Expression of functional prostaglandin D (DP) receptors in human corpus cavernosum smooth muscle.

Prostaglandin D(2) (PGD(2)) binds to specific G-protein coupled receptors (DP) and induces smooth muscle relaxation by stimulating the synthesis of intracellular cAMP. In this study, we examined the role of PGD(2) and DP receptors in regulating human penile smooth muscle contractility. We determined that human corpus cavernosum tissue and smooth muscle cells in culture expressed functional DP receptor and lipocalin-like prostaglandin D synthase by reverse-transcribed polymerase chain reaction (RT-PCR). Functional PGD synthase activity was confirmed by the synthesis of PGD(2) in human corpus cavernosum smooth muscle cells upon addition of exogenous arachidonic acid. Organ bath preparations of human corpus cavernosum tissue strips, contracted with phenylephrine, relaxed in a dose-dependent fashion to either PGD(2) or the DP selective agonist BW245C. Cultures of human corpus cavernosum smooth muscle cells treated with BW245C showed a two-fold increase in cAMP synthesis. These data are consistent with the expression of functional DP receptors in human corpus cavernosum. This suggests the presence of an intact prostanoid autocrine system that may play a role in regulating penile erectile function.

O2-dependent prostanoid synthesis activates functional PGE receptors on corpus cavernosum smooth muscle.

We have previously demonstrated that decreased O2 tension inhibits prostaglandin synthesis from human corpus cavernosum smooth muscle cells in static culture over 8-18 h (R. B. Moreland et al., Molecular Urology 2: 41-47, 1998). In this report, an experimental system was designed that allowed determination of the effects of O2 tension changes over the time frame of physiological penile erection. Human corpus cavernosum smooth muscle cells were cultured on microcarrier beads in enclosed stirrer flasks so that rapid changes of O2 tension could be modulated. After 18 h of equilibration at 30-40 mmHg to simulate blood PO2 at penile flaccidity, O2 tension was increased to 100 mmHg for 1 h and then returned to 30-40 mmHg. Media samples were withdrawn for prostanoid synthesis and cell samples were taken for cAMP determinations. After 18 h of 30-40 mmHg PO2 values, prostanoid synthesis by human corpus cavernosum smooth muscle cells was low (0.1-0.7 pmol/10(6) cells). When PO2 was increased to 100 mmHg, a rapid increase in PGE2 >> PGF2alpha > PGD2 was observed (thromboxane A2 was undetectable), which peaked at 5.7 pmol PGE2/10(6) cells. Increased O2 tension correlated with increased PGE2 and increased intracellular synthesis of cAMP. The prostaglandin G/H synthase inhibitor indomethacin or the E prostanoid (EP2)-selective antagonist AH-6809 each inhibited the O2-tension-dependent increases in cAMP. These data support a role of differential O2 tension in the penis in the smooth muscle synthesis of PGE2, which in turn increases cAMP synthesis via EP2 receptors.

Synthesis of 8-epi-prostaglandin F2alpha by human endothelial cells: role of prostaglandin H2 synthase.

The experiments described in this paper were designed to determine the mechanism underlying the increase in 8-isoprostaglandin F(2alpha) (8-epi-PGF(2alpha)) production by cultured human endothelial cells during reoxygenation following hypoxia. Human umbilical artery endothelial cells were grown on microcarrier beads and exposed to sequential periods of normoxia, hypoxia, and reoxygenation. The amount of 8-epi-PGF(2alpha) in the medium was determined by ELISA. The production of 8-epi-PGF(2alpha) decreased by greater than 90% during hypoxia. Upon reoxygenation 8-epi-PGF(2alpha) production increased linearly for 90 min reaching nearly 3 times normoxic levels. When added to the medium during reoxygenation, neither superoxide dismutase nor Tiron, a cell-permeable superoxide scavenger, inhibited 8-epi-PGF(2alpha) production. However, 8-epi-PGF(2alpha) production was inhibited by catalase. The production of 8-epi-PGF(2alpha) was also inhibited by indomethacin and aspirin. Exogenous hydrogen peroxide stimulated 8-epi-PGF(2alpha) production by normoxic cells, and aspirin inhibited the hydrogen peroxide-mediated increase in 8-epi-PGF(2alpha) production. These results indicate that the reactive oxygen species responsible for 8-epi-PGF(2alpha) synthesis during reoxygenation is hydrogen peroxide and that in endothelial cells 8-epi-PGF(2alpha) synthesis is mediated by prostaglandin H(2) synthase (PGHS). To verify the role of PGHS in 8-epi-PGF(2alpha) synthesis, human PGHS-1 was expressed in COS-7 cells, a PGHS negative cell line that does not synthesize 8-epi-PGF(2alpha). In the presence of exogenous arachidonic acid the COS-7 cells expressing human PGHS-1 produced substantial amounts of PGE(2) and 8-epi-PGF(2alpha). These data indicate that human PGHS-1 can support the synthesis of 8-epi-PGF(2alpha) and that 8-epi-PGF(2alpha) synthesis by cultured human endothelial cells during reoxygenation is dependent on the activity of PGHS-1.

Foot TcPO2 response to lumbar sympathectomy in patients with focal ischemic necrosis.

We prospectively evaluated all patients with superficial foot necrosis of 1-3 cm and transcutaneous oxygen tension (TcPO2) values of <30 mmHg who received a sympathectomy as the primary treatment of their vascular occlusive disease. Preoperatively, and every 2-3 days in the postoperative period, measurement of TcPO2 of the forefoot was performed. Clinical success was defined as healing of the necrosis or healing of a toe amputation and avoidance of a major below-knee/above-knee amputation for 1 year. Ten patients were available for long-term evaluation. During the first 4-5 days, all patients increased their foot TcPO2 and the mean increase (23 mmHg) was significant (p = 0.04). Clinical improvement was marked by an average increase of 29 mmHg by postoperative day 10. In contrast, patients with clinical failure had only an average increase of 5 mmHg in TcPO2 by the same postoperative interval. Preoperative increase in TcPO2 by at least 20 mmHg in response to dependency predicted a favorable response to sympathectomy. In addition, sustained postoperative increases in tissue oxygen levels by postoperative day 10 also favored wound healing.

Photodynamic therapy inactivates extracellular matrix-basic fibroblast growth factor: insights to its effect on the vascular wall.

PURPOSE: Photodynamic therapy (PDT), the light activation of photosensitizer dyes for the production of oxygen and other free radical moieties without the generation of heat, has been shown to inhibit the development of experimentally induced intimal hyperplasia. The host response to PDT, a form of vascular injury that results in complete vascular wall cell eradication, is devoid of inflammation and proliferation and promotes favorable vascular wall healing. These effects do not result in intimal hyperplasia and are suggestive of PDT-induced changes in the extracellular matrix (ECM). As a model to better understand the biologic consequences of PDT on the vascular wall matrix proteins, the effect of PDT was studied on the powerful matrix-resident mitogen basic fibroblast factor (bFGF) in vitro. METHODS: PDT (5 to 200 J/cm2, 100 mW/cm2, 675 nm) was used with the photosensitizer chloroaluminum sulfonated phthalocyanine (5 micrograms/ml) to inactivate bFGF in vitro while 100 J/cm2 of irradiation was administered 24 hours after 5 mg/ml of the photosensitizer was used in vivo. PDT was used on bFGF in solution and on endothelial cell-derived ECM. Enzyme-linked immunosorbent assay was used to quantitate bFGF in solution after PDT treatment or after extraction from the ECM by collagenase and heparin. Functional activity of matrix-associated bFGF was assessed by smooth muscle cell mitogenesis by 3H-thymidine incorporation. To demonstrate the in vivo relevance of these observations, immunohistochemical analysis of PDT-treated rat carotid arteries was undertaken. RESULTS: PDT eliminated detectable levels of bFGF in solution. PDT of ECM significantly reduced matrix-bound bFGF (1.0 +/- 0.6 vs 27.5 +/- 1.3 pg/ml; p < 0.0001). This reduction in bFGF after PDT of the ECM was associated with a decrease in vascular smooth muscle cell mitogenesis (52.4% +/- 4.6%; p < 0.0001) when plated on PDT-treated matrix compared with nontreated matrix. Quantitative replenishment of exogenous bFGF to PDT-treated matrix restored proliferation to baseline levels. PDT of rat carotid arteries demonstrated a loss of bFGF staining compared with control nontreated arteries. CONCLUSIONS: PDT inactivation of matrix-resident bFGF and possibly other bioactive molecules can provide a mechanism by which PDT suppresses smooth muscle cell proliferation in the vessel wall. This free radical-mediated alteration of matrix may contribute to favorable vascular healing when PDT is used for the inhibition of injury-induced intimal hyperplasia.

Effect of hypoxia on steady-state arachidonic acid metabolism in bovine aortic endothelial cells.

At the onset of acute hypoxia, eicosanoid synthesis by bovine aortic endothelial cells (BAEC) markedly decreases, reflecting a decreased release of arachidonic acid from endogenous stores. To determine the cause of decreased arachidonic acid release, we pulse-labeled BAEC with [14C]arachidonic acid for 5 min under normoxic conditions and chased cells for 1 h under normoxic or hypoxic conditions. The 14C incorporation and specific activity (disintegrations per minute per nanomole) of three major arachidonyl molecular species (16:0-20:4, 18:1-20:4, and 18:0-20:4) of each phospholipid class were determined in cells chased under either of the two conditions. There was no relevant difference between normoxic and hypoxic cells in the metabolism of any of the arachidonyl molecular species of diacyl lipids. However, there was a marked decrease (approximately 40%) in the turnover of arachidonyl alkenylacyl phosphatidylethanolamine in the hypoxic cells. From these results, it appears that the source of arachidonic acid supporting constitutive eicosanoid synthesis in BAEC is alkenylacyl phosphatidylethanolamine and that the limiting enzyme activity determining the rate of eicosanoid synthesis is a plasmalogen-specific phospholipase A2.

Transcutaneous partial oxygen pressure changes following skew flap and Burgess-type below-knee amputations.

OBJECTIVE: To evaluate the degree of flap hypoxia following different types of below-knee amputations. DESIGN: Prospective preoperative and postoperative measurements of transcutaneous partial oxygen pressure (TcPo2) at the site of amputation in 10 consecutive patients who underwent a Burgess-type below-knee amputation (group 1) and in 10 consecutive patients who underwent a skew flap amputation (group 2). SETTING: An academic, tertiary care Veterans Affairs medical center. PATIENTS: Individuals with severe arterial occlusive disease of the lower extremity, in many of whom vascular reconstruction has failed. INTERVENTION: Measurements of TcPo2 (in millimeters of mercury). MAIN OUTCOME MEASUREMENT: The decrease in TcPo2 associated with the different "flaps" of a Burgess-type below-knee amputation. RESULTS: In all skin flaps, regardless of the type of amputation, an early postoperative reduction of the TcPo2 was noted. The greatest reduction (11 mm Hg) and persistence at 20 postoperative days were noted in posterior flaps. CONCLUSIONS: Strict conformity to the Burgess-type of below-knee flap design may not provide an optimal incisional blood supply. Consideration should be given to the skew flap technique in patients who require amputation for severe lower limb arterial insufficiency.

Photodynamic therapy of extracellular matrix stimulates endothelial cell growth by inactivation of matrix-associated transforming growth factor-beta.

Photodynamic therapy (PDT), the production of cytotoxic free-radical moieties by light activation of photosensitizer dyes, is a novel approach to inhibit experimental intimal hyperplasia. Local eradication of vascular cells with this method in vivo is followed by expedient reendothelialization, and PDT of extracellular matrix (ECM) in vitro stimulates endothelial cell (EC) growth. This in vitro study explored one possible mechanism underlying these findings by investigating the effects of PDT on matrix-associated transforming growth factor-beta (TGF-beta), a potent inhibitor of EC growth. The ECM deposited by EC on tissue culture plates contained 85.4 +/- 10.2 pg/10 cm2 of TGF-beta, as measured by an ELISA. In contrast, after PDT of ECM, levels of TGF-beta could be barely be detected (0.2 +/- 0.5 pg/10 cm2). The functional consequence of this observation was demonstrated by the finding that PD1 of plates coated with a fibronectin-TGF-beta complex stimulated EC mitogenesis (102.3% +/- 19.3%, p < 0.0005) compared with the untreated control (44.1% +/- 13.5%). The inhibitory effect of ECM-associated TGF-beta on EC was further delineated by blocking its activity with a specific antibody. Whereas the antibody did not affect EC mitogenesis or PDT-treated matrix or matrix-free plates (101% +/- 8.8%, 105.6% +/- 9.8%), EC mitogenesis growing on ECM was significantly enhanced (125.9%, 17.5%, p < 0.05). Finally, SDS-PAGE analysis of PDT-treated TGF-beta in solution demonstrated that the PDT-mediated loss of TGF-beta activity was not associated with changes in its molecular weight. These data demonstrate that increased EC proliferation on PDT-treated matrix is, at least in part, mediated by inactivation of TGF-beta. PDT-removal of this EC growth inhibitor in the intima provides a mechanism by which PDT of the vascular wall could potentiate endothelial regrowth, a factor which may promote proper healing and result in the inhibition of intimal hyperplasia.

Effects of oxygen tension and shear stress on human endothelial cell prostacyclin production.

Under in vivo conditions of ischemia and reperfusion, vascular endothelium (EC) experience concurrent changes in oxygen tension, shear stress, and the local concentration of metabolites. These studies explored the combined effects of shear stress and oxygen tension on EC prostacyclin production. EC grown on microcarrier beads were exposed to 120 min of normoxia and basal shear stress by stirring at 20 rpm. After normoxia, EC were exposed to hypoxia (2% 0(2), 20 rpm), ischemia (2%, 0(2), 5 rpm) or sham ischemia (20% 0(2), 5 rpm). Following hypoxia, EC were reoxygenated (20% 0(2), 20 rpm). After ischemia and sham ischemia, EC were reperfused (20% 0(2), 20 rpm). Minimal accumulation of metabolites occurred during normoxia, hypoxia, and reperfusion. All metabolites were allowed to accumulate in the flasks during ischemia and sham ischemia. Prostacyclin levels were measured by ELISA, and prostaglandin H2 synthase levels in cells were analyzed by immunoblotting. An acute decrease in shear stress decreased prostacyclin production. An acute decrease only in oxygen tension did not alter prostacyclin production significantly. The combined acute decrease in both shear stress and oxygen tension significantly stimulated prostacyclin production for 30 min. By 120 min of ischemia and hypoxia, prostacyclin release was significantly less than sham ischemia. Prostacyclin production after 30 min of reoxygenation was significantly less than that of cells subjected to reperfusion. By 120 min of reperfusion and reoxygenation, there was no significant difference in EC prostacyclin synthesis. These findings suggest that temporal and quantitative aspects of EC prostaglandin synthesis are dependent on both oxygen tension and shear stress.

Prostanoid production in rabbit corpus cavernosum. II. Inhibition by oxidative stress.

PURPOSE: To investigate the effects of reoxygenation following hypoxia on prostanoid production in rabbit penile corpus cavernosum tissue (RCC) in organ culture. MATERIALS AND METHODS: Strips of RCC were incubated in organ culture media under either 21% O2 (control, PO2 approximately 167 mm.Hg) or 0% O2 (hypoxia, PO2 approximately 27 mm.Hg) followed by a reoxygenation period with 21% O2, in the presence or absence of exogenous arachidonate, Tiron or catalase. Prostanoids were measured in collected media by radioimmunoassay. Malondialdehyde levels were measured in RCC following exposure to either control or hypoxia-reoxygenation conditions. RESULTS: Under hypoxic conditions, basal release of prostanoids (PGI2, PGF2 alpha, PGE2 and TXB2) was inhibited. Although this inhibition was reversible upon reoxygenation, the recovery was delayed, requiring at least 2 hours of exposure to 21% oxygen to reestablish prostanoid production. Reoxygenation also caused lipid peroxidation as measured by an increase in malondialdehyde levels. When reoxygenation was done in the presence of exogenous arachidonate, recovery of PGI2 production was complete by 1 hour. Reoxygenation in the presence of a scavenger of reactive oxygen species (Tiron) or catalase significantly improved the recovery rate of PGI2 production. CONCLUSIONS: These results show that reoxygenation of hypoxic tissue generates oxidative stress that interferes with the recovery of prostanoid production by alteration of a biosynthetic point(s) upstream from prostaglandin H synthase (PGHS) including, at least, phospholipid peroxidation.

Differential modulation of vascular endothelial and smooth muscle cell function by photodynamic therapy of extracellular matrix: novel insights into radical-mediated prevention of intimal hyperplasia.

PURPOSE: Photodynamic therapy (PDT) has been demonstrated to inhibit experimental intimal hyperplasia and to lead to expedient reendothelialization but negligible repopulation of the vessel media. The mechanism that underlies the differential ingrowth of cells into PDT-treated vessel segments is not understood. Because the extracellular matrix (ECM) is known to modulate specific cell functions, this study was designed to determine whether PDT of isolated ECM affects the function of endothelial cells (ECs) and smooth muscle cells (SMCs). METHODS: PDT of bovine aortic EC-ECM was performed with chloroaluminum sulfonated phthalocyanine and 675-nm laser light. Control specimens included untreated ECM, ECM-free plates, and ECM exposed to either light or photosensitizer only. Cell function was characterized by attachment, proliferation, and migration of ECs or SMCs that were plated onto identically treated matrixes. RESULTS: SMC attachment (86% +/- 0.4% vs 95% +/- 0.4%), proliferation (46% +/- 0.5% vs 100% +/- 1.4%), and migration (40% +/- 1.0% vs 100% +/- 0.9%) were significantly inhibited after PDT of ECM when compared with untreated ECM (all p < 0.001). In contrast, PDT of ECM significantly enhanced EC proliferation (129% +/- 6.2% vs 100% +/- 6.2%; p < 0.03) and migration (118% +/- 2% vs 100% +/- 0.8; p < 0.01), but did not affect attachment. CONCLUSIONS: This report establishes PDT-induced changes in the ECM with a result of inhibition of SMCs and stimulation of EC functions. It provides insight into how PDT-treated arteries can develop favorable EC repopulation without SMC-derived intimal hyperplasia. These findings may help provide a better understanding of the interactions between cells and their immediate environment in vascular remodeling.

Endogenous reactive oxygen metabolites mediate sublethal endothelial cell dysfunction during reoxygenation.

PURPOSE: Endothelial cells (EC) secrete vasoactive eicosanoids, which maintain organ blood flow. Because EC are a major source of eicosanoids, we studied the effects of reoxygenation on EC prostacyclin production. METHODS: Bovine aortic EC cultures were exposed to 2 hours of normoxia, then 1 hour of hypoxia (PO2 = 10 +/- 3.5 mm Hg), followed by 1.5 hours of reoxygenation in either normal medium or medium plus either superoxide dismutase (SOD, 300 units/ml), catalase (1200 units/ml), allopurinol (5.0 x 10(-4) mol/L), or dinitrophenol (10(-4) mol/L). RESULTS: Prostacyclin production decreased to 40% (p < 0.05) of basal prostacyclin production after 1 hour of hypoxia. EC reoxygenated with control medium recovered to 48% of basal prostacyclin production. EC reoxygenated in SOD resulted in recovery (p < 0.05) to 154% of basal prostacyclin production after 60 minutes. Catalase treatment resulted in recovery to 105% (p < 0.05) of basal prostacyclin production within 30 minutes of reoxygenation. Allopurinol treatment resulted in 77% recovery (p < 0.05) of basal prostacyclin production only during 30 minutes of reoxygenation. Dinitrophenol treatment resulted in significant (> or = 85%, p < 0.05) sustained recovery of basal prostacyclin production at 30, 60, and 90 minutes of experimental reperfusion. CONCLUSIONS: The hypoxia-induced decrease in EC prostacyclin does not recover during reoxygenation. Catalase/SOD allowed return to baseline prostacyclin production during reoxygenation, implicating reactive oxygen metabolites as mediators of decreased eicosanoid biosynthesis. Recovery of prostacyclin production after 60 minutes reoxygenation with dinitrophenol but not allopurinol suggests a mitochondrial origin of the oxygen metabolites responsible for decreased prostacyclin biosynthesis.

Immediate responses of endothelial cells to hypoxia and reoxygenation: an in vitro model of cellular dysfunction.

We have studied endothelial functions and integrity under clinically relevant levels of acute and profound hypoxia. Bovine aortic endothelial cells (EC) grown on microcarrier beads were exposed for 15-min intervals to normoxic (20% O2) or hypoxic (1-2% O2) medium. Control intervals were followed by four hypoxic and then four normoxic intervals for reoxygenation. Prostacyclin release from EC significantly decreased after only 15 min of hypoxia and remained low despite reoxygenation. This decrease in prostacyclin release was not coincident with decreased viable cells (Trypan blue exclusion) or with increased cell lysis (increased lactate dehydrogenase) after hypoxia or reoxygenation. When the medium was supplemented with 30 microM arachidonate (saturating concentration), prostacyclin release still significantly decreased after 30 min of hypoxia but returned to baseline levels by 30 min of reoxygenation. Similar results were obtained for thromboxane B2 release. These data suggest that 1) EC decrease prostacyclin release during acute, profound hypoxia, 2) EC decrease prostaglandin production during hypoxia despite abundant exogenous arachidonate, and 3) recovery of prostaglandin production is dependent on exogenous arachidonate during reoxygenation.

Pentoxifylline therapy for chronic claudication: are patients dependent on therapy?

BACKGROUND: The purpose of this study was to evaluate the efficacy of long-term pentoxifylline therapy. METHODS: Fifty consecutive patients on long-term pentoxifylline therapy entered a weaning program. The standard dose of 400 mg three times a day was reduced to 400 mg twice a day for 1 month, 400 mg daily for an additional month, and then withdrawn completely for 3 months. Subjective evaluation of each patient's symptoms and objective measurements (treadmill testing for initial claudication distance, maximum walking distance, and ankle/brachial indexes) were evaluated during treatment withdrawal. RESULTS: Twenty-seven patients tolerated withdrawal of therapy without symptomatic deterioration. Seven of nine patients who did not tolerate weaning had deterioration of treadmill ICD/MWD parameters; there was a significant absolute decrease (p = 0.016) in MWD from 430 +/- 78 to 221 +/- 23 feet. Patients with an initial low MWD were less likely to tolerate weaning. Sixty percent of the patients with successful weaning had clinically asymptomatic deterioration of treadmill ICD/MWD parameters. CONCLUSIONS: A substantial number of patients who have been on long-term successful pentoxifylline therapy for claudication can be weaned from the drug. Asymptomatic deterioration of treadmill test parameters is not an indication for resuming therapy.

Atrial myxoma with remote metastasis: case report and review of the literature.

The occurrence of a metastatic atrial myxoma after complete resection of the intracardiac lesion is rare. We treated a 66-year-old man who 13 years earlier had undergone resection of a left atrial myxoma, which had been diagnosed during a work-up of symptomatic cerebellar infarcts. He initially had a painless nonpulsatile mass near the radial artery. Surgery was performed to confirm the presumptive diagnosis of calcified ganglion, and subsequently he was found to have a pseudoaneurysm. The pathologic examination identified the tissue as myxoma. The results of echocardiography and cardiac magnetic resonance imaging failed to reveal any residual or new intracardiac tumor. The patient subsequently experienced swelling of the right leg and was found to have a 6 x 10 cm mass in the medial portion of his proximal thigh. At exploration this mass was found to extend from the profunda femoris artery, and it also compressed the femoral vein. Pathologic evaluation based on histologic and electron microscopic features of this lesion confirmed this lesion to be myxoma. Review of the literature reveals that metastatic atrial myxoma is a rare lesion, and such a delayed occurrence after resection of the cardiac lesion has been infrequently reported. This suggests that patients with atrial myxoma may be at risk for the appearance of intravascular metastatic tumor long after resection of their original intracardiac tumor.

Upper extremity claudication 10 years after a Blalock-Taussig shunt treated with a carotid-to-subclavian graft.

A case of progressive upper extremity vascular insufficiency, which occurred 10 years after the construction of a Blalock-Taussig anastomosis, is described. It was successfully managed with a carotid-to-subclavian bypass graft.

Platelet survival and serotonin content after placement of arterial prostheses in dogs: effects of neointimal coverage and high- and low-dose aspirin.

Because prosthetic neointima produces much less prostacyclin (PGI2) than arterial intima and may be more susceptible to cyclooxygenase inhibition, aspirin treatment might enhance surface thrombogenesis. To test this hypothesis, aortic prostheses were placed in eight dogs and measurements of platelet survival and platelet serotonin (5HT) were made under conditions of no treatment and treatment with low-dose (2mg/kg) and high-dose (30 mg/kg) aspirin. These doses equally suppressed platelet function. Measurements were performed preoperatively, 6 to 8 weeks postoperatively (when little neointima was present), and 28 to 32 weeks postoperatively (neointima fully developed). Platelet survival and 5HT levels were markedly reduced 6 to 8 weeks postoperatively and returned to normal at 28 to 32 weeks after implantation. At all times, low-dose aspirin improved platelet survival and this effect was most apparent 6 to 8 weeks postoperatively. Treatment with either aspirin dose decreased platelet 5HT levels at the 28 to 32 week postoperative period but not at other times. At recovery of prostheses, 90% of the luminal surface was covered with endothelialized neointima. Neointimal production of PGI2 was one half to one third that of aortic production. Despite this, low- and high-dose aspirin equally suppressed PGI2 production from both neointima and aorta. Furthermore, aspirin did not increase labeled platelet uptake on neointima. We conclude that (1) aspirin treatment does not render prosthetic neointima thrombogenic and (2) aspirin alters platelet survival and 5HT levels by mechanisms other than inhibition of platelet and neointima cyclooxygenase.

Adult human saphenous vein endothelial cells: assessment of their reproductive capacity for use in endothelial seeding of vascular prostheses.

Autogenous endothelial seeding (AES) of vascular prostheses (VP) using venous endothelial cells (EC) reduces platelet-VP interactions and improves patency rates in small caliber VP in dogs. To conserve patients' veins for use in coronary or limb bypass surgery, human trials of AES should require proof that adequate numbers of EC with the growth capacity to cover VP can be harvested from acceptably small pieces of peripheral vein. EC were isolated from excess saphenous vein segments remaining after coronary bypass surgery by filling veins with 0.1% CLS II collagenase at 37 degrees C for 15 min and removing EC by flushing the veins with culture medium. EC were cultured on fibronectin-coated dishes in medium 199 with 30% human serum and 300 micrograms/ml of endothelial cell growth factor. These cells grew to form confluent monolayers, and were identified as EC by tests for factor VIII antigen. Veins from 53 patients with a mean age of 55.8 +/- 9.8 (SD) years yielded vein segments with an average area of 1.9 +/- 0.6 cm2, from which an average of 5.3 +/- 2.8 X 10(4) cells were removed per cm2 of vein area. EC in culture underwent 14.3 +/- 1.4 population doublings with an average population doubling time of 1.8 +/- 0.3 days (N = 14 cultures), which allowed an 100-fold increase in cell number to occur in 11 to 12 days. These data suggest that the EC available from small vein segments in adult humans have the growth capacity to cover areas comparable in size to the luminal areas of VP commonly used in arterial surgery.