High glucose enhances permeability of human pulmonary microvascular endothelial cells by lipopolysaccharide stimulation in vitro and effect of DDAH/NOS/NO imbalance on its pathogenesis

Abstract

To investigate the damage to endothelial cells incubated in high concentration of glucose challenged by lipopolysaccharide (LPS), and the likely mechanisms of injury. Human pulmonary microvascular endothelial cells (PMVECs) were divided into the following groups: normal glucose group (NG), normal glucose + LPS stimulation group (NGL), high glucose stimulation group (HG), and high glucose + LPS stimulation group (HGL). The cells were incubated with normal glucose (5.5 mmol/L, contained 10% calf serum) or high glucose (33 mmol/L) for 5 days to form a monolayer of cells before LPS stimulation (10 mg/L) for 24 hours. The microfilaments (F-actin) were investigated by immuno-fluorescence, and the number and size change in fenestrae were examined by scanning electron microscopy. The permeability of vascular endothelial cell was assessed by trans-PMVEC horseradish peroxidase (HRP) flux. Western blotting was used to determine the expressions of dimethylarginine dimethylaminohydrolase 2 (DDAH2), inducible nitricoxide synthase (iNOS) and endothelial nitricoxide synthase (eNOS). Nitric oxide (NO) was assessed by Griess method. When stimulated with LPS, cells incubated with high glucose showed obvious microfilament rearrangement, a larger average diameter and increased number of F-actin, as well as higher HRP permeability on the hyperglycemic PMVECs compared with PMVECs cultured with normal glucose [(53.62±6.70)% vs. (23.63±3.92)%, P<0.01]. Furthermore, high glucose down-regulated DDAH2 expression (arbitrary units, AU, 0.33±0.08 vs. 0.77±0.14 , P<0.01) and up-regulated LPS-stimulated iNOS production (1.40±0.29 vs. 1.04±0.09, P<0.01), as well as increased LPS-stimulated nitrite/nitrate and stable NO end products compared with normal (20.36±2.25 μmol/L vs. 7.99±0.33 μmol/L, P<0.01) and reduction of eNOS levels was observed (0.67±0.09 vs. 0.91±0.17, P<0.05). It demonstrated that, in vitro high glucose deteriorate LPS-stimulated F-actin rearrangement and hyperpermeability of an endothelial monolayer, and the worsened imbalance of the NO pathway may lead to endothelial damage in microcirculation.