wetenschapsdag 2023 | 113 Best Abstract Sessie 4 Auteurs K. Rombouts, T. van Merrienboer, N. Bogunovic, J. van der Velden, K. Yeung Abstract titel NUAK1 kinase activity regulates contraction in smooth muscle cells derived from abdominal aortic aneurysm patients Background Abdominal aortic aneurysms (AAA) are defined as a weakening and dilatation of the aortic wall. The aim of this study is to investigate the underlying mechanism of altered in vitro contractility of vascular smooth muscle cells (SMC) derived from AAA patients, compared to control SMC (C-SMC). Methods Contractility of AAA-SMC (n=39) and C-SMC (n=18) was measured upon ionomycin stimulation using Electric Cell-substrate Impedance Sensor. A (phospho)proteomics analysis was performed in AAA-SMC (n=24) and control SMC (n=8). Integrative Inferred Kinase Activity (INKA) analysis was used to calculate kinase activity scores, based on phosphorylation data of either kinases or their substrates. Results AAA-SMC were divided into subgroups based on contraction (AAA-Low contracting: mean: 70,63%, SD: 5,51% (n=8); AAANormal contracting: mean: 83.69%, SD: 3.35% (n=22); AAA-High contracting: mean: 91.72%, SD: 1.22% (n=9)). Protein expression levels of Thrombospondin-1 (r=.21, p= 0.0091), PDZ and LIM domain protein 4 (r=.46, p<0.0001) and ATPase plasma membrane Ca2+ transporting 1 (r=.17, p=0.018) were correlated to SMC contraction, but siRNA mediated knock down of these proteins did not affect SMC contraction. INKA analysis identified NUAK1 kinase activity as potential regulator of SMC contraction, by phosphorylation on 3 amino acids on myosin phosphatase (MYPT1). This was confirmed by a correlation between NUAK1 activity and phosphorylation levels of 2 MYPT1 phosphosites (Ser445 (r=.24, p=0.0056) and Ser910 (r=.52, p<0.0001)). Moreover, NUAK1 protein and RNA expression levels were correlated to SMC contraction and NUAK1 knock down decreased contraction in AAA-SMC, but not in C-SMC. Conclusion Impaired contraction of AAA-SMC is seen compared to C-SMC, and this is regulated by NUAK1 kinase activity and expression. Currently we are performing further experiments to explore how NUAK1 exactly regulates contraction in AAA-SMC. Finding proteins involved in AAA-SMC dysfunction can contribute to novel non-invasive treatment options for prevention and/or stabilization of AAA.
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