A B S T R A C T An in vitro method was used to detect adherence of 51Cr-labeled platelets to monolayers of cultured human endothelial, fibroblast, and smooth muscle cells. Washed platelets did not adhere to untreated or aspirin-treated endothelial monolayers in the absence of thrombin. In contrast, thrombin-induced platelet aggregates adhered to all of the monolayers but adherence to endothelium was significantly less than to the other cells. Additional evidence for adherence of platelets to the endothelium was provided by scanning and transmission electron microscopy. Thrombin-induced platelet adherence to endothelium was inhibited by hirudin. Platelet adherence induced by thrombin was enhanced significantly by treatment of the endothelial monolayer with 1-2 mM aspirin. This increase in adherence was seen even when aspirin-treated platelets were used; adherence values approached those seen with fibroblasts and smooth muscle cells. An aspirin concentration of 0.1 mM was sufficient to block thrombin-induced malonaldehyde production in platelets but it did not interfere with the inhibitory effect of the endothelium against platelet adherence. The effect of aspirin on the endothelium was temporary and inhibitory activity of the endothelium was restored 1 h after aspirin had been removed from the incubation system. The ability of thrombin to cause adherence of platelets to undamaged endothelium, and the potential for aspirin to enhance this adherence have implications for mechanisms which operate in platelet interaction with the blood vessel wall.
When human umbilical vein endothelial cultures were grown in the presence of supplemental arachidonic acid, the cell phospholipids became enriched with arachidonic acid. Prostacyclin (PGI 2 ) accumulated in the medium during supplementation with arachidonic acid. The capacity of these enriched cultures to produce PGI 2 when subsequently incubated with either arachidonic acid or thrombin was reduced by as much as 90%, but release of arachidonic acid from the cell lipids in response to thrombin stimulation was not inhibited. Refractory cultures completely recovered the capacity to form PGI 2 within 18 hours after removal of the medium containing supplemental arachidonic acid. However, recovery was prevented by cycloheximide. When enrichment with arachidonic acid was done in the presence of ibuprofen, a reversible cyclooxygenase inhibitor, PGI 2 did not accumulate in the medium during supplementation, and the subsequent capacity of the cultures to produce PGI 2 in response to thrombin increased by 70% to 240%. By contrast, the capacity of these supplemented cultures to convert added arachidonic acid to PGI 2 did not increase. Therefore, the enhancement in thrombin-stimulated PGI 2 production when the cultures are supplemented with arachidonic acid probably is due to the larger amount of arachidonic acid available in the intracellular lipid substrate pools, rather than to an activation of the PGI 2 synthetic pathway. These findings suggest that changes in the arachidonic acid content of the endothelial cell lipids may modulate the capacity of the endothelium to produce PGI 2 in response to stimulation. (Arteriosclerosis 3:323-331, July/August 1983)
Endothelium was isolated from samples of aorta and vena cava obtained from cadaver donors at the time kidneys were harvested for transplantation. Digestion with collagenase and gentle swabbing were used to free the cells from the Intlmal surface. Low density seeding permitted isolation of individual colonies with typical endothellal morphology. Modified Medium 199 supplemented with 10%-20% human plasma-derived serum and an extract from the bovine hypothalamus (500 ^g/ml) enabled subcultured colonies to grow to confluency when culture surfaces were coated with fibronectin (1 /xg/cm 2 ). The presence of Factor VIII antigen was demonstrated using an indirect immunofluorescence technique. A monoclonal antibody to cultured umbilical vein endothelium, specific for endothelium, reacted with the subcultured cells from the aorta and vena cava. Type IV procollagen, flbronectln, and thrombospondln were identified as labeled proteins secreted by cultures of adult endothelium that had been Incubated with 3 H-prollne and 3 H-glycine. When the cultured endothelium was used In a sodlum-m-periodate stimulated T lymphocyte mltogenic culture system, the endothelium exhibited accessory cell function. Prostacyclln production stimulated by incubation with arachldonlc acid and PGH 2 was variable from vessel to vessel. However, average values were lower than normally seen with cultured primary umbilical vein endothelium. (Arteriosclerosis 4:4-13, January/February 1984) M any discoveries concerning endothelial function have been made in recent years following the development of methods for culturing endothelium. Umbilical vein endothelium has been used by many groups while others have studied endothelium from the bovine aorta, pulmonary artery, or vein. Both qualitative and quantitative differences have been observed in functional assays with endothelial cells from these different sources. We have examined adult endothelium under similar conditions to ascertain its functional characteristics in culture. Received March 19, 1982; revision accepted July 26, 1983. Early attempts to culture endothelium from adult aorta and vena cava were unsuccessful. It was necessary to change the culture conditions that we normally used for primary cultures of umbilical vein endothelium. We previously reported 12 that by modifying the culture conditions we have been able to isolate colonies with endothelial morphology. We have grown sufficient numbers of adult aorta and vena cava endothelium to compare with umbilical vein endothelial cultures. Primary cultures of endothelium from the vena cava and aorta of the same donor, subcultured endothelium from aorta and vena cava samples, primary umbilical vein endothelium and umbilical vein endothelium subcultured with the modification used for adult endothelium have been examined for functional ability by a variety of different assays.
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