Citral relaxes umbilical vessels of normotensive and preeclamptic parturients.

INTRODUCTION: Citral is a low-toxicity monoterpene that has a vasodilator effect on various smooth muscles, and The present study aimed to evaluate its vasorelaxant effect on umbilical vessels of normotensive parturients (NTP) and with preeclampsia parturients (PEP). METHOD: Segments of human umbilical artery (HUA) and vein (HUV) of NTP or PEP were mounted in a bath to record the force of contraction, under tension of 3.0 gf and contracted with the contracting agents: K+ (60 mM), 5 -HT (10 µM) and Ba2+ (1-30 mM). Next, the effect of citral (1-3000 µM) on these contractions and on basal tone was evaluated. RESULTS: In HUA and HUV, citral (1-1000 µM), in NTP condition, inhibited contractions evoked by K+ (IC50 of 413.5 and 271.3, respectively) and by 5-HT (IC50 of 164.8 and 574.3). In the PEP condition, in HUA and HUV, citral also inhibited the contractions evoked by K+ (IC50 of 363.3 and 218.3, respectively) and 5-HT (IC50 of 432.1 and 520.4). At a concentration of 1000 µM, citral completely or almost completely (>90 %) inhibited all contractions. At a concentration of 100-1000 µM, citral, in general, was already able to reduce the contraction induced by 1-3 mM Ba2+ in both AUH and VUH, under NTP and PEP conditions. DISCUSSION: Citral has been shown to be an effective HUA and HUV vasodilator in NTP and PEP. As its toxicity is low, it suggests that this substance can be considered a potential therapeutic agent.

In situ isolation of nuclei or nuclear proteins from adherent cells: a simple, effective method with less cytoplasmic contamination.

BACKGROUND: Isolation of nuclei or nuclear proteins is a prerequisite for western blot, nuclear proteome profiling, and other evaluations of nuclear proteins. Here, we developed a simple method for in situ isolation of nuclei or nuclear proteins by in situ removing the extranuclear part of adherent cells via a classical nonionic detergent triton X-100. RESULTS: First, the feasibility of our method was confirmed by confocal microscopy, atomic force microscopy, scanning electron microscopy, dynamic light scattering, immunofluorescence imaging, and time-lapse dynamic observation. Next, the optimal concentration range (approximately 0.1-1% for ~ 10 min) of triton X-100 and the optimal treatment time (< 30 min) of 0.1-1% Triton X-100 for our method were determined via western blotting of eight extra-/intra-nuclear proteins. Subsequently, the effectiveness, sensitivity, and cytoplasmic contamination of our method were tested by investigating the levels of phosphorylated p65 (a NF-κB subunit) in the nuclei of endothelial or tumor cells treated with/without lipopolysaccharide (LPS) via western blotting and by comparing with a commercial nuclear protein extraction kit (a classical detergent-based method). The data show that compared with the commercial kit our method obtained a higher yield of total nuclear proteins, a higher pP65 level in both control and LPS groups, and much lower content of GAPDH (as a reference for cytoplasmic contamination) in nuclei. CONCLUSIONS: The in situ isolation of nuclei or nuclear proteins from adherent cells in this study is a simple, effective method with less cytoplasmic contamination. This method/strategy has the potential of improving the quality of downstream evaluations including western blotting and proteomic profiling.

Improving prenatal detection of abdominal supraumbilical anomalies: The sonographic examination of fetal anechoic spaces of upper abdomen revisited.

Visualization of the axial plane of the fetal abdomen is mandatory to obtain abdominal biometry in the assessment of fetal growth in the second and third trimesters. The main anatomic landmarks that must be identified in this view include the fetal stomach and the intrahepatic portion of the umbilical vein, which are easily identifiable as they appear anechoic on ultrasound. The gallbladder is the other prominent anechoic structure in this plane. Focused study of the morphological characteristics of, and spatial relationship among, these three anechoic spaces is a simple technique to detect anomalies involving fetal upper abdominal organs. In this review, the sonographic features of those conditions that can be detected using this technique, which was termed the Fetal Examination of the Anechoic Spaces of upper abdomen Technique (FEAST), are classified and illustrated.

In situ isolation of nuclei or nuclear proteins from adherent cells: a simple, effective method with less cytoplasmic contamination

BACKGROUND: Isolation of nuclei or nuclear proteins is a prerequisite for western blot, nuclear proteome profiling, and other evaluations of nuclear proteins. Here, we developed a simple method for in situ isolation of nuclei or nuclear proteins by in situ removing the extranuclear part of adherent cells via a classical nonionic detergent triton X-100. RESULTS: First, the feasibility of our method was confirmed by confocal microscopy, atomic force microscopy, scanning electron microscopy, dynamic light scattering, immunofluorescence imaging, and time-lapse dynamic observation. Next, the optimal concentration range (approximately 0.1-1% for ~ 10 min) of triton X-100 and the optimal treatment time (< 30 min) of 0.1-1% Triton X-100 for our method were determined via western blotting of eight extra-/ intra-nuclear proteins. Subsequently, the effectiveness, sensitivity, and cytoplasmic contamination of our method were tested by investigating the levels of phosphorylated p65 (a NF-κB subunit) in the nuclei of endothelial or tumor cells treated with/without lipopolysaccharide (LPS) via western blotting and by comparing with a commercial nuclear protein extraction kit (a classical detergent-based method). The data show that compared with the commercial kit our method obtained a higher yield of total nuclear proteins, a higher pP65 level in both control and LPS groups, and much lower content of GAPDH (as a reference for cytoplasmic contamination) in nuclei. CONCLUSIONS: The in situ isolation of nuclei or nuclear proteins from adherent cells in this study is a simple, effective method with less cytoplasmic contamination. This method/strategy has the potential of improving the quality of downstream evaluations including western blotting and proteomic profiling.

Clinical Significance of HSCARG for Atherosclerotic Coronary Heart Disease and Reduced ROS-Oxidative Stress in in Vivo and in Vitro Models via p47phox by NF-κB Activity

ABSTRACT Introduction: Coronary heart disease (CHD) is a dynamic process in which there are interactions between endothelial dysfunction, oxidative stress, and inflammatory responses. The aim of the present study was to investigate the function and mechanism of HSCARG in the treatment of CHD. Methods: Male apolipoprotein E/low-density lipoprotein receptor-deficient mice were given a high-fat diet with 21% fat and 0.15% cholesterol for the in vivo model. Human umbilical vein endothelial cells were incubated with angiotensin II for the in vitro model. HSCARG expression was inhibited in patients or mice with CHD. Results: HSCARG reduced oxidative stress in mice with CHD. HSCARG also reduced reactive oxygen species (ROS)-oxidative stress in the in vitro model. HSCARG induced p47phox expression in the in vitro model by NF-κB activity. The regulation of nuclear factor kappa B (NF-κB) activity or p47phox expression participates in the effects of HSCARG in CHD. Conclusion: Altogether, our data indicate that HSCARG reduced ROS-oxidative stress in in vivo and in vitro models of CHD via p47phox by NF-κB activity and may be a clinical target for CHD.

Clinical Significance of HSCARG for Atherosclerotic Coronary Heart Disease and Reduced ROS-Oxidative Stress in in Vivo and in Vitro Models via p47phox by NF-κB Activity.

INTRODUCTION: Coronary heart disease (CHD) is a dynamic process in which there are interactions between endothelial dysfunction, oxidative stress, and inflammatory responses. The aim of the present study was to investigate the function and mechanism of HSCARG in the treatment of CHD. METHODS: Male apolipoprotein E/low-density lipoprotein receptor-deficient mice were given a high-fat diet with 21% fat and 0.15% cholesterol for the in vivo model. Human umbilical vein endothelial cells were incubated with angiotensin II for the in vitro model. HSCARG expression was inhibited in patients or mice with CHD. RESULTS: HSCARG reduced oxidative stress in mice with CHD. HSCARG also reduced reactive oxygen species (ROS)-oxidative stress in the in vitro model. HSCARG induced p47phox expression in the in vitro model by NF-κB activity. The regulation of nuclear factor kappa B (NF-κB) activity or p47phox expression participates in the effects of HSCARG in CHD. CONCLUSION: Altogether, our data indicate that HSCARG reduced ROS-oxidative stress in in vivo and in vitro models of CHD via p47phox by NF-κB activity and may be a clinical target for CHD.

Rapid Fabrication of Microfluidic Devices for Biological Mimicking: A Survey of Materials and Biocompatibility.

Microfluidics is an essential technique used in the development of in vitro models for mimicking complex biological systems. The microchip with microfluidic flows offers the precise control of the microenvironment where the cells can grow and structure inside channels to resemble in vivo conditions allowing a proper cellular response investigation. Hence, this study aimed to develop low-cost, simple microchips to simulate the shear stress effect on the human umbilical vein endothelial cells (HUVEC). Differentially from other biological microfluidic devices described in the literature, we used readily available tools like heat-lamination, toner printer, laser cutter and biocompatible double-sided adhesive tapes to bind different layers of materials together, forming a designed composite with a microchannel. In addition, we screened alternative substrates, including polyester-toner, polyester-vinyl, glass, Permanox® and polystyrene to compose the microchips for optimizing cell adhesion, then enabling these microdevices when coupled to a syringe pump, the cells can withstand the fluid shear stress range from 1 to 4 dyne cm2. The cell viability was monitored by acridine orange/ethidium bromide (AO/EB) staining to detect live and dead cells. As a result, our fabrication processes were cost-effective and straightforward. The materials investigated in the assembling of the microchips exhibited good cell viability and biocompatibility, providing a dynamic microenvironment for cell proliferation. Therefore, we suggest that these microchips could be available everywhere, allowing in vitro assays for daily laboratory experiments and further developing the organ-on-a-chip concept.

IARS2 regulates proliferation, migration, and angiogenesis of human umbilical vein endothelial cells

SUMMARY OBJECTIVE: In this study, we aimed at investigating the role of isoleucyl-tRNA synthetase in the growth, migration, and angiogenesis of human umbilical vein endothelial cells and the underlying molecular mechanism. METHODS: To assess the role of isoleucyl-tRNA synthetase, we silenced isoleucyl-tRNA synthetase in human umbilical vein endothelial cells using lentiviral 2 specific short hairpin RNAs (short hairpin RNAs 1 and 2) and examined silencing efficiency using real time quantitative polymerase chain reaction and western blot analyses. Short hairpin RNAs 1-isoleucyl-tRNA synthetase had greater knockdown efficiency, it was used in the entire downstream analysis. Short hairpin RNAs 1- isoleucyl-tRNA synthetase silencing effects on cell proliferation, cell colony generation, cell migration, as well as angiogenesis were assessed using cell counting kit-8, colony development, cell migration, and angiogenesis tube formation assays, respectively. RESULTS: Compared to the control group, anti-isoleucyl-tRNA synthetase short hairpin RNAs significantly silenced isoleucyl-tRNA synthetase expression in human umbilical vein endothelial cells, and suppressed their proliferation, migration, and angiogenic capacity. To characterize the underlying mechanism, western blot analyses showed that isoleucyl-tRNA synthetase knockdown suppressed phosphorylation of extracellular-regulated kinase ½ and protein-serine- threonine kinase, as well as expression of vascular endothelial growth factor, GSK-3β, and β-catenin. CONCLUSIONS: We have shown, for the first time, the critical role of isoleucyl-tRNA synthetase in human umbilical vein endothelial cells. Our data show that isoleucyl-tRNA synthetase knockdown suppresses human umbilical vein endothelial cell proliferation, migration, and angiogenesis. We have also shown that isoleucyl-tRNA synthetase knockdown suppresses phosphorylation of extracellular-regulated kinase ½ and protein-serine- threonine kinase, as well as expression of vascular endothelial growth factor, GSK-3β, and β-catenin. Together, these data highlight isoleucyl-tRNA synthetase as a potential antitumor anti-angiogenic target.

Intrinsic Angiogenic Potential and Migration Capacity of Human Mesenchymal Stromal Cells Derived from Menstrual Blood and Bone Marrow.

Several therapies are being developed to increase blood circulation in ischemic tissues. Despite bone marrow-derived mesenchymal stromal cells (bmMSC) are still the most studied, an interesting and less invasive MSC source is the menstrual blood, which has shown great angiogenic capabilities. Therefore, the aim of this study was to evaluate the angiogenic properties of menstrual blood-derived mesenchymal stromal cells (mbMSC) in vitro and in vivo and compared to bmMSC. MSC's intrinsic angiogenic capacity was assessed by sprouting and migration assays. mbMSC presented higher invasion and longer sprouts in 3D culture. Additionally, both MSC-spheroids showed cells expressing CD31. mbMSC and bmMSC were able to migrate after scratch wound in vitro, nonetheless, only mbMSC demonstrated ability to engraft in the chick embryo, migrating to perivascular, perineural, and chondrogenic regions. In order to study the paracrine effects, mbMSC and bmMSC conditioned mediums were capable of stimulating HUVEC's tube-like formation and migration. Both cells expressed VEGF-A and FGF2. Meanwhile, PDGF-B was expressed exclusively in mbMSC. Our results indicated that mbMSC and bmMSC presented a promising angiogenic potential. However, mbMSC seems to have additional advantages since it can be obtained by non-invasive procedure and expresses PDGF-B, an important molecule for vascular formation and remodeling.

Berberis microphylla G. Forst (Calafate) Berry Extract Reduces Oxidative Stress and Lipid Peroxidation of Human LDL.

Calafate (Berberis microphylla G. Forst) is a Patagonian barberry very rich in phenolic compounds. Our aim was to demonstrate, through in vitro models, that a comprehensive characterized calafate extract has a protective role against oxidative processes associated to cardiovascular disease development. Fifty-three phenolic compounds (17 of them not previously reported in calafate), were tentatively identified by Ultra-Liquid Chromatography with Diode Array Detector, coupled to Quadrupole-Time of Fly Mass Spectrometry (UHPLC-DAD-QTOF). Fatty acids profile and metals content were studied for the first time, by Gas Chromatography Mass Spectrometry (GC-MS) and Total X-ray Fluorescence (TXRF), respectively. Linolenic and linoleic acid, and Cu, Zn, and Mn were the main relevant compounds from these groups. The bioactivity of calafate extract associated to the cardiovascular protection was evaluated using Human Umbilical Vein Endothelial Cells (HUVECs) and human low density lipoproteins (LDL) to measure oxidative stress and lipid peroxidation. The results showed that calafate extract reduced intracellular Reactive Oxygen Species (ROS) production (51%) and completely inhibited LDL oxidation and malondialdehyde (MDA) formation. These findings demonstrated the potential of the relevant mix of compounds found in calafate extract on lipoperoxidation and suggest a promising protective effect for reducing the incidence of cardiovascular disease.

Endothelium-derived dopamine modulates EFS-induced contractions of human umbilical vessels.

Electrical field stimulation (EFS) induces contractions of both snake aorta and human umbilical cord vessels (HUCV) which were dependent on the presence of the endothelium. This study aimed to establish the nature of the mediator(s) responsible for EFS-induced contractions in HUCV. Rings with or without endothelium from human umbilical artery (HUA) or vein (HUV) were mounted in organ bath chambers containing oxygenated, heated Krebs-Henseleit's solution. Basal release of dopamine (DA), noradrenaline, and adrenaline was measured by LC-MS-MS. Cumulative concentration-response curves were performed with dopamine in the absence and in the presence of L-NAME or of dopamine antagonists. EFS studies were performed in the presence and absence of L-NAME, the α-adrenergic blockers prazosin and idazoxan, and the dopamine antagonists SCH-23390 and haloperidol. Tyrosine hydroxylase (TH) and dopa-decarboxylase (DDC) were studied by immunohistochemistry and fluorescence in situ hybridizations. Basal release of dopamine requires an intact endothelium in both HUA and HUV. TH and DDC are present only in the endothelium of both HUA and HUV as determined by immunohistochemistry. Dopamine induced contractions in HUA only in the presence of L-NAME. Dopamine-induced contractions in HUV were strongly potentiated by L-NAME. The EFS-induced contractions in both HUA and HUV were potentiated by L-NAME and inhibited by the D2-like receptor antagonist haloperidol. The α-adrenergic antagonists prazosin and idazoxan and the D1-like receptor antagonist SCH-23390 had no effect on the EFS-induced contractions of HUA and HUV. Endothelium-derived dopamine is a major modulator of HUCV reactivity in vitro.

Inhibition of miR-383 suppresses oxidative stress and improves endothelial function by increasing sirtuin 1

Previous research has shown that suppression of miR-383 can prevent inflammation of the endothelium, as well as postpone the development of atherosclerosis. However, the role of miR-383 in endothelial cell apoptosis in diabetes remains unclear. The aim of this study was to investigate the function of miR-383 in high glucose-induced apoptosis and oxidative stress in endothelial cells. A series of experiments involving qualitative polymerase chain reaction, cell transfection, luciferase assay, assessment of cell death, detection of catalase and superoxide dismutase concentrations, detection of intracellular reactive oxygen species (ROS), and western blot analysis were performed in this study. We found that miR-383 expression was promoted, while NAD+-dependent deacetylase and sirtuin 1 (SIRT1) expressions were suppressed in the endothelium of the aorta in db/db mice as well as in human umbilical vein endothelial cells, which were treated with high glucose (HG). Increased expression of miR-383 decreased expression of SIRT1, while suppression of miR-383 promoted expression of SIRT1 in human umbilical vein endothelial cells (HUVECs). Furthermore, suppression of miR-383 following transfection with miR-383 suppressor repressed cell death and generation of ROS in HUVECs. SIRT1 knockdown by siRNA-SIRT1 reversed the suppressive effect of miR-383 inhibition on ROS production and cell apoptosis induced by HG treatment. Overall, the findings of our research suggested that suppression of miR-383 repressed oxidative stress and reinforced the activity of endothelial cells by upregulation of SIRT1 in db/db mice, and targeting miR-383 might be promising for effective treatment of diabetes.

Adipose tissue-derived stromal cells' conditioned medium modulates endothelial-mesenchymal transition induced by IL-1ß/TGF-ß2 but does not restore endothelial function.

OBJECTIVES: Endothelial cells undergo TGF-ß-driven endothelial-mesenchymal transition (EndMT), representing up to 25% of cardiac myofibroblasts in ischaemic hearts. Previous research showed that conditioned medium of adipose tissue-derived stromal cells (ASC-CMed) blocks the activation of fibroblasts into fibrotic myofibroblasts. We tested the hypothesis that ASC-CMed abrogates EndMT and prevents the formation of adverse myofibroblasts. MATERIALS AND METHODS: Human umbilical vein endothelial cells (HUVEC) were treated with IL-1ß and TGF-ß2 to induce EndMT, and the influence of ASC-CMed was assessed. As controls, non-treated HUVEC or HUVEC treated only with IL-1ß in the absence or presence of ASC-CMed were used. Gene expression of inflammatory, endothelial, mesenchymal and extracellular matrix markers, transcription factors and cell receptors was analysed by RT-qPCR. The protein expression of endothelial and mesenchymal markers was evaluated by immunofluorescence microscopy and immunoblotting. Endothelial cell function was measured by sprouting assay. RESULTS: IL-1ß/TGF-ß2 treatment induced EndMT, as evidenced by the change in HUVEC morphology and an increase in mesenchymal markers. ASC-CMed blocked the EndMT-related fibrotic processes, as observed by reduced expression of mesenchymal markers TAGLN (P = 0.0008) and CNN1 (P = 0.0573), as well as SM22α (P = 0.0501). The angiogenesis potential was impaired in HUVEC undergoing EndMT and could not be restored by ASC-CMed. CONCLUSIONS: We demonstrated that ASC-CMed reduces IL-1ß/TGF-ß2-induced EndMT as observed by the loss of mesenchymal markers. The present study supports the anti-fibrotic effects of ASC-CMed through the modulation of the EndMT process.

The types of connections between the portal sinus and main portal vein in foetuses / Tipos de conexiones entre el seno portal y la vena porta principal en fetos

In the prenatal period, the three types of connections between the portal sinus and main portal vein have been published in the literature: T, X and H-shaped. The T type is the most frequent in the literature, and the aim of our study is to define the percentage of the connection types during the prenatal period in our population. In this prospective study, 237 women between 20 and 38 weeks of pregnancy without a foetal anomaly or pregnancy-related complications were included, and the precordial veins of the foetuses were examined using a wide-band color Doppler technique. The types of connections were determined by two specialists according to the shape of the colour coded vessels in Doppler examinations. The criteria of Czubalski & Aleksandrowicz (2000) were used. All of the connection types in patients were confirmed using video clips and were stored in the picture archiving and communication system. In 237 patients, the types of connection were determined by the first specialist as 189 foetuses (79.7 %) with the X-shaped or side-to-side connection, 16 foetuses (6.8 %) with the T-shaped or end-to-side type and 32 foetuses (13.5 %) with the H-shaped or parallel-coursed vessels connected with a short segment. The most common types of connections between the portal sinus and main portal vein in foetuses are X shaped or side-to-side, which is contrary to previous studies.

En el período prenatal, se han publicado en la literatura los tres tipos de conexiones entre el seno portal y la vena porta principal: en forma de T, X y H. El tipo T es el más frecuente, y el objetivo de nuestro estudio fue definir el porcentaje de tipos de conexión durante el período prenatal en nuestra población. En este estudio prospectivo, se incluyeron 237 mujeres entre 20 y 38 semanas de embarazo, sin anomalías fetales o complicaciones relacionadas con el embarazo, y se examinaron las venas precordiales de los fetos utilizando una técnica Doppler de banda ancha. Los tipos de conexiones fueron determinados por dos especialistas según la forma de los vasos codificados por color en los exámenes Doppler. Se utilizaron los criterios del estudio de Czubalski & Aleksandrowicz. Todos los tipos de conexión en los pacientes se confirmaron mediante videoclips y se almacenaron en el sistema de comunicación y en archivo de imágenes. En 237 pacientes, el primer especialista determinó en 189 fetos (79,7 %) la conexión en forma de X o de lado a lado; en 16 fetos (6,8 %) la forma de T o Tipo de extremo a lado; y en 32 fetos (13,5 %) los vasos en forma de H o paralelos, conectados con un segmento corto. Los tipos más comunes de conexiones entre el seno portal y la vena porta principal en los fetos son en forma de X o de lado a lado, lo que es contrario a estudios anteriores.

t-Resveratrol Protects against Acute High Glucose Damage in Endothelial Cells.

Postprandial hyperglycemia in diabetic and nondiabetic subjects is associated with endothelial dysfunction. Evidence shows that high glucose generates oxidative stress and a pro-inflammatory state promoting the development of cardiovascular diseases. trans-Resveratrol (t-RV) has been shown to reduce cardiovascular risk. To determine whether t-RV acts as a protector against acute high glucose (AHG)-induced damage, two in vitro models, rat aortic rings (RAR) and human umbilical vein endothelial cells (HUVEC) were used. RAR pretreated with AHG (25 mM D-glucose) for 3 h dramatically decreased the endothelium-dependent relaxation (EDR) induced by acetylcholine in phenylephrine (PE)-precontracted vessels. However, coincubation with t-RV significantly mitigated the damage induced by AHG on EDR. Pretreatment with AHG did not affect the vasodilation induced by sodium nitroprusside. HUVEC treated with t-RV decreased cytotoxicity and reduced radical oxygen species production induced by AHG. Taken together, these results suggest that t-RV can mitigate the AHG-induced EDR damage through a mechanism involving ROS scavenging and probably an increase in the bioavailability of NO.

Effects of gestational hypertension and pre-eclampsia in mRNA expression of fibrinolysis genes in primary cultured human umbilical vein endothelial cells.

Hypertension disorders (HD) and pre-eclampsia (PRE) are leading causes of maternal deaths worldwide. PRE is associated with vascular endothelial dysfunction and with deregulation of the fibrinolysis pathway genes. Fibrinolysis is the fibrin clot hydrolysis process catalyzed by plasmin, a proteolytic enzyme formed from plasminogen. Plasminogen is cleaved by tissue-type (tPA) and urokinase-type (uPA) activators and inhibited by the plasminogen activator inhibitors type-1 (PAI-1) and type-2 (PAI-2). The whole process maintains blood hemostasis. This study aims to assess PAI-1, PAI-2, tPA and uPA mRNA expression in primary cultured human umbilical vein endothelial cells (HUVEC) isolated and cultured from healthy, HD and PRE women. Results show that PAI-1 and PAI-2 mRNA decreased in HD-HUVEC, whereas PAI-1 and uPA decreased in PRE-HUVEC cultures compared to control ones. Notably, the expression ratio between pro- and anti-fibrinolytic actors remained unchanged among the studied groups. It seems that newborn's hemostasis is maintained balanced probably by a compensatory mechanism that involves changes in the fibrinolysis gene expression profile. The real impact of these changes in mRNA expression is unknown, however, it is suggested that these changes could be associated with an increased predisposition to vascular disease development in the progeny.

Maternal insulin therapy does not restore foetoplacental endothelial dysfunction in gestational diabetes mellitus.

Pregnant women diagnosed with gestational diabetes mellitus subjected to diet (GDMd) that do not reach normal glycaemia are passed to insulin therapy (GDMi). GDMd associates with increased human cationic amino acid transporter 1 (hCAT-1)-mediated transport of L-arginine and nitric oxide synthase (NOS) activity in foetoplacental vasculature, a phenomenon reversed by exogenous insulin. Whether insulin therapy results in reversal of the GDMd effect on the foetoplacental vasculature is unknown. We assayed whether insulin therapy normalizes GDMd-associated foetoplacental endothelial dysfunction. Primary cultures of human umbilical vein endothelial cells (HUVECs) from GDMi pregnancies were used to assay L-arginine transport kinetics, NOS activity, p44/42mapk and protein kinase B/Akt activation, and umbilical vein rings reactivity. HUVECs from GDMi or GDMd show increased hCAT-1 expression and maximal transport capacity, NOS activity, and eNOS, and p44/42mapk, but not Akt activator phosphorylation. Dilation in response to insulin or calcitonin-gene related peptide was impaired in umbilical vein rings from GDMi and GDMd pregnancies. Incubation of HUVECs in vitro with insulin (1 nmol/L) restored hCAT-1 and eNOS expression and activity, and eNOS and p44/42mapk activator phosphorylation. Thus, maternal insulin therapy does not seem to reverse GDMd-associated alterations in human foetoplacental vasculature.

Antihypertensive therapy in pre-eclampsia: effects of plasma from nonresponsive patients on endothelial gene expression.

AIM: Over 40% of patients with pre-eclampsia are nonresponsive to antihypertensive therapy, but the underlying mechanisms are unknown. We examined the effects of plasma from nonresponsive and responsive patients on endothelial gene expression. PATIENTS & METHODS: PCR array was performed in human umbilical vein endothelial cells (HUVEC) incubated with plasma from nonresponsive (n = 4) and responsive (n = 4) patients. Gene networks and interactions with antihypertensive drugs used in pre-eclampsia were identified by Ingenuity Pathway Analysis. RESULTS: Nifedipine and hydralazine act by upregulate or downregulate genes found to be downregulated or upregulated in HUVEC incubated with plasma from nonresponsive patients. CONCLUSION: Our novel findings suggest that plasma from nonresponsive and responsive patients evoke different responses in HUVEC, and might advance the pharmacogenomics research in pre-eclampsia.

In vitro T-cell profile induced by BCG Moreau in healthy Brazilian volunteers.

Tuberculosis (TB) remains the world's leading cause of morbidity and mortality. Although Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the only vaccine currently in use, its efficacy is highly variable. It has been suggested that early antigenic presentation is a pivotal event leading to a better immune response in TB vaccine models. To investigate this further, we compared in vitro cell-mediated immune responses in the context of early sensitization with TB (i.e. healthy adults vaccinated with BCG when they were young, HD; n = 25) to those in its absence (i.e., newborns with naïve immunity to TB, UV; n = 10) by challenging mononuclear cells with BCG Moreau. After 48 hours, CD4+ and CD8+ T cells were harvested from both groups and stained for PD-1/CD25/ FOXP3. In addition, supernatants were assayed for a broad range of cytokines using an array system. The HD group showed robust reactivity to Protein Purified Derivative and BCG while the naïve, UV group did not. Similarly, in terms of PD-1 expression and Treg cells (CD4+/CD25high(+)/FOXP3+), only the HD group showed higher levels in CD4 lymphocytes. Otherwise, only the UV group showed expression of CD25dim+ as an activation marker dependent on BCG infection. In terms of cytokines, the HD group showed higher levels of Th1 (IL-2/TNF-α/IFN-γ) and regulatory (IL-10) profiles, with monocytes, but not Tr1 cells, acting as the main source of IL-10. Taken together, our results highlight critical roles of early sensitization with TB in mounting cell-mediated immune responses.

Avaliação ultrassonográfica da involução das estruturas umbilicais extra e intracavitárias em bezerros sadios da raça Nelore concebidos naturalmente e produtos de fertilização in vitro / Ultrasound evaluation of extra- and intra-abdominal umbilical structures involution in healthy Nelore calves products of natural conception or in vitro fertilization

Esse trabalho foi desenvolvido com o objetivo de caracterizar a involução das estruturas umbilicais em bezerros sadios da raça Nelore ao longo dos primeiros 35 dias de vida, e de comparar esse processo em bezerros concebidos por métodos naturais ou por fertilização in vitro (FIV). Quarenta bezerros foram distribuídos em dois grupos (n=20) de acordo com o método de concepção (natural ou FIV) e cada grupo foi composto por dez machos e dez fêmeas. A ultrassonografia (transdutor microconvexo de 7,5 MHz) foi empregada para examinar o conjunto das estruturas remanescentes do cordão umbilical que compõem o umbigo externo e as estruturas abdominais (veia umbilical, artéria umbilical esquerda e ducto alantóide), mensurando-se os seus diâmetros em locais definidos. Os exames foram realizados entre 24 e 36 horas de vida e aos 7, 14, 21, 28 e 35 dias de idade. Testaram-se os efeitos do sexo, da idade e do método de concepção por meio da análise de variâncias de medidas repetidas. O exame ultrassonográfico provou-se adequado para a avaliação das estruturas umbilicais extra e intracavitárias permitindo a caracterização do processo fisiológico de involução das mesmas. No umbigo externo, as veias umbilicais foram observadas como imagem individualizada até os 14 dias de vida e um conjunto de estruturas em processo de atrofia era visualizado após essa idade. No abdômen, a veia e a artéria umbilicais foram visualizadas até os 35 dias de idade e o ducto alantóide somente durante a primeira semana de vida. Essas estruturas apresentaram-se com parede hiperecóica regular e contínua e lúmen homogeneamente anecóico. O diâmetro de todas as estruturas umbilicais estudadas se reduziu continuamente ao longo do primeiro mês de vida (p<0,05), sem efeito do sexo (p>0,05). Comparados aos bezerros concebidos por métodos naturais, os produtos de FIV nasceram com os vasos umbilicais e o ducto alantóide um pouco mais calibrosos (diâmetros 1 a 3 mm maiores). Distintamente dos valores ...

This study was carried out to characterize the involution of the umbilical cord structures in healthy Nelore calves during their first 35 days of life, and to compare this process in calves conceived by natural methods or by in vitro fertilization (IVF). Forty calves were separated in two groups (n=20) according to their conception method (natural or IVF) and each group consisted of ten male and ten female calves. The ultrasound (7.5 MHz micro convex transducer) was used to examine all the remaining structures of the umbilical cord that make the external navel and the abdominal structures (umbilical vein, left umbilical artery and allantoic duct), and their diameters were measured in distinct locations. The examinations were performed between 24 and 36 hours of life and at 7, 14, 21, 28 and 35 days of age. The effects of sex, age and method of conception were tested by repeated measures ANOVA. The ultrasound examination was suitable for evaluation of extra- and intra-abdominal umbilical structures and characterization of its involutive physiological process. Both veins were visualized in the external umbilicus up to 14 days of life and set of structures in process of atrophy were seen after this age. In the abdomen, the artery and the vein could be examined up to 35 days of age, and the allantoic duct only during the first week of life. These structures showed a regular and consistent hyperechoic wall and a homogeneous anechoic lumen. The diameter of all studied structures decreased throughout the first month of life (p<0.05) without any sex effect (p>0.05). The umbilical vessels and the allantoic duct were slightly wider (diameter 1-3 mm larger) in calves conceived by IVF. Differently from the highest values previously demonstrated for Bos taurus calves, we can disclose that in healthy newborn Nelore calves the thickness of the structures which make the external navel should not exceed 2 cm, the diameter of the umbilical vein and artery ...