Permeability Of Microvascular Endothelial Cells Research Articles

Cigarette smoke and Pseudomonas aeruginosa synergistically cause ARDS via impairment of mitophagic flux and NLRP3 inflammasome activation

Itis increasingly recognized that cigarette smoke (CS) increases the incidenceand mortality associated with acute respiratory distress syndrome (ARDS), however, the underlying mechanism(s) for this is unknown. Pseudomonas aeruginosa is a frequent pathogen causing pneumonia, which is the most prevalent cause of ARDS. Whether CS increases prevalence of ARDS in individuals with P. aeruginosa infection is unknown. We have previously shown that CS increases severity of acute lung injury in mice challenged by lipopolysaccharide. In this study, we demonstrated that exposure of mice to CS either briefly (6h) or sub‐acutely (2weeks) suppressed lung bacterial clearance and exacerbated P. aeruginosa (PA103)‐induced lung edema, inflammation and mortality. CS and PA103 also synergistically increased lung microvascular endothelial cell permeability in vitro. To investigate the mechanisms of CS priming for lung injury, we used the clinically relevant double‐hit mouse model of ARDS induced by CS priming, followed by PA103 infection. We found that CS increased mitochondrial fission and mitochondrial oxidativestress and inhibition of mitochondrial fission and oxidative stress prevented CS‐induced increase in endothelial permeability. CS pre‐exposure did not affect PA103‐induced increase in steady‐state mitophagy, but impaired mitophagic fluxin alveolar macrophages and lung endothelial cells; an effect associated with accumulation of fragmented/swollen mitochondria and double‐membraned mitophagosomes in the lungs and NLRP3 inflammasome activation in alveolar macrophages. We further found that inhibition of NLRP3 inflammasome signaling attenuated CS/PA103 double‐hit‐induced lung inflammation and lung edema and improved bacterial clearance, as well as overall animal survival. Our results suggest that CS/PA103 double‐hit injury causes ARDS via enhancement of mitochondrial fission and impairment of mitophagic flux and resultant NLRP3 inflammasome activation. Suppression of mitochondrial fission, improvement of mitophagic flux and inhibition of the inflammasome may be novel targeted therapeutic strategies for smokers with ARDS.Support or Funding InformationCOBREP20GM103652 (Rounds, Lu), R01 HL130230 (Lu), VA Merit Review (Rounds)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The regulation of tight junction protein via PKCα/β for abnormal permeability of brain microvascular endothelial cells exposed to paraquat

Objective: To explore if conventional protein kinase C (cPKC: PKCα and PKCβ) contributes to paraquat (PQ) -induced abnormal permeability of mouse brain microvascular endothelial cells (BMECs) via the regulation of tight junction (TJ) proteins. Methods: The immortalized mouse brain endothelial cell line (bEnd.3) was used to establish a monolayer blood-brain barrier (BBB) model. In order to evaluate the function of the in vitro BBB model, the transendothelial electrical resistance (TEER) and permeability were measured by a Millicell-ERS volt-ohmmeter and sodium fluorescent (Na-FLU) , respectively. MTT assay was used to determine the relative survival rate of cells. The dose-response relationship was determined by treating cells with 0, 50, 100, 200, and 300 μmol/L PQ for 24 hours. The time-response relationship was determined by treating cells with 200 μmol/L PQ for 6, 12, 24, 48, and 72 hours. After the treatment of cells with 0, 100, 200, and 300 μmol/L PQ for 24 hours, the protein and mRNA expression levels of ZO-1, Occludin, and Claudin-5 were measured by immunofluorescence (IF) and quantitative real-time RT-PCR (qRT-PCR) , respectively; the expression of PKCα, PKCβ, phosphorylated (p) -PKCα, and p-PKCβ was determined by Western blot. After the treatment of cells with 200? mol/L PQ for 24 hours following the pretreatment with a classical PKC inhibitor (Go 6983, 1 μmol/L) for 1 hour, the protein expression of ZO-1, Occludin, Claudin-5, p-PKCα, and p-PKCβ was measured by Western blot. Results: The TEER of the bEnd. 3 cells increased gradually with the cell culture time, and reached a peak value of 114.3±6.9 Ω·cm(2) on day 6. According to the permeability analysis by Na-FLU, cell permeability gradually decreased with the cell culture time, and reached 1.7±0.2 cm/min on day 6, suggesting a well-behaved barrier function of cells. Compared with the control group, the survival rates of the bEnd.3 cells were significantly reduced after exposure to 100, 200, or 300 μmol/L PQ for 24 hours (P<0.05) , or after exposure to 200 μmol/L PQ for 6, 12, 24, 48, or 72 hours (P <0.05) , indicating a dose-and time-dependent relationship. The IF and qRT-PCR results showed that the protein and mRNA expression levels of ZO-1, Occludin, and Claudin-5 were significantly reduced with the increase in the concentration of PQ (P<0.05) . The Western blot analysis showed that compared with the control group, cells exposed to PQ had significantly higher protein expression of p-PKCα and p-PKCβ and significantly lower protein expression of ZO-1, Occludin, and Claudin-5 (P<0.05) . Compared with the PQ treatment group, the Go 6983 intervention group had significantly higher protein expression of ZO-1, Occludin, and Claudin-5 and significantly lower protein expression of p-PKCα and p-PKCβ (P<0.05) . Conclusion: By activation of cPKC (PKCα and PKCβ) , PQ reduces the protein and mRNA expression of TJ proteins and enhances the permeability of murine BMECs.

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.

Hemolysis-induced Lung Vascular Leakage Contributes to the Development of Pulmonary Hypertension.

Although hemolytic anemia-associated pulmonary hypertension (PH) and pulmonary arterial hypertension (PAH) are more common than the prevalence of idiopathic PAH alone, the role of hemolysis in the development of PAH is poorly characterized. We hypothesized that hemolysis independently contributes to PAH pathogenesis via endothelial barrier dysfunction with resulting perivascular edema and inflammation. Plasma samples from patients with and without PAH (both confirmed by right heart catheterization) were used to measure free hemoglobin (Hb) and its correlation with PAH severity. A sugen (50 mg/kg)/hypoxia (3 wk)/normoxia (2 wk) rat model was used to elucidate the role of free Hb/heme pathways in PAH. Human lung microvascular endothelial cells were used to study heme-mediated endothelial barrier effects. Our data indicate that patients with PAH have increased levels of free Hb in plasma that correlate with PAH severity. There is also a significant accumulation of free Hb and depletion of haptoglobin in the rat model. In rats, perivascular edema was observed at early time points concomitant with increased infiltration of inflammatory cells. Heme-induced endothelial permeability in human lung microvascular endothelial cells involved activation of the p38/HSP27 pathway. Indeed, the rat model also exhibited increased activation of p38/HSP27 during the initial phase of PH. Surprisingly, despite the increased levels of hemolysis and heme-mediated signaling, there was no heme oxygenase-1 activation. This can be explained by observed destabilization of HIF-1a during the first 2 weeks of PH regardless of hypoxic conditions. Our data suggest that hemolysis may play a significant role in PAH pathobiology.

Mechanisms of Blood Brain Barrier Disruption by Different Types of Bacteria, and Bacterial-Host Interactions Facilitate the Bacterial Pathogen Invading the Brain.

This review aims to elucidate the different mechanisms of blood brain barrier (BBB) disruption that may occur due to invasion by different types of bacteria, as well as to show the bacteria-host interactions that assist the bacterial pathogen in invading the brain. For example, platelet-activating factor receptor (PAFR) is responsible for brain invasion during the adhesion of pneumococci to brain endothelial cells, which might lead to brain invasion. Additionally, the major adhesin of the pneumococcal pilus-1, RrgA is able to bind the BBB endothelial receptors: polymeric immunoglobulin receptor (pIgR) and platelet endothelial cell adhesion molecule (PECAM-1), thus leading to invasion of the brain. Moreover, Streptococcus pneumoniae choline binding protein A (CbpA) targets the common carboxy-terminal domain of the laminin receptor (LR) establishing initial contact with brain endothelium that might result in BBB invasion. Furthermore, BBB disruption may occur by S. pneumoniae penetration through increasing in pro-inflammatory markers and endothelial permeability. In contrast, adhesion, invasion, and translocation through or between endothelial cells can be done by S. pneumoniae without any disruption to the vascular endothelium, upon BBB penetration. Internalins (InlA and InlB) of Listeria monocytogenes interact with its cellular receptors E-cadherin and mesenchymal-epithelial transition (MET) to facilitate invading the brain. L. monocytogenes species activate NF-κB in endothelial cells, encouraging the expression of P- and E-selectin, intercellular adhesion molecule 1 (ICAM-1), and Vascular cell adhesion protein 1 (VCAM-1), as well as IL-6 and IL-8 and monocyte chemoattractant protein-1 (MCP-1), all these markers assist in BBB disruption. Bacillus anthracis species interrupt both adherens junctions (AJs) and tight junctions (TJs), leading to BBB disruption. Brain microvascular endothelial cells (BMECs) permeability and BBB disruption are induced via interendothelial junction proteins reduction as well as up-regulation of IL-1α, IL-1β, IL-6, TNF-α, MCP-1, macrophage inflammatory proteins-1 alpha (MIP1α) markers in Staphylococcus aureus species. Streptococcus agalactiae or Group B Streptococcus toxins (GBS) enhance IL-8 and ICAM-1 as well as nitric oxide (NO) production from endothelial cells via the expression of inducible nitric oxide synthase (iNOS) enhancement, resulting in BBB disruption. While Gram-negative bacteria, Haemophilus influenza OmpP2 is able to target the common carboxy-terminal domain of LR to start initial interaction with brain endothelium, then invade the brain. H. influenza type b (HiB), can induce BBB permeability through TJ disruption. LR and PAFR binding sites have been recognized as common routes of CNS entrance by Neisseria meningitidis. N. meningitidis species also initiate binding to BMECs and induces AJs deformation, as well as inducing specific cleavage of the TJ component occludin through the release of host MMP-8. Escherichia coli bind to BMECs through LR, resulting in IL-6 and IL-8 release and iNOS production, as well as resulting in disassembly of TJs between endothelial cells, facilitating BBB disruption. Therefore, obtaining knowledge of BBB disruption by different types of bacterial species will provide a picture of how the bacteria enter the central nervous system (CNS) which might support the discovery of therapeutic strategies for each bacteria to control and manage infection.

Hsp70 Suppresses Mitochondrial Reactive Oxygen Species and Preserves Pulmonary Microvascular Barrier Integrity Following Exposure to Bacterial Toxins.

Pneumonia is a leading cause of death in children and the elderly worldwide, accounting for 15% of all deaths of children under 5 years old. Streptococcus pneumoniae is a common and aggressive cause of pneumonia and can also contribute to meningitis and sepsis. Despite the widespread use of antibiotics, mortality rates for pneumonia remain unacceptably high in part due to the release of bacterial toxins. Pneumolysin (PLY) is a cholesterol-dependent toxin that is produced by Streptococcus, and it is both necessary and sufficient for the development of the extensive pulmonary permeability edema that underlies acute lung injury. The mechanisms by which PLY disrupts the pulmonary endothelial barrier are not fully understood. Previously, we found that reactive oxygen species (ROS) contribute to the barrier destructive effects of PLY and identified an unexpected but potent role of Hsp70 in suppressing ROS production. The ability of Hsp70 to influence PLY-induced barrier dysfunction is not yet described, and the goal of the current study was to identify whether Hsp70 upregulation is an effective strategy to protect the lung microvascular endothelial barrier from G+ bacterial toxins. Overexpression of Hsp70 via adenovirus-mediated gene transfer attenuated PLY-induced increases in permeability in human lung microvascular endothelial cells (HLMVEC) with no evidence of cytotoxicity. To adopt a more translational approach, we employed a pharmacological approach using geranylgeranylacetone (GGA) to acutely upregulate endogenous Hsp70 expression. Following acute treatment (6 h) with GGA, HLMVECs exposed to PLY displayed improved cell viability and enhanced endothelial barrier function as measured by both Electric Cell-substrate Impedance Sensing (ECIS) and transwell permeability assays compared to control treated cells. PLY promoted increased mitochondrial ROS, decreased mitochondrial oxygen consumption, and increased caspase 3 cleavage and cell death, which were collectively improved in cells pretreated with GGA. In mice, IP pretreatment with GGA 24 h prior to IT administration of PLY resulted in significantly less Evans Blue Dye extravasation compared to vehicle, indicating preserved endothelial barrier integrity and suggesting that the acute upregulation of Hsp70 may be an effective therapeutic approach in the treatment of lung injury associated with pneumonia.

Angiotensin II aggravates lipopolysaccharide induced human pulmonary microvascular endothelial cells permeability in high glucose status

Lung infection is one of the most common infections in diabetes mellitus and is characterized by increased pulmonary microvascular endothelial permeability. Local Angiotensin II (AngII) plays an important role in the pathogenesis of lung diseases. However, whether AngII can aggravate diabetic infectious lung injury is not clear. Therefore, we investigated the effects of AngII on the permeability of human pulmonary microvascular endothelial cells (HPMVECs) challenged by lipopolysaccharide (LPS) in high glucose states in vitro. HPMVECs were divided into five groups: a control group (CON), a high glucose group (HG), an LPS + high glucose group (LH), an LPS + high glucose + AngII group (LHA), and an LPS + high glucose + Losartan group (LHL). The HPMVECs permeability as well as the F-actin levels, cytoskeleton, apoptosis and TNF-α concentrations were evaluated. Compared to the CON group, the HG, LH and LHA groups had significantly higher cellular permeability, cellular apoptosis and TNF-α levels, with more extensive cytoskeletal damage and lower F-actin levels. Additionally, cells in the LHA group exhibited significantly elevated permeability, apoptosis and TNF-α concentrations, lower F-actin levels and more extensive cytoskeletal damage than either the LH or HG group. However, compared to the HG or LH group, the LHL group showed significantly lower cellular permeability, cell apoptosis, cytoskeletal damage and TNF-α concentrations and higher F-actin levels. This study suggests that in a diabetic infectious lung injury cellular model, AngII could aggravate the permeability of HPMVEC via F-actin dynamics and cell apoptosis. Furthermore, blocking the Angiotension II Type 1 Receptor could significantly alleviate the hyperpermeability of HPMVECs.

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

Activation of Epac alleviates inflammation and vascular leakage in LPS-induced acute murine lung injury

Exchange protein directly activated by cAMP (Epac) is an important molecule in cAMP signal transduction, but the effect of Epac on lipopolysaccharide (LPS)-induced acute lung injury (ALI) is unclear. In this study, we treated in vitro and in vivo models with the Epac activator 8CPT to determine the effect and related mechanisms of Epac. The in vitro results indicate that 8CPT inhibits lipopolysaccharide (LPS)-induced tumor necrosis factor-α (TNF-α) release from mouse macrophages (MH-S), whereas the protein kinase A (PKA) activator 6BnZ has no effect. Furthermore, Epac over-expression can significantly suppress TNF-α release from LPS induced MH-S cell, while Epac siRNA can slightly increase TNF-α release. Moreover, 8CPT reduces LPS-induced microvascular permeability in human pulmonary microvascular endothelial cells (HPMVECs), whereas the PKA activator 6BnZ has no effect. In mice with LPS-induced ALI, 8CPT significantly reduces LPS-induced inflammatory cytokine release, neutrophil recruitment, and albumin leakage. LPS simultaneously decreases the Epac but not the PKA levels. However, 8CPT reverses the decreased Epac levels. Furthermore, the mechanism involves the small GTPase Rac1/2 but not the mitogen-activated protein kinase (MAPK) pathway. Thus, Epac activation reduces inflammation and microvascular permeability in LPS-induced lung injury and an Epac activator represents a novel choice for the early therapy of ALI.

The role and mechanism of mesenchymal stem cell in modulating human pulmonary microvascule endothelial cell permeability via paracrine hepatocyte growth factor

Objective: To explore the role and mechanism of mesenchymal stem cell (MSC) in modulating human pulmonary microvascular endothelial cell (HPMECs) permeability via hepatocyte growth factor (HGF). Methods: The study introduced a co-cultured model between HPMECs and human mesenchymal stem cell conditioned media (MSC-CM) collected from MSCs after 24 h hypoxia culture, and meanwhile HGF was neutralized in MSC-CM by anti-HGF antibody respectively, followed by lipopolysaccharide (LPS) stimulation. Finally, the following measurements were performed: the permeability of HPMECs, the protein expression of vascular endothelial cadherin (VE-cadherin), Occludin in HPMECs by Western blot, HPMECs apoptosis by Annexin V-FITC/PI and HPMECs proliferation by 3-(4, 5)-dimethylthiahiazo (-z-y1)-3, 5-di- phenytetrazoliumromide(MTT). Results: Compared to LPS group (4.15±0.88), MSC-CM reduced endothelial paracellular permeability injured by LPS(1.56±0.36, P<0.01), however, the MSC-CM effect was significantly blocked by anti-HGF antibody(3.11±0.74, P<0.05). Furthermore MSC-CM significantly increased the expression of VE-cadherin(0.71±0.05 vs. 0.38±0.19, P<0.05)and Occludin protein(0.96±0.05 vs. 0.51±0.02, P<0.05) in HPMECs, which was significantly blocked by anti-HGF antibody (P<0.05). MSC-CM significantly reduced the number of early apoptotic cells (6.82±1.80 vs. 17.09±1.89, P<0.05). However, the effect of MSC-CM was significantly blocked by neutralizing HGF (12.07±0.98, P<0.01). The cell viability results by MTT assay confirmed that MSC-CM(6.82±1.80, P<0.05)restored cell viability to a greater extent than LPS stimulation only(0.47±0.09), and meanwhile the MSC-CM effect was significantly inhibited by neutralizing HGF from MSC-CM with anti-HGF antibody (0.69±0.29, P<0.05). Conclusion: HGF secreted by MSCs reduces endothelial cell paracelluar permeability induced by LPS, and the possible mechanisms include remodelling of endothelial intercellular adherence junction, promoting endothelial cell proliferation and restraining endothelial cell apoptosis.

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.

Cell-Free Hemoglobin-mediated Increases in Vascular Permeability. A Novel Mechanism of Primary Graft Dysfunction and a New Therapeutic Target.

Cell-free hemoglobin (CFH) is a potent oxidant associated with poor clinical outcomes in a variety of clinical settings. Recent studies suggest that acetaminophen (APAP), a specific hemoprotein reductant, can abrogate CFH-mediated oxidative injury and organ dysfunction. Preoperative plasma CFH levels are independently associated with primary graft dysfunction (PGD) after lung transplant ( 1 ). Our objectives were to determine whether CFH would increase lung vascular permeability in the isolated perfused human lung and whether APAP would limit these effects. Human lungs declined for transplant were inflated and perfused with Dulbecco's modified Eagle medium/5% albumin at a pulmonary artery pressure of 8-12 mm Hg. After steady state was achieved, CFH (100 mg/dl) was added to the perfusate ± APAP (15 μg/ml). Lung permeability was measured by continuous monitoring of lung weight gain and by extravasation of Evans blue dye-labeled albumin from the vasculature into bronchoalveolar lavage. To test the mechanism of increased permeability, human pulmonary microvascular endothelial cells were exposed to CFH (0.5 mg/ml) ± APAP (160 μM) for 24 hours and permeability was assessed by electrical cell-substrate impedance sensing. In the isolated perfused human lung, CFH increased lung permeability over 2 hours compared with control lungs (12% vs. 2% weight gain from baseline, P = 0.03). Increased vascular permeability was confirmed by a 4.8-fold increase in Evans blue dye-labeled albumin in the airspace compared with control lungs. Pretreatment with APAP prevented lung weight gain (P = 0.06 vs. CFH). In human pulmonary microvascular endothelial cells, CFH increased monolayer permeability (P = 0.03 vs. control), and this was attenuated by APAP (P = 0.045 vs. CFH). Circulating CFH increases vascular permeability in the isolated perfused human lung and paracellular permeability in lung microvascular endothelial cells. These effects may explain the association of plasma CFH levels with PGD. The hemoprotein reductant APAP attenuates the effects of CFH and merits further exploration as a potential therapy for PGD prevention.

Triamcinolone Acetonide Suppresses Inflammation and Facilitates Vascular Barrier Function in Human Retinal Microvascular Endothelial Cells.

Diabetic macular edema (DME) is resulted from the retinal microvascular leakage that accompanies the breakdown of blood-retinal barrier. Triamcinolone acetonide (TA) is a therapeutic agent for DME, but since the detailed mechanism of action of TA is not known, part of its action was examined. In vitro model to enhance the permeability of human retinal microvascular endothelial cells (HRMECs) was constructed by using DME-related cytokines such as vascular endothelial growth factor (VEGF), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β). The efficacy of TA and anti-VEGF antibody against retinal permeability was evaluated. Furthermore, the localization of ZO-1 in HRMECs was examined using immunofluorescence staining and the expression level of tight junction proteins (ZO-1, Occludin, and Claudin-5) was examined using immunoblot. TA and anti-VEGF antibody showed inhibitory effects against VEGF-induced permeability enhancement, and TA also inhibited the increase in permeability induced by TNF-α and IL-1β. In addition to the inhibitory effects against cytokine-induced hyperpermeability, TA enhanced the barrier function of HRMECs and reduced the vascular leakage. TA altered the localization of tight junction proteins, but did not increase the upregulation of tight junction proteins. Moreover, the enhancement of barrier function by TA was inhibited by 17-AAG (glucocorticoid receptor inhibitor). It was suggested that TA reinforced the barrier function through the glucocorticoid receptor. In this study, we found that TA suppressed the inflammation caused by VEGF, TNF-α and IL-1β, and decreased the retinal vascular hyperpermeability.

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

VEGF isoforms have differential effects on permeability of human pulmonary microvascular endothelial cells

BackgroundAlternative splicing of Vascular endothelial growth factor-A mRNA transcripts (commonly referred as VEGF) leads to the generation of functionally differing isoforms, the relative amounts of which have potentially significant physiological outcomes in conditions such as acute respiratory distress syndrome (ARDS). The effect of such isoforms on pulmonary vascular permeability is unknown. We hypothesised that VEGF165a and VEGF165b isoforms would have differing effects on pulmonary vascular permeability caused by differential activation of intercellular signal transduction pathways.MethodTo test this hypothesis we investigated the physiological effect of VEGF165a and VEGF165b on Human Pulmonary Microvascular Endothelial Cell (HPMEC) permeability using three different methods: trans-endothelial electrical resistance (TEER), Electric cell-substrate impedance sensing (ECIS) and FITC-BSA passage. In addition, potential downstream signalling pathways of the VEGF isoforms were investigated by Western blotting and the use of specific signalling inhibitors.ResultsVEGF165a increased HPMEC permeability using all three methods (paracellular and transcellular) and led to associated VE-cadherin and actin stress fibre changes. In contrast, VEGF165b decreased paracellular permeability and did not induce changes in VE-cadherin cell distribution. Furthermore, VEGF165a and VEGF165b had differing effects on both the phosphorylation of VEGF receptors and downstream signalling proteins pMEK, p42/44MAPK, p38 MAPK, pAKT and peNOS. Interestingly specific inhibition of the pMEK, p38 MAPK, PI3 kinase and eNOS pathways blocked the effects of both VEGF165a and VEGF165b on paracellular permeability and the effect of VEGF165a on proliferation/migration, suggesting that this difference in cellular response is mediated by an as yet unidentified signalling pathway(s).ConclusionThis study demonstrates that the novel isoform VEGF165a and VEGF165b induce differing effects on permeability in pulmonary microvascular endothelial cells.

Heterogeneity of TRPV4 Expression and Function in Endothelial Cells of Different Vascular Beds

IntroductionThe ubiquitously expressed calcium channel transient receptor potential vanilloid subtype 4 (TRPV4) has been implicated in various endothelial functions. Specifically, TRPV4 activation triggers endothelial barrier failure. In the lung, TRPV4‐mediated pulmonary edema formation has been reported in response to various triggers including acid aspiration and positive pressure ventilation. Thus, TRPV4 is a promising target for preventing or treating lung injury, but its ubiquitous expression requires investigating potential off‐target effects during therapeutic modulation.MethodsThe present study is working toward this aim by comparing TRPV4 expression and function in human endothelial cells from five different vascular beds: pulmonary (HPMEC), cerebral (HCMEC) and adipose (HAMEC) microvascular as well as pulmonary artery (HPAEC) and coronary artery (HCAEC). Response to the TRPV4 agonist GSK1016790A was assessed by ratiometric Fura‐2 imaging of intracellular calcium concentration [Ca2+]i, measuring transendothelial electrical resistance (TEER), and fluorescence imaging of cytoskeletal actin and VE‐cadherin. TRPV4 protein expression was densitometrically quantified by Western blot, normalized to GAPDH for comparison between the five cell types.ResultsAgonist treatment increased [Ca2+]i and decreased TEER in all endothelial cell types studied, while vehicle (dimethylsulfoxide) had no effect. Interestingly, there was strong inverse correlation between the [Ca2+]i response and decrease in TEER amongst the three microvascular cell types, HPMEC, HAMEC and HCMEC, with the [Ca2+]i and reduction of TEER increasing in the order HPMEC &lt; HAMEC &lt; HCMEC. This effect was corroborated by fluorescence imaging that showed TRPV4 agonism caused intercellular adherens junctions to become disrupted and gaps to form within the endothelial monolayer. In some cases, endothelial cells significantly contracted and became rounded, losing surface VE‐cadherin and forming actin stress fibers. The degree of this effect varied between microvascular cells, again in the increasing order HPMEC &lt; HAMEC &lt; HCMEC. Furthermore, these observations correlated with quantification of Western blot showing HCMECs had highest expression of TRPV4 protein. Notably, while macrovascular HPAECs and HCAECs showed quantitatively similar responses as microvascular endothelial cells, they did not fit the linear regression between [Ca2+]i and TEER response.ConclusionThese results demonstrate quantitative heterogeneity in endothelial TRPV4 function in different vascular beds, and identify TRPV4 as a major regulator of microvascular endothelial cell permeability, while confirming the established differences in barrier function between micro‐ and macrovascular endothelial cells.Support or Funding InformationCanadian Institutes of Health Research (CIHR)Ontario Research Fund (ORF)

AM966, an Antagonist of Lysophosphatidic Acid Receptor 1, Increases Lung Microvascular Endothelial Permeability through Activation of Rho Signaling Pathway and Phosphorylation of VE-Cadherin.

Maintenance of pulmonary endothelial barrier integrity is important for reducing severity of lung injury. Lysophosphatidic acid (LPA) regulates cell motility, cytoskeletal rearrangement, and cell growth. Knockdown of LPA receptor 1 (LPA1) has been shown to mitigate lung injury and pulmonary fibrosis. AM966, an LPA1 antagonist exhibiting an antifibrotic property, has been considered to be a future antifibrotic medicine. Here, we report an unexpected effect of AM966, which increases lung endothelial barrier permeability. An electric cell-substrate sensing (ECIS) system was used to measure permeability in human lung microvascular endothelial cells (HLMVECs). AM966 decreased the transendothelial electrical resistance (TEER) value immediately in a dose-dependent manner. VE-cadherin and f-actin double immunostaining reveals that AM966 increases stress fibers and gap formation between endothelial cells. AM966 induced phosphorylation of myosin light chain (MLC) through activation of RhoA/Rho kinase pathway. Unlike LPA treatment, AM966 had no effect on phosphorylation of extracellular signal-regulated kinases (Erk). Further, in LPA1 silencing cells, we observed that AM966-increased lung endothelial permeability as well as phosphorylation of VE-cadherin and focal adhesion kinase (FAK) were attenuated. This study reveals that AM966 induces lung endothelial barrier dysfunction, which is regulated by LPA1-mediated activation of RhoA/MLC and phosphorylation of VE-cadherin.

Dasatinib Inhibits Actin Fiber Reorganization and Promotes Endothelial Cell Permeability through RhoA-Rock Pathway

BackgroundThe balanced translocation of 5′ segment of the breakpoint cluster region (BCR) gene on chromosome 22 to 3′ segment of the Abelson leukemia virus (ABL) gene on chromosome 9 in chronic myeloid leukemia (CML), results in the formation of the BCR-ABL hybrid fusion gene, which has constitutively upregulated tyrosine kinase activity. Imatinib mesylate, a small molecule developed specifically to inhibit BCR-ABL kinase activity, has revolutionized the treatment of CML. Despite the overwhelming success, about 20% of patients develop resistance to imatinib due to point mutations in the BCR-ABL kinase domain. Dasatinib is one of the second generation tyrosine kinase inhibitors (TKI) that was developed to overcome the resistance to imatinib. However, dasatinib inhibits, in addition to BCR-ABL kinase, other tyrosine kinases such as Src family kinases (SFKs), leading to its unique side effects. Clinically, one of the most common non-hematologic side effects of dasatinib is peripheral edema, pleural effusion and pulmonary hypertension and it is seen 20% of patients treated with dasatinib. We investigated the effect of dasatinib on the barrier function of human microvascular endothelial cells in vitro and in vivo.MethodsIn vitro endothelial permeability assays were performed with endothelial cells grown on transwell polycarbonate membranes inserts. FITC-Dextran was added to the upper chamber followed by dasatinib (0-100 ng/ml) or vehicle (DMSO) and at various time intervals, the medium in the lower chamber was collected and the fluorescence intensity was measured. In some experiments, the monolayers were incubated with ROCK1 inhibitor y27632 for 10 minutes before the addition of dasatinib. RhoA activity assay was performed and quantified using the RhoA activation assay kit. Spreading of control and dasatinib treated endothelial cells were quantified by Image J software. Actin cytoskeleton was visualized by Immunocytochemistry. Phosphorylation of cytoskeletal proteins were assessed by Western blot with phospho specific antibodies. In vivo endothelial permeability was measured by quantifying the extravasation of intravenously administered Evans Blue to lung parenchyma in mice.ResultsThe permeability of human microvascular endothelial cells (HMEC1) to FITC-dextran increases in Transwell chambers within 5 minutes following the addition of therapeutic concentrations of dasatinib. These changes in permeability are associated with increased activation of RhoA GTPase and its effector Rho associated coiled-coil kinase I (ROCKI). RhoA inhibitor C3 almost completely inhibits dasatinib-induced increase in permeability. Under similar conditions, imatinib had no effect on permeability and it does not activate RhoA. Since integrin-induced cell spreading suppresses RhoA activation, we examined the effect of dasatinib on cell spreading on fibronectin substrate. Dasatinib impairs endothelial cell spreading in a concentration-dependent manner and induces disorganization of actin fibers. Tyrosine kinases play an essential role in transmitting signals from integrins to RhoA and we examined tyrosine phosphorylation of several cytoskeletal proteins. Dasatinib markedly inhibits tyrosine phosphorylation of p130 Crk associated substrate (p130cas), paxillin and vinculin. These results suggest inhibition of tyrosine phosphorylation of the focal adhesion plaque components by dasatinib may alter the assembly of actin fibers resulting in the activation of RhoA/ROCK pathway. In vitro, consistent with these findings, dasatinib-induced increase in the permeability is blocked by ROCK inhibitor y-23632. In vivo administration of y-27632 significantly inhibits the dasatinib-induced extravasation of Evans Blue to lung parenchyma in mice. Furthermore, dasatinib-induced microvascular permeability is attenuated in ROCK1-deficient mice.ConclusionsOur findings suggest that dasatinib inhibits actin fiber reorganization and promotes endothelial permeability through RhoA-ROCK pathway and ROCK inhibitors could serve as a therapeutic modality to ameliorate the dasatinib-induced pulmonary changes. DisclosuresNo relevant conflicts of interest to declare.

The Andes Virus Nucleocapsid Protein Directs Basal Endothelial Cell Permeability by Activating RhoA.

ABSTRACTAndes virus (ANDV) predominantly infects microvascular endothelial cells (MECs) and nonlytically causes an acute pulmonary edema termed hantavirus pulmonary syndrome (HPS). In HPS patients, virtually every pulmonary MEC is infected, MECs are enlarged, and infection results in vascular leakage and highly lethal pulmonary edema. We observed that MECs infected with the ANDV hantavirus or expressing the ANDV nucleocapsid (N) protein showed increased size and permeability by activating the Rheb and RhoA GTPases. Expression of ANDV N in MECs increased cell size by preventing tuberous sclerosis complex (TSC) repression of Rheb-mTOR-pS6K. N selectively bound the TSC2 N terminus (1 to 1403) within a complex containing TSC2/TSC1/TBC1D7, and endogenous TSC2 reciprocally coprecipitated N protein from ANDV-infected MECs. TSCs normally restrict RhoA-induced MEC permeability, and we found that ANDV infection or N protein expression constitutively activated RhoA. This suggests that the ANDV N protein alone is sufficient to activate signaling pathways that control MEC size and permeability. Further, RhoA small interfering RNA, dominant-negative RhoA(N19), and the RhoA/Rho kinase inhibitors fasudil and Y27632 dramatically reduced the permeability of ANDV-infected MECs by 80 to 90%. Fasudil also reduced the bradykinin-directed permeability of ANDV and Hantaan virus-infected MECs to control levels. These findings demonstrate that ANDV activation of RhoA causes MEC permeability and reveal a potential edemagenic mechanism for ANDV to constitutively inhibit the basal barrier integrity of infected MECs. The central importance of RhoA activation in MEC permeability further suggests therapeutically targeting RhoA, TSCs, and Rac1 as potential means of resolving capillary leakage during hantavirus infections.

Enhancement of endothelial permeability by free fatty acid through lysosomal cathepsin B-mediated Nlrp3 inflammasome activation.

Obesity is an important risk factor for exacerbating chronic diseases such as cardiovascular disease and cancer. High serum level of saturated free fatty acids such as palmitate is an important contributor for obesity-induced diseases. Here, we examined the contribution of inflammasome activation in vascular cells to free fatty acid-induced endothelial dysfunction and vascular injury in obesity. Our findings demonstrated that high fat diet-induced impairment of vascular integrity and enhanced vascular permeability in the myocardium in mice were significantly attenuated by Nlrp3 gene deletion. In microvascular endothelial cells (MVECs), palmitate markedly induces Nlrp3 inflammasome complex formation leading to caspase-1 activation and IL1β production. By fluorescence microscopy and flow cytometry, we observed that such palmitate-induced Nlrp3 inflammasome activated was accompanied by a reduction in inter-endothelial tight junction proteins ZO-1/ZO-2. Such palmitate-induced decrease of ZO-1/ZO-2 was also correlated with an increase in the permeability of endothelial monolayers treated with palmitates. Moreover, palmitate-induced alterations in ZO-1/ZO-2 or permeability were significantly reversed by an inflammasome activity inhibitor, YVAD, or a high mobility group box 1 (HMGB1) activity inhibitor glycyrrhizin. Lastly, blockade of cathepsin B with Ca-074Me significantly abolished palmitate-induced activation of Nlrp3 inflammasomes, down-regulation of ZO-1/ZO-2, and enhanced permeability in MVECs or their monolayers. Together, these data strongly suggest that activation of endothelial inflammasomes due to increased free fatty acids produces HMGB1, which disrupts inter-endothelial junctions and increases paracellular permeability of endothelium contributing to early onset of endothelial injury during obesity.