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.     

Angiogenic role of LYVE-1-positive macrophages in adipose tissue

Here we report the discovery of a characteristic dense vascular network (DVN) in the tip portion of epididymal adipose tissue in adult mice. The DVN is formed by angiogenesis rather than by vasculogenesis, and has functional blood circulation. This DVN and its subsequent branching may provide a new functional route for adipogenesis. The recruitment, infiltration, and accumulation of bone marrow-derived LYVE-1(+) macrophages in the tip region are crucial for the formation of the DVN. Matrix metalloproteinases (MMPs) and the VEGF-VEGFR2 system are responsible not only for the formation of the DVN, but also for the recruitment and infiltration of LYVE-1(+) macrophages into the epididymal adipose tissue tip region. SDF-1, but not the MCP-1-CCR2 system, is a critical factor in recruitment and ongoing retention of macrophages in this area. We also demonstrate that the tip region of epididymal adipose tissue is highly hypoxic, and thus provides a microenvironment conducive to the high expression and enhanced activities of VEGF, VEGFR2, MMPs, and SDF-1 in autocrine and paracrine manners, to create an ideal niche for the recruitment, retention, and angiogenic action of macrophages. These findings shed light on the complex interplay between macrophage infiltration, angiogenesis, and adipogenesis in the tip region of adult epididymal adipose tissue, and provide novel insight into the regulation of alternative outgrowth of adipose tissue.     

Angiopoietin-1 promotes cardiac and skeletal myocyte survival through integrins

Cardiac myocyte loss, regardless of insult, can trigger compensatory myocardial remodeling leading to heart failure. Identifying mediators of cardiac myocyte survival may advance clinical efforts toward myocardial preservation. Angiopoietin-1 limits ischemia-induced cardiac injury. This benefit is ascribed to angiogenesis because the receptor, tie2, is largely endothelial-specific. We propose that direct, non-tie2 interactions of angiopoietin-1 on cardiac myocytes contribute to this cardioprotection. We found that mouse C2C12 skeletal myocytes lack tie2, yet dose-dependently adhered to angiopoietin-1 and angiopoietin-2 similarly to laminin, fibronectin, vitronectin, and more than to collagen-I, -III, and -IV. Adhesion was divalent cation-mediated (Mn2+, Ca2+, not Mg2+), blocked with EDTA/EGTA, RGD-based peptides, and select integrin subunit antibodies. Similar findings were obtained with human skeletal myocytes (HSMs) and freshly isolated rat neonatal cardiac myocytes (NCMs). Furthermore, angiopoietin-1 conferred significant survival advantage exceeding that of most cell matrices, which was not fully explained by differences in cell adhesion. Angiopoietin-1 promoted survival of serum-starved C2C12, HSM, and NCM (MTT, trypan blue) and prevented taxol-induced apoptosis (caspase-3). Immobilized and soluble angiopoietin-1 phosphorylated Akt(S473) and MAPK(p42/44), (not FAK(Y397)) in C2C12 more than in endothelial cells and more than did angiopoietin-2 or cell matrices. EDTA, RGD-based peptides, and some integrin antibodies blocked these responses. Angiopoietin-1 activated HSM and NCM Akt(S473) and MAPK(p42/44) survival pathways. We propose that this novel function contributes to developmental and cardioprotective actions of angiopoietin-1 presently attributed to vascular effects alone. Angiopoietin-1 may prove therapeutically valuable in cardiac remodeling by supporting myocyte viability and preserving pump function. The full text of this article is available online at http://circres.ahajournals.org.     

Interleukin-8 reduces post-surgical lymphedema formation by promoting lymphatic vessel regeneration

Lymphedema is mainly caused by lymphatic obstruction and manifested as tissue swelling, often in the arms and legs. Lymphedema is one of the most common post-surgical complications in breast cancer patients and presents a painful and disfiguring chronic illness that has few treatment options. Here, we evaluated the therapeutic potential of interleukin (IL)-8 in lymphatic regeneration independent of its pro-inflammatory activity. We found that IL-8 promoted proliferation, tube formation, and migration of lymphatic endothelial cells (LECs) without activating the VEGF signaling. Additionally, IL-8 suppressed the major cell cycle inhibitor CDKN1C/p57^KIP2 by downregulating its positive regulator PROX1, which is known as the master regulator of LEC-differentiation. Animal-based studies such as matrigel plug and cornea micropocket assays demonstrated potent efficacy of IL-8 in activating lymphangiogenesis in vivo. Moreover, we have generated a novel transgenic mouse model (K14-hIL8) that expresses human IL-8 in the skin and then crossed with lymphatic-specific fluorescent (Prox1-GFP) mouse. The resulting double transgenic mice showed that a stable expression of IL-8 could promote embryonic lymphangiogenesis. Moreover, an immunodeficient IL-8-expressing mouse line that was established by crossing K14-hIL8 mice with athymic nude mice displayed an enhanced tumor-associated lymphangiogenesis. Finally, when experimental lymphedema was introduced, K14-hIL8 mice showed an improved amelioration of lymphedema with an increased lymphatic regeneration. Together, we report that IL-8 can activate lymphangiogenesis in vitro and in vivo with a therapeutic efficacy in post-surgical lymphedema.     

Matrix metalloproteinase-2 governs lymphatic vessel formation as an interstitial collagenase

Lymphatic dysfunctions are associated with several human diseases, including lymphedema and metastatic spread of cancer. Although it is well recognized that lymphatic capillaries attach directly to interstitial matrix mainly composed of fibrillar type I collagen, the interactions occurring between lymphatics and their surrounding matrix have been overlooked. In this study, we demonstrate how matrix metalloproteinase (MMP)-2 drives lymphatic morphogenesis through Mmp2-gene ablation in mice, mmp2 knockdown in zebrafish and in 3D-culture systems, and through MMP2 inhibition. In all models used in vivo (3 murine models and thoracic duct development in zebrafish) and in vitro (lymphatic ring and spheroid assays), MMP2 blockage or down-regulation leads to reduced lymphangiogenesis or altered vessel branching. Our data show that lymphatic endothelial cell (LEC) migration through collagen fibers is affected by physical matrix constraints (matrix composition, density, and cross-linking). Transmission electron microscopy and confocal reflection microscopy using DQ-collagen highlight the contribution of MMP2 to mesenchymal-like migration of LECs associated with collagen fiber remodeling. Our findings provide new mechanistic insight into how LECs negotiate an interstitial type I collagen barrier and reveal an unexpected MMP2-driven collagenolytic pathway for lymphatic vessel formation and morphogenesis.     

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.     

Biomechanics of a lymphatic vessel.

The principles of mechanics and the current notions of lymphatic physiology are integrated into a simple mathematical model of a lymphatic vessel which establishes a theoretical base for lymph propulsion in the lymphatic system. The model specifically considers the active and passive contractilities of the lymphatics. The model is simulated on a digital computer. The pressure and flow patterns derived from the model are consistent with the currently available experimental data.     

Relationship between LYVE-1, VEGFR-3 and CD44 gene expressions and lymphatic metastasis in gastric cancer

AIM： To investigate the expression levels of lymphatic vessel endothelial hyaluronan receptor-1 （LYVE-1）, vascular endothelial growth factor receptor-3 （VEGFR-3） and CD44 genes and the relationship between their levels and clinicopathological parameters in gastric cancer.METHODS： Tissue samples were obtained from 33 patients （8 females） with gastric cancer. mRNA levels of LYVE-1, VEGFR-3 and CD44 in normal and tumor tissues were quantitatively measured using real time polymerase chain reaction. The results were correlated with lymph node metastasis, histological type and differentiation of the tumor, T-stage, and presence of vascular, perineural and lymphatic invasions. The distribution of molecules in the tissue was evaluated using immunohistochemistry. RESULTS： LYVE-1, CD44 and VEGFR-3 gene expression levels were significantly higher in gastric cancer than in normal tissue. While there was no correlation between gene expressions and clinicopathologic fea- tures such as histologic type, differentiation and stage, gene expression levels were found to be increased in conjunction with positive lymph node/total lymph node ratio and the presence of perineural invasion. A significant correlation was also found between LYVE-1 and CD44 over-expressions and perineural invasion and lymph node positivity in gastric cancers. When the dis- tribution of LYVE-1 antibody-stained lymphatic vessels in tissue was evaluated, lymphatic vessels were located intra-tumorally in 13% and peri-tumorally in 27% of the patients. Moreover, lymph node metastases were also positive in all patients with LYVE-1-staining. CONCLUSION： LYVE-1, VEGFR-3 and CD44 all play an important role in lymphangiogenesis, invasion and metastasis. LYVE-1 is a perfectly reliable lymphatic vessel marker and useful for immunohistochemistry.     

人结直肠黏膜层淋巴管微细分布及超微结构

目的观察人结直肠正常肠壁黏膜层内淋巴管的形态及结构特点,为探讨结直肠癌淋巴道转移的途经提供形态学依据。 方法人结直肠蜡块标本连续切片,通过免疫组化两步法,以鼠抗人D2-40单克隆抗体及鼠抗CD34单克隆抗体为一抗分别标记,光镜下观察抗体标记情况。黏膜层超薄切片电镜下观察淋巴管微观结构。 结果D2-40单克隆抗体免疫组化染色后,在黏膜层,棕黄色显色分布于黏膜固有层与黏膜肌层之间及黏膜肌层内,少量,管壁薄、管腔小,管壁不完整。与CD34染色无重叠。电镜下见黏膜层内少量淋巴管管壁很薄,仅由一层内皮细胞构成,无孔窗,无周细胞,基膜不连续或缺如,内皮细胞间形成端端、重叠或插入连接,管壁外连有锚丝。 结论正常人结直肠黏膜层内有淋巴管存在,存在于固有层与黏膜肌层之间及黏膜肌层内,与黏膜下层淋巴管可能存在通道相联,具备淋巴道转移的解剖学基础。     

淋巴管内皮透明质酸受体1对肺癌转移及预后评价的意义研究

目的检测肺癌患者血清淋巴管内皮透明质酸受体1(LYVE-1)水平,并探讨血清LYVE-1用于判断肺癌患者是否发生淋巴结转移及预测患者预后的临床意义。方法采用酶联免疫法(ELISA)检测57例肺癌患者血清中LYVE-1的水平,同时分析血清LYVE-1水平与患者临床病理特征的关系,通过受试者工作曲线(ROC)分析血清LYVE-1用于判断肺癌患者是否发生淋巴结转移的可行性,Kaplan-Meier法进行生存分析,评价LYVE-1用于预测病人预后的临床意义。结果肺癌患者血清LYVE-1为1625.0±343.0 pg/m L;血清LYVE-1与肺癌患者性别、肿瘤组织学分型、血清CEA和CA125无统计学差异(P0.05),而与TNM分期、淋巴结转移和远处转移情况有关(P0.05);血清LYVE-1用于诊断肺癌患者发生淋巴结转移的曲线下面积为0.728(95%CI:0.685-0.747,P0.05);LYVE-1的为1821 pg/m L时,其诊断的敏感性为79%,特异性为68%;血清LYVE-1大于1821pg/m L的肺癌患者预后要比血清LYVE-1小于1821 pg/m L的肺癌患者预后差,两组总生存时间有统计学差异(P=0.035)。结论血清LYVE-1可用于确定肺癌患者是否发生淋巴结转移,并可用于评估病人预后。     

淋巴管内皮细胞透明质酸受体-1在胃癌组织中的表达及意义

目的:探讨淋巴管内皮细胞透明质酸受体-1(LYVE-1)在胃癌组织中的表达及临床意义．方法:采用组织芯片技术和免疫组化方法对85例胃癌组织和40例正常胃组织中LYVE-1的表达及淋巴管密度(LVD)进行检测．结果:LYVE-1的阳性产物表达于淋巴管内皮细胞胞质中,呈棕黄色染色．胃癌组织中LYVE-1阳性LVD显著高于正常胃组织(7．89±2．14 vs 1．15±1．62,P<0．01);瘤周的LYVE-1阳性LVD明显高于瘤内组织(9．28±1．18 vs 4．75±1．19,P<0．01);低分化(9．21±2．32)、Ⅲ-Ⅳ期(9．46±2．45)、有淋巴结转移(9．37±3．39)和远处转移(9．55±3．50)的胃癌组织中LYVE-1阳性LVD分别比高中分化(7．56±2．24)、Ⅰ-Ⅱ期(7．58±2．36)、无淋巴结转移(7．23±2．74)和远处转移(7．35±2．25)的明显增高(P<0．01, P<0．05)．结论:胃癌组织内及瘤周的LYVE-1阳性LVD明显增高,提示胃癌组织内的新生淋巴管主要位于瘤周,从而促进了肿瘤淋巴道转移．应用组织芯片大规模高效检测临床组织标本是可行的,具有快速、方便、经济、准确的特点．     

Fibroblasts potentiate blood vessel formation partially through secreted factor TIMP-1

During wound repair, new blood vessels form in response to angiogenic signals emanating from injured tissues. Dermal fibroblasts are known to play an important role in wound healing, and have been linked to angiogenesis; therefore, we sought to understand the mechanisms through which these cells control blood vessel formation. Using a three-dimensional angiogenesis assay we demonstrate that dermal fibroblasts enhance the tube-forming potential of endothelial cells, and this augmentation is partially due to secreted factors present in conditioned media. Interestingly, we identified tissue inhibitor of metalloproteinase-1 (TIMP-1) as a factor uniquely secreted by fibroblasts, and addition of exogenous TIMP-1 increased vessel assembly. The enhancing activity of TIMP-1 was matrix metalloproteinase (MMP)-dependent, since a mutant version of TIMP-1 was unable to promote angiogenesis. Consistent with this, chemical inhibition of MMP-2/9 showed a similar increase in angiogenesis, and addition of exogenous MMP-9 blocked the enhancing effect of TIMP-1. We further demonstrated that TIMP-1 inhibits the production of tumstatin, an anti-angiogenic fragment of collagen IV that is produced by MMP-9 cleavage. Our results support the notion that dermal fibroblasts regulate blood vessel formation through multiple mediators, and provide novel evidence that fibroblast-derived TIMP-1 acts on endothelial cells in a pro-angiogenic capacity.     

Neuregulin-1 promotes formation of the murine cardiac conduction system

The cardiac conduction system is a network of cells responsible for the rhythmic and coordinated excitation of the heart. Components of the murine conduction system, including the peripheral Purkinje fibers, are morphologically indistinguishable from surrounding cardiomyocytes, and a paucity of molecular markers exists to identify these cells. The murine conduction system develops in close association with the endocardium. Using the recently identified CCS-lacZ line of reporter mice, in which lacZ expression delineates the embryonic and fully mature conduction system, we tested the ability of several endocardial-derived paracrine factors to convert contractile cardiomyocytes into conduction-system cells as measured by ectopic reporter gene expression in the heart. In this report we show that neuregulin-1, a growth and differentiation factor essential for ventricular trabeculation, is sufficient to induce ectopic expression of the lacZ conduction marker. This inductive effect of neuregulin-1 was restricted to a window of sensitivity between 8.5 and 10.5 days postcoitum. Using the whole mouse embryo culture system, neuregulin-1 was shown to regulate lacZ expression within the embryonic heart, whereas its expression in other tissues remained unaffected. We describe the electrical activation pattern of the 9.5-days postcoitum embryonic mouse heart and show that treatment with neuregulin-1 results in electrophysiological changes in the activation pattern consistent with a recruitment of cells to the conduction system. This study supports the hypothesis that endocardial-derived neuregulins may be the major endogenous ligands responsible for inducing murine embryonic cardiomyocytes to differentiate into cells of the conduction system.     

Angiopoietin-1 improves renal hypoxia in diabetic rats

<正>Objective To investigate the effects of Ang-1 on renal hypoxia.Methods SD diabetic rat model was con-structed by intraperitoneal injection with streptozotocin.Then all the rats were randomly divided into four groups:normal control group (NC group),diabetic group (DM group),Ang-1 treatment group (AV group),and blank vector group (BV group).Eight weeks after the diabetic model was constructed,AV group was treated with Ad-     

Application of microscale culture technologies for studying lymphatic vessel biology

Immense progress in microscale engineering technologies has significantly expanded the capabilities of in vitro cell culture systems for reconstituting physiological microenvironments that are mediated by biomolecular gradients, fluid transport, and mechanical forces. Here, we examine the innovative approaches based on microfabricated vessels for studying lymphatic biology. To help understand the necessary design requirements for microfluidic models, we first summarize lymphatic vessel structure and function. Next, we provide an overview of the molecular and biomechanical mediators of lymphatic vessel function. Then we discuss the past achievements and new opportunities for microfluidic culture models to a broad range of applications pertaining to lymphatic vessel physiology. We emphasize the unique attributes of microfluidic systems that enable the recapitulation of multiple physicochemical cues in vitro for studying lymphatic pathophysiology. Current challenges and future outlooks of microscale technology for studying lymphatics are also discussed. Collectively, we make the assertion that further progress in the development of microscale models will continue to enrich our mechanistic understanding of lymphatic biology and physiology to help realize the promise of the lymphatic vasculature as a therapeutic target for a broad spectrum of diseases.     

血管生成素1在肺部疾病中的作用

血管生成素1属血管生成素家族重要一员,具有抑制内皮细胞凋亡;促进血管出芽、迁徙、趋化;维持血管稳定;防止液体渗漏和减轻局部炎症等作用。血管生成素1具有抑制肿瘤生长的作用;其与肺动脉高压发病有一定关系,但具体作用尚有争议。近年来研究提示其在肺损伤干预中的作用更具有广阔前景,它通过稳定肺微血管内皮和局部抗炎来发挥作用。