Roles of angiopoietin-1 and angiopoietin-2 on airway microvascular permeability in asthmatic patients

BACKGROUND: Vascular endothelial growth factor (VEGF) increases microvascular permeability. Recently, considerable attention has been devoted to the physiologic roles of angiopoietin-1 and angiopoietin-2 as regulatory factors of VEGF. This study was designed to examine the roles of angiopoietin-1 and angiopoietin-2 in controlling airway microvascular permeability in asthma. METHODS: Levels of these angiogenic factors and airway vascular permeability index were examined in 30 asthmatics and 12 control subjects. After 2-week run-in period, all asthmatics were randomly assigned to receive fluticasone propionate (400 mug/d) or montelukast (10 mg) for 12 weeks. RESULTS: VEGF, angiopoietin-1, and angiopoietin-2 levels in induced sputum were significantly higher in asthmatics than in control subjects. We found an inverse correlation between angiopoietin-1 level and vascular permeability index in asthmatics, while there was a positive correlation between angiopoietin-2 level and that index. VEGF and angiopoietin-1 levels were significantly decreased after fluticasone therapy, while VEGF and angiopoietin-2 levels were significantly decreased after montelukast therapy. Although VEGF levels after treatment were different between two groups, vascular permeability index in the montelukast group was the same level as that in the fluticasone group. Moreover, improvement in vascular permeability index after fluticasone therapy was inversely correlated with decrease in angiopoietin-1 level, while that after montelukast therapy was positively correlated with decrease in angiopoietin-2 level. CONCLUSIONS: Angiopoietin-1 and angiopoietin-2 play complementary and coordinated roles in regulating microvascular permeability stimulated by VEGF in asthma. Combination of corticosteroids with leukotriene antagonists might effectively improve plasma leakage and provide a new strategy in treating bronchial asthma.     

Expressional regulation of the angiopoietin-1 and -2 and the endothelial-specific receptor tyrosine kinase Tie2 in adrenal atrophy: a study of adrenocorticotropin-induced repair

Angiopoietin-1 (Ang-1), a newly discovered ligand of the endothelial-specific tyrosine kinase receptor Tie-2, has been found to promote cell survival, vascular maturation, and stabilization, and to function in concert with vascular endothelial growth factor. Adrenal gland has an intense capillary network that regulation remains to be documented. Recently, we demonstrated that vascular endothelial growth factor, and its receptors are expressed in mouse adrenal in vivo, but no detailed study on Ang expression in the adrenal has been reported. The present study shows the expression of Tie2 receptors, Ang-1, and its endogenous antagonist, Ang-2 in mouse adrenal in vivo. Immunohistochemistry disclosed that Tie2 colocalized with platelet-endothelial-cell-adhesion-molecule in endothelial cells from normal mouse adrenal. Daily administration of dexamethasone (DEX) (0.5 mg/100 g body weight.d) for 6 d in mice, decreased steroidogenic function of adrenal as shown by inhibition of the 36-kDa ACTH receptor protein expression, and decreased plasma corticosterone level [control from 465 +/- 35 ng/ml to 114 +/- 18 ng/ml in DEX group (P < 0.001)]. Using semiquantitative RT-PCR, we demonstrate that DEX treatment down regulates Ang-1 mRNA levels by 3- to 4-fold. No significant changes in Ang-2 were detected between control and DEX groups, resulting in an altered Ang-2 to Ang-1 relative ratio. The Tie2 receptor was also found to be down-regulated in DEX group at both mRNA and protein level. ACTH was found to play a causal role in DEX-induced decrease in Ang-1/Tie2 system, because 7 d treatment with long acting 1-39 ACTH (30 IU/kg x d) increased Ang-1, Tie2 expression, and plasma corticosterone back to control levels. These results reinforce the role of ACTH in the regulation of angiogenic factors in adrenal gland and suggest that the Ang/Tie2 system might represent a key player for stabilization of adrenal endothelium.     

Hypoxia and vascular endothelial growth factor acutely up-regulate angiopoietin-1 and Tie2 mRNA in bovine retinal pericytes

The angiopoietin/Tie2 system is a predominant regulator of vascular development. This vascular development appears to be controlled and completed by the coordinated actions of two vascular cells, endothelial cells and their surrounding supporting cells, smooth muscle cells, or pericytes. The role of the angiopoietin/Tie2 system has been studied, but these studies are limited mostly to endothelial cells. In this study, using bovine retinal pericytes (BRP), we investigated the effect of two known angiogenic stimuli, hypoxia and vascular endothelial growth factor (VEGF) treatment, on the regulation of the angiopoietin/Tie2 system. Hypoxia (2% O(2) concentration) was acquired by a hypoxia chamber. Both hypoxia and VEGF (10 ng/ml) treatment significantly increased angiopoietin-1 (Ang1) mRNA expression. This marked augmentation occurred acutely (maximal increase at 2 h) and subsequently decreased. In contrast, angiopoietin-2 (Ang2) mRNA expression was unaltered in BRP upon both treatment. Significant up-regulation of Tie2 mRNA expression was found and lasted up to 12 h. However, using bovine aortic endothelial cells (BAEC), we found that only Ang2 expression, but neither Ang1 nor Tie2, responded to these two angiogenic stimuli, which was consistent with many previous reports. In conclusion, our data suggest that both hypoxia and VEGF treatment differentially regulate the angiopoietin/Tie2 system in the two vascular cells and that, particularly in BRP, the regulation of Ang1, but not Ang2, and Tie2 expression may play an important role in vascular development.     

Basal and angiopoietin-1-mediated endothelial permeability is regulated by sphingosine kinase-1

Endothelial cells (ECs) regulate the barrier function of blood vessels. Here we show that basal and angiopoietin-1 (Ang-1)-regulated control of EC permeability is mediated by 2 different functional states of sphingosine kinase-1 (SK-1). Mice depleted of SK-1 have increased vascular leakiness, whereas mice transgenic for SK-1 in ECs show attenuation of leakiness. Furthermore, Ang-1 rapidly and transiently stimulates SK-1 activity and phosphorylation, and induces an increase in intracellular sphingosine-1-phosphate (S1P) concentration. Overexpression of SK-1 resulted in inhibition of permeability similar to that seen for Ang-1, whereas knockdown of SK-1 by small interfering RNA blocked Ang-1-mediated inhibition of permeability. Transfection with SKS225A, a nonphosphorylatable mutant of SK-1, inhibited basal leakiness, and both SKS225A and a dominant-negative SK-1 mutant removed the capacity of Ang-1 to inhibit permeability. These effects were independent of extracellular S1P as knockdown or inhibition of S1P1, S1P2, or S1P3, did not affect the Ang-1 response. Thus, SK-1 levels in ECs powerfully regulate basal permeability in vitro and in vivo. In addition, the Ang-1-induced inhibition of leakiness is mediated through activation of SK-1, defining a new signaling pathway in the Ang-1 regulation of permeability.     

Mechanisms which mediate the antiapoptotic effects of angiopoietin-1 on endothelial cells

The main objective of this study was to identify molecular mechanisms through which angiopoietin-1 (Ang-1), a ligand for Tie-2 receptors, influences endothelial cell apoptosis. Human umbilical vein endothelial cells were cultured in a medium enriched with 2% fetal bovine serum (FBS) and growth supplements. Apoptosis was induced over 24 h by reducing FBS to 0.1%. Activation of caspase-9, -8, -7, and -3 and the expression of Bcl-2 family proteins, inhibitors of apoptosis (IAPs), cytochrome c, as well as Smac proteins were evaluated with immunoblotting. Ang-1 clearly attenuated serum deprivation-evoked apoptosis, an effect which required Tie-2 receptor activation. Activation of caspase-9, -7, and -3, but not caspase-8, was inhibited by Ang-1. The inhibitory effects of Ang-1 on apoptosis and caspase activation were reversed by a PI-3 kinase inhibitor (wortmannin). Ang-1 exposure upregulated the expression of Survivin but not XIAP (members of IAPs), reduced the cystosolic levels of Smac, but not that of cytochrome c, and had no effect on the expression of Bcl-2 family proteins. This is the first study to report on the mitochondrial mechanisms through which Ang-1 inhibits apoptosis and to investigate the role of the newly discovered Smac. We conclude that Ang-1 inhibits endothelial cell apoptosis through several pathways, which include PI-3 kinase/AKT activation, inhibition of Smac release from the mitochondria, and upregulation of Survivin protein.     

Identification and characterisation of chicken cDNAs encoding the endothelial cell-specific receptor tyrosine kinase Tie2 and its ligands, the angiopoietins

The angiopoietins represent a recently discovered family of angiogenic factors that recognise and specifically bind to the endothelial cell-specific Tie2 receptor tyrosine kinase. The specific biological functions of the angiopoietins have not yet been elucidated, although transgenic technology has shown the critical roles that these proteins have in vascularisation. The chicken vasculature, and more particularly the chorioallantoic membrane, is an important tool in the study of angiogenesis and has been previously used to study the functions of angiogenic factors such as vascular endothelial growth factor. We have undertaken the identification of the chicken orthologs of the angiopoietins and the Tie2 receptor. cDNA clones that encompass the full coding sequence of chicken Tie2 show features typical of this family of receptor tyrosine kinases. Chicken angiopoietin-1 and angiopoietin-2 show a high degree of homology to their human counterparts, 91% and 87% respectively, a reflection of the critical role of this signalling pathway. This revised version was published online in June 2006 with corrections to the Cover Date.     

Tie2 vascular endothelial receptor expression and function in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is generally characterized as a hypervascular tumor of rapid growth. We have previously reported that angiopoietin (Ang), a ligand for Tie2 vascular endothelial-specific receptor tyrosine kinase, may play a role in the progression of human HCC (J Clin Invest 1999;103:341-345) and matrix proteinase expression (Cancer Res 2001;61:2145-2153). However, the role of Tie2 receptor in hepatic oncogenesis is unknown. The Tie2 receptor protein was overexpressed in the neovascular endothelium of 31 of 39 (80%) human HCC tumors by immunohistochemical analysis with significant correlation to cell dedifferentiation and tumor size (P <.05). In vitro expression of a dominant-negative construct, containing a soluble Tie2 ectodomain (sTie2), led to Ang protein interaction, inhibition of endogenous Tie2 phosphorylation in vascular endothelial cells and matrix metalloproteinase 9 (MMP-9) suppression. In conclusion, tumorigenicity with neovascularization was suppressed by in vivo gene transfer and sTie2 expression in a murine HCC model, suggesting a possible role for Tie2 expression in the induction of HCC neovascularization and disease progression. Inhibition of the Ang/Tie2 signal transduction cascade is a promising approach for tumor treatment.     

A nuclease-resistant RNA aptamer specifically inhibits angiopoietin-1-mediated Tie2 activation and function

Tie2 is a receptor tyrosine kinase that is expressed predominantly in the endothelium and plays key roles in both physiological and pathological angiogenesis. The ligands for Tie2, the angiopoietins (Ang), perform opposing functions in vascular maintenance and angiogenesis; Ang1 regulates vascular quiescence, while Ang2 is thought to promote vascular destabilization and facilitate angiogenesis. However, the mechanisms responsible for these differences are not understood. To begin to elucidate the molecular differences between the angiopoietins, we previously developed a specific RNA aptamer inhibitor of Ang2. Here, we used the same iterative in vitro selection process, termed SELEX (Systematic Evolution of Ligands by EXponential enrichment), to screen a library of 2′-fluoro-modified ribonucleotides for Ang1-binding aptamers. After nine rounds of selection, we identified a single clone, ANG9-4, that bound with high affinity to human Ang1 (K _d 2.8 nM) but not Ang2 (K _d  > 1 μM), demonstrating specificity for Ang1. ANG9-4 blocked Ang1-mediated Tie2 phosphorylation and downstream Akt activation. Moreover, ANG9-4 inhibited Ang1-induced endothelial cell survival. Together, these findings demonstrate the feasibility of developing an Ang1-inhibitory aptamer. ANG9-4 and its derivatives may provide useful tools for elucidating the biology of Ang1 and for treating certain angiogenic diseases.     

Role of protein kinase Czeta in thrombin-induced endothelial permeability changes: inhibition by angiopoietin-1

Endothelial cell leakiness is regulated by mediators such as thrombin, which promotes endothelial permeability, and anti-inflammatory agents, such as angiopoietin-1. Here we define a new pathway involved in thrombin-induced permeability that involves the atypical protein kinase C isoform, PKCzeta. Chemical inhibitor studies implicated the involvement of an atypical PKC isoform in thrombin-induced permeability changes in human umbilical vein endothelial cells. Thrombin stimulation resulted in PKCzeta, but not the other atypical PKC isoform, PKClambda, translocating to the membrane, an event known to be critical to enzyme activation. The involvement of PKCzeta was confirmed by overexpression of constitutively active PKCzeta, resulting in enhanced basal permeability. Dominant-negative PKCzeta prevented the thrombin-mediated effects on endothelial cell permeability and inhibited thrombin-induced activation of PKCzeta. Rho activation does not appear to play a role, either upstream or downstream of PKCzeta, as C3 transferase does not block thrombin-induced PKCzeta activation and dominant-negative PKCzeta does not block thrombin-induced Rho activation. Finally, we show that angiopoietin-1 inhibits thrombin-induced PKCzeta activation, Rho activation, and Ca(++) flux, thus demonstrating that the powerful antipermeability action of angiopoietin-1 is mediated by its action on a number of signaling pathways induced by thrombin and implicated in permeability changes.     

Embryonic stem cell tumor model reveals role of vascular endothelial receptor tyrosine phosphatase in regulating Tie2 pathway in tumor angiogenesis

Inhibiting angiogenesis has become an effective approach for treating cancer and other diseases. However, our understanding of signaling pathways in tumor angiogenesis has been limited by the embryonic lethality of many gene knockouts. To overcome this limitation, we used the plasticity of embryonic stem (ES) cells to develop a unique approach to study tumor angiogenesis. Murine ES cells can be readily manipulated genetically; in addition, ES cells implanted subcutaneously in mice develop into tumors that contain a variety of cell types (teratomas). We show that ES cells differentiate into bona fide endothelial cells within the teratoma, and that these ES-derived endothelial cells form part of the functional tumor vasculature. Using this powerful and flexible system, the Angiopoietin/Tie2 system is shown to have a key role in the regulation of tumor vessel size. Endothelial differentiation in the ES teratoma model allows gene-targeting methods to be used in the study of tumor angiogenesis.     

Plasma angiopoietin-1, angiopoietin-2, and angiopoietin receptor tie-2 levels in congestive heart failure

OBJECTIVES: The goal of this research was to test the hypothesis that plasma angiopoietin (Ang-1), its soluble receptor tie-2, and Ang-2 levels would be abnormal in patients with acute and chronic congestive heart failure (CHF) when compared with healthy controls. BACKGROUND: Increased plasma vascular endothelial growth factor (VEGF) in CHF is suggestive of excess angiogenesis-possibly driven by tissue hypoxia. However, other growth factors also have a major role in angiogenesis, such as those of the angiopoietin family (e.g., Ang-1, which exerts its activity via its receptor, tie-2, and Ang-2). METHODS: We recruited 39 patients with acute CHF (mean age 67 +/- 10 years), 40 patients with chronic CHF (mean age 63 +/- 9 years), and 17 healthy controls (mean age 67 +/- 7 years), all in sinus rhythm. Citrated plasma was analyzed for Ang-1, Ang-2, tie-2, and VEGF by enzyme-linked immunosorbent assay. RESULTS: Angiopoietin-2 (p < 0.001), tie-2 (p < 0.05), and VEGF (p < 0.05) levels were all higher in acute CHF compared with controls. The Ang-2 levels were higher in acute CHF compared with chronic CHF (p < 0.001), but there were no significant differences in Ang-1 levels between the groups. The principal significant correlations were between Ang-2 and tie-2 (Spearman, r = 0.407; p < 0.0001) and between Ang-2 and ejection fraction (r = -0.241, p = 0.043). Although only marginally raised, levels of VEGF correlated with both Ang-2 (r = 0.468, p < 0.001) and tie-2 (r = 0.569, p < 0.001). CONCLUSIONS: We have demonstrated abnormal levels of Ang-2 and tie-2, but normal Ang-1, in both CHF patients. These abnormalities may, alongside VEGF, indicate a role for these angiogenic factors in the pathophysiology of CHF.     

Angiopoietin-1 prevents hypertension and target organ damage through its interaction with endothelial Tie2 receptor

AIMS: The endothelium has emerged recently as a therapeutic target in the treatment of hypertension because endothelial dysfunction and subsequent vascular rarefaction cause target organ damage and further elevate blood pressure (BP). It led us to hypothesize that one of the endothelial survival factors, a potent derivative of angiopoietin-1 (cartilage oligomeric matrix protein, COMP-Ang-1), could be a novel class of antihypertensive agents that maintain endothelial integrity and function, thereby preventing the development of hypertension and target organ damage. METHODS AND RESULTS: To study the role of COMP-Ang-1 in preventing hypertension and target organ damage, a COMP-Ang-1 plasmid was electroporated into adductor muscles of 6 weeks old, pre-hypertensive, spontaneously hypertensive rats (SHRs), and the secretion of its expressed protein into the bloodstream was confirmed by western blotting. In comparison with sham and reporter gene transfer, COMP-Ang-1 gene transfer significantly prevented increases in systolic BP and reduced microvascular rarefaction and tissue damage in the heart and kidney. However, overexpression of soluble Tie2 receptor completely abolished these beneficial effects of COMP-Ang-1 gene transfer on SHRs, indicating that expressed COMP-Ang-1 protein has antihypertensive effects in SHRs by binding Tie2 receptors on the vascular endothelium. In particular, COMP-Ang-1 gene-transferred SHRs had significantly higher plasma levels of nitrite than other controls, which was found to be due to that expressed COMP-Ang-1 protein promoted nitrite synthesis by activating endothelial nitric oxide synthase, one of the Tie2 downstream-signalling molecules. CONCLUSION: The present study suggests a new potential of endothelial survival factor, COMP-Ang-1, as an antihypertensive agent that effectively reduces the hypertension-associated cardiovascular and renal damage, as well as prevents the further elevation of BP.     

恙虫病东方体感染对内皮细胞Tie2/Akt/Foxo1通路的影响

目的 研究恙虫病东方体感染对Tie2/Akt/Foxo1通路的影响。方法 本研究应用新建立的小鼠和人脐静脉内皮细胞(HUVEC)严重恙虫病东方体感染模型, 在感染的不同时间点收集小鼠肺脏组织或细胞,用Western blot法检测Tie2/Akt/Foxo1通路蛋白的表达,用RT-PCR法检测炎症因子mRNA的表达。结果 感染后,小鼠肺脏血管生成素2(Ang2)表达升高,而Tie2和磷酸化Tie2(p-Tie2)水平显著下降。感染HUVEC的检测结果支持小鼠实验结果,即Ang2升高,而Tie2和p-Tie2降低;添加重组人源血管生成素1(Ang1)可以部分恢复Tie2的改变。进一步研究发现Tie2信号下游蛋白Akt磷酸化降低、Foxo1去磷酸化增加;Foxo1呈细胞核内积累,HMGB1及IFN-γ等炎症因子表达增加。同时发现恙虫病东方体感染HUVEC后,血管内皮生长因子受体2及钙粘素等细胞通透性相关蛋白表达也同Tie2一样降低。结论恙虫病东方体感染抑制Tie2/Akt/Foxo1信号的活化,而该信号在维持血管正常功能中起重要作用,提示Tie2可能是严重恙虫病治疗的一个潜在靶点。     

血管生成素1基因上调Tie2受体表达促进大鼠急性心肌梗死血管生成的实验研究

目的　通过观察血管生成素 1 (angiopoietin 1 ,Ang1 )基因对急性心肌梗死大鼠心肌Tie2受体表达的影响 ,探讨Ang1基因治疗促进血管生成 ,减少梗死面积的可能机制。方法　结扎左前降支冠状动脉制备大鼠心肌梗死模型 ,心肌内分别直接注射空载质粒或phAng1质粒 ;分别于术后第 3,7,1 4 ,2 8天取材 ,利用RT PCR技术分析心肌组织内Tie2受体表达 ;免疫组化方法计算血管生成以及肌性小动脉生成数量 ,Masson染色法检测心肌梗死面积。结果　Ang1基因治疗组Tie2受体mRNA表达明显高于对照组 ,于术后 7天达到高峰 ,2 8天降至正常水平。术后第 7,1 4和 2 8天 ,Ang1基因治疗组的血管生成及肌性小动脉生成较同期对照组均明显增加 ,梗死面积减小。结论　Ang1基因心肌内注射促进急性心肌梗死血管生成及肌性小动脉生成 ,减小梗死面积 ,其机制可能与促进Tie2受体上调有关。     

Physical association of JAK1 and JAK2 tyrosine kinases with the interleukin 2 receptor beta and gamma chains.

The functional interleukin 2 (IL-2) receptors contain the beta and gamma chains which are necessary for the transduction of cell growth signals. Monoclonal antibodies specific for the beta chain and gamma chain coimmunoprecipitated JAK1 and 114-kDa JAK2 tyrosine kinases, respectively. Tyrosine phosphorylation of JAK1 and JAK2 was induced upon IL-2 stimulation, and IL-2 activated the JAK2 kinase. These results demonstrate that the JAK1 and JAK2 tyrosine kinases are physically associated with the beta chain and gamma chain, respectively, and suggest that regulation of the kinases may be linked to IL-2-induced signal transduction.     

Overexpression of angiopoietin-1 and angiopoietin-2 in hepatocellular carcinoma

BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) is a highly vascular tumor. Angiopoietin-1 and Angiopoietin-2 have been shown to be involved in tumor angiogenesis. We investigated the expression of Angiopoietin-1 and Angiopoietin-2 in HCC. METHODS: The expression of Angiopoietin-1 and Angiopoietin-2 mRNAs in cultured hepatoma cells under hypoxic conditions and in HCC and noncancerous liver tissue was evaluated by real-time PCR. The expression of Angiopoietin-1, Angiopoietin-2, and their receptor Tie-2 in HCC was assessed by immunohistochemistry. The changes in Angiopoietin-1 and Angiopoietin-2 expression were evaluated in relation to tumor differentiation and changes in tumor vascularity. RESULTS: Hypoxic conditions did not up-regulate the expression of Angiopoietin-1 and Angiopoietin-2 mRNAs in hepatoma cells. Increased expression of Angiopoietin-1 and Angiopoietin-2 mRNAs was detected in HCC. Angiopoietin-1 and Angiopoietin-2 were detected in hepatoma cells, hepatic stellate cells, and smooth muscle cells, whereas Tie-2 was detected in endothelial cells, hepatic stellate cells and smooth muscle cells. Increased expression of Angiopoietin-2 and Angiopoietin-2 mRNA was associated with tumor dedifferentiation. The expression of Angiopoietin-1 and Angiopoietin-2 correlated with HCC vascularity. CONCLUSIONS: Our findings indicate that the increased expression of Angiopoietin-1 and Angiopoietin-2 play a critical role in the process of vascular development in HCC.     

Analysis of Tie receptor tyrosine kinase in haemopoietic progenitor and leukaemia cells

We generated a panel of monoclonal antibodies against the extracellular domain of the Tie receptor tyrosine kinase and studied its expression in human haemopoietic and tumour cell lines and in samples from leukaemia patients. Most of the erythroblastic/megakaryoblastic (6/8), 2/7 myeloid and 3/6 B-lymphoblastic leukaemia cell lines were Tie-positive. The erythroblastic/megakaryoblastic leukaemia cell lines also expressed the related Tie-2/Tek gene and, surprisingly, its recently cloned ligand gene angiopoietin-1, which was located in chromosome 8q23.1. In addition, 16% of freshly isolated leukaemia samples were Tie positive. Peripheral blood mononuclear cells were Tie negative, but a few Tie positive cells were found in immunoperoxidase staining of mobilized peripheral blood stem cells. Long-term culture of isolated umbilical cord blood CD34⁺Tie⁺ and CD34⁺Tie⁻cells indicated that the Tie⁺ fraction contained a slightly higher frequency of cobblestone area forming cells (CAFC). Thus, Tie is expressed on haemopoietic progenitor cells and some leukaemic blasts. The coexpression of Tie-2 and angiopoietin-1 in megakaryoblastic leukaemia cell lines suggests the existence of an autocrine ligand/receptor signalling loop in these cells.