Cultured aortic endothelial cells from pigs with von Willebrand disease: in vitro model for studying the molecular defect(s) of the disease.

Abstract

vWd that resembles the classic disease in man has also been described in dogs (5) and pigs (5, 6). The purpose of this communication is to describe the long-term culture and properties of normal and vWd porcine endothelial cells and the use of this in vitro culture system for studying the molecular defect(s) in vWd.MATERIALS AND METHODS Preparation and Culture of Cells. No detectable Willebrand factor or RWF could be demonstrated in the plasma of the vWd pigs that were used as the source of vWd aortas. Willebrand factor was determined by Laurell immunoelectrophoresis (7), and RWF activity was measured by ristocetin-induced platelet aggregation assays.Endothelial cells were obtained from untraumatized, freshly excised normal and vWd porcine aortas (25-35 cm long) by collagenase treatment (0.5 mg/ml, CLS, Worthington Biochemical Corp., Freehold, NJ) for 15-20 min at 37°. Collagenase-treated aortas were rinsed twice with RPMI-1640 medium (GIBCO), and the endothelial cells were removed, in complete medium, by gentle agitation as described for bovine aortic endothelial cells (8). The complete medium consisted of RPMI-1640 medium, 15 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes) buffer, penicillin (100 units/ml), streptomycin (50,ug/ml), neomycin (200 units/ml), 2 mM fresh glutamine, and 20% fetal calf serum or 20% porcine serum (the calf and porcine sera were heat-deactivated at 560 for 30 min). The final complete culture medium was adjusted to pH 7.3. Medium containing neomycin but not penicillin and streptomycin was used for subcultures.Typically, 25-35 cm of aorta yielded 1.5 to 4 X 106 cells in 20 ml of complete medium. Cells were seeded into Falcon T-25 (25 cm2) plastic flasks or Falcon tissue culture petri dishes (35 X 10 mm) at a cell density of no more than 200 cells per cm2. Cell patches rapidly (<1 hr) attached to the substratum and, after 1-2 hr, spread cell patches were rinsed twice with RPMI-1640 medium and fed with complete fresh medium. Medium without penicillin and streptomycin was exchanged at 72-hr intervals, and the cultures were incubated at 370; cultures in unsealed petri dishes were incubated in a 95% air/5% CO2 atmosphere.After 4 t To whom reprint requests should be addressed. 5702The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

Culture-Produced Subendothelium

“…Weiss et al [35] have demon strated that the defect of adhesion of human von Willebrand platelets to a-chymotrypsintreated rabbit SE could be corrected by the addition of F. VIII. We have previously dem onstrated that pretreatment of cultured por cine aortic ECs (damaged by scraping) with monospecific chicken antiporcine F. VIIIR:WF serum completely abolished the interaction of platelets with exposed SE ma terial [8]. We have also demonstrated that the decreased interaction of platelets with SE from cultured von Willebrand porcine aortic ECs may be due to the decreased and/or abnormal distribution of immunologically identifiable F. VIIIR:WF-containing micro filaments in the SE of these von Willebrand ECs [8][9][10], Finally, the localization, by im munofluorescence staining, of F. VIIIR:AG in human vascular SE has recently been re ported [24].…”

“…Platelets were obtained from freshly drawn porcine blood containing ACD (38 m M citric acid -75 mM sodium citrate -125 m M dextrose) as anticoagulant; 1 ml of ACD/8 ml of blood and washed as described previously [8]. The platelet pellet was finally resus pended in modified TBSS at a cell concentration of 4 X 105 platelcts/mm5.…”

“…Aortic EC cultures were established, rou tinely subcultured and maintained as described pre viously [7,8]. Cells were grown on glass covcrslips (10.5 X 50 mm, Wheaton Scientific, Millville, N.J.) placed directly into Falcon T-25 plastic culture flasks prior to seeding of subcultures.…”

Human Pulmonary Endothelial Cells in Culture

“…Most cultured endothelial cells retain the features of endotheliumii in vivo, including an epithelioid shape an(V Weibel-Palade bodies in the cytoplasm (25)(26)(27). Factor VIII antigen is associated with cells fromn human veins (12,15) and with cells from aortas of animials (19,21,24). Angiotensin I convertinig enzyme activity is found in cells from umbilical cord veins (15,16) and also in aortic endothelial cells from various species (20,28,29).…”

“…Endothelial cells have been cultured from veins of human umiibilical cords (12)(13)(14)(15)(16), vena cavae of rabbits (17), andI large arterial vessels of pigs (18)(19)(20) and cows (21)(22)(23)(24). Most cultured endothelial cells retain the features of endotheliumii in vivo, including an epithelioid shape an(V Weibel-Palade bodies in the cytoplasm (25)(26)(27).…”