Browsing by Author "Vega Pizarro, José Luis Eduardo"

Now showing 1 - 5 of 5
  • No Thumbnail Available
    Item
    Equilibrative nucleoside transporters in fetal endothelial dysfunction in diabetes mellitus and hyperglycaemia
    (2009) Westermeier Lafuente, Francisco David; Puebla Aracena, Carlos Alberto; Vega Pizarro, José Luis Eduardo; Farías Jofré, Marcelo Enrique; Escudero Orozco, Carlos Alonso; Casanello Toledo, Paola Cecilia; Sobrevía Luarte, Luis Alberto
    Diabetes mellitus types 1 and 2, and gestational diabetes are characterized by abnormal D-glucose metabolism and hyperglycaemia, and induce foetal endothelial dysfunction with implications in adult life increasing the risk of vascular diseases. Synthesis of nitric oxide (NO) and uptake of L-arginine (i.e. the L-arginine/NO signalling pathway) and adenosine (a vasoactive endogenous nucleoside) by the umbilical vein endothelium is altered in pathological pregnancies, including pregnancies with pre-established diabetes mellitus or in gestational diabetes. The mechanisms underlying these alterations include differential expression of equilibrative nucleoside transporters (ENTs), amino acid transporters and NO synthases (NOS). Modulation of ENTs and NOS expression and activity in endothelium involves several signalling molecules, including protein kinase C, mitogen-activated protein kinases p42 and p44, calcium and phosphatidyl inositol 3 kinase. Elevated extracellular D-glucose and diabetes alters human endothelial function. However, information regarding modulation the transport capacity as well as expression of ENTs is limited. This review focuses on the effect of diabetes mellitus and gestational diabetes, and hyperglycaemia on the reported mechanisms described for transcriptional and posttranscriptional regulation of ENTs, and the potential consequences for foetal endothelial function in these pathologies. Recent available information regarding functional consequences of an abnormal environment on the functionality of the endothelium from microvasculature of the human placenta is mentioned. The available information is scarce, but it could contribute to a better understanding of the cell and molecular basis of the altered vascular endothelial function in this pathological conditions, emphasizing the key role of this type of epithelium in fetal-placental function and the normal foetal development and growth.
  • No Thumbnail Available
    Item
    Gestational diabetes mellitus is associated with NO and PKC-dependent repressor effect of hCHOP-C/EBPalpha on equilibrative nucleoside transporter 1 expression human umbilical vein endothelium
    (2008) Farías Jofré, Marcelo Enrique; Puebla Aracena, Carlos Alberto; Vega Pizarro, José Luis Eduardo; Pastor-Anglaga, M.; Casanello Toledo, Paola Cecilia; Sobrevía Luarte, Luis Alberto
  • Thumbnail Image
    Item
    TGF-beta1 inhibits expression and activity of hENT1 in a nitric oxide-dependent manner in human umbilical vein endothelium
    (2009) Vega Pizarro, José Luis Eduardo; Puebla Aracena, Carlos Alberto; Vásquez, Rodrigo; Farías Jofré, Marcelo Enrique; Alarcón, Julio; Pastor-Anglada, Marçal; Krause Leyton, Bernardo; Casanello Toledo, Paola Cecilia; Sobrevía Luarte, Luis Alberto
    Aims: We studied whether transforming growth factor β1 (TGF-β1) modulates human equilibrative nucleoside transporters 1 (hENT1) expression and activity in human umbilical vein endothelial cells (HUVECs). hENT1-mediated adenosine transport and expression are reduced in gestational diabetes and hyperglycaemia, conditions associated with increased synthesis and release of nitric oxide (NO) and TGF-β1 in this cell type. TGF-β1 increases NO synthesis via activation of TGF-β receptor type II (TβRII), and NO inhibits hENT1 expression and activity in HUVECs. Methods and results: HUVECs (passage 2) were used for experiments. Total and hENT1-mediated adenosine transport was measured in the absence or presence of TGF-β1, NG-nitro-L-arginine methyl ester (L-NAME, NO synthase inhibitor), S-nitroso-N-acetyl-L,D-penicillamine (SNAP, NO donor), and/or KT-5823 (protein kinase G inhibitor) in control cells and cells expressing a truncated form of TGF-β1 receptor type II (TTβRII). Western blot and real-time PCR were used to determine hENT1 protein abundance and mRNA expression. SLC29A1 gene promoter and specific protein 1 (Sp1) transcription factor activity was assayed. Vascular reactivity was assayed in endothelium-intact or -denuded umbilical vein rings. TGF-β1 reduced hENT1-mediated adenosine transport, hENT1 protein abundance, hENT1 mRNA expression, and SLC29A1 gene promoter activity, but increased Sp1 binding to DNA. TGF-β1 effect was blocked by L-NAME and KT-5823 and mimicked by SNAP in control cells. However, TGF-β1 was ineffective in cells expressing TTβRII or a mutated Sp1 consensus sequence. Vasodilatation in response to TGF-β1 and S-(4-nitrobenzyl)-6-thio-inosine (an ENT inhibitor) was endothelium-dependent and blocked by KT-5823 and ZM-241385. Conclusion: hENT1 is down-regulated by activation of TβRII by TGF-β1 in HUVECs, a phenomenon where NO and Sp1 play key roles. These findings comprise physiological mechanisms that could be important in diseases where TGF-β1 plasma level is increased as in gestational diabetic mothers or patients with diabetes mellitus.
  • No Thumbnail Available
    Item
    The transcriptional repression of equilibrative nucleoside transporter 1 by D-glucose rfsponses to transcription factor Sp1 and ZBP-89 in human foetal endothelium
    (2008) Puebla Aracena, Carlos Alberto; Farías Jofré, Marcelo Enrique; Vega Pizarro, José Luis Eduardo; Pastor-Anglada, M.; Casanello Toledo, Paola Cecilia; Sobrevía Luarte, Luis Alberto
    Objectives: Adenosine is a vasodilator in most vascular beds, an effect depending on its extracellular concentration. Uptake of this nucleoside in human umbilical vein endothelial cells (HUVEC) is mainly mediated by human equilibrative nucleoside transporters 1 (hENT1). hENT1 expression and transport activity are reduced in HUVEC exposed to high D-glucose. Since specific mechanisms for these effects of D-glucose are unknown, we examined the role of Sp1 and BP-89 transcription factors on SLC29A1 (for hENT1) promoter activity in response to D-glucose. Methods: HUVEC from normal pregnancies were isolated and exposed (24 h) to 5 mM (normal) or 25 mM (high) D-glucose. Sp1 protein levels were evaluated by western blot in nuclear fractions. Reporter activity of plasmid constructs containing a wild type promoter region of SLC29A1 (-1114 bp to ATG, pGL3-hENT1-1114), or mutations (by PCR) for Sp1 (-815/-801 bp, pGL3-hENT1-1114mutSp1) or ZBP-89 (-992/-969 bp, pGL3-hENT1-1114mutZBP) or both (-1114 bp, pGL3-hENT1-1114mutSp1/ZBP) binding sites were assayed in cells over-expressing Sp1 (using pCGN-Sp1 vector). Results: Nuclear Sp1 abundance was increased, but pGL3-hENT1-1114 transcriptional activity was reduced by high D-glucose. Sp1 over-expression reduced pGL3-hENT1-1114 transcriptional activity in normal or high D-glucose. The effect of high D-glucose or Sp1 over-expression was absent in pGL3-hENT1-1114mutSp1, pGL3-hENT1-1114mutZBP and pGL3-hENT1-1114mutSp1/ZBP cells. Conclusion: A repression of the SLC29A1 promoter activity by Sp1 and ZBP-89 could explain the reduced hENT1 expression and activity exhibited by HUVEC in high extracellular D-glucose. FONDECYT 1070865/1080534/7070249 (Chile), AECI A/5484/06 (Spain). C Puebla andJL Vega hold CONICYT fellowships. M Farías holds CONICYT and PUC-School of Medicine PhD fellowships.
  • No Thumbnail Available
    Item
    Transforming growth factor beta 1 is involved in the inhibitory effect of high D-glucose on human equilibrative nucleosidie transporter 1 in human fetal endothelium
    (2008) Vega Pizarro, José Luis Eduardo; Farías Jofré, Marcelo Enrique; Puebla Aracena, Carlos Alberto; González Ortiz, Marcelo Andrés; Casanello Toledo, Paola Cecilia; Sobrevía Luarte, Luis Alberto
    Objectives: High D-glucose (25 mM) reduces adenosine transport via the human equilibrative nucleoside transporters 1 (hENT1) in human umbilical vein endothelial cells (HUVEC). Interestingly, high D-glucose also increases the release of TGF-b1 from primary cultures of HUVEC leading to altered transport of L-arginine and synthesis of nitric oxide (NO), most likely due to activation of type II TGF-b1 receptors (TbRII). Since NO is involved in the repression of SLC29A1 gene expression in HUVEC, it is likely that D-glucose effect on hENT1 mediated adenosine transport involves TGF-b1 and activation of TbRII in this cell type. Therefore, we have studied whether TGF-b1 is involved in theinhibitory effect of high D-glucose on hENT1 in HUVEC. Methods: Cells were exposed to D-glucose (5-25 mM; 6-24 h) and/or TGF-b1 (0.1-10 ng/ml, 1-24 h) and [3H]adenosine transport (4 mCi/ml, 20 s, 22 C) was measured in absence or presence of nitrobenzylmercapto purine ribose (NBMPR, 1 mM, ENT1 inhibitor). These assays were performed in cells transduced with an adenovirus to induce expression of a truncated form of TbRII (Ad-tTbRII) or with a control adenovirus (Ad-control, empty vector). Results: In cells exposed to normal D-glucose (5 mM), TGF-b1 inhibited hENT1-mediated adenosine transport (~60%), an effect that was dose-dependent reaching a maximal inhibition at 2 ng/ml. The TGF-b1 effect was time-dependent with a significant inhibition of hENT1-mediated adenosine transport at 3 h of incubation, an effect that sustained for further 21 h. The effect of TGF-b1 was absent in cells transduced with Ad-tTbRII. In cells in high D-glucose (25 mM), hENT1-mediated adenosine transport was decreased compared with cells in normal D-glucose. This effect of high D-glucose was blocked in cells transduced with Ad-tTbRII, but it was unaltered in cells transduced with Adcontrol. Conclusion: These results suggest that TGF-b1 could mediate the inhibitory effect of high D-glucose on hENT1 activity by the activation of type II receptor for TGF-b1 in primary cultures of HUVEC.FONDECYT 1070865/1080534/7070249 (Chile), J.L. Vega, C. Puebla and M. González hold a CONICYT PhD fellowships. M. Farías holds CONICYT and PUC-School of Medicine PhD fellowships.