Alain Beretz

Role of cyclic AMP in promoting the thromboresistance of human endothelial cells by enhancing thrombomodulin and decreasing tissue factor activities

1 The effects of forskolin, prostaglandin E1 (PGE1), dibutyryl cyclic AMP (db cyclic AMP), dibutyryl cyclic GMP (db cyclic GMP) and 3‐isobutyl‐1‐methyl‐xanthine (IBMX) were investigated on the expression of tissue factor and thrombomodulin activities on the surface of human saphenous vein endothelial cells (HSVEC) in culture. 2 Forskolin (10−6 to 10−4 m), PGE1 (10−7 to 10−5 m) and db cyclic AMP (10−4 to 10−3 m) caused a concentration‐dependent decrease of cytokine‐induced tissue factor activity. 3 Similar concentrations of forskolin, PGE1 and db cyclic AMP enhanced significantly constitutive thrombomodulin activity and reversed the decrease of this activity caused by interleukin‐1 (IL‐1). 4 IBMX (10−4 m) decreased tissue factor activity and enhanced the effect of forskolin on tissue factor and thrombomodulin activities. 5 Forskolin (10−4 m) decreased the IL‐1‐induced tissue factor mRNA and increased the thrombomodulin mRNA level. IL‐1 did not change the thrombomodulin mRNA level after 2 h of incubation with HSVEC in culture. 6 Dibutyryl cyclic GMP (10−4 m to 10−3 m) did not influence tissue factor or thrombomodulin activity. 7 Our data suggest that elevation of intracellular cyclic AMP levels may participate in the regulation of tissue factor and thrombomodulin expression, thus contributing to promote or restore antithrombotic properties of the endothelium.

Cyclic nucleotide phosphodiesterase inhibitors prevent aggregation of human platelets by raising cyclic amp and reducing cytoplasmic free calcium mobilization

Cyclic nucleotide phosphodiesterase inhibitors (HL-725, RO 15-2041, cilostamide, quercetin and MY-5445) potently inhibit human platelet aggregation induced by ADP. In parallel, PDE inhibitors inhibit the increase in cytoplasmic free Ca2+ evoked by ADP, as measured with the fluorescent probe quin 2. The inhibition of ADP-induced aggregation and rise in [Ca2+]i is potentiated by PGE1 which stimulates adenylate cyclase and is inhibited by adrenaline which inhibits adenylate cyclase. PDE inhibitors increase human platelet cAMP levels in the presence of low concentrations of PGE1. It is suggested that PDE inhibitors prevent platelet aggregation by raising cAMP levels and by subsequent inhibition of cytoplasmic free Ca2+ mobilization.

Endothelial cell seeding: coating Dacron and expanded polytetrafluoroethylene vascular grafts with a biological glue allows adhesion and growth of human saphenous vein endothelial cells.

The establishment of an endothelial lining on vascular grafts to obtain a highly thromboresistant surface in a clinical situation requires optimization of cell collection, quality, adhesion and growth. We have studied the conditions for collection, seeding and growth of human saphenous vein endothelial cells (HSVEC), on Dacron or Gore-Tex expanded polytetrafluoroethylene (PTFE) vascular grafts. Carefully handled veins, as opposed to veins obtained using the usual procedures for coronary bypass graft preparation, yielded a higher rate of successful culture (94% vs 43%) and reached confluence in primary culture sooner (9.4 ± 3 days vs 13.4 ± 4.5 days). HSVEC were seeded at a density of 6x103 cells/cm2 on graft fragments coated with fibronectin (FN) or Transglutine (TGL), a biological glue. There was no HSVEC adhesion on Dacron or PTFE without protein pretreatment of the artificial surface. FN improved HSVEC adhesion but there was no cell growth. Adhesion, doubling time and cell density at confluence on PTFE pretreated with TGL were similar to those on conventional tissue culture polystyrene (TCP) pretreated with TGL or FN. HSVEC adhesion on Dacron pretreated with TGL was lower than on TCP pretreated with TGL; the doubling time was similar but the density at confluence was 40% lower. We conclude that pretreatment of vascular grafts with TGL, besides being an alternative to preclotting of the Dacron graft, allows adhesion and growth to confluence of HSVEC on these surfaces.

Sulfated polysaccharides modulate effects of acidic and basic fibroblast growth factors on repair of injured confluent human vascular endothelium.

Semi-automatic analysis of the repair process of a circular mechanical lesion of confluent human vascular endothelial cells In vitro was used to evaluate the contributions of cell migration and cell proliferation. Standard heparin added to culture medium that contained 30% human serum Induced an Inhibition of cell migration at the lesion margin during the first day after Injury. Several sulfated potysaccharkJes were tested In the presence of 5% human serum. Standard heparin, low molecular weight heparin, or pentosan polysulfate markedly reduced the rate of lesion regeneration. Cell proliferation, measured by3H-thymldlne Incorporation at the lesion margin, and cell migration were both decreased. In contrast, the combination of acidic fibroblast growth factor with a sulfated polysaccharide accelerated the repair process. Basic fibroblast growth factor combined with a sulfated polysaccharide gave a regeneration rate similar to that of the control; however, at 4 days after Injury, the residual lesion was the same when basic fibroblast growth factor was used alone or when It was combined with sulfated polysaccharides. Acidic fibroblast growth factor totally reversed the effects of sulfated polysaccharides on the repair process by enhancing endothelial cell proliferation and allowing endothelial cell migration.

Behavior of confluent endothelial cells after irradiation. Modulation of wound repair by heparin and acidic fibroblast growth factor

Summary— Image analysis was used to study the repair process of a circular mechanical lesion of confluent human endothelial cells in culture after irradiation (10 Gy) prior to wounding. Coverage of denuded areas 48 and 96 h after injury of endothelial cells was identical in control and irradiated cultures, although the labeling index was lowered by 80 to 95% in irradiated cultures. The cell density of non damaged irradiated areas was decreased by 50%. When cultures were submitted to increasing doses of radiation (5.0–30 Gy), the labeling index of the cells diminished rapidly between 0 and 5.0 Gy and reached a plateau at 10 Gy. The decrease in cell density (50% of control at 96 h) was identical at each dose of radiation. Thus cell migration alone could be sufficient for the repair of the lesion, while cell proliferation would mainly maintain the original cell density. The addition of heparin to the culture medium slowed down cell migration and proliferation, but the speed of repair was identical in irradiated and non‐irradiated cultures. Acidic fibroblast growth factor plus heparin accelerated equally the repair process whether the cultures were irradiated or not. In irradiated cultures the presence of acidic fibroblast growth factor and heparin maintained cell density in confluent areas at a level similar to that in non‐irradiated damaged control cultures without addition of mitogens. Thus heparin and acidic fibroblast growth factor play a role in cell proliferation, in the maintenance of the cell monolayer integrity and in restoring a continuous layer by rapid cell migration and elongation after irradiation.

Opposing effects of heparin with TGF‐β or aFGF during repair of a mechanical wound of human endothelium. Influence of cAMP on cell migration

The effects on vascular wound repair in vitro of aFGF and TGF‐β, growth factors having opposite influences on endothelial cell growth and angiogenesis, were studied using as a model a mechanical lesion of confluent endothelium. Modulation by heparin of the activities of these growth factors during the repair process was also examined. Whereas heparin alone inhibited repair by lowering both cell proliferation and cell migration, TGF‐β alone mainly inhibited cell proliferation. When added together, TGF‐β and heparin exerted a combined inhibitory effect resulting in a residual lesion 50% larger than in controls. aFGF alone accelerated lesion coverage and this effect was enhanced by 40% over control values when heparin was added with aFGF. This acceleration was slightly (less than 10%) but consistently diminished by TGF‐β. Cell density in confluent unwounded areas was increased by 40% in the presence of aFGF, but TGF‐β diminished cell density by 20%. A small (30%) increase in intracellular cAMP was measured whenever aFGF was present during the repair process. In comparison, intracellular cAMP inducing agents (forskolin, dbcAMP) accelerated cell migration by 20% during lesion recovery without affecting cell proliferation or density. The present results show that the inhibitory effects of TGF‐β during vascular wound repair are opposed by aFGF. Furthermore, heparin (or heparan sulfates in vivo) modulates growth factors having activating or inhibiting functions and thus plays a regulatory role during the repair process. cAMP‐inducing substances other than growth factors are able to accelerate cell migration.

Mitogenic activity on human arterial smooth muscle cells is increased in the plasma of patients undergoing hemodialysis with cuprophane membranes.

During hemodialysis on cuprophane membranes, platelets are activated and release in plasma alpha-granule-specific substances such as PF4 or platelet-derived growth factor (PDGF). PDGF is the main source of mitogenic activity found in serum. In vitro, it induces the proliferation of smooth muscle cells (SMC) which is known to be involved in the development of atherosclerotic lesions. Atherosclerosis is one of the major complications of uremic patients undergoing chronic hemodialysis. To investigate whether this complication could be due to the dialysis itself, we measured the mitogenic activity in plasma of 10 patients undergoing hemodialysis on cuprophane membrane, using human arterial SMC in culture. Mitogenic activity in plasma increased about 3-fold during dialysis. These results may provide an argument in favor of a contribution of platelet activation and release of mitogenic activity to atherosclerosis in patients dialysed with cuprophane membranes.

Artifact of blood pressure recording using heparin-filled catheter: Effects on blood pressure and coagulation parameters

A usual complication of catheterization procedures of arteries for blood pressure recording is the clogging of catheters due to activation of coagulation. This is usually avoided by filling the catheters with a heparinized saline solution. We have studied rats implanted with four catheters, one of which was used to monitor blood pressure for 6 h. Catheters were filled with 100 IU/ml heparin in saline. Using this standard protocol, approximately 50-200 IU of heparin were injected into the animals. This induces significant anticoagulation. The activated partial thromboplastin time (APTT) increased from 27 s to more than 240 s. We devised a modified low heparin protocol, in which the concentration of heparin in the wash solution of the catheters was lowered from 100 to 0.1 IU/ml, and the pressor transducer was back-perfused with saline solution without heparin. Using this new protocol, no significant modification of the APTT was observed, indicating that only trace amounts of heparin were injected. Subsequent controlled administration of heparin induced a significant decrease in blood pressure. To avoid all effects associated with this unwanted infusion of heparin, mainly a decrease in blood pressure and a major anticoagulant effect, we suggest the use of such low heparin catheterization protocol.