Angiopoietin-1 promotes endothelial differentiation from embryonic stem cells and induced pluripotent stem cells

Angiopoietin-1 (Ang1) plays a crucial role in vascular and hematopoietic development, mainly through its cognate receptor Tie2. However, little is known about the precise role of Ang1 in embryonic stem cell (ESC) differentiation. In the present study, we used COMP-Ang1 (a soluble and potent variant of Ang1) to explore the effect of Ang1 on endothelial and hematopoietic differentiation of mouse ESCs in an OP9 coculture system and found that Ang1 promoted endothelial cell (EC) differentiation from Flk-1(+) mesodermal precursors. This effect mainly occurred through Tie2 signaling and was altered in the presence of soluble Tie2-Fc. We accounted for this Ang1-induced expansion of ECs as enhanced proliferation and survival. Ang1 also had an effect on CD41(+) cells, transient precursors that can differentiate into both endothelial and hematopoietic lineages. Intriguingly, Ang1 induced the preferential differentiation of CD41(+) cells toward ECs instead of hematopoietic cells. This EC expansion promoted by Ang1 was also recapitulated in induced pluripotent stem cells (iPSCs) and human ESCs. We successfully achieved in vivo neovascularization in mice by transplantation of ECs obtained from Ang1-stimulated ESCs. We conclude that Ang1/Tie2 signaling has a pivotal role in ESC-EC differentiation and that this effect can be exploited to expand EC populations.     

Direct chemotactic action of angiopoietin-1 on mesenchymal cells in the presence of VEGF

Angiopoietin-1 (Ang1) and its receptor, Tie2, play an important role in angiogenesis and vessel maturation. We previously reported that overexpression of Ang1 in MCF7 xenograft tumors facilitated vessel stabilization by mural cells, and that cultured SMC express Tie2. Here, we investigated whether Ang1 directly acts as a chemoattractant on mural cells or their precursors. In a Matrigel plug assay, neither Ang1 nor VEGF alone induced angiogenesis but together stimulated infiltration of non-endothelial cells that were CD31-negative, vimentin-positive and also positive for VEGFR-1 and Tie2. While negative for smooth muscle actin, reactivity for desmin suggests that the cells are mural cell precursors. VEGF treatment of cultured smooth muscle cells (SMC) upregulated Tie2 and allowed for Ang1-mediated phosphorylation of Tie2 and the AKT serine-threonine kinase. The combination of Ang1 and VEGF stimulated SMC migration in a Boyden chamber-type assay. In the presence of VEGF, Tie2 is upregulated on mural cells, allowing for a migratory response to Ang1. These findings support the view that Ang1, in concert with VEGF, can act directly on mural cells or their precursors to facilitate their recruitment to new blood vessels. This action may play an important role in vascular stabilization.     

The role of the Angiopoietins in vascular morphogenesis

The Angiopoietin/Tie system acts as a vascular specific ligand/receptor system to control endothelial cell survival and vascular maturation. The Angiopoietin family includes four ligands (Angiopoietin-1, Angiopoietin-2 and Angiopoietin-3/4) and two corresponding tyrosine kinase receptors (Tie1 and Tie2). Ang-1 and Ang-2 are specific ligands of Tie2 binding the receptor with similar affinity. Tie2 activation promotes vessel assembly and maturation by mediating survival signals for endothelial cells and regulating the recruitment of mural cells. Ang-1 acts in a paracrine agonistic manner inducing Tie2 phosphorylation and subsequent vessel stabilization. In contrast, Ang-2 is produced by endothelial cells and acts as an autocrine antagonist of Ang-1-mediated Tie2 activation. Ang-2 thereby primes the vascular endothelium to exogenous cytokines and induces vascular destabilization at higher concentrations. Ang-2 is strongly expressed in the vasculature of many tumors and it has been suggested that Ang-2 may act synergistically with other cytokines such as vascular endothelial growth factor to promote tumor-associated angiogenesis and tumor progression. The better mechanistic understanding of the Ang/Tie system is gradually paving the way toward the rationale exploitation of this vascular signaling system as a therapeutic target for neoplastic and non-neoplastic diseases.     

Tie2 receptor tyrosine kinase, a major mediator of tumor necrosis factor alpha-induced angiogenesis in rheumatoid arthritis

OBJECTIVE: Rheumatoid arthritis (RA) is an inflammatory disease and an angiogenic disease. However, the molecular mechanisms promoting angiogenesis in RA are not clearly identified. Our objective was to study the role of an endothelium-specific receptor tyrosine kinase, Tie2, in angiogenesis of inflammatory arthritis. METHODS: Expression of Tie2 and its ligand, angiopoietin 1 (Ang1), in human synovium was examined by immunohistochemistry and Western blot. A novel synovium vascular window model was established to study the role of Tie2 in angiogenesis in vivo. Primary cultured endothelial cells and synoviocytes were used to study tumor necrosis factor alpha (TNF alpha)-induced Tie2 and Ang1 expression. RESULTS: Tie2 was implicated in pathologic angiogenesis. We observed that Tie2 and Ang1 were elevated in human RA synovium. Using a novel collagen-induced arthritis synovial window model, we demonstrated that Tie2 signaling regulated arthritis angiogenesis in vivo. We also showed that Tie2 mediated TNF alpha-induced angiogenesis in a mouse cornea assay. In addition, we observed that TNF alpha can regulate Tie2 activation in multiple ways that may involve interactions between endothelial cells and synoviocytes. TNF alpha up-regulates Tie2 in endothelial cells through nuclear factor kappa B, and it up-regulates Ang1 in synoviocytes. These findings suggest paracrine regulation of angiogenesis between endothelial cells and synoviocytes. CONCLUSION: This study demonstrates that Tie2 regulates angiogenesis in inflammatory synovium. Tie2 signaling is an important angiogenic mediator that links the proinflammatory cytokine TNF alpha to pathologic angiogenesis.     

Angiopoietins can directly activate endothelial cells and neutrophils to promote proinflammatory responses

Angiopoietin-1 (Ang1) and -2 (Ang2) are endothelial growth factors that bind to the tyrosine kinase receptor Tie2 and contribute to orchestrate blood vessel formation during angiogenesis. Ang1 mediates vessel maturation and integrity by the recruitment of pericytes. In contrast, Ang2 is classically considered as a Tie2 antagonist, counteracting the stabilizing action of Ang1. Inflammation exists in a mutually dependent association with angiogenesis and we have therefore studied the capacity of angiopoietins to modulate proinflammatory activities, namely P-selectin translocation and neutrophil adhesion onto endothelial cells. We observed that both Ang1 and Ang2 increased these biologic activities. Furthermore, combination of Ang1/Ang2 induced an additive effect on neutrophil adhesion but not on P-selectin translocation. In an attempt to clarify this phenomenon, we found that angiopoietins can directly activate neutrophils through Tie2 signaling as well as modulate platelet-activating factor (PAF) synthesis and beta(2) integrin functional up-regulation. Together, our data demonstrate that angiopoietins could promote acute recruitment of leukocytes, which might contribute to facilitate vascular remodeling and angiogenesis.     

Biological action of angiopoietin-2 in a fibrin matrix model of angiogenesis is associated with activation of Tie2

The endothelial cell (EC) specific tyrosine kinase receptor, Tie2, interacts with at least two ligands, angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2). Ang1 stimulates Tie2 receptor autophosphorylation, while Ang2 has been reported to inhibit Ang1-induced Tie2 receptor autophosphorylation. We studied the effects of Ang1 and Ang2 in an in vitro model of angiogenesis. Human ECs (HUVEC), cultured on 3-D fibrin matrices, were treated with conditioned media (CM) from stably transfected cells expressing human Ang1 or Ang2, or with purified recombinant proteins. EC tube formation was measured as a differentiation index (DI), calculated as the ratio of total tube length over residual of EC monolayer. CM from Ang1 overexpressing A10 SMC or HEK293T cells induced profound HUVEC differentiation, resulting in the formation of extensive capillary-like tubes within 48 h (DI: 24.58+/-5.91 and 19.13+/-7.86, respectively) vs. control (DI: 2.73+/-1.68 and 2.15+/-1.45, respectively, both P<0.001). Interestingly, CM from two independent cell lines overexpressing Ang2 also produced a significant increase in EC differentiation (DI: 9.22+/-3.00 and 9.72+/-4.84, both P<0.005 vs. control) although the degree of angiogenesis was significantly less then that seen with Ang1. Addition of Ang1* (a genetically engineered variant of naturally occurring Ang1) or Ang2 also resulted in dose dependent increases in DI, which were blocked by an excess of soluble Tie2 receptor (20 microg/ml). Both Ang1* and Ang2 induced modest increases in [3H]thymidine incorporation into HUVECs (20 and 26%, respectively), which were inhibited by excess soluble Tie2. Although Ang2 was unable to induce significant Tie2 receptor phosphorylation during a 5-min exposure, a 24-h pretreatment with Ang2, followed by brief re-exposure, produced Tie2 phosphorylation in HUVEC comparable to that produced by Ang1*. These results demonstrate for the first time that Ang2 may have a direct role in stimulating Tie2 receptor signaling and inducing in vitro angiogenesis. Our findings suggest that the physiological role of Ang2 is more complex than previously recognized: acting alternately to promote or blunt Tie2 receptor signaling in endothelial cells, depending on local conditions.     

Angiopoietin-2 displays VEGF-dependent modulation of capillary structure and endothelial cell survival in vivo

Modulation of Tie2 receptor activity by its angiopoietin ligands is crucial for angiogenesis, blood vessel maturation, and vascular endothelium integrity. It has been proposed that angiopoietins 1 (Ang1) and 2 (Ang2) are pro- and anti-angiogenic owing to their respective agonist and antagonist signaling action through the Tie2 receptor. The function of Ang2 has remained controversial, however, with recent reports suggesting that in some circumstances, it may be pro-angiogenic. We have examined this issue using the transient ocular microvessel network called the pupillary membrane as a unique in vivo model for studying the effects of vascular regulators. We show that in vivo, in the presence of endogenous vascular endothelial growth factor (VEGF)-A, Ang2 promotes a rapid increase in capillary diameter, remodeling of the basal lamina, proliferation and migration of endothelial cells, and stimulates sprouting of new blood vessels. By contrast, Ang2 promotes endothelial cell death and vessel regression if the activity of endogenous VEGF is inhibited. These observations support a model for regulation of vascularity where VEGF can convert the consequence of Ang2 stimulation from anti- to pro-angiogenic.     

Roles of the receptor tyrosine kinases Tie1 and Tie2 in mediating the effects of angiopoietin-1 on endothelial permeability and apoptosis

Angiopoietin-1 (Ang1) has key roles in development and maintenance of the vascular system. The ligand is a potent inhibitor of vascular leakage and suppresses endothelial apoptosis and vessel regression. Ang1 was originally identified as a ligand for the receptor tyrosine kinase Tie2. Recently however Ang1 has also been found to activate the related tyrosine kinase Tie1. The contribution of Tie1 to mediating the effects of Ang1 on endothelial function is not known. In this study we used an siRNA approach to investigate the relative importance of Tie1 and Tie2 in transducing the effects of Ang1 on monolayer permeability and induction of apoptosis in human endothelial cells. siRNA directed against either Tie1 or Tie2 suppressed expression of each respective receptor by more than 90%. Ang1 inhibited endothelial monolayer permeability and this effect was prevented by suppression of Tie2 expression. In contrast, Ang1 inhibition of permeability was not affected by suppression of Tie1 expression. The ability of Ang1 to inhibit induction of apoptosis in response to serum deprivation was completely blocked by suppression of Tie2 expression, but not diminished by suppression of Tie1 expression. Taken together these data demonstrate that Tie2 mediates the inhibitory effects of Ang1 on endothelial permeability and apoptosis. The data also demonstrates that Tie1 does not transduce anti-apoptotic or anti-permeability effects of Ang1 in endothelial cells.     

Angiopoietin-1 regulates endothelial cell survival through the phosphatidylinositol 3'-Kinase/Akt signal transduction pathway

Angiopoietin-1 (Ang1) is a strong apoptosis survival factor for endothelial cells. In this study, the receptor/second messenger signal transduction pathway for the antiapoptotic effect of Ang1 on human umbilical vein endothelial cells was examined. Pretreatment with soluble Tie2 receptor, but not Tie1 receptor, blocked the Ang1-induced antiapoptotic effect. Ang1 induced phosphorylation of Tie2 and the p85 subunit of phosphatidylinositol 3'-kinase (PI 3'-kinase) and increased PI 3'-kinase activity in a dose-dependent manner. The PI 3'-kinase-specific inhibitors wortmannin and LY294002 blocked the Ang1-induced antiapoptotic effect. Ang1 induced phosphorylation of the serine-threonine kinase Akt at Ser473 in a PI 3'-kinase-dependent manner. Expression of a dominant-negative form of Akt reversed the Ang1-induced antiapoptotic effect. Ang1 mRNA and protein were present in vascular smooth muscle cells but not in endothelial cells. Cultured vascular smooth muscle cells, but not human umbilical vein endothelial cells, secreted Ang1. These findings indicate that the Tie2 receptor, PI 3'-kinase, and Akt are crucial elements in the signal transduction pathway leading to endothelial cell survival induced by the paracrine activity of Ang1.     

Angiopoietin-1 promotes LYVE-1-positive lymphatic vessel formation

Angiopoietin (Ang) signaling plays a role in angiogenesis and remodeling of blood vessels through the receptor tyrosine kinase Tie2, which is expressed on blood vessel endothelial cells (BECs). Recently it has been shown that Ang-2 is crucial for the formation of lymphatic vasculature and that defects in lymphangiogenesis seen in Ang-2 mutant mice are rescued by Ang-1. These findings suggest important roles for Ang signaling in the lymphatic vessel system; however, Ang function in lymphangiogenesis has not been characterized. In this study, we reveal that lymphatic vascular endothelial hyaluronan receptor 1-positive (LYVE-1(+)) lymphatic endothelial cells (LECs) express Tie2 in both embryonic and adult settings, indicating that Ang signaling occurs in lymphatic vessels. Therefore, we examined whether Ang-1 acts on in vivo lymphatic angiogenesis and in vitro growth of LECs. A chimeric form of Ang-1, cartilage oligomeric matrix protein (COMP)-Ang-1, promotes in vivo lymphatic angiogenesis in mouse cornea. Moreover, we found that COMP-Ang-1 stimulates in vitro colony formation of LECs. These Ang-1-induced in vivo and in vitro effects on LECs were suppressed by soluble Tie2-Fc fusion protein, which acts as an inhibitor by sequestering Ang-1. On the basis of these observations, we propose that Ang signaling regulates lymphatic vessel formation through Tie2.     

Interaction between Tie receptors modulates angiogenic activity of angiopoietin2 in endothelial progenitor cells

OBJECTIVE: Ischemia-dependent upregulation of angiopoietin2 (Ang2) led us to hypothesize the potentially proangiogenic Ang2-Tie2 signaling in endothelial progenitor cells (EPCs). Given the well-known vascular destabilizing action of Ang2 in mature endothelium, we investigated the yet unidentified mechanism behind cell-dependent differential activity of Ang2. METHODS AND RESULTS: Both in vitro and in vivo experiments showed that Ang2 promoted angiogenicity of human cord blood-derived EPCs, where Ang2 directly activated Tie2 and its related downstream signaling molecules. However, Ang2 had no such effect in fully differentiated human umbilical vein endothelial cells (HUVECs) under the same condition. Such a cell-dependent Tie2 activation by Ang2 was explained by comparing EPCs and HUVECs, where most Tie2 receptors in EPCs were found to be present unbound to Tie1, whereas those in HUVECs existed as heterocomplexes with Tie1. When Tie2 in HUVECs was prevented from forming heterocomplexes by silencing Tie1 expression, they underwent rapid phosphorylation upon Ang2 treatment, as shown in EPCs. CONCLUSIONS: In contrast with its roles in mature endothelial cells, Ang2 has proangiogenic activities in EPC directly through Tie2 signaling pathway. Such a cell-dependent differential reactivity of Ang2 was for the first time found to be modulated by physical association between Tie1 and Tie2, which inhibited Ang2-mediated Tie2 activation.     

Angiopoietin-1 promotes lymphatic sprouting and hyperplasia

Angiopoietin 1 (Ang1), a ligand for the receptor tyrosine kinase Tie2, regulates the formation and stabilization of the blood vessel network during embryogenesis. In adults, Ang1 is associated with blood vessel stabilization and recruitment of perivascular cells, whereas Ang2 acts to counter these actions. Recent results from gene-targeted mice have shown that Ang2 is also essential for the proper patterning of lymphatic vessels and that Ang1 can be substituted for this function. In order to characterize the effects of the angiopoietins on lymphatic vessels, we employed viral vectors for overexpression of Ang1 in adult mouse tissues. We found that Ang1 activated lymphatic vessel endothelial proliferation, vessel enlargement, and generation of long endothelial cell filopodia that eventually fused, leading to new sprouts and vessel development. Cutaneous lymphatic hyperplasia was also detected in transgenic mice expressing Ang1 in the basal epidermal cells. Tie2 was expressed in the lymphatic endothelial cells and Ang1 stimulation of these cells resulted in up-regulation of vascular endothelial growth factor receptor 3 (VEGFR-3). Furthermore, a soluble form of VEGFR-3 inhibited the observed lymphatic sprouting. Our results reinforce the concept that Ang1 therapy may be useful in settings of tissue edema.     

Tie2 receptor tyrosine kinase,a major mediator of tumor necrosis factor α–induced angiogenesis in rheumatoid arthritis

Objective

Rheumatoid arthritis (RA) is an inflammatory disease and an angiogenic disease. However, the molecular mechanisms promoting angiogenesis in RA are not clearly identified. Our objective was to study the role of an endothelium‐specific receptor tyrosine kinase, Tie2, in angiogenesis of inflammatory arthritis.

Methods

Expression of Tie2 and its ligand, angiopoietin 1 (Ang1), in human synovium was examined by immunohistochemistry and Western blot. A novel synovium vascular window model was established to study the role of Tie2 in angiogenesis in vivo. Primary cultured endothelial cells and synoviocytes were used to study tumor necrosis factor α (TNFα)–induced Tie2 and Ang1 expression.

Results

Tie2 was implicated in pathologic angiogenesis. We observed that Tie2 and Ang1 were elevated in human RA synovium. Using a novel collagen‐induced arthritis synovial window model, we demonstrated that Tie2 signaling regulated arthritis angiogenesis in vivo. We also showed that Tie2 mediated TNFα‐induced angiogenesis in a mouse cornea assay. In addition, we observed that TNFα can regulate Tie2 activation in multiple ways that may involve interactions between endothelial cells and synoviocytes. TNFα up‐regulates Tie2 in endothelial cells through nuclear factor κB, and it up‐regulates Ang1 in synoviocytes. These findings suggest paracrine regulation of angiogenesis between endothelial cells and synoviocytes.

Conclusion

This study demonstrates that Tie2 regulates angiogenesis in inflammatory synovium. Tie2 signaling is an important angiogenic mediator that links the proinflammatory cytokine TNFα to pathologic angiogenesis.

     

Angiopoietin/Tie2系统在血管新生与成熟过程中的表达及意义

血管的新生和成熟需要经过血管内皮细胞与血管周围细胞间相互作用等一系列复杂的生物学过程,这一过程受到多种细胞因子的调控。促血管生成素(angiopoietin,Ang)是近年发现的一个与血管新生与成熟密切相关的家族,包括4个成员,即Ang1、Ang2、Ang3和Ang4,其共同的特异性受体为Tie2。Ang及受体Tie2在生理性血管形成过程中对新生血管的形成、重构及成熟发挥重要的调控作用。另有研究显示,Ang/Tie2系统也参与了缺血后再灌、肿瘤及视网膜病变等多种病理性血管的形成过程。研究Ang/Tie2系统的生物学效应以及其在血管新生与成熟过程中的表达及意义,有助于深入了解相关疾病的发病机制并为疾病的治疗提供新线索。     

Cyclic tensile strain triggers a sequence of autocrine and paracrine signaling to regulate angiogenic sprouting in human vascular cells

Mechanical signals regulate blood vessel development in vivo, and have been demonstrated to regulate signal transduction of endothelial cell (EC) and smooth muscle cell (SMC) phenotype in vitro. However, it is unclear how the complex process of angiogenesis, which involves multiple cell types and growth factors that act in a spatiotemporally regulated manner, is triggered by a mechanical input. Here, we describe a mechanism for modulating vascular cells during sequential stages of an in vitro model of early angiogenesis by applying cyclic tensile strain. Cyclic strain of human umbilical vein (HUV)ECs up-regulated the secretion of angiopoietin (Ang)-2 and PDGF-ββ, and enhanced endothelial migration and sprout formation, whereas effects were eliminated with shRNA knockdown of endogenous Ang-2. Applying strain to colonies of HUVEC, cocultured on the same micropatterned substrate with nonstrained human aortic (HA)SMCs, led to a directed migration of the HASMC toward migrating HUVECs, with diminished recruitment when PDGF receptors were neutralized. These results demonstrate that a singular mechanical cue (cyclic tensile strain) can trigger a cascade of autocrine and paracrine signaling events between ECs and SMCs critical to the angiogenic process.     

Angiopoietin-1 induces endothelial cell sprouting through the activation of focal adhesion kinase and plasmin secretion

Angiopoietin-1 (Ang1) is a strong inducer of endothelial cell sprouting, which is a first step in both angiogenesis and neovascularization. We examined the mechanisms underlying Ang1-induced cell sprouting using porcine pulmonary artery endothelial cells. Ang1 induced the nondirectional and directional migration of endothelial cells mediated through the Tie2 but not the Tie1 receptor. Ang1 induced tyrosine phosphorylation of p125(FAK), and this phosphorylation was dependent on phosphatidylinositol (PI) 3'-kinase activity. Ang1 induced the secretion of plasmin and matrix metalloproteinase-2 (MMP-2), which is inhibited by PI 3'-kinase inhibitors. Ang1 also induced the secretion of small amounts of proMMP-3 and proMMP-9 but not proMMP-1. Ang1 suppressed the secretion of tissue inhibitor of metalloproteinase-2 (TIMP-2), but not of TIMP-1. Addition of alpha(2)-antiplasmin, a combination of TIMP-1 and TIMP-2, or PI 3'-kinase inhibitors inhibited Ang1-induced sprouting activity. Therefore, Ang1-induced sprouting activity in endothelial cells may be accomplished by cytoskeletal changes and secretion of proteinases and may be largely mediated through intracellular PI 3'-kinase activation.     

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。     

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.     

MCP-1 promotes mural cell recruitment during angiogenesis in the aortic ring model

Rings of rat or mouse aorta embedded in collagen gels produce angiogenic outgrowths in response to the injury of the dissection procedure. Aortic outgrowths are composed of branching endothelial tubes and surrounding mural cells. Mural cells emerge following endothelial sprouting and gradually increase during the maturation of the neovessels. Treatment of aortic cultures with angiopoietin-1 (Ang-1), an angiogenic factor implicated in vascular maturation and remodeling, stimulates the mural cell recruitment process. Ang-1 induces expression of many cytokines and chemokines including monocyte chemotactic protein-1 (MCP-1). Inhibition of p38 MAP kinase, a signaling molecule required for mural cell recruitment, blocks Ang1-induced MCP-1 expression. Recombinant MCP-1 dose-dependently increases mural cell number while an anti-MCP-1 blocking antibody reduces it. In addition, antibody mediated neutralization of MCP-1 abrogates the stimulatory effect of Ang-1 on mural cell recruitment. Aortic rings from genetically modified mice deficient in MCP-1 or its receptor CCR2 have fewer mural cells than controls. MCP-1 deficiency also impairs the mural cell recruitment activity of Ang-1. Our studies indicate that spontaneous and Ang1-induced mural cell recruitment in the aortic ring of model of angiogenesis are in part mediated by MCP-1. These results implicate MCP-1 as one of the mediators of mural cell recruitment in the aortic ring model, and suggest that chemokine pathways may contribute to the assembly of the vessel wall during the angiogenesis response to injury.