A B S T R A C T Endothelial cells

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Activation of protein kinase C by phorbol esters is known to accelerate the processing and secretion of the 8/A4 amyloid protein precursor. We have now examined various first messengers that increase protein kinase C activity of target cells for their ability to affect 13/A4 amyloid protein precursor metabolism. Acetylcholine and interleukin 1, which are altered in Alzheimer disease, were shown to increase processing of the 13/A4 amyloid protein precursor via the secretory deavage pathway. Cholinergic agonists stimulated secretion in human glioma and neuroblastoma cells as well as in PC12 cells transfected with the M1 receptor, while interleukin 1 stimulated secretion in human endothelial and glioma cells.Alzheimer disease is characterized by distinct neuropathological lesions, including intracellular neurofibrillary tangles, extracellular parenchymal and cerebrovascular amyloid deposits, and selective cell death that particularly affects cholinergic neurons in the basal forebrain (1). The death of cholinergic neurons is associated with decreased levels of acetylcholine in the cortex and other projection areas. Other biochemical changes that occur in Alzheimer disease include increases in the levels of interleukin 1 (IL-1) in the brain and cerebrospinal fluid (2, 3).The principal component of parenchymal amyloid plaque cores and cerebrovascular amyloid is the 13/A4 amyloid protein (4-6), which is derived from cleavage of a large transmembrane protein, the f3/A4 amyloid protein precursor (APP) (7)(8)(9)(10)(11)(12)(13). APP exists in three major isoforms (APP695, APP751, and APP770), which arise from alternative splicing of a single primary transcript. APP undergoes secretory processing, which leads to release of the extracellular domain of APP into the extracellular space (14). Normal MATERIALS AND METHODS

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Cultured endothelial cells derived from human umbilical veins or bovine aorta produce a potent inhibitor of platelet aggregation. The inhibitor is synthesized from sodium arachidonate or prostaglandin endoperoxides by a microsomal enzyme system. Tranylcypromrine, a specific antagonist of prostacyclin synthetase, suppresses production of the inhibitor by endothelial cells. The inhibitor, which is ether extractable, has been identified using a two-step thin-layer radiochromatographic procedure and a synthetic prostaglandin I2 standard. With this procedure, we have shown that human and bovine endothelial cells convert sodium [3H]arachidonate to radiolabeled prostaglandin I2 and 6-keto-prostaglandin Fla, as well as prostaglandin E2. Thus, endothelial cells may be non-thrombogenic in vivo because they synthesize and release prostaglandin I2, a potent inhibitor of platelet aggregation.Fragments of blood vessel walls acting upon prostaglandin (PG) endoperoxides or arachidonic acid produce an unstable factor that prevents platelet aggregation and release (1, 2). This factor, recently identified as (5Z)-9-deoxy-6,9-a-epoxy A5-PGFIa, (prostacyclin L-glutamine (2 mM). Cells were grown in T-25 or T-75 flasks (Corning) at 370 under 5% C02/95% air until confluence. Both primary cultures and serial passages were utilized. Initial cultures of bovine endothelial cells were kindly provided by Alan Quarfoot and Francois Booyse (13) and were cultured in RPMI 1640 medium containing 10-20% heat-inactivated fetal calf serum and penicillin, streptomycin, and L-glutamine as above. Cells were harvested from culture flasks by treatment with 0.1% collagenase-0.01% EDTA for 10 min at 370, washed three times in buffer A [137 mM NaCl/4 mM KCl/11 mM glucose/10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes), pH 7.5] and finally resuspended in the same buffer at 6 X 106 cells per ml.Culture and Preparation of Other Cells. Human fibroblasts were grown in tissue culture, harvested with trypsin (0.25%) for 10 min, washed similarly, and resuspended in buffer A at the same protein concentration as the endothelial cells. Peripheral blood mixed leukocytes (70-80% neutrophils) were separated from heparin-treated whole blood by dextran sedimentation, washed, and resuspended in-buffer A at a similar protein concentration.Platelet Aggregation Experiments. Platelet-rich plasma (PRP) was prepared from venous blood drawn into 'ho volume of 3.2% trisodium citrate by methods previously described (14) and was kept tightly capped at 220 under 5% C02/95% air until use. Compounds being tested (0.1 ml) were added to 0.4 ml of PRP in the cuvette of a Payton aggregation module (Payton Associates, Buffalo, NY). Aggregating agents used included arachidonic acid (as the sodium salt in 0.1 M Na2COA), adenosine diphosphate (ADP) and collagen (Sigma Chemical Co., St. Louis, MO), and bovine thrombin (Parke Davis Co., Detroit, MI). Synthetic PGI2 (15) final volume of 0.5 ml in an aggregation module cuvette at 370 Abbreviations: PG, prostaglandin; ...

Cultured human endothelial cells synthesize and secrete a protein(s) which has Factor VIII antigen but which lacks Factor VIII clot-promoting activity ( J. Clin. Invest. 52, 2757-2764, 1973). Von Willebrand factor activity has been identified in medium from cultured human endothelial cells. This activity was demonstrated by the ability to correct the defect in platelet adhesiveness of blood obtained from patients with von Willebrand's disease. This activity also supported ristocetin-induced aggregation of washed normal human platelets. The von Willebrand factor activity from cultured endothelial cells has physicochemical and immunologic properties like those of the von Willebrand factor activity and the Factor VIII antigen present in human plasma and the Factor VIII antigen synthesized by human endothelial cells in vitro . Rabbit antibody to chromatographic fractions containing endothelial cell von Willebrand factor inhibits the platelet retention of normal blood in glass bead columns.