Severe pulmonary hypertension in aging female apolipoprotein E-deficient mice is rescued by estrogen replacement therapy

BackgroundApolipoprotein E (ApoE) is a multifunctional protein, and its deficiency leads to the development of atherosclerosis in mice. Patients with pulmonary hypertension (PH) have reduced expression of ApoE in lung tissue. ApoE is known to inhibit endothelial and smooth muscle cell proliferation and has anti-inflammatory and anti-platelet aggregation properties. Young ApoE-deficient mice have been shown to develop PH on high fat diet. The combined role of female sex and aging in the development of PH has not been investigated before. Here, we investigated the development of PH in young and middle-aged (MA) female ApoE-deficient mice and explored the role of exogenous estrogen (E2) replacement therapy for the aging females.MethodsWild type (WT) and ApoE-deficient female mice (Young and MA) were injected with a single intraperitoneal dose of monocrotaline (MCT, 60 mg/kg). Some ApoE-deficient MA female mice that received MCT were also treated with subcutaneous E2 pellets (0.03 mg/kg/day) from day 21 to 30 after MCT injection. Direct cardiac catheterization was performed terminally to record right ventricular systolic pressure (RVSP). Right ventricular (RV), left ventricular (LV), and interventricular septum (IVS) were dissected and weighed. Lung sections were examined using trichrome and immunofluorescence staining. Western blot analyses of lung and RV lysates were performed.ResultsIn WT female mice, the severity of PH was similar between young and MA mice as RVSP was not significantly different (RVSP = 38.2 ± 1.2 in young vs. 40.5 ± 8.3 mmHg in MA, p < 0.05). In ApoE-deficient mice, MA females developed significantly severe PH (RVSP = 63 ± 10 mmHg) compared to young females (RVSP; 36 ± 3 mmHg, p < 0.05 vs. MA female). ApoE-deficient MA females also developed more severe RV hypertrophy compared to young females (RV hypertrophy index (RV/[LV + IVS]) = 0.53 ± 0.06 vs. 0.33 ± 0.01, p < 0.05). ApoE-deficient MA female mice manifested increased peripheral pulmonary artery muscularization and pulmonary fibrosis. E2 treatment of MA female ApoE-deficient mice resulted in a significant decrease in RVSP, reversal of pulmonary vascular remodeling, and RV hypertrophy. In MA female ApoE-deficient mice with PH, only the expression of ERβ in the lungs, but not in RV, was significantly downregulated, and it was restored by E2 treatment. The expression of ERα was not affected in either lungs or RV by PH. GPR30 was only detected in the RV, and it was not affected by PH in MA female ApoE-deficient mice.ConclusionsOur results suggest that only aging female ApoE-deficient but not WT mice develop severe PH compared to younger females. Exogenous estrogen therapy rescued PH and RV hypertrophy in aging female ApoE-deficient mice possibly through restoration of lung ERβ

Pregnancy is associated with decreased cardiac proteasome activity and oxidative stress in mice.

During pregnancy, the heart develops physiological hypertrophy. Proteasomal degradation has been shown to be altered in various models of pathological cardiac hypertrophy. Since the molecular signature of pregnancy-induced heart hypertrophy differs significantly from that of pathological heart hypertrophy, we investigated whether the cardiac proteasomal proteolytic pathway is affected by pregnancy in mice. We measured the proteasome activity, expression of proteasome subunits, ubiquitination levels and reactive oxygen production in the hearts of four groups of female mice: i) non pregnant (NP) at diestrus stage, ii) late pregnant (LP), iii) one day post-partum (PP1) and iv) 7 days post-partum (PP7). The activities of the 26 S proteasome subunits β1 (caspase-like), and β2 (trypsin-like) were significantly decreased in LP (β1∶83.26 ± 1.96%; β2∶74.74 ± 1.7%, normalized to NP) whereas β5 (chymotrypsin-like) activity was not altered by pregnancy but significantly decreased 1 day post-partum. Interestingly, all three proteolytic activities of the proteasome were restored to normal levels 7 days post-partum. The decrease in proteasome activity in LP was not due to the surge of estrogen as estrogen treatment of ovariectomized mice did not alter the 26 S proteasome activity. The transcript and protein levels of RPN2 and RPT4 (subunits of 19 S), β2 and α7 (subunits of 20 S) as well as PA28α and β5i (protein only) were not significantly different among the four groups. High resolution confocal microscopy revealed that nuclear localization of both core (20S) and RPT4 in LP is increased ∼2-fold and is fully reversed in PP7. Pregnancy was also associated with decreased production of reactive oxygen species and ubiquitinated protein levels, while the de-ubiquitination activity was not altered by pregnancy or parturition. These results indicate that late pregnancy is associated with decreased ubiquitin-proteasome proteolytic activity and oxidative stress

Pregnancy is associated with decreased cardiac proteasome activity and oxidative stress in mice.

During pregnancy, the heart develops physiological hypertrophy. Proteasomal degradation has been shown to be altered in various models of pathological cardiac hypertrophy. Since the molecular signature of pregnancy-induced heart hypertrophy differs significantly from that of pathological heart hypertrophy, we investigated whether the cardiac proteasomal proteolytic pathway is affected by pregnancy in mice. We measured the proteasome activity, expression of proteasome subunits, ubiquitination levels and reactive oxygen production in the hearts of four groups of female mice: i) non pregnant (NP) at diestrus stage, ii) late pregnant (LP), iii) one day post-partum (PP1) and iv) 7 days post-partum (PP7). The activities of the 26 S proteasome subunits β1 (caspase-like), and β2 (trypsin-like) were significantly decreased in LP (β1∶83.26 ± 1.96%; β2∶74.74 ± 1.7%, normalized to NP) whereas β5 (chymotrypsin-like) activity was not altered by pregnancy but significantly decreased 1 day post-partum. Interestingly, all three proteolytic activities of the proteasome were restored to normal levels 7 days post-partum. The decrease in proteasome activity in LP was not due to the surge of estrogen as estrogen treatment of ovariectomized mice did not alter the 26 S proteasome activity. The transcript and protein levels of RPN2 and RPT4 (subunits of 19 S), β2 and α7 (subunits of 20 S) as well as PA28α and β5i (protein only) were not significantly different among the four groups. High resolution confocal microscopy revealed that nuclear localization of both core (20S) and RPT4 in LP is increased ∼2-fold and is fully reversed in PP7. Pregnancy was also associated with decreased production of reactive oxygen species and ubiquitinated protein levels, while the de-ubiquitination activity was not altered by pregnancy or parturition. These results indicate that late pregnancy is associated with decreased ubiquitin-proteasome proteolytic activity and oxidative stress

Proteasome activity of 26 S, but not 20 S is reduced in late pregnancy.

<p>Activity of different proteasomal beta subunits of the 26 S (A) and 20 S (B) was measured after initiating the reaction with: Z-LLE-AMC (β1), Boc-LSTR-AMC (β2) and Suc-LLVY-AMC (β5) for non pregnant (NP, black bars), late pregnant (LP, white bars), one day post-partum (PP1, grey bars) and seven days post-partum (PP7, shaded bars). The fluorescence values in arbitrary units are represented as mean ± SEM *p<0.05 vs. NP (n = 4 mice per group) and are normalized to NP levels. The raw proteasome activity values for the NP group are as follows (in nmol/min/mg protein): for the 26 S ATP-dependent activities, β1 was 0.11±0.01, β2 was 0.04±0.01 and β5 was 0.16±0.01, while for the 20 S activity, β1 was 0.21±0.03, β2 was 0.15±0.02 and β5 was 0.12±0.01.</p

Protein levels of RPT4, RPN2, α7, PA28α and β5i are unaffected by pregnancy.

<p>Immunoblotting of whole heart lysates (100 µg) from non pregnant (NP, black bars), late pregnant (LP, white bars), 1 day post-partum (PP1, grey bars) and 7 days post-partum (PP7, shaded bars) with anti-RPN2 (A–B), -RPT4 (C–D), -α7 (E–F), -PA28α (G–H) and -β5i (I–J) antibodies. In (I), the upper 30 kDa band is the β5i containing the pro-peptide. The bar graphs represent the quantification of fluorescent signal intensity normalized to Vinculin. For β5i both bands were taken into consideration in the quantification of protein levels. Vinculin was used as the loading control (n = 4 per group). Values are mean ± SEM in arbitrary units.</p

Increased nuclear labeling of core Subunits and RPT4 in late pregnancy was reversed one week postpartum.

<p>A. Representative single confocal sections of cardiomyocytes dissociated from non pregnant (NP), late pregnant (LP), one day post-partum (PP1) and seven days post-partum (PP7) are co-immunostained with anti-core (green) and -RPT4 (red) antibodies. The nuclear overlay of core and RPT4 are also shown at higher resolution. These results are representative of the labeling pattern observed in myocytes from 3 different animals in each group. B. Quantification of nuclear fluorescence labeling in the four groups mentioned above for core (green bars) and RPT4 (red bars) from at least 20–25 cells per group (n = 3 mice/group). Only the nucleus in the confocal plane of focus was taken into account. **denotes <i>p</i><0.001 <i>vs.</i> NP, #<i>p</i><0.05 <i>vs.</i> LP and ^∧<i>p</i><0.05 <i>vs.</i> PP1.</p

The increased in heart weight of LP mice is reversed post-partum.

<p>Heart weights (HW, A), body weights (BW, B) and HW/BW (C) in non-pregnant (NP, black bars, n = 8), late pregnant (LP, white bars, n = 11), one day post-partum (PP1, grey bars, n = 6) and seven days post-partum (PP7, shaded bars, n = 5) mice. Values are mean ± SEM, ^*p<0.05 and ^**p<0.001 vs. NP, ^#p<0.05 and ^##p<0.001 vs. LP, ^∧p<0.05 and ^∧∧p<0.001 vs. PP1.</p

Transcript levels of proteasome 19S and 20S subunits, as well as the regulatory subunit PA28α, are not modified in late pregnancy.

<p>Relative transcript expression of the cardiac proteasome measured by Real-Time qPCR in non pregnant (NP, black bars), in late pregnancy (LP, white bars), 1 day post-partum (PP1, grey bars) and 7 days post-partum (PP7, shaded bars) for RPN2 and RPT4, which are subunits of 19 S (A–B), β2 and α7, which are subunits of 20 S (C–D) and the proteasome regulatory subunit PA28α (E). GAPDH was used as the internal reference gene (data not shown). Values are mean ± SEM as normalized to NP (n = 3–5 per group).</p