Over the last two years we have collected intriguing new evidence to indicate that ACE is a signal transduction molecule, which is involved in outside-in signalling in as much as the binding of an ACE substrate or an ACE inhibitor to ACE is able to elicit intracellular events.

The aims of this research project are to:
1. elucidate the intracellular events affected by ACE signalling as well as the physiological consequences of their activation.
2. clarify the factors and stimuli determining ACE expression in endothelial cells and as a consequence may affect downstream signalling.

Regulation of the endothelial nitric oxide synthase (eNOS)
Mechanical forces generated at the endothelium by pulsatile blood flow are the physiologically most important stimuli ensuring the continuous release of autacoids such as nitric oxide (NO), prostacyclin and the endothelium-derived hyperpolarizing factor (EDHF), which modulate local vascular tone, cell signaling, and gene expression.
Our group is interested in the mechanisms underlying the regulation of eNOS especially in the regulation of eNOS by its phosphorylation in response to haemodynamic and hormonal stimuli. Indeed, in addition to showing that phosphorylation, independent of a maintained increase in intracellular Ca2+, increases eNOS activity in shear stress-.stimulated cells we have also highlighted the role of Thr495 within the calmodulin binding domain of the enzyme for the initiation of NO production. Phosphorylation also appears to be involved in the phenomenon of eNOS uncoupling, in which the enzyme begins to generate reactive oxygen species rather than NO.
eNOS is also expressed in platelets but the mechanisms regulating the activity of platelet eNOS appear to be distinct from the regulating the enzyme in endothelial cells. Moreover, platelet eNOS-derived NO has the unexpected function of regulating the degranulation of dense granules by a process involving the NO-dependent association of VAMP-3 with synthaxin 2.

Role of cytochrome P450 epoxygenases in the regulation of vascular homeostasis
Our groups interest in cytochrome P450 epoxygenases within the vascular wall began with the race to identify the endothelium-derived hyperpolarizing factor (EDHF) that accounts for the nitric oxide and prostacyclin-independent vasodilatation of some vascular beds. Indeed, a cytochrome P450 epoxygenase of the 2C subfamily could be identified as a coronary EDHF synthase. Over the last 8 years it has become clear that the arachidonic acid-derived products of the CYP 2C epoxygenases (the epoxyeicosatrienoic acids or EETs) are more than simple vasodilators and are able to influence endothelial cell signaling as well as endothelial cell proliferation, migration and angiogenesis.
Although general statements about the impact of cardiovascular disorders on the endothelium and vice versa, cannot adequately address the complexity of the situation, arteriosclerosis is generally associated with a dysregulation of endothelial cell function or "endothelial dysfunction"; roughly defined as the shut down of certain intrinsic endothelial anti-atherogenic mechanisms.
In healthy vessels, the endothelium produces mainly NO and prostacyclin and the vasodilator and growth inhibitory influence predominates. The development of atherosclerosis however, is associated with an apparent decrease in the bioavailability of NO and a concomitant increase in the generation of NO-scavenging oxygen-derived free radicals, such as superoxide anions (O2-). While the enzymatic source of endothelial O2- has been debated intensely over the last few years, it may well turn out that, in addition to the NADH/NADPH oxidase and the uncoupled endothelial NO synthase (eNOS) that cytochrome P450 2C epoxygenases are also physiologically relevant sources of oxygen-derived free radicals. Indeed, an inhibitor of CYP 2C9 restores endothelial function (vasodilatation to acetylcholine) in patients with coronary artery disease.

Relevant publications
Benzing T, Fleming I, Blaukat A, Müller-Esterl W, Busse R. Angiotensin-converting enzyme inhibitor ramiprilat interferes with the sequestration of the B2 kinin receptor within the plasma membrane of native endothelial cells. Circulation. 1999;99:2034-2040.

Kohlstedt K, Shoghi F, Müller-Esterl W, Busse R, Fleming I. CK2 phosphorylates the angiotensin-converting enzyme and regulates its retention in the endothelial cell plasma membrane. Circ Res. 2002;91:749-756.
Kohlstedt K, Brandes RP, Muller-Esterl W, Busse R, Fleming I. Angiotensin-converting enzyme is involved in outside-in signaling in endothelial cells. Circ Res. 2004;94:60-67.

Kohlstedt, K., Busse, R., and Fleming, I. Signalling via the angiotensin-converting enzyme enhances the expression of cyclooxygenase-2 in endothelial cells. Hypertension . 2005. In Press

Ayajiki K, Kindermann M, Hecker M, Fleming I, Busse R. Intracellular pH and tyrosine phosphorylation but not calcium determine shear stress-induced nitric oxide production in native endothelial cells. Circ Res. 1996;78:750-758.

Fleming I, Bara A, Busse R. Calcium signalling and autacoid production in endothelial cells are modulated by changes in tyrosine kinase and phosphatase activity. J Vasc Res. 1996;33:225-234.

Bauersachs J, Bouloumié A, Mülsch A, Wiemer G, Fleming I, Busse R. Vasodilator dysfunction in aged spontaneously hypertensive rats: changes in NO synthase III and soluble guanylyl expression and in superoxide anion production. Cardiovasc Res. 1998;37:772-779.

Fleming I, Bauersachs J, Fisslthaler B, Busse R. Calcium-independent activation of the endothelial nitric oxide synthase in response to tyrosine phosphatase inhibitors and fluid shear stress. Circ Res. 1998;82:686-695.

Fleming I, Bauersachs J, Schäfer A, Scholz D, Aldershvile J, Busse R. Isometric contraction induces the Ca2+-independent activation of the endothelial nitric oxide synthase. Proc Natl Acad Sci USA. 1999;96:1123-1128.

Dimmeler S, Fleming I, Fisslthaler B, Hermann C, Busse R, Zeiher AM. Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature. 1999;399:601-605.

Fleming I, Busse R. Signal transduction of eNOS activation. Cardiovasc Res. 1999;43:532-541.

Fisslthaler B, Dimmeler S, Hermann C, Busse R, Fleming I. Phosphorylation and activation of the endothelial nitric oxide synthase by fluid shear stress. Acta Physiol Scand. 2000;168:81-88.

Fleming I, Fisslthaler B, Dimmeler S, Kemp BE, Busse R. Phosphorylation of Thr495 regulates Ca2+/calmodulin-dependent endothelial nitric oxide synthase activity. Circ Res. 2001;88:e68-e75.

Fleming I, Busse R. Molecular mechanisms involved in the regulation of the endothelial nitric oxide synthase. Am J Physiol Regul Integr Comp Physiol. 2003;284:1-12.

Fleming I, Schulz C, Fichtlscherer B, Kemp BE, Fisslthaler B, Busse R. AMP-activated protein kinase (AMPK) regulates the insulin-induced activation of the nitric oxide synthase in human platelets. Thromb Haemost. 2003;90:863-871.

Lin MI, Fulton D, Babbitt R, Fleming I, Busse R, Pritchard KA, Jr., Sessa WC. Phosphorylation of threonine 497 in endothelial nitric-oxide synthase coordinates the coupling of L-arginine metabolism to efficient nitric oxide production. J Biol Chem. 2003;278:44719-44726.

Lenasi H, Kohlstedt K, Fichtlscherer B, Mülsch A, Busse R, Fleming I. Amlodipine activates the endothelial nitric oxide synthase by altering phosphorylation on Ser1177 and Thr495. Cardiovasc Res. 2003;59:844-853.

Randriamboavonjy V, Schrader J, Busse R, Fleming I. Insulin induces the release of vasodilator compounds from platelets by a nitric oxide-G kinase-VAMP-3-dependent pathway. J Exp Med. 2004;199:347-356.

Fleming I, Busse.R. The physiology of nitric oxide: control and consequences. Curr Med Chem. 2004;3:189-205.

Fleming, I., Mohamed, A., Galle, J., Turchanowa, L., Brandes, R. P., Fisslthaler, B., and Busse, R. Oxidized low density lipoprotein increases superoxide production by endothelial nitric oxide synthase by inhibiting PKCa. Cardiovasc.Res. 2005. In Press.

Bauersachs J, Popp R, Hecker M, Sauer E, Fleming I, Busse R. Nitric oxide attenuates the release of endothelium-derived hyperpolarizing factor. Circulation. 1996;94:3341-3347.

Popp R, Bauersachs J, Hecker M, Fleming I, Busse R. A transferable, b-naphthoflavone-inducible, hyperpolarizing factor is synthesised by native and cultured porcine coronary endothelial cells. J Physiol (Lond ). 1996;497.3:699-709.

Bauersachs J, Fleming I, Scholz D, Popp R, Busse R. Endothelium-derived hyperpolarizing factor but not nitric oxide is reversibly inhibited by brefeldin A. Hypertension. 1997;30:1598-1605.

Popp R, Fleming I, Busse R. Pulsatile stretch in coronary arteries elicits release of endothelium-derived hyperpolarizing factor: a modulator of arterial compliance. Circ Res. 1998;82:696-703.

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.

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.

Bolz SS, Fisslthaler B, Pieperhoff S, de Wit C, Fleming I, Busse R, Pohl U. Antisense oligonucleotides against cytochrome P450 2C8 attenuate EDHF-mediated Ca2+ changes and dilation in isolated resistance arteries. FASEB J. 2000;14:255-260.

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.

Fleming I. Cytochrome P450 2C is an EDHF synthase in coronary arteries. Trends Cardiovasc Med. 2000;10:166-170.

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.

Fisslthaler B, Popp R, Michaelis UR, Kiss L, Fleming I, Busse R. Cyclic Stretch Enhances the Expression and Activity of the Coronary EDHF Synthase. Hypertension. 2001;38:1427-1432.

Fleming I, Fisslthaler B, Michaelis UR, Kiss L, Popp R, Busse R. The coronary EDHF stimulates multiple signalling pathways and proliferation in vascular cells. Pfluger's Arch Eur J Physiol. 2001;442:511-518.

Fleming I. Cytochrome P450 and vascular homeostasis. Circ Res. 2001;89:753-762.

Popp R, Brandes RP, Ott G, Busse R, Fleming I. Dynamic modulation of interendothelial gap junctional communication by 11,12-epoxyeicosatrienoic acid. Circ Res. 2002;90:800-806.

Potente M, Michaelis UR, Fisslthaler B, Busse R, Fleming I. CYP 2C9-induced endothelial cell proliferation involves induction of MAP kinase phosphatase 1, inhibition of the c-Jun N-terminal kinase and upregulation of cyclin D1. J Biol Chem. 2002;277:15671-15676.

Bauersachs J, Christ M, Ertl G, Michaelis UR, Fisslthaler B, Busse R, Fleming I. Cytochrome P450 2C expression and EDHF-mediated relaxation in porcine coronary arteries is increased by cortisol. Cardiovasc Res. 2002;54:669-675.

Busse R, Edwards G, Feletou M, Fleming I, Vanhoutte PM, Weston AH. EDHF: bringing the concepts together. Trends Pharmacol Sci. 2002;23:374-380.

Büssemaker, E., Wallner, C., Fisslthaler, B., and Fleming, I. The Na-K-ATPase is a target for an EDHF displaying characteristics similar to potassium ions in the porcine renal interlobar artery. Br J Pharmacol. 2002;137:647-654.

Hillig T, Krustrup P, Fleming I, Osada T, Saltin B, Hellsten Y. Cytochrome P450 2C9 plays an important role in the regulation of exercise-induced skeletal muscle blood flow and oxygen uptake in humans. J Physiol (Lond ). 2003;546:307-314.

Fisslthaler B, Michaelis UR, Randriamboavonjy V, Busse R, Fleming I. Cytochrome P450 epoxygenases and vascular tone: novel role for HMG Co A reductase inhibitors in the regulation of CYP 2C expression. Biochim Biophys Acta. 2003;1619:332-339.

Michaelis UR, Fisslthaler B, Medhora M, Harder D, Fleming I, Busse R. Cytochrome P450 2C9-derived epoxyeicosatrienoic acids induce angiogenesis via cross-talk with the epidermal growth factor receptor (EGFR). FASEB J. 2003;17:770-772.

Büssemaker E, Popp R, Binder J, Busse R, Fleming I. Characterization of the endothelium-derived hyperpolarizing factor (EDHF) response in the human interlobar artery. Kidney Int. 2003;63:1749-1755.

Potente M, Fisslthaler B, Busse R, Fleming I. 11,12-Epoxyeicosatrienoic acid-induced inhibition of FOXO factors promotes endothelial proliferation by down-regulating p27Kip1. J Biol Chem. 2003;278:29619-29625.

Passauer J, Büssemaker E, Lassig G, Pistrosch F, Fauler J, Gross P, Fleming I. Baseline blood flow and bradykinin-induced vasodilator responses in the human forearm are insensitive to the CYP 2C9 inhibitor sulfaphenazole. Clin Sci (Lond). 2003;105:513-518.

Fichtlscherer S, Dimmeler S, Breuer S, Busse R, Zeiher AM, Fleming I. Inhibition of cytochrome P450 2C9 improves endothelium-dependent, nitric oxide-mediated vasodilatation in patients with coronary artery disease. Circulation. 2004;109:178-183.

Fleming I. Cytochrome P450 epoxygenases as EDHF-synthase(s). Pharmacol Res. 2004;49:525-533.

Michaelis, U. R., Falck, J. R., Schmidt, R., Busse, R., and Fleming, I. Cytochrome P4502C9-derived epoxyeicosatrienoic acids induce the expression of cyclooxygenase-2 in endothelial cells. Arterioscler.Thromb.Vasc.Biol. 2005. In Press

Passauer, J., Pistrosch, F., Lässig, G., Herbrig, K., Büssemaker, E., Gross, P., and Fleming, I. Nitric oxide - and EDHF-mediated arteriolar tone in uremia is unaffected by selective inhibition of vascular cytochrome P450 2C9. Kidney Int. 2005. In Press