Cloned capillary endothelial cells, cultured in tumour-conditioned medium, form capillary tubes. By light and electron microscopy these tubes resemble capillaries in vivo. This first demonstration of angiogenesis in vitro: (1) shows that all the information necessary to develop an entire capillary network in vitro is expressed by one cell type; (2) suggests a mechanism for lumen formation; and (3) offers a possibility of distinguishing between direct and indirect angiogenesis factors.

Capillary endothelial cells from rats, calves, and humans, have been carried in long-term culture. Bovine capillary endothelial cells have been cloned and maintained by serial passage for longer than 8 months. This prolonged culture was accomplished by using tumor-conditioned medium, gelatin-coated plates, and a method of enriching cells However, long-term culture and cloning of capillary endothelial cells have not previously been possible. Del Vecchio et al. (5) reported the isolation of capillary endothelial cells from the adrenal cortex of rats. These cells, however, did not grow and could not be maintained in vitro for more than a few weeks. By modifying Del Vecchio's method and by using tumor-conditioned medium, gelatin-coated plates, and a method of enriching for capillary endothelial cells in primary culture, we have been able to culture capillary endothelial cells obtained from bovine adrenal glands, human adrenal glands, foreskin, and spleen, and rat heart, lung, kidney, and adrenal tissue. We now report long-term culture of human and bovine capillary endothelial cells and the cloning of bovine capillary endothelial cells. bisected and the adrenal cortex was extricated from the capsule. The adrenal cortex was cut into 1-mm pieces. These were centrifuged in phosphate-buffered saline and then incubated in 0.5% collagenase (Worthington) at room temperature for 1 hr in a polyethylene centrifuge tube. This suspension was pipetted with a large-bore pipette against the wall of the centrifuge tube to break the tissue into smaller pieces. The suspension was then passed through a nylon-covered funnel (Nitex Mono Screen Cloth, HC3-110, Tetko, Elmsford, NY). Only capillary segments smaller than 110 ,um passed through the filter. These were centrifuged at 650 rpm for 7 min at 40C and the collagenase was discarded. The pellet was resuspended in 10 ml of Dulbecco's modified Eagle's medium supplemented with 10% calf serum and washed three times. The final pellet of cells was resuspended in 10 ml of Dulbecco's modified Eagle's medium and plated into four gelatinized dishes (60 mm). These plates were incubated at 37°C. Capillary segments and endothelial cell aggregates were the first to adhere to the substratum. Adrenal cortical cells and fibroblasts continued to float, and most were removed by aspirating the supernatant between 1 and 3 hr after plating. After the first aspiration of supernatant, tumor-conditioned medium was added and replaced every 2 days. Capillary fragments usually contained from one to four endothelial cells. As these capillary segments spread onto the coated plastic substratum, each segment became a microcolony with a characteristic appearance that persisted during the next 2-5 days (see Fig. 1 a and b). METHODSCloning. To enrich the primary cultures for bovine capillary endothelial cells and to remove contaminating nonendothelial

Heparin or a heparin fragment administered with cortisone inhibited angiogenesis, caused regression of large tumor masses, and prevented metastases. Oral administration of heparin resulted in the release of non-anticoagulant heparin fragments in the serum which, in the presence of cortisone, had similar anti-angiogenic and antitumor effects. Of all the heparin fragments tested, the most potent inhibition of angiogenesis in the presence of cortisone was provided by a hexasaccharide with a molecular weight of about 1600.

This study investigated whether endothelin-1 (ET-1), a potent vasoconstrictor, which also stimulates cell proliferation, contributes to endothelial dysfunction and atherosclerosis. Apolipoprotein E (apoE)-deficient mice and C57BL͞6 control mice were treated with a Western-type diet to accelerate atherosclerosis with or without ET A receptor antagonist LU135252 (50 mg͞kg͞d) for 30 wk. Systolic blood pressure, plasma lipid profile, and plasma nitrate levels were determined. In the aorta, NO-mediated endotheliumdependent relaxation, atheroma formation, ET receptorbinding capacity, and vascular ET-1 protein content were assessed. In apoE-deficient but not C57BL͞6 mice, severe atherosclerosis developed within 30 wk. Aortic ET-1 protein content (P < 0.0001) and binding capacity for ET A receptors was increased as compared with C57BL͞6 mice. In contrast, NO-mediated, endothelium-dependent relaxation to acetylcholine (56 ؎ 3 vs. 99 ؎ 2%, P < 0.0001) and plasma nitrate were reduced (57.9 ؎ 4 vs. 93 ؎ 10 mol͞liter, P < 0.01). Treatment with the ET A receptor antagonist LU135252 for 30 wk had no effect on the lipid profile or systolic blood pressure in apoE-deficient mice, but increased NO-mediated endothelium-dependent relaxation (from 56 ؎ 3 to 93 ؎ 2%, P < 0.0001 vs. untreated) as well as circulating nitrate levels (from 57.9 ؎ 4 to 80 ؎ 8.3 mol͞liter, P < 0.05). Chronic ET A receptor blockade reduced elevated tissue ET-1 levels comparable with those found in C57BL͞6 mice and inhibited atherosclerosis in the aorta by 31% without affecting plaque morphology or ET receptor-binding capacity. Thus, chronic ET A receptor blockade normalizes NO-mediated endothelial dysfunction and reduces atheroma formation independent of plasma cholesterol and blood pressure in a mouse model of human atherosclerosis. ET A receptor blockade may have therapeutic potential in patients with atherosclerosis.Diseases related to atherosclerosis such as myocardial infarction and stroke account for the majority of deaths in industrialized countries (1). In patients with cardiovascular risk factors such as hypercholesterolemia, hypertension, or aging (2, 3), endothelial dysfunction precedes the development of atherosclerosis and predisposes to the development of structural vascular changes (1, 4). The endothelium releases vasoactive mediators such as NO and endothelin (ET-1), both of which are importantly involved in the regulation of vascular tone (5, 6) and structure (7,8). Endothelial NO synthase (9-11) converts L-arginine into NO and L-citrulline (12) and its expression (13), and the release of NO (14) is reduced in atherosclerosis. In experimental atherosclerosis, inhibition of the L-arginine͞NO pathway accelerates lesion progression in hypercholesterolemic rabbits (15-17) and low density lipoprotein (LDL) receptor-deficient mice (18). Furthermore, superoxide release in atherosclerosis inactivates NO resulting in formation of peroxynitrite (19,20), the production of which is further enhanced by cholesterol (21).In patients with coronary a...

The prototype members of the heparin-binding fibroblast growth factor (FGF) family, acidic FGF (FGF-1) and basic FGF (FGF-2), are among the growth factors that act directly on vascular cells to induce endothelial cell growth and angiogenesis. In vivo, the role of the FGF prototypes in vascular pathology has been difficult to determine. We report here the introduction, by direct gene transfer into porcine arteries, of a eukaryotic expression vector encoding a secreted form of FGF-1. This somatic transgenic model defines gene function in the arterial wall in vivo. FGF-1 expression induced intimal thickening in porcine arteries 21 days after gene transfer, in contrast to control arteries transduced with an Escherichia coli beta-galactosidase gene. Where there was substantial intimal hyperplasia, neocapillary formation was detected in the expanded intima. These findings suggest that FGF-1 induces intimal hyperplasia in the arterial wall in vivo and, through its ability to stimulate angiogenesis in the neointima, FGF-1 could stimulate neovascularization of atherosclerotic plaques. Potentially, gene transfer of FGF-1 could also be used as a genetic intervention to improve blood flow to ischaemic tissues in selected clinical settings.

Platelet-derived growth factor (PDGF) B chain induces cell proliferation in vitro and is associated with arterial lesions that cause cardiovascular disease. However, it has been difficult to document the biological response to PDGF B gene expression in arteries in vivo. To determine the biologic effects of this growth factor in vivo, we have introduced an eukaryotic expression vector plasmid encoding recombinant PDGF B by direct gene transfer into porcine iliofemoral arteries using DNA liposome complexes. Invest. 1993Invest. . 91:1822Invest. -1829

Angiogenesis and tumor growth were inhibited in two different animal models by regional infusion of a partially purified cartilage extract. In rabbits bearing corneal implants of V2 carcinoma and receiving the inhibitor, vascular growth rates were <3% of those in control animals receiving either Ringer's solution or bovine trypsin inhibitor (Trasylol). Subconjunctival B16 melanoma implants-in mice receiving the inhibitor weighed <2.5% of implants in mice receiving Ringer's solution, Trasylol, or albumin. Histplogic study of major organs and standard blood tests revealed no toxic effects in any of the animals. The inhibitor did not retard the growth of either tumor cell type in tissue culture at concentrations as high as 1 mg/ml. These results suggest that the cartilage factor does not interfere with the growth of the tumor cell population directly but that it prevents tumor growth by inhibiting angiogenesis.

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