An efficient,nonenzymatic method for isolation and culture of murine aortic endothelial cells and their response to inflammatory stimuli

Given the utility of murine models and the physiological and pathological significance of the aortic endothelium, we developed a simplified, nonenzymatic method for isolation and culture of murine aortic endothelial cells (MAECs). Aortic explants were initially cultured on fibronectin-coated plastic. Murine aortic endothelial cells migrated from the explants and proliferated. This expansion allowed for cultures to be established from the aortas of one or three mice. Murine aortic endothelial cells were then purified from expanded cultures by fluorescence-activated cell sorting for the uptake of 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate-labeled acetylated low-density lipoprotein. The majority of the cells in expanded cultures were as positive as human umbilical vein endothelial cells labeled in the same way. The most positive half of the labeled MAEC population was placed back in culture, and the cells formed "cobblestone" monolayers at confluence. Smooth muscle alpha-actin, which was present in aortic tissue and to a lesser extent in explant cultures before sorting, was not detected in selected MAECs. Western blotting and immunostaining also demonstrated the presence of the endothelial markers, platelet endothelial cell adhesion molecule-1, factor VIII-related antigen, and Bandeiraea simplicifolia lectin 1 binding. Murine aortic endothelial cells retained expected inflammatory functions: vascular cell adhesion molecule-1 protein was induced by bacterial endotoxin, and NO production was synergistically induced by the combination of endotoxin and interferon-gamma. Our simple, efficient method will facilitate investigations of aortic endothelial cell function in vitro using murine models.     

Nonproteolytic properties of murine alternatively spliced tissue factor: implications for integrin-mediated signaling in murine models

This study was performed to determine whether murine alternatively spliced tissue factor (masTF) acts analogously to human alternatively spliced tissue factor (hasTF) in promoting neovascularization via integrin ligation. Immunohistochemical evaluation of a spontaneous murine pancreatic ductal adenocarcinoma model revealed increased levels of masTF and murine full-length tissue factor (mflTF) in tumor lesions compared with benign pancreas; furthermore, masTF colocalized with mflTF in spontaneous aortic plaques of Ldlr(-/-) mice, indicating that masTF is likely involved in atherogenesis and tumorigenesis. Recombinant masTF was used to perform in vitro and ex vivo studies examining its integrin-mediated biologic activity. Murine endothelial cells (ECs) rapidly adhered to masTF in a β3-dependent fashion. Using adult and embryonic murine ECs, masTF potentiated cell migration in transwell assays. Scratch assays were performed using murine and primary human ECs; the effects of masTF and hasTF were comparable in murine ECs, but in human ECs, the effects of hasTF were more pronounced. In aortic sprouting assays, the potency of masTF-triggered vessel growth was undistinguishable from that observed with hasTF. The proangiogenic effects of masTF were found to be Ccl2-mediated, yet independent of vascular endothelial growth factor. In murine ECs, masTF and hasTF upregulated genes involved in inflammatory responses; murine and human ECs stimulated with masTF and hasTF exhibited increased interaction with murine monocytic cells under orbital shear. We propose that masTF is a functional homolog of hasTF, exerting some of its key effects via β3 integrins. Our findings have implications for the development of murine models to examine the interplay between blood coagulation, atherosclerosis and cancer.     

The role of laminin-1 in the modulation of radiation damage in endothelial cells and differentiation.

Microvascular dysfunction due to endothelial damage is often associated with the ionizing radiation used during cancer therapy. This radiation-induced capillary injury is a major factor in the inhibition of new vessel growth (angiogenesis) and in disease states such as radiation-induced pneumonitis and nephropathy. Many studies have examined the effects of radiation on endothelial cell function; however, little is known regarding the role the basement membrane plays in radiation-induced endothelial cell damage and angiogenesis. Therefore, we examined the effects of gamma radiation on aortic explants, and in vitro on three endothelial cell types (of artery, vein and capillary origin) irradiated with or without the basement membrane glycoprotein laminin-1. As expected, irradiation inhibited angiogenic sprouting of the aortic explants, endothelial cell proliferation, attachment, migration and differentiation in vitro in a dose-dependent manner. However, the effect of radiation on several of these processes in angiogenesis was reduced when the cells were irradiated on laminin-1. To further evaluate the effects of radiation on endothelial cells, we examined the expression of the vascular endothelial cell growth factor (VEGF) kinase domain region receptor in endothelial cells irradiated in the presence and absence of laminin-1. In endothelial cells irradiated on laminin-1, KDR expression increased 2.5-fold over control levels. Therefore, although radiation has a dose-dependent inhibitory effect on processes associated with angiogenesis in vitro, the presence of the basement membrane glycoprotein laminin-1 during irradiation decreases these effects.     

Induction of angiogenesis by murine resistin: putative role of PI3-kinase and NO-dependent pathways

Adipose tissue is a highly active endocrine organ, secreting bioactive molecules, adipokines, into the circulation. Obesity results in dysregulated adipokine secretion, contributing to pathophysiologies associated with this disorder, including insulin resistance and cardiovascular disease.ObjectivesTo establish whether resistin, a novel bioactive molecule produced by murine adipose tissue, and implicated in insulin resistance in rodents, can induce angiogenic responses in aortic tissues and endothelial cells in vitro, and to investigate the signal transduction pathways involved in these responses.ResultsRecombinant murine resistin (5–100 ng ml^? 1) induced sprouting of cellular networks and migration from murine aortic arch explants, primary aortic endothelial cells and in a ‘wound healing’ model utilising murine b.End5 endothelioma cells. The increased migration and sprouting of endothelial cells, due to resistin, were blocked by wortmannin (100 nM) and LY294002 (10 μM), inhibitors of phosphatidylinositol-3-kinase (PI3K), and accompanied by PI3K-dependent phosphorylation of Akt; moreover, while the changes were not associated with altered production of nitric oxide (NO), resistin-induced angiogenic responses were inhibited by IKK Inhibitor X (5 μM), an inhibitor of activation of nuclear factor (NF)-κB.ConclusionsMurine resistin induces endothelial cell migration and sprouting of cellular networks via a mechanism which appears dependent upon PI3K and NF-κB activity, but independent of altered NO production. Resistin may contribute to angiogenic responses sustaining adipose tissue expansion, or in arterial tissues distal to this site.     

Acacetin improves endothelial dysfunction and aortic fibrosis in insulin-resistant SHR rats by estrogen receptors

The aim of the work was to investigate the effects of acacetin on endothelial dysfunction and aortic fibrosis in insulin-resistant SHR rats and explore its mechanism. Seven-week-old male spontaneously hypertensive rats (SHR) were selected to establish a rat model of hypertension with insulin resistance induced by 10% fructose. The nuclear factor kappa B p65 (NF-κB p65) and Collagen I were observed by Immunohistochemistry. Immunofluorescence was used to observe estrogen receptor-alpha (ERα), estrogen receptor-beta (ERβ), and G protein-coupled receptor 30 (GPR30). Western blotting was used to detect interleukin (IL-1β), Arginase 2 (ARG2), Nostrin, endothelial nitric oxide synthase (eNOS), TGF-β, Smad3, ERK pathway proteins such as p-c-Raf, p-MEK1/2, p-ERK, ERK, p-P90RSK and p-MSK1. We found that acacetin did have an improvement on endothelial dysfunction and fibrosis. Meanwhile, it was also found to have a significant effect on the level of estrogen in this model by accident. Then, the experiment of uterine weight gain in mice confirmed that acacetin had a certain estrogen-like effect in vivo and played its role through the estrogen receptors pathway. In vitro experience HUVEC cells were stimulated with 30 mM/L glucose and 100 mM/L NaCl for 24 h to establish the endothelial cell injury model. HUVEC cells were treated with 1 μM/L estrogen receptors antagonist (ICI 182780) for 30 min before administration. Cell experiments showed that acacetin could reduce the apoptosis of HUVEC cells, the levels of inflammatory cytokines and the expression of TGF-β, Collagen I and Smad3 in endothelial cell injury model. After treatment with ICI 182780, the improvement of acacetin was significantly reversed. The results showed that acacetin relieved endothelial dysfunction and reduced the aortic fibrosis in insulin-resistant SHR rats by reducing the release of inflammatory factors and improving vasodilatory function through estrogen signaling pathway.

     

Indomethacin Inhibits Endothelial Cell Proliferation by Suppressing Cell Cycle Proteins and PRB Phosphorylation: A Key to Its Antiangiogenic Action?

Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit angiogenesis in vivo and in vitro, but the mechanism of this action is unclear. Angiogenesis—formation of new capillary vessels—requires endothelial proliferation, migration, and tube formation. It is stimulated by basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The cell cycle is regulated positively by cyclins and negatively by cyclin-dependent kinase inhibitors (CKI) and the retinoblastoma protein (pRb). Since the effects of NSAIDs on cell cycle-regulatory proteins in endothelial cells remain unknown, we examined the effect of indomethacin on bFGF-stimulated endothelial cell proliferation and on cell cycle regulatory proteins in rat primary aortic endothelial cells (RAEC). Indomethacin significantly inhibited basal and bFGF-stimulated endothelial cell proliferation. This inhibition correlated significantly with reduced cyclin D1 and increased p21 protein expression. Furthermore, indomethacin reduced pRb phosphorylation. These findings suggest that indomethacin arrests endothelial cell proliferation essential for angiogenesis by modulating cell cycle protein levels.     

Cell growth factor activity: New quantitative method in cell culture assay

This quantitative method for the assay of growth factor activity in vitro was developed empirically. Based on the empirical equations, it seems logical to express the cell growth rate by cell duplication frequency, f, and the growth factor activity by the normalized cell growth rate: $\text{1}\text{(T-T}\text{min}\text{)}$. The latter is recommended as the basis of defining an arbitrary unit of growth factor activity. For the in vitro growth factor activity assay, the quantitative method based on the empirical equations has two important features: (1) it appears to express linearly the growth factor concentration in assay, and (2) it is not affected by the initial cell counts and the length of cultivation.The view that these two features overcome the apparent defects associated with the expression systems that use the cell count ratio and net cell count is discussed. The quantitative method based on the empirical equations has been verified with experiments and compared with expression systems using the cell count ratio and net cell count. The experiments, which verified the quantitative method based on the empirical equations, were carried out with four selected cell lines: aortic endothelial cells, mouse 3T3 fibroblasts, Walker 256 carcinoma, and Chang's liver cells, using serum as the source of growth factor(s) for all cell lines. Vitreous, retinal extract, and Walker 256 carcinoma extract were also used for aortic endothelial cells.     

Inhibition of prostate tumor angiogenesis by the tumor suppressor CEACAM1

We have previously shown that CEACAM1, a cell-adhesion molecule, acts as a tumor suppressor in prostate carcinoma. Expression of CEACAM1 in prostate cancer cells suppresses their growth in vivo. However, CEACAM1 has no effect on the growth of prostate cancer cells in vitro. This difference suggests that the antitumor effect of CEACAM1 may be due to inhibition of tumor angiogenesis, perhaps by increased secretion of antiangiogenic molecules from the cells. In this study, we have demonstrated that expression of CEACAM1 in DU145 prostate cancer cells induced the production of a factor or factors that specifically blocked the growth of endothelial but not epithelial cells. Conditioned medium from the CEACAM1-expressing cells but not control luciferase-expressing cells inhibited endothelial cell migration up a gradient of stimulatory vascular endothelial growth factor in vitro and inhibited corneal neovascularization induced by basic fibroblast growth factor in vivo. Moreover, conditioned medium from CEACAM1-expressing cells induced endothelial cell apoptosis in vitro. Only medium conditioned by CEACAM1 mutants that were able to suppress tumor growth in vivo could cause endothelial cell apoptosis. These observations suggest that CEACAM1-mediated tumor suppression in vivo is, at least in part, due to the ability of CEACAM1 to inhibit tumor angiogenesis.     

Isolation and characterization of an established endothelial cell line from transgenic mouse hemangiomas

A murine endothelial cell line was isolated from hemangiomas induced by expression of the polyoma early region gene in transgenic mice. After two cell sortings using acetylated low-density lipoprotein with a fluorescent label (Dil-Ac-LDL), a pure population of endothelial cells has been carried for more than 60 passages from the animal. The cells retain endothelial cell properties such as a characteristic cobblestone appearance at confluency, contact-inhibited growth, and active uptake of Ac-LDL. Expression analysis shows that the cells express both the polyoma transgene and the von Willebrand factor, an endothelial cell marker. Subcutaneous injection of the cultured endothelial cells into nontransgenic histocompatible mice or nude mice led to hemangioma formation, and endothelial cells were re-isolated by cell sorting from these secondary hemangiomas. This cell line represents a renewable source of murine endothelial cells derived from transgenic mice that can be studied both in vitro and by reintroduction into a host.     

bcl-2 Gene Prevents Apoptosis of Basic Fibroblast Growth Factor-Deprived Murine Aortic Endothelial Cells

Growth factors such as basic fibroblast growth factor (bFGF) have been found to promote the survival and proliferation of endothelial cells. However, the mechanism by which growth factors control the regeneration and degeneration of the endothelial cells remained poorly understood. In this study, we demonstrated that apoptosis of murine aortic endothelial (MAE) cells was induced by deprivation of bFGF but required new RNA and protein synthesis. Furthermore, enforced expression of bcl-2 gene in MAE cells using gene transfer techniques decreased apoptosis induced by deprivation of bFGF. These findings suggest that bcl-2 interferes with a pathway for endothelial cell death that is induced by deprivation of bFGF.     

Glucocorticoid influence on growth of vascular wall cells in culture

Primary mass cultures and cloned strains of bovine aortic endothelial and smooth muscle cells were investigated with respect to their growth responses to glucocorticoid hormones. The growth of primary endothelial cells was not influenced by glucocorticoid treatment in the absence of fibroblast growth factor (FGF) but was inhibited by about 30% in the presence of FGF; with cloned endothelial cells, glucocorticoids were also growth inhibitory only in the presence of FGF. In contrast, smooth muscle cell growth was inhibited 30%-70% by glucocorticoid treatment in both primary cultures and in the cloned strains in the absence of FGF, and this inhibition was totally abolished by the addition of FGF for both cultures. The corticosteroid influences on cell growth were glucocorticoid specific, concentration dependent, and were observed to be independent of the serum concentration. The results indicate that glucocorticoid hormones have direct and pronounced growth inhibiting effects on aortic smooth muscle cells but only minimal effects on endothelial cells when these components of the vascular wall are analyzed under identical conditions in vitro.     

Inhibition of angiogenic factor- and tumour-induced angiogenesis by gamma linolenic acid.

Angiogenesis, the formation of new blood vessels, is an essential feature of malignant tumour development. Gamma linolenic acid (GLA), a n-6 polyunsaturated fatty acid (PUFA), inhibits the growth and metastasis of a variety of tumour cells, including breast, prostate, pancreatic cancer and hepatoma cells and also has anti-metastatic effects on endothelial cells. In the current study, we tested whether GLA inhibited angiogenesis induced by tumour cells. A rat aortic ring assay and in vitro tube formation of human vascular endothelial cells were used to determine angiogenesis (spontaneous, angiogenic factor- and tumour cells-induced). Inclusion of GLA in this 3-D matrix culture system significantly inhibited angiogenesis from aortic rings in a concentration-dependent manner. The results from tube formation of vascular endothelial cell further confirmed that GLA suppressed angiogenesis. Furthermore, in the cell motility assay (phagokinetic assay and endothelial wounding assay), a significant reduction of the motility of vascular endothelial cells by GLA was seen. It is concluded that gamma linolenic acid inhibits angiogenic factor and tumour-induced angiogenesis in vitro at least in part via its inhibitory effect on the motility of vascular endothelial cells.     

Secretion of SPARC by endothelial cells transformed by polyoma middle T oncogene inhibits the growth of normal endothelial cells in vitro.

Endothelioma cells expressing the polyoma virus middle T oncogene induced hemangiomas in mice by the recruitment of nonproliferating endothelial cells from host blood vessels (Williams et al. 1989). I now report that SPARC, a Ca(2+)-binding glycoprotein that perturbs cell-matrix interactions and inhibits the endothelial cell cycle, is produced by endothelioma cells and is in part responsible for the alterations in the morphology and growth that occur when nontransformed bovine aortic endothelial cells are cocultured with endothelioma cells. Normal endothelial cells cocultured with two different middle T-positive endothelial cell lines, termed End cells, exhibited changes in shape that were accompanied by the formation of cell clusters. Media conditioned by End cells repressed proliferation of normal endothelial cells, but enhanced that of an established line of murine capillary endothelium. Radiolabeling studies revealed no apparent differences in the profile of proteins secreted by aortic or capillary cells cultured in End cell conditioned media. Characterization of proteins produced by End cells led to the identification of type IV collagen, laminin, entactin, and SPARC as major secreted products. Although SPARC did not affect the morphology of End or capillary cells, it was associated with overt changes in the shape of aortic endothelial cells. Moreover, SPARC and a synthetic peptide from SPARC domain II inhibited the incorporation of [3H]thymidine by aortic cells, but had minimal to no effect on the capillary endothelial cell line. The inhibition of growth exhibited by aortic endothelial cells cultured in End cell conditioned media could be partially reversed by antibodies specific for SPARC and SPARC peptides. These studies indicate a potential role for SPARC in the generation of hemangiomas by End cells in vivo, a process that requires normal (host) endothelial cells to disengage from the extracellular matrix, withdraw from the cell cycle, migrate, and reassociate into the disorganized cellular networks that comprise cavernous and capillary hemangiomas.     

Human fibroblast-derived growth factor is a mitogen and chemoattractant for endothelial cells

Human fibroblasts were found to produce a potent mitogen and chemoattractant for fetal bovine aortic endothelial cells. Homogenates from AG1523 and AG1518 foreskin, CCD18Lu lung, and CCD18Co colon fibroblasts produced half-maximal stimulation of endothelial cell growth at concentrations of 1-7 micrograms/ml. The factor was purified from large-scale cultures of the CCD18Co fibroblasts using cation exchange chromatography and heparin-Sepharose chromatography. Such preparations were mitogenic for endothelial cells in vitro at concentrations of about 5-10 ng/ml, and promoted chemotaxis at 0.1-1 ng/ml. Heparinase treatment of the cells prevented the chemotactic response. These properties suggest that the factor may be related to fibroblast growth factor.     

Cytostatic product(s) released by activated macrophages, unrelated to interleukin 1, tumor necrosis factor alpha, and interferon-alpha/beta

Murine peritoneal macrophages activated for cytotoxicity by trehalose dimycolate in vivo and lipopolysaccharide in vitro released cytostatic factor(s) against EMT6 target cells, in 8-hr conditioned medium (CM). The cytostatic factor(s) completely blocked DNA synthesis by EMT6 cells within 16 hr. Other cell lines are less sensitive (P815 and R-L929) or resistant (KB and HT29) to the cytostatic effect of CM. The anti-proliferative activity of CM had a MW greater than 10,000 Da, as judged by ultrafiltration. It was destroyed by proteases and strongly inhibited by P815 cell product(s). Conditioned media from nonactivated macrophages were not cytostatic against EMT6 cells. No relationship was found between cytostatic factor(s) in CM and interleukin 1 (IL-1), tumor necrosis factor alpha (TNF-alpha), and interferon-alpha/beta (IFN-alpha/beta): the growth of EMT6 cells was unaffected by Hu.r.IL-is and Hu.r.TNF-alpha and was only slightly inhibited by IFN-alpha/beta. Furthermore, cytostatic CM contained low levels of TNF and IFN activities. Finally, antibodies raised against murine IFN-alpha/beta had no effect on the cytostatic activity of CM.     

Vascular endothelial growth factor (VEGF)-C synergizes with basic fibroblast growth factor and VEGF in the induction of angiogenesis in vitro and alters endothelial cell extracellular proteolytic activity

Vascular endothelial growth factor-C (VEGF-C) is a recently characterized member of the VEGF family of angiogenic polypeptides. We demonstrate here that VEGF-C is angiogenic in vitro when added to bovine aortic or lymphatic endothelial (BAE and BLE) cells but has little or no effect on bovine microvascular endothelial (BME) cells. As reported previously for VEGF, VEGF-C and basic fibroblast growth factor (bFGF) induced a synergistic in vitro angiogenic response in all three cells lines. Unexpectedly, VEGF and VEGF-C also synergized in the in vitro angiogenic response when assessed on BAE cells. Characterization of VEGF receptor (VEGFR) expression revealed that BME, BAE, and BLE cell lines express VEGFR-1 and -2, whereas of the three cell lines assessed, only BAE cells express VEGFR-3. We also demonstrate that VEGF-C increases plasminogen activator (PA) activity in the three bovine endothelial cell lines and that this is accompanied by a concomitant increase in PA inhibitor-1. Addition of α₂-antiplasmin to BME cells co-treated with bFGF and VEGF-C partially inhibited collagen gel invasion. These results demonstrate, first, that by acting in concert with bFGF or VEGF, VEGF-C has a potent synergistic effect on the induction of angiogenesis in vitro and, second, that like VEGF and bFGF, VEGF-C is capable of altering endothelial cell extracellular proteolytic activity. These observations also highlight the notion of context, i.e., that the activity of an angiogenesis-regulating cytokine depends on the presence and concentration of other cytokines in the pericellular environment of the responding endothelial cell. J. Cell. Physiol. 177:439–452, 1998. © 1998 Wiley-Liss, Inc.     

Functional Effects of FGF-13 on Human Lung Fibroblasts,Dermal Microvascular Endothelial Cells,and Aortic Smooth Muscle Cells

We studied the effects of FGF-13 and FGF-2 on human lung fibroblasts, dermal microvascular endothelial cells, and aortic smooth muscle cells. FGF-13 induced cell growth of lung fibroblasts and aortic smooth muscle cells but had no effect on dermal vascular endothelial cells. FGF-2 induced cell growth in all the three cell types. FGF-13 and FGF-2 had little effect on IL-6 production by lung fibroblasts and aortic smooth muscle cells and substantially enhanced that induced by IL-1α. In contrast, FGF-13 and FGF-2 had little effect on IL-6 production by dermal vascular endothelial cells, either alone or in synergy with IL-1α.