Permeability Of Pulmonary Microvascular Endothelial Cells Research Articles

Mechanisms of pulmonary microvascular endothelial cells barrier dysfunction induced by LPS: The roles of ceramides and the Txnip/NLRP3 inflammasome.

Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) are characterized by pulmonary microvascular endothelial cells (PMVECs) barrier dysfunction and proinflammatory cytokine influx into lung tissue, resulting in pulmonary oedema. Ceramide overproduction is an important mediator of pulmonary hyperinflammation and pulmonary oedema in Acute lung injury (ALI). Ceramides induce NLRP3 inflammasome activation are essential for the hyperinflammatory response. However, the roles and specific mechanisms of ceramide-induced NLRP3 inflammasome activation, proinflammatory cytokine manufacturing and PMVECs barrier dysfunction in ALI are unclear. Herein, pretreatment with the acid sphingomyelinase (ASMase) inhibitor imipramine (but not a neutral sphingomyelinase (NSMase) inhibitor or de novo pathway inhibitor) significantly inhibited ceramide early production in rats with lipopolysaccharide (LPS)-induced ALI; Furthermore, the Txnip/NLRP3 inflammasome activation, proinflammatory cytokine release, increased PMVECs permeability and lung injury were significantly decreased. Verapamil, a Txnip inhibitor, substantially inhibited Txnip/NLRP3 inflammasome activation, proinflammatory cytokine release, increased PMVECs permeability and lung injury in rats with C8-ceramide-induced ALI. In vitro, short hairpin RNA-mediated Txnip silencing significantly inhibited C8-ceramide-induced Txnip/NLRP3 inflammasome activation in NR8383 alveolar macrophages (AMs) and early secretion of the proinflammatory cytokines IL-1β (4-12h) as well as IL-6 and TNF-α at subsequent times (later than 12h). However, C8-ceramide significantly increased the early secretion (within 8h) of the proinflammatory cytokines IL-1β, IL-6 and TNF-α in a co-culture model of NR8383 AMs and PMVECs, and Txnip silencing of NR8383 AMs inhibited the secretion of pro-inflammatory cytokines and reduced cytoskeletal rearrangements, intercellular connection breakage and hyperpermeability in PMVECs. Overall, our results suggest that in LPS-induced ALI, ceramide-mediated Txnip/NLRP3 inflammasome activation in NR8383 AMs leads to early IL-1β release, subsequently inducing PMVECs injury and release of the proinflammatory cytokines IL-6 and TNF-α, ultimately leading to PMVECs barrier dysfunction and ALI.

Protective effects of areca nut polyphenols on hypoxic damage of rat pulmonary microvascular endothelial cells.

To investigate the protective effects of areca nut polyphenols on hypoxic damage of rat pulmonary microvascular endothelial cells (PMVECs). Malondialdehyde and superoxide dismutase (SOD) were used to determine the optimal modeling of lung hypoxic injury cells. CCK-8 method was used to detect cell viability for determining the effective dose of areca nut polyphenols. Rat PMVECs were divided into control group, hypoxia model group and areca nut polyphenols group. BCA method was used to detect the protein concentration of each group, and the oxidative stress level in PMVECs was measured. Western blotting was used to detect the expression of inflammatory and apoptosis-related proteins. Immunofluorescence staining was used to detect the expression of occludin and zonula occludens (ZO) 1. Transwell chamber was used to detect transendothelial electrical resistance, and rhodamine fluorescent dye was used to detect PMVECs barrier permeability. The hypobaric hypoxia-induced cell injury model was established by culturing PMVECs for 48 h at 1% oxygen concentration. The 20 μg/mL areca nut polyphenols significantly reversed the survival rate and the oxidative stress of PMVECs in hypoxia model group (all P<0.05). Areca nut polyphenols had significant inhibitory effect on the up-regulation of inflammation-related proteins, including nuclear factor-κB (NF-κB) and nuclear factor-E2-related factor (Nrf) 2 in hypoxia model group (all P<0.05). And areca nut polyphenols could reduce hypoxia-induced PMVECs apoptosis by down-regulating the expressions of apoptosis-related proteins, including cysteine aspartic acid specific protease (caspase) 3, Bcl-2 associated X protein (Bax) in PMVECs (all P<0.05). In addition, areca nut polyphenols effectively improves the transendothelial electrical resistance and barrier permeability of PMVECs through elevating the expression of occludin and ZO-1 (all P<0.05). Areca nut polyphenols can inhibit the hypoxic damage of PMVECs by reducing oxidative stress and apoptosis down-regulating the expression of inflammatory proteins and reducing membrane permeability.

Highly Pathogenic PRRSV-Infected Alveolar Macrophages Impair the Function of Pulmonary Microvascular Endothelial Cells.

The porcine reproductive and respiratory syndrome virus (PRRSV), especially the highly pathogenic strains, can cause serious acute lung injury (ALI), characterized by extensive hemorrhage, inflammatory cells and serous fluid infiltration in the lung vascular system. Meanwhile, the pulmonary microvascular endothelial cells (PMVECs) are essential for forming the air–blood barrier and keeping the water–salt balance to prevent leakage of circulating nutrients, solutes, and fluid into the underlying tissues. As well, they tightly regulate the influx of immune cells. To determine the possible relationship between the PMVECs’ function changes and lung vascular permeability during PRRSV infection, the PMVECs were co-cultured with HP-PRRSV-inoculated primary pulmonary alveolar macrophages (PAMs) in transwell model, and then the RNA sequencing (RNA-seq) and comprehensive bioinformatics analysis were carried out to characterize the dynamic transcriptome landscapes of PMVECs. In total, 16,489 annotated genes were identified, with 275 upregulated and 270 downregulated differentially expressed genes (DEGs) were characterized at both 18 and 24 h post PRRSV inoculation. The GO terms and KEGG pathways analysis indicated that the immune response, metabolic pathways, cell death, cytokine–cytokine receptor interaction, viral responses, and apoptotic process are significantly regulated upon co-culture with PRRSV-infected PAMs. Moreover, according to the TERR and dextran flux assay results, dysregulation of TJ proteins, including CLDN1, CLDN4, CLDN8, and OCLN, is further confirmed to correlate with the increased permeability of PMVECs. These transcriptome profiles and DEGs will provide valuable clues for further exploring the roles of PMVECs in PRRSV-induced ALI in the future.

Novel HIF-1-target gene isthmin1 contributes to hypoxia-induced hyperpermeability of pulmonary microvascular endothelial cells monolayers.

Hypoxia-induced pulmonary microvascular endothelial cell (PMVEC) monolayers hyperpermeability is vital for vascular leakage, which participates in vascular diseases, such as acute lung injury (ALI) and high-altitude pulmonary edema (HAPE). We previously observed that PMVEC permeability was markedly elevated in hypoxia when cocultured with primary type II alveolar epithelial cells (AECII) in which isthmin1 (ISM1) was highly upregulated. However, whether the upregulation of ISM1 plays a role in hypoxia-induced PMVEC hyperpermeability is unclear. In this study, we assessed the role of AECII-derived ISM1 in hypoxia-induced PMVEC hyperpermeability with an AECII/PMVEC coculture system and uncovered the underlying mechanism whereby hypoxia stimulates ISM1 gene expression. We found that ISM1 gene expression was upregulated in cultured AECII cells exposed to hypoxia (3% O2) and that AECII-derived ISM1 participated in hypoxia-induced hyperpermeability of PMVEC monolayers, as small interference RNA (siRNA)-mediated knockdown of ISM1 in AECII markedly attenuated the increase in PMVEC permeability in coculture system under hypoxia. In addition, we confirmed that ISM1 was regulated by hypoxia-inducible factor-1α (HIF1α) according to the evidence that silencing of HIF1α inhibited the hypoxia-mediated upregulation of ISM1. Mechanismly, overexpression of HIF1α transcriptionally activated ISM1 gene expression by directly binding to the conserved regulatory elements upstream of the ism1 locus. We identified a novel HIF-1-target gene ISM1, which involves in hyperpermeability of pulmonary microvascular endothelial cell monolayers under hypoxia. Our in vitro cell experiments implied that the upregulated ISM1 derived from alveolar epithelium might be a vital modulator in hypoxia-induced endothelial hyperpermeability and thereby implicates with hypoxic pulmonary-related diseases.

Tumor necrosis factor-α requires Ezrin to regulate the cytoskeleton and cause pulmonary microvascular endothelial barrier damage

Acute respiratory distress syndrome (ARDS) is a rapidly progressive disease with unknown pathogenesis. Damage of pulmonary microvascular endothelial cells (PMVECs) caused by inflammatory storm caused by cytokines such as TNF-α is the potential pathogenesis of ARDS. In this study, we examined the role of ezrin and Rac1 in TNF-α-related pathways, which regulates the permeability of PMVECs. Primary rat pulmonary microvascular endothelial cells (RPMVECs) were isolated and cultured. RPMVECs were treated with rat TNF-α (0, 1, 10, 100 ng/ml), and the cell activity of each group was measured using a CCK8 kit. The integrity of endothelial barrier was measured by transendothelial resistance (TEER) and FITC-BSA flux across RPMVECs membranes. Pulldown assay and Western blot was used to detect the activity of RAS-associated C3 botulinum toxin substrate 1 (Rac1) and Ezrin phosphorylation. Short hairpin RNA (shRNA) targeting ezrin and Rac1 was utilized to evaluate the effect of RPMVECs permeability and related pathway. The effects of ezrin and Rac1 on cytoskeleton were confirmed by immunofluorescence. Our results revealed that active Rac1 was essential for protecting the RPMVEC barrier stimulated by TNF-α, while active ezrin could partially destroy the PMVEC barrier by reducing Rac1 activity and regulating the subcellular structure of the cytoskeleton. These findings may be used to create new therapeutic strategies for targeting Rac1 in the treatment of ARDS.

Role of adenosine A2b receptors in pulmonary microvascular endothelial inflammation induced by lipopolysaccharide

To explore the role of the low-affinity A2b adenosine receptors (Adora2b) in pulmonary microvascular endothelial inflammation induced by lipopolysaccharide and its mechanism. Rat pulmonary microvascular endothelial cells (PMVECs) were isolated and cultured in vitro. After serum deprivation for 24 hours, cells were pretreated with Adora2b specific agonist BAY60-6583 (0.1, 1, 10 μmol/L) or Adora2b specific antagonist PSB1115 (1 μmol/L) for 1 hour, respectively, and then challenged with LPS (100 μg/L). Cells without treatment were served as the control group, and those treated with LPS, BAY60-6583 or PSB1115 alone were served as single challenge groups. After incubation with specific drugs for 24 hours, the apoptosis of PMVECs was analyzed by flow cytometry using Annexin V/propidium iodide (PI) technique. The levels of early inflammatory factors in cultured medium were measured using enzyme linked immunosorbent assay (ELISA). The mRNA expressions of chemotactic factors and adhesion molecules were determined by real-time quantitative-polymerase chain reaction (RT-qPCR). Polymorph nuclear neutrophils (PMNs) from venous blood of healthy rats were isolated, and PMN migration through PMVECs monolayer under stimulation of drugs was observed in transwell inserts. The monolayer permeability of PMVECs after adhesion of PMNs was determined by fluorescein isothiocyanate (FITC)-albumin assay. Oxidative stress was detected by DCFH-DA assay. Compared with the control group, more cells entered into the apoptosis stage after LPS challenge. Meanwhile, the levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in cultured medium were significantly increased, as well as the mRNA expressions of chemotactic factors [C-X-C motif chemokine ligand 1 (CXCL-1), CXCL-3 and monocyte chemoattractant protein-1 (MCP-1)] and adhesion molecules [E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1)]. More PMNs migrated through PMVECs following adhesion and the monolayer permeability of PMVECs was rapidly enhanced. The oxidative stress was upregulated. Compared with LPS group, BAY60-6583 pretreatment could dose-dependently decrease the rate of apoptosis, attenuate trans-endothelial migration of PMNs and decrease the endothelial cell barrier leakage. There were significant differences even after incubation of 0.1 μmol/L BAY60-6583 [apoptosis rate: (21.12±2.12)% vs. (27.66±3.57)%, number of migrated PMNs/HP: 260.60±18.24 vs. 290.20±16.48, permeability coefficient (Pd, ×10-6 cm/s): 28.28±2.04 vs. 32.55±2.13, all P < 0.05]. Meanwhile, BAY60-6583 pretreatment also downregulated the levels of early proinflammatory factors in a dose-dependent manner as well as the mRNA expressions of chemotactic factors and adhesion molecules. The statistic difference was significant while treated with 1 μmol/L BAY60-6583 [IL-1β (ng/L): 475.75±63.15 vs. 755.25±67.42, TNF-α (ng/L): 560.25±69.96 vs. 818.75±60.92, CXCL-1 mRNA (2-ΔΔCt): 3.57±0.28 vs. 5.27±0.69, CXCL-3 mRNA (2-ΔΔCt): 4.56±0.48 vs. 7.32±0.54, MCP-1 mRNA (2-ΔΔCt): 2.21±0.31 vs. 3.35±0.21, E-selectin mRNA (2-ΔΔCt): 4.64±0.09 vs. 7.28±0.73, ICAM-1 mRNA (2-ΔΔCt): 4.14±0.30 vs. 5.89±0.25, VCAM-1 mRNA (2-ΔΔCt): 2.23±0.19 vs. 2.92±0.33, all P < 0.05]. Furthermore, pretreatment of 10 μmol/L BAY60-6583 could decrease the oxidative stress [reactive oxygen species (RFU): 629.05±33.10 vs. 781.45±64.59, P < 0.05]. Contrast, PSB1115 pretreatment aggravated apoptosis of PMVECs after LPS incubation [(34.36±4.57)% vs. (27.66±3.57)%], upregulated expressions of proinflammatory and chemotactic factors as well as adhesion molecules [IL-1β (ng/L): 889.00±63.11 vs. 755.25±67.42, TNF-α (ng/L): 939.00±43.44 vs. 818.75±60.92, CXCL-1 mRNA (2-ΔΔCt): 6.66±0.65 vs. 5.27±0.69, CXCL-3 mRNA (2-ΔΔCt): 10.42±0.51 vs. 7.32±0.54, MCP-1 mRNA (2-ΔΔCt): 4.85±0.34 vs. 3.35±0.21, E-selectin mRNA (2-ΔΔCt): 8.42±0.47 vs. 7.28±0.73, ICAM-1 mRNA (2-ΔΔCt): 7.46±0.72 vs. 5.89±0.25, VCAM-1 mRNA (2-ΔΔCt): 4.35±0.26 vs. 2.92±0.33], aggravated trans-endothelial migration of PMNs (cells/HP: 348.40±22.68 vs. 290.20±16.48), enhanced the leakage of PMVECs monolayer [Pd (×10-6 cm/s): 39.65±2.69 vs. 32.55±2.13] and increased oxidative stress in PMVECs [reactive oxygen species (RFU): 847.04±29.26 vs. 781.45±64.59], with statistically significant difference (all P < 0.05). Activation of endothelial Adora2b attenuates LPS-induced pulmonary microvascular inflammation by decreasing the release of early inflammatory factors, downregulating expressions of chemotactic factors and adhesion molecules, attenuating trans-endothelial migration of PMNs and oxidative stress in PMVECs, which suggest endothelial Adora2b is apotential anti-inflammatory target in the treatment of LPS-induced acute lung injury.

Phosphorylated ERM Mediates Lipopolysaccharide Induced Pulmonary Microvascular Endothelial Cells Permeability Through Negatively Regulating Rac1 Activity

IntroductionThe endotoxin lipopolysaccharide (LPS)-induced pulmonary endothelial barrier disruption is a key pathogenesis of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). However, the molecular mechanisms underlying LPS-impaired permeability of pulmonary microvascular endothelial cells (PMVECs) are not fully understood. MethodsRat PMVECs were isolated and monolayered cultured, then challenged with different doses of LPS (0.1mg/L, 1mg/L, and 10mg/L). Trans-endothelial electrical resistance (TER) was utilized to measure the integrity of the endothelial barrier. Ras-related C3 botulinum toxin substrate 1 (Rac1) activity and the phosphorylation of Ezrin/Radixin/Moesin proteins (ERM) were assessed by pulldown assay and Western Blotting. Small interfering RNA (siRNA) inhibition of Rac1 and Moesin were applied to evaluate the effect of PMVEs permeability and related pathway. ResultsLPS induced dose and time-dependent decreases in TER and increase in ERM threonine phosphorylation, while inactivated Rac1 activity in PMVEC. siRNA study demonstrated that both Rac1 and Moesin were involved in the mediation of the LPS-induced hyperpermeability in PMVECs monolayers, and Rac1 and Moesin could regulate each other. ConclusionPhosphorylated ERM mediates LPS induced PMVECs permeability through negatively regulating Rac1 activity.

Phosphorylated ERM Mediates Lipopolysaccharide Induced Pulmonary Microvascular Endothelial Cells Permeability Through Negatively Regulating Rac1 Activity

IntroductionThe endotoxin lipopolysaccharide (LPS)-induced pulmonary endothelial barrier disruption is a key pathogenesis of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). However, the molecular mechanisms underlying LPS-impaired permeability of pulmonary microvascular endothelial cells (PMVECs) are not fully understood. MethodsRat PMVECs were isolated and monolayered cultured, then challenged with different doses of LPS (0.1mg/L, 1mg/L, and 10mg/L). Trans-endothelial electrical resistance (TER) was utilized to measure the integrity of the endothelial barrier. Ras-related C3 botulinum toxin substrate 1 (Rac1) activity and the phosphorylation of Ezrin/Radixin/Moesin proteins (ERM) were assessed by pulldown assay and Western Blotting. Small interfering RNA (siRNA) inhibition of Rac1 and Moesin were applied to evaluate the effect of PMVEs permeability and related pathway. ResultsLPS induced dose and time-dependent decreases in TER and increase in ERM threonine phosphorylation, while inactivated Rac1 activity in PMVEC. siRNA study demonstrated that both Rac1 and Moesin were involved in the mediation of the LPS-induced hyperpermeability in PMVECs monolayers, and Rac1 and Moesin could regulate each other. ConclusionPhosphorylated ERM mediates LPS induced PMVECs permeability through negatively regulating Rac1 activity.

Slit2/Robo4 signaling pathway modulates endothelial hyper-permeability in a two-event in vitro model of transfusion-related acute lung injury

Transfusion-related acute lung injury (TRALI) remains the leading cause of transfusion-related mortality. Endothelium semipermeable barrier function plays a critical role in the pathophysiology of transfusion-related acute lung injury (TRALI). Recently, Roundabout protein 4 (Robo4), interaction with its ligand Slit 2, was appreciated as a modulator of endothelial permeability and integrity. However, not much is known about the role of Slit2/Robo4 signaling pathway in the pathophysiology of TRALI. In this study, the TRALI model was performed by the “two-event” model of polymorphonuclear neutrophils (PMN)-mediated pulmonary microvascular endothelial cells (PMVECs) damage. We investigated the expression of Slit2/Robo4 and VE-cadherin and examined the pulmonary endothelial hyper-permeability in TRALI model. We found that the expression of Slit2/Robo4 and VE-cadherin were significantly decreased in a time-dependent manner, whereas the PMVECs permeability was gradually increased over time in TRALI model. Moreover, the treatment with Slit2-N, an active fragment of Slit2, increased the expression of Slit2/Robo4 and VE-cadherin to protect PMVECs from PMN-mediated pulmonary endothelial hyper-permeability. These results indicate that targeting Slit2/Robo4 signaling pathway may modulate the permeability as well as protect the integrity of endothelial barrier. In addition, Slit2-N appears to be a promising candidate for developing novel therapies against TRALI.

Role of M3 receptor in penehyclidine hydrochloride-induced reduction of increased permeability of human pulmonary microvascular endothelial cells caused by endotoxin: the relationship with MAPK signaling pathway

Objective To evaluate the role of M3 receptor in penehyclidine hydrochloride(PHC)-induced reduction of increased permeability of human pulmonary microvascular endothelial cells(PMVECs)caused by endotoxin and the relationship with mitogen-activated protein kinase(MAPK)signaling pathway. Methods Human PMVECs were seeded in 6-well plates(2 ml/hole)or in culture flasks(4 ml/flask)at the density of 1×105 cells/ml and randomly divided into 6 groups(n=5 each): control group(group C), M3 receptor shRNA transfection group(group shRNA), lipopolysaccharide(LPS)group, penehyclidine plus LPS group(group P+ LPS), LPS plus M3 receptor shRNA transfection group(group LPS+ shRNA)and PHC plus LPS plus M3 shRNA transfection group(group P+ LPS+ shRNA). The cells were transfected with shRNA plasmid containing 2.5 nmol/L M3 receptors in shRNA, LPS+ shRNA and P+ LPS+ shRNA groups.LPS at the final concentration of 0.1 μg/ml was added at 24 h of incubation and then cells were incubated for 1 h in LPS and LPS+ shRNA groups.PHC at the final concentration of 2 μg/ml was added at 24 h of incubation, cells were incubated for 1 h, then LPS at the final concentration of 0.1 μg/ml was added, and cells were incubated for another 1 h in P+ LPS and P+ LPS+ shRNA groups.The permeability of PMVECs was measured using Transwell assay.The expression of phosphorylated p38 MAPK(p-p38 MAPK)and phosphorylated extracellular signal-regulated protein kinase 1/2(p-ERK1/2)was detected by Western blot, the expression of heat shock protein 27(HSP27)using immunofluorescent staining, and the expression of M3receptor mRNA by real-time polymerase chain reaction. Results Compared with group C, M3 receptor mRNA expression was significantly down-regulated in group shRNA, and the permeability of cells was significantly increased, and the expression of p-p38 MAPK, p-ERK1/2, HSP27 and M3 receptor mRNA was up-regulated in group LPS(P<0.05). The permeability of cells was significantly decreased, and the expression of p-p38 MAPK, p-ERK1/2, HSP27 and M3 receptor mRNA was down-regulated in P+ LPS, LPS+ shRNA and P+ LPS+ shRNA groups as compared with group LPS, and in group P+ LPS+ shRNA as compared with group LPS+ shRNA(P<0.05). Conclusion The mechanism by which PHC reduces endotoxin-caused increased permeability of human PMVECs is related to inhibiting activation of MAPK signaling pathway after down-regulating M3 receptor. Key words: Receptor, muscarinic M3; Cholinergic agent; Endothelial cells; Blood capillary; Lung; Endoxemia; p38 MAPKs; Extracellular Signal-Regulated MAP Kinases

Lipopolysaccharide impairs permeability of pulmonary microvascular endothelial cells via Connexin40

The endotoxin lipopolysaccharide (LPS)-induced pulmonary endothelial barrier disruption is a key pathogenesis of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). However, the molecular mechanisms underlying LPS-impaired permeability of pulmonary microvascular endothelial cells (PMVECs) are not fully understood. Gap junctions, particularly Connexin40 (Cx40), are necessary for the maintenance of normal vascular function. In this study, we for the first time investigated the role of Cx40 in LPS-impaired permeability of PMVECs and provided potential therapeutic approaches based on mechanistic findings of Cx40 regulation by LPS stimuli. Rat PMVECs were isolated, cultured and identified with cell morphology, specific markers, ultrastructural characteristics and functional tests. Western blot analysis demonstrated that Cx40 is the major connexin highly expressed in PMVECs. Furthermore, by inhibiting Cx40 in a time-dependent manner, LPS impaired gap junction function and induced permeability injury of PMVECs. The key role of Cx40 decline in mediating detrimental effects of LPS was further confirmed in rescue experiments through Cx40 overexpression. Mechanistically, LPS stress on PMVECs inhibited the protein kinase C (PKC) pathway, which may synergize with the inflammatory nuclear factor kappaB (NFκB) signaling activation in suppressing Cx40 expression level and phosphorylation. Moreover, through pharmacological PKC activation or NFκB inhibition, Cx40 activity in PMVECs could be restored, leading to maintained barrier function under LPS stress. Our findings uncover a previously unrecognized role of Cx40 and its regulatory mechanisms in impaired endothelial integrity under endotoxin and inflammation, shedding light on intervention approaches to improve pulmonary endothelial barrier function in ALI and ARDS.

Role of β-arrestin-1 in penehyclidine hydrochloride-induced inhibition of LPS-caused increase in pulmonary microvascular permeability in human pulmonary microvascular endothelial cells

Objective To evaluate the role of β-arrestin-1 in penehyclidine hydrochloride(PHC)-induced inhibition of lipopolysaccharide(LPS)-caused increase in pulmonary microvascular permeability in human pulmonary microvascular endothelial cells(PMVECs). Methods Human PMVECs were seeded in 6-well plates(2 ml/well)or in culture flasks(4 ml/flask)at the density of 1×105 cells/ml and divided into 5 groups(n=15 each)using a random number table: empty plasmid transfection group(group C), LPS plus empty plasmid transfection group(LPS group), PHC plus LPS plus empty plasmid transfection group(P+ LPS group), LPS plus β-arrestin-1 short hairpin RNA(shRNA)transfection group(LPS+ shRNA group)and PHC plus LPS plus β-arrestin-1 shRNA transfection group(P+ LPS+ shRNA group). In LPS and LPS+ shRNA groups, the cells were transfected with empty plasmid 1.5 μg or with plasmid containing 15 nmol/L β-arrestin-1 shRNA, LPS with the final concentration of 0.1 μg/ml was added at 24 h of incubation, and the cells were then incubated for 1 h. In P+ LPS and P+ LPS+ shRNA groups, the cells were transfected with empty plasmid 1.5 μg or with plasmid containing 15 nmol/L β-arrestin-1 shRNA, PHC with the final concentration of 2 μg/ml was added at 24 h of incubation, LPS with the final concentration of 0.1 μg/ml was added at 1 h of incubation, and the cells were then incubated for 1 h. The cell permeability was measured using Transwell chambers.The expression of heat shock protein(HSP27)was detected by immunofluorescence.The expression of β-arrestin-1, p38 mitogen-activated protein kinase(p38MAPK)and phosphorylated p38MAPK(p-p38MAPK)was detected by Western blot.The ratio of p-p38MAPK/p38MAPK was calculated. Results Compared with group C, the cell permeability was significantly increased, the expression of HSP27 was up-regulated, p-p38MAPK/p38MAPK ratio was increased, and the expression of β-arrestin-1 was down-regulated in LPS, LPS+ shRNA and P+ LPS+ shRNA groups(P 0.05). Compared with group LPS, the cell permeability was significantly decreased, the expression of HSP27 was down-regulated, p-p38MAPK/p38MAPK ratio was decreased, and the expression of β-arrestin-1 was up-regulated in group P+ LPS, and p-p38MAPK/p38MAPK ratio was significantly increased(P 0.05). Compared with group P+ LPS, the cell permeability was significantly increased, the expression of HSP27 was up-regulated, p-p38MAPK/p38MAPK ratio was increased, and the expression of β-arrestin-1 was down-regulated in group P+ LPS+ shRNA(P<0.05). Conclusion The mechanism by which PHC inhibits LPS-induced increase in pulmonary microvascular permeability is totally related to β-arrestin-1 in human PMVECs. Key words: Arrestins; Cholinergic antagonists; Endotoxins; Lung; Capillary permeability

Valproic Acid Treatment Inhibits Vasopermeability and Improves Survival in Rats With Lethal Scald Injury.

The aim of this study was to examine whether administration of valproic acid (VPA), a histone deacetylase inhibitor, inhibits proinflammatory mediators and ameliorate visceral vasopermeability both in a rat model of major burn, and also in rat cultured endothelial cells stimulated with permeability evoking mediators. SD rats were subjected to a 50% TBSA full-thickness scald injury, and treated with either saline or VPA (300 mg/kg) intraperitoneally. Pulmonary vascular endothelial growth factor (VEGF), myeloperoxidase (MPO), pulmonary microvascular permeability, water content, and acetylation of histone H3K9 of lungs were evaluated. In addition, pulmonary microvascular endothelial cells (PMECs) from male SD rats were cultured. With then, MPO, VEGF, histone acetylation, and the permeability of PMECs were investigated. Lethal scald injury resulted in a significant increase in microvascular permeability and water content of lung, accompanied by a significant elevation of the content of VEGF and activity of MPO, and a decrease of histone acetylation. VPA treatment significantly alleviated the microvascular permeability and water content of lung, lowered the levels of VEGF and MPO, and promoted acetylation of histone H3K9 following scald injury. Moreover, VPA reduced permeability of monolayer PMECs subjected to scald serum challenge, reduced the level of MPO and VEGF in supernatants, and promoted acetylation of histone H3K9 in PMECs. These results indicated that VPA can protect pulmonary microvascular endothelial barrier, alleviate proinflammatory mediators-evoked vascular hyperpermeability and tissue edema and improve the survival rate of rats subjected to lethal scald injury.

Septic Murine Pulmonary Microvascular Endothelial Barrier Dysfunction in vitro is Regulated by TIMP3

Sepsis often causes dysfunction of pulmonary microvascular endothelial cells (PMECs) leading to pulmonary edema. Metalloproteinases can regulate endothelial function through processing of cell surface proteins, including adherens junctions proteins, which has been associated with increased permeability. Tissue inhibitor of metalloproteinases 3 (TIMP3) regulates metalloproteinase function in the lung following injury. Thus, we hypothesize that TIMP3 promotes PMEC stability via inhibition of metalloproteinase activity. To test this hypothesis, we used PMECs isolated from WT and Timp3‐/‐mice. We found that TIMP3 levels (mRNA and protein) were decreased in WT PMECs in a time‐dependent fashion under septic conditions (cytomix). Analysis of leak (transendothelial electrical resistance, dextran, and albumin flux) revealed that Timp3‐/‐PMECs had significantly higher permeability under resting conditions (PBS) vs. WT PMECs. Although cytomix treatment significantly increased WT trans‐PMEC permeability, there was no effect on Timp3‐/‐PMEC barrier function. Additionally, the increased basal Timp3‐/‐ PMEC permeability was associated with disrupted surface vascular endothelial‐cadherin localization, which was rescued by treatment with GM6001, a synthetic inhibitor of metalloproteinases. Together, our data suggest that TIMP3 supports normal PMEC barrier function and that septic downregulation of TIMP3 may be an important contributor to septic PMEC barrier dysfunction.

Methods for measurement of permeability coefficient of pulmonary microvascular endothelial cells in rat

To explore an optimal method for the measurement of pulmonary microvascular endothelial cell (PMVEC) permeability coefficient. A monolayer of rat PMVEC model was constructed by culturing a cell suspension on transwell filter or polycarbonate filter membrane. After the state of confluence of cells was affirmed with epithelial volt-ohm meter or inverted microscope, the permeability coefficient was determined by means of transendothelial electrical resistance (TER), fluoresceinisothiocyanate-dextran (Pd), and permeation of Hanks solution (Lp) across monolayers. Meanwhile,changes in PMVEC permeability expressed by the ratio of the observed value and the original value were observed after lipopolysaccharide (LPS) challenge for 0. 5 hour or 2 hours. The cells reached the state of confluence as observed under inverted microscope on the third day post-seeding, and the TER and Pdat this time-point were C(39. 45 ± 3. 96) ( 2 cm2] and [(8. 52 + 0. 50) X 10-6cm/s], respectively. After PMVEC were seeded on transwell filters, the TER increased steadily in a time-dependent manner after seeding of PMVEC, reaching the summit at the fourth day post-seeding C(49. 84 ± 3. 93)f " cm2].Under the natural state, the TER, Pd and Lp of confluent PMVEC monolayers were (49.84 ±3.93) ·.* cm2, (6.15±0.63) X 106 cm/s and (6.80 + 0.62) X10< cm * s-' * cm HZO-', respectively.After PMVEC monolayers were challenged with 10 mg/L LPS for both 0. 5 hour and 2 hours, there was significant decrease in the permeability coefficient as measured by TER (0. 87+ 0. 03, 0.45 0. 04 vs. 1.00+0.08, respectively, both P< 0.05), and an increase in the permeability coefficient measured by Pd (1.33±0. 11, 2.43±0. 14 vs. 1.00+0.10, respectively, both P<0. 05) and the permeability coefficient measured by Lp (1.30± 0.07, 2.38 0.15 vs. 1.00 + 0.11, respectively, both P< 0.05) when compared with the normal group. Three methods, namely TER, Pd and Lp are available to use to assess PMVEC permeability coefficient. The combination of an inverted microscope, TER and Pd enhances the accuracy in determining PMVEC permeability coefficient, and it provides an experimental technique for studying the pathogenesis of acute lung injury in vitro.

Urokinase-type Plasminogen Activator (uPA) Induces Pulmonary Microvascular Endothelial Permeability through Low Density Lipoprotein Receptor-related Protein (LRP)-dependent Activation of Endothelial Nitric-oxide Synthase

Urokinase plasminogen activator (uPA) and PA inhibitor type 1 (PAI-1) are elevated in acute lung injury, which is characterized by a loss of endothelial barrier function and the development of pulmonary edema. Two-chain uPA and uPA-PAI-1 complexes (1-20 nM) increased the permeability of monolayers of human pulmonary microvascular endothelial cells (PMVECs) in vitro and lung permeability in vivo. The effects of uPA-PAI-1 were abrogated by the nitric-oxide synthase (NOS) inhibitor L-NAME (N(D)-nitro-L-arginine methyl ester). Two-chain uPA (1-20 nM) and uPA-PAI-1 induced phosphorylation of endothelial NOS-Ser(1177) in PMVECs, which was followed by generation of NO and the nitrosylation and dissociation of β-catenin from VE-cadherin. uPA-induced phosphorylation of eNOS was decreased by anti-low density lipoprotein receptor-related protein-1 (LRP) antibody and an LRP antagonist, receptor-associated protein (RAP), and when binding to the uPA receptor was blocked by the isolated growth factor-like domain of uPA. uPA-induced phosphorylation of eNOS was also inhibited by the protein kinase A (PKA) inhibitor, myristoylated PKI, but was not dependent on PI3K-Akt signaling. LRP blockade and inhibition of PKA prevented uPA- and uPA-PAI-1-induced permeability of PMVEC monolayers in vitro and uPA-induced lung permeability in vivo. These studies identify a novel pathway involved in regulating PMVEC permeability and suggest the utility of uPA-based approaches that attenuate untoward permeability following acute lung injury while preserving its salutary effects on fibrinolysis and airway remodeling.

Gap junction channel protein connexin40 modulates rabbit pulmonary microvascular endothelial cells permeability in traumatic acute lung injury

Objective To investigate the influence of traumatic acute lung injury (ALI) rabbit serum on gap junction channel ( GJC ) function and connexin40 ( Cx40) expression, and the correlation of GJC and Cx40 with rabbit pulmonary microvascular endothelial cells permeability. Methods Cultured pulmonary microvessel endothelial cells were divided into three groups, control ( Gcontrol) , injured serum (Gserum) , and blocker agent ( Gblocker). GJC function was assessed by scrape-loading and dye transfer techniques. Pulmonary microvessel endothelial cells permeability was measured by Evans blue-labeled albumin transfer, and the expression of Cx40 was measured by Western blotting. Intracellular free calcium concentration was measured by fluo-3am. Results Injured serum decreased GJC function and the level of Cx40,which was aggravated by the GJC blocker (control 726 293. 2 ± 83 684. 1, serum 397 798. 1 ± 87 145. 7, blocker 316 977. 6 ±76 325. 9,n=4,P ＜0. 05). Similarly, pulmonary microvascular endothelial cells permeability was increased significantly in Gserum and Gblocker as compared with Gcontrol (P ＜0. 05). After treatment with injured serum in combination with gap junction blocker in vitro, intracellular free calcium concentration was increased [control (494. 80 ±41.94) nmol/L, serum (569. 80 ±6. 70) nmol/L, blocker (158. 80 ±13. 09) nmol/L, P＜ 0.05]. Conclusion Traumatic ALI rabbit serum inhibits the Cx40 expression and down-regulates GJC function, which contributes to the increase of pulmonary microvascular endothelial cells permeability after ALI This mechanism may participate in the increase of pulmonary vascular permeability and promote the morbidity of ALI after trauma. Key words: Acute lung injury; Connexin40; Pulmonary permeability

Role of myosin light chain kinase in increase in permeability of human pulmonary microvascular endothelial cells induced by mechanical stretch

Objective To evaluate the role of myosin light chain kinase(MLCK)in the increase in the permeability of human pulmonary microvascular endothelial cells(HPMVECs)induced by mechanical stretch.Methods HPMVECs were cultured in vitro and then randomly divided into 3 groups(n = 4 each): mechanical stretch group(group S),ML9 treatment group(group M)and control group(group C).In group S,the monolayer cells and magnetic beads coated with an RGD peptide were incubated in serum-free MCDB131 medium for 2 h,the unbound beads were washed out,and then the cells were exposed to magnetic twisting stimulation(MTS)for 2 h(frequency 3 Hz,intensity 4.2 mT).In group M,the monolayer cells and magnetic beads coated with an RGD peptide were incubated for 2 h in serum-free MCDB131 medium in which MLCK inhibitor ML9(50μmol/L)was added,and the other procedures were the same as in group S.In group C,the cells after washing as in group S were incubated for 2 h without exposing to MTS.Monolayer permeability was detected with FITC-dextran flux in transwell model.The distribution of integrin αVβ3 and actin was detected using the immunofluorescence assay.Results Compared with group C,the permeability of HPMVECs was significantly increased in group S,while no significant change was found in group M.Compared with group S,the permeability of HPMVECs was significantly decreased in group M.In addition,actin polymerized to form stress fiber,and integrin αVβ3 clustered at the end of stress fiber in group S,while actin mainly distributed in the surrounding area of the cell membrane and integrin αVβ3 evenly distributed on the cell surface in group C and M.Conclusion The mechanism by which mechanical stretch induces the increase in the permeability of HPMVECs is related to MLCK activation-mediated stress fiber formation and integrin αVβ3 clustering. Key words: Myosin-light-chain kinase; Capillary permeability; Lung

Alveolar macrophage inducible nitric oxide synthase-dependent pulmonary microvascular endothelial cell septic barrier dysfunction

Inducible nitric oxide (NO) synthase (iNOS) from neutrophils and alveolar macrophages (AM) contributes to the pathophysiology of murine septic acute lung injury (ALI). It is not known if AM iNOS has a direct effect on septic pulmonary microvascular endothelial cell (PMVEC) permeability. We hypothesized that AM iNOS mediates PMVEC permeability in vitro under septic conditions through NO and peroxynitrite. 100,000 confluent PMVEC on cell-culture inserts were co-incubated with iNOS+/+ vs. iNOS−/− AM, in various ratios of AM to PMVEC. PMVEC injury was assessed by trans-PMVEC Evans Blue-labelled albumin flux in the presence or absence of cytomix (equimolar TNF-α, IL-1β and IFN-γ). Cytomix stimulation dose-dependently increased trans-PMVEC EB-albumin flux, which was exaggerated (1.4±0.1% vs. 0.4±0.1% in unstimulated PMVEC, p<0.05) in the presence of iNOS+/+, but not iNOS−/−, AM in the upper compartment. Similarly, iNOS+/+, but not iNOS−/−, AM in the lower compartment also enhanced septic trans-PMVEC albumin leak. The mechanism of iNOS-dependent septic PMVEC permeability was pursued through pharmacologic studies with inhibitors of NOS, and scavengers of NO, superoxide, and peroxynitrite, and treatment of PMVEC with the NO donor, DETA-NONOate. Septic iNOS+/+ AM-dependent trans-PMVEC albumin leak was significantly attenuated by pharmacologic iNOS inhibition (L-NAME and 1400W), and scavenging of either NO (oxyhemoglobin), superoxide (PEG-SOD), or peroxynitrite (FeTPPS). Exogenous NO (DETA-NONOate) had no effect on PMVEC permeability. These data are consistent with a direct role of AM iNOS in septic PMVEC barrier dysfunction, which is likely mediated, in part, through peroxynitrite.

Rolipram and Isoproterenol Reverse Platelet Activating Factor-Induced Increases in Pulmonary Microvascular Permeability and Vascular Resistance

Platelet activating factor (PAF) is an important mediator of pulmonary microvascular endothelial cell (PMVEC) injury in sepsis. Membrane receptors for PAF have been identified on PMVECs and mediate its actions at least in part by protein kinase C activation. Since rolipram, a family IV cyclic AMP phosphodiesterase inhibitor, and isoproterenol, an adenylate cyclase activator, both reverse ischemia-reperfusion-induced lung permeability, we studied the effects of these agents on PAF-induced pulmonary microvascular permeability. The isolated rat lung model was used in which lungs were ventilated and buffer perfused at constant flow while suspended from a force transducer to monitor lung weight along with arterial ( P a) and venous ( P v) pressures. Control lungs ( n = 6) were infused with PAF (40 nmole/kg) via an arterial port and the capillary permeability coefficient ( K f,c) was determined at 0, 15, and 60 min. The remaining lungs were randomized for infusion with either rolipram ( n = 4, 20 μmole/kg) or isoproterenol ( n = 4, 5 μmole/kg) via an arterial port 30 min after injury with PAF. In the rolipram- and isoproterenol-treated groups, the K f,c was determined 15 and 60 min postinfusion with these agents. The control group showed significant elevation in the K f,c and total pulmonary resistance ( R t). At 15 and 60 min, rolipram and isoproterenol reversed PAF injury as shown by the significant improvement in the K f,c and R t. These findings support the concept that increased cyclic AMP is an important mediator in the reversal of PAF-increased PMVEC permeability and pulmonary resistance.