Cannabinoids impact on pregnancy: effects in trophoblast cells

Cannabinoids (CBs) can be classified as: phytocannabinoids, the constituents of the Cannabis sativa plant; synthetic cannabinoids lab-synthesized and the endocannabinoids that are endogenous lipid mediators. Cannabinoid compounds activate cannabinoid receptors – CB1 and CB2. The most prevalent psychoactive phytocannabinoid is Δ9tetrahydrocannabinol (THC), but more than 60 different CBs were already identified in the plant. The best characterized endocannabinoids (eCBs) are anandamide (AEA) and 2arachidonoylglycerol (2-AG), that are involved in several physiological processes including synaptic plasticity, pain modulation, energy homeostasis and reproduction. On the other hand, some synthetic cannabinoids that were initially designed for medical research, are now used as drugs of abuse. During the period of placental development, highly dynamic processes of remodeling occur, involving proliferation, apoptosis, differentiation and invasion of trophoblasts. It is known that a tight control of eCBs levels is required for normal pregnancy progression and that eCBs are involved in trophoblast cells turnover. Therefore, by sharing activation of the same receptors, exposure to exocannabinoids either by recreational or medicinal use may lead to alterations in the eCBs levels and in the endocannabinoid system homeostasis In this work, it was studied the impact of CBs in BeWo trophoblastic cells and in primary cultures of human cytotrophoblasts. Cells were treated for 24 hours with different concentrations of THC, the synthetic cannabinoid WIN‐55,212 (WIN) and 2-AG. Treatment with THC did not affect BeWo cells viability while WIN and 2-AG caused a dose-dependent viability loss. Morphological studies together with biochemical markers indicate that 2-AG is able to induce apoptosis in cytotrophoblasts. On the other hand, morphological studies after acridine orange staining suggest that autophagy may take part in WIN-induced loss of cell viability. All cannabinoids caused a decrease in mitochondrial membrane potential (Δψm) but only 2-AG led to ROS/RNS generation, though no changes in glutathione levels were observed. In addition, ER-stress may be involved in the 2-AG induced-oxidative stress, as preliminary results point to an increase in CCAAT-enhancer-binding protein homologous protein (CHOP) expression. Besides the decrease in cell viability, alterations in cell cycle progression were observed. WIN treatment induced a cell cycle arrest in G0/G1 phase, whereas 2-AG induced a cell cycle arrest in G2/M phase. Here it is reinforced the relevance of cannabinoid signaling in fundamental processes of cell proliferation and cell death in trophoblast cells. Since cannabis-based drugs are the most consumed illicit drugs worldwide and some of the most consumed recreational drugs by pregnant women, this study may contribute to the understanding of the impact of such substances in human reproduction.

The role of non-coding RNA in the development of pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a progressive disease of the small pulmonary arteries, characterised by pulmonary vascular remodelling due to excessive proliferation and resistance to apoptosis of pulmonary artery endothelial cells (PAECs) and pulmonary artery smooth muscle cells (PASMCs). The increased pulmonary vascular resistance and elevated pulmonary artery pressures result in right heart failure and premature death. Germline mutations of the bone morphogenetic protein receptor-2 (bmpr2) gene, a receptor of the transforming growth factor beta (TGF-β) superfamily, account for approximately 75%-80% of the cases of heritable form of PAH (HPAH) and 20% of sporadic cases or idiopathic PAH (IPAH). IPAH patients without known bmpr2 mutations show reduced expression of BMPR2. However only ~ 20% of bmpr2-mutation carriers will develop the disease, due to an incomplete penetrance, thus the need for a ‘second hit’ including other genetic and/or environmental factors is accepted. Diagnosis of PAH occurs most frequently when patients have reached an advanced stage of disease. Although modern PAH therapies can markedly improve a patient’s symptoms and slow the rate of clinical deterioration, the mortality rate from PAH remains unacceptably high. Therefore, the development of novel therapeutic approaches is required for the treatment of this multifaceted disease. Noncoding RNAs (ncRNAs) include microRNAs (miRNAs) and long noncoding RNAs (lncRNAs). MiRNAs are ~ 22 nucleotide long and act as negative regulators of gene ex-pression via degradation or translational inhibition of their target mRNAs. Previous studies showed extensive evidence for the role of miRNAs in the development of PAH. LncRNAs are transcribed RNA molecules greater than 200 nucleotides in length. Similar to classical mRNA, lncRNAs are translated by RNA polymerase II and are generally alternatively spliced and polyadenylated. LncRNAs are highly versatile and function to regulate gene expression by diverse mechanisms. Unlike miRNAs, which exhibit well-defined actions in negatively regulating gene expression via the 3’-UTR of mRNAs, lncRNAs play more diverse and unpredictable regulatory roles. Although a number of lncRNAs have been intensively investigated in the cancer field, studies of the role of lncRNAs in vascular diseases such as PAH are still at a very early stage. The aim of this study was to investigate the involvement of specific ncRNAs in the development of PAH using experimental animal models and cell culture. The first ncRNA we focused on was miR-143, which is up-regulated in the lung and right ventricle tissues of various animal models of PH, as well as in the lungs and PASMCs of PAH patients. We show that genetic ablation of miR-143 is protective against the development of chronic hypoxia induced PH in mice, assessed via measurement of right ventricular systolic pressure (RVSP), right ventricular hypertrophy (RVH) and pulmonary vascular remodelling. We further report that knockdown of miR-143-3p in WT mice via anti-miR-143-3p administration prior to exposure of mice to chronic hypoxia significantly decreases certain indices of PH (RVSP) although no significant changes in RVH and pulmo-nary vascular remodelling were observed. However, a reversal study using antimiR-143-3p treatment to modulate miR-143-3p demonstrated a protective effect on RVSP, RVH, and muscularisation of pulmonary arteries in the mouse chronic hypoxia induced PH model. In vitro experiments showed that miR-143-3p overexpression promotes PASMC migration and inhibits PASMC apoptosis, while knockdown miR-143-3p elicits the opposite effect, with no effects observed on cellular proliferation. Interestingly, miR-143-3p-enriched exosomes derived from PASMCs mediated cell-to-cell communication between PASMCs and PAECs, contributing to the pro-migratory and pro-angiogenic phenotype of PAECs that underlies the pathogenesis of PAH. Previous work has shown that miR-145-5p expression is upregulated in the chronic hypoxia induced mouse model of PH, as well as in PAH patients. Genetic ablation and pharmacological inhibition (subcutaneous injection) of miR-145-5p exert a protective against the de-velopment of PAH. In order to explore the potential for alternative, more lung targeted delivery strategies, miR-145-5p expression was inhibited in WT mice using intranasal-delivered antimiR-145-5p both prior to and post exposure to chronic hypoxia. The decreased expression of miR-145-5p in lung showed no beneficial effect on the development of PH compared with control antimiRNA treated mice exposed to chronic hypoxia. Thus, miR-143-3p modulated both cellular and exosome-mediated responses in pulmonary vascular cells, while the inhibition of miR-143-3p prevented the development of experimental pulmonary hypertension. We focused on two lncRNAs in this project: Myocardin-induced Smooth Muscle Long noncoding RNA, Inducer of Differentiation (MYOSLID) and non-annotated Myolnc16, which were identified from RNA sequencing studies in human coronary artery smooth muscle cells (HCASMCs) that overexpress myocardin. MYOSLID was significantly in-creased in PASMCs from patients with IPAH compared to healthy controls and increased in circulating endothelial progenitor cells (EPCs) from bmpr2 mutant PAH patients. Exposure of PASMCs to hypoxia in vitro led to a significant upregulation in MYOSLID expres-sion. MYOSLID expression was also induced by treatment of PASMC with BMP4, TGF-β and PDGF, which are known to be triggers of PAH in vitro. Small interfering RNA (siR-NA)-mediated knockdown MYOSLID inhibited migration and induced cell apoptosis without affecting cell proliferation and upregulated several genes in the BMP pathway in-cluding bmpr1α, bmpr2, id1, and id3. Modulation of MYOSLID also affected expression of BMPR2 at the protein level. In addition, MYOSLID knockdown affected the BMP-Smad and BMP-non-Smad signalling pathways in PASMCs assessed by phosphorylation of Smad1/5/9 and ERK1/2, respectively. In PAECs, MYOSLID expression was also induced by hypoxia exposure, VEGF and FGF2 treatment. In addition, MYOSLID knockdown sig-nificantly decreased the proliferation of PAECs. Thus, MYOSLID may be a novel modulator in pulmonary vascular cell functions, likely through the BMP-Smad and –non-Smad pathways. Treatment of PASMCs with inflammatory cytokines (IL-1 and TNF-α) significantly in-duced the expression of Myolnc16 at a very early time point. Knockdown of Myolnc16 in vitro decreased the expression of il-6, and upregulated the expression of il-1 and il-8 in PASMCs. Moreover, the expression levels of chemokines (cxcl1, cxcl6 and cxcl8) were sig-nificantly decreased with Myolnc16 knockdown. In addition, Myolnc16 knockdown decreased the MAP kinase signalling pathway assessed by phosphorylation of ERK1/2 and p38 MAPK and inhibited cell migration and proliferation in PASMCs. Thus, Myolnc16 may a novel modulator of PASMCs functions through anti-inflammatory signalling pathways. In summary, in this thesis we have demonstrated how miR-143-3p plays a protective role in the development of PH both in vivo animal models and patients, as well as in vitro cell cul-ture. Moreover, we have showed the role of two novel lncRNAs in pulmonary vascular cells. These ncRNAs represent potential novel therapeutic targets for the treatment of PAH with further work addressing to investigate the target genes, and the pathways modulated by these ncRNAs during the development of PAH.

The role of inflammation in vascular function and pregnancy outcome in the pregnant stroke prone spontaneously hypertensive rat

During pregnancy, the maternal cardiovascular system undergoes major adaptation. One of these changes is a 40-50 % increase in circulating blood volume which requires a systemic remodelling of the vasculature in order to regulate maternal blood pressure and maximise blood supply to the developing placenta and fetus. These changes are broadly conserved between humans and rats making them an appropriate pre-clinical model in which to study the underlying mechanisms of pregnancy-dependent cardiovascular remodelling. Whilst women are normally protected against cardiovascular disease; pregnancy marks a period of time where women are susceptible to cardiovascular complications. Cardiovascular disease is the leading cause of maternal mortality in the United Kingdom; in particular hypertensive conditions are among the most common complications of pregnancy. One of the main underlying pathologies of these pregnancy complications is thought to be a failure of the maternal cardiovascular system to adapt. The remodelling of the uterine arteries, which directly supply the maternal-fetal interface, is paramount to a healthy pregnancy. Failure of the uterine arteries to remodel sufficiently can result in a number of obstetric complications such as preeclampsia, fetal growth restriction and spontaneous pregnancy loss. At present, it is poorly understood whether this deficient vascular response is due to a predisposition from existing maternal cardiovascular risk factors, the physiological changes that occur during pregnancy or a combination of both. Previous work in our group employed the stroke prone spontaneously hypertensive rat (SHRSP) as a model to investigate pregnancy-dependent remodelling of the uterine arteries. The SHRSP develops hypertension from 6 weeks of age and can be contrasted with the control strain, the Wistar Kyoto (WKY) rat. The phenotype of the SHRSP is therefore reflective of the clinical situation of maternal chronic hypertension during pregnancy. We showed that the SHRSP exhibited a deficient uterine artery remodelling response with respect to both structure and function accompanied by a reduction in litter size relative to the WKY at gestational day (GD) 18. A previous intervention study using nifedipine in the SHRSP achieved successful blood pressure reduction from 6 weeks of age and throughout pregnancy; however uterine artery remodelling and litter size at GD18 was not improved. We concluded that the abnormal uterine artery remodelling present in the SHRSP was independent of chronic hypertension. From these findings, we hypothesised that the SHRSP could be a novel model of spontaneously deficient uterine artery remodelling in response to pregnancy which was underpinned by other as yet unidentified cardiovascular risk factors. In Chapter 1 of this thesis, I have characterised the maternal, placental and fetal phenotype in pregnant (GD18) SHRSP and WKY. The pregnant SHRSP exhibit features of left ventricular hypertrophy in response to pregnancy and altered expression of maternal plasma biomarkers which have been previously associated with hypertension in human pregnancy. I developed a protocol for accurate dissection of the rat uteroplacental unit using qPCR probes specific for each layer. This allowed me to make an accurate and specific statement about gene expression in the SHRSP GD18 placenta; where oxidative stress related gene markers were increased in the vascular compartments. The majority of SHRSP placenta presented at GD18 with a blackened ring which encircled the tissue. Further investigation of the placenta using western blot for caspase 3 cleavage determined that this was likely due to increased cell death in the SHRSP placenta. The SHRSP also presented with a loss of one particular placental cell type at GD18: the glycogen cells. These cells could have been the target of cell death in the SHRSP placenta or were utilised early in pregnancy as a source of energy due to the deficient uterine artery blood supply. Blastocyst implantation was not altered but resorption rate was increased between SHRSP and WKY; indicating that the reduction in litter size in the SHRSP was primarily due to late (>GD14) pregnancy loss. Fetal growth was not restricted in SHRSP which led to the conclusion that SHRSP sacrifice part of their litter to deliver a smaller number of healthier pups. Activation of the immune system is a common pathway that has been implicated in the development of both hypertension and adverse pregnancy outcome. In Chapter 2, I proposed that this may be a mechanism of interest in SHRSP pregnancy and measured the pro-inflammatory cytokine, TNFα, as a marker of inflammation in pregnant SHRSP and WKY and in the placentas from these animals. TNFα was up-regulated in maternal plasma and urine from the GD18 SHRSP. In addition, TNFα release was increased from the GD18 SHRSP placenta as was the expression of the pro-inflammatory TNFα receptor 1 (Tnfr1). In order to investigate whether this excess TNFα was detrimental to SHRSP pregnancy, a vehicle-controlled intervention study using etanercept (a monoclonal antibody which works as a TNFα antagonist) was carried out. Etanercept treatment at GD0, 6, 12 and 18 resulted in an improvement in pregnancy outcome in the SHRSP with an increased litter size and reduced resorption rate. Furthermore, there was an improved uterine artery function in GD18 SHRSP treated with etanercept which was associated with an improved uterine artery blood flow over the course of gestation. In Chapter 3, I sought to identify the source of this detrimental excess of TNFα by designing a panel for maternal leukocytes in the blood and placenta at GD18. A population of CD3- CD161+ cells, which are defined as rat natural killer (NK) cells, were increased in number in the SHRSP. Intracellular flow cytometry also identified this cell type as a source of excess TNFα in blood and placenta from pregnant SHRSP. I then went on to evaluate the effects of etanercept treatment on these CD3- CD161+ cells and showed that etanercept reduced the expression of CD161 and the cytotoxic molecule, granzyme B, in the NK cells. Thus, etanercept limits the cytotoxicity and potential damaging effect of these NK cells in the SHRSP placenta. Analysing the urinary peptidome has clinical potential to identify novel pathways involved with disease and/or to develop biomarker panels to aid and stratify diagnosis. In Chapter 4, I utilised the SHRSP as a pre-clinical model to identify novel urinary peptides associated with hypertensive pregnancy. Firstly, a characterisation study was carried out in the kidney of the WKY and SHRSP. Urine samples from WKY and SHRSP taken at pre-pregnancy, mid-pregnancy (GD12) and late pregnancy (GD18) were used in the peptidomic screen. In order to capture peptides which were markers of hypertensive pregnancy from the urinary peptidomic data, I focussed on those that were only changed in a strain dependent manner at GD12 and 18 and not pre-pregnancy. Peptide fragments from the uromodulin protein were identified from this analysis to be increased in pregnant SHRSP relative to pregnant WKY. This increase in uromodulin was validated at the SHRSP kidney level using qPCR. Uromodulin has previously been identified to be a candidate molecule involved in systemic arterial hypertension but not in hypertensive pregnancy thus is a promising target for further study. In summary, we have characterised the SHRSP as the first model of maternal chronic hypertension during pregnancy and identified that inflammation mediated by TNFα and NK cells plays a key role in the pathology. The evidence presented in this thesis establishes the SHRSP as a pre-clinical model for pregnancy research and can be continued into clinical studies in pregnant women with chronic hypertension which remains an area of unmet research need.

Hypertensive disorders of pregnancy and long-term cardiovascular health and outcomes

Introduction The maternal vasculature undergoes significant adaptations during pregnancy to meet the increased metabolic demands of the developing fetus. These adaptations include increased cardiac output and blood volume, as well as reduced systemic vascular resistance. In Hypertensive disorders of pregnancy (HDP) there is an impaired cardiovascular adaptation to pregnancy with effects extending beyond pregnancy. In the present study we aimed to characterize long-term cardiovascular status of women who suffered from HDP. Methods Fifty-eight women who attended at least one post-partum visit and a follow-up visit after at least 5 years from delivery were enrolled in the study. Exclusion criteria included multiple pregnancy, fetal genetic or congenital abnormalities, maternal history of organ transplantation, or chronic renal failure (eGFR≤45ml/min/1.73m2). In the follow-up visit participants underwent a complete cardiovascular assessment including echocardiography and multiparametric vascular function assessment. Results and Discussion Two major cardiovascular events, one stroke and one myocardial infarction, occurred both in women with index-pregnancy complicated by preeclampsia (PE). While not statistically significant, women with HDP-non-PE and PE displayed a trend towards an increased risk of developing composite cardiovascular outcome, and women with PE tended to experience it sooner. Nearly half of the women with a history of HDP, whether PE or HDP-non-PE, developed chronic hypertension. Some women also developed hyperuricemia, chronic kidney disease (CKD), and type 2 diabetes at follow- up, most of them had a previous history of PE. Structural and functional cardiac changes were observed in a few cases, especially among women with PE, and vascular dysfunction was more common in women with a history of HDP compared to those with normotensive pregnancies. Results of the present study adds on literature on long-term cardiovascular impact of HDP and further emphasize the importance of a timely follow-up of women who suffered from HDP and particularly PE.

An Isoflavone From Dipteryx Alata Vogel Is Active Against The In Vitro Neuromuscular Paralysis Of Bothrops Jararacussu Snake Venom And Bothropstoxin I, And Prevents Venom-induced Myonecrosis.

Snakebite is a neglected disease and serious health problem in Brazil, with most bites being caused by snakes of the genus Bothrops. Although serum therapy is the primary treatment for systemic envenomation, it is generally ineffective in neutralizing the local effects of these venoms. In this work, we examined the ability of 7,8,3'-trihydroxy-4'-methoxyisoflavone (TM), an isoflavone from Dipteryx alata, to neutralize the neurotoxicity (in mouse phrenic nerve-diaphragm preparations) and myotoxicity (assessed by light microscopy) of Bothrops jararacussu snake venom in vitro. The toxicity of TM was assessed using the Salmonella microsome assay (Ames test). Incubation with TM alone (200 μg/mL) did not alter the muscle twitch tension whereas incubation with venom (40 μg/mL) caused irreversible paralysis. Preincubation of TM (200 μg/mL) with venom attenuated the venom-induced neuromuscular blockade by 84% ± 5% (mean ± SEM; n = 4). The neuromuscular blockade caused by bothropstoxin-I (BthTX-I), the major myotoxic PLA2 of this venom, was also attenuated by TM. Histological analysis of diaphragm muscle incubated with TM showed that most fibers were preserved (only 9.2% ± 1.7% were damaged; n = 4) compared to venom alone (50.3% ± 5.4% of fibers damaged; n = 3), and preincubation of TM with venom significantly attenuated the venom-induced damage (only 17% ± 3.4% of fibers damaged; n = 3; p < 0.05 compared to venom alone). TM showed no mutagenicity in the Ames test using Salmonella strains TA98 and TA97a with (+S9) and without (-S9) metabolic activation. These findings indicate that TM is a potentially useful compound for antagonizing the neuromuscular effects (neurotoxicity and myotoxicity) of B. jararacussu venom.

Changes In Chromatin Structure In Nih 3t3 Cells Induced By Valproic Acid And Trichostatin A.

Valproic acid (VPA) and trichostatin A (TSA) are known histone deacetylase inhibitors (HDACIs) with epigenetic activity that affect chromatin supra-organization, nuclear architecture, and cellular proliferation, particularly in tumor cells. In this study, chromatin remodeling with effects extending to heterochromatic areas was investigated by image analysis in non-transformed NIH 3T3 cells treated for different periods with different doses of VPA and TSA under conditions that indicated no loss of cell viability. Image analysis revealed chromatin decondensation that affected not only euchromatin but also heterochromatin, concomitant with a decreased activity of histone deacetylases and a general increase in histone H3 acetylation. Heterochromatin protein 1-α (HP1-α), identified immunocytochemically, was depleted from the pericentromeric heterochromatin following exposure to both HDACIs. Drastic changes affecting cell proliferation and micronucleation but not alteration in CCND2 expression and in ratios of Bcl-2/Bax expression and cell death occurred following a 48-h exposure of the NIH 3T3 cells particularly in response to higher doses of VPA. Our results demonstrated that even low doses of VPA (0.05 mM) and TSA (10 ng/ml) treatments for 1 h can affect chromatin structure, including that of the heterochromatin areas, in non-transformed cells. HP1-α depletion, probably related to histone demethylation at H3K9me3, in addition to the effect of VPA and TSA on histone H3 acetylation, is induced on NIH 3T3 cells. Despite these facts, alterations in cell proliferation and micronucleation, possibly depending on mitotic spindle defects, require a longer exposure to higher doses of VPA and TSA.

Resistant Hypertension Revisited: Definition And True Prevalence.

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Low-density Lipoprotein Cholesterol And Radiotherapy-induced Carotid Atherosclerosis In Subjects With Head And Neck Cancer.

Radiotherapy (RT) is a risk factor for accelerated carotid artery atherosclerotic disease in subjects with head and neck cancer. However, the risk factors of RT-induced carotid artery remodeling are not established. This study aimed to investigate the effects of RT on carotid and popliteal arteries in subjects with head and neck cancer and to evaluate the relationship between baseline clinical and laboratory features and the progression of RT-induced atherosclerosis. Eleven men (age = 57.9 ± 6.2years) with head and neck cancer who underwent cervical bilateral irradiation were prospectively examined by clinical and laboratory analysis and by carotid and popliteal ultrasound before and after treatment (mean interval between the end of RT and the post-RT assessment = 181 ± 47 days). No studied subject used hypocholesterolemic medications. Significant increases in carotid intima-media thickness (IMT) (0.95 ± 0.08 vs. 0.87 ± 0.05 mm; p < 0.0001) and carotid IMT/diameter ratio (0.138 ± 0.013 vs. 0.129 ± 0.014; p = 0.001) were observed after RT, while no changes in popliteal structural features were detected. In addition, baseline low-density lipoprotein cholesterol levels showed a direct correlation with RT-induced carotid IMT change (r = 0.66; p = 0.027), while no other studied variable exhibited a significant relationship with carotid IMT change. These results indicate that RT-induced atherosclerosis is limited to the irradiated area and also suggest that it may be predicted by low-density lipoprotein cholesterol levels in subjects with head and neck cancer.

Exposure To Bisphenol-a During Pregnancy Partially Mimics The Effects Of A High-fat Diet Altering Glucose Homeostasis And Gene Expression In Adult Male Mice.

Bisphenol-A (BPA) is one of the most widespread EDCs used as a base compound in the manufacture of polycarbonate plastics. The aim of our research has been to study how the exposure to BPA during pregnancy affects weight, glucose homeostasis, pancreatic β-cell function and gene expression in the major peripheral organs that control energy flux: white adipose tissue (WAT), the liver and skeletal muscle, in male offspring 17 and 28 weeks old. Pregnant mice were treated with a subcutaneous injection of 10 µg/kg/day of BPA or a vehicle from day 9 to 16 of pregnancy. One month old offspring were divided into four different groups: vehicle treated mice that ate a normal chow diet (Control group); BPA treated mice that also ate a normal chow diet (BPA); vehicle treated animals that had a high fat diet (HFD) and BPA treated animals that were fed HFD (HFD-BPA). The BPA group started to gain weight at 18 weeks old and caught up to the HFD group before week 28. The BPA group as well as the HFD and HFD-BPA ones presented fasting hyperglycemia, glucose intolerance and high levels of non-esterified fatty acids (NEFA) in plasma compared with the Control one. Glucose stimulated insulin release was disrupted, particularly in the HFD-BPA group. In WAT, the mRNA expression of the genes involved in fatty acid metabolism, Srebpc1, Pparα and Cpt1β was decreased by BPA to the same extent as with the HFD treatment. BPA treatment upregulated Pparγ and Prkaa1 genes in the liver; yet it diminished the expression of Cd36. Hepatic triglyceride levels were increased in all groups compared to control. In conclusion, male offspring from BPA-treated mothers presented symptoms of diabesity. This term refers to a form of diabetes which typically develops in later life and is associated with obesity.

Cardiac Magnetic Resonance Assessment Of Interstitial Myocardial Fibrosis And Cardiomyocyte Hypertrophy In Hypertensive Mice Treated With Spironolactone.

Nearly 50% of patients with heart failure (HF) have preserved LV ejection fraction, with interstitial fibrosis and cardiomyocyte hypertrophy as early manifestations of pressure overload. However, methods to assess both tissue characteristics dynamically and noninvasively with therapy are lacking. We measured the effects of mineralocorticoid receptor blockade on tissue phenotypes in LV pressure overload using cardiac magnetic resonance (CMR). Mice were randomized to l-nitro-ω-methyl ester (l-NAME, 3 mg/mL in water; n=22), or l-NAME with spironolactone (50 mg/kg/day in subcutaneous pellets; n=21). Myocardial extracellular volume (ECV; marker of diffuse interstitial fibrosis) and the intracellular lifetime of water (τic; marker of cardiomyocyte hypertrophy) were determined by CMR T1 imaging at baseline and after 7 weeks of therapy alongside histological assessments. Administration of l-NAME induced hypertensive heart disease in mice, with increases in mean arterial pressure, LV mass, ECV, and τic compared with placebo-treated controls, while LV ejection fraction was preserved (>50%). In comparison, animals receiving both spironolactone and l-NAME (l-NAME+S) showed less concentric remodeling, and a lower myocardial ECV and τic, indicating decreased interstitial fibrosis and cardiomyocyte hypertrophy (ECV: 0.43 ± 0.09 for l-NAME versus 0.25 ± 0.03 for l-NAME+S, P<0.001; τic: 0.42 ± 0.11 for l-NAME groups versus 0.12 ± 0.05 for l-NAME+S group). Mice treated with a combination of l-NAME and spironolactone were similar to placebo-treated controls at 7 weeks. Spironolactone attenuates interstitial fibrosis and cardiomyocyte hypertrophy in hypertensive heart disease. CMR can phenotype myocardial tissue remodeling in pressure-overload, furthering our understanding of HF progression.

Peripheral P2x7 Receptor-induced Mechanical Hyperalgesia Is Mediated By Bradykinin.

P2X7 receptors play an important role in inflammatory hyperalgesia, but the mechanisms involved in their hyperalgesic role are not completely understood. In this study, we hypothesized that P2X7 receptor activation induces mechanical hyperalgesia via the inflammatory mediators bradykinin, sympathomimetic amines, prostaglandin E2 (PGE2), and pro-inflammatory cytokines and via neutrophil migration in rats. We found that 2'(3')-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate triethylammonium salt (BzATP), the most potent P2X7 receptor agonist available, induced a dose-dependent mechanical hyperalgesia that was blocked by the P2X7 receptor-selective antagonist A-438079 but unaffected by the P2X1,3,2/3 receptor antagonist TNP-ATP. These findings confirm that, although BzATP also acts at both P2X1 and P2X3 receptors, BzATP-induced hyperalgesia was mediated only by P2X7 receptor activation. Co-administration of selective antagonists of bradykinin B1 (Des-Arg(8)-Leu(9)-BK (DALBK)) or B2 receptors (bradyzide), β1 (atenolol) or β2 adrenoceptors (ICI 118,551), or local pre-treatment with the cyclooxygenase inhibitor indomethacin or the nonspecific selectin inhibitor fucoidan each significantly reduced BzATP-induced mechanical hyperalgesia in the rat hind paw. BzATP also induced the release of the pro-inflammatory cytokines tumor necrosis factor α (TNF-α), interleukin (IL)-1β, IL-6 and cytokine-induced neutrophil chemoattractant-1 (CINC-1), an effect that was significantly reduced by A-438079. Co-administration of DALBK or bradyzide with BzATP significantly reduced BzATP-induced IL-1β and CINC-1 release. These results indicate that peripheral P2X7 receptor activation induces mechanical hyperalgesia via inflammatory mediators, especially bradykinin, which may contribute to pro-inflammatory cytokine release. These pro-inflammatory cytokines in turn may mediate the contributions of PGE2, sympathomimetic amines and neutrophil migration to the mechanical hyperalgesia induced by local P2X7 receptor activation.

15-deoxy-Δ12,14-prostaglandin J2 Reduces Albumin-induced Arthritis In Temporomandibular Joint Of Rats.

The aim of this study was to evaluate the peripheral effect of 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) in albumin-induced arthritis in temporomandibular joint (TMJ) of rats. Antigen-induced arthritis (AIA) was generated in rats with methylated bovine serum albumin (mBSA) diluted in complete Freund׳s adjuvant. Pretreatment with an intra-articular injection of 15d-PGJ2 (100 ng/TMJ) before mBSA intra-articular injection (10 µg/TMJ) (challenge) in immunized rats significantly reduced the albumin-induced arthritis inflammation. The results demonstrated that 15d-PGJ2 was able to inhibit plasma extravasation, leukocyte migration and the release of inflammatory cytokines IL-6, IL-12, IL-18 and the chemokine CINC-1 in the TMJ tissues. In addition, 15d-PGJ2 was able to increase the expression of the anti-adhesive molecule CD55 and the anti-inflammatory cytokine IL-10. Taken together, it is possible to suggest that 15d-PGJ2 inhibit leukocyte infiltration and subsequently inflammatory process, through a shift in the balance of the pro- and anti-adhesive properties. Thus, 15d-PGJ2 might be used as a potential anti-inflammatory drug to treat arthritis-induced inflammation of the temporomandibular joint.

Taurine Supplementation Reduces Blood Pressure And Prevents Endothelial Dysfunction And Oxidative Stress In Post-weaning Protein-restricted Rats.

Taurine is a sulfur-containing amino acid that exerts protective effects on vascular function and structure in several models of cardiovascular diseases through its antioxidant and anti-inflammatory properties. Early protein malnutrition reprograms the cardiovascular system and is linked to hypertension in adulthood. This study assessed the effects of taurine supplementation in vascular alterations induced by protein restriction in post-weaning rats. Weaned male Wistar rats were fed normal- (12%, NP) or low-protein (6%, LP) diets for 90 days. Half of the NP and LP rats concomitantly received 2.5% taurine supplementation in the drinking water (NPT and LPT, respectively). LP rats showed elevated systolic, diastolic and mean arterial blood pressure versus NP rats; taurine supplementation partially prevented this increase. There was a reduced relaxation response to acetylcholine in isolated thoracic aortic rings from the LP group that was reversed by superoxide dismutase (SOD) or apocynin incubation. Protein expression of p47phox NADPH oxidase subunit was enhanced, whereas extracellular (EC)-SOD and endothelial nitric oxide synthase phosphorylation at Ser 1177 (p-eNOS) were reduced in aortas from LP rats. Furthermore, ROS production was enhanced while acetylcholine-induced NO release was reduced in aortas from the LP group. Taurine supplementation improved the relaxation response to acetylcholine and eNOS-derived NO production, increased EC-SOD and p-eNOS protein expression, as well as reduced ROS generation and p47phox expression in the aortas from LPT rats. LP rats showed an increased aortic wall/lumen ratio and taurine prevented this remodeling through a reduction in wall media thickness. Our data indicate a protective role of taurine supplementation on the high blood pressure, endothelial dysfunction and vascular remodeling induced by post-weaning protein restriction. The beneficial vascular effect of taurine was associated with restoration of vascular redox homeostasis and improvement of NO bioavailability.

A Decade Of Malaria During Pregnancy In Brazil: What Has Been Done Concerning Prevention And Management.

In Brazil, malaria remains a disease of major epidemiological importance because of the high number of cases in the Amazonian Region. Plasmodium spp infections during pregnancy are a significant public health problem with substantial risks for the pregnant woman, the foetus and the newborn child. In Brazil, the control of malaria during pregnancy is primarily achieved by prompt and effective treatment of the acute episodes. Thus, to assure rapid diagnosis and treatment for pregnant women with malaria, one of the recommended strategy for low transmission areas by World Health Organization and as part of a strategy by the Ministry of Health, the National Malaria Control Program has focused on integrative measures with woman and reproductive health. Here, we discuss the approach for the prevention and management of malaria during pregnancy in Brazil over the last 10 years (2003-2012) using morbidity data from Malaria Health Information System. Improving the efficiency and quality of healthcare and education and the consolidation of prevention programmes will be challenges in the control of malaria during pregnancy in the next decade.

Adipokines: Novel Players In Resistant Hypertension.

Resistant hypertension (RH) is a multifactorial disease, frequently associated with obesity and characterized by blood pressure above goal (140/90 mm Hg) despite the concurrent use of ≥3 antihypertensive drugs of different classes. The mechanisms of obesity-related hypertension include, among others, aldosterone excess and inflammatory adipokines, which have demonstrated a significant role in the pathogenesis of metabolic syndrome and RH. This review aims to summarize recent studies on the role of the adipokines leptin, resistin, and adiponectin in the pathophysiology of RH and target-organ damage associated with this condition. The deregulation of adipokine levels has been associated with clinical characteristics frequently recognized in RH such as diabetes, hyperactivity of sympathetic and renin-angiotensin-aldosterone systems, and vascular and renal damage. Strategies to regulate adipokines may be promising for the management of RH and some clinical implications must be considered when managing controlled and uncontrolled patients with RH.