K K Hamilton

Changes in cytosolic Ca2+ associated with von Willebrand factor release in human endothelial cells exposed to histamine. Study of microcarrier cell monolayers using the fluorescent probe indo-1.

A method for measuring fluorescence in anchored monolayers of human endothelial cells is described and used to demonstrate changes in the cytosolic free-calcium concentration ([Ca2+]c) in these cells exposed to histamine and thrombin; some endothelial responses to both agonists (e.g., mitogenesis) have been suggested to be Ca2+-mediated. Umbilical vein endothelial cells were cultured on microcarriers and loaded with the Ca2+ indicator, indo-1. Enzymatic cell detachment was avoided by monitoring the indo-1 fluorescence ratio (400/480 nm) of a stirred suspension of cell-covered microcarriers. Basal [Ca2+]c was estimated to be 70-80 nM. Thrombin induced a transient dose-dependent increase in [Ca2+]c, which was active-site dependent. Histamine stimulated a dose-dependent increase in [Ca2+]c, which was reversed by removal of histamine and inhibited competitively by the H1-receptor antagonist pyrilamine, but not by the H2-receptor antagonist cimetidine. Furthermore, histamine induced a dose-dependent secretion of von Willebrand factor, which paralleled the rise in [Ca2+]c and was similarly blocked by the H1-receptor antagonist, and which may contribute to platelet deposition at sites of inflammation.

Complement C5b-9 Increases Plasminogen Binding and Activation on Human Endothelial Cells

Deposition of the terminal complement proteins (C5b-9) on human endothelial cells can result in cell lysis or nonlytic alterations of cell function including procoagulant responses. Because regulation of fibrinolysis is a central endothelial function and because C9 contains a carboxyl-terminal lysine similar to other proteins that bind and facilitate activation of plasminogen (PG), the effects of complement injury on PG binding and activation on these cells were investigated. Activation of complement through deposition of C5b67 complexes on endothelial cells resulted in a small increase (approximately 20%) in PG binding. Incorporation of C8 into C5b-8 resulted in no further increase in binding; however, specific 125I-PG binding was increased by approximately 100% after C5b-9 deposition. Moreover, PG was found to bind specifically to C7 and C9. The PG bound to endothelial cells after C5b-9 deposition was readily activated by tissue-type plasminogen activator (TPA). In a cell-free system, complement C9 and a synthetic peptide composed of the 20 carboxyl-terminal amino acids of C9 enhanced PG activation by TPA. Removal of the carboxyl-terminal lysine of C9 abolished the enhancement of PG activation without diminishing PG binding. We conclude that membrane C9 may comprise a binding site for PG and serve to enhance activation of this zymogen by TPA. These findings suggest that immune injury to the endothelium may enhance both the fibrin-generating and fibrinolytic capacity of the vessel wall.

The terminal complement proteins C5b-9 augment binding of high density lipoprotein and its apolipoproteins A-I and A-II to human endothelial cells

Terminal complement protein complexes C5b-9 have been found in human atherosclerotic lesions. Insertion of C5b-9 in the endothelial cell membrane alters permeability, induces membrane vesiculation, and activates secretion. We hypothesized that complement might also alter interactions of the endothelial surface with lipoproteins, particularly high density lipoprotein (HDL), which is reported to inhibit C5b-9-induced hemolysis. We now demonstrate that exposure to C5b-9 increases (by 2- to 50-fold) specific binding of HDL and its apolipoproteins (apo) A-I and A-II to endothelial cells. Binding to cells exposed to antibody, C5b67, and C5b-8 was virtually unchanged. Enhanced binding was also dependent on the number of C5b-9 complexes deposited on the cells. Other agonists that activate endothelial secretion did not augment binding. Calcium was required for full exposure of new binding sites by C5b-9. The C5b-9-induced increase in binding was independent of the increase observed after cholesterol loading. In addition, apo A-I and A-II appear to compete for the same binding sites on untreated and C5b-9-treated cells. In contrast to the data reported for red cells, we were unable to detect significant inhibition of C5b-9-mediated endothelial membrane permeabilization by HDL (up to 1 mg/ml) or by apo A-I (up to 100 micrograms/ml). These data demonstrate that the C5b-9 proteins enhance endothelial binding of HDL and its apoproteins, suggesting that intravascular complement activation may alter cholesterol homeostasis in the vessel wall.

Echocardiographic demonstration of coronary sinus diverticula in patients with Wolff-Parkinson-White syndrome

Coronary sinus (CS) diverticula have been reported to be the anatomic basis of some posteroseptal accessory atrioventricular pathways. During 1 year, 53 patients with Wolff-Parkinson-White syndrome referred to our center for catheter ablation were found to have posteroseptal or left posterior accessory pathways. The accessory pathway was associated with an anomaly of the CS in seven patients (13%), including six diverticula of the CS or its branches and one aneurysmal CS. Four of the CS diverticula were visualized by transesophageal echocardiography. Diverticular appear as echolucent, contractile pouches on the epicardial surface of the posteroseptal or posterior left ventricle that connect to the CS by an isthmus. Previous reports have suggested that accessory pathways associated with CS aneurysms have rapid conduction times and may be associated with an increased risk of rapid ventricular response during atrial fibrillation and sudden death; five of our seven patients had a short preexcited R-R interval in the range of 172 to 225 msec during atrial fibrillation. In summary, many, if not the majority, of CS diverticula associated with preexcitation can be visualized by transesophageal echocardiography.