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(Circulation. 1999;100:541-546.)
© 1999 American Heart Association, Inc.

Basic Science Reports

Protective Effects of Preconditioning in Cultured Rat Endothelial Cells

Effects on Neutrophil Adhesion and Expression of ICAM-1 After Anoxia and Reoxygenation

Philippe Beauchamp, BS; Vincent Richard, PhD; Fabienne Tamion, MD; Françoise Lallemand, BS; Jean-Pierre Lebreton, MD; Hubert Vaudry, PhD; Maryvonne Daveau, PhD; Christian Thuillez, MD, PhD

From IFRMP 23, INSERM E9920 (P.B., V.R., F.T., F.L., C.T.), VACOMED, the Department of Pharmacology, Rouen University Medical School, INSERM U78 (J.-P.L., M.D.), INSERM U413 (H.V.), Rouen, France.

Correspondence to Vincent Richard, Service de Pharmacologie, CHU de Rouen, 76031 Rouen Cedex, France. E-mail vincent.richard{at}chu-rouen.fr

	Abstract

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Background—Preconditioning with brief periods of ischemia protects the coronary endothelium against acute and chronic reperfusion injury, but the mechanisms of this endothelial protection remain unknown. We hypothesized that preconditioning protects endothelial cells through a decreased production of endothelial adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1), leading to a lesser adhesion of neutrophils to the endothelium.

Methods and Results—Cultured rat aortic endothelial cells were subjected to 6-hour anoxia followed by various durations of reoxygenation. Preconditioning was induced by 1-hour anoxia and 1-hour reoxygenation. ICAM-1 gene expression was measured by polymerase chain reaction, and the percentage of cells expressing ICAM-1 was assessed by confocal laser fluorescence microscopy. Anoxia/reoxygenation increased expression of ICAM-1, with a peak occurring after 6 hours of reoxygenation for mRNA and 9 hours for protein. Preconditioning prevented the increase in ICAM-1. Similar reductions were observed with the free radical scavenger N-2 mercaptopropionyl glycine (MPG). The inhibitory effect of preconditioning on ICAM-1 expression was abolished by an inhibitor of protein kinase C, an inhibitor of nitric oxide synthesis, and by MPG but was not affected by an adenosine receptor antagonist. Finally, both preconditioning and MPG partially prevented the increased adhesion of human neutrophils to reoxygenated endothelial cells.

Conclusions—Preconditioning prevented reoxygenation-induced, free radical–mediated expression of ICAM-1 by a mechanism involving activation of protein kinase C and production of nitric oxide and free radicals, and this is associated with a lesser adhesion of neutrophils to endothelial cells. Such prevention of neutrophil adhesion may contribute to the protective effect of preconditioning against reperfusion-induced endothelial injury.

Key Words: endothelium • ischemia • cell adhesion molecules • free radicals

	Introduction

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In addition to protecting myocytes, preconditioning¹ also protects coronary endothelial cells (ECs) against ischemia-reperfusion injury,^{2 3 4 5} but the mechanisms of this protection remain unknown. These mechanisms cannot be easily studied in vivo because of the numerous possible confounding factors that could influence vascular function. Thus investigation of the mechanisms of preconditioning requires an in vitro model of anoxia or ischemia performed in isolated arteries or cultured ECs.

Reperfusion injury to the endothelium is mediated by neutrophils that accumulate early after reperfusion in the previously ischemic cardiac tissue.^{6 7} This adhesion is made possible by the expression of adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1).^{8 9 10 11} Thus the present study was designed to test the hypothesis that preconditioning prevents the increased expression of ICAM-1 and the increased adhesion of neutrophils to cultured ECs subjected to prolonged anoxia followed by reoxygenation (A/R).

	Methods

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Cell Isolation
Rat aortic ECs were prepared from male Wistar rats and grown in culture as described previously.¹² Experiments were performed in confluent EC (3rd to 4th passage). Cell survival was assessed with Trypan blue; characterization of the cells as rat endothelium was verified by immunofluorescence by use of the specific rat vascular EC antibody RECA-1 (Pan endothelium, Euromedex HIS52; Figure 1).

View larger version (62K): [in this window] [in a new window]   Figure 1. Immunostaining of rat ECs examined under confocal laser microscopy. Nuclei were stained red by propidium iodide to identify all present cells; slides were incubated with the use of a Cy2-streptavidin–labeled rat EC antibody (RECA). Green fluorescence demonstrates binding of RECA and thus shows evidence that cells are indeed ECs.

Anoxia/Reoxygenation
Anoxia was performed with the use of an anaerobic chamber (Forma Scientific, model 1029). To induce abrupt anoxia, the cell culture medium was deoxygenated before the experiments by placing it in the chamber for 12 hours. With this technique, the oxygen tension in the anoxic medium was <0.5 mm Hg (ie, >99% decrease in PO₂). During periods of anoxia, cells were kept in an incubator (37°C) within the anaerobic chamber. Reoxygenation was obtained by removing anoxic medium and changing it to a normal, oxygenated culture medium (95 mm Hg) and by returning the EC to the normal cell incubator.

Experimental Protocols
Preconditioning consisted of a 1-hour period of anoxia followed by 1 hour of reoxygenation immediately before prolonged anoxia (see Figure 2). This duration was chosen on the basis that it did not induce any increase in endothelial expression of ICAM-1 or increase neutrophil adhesion. The short duration of reoxygenation between brief and prolonged anoxia (1 hour) was chosen because we wanted to mimic "classic" preconditioning (ie, "first window") for which protective effects rapidly disappear after reperfusion periods of 3 to 6 hours.

View larger version (13K): [in this window] [in a new window]   Figure 2. Experimental protocol.

Prolonged anoxia was set to 6 hours, followed by 0, 1, 3, 6, 9, 12, or 24 hours of reperfusion. The duration of anoxia was chosen on the basis that shorter durations of anoxia were associated with marginal increases in ICAM-1 or neutrophil adhesion (data not shown).

In some experiments, the role of free radicals was assessed with the use of the free radical scavenger N2-mercaptopropionyl glycine (MPG, 10^-5 mol)¹³ added 20 minutes before reoxygenation and throughout the reoxygenation period after prolonged anoxia (Figure 2). The possible additive effect of preconditioning and MPG was also tested by treating preconditioned cells by MPG also given 20 minutes before and throughout reoxygenation. In parallel, the effect of the nuclear factor-B (NFB) inhibitor/free radical scavenger pyrrolidine dithiocarbamate (PDTC, 10^-5 mol) was also tested by use of the same protocol. In the case of PDTC or of the combined preconditioning-MPG treatment, only 1 time point of reoxygenation was assessed (9 hours).

The possible roles of adenosine, protein kinase C, free radicals, and nitric oxide (NO) as mediators of preconditioning were evaluated: cells were preconditioned in the absence or presence of the nonspecific antagonist of adenosine receptors p-sulfophenyl-theophylline (SPT, 10^-5 mol), the inhibitor of protein kinase C chelerythrine (10^-6 mol), the free radical scavenger MPG (10^-5 mol), or the NO synthase inhibitor N^G-nitro-L-arginine (L-NA, 10^-5 mol). All drugs were added to the cell medium during the 2-hour preconditioning period (1 hour of anoxia + 1 hour of reoxygenation). Nonpreconditioned treated cells were incubated with the same compounds for 2 hours before prolonged anoxia.

Assessment of ICAM-1 Gene Expression by Reverse Transcription–Polymerase Chain Reaction
Total RNA extraction was extracted from ECs according to a 1-step method.¹⁴ Reverse transcription (RT) protocols were performed with 2 µg of total RNA in 30 µL (final volume) of reaction buffer. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. Aliquots of the RT reaction were amplified with the following rat ICAM-1 and GAPDH primers: ICAM-1: 5'-CTC ATC CTG CGC TGT CTG GT-3' (sense), 5'-CCG GAG CTG CCT GAC CTC GG-3' (antisense); GAPDH: 5'-TCC ATG ACA ACT TTG GCA TC-3' (sense), 5'-CAT GTC AGA TCC ACC ACG GA-3' (antisense).

The number of polymerase chain reaction (PCR) cycles was 24. Products were separated by electrophoresis gels stained with ethidium bromide, illuminated with UV light, and measured by quantitative scanning densitometry of autoradiographs. All measurements of ICAM-1 mRNA expression are reported as ratio of optical density of the ICAM-1 and GAPDH bands calculated for each experimental sample, in which both ICAM-1 and GAPDH were measured in parallel. Preliminary experiments in which we repeated PCR analysis on the same sample (during determination of the optimal number of PCR cycles) showed a measurement variability of <20%.

Immunofluorescence
The endothelial expression of ICAM-1 was initially assessed by immunofluorescence with a mouse monoclonal anti-rat ICAM-1 antibody (Genzyme, No. 80–2912-01; dilution 1:150) coupled with biotinylated donkey anti-mouse IgG (Jackson ImmunoResearch No. 715–066-171, dilution 1:400), followed by streptavidin fluorescent complex Cy² (Biological Detection System No. PA42001, dilution 1:50). All antibodies were diluted in PBS. Negative control slides were either incubated with the solvent alone or submitted to the full procedure except for the omission of the initial monoclonal antibody. Specimens were viewed with the use of a confocal laser microscope (Leica). Percentage of cells showing positive fluorescence was calculated on each microscopic field. Cells with only focal fluorescence also appeared in some of the negative controls and thus were considered as ICAM-1 negative. Measurements were performed on 10 different fields and averaged.

Immunoperoxidase
In the case of the experiments aimed at studying the mechanisms of the effect of preconditioning on ICAM-1, the expression of ICAM-1 was assessed by use of the immunoperoxidase technique. For this purpose, slides covered with EC were incubated with the first and second antibodies as described above, followed by streptavidin peroxidase complex (Immunotech; dilution 1:200), and the final reaction was achieved by incubation with 3-amino-9-ethylcarbazole (Sigma) and 0.01% H₂O₂ for 30 minutes. Specimens were viewed on an inverted phase contrast microscope (Leica).

Neutrophil Adhesion
Adhesion of neutrophils was assessed with human neutrophils. We chose to use human neutrophils because our preliminary experiments suggested that isolation of rat neutrophils did not yield a sufficient number of cells to perform the adhesion assays. Human neutrophils have previously been shown to adhere to porcine or bovine ECs.^{15 16}

Neutrophils were isolated with the use of Ficoll-Paque (Pharmacia Biotech, specific gravity 1.077 g/mL) and suspended in DMEM with 10% serum. Cell viability, assessed with Trypan blue, was always >96%. The suspension of neutrophils was then incubated with reoxygenated ECs (at 9 hours reoxygenation; ratio of neutrophils/ECs 10:1) under static conditions for 15 minutes at 37°C before removal of nonadherent cells. The cells were then fixed with methanol, stained with May-Grünwald and Giemsa staining, and examined with the use of an inverted phase contrast microscope (Leica). Adherent neutrophils were counted on a minimum of 10 microscopic fields. Results are expressed in number of neutrophils per EC.

Statistics
Results are expressed as mean±SEM and were compared by Student's t test or by ANOVA, followed (when ANOVA showed significant differences) by Tukey test for multiple comparisons. Comparisons of survival were performed with the use of a Pearson ² test. A value of P<0.05 was considered statistically significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Cell Viability
Prolonged anoxia followed by reoxygenation only moderately affected cell survival (control: 96.5±0.5%; A/R 87.8±1.0%; n=3; P<0.05). Preconditioning slightly increased survival as compared with untreated A/R (93.2±0.9%), although this increase was not statistically significant.

ICAM-1 mRNA Synthesis
Figures 3 and 4 show that A/R markedly increased ICAM-1 mRNA, with a peak expression after 6 hours of reoxygenation, after which mRNA decreased and returned to baseline within 12 to 24 hours. This marked increase was significantly reduced by preconditioning. Similarly, the free radical scavenger almost abolished the reoxygenation-induced increase in ICAM-1 mRNA.

View larger version (25K): [in this window] [in a new window]   Figure 3. Expression of ICAM-1 assessed by PCR. Numbers represent duration (hours) of A/R. C indicates control; P, preconditioning; and M, N-2-mercaptopropionyl glycine.

View larger version (19K): [in this window] [in a new window]   Figure 4. Expression of ICAM-1 assessed by PCR. Results are expressed as a ratio of ICAM-1 to GAPDH. PC indicates preconditioning. Values are mean±SEM of 6 experiments. *P<0.05 vs A/R.

ICAM-1 Protein Expression
Figures 5 and 6 show that reoxygenation after prolonged anoxia was associated with a significant increase in the percentage of ICAM-1–positive EC (assessed by immunofluorescence), with a peak occurring after 9 hours of reoxygenation, after which the number of positive cells declined and returned to baseline between 12 and 24 hours. This increased expression was markedly reduced by preconditioning and abolished by MPG, whereas combination of preconditioning and MPG did not further reduce ICAM-1 compared with MPG or preconditioning alone. Increased ICAM-1 expression was also reduced by PDTC, although this effect was less marked than after MPG (72±1%; P<0.05)

View larger version (22K): [in this window] [in a new window]   Figure 5. Immunostaining of rat ECs examined under confocal laser microscopy. Nuclei were stained red by propidium iodide to identify all present cells (both positive and negative for ICAM-1); slides were incubated with the use of a Cy2-streptavidin–labeled rat ICAM-1 antibody. Focal cytoplasmic staining was also visible in negative controls and thus cells showing such focal staining were considered ICAM-1 negative. Left, Anoxia without reoxygenation; center, anoxia followed by 9 hours of reoxygenation; right, preconditioning+anoxia followed by 9 hours of reoxygenation.

View larger version (18K): [in this window] [in a new window]   Figure 6. Percentage of ICAM-1–positive ECs, assessed as described in Figure 5. PC indicates preconditioning. Values are mean±SEM of 4 experiments. *P<0.05 vs A/R.

Adhesion of Neutrophils
Figure 7 shows that A/R induced a 5-fold increase in neutrophil adherence to ECs. This increased neutrophil adhesion was reduced by >40% by preconditioning.

View larger version (14K): [in this window] [in a new window]   Figure 7. Ratio of adhesion of neutrophils to ECs. PC indicates preconditioning. Values are mean±SEM of 4 experiments. *P<0.05 vs A/R.

Mechanisms of Preconditioning
Figure 8 shows the effects on the preconditioning-induced inhibition of ICAM-1 expression of the antagonist of adenosine receptors by SPT, the free radical scavenger MPG, the inhibitor of protein kinase C chelerythrine, or the NO synthase inhibitor L-NA. Chelerythrine, MPG, and L-NA (given during the 2-hour period of preconditioning) but not SPT abolished the effect of preconditioning.

View larger version (19K): [in this window] [in a new window]   Figure 8. Effect of antagonist of adenosine receptor antagonist SPT, inhibitor of protein kinase C chelerythrine, free radical scavenger MPG, or NO synthase inhibitor L-NA on percentage of ICAM-1–positive ECs (assessed by immunoperoxidase) in control (A/R only) or preconditioned ECs. Values are mean±SEM of 6 experiments. *P<0.05 vs control.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The main results of our study, performed in cultured ECs, are that (1) A/R markedly increased the endothelial expression of ICAM-1 as well as neutrophil adhesion to ECs, (2) both ICAM-1 expression and neutrophil adhesion were markedly reduced by preconditioning and by the free radical scavenger MPG, and (3) the mechanisms of the endothelial protective effects of preconditioning appear to involve production of NO and free radicals, together with activation of protein kinase C.

We and others have previously demonstrated that preconditioning may protect ECs against ischemia/reperfusion injury, especially at the level of large and medium-sized coronary arteries.^{2 3 4} Moreover, there is also evidence that reperfusion-induced coronary endothelial injury is due to the adhesion of neutrophils to ECs, secondary to the endothelial expression of neutrophil adhesion molecules such as ICAM-1. In this context, our experiments suggest that the endothelial protective effects of preconditioning may be due in part to a reduced expression of neutrophil adhesion molecules, leading to a reduced adhesion of neutrophils to the endothelium.

Two distinct phases of preconditioning have been described: an early phase, in which effects rapidly disappear after 3 to 6 hours of reperfusion, and a delayed phase ("second window" of preconditioning), appearing after 12 to 24 hours of reperfusion. In the present experiments, the preconditioning protocol consisted of 1-hour anoxia followed by 1 hour of reoxygenation and thus corresponds to the first window of preconditioning;

The present study was performed with aortic ECs. Thus extension of the present results to the coronary circulation must be performed with caution. At the level of the coronary circulation, preconditioning has been shown to induce endothelial protective effects at the level of large or medium-sized arteries² but not at that the level of the microcirculation.¹⁷ Techniques available for rat coronary ECs only allow isolation of cells from the microcirculation, and isolation of a sufficient number of cells from rat epicardial conduit arteries did not appear feasible because of the small size of these arteries. Thus we chose to perform experiments on cells obtained from another conduit artery, that is, the aorta.

Our experiments confirm that A/R increases neutrophil adhesion to ECs. Although several endothelial adhesion molecules may be involved in this adhesion (for example, P and E selectins), there is evidence that reoxygenation or reperfusion-induced increased neutrophil-endothelial interactions are mediated through the interaction between CD18/CD11b and ICAM-1.¹⁸ Indeed, anti–ICAM-1 antibodies prevent endothelial injury after ischemia and reperfusion¹⁰ and markedly reduce neutrophil adhesion in reoxygenated cultured ECs,¹¹ showing that ICAM-1 is essential for adhesion of neutrophils in our model. Thus it is likely that the changes in neutrophil adhesion observed in the present study are related at least in part to the changes in ICAM-1 expression. However, whether other factors, such as expression of selectins and of complement, also contribute to the effect of preconditioning remains to be demonstrated.

In the present study, increased ICAM-1 expression was seen only after 3 to 6 hours of reoxygenation. This time course is in agreement with previous data obtained in cultured ECs¹¹ but contrasts with in vivo data in which increased ICAM-1 expression has been observed after 1 hour of reperfusion.⁸ This could be due to the fact that the kinetics of the cellular changes may be slower in culture conditions. It must be noted, however, that endothelial dysfunction after reperfusion is a long-term phenomenon^{4 19} and that even delayed changes affecting endothelial injury or function may have important long-term consequences, for example, in terms of prevention of spasm or atherosclerosis.

Treatment of ECs with MPG abolished the reoxygenation-induced expression of ICAM-1 and also markedly reduced the increased neutrophil adhesion, suggesting that these events are mediated by a reactive oxygen species, in agreement with previous studies.^{11 20} Indeed, the expression of ICAM-1 is controlled by transcription factors such as NF-B²¹ or AP-1,²² which are themselves activated by free radicals.^{23 24} This is also supported by the fact that in our experiments, the expression of ICAM-1 is also inhibited by PDTC, a putative inhibitor of NF-B. Finally, although we have not measured the production of free radicals in our model and have not tested other free radical scavengers, the results obtained here with MPG, which is a hydroxyl radical scavenger, suggest that hydroxyl radicals are indeed involved in the increased expression of ICAM-1 during reoxygenation. This is in agreement with our in vivo experiments, in which MPG completely prevented coronary endothelial dysfunction after ischemia and reperfusion.²⁵

We found that the adenosine antagonist SPT failed to inhibit the endothelial effect of preconditioning, in contrast with previous experiments in cultured human ECs.⁵ This discrepancy could be due to the different end points measured in the 2 studies, that is, ICAM-1 in the present study and cell death in the study of Zhou et al.⁵ The present study did not use cell death as a primary end point because mortality rate was much lower in our experiments than in those of Zhou et al. This difference in mortality rates could be due to the different cell types used in the 2 studies. Alternatively, the different role of adenosine could be due to species differences. Indeed, adenosine has been shown to play a role in preconditioning in various species (rabbits, dogs, pigs) but not in rats,^{26 27} from which our ECs were obtained.

The inhibitory effect of preconditioning on ICAM-1 expression was abolished by chelerythrine given during preconditioning, suggesting that it is mediated by protein kinase C. There is evidence that the beneficial effects of preconditioning at the level of the myocyte or ECs are mediated by protein kinase C.^{5 28 29} Thus our data extend the role of protein kinase C in preconditioning with regard to its effects on endothelium-neutrophil interactions.

The free radical scavenger MPG also abolished the effect of preconditioning on ICAM-1 expression. This suggests that in addition to being mediators of the expression of ICAM-1 after prolonged anoxia, free radicals (and possibly especially hydroxyl radicals) produced during reperfusion after brief anoxia act as triggers of the protective effect of preconditioning. Such a dual effect of MPG has already been described with delayed preconditioning, especially with regard to its effect on stunning³⁰ or endothelial dysfunction.²⁵ However, the role of free radicals as a trigger for "classic" preconditioning is debated.^{31 32 33 34} Moreover, to the best of our knowledge, no study has assessed the role of free radicals as triggers for the effect of classic preconditioning at the level of the EC.

The effects of preconditioning on ICAM-1 were also abolished by L-NA, suggesting that they require NO. Exogenous administration of NO or stimulation of endogenous production of this factor has been shown to exert potent endothelial protective effects in ischemia/reperfusion.³⁵ Indeed, NO is a potent inhibitor of leukocyte adhesion, at least in part through decreased expression of adhesion molecules.^{36 37} However, this cannot explain how NO produced during preconditioning (ie, before prolonged anoxia) may affect ICAM-1 expression during reoxygenation after prolonged anoxia.

Taken together, our data show that NO, free radicals, and protein kinase C are essential triggers of preconditioning in our model. One possible explanation is that combined production of NO and free radicals during reoxygenation after brief anoxia induces an early activation of protein kinase C, and this leads to a lesser production of free radicals during reoxygenation after prolonged anoxia, possibly through protein kinase C–mediated increased antioxidant defenses.³⁸ Such decreased production of free radicals would then lead to decreased neutrophil adhesion and thus to endothelial protection.

In conclusion, we demonstrated that preconditioning reduced the free radical–mediated expression of endothelial adhesion molecules as well as the increased adhesion of neutrophils to ECs. Given the central role of leukocyte-endothelium interactions in the pathogenesis of vascular injury, identification of the cellular mechanisms of this potent endogenous protective mechanism may lead to the development of new treatments that may protect the vascular wall not only after ischemia/reperfusion but also in the various pathological conditions associated with an increased oxidative stress (such as hypercholesterolemia or hypertension).

	Acknowledgments

 
This work was supported in part by a grant from the Fondation de France. Fabienne Tamion is the recipient of a fellowship from the Groupe de Réflexion sur la Recherche Cardiovasculaire (GRRC).

Received August 5, 1998; revision received March 15, 1999; accepted April 9, 1999.

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