Abstract: The steroid hormone aldosterone activates the mineralocorticoid receptor (MR), a steroid receptor transcription factor that contributes to cardiovascular disease. While MR is best known for its role in enhancing sodium and water reabsorption in the renal epithelia to increase blood volume, clinical trials have demonstrated benefit for MR antagonism disproportionate to its antihypertensiv... read moree effect. Thus antagonism of non-renal MR likely contributes to clinical benefits of these drugs. Endothelial cells compose the innermost layer of the blood vessel and participate in regulation of vasomotor function in health and disease. MR is expressed in endothelial cells, and the literature suggests a role for MR in exacerbating vascular pathology in the presence of numerous cardiovascular risk factors. Estrogen receptor α (ERα), another transcription factor that is activated by estrogen, is also expressed in endothelial cells. Endothelial ERα protects against cardiovascular disease, correlating with the epidemiologic finding that premenopausal women have less cardiovascular disease than age-matched men. We tested the hypothesis that one mechanism for sex differences in cardiovascular disease is that estrogen-activated ERα interferes with the transcriptional effects of aldosterone-activated MR in ECs. First we demonstrated that in HEK293 cells in which we expressed MR and an MRE-luciferase reporter, cotransfection of ERα blocked MR-mediated transcription. The same effect of estrogen- and ERα-mediated inhibition of MR-activated transcription was seen in EAHy926 endothelial cells transfected with the MRE-luciferase reporter. Furthermore, aldosterone-stimulated upregulation of endothelial mRNA and protein of the pro-inflammatory adhesion molecule ICAM-1 was attenuated with coadministration of estrogen. An in vitro leukocyte adhesion assay in which aldosterone is known to promote pro-inflammatory leukocyte adhesion to endothelial cells was likewise inhibited by estrogen administration, suggesting physiologic relevance of ERα-mediated inhibition of MR transcriptional function. Hence in the vascular endothelium, estrogen-activated ERα may achieve its cardioprotective effects in part through inhibition of the harmful effects of aldosterone-stimulated MR. This finding may create novel therapeutic opportunities for targeting the role of endothelial cells in cardiovascular disease. To further investigate the role of endothelial MR in vascular function and disease in vivo, we created a mouse with MR specifically deleted from EC (EC-MR-KO) but with intact leukocyte MR expression and normal renal MR function. Although endothelial cells (EC) express MR, the contribution of EC-MR to blood pressure and resistance vessel function remains unclear. Telemetric blood pressure studies revealed no difference between male EC-MR-KO mice and MR-intact littermate controls in systolic, diastolic, circadian, or salt-sensitive blood pressure or in hypertensive responses to aldosterone/salt or angiotensin II. Vessel myography demonstrated normal vasorelaxation in mesenteric and coronary arterioles from EC-MR-KO mice. After exposure to angiotensin II-induced hypertension, endothelial-dependent relaxation was impaired in coronary and mesenteric arterioles from MR-intact mice. This impairment was prevented specifically in EC-MR-KO mesenteric vessels, which showed increased maximum responsiveness to Ach, compared to MR-intact vessels. These data support that EC-MR plays a role in regulating endothelial function in hypertension. Although there was no effect of EC-MR deletion on mesenteric vasoconstriction, EC-MR-KO coronary arterioles showed decreased constriction to endothelin-1 and thromboxane agonist at baseline and after exposure to hypertension. These data support that EC-MR participates in regulation of vasomotor function in a vascular bed-specific manner that is modulated by risk factors such as hypertension. In addition the literature supports a role for MR in enhancing atherosclerosis. Atherosclerosis is an inflammatory disorder of the vasculature that results in heart attacks and strokes. Healthy mice infused with two weeks of aldosterone show no increase in leukocyte infiltration of the blood vessel, but aldosterone treatment in mice exposed to high fat diet and hyperlipidemia significantly augments leukocyte infiltration, suggesting that MR activation, specifically in the presence of such cardiovascular risk factors, enhances the vascular inflammation characteristic of atherosclerosis. To clarify the role of endothelial MR in atherosclerosis, EC-MR-KO mice were crossed with ApoE-/- mice to generate atherogenic EC-MR-KOApoE-/- mice and MR-intact/ApoE-/- littermates. Mice were fed high fat diet for four weeks and then the aorta was collected and immunohistochemistry was used to assess plaque size and lipid content. A trend towards decreased plaque size in EC-MR-KO/ApoE-/- mice compared to MR-intact/ApoE-/- mice was observed, suggesting endothelial MR may play a role in enhancing plaque development. Future studies will be needed to confirm this finding and to explore additional mechanisms by which EC MR contributes to atherosclerosis.
Thesis (Ph.D.)--Tufts University, 2017.
Submitted to the Dept. of Cell, Molecular & Developmental Biology.
Advisor: Iris Jaffe.
Committee: John Castellot, Lucy Liaw, Richard Karas, and Mike Hill.