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 theoretical account of high blood pressure which may reflect the higher force per unit area and salt sensitiveness associated with climacteric. We antecedently showed in salt-loaded mRen2 female rats that activation of the G protein-coupled estrogen receptor ( GPER ) does non take down blood force per unit area but attenuates nephritic harm. The current survey hypothesized that GPER protects against vascular hurt in this theoretical account. Integral mRen2.Lewis female rats were fed a normal ( 0.5 % Na+) or high salt diet ( HS ; 4 % Na+) for 10 hebdomads and treated with vehicle or the selective GPER agonist G-1 for the last two hebdomads of the survey. Systolic blood force per unit area was measured by tail turnup plethysmography, and aortal subdivisions were mounted on a wire myograph or formalin-fixed for histological analysis. Systolic blood force per unit area increased with HS ( 137 ± 2 mmHg, n=7 to 224 ± 8, n=9 ; P & lt ; 0.001 ) and remained unchanged by G-1 intervention ( 216 ± 4, n=9 ; P & gt ; 0.05 ) . While aortal responsiveness to phenylephrine and acetylcholine were non different between groups ( P & gt ; 0.05 ) , chronic G-1 intervention reduced vasoconstriction to angiotensin II ( P & lt ; 0.05 ) and enhancedex vivoG-1 vasorelaxation ( P & lt ; 0.01 ) in both endothelium-intact and denuded aortal rings. Analysis of aortal subdivisions revealed that while the high salt diet significantly increased the media to lms ratio ( 0.43 ± 0.02, n=6 vs. 0.83 ± 0.05, n=7 ; P & lt ; 0.001 ) , two-week G-1 intervention partly reversed the harm due to HS ( 0.62 ± 0.02, n=6, P & lt ; 0.01 ) . The lessening in aortal thickener was non due to alterations in extracellular matrix proteins, as G-1 did non change collagen or elastin staining. In add-on, there was no grounds of proliferation in the median bed as assessed by staining for PCNA and Ki-67. Interestingly, HS induced an addition in oxidative emphasis in aortal subdivisions, assessed by 4-hydroxynonenal staining, that was ameliorated by G-1 intervention ( ) . In primary cultured aortal smooth musculus cells, HS had no consequence 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 force per unit area. The anti-remodeling actions of this fresh estrogen receptor may happen by cut downing oxidative emphasis in the median bed to antagonize smooth musculus cell hypertrophy. Activation of GPER by estrogen may protect females from vascular harm due to high blood pressure and salt-loading.
Sodium balance plays an built-in function in cardiovascular homeostasis. A high salt diet is considered a major hazard for cardiovascular morbidity and mortality independent of other cardiovascular hazard factors ( Beil et al. Blood Pressure, 1995 ) . Excessive salt consumption has been associated with vascular remodeling, including the reorganisation of the extracellular matrix and hypertrophy and/or hyperplasia of vascular smooth musculus cells. Although vascular remodeling is considered a protective version to a higher wall emphasis, it contributes to the development of high blood pressure by making a thicker, less compliant wall. Aortal stiffness, which contributes to insulate systolic high blood pressure in human topics, is an first-class illustration of the complications presented by vascular remodeling ( Lemarie et al. Journal of Molecular and Cellular Cardiology, 2009 ) .
The ability of estrogen to rarefy vascular reconstructing in injured arterias may at least partially occur via activation of the authoritative steroid receptors ER? and ER? 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 reconstructing were abolished in female ER? smasher mice ( Brouchet et al, Circulation, 2001 ) . Furthermore, in porcine aortal smooth musculus cells, the downregulation of ER? protein degrees by messenger RNA antisense oligomers abrogated the repressive 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 hurt are apparent in both ER? and ER? knockout mice, proposing that another receptor may be necessary ( Iafrati et al. Nat Med. 1997 ; Karas et Al. Proc Natl Acad Sci USA. 1999 ) . The fresh G protein-coupled estrogen receptor ( GPER ) is a membrane-bound receptor linked to acute signaling tracts ( Revankar et al. Science, 2005 ; Thomas et Al. Endocrinology, 2005 ) . Our old surveies showed that GPER activation lowers blood force per unit area in ovariectomized mRen2.Lewis rats and attenuates salt-induced nephritic and cardiac remodeling in integral mRen2.Lewis rats ( Lindsey et al Endocrinology, Jessup et Al, PLOS one, 2010 and Lindsey et al Hypertension ) . Therefore, this fresh receptor may play an of import function in interceding estrogenic effects in the vasculature.
In the current survey, we hypothesized that chronic GPER activation is protective against aortal reconstructing due to salt-sensitive high blood pressure. To prove this hypothesis, we utilized mRen2.Lewis ( mRen2 ) rats, a alone congenic theoretical account of high blood pressure in which HS deeply elevates blood force per unit area in females ( Chappell et al, Am J Physiol Regul Integr Comp Physiol. , 2006 ) . We compared aortal remodeling in high salt-fed rats with or without intervention with the selective GPER agonist G-1. Because estrogen modulates oxidative emphasis to rarefy vascular reconstructing in injured arterias ( Hayashi et Al, Biochem Biophys Bes Commun, 1995 ; Xing et Al. Arteriosclerosis, Thrombosis, and Vascular Biology, 2009 ) and GPER reduces oxidative emphasis 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 emphasis. To farther clarify the cellular mechanisms responsible for vascular remodeling, we utilized primary civilized female rat aortal smooth musculus cells to find the effects of salt on cellular hypertrophy.
All processs were approved by the institutional Animal Care and Use Committee. Hemizygous mRen2.Lewis congenic female rats were obtained from the Wake Forest Hypertension Center engendering settlement. Rats had free entree to nutrient and H2O in a temperature-controlled room ( 22 ± 2°C ) with a 12 hr visible radiation to dark rhythm. At five hebdomads of age, the normal salt diet ( NS, 0.5 % Na ) was switched to high salt ( HS ; 4 % Na ) , as antecedently described ( 1, 2 ) . The selective GPER agonist G-1 ( 400 µg/kg/day ; EMD Chemicals, Gibbstown, NJ ) or vehicle was administered for two hebdomads get downing at 13 hebdomads of age via hypodermic osmotic minipump ( Model 2ML2 ; Alza Corporation, Palo Alto, CA ) . Blood force per unit area was measured via tail turnup plethysmography ( Narco Bio-systems, Houston, TX ) . Animals were indiscriminately assigned to three experimental groups: integral NS ( n=7 ) , integral HS+vehicle ( veh ; n=9 ) , and integral HS+G-1 ( n = 9 ) .
Vascular Reactivity. After forfeit, the upper thoracic aorta was submerged in formol for histology and the lower part used for vascular responsiveness as antecedently described ( Lindsey et al Endocrinology ) . Aortas were equilibrated with 2 g tenseness and the responses to 1 uM phenylephrine, 1 uM acetylcholine, 10 nanometer angiotonin II ( Ang II ) , and 3 uM G-1 were measured.
Histology. Formalin-fixed aortas were embedded in paraffin, cut into 5 um subdivisions, and mounted on slides. To measure aortal wall thickness, slides were stained with hematoxylin and images analyzed utilizing ImagePro package ( XXX company, XXX metropolis, XX province ) . Aortal median country was calculated by the minus of the country of the interior boundary line of the lms ( interior country ) from the country of the outer boundary line of the tunica media ( outer country ) . Collagen staining was performed utilizing picrosirius ruddy and images were taken utilizing a Texas Red fluorescent filter. The median country was selected and the average brightness was recorded for each subdivision. The NovaUltra Orcein Elastin Stain Kit ( IHC World, XXX ) was used for elastin staining. For analysis of oxidative emphasis, subdivisions were immunostained with an antibody against 4-hydroxynonenal as antecedently described ( Lindsey et al Hypertension ) . For analysis of brightfield images, positive staining was identified and the per centum of positive pels in the median country recorded. For all analyses, the norm of four cross-sectional measurings was calculated for each animate being.
Cell Studies. Aortic smooth musculus cells were isolated from big 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 civilization dish incorporating Medium 199 ( Invitrogen, XXX ) and 5 % foetal bovine serum ( Gibco, XXX ) , and cut into little strips. Cells were subcultured up to four transitions. When cells reached 80 % meeting, the medium was switched to Medium 199 without phenol red or foetal bovine serum and Na chloride ( Sigma ) was added to the addition the concentration to 152 millimeters, 160 millimeter, and 182 millimeter.
Add NaCl to do high salt CS medium ( 152, 160, and 182 millimeter ) , Normal medium 142 millimeter
- NO, I find 117 millimeter
Add 0.02 g NaCl to 50 milliliters of normal medium, that makes 152 millimeter
Add 0.045 g NaCl to 50 milliliters of normal medium, that makes 160 millimeter
Add 0.11 g NaCl to 50 milliliters of normal medium, that makes 182 millimeter
After 5 yearss of exposure to high Na medium, cells were harvested for farther experiments. Cells were harvested utilizing trypsin to obtain individual cell suspensions. A sample was taken for finding of cell figure and diameter utilizing XXX cellometer ( Thirty company, XXX metropolis, XXX province ) . Mean cell diameter was determined on 200 indiscriminately chosen cells in each sample. The staying cells were lysed in Thirty with peptidase inhibitor cocktail ( XXX company ) . Cellular protein content was determined in extra utilizing bovid serum albumen as a criterion ( Bio-Rad Protein Assay Kit ) .
As antecedently reported, a high salt diet ( HS ) significantly increased systolic blood force per unit area in integral mRen2 female rats ( Figure 1A ) . Chronic intervention with the selective GPER agonist G-1 for two hebdomads did non act upon blood force per unit area. G-1 did non act upon the aortal response to acetylcholine ( Figure 1B ) or phenylephrine ( Figure 1D ) . However, G-1 interventionin vivoamplified the vasorelaxant response toex vivoapplication of G-1 ( Figure 1C ) and decreased the vasoconstrictive response to Ang II ( Figure 1E ) .
Salt-sensitive high blood pressure in female mRen2 rats significantly increased aortal thickness, as determined by the media/lumen ratio ( Figure 2A-B ) . This remodeling was associated with a important lessening in lumen country but no alteration in the external diameter of the aorta ( Figures 2C-D ) . Chronic G-1 significantly attenuated reconstructing, as evidenced by a reduced media/lumen ratio and an increased lms country, with no alteration in external diameter. The mean measurings for all groups are diagrammatically represented in Figure 2E.
In order to find whether extracellular fibrosis was altered by HS and G-1, aortal subdivisions 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 consequence was non reversed by G-1 ( Figure 4 ) . Elastin Breaks? ? Space between elastin fibres? ? Sections were assessed for proliferation utilizing antibodies against proliferating cell atomic antigen and Ki-67. No grounds of immunostaining was found in the median subdivisions of aorta for these two atomic proteins necessary for cellular proliferation ( informations non shown ) . However, in comparing to aortas from normal salt-fed rats, HS aortas showed a important addition in oxidative emphasis as measured by staining for the lipid peroxidation merchandise 4-HNE ( Figure 5 ) . In add-on, chronic G-1 intervention significantly attenuated 4-HNE staining.
Aortal smooth musculus cells were isolated and cultured in order to find the Effectss of HS and G-1 on cellular hypertrophy.
The present survey demonstrated that estrogen receptor GPER activation attenuated salt-induced addition of aortal wall thickness in mRen2 rats. The mechanism for the G-1 consequence most likely involves antagonizing oxidative emphasis and cut downing vascular smooth musculus cell hypertrophy. This survey showing GPER-induced vascular protection nicely complements our old work demoing similar consequences in the kidney and bosom ( ADD REF ) . Interestingly, GPER’s renoprotective effects were besides associated with a decrease in oxidative emphasis. Furthermore, the good effects in the bosom were likewise independent of changes in the extracellular matrix but straight associated with a decrease in cardiomyocyte size.
Vascular GPER protein is clearly expressed in both endothelial and smooth musculus cells of the aorta ( Lindsey et al, Endocrinology, 2009 ; Dinging et Al, Am J Physiol Cell Physiol, 2009 ; Gros et Al, Hypertension, 2011 ) .
In native vass, the extracellular matrix ( ECM ) is composed largely of collagen, elastin and proteoglycans. These proteins ini¬‚uence cell maps and play an of import function in keeping vas construction by supplying tensile strength ( collagens ) and snap ( elastin ) ( Lemarie et al. Journal of Molecular and Cellular Cardiology, 2009 ) . One of the earliest stairss of vascular remodeling is the reorganisation of the ECM. In the current survey, we have shown that HS increased collagen degrees and suppressed elastin content in the aorta, bespeaking increased stiffness and less snap. Most significantly, the determination that G-1 ameliorates salt-induced collagen addition, without changing salt-induced lessenings in elastin content, suggests that GPER activation protects against the reconstructing procedure via stamp downing collagen degrees, alternatively of elastin. The sum and composing of collagen depends on a balance between synthesis / deposition and debasement. The exact effects of GPER on collagen protein look, debasement, or both remain to be investigated.
Another of import measure during vascular remodeling is the hypertrophy and / or hyperplasia of the VSMCs ( Lemarie et al. Journal of Molecular and Cellular Cardiology, 2009 ) . To turn to this possibility, we used primary civilized ASMCs isolated from female mRen2 rats. We found that both cellular protein content and cell size increased in high salt media ( 160 millimeter and 182 millimeter ) , bespeaking 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 millimeter ) -treated group. However, the cell size has no important difference. One possible account is that the protein started to roll up inside cells, but cells had non begun to enlarge yet. More significantly, we found G-1 abolished the hypertrophy of cells. The hypertrophy appears to ensue from an addition in the rate of protein synthesis and / or a lessening in the rate of protein debasement ( Berk et al, Hypertension, 1989 ; Gu et Al, Hypertension, 1998 ) . Future surveies are required to find the effects of GPER activation on protein look, debasement, 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 surveies have demonstrated that G-1 inhibited serum-stimulated growing in VSMCs missing ER? and ER? ( Haas et al, Circ Res, 2009, ; Ding, Am J Physiol Cell Physiol, 2009 ; Gros et Al, Hypertension, 2011 ) . In our survey, we did non detect any grounds of Ki67 or PCNA staining in the median bed of aortas from any group, although important staining was found in the tunic. These consequences are supported by ourin vitrosurveies which show no alteration in cell figure in response to HS.
The determination that G-1 attenuates aortal thickener and cellular hypertrophy, without changing blood force per unit area, suggests that GPER has protective effects in the cardiovascular system that are independent of blood force per unit area. One possible mechanism is acute additions in oxidative emphasis. Oxidative emphasis is linked to damage within the vasculature and may lend to vascular remodeling ( Hayashi et Al, Biochem Biophys Bes Commun, 1995 ; Xing et Al. Arteriosclerosis, Thrombosis, and Vascular Biology, 2009 ) . To turn to this possibility, we foremost stained 4-HNE, a marker for oxidative emphasis, in the aorta. We found significantly stronger staining of 4-HNE in HS-fed rats. However, this was attenuated by G-1 intervention, proposing that GPER activation attenuates salt-induced oxidative emphasis. To farther confirm this determination, we used tempol, an antioxidant, or G1 to handle ASMCs and measured several hypertrophy parametric quantities. Tempol or G-1 interventions abolished the addition of both cellular protein content and cell size. Broughton et al demonstrated that G1 reduces NADPH-dependent oxidase activity in stray carotid and intracranial arterias of normotensive Sprague-Dawley rats ( Broughton et Al, Am J Physiol Heart Circ Physiol, 2010 ) . Elucidation of the implicit in mechanisms of GPR 30 to rarefy reactive O species within aorta awaits future surveies. Another possibility is that HS stimulates renin-angiotensin system ( RAS ) in mRen2 rats. Ang II increased median thickener of aorta due to VSMC hypertrophy without addition in cell figure ( Owens et al, Circ Res, 1982 & A ; 1983 ) . However, GPER activation may cut down look of the angiotonin II ( Ang II ) AT1 receptor ( AT1R ) and angiotensin-converting enzyme ( ACE ) but increase the look of ACE2. Changes 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 vena transplants ( Wu et al, J Int Med Res, 2011 ) .
In the present survey, the HS medium ( 152, 160, 182 millimeter ) was made by merely adding Na chloride to normal medium ( 142 millimeter ) . This increases both sodium concentration and osmolarity in the medium. It is likely that increased osmolarity plays a important function in salt-induced hypertrophy. Future surveies utilizing Osmitrols are needed to govern out this possibility.
In drumhead, this survey showed a good consequence 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 emphasis and associated cellular hypertrophy.
What about aldosterone?
1.Chappell MC, Westwood BM, and Yamaleyeva LM. Differential effects of sex steroids in immature and elderly female mRen2.Lewis rats: a theoretical account of estrogen and salt-sensitive high blood pressure. Gender medical specialty 5 Suppl A: S65-75, 2008.
2.Lindsey SH, Yamaleyeva LM, Brosnihan KB, Gallagher PE, and Chappell MC. Estrogen receptor GPR30 reduces oxidative emphasis and albuminuria in the salt-sensitive female mRen2.Lewis rat. High blood pressure 58: 665-671, 2011.