Abstract

Intrauterine growth restriction (IUGR) is associated with chronic fetal hypoxia, altered placental vasodilatation and reduced endothelial nitric oxide synthase (eNOS) activity. In human umbilical vein endothelial cells (HUVEC) from pregnancies complicated with IUGR (IUGR cells) and in HUVEC from normal pregnancies (normal cells) cultured under hypoxia l-arginine transport is reduced; however, the mechanisms leading to this dysfunction are unknown. We studied hypoxia effect on l-arginine transport and human cationic amino acid transporters 1 (hCAT-1) expression, and the potential NO and protein kinase C a (PKCa) involvement. Normal or IUGR HUVEC monolayers were exposed (0-24 h) to 5% O 2 (normoxia), and 1 or 2% O 2 (hypoxia). l-Arginine transport and hCAT-1 expression, phosphorylated and total PKCa or eNOS protein and mRNA expression were quantified. eNOS involvement was tested using a siRNA against eNOS (eNOS-siRNA) adenovirus. IUGR cells in normoxia or hypoxia, and normal cells in hypoxia exhibited reduced l-arginine transport, hCAT-1 expression, NO synthesis and eNOS phosphorylation at Serine 1177, effects reversed by calphostin C (PKC inhibitor) and S-nitroso-N-acetyl-l,d-penicillamine (SNAP, NO donor). However, N G-nitro-l-arginine methyl ester (l-NAME, NOS inhibitor) reduced hCAT-1 expression only in normal cells in normoxia. Increased Thr 638-phosphorylated PKCa was exhibited by IUGR cells in normoxia or hypoxia and normal cells in hypoxia. The effects of hypoxia in normal cells were mimicked in eNOS-siRNA transduced cells; however, IUGR phenotype was unaltered by eNOS knockdown. Thus, IUGR- and hypoxia-reduced l-arginine transport could result from increased PKCa, but reduced eNOS activity leading to a lower hCAT-1 expression in HUVEC. In addition, IUGR endothelial cells are either not responsive or maximally affected by hypoxia. These mechanisms could be responsible for placental dysfunction in diseases where fetal endothelium is chronically exposed to hypoxia, such as IUGR. © 2009 Elsevier Ltd. All rights reserved.