Direct regulation of blood pressure by smooth muscle cell mineralocorticoid receptors

Abstract

Hypertension is a cardiovascular risk factor present in over two-thirds of people over age 60 in North America; elevated blood pressure correlates with increased risk of heart attack, stroke and progression to heart and kidney failure. Current therapies are insufficient to control blood pressure in almost half of these patients. The mineralocorticoid receptor (MR), acting in the kidney, is known to regulate blood pressure through aldosterone binding and stimulation of sodium retention. However, recent studies support the concept that the MR also has extrarenal actions and that defects in sodium handling alone do not fully explain the development of hypertension and associated cardiovascular mortality. We and others have identified functional MR in human vascular smooth muscle cells (SMCs), suggesting that vascular MR might directly regulate blood pressure. Here we show that mice with SMC-specific deficiency of the MR have decreased blood pressure as they age without defects in renal sodium handling or vascular structure. Aged mice lacking MR in SMCs (SMC-MR) have reduced vascular myogenic tone, agonist-dependent contraction and expression and activity of L-type calcium channels. Moreover, SMC-MR contributes to angiotensin II–induced vascular oxidative stress, vascular contraction and hypertension. This study identifies a new role for vascular MR in blood pressure control and in vascular aging and supports the emerging hypothesis that vascular tone contributes directly to systemic blood pressure.

Fig 1. Lower blood pressure in aged mice with SMC-specific MR deletion

(a) PCR assay amplifies the LoxP flanked MR allele (lower band) or the exon 5 and 6 excised MR allele (upper band) from tissue DNA from Tam-induced MR^f/f/SMA-Cre-ER^T2+ (Cre+) and MR^f/f/SMA-Cre-ER^T2− (Cre−) littermates. HipC=Hippocampus, Blad=bladder. (b) PCR assay for MR excision on DNA from whole or denuded (adventitia and endothelial cells removed) aortas. (c) Expression of MR (Nr3c2 gene) mRNA in freshly isolated aortic SMC from Cre− and Cre+ adult mice by QRT-PCR. (d) 24 hour average systolic BP measured telemetrically in mice from 4 to 10 months of age. Age dependent increase for <7 month- versus >7 month-old mice: P < 0.001 for all mice, P < 0.01 for Cre− and P < 0.05 for Cre+. (e) Vasoconstriction responses of mesenteric resistance arteries from adult (3–4 month-old) mice and aged (>9 month-old) mice, in response to potassium chloride (KCl), phenylephrine (PE), and TP agonist U46619. For all figures grey represents Cre− mice and black represents Cre+ mice. *P < 0.05 Cre− adult vs. Cre+ adult, **P < 0.01 Cre− aged vs. Cre+ aged; ***P < 0.001 Cre− vs. Cre+. ^P < 0.05 Cre− aged vs. Cre+ aged; #P < 0.05 Cre− adult vs. Cre− aged; ##P < 0.001 adult vs. aged.

Fig 2. Intact renal MR function in SMC-MR-deficient mice

Systolic BP measured by telemetry in adult (3–4 month-old) (a, c) and aged (>9 month-old) (b, d) Cre− and Cre+ mice. (a, b) Average systolic BP for mice on normal chow (0.3% sodium), high sodium (6%), and low sodium (0.02%) diets. (c, d) Mineralocorticoid/salt-induced hypertension in Cre− and Cre+ mice. Average systolic BP of Cre− and Cre+ mice before and after aldosterone infusion combined with 1% NaCl in drinking water. (e) 24 hour urinary sodium measured from aged (>9 months) mice fed a normal diet (day 0) and during day 1 or day 2 of sodium restriction. (f) Fractional excretion of sodium (FENa%). For all figures grey represents Cre− mice and black represents Cre+ mice. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 3. Normal vascular structure with decreased tone in mice with SMC-specific MR deletion

(a) Elastin and (b) trichrome staining of representative thoracic aorta sections from 3, 9, and 18 month-old Cre− and Cre+ littermates. Medial area and collagen content are quantified on right. Scale bar = 100 μm (c) Passive vessel area, lumen area and distensibility in cannulated mesenteric resistance arteries (MRA) from aged (>9 months-old) Cre− and Cre+ mice over a range of intraluminal pressures. (d) Passive and active diameters of cannulated MRA from aged Cre− and Cre+ mice over a range of intraluminal pressures. P < 0.001 for active versus passive tone. Average spontaneous tone at 70 mmHg is calculated as the percent decrease in active lumen diameter from the passive diameter and compared on the right. (e) Mesenteric SMC patch clamp studies: Upper panels show representative whole cell K⁺ current recordings for Cre− and Cre+ mesenteric SMC. BKCa component is isolated by inhibition with the BKCa inhibitor iberiotoxin (IBTX; 10⁻⁷M) and voltage-gated K⁺ channels (Kv) by inhibition with 4-aminopyridine (4-AP; 10⁻³M). Middle panels show group data: circles=total K+ current; squares=after inhibition with IBTX; and triangles=after inhibition with both IBTX and 4-AP. Lower panels show responses to the BKCa activator, NS1619 presented as percent of basal current. Stimulatory effect of NS1619 is reversed by IBTX. (f) Expression of BKCaα (Kcnma1 gene), BKCaβ (Kcnmb1 gene), and Cav1.2 (Cacna1c gene) mRNA in aortas isolated from Cre− and Cre+ aged mice by QRT-PCR. (g) Contractile response of pressurized MRA (at 70 mmHg) to PE or L-type calcium channel activation with BayK8644 is represented as percent decrease in MRA diameter upon agonist treatment of aged vessels. For all figures grey represents Cre− and black represents Cre+. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 4. SMC-MR contributes to the hypertensive, contractile, and oxidative stress response to AngII

Average 24 hour systolic BP measured by telemetry in (a) adult (3–4 month-old) and (b) aged (>9 month-old) Cre− and Cre+ mice during seven day AngII infusion. P < 0.001 for Cre− adult versus Cre− aged mice. Contraction of mesenteric resistance arteries from (c) adult and (d) aged Cre− and Cre+ mice, to AngII treatment. Superoxide production in carotid arteries from (e) adult and (f) aged Cre− and Cre+ mice, quantified with dihydroethidium (DHE) staining following 30min ex vivo treatment with vehicle or AngII. Scale bar = 200 μm. For all figures grey represent Cre− mice and black represent Cre+ mice. #P < 0.05 versus no AngII. *P < 0.05, **P < 0.01, ***P < 0.001 Cre+ versus Cre− or as indicated.