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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.)

Remission plasma samples of some patients with chronic relapsing thrombotic thrombocytopenic purpura (TTP) contain unusually large von Willebrand factor (vWF) multimers similar to those produced by normal human endothelial cells in culture. The infusion of the cryosupernatant fraction of normal plasma is as effective as normal fresh-frozen plasma (FFP) in the treatment or prevention of TTP episodes in patients with the chronic relapsing form of TTP. Three patients with chronic relapsing TTP during remission have unusually large vWF multimers present in their plasma. Two of the patients were transfused once with FFP, one of the two received cryosupernatant on three occasions, and the third patient was studied before and immediately after plasma exchange. Unusually large vWF multimers decreased or disappeared from patient plasma samples within 1/2 to 1 1/2 hours following the transfusion of FFP (on two occasions) or cryosupernatant (on two of three occasions), and immediately after plasma exchange (on one occasion). The patient who received cryosupernatant was studied serially after the infusions. Unusually large vWF multimers returned to her plasma within ten to 24 hours and persisted thereafter. Unusually large vWF multimers did not disappear from patient remission plasma samples, or from the culture medium removed from normal human endothelial cells, when these fluids were incubated in vitro with either normal FFP or cryosupernatant. We conclude that an activity in FFP, and its cryosupernatant fraction, promoted the rapid in vivo disappearance of unusually large vWF multimers from the plasma of two patients with chronic relapsing TTP in remission, and plasma exchange reversed the abnormality in a third patient who was in partial remission. Neither FFP nor cryosupernatant directly converted unusually large multimers to smaller vWF forms in vitro in the fluid phase. These results indicate that an activity in the cryosupernatant fraction of normal plasma is involved in vivo in controlling the metabolism of unusually large vWF multimers, and that this process is defective in some chronic relapsing TTP patients.

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.

A platelet-agglutinating factor has been detected in the plasma of some patients during episodes of thrombotic thrombocytopenic purpura (TTP).1 , 2 Agglutination induced in vitro by this plasma fac...

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.

Plasma VIII:von Willebrand factor antigen (VIII:vWF) levels were elevated approximately two- to eightfold in seven patients (three adults and four children) during acute episodes of thrombocytopenia, renal failure, and hemolytic anemia (the hemolytic-uremic syndrome, HUS). In all seven patients, there was an alteration in plasma VIII:vWF patterns during these acute HUS episodes, so that the largest VIII:vWF forms were relatively decreased. Plasma VIII:vWF multimer patterns returned to normal, or nearly to normal, as platelet counts returned to preexisting levels, even in the patients whose recovery of renal function was incomplete and whose plasma VIII:vWF antigen level remained above normal. The sister of one of the HUS patients had a similar clinical prodrome (gastroenteritis) that was not followed by thrombocytopenia or renal failure and was not accompanied by an elevated level or abnormal forms of plasma VIII:vWF. These results suggest that an alteration in VIII:vWF metabolism, distribution, or interaction with platelets is associated with acute HUS episodes. In contrast to patients with chronic relapsing thrombotic thrombocytopenic purpura, none of the HUS patients (either during or after the acute HUS episodes) had a defect in the conversion of unusually large VIII:vWF multimers derived from endothelial cells to the VIII:vWF forms found in normal plasma.

As a 51-year-old woman recovered from an initial acute episode of thrombotic thrombocytopenic purpura (TTP), her plasma was found to contain unusually large von Willebrand factor (vWF) multimers. Clinical, hematological, and vWF studies of her siblings and children were normal. The unusually large vWF forms were presumably derived from endothelial cells, persisted in her plasma after recovery, and were associated with recurrent episodes of TTP during the subsequent 6 months. After the last episode of relapse they disappeared from her plasma following 3 1/2 weeks of therapy with prednisone and did not return during 17 months of treatment with prednisone and/or azathioprine. She is now receiving no drugs, has normal plasma vWF forms, and has not had any more episodes of TTP. We conclude that our patient had an acquired defect in the conversion of unusually large vWF multimers derived from endothelial cells to the somewhat smaller vWF forms usually present in circulation. The defect may have been immune-mediated, because it was eliminated during therapy with immunosuppressive drugs.

Methylisobutylxanthine (MIX) raised cAMP levels and inhibited prostacyclin synthesis and arachidonic acid release in endothelial cells from both pig aorta and human umbilical vein. These effects were reversible and dose dependent on MIX concentrations. Dibutyryl cAMP (3 mM) alone did not inhibit prostacyclin synthesis or arachidonic acid release. When added with MIX, dibutyryl cAMP did not enhance the inhibition elicited by MIX. MIX inhibited the formation of lysophospholipids, 1,2-diacylglycerol and phosphatidic acid in bradykinin-stimulated pig endothelial cells, suggesting that the inhibition of prostacyclin synthesis resulted from an apparent inhibition of both phospholipase A2 and phospholipase C. Other phosphodiesterase inhibitors, theophylline and mopidamole, also raised cAMP levels and inhibited arachidonic acid release. However, there was no correlation between cAMP levels and these inhibitions. Forskolin, an adenylate cyclase activator, elevated intracellular cAMP levels with no apparent inhibition on prostacyclin synthesis. We conclude that the inhibitory effect of MIX on phospholipase A2 and phospholipase C is probably through mechanisms other than the elevation of the cAMP level.