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Diabetic cardiomyopathy is a distinct form of heart disease that represents a major cause of death and disability in diabetic patients, particularly, the more prevalent type 2 diabetes patient population. In the current study, we investigated whether administration of recombinant adeno-associated viral vectors carrying a constitutively active phosphoinositide 3-kinase (PI3K)(p110α) construct (rAAV6-caPI3K) at a clinically relevant time point attenuates diabetic cardiomyopathy in a preclinical type 2 diabetes (T2D) model. T2D was induced by a combination of a high-fat diet (42% energy intake from lipid) and low-dose streptozotocin (three consecutive intraperitoneal injections of 55 mg/kg body wt), and confirmed by increased body weight, mild hyperglycemia, and impaired glucose tolerance (all P < 0.05 vs. nondiabetic mice). After 18 wk of untreated diabetes, impaired left ventricular (LV) systolic dysfunction was evident, as confirmed by reduced fractional shortening and velocity of circumferential fiber shortening (Vcfc, all P < 0.01 vs. baseline measurement). A single tail vein injection of rAAV6-caPI3K gene therapy (2×1011vector genomes) was then administered. Mice were followed for an additional 8 wk before end point. A single injection of cardiac targeted rAAV6-caPI3K attenuated diabetes-induced cardiac remodeling by limiting cardiac fibrosis (reduced interstitial and perivascular collagen deposition, P < 0.01 vs. T2D mice) and cardiomyocyte hypertrophy (reduced cardiomyocyte size and Nppa gene expression, P < 0.001 and P < 0.05 vs. T2D mice, respectively). The diabetes-induced LV systolic dysfunction was reversed with rAAV6-caPI3K, as demonstrated by improved fractional shortening and velocity of circumferential fiber shortening (all P < 0.05 vs pre-AAV measurement). This cardioprotection occurred in combination with reduced LV reactive oxygen species (P < 0.05 vs. T2D mice) and an associated decrease in markers of endoplasmic reticulum stress (reduced Grp94 and Chop, all P < 0.05 vs. T2D mice). Together, our findings demonstrate that a cardiac-selective increase in PI3K(p110α), via rAAV6-caPI3K, attenuates T2D-induced diabetic cardiomyopathy, providing proof of concept for potential translation to the clinic.NEW & NOTEWORTHY Diabetes remains a major cause of death and disability worldwide (and its resultant heart failure burden), despite current care. The lack of existing management of heart failure in the context of the poorer prognosis of concomitant diabetes represents an unmet clinical need. In the present study, we now demonstrate that delayed intervention with PI3K gene therapy (rAAV6-caPI3K), administered as a single dose in mice with preexisting type 2 diabetes, attenuates several characteristics of diabetic cardiomyopathy, including diabetes-induced impairments in cardiac remodeling, oxidative stress, and function. Our discovery here contributes to the previous body of work, suggesting the cardioprotective effects of PI3K(p110α) could be a novel therapeutic approach to reduce the progression to heart failure and death in diabetes-affected patients.
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Classe I de Fosfatidilinositol 3-Quinases/genética , Diabetes Mellitus Tipo 2/complicações , Cardiomiopatias Diabéticas/terapia , Terapia Genética/métodos , Animais , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Dependovirus/genética , Dependovirus/metabolismo , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Tipo 2/etiologia , Cardiomiopatias Diabéticas/etiologia , Cardiomiopatias Diabéticas/patologia , Dieta Hiperlipídica/efeitos adversos , Estresse do Retículo Endoplasmático , Fibrose , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Masculino , Camundongos , Miocárdio/metabolismo , Espécies Reativas de Oxigênio , Remodelação VentricularRESUMO
The formyl peptide receptor (FPR) family are a group of G-protein coupled receptors that play an important role in the regulation of inflammatory processes. It is well-established that activation of FPRs can have cardioprotective properties. Recently, more stable small-molecule FPR1/2 agonists have been described, including both Compound 17b (Cmpd17b) and Compound 43 (Cmpd43). Both agonists activate a range of signals downstream of FPR1/2 activation in human-engineered FPR-expressing cells, including ERK1/2 and Akt. Importantly, Cmpd17b (but not Cmpd43) favours bias away from intracellular Ca2+ mobilisation in this context, which has been associated with greater cardioprotection in response to Cmpd17b over Cmpd43. However, it is unknown whether these FPR agonists impact vascular physiology and/or elicit vasoprotective effects in the context of diabetes. First, we localized FPR1 and FPR2 receptors predominantly in vascular smooth muscle cells in the aortae of male C57BL/6 mice. We then analysed the vascular effects of Cmpd17b and Cmpd43 on the aorta using wire-myography. Cmpd17b but not Cmpd43 evoked a concentration-dependent relaxation of the mouse aorta. Removal of the endothelium or blockade of endothelium-derived relaxing factors using pharmacological inhibitors had no effect on Cmpd17b-evoked relaxation, demonstrating that its direct vasodilator actions were endothelium-independent. In aortae primed with elevated K+ concentration, increasing concentrations of CaCl2 evoked concentration-dependent contraction that is abolished by Cmpd17b, suggesting the involvement of the inhibition of Ca2+ mobilisation via voltage-gated calcium channels. Treatment with Cmpd17b for eight weeks reversed endothelial dysfunction in STZ-induced diabetic aorta through the upregulation of vasodilator prostanoids. Our data indicate that Cmpd17b is a direct endothelium-independent vasodilator, and a vasoprotective molecule in the context of diabetes.
Assuntos
Anexina A1/metabolismo , Diabetes Mellitus Experimental/tratamento farmacológico , Substâncias Protetoras/uso terapêutico , Bibliotecas de Moléculas Pequenas/uso terapêutico , Animais , Aorta/metabolismo , Glicemia/metabolismo , Diabetes Mellitus Experimental/sangue , Masculino , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Substâncias Protetoras/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Formil Peptídeo/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Estreptozocina , Vasodilatadores/farmacologiaRESUMO
Early maternal vascular adaptations to pregnancy are predominantly driven by changes in vascular tone, reactivity, and remodeling. Failure of the maternal systemic vasculature to adapt sufficiently can lead to serious complications of pregnancy. The hormone relaxin is widely recognized for its contribution to the essential renal and systemic hemodynamic adaptations in early pregnancy through direct actions on the maternal vasculature. Studies in relaxin gene knockout mice revealed that endogenous relaxin is not only a "pregnancy hormone" but has pleiotropic actions in various tissues in males and non-pregnant females. There is strong interest in relaxin's actions in the vasculature and its utility in the treatment of vascular diseases. Relaxin treatment in rodents for 2-5 days or acute intravenous injection enhances endothelium-dependent relaxation and decreases myogenic tone in resistance arteries. These vascular actions are prolonged, even in the absence of circulating relaxin, and are underpinned by the production of endothelium-derived relaxing factors including nitric oxide, endothelium-derived hyperpolarization, and prostacyclin. Relaxin is also capable of remodeling the vascular wall in a variety of blood vessels in disease conditions. Lessons learned in pregnancy research have aided studies investigating the potential therapeutic potential of relaxin in cardiovascular disease.
Assuntos
Gravidez , Relaxina/fisiologia , Animais , Vasos Sanguíneos/efeitos dos fármacos , Doenças Cardiovasculares/tratamento farmacológico , Feminino , Hemodinâmica/efeitos dos fármacos , Humanos , Masculino , Relaxina/deficiência , Relaxina/uso terapêutico , Vasodilatação/efeitos dos fármacosRESUMO
Preeclampsia is a pregnancy-specific disorder, primarily characterized by new-onset hypertension in combination with a variety of other maternal or fetal signs. The pathophysiological mechanisms underlying the disease are still not entirely clear. Systemic maternal vascular dysfunction underlies the clinical features of preeclampsia. It is a result of oxidative stress and the actions of excessive anti-angiogenic factors, such as soluble fms-like tyrosine kinase, soluble endoglin, and activin A, released by a dysfunctional placenta. The vascular dysfunction then leads to impaired regulation and secretion of relaxation factors and an increase in sensitivity/production of constrictors. This results in a more constricted vasculature rather than the relaxed vasodilated state associated with normal pregnancy. Currently, the only effective "treatment" for preeclampsia is delivery of the placenta and therefore the baby. Often, this means a preterm delivery to save the life of the mother, with all the attendant risks and burdens associated with fetal prematurity. To lessen this burden, there is a pressing need for more effective treatments that target the maternal vascular dysfunction that underlies the hypertension. This review details the vascular effects of key drugs undergoing clinical assessment as potential treatments for women with preeclampsia.
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STUDY QUESTION: What is the association between placental formyl peptide receptor 2 (FPR2) and trophoblast and endothelial functions in pregnancies affected by foetal growth restriction (FGR)? SUMMARY ANSWER: Reduced FPR2 placental expression in idiopathic FGR results in significantly altered trophoblast differentiation and endothelial function in vitro. WHAT IS KNOWN ALREADY: FGR is associated with placental insufficiency, where defective trophoblast and endothelial functions contribute to reduced feto-placental growth. STUDY DESIGN, SIZE, DURATION: The expression of FPR2 in placental tissues from human pregnancies complicated with FGR was compared to that in gestation-matched uncomplicated control pregnancies (n = 25 from each group). Fpr2 expression was also determined in placental tissues obtained from a murine model of FGR (n = 4). The functional role of FPR2 in primary trophoblasts and endothelial cells in vitro was assessed in diverse assays in a time-dependent manner. PARTICIPANTS/MATERIALS, SETTING, METHODS: Placentae from third-trimester pregnancies complicated by idiopathic FGR (n = 25) and those from gestation-matched pregnancies with appropriately grown infants as controls (n = 25) were collected at gestation 27-40 weeks. Placental tissues were also collected from a spontaneous CBA/CaH × DBA/2 J murine model of FGR. Placental FPR2/Fpr2 mRNA expression was determined by real-time PCR, while protein expression was examined by immunoblotting and immunohistochemistry. siRNA transfection was used to silence FPR2 expression in primary trophoblasts and in human umbilical vein endothelial cells (HUVEC), and the quantitation of cytokines, chemokines and apoptosis was performed following a cDNA array analyses. Functional effects of trophoblast differentiation were measured using HCGB/ß-hCG and syncytin-2 expression as well as markers of apoptosis, tumour protein 53 (TP53), caspase 8, B cell lymphoma 2 (BCL2) and BCL associated X (BAX). Endothelial function was assessed by proliferation, network formation and permeability assays. MAIN RESULTS AND THE ROLE OF CHANCE: Placental FPR2/Fpr2 expression was significantly decreased in FGR placentae (n = 25, P < 0.05) as well as in murine FGR placentae compared to controls (n = 4, P < 0.05). FPR2 siRNA (siFPR2) in term trophoblasts significantly increased differentiation markers, HCGB and syncytin-2; cytokines, interleukin (IL)-6, CXCL8; and apoptotic markers, TP53, caspase 8 and BAX, but significantly reduced the expression of the chemokines CXCL12 and its receptors CXCR4 and CXCR7; CXCL16 and its receptor, CXCR6; and cytokine, IL-10, compared with control siRNA (siCONT). Treatment of HUVECs with siFPR2 significantly reduced proliferation and endothelial tube formation, but significantly increased permeability of HUVECs. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Reduced expression of placental FPR2/Fpr2 was observed in the third trimester at delivery after development of FGR, suggesting that FPR2 is associated with FGR pregnancies. However, there is a possibility that the decreased placental FPR2 observed in FGR may be a consequence rather than a cause of FGR, although our in vitro functional analyses using primary trophoblasts and endothelial cells suggest that FPR2 may have a direct or indirect regulatory role on trophoblast differentiation and endothelial function in FGR. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study linking placental FPR2 expression with changes in the trophoblast and endothelial functions that may explain the placental insufficiency observed in FGR. STUDY FUNDING/COMPETING INTERESTS: P.M. and P.R.E. received funding from the Australian Institute of Musculoskeletal Science, Western Health, St. Albans, Victoria 3021, Australia. M.L. is supported by a Career Development Fellowship from the National Health and Medical Research Council (NHMRC; Grant no. 1047025). Monash Health is supported by the Victorian Government's Operational Infrastructure Support Programme. The authors declare that there is no conflict of interest in publishing this work.
Assuntos
Retardo do Crescimento Fetal/metabolismo , Placenta/metabolismo , Receptores de Formil Peptídeo/metabolismo , Receptores de Lipoxinas/metabolismo , Apoptose/genética , Apoptose/fisiologia , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Feminino , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Gravidez , Primeiro Trimestre da Gravidez , Terceiro Trimestre da Gravidez , Receptores de Formil Peptídeo/genética , Receptores de Lipoxinas/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Trofoblastos/citologia , Trofoblastos/metabolismoRESUMO
The peptide hormone relaxin has numerous roles both within and independent of pregnancy and is often thought of as a "pleiotropic hormone." Relaxin targets several tissues throughout the body, and has many functions associated with extracellular matrix remodeling and the vasculature. This review considers the potential therapeutic applications of relaxin in cervical ripening, in vitro fertilization, preeclampsia, acute heart failure, ischemia-reperfusion, and cirrhosis. We first outline the animal models used in preclinical studies to progress relaxin into clinical trials and then discuss the findings from these studies. In many cases, the positive outcomes from preclinical animal studies were not replicated in human clinical trials. Therefore, the focus of this review is to evaluate the various animal models used to develop relaxin as a potential therapeutic and consider the limitations that must be addressed in future studies. These include the use of human relaxin in animals, duration of relaxin treatment, and the appropriateness of the clinical conditions being considered for relaxin therapy.
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Relaxina/farmacologia , Relaxina/fisiologia , Animais , Modelos Animais de Doenças , Feminino , Insuficiência Cardíaca/tratamento farmacológico , Humanos , Hepatopatias/tratamento farmacológico , Gravidez , Relaxina/uso terapêutico , Traumatismo por Reperfusão/tratamento farmacológicoRESUMO
Preeclampsia affects up to 8% of pregnancies worldwide and is a leading cause of both maternal and fetal morbidity and mortality. Our current understanding of the cause(s) of preeclampsia is far from complete, and the lack of a single reliable animal model that recapitulates all aspects of the disease further confounds our understanding. This is partially due to the heterogeneous nature of the disease, coupled with our evolving understanding of its etiology. Nevertheless, animal models are still highly relevant and useful tools that help us better understand the pathophysiology of specific aspects of preeclampsia. This review summarizes the various types and characteristics of animal models used to study preeclampsia, highlighting particular features of these models relevant to clinical translation. This review points out the strengths and limitations of these models to illustrate the importance of using the appropriate model depending on the research question.
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Pressão Sanguínea , Pré-Eclâmpsia/fisiopatologia , Pesquisa Translacional Biomédica/métodos , Animais , Biomarcadores/sangue , Modelos Animais de Doenças , Feminino , Humanos , Circulação Placentária , Pré-Eclâmpsia/sangue , Pré-Eclâmpsia/etiologia , Gravidez , Fatores de Risco , Transdução de Sinais , Especificidade da EspécieRESUMO
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.
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Colo do Útero/metabolismo , Matriz Extracelular/metabolismo , Metaloproteinases da Matriz/metabolismo , Relaxina/genética , Animais , Feminino , Gelatinases/genética , Gelatinases/metabolismo , Metaloproteinases da Matriz/genética , Camundongos , Camundongos Knockout , Gravidez , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Peptídeos/genética , Receptores de Peptídeos/metabolismo , Relaxina/metabolismoRESUMO
A correlation exists between the extent of pericardial adipose and atrial fibrillation (AF) risk, though the underlying mechanisms remain unclear. Selected adipose depots express high levels of aromatase, capable of converting androgens to estrogens - no studies have investigated aromatase occurrence/expression regulation in pericardial adipose. The Women's Health Initiative reported that estrogen-only therapy in women elevated AF incidence, indicating augmented estrogenic influence may exacerbate cardiac vulnerability. The aim of this study was to identify the occurrence of pericardial adipose aromatase, evaluate the age- and sex-dependency of local cardiac steroid synthesis capacity and seek preliminary experimental evidence of a link between pericardial adipose aromatase capacity and arrhythmogenic vulnerability. Both human atrial appendage and epicardial adipose exhibited immunoblot aromatase expression. In rodents, myocardium and pericardial adipose aromatase expression increased >20-fold relative to young controls. Comparing young, aged and aged-high fat diet animals, a significant positive correlation was determined between the total aromatase content of pericardial adipose and the occurrence/duration of triggered atrial arrhythmias. Incidence and duration of arrhythmias were increased in hearts perfused with 17ß-estradiol. This study provides novel report of pericardial adipose aromatase expression. We show that aromatase expression is remarkably upregulated with aging, and aromatase estrogen conversion capacity significantly elevated with obesity-related cardiac adiposity. Our studies suggest an association between adiposity, aromatase estrogenic capacity and atrial arrhythmogenicity - additional investigation is required to establish causality. The potential impact of these findings may be considerable, and suggests that focus on local cardiac steroid conversion (rather than systemic levels) may yield translational outcomes.
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Tecido Adiposo/metabolismo , Envelhecimento/patologia , Aromatase/metabolismo , Arritmias Cardíacas/terapia , Obesidade/terapia , Pericárdio/patologia , Pesquisa Translacional Biomédica , Animais , Arritmias Cardíacas/enzimologia , Arritmias Cardíacas/patologia , Estradiol/farmacologia , Estrogênios/biossíntese , Feminino , Átrios do Coração/efeitos dos fármacos , Átrios do Coração/patologia , Humanos , Masculino , Camundongos , Obesidade/enzimologia , Obesidade/patologia , RatosRESUMO
BACKGROUND: Short-term IV sRLX (recombinant human relaxin-2) infusion enhances endothelium-dependent relaxation in mesenteric arteries. This is initially underpinned by increased NO followed by a transition to prostacyclin. The effects of short-term IV sRLX treatment on pressure-induced myogenic tone and vascular remodeling in these arteries are unknown. Therefore, we investigated the effects of sRLX infusion on pressure-induced myogenic tone and passive mechanical wall properties in mesenteric arteries. METHODS: Mesenteric artery myogenic tone and passive mechanics were examined after 48-hours and 10-days infusion of sRLX. Potential mechanisms of action were assessed by pressure myography, qPCR, and Western blot analysis. RESULTS: Neither 48-hours nor 10-days sRLX treatment had significant effects on myogenic tone, passive arterial wall stiffness, volume compliance, or axial lengthening. However, in 48-hours sRLX -treated rats, incubation with the NO synthase blocker L-NAME significantly increased myogenic tone (P<.05 vs placebo), demonstrating an increased contribution of NO to the regulation of myogenic tone. eNOS dimerization, but not phosphorylation, was significantly upregulated in the arteries of sRLX -treated rats. CONCLUSION: In mesenteric arteries, 48-hours sRLX treatment upregulates the role of NO in the regulation of myogenic tone by enhancing eNOS dimerization, without altering overall myogenic tone or vascular remodeling.
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Artérias Mesentéricas/efeitos dos fármacos , Tono Muscular/efeitos dos fármacos , Relaxina/farmacologia , Remodelação Vascular/efeitos dos fármacos , Animais , Óxido Nítrico/fisiologia , Óxido Nítrico Sintase Tipo III/metabolismo , Multimerização Proteica , Ratos , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/farmacologia , Relaxina/administração & dosagem , Fatores de TempoRESUMO
The peptide relaxin has gained considerable attention as a new vasoactive drug, largely through its beneficial therapeutic effects in cardiovascular disease. In this study, we tested the hypothesis that relaxin treatment alleviates systemic vascular dysfunction characteristic of hypertensive diseases of pregnancy. We investigated vascular effects and mechanisms of relaxin action in (i) pregnant relaxin-deficient (Rln-/-) mice with enhanced responses to angiotensin II (AngII) and (ii) arteries pre-incubated ex vivo in trophoblast conditioned media (TCM) to induce endothelial dysfunction. Pregnant Rln-/- mice received 0.5 µg/h recombinant human H2 relaxin (rhRLX: n = 5) or placebo (20 nM sodium acetate; n = 7) subcutaneously via osmotic minipumps for 5 days prior to gestational day 17.5. This treatment protocol significantly reduced AngII-mediated contraction of mesenteric arteries and increased plasma 6-keto prostaglandin F1α. These vascular effects were endothelium independent and likely involve smooth muscle-derived vasodilator prostanoids. In the second study, mesenteric arteries were incubated ex vivo for 24 h at 37°C in TCM, which contained high levels of soluble Flt-1 (>20 ng/ml) and soluble Eng (>1 ng/ml). TCM incubation caused significant reduction in endothelium-dependent relaxation and increased sensitivity to AngII. Co-incubation of arteries with rhRLX for 24 h (n = 6-16/treatment) prevented endothelial dysfunction but had no effect on AngII-mediated contraction. In conclusion, relaxin treatment prevents and/or reverses vascular dysfunction in mesenteric arteries, but acts through different vascular pathways depending on duration of relaxin treatment and type of vascular dysfunction. Overall, our data suggest that relaxin is a potential therapeutic to alleviate maternal systemic vascular dysfunction associated with hypertensive diseases in pregnant women.
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Angiotensina II/farmacologia , Endotélio Vascular/efeitos dos fármacos , Relaxina/farmacologia , Vasoconstrição/fisiologia , Animais , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Camundongos Knockout , GravidezRESUMO
Pre-eclampsia (PE) is a leading cause of maternal and fetal death, characterised by an imbalance of placental growth factors and hypertension at >20 weeks gestation. Impaired maternal systemic vascular adaptations and fetal growth restriction are features of both PE and pregnant relaxin-deficient (Rln-/-) mice. The aim of the present study was to investigate whether these phenotypes in Rln-/- mice are associated with abnormal placental growth factor expression, increased soluble fms-like tyrosine kinase-1 (sFlt-1), proteinuria and/or hypertension during pregnancy. In addition, we examined relaxin and relaxin receptor (relaxin/insulin like family peptide receptor 1 (RXFP1)) mRNA expression in placentas of women with PE. There was no significant difference in placental vascular endothelial growth factor A (VegfA) and placenta growth factor (Plgf) gene expression between Rln-/- and wild-type mice. Circulating plasma sFlt-1 concentrations in pregnant mice of both genotypes and ages were increased compared with non-pregnant mice but were lower in younger pregnant Rln-/- mice compared with aged-matched Rln+/+ mice. Aged pregnant Rln-/- mice had higher urinary albumin:creatinine ratios compared with age-matched Rln+/+ mice, indicative of proteinuria. Systolic and diastolic blood pressures did not differ between genotypes. In addition, PE in women was not associated with altered placental mRNA expression of RLN2 or RXFP1 at term. Overall, the data demonstrate that pregnant Rln-/- mice do not have the typical characteristics of PE. However, these mice show evidence of proteinuria, but we suggest that this results from systemic renal vascular dysfunction before pregnancy.
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Placenta/metabolismo , Pré-Eclâmpsia/metabolismo , Proteinúria/metabolismo , Relaxina/metabolismo , Animais , Pressão Sanguínea/fisiologia , Feminino , Humanos , Camundongos , Camundongos Knockout , Fator de Crescimento Placentário/genética , Fator de Crescimento Placentário/metabolismo , Pré-Eclâmpsia/genética , Gravidez , Proteinúria/genética , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Peptídeos/genética , Receptores de Peptídeos/metabolismo , Relaxina/genética , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The peptide hormone relaxin is recognized for its connective tissue remodeling actions in the reproductive tract during pregnancy and parturition, but it also has vascular remodeling actions independent of pregnancy. Recombinant human relaxin (serelaxin) treatment in male and non-pregnant female rodents enhances passive arterial compliance in the renal vasculature. This review focuses on serelaxin's actions on passive mechanical wall properties in small arteries and highlights the diversity of responses to serelaxin treatment in rodents. Different experimental approaches (duration of serelaxin treatment, rat strain, age) and animal models of disease (obesity, hypertension) will be considered. Most studies in young rodents demonstrate that serelaxin treatment fails to alter passive compliance in resistance-size arteries (mesenteric and femoral arteries and cerebral parenchymal arterioles), suggesting that serelaxin's beneficial effects are minimal in healthy animals. Short-term serelaxin treatment (5d) in aged, obese, and spontaneously hypertensive rats (SHRs) is largely without effect on passive mechanical wall properties. However, a longer duration of serelaxin treatment in SHRs (14d) enhances passive compliance in large muscular arteries as well as resistance-size arteries. In conclusion, serelaxin is capable of vascular remodeling. Its actions are vascular bed-dependent, more prominent in disease, and likely requires a longer duration of treatment to be effective.
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Artérias/efeitos dos fármacos , Complacência (Medida de Distensibilidade)/efeitos dos fármacos , Relaxina/uso terapêutico , Animais , Artérias/fisiologia , Fenômenos Biomecânicos/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Rim/irrigação sanguínea , Ratos , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Relaxina/farmacologia , Projetos de Pesquisa , Fatores de TempoRESUMO
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.
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Adaptação Fisiológica/fisiologia , Angiotensina II/metabolismo , Artérias Mesentéricas/fisiologia , Prenhez , Relaxina/metabolismo , Animais , Feminino , Regulação da Expressão Gênica/fisiologia , Camundongos , Camundongos Knockout , Gravidez , Prenhez/fisiologia , Receptores de Angiotensina/fisiologia , Relaxina/genética , Artéria Uterina/fisiologia , Vasodilatação/fisiologiaRESUMO
BACKGROUND: Extensive 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. METHODS: Normal 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. RESULTS: Microarray 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. CONCLUSIONS: Relaxin 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.
Assuntos
Neovascularização Fisiológica/genética , Relaxina/fisiologia , Animais , Proliferação de Células , Endométrio/citologia , Endométrio/metabolismo , Feminino , Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Fisiológica/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Gravidez , Relaxina/farmacologia , Células Estromais , Útero/citologia , Útero/metabolismoRESUMO
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.
Assuntos
Antígenos CD/metabolismo , Fármacos Cardiovasculares/uso terapêutico , Endotélio Vascular/efeitos dos fármacos , Metformina/uso terapêutico , Pré-Eclâmpsia/tratamento farmacológico , Pré-Eclâmpsia/prevenção & controle , Receptores de Superfície Celular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Biomarcadores/metabolismo , Fármacos Cardiovasculares/farmacologia , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Endoglina , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiopatologia , Feminino , Humanos , Técnicas In Vitro , Metformina/farmacologia , Placenta/efeitos dos fármacos , Placenta/metabolismo , Pré-Eclâmpsia/metabolismo , Pré-Eclâmpsia/fisiopatologia , Gravidez , Resultado do Tratamento , Vasodilatação/efeitos dos fármacosRESUMO
Diabetes-induced endothelial dysfunction is a critical initiating factor in the development of cardiovascular complications. Treatment with relaxin improves tumour necrosis factor α-induced endothelial dysfunction by enhancing endothelial nitric oxide synthase (eNOS) activity and restoring superoxide dismutase 1 protein in rat aortic rings ex vivo. It is, therefore, possible that relaxin treatment could alleviate endothelial dysfunction in diabetes. This study aimed to test the hypothesis that serelaxin (recombinant human relaxin-2) prevents high glucose-induced vascular dysfunction in the mouse aorta. Abdominal aortae were isolated from C57BL/6 male mice and incubated in M199 media for 3days with either normal glucose (5.5mM) or high glucose (30mM), and co-incubated with placebo (20mM sodium acetate) or 10nM serelaxin at 37°C in 5% CO2. Vascular function was analysed using wire-myography. High glucose significantly reduced the sensitivity to the endothelium-dependent agonist, acetylcholine (ACh) (pEC50; normal glucose=7.66±0.10 vs high glucose=7.29±0.10, n=11-12, P<0.05) and the contraction induced by NOS inhibitor, L-NAME (200µM) (normal glucose=59.9±8.3% vs high glucose=38.7±4.3%, n=6, P<0.05), but had no effect on the endothelium-independent agonist, sodium nitroprusside (SNP)-mediated relaxation. Treatment with serelaxin restored endothelial function (pEC50; 7.83±0.11, n=11) but not NO availability. The presence of the cyclooxygenase (COX) inhibitor, indomethacin (1µM) (pEC50; control=7.29±0.10 vs indo=7.74±0.18, n=6-12, P<0.05) and a superoxide dismutase mimetic, tempol (10µM) (pEC50; control=7.29±0.10 vs tempol=7.82±0.05, n=6-12, P<0.01) significantly improved sensitivity to ACh in high glucose treated aortae, but had no effect in serelaxin treated aortae. This suggests that high glucose incubation alters the superoxide and COX-sensitive pathway, which was normalized by co-incubation with serelaxin. Neither high glucose incubation nor serelaxin treatment had an effect on cyclooxygenase 1 and 2 (Ptgs1, Ptgs2), prostacyclin synthase (PTGIS) and receptor (Ptgir) as well as thromboxane A2 receptor (Tbxa2r) mRNA expression. Importantly, production of prostacyclin was significantly (P<0.05) attenuated in high glucose treated aortae, which was prevented by serelaxin treatment. Our data show that serelaxin treatment for 3 days restores high glucose-induced endothelial dysfunction by ameliorating vasodilator prostacyclin production and possibly through the reduction of superoxide in the mouse aorta.
Assuntos
Aorta Abdominal/efeitos dos fármacos , Aorta Torácica/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Epoprostenol/metabolismo , Relaxina/farmacologia , Acetilcolina/farmacologia , Animais , Aorta Abdominal/fisiologia , Aorta Torácica/fisiologia , Óxidos N-Cíclicos/farmacologia , Inibidores de Ciclo-Oxigenase/farmacologia , Glucose/farmacologia , Humanos , Técnicas In Vitro , Indometacina/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nitroprussiato/farmacologia , Proteínas Recombinantes/farmacologia , Marcadores de Spin , Vasodilatadores/farmacologiaRESUMO
Increased vascular stiffness and reduced endothelial nitric oxide (NO) bioavailability are characteristic of diabetes. Whether these are evident at a more moderate levels of hyperglycaemia has not been investigated. The objectives of this study were to examine the association between the level of glycaemia and resistance vasculature phenotype, incorporating both arterial stiffness and endothelial function. Diabetes was induced in male Sprague Dawley rats with streptozotocin (STZ; 55mg/kg i.v.) and followed for 8 weeks. One week post STZ, diabetic rats were allocated to either moderate (â¼20mM blood glucose, 6-7U/insulins.c. daily) or severe hyperglycaemia (â¼30mM blood glucose, 1-2U/insulins.c. daily as required). At study end, rats were anesthetized, and the mesenteric arcade was collected. Passive mechanical wall properties were assessed by pressure myography. Responses to the endothelium-dependent vasodilator acetylcholine (ACh) were assessed using wire myography. Our results demonstrated for the first time that mesenteric arteries from both moderate and severely hyperglycaemic diabetic rats exhibited outward hypertrophic remodelling and increased axial stiffness compared to arteries from non-diabetic rats. Secondly, mesenteric arteries from severely (â¼30mM blood glucose), but not moderately hyperglycaemic (â¼20mM blood glucose) rats exhibit a significant reduction to ACh sensitivity compared to their non-diabetic counterparts. This endothelial dysfunction was associated with significant reduction in endothelium-derived hyperpolarisation and endothelium-dependent NO-mediated relaxation. Interestingly, endothelium-derived nitroxyl (HNO)-mediated relaxation was intact. Therefore, moderate hyperglycaemia is sufficient to induce adverse structural changes in the mesenteric vasculature, but more severe hyperglycaemia is essential to cause endothelial dysfunction.
Assuntos
Glicemia/metabolismo , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Tipo 1/complicações , Angiopatias Diabéticas/etiologia , Endotélio Vascular/fisiopatologia , Artérias Mesentéricas/fisiopatologia , Remodelação Vascular , Rigidez Vascular , Animais , Biomarcadores/sangue , Glicemia/efeitos dos fármacos , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/tratamento farmacológico , Angiopatias Diabéticas/sangue , Angiopatias Diabéticas/fisiopatologia , Relação Dose-Resposta a Droga , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Hemoglobinas Glicadas/metabolismo , Hipoglicemiantes/administração & dosagem , Insulina/administração & dosagem , Masculino , Artérias Mesentéricas/efeitos dos fármacos , Artérias Mesentéricas/metabolismo , Óxido Nítrico/metabolismo , Óxidos de Nitrogênio/metabolismo , Ratos Sprague-Dawley , Índice de Gravidade de Doença , Resistência Vascular , Vasodilatação , Vasodilatadores/farmacologiaRESUMO
The vascular effects of exogenous relaxin (Rln) treatment are well established and include decreased myogenic reactivity and enhanced relaxation responses to vasodilators in small resistance arteries. These vascular responses are reduced in older animals, suggesting that Rln is less effective in mediating arterial function with aging. The present study investigated the role of endogenous Rln in the aorta and the possibility that vascular dysfunction occurs more rapidly with aging in Rln-deficient (Rln(-/-)) mice. We compared vascular function and underlying vasodilatory pathways in the aorta of male wild-type (Rln(+/+)) and Rln(-/-) mice at 4 and 16 mo of age using wire myography. Superoxide production, but not nitrotyrosine or NADPH oxidase expression, was significantly increased in the aorta of young Rln(-/-) mice, whereas endothelial nitric oxide (NO) synthase and basal NO availability were both significantly decreased compared with Rln(+/+) mice. In the presence of the cyclooxygenase inhibitor indomethacin, sensitivity to ACh was significantly decreased in young Rln(-/-) mice, demonstrating altered NO-mediated relaxation that was normalized in the presence of a membrane-permeable SOD or ROS scavenger. These vascular phenotypes were not exacerbated in old Rln(-/-) mice and, in most cases, did not differ significantly from old Rln(+/+) mice. Despite the vascular phenotypes in Rln(-/-) mice, endothelium-dependent and -independent vasodilation were not adversely affected. Our data show a role for endogenous Rln in reducing superoxide production and maintaining NO availability in the aorta but also demonstrate that Rln deficiency does not compromise vascular function in this artery or exacerbate endothelial dysfunction associated with aging.
Assuntos
Aorta/metabolismo , Óxido Nítrico/metabolismo , Relaxina/deficiência , Superóxidos/metabolismo , Vasodilatação , Fatores Etários , Animais , Aorta/efeitos dos fármacos , Inibidores de Ciclo-Oxigenase/farmacologia , Relação Dose-Resposta a Droga , Genótipo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase Tipo III/metabolismo , Fenótipo , Receptores Acoplados a Proteínas G/metabolismo , Relaxina/genética , Fatores Sexuais , Transdução de Sinais , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologiaRESUMO
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.