Abstract
Background:
Endothelial cells are the main source for nitric oxide (NO) production in the vasculature that regulates blood pressure and vascular tone. Studies suggest that the mechanical properties of endothelial cells, characterized by increased stiffness, predicts future cardiovascular events, and is linked with impaired endothelial cell derived NO production. Recent evidence suggests that Epithelial Sodium Channel (ENaC) and the endothelial glycocalyx (eGC) are potential players influencing endothelial cell stiffness (ECS). ENaC consists of four subunits (α, β, γ, and δ). Emerging evidence suggests that ENaC in the vasculature mediates vascular tone and regulates blood pressure. On the other side, the eGC is a carbohydrate rich layer on the luminal surface of endothelial cells. eGC comprises of glycoproteins with carbohydrate side chains and membrane bound proteoglycans with glycosaminoglycans (GAGs) side chains. GAGs like heparan sulfate (HS) and hyaluronic acid (HA) play a major role in NO production. Decreased NO production is linked with increased ECS and this is an important determinant of hypertension and endothelial dysfunction. However, how ENaC and the eGC mediate ECS and this impairs NO production are still poorly understood.
Hypotheses:
1) ENaC and the eGC act synergistically to contribute to increased ECS. 2) Increased F-actin cell cytoskeleton associates with increased ECS. 3) ECS is characterized by decreased NO production.
Methods:
To challenge the hypothesis, protocols were developed to increase and decrease ENaC expression and change the eGC. ENaC expression was increased in response to aldosterone and laminar shear stress (LSS) in human umbilical vein endothelial cells (HUVECs) and mouse myocardial endothelial (MyEnd) cells. Further, ENaC expressions were increased following overexpression of ENaC subunits and decreased following knockdown of ENaC subunits in HUVECs. The increase and decrease in ENaC expressions were detected through Western blot semi-quantitative analysis. On the other hand, the eGC was decreased in response to heparinase III (Hep III) and following knockdown of Hyaluronan synthase 2 (HAS2) and increased following overexpression of HAS2 in HUVECs. The increase and decrease in eGC were confirmed by confocal microscopy. These protocols were then utilized to measure Young’s modulus changes through the Atomic Force Microscopy (AFM). Here, Young’s modulus is an indicator of the mechanical properties of cells and was used to assess ECS. Furthermore, changes in F-actin cytoskeleton were detected through confocal microscopy. Finally, changes in intracellular NO production were detected through fluorescence microscopy.
Results:
Aldosterone (10 nM, 24 hrs) increased α and δ-ENaC (human specific) protein expression in HUVECs, and α-ENaC protein expression in MyEnd cells under static conditions. The result showed that LSS (10 dyn/cm², 24 hrs) alone increases δ-ENaC protein expression in HUVECs, and γ-ENaC protein expression in MyEnd cells. It was also evident that aldosterone increased β-ENaC protein expression in HUVECs, while α and β-ENaC in MyEnd cells in response to aldosterone under LSS. Further, the results indicated that Hep III and knockdown of HAS2 decreased eGC, and overexpression of HAS2 increased eGC. The results also showed that the increased ENaC expression and the degraded eGC were associated with an increased Young's modulus that is an indicator of elevated ECS. The upregulated ENaC expression, degraded eGC, and increased stiffness coincided with increased F-actin in response to aldosterone and Hep III. On the other hand, HAS2 siRNA transfection did not increase F-actin while HAS2 plasmid transfection decreased F-actin expression. Furthermore, the current study demonstrated that increased ECS associates with reduced NO level in response to aldosterone and Hep III, indicating that the cell’s ability to produce NO was decreased in response to aldosterone and Hep III treatment.
Main conclusion:
This dissertation provided evidence that ENaC and the eGC have synergistic effects on ECS. The increased ECS coincides with increased F-actin, and lower NO contents, a hallmark of endothelial dysfunction and hypertension. This study help understands the physiology and pathophysiology of vascular function with new clinical implications for cardiovascular preventive strategies and treatment against increased blood pressure.