RESUMO
BACKGROUND: Pelvic floor muscles (PFM) and rectus abdominis muscles (RAM) of pregnant diabetic rats exhibit atrophy, co-localization of fast and slow fibers and an increased collagen type I/III ratio. However, the role of similar PFM or RAM hyperglycemic-related myopathy in women with gestational diabetes mellitus (GDM) remains poorly investigated. This study aims to assess the frequency of pelvic floor muscle disorders and pregnancy-specific urinary incontinence (PS-UI) 12 months after the Cesarean (C) section in women with GDM. Specifically, differences in PFM/RAM hyperglycemic myopathy will be evaluated. METHODS: The Diamater is an ongoing cohort study of four groups of 59 pregnant women each from the Perinatal Diabetes Research Centre (PDRC), Botucatu Medical School (FMB)-UNESP (São Paulo State University), Brazil. Diagnosis of GDM and PS-UI will be made at 24-26 weeks, with a follow-up at 34-38 weeks of gestation. Inclusion in the study will occur at the time of C-section, and patients will be followed at 24-48 h, 6 weeks and 6 and 12 months postpartum. Study groups will be classified as (1) GDM plus PS-UI; (2) GDM without PS-UI; (3) Non-GDM plus PS-UI; and (4) Non-GDM without PS-UI. We will analyze relationships between GDM, PS-UI and hyperglycemic myopathy at 12 months after C-section. The mediator variables to be evaluated include digital palpation, vaginal squeeze pressure, 3D pelvic floor ultrasound, and 3D RAM ultrasound. RAM samples obtained during C-section will be analyzed for ex-vivo contractility, morphological, molecular and OMICS profiles to further characterize the hyperglycemic myopathy. Additional variables to be evaluated include maternal age, socioeconomic status, educational level, ethnicity, body mass index, weight gain during pregnancy, quality of glycemic control and insulin therapy. DISCUSSION: To our knowledge, this will be the first study to provide data on the prevalence of PS-UI and RAM and PFM physical and biomolecular muscle profiles after C-section in mothers with GDM. The longitudinal design allows for the assessment of cause-effect relationships between GDM, PS-UI, and PFMs and RAMs myopathy. The findings may reveal previously undetermined consequences of GDM.
Assuntos
Diabetes Gestacional/fisiopatologia , Doenças Musculares/fisiopatologia , Incontinência Urinária/fisiopatologia , Adulto , Brasil , Cesárea , Estudos de Coortes , Feminino , Idade Gestacional , Ganho de Peso na Gestação , Humanos , Idade Materna , Contração Muscular/fisiologia , Força Muscular/fisiologia , Palpação , Diafragma da Pelve/fisiopatologia , Período Pós-Parto , Gravidez , Reto do Abdome/fisiopatologia , VaginaAssuntos
Vasos Sanguíneos/efeitos dos fármacos , Diabetes Mellitus/tratamento farmacológico , Angiopatias Diabéticas/tratamento farmacológico , Hipoglicemiantes/uso terapêutico , Resistência à Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Animais , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/fisiopatologia , Diabetes Mellitus/epidemiologia , Diabetes Mellitus/metabolismo , Diabetes Mellitus/fisiopatologia , Angiopatias Diabéticas/epidemiologia , Angiopatias Diabéticas/metabolismo , Angiopatias Diabéticas/fisiopatologia , Humanos , Hipoglicemiantes/efeitos adversos , Células Secretoras de Insulina/metabolismo , Fatores de Risco , Resultado do TratamentoRESUMO
The human insulin receptor (IR) exists in two isoforms that differ by the absence (IR-A) or the presence (IR-B) of a 12-amino acid segment encoded by exon 11. Both isoforms are functionally distinct regarding their binding affinities and intracellular signalling. However, the underlying mechanisms related to their cellular functions in several tissues are only partially understood. In this review, we summarize the current knowledge in this field regarding the alternative splicing of IR isoform, tissue-specific distribution and signalling both in physiology and disease, with an emphasis on the human placenta in gestational diabetes mellitus (GDM). Furthermore, we discuss the clinical relevance of IR isoforms highlighted by findings that show altered insulin signalling due to differential IR-A and IR-B expression in human placental endothelium in GDM pregnancies. Future research and clinical studies focused on the role of IR isoform signalling might provide novel therapeutic targets for treating GDM to improve the adverse maternal and neonatal outcomes.
Assuntos
Diabetes Gestacional/metabolismo , Diabetes Gestacional/patologia , Insulina/metabolismo , Receptor de Insulina/metabolismo , Feminino , Humanos , Gravidez , Isoformas de Proteínas , Transdução de SinaisRESUMO
To sustain tumor growth, the cancer cells need to adapt to low levels of oxygen (i.e., hypoxia) in the tumor tissue and to the tumor-associated acidic microenvironment. In this phenomenon, the activation of the sodium/proton exchanger 1 (NHE1) at the plasma membrane and the hypoxia-inducible factor (HIF) are critical for the control of the intracellular pH (pHi) and for hypoxia adaptation, respectively. Interestingly, both of these mechanisms end in sustaining cancer cell proliferation. However, regulatory mechanisms of pHi in human ovary tissue and in malignant ascites are unknown. Additionally, a potential role of NHE1 in the modulation of H(+) efflux in human ovarian cancer cells is unknown. In this review, we discussed the characteristics of tumor microenvironment of primary human ovarian tumors and tumor ascites, in terms of pHi regulatory mechanisms and oxygen level. The findings described in the literature suggest that NHE1 may likely play a role in pHi regulation and cell proliferation in human ovarian cancer, potentially involving HIF2α activation. Since ovarian cancer is the fifth cause of prevalence of women cancer in Chile and is usually of late diagnosis, i.e., when the disease jeopardizes peritoneal cavity and other organs, resulting in reduced patient survival, new efforts are required to improve patient-life span and for a better understanding of the pathophysiology of the disease. The potential advantage of the use of amiloride and amiloride-derivatives for cancer treatment in terms of NHE1 expression and activity is also discussed as a therapeutic approach in human ovarian cancer.
Assuntos
Neoplasias Ovarianas/metabolismo , Animais , Proliferação de Células/fisiologia , Feminino , Humanos , Concentração de Íons de Hidrogênio , Oxigênio/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Microambiente Tumoral/fisiologiaRESUMO
INTRODUCTION: Maternal hypercholesterolemia and hypertriglyceridemia during pregnancy is correlated with fetoplacental endothelial dysfunction and atherosclerotic lesions in fetal arteries. Few studies have reported the distribution of the concentrations of maternal total cholesterol (TCh), lipoprotein cholesterol and triglycerides during pregnancy. Therefore, we determined maternal lipid concentration during pregnancy and established the percentiles over which fetoplacental endothelial dysfunction is observed. METHODS: A lipoprotein profile was determined for 249 pregnant Chilean women in each trimester of pregnancy in cross-sectional and longitudinal lipid determination studies. Distribution percentiles for TCh, high-, low- and very-low-density lipoprotein (HDL, LDL, and vLDL, respectively) cholesterol and triglycerides were estimated. The reactivity of human umbilical vein rings to the calcitonin gene-related peptide (0.1-1000 nmol/L, 5 min) and sodium nitroprusside (10 µmol/L, 5 min) was measured (wire myography) in KCl-preconstricted vessels. RESULTS: Maternal lipoproteins and triglyceride concentrations increased over time from preconception to the 3rd trimester of pregnancy. Newborn umbilical blood lipoprotein and triglyceride concentrations were lower than those in maternal circulation. Changes in maternal HDL correlated with newborn HDL concentration; however, no correlation between maternal lipoprotein concentrations and newborn weight was found. Maternal TCh and LDL concentrations were inversely correlated with the maximal dilation, but the >75th percentile of maternal TCh and LDL concentrations (>291 and >169 mg/dL, respectively) correlated with reduced calcitonin gene-related peptide sensitivity of the vein rings. DISCUSSION AND CONCLUSION: We identified percentiles for maternal TCh and LDL concentrations over which abnormal endothelium-dependent human fetoplacental vascular response is observed.
Assuntos
LDL-Colesterol/sangue , Veias Umbilicais/fisiologia , Adolescente , Adulto , Peptídeo Relacionado com Gene de Calcitonina/farmacologia , HDL-Colesterol/sangue , Estudos Transversais , Feminino , Humanos , Estudos Longitudinais , Nitroprussiato/farmacologia , Gravidez , Estudos Retrospectivos , Triglicerídeos/sangue , Veias Umbilicais/efeitos dos fármacos , Vasodilatadores/farmacologia , Adulto JovemRESUMO
OBJECTIVE: Human foetal development and growth in an environment of maternal obesity associates with high risk of cardiovascular disease and adverse neonatal outcome. We studied whether supraphysiological gestational weight gain results in human fetoplacental endothelial dysfunction and altered fetoplacental vascular reactivity. METHODS: Primary cultures of human umbilical vein endothelial cells (HUVECs) and umbilical vein rings were obtained from pregnant women (112 total of patients recruited, 7 patients dropped out) exhibiting prepregnancy normal weight that ended with a physiological (pGWG (n=67), total weight gain 11.5-16 kg, rates of weight gain ≤0.42 kg per week) or supraphysiological (spGWG (n=38), total weight gain >16 kg, rates of weight gain >0.42 kg per week) gestational weight gain (reference values from US Institute of Medicine guidelines). Vascular reactivity to insulin (0.1-1000 nmol l(-1), 5 min) in KCl-preconstricted vein rings was measured using a wire myograph. Protein levels of human equilibrative nucleoside transporter 1 (hENT1), total and Ser(1177)- or Thr(495)-phosphorylated endothelial nitric oxide synthase (eNOS) were detected by western blot or immunofluorescence, and adenosine transport (0-250 µmol l(-1) adenosine, 2 µCi ml(-1) [(3)H]adenosine, 20 s, 25 °C) was measured in the presence or absence of 1 µmol l(-1) nitrobenzylthioinosine (hENT1 inhibitor) or 10 µmol l(-1) chlorpromazine (CPZ, endocytosis inhibitor) in HUVECs. RESULTS: spGWG associates with reduced NOS activity-dependent dilation of vein rings (P=0.001), lower eNOS expression and higher Thr(495) (P=0.044), but unaltered Ser(1177)eNOS phosphorylation. hENT1-adenosine maximal transport activity was reduced (P=0.041), but the expression was increased (P=0.001) in HUVECs from this group. CPZ increased hENT1-adenosine transport (P=0.040) and hENT1 plasma membrane accumulation only in cells from pGWG. CONCLUSION: spGWG in women with a normal prepregnancy weight causes lower fetoplacental vascular reactivity owing to the downregulation of eNOS activity and adenosine transport in HUVECs. Maternal spGWG is a detrimental condition for human fetoplacental endothelial function and reducing these alterations could result in a better neonate outcome.
Assuntos
Adenosina/metabolismo , Endotélio Vascular/metabolismo , Óxido Nítrico/metabolismo , Obesidade/fisiopatologia , Placenta/irrigação sanguínea , Efeitos Tardios da Exposição Pré-Natal/fisiopatologia , Aumento de Peso , Adulto , Transporte Biológico , Chile , Feminino , Humanos , Recém-Nascido , Obesidade/complicações , Circulação Placentária , Gravidez , Gestantes , Transdução de Sinais , Veias Umbilicais/citologiaRESUMO
INTRODUCTION: Preeclampsia is associated with impaired placental vasodilation and reduced endothelial nitric oxide synthase (eNOS) activity in the foetoplacental circulation. Adenosine and insulin stimulate vasodilation in endothelial cells, and this activity is mediated by adenosine receptor activation in uncomplicated pregnancies; however, this activity has yet to be examined in preeclampsia. Early onset preeclampsia is associated with severe placental vasculature alterations that lead to altered foetus growth and development, but whether late-onset preeclampsia (LOPE) alters foetoplacental vascular function is unknown. METHODS: Vascular reactivity to insulin (0.1-1000 nmol/L, 5 min) and adenosine (1 mmol/L, 5 min) was measured in KCl-preconstricted human umbilical vein rings from normal and LOPE pregnancies using a wire myograph. The protein levels of human cationic amino acid transporter 1 (hCAT-1), adenosine receptor subtypes, total and Ser¹¹77- or Thr495-phosphorylated eNOS were detected via Western blot, and L-arginine transport (0-1000 µmol/L L-arginine, 3 µCi/mL L-[³H]arginine, 20 s, 37 °C) was measured in the presence or absence of insulin and adenosine receptor agonists or antagonists in human umbilical vein endothelial cells (HUVECs) from normal and LOPE pregnancies. RESULTS: LOPE increased the maximal L-arginine transport capacity and hCAT-1 and eNOS expression and activity compared with normal conditions. The A(2A) adenosine receptor (A(2A)AR) antagonist ZM-241385 blocked these effects of LOPE. Insulin-mediated umbilical vein ring relaxation was lower in LOPE pregnancies than in normal pregnancies and was restored using the A(2A)AR antagonist. DISCUSSION AND CONCLUSIONS: The reduced foetoplacental vascular response to insulin may result from A(2A)AR activation in LOPE pregnancies.
Assuntos
Arginina/metabolismo , Endotélio Vascular/metabolismo , Resistência à Insulina , Insulina/metabolismo , Pré-Eclâmpsia/metabolismo , Receptor A2A de Adenosina/metabolismo , Veias Umbilicais/metabolismo , Adenosina/metabolismo , Antagonistas do Receptor A2 de Adenosina/farmacologia , Adulto , Transporte Biológico/efeitos dos fármacos , Células Cultivadas , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/patologia , Feminino , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Hipoglicemiantes/uso terapêutico , Técnicas In Vitro , Insulina/farmacologia , Pré-Eclâmpsia/tratamento farmacológico , Pré-Eclâmpsia/patologia , Gravidez , Terceiro Trimestre da Gravidez , Receptor A2A de Adenosina/química , Transdução de Sinais/efeitos dos fármacos , Veias Umbilicais/efeitos dos fármacos , Veias Umbilicais/patologia , Adulto JovemRESUMO
Gestational diabetes mellitus (GDM) is a diseases that alters human placenta macro and microvascular reactivity as a result of endothelial dysfunction. The human placenta is a highly vascularized organ which lacks innervation, so blood flux is governed by locally released vasoactive molecules, including the endogenous nucleoside adenosine and the free radical nitric oxide (NO). Altered adenosine metabolism and uptake by the endothelium leads to increased NO synthesis which then turns-off the expression of genes coding for a family of nucleoside membrane transporters belonging to equilibrative nucleoside transporters, particularly isoforms 1 (hENT1) and 2 (hENT2). This mechanism leads to increased extracellular adenosine and, as a consequence, activation of adenosine receptors to further sustain a tonic activation of NO synthesis. This is a phenomenon that seems operative in the placental macro and microvascular endothelium in GDM. We here summarize the findings available in the literature regarding these mechanisms in the human feto-placental circulation. This phenomenon is altered in the feto-placental vasculature, which could be crucial for understanding GDM deleterious effects in fetal growth and development.
Assuntos
Adenosina/metabolismo , Diabetes Gestacional/metabolismo , Endotélio Vascular/metabolismo , Proteínas de Transporte de Nucleosídeo Equilibrativas/metabolismo , Placenta/irrigação sanguínea , Receptores Purinérgicos P1/metabolismo , Sistemas do Segundo Mensageiro , Proteínas de Transporte de Nucleosídeo Equilibrativas/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Microvasos/metabolismo , Óxido Nítrico/metabolismo , Placenta/metabolismo , Circulação Placentária , GravidezRESUMO
The main vasodilator in the placenta is nitric oxide (NO), which is synthesized by endothelial NO synthase (eNOS). Arginase-2 competes with eNOS for l-arginine, and its activity has been related with vascular dysfunction. Recently, we showed that hypoxia induces arginase-2, and decreases eNOS activity in human umbilical vein endothelial cells (HUVEC). However there is evidence that vascular responses to hypoxia are not similar throughout the placental vascular tree. We studied whether arginase-2 plays a role controlling vascular tone in human umbilical vessels, and the changes in the expression of arginase-2 and eNOS proteins by hypoxia in endothelial cells from umbilical arteries (HUAEC) and veins (HUVEC). In isolated umbilical vessels the presence of eNOS and arginase-2 was determined in the endothelium, and the NO-dependent vasoactive responses in the presence and absence of S-(2-boronoethyl)-L-cysteine (BEC, arginase inhibitor) were studied. Additionally, HUAEC and HUVEC were exposed (0-24 h) to hypoxia (2% O2) or normoxia (5% O2), and protein levels of eNOS (total and phosphorylated at serine-1177) and arginase-2 were determined. In umbilical arteries and veins arginase-2 and eNOS were detected mainly at the endothelium. BEC induced a higher concentration-dependent relaxation in umbilical arteries than veins, and these responses were NOS-dependent. In HUAEC exposed to hypoxia there were no changes in eNOS and arginase-2 levels, however there was a significant increase of p-eNOS. In contrast, HUVEC showed an increase in arginase-2 and a reduction of p-eNOS in response to hypoxia. These results show that arginases have a vascular role in placental vessels counteracting the NOS-dependent relaxation, which is differentially regulated in placental artery and vein endothelial cells.
Assuntos
Arginase/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Gravidez/metabolismo , Artérias Umbilicais/enzimologia , Veias Umbilicais/enzimologia , Vasodilatação , Células Endoteliais/enzimologia , Endotélio Vascular/enzimologia , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Hipóxia/enzimologia , MiografiaRESUMO
Hypoxia relates with altered placental vasodilation, and in isolated endothelial cells, it reduces activity of the endothelial nitric oxide synthase (eNOS) and l-arginine transport. It has been reported that arginase-2 expression, an alternative pathway for l-arginine metabolism, is increased in adult endothelial cells exposed to hypoxia as well as in pre-eclamptic placentae. We studied in human umbilical vein endothelial cells (HUVEC) whether hypoxia-reduced NO synthesis results from increased arginase-mediated l-arginine metabolism and changes in subcellular localization of eNOS and arginase-2. In HUVEC exposed (24 h) to 5% (normoxia) or 2% (hypoxia) oxygen, l-arginine transport kinetics, arginase activity (urea assay), and NO synthase (NOS) activity (l-citrulline assay) were determined. Arginase-1, arginase-2 and eNOS expression were determined by RT-PCR and Western blot. Subcellular localization of arginase-2 and eNOS were studied using confocal microscopy and indirect immunofluorescence. Experiments were done in absence or presence of S-(2-boronoethyl)-l-cysteine-HCl (BEC, arginase inhibitor) or N(G)-nitro-l-arginine methyl ester (l-NAME). Hypoxia-induced reduction in eNOS activity was associated with a reduction in eNOS phosphorylation at Serine-1177 and increased phosphorylation at Threonine-495. This was paralleled with an induction in arginase-2 expression and activity, and decreased l-arginine transport. In hypoxia the arginase inhibition, restored NO synthesis and l-arginine transport, without changes in the eNOS post-translational modification status. Hypoxia increased arginase-2/eNOS colocalization, and eNOS redistribution to the cell periphery. Altogether these data reinforce the thought that eNOS cell location, post-translational modification and substrate availability are important mechanisms regulating eNOS activity. If these mechanisms occur in pregnancy diseases where feto-placental oxygen levels are reduced remains to be clarified.
Assuntos
Arginase/biossíntese , Células Endoteliais da Veia Umbilical Humana/enzimologia , Hipóxia/metabolismo , Óxido Nítrico Sintase Tipo III/biossíntese , Adulto , Arginase/antagonistas & inibidores , Arginina/metabolismo , Ácidos Borônicos/farmacologia , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Microscopia Confocal , NG-Nitroarginina Metil Éster/farmacologia , Fosforilação , Gravidez , Processamento de Proteína Pós-Traducional , Frações Subcelulares/enzimologiaRESUMO
Human endothelial dysfunction is a common feature in many diseases of pregnancy, such as gestational diabetes (GD). Metabolic changes include abnormal synthesis of nitric oxide (NO) and abnormal membrane transport of l-arginine and adenosine in primary cultures of human umbilical vein (HUVEC, macrovascular) and placental microvillus (hPMEC, microvascular) endothelial cells. These alterations are associated with modifications in the expression and activity of endothelial (eNOS) and inducible (iNOS) NO synthases, respectively, an effect that is maintained at least up to passage 5 in culture. HUVEC and hPMEC exhibit expression and activity of the human cationic amino acid transporter 1 (hCAT-1), equilibrative nucleoside transporters 1 (hENT1) and hENT2, as well as the corresponding SLC7A1, SLC29A1 and SLC29A2 gene promoter activities. Altered gene expression results from increased NO level, protein kinase C, mitogen-activated protein kinases, and hCHOP-C/EBPα transcription factor activation. Reduced ENT-mediated adenosine transport in GD is associated with stimulation of the l-arginine/NO pathway, and mainly due to reduced expression and activity of hENT1. In addition, hENT2 activity seems able to restore the reduced adenosine transport in GD. Additionally, insulin exerts a differential modulation of endothelial cells from macrocirculation compared with microcirculation, possibly due to expression of different insulin receptor isoforms. It is suggested that a common functional characteristic leading to changes in the bioavailability of adenosine and metabolism of l-arginine is evidenced by human fetal micro and macrovascular endothelium in GD.
Assuntos
Diabetes Gestacional/fisiopatologia , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiopatologia , Microvasos/fisiopatologia , Placenta/irrigação sanguínea , Placenta/fisiopatologia , Diabetes Gestacional/metabolismo , Endotélio Vascular/citologia , Feminino , Humanos , Microvasos/citologia , Microvasos/metabolismo , Placenta/citologia , Placenta/metabolismo , GravidezRESUMO
Extracellular adenosine removal is via human equilibrative nucleoside transporters 1 (hENT1) and 2 (hENT2) in the endothelium, thus regulating adenosine-induced revascularization and angiogenesis. Since human endothelial progenitor cells (hEPCs) promote revascularization, we hypothesize differential expression of nucleoside transporters in hEPCs. hEPCs were cultured 3 (hEPC-3d) or 14 (hEPC-14d) days. RT-PCR for prominin 1, CD34, octamer-4, kinase insert domain receptor, oxidized low-density lipoprotein (lectin-like) receptor 1 and tyrosine endothelial kinase was used to evaluate phenotypic differentiation. Flow cytometry was used to estimate CD34(+)/KDR(-) (non-differentiated), CD34(-)/KDR(+) (differentiated) or CD34(+)/KDR(+) (mixed) cell populations. Adenosine transport was measured in absence or presence of sodium, S-(4-nitrobenzyl)-6-thio-inosine (NBTI, 1-10 µM), inosine, hypoxanthine or guanine (0.1-5 mM), hENTs protein abundance by western blot, and hENTs, hCNT1, hCNT2 and hCNT3 mRNA expression by real time RT-PCR. hEPC-3d cells were CD34(+)/KDR(-) compared with hEPC-14d cells that were CD34(-)/KDR(+). hEPC-3d cells exhibit hENT1-like adenosine transport (NBTI-sensitive, Na(+)-independent), which is absent in hEPC-14d cells. hEPC-14d cells exhibit two transport components: component 1 (NBTI insensitive, Na(+)-independent) and component 2 (NBTI insensitive, Na(+)-dependent, Hill coefficient â¼1.8), the latter resembling CNT3-like transport. hEPC-3d cells express hENT1 protein and mRNA, which is reduced (â¼90%) in hEPC-14d cells, but instead only hCNT3 mRNA is expressed in this cell type. hENT2, hCNT1 and hCNT2 were undetectable in hEPCs. Thus, hEPCs exhibit a differential expression of hENT1 and hCNT3 functional nucleoside transporters, which could be related with its differentiation stage.
Assuntos
Células Endoteliais/fisiologia , Transportador Equilibrativo 1 de Nucleosídeo/biossíntese , Transportador Equilibrativo 2 de Nucleosídeo/biossíntese , Células-Tronco/fisiologia , Adenosina/metabolismo , Transporte Biológico , Western Blotting , Diferenciação Celular/fisiologia , Células Endoteliais/metabolismo , Transportador Equilibrativo 1 de Nucleosídeo/genética , Transportador Equilibrativo 2 de Nucleosídeo/genética , Humanos , Cinética , RNA/química , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células-Tronco/metabolismo , Tioinosina/análogos & derivados , Tioinosina/farmacologiaRESUMO
Workshops are an important part of the annual meeting of the International Federation of Placenta Associations (IFPA). At IFPA Meeting 2009 diverse topics were discussed in twelve themed workshops. Topics covered included: immune response to pregnancy; signaling between fetus and placenta; bioactive lipids in placenta; placenta in agricultural species; epigenetics and placentation; trophoblast deportation; glucocorticoids and placental function; endothelium; placental transport; genes and placenta; uteroplacental blood flow and placental stem cells. This report is a full summary of the various topics covered.
Assuntos
Placenta/fisiologia , Animais , Congressos como Assunto , Feminino , Troca Materno-Fetal , GravidezRESUMO
Up regulation of the transforming growth factor-beta 1 (TGF-beta1) axis has been recognized as a pathogenic event for progression of glomerulosclerosis in diabetic nephropathy. We demonstrate that glomeruli isolated from diabetic rats accumulate up to sixfold more extracellular adenosine than normal rats. Both decreased nucleoside uptake activity by the equilibrative nucleoside transporter 1 and increased AMP hydrolysis contribute to raise extracellular adenosine. Ex vivo assays indicate that activation of the low affinity adenosine A2B receptor subtype (A2BAR) mediates TGF-beta1 release from glomeruli of diabetic rats, a pathogenic event that could support progression of glomerulopathy when the bioavailability of adenosine is increased.
Assuntos
Adenosina/metabolismo , Nefropatias Diabéticas/metabolismo , Glomérulos Renais/metabolismo , Receptor A2B de Adenosina/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Monofosfato de Adenosina/metabolismo , Animais , Disponibilidade Biológica , Transportador Equilibrativo 1 de Nucleosídeo/metabolismo , Hidrólise , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Intrauterine growth restriction (IUGR) is associated with chronic fetal hypoxia, altered placental vasodilatation and reduced endothelial nitric oxide synthase (eNOS) activity. In human umbilical vein endothelial cells (HUVEC) from pregnancies complicated with IUGR (IUGR cells) and in HUVEC from normal pregnancies (normal cells) cultured under hypoxia l-arginine transport is reduced; however, the mechanisms leading to this dysfunction are unknown. We studied hypoxia effect on l-arginine transport and human cationic amino acid transporters 1 (hCAT-1) expression, and the potential NO and protein kinase C alpha (PKCalpha) involvement. Normal or IUGR HUVEC monolayers were exposed (0-24h) to 5% O(2) (normoxia), and 1 or 2% O(2) (hypoxia). l-Arginine transport and hCAT-1 expression, phosphorylated and total PKCalpha or eNOS protein and mRNA expression were quantified. eNOS involvement was tested using a siRNA against eNOS (eNOS-siRNA) adenovirus. IUGR cells in normoxia or hypoxia, and normal cells in hypoxia exhibited reduced l-arginine transport, hCAT-1 expression, NO synthesis and eNOS phosphorylation at Serine(1177), effects reversed by calphostin C (PKC inhibitor) and S-nitroso-N-acetyl-l,d-penicillamine (SNAP, NO donor). However, N(G)-nitro-l-arginine methyl ester (l-NAME, NOS inhibitor) reduced hCAT-1 expression only in normal cells in normoxia. Increased Thr(638)-phosphorylated PKCalpha was exhibited by IUGR cells in normoxia or hypoxia and normal cells in hypoxia. The effects of hypoxia in normal cells were mimicked in eNOS-siRNA transduced cells; however, IUGR phenotype was unaltered by eNOS knockdown. Thus, IUGR- and hypoxia-reduced l-arginine transport could result from increased PKCalpha, but reduced eNOS activity leading to a lower hCAT-1 expression in HUVEC. In addition, IUGR endothelial cells are either not responsive or maximally affected by hypoxia. These mechanisms could be responsible for placental dysfunction in diseases where fetal endothelium is chronically exposed to hypoxia, such as IUGR.
Assuntos
Arginina/metabolismo , Células Endoteliais/enzimologia , Retardo do Crescimento Fetal/metabolismo , Hipóxia/metabolismo , Óxido Nítrico/biossíntese , Veias Umbilicais/citologia , Adulto , Transportador 1 de Aminoácidos Catiônicos/genética , Transportador 1 de Aminoácidos Catiônicos/metabolismo , Células Cultivadas , Células Endoteliais/citologia , Feminino , Humanos , Modelos Biológicos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Fosforilação/fisiologia , Gravidez , Proteína Quinase C-alfa/genética , Proteína Quinase C-alfa/metabolismo , RNA Mensageiro/metabolismo , Vasodilatação/fisiologia , Adulto JovemRESUMO
Pre-eclampsia is associated with elevated maternal blood pressure and proteinuria, altered fetal growth, and increased plasma adenosine concentration in the mother and the fetus. Human equilibrative nucleoside transporters 1 (hENT1) and hENT2 are crucial to maintain physiological plasma levels of adenosine, thus modulating its several biological effects through adenosine receptor activation. However, it is unknown whether hENTs and adenosine receptors are expressed in human placental microvascular endothelium (hPMEC). To assay whether the increased fetal plasma adenosine concentration in pre-eclampsia results from altered hENT-mediated transport, and the potential involvement of adenosine receptors in this phenomenon, we investigated hENTs and A2A and A2B adenosine receptors expression and function in hPMEC. Cells were isolated and cultured from normal pregnancies (n=17) or pre-eclampsia with adequate-for-gestational age fetuses (n=7). hENT1, hENT2, A2A and A2B adenosine receptors were expressed and functional in hPMEC. Extracellular adenosine concentration was higher (4-fold) in pre-eclampsia versus normal pregnancies. hPMEC from pre-eclampsia exhibit increased total transport (hENT1+hENT2), and maximal velocity (Vmax) for hENT2- (2-fold), but reduced Vmax for hENT1-mediated adenosine transport (75%), with no changes in apparent Km. hENT2 expression was increased (4.5-fold), but hENT1 protein abundance was reduced (80%) in pre-eclampsia. Equally, A2A expression was reduced (50-80%) in pre-eclampsia. CGS-21680 (A2A agonist) did not alter hENTs expression or activity, but ZM-241385 (A2A antagonist) blocked pre-eclampsia effects and increased hENT1-mediated transport in normal pregnancies. Thus, A2B adenosine receptors may differentially modulate hENTs in hPMEC, which could be a mechanism attempting to re-establish physiological extracellular adenosine levels in pre-eclampsia.
Assuntos
Células Endoteliais/fisiologia , Transportador Equilibrativo 1 de Nucleosídeo/biossíntese , Transportador Equilibrativo 2 de Nucleosídeo/biossíntese , Placenta/citologia , Pré-Eclâmpsia/fisiopatologia , Receptor A2B de Adenosina/fisiologia , Adenosina/análogos & derivados , Adenosina/metabolismo , Adenosina/farmacologia , Agonistas do Receptor A2 de Adenosina , Antagonistas do Receptor A2 de Adenosina , Adulto , Transportador Equilibrativo 1 de Nucleosídeo/fisiologia , Transportador Equilibrativo 2 de Nucleosídeo/fisiologia , Feminino , Regulação da Expressão Gênica , Humanos , Fenetilaminas/farmacologia , Gravidez , Receptor A2A de Adenosina/biossíntese , Triazinas/farmacologia , Triazóis/farmacologiaRESUMO
Preeclampsia is a human syndrome characterized by elevated maternal blood pressure and proteinuria. It is the main cause of maternal morbidity and mortality, and fetal metabolic disturbances in developed and developing countries. Fetal complications in preeclampsia have been related with lower placental blood flow. The placenta lacks of innervation, thus vascular tone regulation depends on endothelial release of vasoactive molecules such as adenosine and nitric oxide (NO). Information about NO synthesis and its action in the feto-placental vasculature in preeclamptic pregnancies is controversial mainly due to the use of different methodological approaches, severity of the disease and cell type that had been analyzed. A high plasma level of adenosine has been reported in umbilical vein from preeclampsia compared with normal pregnancies. Since this nucleoside is mainly involved in the regulation of vascular tone and angiogenesis, perhaps through the modulation of potassium channels, it is suggested that it would be involved in the maintenance of normal feto-placental function. In this review we hypothesize a potential adenosine-mediated, NO-dependent mechanism accounting for the feto-placental reduced blood flow exhibited in preeclampsia. We summarize the potential mechanisms involved in the modulation of inducible NO synthase expression and activity in preeclampsia, emphasizing metabolic alterations in the placenta microvascular endothelial function. The involvement of adenosine, nucleoside membrane transporters and adenosine receptors, nuclear factor kappa B (NF-kappaB), potassium channels and angiogenesis, are also discussed regarding abnormal endothelial function in preeclampsia.