Asian Cardiovasc Thorac Ann 1999;7:101-105
© 1999 Asia Publishing EXchange Pte Ltd
Anoxia-Reoxygenation in Cultured Endothelial Cells: Effects of Fructose Diphosphate and Captopril
Yu Shi Qiang, MD,
Liu Wei Yong, MD,
Wang Gang, MD,
Ren Yu Shen, MD
Department of Cardiovascular Surgery
Xijing Hospital
Fourth Military Medical University
Xian, People's Republic of China
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For reprint information contact: Yu Shi Qiang, MD Tel: 86 29 337 3938 Fax: 86 29 337 3816 Department of Cardiovascular Surgery, Xijing Hospital, Fourth Military Medical University, Xian 710032, People's Republic of China.
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Abstract
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Cultured rat aortic endothelial cells were morphologically and immunologically characterized before incubation under anoxic conditions for 120 minutes. Cell samples were reoxygenated for 10, 30, and 60 minutes as a model of anoxia-reperfusion injury. The effects of anoxia-reoxygenation were evaluated by measurements of membrane microviscosity, intracellular Ca2+ content, release of 51Cr, and uptake of trypan blue. Membrane microviscosity decreased from 2.03 ± 0.17 poise before anoxia to 1.72 ± 0.22 poise after 120 minutes of anoxia, with a further decrease to 1.54 ± 0.29 poise after 60 minutes of reoxygenation. Release of 51Cr correlated negatively with the decrease in membrane microviscosity and rose from 7.14% ± 0.4% to 12.16% ± 2.79% after anoxia and to 27.17% ± 2.59% after 60 minutes of reoxygenation. Intracellular Ca2+ content and uptake of trypan blue showed no noticeable change during anoxia but they increased significantly during reoxygenation. Addition of fructose-1,6-diphosphate to the anoxic incubation medium partly prevented the change in microviscosity and significantly reduced the release of 51Cr and the uptake of Ca2+ and trypan blue. Captopril exerted similar but less potent effects to those of fructose-1,6-diphosphate.
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Introduction
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Endothelial cells are an important component of blood vessels and when injured they effect substance exchange between the blood and tissues as well as the metabolism of vasoactive agents. In this experimental study, cultured endothelial cells were used as a model for anoxia-reperfusion injury in which changes in membrane fluidity, rate of 51Cr release, uptake of trypan blue, and intracellular Ca2+ content were studied. The effects of fructose-1,6-diphosphate (FDP) and captopril on cultured endothelial cells were also investigated.
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Materials and Methods
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Endothelial cells from the aortic walls of inbred Sprague-Dawley rats were prepared for culture under sterile conditions and cultured in MEM culture medium (Gibco BRL, Grand Island, NY, USA) containing 10% calf serum, as described by Zhang and colleagues.1 The cultured cells were identified as endothelial cells by their monolayer arrangement and a positive reaction to factor VIII in immunofluorescence tests. The 6th to 12th generations of the cultured endothelial cells that had shown steady and stable growth were selected for the experiments. Samples of cultured endothelial cells (approximately 1 x 106 cells) were washed three times with 3 mL of phosphate-buffered saline solution before adding 3 mL of calcium-free magnesium-free Hanks (D-Hanks) fluid. The samples were placed in a pure nitrogen environment at 37°C for 120 minutes, followed by reoxygenation. Ten samples were collected at each time point: before anoxia; immediately after 120 minutes of anoxia; and at 10, 30, and 60 minutes after reoxygenation. In control experiments, no drugs were added during anoxia-reoxygenation. Additional experiments were carried out with either 5 g•L–1 FDP or 0.5 mg•L–1 captopril added to the D-Hanks solution during anoxia-reoxygenation.
The membrane fluidity of the cultured endothelial cells was determined after resuspension with pancreatin as described by Prendergast and colleagues2 followed by 3 washes with 3 mL phosphate-buffered saline at 37°C. The cultured endothelial cells were incubated at 37°C for 30 minutes with 1 mL phosphate-buffered saline containing 2 x 10–6 M 1,6-phenylhexa-1,3,5-triene (DPH; Sigma Chemical Co., St. Louis, MO, USA). Membrane microviscosity was calculated by measurements of fluorescence intensity at 0–0, 0–90, 90–90, and 90–0, using a Hitachi 800 spectrophotometer (Hitachi Corp., Tokyo, Japan) with thermostatic polarized fluorescent light. DPH, a fluorescence probe, was incorporated into the hydrocarbon chain of the membrane lipids, rendering them capable of emitting fluorescent light. Fluorescence polarization can demonstrate the fluidity of membrane lipid domains and quantify their mobility because of the approximate parallelism between the long axis of DPH and that of the lipid chain. Membranes showed appropriate fluidity under normal condition so that substance exchange was guaranteed. Abnormalities due to an increase or decrease in membrane fluidity can be quantified by the following equations: correction factor G = I90–0/I90–90 (where I is fluorescence intensity); polarization P = (I0–0 – GI0–90)/(I0–0 + GI0–90); microviscosity = 2P/(0.46 – P). The higher the value of P, the greater the microviscosity and the lower the membrane fluidity.
To determine the rate of 51Cr release, the culture medium in the sample bottles (n = 10) was discarded and the cells were washed three times with 3 mL phosphate-buffered saline at 37°C. The cells were incubated in 5% CO2 and 95% air at 37°C for 16 to 18 hours in 3 mL culture medium containing 1 µCi•mL–1 of 51Cr-labelled sodium chromate so that chromium might be incorporated into the cells with an equilibrium between its association and dissociation with cellular proteins. The anoxia-reperfusion injury model was established and endothelial cells with no chromium were used as controls. The intensity of gamma-ray emission over one minute was measured in the supernatant, the endothelial cells, and the controls using a single-channel gamma energy spectrum analyzer. The rate of 51Cr release was calculated from the following equation: 51Cr release (%) = (supernatant – control)/(supernatant + cells – control) x 100%.
The uptake of trypan blue was measured in endothelial cells after resuspension with pancreatin in 2.5 mL culture medium containing 0.5 mL of 4% w/v trypan blue solution. The cells were incubated for 3 minutes at 37°C in a CO2 incubator (Sheldon Mfg., Inc., Corelius, OR, USA). The number of stained and unstained cells were counted. Uptake of trypan blue is a good indicator of the mortality rate of endothelial cells because trypan blue is unable to pass through viable cell membranes. The uptake rate of trypan blue was calculated as follows: uptake of trypan blue (%) = no. of stained cells/(no. of stained + unstained cells) x 100%.
To determine intracellular calcium content, the endothelial cells were washed three times with 3 mL of deionized water and 0.4 mL of 6% w/v NaOH solution was added to digest the cells. After complete digestion, 0.1 mL of the digested solution was taken for measurement of intracellular Ca2+ using a Hitachi 250 atomic absorption spectrometer (Hitachi Corp., Tokyo, Japan). A second 0.1-mL sample of the digested cell solution was taken for protein measurement by the method of Lowry3. Calcium content was expressed as µg•mg–1 protein.
All results are expressed as mean ± standard deviation. Analysis of variance and the Student t test were used for correlation of means.
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Results
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After anoxia and reoxygenation, endothelial cell membrane microviscosity decreased from a mean of 2.03 ± 0.17 poise before anoxia to 1.72 ± 0.22 poise after 120 minutes of anoxia (p < 0.01), with a further decrease to 1.54 ± 0.29 poise (p < 0.01) after 60 minutes of reoxygenation (Figure 1
), while the release of 51Cr increased from 7.14% ± 0.4% to 12.16% ± 2.79% after anoxia and to 27.17% ± 2.59% after 60 minutes of reoxygenation (Figure 2) and the differences compared with the level before anoxia were highly significant (p < 0.01). There was a close negative correlation between membrane microviscosity and the rate of 51Cr release (r = –0.86). No significant changes in intracellular Ca2+ content or the uptake rate of trypan blue were observed after 120 minutes of anoxia but they increased significantly (p < 0.01) during reoxygenation (Figures 3 and 4).
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Figure 1. Microviscosity of rat aortic endothelial cell membranes during anoxia and reoxygenation in untreated (control), fructose-1, 6-diphosphate-treated (FDP), and captopril-treated samples.
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Figure 2. Release of 51Cr from rat aortic endothelial cells during anoxia and reoxygenation in untreated (control), fructose-1,6-diphosphate-treated (FDP), and captopril-treated samples.
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Figure 3. Calcium content of rat aortic endothelial cells during anoxia and reoxygenation in untreated (control), fructose-1,6-diphosphate-treated (FDP), and captopril-treated samples.
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Figure 4. Uptake of trypan blue by rat aortic endothelial cells during anoxia and reoxygenation in untreated (control), fructose-1,6-diphosphate-treated (FDP), and captopril-treated samples.
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The endothelial cell membrane microviscosity remained relatively stable and close to the normal level when FDP was present during anoxia-reperfusion (Figure 1
). There was a significant difference between the control and FDP-treated samples (p < 0.01). Captopril exerted similar effects to FDP and there was a significant difference (p < 0.01) between the microviscosity of the control and captopril-treated samples but no statistically significant difference was found between the microviscosity of the FDP-treated and captopril-treated cells (Figure 1). The rate of 51Cr release in the control samples progressively increased during reoxygenation and the increase was most notable 60 minutes after reoxygenation, whereas the rate of 51Cr release in the FDP-treated samples rose slightly (Figure 2) but remained significantly lower than the rate in untreated cells (p < 0.01). Captopril also reduced the rate of 51Cr release (p < 0.01) but the effect was not as marked as that of FDP (Figure 2) and the differences between these two agents was significant (p < 0.01). After 120 minutes of anoxia, no significant difference was observed in the cells treated with FDP or captopril compared with the untreated samples with respect to intracellular Ca2+ or the uptake of trypan blue (p > 0.05). However, during reoxygenation (Figures 3 and 4), FDP and captopril reduced intracellular Ca2+ accumulation and the entry of trypan blue into endothelial cells (p < 0.01). No significant difference was found between the protective effects of the two agents on these parameters (p > 0.05).
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Discussion
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The use of cultured cells as experimental models has increased in the past decade. Our choice of this model for studying anoxia-reperfusion injury was based on the advantage of freedom from neural and hormonal influences that are difficult to control in blood vessels both in vitro and in vivo. The physical and chemical environment can be better controlled in cultured endothelial cells for observation of transmembrane movements of ions and molecules, the biochemical, metabolic, and functional changes, and the effects of potential protective agents.
This study showed that membrane fluidity and 51Cr release increased during anoxia and occurred before calcium overload. Intracellular Ca2+ content rose sharply after 30 minutes of reoxygenation, to twice the level before reoxygenation, and continued to rise as reoxygenation continued. Our previous study on anoxia-reoxygenation injury in cultured myocardial cells showed similar changes that suggested the alterations in calcium content correlated closely with reperfusion injury.4 The main reason for the sharp rise in intracellular Ca2+ during anoxia-reoxygenation was probably the anoxia-induced disturbance of energy metabolism and increased membrane permeability, which might result in decreased activity of the membrane ion pump and increased inflow of excessive oxygen and calcium.5 In addition, oxygen free radicals may cause peroxidation of membrane lipids and accelerate membrane injury, including destruction of the function and structure of sarcoplasmic reticulum and mitochondria, leading to calcium accumulation.6 Kitakaze and colleagues7 also concluded that calcium overload occurred as a result of peroxidation of membrane lipids and degradation of membrane phospholipids. Therefore, calcium overload was the result of cell injury that in turn contributed to the death of endothelial cells.
The cell membrane is a liquid bilaminar membrane composed of lipoproteins and appropriate fluidity is required for normal cellular activity. Anoxia reduces intracellular adenosine triphosphate and the activity of phosphofructokinase and the production of free radicals during reoxygenation can cause peroxidation of the membrane lipids. These factors induce structural and functional alterations in the cell membrane causing changes in membrane fluidity.8 When the membranes of different cells undergo peroxidation, the changes in membrane fluidity vary. Ahmed Ibrahim and colleagues9 found that ischemia-reperfusion injury to intestinal mucosal membranes caused no significant change in lipid composition but increased membrane fluidity. On the other hand, Bagchi and colleagues10 noted that hepatic lipid peroxidation decreased membrane fluidity.
The rate of 51Cr release, an indicator of membrane injury, increased during anoxia and reoxygenation in this study and correlated closely with membrane microviscosity.11 This finding suggests that there was an increase in membrane fluidity as well as changes in membrane structure and function after 120 minutes of anoxia in rat aortic endothelial cells. The increase in the rate of 51Cr release was most notable after 60 minutes of reoxygenation and the degree of change was greater than that of membrane fluidity, which indicates that changes in membrane structure and function continue for up to 60 minutes after reoxygenation. In these experiments, membrane fluidity and functional changes did not correlate with changes in intracellular Ca2+ content or the uptake of trypan blue, which showed no noticeable change after 120 minutes anoxia but increased progressively after reoxygenation. These findings indicate that calcium accumulation and cellular necrosis or loss of function occurred mainly in the reoxygenation period. Modifications by FDP and captopril of endothelial cell changes during anoxia-reoxygenation were highly significant. The inhibitory effect of captopril on indices of cell damage was less potent than that of FDP, especially in terms of the inhibition of 51Cr release.
FDP is an intermediate product as well as a substrate in the metabolism of carbohydrates. It can enhance anaerobic glycolysis and increase the synthesis of adenosine triphosphate. It supplements the energy necessary for cellular metabolism during anoxia and thus compensates for oxygen debt. This keeps the cell membrane stable and provides a basis for protection of endothelial cells against anoxia-reoxygenation injury. Captopril is an inhibitor of angiotensin transferase, which suppresses the generation of angiotensin II and the degradation of bradykinin and speeds up the production of prostacyclin.12 In addition, the hydrosulfide group in captopril is a nonspecific scavenger of free radicals, which exerts a protective effect on endothelial cells against anoxia-reoxygenation injury. The fact that its protective effect against anoxia-reoxygenation injury is less potent than that of FDP may be due to their different mechanisms of action.
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References
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Abstract
Introduction
