Iron-induced remodeling in cultured rat pulmonary artery endothelial cells

Abstract

Although iron is known to be a component of the pathogenesis and/or maintenance of acute lung injury (ALI) in experimental animals and human subjects, the majority of these studies have focused on disturbances in iron homeostasis in the airways resulting from exposure to noxious gases and particles. Considerably less is known about the effect of increased plasma levels of redox-reactive non-transferrin bound iron (NTBI) and its impact on pulmonary endothelium. Plasma levels of NTBI can increase under various pathophysiological conditions, including those associated with ALI, and multiple mechanisms are in place to affect the [Fe(2+)]/[Fe(3+)] redox steady state. It is well accepted, however, that intracellular transport of NTBI occurs after reduction of [Fe(3+)] to [Fe(2+)] (and is mediated by divalent metal transporters). Accordingly, as an experimental model to investigate mechanisms mediating vascular effects of redox reactive iron, rat pulmonary artery endothelial cells (RPAECs) were subjected to pulse treatment (10 min) with [Fe(2+)] nitriloacetate (30 μM) in the presence of pyrithione, an iron ionophore, to acutely increase intracellular labile pool of iron. Cellular iron influx and cell shape profile were monitored with time-lapse imaging techniques. Exposure of RPAECs to [Fe(2+)] resulted in: (i) an increase in intracellular iron as detected by the iron sensitive fluorophore, PhenGreen; (ii) depletion of cell glutathione; and (iii) nuclear translocation of stress-response transcriptional factors Nrf2 and NFkB (p65). The resulting iron-induced cell alterations were characterized by cell polarization and formation of membrane cuplike and microvilli-like projections abundant with ICAM-1, caveolin-1, and F-actin. The iron-induced re-arrangements in cytoskeleton, alterations in focal cell-cell interactions, and cell buckling were accompanied by decrease in electrical resistance of RPAEC monolayer. These effects were partially eliminated in the presence of N,N'-bis (2-hydroxybenzyl) ethylenediamine-N,N'-diacetic acid, an iron chelator, and Y27632, a Rho-kinase inhibitor. Thus acute increases in labile iron in cultured pulmonary endothelium result in structural remodeling (and a proinflammatory phenotype) that occurs via post-transcriptional mechanisms regulated in a redox sensitive fashion.

Targeting the Metastasis Suppressor, NDRG1, Using Novel Iron Chelators: Regulation of Stress Fiber-Mediated Tumor Cell Migration via Modulation of the ROCK1/pMLC2 Signaling Pathway

“…In agreement with our study, it has been reported in rat pulmonary artery endothelial cells that F-actin polymerization and cellular remodeling can be induced by iron supplementation (Gorbunov et al, 2012). Furthermore, iron-induced rearrangements of the cytoskeleton can be partially eliminated in the presence of the iron chelator, N,N9-bis-(2-hydroxybenzyl)-ethylenediamine-N,N9-diacetic acid (Gorbunov et al, 2012).…”

“…Furthermore, iron-induced rearrangements of the cytoskeleton can be partially eliminated in the presence of the iron chelator, N,N9-bis-(2-hydroxybenzyl)-ethylenediamine-N,N9-diacetic acid (Gorbunov et al, 2012).…”

A new fluorescent dye accumulation assay for parallel measurements of the ABCG2, ABCB1 and ABCC1 multidrug transporter functions

“…PhenGreen diacetate (PGD) is a hydrophobic, cell permeable molecule, which inside the cells is cleaved by cellular esterases into fluorescent PhenGreen (PG), and this hydrophilic product is accumulated inside the cells. Interaction of PG with various metal ions results in the quenching of PG fluorescence, thus allows the quantitative estimation of cellular metal concentrations [ 20 – 22 ].…”

“…The green fluorescence of PG is variably quenched in the presence of divalent metal ions, especially by heavy metal ions [ 19 ]. Therefore, this PGD loading and PG fluorescence measurement technology has been applied for the determination of iron or cadmium ions in various cellular systems [ 20 – 22 ].…”

Pulmonary Arterial Hypertension: Iron Matters

“…In another study, use of plumbagin, an iron chelator, ( Padhye et al, 2012 ) was found to limit proliferation in human PASMCs and decrease distal pulmonary artery remodeling in a rat model of PAH ( Courboulin et al, 2012 ). Additionally, iron was found to induce remodeling in cultured rat PAECs ( Gorbunov et al, 2012 ). Together, these studies indicate a role for iron availability in proliferation and remodeling.…”