Ang-1/Tie2系统与病理性血管形成的关系

血管生成素1(Ang-1)是继血管内皮生长因子(VEGF)之后,人们发现的又一重要的促血管生成因子。血管生成素家族包括Ang-l、Ang-2、Ang-3、Ang-4四种分子。其共同的特异性受体为Tie-2。目前对Ang-1/Tie2系统参与新生血管形成、促进血管成熟、抑制血管渗漏及炎症的作用研究相对深入,在创伤后修复、缺血后再通、肿瘤、糖尿病并发症及子宫内膜异位等多种病理性血管形成过程中起重要作用。     

Cell Type-Specific Expression of Angiopoietin-1 and Angiopoietin-2 Suggests a Role in Glioblastoma Angiogenesis

Glioblastomas are highly vascular tumors which overexpress the angiogenesis factor vascular endothelial growth factor (VEGF). VEGF and its receptors, VEGF-R1 and VEGF-R2, have been shown to be necessary for embryonic angiogenesis as well as for tumor angiogenesis. Recently, the angiopoietin/Tie2 receptor system has been shown to exert functions in the cardiovascular system that are distinct from VEGF but are also critical for normal vascular development. To assess the potential role of Tie2 and its ligands angiopoietin-1 and angiopoietin-2 in tumor vascularization, we analyzed their expression pattern in human gliomas. Tie-2 was up-regulated in tumor endothelium compared to normal human brain tissue. We further observed cell type-specific up-regulation of the message for both angiopoietin-1 and angiopoietin-2 in gliomas. Whereas Ang-1 mRNA was expressed in tumor cells, Ang-2 mRNA was detected in endothelial cells of a subset of glioblastoma blood vessels. Small capillaries with few periendothelial support cells showed strong expression of Angiopoietin-2, whereas larger glioblastoma vessels with many periendothelial support cells showed little or no expression. Although the function of Tie2 and its ligands in tumor angiogenesis remains a subject of speculation, our findings are in agreement with a recently proposed hypothesis that in the presence of VEGF, local production of Ang-2 might promote angiogenesis.     

大鼠脑缺血后血管内皮生长因子和血管生成素的表达变化及其作用

目的 探讨大鼠脑缺血后血管内皮生长因子(VEGF)和血管生成素(angiopoietin,Ang)的表达变化及其在血管生成和血管通透性变化中的作用.方法 选择160只雄性SD大鼠,采用随机区组法分为假手术组,缺血2h、6h、12h、1d、3d、7d和14d组,用线栓法制作大脑中动脉阻塞脑缺血损伤模型,分别于脑缺血后不同时间点处死大鼠.通过逆转录聚合酶链反应(RT-PCR)、Western blot及免疫组织化学方法检测大鼠脑缺血后不同时间点VEGF、Ang-1及Ang-2 mRNA和蛋白的表达变化.CD31标记鼠脑缺血后不同时间点的血管数量.Evans blue检测血脑屏障通透性.结果 大鼠脑缺血后VEGF mRNA表达逐渐增高,12 h达第一个高峰(0.7249 ±0.1933,P＜0.01),7d达第二个高峰(0.5264±0.1519,P＜0.01);VEGF蛋白表达也逐渐增高,于12 h达高峰(1.1017±0.1302,P＜0.01).Ang-2 mRNA和蛋白在脑缺血组织也逐渐增高,均12 h达高峰(0.6747±0.2416,P＜0.01;1.1197±0.1780,P＜0.01).与之相反,Ang-1 mRNA和蛋白表达逐渐降低,分别于3 d(0.3220±0.1427,P＜0.01)和1 d(0.1298±0.0293,P＜0.01)达最低水平,这些因子在脑缺血后的表达促进血管生成.脑缺血后血管通透性逐渐增加,EB含量在缺血1d达高峰[(6.219±0.887) μg/g,P＜0.01].结论 大鼠脑缺血后VEGF、Ang-1和Ang-2共同协调作用促进血管生成,在组织损伤修复过程中发挥着重要的作用.     

Sonic hedgehog inversely regulates the expression of angiopoietin-1 and angiopoietin-2 in fibroblasts

Nerves and blood vessels have similar branching patterns and use common morphogenic molecules during development. Recent studies show that sonic hedgehog (Shh), a traditional neurogenic morphogen, is required for embryonic arterial differentiation and can induce angiogenesis. We investigated whether Shh regulates the expression of angiogenic factors. Using NIH3T3 embryonic fibroblast cells, we demonstrated that Shh increased the mRNA levels of angiopoietin-1 (Ang-1), a secreted ligand that regulates endothelial interaction with mural cells (pericytes and smooth muscle cells) and promotes blood vessel maturation. In contrast, Shh decreased mRNA levels of angiopoietin-2 (Ang-2), a negative modulator of Ang-1. By contrast, Shh did not change the expression of vascular endothelial growth factor (VEGF) mRNA, a potent endothelial mitogen. The effect of Shh appeared to be cell-type specific as the addition of Shh to neural progenitor cells or neurons did not alter Ang-1, Ang-2 or VEGF mRNA levels. The addition of cyclopamine, an inhibitor of Shh signaling, to NIH3T3 cells, suppressed the regulation of Ang-1 and Ang-2 mRNA levels in the presence of Shh. Collectively, our results suggest that Shh may contribute to blood vessel growth, maturation and stabilization in a neurovascular network by reciprocally regulating the vascular morphogens Ang-1 and Ang-2 in a cell-type-specific manner.     

Engeletin protects against cerebral ischemia/reperfusion injury by modulating the VEGF/vasohibin and Ang-1/Tie-2 pathways

Engeletin is a natural derivative of Smilax glabra rhizomilax that exhibits anti-inflammatory activity and suppresses lipid peroxidation. In the present study, we sought to elucidate the mechanistic basis for the neuroprotective and pro-angiogenic activity of engeltin in a human umbilical vein endothelial cells (HUVECs) oxygen-glucose deprivation and reoxygenation (OGD/R) model system and a middle cerebral artery occlusion (MCAO) rat model of cerebral ischemia and reperfusion injury. These analyses revealed that engeletin (10, 20, or 40 mg/kg) was able to reduce the infarct volume, increase cerebral blood flow, improve neurological function, and bolster the expression of vascular endothelial growth factor (VEGF), vasohibin-2 (Vash-2), angiopoietin-1 (Ang-1), phosphorylated human angiopoietin receptor tyrosine kinase 2 (p-Tie2), and platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) in MCAO rats. Similarly, engeletin (100, 200, or 400 nM) markedly enhanced the migration, tube formation, and VEGF expression of HUVECs in an OGD/R model system, while the VEGF receptor (R) inhibitor axitinib reversed the observed changes in HUVEC tube formation activity and Vash-2, VEGF, and CD31 expression. These data suggested that engeletin exhibited significant neuroprotective effects against cerebral ischemia and reperfusion injury in rats, and improved cerebrovascular angiogenesis by modulating the VEGF/vasohibin and Ang-1/Tie-2 pathways.     

Expressional regulation of angiopoietin-1 and -2 and the tie-1 and -2 receptor tyrosine kinases during cutaneous wound healing: a comparative study of normal and impaired repair

It has become evident that a closely regulated presence of vascular endothelial growth factor (VEGF) and angiopoietin (Ang) factors determines the fate of blood vessel formation during angiogenesis. As angiogenesis is central to a normal wound-healing process, we investigated the regulation of Ang-1 and -2 and the related tyrosine kinase with immunoglobulin and epidermal growth factor homology (Tie)-1 and -2 receptors during normal repair in Balb/c mice and diabetes-impaired wound healing conditions in genetically diabetic (db/db) mice. For both normal and impaired healing conditions, we observed a constitutive expression of Ang-1, which was paralleled by an increase of Ang-2 upon injury. Whereas the observed Ang-2 expression declines from Day 7 after injury in control mice, diabetic-impaired healing was characterized by still increasing amounts of Ang-2 at these time points. Furthermore, Tie-1 was strongly induced during repair with a prolonged expression in diabetic mice, whereas Tie-2 expression was constitutive during normal repair but completely absent in diabetes-impaired healing. The overexpression of Ang-2 in the presence of markedly reduced VEGF in wounds of diabetic mice was associated with a dramatic decrease in endothelial cell numbers compared with normal healing as assessed by analysis of the endothelium-specific markers CD31 and von Willebrand factor, whereas the lymphatic endothelium remained stable as determined by expression of VEGF receptor-3 (VEGFR-3/Flt-4).     

Down-regulation of angiopoietin-1 expression in menorrhagia

Angiogenesis is an essential component of endometrial repair and regeneration following menses. Perturbation of this process is associated with menorrhagia, a common gynecological disorder that results in excessive menstrual bleeding. Angiopoietin-1 (Ang-1) promotes vascular maturation via the Tie-2 receptor, while angiopoietin-2 (Ang-2) is its natural antagonist that destabilizes vessels and initiates neovascularization in the presence of vascular endothelial growth factor. To test the hypothesis that menorrhagia arises as a result of poor signal for vascular maturation, we have examined the expression of Ang-1, Ang-2, and Tie-2 in endometrium throughout the menstrual cycle from 30 normal women and 28 patients with menorrhagia. Ribonuclease protection assay and Western blot analysis showed Ang-2 expression was consistently higher than Ang-1 in normal endometrium throughout the cycle. However, with menorrhagia Ang-1 mRNA and protein were not detected or down-regulated, while Ang-2 was observed at similar levels in both normal and menorrhagic endometrium resulting in a greater than a 50% decrease in the ratio of Ang-1 to Ang-2 protein. In situ hybridization and immunohistochemical studies supported these findings and revealed cyclical changes in the expression of Ang-1 and Ang-2. These results suggest that the angiopoietin/Tie-2 system promotes vascular remodeling in endometrium and loss of normal Ang-1 expression may contribute to the excessive blood loss observed in menorrhagia.     

Increased Tie2 Expression, Enhanced Response to Angiopoietin-1, and Dysregulated Angiopoietin-2 Expression in Hemangioma-Derived Endothelial Cells

Infantile hemangiomas are endothelial tumors that grow rapidly in the first year of life and regress slowly during early childhood. Although hemangiomas are well-known vascular lesions, little is known about the mechanisms that cause the excessive endothelial cell proliferation in these most common tumors of infancy. To investigate the molecular basis of hemangioma, we isolated endothelial cells from several proliferative-phase lesions and showed that these cells are clonal and exhibit abnormal properties in vitro (E. Boye, Y. Yu, G. Paranya, J. B. Mulliken, B. R. Olsen, J. Bischoff: Clonality and altered behavior of endothelial cells from hemangiomas. J Clin Invest 2001, 107:745-752). Here, we analyzed mRNA expression patterns of genes required for angiogenesis, including members of the vascular endothelial growth factor (VEGF)/VEGF receptor family and the angiopoietin/Tie family, in hemangioma-derived and normal endothelial cells. KDR, Flt-1, Tie1, Tie2, and angiopoietin-2 (Ang2) were strongly expressed in cultured hemangioma-derived endothelial cells and in hemangioma tissue. In contrast, there was little expression of angiopoietin-1 (Ang1) or VEGF. We found Tie2 mRNA and protein up-regulated with a concomitant increase in cellular responsiveness to Ang1 in most hemangioma-derived endothelial cells. Ang2 mRNA was down-regulated in response to serum in hemangioma-derived endothelial cells, but not in normal endothelial cells, suggesting altered regulation. These findings implicate Tie2 and its ligands Ang1 and Ang2 in the pathogenesis of hemangioma.     

Proangiogenic cytokines as hypoxia-dependent factors stimulating migration of human hepatic stellate cells

Pathological angiogenesis is associated with the fibrogenic progression of chronic liver diseases. Experimental data suggest that hypoxia and vascular endothelial growth factor (VEGF) may stimulate proliferation and synthesis of type I collagen in activated, myofibroblast-like rat hepatic stellate cells (HSC/MFs). In this study, we investigated whether hypoxia, recombinant VEGF, or angiopoietin 1 (Ang-1) may affect other crucial profibrogenic features. In human HSC/MFs, which constitutively express VEGF receptor-1 and -2 (VEGFR-1, VEGFR-2) and the Ang-1 receptor Tie-2, exposure to hypoxia, VEGF, or Ang-1 resulted in a Ras/Erk-dependent stimulation of chemokinesis and chemotaxis. Migration of human HSC/MFs under hypoxic conditions involved up-regulation of VEGF-A, Ang-1, and related receptors and was mainly dependent on VEGFR-2 (Flk-1). In specimens from either cirrhotic rat livers or from patients with hepatitis C virus-related cirrhosis, HSC/MFs expressed proangiogenic factors and related receptors in areas of active fibrogenesis (ie, at the leading or lateral edge of developing incomplete fibrotic septa). Data presented herein suggest that VEGF and Ang-1 may contribute to fibrogenesis by acting as hypoxia-inducible, autocrine, and paracrine factors able to recruit myofibroblast-like cells. Moreover, HSC/MFs, in addition to their established profibrogenic role, may also contribute to neoangiogenesis during chronic hepatic wound healing.     

血管生成素对糖尿病大鼠股骨头血管新生及渗漏的影响

目的　探讨血管生成素1(angi.Poietin-1,Ang-1)对糖尿病大鼠股骨头微血管新生及渗漏的影响。 方法　建立速发型链脲佐菌素（streptozotocin,STZ）糖尿病大鼠模型,随机分为正常5周组　(CON1)、10周组(CON2)及15周组(CON3),糖尿病5周组（DM1）、10周组（DM2）及15周组（DM3）,每组　10只。墨汁灌注观测股骨头微血管密度;摘取模型动物股骨头组织,免疫组化分析凝血因子Ⅷ（FⅧ）表达;原位杂交分析血管内皮生长因子（VEGFmRNA）表达强度;RT-PCR分析Ang-1的mRNA表达。 结果　糖尿病大鼠股骨头随病程发展,Ang-1、FⅧ因子表达上升,与正常组相比有显著性差异（P<0.01）。　VEGFmRNA表达量均高于正常组（P<0.01）;微血管密度加大,显示血管增生、渗漏。 结论　糖尿病股骨头　Ang-1与VEGFmRNA相互协同或拮抗分别促进微血管增生、抗血管渗漏。表达于血管内皮细胞的FⅧ及VEGFmRNA与微血管密度（MVD）变化存在正相关。     

Localization of Ang-1, -2, Tie-2, and VEGF expression at endothelial-pericyte interdigitation in rat angiogenesis

Endothelial cells and pericytes play critical role in angiogenesis, which is controlled, in part, by the angiopoietin (Ang)/Tie-2 system and vascular endothelial growth factor (VEGF). Here, we investigated Ang, Tie-2, and VEGF expression within endothelial cells and pericyte interdigitations (EPI), which consist of cytoplasmic projections of pericytes and corresponding endothelial indentations. After subcutaneous implantation of a thermoreversible gelation polymer disc in rats, the capillary density was low on day 5, increased to a peak on day 7, and then decreased on days 10-20. A small number of EPI were observed on day 5, then increased sharply to a peak on day 10, but had decreased on day 20. Light and electron microscopy immunohistochemical and RNA in situ hybridization analyses revealed that Tie-2 localized at endothelial cells, and Ang-2 localized at endothelial cells and pericytes, while Ang-1 and VEGF localized at pericytes, and Ang-1 was most intensely observed at EPI of pericytes. Conventional quantitative RT-PCR and Western blot analyses revealed that the level of Ang-1 was low on days 5-7, then increased on days 10-20, while the level of VEGF was high on days 5-10, but had decreased on day 20. The level of Ang-2 remained high and Tie-2 remained at the level of the control on days 5-20. The present study showed that the angiogenic phase might be initiated by increases in Ang-2 and VEGF, while the microvessel maturation phase might be initiated by a relative increase in Ang-1 and a decrease in VEGF. Moreover, EPI might serve as a pathway for the Ang-1/Tie-2 system, with VEGF promoting pericyte recruitment for microvascular integrity.     

Clinical relevance of angiopoietin-1, angiopoietin-2, and their receptor Tie-2 expression in acute myeloid leukemia

The angiogenic-related factors: angiopoietin-1 and -2 and their receptor Tie-2 have wide-ranging effects on tumor behavior that includes angiogenesis and, inflammation. These multifaceted pathways present a potential target in developing novel inhibition strategies for cancer therapy. The present work aimed at detecting the prevalence of expression of: angiopoietin-1, angiopoietin-2, and their receptor Tie-2 in 56 Egyptian de novo acute myeloid leukemia (AML) patients by conventional RT-PCR to verify the prognostic impact of their expression on the response to induction chemotherapy. Thirty age- and sex-matched healthy volunteers were subjected to the same analysis as a control group. High expression of angiopoietin-1 (Ang-1) was detected in the patient group but not the control group. AML patients expressing angiopoietin-2 (Ang-2) either solely or in combination with high Ang-1 and/or Tie-2 showed unfavorable response to induction chemotherapy; either failed induction or death during induction. These data provide evidence that the alternation of angiopoietin balance in favor of Ang-2 may play a critical role in the pathophysiology of AML. Furthermore, positive pre-therapeutic expression of Ang-2 indicates valiable unfavorable prognostic marker in AML patients and may be used as a prognostic tool in the risk-adaptive management of AML.     

Clinical relevance of angiopoietin-1, angiopoietin-2, and their receptor Tie-2 expression in acute myeloid leukemia

The angiogenic-related factors, angiopoietin-1 and angiopoietin-2 and their receptor Tie-2, have wide-ranging effects on tumor behavior that includes angiogenesis and inflammation. These multifaceted pathways present a potential target in developing novel inhibition strategies for cancer therapy. The present work aimed at detecting the prevalence of expression of angiopoietin-1, angiopoietin-2, and their receptor Tie-2 in 56 Egyptian de novo acute myeloid leukemia (AML) patients by conventional RT-PCR to verify the prognostic impact of their expression on the response to induction chemotherapy. Thirty age- and sex-matched healthy volunteers were subjected to the same analysis as a control group. High expression of Ang-1 was detected in the patient group but not the control group. AML patients expressing Ang-2 either solely or in combination with high Ang-1 and/or Tie-2 showed unfavorable response to induction chemotherapy, either failed induction or death during induction. These data provide evidence that the alternation of angiopoietin balance in favor of Ang-2 may play a critical role in the pathophysiology of AML. Furthermore, positive pre-therapeutic expression of Ang-2 indicates viable unfavorable prognostic marker in AML patients and may be used as a prognostic tool in the risk-adaptive management of AML.     

Direct actions of angiopoietin-1 on human endothelium: evidence for network stabilization, cell survival, and interaction with other angiogenic growth factors

Angiopoietin-1 (Ang-1) is a recently described angiogenic protein that activates the endothelial Tie 2 receptor. Disruption of the Ang-1 gene shows that it has an indispensable role in blood vessel development, but it is not clear what specific effects, if any, Ang-1 has on endothelial cell (EC) phenotypes. Here, we show that Ang-1 dose-dependently stabilizes HUVEC network organization for up to 48 hours; this action of Ang-1 is dependent on Tie-2 receptor activation, because a soluble form of the Tie2-, but not the Tie1-receptor, completely blocks the effects of Ang-1. Moreover, we show that Ang-1 potentiates the actions of other angiogenic growth factors. Ang-1 markedly increases the survival of vascular networks (up to 96 hours) exposed to either vascular endothelial growth factor or endothelial cell growth supplement, a form of acidic fibroblast growth factor. In addition, Ang-1 prevents apoptotic death in HUVEC triggered by withdrawal of endothelial cell growth supplement. Collectively, these data are consistent with the idea that Ang-1 directly acts on human EC and interacts with other angiogenic molecules to stabilize vascular structures by promoting the survival of differentiated ECs.     

Retinal angiogenesis is mediated by an interaction between the angiotensin type 2 receptor,VEGF, and angiopoietin

There is evidence that angiotensin II, vascular endothelial growth factor (VEGF), angiopoietins, and their cognate receptors participate in retinal angiogenesis. We investigated whether angiotensin type 2-receptor blockade (AT2-RB) reduces retinal angiogenesis and alters the expression of VEGF/VEGF-R2 and angiopoietin-Tie2. Retinopathy of prematurity (ROP) was induced in Sprague Dawley (SD) rats by exposure to 80% oxygen from postnatal (P) days 0 to 11, followed by 7 days in room air. ROP shams were in room air from P0-18. A group of ROP rats received the AT2-RB, PD123319, by mini-osmotic pump (5 mg/kg/day) from P11-18 (angiogenesis period). Evaluation of the retinal status of the AT2 receptor indicated that this receptor, as assessed by real-time PCR, immunohistochemistry, and in vitro autoradiography, was present in the retina, was more abundant than the AT1 receptor in the neonatal retina, and was increased in the ROP model. AT2-RB reduced retinal angiogenesis. VEGF and VEGF-R2 mRNA were increased in ROP and localized to blood vessels, ganglion cells, and the inner nuclear layer, and were decreased by PD123319. Angiopoietin2 and Tie2, but not angiopoietin1 mRNA were increased with ROP, and angiopoietin2 was reduced with PD123319. This study has identified a potential retinoprotective role for AT2-RB possibly mediated via interactions with VEGF- and angiopoietin-dependent pathways.     

Time course of changes in angiogenesis-related factors in denervated muscle

Aim: Denervation leads to capillary regression in skeletal muscle. To gain insight into the regulation of this process, we investigated the time course of changes in capillary supply and gene expression of angiogenesis‐related factors during muscle denervation. Method: Female mice underwent surgery to transect the sciatic nerve, and then the gastrocnemius muscles were isolated at 12 h, 1, 3, 5, 10, 20, or 30 days after surgery. The capillary supply was assessed by immunohistochemistry using anti‐PECAM‐1/CD31 antibody. The mRNA levels for angiogenesis‐related factors were analysed using a real‐time polymerase chain reaction. Results: We found that the capillary‐to‐fibre ratio began to decrease 10 days after muscle denervation and decreased by 52% after 30 days. The levels of mRNA for vascular endothelial growth factor (VEGF), its receptors [fms‐like tyrosine kinase (Flt‐1) and a kinase insert domain‐containing receptor/fetal liver kinase‐1 (KDR/Flk‐1)], angiopoietin‐1 and angiopoietin‐2 of denervated muscle were immediately down‐regulated after 12 h and remained lower than control muscle until 30 days after muscle denervation. The levels of mRNA for the VEGF receptor, neuropilin‐1, angiopoietin receptor and Tie‐2 decreased within 12–24 h, but returned to near those of control muscle after 10–20 days, and again decreased after 30 days. Conclusions: These findings suggest that denervation‐induced capillary regression may be associated with down‐regulation of VEGF and angiopoietin signalling.     

Angiopoietin-2 is implicated in the regulation of tumor angiogenesis

We addressed the effect of angiopoietin expression on tumor growth and metastasis. Overexpression of angiopoietin-2 (Ang-2) in Lewis lung carcinoma and TA3 mammary carcinoma cells inhibited their ability to form metastatic tumors and prolonged the survival of mice injected with the corresponding transfectants. In contrast, angiopoietin-1 (Ang-1) overexpression had no detectable effect on the ability of either tumor type to disseminate. Tumors derived from Ang-2-overexpressing cells displayed aberrant angiogenic vessels that took the form of vascular cords or aggregated vascular endothelial cells with few associated smooth muscle cells. These vascular cords or aggregates were accompanied by endothelial and tumor cell apoptosis, suggesting that an imbalance in Ang-2 expression with respect to Ang-1 and vascular endothelial growth factor may disrupt angiogenesis and tumor survival in vivo. Our observations suggest that Ang-2 may play an important role in regulating tumor angiogenesis.