Antiviral Therapy 2014; 19: (doi: /IMP2752)

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1 Antivirl Therpy 214; 19: (doi: /IMP2752) Originl rticle Impct of drunvir, tznvir nd lopinvir oosted with ritonvir on cultured humn endothelil cells: eneficil effect of prvsttin Mrtine Auclir 1,2,3, Puline Afonso 1,2,3, Emilie Cpel 1,2,3, Mrtine Cron-Derle 1,2,3, Jcqueline Cpeu 1,2,3,4 * 1 INSERM, UMR_S 938, CDR Sint Antoine, F-7512, Pris, Frnce 2 Soronne Universités UPMC Univ Pris 6, UMR_S 938, F-7512, Pris, Frnce 3 ICAN, Institute of Crdiometolism nd Nutrition, Pris, Frnce 4 AP-HP, Hopitl Tenon, Deprtment of Biochemistry, F-752, Pris, Frnce *Corresponding uthor e-mil: jcqueline.cpeu@inserm.fr Bckground: HIV-infected ptients dministered long-term ritonvir-oosted protese inhiitors (PIs) re t greter risk for developing crdiovsculr diseses. Endothelil dysfunction is n inititing event in HIV-ssocited therosclerosis. Cultured endothelil cells cn e used s model to compre the endothelil toxicity of different PIs. Methods: We compred the effect of drunvir (), drunvir/ritonvir (/r), lopinvir/ritonvir () nd tznvir/ritonvir (), used t cliniclly relevnt concentrtions, on humn coronry rtery endothelil cell vsculr function, oxidtive stress, inflmmtion nd senescence, nd studied the effect of prvsttin on PIinduced ltertions. Results: Vsculr endothelil cell function, evluted y the expression of endothelil nitric oxide synthse nd the production of nitric oxide nd endothelin-1, ws unffected y or /r, ut ltered y or. or /r did not lter, or mildly induced oxidtive stress nd inflmmtion (phosphoryltion of p65/rela-nfkb, secretion of IL-6 nd IL-8), while nd induced mrked increse. Secretion of sicam or svcam, indictive of ltered cell integrity, ws not or wekly ltered y or /r, ut incresed y 2 3-fold y or. Similr results were oserved regrding senescence mrkers: SA--glctosidse ctivtion nd overexpression of phospho-p53, p16 ink4, p21 WAF-1 nd prelmin A. Prvsttin could, in prt, reverse PI-induced dverse effects. Conclusions: Ritonvir-oosted PIs differentilly induced vsculr endothelil cell dysfunction, rective oxygen species production, inflmmtion nd senescence with no effect or mild effect of /r, n intermedite effect of, nd stronger effect of. Sttins could, in prt, protect the cells from PI-induced endothelil dysfunction. Introduction Protese inhiitors (PIs) re widely used to control HIV infection; however, PI-sed therpies re known to increse crdiovsculr risk in HIV-infected ptients [1 4]. Among currently used PIs, lopinvir (LPV) oosted with ritonvir () hs een ssocited with greter risk of crdiovsculr disese [4,5]. Atznvir (ATV) hs not een ssocited with n incresed risk of myocrdil infrction in the D:A:D study [6] ut, oosted with ritonvir (), it exerted more therogenic lipid profile thn nevirpine in tretmentnive ptients [7]. Up to now, no dt re ville on potentil crdiovsculr risk exerted y drunvir (). -sed therpies re considered s sfe nd well-tolerted t the metolic level [8 1]. In ddition to PI-sed therpies, the incresed risk of premture myocrdil infrction hs een ttriuted to HIV infection-relted fctors (CD4 ndir, CD8 level, HIV virl lod) nd to other clssic crdiovsculr risk fctors [3 5,11 13]. Antiretrovirl therpy my promote premture crdiovsculr disese through endothelil dysfunction either indirectly, vi PI-induced metolic disturnces [5,14 17] or directly, vi ltertions of vsculr endothelil cells [18 22]. The endothelium normlly exerts numer of vsoprotective effects such s vsodiltion, suppression of smooth muscle cell growth nd inhiition of inflmmtory responses. Mny of these effects re lrgely medited y nitric 214 Interntionl Medicl Press (print) (online) 773

2 M Auclir et l. oxide (NO), the most potent endogenous vsodiltor tht opposes the effect of endothelium-derived vsoconstrictors such s endothelin-1. A defect in the production or ctivity of NO leds to endothelil dysfunction, which is n erly mrker for therosclerosis nd cn e detected in vivo efore structurl chnges to the vessel re pprent [23]. In vitro, numer of endothelil functions cn lso e evluted s the production of NO nd endothelin-1, oxidtive stress nd the relese of proinflmmtory cytokines. Regrding PIs, it hs een previously shown tht short-term tretment with ritonvir (RTV; h), lone or in ssocition with LPV, directly ltered endothelil cell function [22,24] y triggering oxidtive stress in humn [25] nd porcine endothelil cells [26 28]. Otherwise, long-term tretment with RTV or induced inflmmtion nd premture senescence in cultured humn endothelil cells together with oxidtive stress [29]. The tretment with the comintion rought controversil dt: 4-week tretment of helthy sujects with or did not impir endothelil function [3,31], wheres switching to tretment did not improve endothelil dysfunction in HIV-infected ptients [32]. To our knowledge, the impct of or /r on cultured humn endothelil cells hs never een evluted. The risk of premture crdiovsculr events might lso result from the ccelerted iologicl geing imposed y ntiretrovirl therpy nd/or HIV itself [33 35]. Indeed, vsculr endothelil cell dysfunction is feture of the humn physiologicl geing process [36,37], nd syndromes of premture geing re ssocited with precocious crdiovsculr disese. The most striking findings re oserved in progeroid syndromes linked to moleculr ltertions in the prelmin A mturtion process [38 41], in which ltertion of vsculr cells is ovious. Importntly, some PIs, ut not ll, cn induce the ccumultion of frnesylted prelmin A y inhiiting the metlloprotese ZPM- STE24, involved in the prelmin A mturtion process. The ility of the different PIs to inhiit this enzyme decresed from LPV, to RTV nd then to ATV, eing devoid of ny effect even t high concentrtions [42,43]. Our ojective ws to compre the long-term effect of (used lone or in comintion with oosting concentrtion of RTV), or on cultured humn endothelil cells. We evluted the possile role of frnesylted prelmin A ccumultion, y treting the PI-treted cells with prvsttin, since sttins inhiit the synthesis of the frnesyl nchor nd therefore decrese the ccumultion of frnesylted prelmin A [44], s previously shown in endothelil cells treted with or RTV [29]. Methods Cell culture nd tretment Humn coronry rtery endothelil cells (HCAECs; PromoCell, Heidelerg, Germny) were cultured s previously descried [29]. They were exposed during 2 dys to cliniclly relevnt concentrtions of (11.8 mmol/l), /r (11.8/.8 mmol/l) [45], (15.9/1.4 mmol/l) [46], (7.4/1.3 mmol/l) [47] or to the solvent (.1% dimethyl sulfoxide []). The tretment with prvsttin (25 mmol/l) ws performed during the lst 3 dys. ws otined from Jnssen-Cilg (Issy-les-Moulineux, Frnce) nd the other PIs purchsed from Snt Cruz Biotechnology, Inc. (Snt Cruz, CA, USA). Western lotting Whole cell lystes were sujected to SDS/PAGE nd western lotting. We used ntiodies ginst endothelil nitric oxide synthse (e-nos; SC-653), endothelin-1 (ET-1, SC-21625) nd prelmin A (SC-6214) from Snt Cruz Biotechnology, Inc. Antiodies ginst NF-kB p65/rela (#3987) nd phospho-nf-kb p65/ RelA (ser 536; 333) were from Cell Signling Technology, Inc. (Dnvers, MA, USA). Antiodies ginst p53 (clone DO-1, 8645) nd nti-phospho(s15)-p53 (38497) were from Acm (Cmridge, UK). Antiodies ginst p16 INK4 (551144) nd p21 WAF-1 (556431) were from BD Biosciences (Sn Jose, CA, USA). Betctin (A-5441; Sigm-Aldrich, Sint Louis, MO, USA) ws used s n index of the cellulr protein content. NO production NO production ws ssessed with the cell-perment NO indictor 4-mino-5-methylmino-2,7 - difluorofluorescein dicette (DAF-FM; D23844; Moleculr Proes, Life Technologies, Crlsd, CA, USA). Cells were cultured in 96-well pltes, wshed nd incuted with DAF-FM (12.5 mmol/l) or Hoechst (.1 mg/ml) in DMEM without FBS for 3 min t 37 C in the drk. Quntifiction ws performed with plte fluorescence reder (Infinite M2; Tecn-Frnce, Trppes, Frnce) t 515 nm (DAF-FM) nd 46 nm (Hoechst 33258), respectively. NO production ws lso indirectly determined y e-nos protein expression. Oxidtive stress nd inflmmtion The production of rective oxygen species ws indirectly mesured y the oxidtion of CM- H 2 DCFDA derivtives (5-[nd 6]-chloromethyl-2,7 - dichlorodihydrofluorescein dicette, cetyl ester) nd the reduction of nitrolue tetrzolium (NBT) s descried [29]. Secretion of interleukin (IL)-6 nd IL-8 ws nlysed in 24-h culture superntnts y Luminex Interntionl Medicl Press

3 Effect of HIV protese inhiitors on endothelil cells technology [29]. Inflmmtion ws lso determined y the protein expression of the phosphorylted form (serine 536) of the p65/rela suunit of NF-kB. Senescence Cell senescence ws evluted y the senescencessocited (SA)--glctosidse ctivity, the protein expression of the senescence mrkers phospho-p53, p21 WAF-1 nd p16 INK4, nd the ccumultion of prelmin A, s descried previously [29,34]. Sttisticl nlysis The experiments were repeted 3 8. Results re expressed s men ±sem. Sttisticl significnce ws determined using ANOVA nd the Kruskl Wllis non-prmetric test, followed y Fisher protected lest significnt difference test for pir-wise differences. P-vlues were clculted reltive to cells cultured with.1%. P-vlues of less thn.5 were considered significnt. Results Endothelil cell dysfunction used lone or in comintion with ritonvir (/r) did not decrese the protein expression of e-nos (Figure 1A) or the production of NO y endothelil cells, mesured y the fluorescent NO indictor DAF-FM dicette (Figure 1B). In ddition, or /r did not lter the sl level of cellulr or secreted ET-1 (Figure 1A). ctivted ll mrkers of endothelil cell vsculr dysfunction. It decresed e-nos protein expression (y 3-fold) nd incresed cellulr nd secreted ET-1 y 2 3- fold (Figure 1A nd 1B). lso dversely modified vsculr endothelil cell functions: its effect ws consistently lower thn tht of nd higher thn tht of /r ( 2-fold). Regrding the relese of the dhesion molecules sicam nd svcam (Figure 1C), which re mrkers of ltered endothelil cell integrity, hd no effect on the secretion of sicam or svcam, wheres /r modertely incresed their secretion (-fold increse), compred with the effect of (4 6-fold increse) or (3 4-fold increse). Regrding oxidtive stress evluted y CM- H 2 DCFDA oxidtion nd NBT reduction, did not lter the oxidtive stress mrkers (Figure 2A), while /r modertely incresed CM-H 2 DCFDA oxidtion (y 2-fold), nd hd no effect on NBT reduction. By contrst, or incresed the two mrkers of oxidtive stress y, respectively, 6.5- nd 5-fold (CM- H 2 DCFDA) nd 3-fold (NBT). With regrd to inflmmtion, or /r did not modify the phosphoryltion stte of the p-65/ RelA suunit of NF-kB s compred with control or -treted cells (Figure 2B). In ddition, or /r hd low, or hd no effect on the secretion of IL-6 nd IL-8 (Figure 2C). mrkedly incresed p65/rela NF-kB phosphoryltion (y 3-fold) nd cytokine secretion (y 3 4-fold). lso incresed inflmmtion ut in most cses t lower level thn (Figure 2B nd 2C). Endothelil cell senescence or /r hd no effect on the cell cycle rrest mrkers phospho-p53, p21 WAF-1 nd p16 INK4, nd did not induce prelmin A ccumultion (Figure 3A). The comintion /r modertely incresed SA--glctosidse ctivity (y 1.6-fold), wheres or hd stronger effect (3.2- nd 2.8-fold increse; Figure 3B), consistent with the - nd -incresed protein expression of cell cycle rrest mrkers nd prelmin A (Figure 3A). Effect of prvsttin on PI-induced endothelil cell dysfunction nd senescence We then studied the effect of prvsttin on PI-induced endothelil cell dysfunctions. PI-induced HCAEC dysfunction ws improved y prvsttin, s shown y the normlized production of NO, sicam or svcam in - or -treted cells (Figure 4A). In ddition, prvsttin decresed the effect of nd on oxidtive stress mrkers y fold, nd on inflmmtion nd senescence mrkers y fold (Figure 4B nd 4C). Discussion We compred the effect of, /r, nd on cultured humn endothelil cells. All drug comintions were used t concentrtions ner to the mximum concentrtion mesured in the ptients serum [45 47]. The PI comintions differentilly ffected endothelil functions nd induced senescence. A continuous incution of HCAECs for 2 dys with or decresed e-nos protein expression nd NO production, cused the secretion of ET-1, nd induced oxidtive stress nd inflmmtion. The ility of PIs to enhnce the secretion of IL-6 nd IL-8 led us to evlute their ility to increse the phosphoryltion of the NF-kB suunit, p65/rel A on serine 536. The phosphoryltion of p65/rel A, though medited independently of IkB, is importnt for the expression of IL-6 nd IL-8 [48,49]. nd lso ltered endothelil cell integrity, s shown y the incresed relese of sicam-1 nd svcam-1, nd induced senescence. PI-induced endothelil cell dysfunction nd senescence could regress in the presence of prvsttin. Antivirl Therpy

4 M Auclir et l. Figure 1. PI effect on endothelil cell functions A.4 e-nos Cellulr ET-1 Secreted ET-1 e-nos Cellulr ET-1 Secreted ET-1 β-ctin Protein/β-ctin, AU /r /r B C.4.2 sicam DAF-FM/DNA, fluorescence ng/µg protein/24 h svcam /r /r Humn coronry rtery endothelil cells were cultured for 2 dys with the indicted protese inhiitors (PIs). (A) Protein expression of cellulr nd/or secreted endothelil nitric oxide synthse (e-nos), endothelin-1 (ET-1) nd -ctin. Representtive lots (performed in triplicte) re shown. Quntifiction ws performed reltive to -ctin nd expressed s ritrry units (AU). (B) Nitric oxide (NO) production ws evluted y the fluorescence of 4-mino-5-methylmino-2,7 - difluorofluorescein dicette (DAF-FM) normlized to DNA (Hoechst fluorescence). (C) Solule ICAM nd VCAM ws evluted in 24 h culture superntnt y Luminex technology nd expressed s ng/mg protein/24 h. Results re the men ±sem of 3 5 experiments. P<.5 versus dimethyl sulfoxide ()-incuted cells., ritonvir-oosted tznvir; /r, ritonvir-oosted drunvir;, ritonvir-oosted lopinvir. A 2-dy tretment of HCAECs with hd no incidence on endothelil cell function. When ws ssocited with low concentrtion of RTV (/r), it exerted no, or little effect on endothelil function ( 2-fold chnge of some, ut not ll, mrkers). This low-grde toxicity could proly result from the toxicity of the ooster, even t this low concentrtion [5]. Even if we hve not directly tested the toxicity of RTV used lone t low concentrtions, it cn e expected tht its toxicity will increse with incresing concentrtions, ecoming importnt t 7.5 mmol/l, s previously reported [29]. The higher toxicity of compred with /r on endothelil cell function my e explined in prt y higher impct of the ooster, which ws used t 1.6-fold higher concentrtion in versus /r (1.3 versus.8 mmol/l). Indeed, RTV used lone in porcine nd humn coronry rtery endothelil cells (24 h, 15 3 mmol/l) cn decrese e-nos expression [27], nd increse oxidtive stress [21,24,28]. Incresed ET-1 relese nd decresed NO production ws suspected to medite the dverse effects of HIV drugs on endothelil cell function [2,25,51,52] Interntionl Medicl Press

5 Effect of HIV protese inhiitors on endothelil cells Figure 2. PI effect on mrkers of oxidtive stress nd inflmmtion A 3. CM-H 2 DCFDA/DNA, fluorescence NBT reduction, OD/µg protein /r 5 /r B p-p65/reia p65/reia NF-κB /r p-p65/reia/p65reia NF-κB, AU C IL-6 3. IL-8 /r ng/µg protein/24 h.9.6 ng/µg protein/24 h /r /r Humn coronry rtery endothelil cells were cultured for 2 dys with the indicted protese inhiitors (PIs). (A) Rective oxygen species production ws ssessed y the oxidtion of CM-H 2 DCFDA derivtives or the reduction of nitrolue tetrzolium (NBT). Results re normlized to the DNA or protein content, respectively, nd expressed s men ±sem of 3 1 experiments. (B) The ctivtion of NF-kB ws evluted y the phosphoryltion of the p65/rela suunit. Quntifiction ws performed versus totl p65/ RelA NF-kB nd expressed s ritrry units (AU). Representtive lots (performed in triplicte) re shown. (C) The secretion of interleukin (IL)-6 nd IL-8 in 24 h culture superntnts ws mesured y Luminex technology. Results re normlized to the cell protein content nd re the men ±sem of 3 8 experiments performed in triplicte. P<.5 versus dimethyl sulfoxide ()-incuted cells., ritonvir-oosted tznvir; /r, ritonvir-oosted drunvir;, ritonvir-oosted lopinvir. Antivirl Therpy

6 M Auclir et l. Figure 3. PI effect on senescence mrkers A p p-p53/p53 p-p53 p21 WAF p16 INK4 Prelmin A β-ctin AU /r /r B p21 WAF-1 p16 INK4.8 SA-β-gl AU OD ph 6/OD ph /r /r Humn coronry rtery endothelil cells were cultured for 2 dys with the indicted protese inhiitors (PIs). (A) Senescence ws evluted y the phosphoryltion stte of p53 (pp53/p53) nd the expression of p21 WAF-1, p16 INK4 nd prelmin A. Quntifiction ws performed reltive to -ctin. Results re expressed s ritrry units (AU) nd re the men ±sem of 3 8 experiments. Representtive lots (performed in triplicte) re shown. (B) Senescence-ssocited -glctosidse (SA--gl) ctivity. Blue X-gl stining ws evluted t 63 nm. Results re the men ±sem of 3 8 experiments performed in triplicte. P<.5 versus dimethyl sulfoxide ()- incuted cells., ritonvir-oosted tznvir; /r, ritonvir-oosted drunvir;, ritonvir-oosted lopinvir. However, n effect linked to ATV is lso possile, since in the sence of RTV it cn promote senescence of humn mesenchyml stem cells [53]. Moreover, ATV, s LPV nd RTV, could inhiit ZMPSTE24, even if the level of inhiition ws lower thn oserved with the two other PIs, while ws devoid of ny inhiitory effect [54]. This in vitro toxicity contrsts with the in vivo clinicl dt since tretment with ATV or ws not ssocited with n incresed risk of myocrdil infrction in HIV-infected ptients [6]. It could e proposed tht the incresed level of free iliruin generlly oserved in ptients receiving ATV might exert eneficil nti-oxidnt effects. Indeed, dietic ptients with Gilert syndrome nd incresed iliruin level presented with lower prevlence of vsculr complictions s well s reduced levels of mrkers of oxidtive stress nd inflmmtion s compred to dietic ptients without Gilert syndrome [55]. Otherwise, mrkedly ltered endothelil cell function: it impired NO production, incresed the secretion of ET-1 nd dhesion molecules, nd induced oxidtive stress nd inflmmtion. It lso induced premture senescence. These results gree with our previous studies [29], even if shorter incution time (2- versus 3-dy) Interntionl Medicl Press

7 Effect of HIV protese inhiitors on endothelil cells Figure 4. Effect of prvsttin on PI-induced endothelil cell dysfunctions A.4 Minus prvsttin.2 Plus prvsttin sicam svcam DAF-FM/DNA, fluorescence ng/µg protein/24 h B /r 3.5 /r /r CM-H 2 DCFDA/DNA, fluorescence NBT reduction, OD/µg protein C IL-6 /r 3. IL-8 1. /r SA-β-gl ng/µg protein/24 h OD ph6/od ph /r /r /r Humn coronry rtery endothelil cells were cultured for 2 dys with the indicted protese inhiitors (PIs). Prvsttin (25 mmol/l) ws dded for the lst 3 dys of incution. (A) Nitric oxide (NO) production ws evluted with the fluorescent indictor 4-mino-5-methylmino-2,7 -difluorofluorescein dicette (DAF-FM), nd sicam nd svcam secretion y Luminex technology. (B) Oxidtive stress ws evluted s in the footnote of Figure 2A. (C) Inflmmtion (interleukin [IL]-6 nd IL-8) nd senescence (SA--glctosidse [SA--gl] ctivity) mrkers were evluted s in the footnotes of Figures 2 nd 3, respectively. The results re the men ±sem of four seprte experiments performed in triplicte. P<.5 versus dimethyl sulfoxide ()-incuted cells. P<.5 versus the respective control or PI-treted cells. Antivirl Therpy

8 M Auclir et l. nd other concentrtions of LPV (15.9 versus 1. mmol/l) nd RTV (1.4 versus 2. mmol/l) hve een tested. Sttins re lipid-lowering drugs widely used for the tretment nd prevention of crdiovsculr disese. They disply dditionl cholesterol-independent or pleiotropic effects on vrious spects of crdiovsculr disese, including improving endothelil function, decresing vsculr inflmmtion nd enhncing plque stility [56]. Sttins hve lso een shown to decrese oxidtive stress [57]. In the present study, eneficil effect of prvsttin ws oserved on ll PI-induced endothelil cell dysfunctions; vsculr dysfunction, inflmmtion, oxidtive stress nd lso on PIinduced senescence. This effect could e relted to the ility of sttins to decrese the PI-induced ccumultion of frnesylted prelmin A, y impeding the synthesis of the frnesyl nchor, nd therefore decresing prelmin A toxicity nd ility to induce cell senescence [29,58]. In our previous study we lso reported the eneficil effect of n nti-oxidnt tretment [29]. These dt led us to propose tht the PI-induced frnesylted prelmin A ccumultion is the initil toxic event leding to incresed oxidtive stress, inflmmtion nd to senescence. The ility of the different PI comintions to lter endothelil cell functions, s shown here, correlted with their efficiency to inhiit ZMPSTE24 [54]. This study hs limittions. We hve not evluted humn endothelil cell smples from HIV-infected ptients treted with these PI comintions. However, in our previous study senescence mrkers hve een detected in peripherl lood mononucler cells from HIV-infected ptients under ritonvir-oosted PIs, nd their level ws lower when the ptients were co-treted with sttins [29]. In conclusion, we report here tht /r, nd differentilly ffected vsculr endothelil cell function, cell integrity nd induced senescence. The effect of ech PI comintion on endothelil cells might in prt result from the concentrtion of the ritonvir oost nd from their ility to inhiit ZMPSTE24. Even if these in vitro studies cnnot e trnslted directly to the clinics, they suggest tht even t oosting concentrtions, RTV could dversely ffect endothelium. Whether nother CYP3A4 inhiitor, such s coicistt, structurlly relted ut devoid of ny inhiitory effect on the HIV protese, will exert or not n effect on ZMPSTE24 is difficult to infer nd requires dditionl experiments. Importntly, dding sttin to long-term PI-treted cells could diminish endothelil cell dysfunction nd dely senescence, which is importnt in the clinicl use. Acknowledgements This work ws supported y grnt from Jnssen-Cilg nd y INSERM. We thnk N Brunel nd B Lescure, Institut de Recherche en Snté Sint-Antoine-IFR 65, for their expertise in Technology (Luminex). Disclosure sttement This work ws supported y grnt from Jnssen- Cilg. 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