Endothelial Mineralocorticoid Receptors Differentially Contribute to Coronary and Mesenteric Vascular Function Without Modulating Blood Pressure

Abstract

Arteriolar vasoreactivity tightly regulates tissue-specific blood flow and contributes to systemic blood pressure (BP) but becomes dysfunctional in the setting of cardiovascular disease. The mineralocorticoid receptor (MR) is known to regulate BP via the kidney and by vasoconstriction in smooth muscle cells. Although endothelial cells (EC) express MR, the contribution of EC-MR to BP and resistance vessel function remains unclear. To address this, we created a mouse with MR specifically deleted from EC (EC-MR knockout [EC-MR-KO]) but with intact leukocyte MR expression and normal renal MR function. Telemetric BP studies reveal no difference between male EC-MR-KO mice and MR-intact littermates in systolic, diastolic, circadian, or salt-sensitive BP or in the hypertensive responses to aldosterone±salt or angiotensin II±l-nitroarginine methyl ester. Vessel myography demonstrated normal vasorelaxation in mesenteric and coronary arterioles from EC-MR-KO mice. After exposure to angiotensin II-induced hypertension, impaired endothelial-dependent relaxation was prevented in EC-MR-KO mice in mesenteric vessels but not in coronary vessels. Mesenteric vessels from angiotensin II-exposed EC-MR-KO mice showed increased maximum responsiveness to acetylcholine when compared with MR-intact vessels, a difference that is lost with indomethacin+l-nitroarginine methyl ester pretreatment. 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, coronary arterioles from EC-MR-KO mice showed decreased constriction to endothelin-1 and thromboxane agonist at baseline and also after exposure to hypertension. These data support that EC-MR participates in regulation of vasomotor function in a vascular bed-specific manner that is also modulated by risk factors, such as hypertension.

Keywords: blood pressure; endothelial cells; endothelin-1; hypertension; receptors, mineralocorticoid.

Conflict of interest statement

Disclosures: The authors declare no conflicts of interest.

© 2015 American Heart Association, Inc.

Figures

Figure 1. A mouse model with MR deleted specifically from EC and not leukocytes

(A–C) MR genomic DNA was amplified with primers specific for the LoxP MR or recombined MR. (A) The MR gene is completely recombined in primary cultured mouse ECs only from Cre+ mice. (B) MR recombination occurs in Cre+ aorta, carotid, coronary, and mesenteric vessels. (C) MR recombination occurs only in EC-containing tissues in VE-cad-Cre+ mice and not in lymph nodes or splenic leukocytes (Leuk.) (D) MR mRNA is reduced in primary ECs cultured from mouse lungs (mouse lung endothelial cells, MLECs) and hearts (MHECs) but not in leukocytes from EC-MR-KO mice. Number of animals per group is indicated in parentheses. *p

Figure 2. EC-MR does not contribute to basal, salt sensitive, aldosterone/salt-enhanced, or angiotensin-enhanced BP

(A) 24-hour ambulatory blood pressure in healthy 6-month old mice. (B) Average systolic blood pressure (SBP) at 4, 6 and 8 months of age in MR-intact and EC-MR-KO mice. (C) Average SBP in 5–6 month old mice on days 3–5 of normal, low, or high salt diets. *p+) on normal and low-salt diet. *p<0.05 versus normal diet. (E–F) SBP assessment in hypertension models in 7–8 month old mice. (E) SBP on day 4 of aldosterone infusion; day 9 of aldosterone+1% NaCl in water.*p<0.05 versus baseline; #p<0.05 versus aldosterone alone. (F) BP on day 7 of AngII infusion; and on day 14 of AngII+L-NAME.*p<0.05 versus baseline; #p<0.05 versus AngII alone. Number of animals per group is indicated in parentheses.

Figure 3. EC-MR does not contribute to mesenteric or coronary vasodilation

Sodium nitroprusside (SNP) and acetylcholine (Ach) dose-response curves were assessed by wire myography in (A) Mesenteric arterioles pre-constricted with PE (1 μM) and (B) Coronary arterioles pre-constricted with U46619 (0.1–0.3 μM). Number of animals per group is indicated in parentheses.

Figure 4. EC-MR deletion modulates mesenteric endothelial-dependent relaxation after exposure to AngII-induced hypertension

Mesenteric arterioles were isolated from untreated mice (A) or mice exposed to 14-day AngII infusion (B), and the change in vessel diameter after pre-constriction with PE (100 nM) was assessed by pressure myography. Relaxation-response curves are shown to sodium nitroprusside (SNP), Acetylcholine (Ach), Ach+L-NAME+indomethacin (Indo), or Ach+L-NAME+Indo+Apamin+TRAM34. Number of animals per group is indicated in parentheses. *p

Figure 5. EC-MR differentially regulates vasoconstriction depending on the vascular bed and the contractile agonist

Dose-response curves for contractile force were recorded by wire myography in (A) mesenteric arterioles in response to KCl, endothelin-1, the thromboxane agonist U46619, phenylephrine (PE), or a single dose of AngII; and in (B) coronary arterioles (normalized to length of the vessel segment) to KCl, endothelin-1, and U46619. No graph is included for PE and AngII as coronary arterioles did not respond to those agonists. Number of animals per group is indicated in parentheses. *p

Figure 6. Decreased coronary vasoconstriction to endothelin-1 and U46619 after hypertension and decreased EC endothelin-B receptor mRNA in EC-MR-KO mice

Coronary vasoconstriction was assessed by wire myography after 14 day AngII infusion to a dose escalation of (A) endothelin-1 and (B) thromboxane agonist U46619. *p