RESUMO
We hypothesize that intrauterine hypoxia (HPX) alters the mitochondrial phenotype in fetal hearts contributing to developmental programming. Pregnant guinea pigs were exposed to normoxia (NMX) or hypoxia (HPX, 10.5% O2), starting at early [25 days (25d), 39d duration] or late gestation (50d, 14d duration). Near-term (64d) male and female fetuses were delivered by hysterotomy from anesthetized sows, and body/organ weights were measured. Left ventricles of fetal hearts were excised and frozen for measurement of expression of complex (I-V) subunits, fusion (Mfn2/OPA1) and fission (DRP1/Fis1) proteins, and enzymatic rates of I and IV from isolated mitochondrial proteins. Chronic HPX decreased fetal body weight and increased relative placenta weight regardless of timing. Early-onset HPX increased I, III, and V subunit levels, increased complex I but decreased IV activities in males but not females (all P < 0.05). Late-onset HPX decreased (P < 0.05) I, III, and V levels in both sexes but increased I and decreased IV activities in males only. Both HPX conditions decreased cardiac mitochondrial DNA content in males only. Neither early- nor late-onset HPX had any effect on Mfn2 levels but increased OPA1 in both sexes. Both HPX treatments increased DRP1/Fis1 levels in males. In females, early-onset HPX increased DRP1 with no effect on Fis1, whereas late-onset HPX increased Fis1 with no effect on DRP1. We conclude that both early- and late-onset HPX disrupts the expression/activities of select complexes that could reduce respiratory efficiency and shifts dynamics toward fission in fetal hearts. Thus, intrauterine HPX disrupts the mitochondrial phenotype predominantly in male fetal hearts, potentially altering cardiac metabolism and predisposing the offspring to heart dysfunction.
Assuntos
Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo II de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Hipóxia Fetal/enzimologia , Mitocôndrias Cardíacas/enzimologia , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Miócitos Cardíacos/enzimologia , Animais , Hipóxia Celular , Respiração Celular , Modelos Animais de Doenças , Dinaminas/genética , Dinaminas/metabolismo , Complexo I de Transporte de Elétrons/genética , Complexo II de Transporte de Elétrons/genética , Complexo III da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Feminino , Hipóxia Fetal/genética , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Idade Gestacional , Cobaias , Masculino , Mitocôndrias Cardíacas/genética , Dinâmica Mitocondrial , ATPases Mitocondriais Próton-Translocadoras/genética , Fatores SexuaisRESUMO
This study was designed to determine the role of the MEK/ERK1/2 and PI3K/Akt pathways in cortisol production and endothelial nitric oxide synthase (eNOS) phosphorylation (peNOS) in the ovine fetal adrenal in response to long-term hypoxia (LTH). Pregnant ewes were maintained at high altitude (3820 m) for the last 100 days of gestation (dGa). At 138 to 142 dGa, fetal adrenal cortical cells (FACs) were collected from LTH and age-matched normoxic fetuses. Cortisol production and peNOS were measured in response to pretreatment with the MEK/ERK1/2 pathway inhibitor UO126 (UO) and adrenocorticotropic hormone (ACTH) stimulation. UO126 reduced ACTH-stimulated cortisol in both normoxic and LTH FACs. UO126 alone or in combination with ACTH reduced peNOS in the normoxic group, while ACTH alone or ACTH + UO inhibited peNOS in LTH FACs. Additionally, cortisol was measured in response to pretreatment with UO and treatment with 22R-hydroxycholesterol (22R-OHC) or water-soluble cholesterol (WSC) with and without ACTH stimulation. UO126 had no effect on 22R-OHC-treated cells, but reduced cortisol in cells treated with WSC and/or ACTH. Cortisol and peNOS were also measured in response to pretreatment with PI3K/Akt pathway inhibitor Wortmannin (WT) and ACTH stimulation. Wortmannin further increased cortisol under ACTH-stimulated conditions and, like ACTH, reduced peNOS in LTH but not normoxic FACs. Together, these data suggest that in LTH FACs MEK/ERK1/2 does not regulate peNOS but that UO acts downstream from eNOS, possibly at cholesterol transport, to affect cortisol production in LTH FACs, while the PI3K/Akt pathway, along with ACTH, regulates peNOS and plays a role in the fetal adaptation to LTH in FACs.
Assuntos
Córtex Suprarrenal/efeitos dos fármacos , Córtex Suprarrenal/enzimologia , Hormônio Adrenocorticotrópico/farmacologia , Hipóxia Fetal/enzimologia , Hidrocortisona/biossíntese , Óxido Nítrico Sintase Tipo III/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Adaptação Fisiológica , Córtex Suprarrenal/embriologia , Córtex Suprarrenal/fisiopatologia , Altitude , Animais , Células Cultivadas , Modelos Animais de Doenças , Feminino , Hipóxia Fetal/embriologia , Hipóxia Fetal/fisiopatologia , Idade Gestacional , Hidroxicolesteróis/farmacologia , Fosfatidilinositol 3-Quinase/metabolismo , Fosforilação , Gravidez , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ovinos , Transdução de Sinais/efeitos dos fármacos , Fatores de TempoRESUMO
Preterm brain injury is partly associated with hypoxia-ischemia starting before birth. Excessive nitric oxide production during HI may cause nitrosative stress, leading to cell membrane and mitochondrial damage. We therefore tested the hypothesis that therapy with a new, selective neuronal nitric oxide synthase (nNOS) inhibitor, JI-10 (0.022mg/kg bolus, n=8), given 30min before 25min of complete umbilical cord occlusion was protective in preterm fetal sheep at 101-104day gestation (term is 147days), compared to saline (n=8). JI-10 had no effect on fetal blood pressure, heart rate, carotid and femoral blood flow, total EEG power, nuchal activity, temperature or intracerebral oxygenation on near-infrared spectroscopy during or after occlusion. JI-10 was associated with later onset of post-asphyxial seizures compared with saline (p<0.05), and attenuation of the subsequent progressive loss of cytochrome oxidase (p<0.05). After 7days recovery, JI-10 was associated with improved neuronal survival in the caudate nucleus (p<0.05), but not the putamen or hippocampus, and more CNPase positive oligodendrocytes in the periventricular white matter (p<0.05). In conclusion, prophylactic nNOS inhibition before profound asphyxia was associated with delayed onset of seizures, slower decline of cytochrome oxidase and partial white and gray matter protection, consistent with protection of mitochondrial function.
Assuntos
Inibidores Enzimáticos/farmacologia , Hipóxia Fetal/complicações , Fármacos Neuroprotetores/farmacologia , Óxido Nítrico Sintase Tipo I/antagonistas & inibidores , Efeitos Tardios da Exposição Pré-Natal/prevenção & controle , Animais , Paralisia Cerebral/etiologia , Paralisia Cerebral/prevenção & controle , Modelos Animais de Doenças , Feminino , Hipóxia Fetal/enzimologia , Feto , Hipóxia-Isquemia Encefálica/fisiopatologia , Gravidez , Efeitos Tardios da Exposição Pré-Natal/etiologia , Convulsões/etiologia , Convulsões/prevenção & controle , OvinosRESUMO
Experimental studies indicate that hypoxia to the fetus, a common occurrence in many birth complications in humans, results in long-term disturbances of the central dopaminergic (DA) systems that persist in adulthood. Because dysregulation of DA systems is involved in the pathophysiology of many neurological and psychiatric disorders, we investigated the effects of perinatal hypoxia on the mesencephalic DA neurons of the human neonate using immunohistochemistry. We studied the expression of tyrosine hydroxylase (TH), the first and rate-limiting enzyme in catecholamine synthesis, in substantia nigra, and ventral tegmental area of 18 neonates in relation to the age and severity/duration of hypoxic injury estimated by neuropathological criteria. In severe/abrupt perinatal hypoxia, intense TH staining was observed in substantia nigra, ventral tegmental area, and, surprisingly, in the nonpreganglionic Edinger-Westphal nucleus. In severe/prolonged hypoxia, there was a striking reduction or even absence of TH immunoreactivity in all the mesencephalic nuclei. These observations suggest that at early states of perinatal hypoxia, there is a massive increase in dopamine synthesis and release that is followed by feedback blockage of dopamine synthesis through inhibition of TH by the end product dopamine. Early dysregulation of DA neurotransmission could predispose infant survivors of severe perinatal hypoxia to dopamine-related neurological and/or cognitive deficits later in life.
Assuntos
Neurônios Dopaminérgicos/enzimologia , Hipóxia Fetal/enzimologia , Regulação Enzimológica da Expressão Gênica , Mesencéfalo/enzimologia , Mesencéfalo/patologia , Tirosina 3-Mono-Oxigenase/biossíntese , Neurônios Dopaminérgicos/patologia , Feminino , Hipóxia Fetal/patologia , Humanos , Recém-Nascido , Masculino , Estudos Prospectivos , Fatores de Tempo , Tirosina 3-Mono-Oxigenase/químicaRESUMO
Exposure to chronic hypoxia during gestation predisposes infants to neonatal pulmonary hypertension, but the underlying mechanisms remain unclear. Here, we test the hypothesis that moderate continuous hypoxia during gestation causes changes in the rho-kinase pathway that persist in the newborn period, altering vessel tone and responsiveness. Lambs kept at 3,801 m above sea level during gestation and the first 2 wk of life were compared with those with gestation at low altitude. In vitro studies of isolated pulmonary arterial rings found a more forceful contraction in response to KCl and 5-HT in high-altitude compared with low-altitude lambs. There was no difference between the effects of blockers of various pathways of extracellular Ca(2+) entry in low- and high-altitude arteries. In contrast, inhibition of rho-kinase resulted in significantly greater attenuation of 5-HT constriction in high-altitude compared with low-altitude arteries. High-altitude lambs had higher baseline pulmonary artery pressures and greater elevations in pulmonary artery pressure during 15 min of acute hypoxia compared with low-altitude lambs. Despite evidence for an increased role for rho-kinase in high-altitude arteries, in vivo studies found no significant difference between the effects of rho-kinase inhibition on hypoxic pulmonary vasoconstriction in intact high-altitude and low-altitude lambs. We conclude that chronic hypoxia in utero results in increased vasopressor response to both acute hypoxia and serotonin, but that rho-kinase is involved only in the increased response to serotonin.
Assuntos
Hipóxia Fetal/complicações , Síndrome da Persistência do Padrão de Circulação Fetal/etiologia , Artéria Pulmonar/enzimologia , Vasoconstrição , Quinases Associadas a rho/metabolismo , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Altitude , Animais , Animais Recém-Nascidos , Pressão Arterial , Bloqueadores dos Canais de Cálcio/farmacologia , Doença Crônica , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Hipóxia Fetal/enzimologia , Hipóxia Fetal/fisiopatologia , Idade Gestacional , Humanos , Recém-Nascido , Síndrome da Persistência do Padrão de Circulação Fetal/enzimologia , Síndrome da Persistência do Padrão de Circulação Fetal/fisiopatologia , Inibidores de Proteínas Quinases/farmacologia , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/fisiopatologia , Serotonina/farmacologia , Ovinos , Fatores de Tempo , Vasoconstrição/efeitos dos fármacos , Vasoconstritores/farmacologia , Quinases Associadas a rho/antagonistas & inibidoresRESUMO
The aim of this study is to quantify the effect of intrauterine hypoxia (HPX) and the role of nitric oxide (NO) on the apoptotic enzyme, caspase 3, and DNA fragmentation in fetal heart and brain. Hypoxia and NO are important regulators of apoptosis, although this has been little studied in the fetal organs. We investigated the effect of intrauterine HPX on apoptosis and the role of NO in both fetal hearts and brains. Pregnant guinea pigs were exposed to room temperature (N = 14) or 10.5% O2 (N = 12) for 14 days prior to term (term = 65 days) and administered water or L-N6-(1-iminoethyl)-lysine (LNIL), an inducible nitric oxide synthase (iNOS) inhibitor, for 10 days. Fetal hearts and brains were excised from anesthetized near-term fetuses for study. Chronic HPX decreased pro- and active caspase 3, caspase 3 activity, and DNA fragmentation levels in fetal hearts compared with normoxic controls. L-N6-(1-iminoethyl)-lysine prevented the HPX-induced decrease in caspase 3 activity but did not alter DNA fragmentation levels. In contrast, chronic HPX increased both apoptotic indices in fetal brains, which were inhibited by LNIL. Thus, the effect of HPX on apoptosis differs between fetal organs, and NO may play an important role in modulating these effects.
Assuntos
Encéfalo/metabolismo , Caspase 3/metabolismo , Fragmentação do DNA , Hipóxia Fetal/metabolismo , Miocárdio/metabolismo , Neurônios/metabolismo , Animais , Apoptose , Encéfalo/embriologia , Encéfalo/enzimologia , Feminino , Hipóxia Fetal/enzimologia , Cobaias , Coração/embriologia , Miocárdio/enzimologia , Proteínas do Tecido Nervoso/metabolismo , Neurônios/enzimologia , Especificidade de Órgãos , GravidezRESUMO
Several experimental models on adult and newborn animals showed that in cerebral hypoxic-ischemic conditions similar to clinical states the main source of the excessive production of free oxygen radicals is the highly activated xanthine oxidase (XO) enzyme reaction. Long before this data were available, it became known that the main role of allopurinol (AP) is the inhibition of XO. On the basis of these results, many therapeutic trials with AP were performed both in experimental and clinical studies of ischemia and reperfusion. However, it has been shown that only preventive administration of AP has favorable effects. The explanation for the poor results of AP treatment in human fetal brain damage (FBD) cases is that the drug was applied postnatally. The clinical studies performed in healthy laboring mothers whose deliveries were complicated with FBD showed that placental transfer after prenatal administration of AP may be effective in protecting newborns at increased risk of hypoxic-ischemic cerebral damage. Further controlled trials are required to determine if the prophylactic use of the drug might prevent hypoxic-ischemic injuries when the drug is administered immediately prior to impending fetal hypoxia, or even in deliveries at risk of developing hypoxia.
Assuntos
Alopurinol/uso terapêutico , Lesões Encefálicas/prevenção & controle , Hipóxia Fetal/tratamento farmacológico , Hipóxia-Isquemia Encefálica/tratamento farmacológico , Xantina Oxidase/antagonistas & inibidores , Alopurinol/administração & dosagem , Animais , Animais Recém-Nascidos , Lesões Encefálicas/tratamento farmacológico , Lesões Encefálicas/enzimologia , Lesões Encefálicas/etiologia , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/uso terapêutico , Feminino , Hipóxia Fetal/complicações , Hipóxia Fetal/enzimologia , Humanos , Hipóxia-Isquemia Encefálica/complicações , Hipóxia-Isquemia Encefálica/enzimologia , Recém-Nascido , Gravidez , Traumatismo por Reperfusão/tratamento farmacológicoRESUMO
OBJECTIVE: The purpose of this study was to investigate the impact of chronic hypoxia on the nitric oxide synthase isoenzymes in specific brain structures. STUDY DESIGN: Time-mated pregnant guinea pigs were exposed to 10.5% molecular oxygen for 14 days (animals with chronic fetal hypoxia; HPX) or room air (control animals; NMX); L-N6-(1-iminoethyl)-lysine (L-NIL; an inducible nitric oxide synthase inhibitor, 1 mg/kg/d) was administered to HPX group for 14 days (L-NIL + HPX). Fetal brains were harvested at term. Multilabeled immunofluorescence was used to generate a brain injury map. Laser capture microdissection and quantitative polymerase chain reaction were applied; cell injury markers, apoptosis activation, neuron loss, total nitric oxide, and the levels of individual nitric oxide synthase isoenzymes were quantified. RESULTS: Chronic hypoxia causes selective fetal brain injury rather than global. Injury is associated with differentially affected nitric oxide synthases in both neurons and glial cells, with inducible macrophage-type nitric oxide synthase up-regulated at all injury sites. L-NIL attenuated the injury, despite continued hypoxia. CONCLUSION: These studies demonstrate that chronic hypoxia selectively injures the fetal brain in part by the differential regulation of nitric oxide synthase isoenzymes in an anatomic- and cell-specific manner.
Assuntos
Lesões Encefálicas/enzimologia , Hipóxia Fetal/enzimologia , Óxido Nítrico Sintase/metabolismo , Complicações na Gravidez , Animais , Lesões Encefálicas/etiologia , Doença Crônica , Feminino , Hipóxia Fetal/complicações , Cobaias , Isoenzimas/metabolismo , GravidezRESUMO
In the developing fetus, cerebral arteries (CA) show striking differences in signal transduction mechanisms compared with the adult, and these differences are magnified in response to high-altitude long-term hypoxia (LTH). In addition, in the mature organism, cerebrovascular acclimatization to LTH may be associated with several clinical problems, the mechanisms of which are unknown. Because PKC plays a key role in regulating CA contractility, in fetal and adult cerebral arteries, we tested the hypothesis that LTH differentially regulates the PKC-mediated Ca(2+) sensitization pathways and contractility. In four groups of sheep [fetal normoxic (FN), fetal hypoxic (FH), adult normoxic (AN), and adult hypoxic (AH)], we examined, simultaneously, responses of CA tension and intracellular Ca(2+) concentration and measured CA levels of PKC, ERK1/2, RhoA, 20-kDa myosin light chain, and the 17-kDa PKC-potentiated myosin phosphatase inhibitor CPI-17. The PKC activator phorbol 12,13-dibutyrate (PDBu) produced robust contractions in all four groups. However, PDBu-induced contractions were significantly greater in AH CA than in the other groups. In all CA groups except AH, in the presence of MEK inhibitor (U-0126), the PDBu-induced contractions were increased a further 20-30%. Furthermore, in adult CA, PDBu led to increased phosphorylation of ERK1, but not ERK2; in fetal CA, the reverse was the case. PDBu-stimulated ERK2 phosphorylation also was significantly greater in FH than FN CA. Also, although RhoA/Rho kinase played a significant role in PDBu-mediated contractions of FN CA, this was not the case in FH or either adult group. Also, whereas CPI-17 had a significant role in adult CA contractility, this was not the case for the fetus. Overall, in ovine CA, the present study demonstrates several important maturational and LTH acclimatization changes in PKC-induced contractile responses and downstream pathways. The latter may play a key role in the pathophysiologic disorders associated with acclimatization to high altitude.
Assuntos
Aclimatação , Envelhecimento , Sinalização do Cálcio , Artérias Cerebrais/enzimologia , Hipóxia Fetal/enzimologia , Hipóxia/enzimologia , Proteína Quinase C/metabolismo , Vasoconstrição , Fatores Etários , Animais , Sinalização do Cálcio/efeitos dos fármacos , Artérias Cerebrais/efeitos dos fármacos , Artérias Cerebrais/embriologia , Artérias Cerebrais/crescimento & desenvolvimento , Artérias Cerebrais/fisiopatologia , Modelos Animais de Doenças , Ativação Enzimática , Ativadores de Enzimas/farmacologia , Hipóxia Fetal/fisiopatologia , Idade Gestacional , Hipóxia/fisiopatologia , MAP Quinase Quinase Quinases/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Cadeias Leves de Miosina/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Ovinos , Fatores de Tempo , Vasoconstrição/efeitos dos fármacos , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
RATIONALE: Epidemiological studies demonstrate a clear association of adverse intrauterine environment with an increased risk of ischemic heart disease in adulthood. Hypoxia is a common stress to the fetus and results in decreased protein kinase C epsilon (PKCepsilon) expression in the heart and increased cardiac vulnerability to ischemia and reperfusion injury in adult offspring in rats. OBJECTIVES: The present study tested the hypothesis that fetal hypoxia-induced methylation of cytosine-phosphate-guanine dinucleotides at the PKCepsilon promoter is repressive and contributes to PKCepsilon gene repression in the heart of adult offspring. METHODS AND RESULTS: Hypoxic treatment of pregnant rats from days 15 to 21 of gestation resulted in significant decreases in PKCepsilon protein and mRNA in fetal hearts. Similar results were obtained in ex vivo hypoxic treatment of isolated fetal hearts and rat embryonic ventricular myocyte cell line H9c2. Increased methylation of PKCepsilon promoter at SP1 binding sites, -346 and -268, were demonstrated in both fetal hearts of maternal hypoxia and H9c2 cells treated with 1% O(2) for 24 hours. Whereas hypoxia had no significant effect on the binding affinity of SP1 to the unmethylated sites in H9c2 cells, hearts of fetuses and adult offspring, methylation of both SP1 sites reduced SP1 binding. The addition of 5-aza-2'-deoxycytidine blocked the hypoxia-induced increase in methylation of both SP1 binding sites and restored PKCepsilon mRNA and protein to the control levels. In hearts of both fetuses and adult offspring, hypoxia-induced methylation of SP1 sites was significantly greater in males than in females, and decreased PKCepsilon mRNA was seen only in males. In fetal hearts, there was significantly higher abundance of estrogen receptor alpha and beta isoforms in females than in males. Both estrogen receptor alpha and beta interacted with the SP1 binding sites in the fetal heart, which may explain the sex differences in SP1 methylation in the fetal heart. Additionally, selective activation of PKCepsilon restored the hypoxia-induced cardiac vulnerability to ischemic injury in offspring. CONCLUSIONS: The findings demonstrate a direct effect of hypoxia on epigenetic modification of DNA methylation and programming of cardiac PKCepsilon gene repression in a sex-dependent manner, linking fetal hypoxia and pathophysiological consequences in the hearts of adult offspring.
Assuntos
Proteína Quinase C-épsilon/genética , Animais , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Western Blotting , Metilação de DNA/genética , Metilases de Modificação do DNA/antagonistas & inibidores , Decitabina , Epigênese Genética/genética , Feminino , Coração Fetal/fisiopatologia , Hipóxia Fetal/enzimologia , Hipóxia Fetal/genética , Regulação Enzimológica da Expressão Gênica , Masculino , Metilação , Gravidez , Regiões Promotoras Genéticas/genética , Proteína Quinase C-épsilon/deficiência , RNA Mensageiro/genética , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição Sp1/antagonistas & inibidores , Fator de Transcrição Sp1/efeitos dos fármacos , Fator de Transcrição Sp1/metabolismoRESUMO
The present study tested the hypothesis that protein kinase C (PKC) epsilon plays a key role in the sex dichotomy of heart susceptibility to ischemia and reperfusion injury in adult offspring resulting from prenatal hypoxic exposure. Time-dated pregnant rats were divided between normoxic and hypoxic (10.5% O(2) on days 15-21 of gestation) groups. Hearts of 3-month-old progeny were subjected to ischemia and reperfusion (I/R) injury in a Langendorff preparation. Preischemic values of left ventricle (LV) function were the same between control and hypoxic animals. Prenatal hypoxia significantly decreased postischemic recovery of LV function and increased cardiac enzyme release and infarct size in adult male, but not female, rats. This was associated with significant decreases in PKC(epsilon) and phospho-PKC(epsilon) levels in the LV of the male, but not female, rats. The PKC(epsilon) translocation inhibitor peptide (PKC(epsilon)-TIP) significantly decreased phospho-PKC(epsilon) in control male rats to the levels found in the hypoxic animals and abolished the difference in I/R injury observed between the control and hypoxic rats. In females, PKC(epsilon)-TIP inhibited PKC(epsilon) phosphorylation and decreased postischemic recovery of LV function equally well in both control and hypoxic animals. PKC(epsilon)-TIP had no effect on PKCdelta activation in either male or female hearts. The results demonstrated that prenatal hypoxia caused an increase in heart susceptibility to ischemia and reperfusion injury in offspring in a sex-dependent manner, which was due to fetal programming of PKC(epsilon) gene repression resulting in a down-regulation of PKC(epsilon) function in the heart of adult male offspring.
Assuntos
Hipóxia Fetal/enzimologia , Predisposição Genética para Doença , Isquemia Miocárdica/enzimologia , Traumatismo por Reperfusão Miocárdica/enzimologia , Proteínas da Gravidez/fisiologia , Efeitos Tardios da Exposição Pré-Natal/enzimologia , Proteína Quinase C-épsilon/fisiologia , Caracteres Sexuais , Fatores Etários , Animais , Regulação para Baixo/genética , Feminino , Hipóxia Fetal/genética , Masculino , Isquemia Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/genética , Gravidez , Proteínas da Gravidez/genética , Efeitos Tardios da Exposição Pré-Natal/genética , Proteína Quinase C-épsilon/genética , Distribuição Aleatória , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: Chronic hypoxia in utero (CHU) is one of the most common insults to fetal development and may be associated with poor cardiac recovery from ischaemia-reperfusion injury, yet the effects on normal cardiac mechanical performance are poorly understood. METHODS: Pregnant female wistar rats were exposed to hypoxia (12% oxygen, balance nitrogen) for days 10-20 of pregnancy. Pups were born into normal room air and weaned normally. At 10 weeks of age, hearts were excised under anaesthesia and underwent retrograde 'Langendorff' perfusion. Mechanical performance was measured at constant filling pressure (100 cm H2O) with intraventricular balloon. Left ventricular free wall was dissected away and capillary density estimated following alkaline phosphatase staining. Expression of SERCA2a and Nitric Oxide Synthases (NOS) proteins were estimated by immunoblotting. RESULTS: CHU significantly increased body mass (P < 0.001) compared with age-matched control rats but was without effect on relative cardiac mass. For incremental increases in left ventricular balloon volume, diastolic pressure was preserved. However, systolic pressure was significantly greater following CHU for balloon volume = 50 microl (P < 0.01) and up to 200 microl (P < 0.05). For higher balloon volumes systolic pressure was not significantly different from control. Developed pressures were correspondingly increased relative to controls for balloon volumes up to 250 microl (P < 0.05). Left ventricular free wall capillary density was significantly decreased in both epicardium (18%; P < 0.05) and endocardium (11%; P < 0.05) despite preserved coronary flow. Western blot analysis revealed no change to the expression of SERCA2a or nNOS but immuno-detectable eNOS protein was significantly decreased (P < 0.001) in cardiac tissue following chronic hypoxia in utero. CONCLUSION: These data offer potential mechanisms for poor recovery following ischaemia, including decreased coronary flow reserve and impaired angiogenesis with subsequent detrimental effects of post-natal cardiac performance.
Assuntos
Hipóxia Fetal/embriologia , Miocárdio/enzimologia , Óxido Nítrico Sintase Tipo III/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Animais , Capilares/metabolismo , Capilares/patologia , Circulação Coronária/fisiologia , Vasos Coronários/patologia , Feminino , Hipóxia Fetal/enzimologia , Hipóxia Fetal/patologia , Hipóxia Fetal/fisiopatologia , Técnicas In Vitro , Masculino , Contração Miocárdica/fisiologia , Traumatismo por Reperfusão Miocárdica/congênito , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miocárdio/patologia , Gravidez , Efeitos Tardios da Exposição Pré-Natal , Ratos , Ratos Wistar , Pressão Ventricular/fisiologiaRESUMO
Intrauterine hypoxia impacts fetal growth and organ function. Inducible nitric oxide synthase (iNOS) and neuronal NOS (nNOS) expression was measured to assess the response of fetal hearts to hypoxic (HPX) stress. Pregnant guinea pigs were housed in a hypoxic chamber (10.5% O2 for 14 d, n = 17) or room air [normoxic (NMX), n = 17]. Hearts of anesthetized near-term fetuses were removed. mRNA [hypoxia-inducible factor, (HIF)-1alpha, 1beta, 2alpha, 3alpha, iNOS, and nNOS] and protein levels (HIF-1alpha, iNOS, and nNOS) of fetal cardiac left ventricles were quantified by real time polymerase chain reaction (PCR) and Western analysis, respectively. Cardiac nitrite/nitrate levels were measured in the presence/absence of L-N6-(1-iminoethyl)-lysine (L-NIL), an iNOS inhibitor, administered to pregnant sows. Hypoxia significantly increased fetal cardiac HIF-1alpha and -2alpha mRNA, HIF-1alpha protein but not HIF-3alpha or -1beta mRNA levels. Hypoxia increased both iNOS mRNA (by 5x) and protein (by 23%) levels but had no effect on nNOS levels. Nitrite/nitrate levels were increased in HPX hearts by 2.5x and decreased with L-NIL by 67 +/- 14%. Thus, up-regulation of iNOS-derived nitric oxide (NO) generation is an important mechanism by which fetal hearts respond to chronic hypoxic stress.
Assuntos
Hipóxia Fetal/enzimologia , Coração/embriologia , Miocárdio/enzimologia , Óxido Nítrico Sintase Tipo II/biossíntese , Óxido Nítrico/metabolismo , Animais , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Doença Crônica , Modelos Animais de Doenças , Indução Enzimática , Inibidores Enzimáticos/farmacologia , Feminino , Hipóxia Fetal/embriologia , Idade Gestacional , Cobaias , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Lisina/análogos & derivados , Lisina/farmacologia , Nitratos/metabolismo , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/genética , Nitritos/metabolismo , Gravidez , RNA Mensageiro/metabolismo , Regulação para CimaRESUMO
Growing evidence indicates that nitrite, NO2-, serves as a circulating reservoir of nitric oxide (NO) bioactivity that is activated during physiological and pathological hypoxia. One of the intravascular mechanisms for nitrite conversion to NO is a chemical nitrite reductase activity of deoxyhemoglobin. The rate of NO production from this reaction is increased when hemoglobin is in the R conformation. Because the mammalian fetus exists in a low-oxygen environment compared with the adult and is exposed to episodes of severe ischemia during the normal birthing process, and because fetal hemoglobin assumes the R conformation more readily than adult hemoglobin, we hypothesized that nitrite reduction to NO may be enhanced in the fetal circulation. We found that the reaction was faster for fetal than maternal hemoglobin or blood and that the reactions were fastest at 50-80% oxygen saturation, consistent with an R-state catalysis that is predominant for fetal hemoglobin. Nitrite concentrations were similar in blood taken from chronically instrumented normoxic ewes and their fetuses but were elevated in response to chronic hypoxia. The findings suggest an augmented nitrite reductase activity of fetal hemoglobin and that the production of nitrite may participate in the regulation of vascular NO homeostasis in the fetus.
Assuntos
Sangue Fetal/enzimologia , Hemoglobinas/metabolismo , Óxido Nítrico/sangue , Nitrito Redutases/sangue , Nitritos/sangue , Animais , Biocatálise , Doença Crônica , Modelos Animais de Doenças , Ditionita/química , Feminino , Hipóxia Fetal/enzimologia , Hemoglobinas/química , Concentração de Íons de Hidrogênio , Hipóxia/enzimologia , Cinética , Metemoglobina/metabolismo , Oxigênio/sangue , Gravidez , Conformação Proteica , OvinosRESUMO
Intrauterine asphyxia is one of the major contributors for perinatal death, mental and physical disorders of surviving children. Brain-derived neurotrophic factor (BDNF) provides a promising solution to hypoxic injury due to its survival-promoting effects. In an attempt to identify possible molecular mechanisms underlying the neuroprotective role of BDNF, we studied extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase (PI-3-K) and p38 mitogen-activated protein kinase (MAPK) pathways. We demonstrated that BDNF protected cortical neurons against hypoxic injury in vitro via activation of both the ERK and PI-3-K pathways but not the p38 MAPK pathway. We also showed that both hypoxic stimuli and exogenous BDNF treatment phosphorylated the cyclic AMP response element-binding protein (CREB) and that CREB phosphorylation induced by BDNF was mediated via the ERK pathway in cultured cortical neurons.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/farmacologia , Encéfalo/enzimologia , Hipóxia Fetal/enzimologia , Hipóxia-Isquemia Encefálica/enzimologia , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Animais , Encéfalo/fisiopatologia , Células Cultivadas , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/enzimologia , Córtex Cerebral/fisiopatologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/efeitos dos fármacos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Feminino , Hipóxia Fetal/fisiopatologia , Hipóxia-Isquemia Encefálica/tratamento farmacológico , Hipóxia-Isquemia Encefálica/fisiopatologia , Neurônios/efeitos dos fármacos , Neurônios/enzimologia , Fármacos Neuroprotetores/farmacologia , Fosforilação/efeitos dos fármacos , Gravidez , RatosRESUMO
OBJECTIVE: Glutathione is a natural antioxidant in the fetus and adult. We sought to determine whether maternal hypoxia alters glutathione levels in fetal organs as an adaptive response to the reduced oxygenation. STUDY DESIGN: Timed pregnant guinea pigs were housed in either a Plexiglas chamber containing 10.5% O(2) from 46 to 60 days gestation (HPX, n=6) or in room air, as the normoxic control (NMX, n=5). Pregnant guinea pigs were anesthetized at near term ( approximately 60 days, term=65 days) and liver, lungand kidney were excised from anesthetized fetuses and stored frozen (-80 degrees C) prior to sample processing. Using the hypoxia marker, pimonidazole, we measured a hypoxia-induced increase in stained cells of fetal liver compared to no change in either the lung or kidney. To measure the effect of hypoxia among different organs, total glutathione (GSH) content and protein levels of gamma-glutamyl cysteine synthetase (gamma-GCS) were measured from the same organs. RESULTS: Maternal hypoxia increased (P<0.05) total glutathione levels by 121% in the fetal liver but had no effect in either fetal lung or kidney. Chronic hypoxia increased (P<0.05) gamma-GCS protein levels in all three fetal organs studied. CONCLUSION: These results demonstrate that the fetal response to maternal hypoxia may be organ specific. The increase in fetal liver glutathione via upregulation of gamma-GCS may be an important adaptive response to prolonged hypoxic stress.
Assuntos
Hipóxia Fetal/metabolismo , Glutamato-Cisteína Ligase/biossíntese , Glutationa/metabolismo , Fígado/metabolismo , Animais , Western Blotting , Peso Corporal/fisiologia , Feminino , Hipóxia Fetal/sangue , Hipóxia Fetal/enzimologia , Feto , Cobaias , Imuno-Histoquímica , Rim/enzimologia , Rim/metabolismo , Ácido Láctico/sangue , Fígado/embriologia , Fígado/enzimologia , Pulmão/enzimologia , Pulmão/metabolismo , Tamanho do Órgão/fisiologia , Gravidez , Ácido Pirúvico/sangueRESUMO
Complications during pregnancy and birth asphyxia lead to brain injury, with devastating consequences for the neonate. In this paper we present evidence that the steroid environment during pregnancy and at birth aids in protecting the fetus and neonate from asphyxia-induced injury. Earlier studies show that the placental progesterone production has a role in the synthesis and release of neuroactive steroids or their precursors into the fetal circulation. Placental precursor support leads to remarkably high concentrations of allopregnanolone in the fetal brain and to a dramatic decline with the loss of the placenta at birth. These elevated concentrations influence the distinct behavioral states displayed by the late gestation fetus and exert a suppressive effect that maintains sleep-like behavioral states that are present for much of fetal life. This suppression reduces CNS excitability and suppresses excitotoxicity. With the availability of adequate precursors, mechanisms within the fetal brain ultimately control neurosteroid levels. These mechanisms respond to episodes of acute hypoxia by increasing expression of 5alpha-reductase and P450scc enzymes and allopregnanolone synthesis in the brain. This allopregnanolone response, and potentially that of other neurosteroids including 5alpha-tetrahydrodeoxycorticosterone (TH-DOC), reduces hippocampal cell death following acute asphyxia and suggests that stimulation of neurosteroid production may protect the fetal brain. Importantly, inhibition of neurosteroid synthesis in the fetal brain increases the basal cell death suggesting a role in controlling developmental processes late in gestation. Synthesis of neurosteroid precursors in the fetal adrenal such as deoxycorticosterone (DOC), and their conversion to active neurosteroids in the fetal brain may also have a role in neuroprotection. This suggests that the adrenal glands provide precursor DOC for neurosteroid synthesis after birth and this may lead to a switch from allopregnanolone alone to neuroprotection mediated by allopregnanolone and TH-DOC.
Assuntos
Animais Recém-Nascidos/metabolismo , Feto/metabolismo , Recém-Nascido/metabolismo , Neurotransmissores/metabolismo , Complicações na Gravidez/fisiopatologia , Esteroides/metabolismo , Animais , Química Encefálica/fisiologia , Encefalopatias/congênito , Feminino , Hipóxia Fetal/enzimologia , Hipóxia Fetal/metabolismo , Feto/irrigação sanguínea , Feto/enzimologia , Humanos , Placenta/fisiologia , Gravidez , Complicações na Gravidez/metabolismo , Pregnanolona/metabolismoRESUMO
We previously found that prenatal hypoxia induces a significant increase in the levels of active Caspase 3 at 60 min post-hypoxia (p-h) and in the number of TUNEL-positive pyknotic cells, which peaks at 6h p-h. The aim of this work was to study alterations in MAPKs pathways and the effect of specific inhibitors of the JNK (SP600125) and p38 (SB203580) pathways following acute hypoxia in chick optic lobe at embryonic day (ED) 12. To this end, JNK, p38 and ERK1-2 protein kinase expression levels were determined by Western blot in both their active and inactive forms, evaluated at successive p-h times. At 10 and 30 min p-h the P-JNK/JNK ratio was 1.912+/-0.341 and 1.920+/-0.304, respectively. Concomitantly, at 0 min p-h the P-p38/p38 ratio was 1.657+/-0.203. Lastly, the P-ERK/ERK ratio proving non-significant throughout. When inhibitors for JNK and p38 were used, we observed a decrease in the values of active Caspase 3 at 60 min p-h, which correlated with the control values in the parameters of TUNEL-positive cells at 6h p-h. Analysis for P-ATF-2 demonstrated an increase in hypoxic embryos compared to control ones which was reverted in a dose-dependent manner with the use of both inhibitors. All these results indicate that at ED 12, acute hypoxia might be differentially activating JNK and p38 pathways, without affecting the ERK pathway, which in turn would be activating Caspase 3, thus leading to cell death by apoptosis. Furthermore, JNK and p38 activation precede in time the programmed cell death induced by hypoxia.
Assuntos
Apoptose/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/biossíntese , Hipóxia Fetal/enzimologia , Hipóxia Fetal/patologia , Proteínas Quinases JNK Ativadas por Mitógeno/biossíntese , Proteínas Quinases p38 Ativadas por Mitógeno/biossíntese , Animais , Apoptose/genética , Western Blotting , Caspase 3/biossíntese , Caspase 3/genética , Embrião de Galinha , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/genética , Hipóxia Fetal/genética , Marcação In Situ das Extremidades Cortadas , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
OBJECTIVE: The purpose of the present study was to quantify the effect of chronic hypoxia on endothelial nitric oxide synthase (eNOS) gene and protein expression of fetal coronary artery segments and cardiac tissue of fetal guinea pig hearts. METHODS: Time-mated pregnant guinea pigs (term = 65 days) were housed in room air (NMX, n = 6) or in a hypoxic chamber containing 10.5% O2 for 14 days (HPX14, n = 6). At near term (60 days gestation), fetuses were excised from anesthetized animals via hysterotomy and hearts were removed and weighed. Both coronary artery segments and cardiac ventricle were excised from the same hearts, frozen, and stored at -80 C until ready for study. eNOS mRNA was quantified using real-time polymerase chain reaction (PCR) based on SYBR Green I labeling (BioRad Laboratories, Hercules, CA) using eNOS primers obtained from GeneBank normalized to 18S. eNOS proteins were quantified by Western immunoblotting using eNOS antibody (1:200) and normalized to normoxic controls. eNOS cell-specific localization in the fetal guinea pig heart was performed by double immunofluorescence staining. RESULTS: Both coronary artery endothelial cells (EC) and cardiomyocytes (CM) but not vascular smooth muscle cells of normoxic hearts exhibited positive immunostaining of eNOS protein. Chronic hypoxia significantly (P < .05) increased both eNOS mRNA and protein levels of coronary artery segments (by 210.6% and 51.4%, respectively) but decreased (P < .05) mRNA and protein of cardiac tissue (by 50.0% and 40.6%, respectively) in the same hearts. CONCLUSIONS: Chronic fetal hypoxia, after 14 days, induces sustained changes in eNOS gene and eNOS protein expression that differ between coronary and cardiac tissue in the fetal guinea pig heart. This study suggests that while the functional roles of altered eNOS expression in hypoxic fetal hearts remain unclear, the site at which eNOS expression is altered may be important in the adaptive response of the fetal heart to hypoxia.
Assuntos
Vasos Coronários/enzimologia , Coração Fetal/enzimologia , Hipóxia Fetal/enzimologia , Óxido Nítrico Sintase Tipo III/biossíntese , Óxido Nítrico Sintase Tipo III/genética , Animais , Western Blotting , Feminino , Imunofluorescência , Expressão Gênica , Cobaias , Modelos Animais , Reação em Cadeia da Polimerase , Gravidez , RNA Mensageiro/metabolismoRESUMO
The present study was undertaken to determine (1) if hypoxia-induced down-regulation of placental 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2; encoded by HSD11B2 gene) activity and protein in human trophoblast cells during in vitro differentiation was mediated at the level of HSD11B2 gene transcription; and (2) whether the reduced placental 11beta-HSD2 in pregnancies complicated with fetal growth restriction (FGR) was a consequence of intrinsic abnormalities in trophoblast cells. Trophoblast cells were isolated from uncomplicated pregnancies and those complicated with FGR at term, and cultured for up to 72 h under normoxic (20% oxygen) or hypoxic (1% oxygen) conditions. Under normoxia, 11beta-HSD2 activity and protein increased progressively over the 72 h culture period, which was accompanied by a corresponding rise in 11beta-HSD2 mRNA. As demonstrated previously, hypoxia blocked the increase in levels of both 11beta-HSD2 activity and protein within the first 24h. In contrast, although hypoxia also prevented the rise in 11beta-HSD2 mRNA, it did not do so until 48 h. This time-dependent effect of hypoxia on placental 11beta-HSD2 activity/protein and mRNA suggests a dual mechanism of action whereby hypoxia may induce a rapid down-regulation of 11beta-HSD2 protein synthesis, which occurs initially at the level of translation, and later extends to the level of transcription. Indeed, transient transfection studies demonstrated that hypoxia diminished HSD11B2 promoter activity. When trophoblast cells isolated from FGR placentas were cultured and allowed to differentiate under the same conditions, they not only exhibited a similar pattern of 11beta-HSD2 activity and mRNA expression but also responded to hypoxia similarly to those from normal placentas. This suggests that the reduced placental 11beta-HSD2 in FGR is not due to intrinsic abnormalities in trophoblast cells, but likely a result of extrinsic factors associated with FGR.