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Eicosapentaenoic Acid ProtectsEndothelial Cell Function Injured byHypoxia/Reoxygenation

IKUO MORITA, YOU-WEI ZHANG, AND SEI-ITSU MUROTA

Department of Cellular Physiological Chemistry, Graduate School,Tokyo Medical and Dental University, Bunkyo-ku, Tokyo, Japan

ABSTRACT: Eicosapentaenoic acid (EPA) may protect against atherosclerosis byimproving lipid metabolism and modulating vascular cell function. Ischemia/reperfusion injury is one risk factor for atherosclerosis. We investigated if EPAcould improve hypoxia/reoxygenation (H/R)-induced endothelial cell dysfunc-tion of gap junctional intercellular communication (GJIC). GJIC in humanumbilical vascular endothelial cells (HUVECs) was measured using a pho-tobleaching technique. Results demonstrated that H (24h)/R 2h) induced aGJIC reduction in HUVECs; however, it was inhibited by EPA pretreatment.H/R produced reactive oxygen species, but it was not affected by EPA, and itcontributed little to GJIC dysfunction. By contrast, tyrosine kinase activatedby H/R was inhibited by EPA pretreatment, and tyrosine kinase inhibitors alsoabolished H/R-induced GJIC reduction. The protective effects of EPA on theH/R-induced GJIC reduction was also observed in cells treated with tyrosinephosphatase inhibitor. These data indicate the EPA improves H/R-inducedendothelial dysfunction through inhibition of tyrosine kinase activation, and itcould lead to prevention of progression and/or initiation of atherosclerosis.

KEYWORDS: eicosapentaenoic acid; EPA; endothelial cell function; hypoxia;reoxygenation; tyrosine kinase

INTRODUCTION

Eicosapentaenoic acid (EPA), n-3 polyunsaturated fatty acid, may protect againstatherosclerosis by improving lipid metabolism and modulating vascular cell func-tion. Ischemia/reperfusion injury is one risk factor for atherosclerosis. In this study,we investigated whether EPA could improve hypoxia/reoxygenation (H/R)-inducedendothelial cell dysfunction of gap junctional intercellular communication (GJIC).The results demonstrated that H(24 hours)/R(2 hours) induced a GJIC reduction inhuman umbilical vein endothelial cells (HUVECs); however, it was inhibited by EPApretreatment (3 mg/ml for 2 days). H/R produced reactive oxygen species, and it waspartly inhibited by EPA, but contributed little to GJIC dysfunction. By contrast, ty-rosine kinase activated by H/R was inhibited by EPA pretreatment, and tyrosine ki-

Address for correspondence: Dr. Ikuo Morita, Department of Cellular Physiological Chemis-try, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo113-8549, Japan. Voice: +81-5803-5575; fax: +81-3-5803-0212.

morita.cell@tmd.ac.jp

395MORITA et al.: ENDOTHELIAL CELL FUNCTION

nase inhibitors also abolished H/R-induced GJIC reduction. The protective effect ofEPA on H/R-induced GJIC reduction was also observed in cells treated with tyrosinephosphatase inhibitor. These data indicate that EPA improves H/R-induced endothe-lial dysfunction through inhibition of tyrosine kinase activation, and it would lead toprevent progression or/and initiation of atherosclerosis.

MATERIAL AND METHODS

Isolation and cultivation of HUVECs have previously been reported,1 and a mod-ified in vitro H/R model was adopted.1,2 A cell-coupling assay was performed as pre-viously described, using the gap fluorescence recovery after photobleaching (gap-FRAP) technique.2,3 Tyrosine kinase activation was estimated by examining the in-tracellular production of tyrosine phosphorylated proteins using a modified proce-dure.2 To measure reactive oxygen species in living cells, cells were washed twicewith phosphate-buffered saline and cultured in medium with 5–10 mM CDCFDA for20 minutes. Medium was then washed out, and cells were rinsed three times withPBS to remove extracellular CDCFDA. Generation of reactive oxygen species wasthen measured with an ACAS 570 laser cytometer.

RESULTS AND DISCUSSION

As previously reported,2 the GJIC in control cells was not affected by 24 hoursof hypoxia alone. After 2 hours of reoxygenation after hypoxia, the GJIC in controlcells was significantly inhibited, whereas in EPA–pretreated cells this inhibition wasabrogated. After 4 hours of reoxygenation, the GJIC abnormality in control cells wasrestored, and after 6 hours of reoxygenation it nearly returned to normal. By contrast,no obvious reduction in GJIC was observed in EPA cells even after 12 hours of re-oxygenation. These data suggest that H/R induced a temporal GJIC reduction thatpeaked after 2 hours of reoxygenation, and EPA pretreatment prevented this GJICabnormality.

Studies have suggested that reactive oxygen species5 and tyrosine kinaseactivation6 are involved in H/R injuries. To understand how EPA protected H/R-injured GJIC, we examined the production of reactive oxygen species and activationof tyrosine kinase. As illustrated in TABLE 1a, generation of reactive oxygen speciesreached a peak after 30 minutes of reoxygenation; 2 hours later it significantly de-creased, and after 4 hours of reoxygenation it nearly returned to normal. EPA and thereactive oxygen species inhibitor DMSO both significantly inhibited reactive oxy-gen species production, whereas the tyrosine kinase inhibitor genistein did not showany effect. Tyrosine kinase activation was then investigated. The data in TABLE 1bdemonstrate that tyrosine kinase was maximally activated after 2 hours of reoxygen-ation, and 4 hours later it nearly returned to normal. The time-course activation oftyrosine kinase was consistent with that of GJIC reduction. Pretreatment with EPAor genistein completely inhibited this tyrosine kinase activation, whereas DMSOshowed no effect.

Next, to determine how GJIC was inhibited by H/R, we examined the effects ofinhibitors of both reactive oxygen species and tyrosine kinase on H/R-injured GJIC.

396 ANNALS NEW YORK ACADEMY OF SCIENCES

Reactive oxygen species inhibitors DMSO and SOD did not protect GJIC reduction;however, EPA and the tyrosine kinase inhibitor genistein inhibited it. These data in-dicate that tyrosine kinase activation might be the reason for H/R to injure GJIC, andEPA protected this injury by blocking tyrosine kinase activation. To further confirmthis, we examined the effects of EPA on tyrosine kinase activator vanadate-inducedtyrosine kinase activation and GJIC dysfunction. As illustrated in TABLE 2a, vana-date enhanced tyrosine kinase activation at H/R; however, EPA pretreatment attenu-ated this increase in tyrosine kinase activation. Similarly, GJIC was further inhibitedin the presence of vanadate in control cells at H/R, whereas in EPA- pretreated cellsthis reduction in GJIC was significantly prevented (TABLE 2b). These data suggest

TABLE 1. Production of reactive oxygen species (ROS) and activation of tyrosinekinase (TK)

a. Normoxia

Reoxygenation

0.5 h 2 h 4 h 6 h

Control 100 248 148 115 118

EPA 95 176 135 104 102

DMSO 98 174 106 94 86

Gnistein 102 239 139 114 118

b. Normoxia

Reoxygenation

0 h 2 h 4 h 6 h

Control 100 110 197 106 97

EPA 97 102 113 103 105

DMSO 102 112 184 96 93

Gnistein 98 5 112 93 86

NOTE: The amount of ROS or TK activation of control cells at normoxia was regarded as100%. a, production of ROS; b, TK activation.

TABLE 2. Effects of eicosapentaenoic acid (EPA) on vandate-induced (tyrosinekinase (TK) and gap junctional intercellular communication

NOTE: a, TK activation. Control cells at normoxia were regarded as 100%. b, GJIC. Valuesrepresent mean ± SEM from 3 to 6 separate experiments; ( ) means the analyzed cell number. **p<0.01 vs. norm0xia control.

a. Control EPA Vanadate Vanadate-EPA

Normoxia 100 96 160 123

Reoxygenation 2 h 145 96 197 129

b. Control EPA Vanadate Vanadate-EPA

Normoxia 96.59 ± 2.84(20)

93.52 ± 3.84(31)

76.61 ± 2.27(46)**

85.10 ± 3.46(51)

Reoxygenation 2 h 77.13 ± 3.40(35)**

97.34 ± 3.19(27)

57.74 ± 2.78(23)**

87.68 ± 2.78(60)

397MORITA et al.: ENDOTHELIAL CELL FUNCTION

that EPA protected against H/R-induced GJIC reduction through inhibition of ty-rosine kinase activation.

The present study shows that pretreatment with EPA inhibited H/R-induced tem-poral GJIC injury by inhibition of tyrosine kinase activation, but not reactive oxygenspecies production.

REFERENCES

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