In vivo time-course of the angiogenic response induced by multiple myeloma plasma cells in the chick embryo chorioallantoic membrane

In this study, we set out to make a fine characterization of the angiogenic response induced by plasma cells obtained from patients with active-multiple myeloma (MM), in comparison with cells obtained from patients with non-active MM and benign lesions such as monoclonal gammopathy of undetermined significance (MGUS), in the chick embryo chorioallantoic membrane (CAM) assay. To achieve this we investigated the time-course of the angiogenic response induced by gelatin sponges soaked in the cell suspensions and implanted on the CAM surface from day 8 to day 12 of incubation by evaluating the number of vessels, of the vessel bifurcation and the intervascular distance at 24, 48, 72 and 96 h after the implants. The results show that plasma cell suspensions obtained from patients with active MM induce a vasoproliferative response that was significantly higher than that induced by cell suspensions obtained from patients with non-active MM or with MGUS, which is also a function of the day of implantation. In fact, implants made from day 8 to day 10 induce a strong angiogenic response, whereas those made from day 11 to day 12 do not. This finding might depend on the fact that CAM endothelium exhibits an intrinsically high mitotic rate until day 10. Thereafter, the endothelial mitotic index declines rapidly, and consequently cell suspensions implanted on the CAM of successively older embryos are not able to induce a vasoproliferative response in parallel with the reduced rates of growth of the CAM's endothelial cells.     

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. Anat Rec 268:85–89, 2002. © 2002 Wiley‐Liss, Inc.     

不同鼻咽癌细胞株对鸡胚尿囊膜模型血管生成影响的比较研究

目的比较不同鼻咽癌细胞株在鸡胚尿囊膜模型上促进血管生成能力的差异。方法选用6日龄鸡胚,开窗后分别种植5-8F、6-10B及CNE-2三种鼻咽癌细胞株,三种细胞株细胞数为2×106/鸡胚者各一组,细胞数为5×105/鸡胚者各一组,另一组为对照组（PBS）,每组10个鸡胚。孵育6日后,统计分析新生血管数及血管面积/鸡胚面积比。结果种植细胞数为5×105/鸡胚时,5-8F、6-10B细胞部分鸡胚有移植瘤形成,CNE-2细胞鸡胚均未见成瘤。种植细胞数为2×106/鸡胚时,三种鼻咽癌细胞株均可100%成瘤,其新生血管数依次递减,分别为（38.7±2.50）、（33.5±4.43）、（29.7±2.71）,差异有统计学意义（P〈0.05）;血管面积/鸡胚面积比分别为（22.2±2.18）%、（18.7±2.45）%、（16.9±2.62）%,均高于对照组的（9.5±1.86）%,差异有统计学意义（P〈0.05）,且5-8F面积比高于其他两种细胞株（P〈0.05）。结论鼻咽癌5-8F、6-10B及CNE-2三种细胞株在鸡胚尿囊膜模型上血管生成能力依次减弱,实验研究可根据实际情况选用。     

Fibrin degradation and angiogenesis: quantitative analysis of the angiogenic response in the chick chorioallantoic membrane

Fibrin deposition and removal is a feature common to major pathological processes such as wound healing, chronic inflammation and tumour invasion: processes involving the ingrowth of new blood vessels. Low molecular weight fibrin degradation products (MW less than 50,000) are now shown to induce angiogenesis in the chick chorioallantoic membrane (CAM). This effect has also been shown by new quantitative assays to be associated with stimulation of both DNA and protein synthesis. Autoradiography indicates that all cell types in the CAM are stimulated to divide, and it is proposed that fibrin degradation products are a pathological growth factor.     

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.     

Angiotensinogen impairs angiogenesis in the chick chorioallantoic membrane

Angiotensinogen shares with other members of the serine protease inhibitor (serpin) family antiangiogenic properties. Angiotensinogen inhibits in vitro endothelial cell proliferation, and is antiangiogenic in ovo in the chick chorioallantoic membrane assay. The cellular mode of action of angiotensinogen has been studied by applying purified human angiotensinogen or Chinese hamster ovary cells producing recombinant angiotensinogen onto the developing chorioallantoic membrane. Vessel density of the control and angiotensinogen-treated areas was quantitated by using Sambucus nigra lectin, a specific endothelial cell marker. After 48 h of angiotensinogen treatment by either applying purified angiotensinogen or angiotensinogen-producing Chinese hamster ovary cells, there was a 70% decrease in mesodermic vessel density in comparison to the control sections. Angiotensinogen treatment induced a strong decrease in endothelial cell proliferation of the chorioallantoic membrane vasculature, as shown by incorporation of bromo-deoxyuridine. Two days after local angiotensinogen treatment, increased apoptosis of endothelial cells of mesodermal blood vessels was detected by transferase-mediated deoxyuridine triphosphate nick end labeling assay. As assessed by in situ hybridization, the gene expression pattern of the main vascular growth factors and their receptors was not altered by angiotensinogen. Angiotensinogen, therefore, impairs angiogenesis without altering the expression level of vascular growth factors through the induction of apoptosis and decreased endothelial cell proliferation.     

Altered ratios of pro‐ and anti‐angiogenic VEGF‐A variants and pericyte expression of DLL4 disrupt vascular maturation in infantile haemangioma

Infantile haemangioma (IH), the most common neoplasm in infants, is a slowly resolving vascular tumour. Vascular endothelial growth factor A (VEGF‐A), which consists of both the pro‐ and anti‐angiogenic variants, contributes to the pathogenesis of IH. However, the roles of different VEGF‐A variants in IH progression and its spontaneous involution is unknown. Using patient‐derived cells and surgical specimens, we showed that the relative level of VEGF‐A₁₆₅b was increased in the involuting phase of IH and the relative change in VEGF‐A isoforms may be dependent on endothelial differentiation of IH stem cells. VEGFR signalling regulated IH cell functions and VEGF‐A₁₆₅b inhibited cell proliferation and the angiogenic potential of IH endothelial cells in vitro and in vivo. The inhibition of angiogenesis by VEGF‐A₁₆₅b was associated with the extent of VEGF receptor 2 (VEGFR2) activation and degradation and Delta‐like ligand 4 (DLL4) expression. These results indicate that VEGF‐A variants can be regulated by cell differentiation and are involved in IH progression. We also demonstrated that DLL4 expression was not exclusive to the endothelium in IH but was also present in pericytes, where the expression of VEGFR2 is absent, suggesting that pericyte‐derived DLL4 may prevent sprouting during involution, independently of VEGFR2. Angiogenesis in IH therefore appears to be controlled by DLL4 within the endothelium in a VEGF‐A isoform‐dependent manner, and in perivascular cells in a VEGF‐independent manner. The contribution of VEGF‐A isoforms to disease progression also indicates that IH may be associated with altered splicing. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Angiogenic response induced by acellular femoral matrix in vivo

We investigated the angiogenic response induced by acellular femoral matrices implanted in vivo on to the chick embryo chorioallantoic membrane (CAM), a useful model for such investigation. The results showed that acellular matrices were able to induce a strong angiogenic response, comparable with that of fibroblast growth factor-2 (FGF-2), a well-known angiogenic cytokine. The angiogenic response was further increased when exogenous FGF-2 or transforming growth factor beta-1 (TGF-beta1) was added to the matrices and inhibited by the addition of anti-FGF-2 or anti-TGF-beta1 antibodies. The response may be considered to be dependent on a direct angiogenic effect exerted by the matrices, and also in part by the presence of FGF-2 and TGF-beta1 in the acellular matrices.     

Glycoconjugate expression in the chick embryonic chorioallantoic membrane: Comparisons of the chorionic ectoderm and allantoic endoderm

Background: The chorioallantoic membrne (CAM) of the chick embryo expands during embryogenesis to meet the increased oxygen demands during growth and differentiation. Temporal and spatial glycosylation patterns of CAM ectodermal and endodermal proteins likely contribute to differentiation of the functional attributes of the CAM. Methods: Using lectins for light and electron microscopic observations, we studied the patterns of glycoconjugate expression on the ectoderm and endoderm of the chorioallantoic membrane (CAM) of the chick at days 4.5, 5.0, 6.0, and 10 of morphogenesis. For light microscopy, samples of unfixed CAM were incubated with the following FITC lectins: Con A, DBA, GSA-I, GSA-II, PNA, SBA, UEA-I, and WGA. Results: All lectins, except GSA-I and -II, gave positive results. The positive lectins, labeled with HRP, served to ultrastructurally localize PNA, SBA, and WGA, but not DBA binding to the luminal surface of the endoderm. UEA-I and Con A bound similarly except on day 10 when UEA-I no longer bound. On the ectodermal surface, only WGA bound at all times studied. PNA and SBA binding were present from days 5.0 to 6.0 but absent at days 4.5 and 10. DBA binding occurred through day 5.0 but was absent thereafter. UEA-I bound to the ectoderm at days 4.5, 5.0, and 10 but not days 5.5 and 6.0 Con A bound only on days 5.0 and 10. Conclusion: That the ultrastructurally similar ectoderm and endoderm of the CAM display functional differences conforms to the hypothesis that differential expression of glycoconjugate microdoains likely contributes to such functional specialization. © 1995 Wiley-Liss, Inc.     

胰岛素对人肝癌HepG2细胞体外诱导人脐静脉内皮细胞血管形成能力的影响

目的:探讨胰岛素对人肝癌细胞系HepG2体外诱导人脐静脉内皮细胞(HUVECs)血管形成能力的影响及其可能机制。方法:用含不同浓度胰岛素的完全培养基预培养肝癌细胞系HepG2制备条件培养基;应用预铺Matrigel基质胶的Transwell小室检测不同组条件培养基对HUVECs迁移能力的影响;运用CCK-8方法及EdU细胞增殖实验检测不同组条件培养基对HUVECs增殖能力的影响;运用内皮细胞成管实验检测不同组条件培养基对HUVECs成血管能力的影响;同时应用RT-PCR检测不同胰岛素浓度培养的HepG2细胞中血管内皮生长因子(VEGF)121、VEGF165、环氧化酶2(COX-2)的转录水平。结果:在一定浓度范围内,HepG2细胞对HUVECs的侵袭迁移能力、增殖能力的影响,对HUVECs的成血管能力的影响,对HepG2细胞VEGF121、VEGF165、COX-2转录水平的影响,均分别与胰岛素浓度呈正相关。结论:在一定浓度范围内,胰岛素可能通过上调HepG2细胞中VEGF121、VEGF165、COX-2的表达水平促进HepG2细胞诱导HUVECs血管形成能力。     

Behavior of the P1.HTR mastocytoma cell line implanted in the chorioallantoic membrane of chick embryos

The P1.HTR cell line includes highly transfectable cells derived from P815 mastocytoma cells originating from mouse breast tissue. Despite its widespread use in immunogenic studies, no data are available about the behavior of P1.HTR cells in the chick embryo chorioallantoic membrane model. The objective of the present investigation was to study the effects of P1.HTR cells implanted on the chorioallantoic membrane of chick embryos. We inoculated P1.HTR cells into the previously prepared chick embryo chorioallantoic membrane and observed the early and late effects of these cells by stereomicroscopy, histochemistry and immunohistochemistry. A highly angiotropic and angiogenic effect occurred early after inoculation and a tumorigenic potential with the development of mastocytoma keeping well mast cells immunophenotype was detected later during the development. The P1.HTR mastocytoma cell line is a good tool for the development of the chick embryo chorioallantoic membrane mastocytoma model and also for other studies concerning the involvement of blood vessels. The chick embryo chorioallantoic membrane model of mastocytoma retains the mast cell immunophenotype under experimental conditions and could be used as an experimental tool for in vivo preliminary testing of antitumor and antivascular drugs.     

Osteogenic protein‐1, a bone morphogenetic protein,induces angiogenesis in the chick chorioallantoic membrane and synergizes with basic fibroblast growth factor and transforming growth factor‐β1

Capillary invasion is a vital regulatory signal during bone morphogenesis that is influenced by angiogenic molecules such as fibroblast growth factor (FGF) and some members of the transforming growth factor‐β (TGF‐β) superfamily, including TGF‐βs themselves. Bone morphogenetic proteins (BMPs), which are members of the TGF‐β superfamily, have previously not been shown to possess direct angiogenic properties. Osteogenic protein‐1 (OP‐1; BMP‐7) is a potent regulator of cartilage and bone differentiation in vivo. The osteogenic and angiogenic properties of OP‐1 at both ortho‐ and heterotopic sites in adult chacma baboons (Papio ursinus) are enhanced synergistically by the simultaneous application of relatively low doses of TGF‐β1. The single application of relatively high doses of TGF‐β1 (20 ng), and bFGF (500 ng) or relatively low (100 ng) and high (1,000 ng) doses of OP‐1 in the chick chorioallantoic membrane (CAM) assay elicited a prominent and (for OP‐1) dose‐dependent angiogenic response. The binary application of a relatively low dose of OP‐1 (100 ng) with a relatively low dose of bFGF (100 ng) or with a relatively low (5 ng) or high (20 ng) dose of TGF‐β1 resulted in a synergistic enhancement of the angiogenic response. The angiogenic effect of the relatively low doses of the combined morphogens was distinctly more pronounced than that of the single application of the relatively high doses of the respective factors. The present findings suggest that these morphogens may be deployed in binary combination in order to accentuate experimental angiogenesis. The cooperative interaction of the different morphogens in the CAM assay may provide important biological clues towards the control of clinical angiogenesis. Anat Rec 259:97‐107, 2000. © 2000 Wiley‐Liss, Inc.     

Granulocyte colony-stimulating factor promotes tumor angiogenesis via increasing circulating endothelial progenitor cells and Gr1+CD11b+ cells in cancer animal models

Recombinant granulocyte colony-stimulating factor (G-CSF) is used for cancer patients with myelosuppression induced by chemotherapy. G-CSF has been reported to progress tumor growth and angiogenesis, but the precise mechanism of tumor angiogenesis activated by G-CSF has not been fully clarified. N-terminal-mutated recombinant human G-CSF administration increased WBCs and neutrophils in peripheral blood and reduced bone marrow stromal cell-derived factor-1 in mice, indicating its biological relevance. Mice were inoculated with Lewis lung carcinoma cells (LLCs) or KLN205 cells and treated with G-CSF. G-CSF accelerated tumor growth and intratumoral vessel density, while it did not accelerate proliferation of LLCs, KLN205 cells or human umbilical vein endothelial cells in vitro. In the absence of tumors, G-CSF did not increase circulating cells that displayed phenotypic characteristics of endothelial progenitor cells (EPCs). In the presence of tumors, G-CSF increased circulating EPCs. In addition, G-CSF treatment increased immune suppressor and endothelial cell-differentiating Gr1+CD11b+ cells in tumor-bearing mice. We conclude that G-CSF promotes tumor growth by activating tumor angiogenesis via increasing circulating EPCs and Gr1+CD11b+ cells in cancer animal models.     

Tumor angiogenesis in the bone marrow of multiple myeloma patients and its alteration by thalidomide treatment

Angiogenesis in solid tumors is important to tumor growth, invasion and metastasis. Recently, it has been suggested that angiogenesis plays a certain role in the development of hematopoietic malignancies, including leukemia and multiple myeloma. We evaluated tumor angiogenesis in the bone marrow (BM) of multiple myeloma (MM) patients by calculating microvessel density (MVD) in needle-biopsy specimens obtained from 51 cases of untreated MM or monoclonal gammopathy of undetermined significance (MGUS). The MVD in the BM of donors for transplantation and patients with non-hematological diseases was calculated as a control. There was an obvious increase in MVD in the BM of MM patients, and the MVD correlated with the grade of myeloma cell invasion of the BM in the untreated MM cases. It was recently reported that thalidomide might be effective for the treatment of MM. We assessed the effect of thalidomide on angiogenesis in BM treatment of 11 patients with refractory MM. The concentration of M-protein in the serum or urine of seven of the 11 patients was reduced by at least 30% after thalidomide treatment, and MVD in the BM decreased in three of these seven cases in response to thalidomide. Increased plasma concentrations of basic fibroblast growth factor (FGF-2) and vascular endothelial growth factor (VEGF) were observed in all 11 cases before thalidomide administration and both levels were reduced after treatment with thalidomide. Augmented angiogenesis in the bone marrow of MM patients was confirmed in the present study. It seems that thalidomide is effective in the treatment of MM through the impairment of angiogenesis by decreasing FGF-2 and VEGF production. This is the first report on pathological evidence in the bone marrow of MM before and after thalidomide treatment, in Japan.     

联合应用血管内皮生长因子及血小板衍生生长因子BB干预骨髓间充质干细胞的血管化及增殖能力

背景：组织工程骨为修复极限骨缺损提供了新的选择,但只有先形成完善的功能性血管网,保证稳定的成骨和骨整合,才能取得良好的治疗效果。因此,血管化可以说是组织工程骨面临的最大挑战与难题。 目的：探讨体外联合应用血管内皮生长因子(vascular endothelial growth factor,VEGF)和血小板衍生生长因子BB(platelet-derived growth factor-BB,PDGF-BB)对骨髓间充质干细胞增殖和成血管能力的影响。 方法：体外分离培养SD大鼠骨髓间充质干细胞,分别用不同质量浓度VEGF(20,40,60,80,100,120 μg/L)、PDGF-BB(20,40,60,80,100,120 μg/L)联合干预以及100 μg/L VEGF单独干预、100 μg/L PDGF-BB单独干预,CCK-8实验检测2种细胞因子促进细胞增殖的最佳质量浓度,然后在第7天和第14天通过RT-PCR方法检测血管生成素1、缺氧诱导因子1α、肝细胞生长因子、胰岛素样生长因子等相关成血管基因的表达量。 结果与结论：①加入生长因子后,细胞增殖能力明显提高,联合作用效果更优,最佳组合为80 μg/L VEGF+   80 μg/L PDGF-BB;②VEGF、PDGF-BB都可以促进血管生成素1、缺氧诱导因子1α、肝细胞生长因子和胰岛素样生长因子的mRNA表达,联合应用时效果最佳;③缺氧诱导因子1α、肝细胞生长因子 mRNA表达随时间延长有所升高,差异有显著性意义(P < 0.05);而血管生成素1、胰岛素样生长因子 mRNA表达量随时间延长有所降低,差异有显著性意义(P < 0.05);④体外实验结果证明,当VEGF和PDGF-BB质量浓度均为80 μg/L时,能够持续稳定促进整个血管形成过程,且促进作用优于单独一种生长因子。ORCID: 0000-0003-1918-579X(何惠宇) 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

Membrane fragments from cultured endothelial cells for use in screening anti-FGF receptor antibodies

Summary Endothelial cell properties may be studied by either detecting their functional activity or immunologic reactivity. Antibodies against the fibroblast growth factor (FGF) receptor may help delineate signal transduction pathways involved in the proliferation of endothelial cells and angiogenesis. This paper describes membrane fragment preparation from cultured coronary venular endothelial cells for obtaining intact FGF receptors for the assay of the anti-receptor antibodies.     

Plasma levels and skin-eosinophil-expression of vascular endothelial growth factor in patients with chronic urticaria

Background:  Although chronic urticaria (CU) is often regarded as autoimmune in nature, only less than 50% of sera from CU patients contain histamine-releasing autoantibodies. This suggests that other factors may contribute to its pathogenesis. We evaluated the possible involvement of vascular endothelial growth factor (VEGF), one of the major mediators of vascular permeability, in CU.
Methods:  Eighty consecutive adult patients with CU and 53 healthy subjects were studied. VEGF and prothrombin fragment F₁₊₂ were measured by enzyme immunoassays. Autologous plasma skin test (APST) was performed in CU patients and, in six of them, skin biopsy specimens were taken from wheals to evaluate the immunohistochemical expression of VEGF and eosinophil cationic protein (ECP).
Results:  Plasma VEGF concentrations were higher in CU patients (8.00 ± 0.90 pmol/l) than in controls (0.54 ± 0.08 pmol/l) ( P  =   0.0001) and tended to parallel both the severity of CU and to correlate with F₁₊₂ levels. APST was positive in 85.1% of patients. VEGF concentration was significantly higher in APST-positive than in APST-negative patients ( P  =   0.0003). Immunohistochemically, all specimens from patients with CU showed a strong expression of VEGF ( P  =   0.002) that colocalized with ECP, a classic eosinophil marker.
Conclusions:  VEGF plasma levels are elevated in CU and parallel the disease severity. This supports a possible role of this molecule in CU pathophysiology. Eosinophils are the main cellular source of VEGF in CU lesional skin.