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Phospholipases C (PLCs) are ubiquitous enzymes which play key roles in the response of cells to extracellular agonists. Endothelial cells are involved in myriad normal and pathophysiologic functions. Although it is known that agonists activate PLCs in endothelial cells, second messengers form, and cellular responses ensue, more knowledge is needed about the specific types of PLCs in these cells. To this end, cytosolic PLCs from porcine aortic endothelial cells were partially purified by ammonium sulfate fractionation and column chromatography on DEAE-Sepharose CL-6B and heparin-agarose. Three PLC isozymes immunologically similar to bovine brain PLC-beta, PLC-gamma, and PLC-delta were identified. The relative levels of PLC activities in the cytosol were: PLC-beta, 50%; PLC-gamma, 44%; PLC-delta, 6%. The level of PLC-beta activity in porcine endothelial cells appeared higher than the levels reported for several established cell lines, suggesting that this enzyme may play a specific role in endothelial cell function. Elution profiles of PLC activity with phosphatidylinositol 4,5-bisphosphate (Ptdlns(4,5)P2) as substrate were similar to those with phosphatidylinositol (Ptdlns) as substrate, indicating that cytosolic PLCs hydrolyze both Ptdlns and Ptdlns(4,5)P2 and no Ptdlns(4,5)P2-specific PLC was present in the cytosol. The catalytic properties of the partially purified PLC isozymes from porcine endothelial cells were similar to their counterparts from bovine brain. These include the dependence of hydrolysis of Ptdlns on Ca2+, the optimal Ca2+ concentrations for the hydrolysis of Ptdlns and Ptdlns(4,5)P2, the pH optima, and the stimulatory effects of deoxycholate.

Purpose: The purpose of this study was to explore new strategies for enhancing specific cell type attachment to biomaterials using immobilized lectins for cell surface glycoconjugates. The lectin Ulex europaeus I (UEA I) has a high affinity for human vascular endothelial cell surface glycoconjugates. Methods: UEA I was covalently bound to polyethylene terephthalate (Dacron) with the cross-linking agent 1-ethyl-3-(dimethylaminopropyl) carbodiimide hydrochloride to achieve oligosaccharide-mediated endothelial cell attachment to this otherwise nonadherent surface. Results: Experiments with radiolabeled UEA I demonstrated covalent linkage of as much as 1.35 μg/cm 2 . The lectin binding site is available after the reaction, as demonstrated in experiments a neoglycoprotein. Adhesion studies reveal a 100-fold increase in endothelial cell attachment for the UEA I/polyethylene terephthalate surface (99.7 ± 29.6 cells/high-power field) when compared with untreated (0.7 ± 0.5), crosslinking agent (0.4 ± 0.3), and denatured UEA I (1.2 ± 1.1) control groups. Five vascular endothelial cell lines adhered to the UEA I/polyethylene terephthalate surface, whereas monocytes, smooth muscle cells, and fibroblasts did not. Conclusion: These results imply new strategies for endothelialization of prosthetic grafts and promotion of selective cell adherence to biomaterials, with emphasis on carbohydrate interactions. Moreover, this experimental system offers a model for exploring the biologic significance of the endothelial cell-UEA I ligand. (J VASC SURG 1993;18:486-94.)

Methylcholanthrene transformed mouse fibroblast cells can be induced to synthesize prostaglandins by a short term incubation with various vasoactive agents including serum, bradykinin and thrombin or in response to mechanical detachment from the culture dish. The ability of the cells to synthesize prostaglandins upon stimulation changes during growth of the culture on the dish; the response is maximal on the first day after inoculation and decreased sharply thereafter. Feeding of the cells with fresh growth medium enhances prostaglandin production induced by all stimuli. The difference in the cell response during growth is probably not due to change of prostaglandin synthetase activity since the specific enzyme activities assayed with microsomal preparations of cells harvested from the first and third day culture are similar. However, analysis of the cellular content of arachidonic acid after saponification of the total lipid extract of cells harvested at different days of growth reveals that the level of arachidonic acid per cell during growth is parallel to the response to stimuli. It is maximal on the first day and decreases sharply on the second day and stays low on the third day. Our study suggests that the level of arachidonic acid in the cell governs the extent of prostaglandin synthesis upon stimulation.

We have discovered that bradykinin is effective in causing the synthesis of prostacyclin in endothelial cells cultured from calf and pig aorta and human umbilical cord vein. Maximal stimulation is attained at 10 ng bradykinin per ml with a 10 fold increase of PGI 2 as assayed by radioimmunoassay for 6-k-PGF 1α . Ionophore A23187, trypsin, and the precursor arachidonic acid are also stimulatory. We have confirmed the study of Weksler et al ( J. Clin. Invest., 62 , 923, 1978) and Czervionke et al ( Thrombosis Research, 14 , 781, 1979) that thrombin is effective in stimulating PGI 2 synthesis in endothelial cells from human umbilical cord vein. However, we found that cells from calf or pig aorta are not stimulated as well by thrombin. Thus, there appears to be a difference in the response to thrombin between these cells. When calf cells were grown in the presence of [ 3 H]arachidonic acid, the radioactivity is incorporated mainly into the phospholipids. Bradykinin, ionophore A23187, and trypsin stimulated the release of radioactive materials into medium from [ 3 H]arachidonic acid labeled calf cells. Arachidonic acid is the major radioactive substance released and PGI 2 is the major known arachidonic acid metabolite formed.

Graft thrombosis soon after reconstruction remains a major obstacle to the use of reversed vein grafts in infrapopliteal reconstruction. Our clinical experience with in situ vein grafts corroborates Leather's results by demonstrating an overall graft patency of 95% below the knee at 1 year and 94% in the infrapopliteal group. It has been postulated that this improved early patency rate of in situ vein grafts is the result of more optimal preservation of the endothelium of the vein graft. To investigate this hypothesis, human saphenous veins were handled by an in situ and a reversed technique. The intact vein segments were then tested for luminal production of prostacyclin and thromboxane A 2 and fixed for scanning electron microscopic analysis of the surface morphology. This study demonstrated that endothelial cell prostacyclin release is enhanced in human in situ vein segments but not in reversed vein segments. In addition, luminal production of thromboxane A 2 is significantly greater in the reversed than in the in situ vein segments. These findings are associated with marked endothelial structural damage in the reversed veins and minimal endothelial disruption in the situ veins. Therefore the ratio of the antiaggregatory vasodilator prostacyclin to the proaggregatory vasoconstrictor thromboxane A 2 is significantly more favorable for the in situ vein segment than for the reversed vein segment. The observed excellent early patency of the situ vein grafts in our poor-risk patient population may in part be the result of this favorable balance of prostacyclin and thromboxane A 2 and the more optimally preserved endothelial morphology. (J VASC SURG 1984;1:149-59.)

The plasma of a 63-year-old patient with an initial acute, fatal episode of thrombotic thrombocytopenic purpura (TTP) contained agglutinated platelets and a factor VIII-related von Willebrand factor (vWF) antigen level that was elevated sevenfold above normal. Unusually large vWF multimers derived from endothelial cells were detected in her plasma at the on-set of the TTP episode. This is the first patient in whom vWF abnormalities indicative of in vivo endothelial cell damage or perturbation have been found during an acute episode of TTP.