Abstract

Cell-to-cell communication plays an important role in multicellular tissues. It is also pivotal in the modulation of vessel functions, such as the regulation of vessel tone. Heterocellular communication between endothelial cells (EC) and vascular smooth muscle cells (VSMC) via myoendothelial gap junctions (MEGJs) represents an important communication pathway in resistance arteries and arterioles. Myoendothelial junctions (MEJ) are, however, not only the site of direct cell communication via gap junctions but also represent a signaling microdomain critical for localizing, concentrating, and organizing cell-signaling components and regulating various vascular biological processes such as endothelium dependent hyperpolarization (EDH) of vascular smooth muscle. Therefore, a better understanding of MEJ’s physiological functions allows new insights into regulation of vessel function. Up to now, little is known, however, about the regulation for MEJ formation. The 5’-adenosine monophosphate-activated protein kinase (AMPK) is not only one of the most important enzymes controlling cell catabolic metabolism, but has been shown to influence vascular tone, thereby augmenting blood and oxygen supply as required for catabolic pathways. It has not yet been studied, however, whether AMPK could also affect MEJ. Thus, the aim of this project was to elucidate the impact of AMPK on MEJ dynamics and its potential mechanism of action. We studied isolated small mesenteric and skeletal muscle resistance arteries of mice using a pressure myograph system developed in our laboratory. Using confocal and two photon excitation fluorescence microscopy we identified the internal elastic lamina, which in these vessels exhibits small holes that allow direct contact between endothelial and smooth muscle cells. On average, we found that in about 35% of the holes, structures with a bright actin core as assessed by using F-actin fluorescence in LifeAct mice or by staining with phalloidin which we could define as sites of MEJ. These structures showed expression of Cx37 and Cx43 suggesting that MEGJs were also localized in these structures. We found that endoplasmic reticulum (ER) was enriched around MEJs which is consistent with MEJs acting as active signaling domains. Over observation time of up to 2 hours, we found that the number and localization of these MEJ varied, while they always showed expression in the area of holes of IEL only. Thus, for the first time we could investigate MEJ dynamics. AMPK negatively regulates MEJ expression, since in AMPK α1, but not in α2 knockout (KO) mice, the number of MEJ was significantly increased. In accordance, in wild type mice, incubation with the AMPK inhibitory compound C (CC) significantly augmented the number of MEJ while the AMPK stimulator A76 did not further decrease them during the observation time. Furthermore, we found that the KO of AMPK α1, in intact mice as well as in cultured human and porcine smooth muscle cells, went along with an increase in PAI-1 expression. Accordingly, incubation of arteries with exogenous PAI-1 also increased the number of MEJ. The negative effect of AMPK on PAI-1 could be explained by enhanced expression of the silencer of the PAI-1 gene, small heterodimer partner (SHP). PAI-1 expression in arteries from AMPK α2 KO mice was unchanged compared to arteries from WT mice. Since MEJ are also involved in EDH, we studied whether the expression of MEJ correlated with EDH induced vasodilation. The higher amount of MEJs in α1 KO mice went indeed along with a left shift of acetylcholine (ACh) -induced dilation dose effect curve. In summary, this work describes for the first time a role of AMPK as a potent modulator of MEJ dynamics. This effect is selectively mediated by the α1 subunit of AMPK which is probably mediated by controlling PAI-1 expression. Our data also for the first time demonstrate a dynamic regulation of MEJ expression in intact blood vessels. Since MEJ represent a gateway for the communication between EC and VSMC and are involved in EDH, our observations may point towards a new target with therapeutic potential in small resistance vessels.

Abstract