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BACKGROUND: The antifibrotic effects of recombinant human relaxin (RLX) in the kidney are dependent on an interaction between its cognate receptor (RXFP1) and the angiotensin type 2 receptor (AT(2)R) in male models of disease. Whether RLX has therapeutic effects, which are also mediated via AT(2)R, in hypertensive adult and aged/reproductively senescent females is unknown. Thus, we determined whether treatment with RLX provides cardiorenal protection via an AT(2)R-dependent mechanism in adult and aged female stroke-prone spontaneously hypertensive rats (SHRSPs). METHODS: In 6-month-old (6MO) and 15-month-old ([15MO]; reproductively senescent) female SHRSP, systolic BP (SBP), GFR, and proteinuria were measured before and after 4 weeks of treatment with vehicle (Veh), RLX (0.5 mg/kg per day s.c.), or RLX+PD123319 (AT(2)R antagonist; 3 mg/kg per day s.c.). Aortic endothelium–dependent relaxation and fibrosis of the kidney, heart, and aorta were assessed. RESULTS: In 6MO SHRSP, RLX significantly enhanced GFR by approximately 25% (P=0.001) and reduced cardiac fibrosis (P=0.01) as compared with vehicle-treated counterparts. These effects were abolished or blunted by PD123319 coadministration. In 15MO females, RLX reduced interstitial renal (P=0.02) and aortic (P=0.003) fibrosis and lowered SBP (13±3 mm Hg; P=0.04) relative to controls. These effects were also blocked by PD123319 cotreatment (all P=0.05 versus RLX treatment alone). RLX also markedly improved vascular function by approximately 40% (P

Loss of ovarian hormones following menopause contributes to the rise in cardiovascular risk with age. Estrogen plays a protective role against hypertension and end-organ damage by modulating the depressor actions of the AT 2 R (angiotensin type 2 receptor). Our aim was to determine whether estrogen replacement in aged female mice can lower arterial pressure, improve endothelial function, and reduce organ fibrosis via an AT 2 R-mediated mechanism. Mean arterial pressure was measured via radiotelemetry in ovary-intact adult (3–4-month-old), aged (16–18-month-old; reproductively senescent) and aged–17β-estradiol (E 2 )–treated (3 µg/day SC) female mice, which were administered vehicle, Ang II (angiotensin II; 600 ng/[kg·min] SC) or Ang II+PD123319 (AT 2 R antagonist; 3 mg/[kg·day SC). On day 21 of treatment, aortic endothelium-dependent relaxation and cardiac and renal tissue (fibrosis and gene expression) were analyzed. Basal mean arterial pressure was lower in E 2 -treated aged mice (89±1 mm Hg, n=20) relative to aged controls (94±1 mm Hg; n=18, P =0.002). The Ang II pressor response was enhanced by ≈20 mm Hg in aged compared with adult females ( P =0.01). E 2 -treatment reduced the Ang II pressor response in aged females ( P =0.002), an effect that was reversed by PD123319 in the aged E 2 –Ang II group ( P =0.0009). E 2 -treatment increased renal AT 2 R (≈6-fold; P P 2 -treatment in aged female mice. In conclusion, estrogen replacement in aged females may reduce arterial pressure to levels observed in adult females, via an AT 2 R-mediated renal mechanism.

Postmenopausal women have a greater risk for cardiovascular disease (CVD) than premenopausal women. Cardiovascular protection in young women may be attributed to an interaction between female sex-hormones and the angiotensin type 2 receptor (AT2R) of the blood-pressure system, a mechanism lost with age. Hormone replacement in aged females with CVD, may therefore restore the cardiovascular protective effects of the AT2R. The findings of this thesis demonstrate that estrogen and relaxin treatment in aged females improved blood-pressure and kidney function, and reversed organ-injury by enhancing AT2R function. Thus, targeting the AT2R may be a novel therapeutic strategy for treating CVD in postmenopausal women.

BACKGROUND: Recombinant human relaxin-2 (serelaxin), which has organ-protective actions mediated via its cognate G protein–coupled receptor relaxin family peptide receptor 1 (RXFP1), has emerged as a potential agent to treat fibrosis. Studies have shown that serelaxin requires the angiotensin II (AngII) type 2 receptor (AT(2)R) to ameliorate renal fibrogenesis in vitro and in vivo. Whether its antifibrotic actions are affected by modulation of the AngII type 1 receptor (AT(1)R), which is expressed on myofibroblasts along with RXFP1 and AT(2)R, is unknown. METHODS: We examined the signal transduction mechanisms of serelaxin when applied to primary rat renal and human cardiac myofibroblasts in vitro, and in three models of renal- or cardiomyopathy-induced fibrosis in vivo. RESULTS: The AT(1)R blockers irbesartan and candesartan abrogated antifibrotic signal transduction of serelaxin via RXFP1 in vitro and in vivo. Candesartan also ameliorated serelaxin’s antifibrotic actions in the left ventricle of mice with cardiomyopathy, indicating that candesartan’s inhibitory effects were not confined to the kidney. We also demonstrated in a transfected cell system that serelaxin did not directly bind to AT(1)Rs but that constitutive AT(1)R–RXFP1 interactions could form. To potentially explain these findings, we also demonstrated that renal and cardiac myofibroblasts expressed all three receptors and that antagonists acting at each receptor directly or allosterically blocked the antifibrotic effects of either serelaxin or an AT(2)R agonist (compound 21). CONCLUSIONS: These findings have significant implications for the concomitant use of RXFP1 or AT(2)R agonists with AT(1)R blockers, and suggest that functional interactions between the three receptors on myofibroblasts may represent new targets for controlling fibrosis progression.

It is well established that there are pronounced sex differences in the physiological regulation of arterial pressure and vascular function. Longitudinal studies have demonstrated that in premenopausal women, arterial pressure is lower than age-matched males. In part, sex differences in the regulation of arterial pressure may be underpinned by an enhanced role for the angiotensin type 2 (AT2) receptor in females compared with males. Thus the protective actions of the AT2 receptor likely confer benefits in terms of protection from hypertension and cardiovascular disease in women of reproductive age. Moreover, the AT2 receptor may contribute to the normal cardiovascular adaptations that occur during pregnancy, which enable blood volume to increase without a concomitant increase in blood pressure. Conversely, evidence now suggests that defects in AT2 receptor expression may contribute to the pathophysiology of pregnancy-induced hypertension and postmenopausal hypertension.

The angiotensin type 2 receptor (AT 2 R) mediates cardiovascular and renal protection in females of reproductive age, a mechanism that is lost with advancing age. Recently, we demonstrated that the nitric oxide-promoting and transforming growth factor-β1 inhibitory actions of relaxin (RLX) involve an interaction between the relaxin-insulin like family peptide receptor 1 (RXFP1) and the AT 2 R. In the present study, we aimed to determine whether RLX induces cardio-renal protection and slows the progression of end-organ damage, via an AT 2 R-dependent mechanism. In female stroke prone-spontaneously hypertensive rats (SP-SHR) at 6 (young) and 14-15 (aged reproductively senescent) months of age arterial pressure, cardiac function, glomerular filtration rate (GFR) and proteinuria were measured before and after 4 weeks of treatment with vehicle (sodium acetate s.c.), RLX (serelaxin; 0.5mg/kg/day s.c.) or RLX+PD123319 (AT 2 R antagonist; 3mg/kg/day s.c). In addition, aortic endothelium-dependent relaxation in response to acetylcholine was assessed and cardiac and renal tissues were histologically analyzed for fibrosis. An age-related decline in GFR and increases in arterial pressure, proteinuria and cardiac and renal fibrosis were observed (all P2 R. Thus, targeting the RXFP1-AT 2 R axis may be a therapeutic option for the treatment of cardiovascular disease and associated end-organ damage in hypertensive women.

Loss of estrogen (E 2 ) following menopause contributes to the sharp rise in cardiovascular risk with age. E 2 is postulated to play a protective role against hypertension and end-organ damage by counterbalancing the pressor actions of the RAS and enhancing the depressor RAS pathways. The aim was to determine whether E 2 replacement in aged females can lower arterial pressure and improve endothelial function via an AT 2 R-mediated mechanism. MAP was measured via telemetry in ovary-intact adult (4-month old), aged (17-month old) and aged+E 2 (3 μg/day sc) FVB/N female mice, which were co-treated with vehicle, AngII (600 ng/kg/min sc) or AngII+PD (PD123319, AT 2 R antagonist; 3 mg/kg/day sc). On day 21 of treatment, endothelium-dependent relaxation in response to acetylcholine was assessed in aortic vessels. Cardiac and renal, tissue fibrosis and gene expression of E 2 receptors and RAS components were also analysed. Basal MAP was lower in E 2 -treated aged mice (90±1 mmHg, n=20) relative to adult (94±1 mmHg, n=10) and aged controls (94±1 mmHg; n=21, both P2 treatment reduced the AngII pressor response in aged females (Figure). Moreover, the attenuated pressor response observed in the aged E 2 +AngII group was abolished by co-infusion with PD (Figure). Endothelial function was markedly impaired with age (~23% reduced maximal vasodilation, P=0.03) and worsened by AngII infusion (~38%, P=0.001), however E 2 did not significantly improve the response in any group. In conclusion, E 2 replacement may reinstate cardio-protection in aged females via an AT 2 R-mediated mechanism.

Background Animal models have become valuable experimental tools for understanding the pathophysiology and therapeutic interventions in cardiovascular disease. Yet to date, few studies document the age- and sex-related differences in arterial pressure, circadian rhythm, and renal function in normotensive mice under basal conditions, across the life span. We hypothesized that mice display similar sex- and age-related differences in arterial pressure and renal function to humans. Methods Mean arterial pressure (MAP) and circadian rhythm of arterial pressure were measured over 3 days via radiotelemetry, in 3- and 5-month-old (adult) and 14- and 18-month-old (aged) FVB/N and in 5-month-old (adult) C57BL/6 male and female normotensive mice. In FVB/N mice, albuminuria from 24-h urine samples as well as body, heart, and kidney weights were measured at each age. Results Twenty-four-hour MAP was greater in males than females at 3, 5, and 14 months of age. A similar sex difference in arterial pressure was observed in C57BL/6 mice at 5 months of age. In FVB/N mice, 24-h MAP increased with age, with females displaying a greater increase between 3 and 18 months of age than males, such that MAP was no longer different between the sexes at 18 months of age. A circadian pattern was observed in arterial pressure, heart rate, and locomotor activity, with values for each greater during the active (night/dark) than the inactive (day/light) period. The night-day dip in MAP was greater in males and increased with age in both sexes. Albuminuria was greater in males than females, increased with age in both sexes, and rose to a greater level in males than females at 18 months of age. Conclusions Arterial pressure and albuminuria increase in an age- and sex-specific manner in mice, similar to patterns observed in humans. Thus, mice represent a useful model for studying age and sex differences in the regulation of arterial pressure and renal disease. Understanding the mechanisms that underlie the pathophysiology of cardiovascular disease may lead to new and better-tailored therapies for men and women.