[Efficacy of using rivaroxaban for treatment of heat-induced thrombosis after endovenous laser ablation]. / Effektivnost' primeneniya rivaroksabana dlya lecheniya termoindutsirovannykh trombozov posle endovenoznoi lazernoi koagulyatsii.

AIM: The study was aimed at assessing efficacy of using rivaroxaban for treatment of endothermal heat-induced thrombosis (EHIT) after endovenous laser ablation (EVLA) of saphenous veins. MATERIAL AND METHODS: Our prospective study included a total of 1,326 patients subjected to 1,514 EVLAs. In 1,091 (72.1%) cases the great saphenous vein (GSV) was ablated, in 124 (8.2%) cases the anterior accessory vein (AAV) was treated and in 299 (19.7%) cases the small saphenous vein (SSV) was treated. Heat-induced thrombosis developed in 21 (1.4%) cases: in 19 cases in the basin of the great saphenous vein and in 2 cases in the anterior accessory saphenous vein. No heat-induced thromboses in the basin of the small saphenous vein were observed. In 9 (0.6%) cases there was class 1 EHIT (according to the Kabnick classification), class 2 EHIT was noted in 10 (0.7%) cases and class 3 EHIT was observed in 2 (0.1%) cases. All patients with EHIT were given rivaroxaban: patients with class 1 EHIT received it at a single daily dose of 20 mg, patients with class 2 and 3 EHIT - at a dose of 15 mg twice daily. In one (4.8%) case the drug had to be discontinued on day two due to the development of dyspeptic events. All patients were found to have complete regression of the heat-induced thrombus within 6-25 days. No cases of clinical manifestations of pulmonary artery thromboembolism were observed. A conclusion was drawn that in clinical practice EHIT is an important and insufficiently studied problem. Rivaroxaban may be used as an oral agent for treatment of heat-induced thromboses after EVLA. Further studies are required to examine its efficacy and safety profile.

Ageing is a process where the growth effect of neuronal noradrenaline changes progressively in favour of the flow mediated, neurodegenerative and inflammatory effect of plasma noradrenaline.

The noradrenaline stimulus has two components, one excitor, the other inhibitory. Neuronal noradrenaline is the excitor component and plasma noradrenaline is the inhibitory. The balance of effect between the two, the noradrenergic balance, is the controlled variable of the sympathetic system and determines the effect of noradrenaline. Neuronal noradrenaline stimulates tissues by diffusion from their sympathetic nerve endings, plasma noradrenaline does so by diffusion from their microcirculations. Changes in microcirculatory flow, by altering the flow mediated effect of plasma noradrenaline, are mainly responsible for altering the noradrenergic balance in the peripheral tissues; changes in CSF flow are speculated to be mainly responsible for doing the same in the brain, by altering the balance between synaptic noradrenaline in the brain and nonsynaptic noradrenaline in the subarachnoid CSF. When plasma noradrenaline alters the noradrenergic balance it triggers afferent sympathetic activity that alerts hypothalamic neurons to the event and they restore the balance and tissue homeostasis, within milliseconds, by adjusting the level of efferent sympathetic activity they project back to the affected tissue. Because the restoration is so rapid the effect of plasma noradrenaline is normally unobservable and dismissed as not having occurred. Because the hypothalamus is not involved with the responses of isolated canine lateral saphenous vein segments to noradrenaline, the effects of plasma noradrenaline in that preparation are not countered by reactive efferent activity and, consequently, are readily apparent in it. Quantitatively, they have been found to be a function of microcirculatory flow and noradrenaline concentration and, qualitatively, to be inhibitory, dilator, pro inflammatory and neurodegenerative. In life, due to a progressive increase in plasma noradrenaline concentration and, more so, in microcirculatory flow, the noradrenergic balance moves progressively in favour of the neurodegenerative and inflammatory effects of plasma noradrenaline. These observations are the basis of an hypothesis that ageing is caused by a genetically programmed shift in balance away from the growth and anti-inflammatory effects of neuronal noradrenaline, early in life, towards the neurodegenerative and pro-inflammatory effects of plasma noradrenalin, later in life. Death is believed to occur when plasma noradrenaline has damaged the structure of the sympathetic system so much that it can no longer create the minimum quantity of neurotransmitter needed to maintain the level of noradrenergic balance and homeostasis necessary for life.

[THE RISK OF OCCURRENCE OF THE VARICOSE DISEASE RECURRENCE, DEPENDING ON DIAMETER OF VARICOSE--CHANGED VEINS OF LOWER EXTREMITIES WHILE PERFORMANCE OF THE FOAM SCLEROTHERAPY].

The results of treatment of 139 patients, to whom for the varicose disease of lower extremities there was conducted a foam sclerotherapy of trunks and tributaries of big subcutaneous vein, small subcutaneous vein and/or nonsaphenous veins with signs of a valvular insufficiency under ultrasonographic control were presented. Complex of measures for prophylaxis of the disease recurrences and complications was elaborated.

[EFFICACY OF SURGICAL TREATMENT OF VARICOSE DISEASE, DEPENDING ON ADSORPTION-RHEOLOGIC PROPERTIES OF BLOOD].

Physico-chemical adsorption-rheological properties of venous blood in patients, suffering varicose disease of the lower extremities, and their impact on efficacy of various methods of surgical treatment were studied. Conduction of endovasal laser coagulation in combination with crossectomy have promoted enhancement of operative treatment efficacy in patients in initial terms of observation (in 1 week), in 1 month a complete occlusion of the vein was noted more rarely. Efficacy of a small--power laser ablation with irradiation power of 10 W and less in 4 weeks postoperatively is higher, than of surgical treatment with a laser irradiation power 15 W. In a varicose disease of the lower extremities there were observed the raising of the blood volume toughness, superficial relaxation and superficial stress on background of reduction of the toughness--elasticity module, superficial toughness and superficial elasticity. Crossectomy conduction did not influence the integral dynamics of adsorption--rheological properties of venous blood, but in 1 month after endovasal laser coagulation a normalization of physicchemical parameters of blood was noted. Application of laser irradiation of the 10 W power and less promotes inhibition of the relaxation properties of venous blood; a prognostic meaning owes initial value of the blood volume toughness.

Comparing cold and warm tumescent anesthesia for pain perception during and after the endovenous laser ablation procedure with 1470 nm diode laser.

OBJECTIVE: The aim of this study was to compare the pain perception and side effects during and after endovenous laser ablation with a 1470 nm diode laser using cold or room temperature tumescence anesthesia. METHODS: One hundred and one patients were randomly assigned in two groups. Group A received room temperature (+24℃) and Group B received cold (+4℃) tumescence fluid, which was used for local anesthesia in the track of great saphenous vein. A visual analog score was recorded immediately after the procedure. Patients were asked to register pain scores and the amount of pain medication consumed during the week. RESULTS: There was no significant difference concerning gender, age, Clinical Etiological Anatomical Pathological Classification, body mass index, or diameter of the treated vein. In Group A, the mean linear endovenous energy density was 59.5 J/cm and in Group B, it was 60.4 J/cm. The average visual analog score after the endovenous laser ablation procedure in Group A was 5 and in Group B was 2. Third day after the procedure, the average visual analog score in Group A was 3 and in Group B was 1. Patients in Group B needed significantly less analgesics compared with patients in Group A (p<0.05). The most frequent side effects in both groups were ecchymosis, induration, and minor paraesthesia, all of which were more common in Group A (p < 0.001). CONCLUSIONS: To date, most published endovenous laser ablation series describe the use of room temperature tumescence fluid infiltration of the perivenous stroma for tumescent analgesia and protection against thermal injury to the nearby structures. We describe an alternative technique using cold tumescence fluid infiltration, which is equally effective as, but safer than, room temperature tumescence fluid infiltration, and which yields better visual analog scores.

Lower-limb veins are thicker and vascular reactivity is decreased in a rat PCOS model: concomitant vitamin D3 treatment partially prevents these changes.

Polycystic ovary syndrome (PCOS) causes vascular damage to arteries; however, there are no data for its effect on veins. Our aim was to clarify the effects of dihydrotestosterone (DHT)-induced PCOS both on venous biomechanics and on pharmacological reactivity in a rat model and to test the possible modulatory role of vitamin D3 (vitD). PCOS was induced in female Wistar rats by DHT treatment (83 µg/day, subcutaneous pellet). After 10 wk, the venous biomechanics, norepinephrine (NE)-induced contractility, and acetylcholine-induced relaxation were tested in saphenous veins from control animals and from animals treated with DHT or DHT with vitD using pressure angiography. Additionally, the expression levels of endothelial nitric oxide synthase (eNOS) and cyclooxygenase (COX-2) were measured using immunohistochemistry. Increased diameter, wall thickness, and distensibility as well as decreased vasoconstriction were detected after the DHT treatment. Concomitant vitD treatment lowered the mechanical load on the veins, reduced distensibility, and resulted in vessels that were more relaxed. Although there was no difference in the endothelial dilation tested using acetylcholine (ACh), the blocking effect of N(G)-nitro-l-arginine methyl ester (l-NAME) was lower and was accompanied by lower COX-2 expression in the endothelium after the DHT treatment. Supplementation with vitD prevented these alterations. eNOS expression did not differ among the three groups. We conclude that the hyperandrogenic state resulted in thicker vein walls. These veins showed early remodeling and altered vasorelaxant mechanisms similar to those of varicose veins. Alterations caused by the chronic DHT treatment were prevented partially by concomitant vitD administration.

Secondary revascularization after CABG surgery.

CABG surgery is an effective way to improve symptoms and prognosis in patients with advanced coronary atherosclerotic disease. Despite multiple improvements in surgical technique and patient treatment, graft failure after CABG surgery occurs in a time-dependent fashion, particularly in the second decade after the intervention, in a substantial number of patients because of atherosclerotic progression and saphenous-vein graft (SVG) disease. Until 2010, repeat revascularization by either percutaneous coronary intervention (PCI) or surgical techniques was performed in these high-risk patients in the absence of specific recommendations in clinical practice guidelines, and within a culture of inadequate communication between cardiac surgeons and interventional cardiologists. Indeed, some of the specific technologies developed to reduce procedural risk, such as embolic protection devices for SVG interventions, are largely underused. Additionally, the implementation of secondary prevention, which reduces the need for reintervention in these patients, is still suboptimal. In this Review, graft failure after CABG surgery is examined as a clinical problem from the perspective of holistic patient management. Issues such as the substrate and epidemiology of graft failure, the choice of revascularization modality, the specific problems inherent in repeat CABG surgery and PCI, and the importance of secondary prevention are discussed.

Endovenous laser ablation: a review of mechanisms of action.

BACKGROUND: The aim of this article is to summarize and review the proposed theories on the laser action during endovenous ablation. METHODS: Laser mechanics and laser-tissue interaction are summarized from articles found in literature. Several theories, like the "steam bubble theory," the "direct contact theory," the "heat pipe," and "direct light energy absorption" are discussed. RESULTS: The laser light emitted intraluminally can be absorbed, scattered, or reflected. Reflection is negligible in the near-infrared spectrum. By combining absorption and scattering, the optical extinction of different wavelengths related to different biological tissues can be determined. The direct contact of the fiber tip and the vein wall may be a way of destroying the vein wall, but results in ulcerations and perforations of the vein wall. Avoiding this contact, and allowing direct light absorption into the vein wall, results in a more homogenous vein wall destruction. If the energy is mainly absorbed by the intraluminal blood, the laser fiber will act as a heat pipe. Histological studies show that a more circumferential vein wall destruction can be obtained when the vein is emptied of its intraluminal blood. The use of tumescent liquid reinforces spasm of the vein and protects the perivenous tissue. CONCLUSION: Several factors play an important role in the mechanism of endovenous laser ablation. Direct energy absorption by the vein wall is the most efficient mechanism. It is important to empty the vein of its intraluminal blood and to inject tumescent liquid around the vein.

Protandim attenuates intimal hyperplasia in human saphenous veins cultured ex vivo via a catalase-dependent pathway.

Human saphenous veins (HSVs) are widely used for bypass grafts despite their relatively low long-term patency. To evaluate the role of reactive oxygen species (ROS) signaling in intima hyperplasia (IH), an early stage pathology of vein-graft disease, and to explore the potential therapeutic effects of up-regulating endogenous antioxidant enzymes, we studied segments of HSV cultured ex vivo in an established ex vivo model of HSV IH. Results showed that HSV cultured ex vivo exhibit an ~3-fold increase in proliferation and ~3.6-fold increase in intimal area relative to freshly isolated HSV. Treatment of HSV during culture with Protandim, a nutritional supplement known to activate Nrf2 and increase the expression of antioxidant enzymes in several in vitro and in vivo models, blocks IH and reduces cellular proliferation to that of freshly isolated HSV. Protandim treatment increased the activity of SOD, HO-1, and catalase 3-, 7-, and 12-fold, respectively, and decreased the levels of superoxide (O(2)(•-)) and the lipid peroxidation product 4-HNE. Blocking catalase activity by cotreating with 3-amino-1,2,4-triazole abrogated the protective effect of Protandim on IH and proliferation. In conclusion, these results suggest that ROS-sensitive signaling mediates the observed IH in cultured HSV and that up-regulation of endogenous antioxidant enzymes can have a protective effect.

Arterial pO2 stimulates intimal hyperplasia and serum stimulates inward eutrophic remodeling in porcine saphenous veins cultured ex vivo.

Ex vivo culture of arteries and veins is an established tool for investigating mechanically induced remodeling. Porcine saphenous veins (PSV) cultured ex vivo with a venous mechanical environment, serum-supplemented cell-culture medium and standard cell-culture conditions (5% CO2 and 95% balance air ~140 mmHg pO2) develop intimal hyperplasia (IH), increased cellular proliferation, decreased compliance and exhibit inward eutrophic remodeling thereby suggesting that nonmechanical factors stimulate some changes observed ex vivo. Herein we explore the contribution of exposure to greater than venous pO2 and serum to these changes in cultured veins. Removing serum from culture medium did not inhibit development of IH, but did reduce cellular proliferation and inward eutrophic remodeling. In contrast, veins perfused using reduced pO2 (75 mmHg) showed reduced IH. Among the statically cultured vessels, veins cultured at arterial pO2 (95 mmHg) and above showed IH as well as increase in proliferation and vessel weight compared to fresh veins; veins cultured at venous pO2 did not. Taken together, these data suggest that exposure of SV to arterial pO2 stimulates IH and cellular proliferation independent of changes in the mechanical environment, which might provide insight into the etiology of IH in SV used as arterial grafts.

Histopathological findings in varicose veins following bipolar radiofrequency-induced thermotherapy--results of an ex vivo experiment.

OBJECTIVE: To describe the histopathological changes of the vein wall caused by bipolar radiofrequency-induced thermotherapy (RFITT), and to examine influence of power and application time on the histopathological changes. MATERIAL AND METHODS: Twenty vein specimens atraumatically extracted near the saphenofemoral junction were treated by RFITT ex vivo. RFITT was applied with fixed (2 seconds) and varied (up to an impedance of 400 Ω) application time and increasing power (5, 10, 15, 20, 25 W). Specimens were processed histologically. RESULTS: RFITT induced coagulation of collagen bundles, shrinking of muscles, splitting and necrosis of the vein wall. Circumferential necrosis of intima and media was reached by a power of 20-25 W and an application time up to an impedance of 400 Ω. Only heterogeneous necrosis of intima and media in parts of the vessel was reached by lower power with long application time. CONCLUSION: A homogeneous necrosis of the circumferential vein wall needs high power and sufficient application time.

c-Jun regulates shear- and injury-inducible Egr-1 expression, vein graft stenosis after autologous end-to-side transplantation in rabbits, and intimal hyperplasia in human saphenous veins.

Coronary artery bypass graft failure represents an unsolved problem in interventional cardiology and heart surgery. Late occlusion of autologous saphenous vein bypass grafts is a consequence of neointima formation underpinned by smooth muscle cell (SMC) migration and proliferation. Poor long term patency and the lack of pharmacologic agents that prevent graft failure necessitate effective alternative therapies. Our objective here was to evaluate the effect of targeted inhibition of the bZIP transcription factor c-Jun on intimal hyperplasia in human saphenous veins and vein graft stenosis after autologous end-to-side transplantation. DNAzymes targeting c-Jun attenuated intimal hyperplasia in human saphenous vein explants. Adenovirus-forced c-Jun expression stimulated SMC proliferation, proliferating cell nuclear antigen, and MMP-2 expression. c-Jun DNAzymes abrogated Adeno-c-Jun-inducible SMC growth and wound repair and reduced intimal thickening in jugular veins of New Zealand white rabbits 4 weeks after autologous end-to-side transplantation to carotid arteries. Conversely, in a DNAzyme-free setting, Adeno-c-Jun potentiated neointima formation in the veins compared with Adeno-LacZ. Inducible c-Jun expression is ERK1/2- and JNK-dependent but p38-independent. Injury- and shear-inducible c-Jun controls early growth response-1. These data demonstrate that strategies targeting c-Jun may be useful for the prevention of vein graft stenosis. Control of one important shear-responsive transcription factor by another indicates the existence of transcriptional amplification mechanisms that magnify the vascular response to cell injury or stress through inducible transcriptional networks.

Leoligin, the major lignan from Edelweiss, inhibits intimal hyperplasia of venous bypass grafts.

AIMS: Despite the lower patency of venous compared with arterial coronary artery bypass grafts, approximately 50% of grafts used are saphenous vein conduits because of their easier accessibility. In a search for ways to increase venous graft patency, we applied the results of a previous pharmacological study screening for non-toxic compounds that inhibit intimal hyperplasia of saphenous vein conduits in organ cultures. Here we analyse the effects and mechanism of action of leoligin [(2S,3R,4R)-4-(3,4-dimethoxybenzyl)-2-(3,4-dimethoxyphenyl)tetrahydrofuran-3-yl]methyl (2Z)-2-methylbut-2-enoat, the major lignan from Edelweiss (Leontopodium alpinum Cass.). METHODS AND RESULTS: We found that leoligin potently inhibits vascular smooth muscle cell (SMC) proliferation by inducing cell cycle arrest in the G1-phase. Leoligin induced cell death neither in SMCs nor, more importantly, in endothelial cells. In a human saphenous vein organ culture model for graft disease, leoligin potently inhibited intimal hyperplasia, and even reversed graft disease in pre-damaged vessels. Furthermore, in an in vivo mouse model for venous bypass graft disease, leoligin potently inhibited intimal hyperplasia. CONCLUSION: Our data suggest that leoligin might represent a novel non-toxic, non-thrombogenic, endothelial integrity preserving candidate drug for the treatment of vein graft disease.

Effects of dietary L-arginine on structure and function of flow-restricted vein grafts.

PURPOSE: Experiments were designed to determine effects of dietary supplementation with L -arginine on structure and function of flow-restricted vein grafts. METHODS: Saphenous veins were placed as bilateral interposition grafts in femoral arteries of two groups of adult male mongrel dogs; one group was maintained on a normal diet (control), the other group supplemented with L -arginine (200 mg/kg per day) beginning 1 week before surgery. In each dog, flow was reduced by 50% in one graft by placing an adjustable clamp on the artery distal to the distal anastomosis. Plasma amino acids and oxidized products of nitric oxide (NO(x )) were measured before and after L -arginine feeding. At postoperative week 4, grafts were removed and prepared for organ chamber studies to determine functions of the endothelium or smooth muscle and for histology. RESULTS: Plasma L -arginine increased within 3 hours after feeding and increased from 141 +/- 8 nmol/mL to 169 +/- 11 nmol/mL (n = 6) after 5 weeks of supplementation. Plasma ornithine and citrulline paralleled arginine, whereas circulating NO(x ) was unchanged. Maximal contractions to 60 mmol/L KCl were reduced in grafts from L -arginine-fed dogs. Endothelium-dependent relaxations to the calcium ionophore A23187 and relaxations of the smooth muscle NO were reduced in grafts from L -arginine-fed dogs. Neointimal hyperplasia was increased in grafts with reduced flow and not affected by arginine feeding. CONCLUSIONS: Dietary supplementation with L -arginine did not increase plasma NO in dogs with peripheral vein grafts or increase endothelium-dependent relaxations in control or flow-restricted grafts. Therefore, dietary supplementation with L -arginine may not improve long-term functions of flow-restricted peripheral bypass grafts.

Contribución del neuropéptido Y a la fisiología de la co-transmisión simpática humana: estudios en biopsias de vena safena / Neuropeptide Y contribution to the physiology of human sympathetic cotransmission: studies in saphenous vein biopsies

Background: It is known that the sympathetic varicosities co-store and co-release norepinephrine (NE) together with adenosine S-triphosphate (ATP) and neuropeptide Y (NPY). Aim: To describe the chemical characterisation of stored and released NPY from the varicosities of sympathetic nerve terminals surrounding segments of the human saphenous vein, and the vasomotor activity of rings electrically depolarized or contracted by the exogenous application of the co-transmitters. Material and methods: Saphenous vein tissues were obtained from patients undergoing elective cardiac revascularization surgery. Results: The chromatographic profile of NPY extracted from biopsies is identical to a chemical standard of human NPY. Upon electrical depolarisation of the perivascular sympathetic nerve terminals, we demonstrated the release of NPY to the superfusion media, which did not exceed a 1percent of its stored content. The release of the peptide is sensitive to guanethidine, and to extracellular calcium, suggesting that the mechanism of its release is exocytotic in nature. The electrically evoked release of NPY is dependent on the frequency and duration of the electrical pulses. Phenoxybenzamine reduces the electrically evoked release of NPY. Exogenous application of NE and ATP contract saphenous vein rings; the simultaneous application of NE plus ATP causes a synergic response, effect which is further potentiated by the joint co-application of 10 nM NPY. Conclusions: Present results highlight the role of NPY as a sympathetic co-transmitter in the regulation of human vascular tone

Diagnosis and treatment in the management of chronic venous insufficiency.

Chronic venous insufficiency (CVI) is caused mainly by an alteration in the elasticity of venous walls and the dysfunction of venous valves. The diagnosis and treatment for CVI management are discussed in this paper.

Influence of angioscopic vein graft preparation on development of neointimal hyperplasia in an organ culture model of human saphenous vein.

PURPOSE: Angioscopy for in situ vein graft preparation has been criticized on the basis that the trauma of instrumentation may predispose to accelerated intimal hyperplasia, jeopardizing patency rates following infrainguinal revascularization. The aim of this study was to assess the effects of angioscopic preparation on endothelial integrity and smooth muscle cell (SMC) behavior in an established organ culture model of human saphenous vein (HSV). METHODS: HSV was harvested from 12 patients during bypass surgery before and after angioscopic preparation. Endothelial integrity was evaluated by immunohistochemical staining with JC-70 and scanning electron microscopy (SEM); remaining segments of pre- and postangioscopy vein were maintained in culture for 14 days in medium supplemented with 30% fetal calf serum. Viability was confirmed by measurement of tissue adenosine triphosphate on day 14 and thickness of the neointima was measured by computerized image analysis of histologic sections. Monoclonal antibodies to proliferating cell nuclear antigen (PCNA) were used as an immunohistochemical marker for proliferating SMCs. RESULTS: There was a significant reduction in the percentage staining by JC-70 (71.3% versus 20.4%) in pre- versus postangioscopy vein (p = 0.002 by Wilcoxon's rank test; n = 12). This was supported by SEM images. Despite this, there were no significant differences between the pre- and postangioscopy HSVs after 14 days of culture with respect to neointimal thickness (61 versus 56 microns) and staining with PCNA (4.80 versus 4.08 nuclei per 10 microns), all according to Wilcoxon's rank test. CONCLUSIONS: Angioscopic vein graft preparation is associated with endothelial cell loss but does not induce additional neointimal hyperplasia in HSV in vitro. These results suggest that angioscopic manipulation does not alter SMC behavior.

[Suicide by severing blood vessels using local anesthesia: reduced risk in extraction of HIV infected tissue using a stomacher]. / Suicid durch Eröffnen von Gefässen unter Lokalanästhesie: risikoarme Extraktion von HIV-infiziertem Gewebe mittels Stomacher.

Authors report an unusual suicide undertaken by a medical person who after applying the local anesthetic lidocaine, opened a leg vein with a scalpel. The cause of the suicide was presumed advanced AIDS symptoms with a progressive Kaposi sarcoma and Non-Hodgkin lymphoma. For the toxicological investigation of the tissue we used the stomacher extraction system, a method not widely known in Forensic science, however, recommended for the extraction of all infectious tissues.

Prelining of polytetrafluoroethylene grafts with cultured human endothelial cells isolated from varicose veins.

Prelining graft material with autologous functioning endothelial cells might be one of the ultimate requirements to obtain a biocompatible surface. Accordingly, endothelial cells from stripped varicose veins were enzymatically harvested and grown on a fibronectin matrix. Proliferation was investigated in defined medium supplemented with various concentrations of endothelial cell growth supplement (ECGS) (25, up to 150 micrograms/ml) and heparin (10(-8), up to 10(-5)mol/L): optimal growth required both 150 micrograms/ml of ECGS and 10(-5)mol/L heparin. Under these conditions, cell culture achieved cell densities at a confluence of 1.2 +/- 1.1 10(5) cells/cm2 with a doubling time of 1 day. During subcultivation cultured cells consistently exhibited characteristic cobblestone morphology and immunofluorescent staining for factor VIII-related antigen, whereas prostacyclin production determined by enzyme-linked immunosorbent assay for 6-keto-prostaglandin F1 alpha reached 21.1 +/- 1.2 ng/10(6) cells after 15-minute stimulation with 1 U/ml of thrombin. Heparin-containing culture medium-endothelial cell interactions were particularly studied, and with iodine 125-heparin, binding was demonstrated with an apparent dissociation constant (Kd) of 0.36 +/- 0.04 mumol/L. A cold storage technique at -80 degrees C was sought, and freezed cells were used to coat in vitro polytetrafluoroethylene grafts. Protein-treated material allowed cell attachment and growth to a confluent monolayer as assayed by light and scanning electron microscopy. These data validate the feasibility of prelining grafts in vitro with autologous functioning endothelial cells. This approach may be useful in improving the performance of small-caliber vascular grafts according to prostacyclin production and surface-bound heparin of these cells.