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Activation of the G protein-coupled estrogen receptor ameliorates salt-induced vascular remodeling
The mRen2.Lewis (mRen2) female rat is an estrogen- and salt-sensitive model of hypertension which may reflect the higher pressure and salt sensitivity associated with menopause. We previously showed in salt-loaded mRen2 female rats that activation of the G protein-coupled estrogen receptor (GPER) does not lower blood pressure but attenuates renal damage. The current study hypothesized that GPER protects against vascular injury in this model. Intact mRen2.Lewis female rats were fed a normal (0.5% Na+) or high salt diet (HS; 4% Na+) for 10 weeks and treated with vehicle or the selective GPER agonist G-1 for the last two weeks of the study. Systolic blood pressure was measured by tail cuff plethysmography, and aortic sections were mounted on a wire myograph or formalin-fixed for histological analysis. Systolic blood pressure increased with HS (137 ± 2 mmHg, n=7 to 224 ± 8, n=9; P<0.001) and remained unchanged by G-1 treatment (216 ± 4, n=9; P>0.05). While aortic reactivity to phenylephrine and acetylcholine were not different between groups (P>0.05), chronic G-1 treatment reduced vasoconstriction to angiotensin II (P<0.05) and enhanced ex vivo G-1 vasorelaxation (P<0.01) in both endothelium-intact and denuded aortic rings. Analysis of aortic sections revealed that while the high salt diet significantly increased the media to lumen ratio (0.43 ± 0.02, n=6 vs. 0.83 ± 0.05, n=7; P<0.001), two-week G-1 treatment partially reversed the damage due to HS (0.62 ± 0.02, n=6, P<0.01). The decrease in aortic thickening was not due to changes in extracellular matrix proteins, as G-1 did not alter collagen or elastin staining. In addition, there was no evidence of proliferation in the medial layer as assessed by staining for PCNA and Ki-67. Interestingly, HS induced an increase in oxidative stress in aortic sections, assessed by 4-hydroxynonenal staining, that was ameliorated by G-1 treatment ( ). In primary cultured aortic smooth muscle cells, HS had no effect on cell proliferation but increased cellular protein content. Treatment with G-1 or the antioxidant Tempol reversed HS-induced cellular hypertrophy. We conclude that GPER exerts vascular protective actions in the aorta of salt-loaded mRen2 females independently of blood pressure. The anti-remodeling actions of this novel estrogen receptor may occur by reducing oxidative stress in the medial layer to counteract smooth muscle cell hypertrophy. Activation of GPER by estrogen may protect females from vascular damage due to hypertension and salt-loading.
Sodium balance plays an integral role in cardiovascular homeostasis. A high salt diet is considered a major risk for cardiovascular morbidity and mortality independent of other cardiovascular risk factors (Beil et al. Blood Pressure, 1995). Excessive salt intake has been associated with vascular remodeling, including the reorganization of the extracellular matrix and hypertrophy and/or hyperplasia of vascular smooth muscle cells. Although vascular remodeling is considered a protective adaptation to a higher wall stress, it contributes to the development of hypertension by creating a thicker, less compliant wall. Aortic stiffness, which contributes to isolated systolic hypertension in human subjects, is an excellent example of the complications presented by vascular remodeling (Lemarie et al. Journal of Molecular and Cellular Cardiology, 2009).
The ability of estrogen to attenuate vascular remodeling in injured arteries may at least partly occur via activation of the classic steroid receptors ERα and ERβ in VSMC and endothelial cells (Xing et al. Arteriosclerosis, Thrombosis, and Vascular Biology, 2009). Brouchet et al. showed that estrogen’s protective effects on vascular remodeling were abolished in female ERα knockout mice (Brouchet et al, Circulation, 2001). Moreover, in porcine aortic smooth muscle cells, the downregulation of ERβ protein levels by mRNA antisense oligomers abrogated the inhibitory effects of estrogen on mitogen-activated protein kinase (MAPK) phosphorylation, migration, and proliferation (Geraldes et al. Circ Res, 2003; Xing et al. Am J Physiol., 2007). However, the protective effects of estrogen on vascular injury are evident in both ERα and ERβ knockout mice, suggesting that another receptor may be necessary (Iafrati et al. Nat Med. 1997; Karas et al. Proc Natl Acad Sci USA. 1999). The novel G protein-coupled estrogen receptor (GPER) is a membrane-bound receptor linked to acute signaling pathways (Revankar et al. Science, 2005; Thomas et al. Endocrinology, 2005). Our previous studies showed that GPER activation lowers blood pressure in ovariectomized mRen2.Lewis rats and attenuates salt-induced renal and cardiac remodeling in intact mRen2.Lewis rats (Lindsey et al Endocrinology, Jessup et al, PLOS one, 2010 and Lindsey et al Hypertension). Therefore, this novel receptor may play an important role in mediating estrogenic effects in the vasculature.
In the current study, we hypothesized that chronic GPER activation is protective against aortic remodeling due to salt-sensitive hypertension. To test this hypothesis, we utilized mRen2.Lewis (mRen2) rats, a unique congenic model of hypertension in which HS profoundly elevates blood pressure in females (Chappell et al, Am J Physiol Regul Integr Comp Physiol., 2006). We compared aortic remodeling in high salt-fed rats with or without treatment with the selective GPER agonist G-1. Because estrogen modulates oxidative stress to attenuate vascular remodeling in injured arteries (Hayashi et al, Biochem Biophys Bes Commun, 1995; Xing et al. Arteriosclerosis, Thrombosis, and Vascular Biology, 2009) and GPER reduces oxidative stress in the kidneys of female mRen2 rats (Lindsey et al. Hypertension, 2011), we further determined whether the effects of HS and G-1 were mediated by modulating oxidative stress. To further elucidate the cellular mechanisms responsible for vascular remodeling, we utilized primary cultured female rat aortic smooth muscle cells to determine the effects of salt on cellular hypertrophy.
All procedures were approved by the institutional Animal Care and Use Committee. Hemizygous mRen2.Lewis congenic female rats were obtained from the Wake Forest Hypertension Center breeding colony. Rats had free access to food and water in a temperature-controlled room (22 ± 2°C) with a 12 hour light to dark cycle. At five weeks of age, the normal salt diet (NS, 0.5% Na) was switched to high salt (HS; 4% Na), as previously described (1, 2). The selective GPER agonist G-1 (400 µg/kg/day; EMD Chemicals, Gibbstown, NJ) or vehicle was administered for two weeks beginning at 13 weeks of age via subcutaneous osmotic minipump (Model 2ML2; Alza Corporation, Palo Alto, CA). Blood pressure was measured via tail cuff plethysmography (Narco Bio-systems, Houston, TX). Animals were randomly assigned to three experimental groups: intact NS (n=7), intact HS+vehicle (veh; n=9), and intact HS+G-1 (n = 9).
Vascular Reactivity. After sacrifice, the upper thoracic aorta was submerged in formalin for histology and the lower portion used for vascular reactivity as previously described (Lindsey et al Endocrinology). Aortas were equilibrated with 2 g tension and the responses to 1 uM phenylephrine, 1 uM acetylcholine, 10 nM angiotensin II (Ang II), and 3 uM G-1 were measured.
Histology. Formalin-fixed aortas were embedded in paraffin, cut into 5 um sections, and mounted on slides. To evaluate aortic wall thickness, slides were stained with hematoxylin and images analyzed using ImagePro software (XXX company, XXX city,XX state). Aortic medial area was calculated by the subtraction of the area of the inner border of the lumen (inner area) from the area of the outer border of the tunica media (outer area). Collagen staining was performed using picrosirius red and images were taken using a Texas Red fluorescent filter. The medial area was selected and the mean luminosity was recorded for each section. The NovaUltra Orcein Elastin Stain Kit (IHC World, XXX) was used for elastin staining. For analysis of oxidative stress, sections were immunostained with an antibody against 4-hydroxynonenal as previously described (Lindsey et al Hypertension). For analysis of brightfield images, positive staining was identified and the percent of positive pixels in the medial area recorded. For all analyses, the average of four cross-sectional measurements was calculated for each animal.
Cell Studies. Aortic smooth muscle cells were isolated from adult female Lewis rats by explant method. Aortas were excised, cleaned of fat, cut longitudinally, scraped of endothelial cells, laid luminal side down in a cell culture dish containing Medium 199 (Invitrogen, XXX) and 5% fetal bovine serum (Gibco, XXX), and cut into small strips. Cells were subcultured up to four passages. When cells reached 80% confluence, the medium was switched to Medium 199 without phenol red or fetal bovine serum and sodium chloride (Sigma) was added to the increase the concentration to 152 mM, 160 mM, and 182 mM.
Add NaCl to make high salt CS medium (152, 160, and 182 mM), Normal medium 142 mM
- NO, I find 117 mM
Add 0.02 g NaCl to 50 ml of normal medium, that makes 152 mM
Add 0.045 g NaCl to 50 ml of normal medium, that makes 160 mM
Add 0.11 g NaCl to 50 ml of normal medium, that makes 182 mM
After 5 days of exposure to high sodium medium, cells were harvested for further experiments. Cells were harvested using trypsin to obtain single cell suspensions. A sample was taken for determination of cell number and diameter using XXX cellometer (XXX company, XXX city, XXX state). Mean cell diameter was determined on 200 randomly chosen cells in each sample. The remaining cells were lysed in XXX with protease inhibitor cocktail (XXX company). Cellular protein content was determined in duplicate using bovine serum albumin as a standard (Bio-Rad Protein Assay Kit).
As previously reported, a high salt diet (HS) significantly increased systolic blood pressure in intact mRen2 female rats (Figure 1A). Chronic treatment with the selective GPER agonist G-1 for two weeks did not influence blood pressure. G-1 did not influence the aortic response to acetylcholine (Figure 1B) or phenylephrine (Figure 1D). However, G-1 treatment in vivo amplified the vasorelaxant response to ex vivo application of G-1 (Figure 1C) and decreased the vasoconstrictor response to Ang II (Figure 1E).
Salt-sensitive hypertension in female mRen2 rats significantly increased aortic thickness, as determined by the media/lumen ratio (Figure 2A-B). This remodeling was associated with a significant decrease in lumen area but no change in the external diameter of the aorta (Figures 2C-D). Chronic G-1 significantly attenuated remodeling, as evidenced by a decreased media/lumen ratio and an increased lumen area, with no change in external diameter. The average measurements for all groups are graphically represented in Figure 2E.
In order to determine whether extracellular fibrosis was altered by HS and G-1, aortic sections were analyzed for collagen and elastin content. Figure 3 shows that picrosirius red staining was similar in all groups (Figure 3). Elastin staining was significantly decreased by HS but this effect was not reversed by G-1 (Figure 4). Elastin Breaks?? Space between elastin fibers?? Sections were assessed for proliferation using antibodies against proliferating cell nuclear antigen and Ki-67. No evidence of immunostaining was found in the medial sections of aorta for these two nuclear proteins necessary for cellular proliferation (data not shown). However, in comparison to aortas from normal salt-fed rats, HS aortas showed a significant increase in oxidative stress as measured by staining for the lipid peroxidation product 4-HNE (Figure 5). In addition, chronic G-1 treatment significantly attenuated 4-HNE staining.
Aortic smooth muscle cells were isolated and cultured in order to determine the Effects of HS and G-1 on cellular hypertrophy.
The present study demonstrated that estrogen receptor GPER activation attenuated salt-induced increase of aortic wall thickness in mRen2 rats. The mechanism for the G-1 effect most likely involves counteracting oxidative stress and reducing vascular smooth muscle cell hypertrophy. This study demonstrating GPER-induced vascular protection nicely complements our previous work showing similar results in the kidney and heart (ADD REF). Interestingly, GPER’s renoprotective effects were also associated with a reduction in oxidative stress. Moreover, the beneficial effects in the heart were similarly independent of alterations in the extracellular matrix but directly associated with a reduction in cardiomyocyte size.
Vascular GPER protein is clearly expressed in both endothelial and smooth muscle cells of the aorta (Lindsey et al, Endocrinology, 2009; Ding et al, Am J Physiol Cell Physiol, 2009; Gros et al, Hypertension, 2011).
In native vessels, the extracellular matrix (ECM) is composed mostly of collagen, elastin and proteoglycans. These proteins in¬‚uence cell functions and play an important role in maintaining vessel structure by providing tensile strength (collagens) and elasticity (elastin) (Lemarie et al. Journal of Molecular and Cellular Cardiology, 2009). One of the earliest steps of vascular remodeling is the reorganization of the ECM. In the current study, we have shown that HS increased collagen levels and suppressed elastin content in the aorta, indicating increased stiffness and less elasticity. Most importantly, the finding that G-1 ameliorates salt-induced collagen increase, without altering salt-induced decreases in elastin content, suggests that GPER activation protects against the remodeling process via suppressing collagen levels, instead of elastin. The amount and composition of collagen depends on a balance between synthesis / deposition and degradation. The exact effects of GPER on collagen protein expression, degradation, or both remain to be investigated.
Another important step during vascular remodeling is the hypertrophy and / or hyperplasia of the VSMCs (Lemarie et al. Journal of Molecular and Cellular Cardiology, 2009). To address this possibility, we used primary cultured ASMCs isolated from female mRen2 rats. We found that both cellular protein content and cell size increased in high salt media (160 mM and 182 mM), indicating that high salt induces cellular hypertrophy. The cellular protein content of 152 mM media-treated cells is significantly higher than that of normal media (142 mM)-treated group. However, the cell size has no significant difference. One possible explanation is that the protein started to accumulate inside cells, but cells had not begun to enlarge yet. More importantly, we found G-1 abolished the hypertrophy of cells. The hypertrophy appears to result from an increase in the rate of protein synthesis and / or a decrease in the rate of protein degradation (Berk et al, Hypertension, 1989; Gu et al, Hypertension, 1998). Future studies are required to determine the effects of GPER activation on protein expression, degradation, or both.
We further looked into the possibility of salt-induced proliferation in ASMCs. Although it has been shown that GPER induced the activation of MAPK signaling and cellular hyperplasia in VSMCs, other studies have demonstrated that G-1 inhibited serum-stimulated growth in VSMCs lacking ERα and ERβ (Haas et al, Circ Res, 2009,; Ding, Am J Physiol Cell Physiol, 2009; Gros et al, Hypertension, 2011). In our study, we did not observe any evidence of Ki67 or PCNA staining in the medial layer of aortas from any group, although significant staining was found in the adventitia. These results are supported by our in vitro studies which show no change in cell number in response to HS.
The finding that G-1 attenuates aortic thickening and cellular hypertrophy, without altering blood pressure, suggests that GPER has protective effects in the cardiovascular system that are independent of blood pressure. One possible mechanism is acute increases in oxidative stress. Oxidative stress is linked to damage within the vasculature and may contribute to vascular remodeling (Hayashi et al, Biochem Biophys Bes Commun, 1995; Xing et al. Arteriosclerosis, Thrombosis, and Vascular Biology, 2009). To address this possibility, we first stained 4-HNE, a marker for oxidative stress, in the aorta. We found significantly stronger staining of 4-HNE in HS-fed rats. However, this was attenuated by G-1 treatment, suggesting that GPER activation attenuates salt-induced oxidative stress. To further confirm this finding, we used tempol, an antioxidant, or G1 to treat ASMCs and measured several hypertrophy parameters. Tempol or G-1 treatments abolished the increase of both cellular protein content and cell size. Broughton et al demonstrated that G1 reduces NADPH-dependent oxidase activity in isolated carotid and intracranial arteries of normotensive Sprague-Dawley rats (Broughton et al, Am J Physiol Heart Circ Physiol, 2010). Elucidation of the underlying mechanisms of GPR 30 to attenuate reactive oxygen species within aorta awaits future studies. Another possibility is that HS stimulates renin-angiotensin system (RAS) in mRen2 rats. Ang II increased medial thickening of aorta due to VSMC hypertrophy without increase in cell number (Owens et al, Circ Res, 1982 & 1983). However, GPER activation may reduce expression of the angiotensin II (Ang II) AT1 receptor (AT1R) and angiotensin-converting enzyme (ACE) but increase the expression of ACE2. Alterations in ACE and ACE2 may increase the ratio of Ang-(1-7) to Ang II in tissues (Lindsey et al, Gender Medicine, 2011). Ang-(1-7) inhibited vascular remodeling in rat jugular vein grafts (Wu et al, J Int Med Res, 2011).
In the present study, the HS medium (152, 160, 182 mM) was made by simply adding sodium chloride to normal medium (142 mM). This increases both sodium concentration and osmolarity in the medium. It is likely that increased osmolarity plays a significant role in salt-induced hypertrophy. Future studies using mannitol are needed to rule out this possibility.
In summary, this study showed a beneficial effect of the GPER agonist G-1 in salt- and pressure-induced vascular remodeling. These protective effects of G-1 may be due to suppression of oxidative stress and associated cellular hypertrophy.
What about aldosterone?
1.Chappell MC, Westwood BM, and Yamaleyeva LM. Differential effects of sex steroids in young and aged female mRen2.Lewis rats: a model of estrogen and salt-sensitive hypertension. Gender medicine 5 Suppl A: S65-75, 2008.
2.Lindsey SH, Yamaleyeva LM, Brosnihan KB, Gallagher PE, and Chappell MC. Estrogen receptor GPR30 reduces oxidative stress and proteinuria in the salt-sensitive female mRen2.Lewis rat. Hypertension 58: 665-671, 2011.