Bradykinin-stimulated calcium influx in cultured bovine aortic endothelial cells.

Bradykinin (BK)-stimulated release of endothelium-derived relaxing factor has been linked to a rise in cytosolic Ca2+ concentration and a change of K+ permeability of the endothelial cell. In the present study, measurement of BK-induced changes in fura-2 fluorescence and 86Rb+ efflux were used to monitor changes in cytosolic Ca2+ and K+ permeability in cultured bovine aortic endothelial cells. In the presence of normal extracellular Ca2+, BK induced a fourfold increase in cytosolic Ca2+, which peaked at 20 s and declined within 1 min to a value that was 50% of the peak level. Subsequently, cytosolic Ca2+ decreased and approached basal levels within 8 min. In the absence of Ca2+, BK produced a 1.5- to 2-fold increase in cytosolic Ca2+ that peaked within 20 s and declined to basal levels within 2 min. Addition of Ca2+ to the Ca-free reaction buffer 3-5 min after addition of BK resulted in a two-to threefold increase in cytosolic Ca2+ that declined slowly back to basal levels. Thus Ca2+ influx can occur in response to BK at a time when there is minimal elevation of cytosolic Ca2+ above the resting level. Under all conditions tested, 86Rb+ efflux paralleled changes in the cytosolic Ca2+, suggesting that efflux occurred through Ca2+-activated K+ channels. Isosmotic substitution of Na+ with N-methyl-D-glucamine did not affect the BK-stimulated changes in cytosolic Ca2+ or 86Rb+ efflux, suggesting that Na+-Ca2+ exchange plays little role in the BK response. These results suggest that BK stimulates Ca2+ influx via a BK receptor-operated channel or a channel activated by some internal messenger other than Ca2+.

Bradykinin-induced increases in cytosolic calcium and ionic currents in cultured bovine aortic endothelial cells.

The goal of the present study was to determine if voltage-sensitive calcium channels are present in bovine aortic endothelial cell plasmalemma and if they contribute to the rise in cytosolic calcium produced by bradykinin. After bradykinin (100 nM) exposure, endothelial cell associated fura-2 fluorescence peaked within 10-20 seconds and then declined to a steady level 2- to 3-fold above resting values. Pretreatment with lanthanum (20 microM) abolished the steady level produced by bradykinin but had little effect on the initial, transient rise in cytosolic calcium. Chelation of extracellular calcium with EGTA before addition of bradykinin resulted in a substantial decrease in the fura-2 transient and elimination of the long-lasting component. Nimodipine (3 microM) and nitrendipine (1 microM) were without effect on either phase of the bradykinin-induced response. Moreover, elevation of extracellular potassium failed to produce a rise in intracellular calcium. With the use of the tight seal technique to voltage clamp the cells, inwardly rectifying and calcium-activated potassium currents were found to exist in the endothelial cells. Addition of bradykinin (100 nM) elicited a calcium-activated potassium current that was eliminated in the absence of intracellular potassium. No voltage-sensitive calcium currents were activated when the cells were exposed to 10 mM or 110 mM calcium chloride in the presence or absence of bradykinin. The binding of [3H](+)PN200-110 to endothelial cell membrane preparations was 1-3 orders of magnitude lower than that observed in PC-12, GH3, or BC3H1 cell membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Seeding of arteriovenous prostheses with homologous endothelium. A preliminary report.

Endothelial cell seeding of expanded polytetrafluoroethylene (e-PTFE) arteriovenous prostheses was performed to compare seeding with homologous vs. autologous cells and to study the effect of homologous seeding with a larger vs. a smaller number of cells. Sixteen dogs were randomly assigned to four equal groups: I, control; II, light homologous seeded; III, heavy homologous seeded; and IV, autologous seeded. Bilateral femoral arteriovenous loop grafts were inserted in all. Efficiency of seeding was assessed by cell counts of instilled vs. retained cells. All grafts remained patent and were harvested approximately 8 weeks after implantation. Samples were evaluated grossly by scanning electron microscopy and light microscopy. There was a direct correlation between number of cells instilled and number retained for each graft; group III received and retained the largest number (p less than 0.001 and p less than 0.05, respectively). The amount of thrombus deposition on the lumen of all grafts was grossly the same. Endothelium was demonstrated in samples obtained from the midgraft of groups III and IV; in contrast no endothelium was seen in groups I and II. The percentage of endothelialized surface was not determined. No immunologic cellular reaction was detected in any of the samples. We conclude that in the animal laboratory it is possible to seed e-PTFE arteriovenous prostheses successfully with homologous cells and to improve the efficiency of seeding by implanting a larger number of cells obtained from an endothelial cell bank. The potential applications of this technique to the clinical field are discussed herein.

Hemodynamic and morphologic features of arteriovenous angioaccess loop grafts.

Bilateral femoral A-VLG (5 acute, 5 chronic) were implanted in 10 dogs (e-PTFE, 6 mm ID, 25 cm length). Acute studies included measurements of cardiac output (CO) and systemic vascular resistance (SVR). Blood pressure and pressure waveforms in the graft were obtained by retrograde catheterization and pull-back readings. Chronic dogs were sacrificed 8 wks postimplant; samples were obtained following a standardized protocol and studied by light microscopy (LM) and scanning electron microscopy (SEM). Hemodynamic data show a rise in CO, a decline in SVR and a high flow through each graft. Pull-back readings show a gradual drop in pressure and loss of pulsatility from the arterial to the venous side of the graft. LM studies show IH primarily in the proximal vein. SEM showed limited pannus ingrowth endothelium close to the anastomoses and otherwise a thrombus layer throughout the lumen. A-VLG are associated with high flow, pressure drop and loss of pulsatility. Proximal vein IH is a reproducible lesion. We conclude there is severe hemodynamic stress from disturbed flow and high flow velocity and it plays a critical role in the development of venous intimal hyperplasia in AV loop grafts.

Response of cultured endothelial cells to steady flow.

A system has been developed for subjecting endothelial cell monolayers to prolonged steady flow while maintaining normal culture conditions. Cloned bovine endothelial cells were grown to confluence on one wall of a square glass tube which was then incorporated in the flow circuit. Flow rates of 19-21 ml/min were sustained for periods of 6-45 hr, subjecting the cells along the center line of the wall of the tube to a maximum shear stress of 34 dyn/cm2. The cells in all the experiments remained attached and viable when subjected to this shear stress. Photographic data from experimental runs were qualitatively assessed for changes in cell morphology, confluence, and orientation and were compared to data from matched stationary controls. Five experiments were chosen for quantitative morphometric analysis. In three experiments, the cells showed elongation with their long axes aligned with the direction of flow in 6.5, 21, and 22 hr. In the other experiments, either the cells formed a swirling pattern or no change in morphology was apparent. Although cell shape (form) changed in response to shear stress, cell area remained unaffected by exposure to flow.

Behavior of endothelial cells cultured on Silastic and Dacron velour under flow conditions in vitro: implications for prelining vascular grafts with cells.

Tissue-cultured bovine aortic endothelial cells were subjected to flow in an in vitro circulatory loop designed to simulate the flow and pressure conditions in the aorta. The cells were cultured under stationary conditions on tubes of fibronectin-coated Silastic or of Dacron velour. The tubes were then added to the flow loop, which circulated complete tissue culture medium in a pulsatile mode. Light microscopy and cell counts showed that the cells not only remained adherent for up to 2 weeks under flow conditions, but also underwent hypertrophy and proliferation in response to the flow regimen.

Performance of tissue cultured endothelial cells in a mock circulatory loop.

Tissue cultured bovine aortic endothelial cells were subjected to flow in an in vitro circulatory loop which was designed to simulate the flow and pressure conditions in the aorta. The cells were cultured to confluence under stationary conditions on a tube consisting of microfabric backed with polyurethane. The tube with the cultured cells was then added to the flow loop which circulated complete tissue culture medium in a pulsatile mode. Light microscopy, and scanning and transmission electron microscopy showed that the cells not only remained adherent for 2 week periods under flow conditions, but also underwent hypertrophy and proliferation in response to the flow regimen.

Human smooth muscle cells cultured from atherosclerotic plaques and uninvolved vessel wall.

Smooth muscle cells (SMC) were cultured from atherosclerotic plaques and uninvolved arteries to determine if differences exist between growth characteristics or ultrastructure of the cultured cells. Eighteen aortic punch biopsies provided the uninvolved tissue, and 58 carotid plaques provided the atherosclerotic tissue. Eighty percent of the samples yielded viable cultured cells, which reached a maximum population doubling time during log phase growth of 72 h (seeding density = 1.0 x 10(4) cells/cm2, 2nd passage). Growth characteristics of both normal and plaque-derived cells were the same in vitro. Growth rate declined with time in culture, and cell division ceased by the 5th or 6th passage. In culture, spindle shaped cells formed the "hill and valley" configuration typical of SMC. Plaque-derived SMC were ultrastructurally similar to SMC from uninvolved vessel wall. Proliferative potential did not vary with age or sex, with method of culture, or with whether the cells were plaque derived or not.