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Unfractionated heparin ameliorates pulmonary microvascular endothelial barrier dysfunction via microtubule stabilization in acute lung injury.

ABSTRACT: 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.

SUBMITTER: Mu S

PROVIDER: S-EPMC6238311 | biostudies-other | 2018 Nov

REPOSITORIES: biostudies-other

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Unfractionated heparin ameliorates pulmonary microvascular endothelial barrier dysfunction via microtubule stabilization in acute lung injury.

Mu Shengtian S Liu Yina Y Jiang Jing J Ding Renyu R Li Xu X Li Xin X Ma Xiaochun X

Respiratory research 20181115 1

<h4>Background</h4>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.<h4>Methods</h4>Here, we investigated the effects and underlying mechanisms of UFH on lipopolysaccharide ...[more]

PMID: 30442128

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Project description:Sepsis is characterized by injury of pulmonary microvascular endothelial cells (PMVEC) leading to barrier dysfunction. Multiple mechanisms promote septic PMVEC barrier dysfunction, including interaction with circulating leukocytes and PMVEC apoptotic death. Our previous work demonstrated a strong correlation between septic neutrophil (PMN)-dependent PMVEC apoptosis and pulmonary microvascular albumin leak in septic mice in vivo; however, this remains uncertain in human PMVEC. Thus, we hypothesize that human PMVEC apoptosis is required for loss of PMVEC barrier function under septic conditions in vitro. To assess this hypothesis, human PMVECs cultured alone or in coculture with PMN were stimulated with PBS or cytomix (equimolar interferon γ, tumor necrosis factor α, and interleukin 1β) in the absence or presence of a pan-caspase inhibitor, Q-VD, or specific caspase inhibitors. PMVEC barrier function was assessed by transendothelial electrical resistance (TEER), as well as fluoroisothiocyanate-labeled dextran and Evans blue-labeled albumin flux across PMVEC monolayers. PMVEC apoptosis was identified by (1) loss of cell membrane polarity (Annexin V), (2) caspase activation (FLICA), and (3) DNA fragmentation [terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)]. Septic stimulation of human PMVECs cultured alone resulted in loss of barrier function (decreased TEER and increased macromolecular flux) associated with increased apoptosis (increased Annexin V, FLICA, and TUNEL staining). In addition, treatment of septic PMVEC cultured alone with Q-VD decreased PMVEC apoptosis and prevented septic PMVEC barrier dysfunction. In septic PMN-PMVEC cocultures, there was greater trans-PMVEC macromolecular flux (both dextran and albumin) vs. PMVEC cultured alone. PMN presence also augmented septic PMVEC caspase activation (FLICA staining) vs. PMVEC cultured alone but did not affect septic PMVEC apoptosis. Importantly, pan-caspase inhibition (Q-VD treatment) completely attenuated septic PMN-dependent PMVEC barrier dysfunction. Moreover, inhibition of caspase 3, 8, or 9 in PMN-PMVEC cocultures also reduced septic PMVEC barrier dysfunction whereas inhibition of caspase 1 had no effect. Our data demonstrate that human PMVEC barrier dysfunction under septic conditions in vitro (cytomix stimulation) is clearly caspase-dependent, but the mechanism differs depending on the presence of PMN. In isolated PMVEC, apoptosis contributes to septic barrier dysfunction, whereas PMN presence enhances caspase-dependent septic PMVEC barrier dysfunction independently of PMVEC apoptosis.

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Unfractionated heparin ameliorates pulmonary microvascular endothelial barrier dysfunction via microtubule stabilization in acute lung injury.

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Unfractionated heparin ameliorates pulmonary microvascular endothelial barrier dysfunction via microtubule stabilization in acute lung injury.

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