Relaxin Deficiency Research Articles

Ocular Phenotype of Relaxin Gene Knockout (Rln-/-) Mice

ABSTRACT Purpose: To test if relaxin deficiency affects ocular structure and function we investigated expression of relaxin (Rln) and RXFP receptors (Rxfp1, Rxfp2), and compared ocular phenotypes in relaxin gene knockout (Rln−/− ) and wild type (Rln+/+ ) mice. Materials and Methods: Rln, Rxfp1 and Rxfp2 mRNA expression was detected in ocular tissues of Rln+/+ mice using RT-PCR. The eyes of 11 Rln−/− and 5 Rln+/+ male mice were investigated. Corneal and retinal thickness was assessed using optical coherence tomography. Intraocular pressure was measured using a rebound tonometer. Retinal, choroidal and sclera morphology and thickness were evaluated histologically. Eyes were collected and fixed for immunofluorescence staining or used for RNA extraction to evaluate mRNA expression using real-time PCR. Results: Rln mRNA was expressed only in the retina, whereas Rxfp1 transcripts were detected in the retina, cornea and sclera/choroid. Rxfp2 was only present in the cornea. None of these genes were expressed in the lacrimal gland, eyelid or lens. Intraocular pressure was higher and central cornea of Rln−/− mice was significantly thicker and had significantly larger endothelial cells and a lower endothelial cell density than Rln+/+ mice. Immunohistochemistry demonstrated no significant difference in AQP3 and AQP5 staining in the cornea or other regions between wildtype and Rln−/− mice. mRNA expression of Aqp4 was significantly higher in Rln−/− than in Rln+/+ corneas, whereas Col1a2, Mmp9, Timp1 and Timp2 were significantly decreased. Expression of Aqp1, Aqp4, Aqp5, Vim and Tjp1 was significantly decreased in Rln−/− compared to Rln+/+ uvea. No significant differences in these genes were detected in the retina. Retinal, choroidal and scleral thicknesses were not different and morphology appeared normal. Conclusion: The findings indicate that loss of Rln affects expression of several genes in the uvea and cornea and results in thicker corneas with altered endothelial cells. Many of the gene changes suggest alterations in extracellular matrix and fluid transport between cells.

Relaxin Deficiency Leads to Uterine Artery Dysfunction During Pregnancy in Mice.

The uterine vasculature undergoes profound adaptations in response to pregnancy. Augmentation of endothelial vasodilator function and reduced smooth muscle reactivity are factors contributing to uterine artery adaptation and are critical for adequate placental perfusion. The peptide hormone relaxin has an important role in mediating the normal maternal renal vascular adaptations during pregnancy through a reduction in myogenic tone and an increase in flow-mediated vasodilation. Little is known however about the influence of endogenous relaxin on the uterine artery during pregnancy. We tested the hypothesis that relaxin deficiency increases myogenic tone and impairs endothelial vasodilator function in uterine arteries of late pregnant relaxin deficient (Rln−/−) mice. Reactivity of main uterine arteries from non-pregnant and late pregnant wild-type (Rln+/+) and Rln−/− mice was studied using pressure and wire myography and changes in gene expression explored using PCR. Myogenic tone was indistinguishable in arteries from non-pregnant mice. In late pregnancy uterine artery myogenic tone was halved in Rln+/+ mice (P < 0.0001), an adaptation that failed to occur in arteries from pregnant Rln−/− mice. The role of vasodilator prostanoids in the regulation of myogenic tone was significantly reduced in arteries of pregnant Rln−/− mice (P = 0.02). Agonist-mediated endothelium-dependent vasodilation was significantly impaired in non-pregnant Rln−/− mice. With pregnancy, differences in total endothelial vasodilator function were resolved, although there remained an underlying deficiency in the role of vasodilator prostanoids and alterations to the contributions of calcium-activated K+ channels. Fetuses of late pregnant Rln−/− mice were ~10% lighter (P < 0.001) than those of Rln+/+ mice. In conclusion, relaxin deficiency is associated with failed suppression of uterine artery myogenic tone in pregnancy, which likely contributes to reduced uteroplacental perfusion and fetal growth restriction.

Abnormal extracellular matrix remodelling in the cervix of pregnant relaxin-deficient mice is not associated with reduced matrix metalloproteinase expression or activity

Relaxin regulates cervical extracellular matrix (ECM) remodelling during pregnancy by modifying collagen and other ECM molecules by unknown mechanisms. We hypothesised that abnormal collagen remodelling in the cervix of pregnant relaxin-deficient (Rln1-/-) mice is due to excessive collagen (Col1a1 and Col3a1) and decreased matrix metalloproteinases (Mmp2, Mmp9, Mmp13 and Mmp7) and oestrogen receptors (Esr1 and Esr2). Quantitative polymerase chain reaction, gelatinase zymography, MMP activity assays and histological staining evaluated changes in ECM in pregnant wildtype (Rln1+/+) and Rln1-/- mice. Cervical Col1a1, Col3a1 and total collagen increased in Rln1-/- mice and were higher at term compared with Rln1+/+ mice. This was not correlated with a decrease in gelatinase (Mmp2, Mmp9) expression or activity, Mmp7 or Mmp13 expression, which were all significantly higher in Rln1-/- mice. In late pregnancy, circulating MMP2 and MMP9 were unchanged. Esr1 expression was highest in Rln1+/+ and Rln1-/- mice in late pregnancy, coinciding with a decrease in Esr2 in Rln1+/+ but not Rln1-/- mice. The relaxin receptor (Rxfp1) decreased slightly in late-pregnant Rln1+/+ mice, but was significantly higher in Rln1-/- mice. In summary, relaxin deficiency results in increased cervical collagen in late pregnancy, which is not explained by a reduction in Mmp expression or activity or decreased Rxfp1. However, an imbalance between Esr1 and Esr2 may be involved.

Relaxin contributes to the regulation of arterial pressure in adult female mice.

Relaxin is increasingly being recognized as a potent vasodilatory and antifibrotic hormone. Given that relaxin is present in the circulation during the luteal phase of the menstrual cycle and during pregnancy, when arterial pressure is lowest in women, relaxin may contribute to the relative cardiovascular protection observed in premenopausal women as compared with age-matched men and postmenopausal women. In the present study, we investigated the contribution of relaxin to the normal regulation of arterial pressure in adult female and male mice and during pregnancy. Mean arterial pressure (MAP) was measured via radiotelemetry in 14-week-old male and female wild-type (WT; C67BL/6xSv129) and relaxin knockout (KO) mice. Thereafter, female mice were time-mated with a (non-telemetered) male of the same genotype and MAP was measured throughout gestation. Basal MAP was ∼10 mmHg lower in WT females than males (P<0.05). Relaxin deficiency increased basal MAP in females (P<0.05 vs WT female), but not males. As expected, MAP decreased during gestation in WT mice. Conversely, in relaxin KO mice, arterial pressure increased during mid and late gestation (P<0.05 as compared with WT). Moreover, relaxin deficiency impaired gestational weight gain and reduced litter size. This is the first study to (i) demonstrate that relaxin contributes to the sexual dimorphism of arterial pressure in mice and (ii) document the changes in the arterial pressure profile of pregnant relaxin KO mice. Understanding the mechanisms that underlie the regulation of arterial pressure in premenopausal females may uncover new strategies to treat hypertension in women (non-pregnant and pregnant) and men.

Relaxin deficiency results in increased expression of angiogenesis- and remodelling-related genes in the uterus of early pregnant mice but does not affect endometrial angiogenesis prior to implantation.

BackgroundExtensive uterine adaptations, including angiogenesis, occur prior to implantation in early pregnancy and are potentially regulated by the peptide hormone relaxin. This was investigated in two studies. First, we took a microarray approach using human endometrial stromal (HES) cells treated with relaxin in vitro to screen for target genes. Then we aimed to investigate whether or not relaxin deficiency in mice affected uterine expression of representative genes associated with angiogenesis and uterine remodeling, and also blood vessel proliferation in the pre-implantation mouse endometrium.MethodsNormal HES cells were isolated and treated with recombinant human relaxin (10 ng/ml) for 24 h before microarray analysis. Reverse transcriptase PCR was used to analyze gene expression of relaxin and its receptor (Rxfp1) in ovaries and uteri; quantitative PCR was used to analyze steroid receptor, angiogenesis and extracellular matrix remodeling genes in the uteri of wild type (Rln+/+) and Rln-/- mice on days 1-4 of pregnancy. Immunohistochemistry localized endometrial endothelial cell proliferation and mass spectrometry measured steroid hormones in the plasma.ResultsMicroarray analysis identified 63 well-characterized genes that were differentially regulated in HES cells after relaxin treatment. Expression of some of these genes was increased in the uterus of Rln+/+ mice by day 4 of pregnancy. There was significantly higher vascular endothelial growth factor A (VegfA), estrogen receptor 1 (Esr1), progesterone receptor (Pgr), Rxfp1, egl-9 family hypoxia-inducible factor 1 (Egln1), hypoxia inducible factor 1 alpha (Hif1α), matrix metalloproteinase 14 (Mmp14) and ankryn repeat domain 37 (Ankrd37) in Rln-/- compared to Rln+/+ mice on day 1. Progesterone receptor expression and plasma progesterone levels were higher in Rln-/- mice compared to Rln+/+ mice. However, endometrial angiogenesis was not advanced as pre-implantation endothelial cell proliferation did not differ between genotypes.ConclusionsRelaxin treatment modulates expression of a variety of angiogenesis-related genes in HES cells. However, despite accelerated uterine gene expression of steroid receptor, progesterone and angiogenesis and extracellular matrix remodeling genes in Rln-/- mice, there was no impact on angiogenesis. We conclude that although relaxin deficiency results in phenotypic changes in the pre-implantation uterus, endogenous relaxin does not play a major role in pre-implantation angiogenesis in the mouse uterus.Electronic supplementary materialThe online version of this article (doi:10.1186/s12958-016-0148-y) contains supplementary material, which is available to authorized users.

Relaxin deficiency attenuates pregnancy-induced adaptation of the mesenteric artery to angiotensin II in mice.

Pregnancy is associated with reduced peripheral vascular resistance, underpinned by changes in endothelial and smooth muscle function. Failure of the maternal vasculature to adapt correctly leads to serious pregnancy complications, such as preeclampsia. The peptide hormone relaxin regulates the maternal renal vasculature during pregnancy; however, little is known about its effects in other vascular beds. This study tested the hypothesis that functional adaptation of the mesenteric and uterine arteries during pregnancy will be compromised in relaxin-deficient (Rln(-/-)) mice. Smooth muscle and endothelial reactivity were examined in small mesenteric and uterine arteries of nonpregnant (estrus) and late-pregnant (day 17.5) wild-type (Rln(+/+)) and Rln(-/-) mice using wire myography. Pregnancy per se was associated with significant reductions in contraction to phenylephrine, endothelin-1, and ANG II in small mesenteric arteries, while sensitivity to endothelin-1 was reduced in uterine arteries of Rln(+/+) mice. The normal pregnancy-associated attenuation of ANG II-mediated vasoconstriction in mesenteric arteries did not occur in Rln(-/-) mice. This adaptive failure was endothelium-independent and did not result from altered expression of ANG II receptors or regulator of G protein signaling 5 (Rgs5) or increases in reactive oxygen species generation. Inhibition of nitric oxide synthase with l-NAME enhanced ANG II-mediated contraction in mesenteric arteries of both genotypes, whereas blockade of prostanoid production with indomethacin only increased ANG II-induced contraction in arteries of pregnant Rln(+/+) mice. In conclusion, relaxin deficiency prevents the normal pregnancy-induced attenuation of ANG II-mediated vasoconstriction in small mesenteric arteries. This is associated with reduced smooth muscle-derived vasodilator prostanoids.

Differential effects of relaxin deficiency on vascular aging in arteries of male mice.

Exogenous treatment with the naturally occurring peptide relaxin increases arterial compliance and reduces vascular stiffness. In contrast, relaxin deficiency reduces the passive compliance of small renal arteries through geometric and compositional vascular remodeling. The role of endogenous relaxin on passive mechanical wall properties in other vascular beds is unknown. Importantly, no studies have investigated the effects of aging in arteries of relaxin-deficient mice. Therefore, we tested the hypothesis that mesenteric and femoral arteries stiffen with aging, and this is exacerbated with relaxin deficiency. Male wild-type (Rln (+/+)) and relaxin knockout (Rln (-/-)) mice were aged to 3, 6, 12, 18, and 23 months. Passive mechanical wall properties were assessed by pressure myography. In both genotypes, there was a significant increase in circumferential stiffening in mesenteric arteries with aging, whereas in the femoral artery, aging reduced volume compliance. This was associated with a reduced ability of the artery to lengthen with aging. The predominant phenotype observed in Rln (-/-) mice was reduced volume compliance in young mice in both mesenteric and femoral arteries. In summary, aging induces circumferential stiffening in mesenteric arteries and axial stiffening in femoral arteries. Passive mechanical wall properties of Rln (-/-) mouse arteries predominantly differ at younger ages compared with Rln (+/+) mice, suggesting that a lack of endogenous relaxin only has a minor effect on vascular aging.

Enhanced Uterine Artery Stiffness in Aged Pregnant Relaxin Mutant Mice Is Reversed with Exogenous Relaxin Treatment1

Pregnancy is associated with a progressive remodeling of the uterine artery. This adaptation is influenced by local and systemic pregnancy-dependent factors. We recently demonstrated that the peptide hormone relaxin mediates uterine artery remodeling in late pregnant rats. The objective of this study in relaxin gene knockout (Rln(-/-)) mice was to test the hypothesis that relaxin deficiency throughout pregnancy disrupts uterine artery remodeling, an effect that is exacerbated by aging and reversed with relaxin treatment. Passive mechanical wall properties and extracellular matrix components were measured using pressure myography, quantitative PCR, and zymography in uterine arteries from pregnant wild-type (Rln(+/+)) and Rln(-/-) mice aged 5 and 8 mo on Days 12.5 and 17.5 pregnancy. In a second study, 8-mo-old Rln(-/-) mice received either placebo or human recombinant relaxin subcutaneously for 5 days from Day 12.5 pregnancy. Relaxin deficiency in pregnancy did not alter uterine artery remodeling in young mice. However, remodeling was impaired in older pregnant Rln(-/-) mice, resulting in significantly stiffer uterine arteries. Uterine arteries of aged Rln(-/-) pregnant mice had increased expression of elastin, whereas several matrix metalloproteinases and cell adhesion molecules were decreased relative to Rln(+/+) mice. Fetal weight was also significantly reduced in Rln(-/-) mice in late pregnancy in both young and old dams, whereas placental weight was unchanged. Arterial stiffness and reduced fetal weight were reversed after relaxin treatment. In conclusion, relaxin deficiency compromises uterine artery remodeling in older pregnant females, increasing the risk of pregnancy complications such as hypertension and intrauterine growth restriction.

221. Localisation of relaxin receptors (Rxfp1) in the uterine artery and the effects of blocking circulating relaxin on passive mechanical wall properties in the uterine artery of late pregnant rats

During pregnancy, the uteroplacental circulation undergoes dramatic alterations to allow for the large increase in blood flow to the feto-placental unit. These alterations are achieved through several mechanisms including structural changes in the uterine artery wall and endothelium-dependent vasodilation. Small renal arteries of relaxin-deficient mice and rats have enhanced myogenic reactivity and decreased passive compliance, and are relatively vasoconstricted (Novak et al. 2001, 2006). To date, no study has identified relaxin receptors (Rxfp1) in arteries or investigated the effects of relaxin deficiency in pregnancy on uterine artery function. The aims of this current study were to: 1) localise Rxfp1 in the uterine arteries, 2) measure myogenic reactivity in small uterine arteries after relaxin treatment, and 3) test the hypothesis that blocking circulating relaxin in late pregnancy will increase uterine artery wall stiffness. We demonstrated that Rxfp1 is expressed in the uterine arteries of pregnant mice and rats. Brightfield immunohistochemistry and immunofluorescence using antibodies specific for rat Rxfp1, α-smooth muscle actin and CD31 localised Rxfp1 protein predominantly to the vascular smooth muscle in the uterine artery of pregnant rats. Administration of recombinant human H2 relaxin (4 ug/h) for 6 h or 5 days in intact and ovariectomised rats reduced myogenic reactivity of small uterine arteries in vitro. Pregnant rats were treated with a monoclonal antibody against circulating relaxin (MCA1) or control (MCAF) for 3 days (Days 17–19) and uterine arteries were mounted on a pressure myograph to assess passive mechanical wall properties. Neutralising circulating relaxin in late pregnancy resulted in a significant increase in uterine artery wall stiffness. These data demonstrate that relaxin acts on the vascular smooth muscle cells in the uterine artery and may be involved in the pregnancy-specific vascular remodelling of uterine arteries to increase vasodilation and blood flow to the uterus and placenta. (1) Novak J et al. (2001). J Clin Invest 107: 1469–75 (2) Novak J et al. (2006). FASEB J 20: 2352–62

Relaxin Plays an Important Role in the Regulation of Airway Structure and Function

Relaxin is a reproductive hormone with pleiotropic actions. In addition to airway fibrosis, relaxin deficiency results in airway structural changes (epithelial thickening) and increased lung recoil, suggesting that relaxin may impact other aspects of airway/lung structure and function beyond its ability to regulate collagen turnover. Furthermore, these structural changes associated with relaxin deficiency show marked similarity to the structural changes seen in asthma. The current study investigated the broader role of relaxin in regulating airway structure and function and examined the relationship between airway inflammation, structural changes, and airway hyperresponsiveness (AHR) using an ovalbumin (OVA)-induced model of allergic airways disease (AAD). The model of AAD was applied to 12-month-old relaxin-deficient (Rln(-/-)) mice with established airway fibrosis and age-matched wild-type (Rln(+/+)) controls. OVA-treated Rln(+/+) mice (induced inflammation) developed increased epithelial thickening (P < 0.05) and AHR (P < 0.05) but not airway fibrosis, compared with saline-treated Rln(+/+) controls. Saline-treated Rln(-/-) mice had significantly increased lung collagen deposition (existing fibrosis) and epithelial thickening and remarkably were found to have increased AHR that was equivalent to that in OVA-treated Rln(+/+) mice (all P < 0.05 vs. saline-treated Rln(+/+) controls). OVA-treated Rln(-/-) mice (existing fibrosis and induced inflammation) had increased airway/lung fibrosis (P < 0.05) but equivalent airway inflammation and AHR compared with OVA-treated Rln(+/+) animals. These findings demonstrate for the first time a role for relaxin in the regulation of airway responses using Rln(-/-) mice and suggest that airway fibrosis and/or epithelial thickening can result in increased AHR equivalent to that induced by airway inflammation in AAD.

The Relaxin Gene Knockout Mouse: A Model of Progressive Scleroderma

Relaxin is a peptide hormone with anti-fibrotic properties. To investigate the long-term effects of relaxin deficiency on the ageing skin, we compared structural changes in the skin of ageing relaxin-deficient (RLX-/-) and normal (RLX+/+) mice, by biochemical, histological, and magnetic resonance imaging analyses. Skin biopsies from RLX+/+ and RLX-/- mice were obtained at different ages and analyzed for changes in collagen expression and distribution. We demonstrated an age-related progression of dermal fibrosis and thickening in male and female RLX-/- mice, associated with marked increases in types I and III collagen. The increased collagen was observed primarily in the dermis of RLX-/- mice by 1 mo of age, and eventually superseded the hypodermal layer. Additionally, fibroblasts from the dermis of RLX-/- mice were shown to produce increased collagen in vitro. Recombinant human gene-2 (H2) relaxin treatment of RLX-/- mice resulted in the complete reversal of dermal fibrosis, when applied to the early onset of disease, but was ineffective when applied to more established stages of dermal scarring. These combined findings demonstrate that relaxin provides a means to regulate excessive collagen deposition in disease states characterized by dermal fibrosis and with our previously published work demonstrate the relaxin-null mouse as a model of progressive scleroderma.

201. Effects of relaxin deficiency on matrix metalloproteinase expression in the cervix and vagina of pregnant mice

The major functions of relaxin are associated with female reproductive physiology, especially the regulation of biochemical processes involved in the remodelling of the reproductive tract at term. Studies in relaxin deficient mice (Rlx–/–) demonstrate that although females give birth to live young without apparent dystocia, they have abnormal cervices and vaginae. This phenotype is attributed to an increase in stromal collagen, but the mechanism(s) by which relaxin regulates extracellular matrix (ECM) production in reproductive tissues is poorly understood. In this study, we assessed the expression of matrix metalloproteinases (MMPs) in the cervix and vagina of pregnant wild-type (Rlx+/+) and Rlx–/– mice. Tissues were obtained from adult C57/Blk6J Rlx+/+ mice on days 7.5, 14.5, 17.5, 18.5 pc and Rlx–/– littermates on days 7.5, 14.5 and 18.5 pc. Real-time PCR using dual-labelled fluorogenic probes was performed in an Opticon 2 cycler (MJ Research) to quantify MMP-2, -3, -7, -9 and -13 gene expression. In the cervix and vagina of Rlx+/+ mice, only MMP-2 mRNA levels were significantly higher at term compared with earlier stages of gestation. There were significant decreases in MMP-7 and -13 expression at term, but no change in MMP-3 and -9. In contrast, MMP-3, -7, -9 and -13 mRNA levels were significantly higher in the cervix and vagina of late pregnant Rlx–/– mice. The expression of MMP-2 did not differ between Rlx+/+ and Rlx–/– mice at term. Despite the higher expression of the majority of MMPs we examined in Rlx–/– mice, there was no histological evidence of increased ECM degradation in the cervix and vagina in late gestation. Although previous in vitro studies suggest that relaxin positively regulates MMP activity, our data demonstrate that relaxin deficiency does not result in decreased MMP expression in the mouse cervix and vagina in vivo.

Relaxin modulates cardiac fibroblast proliferation, differentiation, and collagen production and reverses cardiac fibrosis in vivo.

Cardiac fibrosis is a key component of heart disease and involves the proliferation and differentiation of matrix-producing fibroblasts. The effects of an antifibrotic peptide hormone, relaxin, in inhibiting this process were investigated. We used rat atrial and ventricular fibroblasts, which respond to profibrotic stimuli and express the relaxin receptor (LGR7), in addition to two in vivo models of cardiac fibrosis. Cardiac fibroblasts, when plated at low density or stimulated with TGF-beta or angiotensin II (Ang II), accelerated fibroblast differentiation into myofibroblasts, as demonstrated by significantly increased alpha-smooth muscle actin expression, collagen synthesis, and collagen deposition (by up to 95% with TGF-beta and 40% with Ang II; all P < 0.05). Fibroblast proliferation was significantly increased by 10(-8) m and 10(-7) m Ang II (63-75%; P < 0.01) or 0.1-1 microg/ml IGF-I (27-40%; P < 0.05). Relaxin alone had no marked effect on these parameters, but it significantly inhibited Ang II- and IGF-I-mediated fibroblast proliferation (by 15-50%) and Ang II- and TGF-beta-mediated fibroblast differentiation, as detected by decreased expression of alpha-smooth muscle actin (by 65-88%) and collagen (by 60-80%). Relaxin also increased matrix metalloproteinase-2 expression in the presence of TGF-beta (P < 0.01) and Ang II (P < 0.05). Furthermore, relaxin decreased collagen overexpression when administered to two models of established fibrotic cardiomyopathy, one due to relaxin deficiency (by 40%; P < 0.05) and the other to cardiac-restricted overexpression of beta2-adrenergic receptors (by 58%; P < 0.01). These coherent findings indicate that relaxin regulates fibroblast proliferation, differentiation, and collagen deposition and may have therapeutic potential in diseased states characterized by cardiac fibrosis.

272.Decreased expression of oestrogen receptor β in the reproductive tract of pregnant relaxin-deficient (Rlx - / - ) mice

The peptide hormone relaxin (RLX) is reported to directly affect uterine oestrogen receptors (ERs) in the rat (1). Treatment of immature ovariectomised rats with porcine RLX causes a decrease in uterine ERβ mRNA levels within 6 h. However, RLX has no effect on ERα expression. As both ERβ1 and ERβ2 inhibit ER-mediated transcriptional activity, this RLX-induced downregulation in ERβ could be a prerequisite for oestrogen to exert its effects on target tissues. The aim of the current study was to use relaxin-deficient (Rlx–/–) pregnant mice to investigate if relaxin deficiency results in alterations in either ERβ or ERα mRNA expression in reproductive tissues. Cervix and vagina tissues were obtained from adult C57/Blk6J wild-type mice at five stages of gestation (Days 7.5, 10.5, 14.5, 17.5, 18.5 pc) and Rlx–/– littermates on Days 7.5, 14.5 and 18.5 pc. Q-PCR with TaqMan probes in the Opticon 2 thermal cycler (MJ Research, GeneWorks) was used to quantify ERα and ERα gene expression. ERα mRNA levels were significantly (P &lt; 0.05; ANOVA) increased in the cervix/vagina on Days 17.5 and 18.5 pc in Rlx+/+ mice. The increase in ERα in Rlx+/+ mice was negatively correlated with a significant decrease in ERβ expression from Day 14.5 pc. In contrast, there was no decrease in ERβ expression in the cervix/vagina of Rlx–/– mice; ERβ mRNA levels were significantly (P &lt; 0.05) higher compared to Rlx+/+ mice on Days 14.5 or 18.5 pc. However, there was no corresponding reduction in ERα expression in the cervix/vagina of the Rlx–/– mice, so that ERα mRNA levels were still elevated at term despite the maintenance of high ERα expression. In summary, these data show changes in ERα expression in the cervix/vagina of relaxin-deficient mice, which may subsequently affect ERα-mediated transcriptional activity. (1) Pillai et al. (2002) Biol. Reprod. 67, 1919–1926.

Relaxin deficiency in mice is associated with an age-related progression of pulmonary fibrosis.

Relaxin (RLX) is a peptide hormone with known antifibrotic properties. However, its significance in the lung and its role as a therapeutic agent against diseases characterized by pulmonary fibrosis are yet to be established. In this study, we examined age-related structural and functional changes in the lung of relaxin-deficient mice. Lung tissues of male and female RLX knockout (-/-) and RLX wild-type (+/+) mice at various ages were analyzed for changes in collagen expression and content. We demonstrate an age-related progression of lung fibrosis in RLX -/- mice with significantly increased tissue wet weight, collagen content and concentration, alveolar congestion, and bronchiole epithelium thickening. The increased fibrosis was associated with significantly altered peak expiratory flow and lung recoil (lung function) in RLX -/- mice. Treatment of RLX -/- mice with relaxin in early and developed stages of fibrosis resulted in the reversal of collagen deposition. Organ bath studies showed that precontracted lung strips relaxed in the presence of relaxin. Together, these data indicate that relaxin may provide a means to regulate excessive collagen deposition in diseased states characterized by pulmonary fibrosis.