内皮细胞的肝细胞生长因子表达的影响

目的探讨晚期糖基化终产物(AGEs)对人脐静脉内皮细胞的肝细胞生长因子(HGF)mRNA及蛋白表达的影响。方法体外培养人脐静脉内皮细胞,予不同浓度(100mg/L、200mg/L、400mg/L)的AGEs刺激24h及400mg/LAGEs作用6h、12h、24h及48h,采用RT-PCR及免疫细胞化学法检测内皮细胞HGFmRNA及蛋白的表达水平。结果在一定范围内随着AGEs浓度增加,内皮细胞HGF表达逐渐增高;AGEs早期作用内皮细胞,促进HGFmR-NA及蛋白的表达,随着AGEs的持续作用,HGF表达减弱。结论随着AGEs作用时间的延长,HGF对受损内皮细胞的修复作用先增强后减弱。