Hydroxyurea downregulates endothelin-1 gene expression and upregulates ICAM-1 gene expression in cultured human endothelial cells

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1 The Phrmcogenomics Journl (23) 3, & 23 Nture Pulishing Group All rights reserved X/3 $25. ORIGINAL ARTICLE Hydroxyure downregultes endothelin-1 gene expression nd upregultes ICAM-1 gene expression in cultured humn endothelil cells M Brun 1,2,3 S Bourdoulous 1,4,5 PO Courud 1,4,5 J Elion 1,3,6 R Krishnmoorthy 1,3,6 C Lpoumeroulie 1,3,6 1 Institut Ntionl de l Snté et de l Recherche Médicle (INSERM), Frnce; 2 Université des Antilles et de l Guyne, Fculté de Medeciné UMR458, Frnce; 3 Institut Clude Bernrd, IFR2/UMR458 Biologie du developpement, Frnce; 4 Université Pris 5, René Descrtes Fculte de Medecine Cochin, Port Royl, Frnce; 5 Institut Cochin, UMR 567 Mldies Infectieuses, Frnce; 6 Université Pris 7, Denis Diderot Fculté de Médecine Xvier Bicht, Frnce Correspondence: C Lpouméroulie, UMR 458, Hôpitl Roert Deré, 48 Bd Sérurier, 7519 Pris, Frnce. Tel: , Fx: E-mil: lpoumer@idf.inserm.fr ABSTRACT The clinicl efficcy of orl hydroxyure (HU) in dults nd children with sickle cell nemi (SCA) cnnot solely e explined y its ility to enhnce fetl hemogloin (HF) expression. Since incresed dherence of sickle red lood cells to vsculr endothelium is possile contriuting fctor to vsoocclusive crisis (VOC), we explored the effect of HU on humn endothelil cell (EC) lines (TrHBMEC nd EA-hy 926). We demonstrted tht HU, in dose-dependent nd reversile mnner, significntly decresed (up to threefold) the relese of endothelin-1 (ET-1), vsoconstrictor peptide through downregultion (up to three-fold) of ET-1 gene expression. This finding is of therpeutic relevnce s SCA ptients exhiit elevted serum levels of ET-1 during episodes of VOC nd levels correlte with disese severity. Unexpectedly, HU upregulted (up to three-fold) the expression of memrne-ound intercellulr cell dhesion molecule 1 (micam-1) nd its solule form (sicam-1) with prllel increse in ICAM-1 mrna expression. Although ICAM-1 does not pper to e involved in the sickle cell dhesion to vsculr endothelium, it my excerte vso-occlusion y promoting leukocyte dhesion. The HU-induced increse in micam-1 my pper inconsistent with the clinicl enefits confered y HU. However, oth the increse in sicam-1- nd HU-induced leukocyte reduction in ptients, my counterct the potentilly detrimentl effect of elevted micam-1 expression. Also HU reduces the expression of vsculr cell dhesion molecule (VCAM-1) on EC. Since HU reduces the very lte ntigen 4-positive reticulocytes in SCA ptients, lignd for VCAM-1, HU-induced downregultion of VCAM-1 on EC will very likely decrese the reticulocyte endothelium dhesion. Thus, HU, prt from inducing HF expression in the red cell, lso ffects the expression profile of EC comprtment. The Phrmcogenomics Journl (23) 3, doi:1.138/sj.tpj Keywords: sickle cell nemi; hydroxyure; endothelium Received: 8 Octoer 22 Revised: 7 Mrch 23 Accepted: 2 Mrch 23 INTRODUCTION Hydroxyure (HU) is n inhiitor of rionucleotide reductse, rte-limiting enzyme, involved in the conversion of rionucleotides into deoxyrionucleotides. Thus, HU ffects cells tht re ctively synthesizing DNA. HU hs long een used to tret vriety of neoplstic disorders including chronic grnulocyte leukemi, hed nd neck cncer nd polycythemi ver. In ddition to inhiiting rionucleotide reductse, HU ffects gene expression, most notly of the humn fetl gloin genes. HU enhnces the expression of fetl hemogloin (HF) in cell culture nd primte models. 1,2 As sickle cell nemi (SCA) ptients

2 216 Downregultion of endothelin-1 nd upregultion of ICAM-1 gene expressions with high levels of HF experience less severe form of the disese, HU therpy ws tested in severl clinicl trils in ptients with SCA to induce HF expression. In clinicl trils, HU therpy hd mny positive clinicl nd iologicl enefits. The clinicl enefits include lrge reduction in the frequency of hospitliztions, pin episodes, cute chest syndromes (ACS) nd lood trnsfusions. 3 Biologiclly, HU therpy incresed HF levels, F reticulocyte count, F-cell count, men corpusculr volume, red lood cell (RBC) survivl nd deformility, oxygen ffinity, ction content nd hydrtion. Also, HU decresed the men corpusculr H concentrtion, hemolysis, numer of irreversily sickled red cells, KCl cotrnsport 4 8 expression of red cell dhesion molecules nd their in vitro dhesion. 9 Although the efficcy of HU therpy in SCA ws elieved to e medited primrily through the induction of HF expression, the improvement in clinicl symptoms preceding ny significnt increse in HF rises the possiility tht HU my lso ct through other mechnisms. 1 In SCA ptients treted with HU, sickle cell endothelil cell (EC) dherence decresed prior to ny significnt increse in HF. 11 This suggests tht HU my ffect the RBC nd/or EC surfce. Very few studies hve explored the effect of HU on EC function. 9 ECs ply mjor role in vsodhesion of vriety of lood cells vi cell surfce dhesion molecules, such s intercellulr cell dhesion molecule 1 (ICAM-1) nd vsculr cell dhesion molecule 1 (VCAM-1). RBCs from SCA ptients (SS RBCs) hve the predilection to dhere to the endothelil surfce s compred to the RBCs from norml sujects (AA RBCs). 12 Such norml RBC endothelil dherence is elieved to initite pinful vso-occlusive crisis (VOC), physiopthologic hllmrk of the disese. ECs respond to numerous extrcellulr signls such s vsoctive fctors (ET-1 nd NO), proinflmmtory cytokines (TNF, interleukin 1 nd IFNg), oxidnts nd hypoxi. 13 Vritions in these signls cn ffect the EC chrcteristics nd therey contriute to the unpredictle occurrences of VOC. The resulting endothelil injury, indicted y the presence of ctivted ECs circulting in the lood of SCA ptients, 14 could contriute to dditionl complictions. Here, we evlute the effects of HU on vsculr ECs. RESULTS HU Reduces ET-1 mrna nd Peptide Expression TrHBMEC nd EA-hy 926 cells were exposed to HU t concentrtion (25 mm) similr to the in vivo HU levels of treted SCA ptients. Experiments were performed oth in the sence nd in the presence of 1 U/ml of TNF nd IFNg ( concentrtion tht induces the expression of dhesion molecules, ut does not ffect the synthesis nd relese of endothelin-1, ET-1). The presence of proinflmmtory cytokines llowed simultion of the in vivo inflmmtory sttus of SCA ptients. Under sl culture conditions, the mount of ET-1 peptide relesed into the medium ws comprle for the two endothelil cell types: pg/ml for TrHBMEC, pg/ml for EA-hy 926 (Figure 1) nd ws not significntly ltered y the presence of proinflmmtory cytokines. The presence of HU significntly reduced ET-1 peptide relese from TrHBMEC ( pg/ml) nd EA-hy 926 cells ( pg/ml) nd the mgnitude of the reduction ws similr in the presence of cytokines ( , pg/ml, respectively). In the presence of HU (48 h exposure), there ws lso decrese in ET-1 mrna s ssessed y the rel-time quntittive PCR (Figure 2). In TrHBMEC cells, there ws significnt reduction in ET-1 mrna level ( % of the sl vlue), wheres reduction ws greter ( % of the sl vlue) when HU tretment ws pplied in the presence of two inflmmtory cytokines (Figure 2). ET-l (pg/ml) ET-l (pg/ml) Figure 1 Effect of HU tretment on ET-1 relese. Anlyzed y ELISA. Results re the men7sd in picogrm of ET-1/ml for 1 5 cells of four to five independent experiments in duplicte. Control: nontreted cells, HU: cells treted with HU 25 mm for 48 h, Cyto: cells treted with TNF nd IFNg t 1 U/ml for 48 h, HU + Cyto: comintion of HU nd Cyto. () TrHBMEC cells, () EA hy 926 cells. *Mens P vlueo.5, **Po.1. The Phrmcogenomics Journl

3 Downregultion of endothelin-1 nd upregultion of ICAM-1 gene expressions Cytokines + Cytokines ET-l (% residul expression) ET-l (pgml) 1 5 ET-l (% residul expression) Control HU HU+Cyto ET-l (% residul expression) HU (µm) - Cytokines + Cytokines Control HU HU+Cyto Figure 2 Effect of HU on ET-1 gene expression. Anlyzed y reltime quntittive RT-PCR (TqMn 77). Results re expressed s percentge of residul ET-1 gene expression compred to nontreted cells for HU- nd cytokine-treted cells for HU+cyto. The reference gene used ws the TBP gene. Results re men77sd of five independent experiments in duplicte. Control: nontreted cells/cyto-treted cells, HU: cells treted with HU 25 mm for 48 h, HU + Cyto: cells treted with TNF nd IFNg t 1 U/ml nd HU 25 mm for 48 h. () TrHBMEC cells nd () EA hy 926 cells. *Mens P vlueo HU (µm) Figure 3 Dose response effect of HU on ET-1 relese nd expression on TrHBMEC cells. Cells were incuted for 48 h with vrious concentrtions of HU ( lm) in the presence (+ cytokines) or sence ( cytokines) of TNF, IFNc t 1 U/ml. ET-1 concentrtions were nlyzed y ELISA for ET-1 relese nlysis () nd ET-1 expression y rel-time RT PCR (). Results re men7sd of t lest three independent experiments. Although trend towrds decrese ws oserved for EA-hy 926 cells with residul mrna level of % of the sl vlue with HU lone difference ws noted in the presence of oth HU nd cytokines. The decrese ws much less pronounced (only % of the sl vlue) in these cells. Such differences my represent the difference in cell lines. To explore if ET-1 expression is HU dose dependent, the TrHBMEC cells were incuted with HU t concentrtions from 62.5 to 5 mm (eyond 5 mm, HU ws found to e consistently lethl for endothelil cells). Both the decrese in ET-1 peptide relese (Figure 3) nd mrna undnce (Figure 3) were found to e HU dose dependent. The decrese in mrna ws much more prominent thn the peptide relese for HU concentrtions of 25 mm or lower, especilly in the presence of inflmmtory cytokines. HU Effect on ET-1 mrna Expression is Reversile To study whether the effect of HU on ET-1 peptide relese nd mrna concentrtion ws reversile, EA-hy 926 cells were incuted for 48 h with HU (25 mm) nd then in HUfree DMEM growth medium oth with nd without cytokines (Figure 4). Since TrHBMEC cells strted to detch from 72 h onwrds nd very few cells remined dhered fter 96 h tretment with HU, reversile effect of HU ws tested only with EA-hy 926 cells. Nevertheless, the overll

4 218 Downregultion of endothelin-1 nd upregultion of ICAM-1 gene expressions 1 15 ET-l (% residul expression) ET-l (pg/ml) h +HU 48h -HU 96h 1 48h +HU 48h -HU 96h 15 ET-l (% residul expression) ET-l (pg/ml) h HU+Cyto Cyto 96h 48h Figure 4 Reversiility of ET-1 expression decrese in EA-hy 926 cells. Anlyzed y rel-time RT-PCR. Cells were treted for 48 h with HU 25 lm, then the culture medium ws replced y fresh medium free of HU for 48 dditionl hours for totl experiment time of 96 h. () Anlysis in sl conditions nd () Anlysis in inflmmtion conditions, TNF, IFNc t 1 U/ml: cyto. Results re men7sd of t lest three independent experiments. *Mens P vlueo.5 etween HU + Cyto nd Cyto. trend in expression of ET-1 mrna in TrHBMEC cells ws similr to tht of EA-hy 926 cells up to 72 h (dt not shown). After exposure of cells to HU (without cytokines) for 48h, the mount of ET-1 mrna ws % of the sl level ut rose to % 48 h fter the removl of HU from the medium. Exposure of cells to HU in the presence of cytokines resulted in decrese of ET-1 mrna ( % of the sl level) nd fter its removl, recovery ws lmost complete ( % of control) (Figure 4). With the sme experimentl setup, we exmined the sttus of ET-1 peptide relese into the culture superntnt. Under sl conditions (control), ET-1 secretion ws 48h HU+Cyto Cyto 96h 48h Figure 5 Reversiility of ET-1 synthesis decrese in EA hy 926 cells. Anlyzed y ELISA. Cells were treted for 48 h with HU 25 lm, then the culture medium ws replced y fresh medium free of HU for 48 dditionl hours for totl experiment time of 96 h. () Anlysis in sl conditions nd () Anlysis in inflmmtion conditions, TNF, IFNc t 1 U/ml: cyto. Results re men in picogrm of ET-1/ml for 1 5 cells7sd of t lest three independent experiments pg/ml nd in the presence of HU, it decresed to pg/ml (Figure 5). At 48 h fter the removl of HU, the ET-1 peptide relese remined low ( pg/ml). A similr trend ws noted in the presence of cytokines (Figure 5). Thus, HU inhiited synthesis nd relese of ET-1 peptide in dose-dependent mnner from two different endothelil cell lines oth in the presence nd sence of proinflmmtory cytokines. Our dt further demonstrted tht the The Phrmcogenomics Journl

5 Downregultion of endothelin-1 nd upregultion of ICAM-1 gene expressions 219 downregultion of ET-1 y HU occurs t the trnscriptionl level. HU Upregultes Memrne-Bound ICAM-1 Expression Flow cytometry ws used to investigte the effect of HU on the expression of memrne-ound ICAM-1 (micam-1) y TrHBMEC nd EA-hy 926 cells. The results re sed on the nlysis of 5 events. In the presence of HU lone, TrHBMEC cells exhiited slight increse in micam-1 expression with men fluorescence index (MFI) of 8.1 s compred to the sl vlue of MFI 2.6 (Figure 6). In the presence of proinflmmtory cytokines, there ws lrge increse in micam-1 expression (MFI 24.1). These cells were incuted with oth HU nd cytokines, nd synergistic effect on the expression of micam-1 ws oserved (MFI 79). The effect of HU on EA-hy 926 cells ws similr to tht oserved for TrHBMEC cells (Figure 6). HU Enhnces Relese of Solule ICAM-1 In the presence of proinflmmtory cytokines, HU induced two-fold increse in the relese of solule ICAM-1 (sicam- 1) into the culture superntnt ( vs ng/ml in the presence of cytokines lone) (Figure 6). Without cytokines, sicam-1 ws not detectle in the superntnt of TrHBMEC cells. sicam-1 relese from control nd HUtreted EA-hy 926 cells ws indistinguishle (dt not shown). HU Provokes Sustined Induction of ICAM-1 mrna Expression ICAM-1 mrna ws nlyzed y rel-time quntittive PCR. Results re reported s men7sd fold induction in HUtreted cells compred to control. Exposure of cells for 24 h to HU lone did not modify the ICAM-1 mrna level (1.17.3). However, in the presence of cytokines nd HU, ICAM-1 mrna undnce incresed up to 5.6-fold, similr MFI 5 MFI 25 1 c 3 d 2 sicam-l (pg/ml) 2 1 IL-(pg/ml) 1 Cytokines HU+Cytokines NT HU Cyto HU+Cyto Figure 6 Anlysis of the effect of HU on micam-1, sicam-1 nd IL-6 relese. Control: nontreted cells, HU: cells treted with HU 25 lm for 48 h, Cyto: cells treted with TNF nd IFNc t 1 U/ml for 48 h, HU + Cyto: comintion of HU nd Cyto. () FACS nlysis of micam- 1 expression in TrHBMEC cells; the results re expressed s the MFI for one representtive experiment of three. () Sme conditions s () ut in EA hy 926 cells. (c) ELISA nlysis of sicam-1 relese into the culture superntnt of TrHBMEC cells. Results re mens in picogrm of ET-1/ml for 1 5 cells7sd of six independent experiments. No sicam-1 ws detectle in the culture superntnt of unstimulted cells. (d) ELISA nlysis of IL-6 relese into the culture superntnt of TrHBMEC cells. Results re mens in nnogrm of ET-1/ml for 1 5 cells7sd of three independent experiments. *Mens P vlueo.5.

6 22 Downregultion of endothelin-1 nd upregultion of ICAM-1 gene expressions to vlues otined for cytokines lone ( ) (Figure 7). Exposing the cells for 48 h to HU lone, led to fold increse in ICAM-1 mrna. Under the sme conditions, the cytokines lone cused fold increse in the ICAM-1 mrna, wheres in comintion with HU, the increse ws fold. In seprte time-course experiments, we oserved tht the mgnitude of increse in ICAM-1 mrna y cytokines nd HU peked t 24 nd 48 h, respectively. In ddition, we noted tht HU provoked sustined increse in ICAM-1 mrna undnce ut only in the presence of proinflmmtory cytokines. ICAM-1 (fold induction / control) ICAM-1 (fold induction / control) Figure 7 Anlysis of the effect of HU on ICAM-1 gene expression in TrHBMEC cells. Anlyzed y rel-time RT-PCR. Control: nontreted cells, HU: cells treted with HU 25 lm, Cyto: cells treted with TNF nd IFNc t 1 U/mlfor 48 h, HU + Cyto: comintion of HU nd Cyto. The mplifiction frgment is locted on the extrcellulr domin of ICAM-1 sequence. () At 24 h of HU tretment nd () 48 h of HU tretment. Results re mens7sd of fold induction/ nontreted cells of three independent experiments. HU Decreses Relese of IL-6 A slight decrese in the relese of IL6 ws oserved in HUtreted cells ( pg/ml) s compred to nontreted cells ( pg/ml) (Figure 6d). In the presence of proinflmmtory cytokines nd HU, the relese of IL-6 ( pg/ml) ws significntly lower thn tht y cells treted with cytokines lone ( pg/ml). HU Downregultes the Memrne-ound VCAM-1 Expression without Affecting svcam-1 Relese The mximl expression of memrne-ound VCAM-1 (mvcam-1) in the presence of HU nd cytokines ws etween 12 nd 24 h (dt not shown). A 24 h exposure of TrHBMEC cells to HU lone (MFI.3) did not modify the expression of mvcam-1. In the presence of proinflmmtory cytokines, 5% of the cells ecme positive for mvcam-1 (MFI 4.1), s ssessed y the flow cytometry. Addition of HU to cytokine-treted cells reduced the MFI to 2.9 nd the percentge of mvcam-1- positive cells to 36% (Figure 8). A 24 h exposure of TrHBMEC cells to only HU did not modify the relese of svcam-1 into the culture superntnt ( ng/ml s compred to ng/ml for the untreted control). Cytokines incresed the level of svcam-1 relese ( ng/ml), ut the presence of HU in ddition to cytokines hd no significnt effect ( ng/ml) (Figure 8). VCAM-1 mrna Expression is Downregulted y HU VCAM-1 mrna ws studied y rel-time quntittive PCR. Results re mens7sd of fold induction s compred to control. ECs exposed for 24 h to proinflmmtory cytokines produced lrge increse (25-fold) in VCAM-1 mrna undnce (Figure 9). When these cells were treted with HU nd cytokines together, the mgnitude of increse in VCAM-1 mrna ws limited to 15-fold. The effects of vrious concentrtions of HU (62.5, 125, 25 nd 5 mm) in the presence of cytokines were tested nd the decrese in VCAM-1 mrna undnce ws HU dose dependent (Figure 9). Production of NO nd Expression of NO Synthse Only trce mounts of NO derivtives were detected in the culture superntnt in ll the experimentl conditions. The constitutively synthesized endothelil NO synthse (enos) did not vry etween the different culture conditions (42759 pg/ml for untreted control, pg/ml for HU treted cells, pg/ml for cytokines treted cells nd pg/ml for HU- nd cytokine-treted cells). We could not detect the inducile NOS (inos) mrna in these experiments, lthough the control mrna from A549 cell line provided the expected positive signl. Effect of HU on Endothelil Cell Cycle Chnges The cytosttic effect of HU ws exmined under our experimentl conditions. Both the TrHBMEC nd EA-hy 926 cells were llowed to rech confluent monolyer efore their exposure to vrious gent. A significnt The Phrmcogenomics Journl

7 Downregultion of endothelin-1 nd upregultion of ICAM-1 gene expressions % 3 MFI % VCAM-l (fold induction / control) svcam-l(ng/ml) 1 5 VCAM-l (fold induction / control) HU (µm) Figure 8 Anlysis of the effect of HU on mvcam-1 nd svcam-1 relese. Control: nontreted cells, HU: cells treted with HU 25 lm for 24 h, Cyto: cells treted with TNF nd IFNc t 1 U/ml for 24 h, HU+Cyto: comintion of HU nd Cyto. () FACS nlysis of mvcam-1 expression in TrHBMEC cells (numers ove columns re percentge of positive cells) nd the results re expressed s MFI for one representtive experiment mong three. () ELISA nlysis of svcam-1 relese into the culture superntnt of TrHBMEC cells. Results re mens in picogrm of ET-1/ml for 1 5 cells7sd of three independent experiments. Figure 9 Anlysis of the effect of HU on VCAM-1 expression in TrHBMEC cells. Anlyzed y rel-time RT-PCR. Control: nontreted cells, HU: cells treted with HU 25 lm, Cyto: cells treted with TNF nd IFNc t 1 U/ml for 24 h, HU + Cyto: comintion of HU nd Cyto. The mplifiction frgment is locted on the extrcellulr domin of VCAM-1 sequence. () At 24 h of HU tretment. () Dose-response effect of HU on VCAM-1 gene expression. Results re mens7sd of fold induction/nontreted cells of three independent experiments. proportion of these cells (58 62% of TrHBMEC nd 65 68% of EA-hy 926 cells) were in G/G1 phse with much less numer in G2/M (22 27% of TrHBMEC cells nd 13 16% of EA-hy 926 cells) nd in S phse (11 2% of TrHBMEC nd 16 22% of EA-hy 926 cells). The presence of cytokines for 24 nd 48 h did not lter the proportions, wheres exposure of these cells to HU cused reduction in the proportion of cells t G/G1 phse (22 32% t 24 nd 22 23% t 48 h for oth TrHBMEC nd EA-hy 926 cells). Prllel to such decrese in G/G1 phse cells, there ws n increse in the proportion of cells in S phse (2 31% oth t 24 nd 48 h) with no ltertion in the proportion of G2/M-phse cells. DISCUSSION HU Downregultes ET-1 Expression t the mrna nd Protein Levels ET-1, potent long-cting meditor of vsoconstriction nd inflmmtion, is produced y endothelil nd vsculr

8 222 Downregultion of endothelin-1 nd upregultion of ICAM-1 gene expressions smooth muscle cells in response to hypoxi nd sher stress. Other stimuli such s TNF, proinflmmtory cytokine, induces ET-1 relese from vriety of ECs in time nd concentrtion-dependent mnner ccompnied y corresponding increse in the trnscriptionl rte of the ET-1 gene. 18 Interestingly, ET-1 mrna production nd ET-1 peptide relese increse when ECs from ovine pulmonry rtery were exposed to plsm from ptients with ACS. 19 Similrly sickle erythrocytes specificlly stimulte the expression of the ET-1 gene nd production of the peptide in humn ECs in culture. 2 In niml models ET-1: (i) cuses ccumultion of leukocytes in the pulmonry microvsculture, 21 (ii) increses vsculr permeility in the presence of leukocytes in perfusion 22 nd (iii) results in time- nd dose-dependent sequentil entrpment of pltelets nd neutrophils in the pulmonry circultion. 23 ET-1 ctivtes the Grdos chnnel in mouse erythrocytes. 24 This chnnel ctivtion, in the context of sickle red cell, is expected to dehydrte these cells. Importnt clinicl findings include significntly elevted levels of ET-1 in SCA ptients during the episodes of pinful crisis 25,26 nd ACS. Also, the extent of ET-1 increse correltes with disese severity. 19 These experimentl nd clinicl oservtions suggest tht ET-1 might ply n importnt role in the cycle of ischemi nd inflmmtion tht initites nd sustins pinful crisis in sickle cell ptients. Locl ET-1 levels in the microvsculture re proly much higher thn those in the systemic circultion, nd my contriute to the prolonged locl vsospsm nd inflmmtion nd thus to vso-occlusive events, the hllmrk of the sickle cell disese. Our finding tht HU significntly suppresses ET-1 gene expression nd peptide relese in dose-dependent mnner from ECs (Figure 3 nd ) indictes one of the wys in which HU exerts eneficil effects in SCA. The effect of HU on the downregultion ET-1 expression (further supported y our unpulished oservtions tht SCA ptients under HU hve significntly lower plsm ET-1 levels) if confirmed in vivo, might offer novel therpeutic strtegies for sickle cell disese. Furthermore, the two-fold reduction in IL-6 production y the HU-exposed ECs (Figure 6d) my stem from the suppression of ET-1 expression. Previous studies show tht ET-1 cn induce IL-6 expression in humn endothelil cells. 27 Alterntively, HU my hve direct effect on IL-6 expression. Induced secretion of IL-6 y ECs is ssocited with incresed cell permeility, genertion of rective oxygen species nd trnsmigrtion of monocytes through endothelium 28 presumptively cusl for vsoendothelil injury. Agin the eneficil effects of HU in SCA my e medited through its effect on IL-6 either directly or through suppression of ET-1 expression. We lso exmined the effect of HU on the expression of NO, n importnt component of the vsomotor regultion. HU is elieved to e chemicl donor of NO, 29,3 potent vsodiltor, nd NO seems to provide vriety of eneficil effects in SCA: (i) low-dose inhltion of NO increses the oxygen ffinity of sickle red cells oth in vivo nd in vitro, 31 (ii) NO inhltion t 2 ppm seems to provide rpid protection ginst severe hypoxic stress in trnsgenic mice model for SCD, 32 (iii) in SCA ptients with ACS, inhled NO improves ventiltion perfusion mtching 33 nd (iv) SCA ptients with elevted levels of NO metolites report lower pin scores. 34 NOS tightly regultes the NO expression in endothelil cells. We did not oserve ny ltertion in the level of NO metolites or chnges in the endothelil expression of NOS either t trnscriptionl or protein level in HU-treted ECs, which suggests tht the HU effect my not e medited y this pthwy. HU Upregultes micam-1 Expression nd sicam-1 Relese (micam-1 or CD54) is cell surfce glycoprotein of the immunogloulin superfmily with multiple immune response-relted functions, which include cell cell interctions nd leukocyte dhesion to vsculr endothelil cells. The ltter event initites trnsmigrtory egress of leukocytes from the vsculture. ICAM-1 performs these functions through its ility to ind 2 integrins such s LFA-1 nd Mc-1. Expressed constitutively in severl cell types including vsculr EC, the level of expression of micam-1 is mrkedly upregulted y inflmmtory stimuli such s TNF nd IFNg. 35 micam-1 lso cts s mjor cell-surfce receptor for rhinovirus nd Plsmodium flciprum. 36 In ddition, ICAM-1/LFA-1 interction seems to promote HIV-1 infectivity. 37 An sicam-1 contining most of the extrcellulr portion of micam-1 is detectle in norml plsm. The sicam-1 level my reflect the expression sttus of micam-1 on endothelil cells therey indicting the degree of inflmmtory process nd/or EC ctivtion. 38 Elevted serum sicam-1 levels hve een noted in vrious immune nd inflmmtory disorders including SCA. 39 Indeed, sickle RBCs hve een shown to induce micam-1 expression y endothelil cells in culture oth in sttionry nd flow studies. 4 Under flow conditions, upregultion of micam- 1 involves oth trnscriptionl nd trnsltionl control. sicam-1 levels re higher (two- to three-fold) in children with SCA thn in ethniclly mtched controls. 39 Our in vitro experimentl finding tht HU enhnces the expression of micam-1 y endothelil cells in culture (Figure 6 nd ) does not pper to e consistent with the significnt clinicl enefits conferred y this drug in SCA ptients. The strongest correltion ws found etween totl white cell count nd severity of crisis rther thn with erythrocyte-relted prmeters. Although there is no evidence showing tht ICAM-1 is directly involved in sickle cell dhesion to endothelil cells, it is plusile tht leukocyte endothelium dhesion my initite the sequestrtion nd entrpment of RBCs resulting in the ostruction of the microvsculr lumen. Accordingly HU-enhnced m ICAM- 1 expression would e expected to ggrvte rther thn to ttenute the sickle cell crisis. An explntion for this prdox my come from the HU-induced increse in sicam- 1 (Figure 6c). Indeed sicam-1 nd solule selectins hve een shown to suppress significntly the neutrophil endothelil dhesion in vitro. 41 Thus, the eneficil effect of HU my e medited through enhnced relese of solule The Phrmcogenomics Journl

9 Downregultion of endothelin-1 nd upregultion of ICAM-1 gene expressions 223 dhesive receptors, which compete with the memrneound receptor for leukocytes nd therey minimize the vsculr dhesion. How sicam-1 is generted is still uncler. Two possiilities re: (i) differentil splicing of ICAM-1 mrna 42 nd (ii) proteolytic clevge of micam Distinct mrna species encoding sicam-1 hve een identified in severl humn cell lines, which support the first possiility. However, nd in support of the second possiility, the genertion of sicam-1 from neutrophil micam-1 y elstse hs een demonstrted. 44 Whtever the mechnism, the HU-induced enhncement of the relese/synthesis of solule ICAM-1 my prticipte in the eneficil effects of this drug. Thus, trgeted ntidhesion therpies my ecome useful lterntive in the tretment of SCA. HU Downregultes VCAM-1 Expression VCAM-1, like the ICAM-1, elongs to the immunogloulin superfmily nd exists in oth memrne-ound (mvcam-1) nd solule (svcam-1) forms. VCAM-1 whose expression in ECs is upregulted y proinflmmtory cytokines is the receptor for the very lte ctivtion ntigen 4 (VLA-4, 4 1 ), 45 expressed, only on the sickle erythrocyte memrnes. 46 ECs stimulted y cytokines exhiit incresed levels of surfce dhesion molecules tht could e specificlly locked y ntiodies to VLA-4 nd VCAM-1. Adhesion results in morphologicl dmge to endothelium, the extent of which correltes with the numer of dherent sickle erythrocytes. 47 Similrly, hypoxi significntly nd specificlly increses sickle erythrocyte dhesion to ortic nd retinl cpillry ECs without hving ny effect on norml erythrocytes. 48 The enhnced dhesion of sickle erythrocytes under hypoxi is ccompnied y upregultion of expression of oth VCAM-1 nd ICAM-1. 4 The expression of these surfce dhesion molecules is downregulted in vivo in mouse model y sulfslzine, powerful inhiitor of ctivtion of nucler fctor kpp B (NFkB). 49 In SCA ptients, significntly high concentrtion of svcam-1 hs een reported; however, HU tretment does not seem to ffect the svcam-1 level. 5 Our experimentl oservtion tht HU downregultes VCAM-1 gene expression nd mvcam-1 production in dose-dependent mnner, especilly in the presence of cytokines (Figures 8, nd 9 nd 9), contrsts with wht ws oserved for ICAM-1 expression. However, HU did not promote the relese of svcam-1. Such downregultion of mvcam-1, lthough modest, could contriute to the ttenution of SS RBC endothelium dhesion. The moleculr mechnisms y which HU differentilly regultes the ICAM-1 nd VCAM-1 gene expression re not cler, lthough the promoters of oth genes hve, inding sites for severl trnscription fctors including NFkB, AP-1, AP-2, AP-3, nd GATA in their 5 regultory regions. 51,52 Interestingly, HU prevented the inding of NFkB to the HIV- 1 LTR region, which might explin the downregultion of VCAM-1, ut not the upregultion of ICAM-1. HU nd Mlri ICAM-1 is the mjor cell-surfce receptor on vsculr endothelil cells for Plsmodium flciprum-infected RBC. 53 As HU upregultes micam-1 expression, it my promote RBC endothelium dhesion nd promote the progression of mlri. Hence, the dministrtion of HU to ptients from endemic res for mlri wrrnts cution. Alterntively, s discussed ove for SCA, the enhnced relese of sicam-1 my tend to mitigte such effects nd eventully e eneficil. At present, cytodherence experiments re underwy to clrify the vlue of using HU in res endemic for mlri. HU nd Cell Cycle Chnges Given the fct tht HU is S-phse locking gent, the ove-discussed HU-induced chnges in EC phenotype nd gene expression could e the result of cell cycle chnges. To explore this possiility, cell cycle studies were performed. In our experimentl conditions round 2 3% of the cells re rrested t the G1/S order nd the oserved chnges in the expression sttus of ECs could e ecuse of the ccumultion of such cell supopultion. However, the proinflmmtory cytokines did not hve ny effect on the proportion of cells in S phse, ut they do ffect the expression sttus of cell surfce nd solule dhesion molecules (ICAM-1 nd VCAM-1) of ECs. This suggests tht cell cycle chnges lone cnnot explin the chnges in the expression of these molecules. Whtever the mechnism of ction of HU, either through its ction on gene expression or on cell cycle or oth, this study provides further insight into the effect of HU on endothelil cells. Deciphering the trnscriptionl signture of HU in ECs my shed further light on the mechnism of ction of HU nd therey provide clues to other tretment options for SCA. METHODS Endothelil Cell Culture Two types of ECs were studied nd were mintined t 371C with 5% cron dioxide in humidified incutor. Trnsformed humn one mrrow endothelil cells (TrHBMEC), kindly provided y B Weskler, were cultured on geltin-coted Petri dishes s previously descried. 15 For this cell line, ll experiments were crried out etween pssge 2 nd 24. The cell line EA-hy 926 (epithelil/ endothelil hyrid cell line), gift from CS Edgell, were cultured under the experimentl conditions descried previously. 16 Both cell types were grown until confluence nd then the sl medium ws replced y medium contining HU t vrious concentrtions (62.5, 125, 25 nd 5 mm) in the presence or sence of mixture of TNF nd IFNg (R&D System, Aingdon, UK) ech t 1 U/ml nd incuted for 48 h, unless otherwise indicted. Flow Cytometry for Cell Cycle Anlysis TrHBMEC cells nd EA-hy 926 were grown s descried ove nd treted with 25 mm HU for 24 nd 48 h in three independent experiments oth in the presence nd sence of proinflmmtory cytokines (TNF nd IFN t 1 U/ml).

10 224 Downregultion of endothelin-1 nd upregultion of ICAM-1 gene expressions Cells were fixed with 1 ml of ice-cold 7% ethnol for 1 min t 41C. Cells were spun for 5 min t 2 rpm nd then 1 ml of PBS RNAse A (Sigm, Sint Louis, MO, USA) t 2 mg/ml ws dded nd cells were incuted for 3 min t room temperture. Then, 2 ml of PBS propidium iodide (Sigm, Sint Louis, MO, USA) t 75 mg/ml ws dded nd cells were incuted for 15 min t room temperture. A totl of 1 cells were nlyzed in n Epics-Elite flow cytometer (Coulter Electronic, Hileh, FL, USA). ET-1, sicam-1, IL-6 nd svcam-1 Assy Culture superntnts were spun for 1 min t 1 g t 41C, frozen t 21C in severl liquots (5 ml) until ssy for ET-1, svcam, IL-6 nd sicam y ELISA (commercilly ville kits BBE5, BBE3, D65, nd BBE 1B ssy Kit, R&D System, Aingdon, UK). The results for the superntnt from plted cells re expressed in pg/ml for ET-1 nd in ng/ml for sicam-1, IL-6 nd svcam-1. RNA Extrction nd Reverse Trnscription Totl RNA ws extrcted from the cultured cells using commercil kit (RNesy, Qigen, Vlenci, CA, USA). Briefly, 5 ml of the RNA preprtion ws mixed with 1 U of RQ1 RNAse free DNAse (Promeg, Mdison, WI, USA) nd incuted for 3 min t 371C in the presence of DNAse uffer. Aliquots of 3 mg of RNA were nneled to 8 pg of rndom primer mixture (Life Technologies, Grnd Islnd, NY, USA) t 71C for 1 min nd then the tues were plced in ice. The reverse trnscription rection in finl volume of 5 ml, contining 1 ml of 5 RT uffer, 8 ml of 1 mm dithiothreitol, 1 ml (4 U) of RNAsin (Promeg, Mdison, WI, USA) nd 4 ml of25mm dntps, ws initited y dding 2 U of Superscript II reverse trnscriptse from Moloney Murine Leukemi Virus (Life Technologies Grnd Islnd, NY, USA) nd incuted for 1 h t 371C. The rection ws stopped y incuting the rection mixture t 71C for 1 min. The tues were stored t 21C until the quntittive PCR ssy. Rel-Time Quntittive PCR Assy for ET-1, ICAM-1 nd VCAM-1 mrna To evlute the expression of ET-1, ICAM-1 nd VCAM-1 mrna levels under different experimentl conditions, reltime quntittive PCR ws performed using n ABI PRISM 77 Sequence Detector (PE Applied Biosystem, Foster City, CA, USA). The fluorogenic Tqmn proes nd the primers were designed using the Primer Express Softwre 1.7 (PE Applied Biosystem, Foster City, CA, USA). The proes consisted of n oligonucleotide with 5 FAM (6-croxyfluorescein) reporter dye nd 3 TAMRA (6-croxytetrmethylrhodmine) quencher dye. A frgment of 115 p extending from the untrnslted 5 region of the ET-1 mrna to the 3 end of the coding region ws mplified using the following primers: forwrd het-1(a) 5 -ACGGCGGGGAGAAACC-3 nd reverse het-1(b) 5 -AT- GATGTCCAGGTGGCAGAAG-3. For ICAM-1, frgment of 12 p corresponding to the extrcellulr domin ws mplified using the following primers: forwrd hicam-1 (A) 5 -GCAATGTGCAAGAAGATAGCCA-3 nd reverse hi- CAM-1 (B) 5 -GGGCAAGACCTCAGGTCATGT-3. A 125 p frgment of the region encoding extrcellulr domin of VCAM-1 ws mplified using the following primers: hvcam-1 (A) 5 -GAGTACGCAAACACTTTATGTCAATGT-3 nd reverse hvcam-1 (B) 5 -CTCGTCCTTTCGGGACCG-3. The qulity of the PCR products ws checked y size nlysis nd y direct nucleotide sequencing. To normlize the quntittive dt, TATA inding protein (TBP) mrna ws used s n internl control (detiled elow) with the following primers: forwrd TBP primer: 5 -CAC- GAACCACGGCACTGATT-3 nd reverse TBP primer: 5 - TTTTCTTGCTGCCAGTCTGGAC-3, which provide n mplicon of 88 p. Sequences of the fluorogenic proes re s follows: ET-1 proe 5 -(FAM)-TGCTCCCTGCTCGTCCCTGATGGATA-3 - (TAMRA); TBP proe 5 -(FAM)-TGTGCACAGGAGCCAA- GAGTGAAGA-3 -(TAMRA); VCAM-1 proe 5 -(FAM)- AACCGTCTTGGTCAGCCCTTCCTCC-3 -(TAMRA); ICAM-1 proe 5 -(FAM)-CAATGTGCTATTCAAACTGCCCTGATGGG- 3 -(TAMRA). cdna smples were diluted three-fold with the Mster Mix (PE Applied Biosystem, Foster City, CA, USA) contining MgCl 2, PCR uffer, dntp, AmpErse UNG nd AmpliTq Gold DNA polymerse to finl volume of 25 ml nd were run for rel-time quntittive PCR under the following therml conditions: initilly 2 min t 51C nd 1 min t 951C nd then 45 cycles of 15 s t 951C nd 1 min t 61C. Normliztion of the Quntittive PCR Dt Prior to quntittive nlysis of the smples, stndrd curve for threshold cycle (C T ) vs mount of RNA (for ech cell type) ws estlished to ccount for the differences in PCR efficiency etween the test ET-1, ICAM-1, VCAM-1 mrna nd the control (TBP) mrna. For this purpose, RNA concentrtions of 1, 1, 1 pg nd 1 ng (diluted with solution of trna to keep the totl RNA concentrtion constnt) were used. Slopes of the stndrd curves generted were lwys etween 3.2 nd 3.6 corresponding to PCR efficiency of 85 9% with correltion coefficient consistently higher thn.9. The vritions in C t for TBP mrna did not exceed one etween experiments. For ech smple, the test/tbp mrna rtio ws clculted nd the results re given s the percentge of reltive expression in treted vs nontreted cells. Flow Cytometry Anlysis for micam-1 nd mvcam-1 Expression Cells (finl cell density of cells/ml) were incuted for 2 h t 41C in 1 ml PBS contining 3% fetl clf serum (FCS) oth in the presence nd sence of 1 mg of ICAM-1 nd VCAM-1 ntiodies (R&D System, Aingdon, UK). Then the cells were wshed, resuspended in the sme uffer in the presence of 1 ml FITC-leled got nti-mouse Igs nd incuted t 41C for 1 h in the drk. After three wshes with PBS contining 3% of FCS, the cells were fixed with 5 ml of 4% prformldehyde nd totl of 5 cells were The Phrmcogenomics Journl

11 Downregultion of endothelin-1 nd upregultion of ICAM-1 gene expressions 225 nlyzed in n Epics-Elite flow cytometer (Coulter Electronic, Hileh, FL, USA). Results re expressed s MFI. NO nd NO Synthse Both nitrites nd nitrtes, s indirect mesures of NO, were ssessed in the culture superntnts using the Griess rection. 17 The enos in cell lystes were determined y ELISA using commercil kit (R&D System, Aingdon, UK). To explore the expression of the inos gene, we crried out rel-time quntittive PCR with Syer Green proe using the following primers : forwrd primer inos (c) 5 -GGACCA- CATCTACCAGGAGGAG-3 nd reverse primer inos (d) 5 - CCTGAACATAGACCTTGGGCT-3, which llowed mplifiction of 111 p frgment encompssing the sequence of exon 25. Sttistics Sttisticl nlysis ws performed with the nonprmetric Mnn Whitney test using GrphPd Prism (Grph Pd Softwre, Sn Diego, CA, USA) nd difference etween the groups ws considered significnt when Po.5. ACKNOWLEDGEMENTS We re very grteful to Bette B Weskler from Cornell University Medicl College, NY, USA nd Cor-Jen S Edgell from University of North Crolin, Chpel Hill, USA for providing the TrHBMEC nd EA-hy 926 cell lines, respectively. We thnk Mry Dominique Hrdy- Dessources for IL-6 experiments (Gudeloupe, FWI). We lso thnk Hélène Cvé nd Cécile Acquviv (Service de Biochimie Génétique, Hôspitl Roert Deré, Pris, Frnce) for technicl ssistnce for rel-time quntittive PCR. Mnuel Brun is recipient of Doctorl fellowship from the French Ministère de l recherche et de l Technologie. DUALITY OF INTEREST None declred. REFERENCES 1 Dover GJ, Humphries RK, Moore JG, Ley TJ, Young NS, Chrche S et l. Hydroxyure induction of hemogloin F production in sickle cell disese: reltionship etween cytotoxicity nd F cell production. Blood 1986; 67: Miller BA, Pltt O, Hope S, Dover G, Nthn DG. Influence of hydroxyure on fetl hemogloin production in vitro. 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