Maria Luiza M. Barreto-Chaves

Thyroid hormone stimulates NO production via activation of the PI3K/Akt pathway in vascular myocytes

AIMS Thyroid hormone (TH) rapidly relaxes vascular smooth muscle cells (VSMCs). However, the mechanisms involved in this effect remain unclear. We hypothesize that TH-induced rapid vascular relaxation is mediated by VSMC-derived nitric oxide (NO) production and is associated with the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signalling pathway. METHODS AND RESULTS NO levels were determined using a NO-specific fluorescent dye (DAF-2) and nitrite (NO2-) levels. Expression of NO synthase (NOS) isoforms and proteins of the PI3K/Akt pathway was determined by both western blotting and immunocytochemistry. Myosin light chain (MLC) phosphorylation levels were also investigated by western blotting. Exposure of cultured VSMCs from rat thoracic aortas to triiodothyronine (T3) resulted in a significant decrease of MLC phosphorylation levels. T3 also induced a rapid increase in Akt phosphorylation and increased NO production in a dose-dependent manner (0.001-1 microM). VSMCs stimulated with T3 for 30 min showed an increase in the expression of all three NOS isoforms and augmented NO production, effects that were prevented by inhibitors of PI3K. Vascular reactivity studies showed that vessels treated with T3 displayed a decreased response to phenylephrine, which was reversed by NOS inhibition. These data suggest that T3 treatment induces greater generation of NO both in aorta and VSMCs and that this phenomenon is endothelium independent. In addition, these findings show for the first time that the PI3K/Akt signalling pathway is involved in T3-induced NO production by VSMCs, which occurs with expressive participation of inducible and neuronal NOS. CONCLUSION Our data strongly indicate that T3 causes NO-dependent rapid relaxation of VSMC and that this effect is mediated by the PI3K/Akt signalling pathway.

Ecto-nucleotide pyrophosphatase/phosphodiesterase as part of a multiple system for nucleotide hydrolysis by platelets from rats: Kinetic characterization and biochemical properties

In this study, we describe an ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP) activity in rat platelets. Using p-nitrophenyl 5′-thymidine monophosphate (p-Nph-5′-TMP) as a substrate for E-NPP, we demonstrate an enzyme activity that shares the major biochemical properties described for E-NPPs: alkaline pH dependence, divalent cation dependence and blockade of activity by metal ion chelator. Km and Vmax values for p-Nph-5′-TMP hydrolysis were found to be 106 ± 18 µM and 3.44 ± 0.18 nmol p-nitrophenol/min/mg (mean ± SD, n = 5). We hypothesize that an E-NPP is co-localized with an ecto-nucleoside triphosphate diphosphohydrolase and an ecto-5′-nucleotidase on the platelet surface, as part of a multiple system for nucleotide hydrolysis, since they can act under distinct physiological conditions and can be differently regulated. Thus, 0.25 mM suramin inhibited p-Nph-5′-TMP, ATP and ADP hydrolysis, while 0.5 mM AMP decreased only p-Nph-5′-TMP hydrolysis. Besides, 5.0, 10 and 20 mM sodium azide just inhibited ATP and ADP hydrolysis. Angiotensin II (5.0 and 10 nM) affected only ADP hydrolysis. Gadolinium chloride (0.2 and 0.5 mM) strongly inhibited the ATP and ADP hydrolysis. The E-NPP described here represents a novel insight into the control of platelet purinergic signaling.

Circulating renin-angiotensin system and catecholamines in childhood: is there a role for birthweight?

There have been only a few reports on the sympathoadrenal and renin-angiotensin systems in children of small gestational age. The purpose of the present study was to investigate plasma levels of ACE (angiotensin-converting enzyme) activity, angiotensin and catecholamines in 8- to 13-year-old children and to determine whether there are correlations between the components of these systems with both birthweight and BP (blood pressure) levels. This clinical study included 66 children (35 boys and 31 girls) in two groups: those born at term with an appropriate birthweight [AGA (appropriate-for-gestational age) group, n=31] and those born at term but with a small birthweight for gestational age [SGA (small-for-gestational age) group, n=35]. Concentrations of angiotensin, catecholamines and ACE activity were determined in plasma. Circulating noradrenaline levels were significantly elevated in SGA girls compared with AGA girls (P=0.036). In addition, angiotensin II and ACE activity were higher in SGA boys (P=0.024 and P=0.050 respectively). There was a significant association of the circulating levels of both angiotensin II and ACE activity with BP levels in our study population. Although the underlying mechanisms that link restricted fetal growth with later cardiovascular events are not fully understood, the findings in the present study support the link between low birthweight and overactivity of both sympathoadrenal and renin-angiotensin systems into later childhood.

Cardiac angiotensin II type I and type II receptors are increased in rats submitted to experimental hypothyroidism

This study assessed the behaviour of angiotensin II (Ang II) receptors in an experimental hypothyroidism model in male Wistar rats. Animals were subjected to thyroidectomy and resting for 14 days. The alteration of cardiac mass was evaluated by total heart weight (HW), right ventricle weight (RVW), left ventricle weight (LVW), ratio of HW, RVW and LVW to body weight (BW) and atrial natriuretic factor (ANF) expression. Cardiac and plasma Ang II levels and serum T3 and T4 were determined. The mRNA and protein levels of Ang II receptors were investigated by RT‐PCR and Western blotting, respectively. Functional analyses were performed using binding assays. T3 and T4 levels and the haemodynamic parameters confirmed the hypothyroid state. HW/BW, RVW/BW and LVW/BW ratios and the ANF expression were lower than those of control animals. No change was observed in cardiac or plasma Ang II levels. Both AT1/AT2 mRNA and protein levels were increased in the heart of hypothyroid animals due to a significant increase of these receptors in the RV. Experiments performed in cardiomyocytes showed a direct effect promoted by low thyroid hormone levels upon AT1 and AT2 receptors, discarding possible influence of haemodynamic parameters. Functional assays showed that both receptors are able to bind Ang II. Herein, we have identified, for the first time, a close and direct relation of elevated Ang II receptor levels in hypothyroidism. Whether the increase in these receptors in hypothyroidism is an alternative mechanism to compensate the atrophic state of heart or whether it may represent a potential means to the progression of heart failure remains unknown.

Angiotensin type 1 (AT1) and type 2 (AT2) receptors mediate the increase in TGF-β1 in thyroid hormone-induced cardiac hypertrophy

Increased thyroid hormone (TH) levels are known to induce cardiac hypertrophy. Some studies have provided evidence for a functional link between angiotensin II (ANG II) and transforming growth factor β1 (TGF-β1) in the heart, both being able to also induce cardiac hypertrophy. However, the contribution of this growth factor activated directly by TH or indirectly by ANG II in cardiac hypertrophy development remains unknown. To analyze the possible role of TGF-β1 in cardiac hypertrophy induced by TH and also to evaluate if the TGF-β1 effect is mediated by ANG II receptors, we employed Wistar rats separated into control, hypothyroid (hypo) and hyperthyroid (T4 − 10) groups combined or not with ANG II receptor blockers (losartan or PD123319). Serum levels of T3 and T4, systolic pressure and heart rate confirmed the thyroid state of the groups. The T4 − 10 group presented a significant increase in cardiac TGF-β1 levels; however, TGF-β1 levels in the hypo group did not change in relation to the control. Inhibition of the increase in cardiac TGF-β1 levels was observed in the groups treated with T4 in association with losartan or PD123319 when compared to the T4 − 10 group. These results demonstrate for the first time the TH-modulated induction of cardiac TGF-β1 in cardiac hypertrophy, and that this effect is mediated by ANG II receptors.

Tissue-specific modulation of angiotensin-converting enzyme (ACE) in hyperthyroidism

We have previously demonstrated the interaction between the RAS and thyroid hormones (TH). The present study was designed to determine the role of TH in the local regulation of ACE activity and expression in different tissues. Adult male Wistar rats were randomized into three groups: T4-25 and T4-100 (0.025 and 0.100mg/kg of body weight/day of l-thyroxine for 14 days, respectively) and control. Hemodynamic parameters as well as cardiac and renal hypertrophy were evaluated. ACE activity and mRNA levels were determined by Fluorimetric and Northern blot assays, respectively. Both doses increased SBP and HR, as well as inducing cardiac and renal hypertrophy. Pulmonary and serum ACE levels were comparable among the groups. Both doses promoted increased renal ACE activity and expression but surprisingly ACE was diminished in the heart in both hyperthyroid groups. This change was mediated by a tissue-specific transcription mechanism.

Hypothyroidism changes adenine nucleotide hydrolysis in synaptosomes from hippocampus and cerebral cortex of rats in different phases of development

The influence of the thyroid hormones on the normal function of the mammalian central nervous system depends on the brain region and on the developmental stage. Adenine nucleotides and their products also affect the brain function; ATP is an excitatory neurotransmitter, and adenosine has inhibitory effects on neurotransmission. Thus, this study aimed to evaluate the effects of hypothyroidism on the hydrolysis of ATP to adenosine in hippocampal and cortical synaptosomes and blood serum of rats during different phases of development. Rats aged 60 and 420 days old were divided into three groups: control, sham‐operated and hypothyroid. Hypothyroidism was induced in these rats by thyroidectomy and methimazole (0.05%) added to their drinking water for 14 days. Neonatal hypothyroidism was induced by adding 0.02% methimazole in the drinking water from day 9 of gestation, and continually until 14 days old. Hypothyroidism increased the AMP hydrolysis in both hippocampus and cerebral cortex synaptosomes of rats in all aged tested. In blood serum, thyroid hormones deficiency increased the AMP hydrolysis in 14‐day‐old rats and the hydrolysis of ATP, ADP and AMP in 60‐day‐old rats; however, no alteration was observed in 420‐day‐old rats. Thus, our results suggest the involvement of the 5′‐nucleotidase in synaptic function control in hypothyroidism throughout brain development.

Redox-sensitive prosurvival and proapoptotic protein expression in the myocardial remodeling post-infarction in rats

In this study, we investigated the oxidative stress influence in some prosurvival and proapoptotic proteins after myocardial infarction (MI). Male Wistar rats were divided in two groups: Sham-operated (control) and MI. MI was induced by left coronary artery occlusion. 28-days after surgery, echocardiographic, morphometric, and hemodynamic parameters were evaluated. Redox status (reduced to oxidized glutathione ratio, GSH/GSSG) and hydrogen peroxide levels (H2O2) were measured in heart tissue. The p-ERK/ERK, p-Akt/Akt, p-mTOR/mTOR and p-GSK-3β/GSK-3β ratios, as well as apoptosis-inducing factor (AIF) myocardial protein expression were quantified by Western blot. MI group showed an increase in cardiac hypertrophy (23%) associated with a decrease in ejection fraction (38%) and increase in left ventricular end-diastolic pressure (82%) when compared to control, characterizing ventricular dysfunction. Redox status imbalance was seen in MI animals, as evidenced by the decrease in the GSH/GSSG ratio (30%) and increased levels of H2O2 (45%). This group also showed an increase in the ERK phosphorylation and a reduction of Akt and mTOR phosphorylation when compared to control. Moreover, we showed a reduction in the GSK-3β phosphorylation and an increase in AIF protein expression in MI group. Taken together, our results show increased H2O2 levels and cellular redox imbalance associated to a higher p-ERK and AIF immunocontent, which would contribute to a maladaptive hypertrophy phenotype.

MiRNA-208a and miRNA-208b are triggered in thyroid hormone-induced cardiac hypertrophy - role of type 1 Angiotensin II receptor (AT1R) on miRNA-208a/α-MHC modulation

Hyperthyroidism promotes cardiac hypertrophy and the Angiotensin type 1 receptor (AT1R) has been demonstrated to mediate part of this response. Recent studies have uncovered a potentially important role for the microRNAs (miRNAs) in the control of diverse aspects of cardiac function. Then, the objective of the present study was to investigate the action promoted by hyperthyroidism on β-MHC/miR-208b expression and on α-MHC/miR-208a expression, as well as the possible contribution of the AT1R in this event. The findings of this study confirmed that AT1R is a key mediator of the cardiac hypertrophy induced by hyperthyroidism. Additionally, we demonstrated that like β-MHC, miR-208b was down-regulated in the hyperthyroid group. Similarly, like the expression of its host gene, α-MHC, miR-208a expression was up-regulated in response to hyperthyroidism. Finally, our data suggest for the first time that AT1R mediates the hyperthyroidism-induced increase on cardiac miRNA-208a/α-MHC levels, while does not influence on the reduction of miRNA-208b/β-MHC levels.

Reactive oxygen and nitrogen species balance in the determination of thyroid hormones-induced cardiac hypertrophy mediated by renin–angiotensin system

Role of reactive oxygen species (ROS)/nitric oxide (NO) balance and renin-angiotensin system in mediating cardiac hypertrophy in hyperthyroidism was evaluated in an in vivo and in vitro experimental model. Male Wistar rats were divided into four groups: control, thyroid hormone, vitamin E (or Trolox, its hydrosoluble analogue), thyroid hormone+vitamin E. Angiotensin II receptor (AT1/AT2) gene expression, immunocontent of AT1/AT2 receptors, angiotensinogen, NADPH oxidase (Nox2), and nitric oxide synthase isoforms, as well as ROS concentration (hydrogen peroxide and superoxide anion) were quantified in myocardium. Thyroid hormone increased ROS and NO metabolites, iNOS, nNOS and eNOS isoforms and it was accompanied by cardiac hypertrophy. AT1/AT2 expression and the immunocontent of angiotensinogen and Nox2 were enhanced by thyroid hormone. Antioxidants reduced ROS levels, Nox2, AT1/AT2, NOS isoforms and cardiac hypertrophy. In conclusion, ROS/NO balance may play a role in the control of thyroid hormone-induced cardiac hypertrophy mediated by renin-angiotensin system.