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Howard Florey Institute (E.D.L., C.Z., G.W.T., R.A.D.B., C.S.S.), University of Melbourne, Parkville, Victoria 3010, Australia; Baker Heart Research Institute (E.D.L., X.L.M., X.-J.D.), Melbourne, Victoria 8008, Australia; Department of Immunology (S.G.R., M.L.K.T.), Murdoch Children’s Research Institute, Royal Children’s Hospital, Parkville, Victoria 3052, Australia; and Department of Nephrology (T.D.H), Royal Melbourne Hospital, Parkville, Victoria 3050, Australia
Address all correspondence and requests for reprints to: Chrishan Samuel, Ph.D., Howard Florey Institute, Gate 11, University of Melbourne, Parkville, Victoria 3010, Australia. E-mail: c.samuel{at}hfi.unimelb.edu.au.
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These combined findings are remarkably consistent with recent studies in genetically modified mice, which have shown that gender can influence the development of fibrotic phenotypes (13), signifying the impact of gender-related factors such as sex hormones on the progression of disease. Limited studies have suggested that androgens in male mice may promote the progression and severity of fibrosis (14). Alternatively, a large number of studies have suggested that estrogens play a key role in protecting several organs from the pathogenesis of diseased states (15), implying that estrogens in female Rln1–/– mice may partially compensate for the absence of relaxin. Thus, further studies are required to explore the mechanism for gender differences in this Rln1–/– mouse model.
Clinical and experimental studies have revealed a gender difference in the progression of a fibrotic and hypertrophic pathophysiology. It has been well-documented that compared with premenopausal women, men and postmenopausal women are at a higher risk from cardiovascular, renal, and pulmonary diseases, suggestive of a possible protective role of estrogen in the heart (13, 14, 15, 16, 17), kidney (18), and lung (19), respectively. Protection of the cardiovascular, renal, and pulmonary systems has been associated with estrogen replacement therapy (ERT). Estrogen and its metabolites have been shown to attenuate cardiac (13, 14, 15, 16, 17) and renal (20, 21) fibrosis by inhibiting fibroblast and mesangial cell proliferation and collagen synthesis. However, there are contradictory reports showing that estrogen can promote fibrogenesis in a model of lung disease (22), suggesting differential effects of the sex steroid in nonreproductive organs. Taken together, these studies suggest that estrogen may modulate cardiac, renal, and pulmonary pathophysiology.
Based on these findings, we hypothesize that female hormones, particularly estrogen, compensate for the loss of relaxin and protect female mice from the resulting fibrotic phenotype observed in male Rln1–/– mice (10, 11, 12). Given the similarities in the effects of relaxin and estrogen on cell proliferation and fibrogenesis, we chose to assess the effects of relaxin and estrogen deficiency in addition to ERT on the progression of cardiac, renal, and airway/lung fibrosis and hypertrophy in sham-operated and ovariectomized female Rln1+/+ and Rln1–/– mice. Our combined findings demonstrate differential and beneficial effects of ERT, depending on the organ it is applied to.
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Ovariectomy and ERT
To investigate the effects of hormonal deficiency on adult female mice, 4-wk-old Rln1+/+ (n = 24) and Rln1–/– (n = 24) animals were bilaterally ovariectomized (23). The mice were then maintained as described above until 9 or 12 months of age (n = 8 mice per genotype and time point). The time points indicated were chosen to match the age at which male Rln1–/– mice developed cardiac (10), renal (12), and pulmonary (11) fibrosis.
To examine the effects of ERT in Rln1+/+ and Rln1–/– mice, a subgroup of ovariectomized animals (n = 8 per genotype) received sc implanted 90-d release pellets of 17ß-estradiol (2.5 mg, equivalent to 27.8 µg/d; Innovative Research of America, Sarasota, FL) from 9 to 12 months of age, based on a therapeutic dose used in a previous study (24). A separate group of age-matched Rln1+/+ and Rln1–/– mice (n = 5 per genotype and time point) underwent sham operations, as controls.
Tissue collection
All mice were weighed and killed at 9 or 12 months of age. The uterus of each animal was carefully dissected out and weighed. Success of ovariectomy was verified by uterine atrophy, whereas success of ERT was confirmed by the reversal of uterine atrophy (by weight). The heart, lungs, and kidneys were rapidly excised, washed in ice-cold PBS, blotted dry, and weighed. The heart was dissected into left ventricles (LVs) and right ventricles (RVs) and atria. The kidney was further separated into the cortex and medulla. Tissues were either snap frozen in liquid nitrogen and stored at –80 C for hydroxyproline analysis, fixed in 4% paraformaldehyde for histological analysis, or placed in Trizol reagent (Invitrogen, Carlsbad, CA) and stored at –80 C for RNA extraction and analysis.
Collagen concentration
Atria, the RV, and a portion of the LV, kidney cortex, kidney medulla, and lung were lyophilized to dry weight and treated as described before (25) to determine their hydroxyproline contents. Hydroxyproline values were converted to collagen content by multiplying by 6.94 (based on hydroxyproline representing approximately 14.4% of the amino acid composition of collagen, in most mammalian tissues) and expressed as a proportion of dry tissue weight (collagen concentration), respectively.
Histology
For histological analysis, paraformaldehyde-fixed tissues (LV, kidney, lung) were paraffin embedded, cut into 5-µm sections, and treated as follows. Sections of the LV were stained with Masson trichrome and the density of collagen (blue staining) quantified with a computerized morphometry system (Optimas Bioscan, Edmonds, WA) (4, 10). Images of the LV were collected, digitized, and the percentage area stained was quantified as described previously (4, 10), using 15–20 fields from each tissue section for analysis. Results were expressed as the average percentage of total area stained per field (collagen content). Sections of kidney tissues were stained with Picrosirius red to examine differences in glomerular and interstitial collagen deposition in each group, as described before (12). Sections were also stained with Masson trichrome for morphometric analysis of airway collagen deposition for each experimental group. Morphometric analysis of collagen thickness was performed on at least five bronchi with a diameter of 150–350 µm from each representative section, as described before (26) using Image Pro-Discovery software (Media Cybernetics, Silver Spring), which was calibrated with a reference micrometer slide. The mean ± SE airway collagen thickness was then determined for each section.
Assessment of cardiac and renal hypertrophy and airway smooth muscle (SM) thickening
Body weight (BW), total heart, ventricular (LV and RV), and atrial weights were measured and expressed as absolute values. Ventricle myocyte cross-sectional area was also measured from the paraformaldehyde-fixed sections. An average of 70–100 cells from randomly selected fields in the LV of each heart was calculated. Total kidney and lung weights were also measured.
For further assessment of organ hypertrophy, the following methods were employed.
Cardiac hypertrophy.
Total RNA was extracted and quantified from the atria using Trizol reagent as described before (4). Total RNA (1 µg) from each sample was used for the RT reaction using a kit from Promega (Annandale, NSW, Australia). The expression pattern of hypertrophy-related genes encoding atrial natriuretic peptide (ANP) and ß-myosin heavy chain (MHC), as well as 
-MHC, was evaluated using SYBR Green real-time quantitative PCR kits (Invitrogen) and specific primers (27) or primers designed from known mouse sequences. All PCRs were carried out with an ABI Prism 7500 system (Applied Biosystems, Foster City, CA). Expression levels of ANP, -MHC, and ß-MHC were normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA. Results were expressed as fold increase over the sham-operated Rln1+/+ group.
Renal hypertrophy.
Morphometric estimation of glomerular volume (Vg) and density were used to estimate renal hypertrophy, as described previously (28). Glomerular profile area (Ag) was measured by point-counting using an ocular grid with 25 µm between each grid at tissue level. The glomerular cross sectional area (Ag) was calculated using the following formula: Ag = (antilog 
log10 Pi/n) x k2, where Pi is the number of points falling on each profile, n is the number of profiles sampled (n = 19–28), and k is the distance between points. The formula Vg = B/k (Ag)3/2, where B = 1.38 is the shape coefficient for spheres and k = 1.1 is a size distribution coefficient, was used to estimate Vg. Tubulointerstitial hypertrophy was examined indirectly by measuring the average density of glomerular profiles in a minimum of three low-power fields (x10) in each group.
Airway SM thickening.
SM thickness was measured by morphometric analysis, as described above for measurement of airway collagen thickness.
Western blot analysis of -SM actin (-SMA)
-SMA expression was used to assess any changes in airway SM accumulation and fibroblast differentiation into myofibroblasts after ovariectomy and ERT from each group of 12-month-old mice (n = 3 samples per group). Total protein was extracted and quantified from the lung samples as described before (6). An equal amount of total protein was then electrophoresed as described before (6). Western blot analysis was performed with a monoclonal antibody to -SMA (M0851; 1:750 dilution; Dako Corp., Carpinteria, CA) and appropriate secondary antibody as described previously (6). Densitometry of the -SMA bands was performed using Bio-Rad GS710 Calibrated Imaging Densitometer and Quantity-One software (Bio-Rad, Hercules, CA).
Statistical analysis
All data were analyzed in GraphPad Prism (version 4) by two-way ANOVA, using a Bonferroni post hoc test, to examine the significance of genotype (Rln1+/+, Rln1–/–), treatment (sham, ovariectomy, ovariectomy + ERT), and the interaction between genotype and treatment. Furthermore, with the 9-month data, a Student’s t test was used to evaluate the significance of ovariectomy within each genotype, and with the 12-month data, a one-way ANOVA, using the Bonferroni post hoc test, was used to evaluate the significance of treatment within each genotype. All data are expressed as the mean + SEM, with P < 0.05 described as statistically significant.
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. Uterine weight was used to demonstrate effectiveness of the ovariectomy. Ovariectomized mice of both genotypes had an atrophied uterus (
50 mg) that was reversed by ERT.
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With respect to the 12-month data, unlike their Rln1+/+ counterparts, Rln1–/– mice experienced a significant gain in BW after ovariectomy compared with that measured from sham-operated Rln1–/– mice and ovariectomized Rln1+/+ mice (both P < 0.05). For this reason, organ weights were given as absolute values rather than as a ratio to BW. Similar to that seen at 9 months, a significant increase in the heart (P < 0.01), LV (P < 0.05), and RV (P < 0.05) weight, but not lung or kidney weight, was observed in ovariectomized Rln1–/– mice, compared with that measured in age-matched ovariectomized Rln1+/+ mice (Table 1). Interestingly, the most striking increase in cardiac chamber weight was observed in the atria of both groups of mice, whereby a 2.5-fold (P < 0.01) and 6.5-fold (P < 0.01) increase in atrial weight was observed in ovariectomized Rln1+/+ and Rln1–/– mice, respectively. ERT was able to significantly decrease (P < 0.05) the BW of Rln1–/– mice to that seen in sham-operated animals. Furthermore, ERT significantly restored (P < 0.01) atrial weights in both groups of mice, as well as LV and RV weights in Rln1–/– mice. ERT, however, significantly increased (by 20%; P < 0.05) the kidney weight of ovariectomized Rln1–/– mice (Table 1). From these findings, an interaction between genotype and treatment was specifically associated with the changes in atrial weight (hypertrophy) observed.
Effects of relaxin and 17ß-estradiol on cardiac collagen deposition and hypertrophy
Biochemical analysis of cardiac tissues from Rln1+/+ and Rln1–/– mice demonstrated no significant differences in collagen content or concentration in the LV (Fig. 1A), RV, and atria (data not shown) among the groups (sham-operated, ovariectomized, or ovariectomized + ERT animals) at either 9 or 12 months of age. These findings were also confirmed by quantitative histological analysis of Masson trichrome-stained cardiac tissue sections (data not shown).
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). However, the relatively mild degree of hypertrophy in the LV was not reflected by significant changes in LV myocyte size (Table 1). We thus performed real-time PCR analysis of hypertrophy-related genes in the atria that had the most marked increase in weight. Atrial levels of ANP were significantly elevated in all ovariectomized mice compared with that in sham-operated mice (by 90% in Rln1+/+ mice and by 170% in Rln1–/– mice; both P < 0.01, Fig. 1B). Similarly, compared with sham-operated mice, ß-MHC expression was increased with ovariectomy (by 3.8-fold in Rln1+/+ mice and 5.6-fold in Rln1–/– mice; both P < 0.05, Fig 1B), whereas ovariectomy decreased -MHC by 80% in Rln1+/+ mice and by 40% in Rln1–/– mice (both P < 0.05, Fig 1B). ERT significantly suppressed the expression of both ANP and ß-MHC in both groups of ovariectomized mice (P < 0.05, Fig 1B). Although levels of -MHC were somewhat restored in both groups of ovariectomized animals with ERT, this was not statistically significant. Furthermore, in accordance with our findings on atrial weight, an interaction between genotype and treatment was specifically associated with changes in the levels of ANP mRNA measured.
Effects of relaxin and 17ß-estradiol on renal collagen deposition and hypertrophy
No detectable changes in collagen concentration were observed in the kidney cortex (Fig. 2A) and medulla (Fig. 2B), between the groups studied at either 9 or 12 months of age. These findings were also confirmed by quantitative histological analysis of Picrosirius red-stained renal tissue sections (data not shown).
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Effects of relaxin and 17ß-estradiol on airway/lung collagen deposition and SM thickening
In contrast to our findings in the heart and kidney, collagen concentration was significantly increased in the lung of sham-operated Rln1–/– mice (by 21%; P < 0.05) and ovariectomized Rln1+/+ mice at 12 months of age (by 17%; P < 0.05), compared with levels measured in age-matched sham-operated Rln1+/+ animals (Fig. 3A). An even greater increase in lung collagen concentration was measured in ovariectomized Rln1–/– mice at 12 months (by 36%; P < 0.05) compared with that measured in ovariectomized Rln1+/+ animals (Fig. 3A). ERT had no marked effect on lung collagen concentration when administered to 12-month-old Rln1+/+ animals but was able to significantly decrease lung collagen concentration (by up to 20%; P < 0.05) in age-matched Rln1–/– mice (Fig. 3A), compared with that measured in the lungs of ovariectomized Rln1–/– animals.
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) showed that the increased collagen in ovariectomized animals was predominantly detected in the subepithelial basement membrane (BM) layer of the airway wall and in the adventitia surrounding the airway structures. Morphometric analysis of the total area of subepithelial collagen deposition in the BM region of lung sections (Fig. 3C) revealed a significant increase in collagen deposition in the BM layer of 12-month-old sham-operated Rln1–/– mice (P < 0.05) and ovariectomized Rln1+/+ mice (P < 0.05), compared with that measured in sham-operated Rln1+/+ animals. A trend toward a further increase in subepithelial collagen deposition was also detected in ovariectomized Rln1–/– mice (by 16%), compared with that measured in sham-treated Rln1–/– mice and ovariectomized Rln1+/+ animals (Fig. 3C). Although having no effect on BM collagen staining in Rln1+/+ mice, ERT significantly decreased BM collagen (by 32%; P < 0.05) in Rln1–/– animals, compared with that measured in ovariectomized Rln1–/– mice (Fig. 3C). These combined findings suggested that airway fibrosis in the lung was susceptible to the influence of relaxin and estrogen and that estrogen was able to partially protect the lung from the progression of disease in the absence of relaxin.
Airway SM thickness was generally increased in the bronchi of ovariectomized Rln1+/+ (by 78%; P < 0.05) and Rln1–/– (by 109%; P < 0.05) mice by 12 months of age, compared with that measured in their respective sham-operated animals (Fig. 3D). ERT did not have any effect on airway SM thickness in ovariectomized Rln1+/+ mice but was able to significantly decrease airway SM thickening in ovariectomized Rln1–/– animals to that measured in sham-operated controls (P < 0.05; Fig. 3D).
Effects of relaxin and 17ß-estradiol on -SMA in the airway/lung
Based on the increased airway fibrosis and airway SM thickening measured in the lung, in the absence of relaxin and estrogen, the effects of ovariectomy and ERT on -SMA (which is expressed by airway SM cells and myofibroblasts) was used to detect changes in SM accumulation and lung fibroblast differentiation. Low levels of -SMA expression were observed in sham-operated animals of both genotypes (Fig. 3E). However, in ovariectomized mice, -SMA was markedly up-regulated by 10- to 11-fold (P < 0.01), compared with that measured in their respective sham-operated animals (Fig. 3E). ERT was able to significantly reduce -SMA expression by approximately 65% (P < 0.01) compared with that measured in ovariectomized mice (Fig. 3E); however, the levels of -SMA in mice given ERT were still significantly higher (P < 0.05) than that measured in their respective sham-operated animals.
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Previous studies have demonstrated that estrogen and its various bioactive metabolites can remodel the matrix and attenuate cardiac fibrosis (13, 14, 15, 16, 17). Likewise, previous studies have demonstrated that estrogen and its various bioactive metabolites can reduce renal collagen synthesis in vitro and ameliorate renal damage in some (20, 21), albeit not all (18), models of renal disease. In our study, however, a dual deficiency of relaxin and estrogen did not lead to the development of cardiac or renal fibrosis. Indeed, findings from this study suggest that estrogens appear to play a less prominent role in the natural history of age-dependent fibrosis. Importantly, in the context of our work, Baylis (33) showed that both intact and ovariectomized female rats were protected from age-related glomerular damage. Consistent with this, ovariectomy and ERT in female Rln1+/+ and Rln1–/– mice had no effect on renal collagen levels, thereby suggesting that estrogen does not compensate for the loss of relaxin and protect the kidney from the progression of fibrosis. These combined findings suggest that other gender-specific factors are more likely to play an essential role in the progression of cardiac and renal fibrosis.
Growing evidence suggests that androgens may significantly contribute to the gender-associated differences in cardiovascular (13, 14) and renal (33, 34) pathology. Indeed, two recent studies demonstrated that gender-related differences in two transgenic murine models of cardiac fibrosis and hypertrophy were diminished after castration in males and increased with chronic testosterone infusion after ovariectomy in females, suggesting that sex differences could be more dependent on prevailing levels of testicular hormones rather than on the cardiac protection by ovarian hormones (35, 36). Similarly, castration of male rats was protective of age-related renal fibrosis, suggesting that androgens, rather than estrogen, are the relevant risk factor (33). These findings suggest that higher levels of androgens may contribute to the fibrosis measured in the heart (10) and kidneys (12) of male Rln1–/– mice, whereas female Rln1–/– mice may be somewhat protected from the progression of disease by lacking such contributing factors. Future studies in our laboratory will investigate the effects of other sex steroids (such as androgens) to address the gender-biased differences in the fibrotic and hypertrophic phenotypes observed in the Rln1–/– model.
Interestingly, airway fibrosis in the lung appeared to be regulated by relaxin and estrogen. Additionally, estrogen appeared to partially protect female Rln1–/– mice from the progression of airway fibrosis (by decreasing lung collagen levels) and compensate for the absence of relaxin. Airway fibrosis in ovariectomized female Rln1–/– mice was equivalent to that measured in male Rln1–/– animals (11) and significantly reversed by ERT, confirming that both relaxin and estrogen play a synergistic role in the progression of airway fibrosis in the ageing female lung. These findings are consistent with the antifibrotic effects of relaxin (9, 26) and estrogen (19, 37, 38, 39) on other models of lung disease and highlight the potential of ERT for the treatment of lung disorders characterized by increase airway remodeling and collagen accumulation. Both hormones have been shown to inhibit airway fibrosis through the inhibition of key profibrotic factors, such as TGF-ß, angiotensin II, and endothelin-1 (9, 38, 39), inhibition of fibroblast proliferation (40), and, in our study, through attenuation of fibroblast differentiation.
Surprisingly, ovariectomy of Rln1–/– mice was associated with a significant although moderate cardiac hypertrophy, which was reflected by significant increases in cardiac chamber weights, compared with that measured in age-matched ovariectomized Rln1+/+ mice. This increase in organ weight was restricted to the heart but not to other organs of 9- and 12-month-old ovariectomized Rln1–/– mice, implying organ-specific effects of relaxin and estrogen deficiency. In 9-month-old Rln1–/– mice, the increased cardiac chamber weight observed was not associated with an increase in BW, whereas in 12-month-old Rln1–/– mice, a 12% increase in BW was observed, which was very likely a novel phenotype resulting from the dual deficiency of relaxin and estrogen. These combined findings suggest that the organ-specific effects of relaxin were independent of body/growth rate. Interestingly, a remarkable increase in atrial weight was observed in ovariectomized Rln1+/+, which was further exaggerated in Rln1–/– mice, highlighting the synergism between relaxin and estrogen in the atria. These findings were complemented by a marked increase in the hypertrophy-related genes, ANP and ß-MHC, in the atria of Rln1+/+ mice and to a significantly greater extent in the atria of Rln1–/– animals, confirming a synergistic and protective action of both relaxin and estrogen in the hypertrophic response observed in the atria.
Airway SM thickening was also observed in ovariectomized Rln1+/+ animals and to a somewhat greater extent in Rln1–/– mice, again confirming the protective effects of relaxin and estrogen in the lung. Unlike our findings in the heart, the changes measured in airway SM thickening did not reflect an additive or synergistic effect of relaxin and estrogen deficiency. As expected, the increase in subepithelial airway SM thickening, which was also associated with an increase in subepithelial airway fibrosis, was not severe enough to induce significant alterations in the lung weight of ovariectomized mice. In both the atria and lung, ERT was able to markedly, although not fully, decrease hypertrophy/thickening to that measured in sham-operated control animals, suggesting that other factors may also play a role in this process.
Multiple pathways are involved in cardiac (41) and airway SM (42) hypertrophy, thus making it possible that the antihypertrophic effects of relaxin and estrogen may be mediated by one or several of these pathways. Recently, it was shown that atrial and LV hypertrophy in spontaneously hypertensive rats was associated with elevated levels of myocardial relaxin mRNA and peptide (43). In vitro studies by our group have also demonstrated that relaxin can blunt cardiomyocyte (CM) hypertrophy evoked by fibroblast-conditioned media (Moore, X. L., and X.-J. Du, unpublished data). From these studies on relaxin and separately on estrogen (44), it has been postulated that both hormones potentially mediate their antihypertrophic effects via the likely inhibition of the MAPK and ERK pathways.
The organ-specific phenotypes observed by the dual deficiency of relaxin and estrogen in addition to ERT may correlate to differences in the expression pattern and/or subtypes of their respective receptors in the heart and lung. High affinity binding sites for relaxin have been well documented in the cardiac atrium (45, 46), consistent with RXFP1 (LGR7) expression in atrial cells (6) and tissues (47). The lung has also emerged as a target organ for relaxin, with RXFP1 expression being identified by RT-PCR (11) and immunostaining in the bronchial epithelium (Royce, S. G., C. S. Samuel, and M. L. K. Tang, unpublished data). Similarly, estrogen receptor (ER) ß is highly expressed in the rodent atria, compared with other chambers of the heart (48) and in the mouse (49) and human (50) lung. Both ERß (51, 52) and ER (53) have been shown to be involved in protecting the rodent heart against cardiac disease. Therefore, a plausible explanation as to why ERT sensitivity was increased in the heart and lung of Rln1–/– mice may be due to alterations in the expression pattern of ER and/or ERß in these organs. This is supported by a previous study (54) that demonstrated an altered expression of ER in the myometrium of Rln1–/– mice. Further studies are required to determine the signaling mechanisms by which relaxin and estrogen promote their protective effects and to determine which of these actions are mediated via separate and synergistic pathways.
In conclusion, this study demonstrates that cardiac hypertrophy, airway fibrosis in the lung and airway SM thickening are susceptible to the dual regulation by relaxin and estrogen, whereby a deficiency of both hormones in female mice resulted in maximal increases in organ fibrosis and SM thickening or hypertrophy. ERT at a therapeutic dose was able to significantly normalize the pathologies of these organs, confirming that estrogen may be able to compensate for the absence of relaxin and does, in fact, protect the heart and lung from the progression of hypertrophy and/or fibrosis. In contrast, neither ovariectomy nor ERT had any significant effects in the kidney of female Rln1–/– mice, suggesting that estrogen does not play a role in renal homeostasis. These novel findings extend our understanding of the biological actions of relaxin and estrogen and may have implications in the increased risk of diseases to these organs after menopause.
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Acknowledgments |
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Footnotes |
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A portion of this work was presented in abstract form at the 88th Annual Meeting of The Endocrine Society, Boston, MA (June 2006).
All authors have nothing to declare.
First Published Online August 24, 2006
Abbreviations: Ag, Glomerular profile area; ANP, atrial natriuretic peptide; BM, basement membrane; BW, body weight; CM, cardiomyocyte; ER, estrogen receptor; ERT, estrogen replacement therapy; LV, left ventricle; MHC, myosin heavy chain; RV, right ventricle; SM, smooth muscle; -SMA, -SM actin; Vg, glomerular volume.
Received April 21, 2006.
Accepted for publication August 14, 2006.
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and ß in human lung tissue and cell lines. Lung Cancer 37:153–159[CrossRef][Medline] agonist 16-LE2 inhibits cardiac hypertrophy and improves hemodynamic function in estrogen-deficient spontaneously hypertensive rats. Cardiovasc Res 67:604–612 expression, but not relaxin receptors (LGR7), in the myometrium of late pregnant relaxin gene knockout mice. Endocrinology 144:4272–4275This article has been cited by other articles:
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  D. S. Hydock, C.-Y. Lien, C. M. Schneider, and R. Hayward Effects of voluntary wheel running on cardiac function and myosin heavy chain in chemically gonadectomized rats Am J Physiol Heart Circ Physiol, December 1, 2007; 293(6): H3254 - H3264. [Abstract] [Full Text] [PDF]
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 M. A. Carey, J. W. Card, J. W. Voltz, D. R. Germolec, K. S. Korach, and D. C. Zeldin The impact of sex and sex hormones on lung physiology and disease: lessons from animal studies Am J Physiol Lung Cell Mol Physiol, August 1, 2007; 293(2): L272 - L278. [Abstract] [Full Text] [PDF]
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