International Journal of Cardiology 177 (2014) 140–141
Contents lists available at ScienceDirect
International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard
Letter to the Editor
Response to letter by Tsikas et al. Chintan N. Koyani a, Werner Windischhofer b, Christine Rossmann a, Frank R. Heinzel c, Wolfgang Sattler a, Ernst Malle a,⁎ a b c
Institute of Molecular Biology and Biochemistry, Medical University of Graz, Austria Department of Pediatrics and Adolescence Medicine, Research Unit of Osteological Research and Analytical Mass Spectrometry, Medical University of Graz, Austria Department of Internal Medicine, Division of Cardiology, Medical University of Graz, Austria
a r t i c l e
i n f o
Article history: Received 6 September 2014 Accepted 20 September 2014 Available online 28 September 2014 Keywords: Cardiomyocytes 15d-PGJ2 PGD2 receptor DP2 Reactive oxygen species Therapeutic target
Dear Editor, We appreciate the interest of Tsikas et al. [1] in our manuscript [2] and the opportunity to clarify theoretical and experimental aspects describing the effects of 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2) on cardiomyocyte function. We agree that concentrations of 15d-PGJ2 (a degradation product of prostaglandin D2 [PGD2]) used in most in vitro studies [3–5] including ours (Figs. 1–6 [2]) are higher than occurring in vivo. In our study, HL1 cells (an immortalized cardiomyocyte cell line [6]) required such a high 15d-PGJ2 concentration (15 μM, Figs. 1–6 [2]) while in primary murine cardiomyocytes downstream signaling and induction of apoptosis were observed at much lower 15d-PGJ2 concentrations (10–50 nM; Fig. 7B–C [2]). These data suggest that primary cardiomyocytes respond to 15d-PGJ2 treatment at more physiologically relevant concentrations. Another point worth mentioning is avid binding of 15d-PGJ2 to cardiomyocyte or hepatic stellate cell culture medium constituents [7–9], which significantly reduces bioavailability of the active 15d-PGJ2 compound. ⁎ Corresponding author at: Medical University of Graz, Institute of Molecular Biology and Biochemistry, Harrachgasse 21, A-8010 Graz, Austria. Tel.: + 43 316 380 4208; fax: + 43 316 380 9615. E-mail address: [email protected] (E. Malle).
http://dx.doi.org/10.1016/j.ijcard.2014.09.111 0167-5273/© 2014 Elsevier Ireland Ltd. All rights reserved.
As the authors [1] correctly point out, the use of N-acetyl-L-cysteine (NAC) as reactive oxygen species (ROS) scavenger is questionable due to the formation of thioethers between NAC and 15d-PGJ2. To overcome this problem we have used pyrrolidine dithiocarbamate and Tempol (Fig. 1E [2]), two compounds that reduced DCF fluorescence (an indicator of ROS formation) more efficiently than NAC. Cyclopentenone PGs, such as 15d-PGJ2, are characterized by a highly reactive electrophilic carbonyl group in the prostane ring, which can readily adduct thiol-containing compounds including reduced glutathione (GSH) or cysteine [10]. Thioether formation between 15d-PGJ2 and GSH or cysteine reaches steady state levels after 2–3 h under in vitro conditions [11,12]. In contrast, most PGs exert their effects within few minutes [3]. In line, we report that 15d-PGJ2-mediated cellular signaling cascades are activated 2.5 min post treatment (Fig. 1D [2]). This difference in kinetics suggests that the effects we observed are independent of thiolation reactions. Using GC–MS/MS technology, Tsikas et al. [1,13] determined 15dPGJ2 levels in urine of healthy donors in the pM range. It remains to be evaluated whether these findings can be extrapolated to 15d-PGJ2 levels in serum/plasma or organs/tissues. In light of local macrophage inflammasome activation that can result in an ‘eicosanoid storm’ [14] (including PGD2, the parent eicosanoid of 15d-PGJ2), it is conceivable that such an event could generate a rapid local increase of PG concentrations. 15d-PGJ2 can exert its biological effects via receptor-independent or -dependent pathways. The latter include peroxisome proliferatoractivated-receptor γ (PPARγ) [10] and PGD2 receptors (DP1 and DP2) [7,15]. The Powell group [15] has unambiguously shown that PPARγmediated activation requires high 15d-PGJ2 concentrations while DP2 receptor-mediated activation in eosinophils is favored at low (1–10 nM) concentrations. In line, our results demonstrate that low 15d-PGJ2 concentrations (10–50 nM) activated DP2-dependent but PPARγ-independent signaling cascades in primary cardiomyocytes (Fig. 7 [2]). In HL-1 cells, 15d-PGJ2 failed to activate PPARγ-dependent signaling even at high concentrations (10–30 μM, Figs. 1 and 4 [2]). The low 15d-PGJ2 concentrations used in our study are in line with the assumption that low levels of this compound would be pro- rather than anti-inflammatory [16]. It is currently not clear whether the urinary 15(S)-8-iso-PGF/15dPGJ2 ratio, as mentioned by Tsikas et al. [1], might be considered a surrogate marker for the generation of 15d-PGJ2-mediated oxidative stress. However, a series of published articles revealed an association of 15d-
C.N. Koyani et al. / International Journal of Cardiology 177 (2014) 140–141
PGJ 2 with ROS production in different cellular systems at lower (95–160 nM) [17] as well as higher concentrations (1–20 μM) [5,18,19]. We agree with Tsikas et al. [1] on their view that direct therapeutic targeting of 15d-PGJ2 would be a difficult task. Nevertheless, upstream interference can be achieved by modulation of COX-1/2-dependent pathways, e.g. by aspirin in a murine model of myocardial infarction [20]. This is supported by findings in COX-1 knockout mice, which are protected against pathological effects of high concentrations of PGs that are released in response to inflammasome activation [14]. Pharmacological targeting of lipocalin-type PGD synthase, a powerful marker for coronary artery diseases in humans [21], might represent another indirect approach to impair PGD2 levels. Moreover, we present a novel therapeutic alternative to overcome 15d-PGJ2-induced cardiomyocyte injury by DP2 antagonism. In conclusion, DP2 antagonism may represent a pharmacological strategy to alleviate cellular/organ dysfunction induced by PGD2, its metabolites including 15d-PGJ2 or other hitherto unidentified DP2 agonists. Conflict of interest The authors report no relationship that could be constructed as a conflict of interest. References [1] Tsikas D, Niemann J, Beckmann B. 15-deoxy-delta12,14-PGJ2: an interesting but unapproachable pharmacological target? Int J Cardiol 2014. http://dx.doi.org/10.1016/ j.ijcard.2014.08.145. [2] Koyani CN, Windischhofer W, Rossmann C, Jin G, Kickmaier S, Heinzel FR, et al. 15deoxy-delta12,14-prostaglandin J2 promotes inflammation and apoptosis in cardiomyocytes via the DP2/MAPK/TNFalpha axis. Int J Cardiol 2014;173:472–80. [3] Bell-Parikh LC, Ide T, Lawson JA, McNamara P, Reilly M, FitzGerald GA. Biosynthesis of 15-deoxy-delta, 12,14-PGJ2 and the ligation of PPARgamma. J Clin Invest 2003; 112:945–55. [4] Kim EH, Surh YJ. 15-deoxy-delta12,14-prostaglandin J2 as a potential endogenous regulator of redox-sensitive transcription factors. Biochem Pharmacol 2006;72: 1516–28. [5] Kitz K, Windischhofer W, Leis HJ, Huber E, Kollroser M, Malle E. 15-deoxydelta12,14-prostaglandin J2 induces Cox-2 expression in human osteosarcoma cells through MAPK and EGFR activation involving reactive oxygen species. Free Radic Biol Med 2011;50:854–65.
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[6] Claycomb WC, Lanson Jr NA, Stallworth BS, Egeland DB, Delcarpio JB, Bahinski A, et al. HL-1 cells: a cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte. Proc Natl Acad Sci U S A 1998;95: 2979–84. [7] Rajakariar R, Hilliard M, Lawrence T, Trivedi S, Colville-Nash P, Bellingan G, et al. Hematopoietic prostaglandin D2 synthase controls the onset and resolution of acute inflammation through PGD2 and 15-deoxy-delta12,14 PGJ2. Proc Natl Acad Sci U S A 2007;104:20979–84. [8] Alves C, de Melo N, Fraceto L, de Araujo D, Napimoga M. Effects of 15d-PGJ(2)-loaded poly(D, L-lactide-co-glycolide) nanocapsules on inflammation. Br J Pharmacol 2011; 162:623–32. [9] Hagens WI, Mattos A, Greupink R, de Jager-Krikken A, Reker-Smit C, van LoenenWeemaes A, et al. Targeting 15d-prostaglandin J2 to hepatic stellate cells: two options evaluated. Pharm Res 2007;24:566–74. [10] Straus DS, Glass CK. Cyclopentenone prostaglandins: new insights on biological activities and cellular targets. Med Res Rev 2001;21:185–210. [11] Cox B, Murphey LJ, Zackert WE, Chinery R, Graves-Deal R, Boutaud O, et al. Human colorectal cancer cells efficiently conjugate the cyclopentenone prostaglandin, prostaglandin J2, to glutathione. Biochim Biophys Acta 2002;1584:37–45. [12] Cheron A, Peltier J, Perez J, Bellocq A, Fouqueray B, Baud L. 15-deoxy-delta12,14prostaglandin J2 inhibits glucocorticoid binding and signaling in macrophages through a peroxisome proliferator-activated receptor gamma-independent process. J Immunol 2004;172:7677–83. [13] Tsikas D, Stichtenoth DO. Dietary eicosapentaenoic acid (EPA) to produce antileukemic cyclopentenone prostaglandin J3? Blood 2012;119:2967–8. [14] von Moltke J, Trinidad NJ, Moayeri M, Kintzer AF, Wang SB, van Rooijen N, et al. Rapid induction of inflammatory lipid mediators by the inflammasome in vivo. Nature 2012;490:107–11. [15] Monneret G, Li H, Vasilescu J, Rokach J, Powell WS. 15-deoxy-delta12,14-prostaglandins D2 and J2 are potent activators of human eosinophils. J Immunol 2002;168: 3563–9. [16] Powell WS. 15-Deoxy-delta, 12,14–PGJ2: endogenous PPARgamma ligand or minor eicosanoid degradation product? J Clin Invest 2003;112:828–30. [17] Wasinger C, Künzl M, Minichsdorfer C, Höller C, Zellner M, Hohenegger M. Autocrine secretion of 15d-PGJ2 mediates simvastatin induced apoptotic burst in human metastatic melanoma cells. Br J Pharmacol 2014. http://dx.doi.org/10.1111/bph.12871. [18] Ho TC, Chen SL, Yang YC, Chen CY, Feng FP, Hsieh JW, et al. 15-deoxy-delta12,14 prostaglandin J2 induces vascular endothelial cell apoptosis through the sequential activation of MAPKS and p53. J Biol Chem 2008;283:30273–88. [19] Lim HJ, Lee KS, Lee S, Park JH, Choi HE, Go SH, et al. 15d-PGJ2 stimulates HO-1 expression through p38 MAP kinase and Nrf-2 pathway in rat vascular smooth muscle cells. Toxicol Appl Pharmacol 2007;223:20–7. [20] Qiu H, Liu JY, Wei D, Li N, Yamoah EN, Hammock BD, et al. Cardiac-generated prostanoids mediate cardiac myocyte apoptosis after myocardial ischaemia. Cardiovasc Res 2012;95:336–45. [21] Inoue T, Eguchi Y, Matsumoto T, Kijima Y, Kato Y, Ozaki Y, et al. Lipocalin-type prostaglandin D synthase is a powerful biomarker for severity of stable coronary artery disease. Atherosclerosis 2008;201:385–91.
Contents lists available at ScienceDirect
International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard
Letter to the Editor
Response to letter by Tsikas et al. Chintan N. Koyani a, Werner Windischhofer b, Christine Rossmann a, Frank R. Heinzel c, Wolfgang Sattler a, Ernst Malle a,⁎ a b c
Institute of Molecular Biology and Biochemistry, Medical University of Graz, Austria Department of Pediatrics and Adolescence Medicine, Research Unit of Osteological Research and Analytical Mass Spectrometry, Medical University of Graz, Austria Department of Internal Medicine, Division of Cardiology, Medical University of Graz, Austria
a r t i c l e
i n f o
Article history: Received 6 September 2014 Accepted 20 September 2014 Available online 28 September 2014 Keywords: Cardiomyocytes 15d-PGJ2 PGD2 receptor DP2 Reactive oxygen species Therapeutic target
Dear Editor, We appreciate the interest of Tsikas et al. [1] in our manuscript [2] and the opportunity to clarify theoretical and experimental aspects describing the effects of 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2) on cardiomyocyte function. We agree that concentrations of 15d-PGJ2 (a degradation product of prostaglandin D2 [PGD2]) used in most in vitro studies [3–5] including ours (Figs. 1–6 [2]) are higher than occurring in vivo. In our study, HL1 cells (an immortalized cardiomyocyte cell line [6]) required such a high 15d-PGJ2 concentration (15 μM, Figs. 1–6 [2]) while in primary murine cardiomyocytes downstream signaling and induction of apoptosis were observed at much lower 15d-PGJ2 concentrations (10–50 nM; Fig. 7B–C [2]). These data suggest that primary cardiomyocytes respond to 15d-PGJ2 treatment at more physiologically relevant concentrations. Another point worth mentioning is avid binding of 15d-PGJ2 to cardiomyocyte or hepatic stellate cell culture medium constituents [7–9], which significantly reduces bioavailability of the active 15d-PGJ2 compound. ⁎ Corresponding author at: Medical University of Graz, Institute of Molecular Biology and Biochemistry, Harrachgasse 21, A-8010 Graz, Austria. Tel.: + 43 316 380 4208; fax: + 43 316 380 9615. E-mail address: [email protected] (E. Malle).
http://dx.doi.org/10.1016/j.ijcard.2014.09.111 0167-5273/© 2014 Elsevier Ireland Ltd. All rights reserved.
As the authors [1] correctly point out, the use of N-acetyl-L-cysteine (NAC) as reactive oxygen species (ROS) scavenger is questionable due to the formation of thioethers between NAC and 15d-PGJ2. To overcome this problem we have used pyrrolidine dithiocarbamate and Tempol (Fig. 1E [2]), two compounds that reduced DCF fluorescence (an indicator of ROS formation) more efficiently than NAC. Cyclopentenone PGs, such as 15d-PGJ2, are characterized by a highly reactive electrophilic carbonyl group in the prostane ring, which can readily adduct thiol-containing compounds including reduced glutathione (GSH) or cysteine [10]. Thioether formation between 15d-PGJ2 and GSH or cysteine reaches steady state levels after 2–3 h under in vitro conditions [11,12]. In contrast, most PGs exert their effects within few minutes [3]. In line, we report that 15d-PGJ2-mediated cellular signaling cascades are activated 2.5 min post treatment (Fig. 1D [2]). This difference in kinetics suggests that the effects we observed are independent of thiolation reactions. Using GC–MS/MS technology, Tsikas et al. [1,13] determined 15dPGJ2 levels in urine of healthy donors in the pM range. It remains to be evaluated whether these findings can be extrapolated to 15d-PGJ2 levels in serum/plasma or organs/tissues. In light of local macrophage inflammasome activation that can result in an ‘eicosanoid storm’ [14] (including PGD2, the parent eicosanoid of 15d-PGJ2), it is conceivable that such an event could generate a rapid local increase of PG concentrations. 15d-PGJ2 can exert its biological effects via receptor-independent or -dependent pathways. The latter include peroxisome proliferatoractivated-receptor γ (PPARγ) [10] and PGD2 receptors (DP1 and DP2) [7,15]. The Powell group [15] has unambiguously shown that PPARγmediated activation requires high 15d-PGJ2 concentrations while DP2 receptor-mediated activation in eosinophils is favored at low (1–10 nM) concentrations. In line, our results demonstrate that low 15d-PGJ2 concentrations (10–50 nM) activated DP2-dependent but PPARγ-independent signaling cascades in primary cardiomyocytes (Fig. 7 [2]). In HL-1 cells, 15d-PGJ2 failed to activate PPARγ-dependent signaling even at high concentrations (10–30 μM, Figs. 1 and 4 [2]). The low 15d-PGJ2 concentrations used in our study are in line with the assumption that low levels of this compound would be pro- rather than anti-inflammatory [16]. It is currently not clear whether the urinary 15(S)-8-iso-PGF/15dPGJ2 ratio, as mentioned by Tsikas et al. [1], might be considered a surrogate marker for the generation of 15d-PGJ2-mediated oxidative stress. However, a series of published articles revealed an association of 15d-
C.N. Koyani et al. / International Journal of Cardiology 177 (2014) 140–141
PGJ 2 with ROS production in different cellular systems at lower (95–160 nM) [17] as well as higher concentrations (1–20 μM) [5,18,19]. We agree with Tsikas et al. [1] on their view that direct therapeutic targeting of 15d-PGJ2 would be a difficult task. Nevertheless, upstream interference can be achieved by modulation of COX-1/2-dependent pathways, e.g. by aspirin in a murine model of myocardial infarction [20]. This is supported by findings in COX-1 knockout mice, which are protected against pathological effects of high concentrations of PGs that are released in response to inflammasome activation [14]. Pharmacological targeting of lipocalin-type PGD synthase, a powerful marker for coronary artery diseases in humans [21], might represent another indirect approach to impair PGD2 levels. Moreover, we present a novel therapeutic alternative to overcome 15d-PGJ2-induced cardiomyocyte injury by DP2 antagonism. In conclusion, DP2 antagonism may represent a pharmacological strategy to alleviate cellular/organ dysfunction induced by PGD2, its metabolites including 15d-PGJ2 or other hitherto unidentified DP2 agonists. Conflict of interest The authors report no relationship that could be constructed as a conflict of interest. References [1] Tsikas D, Niemann J, Beckmann B. 15-deoxy-delta12,14-PGJ2: an interesting but unapproachable pharmacological target? Int J Cardiol 2014. http://dx.doi.org/10.1016/ j.ijcard.2014.08.145. [2] Koyani CN, Windischhofer W, Rossmann C, Jin G, Kickmaier S, Heinzel FR, et al. 15deoxy-delta12,14-prostaglandin J2 promotes inflammation and apoptosis in cardiomyocytes via the DP2/MAPK/TNFalpha axis. Int J Cardiol 2014;173:472–80. [3] Bell-Parikh LC, Ide T, Lawson JA, McNamara P, Reilly M, FitzGerald GA. Biosynthesis of 15-deoxy-delta, 12,14-PGJ2 and the ligation of PPARgamma. J Clin Invest 2003; 112:945–55. [4] Kim EH, Surh YJ. 15-deoxy-delta12,14-prostaglandin J2 as a potential endogenous regulator of redox-sensitive transcription factors. Biochem Pharmacol 2006;72: 1516–28. [5] Kitz K, Windischhofer W, Leis HJ, Huber E, Kollroser M, Malle E. 15-deoxydelta12,14-prostaglandin J2 induces Cox-2 expression in human osteosarcoma cells through MAPK and EGFR activation involving reactive oxygen species. Free Radic Biol Med 2011;50:854–65.
141
[6] Claycomb WC, Lanson Jr NA, Stallworth BS, Egeland DB, Delcarpio JB, Bahinski A, et al. HL-1 cells: a cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte. Proc Natl Acad Sci U S A 1998;95: 2979–84. [7] Rajakariar R, Hilliard M, Lawrence T, Trivedi S, Colville-Nash P, Bellingan G, et al. Hematopoietic prostaglandin D2 synthase controls the onset and resolution of acute inflammation through PGD2 and 15-deoxy-delta12,14 PGJ2. Proc Natl Acad Sci U S A 2007;104:20979–84. [8] Alves C, de Melo N, Fraceto L, de Araujo D, Napimoga M. Effects of 15d-PGJ(2)-loaded poly(D, L-lactide-co-glycolide) nanocapsules on inflammation. Br J Pharmacol 2011; 162:623–32. [9] Hagens WI, Mattos A, Greupink R, de Jager-Krikken A, Reker-Smit C, van LoenenWeemaes A, et al. Targeting 15d-prostaglandin J2 to hepatic stellate cells: two options evaluated. Pharm Res 2007;24:566–74. [10] Straus DS, Glass CK. Cyclopentenone prostaglandins: new insights on biological activities and cellular targets. Med Res Rev 2001;21:185–210. [11] Cox B, Murphey LJ, Zackert WE, Chinery R, Graves-Deal R, Boutaud O, et al. Human colorectal cancer cells efficiently conjugate the cyclopentenone prostaglandin, prostaglandin J2, to glutathione. Biochim Biophys Acta 2002;1584:37–45. [12] Cheron A, Peltier J, Perez J, Bellocq A, Fouqueray B, Baud L. 15-deoxy-delta12,14prostaglandin J2 inhibits glucocorticoid binding and signaling in macrophages through a peroxisome proliferator-activated receptor gamma-independent process. J Immunol 2004;172:7677–83. [13] Tsikas D, Stichtenoth DO. Dietary eicosapentaenoic acid (EPA) to produce antileukemic cyclopentenone prostaglandin J3? Blood 2012;119:2967–8. [14] von Moltke J, Trinidad NJ, Moayeri M, Kintzer AF, Wang SB, van Rooijen N, et al. Rapid induction of inflammatory lipid mediators by the inflammasome in vivo. Nature 2012;490:107–11. [15] Monneret G, Li H, Vasilescu J, Rokach J, Powell WS. 15-deoxy-delta12,14-prostaglandins D2 and J2 are potent activators of human eosinophils. J Immunol 2002;168: 3563–9. [16] Powell WS. 15-Deoxy-delta, 12,14–PGJ2: endogenous PPARgamma ligand or minor eicosanoid degradation product? J Clin Invest 2003;112:828–30. [17] Wasinger C, Künzl M, Minichsdorfer C, Höller C, Zellner M, Hohenegger M. Autocrine secretion of 15d-PGJ2 mediates simvastatin induced apoptotic burst in human metastatic melanoma cells. Br J Pharmacol 2014. http://dx.doi.org/10.1111/bph.12871. [18] Ho TC, Chen SL, Yang YC, Chen CY, Feng FP, Hsieh JW, et al. 15-deoxy-delta12,14 prostaglandin J2 induces vascular endothelial cell apoptosis through the sequential activation of MAPKS and p53. J Biol Chem 2008;283:30273–88. [19] Lim HJ, Lee KS, Lee S, Park JH, Choi HE, Go SH, et al. 15d-PGJ2 stimulates HO-1 expression through p38 MAP kinase and Nrf-2 pathway in rat vascular smooth muscle cells. Toxicol Appl Pharmacol 2007;223:20–7. [20] Qiu H, Liu JY, Wei D, Li N, Yamoah EN, Hammock BD, et al. Cardiac-generated prostanoids mediate cardiac myocyte apoptosis after myocardial ischaemia. Cardiovasc Res 2012;95:336–45. [21] Inoue T, Eguchi Y, Matsumoto T, Kijima Y, Kato Y, Ozaki Y, et al. Lipocalin-type prostaglandin D synthase is a powerful biomarker for severity of stable coronary artery disease. Atherosclerosis 2008;201:385–91.
