Vascular complications of type 2 diabetes: tyrosine phosphorylation of the endothelial nitric oxide synthase (eNOS)
Final Report Abstract
Up to 80% of deaths in patients with diabetes are due to the associated cardiovascular complications. Endothelial dysfunction is an early event in disease development and is characterised by the decreased production of endothelium-derived nitric oxide (NO). The activity of the endothelial NO synthase (eNOS) is regulated by Ca2+-dependent and – independent processes; particularly by its post-translational modification by phosphorylation. At the initiation of this project we had identified a novel regulatory tyrosine residue within eNOS (Tyr657; human sequence) that attenuates enzyme activity and linked this event with the activation of the redox- and insulin-sensitive proline-rich tyrosine kinase (PYK2). To assess the physiological role of this residue in vivo a knock-in mouse was generated in which Tyr656 (mouse sequence) was replaced with phenylalanine. The resulting Y656F-eNOS mice demonstrated a mildly hypotensive phenotype and both in situ assessment of vasodilatation and in vitro analysis of aortic tone revealed an enhanced response to acetylcholine. Most significantly the animals were resistant to angiotensin II-induced endothelial dysfunction. To determine the relevance of eNOS tyrosine phosphorylation in the development of cardiovascular disease the Y656F-eNOS mice were crossed into the ApoE-/- background and atherosclerosis studied following the feeding of a high cholesterol diet and following partial carotid artery ligation. Preventing the tyrosine phosphorylation of eNOS resulted in attenuated plaque formation in the aorta, aortic arch and carotid arteries and markedly reduced the endothelial dysfunction assessed in carotid arteries 7 days after ligation. The attenuated plaque formation was associated with decreased neutrophil recruitment. At the molecular level studies are focussed on determining alterations in O2- /H2O2 and NO production by eNOS and the potential interplay between the tyrosine phosphorylation and S-glutathionylation of eNOS. The Tyr656 site is found in the FMN-binding domain of eNOS – a region in which critical changes in enzyme conformation take place during electron transport. The finding that the mutation of Tyr656 was able to increase NO output in healthy animals was surprising given the large reserve of NO production evident in wild-type mice. Most surprising was the apparent protection against the development of endothelial dysfunction following the infusion of angiotensin II – which indicates that targeting the pathways resulting in eNOS tyrosine phosphorylation may be potentially targetable for a therapeutic approach. To date we have only identified Src and PYK2 as being able to phosphorylate this site and available PYK2 inhibitors are able to prevent the H2O2 induced stunning of the endothelium in vitro. PYK2 is however closely linked to the focal adhesion kinase FAK and currently available inhibitors tend to inhibit both kinases – a major disadvantage given the important role of FAK in cellular processes. Therefore proteomic studies are ongoing to determine the components of the eNOS signalosome and how the phosphorylation of Tyr657 affects its components, and to identify additional kinases and tyrosine phosphatase that affect the phosphorylation state of this site. Taken together, the identification of a novel residue within eNOS, the phosphorylation of which has the potential to affect endothelial dysfunction is of great potential clinical relevance.
Publications
-
cAMP phosphodiesterase inhibitors increase nitric oxide production by modulating dimethylarginine dimethylaminohydrolases. Circulation. 2011;123:1194-204
Pullamsetti SS, Savai R, Schaefer MB, Wilhelm J, Ghofrani HA, Weissmann N, Schudt C, Fleming I, Mayer K, Leiper J, Seeger W, Grimminger F, Schermuly RT
-
Nitric oxide-induced activation of the AMP-activated protein kinase 2 subunit attenuates IkB kinase activity and inflammatory responses in endothelial cells. PLoS ONE. 2011;6:e20848
Bess E, Fisslthaler B, Frömel T, Fleming I
-
DNA repair deficiency of the nucleotide excision repair system contributes to the progression of age-related vascular dysfunction. Circulation. 2012;126:468-78
Durik M, Kavousi M, van der Pluijm I, Isaacs A, Cheng C, Verdonk K, Loot AE, Oeseburg H, Bhaggoe U, Leijten F, van Veghel R, de Vries R, Rudez G, Brandt R, Ridwan Y, van Deel E, de Boer M, Tempel D, Fleming I, Mitchell GF, Verwoert GC, Tarasov KV, Uitterlinden AG, Hofman A, Duckers HJ, van Duijn CM, Oostra BA, Witteman J, Duncker DJ, Danser AHJ, Hoeijmakers J, Roks AJM
-
Leptin potentiates endothelium-dependent relaxation by inducing endothelial expression of neuronal nitric oxidase synthase (nNOS). Arterioscler Thromb Vasc Biol. 2012;32:1605-12
Benkhoff S, Loot AE, Pierson I, Sturza A, Kohlstedt K, Fleming I, Shimokawa H, Grisk O, Brandes RP, Schröder K
-
Monoamine oxidases are mediators of endothelial dysfunction in the mouse aorta. Hypertension. 2013;62:140-6
Sturza A, Leisegang M, Babelova A, Schröder K, Benkhoff S, Loot AE, Fleming I, Schulz R, Muntean D, Brandes RP
-
The atherosusceptible endothelium: endothelial phenotypes in complex hemodynamic shear stress regions in vivo. Cardiovasc Res. 2013;99:315-27
Davies PF, Civelek M, Fang Y, Fleming I
-
Role of secreted modular calcium binding protein 1 (SMOC1) in transforming growth factor β signaling and angiogenesis. Cardiovasc Res. 2015
Awwad K, Hu J, Shi L, Mangels N, Abdel Malik R, Zippel N, Fisslthaler B, Eble JA, Pfeilschifter J, Popp R, Fleming I
