Norma Adragna (Advisor), David Cool (Committee Member), Mauricio Di Fulvio (Committee Member), Peter Lauf (Committee Member), Karen Luchin (Other), Mill Miller (Other), Lawrence Prochaska (Committee Chair)
Doctor of Philosophy (PhD)
Atherosclerosis and high circulating levels of oxidized low density lipoproteins (oxLDL) are considered among the most important risk factors for the occurrence and development of cardiovascular disease (CVD). During the atherosclerotic lesion repair, phenotypic transition of vascular smooth muscle cells (VSMCs) from contractile to synthetic states plays a central role. In this process, enhanced proliferation/migration of VSMCs, from the tunica media to the intima, is required to sustain blood vessel endothelium integrity, and for inducing vessel wall remodeling in response to injury. At the molecular level, the activity of electroneutral potassium-chloride cotransporters or KCCs, is necessary to: a) allow changes in cell volume (key prerequisites to coordinate cell proliferation/migration) and b) sustain normal cardiovascular function. Stimulation of the adipokine apelin and its receptor APJ (apelin/APJ) signaling pathway has been shown to protect against atherosclerotic lesion formation by lowering blood pressure levels and promoting vasodilation. Upon binding to APJ, apelin promotes nitric oxide (NO)-mediated vasodilation, and cell proliferation via the PI3K/Akt and MAPK pathways. Apelin/APJ exert its action through the same signaling pathways regulating the KCCs. However, the mechanisms of KCC regulation by apelin/APJ remain to be determined. Thus, we hypothesized that KCC expression and activity play an important role during VSMCs' phenotypic transition and could be involved in the apelin/APJ cardioprotective effects. In addition, it is possible that the apelin-mediated effect on KCC activity could also be dependent on factors affecting the transporter, such as, serum, ionic strength, osmolality, cell proliferation and migration. This hypothesis will be tested using rat aortic VSMCs that were immunologically validated by specific markers. KCC activity was measured by atomic absorption spectrophotometry using rubidium as a potassium (K+) congener. KCC expression and transport activity were characterized with respect to the VSMC phenotypes, in the presence or absence of apelin and corresponding inhibitors of the signaling pathways, oxLDL and as a function of the various aforementioned physiological factors. While markers for the contractile VSMC phenotype are well known, they are less defined for the early and late synthetic ones. In the present study, we showed a decrease in expression levels of cytoskeletal proteins like a-actin, desmin and vimentin in late synthetic VSMCs compared to early states suggesting that the latter show similar characteristics to the contractile phenotype. In addition, KCC1 and KCC4 protein expression and overall KCC activity increased in late vs early synthetic VSMCs. The ion binding affinity (Km) for late and early synthetic VSMCs was similar for K+ whereas it decreased for Cl-. Whether the increase in KCC activity in late synthetic VSMCs is due to turnover changes of pre-existing KCCs or enhanced membrane trafficking/insertion of de novo synthesized KCC protein remains to be studied. Using selective inhibitors, we showed that apelin-mediated activation of KCCs occurs through the NO/sGC/PKG pathway in contractile, and by PI3K/Akt and MAPK dependent pathway(s) in synthetic VSMCs. Furthermore, apelin rescued the inhibition of KCC induced by oxLDL in contractile VSMCs. We also showed that the apelin-mediated activation of KCCs is dependent on extracellular sodium, osmolality, presence of serum in the growth media, VSMCs phenotype (contractile vs synthetic), and passage number. Altogether, our results identify apelin/APJ as an important modulator of KCC activity to sustain cell volume regulation and overall vascular function.
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