Bárbara Fuenzalida

Insulin requires normal expression and signaling of insulin receptor A to reverse gestational diabetes-reduced adenosine transport in human umbilical vein endothelium

Reduced adenosine uptake via human equilibrative nucleoside transporter 1 (hENT1) in human umbilical vein endothelial cells (HUVECs) from gestational diabetes mellitus (GDM) is reversed by insulin by restoring hENT1 expression. Insulin receptors A (IR‐A) and B (IR‐B) are expressed in HUVECs, and GDM results in higher IR‐A mRNA expression vs. cells from normal pregnancies. We studied whether the reversal of GDM effects on transport by insulin depends on restoration of IR‐A expression.We specifically measured hENT1 expression [mRNA, protein abundance, SLC29A1 (for hENT1) promoter activity] and activity (adenosine transport kinetics) and the role of IR‐A/IR‐B expression and signaling [total and phosphorylated 42 and 44 kDa mitogen‐activated protein kinases (p44/42mapk) and Akt] in IR‐A, IR‐B, and IR‐A/B knockdown HUVECs from normal (n = 33) or GDM (n = 33) pregnancies. GDM increases IR‐A/IR‐B mRNA expression (1.8‐fold) and p44/ 42mapk:Akt activity (2.7‐fold) ratios. Insulin reversed GDM‐reduced hENT1 expression and maximal transport capacity(Vmax/Km),andGDM‐increased IR‐A/IR‐B mRNA expression and p44/42mapk:Akt activity ratios to values in normal pregnancies. Insulins effect was abolished in IR‐A or IR‐A/B knockdown cells. Thus, insulin requires normal IR‐A expression and p44/42mapk/Akt signaling to restore GDM‐reduced hENT1 expression and activity in HUVECs. This could be a protective mechanism for the placental macrovascular endothelial dysfunction seen in GDM.‐Westermeier, F., Salomon, C., Farías, M., Arroyo, P., Fuenzalida, B., Sáez, T., Salsoso, R., Sanhueza, C., Guzmán‐Gutiérrez, E., Pardo, F., Leiva, A., Sobrevia, L. Insulin requires normal expression and signaling of insulin receptor A to reverse gestational diabetes‐reduced adenosine transport in human umbilical vein endothelium. FASEB J. 29, 37–49 (2015). www.fasebj.org

Role of Insulin and Adenosine in the Human Placenta Microvascular and Macrovascular Endothelial Cell Dysfunction in Gestational Diabetes Mellitus

Microvascular and macrovascular endothelial function maintains vascular reactivity. Several diseases alter endothelial function, including hypertension, obesity, and diabetes mellitus. In addition, micro‐ and macrovascular endothelial dysfunction is documented in GDM with serious consequences for the growing fetus. Increased l‐arginine uptake via hCAT‐1 and NO synthesis by eNOS is associated with GDM. These alterations are paralleled by activation of purinergic receptors and increased umbilical vein, but not arteries blood adenosine accumulation. GDM associates with NO‐reduced adenosine uptake in placental endothelium, suggested to maintain and/or facilitate insulin vasodilation likely increasing hCAT‐1 and eNOS expression and activity. It is proposed that increased umbilical vein blood adenosine concentration in GDM reflects a defective metabolic state of human placenta. In addition, insulin recovers GDM‐alterations in hCAT‐1 and eNOS in human micro‐ and macrovascular endothelium, and its biological actions depend on preferential activation of insulin receptors A and B restoring a normal‐like from a GDM‐like phenotype. We summarized existing evidence for a potential role of insulin/adenosine/micro‐ and macrovascular endothelial dysfunction in GDM. These mechanisms could be crucial for a better management of the mother, fetus and newborn in GDM pregnancies.

Nitric Oxide is a Central Common Metabolite in Vascular Dysfunction Associated with Diseases of Human Pregnancy

Preeclampsia (PE), gestational diabetes mellitus (GDM), and maternal supraphysiological hypercholesterolaemia (MSPH) are pregnancy-related conditions that cause metabolic disruptions leading to alterations of the mother, fetus and neonate health. These syndromes result in fetoplacental vascular dysfunction, where nitric oxide (NO) plays a crucial role. PE characterizes by abnormal increase in the placental blood pressure and a negative correlation between NO level and fetal weight, suggesting that increased NO level and oxidative stress could be involved. GDM courses with macrosomia along with altered function of the fetal cardiovascular system and fetoplacental vasculature. Even when NO synthesis in the fetoplacental vasculature is increased, NO bioavailability is reduced due to the higher oxidative stress seen in this disease. In MSPH, there is an early development of atherosclerotic lesions in fetal and newborn arteries, altered function of the fetoplacental vasculature, and higher markers of oxidative stress in fetal blood and placenta, thus, vascular alterations related with NO metabolism occur as a consequence of this syndrome. Potential mechanisms of altered NO synthesis and bioavailability result from transcriptional and post-translational NO synthases (NOS) modulation, including phosphorylation/dephosphorylation cycles, coupling/uncoupling of NOS, tetrahydrobiopterin bioavailability, calcium/calmodulin-NOS and caveolin-1-NOS interaction. Additionally, oxidative stress also plays a role in the reduced NO bioavailability. This review summarizes the available information regarding lower NO bioavailability in these pregnancy pathologies. A common NO-dependent mechanism in PE, GDM and MSPH contributing to fetoplacental endothelial dysfunction is described.

Insulin Is a Key Modulator of Fetoplacental Endothelium Metabolic Disturbances in Gestational Diabetes Mellitus.

Gestational diabetes mellitus (GDM) is a disease of the mother that associates with altered fetoplacental vascular function. GDM-associated maternal hyperglycaemia result in fetal hyperglycaemia, a condition that leads to fetal hyperinsulinemia and altered L-arginine transport and synthesis of nitric oxide, i.e., endothelial dysfunction. These alterations in the fetoplacental endothelial function are present in women with GDM that were under diet or insulin therapy. Since these women and their newborn show normal glycaemia at term, other factors or conditions could be altered and/or not resolved by restoring normal level of circulating D-glucose. GDM associates with metabolic disturbances, such as abnormal handling of the locally released vasodilator adenosine, and biosynthesis and metabolism of cholesterol lipoproteins, or metabolic diseases resulting in endoplasmic reticulum stress and altered angiogenesis. Insulin acts as a potent modulator of all these phenomena under normal conditions as reported in primary cultures of cells obtained from the human placenta; however, GDM and the role of insulin regarding these alterations in this disease are poorly understood. This review focuses on the potential link between insulin and endoplasmic reticulum stress, hypercholesterolemia, and angiogenesis in GDM in the human fetoplacental vasculature. Based in reports in primary culture placental endothelium we propose that insulin is a factor restoring endothelial function in GDM by reversing ERS, hypercholesterolaemia and angiogenesis to a physiological state involving insulin activation of insulin receptor isoforms and adenosine receptors and metabolism in the human placenta from GDM pregnancies.

Dexmedetomidine protects the heart against ischemia-reperfusion injury by an endothelial eNOS/NO dependent mechanism.

The alpha2-adrenergic receptor agonist Dexmedetomidine (Dex) is a sedative medication used by anesthesiologists. Dex protects the heart against ischemia-reperfusion (IR) and can also act as a preconditioning mimetic. The mechanisms involved in Dex-dependent cardiac preconditioning, and whether this action occurs directly or indirectly on cardiomyocytes, still remain unclear. The endothelial nitric oxide synthase (eNOS)/nitric oxide (NO) signaling pathway and endothelial cells are known to play key roles in cardioprotection against IR injury. Therefore, the aims of this work were to evaluate whether the eNOS/NO pathway mediates the pharmacological cardiac effect of Dex, and whether endothelial cells are required in this cardioprotective action. Isolated adult rat hearts were treated with Dex (10nM) for 25min and the dimerization of eNOS and production of NO were measured. Hearts were then subjected to global IR (30/120min) and the role of the eNOS/NO pathway was evaluated. Dex promoted the activation of eNOS and production of NO. Dex reduced the infarct size and improved the left ventricle function recovery, but this effect was reversed when Dex was co-administered with inhibitors of the eNOS/NO/PKG pathway. In addition, Dex was unable to reduce cell death in isolated adult rat cardiomyocytes subjected to simulated IR. Cardiomyocyte death was attenuated by co-culturing them with endothelial cells pre-treated with Dex. In summary, our results show that Dex triggers cardiac protection by activating the eNOS/NO signaling pathway. This pharmacological effect of Dex requires its interaction with the endothelium.

Akt/mTOR Role in Human Foetoplacental Vascular Insulin Resistance in Diseases of Pregnancy.

Insulin resistance is characteristic of pregnancies where the mother shows metabolic alterations, such as preeclampsia (PE) and gestational diabetes mellitus (GDM), or abnormal maternal conditions such as pregestational maternal obesity (PGMO). Insulin signalling includes activation of insulin receptor substrates 1 and 2 (IRS1/2) as well as Src homology 2 domain-containing transforming protein 1, leading to activation of 44 and 42 kDa mitogen-activated protein kinases and protein kinase B/Akt (Akt) signalling cascades in the human foetoplacental vasculature. PE, GDM, and PGMO are abnormal conditions coursing with reduced insulin signalling, but the possibility of the involvement of similar cell signalling mechanisms is not addressed. This review aimed to determine whether reduced insulin signalling in PE, GDM, and PGMO shares a common mechanism in the human foetoplacental vasculature. Insulin resistance in these pathological conditions results from reduced Akt activation mainly due to inhibition of IRS1/2, likely due to the increased activity of the mammalian target of rapamycin (mTOR) resulting from lower activity of adenosine monophosphate kinase. Thus, a defective signalling via Akt/mTOR in response to insulin is a central and common mechanism of insulin resistance in these diseases of pregnancy. In this review, we summarise the cell signalling mechanisms behind the insulin resistance state in PE, GDM, and PGMO focused in the Akt/mTOR signalling pathway in the human foetoplacental endothelium.

Adenosine receptors: modulators of lipid availability that are controlled by lipid levels

Adenosine as well as agonists and antagonists for the four adenosine receptor subtypes (A1R, A2AR, A2BR and A3R) play a role in several key physiological and pathophysiological processes, including the regulation of vascular tone, thrombosis, immune response, inflammation, and angiogenesis. This review focuses on the adenosine-mediated regulation of lipid availability in the cell and in the systemic circulation as well in humans and animal models. Therefore, adenosine, mainly by acting on A1R, inhibits lipolysis activity, leading to reduction of the circulating fatty acid levels. This nucleoside can also participate in the early development of atherosclerosis by inhibiting the formation of foam cells via stimulation of cholesterol efflux through A2AR expressed on macrophages and reduction of the inflammatory process by activating A2AR and A2BR. Adenosine also appears to modulate intracellular cholesterol availability in Niemann-Pick type C1 disease and Alzheimer disease via A2AR and A3, respectively. Remarkably, the role of adenosine receptors in the regulation of plasma total cholesterol and triglyceride levels has been studied in animal models. Thus, an anti-atherogenic role for A2BR as well as a pro-atherogenic role of A2AR and A1 have been proposed; A3R has not been shown to participate in the control of lipid levels or the development of atherosclerosis. Surprisingly, and despite the role of A2A in the inhibition of foam cell formation among isolated cells, this receptor appears to be pro-atherogenic in mice. Remarkably, the role of adenosine receptors in human dyslipidaemia and atherosclerosis must to be elucidated. Additionally, it has been reported that increased lipid levels impair the effects of adenosine/adenosine receptors in controlling vascular tone, and we speculate on the possibility that this impairment could be due to alterations in the composition of the membrane microdomains where the adenosine receptors are located. Finally, a possible role for adenosine/adenosine receptors in the phenomena of dyslipidaemia in pregnancy has been proposed.

Role for Tetrahydrobiopterin in the Fetoplacental Endothelial Dysfunction in Maternal Supraphysiological Hypercholesterolemia

Maternal physiological hypercholesterolemia occurs during pregnancy, ensuring normal fetal development. In some cases, the maternal plasma cholesterol level increases to above this physiological range, leading to maternal supraphysiological hypercholesterolemia (MSPH). This condition results in endothelial dysfunction and atherosclerosis in the fetal and placental vasculature. The fetal and placental endothelial dysfunction is related to alterations in the L-arginine/nitric oxide (NO) pathway and the arginase/urea pathway and results in reduced NO production. The level of tetrahydrobiopterin (BH4), a cofactor for endothelial NO synthase (eNOS), is reduced in nonpregnant women who have hypercholesterolemia, which favors the generation of the superoxide anion rather than NO (from eNOS), causing endothelial dysfunction. However, it is unknown whether MSPH is associated with changes in the level or metabolism of BH4; as a result, eNOS function is not well understood. This review summarizes the available information on the potential link between MSPH and BH4 in causing human fetoplacental vascular endothelial dysfunction, which may be crucial for understanding the deleterious effects of MSPH on fetal growth and development.

Tetrahydrobiopterin Role in human umbilical vein endothelial dysfunction in maternal supraphysiological hypercholesterolemia

Maternal physiological hypercholesterolemia (MPH) allows a proper foetal development; however, maternal supraphysiological hypercholesterolemia (MSPH) associates with foetal endothelial dysfunction and early development of atherosclerosis. MSPH courses with reduced endothelium-dependent dilation of the human umbilical vein due to reduced endothelial nitric oxide synthase activity compared with MPH. Whether MSPH modifies the availability of the nitric oxide synthase cofactor tetrahydrobiopterin is unknown. We investigated whether MSPH-associated lower umbilical vein vascular reactivity results from reduced bioavailability of tetrahydrobiopterin. Total cholesterol <7.2mmol/L was considered as maternal physiological hypercholesterolemia (n=72 women) and ≥7.2mmol/L as MSPH (n=35 women). Umbilical veins rings were used for vascular reactivity assays (wire myography), and primary cultures of human umbilical vein endothelial cells (HUVECs) to measure nitric oxide synthase, GTP cyclohydrolase 1, and dihydrofolate reductase expression and activity, as well as tetrahydrobiopterin content. MSPH reduced the umbilical vein rings relaxation caused by calcitonine gene-related peptide, a phenomenon partially improved by incubation with sepiapterin. HUVECs from MSPH showed lower nitric oxide synthase activity (l-citrulline synthesis from l-arginine) without changes in its protein abundance, as well as reduced tetrahydrobiopterin level compared with MPH, a phenomenon reversed by incubation with sepiapterin. Expression and activity of GTP cyclohydrolase 1 was lower in MSPH, without changes in dihydrofolate reductase expression. MSPH is a pathophysiological condition reducing human umbilical vein reactivity due to lower bioavailability of tetrahydrobiopterin leading to lower NOS activity in the human umbilical vein endothelium.

Maternal supraphysiological hypercholesterolemia associates with endothelial dysfunction of the placental microvasculature

Maternal physiological or supraphysiological hypercholesterolemia (MPH, MSPH) occurs during pregnancy. MSPH is associated with foetal endothelial dysfunction and atherosclerosis. However, the potential effects of MSPH on placental microvasculature are unknown. The aim of this study was to determine whether MSPH alters endothelial function in the placental microvasculature both ex vivo in venules and arterioles from the placental villi and in vitro in primary cultures of placental microvascular endothelial cells (hPMEC). Total cholesterol < 280 mg/dL indicated MPH, and total cholesterol ≥280 mg/dL indicated MSPH. The maximal relaxation to histamine, calcitonin gene-related peptide and adenosine was reduced in MSPH venule and arteriole rings. In hPMEC from MSPH placentas, nitric oxide synthase (NOS) activity and L-arginine transport were reduced without changes in arginase activity or the protein levels of endothelial NOS (eNOS), human cationic amino acid 1 (hCAT-1), hCAT-2A/B or arginase II compared with hPMEC from MPH placentas. In addition, it was shown that adenosine acts as a vasodilator of the placental microvasculature and that NOS is active in hPMEC. We conclude that MSPH alters placental microvascular endothelial function via a NOS/L-arginine imbalance. This work also reinforces the concept that placental endothelial cells from the macro- and microvasculature respond differentially to the same pathological condition.