Claudio Aguayo

Inhibition of Nitrobenzylthioinosine-Sensitive Adenosine Transport by Elevated d-Glucose Involves Activation of P2Y2 Purinoceptors in Human Umbilical Vein Endothelial Cells

Chronic incubation with elevated d-glucose reduces adenosine transport in endothelial cells. In this study, exposure of human umbilical vein endothelial cells to 25 mmol/L d-glucose or 100 &mgr;mol/L ATP, ATP-&ggr;-S, or UTP, but not ADP or &agr;,&bgr;-methylene ATP, reduced adenosine transport with no change in transport affinity. Inhibition of transport by d-glucose, ATP, and ATP-&ggr;-S was associated with reduced maximal binding, with no changes in the apparent dissociation constant for nitrobenzylthioinosine (NBMPR). A significant reduction (≈60±10%, P <0.05; n=6) in the number of human equilibrative NBMPR-sensitive nucleoside transporters (hENT1s) per cell (1.8±0.1×106 in 5 mmol/L d-glucose) and in hENT1 mRNA levels was observed in cells exposed to d-glucose or ATP-&ggr;-S. Incubation with elevated d-glucose, but not with d-mannitol, increased the ATP release by 3±0.2-fold. The effects of d-glucose and nucleotides on the number and activity of hENT1 and hENT1 mRNA were blocked by reactive blue 2 (nonspecific P2Y purinoceptor antagonist), suramin (G&agr;s protein inhibitor), or hexokinase but not by pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid (nonselective P2 purinoceptor antagonist). Our findings demonstrate that inhibition of adenosine transport via hENT1 in endothelial cells cultured in 25 mmol/L d-glucose could be due to stimulation of P2Y2 purinoceptors by ATP, which is released from these cells in response to d-glucose. This could be a mechanism to explain in part the vasodilatation observed in the early stages of diabetes mellitus or in response to d-glucose infusion.

Rapid Stimulation of L-Arginine Transport by D-Glucose Involves p42/44 mapk and Nitric Oxide in Human Umbilical Vein Endothelium

Abstract— d-Glucose infusion and gestational diabetes induce vasodilatation in humans and increase l-arginine transport and nitric oxide (NO) synthesis in human umbilical vein endothelial cells. High d-glucose (25 mmol/L, 2 minutes) induced membrane hyperpolarization and an increase of l-arginine transport (Vmax 6.1±0.7 versus 4.4±0.1 pmol/&mgr;g protein per minute) with no change in transport affinity (Km 105±9 versus 111±16 &mgr;mol/L). l-[3H]Citrulline formation and intracellular cGMP, but not intracellular Ca2+, were increased by high d-glucose. The effects of d-glucose were mimicked by levcromakalim (ATP-sensitive K+ channel blocker), paralleled by p42/p44mapk and Ser1177–endothelial NO synthase phosphorylation, inhibited by NG-nitro-l-arginine methyl ester (L-NAME; NO synthesis inhibitor), glibenclamide (ATP-sensitive K+ channel blocker), KT-5823 (protein kinase G inhibitor), PD-98059 (mitogen-activated protein kinase kinase 1/2 inhibitor), and wortmannin (phosphatidylinositol 3-kinase inhibitor), but they were unaffected by calphostin C (protein kinase C inhibitor). Elevated d-glucose did not alter superoxide dismutase activity. Our findings demonstrate that the human fetal endothelial l-arginine/NO signaling pathway is rapidly activated by elevated d-glucose via NO and p42/44mapk. This could be determinant in pathologies in which rapid fluctuations of plasma d-glucose may occur and may underlie the reported vasodilatation in early stages of diabetes mellitus.

Regulation of adenosine transport by D-glucose in human fetal endothelial cells: involvement of nitric oxide, protein kinase C and mitogen-activated protein kinase

1 The effects of elevated D‐glucose on adenosine transport were investigated in human cultured umbilical vein endothelial cells isolated from normal pregnancies. 2 Elevated D‐glucose resulted in a time‐ (8‐12 h) and concentration‐dependent (half‐maximal at 10 ± 2 mM) inhibition of adenosine transport, which was associated with a reduction in the Vmax for nitrobenzylthioinosine (NBMPR)‐sensitive (es) saturable nucleoside with no significant change in Km. D‐Fructose (25 mM), 2‐deoxy‐D‐glucose (25 mM) or D‐mannitol (20 mM) had no effect on adenosine transport. 3 Adenosine transport was inhibited following incubation of cells with the protein kinase C (PKC) activator phorbol 12‐myristate 13‐acetate (PMA; 100 nM, 30 min to 24 h). D‐Glucose‐induced inhibition of transport was abolished by calphostin C (100 nM, an inhibitor of PKC), and was not further reduced by PMA. 4 Increased PKC activity in the membrane (particulate) fraction of endothelial cells exposed to D‐glucose or PMA was blocked by calphostin C but was unaffected by NG‐nitro‐L‐arginine methyl ester (L‐NAME; 100 μM, an inhibitor of nitric oxide synthase (NOS)) or PD‐98059 (10 μM, an inhibitor of mitogen‐activated protein kinase kinase 1). 5 D‐Glucose and PMA increased endothelial NOS (eNOS) activity, which was prevented by calphostin C or omission of extracellular Ca2+ and unaffected by PD‐98059. 6 Adenosine transport was inhibited by S‐nitroso‐N‐acetyl‐l,D‐penicillamine (SNAP; 100 μM, an NO donor) but was increased in cells incubated with L‐NAME. The effect of SNAP on adenosine transport was abolished by PD‐98059. 7 Phosphorylation of mitogen‐activated protein kinases p44mapk (ERK1) and p42mapk (ERK2) was increased in endothelial cells exposed to elevated D‐glucose (25 mM for 30 min to 24 h) and the NO donor SNAP (100 μM, 30 min). The effect of D‐glucose was blocked by PD‐98059 or L‐NAME, which also prevented the inhibition of adenosine transport mediated by elevated D‐glucose. 8 Our findings provide evidence that D‐glucose inhibits adenosine transport in human fetal endothelial cells by a mechanism that involves activation of PKC, leading to increased NO levels and p42‐p44mapk phosphorylation. Thus, the biological actions of adenosine appear to be altered under conditions of sustained hyperglycaemia.

Mesenchymal Stem Cell-Derived Extracellular Vesicles Promote Angiogenesis: Potencial Clinical Application

Mesenchymal stem cells (MSCs) are adult multipotent stem cells that are able to differentiate into multiple specialized cell types including osteocytes, adipocytes, and chondrocytes. MSCs exert different functions in the body and have recently been predicted to have a major clinical/therapeutic potential. However, the mechanisms of self-renewal and tissue regeneration are not completely understood. It has been shown that the biological effect depends mainly on its paracrine action. Furthermore, it has been reported that the secretion of soluble factors and the release of extracellular vesicles, such as exosomes, could mediate the cellular communication to induce cell-differentiation/self-renewal. This review provides an overview of MSC-derived exosomes in promoting angiogenicity and of the clinical relevance in a therapeutic approach.

Insulin Reverses D-Glucose–Increased Nitric Oxide and Reactive Oxygen Species Generation in Human Umbilical Vein Endothelial Cells

Vascular tone is controlled by the L-arginine/nitric oxide (NO) pathway, and NO bioavailability is strongly affected by hyperglycaemia-induced oxidative stress. Insulin leads to high expression and activity of human cationic amino acid transporter 1 (hCAT-1), NO synthesis and vasodilation; thus, a protective role of insulin on high D-glucose–alterations in endothelial function is likely. Vascular reactivity to U46619 (thromboxane A2 mimetic) and calcitonin gene related peptide (CGRP) was measured in KCl preconstricted human umbilical vein rings (wire myography) incubated in normal (5 mmol/L) or high (25 mmol/L) D-glucose. hCAT-1, endothelial NO synthase (eNOS), 42 and 44 kDa mitogen-activated protein kinases (p42/44mapk), protein kinase B/Akt (Akt) expression and activity were determined by western blotting and qRT-PCR, tetrahydrobiopterin (BH4) level was determined by HPLC, and L-arginine transport (0–1000 μmol/L) was measured in response to 5–25 mmol/L D-glucose (0–36 hours) in passage 2 human umbilical vein endothelial cells (HUVECs). Assays were in the absence or presence of insulin and/or apocynin (nicotinamide adenine dinucleotide phosphate-oxidase [NADPH oxidase] inhibitor), tempol or Mn(III)TMPyP (SOD mimetics). High D-glucose increased hCAT-1 expression and activity, which was biphasic (peaks: 6 and 24 hours of incubation). High D-glucose–increased maximal transport velocity was blocked by insulin and correlated with lower hCAT-1 expression and SLC7A1 gene promoter activity. High D-glucose–increased transport parallels higher reactive oxygen species (ROS) and superoxide anion (O2 •–) generation, and increased U46619-contraction and reduced CGRP-dilation of vein rings. Insulin and apocynin attenuate ROS and O2 •– generation, and restored vascular reactivity to U46619 and CGRP. Insulin, but not apocynin or tempol reversed high D-glucose–increased NO synthesis; however, tempol and Mn(III)TMPyP reversed the high D-glucose–reduced BH4 level. Insulin and tempol blocked the high D-glucose–increased p42/44mapk phosphorylation. Vascular dysfunction caused by high D-glucose is likely attenuated by insulin through the L-arginine/NO and O2 •–/NADPH oxidase pathways. These findings are of interest for better understanding vascular dysfunction in states of foetal insulin resistance and hyperglycaemia.

Fetal endothelium dysfunction is associated with circulating maternal levels of sE-selectin, sVCAM1, and sFlt-1 during pre-eclampsia

Objectives. To evaluate the association between endothelial activation markers in the maternal circulation with nitric oxide (NO) synthesis in human umbilical endothelial cells. Study design. This is a case-control study of normal and pre-eclamptic pregnancies. The levels of sE-selectin, soluble vascular cell adhesion molecule 1 (sVCAM-1), and soluble fms-like tyrosine kinase 1 (sFlt-1) were measured by enzyme-linked immunosorbent assay, and histamine-induced NO synthesis was detected by fluorometric examination of the human umbilical vein endothelial cells (HUVECs) isolated from normal and pathological pregnancies. Results. Mothers with severe pre-eclamptic pregnancies have premature and smaller babies than mothers with normal pregnancies (P < 0.05); they also have high maternal plasma levels of sVCAM-1 (∼2-fold), sFlt-1 (∼2.5-fold), and lower (∼70%) histamine-stimulated NO synthesis in HUVECs. A positive relationship between systolic blood pressure (SBP) and plasma levels of sE-selectin, sVCAM-1, and sFlt-1 was demonstrated. Moreover, levels of sE-selectin, sVCAM-1, and sFlt-1 were negatively associated with newborn weight (NBW), gestational age at delivery, and NO synthesis. Women with high E-selectin (>63 ng/ml), VCAM-1 (>752 ng/ml), and sFlt-1 (>15204 pg/ml) showed high risk (∼2-fold) for preterm delivery and very preterm delivery, or fetal weight <1500 g (∼1.5-fold) compared with women with low levels. Conclusions. High circulating levels of maternal endothelial dysfunction markers present in pre-eclampsia are associated with decreased NO synthesis in fetal endothelium.

Role of Extracellular Vesicles and microRNAs on Dysfunctional Angiogenesis during Preeclamptic Pregnancies

Preeclampsia is a syndrome characterized by hypertension during pregnancy, which is a leading cause of morbidity and mortality in both mother and newborn in developing countries. Some advances have increased the understanding of pathophysiology of this disease. For example, reduced utero-placental blood flow associated with impaired trophoblast invasion may lead to a hypoxic placenta that releases harmful materials into the maternal and feto-placental circulation and impairs endothelial function. Identification of these harmful materials is one of the hot topics in the literature, since these provide potential biomarkers. Certainty, such knowledge will help us to understand the miscommunication between mother and fetus. In this review we highlight how placental extracellular vesicles and their cargo, such as small RNAs (i.e., microRNAs), might be involved in endothelial dysfunction, and then in the angiogenesis process, during preeclampsia. Currently only a few reports have addressed the potential role of endothelial regulatory miRNA in the impaired angiogenesis in preeclampsia. One of the main limitations in this area is the variability of the analyses performed in the current literature. This includes variability in the size of the particles analyzed, and broad variation in the exosomes considered. The quantity of microRNA targets genes suggest that practically all endothelial cell metabolic functions might be impaired. More studies are required to investigate mechanisms underlying miRNA released from placenta upon endothelial function involved in the angiogenenic process.

Modulation of adenosine transport by insulin in human umbilical artery smooth muscle cells from normal or gestational diabetic pregnancies

1 Adenosine transport was measured in human cultured umbilical artery smooth muscle cells, isolated from non‐diabetic or gestational diabetic pregnancies, under basal conditions and after pretreatment in vitro with insulin. 2 Adenosine transport in non‐diabetic smooth muscle cells was significantly increased by insulin (half‐maximal stimulation at 0.33 ± 0.02 nm, 8 h) and characterized by a higher maximal rate (Vmax) for nitrobenzylthioinosine (NBMPR)‐sensitive (es) saturable nucleoside transport (17 ± 5 vs. 52 ± 12 pmol (μg protein)−1 min−1, control vs. insulin, respectively) and maximal binding sites (Bmax) for [3H]NBMPR (0.66 ± 0.07 vs. 1.1 ± 0.1 fmol (μg protein)−1, control vs. insulin, respectively), with no significant changes in Michaelis‐Menten (Km) and dissociation (Kd) constants. 3 In contrast, in smooth muscle cells from diabetic pregnancies, where the values of Vmax for adenosine transport (59 ± 4 pmol (μg protein)−1 min−1) and Bmax for [3H]NBMPR binding (1.62 ± 0.16 fmol (μg protein)−1) were significantly elevated by comparison with non‐diabetic cells, insulin treatment (1 nm, 8 h) reduced the Vmax for adenosine transport and Bmax for [3H]NBMPR binding to levels detected in non‐diabetic cells. 4 In non‐diabetic cells, the stimulatory effect of insulin on adenosine transport was mimicked by dibutyryl cGMP (100 nm) and reduced by inhibitors of phosphatidylinositol 3‐kinase (10 nm wortmannin), nitric oxide synthase (100 μmNG‐nitro‐l‐arginine methyl ester, l‐NAME) or protein synthesis (1 μm cycloheximide), whereas inhibition of adenylyl cyclase (100 μm SQ‐22536) had no effect. 5 Wortmannin or SQ‐22536, but not l‐NAME or cycloheximide, attenuated the inhibitory action of insulin on the diabetes‐induced stimulation of adenosine transport. 6 Protein levels of inducible NO synthase (iNOS) were similar in non‐diabetic and diabetic cells, but were increased by insulin (1 nm, 8 h) only in non‐diabetic smooth muscle cells. 7 Our results suggest that adenosine transport via the es nucleoside transporter is modulated differentially by insulin in either cell type. Insulin increased adenosine transport in non‐diabetic cells via NO and cGMP, but inhibited the diabetes‐elevated adenosine transport via activation of adenylyl cyclase, suggesting that the biological actions of adenosine may be altered under conditions of sustained hyperglycaemia in uncontrolled diabetes.

Endothelium Trans Differentiated from Wharton's Jelly Mesenchymal Cells Promote Tissue Regeneration: Potential Role of Soluble Pro-Angiogenic Factors

Background Mesenchymal stem cells have a high capacity for trans-differentiation toward many adult cell types, including endothelial cells. Feto-placental tissue, such as Whartons jelly is a potential source of mesenchymal stem cells with low immunogenic capacity; make them an excellent source of progenitor cells with a potential use for tissue repair. We evaluated whether administration of endothelial cells derived from mesenchymal stem cells isolated from Whartons jelly (hWMSCs) can accelerate tissue repair in vivo. Methods Mesenchymal stem cells were isolated from human Whartons jelly by digestion with collagenase type I. Endothelial trans-differentiation was induced for 14 (hWMSC-End14d) and 30 (hWMSC-End30d) days. Cell phenotyping was performed using mesenchymal (CD90, CD73, CD105) and endothelial (Tie-2, KDR, eNOS, ICAM-1) markers. Endothelial trans-differentiation was demonstrated by the expression of endothelial markers and their ability to synthesize nitric oxide (NO). Results hWMSCs can be differentiated into adipocytes, osteocytes, chondrocytes and endothelial cells. Moreover, these cells show high expression of CD73, CD90 and CD105 but low expression of endothelial markers prior to differentiation. hWMSCs-End express high levels of endothelial markers at 14 and 30 days of culture, and also they can synthesize NO. Injection of hWMSC-End30d in a mouse model of skin injury significantly accelerated wound healing compared with animals injected with undifferentiated hWMSC or injected with vehicle alone. These effects were also observed in animals that received conditioned media from hWMSC-End30d cultures. Conclusion These results demonstrate that mesenchymal stem cells isolated from Whartons jelly can be cultured in vitro and trans-differentiated into endothelial cells. Differentiated hWMSC-End may promote neovascularization and tissue repair in vivo through the secretion of soluble pro-angiogenic factors.

Nitric Oxide, cGMP and cAMP Modulate Nitrobenzylthioinosine-Sensitive Adenosine Transport in Human Umbilical Artery Smooth Muscle Cells from Subjects with Gestational Diabetes

Adenosine transport was characterized in human umbilical artery smooth muscle cells isolated from non‐diabetic and diabetic pregnant subjects. Transport of adenosine was mediated by a Na+‐independent transport system inhibited by nanomolar concentrations of nitrobenzylthioinosine (NBMPR) in both cell types. Diabetes increased adenosine transport, an effect that was associated with a higher maximal velocity (Vmax) for NBMPR‐sensitive (es) saturable nucleoside transport (18 ± 2 vs. 61 ± 3 pmol (μg protein)‐1 min‐1, P < 0.05) and the maximal number of binding sites (Bmax) for specific [3H]NBMPR binding (74 ± 4 vs. 156 ± 10 pmol (μg protein)‐1, P < 0.05), with no significant changes in the Michaelis‐Menten (Km) and dissociation (Kd) constants, respectively. Adenosine transport was unaltered by inhibition of nitric oxide (NO) synthase (with 100 μM NG‐nitro‐L‐arginine methyl ester, L‐NAME) or protein synthesis (with 1 μM cycloheximide), but was increased by inhibition of adenylyl cyclase activity (with 100 μM, SQ‐22536) in non‐diabetic cells. Diabetes‐induced adenosine transport was blocked by L‐NAME and associated with an increase in L‐[3H]citrulline formation from L‐[3H]arginine and intracellular cGMP, but with a decrease in intracellular cAMP compared with non‐diabetic cells. Expression of inducible NO synthase (iNOS) was unaltered by diabetes. Dibutyryl cGMP (dbcGMP) increased, but dibutyryl cAMP (dbcAMP) decreased, adenosine transport in non‐diabetic cells. dbcGMP or the NO donor S‐nitrosoacetylpenicillamine (SNAP, 100 μM) did not alter the diabetes‐elevated adenosine transport. However, activation of adenylyl cyclase with forskolin (1 μM), directly or after incubation of cells with dbcAMP, inhibited adenosine transport in both cell types. Our findings provide the first evidence that adenosine transport in human umbilical artery smooth muscle cells is mediated by the NBMPR‐sensitive transport system es, and that its activity is upregulated by gestational diabetes.