Laura J. Parry

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.

Increased myocardial collagen and ventricular diastolic dysfunction in relaxin deficient mice: a gender-specific phenotype

OBJECTIVE To investigate cardiac phenotypes in mice deficient in the peptide hormone relaxin by gene targeting. METHODS Echocardiography and cardiac catheterization were performed on male and female relaxin deficient (Rlx(-/-)) mice as well as heterozygous (Rlx(+/-)) and wildtype (Rlx(+/+)) littermates aged between 8 and 24 months. Collagen expression and content in the heart were analysed by real-time PCR, hydroxyproline assay and histology. RESULTS Heart rate, blood pressures, left ventricular (LV) dimensions, fractional shortening and maximal and minimal dP/dt did not differ significantly between the three genotypes of either gender at any age. However, 8-10-month-old Rlx(-/-) males exhibited a greater transmitral flow velocity (A-wave) at the late LV diastolic phase. Male Rlx(-/-) mice aged between 12 and 24 months had significantly higher LV end-diastolic pressures, a 30% increase in atrial weight and 10-30% increases in lung and liver weights. Male mice also showed an age-dependent increase (P<0.01) in LV collagen content that was more pronounced in Rlx(-/-) than control littermates (P<0.01). Procollagen type-1 expression was also significantly higher in the LV of Rlx(-/-) males compared with either Rlx(+/-) or Rlx(+/+) males at 6, 9 and 12 months of age. Age-matched female Rlx(-/-) mice did not display any of these cardiac phenotypes seen in Rlx(-/-) males. CONCLUSIONS Male Rlx(-/-) mice had impeded LV diastolic filling and increased atrial weights, most likely due to an increase in ventricular collagen content and chamber stiffness. These phenotypes in the Rlx(-/-) males were not observed in Rlx(-/-) females, indicating the importance of other gender-related factors in cardiovascular function.

Geographic Affinity, Cuticular Hydrocarbons and Colony Recognition in the Australian Meat Ant Iridomyrmex purpureus

Correspondence to: M.L. Thomas The defence of nests from intruders is critical for maintaining the integrity and survival of social insect colonies. Using a sampling method that attempts to minimise genetic influences, we examined whether geographic affinity (the distance between nests) affects non-nestmate recognition. Experimentally staged conflicts between ’chilled’ workers of Iridomyrmex purpureus from different nests revealed that individuals reacted more aggressively towards intruders from nests further away than from neighbouring nests. Further bioassays using filter papers coated with extracted cuticular hydrocarbons revealed a similar pattern. These experiments, which were conducted blind, demonstrate that recognition between conspecific nonnestmates is mediated by these chemicals. However, other cues may also be important since ants responded more aggressively in bioassays using chilled workers than those using only cuticular hydrocarbons. Non-nestmate aggression in I. purpureus was also influenced by the density of surrounding conspecific nests; workers from sites with a higher density of nests tended to be more aggressive than those from less dense sites. These data suggest that past interactions to some extent dictate workers’ reactions to non-nestmate intruders.

Evidence for local relaxin ligand-receptor expression and function in arteries

Relaxin is a 6 kDa protein hormone produced by the corpus luteum and secreted into the blood during pregnancy in rodents and humans. Growing evidence indicates that circulating relaxin causes vasodilatation and increases in arterial compliance, which may be among its most important actions during pregnancy. Here we investigated whether there is local expression and function of relaxin and relaxin receptor in arteries of nonpregnant females and males. Relaxin‐1 and its major receptor, Lgr7, mRNA are expressed in thoracic aortas, small renal and mesenteric arteries from mice and rats of both sexes, as well as in small renal arteries from female tammar wallabies (an Australian marsupial). Using available antibodies for rat and mouse Lgr7 receptor and rat relaxin, we also identified protein expression in arteries. Small renal arteries isolated from relaxin‐1 gene‐deficient mice demonstrate enhanced myogenic reactivity and decreased passive compliance relative to wild‐type (WT) and heterozygous mice. Taken together, these findings reveal an arterial‐derived, relaxin ligand‐receptor system that acts locally to regulate arterial function.—Novak, J., Parry, L. J., Matthews, J. E., Kerchner, L. J., Indovina, K., Hanley‐Yanez, K., Doty, K. D., Debrah, D. O., Shroff, S. G., Conrad, K. P. Evidence for local relaxin ligand‐receptor expression and function in arteries. FASEB J. 20, 2352–2362 (2006)

Strain‐specific genetics, anatomy and function of enteric neural serotonergic pathways in inbred mice

Serotonin (5‐HT) powerfully affects small intestinal motility and 5‐HT‐immunoreactive (IR) neurones are highly conserved between species. 5‐HT synthesis in central neurones and gastrointestinal mucosa depends on tissue‐specific isoforms of the enzyme tryptophan hydroxylase (TPH). RT‐PCR identified strain‐specific expression of a polymorphism (1473C/G) of the tph2 gene in longitudinal muscle–myenteric plexus preparations of C57Bl/6 and Balb/c mice. The former expressed the high‐activity C allele, the latter the low‐activity G allele. Confocal microscopy was used to examine close contacts between 5‐HT‐IR varicosities and myenteric neurones immunoreactive for neuronal nitric oxide synthase (NOS) or calretinin in these two strains. Significantly more close contacts were identified to NOS‐ (P < 0.05) and calretinin‐IR (P < 0.01) neurones in C57Bl/6 jejunum (NOS 1.6 ± 0.3, n= 52; calretinin 5.2 ± 0.4, n= 54), than Balb/c jejunum (NOS 0.9 ± 0.2, n= 78; calretinin 3.5 ± 0.3, n= 98). Propagating contractile complexes (PCCs) were identified in the isolated jejunum by constructing spatiotemporal maps from video recordings of cannulated segments in vitro. These clusters of contractions usually arose towards the anal end and propagated orally. Regular PCCs were initiated at intraluminal pressures of 6 cmH2O, and abolished by tetrodotoxin (1 μm). Jejunal PCCs from C57Bl/6 mice were suppressed by a combination of granisetron (1 μm, 5‐HT3 antagonist) and SB207266 (10 nm, 5‐HT4 antagonist), but PCCs from Balb/c mice were unaffected. There were, however, no strain‐specific differences in sensitivity of longitudinal muscle contractions to exogenous 5‐HT or blockade of 5‐HT3 and 5‐HT4 receptors. These data associate a genetic difference with significant structural and functional consequences for enteric neural serotonergic pathways in the jejunum.

Localization of relaxin receptors in arteries and veins, and region-specific increases in compliance and bradykinin-mediated relaxation after in vivo serelaxin treatment

Relaxin is a potent vasodilator of small resistance arteries and modifies arterial compliance in some systemic vascular beds, yet receptors for relaxin, such as RXFP1, have only been localized to vascular smooth muscle. This study first aimed to localize RXFP1 in rat arteries and veins from different organ beds and determine whether receptors are present in endothelial cells. We then tested the hypothesis that region‐specific vascular effects of relaxin may be influenced by the cellular localization of RXFP1 within different blood vessels. The aorta, vena cava, mesenteric artery, and vein had significantly higher (P<0.05) RXFP1 immunostaining in endothelial cells compared with vascular smooth muscle, whereas the femoral artery and vein and small pulmonary arteries had higher (P<0.01) RXFP1 immunostaining in the vascular smooth muscle. Male rats were treated subcutaneously with recombinant human relaxin‐2 (serelaxin; 4 μg/h) for 5 d; vasodilation and compliance in mesenteric and femoral arteries and veins were compared with placebo controls. Serelaxin significantly (P=0.04) reduced wall stiffness and increased volume compliance in mesenteric arteries but not in the other vessels examined. This was associated with changes in geometrical properties, and not compositional changes in the extracellular matrix. Serelaxin treatment had no effect on acetylcholine‐mediated relaxation but significantly (P< 0.001) enhanced bradykinin (BK)‐mediated relaxation in mesenteric arteries, involving enhanced nitric oxide but not endothelium‐derived hyperpolarization or vasodilatory prostanoids. In conclusion, there is differential distribution of RXFP1 on endothelial and smooth muscle across the vasculature. In rats, mesenteric arteries exhibit the greatest functional response to chronic serelaxin treatment.—Jelinic, M., Leo, C‐H., Post Uiterweer, E. P., Sandow, S. L., Gooi, J. H., Wlodek, M. E., Conrad, K. P., Parkington, H., Tare, M., Parry, L. J. Localization of relaxin receptors in arteries and veins, and region‐specific increases in compliance and bradykinin‐mediated relaxation after in vivo serelaxin treatment. FASEB J. 28, 275–287 (2014). www.fasebj.org

Structure and expression of the rat relaxin-like factor (RLF) gene

The relaxin‐like factor (RLF) is a novel member of the insulin‐IGF‐relaxin family of growth factors and hormones, and its mRNA is expressed very specifically in the Leydig cells of the testis and in the theca and luteal cells of the ovary. Here we report the cloning of the RLF gene and cDNA from the rat. The 0.8kb mRNA is produced from a small gene comprising two exons situated less than 1 kb downstream of the gene for the signalling factor JAK3. Northern hybridization confirms high RLF mRNA expression in the adult rat testis, and low expression in the ovary, but in no other tissues examined. Northern analysis of fetal and neonatal gonadal tissues showed that RLF mRNA is highly upregulated in the testes of day 19 embryos, but not in later neonatal stages, nor in any ovarian tissue from this period. This would indicate that RLF is a marker for the mature fetal as well as the adult‐type Leydig cell, but is not expressed in premature, precursor, or dedifferentiated Leydig cells of either cell type. Finally, RNA was analysed from the testes of rats which had been treated with ethylene dimethane sulfonate (EDS), an alkylating agent that specifically destroys rat Leydig cells. RLF mRNA was absent from the acutely treated testes, but became detectable between 15 and 20 days post‐treatment, concomitant with the repopulation of the testes by new Leydig cells. Continuous testosterone substitution of EDS‐treated rats suppressed the production of gonadotropins, and LH‐dependent Leydig cell differentiation, with the result that RLF mRNA remained undetectable throughout the study period. In conclusion, RLF is a very specific marker for the mature Leydig cell phenotype in both the adult‐type and fetal Leydig cell populations of the rat testis. Mol. Reprod. Dev. 54:319–325, 1999.

Metformin as a prevention and treatment for preeclampsia: effects on soluble fms-like tyrosine kinase 1 and soluble endoglin secretion and endothelial dysfunction

BACKGROUND Preeclampsia is associated with placental ischemia/hypoxia and secretion of soluble fms-like tyrosine kinase 1 and soluble endoglin into the maternal circulation. This causes widespread endothelial dysfunction that manifests clinically as hypertension and multisystem organ injury. Recently, small molecule inhibitors of hypoxic inducible factor 1α have been found to reduce soluble fms-like tyrosine kinase 1 and soluble endoglin secretion. However, their safety profile in pregnancy is unknown. Metformin is safe in pregnancy and is also reported to inhibit hypoxic inducible factor 1α by reducing mitochondrial electron transport chain activity. OBJECTIVE The purposes of this study were to determine (1) the effects of metformin on placental soluble fms-like tyrosine kinase 1 and soluble endoglin secretion, (2) to investigate whether the effects of metformin on soluble fms-like tyrosine kinase 1 and soluble endoglin secretion are regulated through the mitochondrial electron transport chain, and (3) to examine its effects on endothelial dysfunction, maternal blood vessel vasodilation, and angiogenesis. STUDY DESIGN We performed functional (in vitro and ex vivo) experiments using primary human tissues to examine the effects of metformin on soluble fms-like tyrosine kinase 1 and soluble endoglin secretion from placenta, endothelial cells, and placental villous explants. We used succinate, mitochondrial complex II substrate, to examine whether the effects of metformin on soluble fms-like tyrosine kinase 1 and soluble endoglin secretion were mediated through the mitochondria. We also isolated mitochondria from preterm preeclamptic placentas and gestationally matched control subjects and measured mitochondrial electron transport chain activity using kinetic spectrophotometric assays. Endothelial cells or whole maternal vessels were incubated with metformin to determine whether it rescued endothelial dysfunction induced by either tumor necrosis factor-α (to endothelial cells) or placenta villous explant-conditioned media (to whole vessels). Finally, we examined the effects of metformin on angiogenesis on maternal omental vessel explants. RESULTS Metformin reduced soluble fms-like tyrosine kinase 1 and soluble endoglin secretion from primary endothelial cells, villous cytotrophoblast cells, and preterm preeclamptic placental villous explants. The reduction in soluble fms-like tyrosine kinase 1 and soluble endoglin secretion was rescued by coadministration of succinate, which suggests that the effects of metformin on soluble fms-like tyrosine kinase 1 and soluble endoglin were likely to be regulated at the level of the mitochondria. In addition, the mitochondrial electron transport chain inhibitors rotenone and antimycin reduced soluble fms-like tyrosine kinase 1 secretion, which further suggests that soluble fms-like tyrosine kinase 1 secretion is regulated through the mitochondria. Mitochondrial electron transport chain activity in preterm preeclamptic placentas was increased compared with gestation-matched control subjects. Metformin improved features of endothelial dysfunction relevant to preeclampsia. It reduced endothelial cell messenger RNA expression of vascular cell adhesion molecule 1 that was induced by tumor necrosis factor-α (vascular cell adhesion molecule 1 is an inflammatory adhesion molecule up-regulated with endothelial dysfunction and is increased in preeclampsia). Placental conditioned media impaired bradykinin-induced vasodilation; this effect was reversed by metformin. Metformin also improved whole blood vessel angiogenesis impaired by fms-like tyrosine kinase 1. CONCLUSION Metformin reduced soluble fms-like tyrosine kinase 1 and soluble endoglin secretion from primary human tissues, possibly by inhibiting the mitochondrial electron transport chain. The activity of the mitochondrial electron transport chain was increased in preterm preeclamptic placenta. Metformin reduced endothelial dysfunction, enhanced vasodilation in omental arteries, and induced angiogenesis. Metformin has potential to prevent or treat preeclampsia.

Cardio‐renal and metabolic adaptations during pregnancy in female rats born small: implications for maternal health and second generation fetal growth

Non‐technical summary  Low weight at birth, or being born small for gestational age, is associated with increased risk of a number of adult diseases, including cardiovascular and kidney disease and diabetes. Generally, low birth weight males have a greater risk of developing such diseases but females do present with subtle changes in organ structure and function that might render them susceptible to lifestyle challenges. We show, for the first time, that low birth weight females have largely normal cardiovascular and kidney adaptations to pregnancy but they do develop altered glucose control. We have shown that their own fetuses are growth restricted suggesting that low birth weight and risk of disease development can be passed on to subsequent generations. These results warrant close monitoring of pregnant women who were born small and shape future studies to focus on therapeutic strategies to minimize the transmission of low birth weight and adult disease risk.

ESTIMATION OF PREGNANCY RATES AND REPRODUCTIVE FAILURE IN NEW ZEALAND FUR SEALS (ARCTOCEPHALUS FORSTERI)

Abstract Progesterone concentrations after implantation and the observation of live births were used to investigate the reproductive performance and timing of reproductive failure in New Zealand fur seals (Arctocephalus forsteri). Progesterone concentrations in females that gave birth were relatively low during early active gestation (8.6 ± 0.9 ng/ml) and increased significantly toward the end of the 1st trimester (14.9 ± 0.9 ng/ml). In contrast, progesterone concentrations in females that did not give birth remained low. Estimated pregnancy rates and live births varied significantly between years. In 2000, the overall reproductive success of mature females was low (25.7%) and 42.3% of reproductive failure occurred at or before implantation. In 2001 and 2002, reproductive rates were higher (56.5% and 63.5%, respectively) and reproductive failure was greatest (70% and 89.5%, respectively) in mid- to late active gestation. Reproductive failure during active gestation appears to be the most significant stage in determining reproductive success; however, in years of low reproductive success, failure before or at implantation also contributed significantly to reduced reproductive rates. The finding that significant fetal mortality occurs in late gestation reinforces the caution that the use of pregnancy rates, without consideration for the stage of gestation at which measurements were taken, can positively bias estimates of reproductive rates.