Aquaporin-1 expression in the chick embryo chorioallantoic membrane

The chick embryo chorioallantoic membrane (CAM) is commonly used in vivo to study both angiogenesis and anti-angiogenesis. Rapid membrane water transport is mediated by a family of molecular water channels, called aquaporins (AQPs), which have been identified in the epithelial and endothelial cells of higher vertebrates. AQP1, expressed in adsorptive and secretory epithelia, is also expressed in endothelial cells of capillaries and arteries. Its mRNA has been found in vascular smooth muscle cells (VSMCs) of arteries and capillaries, as well as in a subset of VSMCs of human atherosclerotic plaques. This study investigated the developmental expression of AQP1 in the chick CAM by Western blot and immunohistochemistry. Western blot results show that a major nonglycosylated band was observed with electrophoretic mobility of approximately 28 kDa in the three developmental stages examined. Immunohistochemistry data demonstrate that AQP1 was clearly expressed in the ectodermal and endodermal epithelia, the vascular endothelium, and the VSMCs. Because little information is available on the behavior of microvessel AQP1 during angiogenesis in normal and pathological conditions, our data relative to the pattern of expression of AQP1 in CAM blood vessels in normal conditions may be considered a useful tool to further investigate its modifications in several experimental conditions implying a stimulation or an inhibition of angiogenesis in the CAM assay.     

Localization and expression of the aquaporin-1 water channel in mesangial cells in the human glomerulus

The expression and localization of the aquaporin-1 (AQP1) water channel were examined in the glomeruli of the human kidney. A ribonuclease protection assay showed the expression of AQP1 mRNA in human glomeruli but not in rat glomeruli. Western blot analysis revealed 28 kDa and 35 kDa bands corresponding to unglycosylated and glycosylated AQP1 proteins in human glomeruli. Immunoreactive AQP1 was demonstrated almost exclusively in the mesangium in the human glomeruli by immunohistochemistry. The endothelium of glomerular capillaries was only partly immunostained while podocytes and Bowman's capsule epithelia were not immunolabeled. Immunoelectron microscopy localized the immunoreactive AQP1 on the plasma membrane of mesangial cells in human glomeruli. The immouno-gold labeling was dense on the projections of mesangial cells protruding to the glomerular capillary lumen or to endothelial cells, but was sparse on other parts of the mesangial cell surface. No immunoreactivity for AQP1 was demonstrated in rat glomeruli. This study showed the distinct localization of AQP1 in the mesangial cells of human glomeruli, suggesting its role in water movement through these cells.     

Aquaporins in the digestive system

Fluid transfer such as secretion and absorption is one of the major functions of the digestive system. Aquaporins are water channel proteins providing water transfer across the cellular membrane. At least six aquaporin isoforms are expressed in the digestive system. Aquaporin-1 (AQP1) is widely distributed in endothelial cells of capillaries and small vessels as well as in the central lacteals in the small intestine. AQP1 is also present in the duct system in the pancreas, liver, and bile duct. AQP3 is mainly expressed in the epithelia of the upper digestive tract from the oral cavity to the stomach and of the lower digestive tract from the distal colon to the anus. AQP4 is present in the parietal cells of the stomach and in the intestinal epithelia. AQP5 is expressed in acinar cells of the salivary, pyloric, and duodenal glands. AQP8 is expressed in the intestinal epithelia, salivary glands, pancreas, and liver. AQP9 is present in the liver and intestinal goblet cells. Aquaporins have important roles in the digestive system, such as AQP5 in saliva secretion, as shown by the studies on AQP5-null mice. In addition, water transfer across the digestive epithelia seems to occur not only via aquaporins but also via other transporter or channel systems.     

基因重组纤溶酶原 Kringle 5 对血管生成抑制作用的研究

目的 研究基因重组纤溶酶原 (Kringle 5, K5) 对小鼠的血管生成的抑制作用。 方法 以体外 培养的小鼠内皮细胞为研究对象, 利用划痕实验和黏附能力测定实验观察重组 K5 对内皮细胞迁移、 黏附 能力的影响; 利用体外血管生成体系、 创伤愈合和鸡胚尿囊膜 ( chicken embryo allantoic membrane, CAM) 血管生成能力实验, 检测 K5 对血管生成作用的影响。 结果 200 nmol / L K5 显著抑制内皮细胞的迁移、 黏 附以及新生血管的生长长度。 并且 K5 在 2 ~ 18 mg / kg 浓度范围内对小鼠创伤愈合具有明显的抑制作用。 此 外, 6、 12 与 25 mg / L K5 显著抑制 CAM 血管生成, 25 mg / L K5 的抑制作用最强。 结论 K5 能明显抑制血 管生成, 为临床抑制血管生成治疗提供理论依据。     

Chorioallantoic membrane capillary bed: A useful target for studying angiogenesis and anti-angiogenesis in vivo

The chick embryo chorioallantoic membrane (CAM) is an extraembryonic membrane that is commonly used in vivo to study both angiogenesis and anti-angiogenesis. This review 1) summarizes the current knowledge about the structure of the CAM's capillary bed; 2) discusses the controversy about the existence of a single blood sinus or a capillary plexus underlying the chorionic epithelium; 3) describes a new model of the CAM vascular growth, namely the intussusceptive mode; 4) reports findings regarding the role played by endogenous fibroblast growth factor-2 in CAM vascularization; and 5) addresses the use and limitations of the CAM as a model for studying angiogenesis and anti-angiogenesis. Anat Rec 264:317–324, 2001. © 2001 Wiley-Liss, Inc.     

Immunohistochemical Localization of the Aquaporins AQP1, AQP3, AQP4, and AQP5 in the Mouse Respiratory System

Aquaporins are membrane water channel proteins that function mainly in water transfer across cellular membranes. In our present study, we investigated the immunohistochemical distribution of aquaporin 1 (AQP1), AQP3, AQP4, and AQP5 in the mouse respiratory system by immunofluorescence, immunoperoxidase, and immunoelectron microscopy. AQP3, AQP4, and AQP5 are expressed in epithelial cells, whereas AQP1 is expressed in subepithelial connective tissues and capillaries. In the airway surface epithelia from the nasal cavity to the intrapulmonary bronchioles, AQP5 was found to be mainly localized to the luminal side and both AQP3 and AQP4 to the abluminal side. In the alveolar epithelium, AQP5 is localized to the apical membranes of both type I and type II alveolar cells. Compared with the previous studies on the rat respiratory system, in which AQP5 is restricted to the alveolar type I cells and absent from the airway surface epithelia, we found that AQP5 in the mouse is much more widely distributed throughout the surface epithelia. These results suggest that AQP5 has a critical role in water-handling, such as the maintenance of airway surface liquid and clearance of alveolar fluid in the mouse respiratory system.     

A critical analysis of current in vitro and in vivo angiogenesis assays

The study of angiogenesis has grown exponentially over the past 40 years with the recognition that angiogenesis is essential for numerous pathologies and, more recently, with the advent of successful drugs to inhibit angiogenesis in tumours. The main problem with angiogenesis research remains the choice of appropriate assays to evaluate the efficacy of potential new drugs and to identify potential targets within the angiogenic process. This selection is made more complex by the recognition that heterogeneity occurs, not only within the endothelial cells themselves, but also within the specific microenvironment to be studied. Thus, it is essential to choose the assay conditions and cell types that most closely resemble the angiogenic disease being studied. This is especially important when aiming to translate data from in vitro to in vivo and from preclinical to the clinic. Here we critically review and highlight recent advances in the principle assays in common use including those for endothelial cell proliferation, migration, differentiation and co-culture with fibroblasts and mural cells in vitro , vessel outgrowth from organ cultures and in vivo assays such as chick chorioallantoic membrane (CAM), zebrafish, sponge implantation, corneal, dorsal air sac, chamber and tumour angiogenesis models. Finally, we briefly discuss the direction likely to be taken in future studies, which include the use of increasingly sophisticated imaging analysis systems for data acquisition.     

抗人VEGF单抗的研制及对肿瘤生长及转移的抑制作用

为了确定人血管内皮细胞生长因子在肿瘤生长及转移中的作用。方法：利用ＤＮＡ重组技术表达人ＶＥＧＦ,以其为抗原免疫Ｂａｌｂ／ｃ小鼠,应用杂交瘤技术获得１４株稳定分泌抗ＶＥＧＦ单抗的杂交瘤细胞株。结果ＥＬＩＳＡ测定所分泌的单抗只与ＶＥＧＦ特异结合,与ＥＧＦ、ＦＧＦ、ＰＤＧＦ、ＴＧＦα及ＴＮＦ等均无交叉反应。     

Current methods for assaying angiogenesis in vitro and in vivo

Angiogenesis, the development of new blood vessels from an existing vasculature, is essential in normal developmental processes and in numerous pathologies, including diabetic retinopathy, psoriasis and tumour growth and metastases. One of the problems faced by angiogenesis researchers has been the difficulty of finding suitable methods for assessing the effects of regulators of the angiogenic response. The ideal assay would be reliable, technically straightforward, easily quantifiable and, most importantly, physiologically relevant. Here, we review the advantages and limitations of the principal assays in use, including those for the proliferation, migration and differentiation of endothelial cells in vitro, vessel outgrowth from organ cultures and in vivo assays such as sponge implantation, corneal, chamber, zebrafish, chick chorioallantoic membrane (CAM) and tumour angiogenesis models.     

The role of eosinophil major basic protein in angiogenesis

Background:  Eosinophil-derived major basic protein (MBP) plays an active role in allergic inflammation and tissue remodelling. However, its role in angiogenesis has not been established as yet. Therefore our objective was to investigate whether MBP exhibits any direct pro-angiogenic effects.
Methods:  Rat aortic endothelial cells and human umbilical vascular endothelial cells were cultured with different concentrations of MBP and their viability (Trypan blue exclusion test), proliferation (thymidine incorporation) and capillary-like structure formation (matrigel assay) were investigated in vitro . The angiogenic activity of MBP was then tested in vivo using the chick chorio allantoic membrane (CAM) assay.
Results:  Subcytotoxic concentrations of MBP induce endothelial cell proliferation and enhance the pro-mitogenic effect of vascular endothelial growth factor (VEGF), but do not affect their VEGF release. MBP promotes capillarogenesis by endothelial cells seeded on matrigel and sprouting formation in the CAM assay. Furthermore, we have shown that the pro-angiogenic effect of MBP is not due to its cationic charge since stimulation of the CAMs with the synthetic polycation, poly- l -arginine does not induce any angiogenic effects.
Conclusions:  These data demonstrate that MBP has pro-angiogenic effects in vitro and in vivo , providing a novel mechanism whereby MBP can participate in tissue inflammation and remodelling in atopic diseases.     

Localisation of Placenta Growth Factor (PlGF) in Human Term Placenta

Abstract

Placenta growth factor (PlGF) is a growth factor which belongs to the vascular endothelial growth factor (VEGF) family and is known to bind to the fms-like tyrosine kinase receptor (flt-1). Using Western blot analysis a 50 kDa band was identified in placental protein extract which corresponded to PlGF homodimer. Immunoreactive PlGF was localised to the vasculosyncytial membrane and in the media of large blood vessels of the placental villi, while staining within the mesenchyme was weak and diffuse. There was moderate staining for PlGF in discrete cells in the chorion and no staining in the epithelial layer of the amnion. The maternal decidual cells showed strong staining for PlGF immunoreactive protein. PlGF mRNA was predominantly expressed by the vasculosyncytial membrane of villous trophoblast, whilst there was no apparent expression of PlGF mRNA within the villous mesenchyme. These results suggest that PlGF may be an important paracrine factor for vascular endothelial cells in placental angiogenesis and an autocrine mediator of trophoblast function.     

Coeliac disease-specific autoantibodies targeted against transglutaminase 2 disturb angiogenesis

Coeliac disease is characterized by immunoglobulin-A (IgA)-class autoantibodies targeted against transglutaminase 2 (TG2), a multi-functional protein also with a role in angiogenesis. These antibodies are present in patient serum but are also found bound to TG2 below the epithelial basement membrane and around capillaries in the small intestinal mucosa. Based on these facts and the information that the mucosal vasculature of coeliac patients on a gluten-containing diet is disorganized, we studied whether the coeliac disease-specific autoantibodies targeted against TG2 would disturb angiogenesis. The effects of coeliac disease-specific autoantibodies on in vitro angiogenesis were studied in angiogenic cell cultures. The binding of the antibodies to cells, endothelial sprouting, migration of both endothelial and vascular mesenchymal cells, the integrity of the actin cytoskeleton in both cell types and the differentiation of vascular mesenchymal cells were recorded. In vitro, IgA derived from coeliac disease patients on a gluten-containing diet binds to surface TG2 on endothelial and vascular mesenchymal cells and this binding can be inhibited by the removal of TG2. In addition, coeliac disease-specific autoantibodies targeting TG2 disturb several steps of angiogenesis: endothelial sprouting and the migration of both endothelial and vascular mesenchymal cells. Furthermore, the autoantibodies cause disorganization of the actin cytoskeleton in both capillary cell types that account most probably for the defective cellular migration. We conclude that coeliac disease-specific autoantibodies recognizing TG2 inhibit angiogenesis in vitro. This disturbance of the angiogenic process could lead in vivo to the disruption of the mucosal vasculature seen in coeliac disease patients on a gluten-containing diet.     

Transient expression of insulin-like growth factor I immunoreactivity by vascular cells during angiogenesis

The present study was undertaken to investigate whether vascular cells show insulin-like growth factor I (IGF-I; somatomedin C) immunoreactivity under normal conditions and/or during angiogenesis in humans and animals, as the trophic peptide IGF-I is considered important for cell growth and differentiation. In adult animals normal blood vessels, i.e., arteries, veins, and capillaries, did not show any IGF-I immunoreactivity. In newborn animals every vascular cell showed IGF-I immunoreactivity; the frequency and intensity thereafter decreased and eventually vanished as the animals approached maturity. Injury of a tissue or organ rapidly induced extensive blood vessel formation and such new blood vessels transiently expressed IGF-I immunoreactivity. Endothelial cells in budding capillaries showed distinct cytoplasmic IGF-I immunoreactivity, as did endothelial cells, smooth muscle cells, and fibroblast in newly formed arteries and veins. In biopsies of human tissue, transient IGF-I immunoreactivity was evident in vascular cells during angiogenesis after injury, as it also was in granulation tissue, skin wounds, and scar capsules around implants. Increased IGF-I immunoreactivity was further demonstrated in vascular cells in biopsies from patients with other changes involving blood vessel formation, e.g., nasal polyps, and in specimens from patients with arteritis, tendonitis, synovitis, Wegener's granulomatosis, idiopathic midline destructive disease, neurofibromatosis (von Recklinghausen's disease), and muscular dystrophy. It is concluded that during angiogenesis, obviously irrespective of inducing factors and mechanisms, vascular wall cells transiently show IGF-I immunoreactivity.     

Expression and localization of placenta growth factor and PlGF receptors in human meningiomas

It has previously been suggested that in human brain tumours, endothelial cell proliferation during angiogenesis is regulated by a paracrine mechanism involving vascular endothelial growth factor (VEGF) and its receptors (VEGF receptor 1 and VEGF receptor 2). The mechanism of growth factor up-regulation is based on hypoxic activation of mRNA expression and mRNA stabilization and genetic events, leading to an increase of growth factor gene expression. The role of the other newly discovered VEGF family members with a high specificity for endothelial cells in the pathogenesis of glial neoplasms is unknown. To investigate which other members of the VEGF family are overexpressed in human brain tumours, the mRNA levels of placenta growth factor (PlGF), VEGF-A, and VEGF-B genes were determined by northern blot analysis in surgically obtained human meningiomas. In the 16 meningiomas examined, the mRNA for PlGF was highly expressed in four tumours and VEGF-A mRNA was highly abundant in three tumour samples. There was no close correlation between PlGF mRNA levels and VEGF-A expression levels. VEGF-B mRNA was abundantly expressed in all tumour samples at uniform levels. In a PlGF-positive tumour sample, immunoreactive VEGFR-1 and VEGFR-2 were detected in endothelial cells of the blood vessels. PlGF protein was detectable in most but not all capillaries of the tumour. PlGF is thus highly up-regulated in a subset of human meningiomas and may therefore have functions, in some tumour vessels, connected to endothelial cell maturation and tube formation. These findings suggest that PlGF, in addition to VEGF-A, may be another positive factor in tumour angiogenesis in human meningiomas. Copyright © 1999 John Wiley & Sons, Ltd.     

VEGF-A/HGF induce Prox-1 expression in the chick embryo chorioallantoic membrane lymphatic vasculature

Vascular endothelial growth factor-A (VEGF-A) and hepatocyte growth factor (HGF) are well known angiogenesis inductors and promoters in normal and pathologic conditions. Recent data showed that VEGF-A and HGF could also influence lymphangiogenesis but this matter has not been completely elucidated. Administration of VEGF-A and HGF in combination has been used to improve the angiogenic response in different experimental models, but their effects on lymphangiogenesis have not been investigated. The aim of this study was to characterize blood and lymphatic vascular response to VEGF-A/HGF administration. To this purpose, we built a pBlast VEGF-A/HGF combination suitable for in vivo research. By using as an experimental in vivo model the chick embryo chorioallantoic membrane (CAM) assay, we applied pBlast VEGF-A/HGF combination for 7 days. Results showed that VEGF-A/HGF combination was able to induce a strong angiogenic response and the expression of Prox-1 in the lymphatic endothelial cells of the CAM. The possible mechanisms involved have been speculated.     

直流电场对鸡胚绒毛尿囊膜血管生成作用的研究

目的：探讨直流电场（electric fields．EF）对血管生成的影响及作用机制。方法：于发育第8天的鸡胚绒毛尿囊膜（chick embryo chorioallantoic membrane，CAM）上植入明胶海绵载体，其中加载不同强度EF，计数长入载体的血管和载体内的微血管密度（microvascular densily．MVD）。结果：150mV／mm、200mV／mm组在CAM上长入载体边缘的血管数和MVD与对照组间的有显著差别（p〈0．01）。结论：强度为150mV／mm、200mV／mm的直流电场能促进CAM的血管生成。     

Ultrastructural study on endothelial cells in splenic autotransplantation

The splenic tissue of the rat was autotransplanted into the greater omentum to investigate the angiogenic process during splenic regeneration. Chronological observations showed the following two stages. Initially surviving subcapsular sinus endothelial cells migrated to form a preliminary vascular network, which anastomosed with ingrowing omental capillaries to form a circulatory system. In this way, splenic autotransplants would not undergo necrosis and would rapidly form endothelial and reticular cells. At a later stage, the preliminary vascular network differentiated into arteries (arterioles, capillaries), splenic sinuses, and veins. Marked morphologic changes occurred in the endothelial cells, such as vacuole formation, fenestration, and the condensation of intraendothelial microfilaments, resulting in interendothelial slits. These results showed that the formation of a preliminary vascular network caused distinct splenic regeneration and that splenic autotransplantation is a suitable model to study angiogenesis in vivo.     

Promotion of angiogenesis by low energy laser irradiation

The effect of low energy laser (He-Ne) irradiation (LELI) on the process of angiogenesis in the infarcted rat heart and in the chick chorioallantoic membrane (CAM), as well as the proliferation of endothelial cells in tissue culture, was investigated. Formation of new blood vessels in the infarcted rat heart was monitored by counting proliferating endothelial cells in blood vessels. In the CAM model, defined areas were laser-irradiated or nonirradiated and blood vessel density was recorded in each site in the CAM at various time intervals. Laser irradiation caused a 3.1-fold significant increase in newly formed blood vessels 6 days post infarction, as compared with nonirradiated rats. In the CAM model, a slight inhibition of angiogenesis up to 2 days post irradiation and a significant enhancement of angiogenesis in the laser-irradiated foci as compared with control nonirradiated spots were evident. The LELI caused a 1.8-fold significant increase in the rate of proliferation in endothelial cells in culture over nonirradiated cells. It is concluded that LELI can promote the proliferation of endothelial cells in culture, which may partially explain the augmentation of angiogenesis in the CAM model and in the infarcted heart. These results may have clinical significance by offering therapeutic options to ameliorate angiogenesis in ischemic conditions.     

Nestin expression by newly formed human blood vessels

Nestin is a type VI intermediate filament protein originally described in neural stem cells. Here we report that immature endothelial cells generated in the course of angiogenesis express nestin. Endothelial cells of embryonic capillaries destined to vascularize growing organs also express this intermediate filament protein. Whereas nestin was sporadically expressed in mature adult human endothelial cells sporadically express nestin, this protein was consistently expressed in adult angiogenic vasculature. Nestin expression was also detected in capillaries of the corpus luteum, which replenishes itself by angiogenesis. Nestin-immunoreactive vessels were also observed in the infarcted hearts where transient ischemia triggered regeneration accompanied with neovascularization of the myocardium. Nestinpositive endothelial cells lined vessels nourishing solid growing tumors, including melanoblastomas and glioblastomas. Our data provide definitive evidence that endothelial precursors express the neural stem cell marker nestin and that this protein participates in formation of the cytoskeleton of newly formed endothelial cells. Because nestin expression was recognized under all conditions of vascular development, nestin represents a novel and reliable marker of neovascularization.