Brazilian Journal of Medical and Biological Research (2006) 39: 1189-1196 Endothelial cell activation and hypoxia 1189 ISSN 0100-879X The blockade of cyclooxygenases-1 and -2 reduces the effects of hypoxia on endothelial cells M.A. Gloria1, 1Laboratório de Imunologia Clínica e Experimental, Divisão de Nefrologia, M.A. Cenedeze1, Universidade Federal de São Paulo, Hospital do Rim e Hipertensão, A. Pacheco-Silva1 Fundação Oswaldo Ramos, São Paulo, SP, Brasil and N.O.S. Câmara1,2 2Laboratório de Imunobiologia de Transplantes, Departamento de Imunologia, Instituto de Ciências Biomédicas IV, Universidade de São Paulo, São Paulo, SP, Brasil Abstract Correspondence Hypoxia activates endothelial cells by the action of reactive oxygen Key words N.O.S. Câmara species generated in part by cyclooxygenases (COX) production • Endothelial cell Laboratório de Imunologia Clínica enhancing leukocyte transmigration. We investigated the effect of • Hypoxia e Experimental specific COX inhibition on the function of endothelial cells exposed • Indomethacin Divisão de Nefrologia, UNIFESP to hypoxia. Mouse immortalized endothelial cells were subjected to • Cyclooxygenase Rua Botucatu, 740 • Heme oxygenase 1 04023-900 São Paulo, SP 30 min of oxygen deprivation by gas exchange. Acridine orange/ Brasil ethidium bromide dyes and lactate dehydrogenase activity were used Fax: +55-11-5573-9652 to monitor cell viability. The mRNA of COX-1 and -2 was amplified E-mail: [email protected] and semi-quantified before and after hypoxia in cells treated or not with indomethacin, a non-selective COX inhibitor. Expression of Research supported by CAPES and RANTES (regulated upon activation, normal T cell expressed and FAPESP (Nos. 04/08311-6 and secreted) protein and the protective role of heme oxygenase-1 (HO-1) 04/13826-5). were also investigated by PCR. Gas exchange decreased partial oxy- gen pressure (PaO2) by 45.12 ± 5.85% (from 162 ± 10 to 73 ± 7.4 mmHg). Thirty minutes of hypoxia decreased cell viability and en- Received August 19, 2005 hanced lactate dehydrogenase levels compared to control (73.1 ± 2.7 Accepted May 31, 2006 vs 91.2 ± 0.9%, P < 0.02; 35.96 ± 11.64 vs 22.19 ± 9.65%, P = 0.002, respectively). COX-2 and HO-1 mRNA were up-regulated after hy- poxia. Indomethacin (300 µM) decreased COX-2, HO-1, hypoxia- inducible factor-1α and RANTES mRNA and increased cell viability after hypoxia. We conclude that blockade of COX up-regulation can ameliorate endothelial injury, resulting in reduced production of chemokines. Introduction from recipients with delayed graft function are more associated with chronic allograft Acute renal failure, a clinical feature of nephropathy and consequently with a re- ischemia and reperfusion injury (IRI), re- duced survival rate (2,3). mains a major cause of morbidity and mor- The cellular mechanisms involved in IRI tality in hospitalized patients (1). In the trans- include ATP depletion, impaired calcium plantation area, IRI is closely related to early regulation, generation of reactive oxygen and long-term graft outcomes. Indeed, grafts species (ROS), and the production of pro- Braz J Med Biol Res 39(9) 2006 1190 M.A. Gloria et al. inflammatory cytokines/chemokines. All ucts of heme metabolism catalyzed by HO-1 these mediators ultimately contribute to ac- (reviewed in Ref. 13). The enhanced HO-1 tivation of endothelial cells (4,5). Endotheli- expression observed during hypoxia is due al cell activation is associated with pro-co- to activation of the transcription factor, hy- agulant and pro-inflammatory phenotypes. poxia-inducible factor (HIF)-1α. HIF-1α is The up-regulation of class II molecules of the a heterodimeric peptide involved not only in major histocompatibility complex, together the sensing, but also in the adapting of cells with secretion of chemokines, promotes leu- to changes in O2 levels. It is well established kocyte adhesion and further transmigration of that HIF-1 expression and activation corre- leukocytes through the endothelial barrier into late with tumor progression, cell apoptosis, the tissue. Regulated upon activation, normal resistance to cancer treatment, and angio- T cell expressed and secreted (RANTES) pro- genesis (14-16). tein is a key molecule involved in these events On the basis of these considerations, we which is secreted by endothelial cells and hypothesize that the beneficial effects ob- activated leukocytes (6,7). served with COX blockade in ischemic or- Cyclooxygenase (COX) is the key en- gan injuries might be mediated in part by the zyme implicated in the generation of prosta- amelioration of the response of endothelial glandins from arachidonic acid (8), a pro- cells to hypoxia. We addressed this question cess that promotes the generation of ROS by investigating the effect of specific COX (9). Both isoforms, COX-1 and COX-2, par- blockade (non-selective inhibition of COX- ticipate in the endothelial cell activation that 1 and COX-2) in an in vitro model of gas- follows IRI (9,10). We have shown that eous hypoxia. We anticipated that pre-treat- COX blockade in the murine model of IRI ment with indomethacin would promote bet- leads to better organ function and preserves ter endothelial cell viability and down-regu- the parenchymal architecture (11,12). The lation of COX-2 and RANTES messenger mechanisms underlying this beneficial ef- RNA (mRNA) in the presence of hypoxia. In fect are not completely understood. addition, COX inhibition was also associ- It has become apparent that the balance ated with a reduced expression of HO-1 and between the injury factors and the tissue HIF-1α gene transcripts under hypoxic con- responses that follow the initial damage is a ditions. determinant of organ outcome. The tissue can protect itself from injury by up-regulat- Material and Methods ing a group of genes with anti-apoptotic and anti-inflammatory properties, intended to Cells promote and maintain the physiological func- tions of the organs. Heme oxygenase-1 (HO- Mouse immortalized endothelial cells 1) is one of the recently described protective were purchased from the American Type of genes. The mechanisms underlying its role Culture Collection (ATCC, CRL-2167, Ma- are not clear, but probably rely on the ability nassas, VA, USA) and cultivated in Dul- of this gene to protect endothelial cells from becco’s modified Eagle’s medium (Gibco, undergoing apoptosis, to inhibit the pro- Carlsbald, CA, USA) supplemented with 5% inflammatory phenotype of activated mono- fetal bovine serum (Cultilab, Campinas, SP, cytes/macrophages, to promote vasodilata- Brazil), HEPES, and penicillin/streptomy- tion, to suppress platelet activation, and to cin. All cultures were maintained in 25-cm2 block smooth cell proliferation. These ef- polystyrene bottles in an incubator at 37ºC fects occur via generation of carbon monox- and humidified with a gaseous mixture of ide, biliverdin and free iron, the end prod- 95% air and 5% carbon dioxide. Braz J Med Biol Res 39(9) 2006 Endothelial cell activation and hypoxia 1191 Gas hypoxia and considered to be viable when they were stained green and nonviable when they were Each culture bottle was capped with a stained orange. Results are reported as per- silicone cork and sealed with paraffin. We cent viable cells. used two 30 x 7-mm needles to transfix the Cell viability was also measured by lac- cork to provide an access to infuse the gas- tate dehydrogenase (LDH) leakage through eous mixture containing 95% nitrogen and the membrane into the medium. This assay, 5% carbon dioxide. Three to 4 liters per which strongly correlates with the number minute of this gaseous mixture were infused of lysed cells, was based on the reduction of for 30 min, reducing the partial oxygen pres- nicotinamide-adenine dinucleotide (NAD) sure (PaO2, 162 ± 10 mmHg) in the culture to NADH by LDH (18). LDH activity in the medium (17). PaO2 was measured with a supernatant and in the cell homogenate was potentiometric electrode in a blood gas ana- determined by measuring absorbance at 340 lyzer. Cells were then harvested for the ex- nm. Cytotoxicity was reported as percent periments. LDH released, corresponding to the ratio between LDH activity in the supernatant and Drugs total LDH activity. Throughout the entire reading, the samples were maintained under Indomethacin (Sigma, St. Louis, MO, shaking and at a constant temperature of USA) was used at two different concentra- 25ºC. tions, 100 and 300 µM, for 72 h prior to the gaseous hypoxia. Controls included cells mRNA expression not treated with indomethacin or treated with the vehicle (DMSO, 10 and 30 µL), sub- RNA was extracted from endothelial cells jected or not to hypoxia. The cells were exposed or not to gaseous hypoxia, and pre- studied immediately after hypoxia, with no treated or not with indomethacin, using TRI- reperfusion. REAGENT™ (Sigma), according to the manufacturer’s protocol. RNA concentra- Protocol of cell injury tion was quantified with a spectrophotom- eter. Samples with an A260/A280 ratio above Cell viability was determined by the ex- 1.7 were considered to be free of DNA and clusion method using two fluorescent dyes. proteins. mRNA was reverse transcribed into After trypsinization the cells were resus- complementary DNA using expand reverse pended in 1 mL PBS. A solution of acridine polymerase (Boehringer Mannheim, East orange (100 µg/mL) and ethidium bromide Sussex, UK). The mRNA of COX-1, COX- (100 µg/mL) was added to the 10-µL cell 2, HO-1, HIF-1α, RANTES, and ß-actin sample. Ethidium bromide and acridine or- was amplified using specific primer se- ange excite orange and green fluorescence, quences: COX-1 280 bp, sense: 5' GGT GGA respectively, when they are intermingled GGT AGG AAC TTT GAC 3', anti-sense: 5' within the DNA. Ethidium bromide is elimi- GAG CCC CCA TCT CTA TCA TAC 3'; nated when the plasma membrane is intact COX-2: 448 bp, sense: 5' CAT TGA AGA and therefore only acridine orange crosses CCA GGA GTA 3', anti-sense: 5' CCA GTA the membrane, leading to green fluorescence.