1 The role of the endothelium in the vasomotor control of human veins in the lower extremity is little understood. We tested the hypothesis that the production of relaxing and contracting factors is altered in endothelial cells from varicose saphenous veins which may predispose to the decreased vessel tone observed in primary varicosis. 2 We determined the intracellular accumulation of guanosine 3':5'-cyclic monophosphate cyclic GMP; a measure of nitric oxide production) and the release of endothelin and prostacyclin (measured as its stable metabolite 6-keto-prostaglandin F 1a ) from cultured cells derived from the long saphenous veins of patients with primary varicosis (Varicose saphena group, n=27) or from patients undergoing coronary artery bypass surgery (Healthy saphena group, n=22). In addition, levels of endothelin, angiotensin II, bradykinin, cyclic GMP and cyclic AMP in plasma from patients with primary varicosis and healthy volunteers (n=8 ± 11 in each group) were determined. 3 Although basal cyclic GMP levels were similar, more cyclic GMP accumulated in response to histamine (1 ± 100 mmol l 71 ) in cells from varicose saphenous veins (0.75+0.1 pmol per well) than in cells from veins without varicosis (0.27+0.05 pmol per well). Furthermore, the relaxant potency of nitroprusside (1 nmol l 71 ± 300 mmol l 71 ) in vitro was higher for varicose veins (mean EC 50 =5.9 m mol l 71 ; n=8) than healthy veins (mean EC 50 =20.0 mmol l 71 ; n=7). 4 The production of prostacyclin was signi®cantly less in cells from varicose than healthy saphenous veins (66+8.7 and 121+20.1 nmol g 71 protein), but the production of endothelin was similar in both groups. Prostacyclin (3 nmol l 71 ± 30 mmol l 71 ) consistently contracted rings of varicose saphenous vein in vitro with a mean EC 50 value of 10 ± 20 mmol l 71 (n=7); the maximum tension generated was *50% of that of a completely depolarizing solution of K + (120 mmol l 71 ). 5 In plasma from patients with varicose veins, levels of cyclic GMP were higher than in healthy controls (9.2+0.03 and 7.2+0.02 nmol l 71 ), levels of angiotensin II were lower (81+11.5 and 147+21.7 pmol l 71 ), and levels of endothelin, cyclic AMP, and bradykinin were not di erent. 6 It is concluded that endothelial cells from diseased saphenous veins secrete less constrictor mediators than cells from healthy veins and that in diseased veins the nitric oxide/cyclic GMP system is upregulated which may shift the balance of vasoactive factors towards vasodilatation and contribute to the development of primary varicosis.

A rapid and reliable harvest and culture technique was developed to provide a sufficient number of autologous endothelial cells for the confluent in vitro lining of cardiovascular prostheses. Enzymatic endothelial cell detachment was achieved by the in situ application of collagenase to short vessel segments. This harvest technique resulted in a complete lack of contaminating smooth muscle cells in all of 124 cultures from nonhuman primates and 13 cultures from human adults. The use of a microgrid technique enabled the daily in situ quantification of available endothelial cells. To assess ideal plating densities after passage the population doubling time was continuously related to the cell density. Surprisingly, a low plating density of 1.5 X 10(3) endothelial cells/cm2 achieved 43% shorter cell cycles than the usual plating density of 1.0 X 10(4) endothelial cells/cm2. Moreover, low density plating enabled mass cultures after one single cell passage, thereby reducing the cell damaging effect of trypsin. When the growth characteristics of endothelial cells from five anatomically different vessel sites were compared, the external jugular vein--which would be easily accessible and dispensable in each patient--proved to be an excellent source for endothelial cell cultures. By applying in situ administration of collagenase, low density plating and microgrid follow-up to adult human saphenous vein endothelial cells, 14,000,000 first passage endothelial cells--sufficient for the in vitro lining of long vascular prostheses--were obtained 26.2 days after harvest. (95% confidence interval:22.3 to 32.2 days).

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