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Adrenomedullin deficiency potentiates hyperoxic injury in fetal human pulmonary microvascular endothelial cells.

ABSTRACT: Bronchopulmonary dysplasia (BPD) is a chronic lung disease of premature infants that is characterized by alveolar simplification and decreased lung angiogenesis. Hyperoxia-induced oxidative stress and inflammation contributes to the development of BPD in premature infants. Adrenomedullin (AM) is an endogenous peptide with potent angiogenic, anti-oxidant, and anti-inflammatory properties. Whether AM regulates hyperoxic injury in fetal primary human lung cells is unknown. Therefore, we tested the hypothesis that AM-deficient fetal primary human pulmonary microvascular endothelial cells (HPMEC) will have increased oxidative stress, inflammation, and cytotoxicity compared to AM-sufficient HPMEC upon exposure to hyperoxia. Adrenomedullin gene (Adm) was knocked down in HPMEC by siRNA-mediated transfection and the resultant AM-sufficient and -deficient cells were evaluated for hyperoxia-induced oxidative stress, inflammation, cytotoxicity, and Akt activation. AM-deficient HPMEC had significantly increased hyperoxia-induced reactive oxygen species (ROS) generation and cytotoxicity compared to AM-sufficient HPMEC. Additionally, AM-deficient cell culture supernatants had increased macrophage inflammatory protein 1? and 1?, indicating a heightened inflammatory state. Interestingly, AM deficiency was associated with an abrogated Akt activation upon exposure to hyperoxia. These findings support the hypothesis that AM deficiency potentiates hyperoxic injury in primary human fetal HPMEC via mechanisms entailing Akt activation.

SUBMITTER: Zhang S

PROVIDER: S-EPMC4558361 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature

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Adrenomedullin deficiency potentiates hyperoxic injury in fetal human pulmonary microvascular endothelial cells.

Zhang Shaojie S Patel Ananddeep A Moorthy Bhagavatula B Shivanna Binoy B

Biochemical and biophysical research communications 20150718 4

Bronchopulmonary dysplasia (BPD) is a chronic lung disease of premature infants that is characterized by alveolar simplification and decreased lung angiogenesis. Hyperoxia-induced oxidative stress and inflammation contributes to the development of BPD in premature infants. Adrenomedullin (AM) is an endogenous peptide with potent angiogenic, anti-oxidant, and anti-inflammatory properties. Whether AM regulates hyperoxic injury in fetal primary human lung cells is unknown. Therefore, we tested the ...[more]

PMID: 26196743

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Project description:ObjectivesAcute respiratory distress syndrome represents the devastating result of acute lung injury, with high mortality. Limited methods are available for rehabilitation of lungs affected by acute respiratory distress syndrome. Our laboratory has demonstrated rehabilitation of sepsis-injured lungs via normothermic ex vivo and in vivo perfusion with Steen solution (Steen). However, mechanisms responsible for the protective effects of Steen remain unclear. This study tests the hypothesis that Steen directly attenuates pulmonary endothelial barrier dysfunction and inflammation induced by lipopolysaccharide.MethodsPrimary pulmonary microvascular endothelial cells were exposed to lipopolysaccharide for 4 hours and then recovered for 8 hours in complete media (Media), Steen, or Steen followed by complete media (Steen/Media). Oxidative stress, chemokines, permeability, interendothelial junction proteins, and toll-like receptor 4-mediated pathways were assessed in pulmonary microvascular endothelial cells using standard methods.ResultsLipopolysaccharide treatment of pulmonary microvascular endothelial cells and recovery in Media significantly induced reactive oxygen species, lipid peroxidation, expression of chemokines (eg, chemokine [C-X-C motif] ligand 1 and C-C motif chemokine ligand 2) and cell adhesion molecules (P-selectin, E-selectin, and vascular cell adhesion molecule 1), permeability, neutrophil transmigration, p38 mitogen-activated protein kinase and nuclear factor kappa B signaling, and decreased expression of tight and adherens junction proteins (zonula occludens-1, zonula occludens-2, and vascular endothelial-cadherin). All of these inflammatory pathways were significantly attenuated after recovery of pulmonary microvascular endothelial cells in Steen or Steen/Media.ConclusionsSteen solution preserves pulmonary endothelial barrier function after lipopolysaccharide exposure by promoting an anti-inflammatory environment via attenuation of oxidative stress, toll-like receptor 4-mediated signaling, and conservation of interendothelial junctions. These protective mechanisms offer insight into the advancement of methods for in vivo lung perfusion with Steen for the treatment of severe acute respiratory distress syndrome.

Project description:BackgroundPrevious experiments revealed phospholipid scramblase 4 (PLSCR4) mRNA to be significantly increased in a lipopolysaccharide (LPS)-induced acute respiratory distress syndrome (ARDS) model of human pulmonary microvascular endothelial cells (HPMECs); however, the effect of PLSCR4 and its mechanism have not been reported to date. The PLSCR family is thought to mediate the transmembrane movement of phospholipids (PS), and has been found to be involved in pyroptosis through combing with gasdermin D (GSDMD). We therefore speculated that PLSCR4 may contribute to cell death via pyroptosis.MethodsTo investigate the effect and mechanism of PLSCR4 in ARDS, we constructed an in vitro model of LPS-induced ARDS in HPMECs transfected with PLSCR4 small interfering RNA (siRNA) or scramble siRNA (sc siRNA). After 4 h of LPS stimulation, western blotting, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), tracer flux assays, and fluorescence assays were used to study the relationship between PLSCR4 and pyroptosis with regards to their impact on ARDS. We also established an ARDS mouse model which was pretreated with a liquid complex of PLSCR4 siRNA/sc siRNA-lipofectamine 2000 through the fundus venous plexus. Finally, we used DNA pull-down and protein profiling to study the potential transcription factor of PLSCR4.ResultsIt was found that when the expression of PLSCR4 was elevated, the concentration of interleukin 1 beta (IL-1β) and IL-18 decreased, along with barrier damage (P<0.05). Furthermore, HPMEC injury was reduced with more distribution of PS and N-terminal cleavage product (GSDMD-NT) of GSDMD on the external side of cell membrane. However, the pyroptosis-relevant proteins of GSDMD and caspase-1 were not obviously changed (P<0.05); we further found that when PLSCR4 was depressed, the lung injury was aggravated in the mice. In the DNA pull-down assay, P62280 remarkably increased, which suggested that P62280 might be the transcription factor for PLSCR4.ConclusionsPLSCR4 alleviated pyroptosis by transporting PS to the outside of the membrane, blocking the formation of pyroptosis pores composed of GSDMD. Moreover, P62280 might be the transcription factor of PLSCR4. These insults may provide useful insights into the clinical treatment of ARDS.

Project description:BACKGROUND:Endothelial barrier dysfunction is central to the pathogenesis of sepsis-associated acute lung injury (ALI). Microtubule (MT) dynamics in vascular endothelium are crucial for the regulation of endothelial barrier function. Unfractionated heparin (UFH) possesses various biological activities, such as anti-inflammatory activity and endothelial barrier protection during sepsis. METHODS:Here, we investigated the effects and underlying mechanisms of UFH on lipopolysaccharide (LPS)-induced endothelial barrier dysfunction. C57BL/6 J mice were randomized into vehicle, UFH, LPS and LPS?+?UFH groups. Intraperitoneal injection of 30 mg/kg LPS was used to induce sepsis. Mice in the LPS?+?UFH group received intravenous UFH 0.5 h prior to LPS injection. Human pulmonary microvascular endothelial cells (HPMECs) were cultured for analyzing the effects of UFH on LPS-induced and nocodazole-induced hyperpermeability, F-actin remodeling, and LPS-induced p38 MAPK activation. RESULTS:UFH pretreatment significantly attenuated LPS-induced pulmonary histopathological changes, and increased the lung W/D ratio and Evans blue accumulation in vivo. Both in vivo and in vitro studies showed that UFH pretreatment blocked the LPS-induced increase in guanine nucleotide exchange factor (GEF-H1) expression and myosin phosphatase target subunit 1 (MYPT1) phosphorylation, and microtubule (MT) disassembly in LPS-induced ALI mouse model and human pulmonary microvascular endothelial cells (HPMECs). These results suggested that UFH ameliorated LPS-induced endothelial barrier dysfunction by inhibiting MT disassembly and GEF-H1 expression. In addition, UFH attenuated LPS-induced hyperpermeability of HPMECs and F-actin remodeling. In vitro, UFH pretreatment inhibited LPS-induced increase in monomeric tubulin expression and decrease in tubulin polymerization and acetylation. Meanwhile, UFH ameliorates nocodazole-induced MTs disassembly and endothelial barrier dysfunction.Additionally, UFH decreased p38 phosphorylation and activation, which was similar to the effect of the p38 MAPK inhibitor, SB203580. CONCLUSIONS:UFH exert its protective effects on pulmonary microvascular endothelial barrier dysfunction via microtubule stabilization and is associated with the p38 MAPK pathway.

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Adrenomedullin deficiency potentiates hyperoxic injury in fetal human pulmonary microvascular endothelial cells.

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Adrenomedullin deficiency potentiates hyperoxic injury in fetal human pulmonary microvascular endothelial cells.

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