Busse Beate Fisslthaler, Rüdiger Popp, U. Ruth ... - Hypertension

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Figure 8. Effect of tyrosine kinase inhibitors on the stretchinduced tyrosine phosphorylation of endothelial cells and the expression of CYP 2C. A and B, Immunohistochemical staining of tyrosine-phosphorylated proteins (A) and CYP 2C (B) in porcine coronary endothelial cells maintained under static conditions and after exposure to cyclic stretch (6%, 1 Hz) in the absence and presence of herbimycin (Herbi, 5 �mol/L) and PP1 (10 �mol/L). The results presented are representative of data obtained in 2 further experiments, and the bar represents 20 �m. C, Cultured coronary endothelial cells were either maintained under static conditions (S) or exposed to cyclic stretch (C; 6%, 1 Hz, 18 hours) in the absence and presence of Herbi (5 �mol/L). Thereafter, RNA was isolated for RT-PCR as described. The results presented are representative of data obtained in 2 further experiments. protein in native and cultured endothelial cells. The effects of stretch on CYP 2C appear to be 2-fold, inasmuch as the acute application of moderate levels of cyclic stretch enhanced enzyme activity and EET production, whereas chronic stretch enhanced the expression of CYP 2C mRNA and protein. Native and cultured endothelial cells express several CYP isozymes, including the hydroxylases CYP 3A and CYP 2B and the arachidonic acid epoxygenases CYP 2J and CYP Fisslthaler et al Cyclic Stretch and Endothelial CYP 2C Expression 1431 2C. 4,6,8,9 Of these isozymes, cultured endothelial cells constitutively express CYP 3A and CYP 2J, whereas CYP 2C levels are highly variable, and often no signal is detectable with the use of RT-PCR. For example, although native porcine coronary artery endothelial cells clearly express CYP 2C protein, CYP 2C mRNA and protein are barely detectable in primary cultures of these cells when maintained under static conditions. 4,6 Because pulsatile stretch has been reported to activate the EDHF synthase/CYP 2C in porcine coronary arteries, 3 we postulated that cyclic stretch may regulate the activity and expression of CYP 2C in a manner analogous to the regulation of eNOS activity and expression by fluid shear stress. The results obtained clearly indicate that cyclic stretch regulates the expression of CYP 2C mRNA and protein in both native and cultured endothelial cells. Thus, it is tempting to speculate that cyclic stretch maintains the expression of EDHF synthase in vivo and that its decreased expression in vitro is attributable to the lack of this continuous hemodynamic stimulus. Although endotheliumdependent hyperpolarization of coronary artery smooth muscle cells was not investigated in the present study, we have previously demonstrated that the generation of EETs by endothelial cells pretreated with either nifedipine 6 or �-naphthoflavone, 4 which were similar to those measured in stretch-stimulated endothelial cells, was sufficient to potentiate the endothelium-dependent hyperpolarization of porcine coronary artery smooth muscle cells. The application of cyclic stretch to a variety of different cells is reported to activate numerous signal transduction cascades, including the Janus kinase/signal transducer(s) and activator(s) of transcription 10 and Ras/Raf/extracellular signal–regulated kinase 1/2 pathways, 11 as well as the epidermal growth factor receptor tyrosine kinase, 12 the c-Jun NH 2terminal, 11 and p38 MAP kinases. 12 The mechanism by which pulsatile stretch increases CYP expression in endothelial cells remains to be fully elucidated. In the present study, we observed that the PI 3-K/Akt/mTOR signaling pathway was rapidly activated in stretched endothelial cells, but neither wortmannin nor rapamycin was found to significantly inhibit the induction of CYP 2C protein or the stretch-induced generation of EETs. Additional signaling molecules rapidly activated after the application of cyclic stretch were tyrosine kinases and p38 MAP kinase (authors’ unpublished data, 2001). However, it was not possible to link either of these kinases to the stretch-induced increase in CYP 2C expression. Part of the difficulty in elucidating the pathway determining the stretch-induced expression of CYP 2C was due to the fact that SB203580 and SB220025, 2 specific inhibitors of p38 MAP kinase, and the tyrosine kinase inhibitor herbimycin enhanced the basal expression of CYP 2C in endothelial cells maintained under static conditions. However, each of these inhibitors prevented the stretch-induced activation of the respective kinases in coronary endothelial cells. Because many CYP substrates increase the expression of the enzymes responsible for their metabolism, 13 it is likely that the pharmacological inhibitors may be at least partially metabolized by CYP 2C. Indeed, our previous observation, ie, that the Ca 2� antagonist nifedipine enhances the expression and the activity of the EDHF synthase/CYP 2C in native and Downloaded from http://hyper.ahajournals.org/ by guest on January 29, 2013
1432 Hypertension December 2001 cultured endothelial cells, 6 may be attributable to a similar phenomenon. The functional consequences of endothelial CYP 2C/ EDHF synthase regulation by cyclic stretch are potentially wide reaching, because in addition to activating Ca 2� - dependent K � channels and modulating arterial tone, the EETs regulate multiple endothelial and smooth muscle signaling pathways and may promote both endothelial cell proliferation 14 and angiogenesis. 15 Moreover, EDHF synthase has recently been shown to generate physiologically relevant amounts of free radicals, including O 2 �7 ; thus, the activation of CYP 2C in endothelial cells may participate in the stretch-induced generation of O 2 � , which has, until now, been attributed to the activation of NADPH oxidase. 16 Acknowledgments This work was supported by the Deutsche Forschungsgemeinschaft (FI 830/1-1) and Institut de Recherches Internationales Servier. The authors are indebted to Isabel Winter, Stergiani Hauk, and Sina Bätz for expert technical assistance. References 1. Busse R, Fleming I. Pulsatile stretch and shear stress: physical stimuli determining the production of endothelium-derived relaxing factors. J Vasc Res. 1998;35:73–84. 2. Fleming I. Cytochrome P450 2C is an EDHF synthase in coronary arteries. Trends Cardiovasc Med. 2000;10:166–170. 3. Popp R, Fleming I, Busse R. Pulsatile stretch elicits the release of the endothelium-derived hyperpolarizing factor from isolated coronary arteries: a modulator of arterial compliance. Circ Res. 1998;82:696–703. 4. Fisslthaler B, Popp R, Kiss L, Potente M, Harder DR, Fleming I, Busse R. Cytochrome P450 2C is an EDHF synthase in coronary arteries. Nature. 1999;401:493–497. 5. Popp R, Bauersachs J, Hecker M, Fleming I, Busse R. A transferable, �-naphthoflavone-inducible, hyperpolarizing factor is synthesised by native and cultured porcine coronary endothelial cells. J Physiol (Lond). 1996;497:3:699–709. 6. Fisslthaler B, Hinsch N, Chataigneau T, Popp R, Kiss L, Busse R, Fleming I. Nifedipine increases cytochrome P4502C expression and EDHF-mediated responses in coronary arteries. Hypertension. 2000;36: 270–275. 7. Fleming I, Michaelis UR, Bredenkotter D, Fisslthaler B, Dehghani F, Brandes RP, Busse R. Endothelium-derived hyperpolarizing factor synthase (cytochrome P450 2C9) is a functionally significant source of reactive oxygen species in coronary arteries. Circ Res. 2001;88:44–51. 8. Lin JHC, Kobari Y, Zhu Y, Stemerman MB, Pritchard KA Jr. Human umbilical vein endothelial cells express P450 2C8 mRNA: cloning of endothelial P450 epoxygenase. Endothelium. 1996;4:219–229. 9. Node K, Huo Y, Ruan X, Yang B, Spiecker M, Ley K, Zeldin DC, Liao JK. Anti-inflammatory properties of cytochrome P450 epoxygenasederived eicosanoids. Science. 1999;285:1276–1279. 10. Pan J, Fukuda K, Saito M, Matsuzaki J, Kodama H, Sano M, Takahashi T, Kato T, Ogawa S. Mechanical stretch activates the JAK/STAT pathway in rat cardiomyocytes. Circ Res. 1999;84:1127–1136. 11. MacKenna DA, Dolfi F, Vuori K, Ruoslahti E. Extracellular signalregulated kinase and c-Jun NH 2-terminal kinase activation by mechanical stretch is integrin dependent and matrix specific in rat cardiac fibroblasts. J Clin Invest. 1998;101:301–310. 12. Kudoh S, Komuro I, Hiroi Y, Zou Y, Harada K, Sugaya T, Takekoshi N, Murakami K, Kadowaki T, Yazaki Y. Mechanical stretch induces hypertrophic responses in cardiac myocytes of angiotensin II type 1a receptor knockout mice. J Biol Chem. 1998;273:24037–24043. 13. Guengerich FP. Human cytochrome P450 enzymes. In: Ortiz de Montellano PR, ed. Cytochrome P450: Structure, Mechanism, and Biochemistry. New York, NY: Plenum Press; 1995. 14. Fleming I, Fisslthaler B, Michaelis UR, Kiss L, Popp R, Busse R. The coronary endothelium-derived hyperpolarizing factor (EDHF) stimulates multiple signalling pathways and proliferation in vascular cells. Pflugers Arch. 2001;442:511–518.. 15. Munzenmaier DH, Harder DR. Cerebral microvascular endothelial cell tube formation: role of astrocytic epoxyeicosatrienoic acid release. Am J Physiol. 2000;278:H1163–H1167. 16. Hishikawa K, Oemar BS, Yang ZH, Luscher TF. Pulsatile stretch stimulates superoxide production and activates nuclear factor-�B in human coronary smooth muscle. Circ Res. 1997;81:797–803. Downloaded from http://hyper.ahajournals.org/ by guest on January 29, 2013
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