Endothelial cells from human blood vessels were cultured in vitro, with doubling times of 17 to 21 hours for 42 to 79 population doublings. Cloned human endothelial cell strains were established for the first time and had similar proliferative capacities. This vigorous cell growth was achieved by addition of heparin to culture medium containing reduced concentrations of endothelial cell growth factor. The routine cloning and long-term culture of human endothelial cells will facilitate studying the human endothelium in vitro.

The purpose of this study was to characterize the permeability characteristics of an in vitro endothelial cell monolayer system and relate this information to available in vivo data. We cultured bovine fetal aortic endothelial cells on fibronectin-coated polycarbonate filters and confirmed that our system was similar to others in the literature with regard to morphological appearance, transendothelial electrical resistance, and the permeability coefficient for albumin. We then compared our system with in vivo endothelium by studying the movement of neutral and negatively charged radiolabeled dextran tracers across the monolayer and by using electron microscopy to follow the pathways taken by native ferritin. There were a number of differences. The permeability of our monolayer was 10-100 times greater than seen in intact endothelium, there was no evidence of "restricted" diffusion or charge selectivity, and ferritin was able to move freely into the subendothelial space. The reason for these differences appeared to be small (0.5-2.0 micron) gaps between 5 and 10% of the endothelial cells. Although the current use of cultured endothelial cells on porous supports may provide useful information about the interaction of macromolecules with the endothelium, there appear to be differences in the transendothelial permeability characteristics of these models and in vivo blood vessels.

PV701 is a naturally-attenuated, non-recombinant, oncolytic strain of Newcastle disease virus that displays preclinical intravenous (IV) efficacy. PV701 is selective at killing human cancer cells versus normal human cells based on tumor specific defects in the interferon (IFN)-mediated antiviral response. This oncolytic virus displays a broad spectrum of antitumor activity in vitro and in vivo. Preclinical models successfully predicted key clinical parameters including the mechanism of toxicity, two complementary strategies (desensitization and slow infusion) to reduce toxicity, and the starting dose for phase 1 trials. In three phase 1 trials of 114 patients using IV administration of PV701, Wellstat Biologics Corporation has evaluated the effects of dose, schedule, and infusion rate for PV701. Three general classes of side effects were seen: flu-like symptoms; tumor-site-specific adverse events (AEs); and infusion reactions. The first PV701 dose desensitized the patient to the side effects of further doses, allowing a marked increase in the maximum tolerated dose for subsequent doses compared to the first dose. Tumor responses were first noted at the higher doses achieved using desensitization. In the most recent phase 1 trial of 19 patients at Hamilton, Ontario, that employed desensitization, high repeat doses, and a slower infusion rate (Hamilton Regimen), there were six responses (4 major; 2 minor) and a total of six patients with survival for at least 2 years. In addition, patient tolerability improved using the Hamilton Regimen compared to IV bolus dosing used previously. Phase 2 studies of this novel biologic agent are about to begin.

A cloned strain of bovine vascular endothelial cells with a finite in vitro lifespan was treated with benzo(a)pyrene (BP) after approximately 75% of its lifespan was completed. Untreated cultures of this strain senesced upon serial subcultivation and contained large, nondividing cells. In three out of seven trials, BP treatment produced transformed lines with altered phenotypic characteristics. The transformed cells appeared in the cultures concomitant with the senescence of the parent cells. All transformed cell lines examined exhibited indefinite lifespans and altered karyotypes. Two of the lines retained most of the characteristics of normal endothelial cells, except that one became aneuploid and the other polyploid. Neither of these lines formed tumors when inoculated into nude mice. The remaining two lines retained mostly diploid karyotypes, but a high percentage of cells contained Robertsonian translocations. In one line cell volume was markedly reduced. In addition, these lines grew in multilayers, were anchorage independent, and proliferated in medium containing 0.5% serum. When 10(7) cells of these lines were injected into nude mice, tumors appeared within 1 week and were identified as malignant hemangioendotheliomas of bovine origin.

This report describes a new in vitro method for measuring the diffusional permeability of an endothelial monolayer and its use in investigating the modulation of permeability by various agents, e.g., isoproterenol, propranolol, dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP), and cytochalasin D. To determine permeability, tracers of different molecular weights were applied simultaneously on a chromatography column containing confluent endothelial cells cultured on porous microcarrier beads. The Sangren-Sheppard model was used to determine the permeability of the endothelial monolayer from the tracer elution profiles. For six radiolabeled tracers the mean (+/- SD) permeabilities (cm/s x 10(-5)) in order of increasing tracer molecular weight were [3H]water, 82.0 +/- 28.8; [14C]urea, 49.5 +/- 9.5; [14C]mannitol, 13.3 +/- 4.7; [14C]-sucrose, 14.1 +/- 2.5; [3H]polyethylene glycol (900 mol wt), 4.80 +/- 1.61; and [3H]polyethylene glycol (4,000 mol wt), 1.97 +/- 1.01. These permeabilities deviate less from in vivo values than those obtained in other in vitro systems and are 10 times higher than in vivo estimates. The values were reproducible for up to the 4 h tested. Modulation of endothelial monolayer permeability was studied in a separate series of experiments. The beta-adrenergic agonist isoproterenol (10(-6) M) decreased the permeability to mannitol by 36% and to polyethylene glycol (900 mol wt) by 49%; in both instances the decrease in permeability was reversed by propranolol. Propranolol alone had no effect. Dibutyryl cAMP (10(-3) M) decreased the permeability to mannitol by 40% and to polyethylene glycol by 47%; permeability returned to base line when dibutyryl cAMP was removed. Cytochalasin D (1 microgram/ml) increased permeability by 350% for mannitol and 380% for polyethylene glycol; the permeability change was reversed after removal of cytochalasin D. The results indicate that cell-column chromatography is a powerful method that can be used to characterize the permeability of endothelial monolayers and to investigate permeability changes produced by various agents.