Human Lung Endothelial Cells Research Articles

Caught The Flu? Exploring the novel role of high mobility group box 1 protein in severe influenza infection

Severe influenza infections are a common cause of acute respiratory distress syndrome (ARDS). ARDS is characterized by acute lung injury as well as pulmonary edema or “leakage,” which is caused by endothelial dysfunction and subsequent alveolar flooding from circulating blood vessels. High mobility group box 1 (HMGB1) is a protein released from lung endothelial cells that acts as a proinflammatory cytokine during bacterial and viral infections. However, the effect of HMGB1 on the endothelium during influenza viral infection is unknown. This study demonstrates that HMGB1 released from primary human microvascular lung endothelial cells (HLMECs) during influenza infection increases endothelial activation and apoptosis, while also disrupting adherens junctions between cells. Sequestering extracellular HMGB1 using the small molecule glycyrrhizin (GCZ) attenuates these effects, suggesting that HMGB1 signaling occurs in a paracrine manner. Inhibition of the Toll-like receptor (TLR) 4 pathway, a downstream effector of HMGB1, with peptide P5779 did not substantially decrease endothelial apoptosis. Therefore, HMGB1 likely elicits its effects on the endothelium in a TLR4-independent manner. These results will elucidate a partial mechanism that potentially explains ARDS symptoms seen in patients with severe influenza infections.

A novel role of human lung endothelial cells in allergic airway disease by producing and responding to IL-33

Abstract Allergic asthma affects more than 300 million people worldwide and is characterized by airway hypersensitivity and eosinophilia. A prevalent feature of allergic asthma is increased serum IL-33 levels, and asthma GWAS have demonstrated that SNPs in the IL-33 locus are significantly associated with disease. While IL-33 and its downstream type 2 responses have been extensively studied in murine models of asthma, much less is known about the regulation of human IL33. Analysis of the mouse and human IL33 loci reveals little genomic conservation between the two species in non-coding regions. We generated a novel BAC transgenic mouse strain containing the human IL-33 locus with a fluorescent reporter to interrogate the regulation and expression of human IL-33. Surprisingly, the mice expressed human IL-33 primarily in endothelial cells, whereas murine IL-33 was expressed primarily in epithelial cells. These results mirror the expression profiles of IL-33 in primary human lung cells from the LungGENS database, thus demonstrating that our novel mouse model faithfully replicates human IL-33 expression. To understand how human IL-33 in the lung is regulated and expressed during inflammation, we examined BAC transgenic mice challenged with either house dust mite extract (HDM) or poly(I:C). In contrast to murine IL-33, expression of human IL-33 was reduced during allergic inflammation. We tested whether IL-33 is able to autoregulate itself via a negative feedback loop. Indeed, IL-33 administration downregulated the expression of human IL-33 in lung endothelial cells. Together, these data emphasize a distinct and novel role in humans for lung endothelial cells in allergic airway disease by producing and responding to IL-33.

The Role of Cytoplasmic Deacetylase, SIRT2, in Stabilizing Lung Endothelial Barrier Function

Lung microvascular endothelial functional and structural integrity is essential for maintenance of tissue‐fluid homeostasis and normal function of the respiratory system. Disruption of the pulmonary endothelial barrier leads to pulmonary edema, acute lung injury, and respiratory failure. Adherens junction (AJ) proteins, actin cytoskeleton, and microtubules are major components required for maintenance of barrier integrity. Post‐translational protein modifications of AJ constituents, such as phosphorylation, nitration, and nitrosylation, have been well known to regulate endothelial barrier function. More recently, lysine acetylation/deacetylation has emerged as an important and physiologically significant posttranslational protein modification in physiological and pathological conditions. However, its role in endothelial barrier function remains largely unknown. Sirtuins (SIRTs) are NAD+‐dependent deacetylases, and critical regulators of energy metabolism and oxidative stress response in different cell types. We studied the function of SIRT2, the only cytosolic sirtuin family member, in regulating pulmonary endothelial barrier function during inflammatory response. We showed that SIRT2 is the most abundant sirtuin in human lung endothelial cells. Importantly, SIRT2 protein level is significantly increased in lung endothelial cells upon challenging mice with a sublethal dose of lipopolysaccharides, a gram‐negative bacteria endotoxin which causes systemic inflammatory responses resulting in acute lung injury. Increase in SIRT2 protein level was also observed in human lung microvascular endothelial cells (HLMVEC) upon challenge with permeability‐increasing agents such as human α‐thrombin and TNF‐α, further emphasizing the potentially important role of SIRT2 in regulating adaptation of endothelial cells to inflammatory responses. To determine the role of SIRT2 activity in the maintenance of lung endothelial barrier function, we knocked down SIRT2 in HLMVECs and assessed changes in endothelial permeability upon challenge with α‐thrombin. We found that SIRT2 knock down significantly augmented α‐thrombin‐induced permeability increases as compared to control siRNA‐treated cells, suggesting that SIRT2 activity may play a critical role in regulating endothelial barrier integrity. Furthermore, treatment of endothelial cells with AGK2, a SIRT2‐selective inhibitor, significantly decreased assembly of endothelial junctions due to increased accumulation of VE‐cadherin in the cytoplasm. Our data suggest that SIRT2 plays a critical role in the maintenance of endothelial barrier function under basal conditions and upon inflammatory response.Support or Funding InformationXiaoyan Yang : T32HL007829‐23, AHA 17SDG33410608.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Endothelial immune activation programmes cell-fate decisions and angiogenesis by inducing angiogenesis regulator DLL4 through TLR4-ERK-FOXC2 signalling.

The mechanisms by which bacteria alter endothelial cell phenotypes and programme inflammatory angiogenesis remain unclear. In lung endothelial cells, we demonstrate that toll-like receptor 4 (TLR4) signalling induces activation of forkhead box protein C2 (FOXC2), a transcriptional factor implicated in lymphangiogenesis and endothelial specification, in an extracellular signal-regulated kinase (ERK)-dependent manner. TLR4-ERK-FOXC2 signalling regulates expression of the Notch ligand DLL4 and signals inflammatory angiogenesis in vivo and in vitro. Our work reveals a novel link between endothelial immune signalling (TLR pathway) and a vascular transcription factor, FOXC2, that regulates embryonic vascular development. This mechanism is likely to be relevant to pathological angiogenesis complicating inflammatory diseases in humans. Endothelial cells (ECs) mediate a specific and robust immune response to bacteria in sepsis through the activation of toll-like receptor (TLR) signalling. The mechanisms by which bacterial ligands released during sepsis programme EC specification and altered angiogenesis remain unclear. We postulated that the forkhead box protein C2 (FOXC2) transcriptional factor directs EC cell-fate decisions and angiogenesis during TLR signalling. In human lung ECs, lipopolysaccharide (LPS) induced ERK phosphorylation, FOXC2, and delta-like 4 (DLL4, the master regulator of sprouting angiogenesis expression) in a TLR4-dependent manner. LPS-mediated ERK phosphorylation resulted in FOXC2-ERK protein ligation, ERK-dependent FOXC2 serine and threonine phosphorylation, and subsequent activation of DLL4 gene expression. Chemical inhibition of ERK or ERK-2 dominant negative transfection disrupted LPS-mediated FOXC2 phosphorylation and transcriptional activation of FOXC2. FOXC2-siRNA or ERK-inhibition attenuated LPS-induced DLL4 expression and angiogenic sprouting in vitro. In vivo, intraperitoneal LPS induced ERK and FOXC2 phosphorylation, FOXC2 binding to DLL4 promoter, and FOXC2/DLL4 expression in the lung. ERK-inhibition suppressed LPS-induced FOXC2 phosphorylation, FOXC2-DLL4 promoter binding, and induction of FOXC2 and DLL4 in mouse lung ECs. LPS induced aberrant retinal angiogenesis and DLL4 expression in neonatal mice, which was attenuated with ERK inhibition. FOXC2+/- mice treated with LPS showed a mitigated increase in FOXC2 and DLL4 compared to FOXC2+/+ mice. These data reveal a new mechanism (TLR4-ERK-FOXC2-DLL4) by which sepsis-induced EC TLR signalling programmes EC specification and altered angiogenesis.

Human lung endothelial cells produce IL-33 and TSLP simultaneously after dsRNA stimulation

Human lung endothelial cells produce IL-33 and TSLP simultaneously after dsRNA stimulation

Novel Mechanism for Nicotinamide Phosphoribosyltransferase Inhibition of TNF-α-mediated Apoptosis in Human Lung Endothelial Cells.

Nicotinamide phosphoribosyltransferase (NAMPT) exists as both intracellular NAMPT and extracellular NAMPT (eNAMPT) proteins. eNAMPT is secreted into the blood and functions as a cytokine/enzyme (cytozyme) that activates NF-κB signaling via ligation of Toll-like receptor 4 (TLR4), further serving as a biomarker for inflammatory lung disorders such as acute respiratory distress syndrome. In contrast, intracellular NAMPT is involved in nicotinamide mononucleotide synthesis and has been implicated in the regulation of cellular apoptosis, although the exact mechanisms for this regulation are poorly understood. We examined the role of NAMPT in TNF-α-induced human lung endothelial cell (EC) apoptosis and demonstrated that reduced NAMPT expression (siRNA) increases EC susceptibility to TNF-α-induced apoptosis as reflected by PARP-1 cleavage and caspase-3 activation. In contrast, overexpression of NAMPT served to reduce degrees of TNF-α-induced EC apoptosis. Inhibition of nicotinamide mononucleotide synthesis by FK866 (a selective NAMPT enzymatic inhibitor) failed to alter TNF-α-induced human lung EC apoptosis, suggesting that NAMPT-dependent NAD+ generation is unlikely to be involved in regulation of TNF-α-induced EC apoptosis. We next confirmed that TNF-α-induced EC apoptosis is attributable to NAMPT secretion into the EC culture media and subsequent eNAMPT ligation of TLR4 on the EC membrane surface. Silencing of NAMPT expression, direct neutralization of secreted eNAMPT by an NAMPT-specific polyclonal antibody (preventing TLR4 ligation), or direct TLR4 antagonism all served to significantly increase EC susceptibility to TNF-α-induced EC apoptosis. Together, these studies provide novel insights into NAMPT contributions to lung inflammatory events and to novel mechanisms of EC apoptosis regulation.

Thrombin stimulates increased plasminogen activator inhibitor-1 release from liver compared to lung endothelium

BackgroundPlasminogen activator inhibitor-1 (PAI-1) is a major regulator of the fibrinolytic system, covalently binding to tissue plasminogen activator and blocking its activity. Fibrinolysis shutdown is evident in the majority of severely injured patients in the first 24 h and is thought to be due to PAI-1. The source of this PAI-1 is thought to be predominantly endothelial cells, but there are known organ-specific differences, with higher levels thought to be in the liver. Thrombin generation is also elevated in injured patients and is a potent stimulus for PAI-1 release in human umbilical endothelial cells. We hypothesize that thrombin induces liver endothelial cells to release increased amounts of PAI-1, versus pulmonary endothelium, consisting of both stored PAI-1 and a larger contribution from de novo PAI-1 synthesis. MethodsHuman liver sinusoidal endothelial cells (LSECs) and human microvascular lung endothelial cells (HMVECs) were stimulated in vitro ± thrombin (1 and 5 IU/mL) for 15-240 min, the supernatants were collected, and PAI-1 was measured by enzyme-linked immunosorbent assays. To elucidate the PAI-1 contribution from storage versus de novo synthesis, cycloheximide (10 μg/mL) was added before thrombin in separate experiments. ResultsWhile both LSECs and HMVECs rapidly stimulated PAI-1 release, LSECs released more PAI-1 than HMVECs in response to high-dose thrombin, whereas low-dose thrombin did not provoke immediate release. LSECs continued to release PAI-1 over the ensuing 240 min, whereas HMVECs did not. Cycloheximide did not inhibit early PAI-1 release from LSECs but did at the later time points (30-240 min). ConclusionsThrombin elicits increased amounts of PAI-1 release from liver endothelium compared with lung, with a small presynthesized stored contribution and a later, larger increase in PAI-1 release via de novo synthesis. This study suggests that the liver may be an important therapeutic target for inhibition of the hypercoagulable surgical patient and the associated complications that result.

Sphingolipid regulation of lung epithelial cell mitophagy and necroptosis during cigarette smoke exposure.

The mechanisms by which lung structural cells survive toxic exposures to cigarette smoke (CS) are not well defined but may involve proper disposal of damaged mitochondria by macro-autophagy (mitophagy), processes that may be influenced by pro-apoptotic ceramide (Cer) or its precursor dihydroceramide (DHC). Human lung epithelial and endothelial cells exposed to CS exhibited mitochondrial damage, signaled by phosphatase and tensin homolog-induced putative kinase 1 (PINK1) phosphorylation, autophagy, and necroptosis. Although cells responded to CS by rapid inhibition of DHC desaturase, which elevated DHC levels, palmitoyl (C16)-Cer also increased in CS-exposed cells. Whereas DHC augmentation triggered autophagy without cell death, the exogenous administration of C16-Cer was sufficient to trigger necroptosis. Inhibition of Cer-generating acid sphingomyelinase reduced both CS-induced PINK1 phosphorylation and necroptosis. When exposed to CS, Pink1-deficient ( Pink1-/-) mice, which are protected from airspace enlargement compared with wild-type littermates, had blunted C16-Cer elevations and less lung necroptosis. CS-exposed Pink1-/- mice also exhibited significantly increased levels of lignoceroyl (C24)-DHC, along with increased expression of Cer synthase 2 ( CerS2), the enzyme responsible for its production. This suggested that a combination of high C24-DHC and low C16-Cer levels might protect against CS-induced necroptosis. Indeed, CerS2-/- mice, which lack C24-DHC at the expense of increased C16-Cer, were more susceptible to CS, developing airspace enlargement following only 1 month of exposure. These results implicate DHCs, in particular, C24-DHC, as protective against CS toxicity by enhancing autophagy, whereas C16-Cer accumulation contributes to mitochondrial damage and PINK1-mediated necroptosis, which may be amplified by the inhibition of C24-DHC-producing CerS2.-Mizumura, K., Justice, M. J., Schweitzer, K. S., Krishnan, S., Bronova, I., Berdyshev, E. V., Hubbard, W. C., Pewzner-Jung, Y., Futerman, A. H., Choi, A. M. K., Petrache, I. Sphingolipid regulation of lung epithelial cell mitophagy and necroptosis during cigarette smoke exposure.

Vasculotide reduces pulmonary hyperpermeability in experimental pneumococcal pneumonia

BackgroundCommunity-acquired pneumonia (CAP) is a significant cause of morbidity and mortality worldwide. Despite effective antimicrobial therapy, CAP can induce pulmonary endothelial hyperpermeability resulting in life-threatening lung failure due to an exaggerated host-pathogen interaction. Treatment of acute lung injury is mainly supportive because key elements of inflammation-induced barrier disruption remain undetermined. Angiopoietin-1 (Ang-1)-mediated Tie2 activation reduces, and the Ang-1 antagonist Ang-2 increases, inflammation and endothelial permeability in sepsis. Vasculotide (VT) is a polyethylene glycol-clustered Tie2-binding peptide that mimics the actions of Ang-1. The aim of our study was to experimentally test whether VT is capable of diminishing pneumonia-induced lung injury.MethodsVT binding and phosphorylation of Tie2 were analyzed using tryptophan fluorescence spectroscopy and phospho-Tie-2 enzyme-linked immunosorbent assay. Human and murine lung endothelial cells were investigated by immunofluorescence staining and electric cell-substrate impedance sensing. Pulmonary hyperpermeability was quantified in VT-pretreated, isolated, perfused, and ventilated mouse lungs stimulated with the pneumococcal exotoxin pneumolysin (PLY). Furthermore, Streptococcus pneumoniae-infected mice were therapeutically treated with VT.ResultsVT showed dose-dependent binding and phosphorylation of Tie2. Pretreatment with VT protected lung endothelial cell monolayers from PLY-induced disruption. In isolated mouse lungs, VT decreased PLY-induced pulmonary permeability. Likewise, therapeutic treatment with VT of S. pneumoniae-infected mice significantly reduced pneumonia-induced hyperpermeability. However, effects by VT on the pulmonary or systemic inflammatory response were not observed.ConclusionsVT promoted pulmonary endothelial stability and reduced lung permeability in different models of pneumococcal pneumonia. Thus, VT may provide a novel therapeutic perspective for reduction of permeability in pneumococcal pneumonia-induced lung injury.

The Splicing Factor hnRNPA1 Regulates Alternate Splicing of the MYLK Gene.

Profound lung vascular permeability is a cardinal feature of acute respiratory distress syndrome (ARDS) and ventilator-induced lung injury (VILI), two syndromes known to centrally involve the nonmuscle isoform of myosin light chain kinase (nmMLCK) in vascular barrier dysregulation. Two main splice variants, nmMLCK1 and nmMLCK2, are well represented in human lung endothelial cells and encoded by MYLK, and they differ only in the presence of exon 11 in nmMLCK1, which contains critical phosphorylation sites (Y464 and Y471) that influence nmMLCK enzymatic activity, cellular translocation, and localization in response to vascular agonists. We recently demonstrated the functional role of SNPs in altering MYLK splicing, and in the present study we sought to identify the role of splicing factors in the generation of nmMLCK1 and nmMLCK2 spliced variants. Using bioinformatic in silico approaches, we identified a putative binding site for heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), a recognized splicing factor. We verified hnRNPA1 binding to MYLK by gel shift analyses and that hnRNPA1 gene and protein expression is upregulated in mouse lungs obtained from preclinical models of ARDS and VILI and in human endothelial cells exposed to 18% cyclic stretch, a model that reproduces the excessive mechanical stress observed in VILI. Using an MYLK minigene approach, we established a direct role of hnRNPA1 in MYLK splicing and in the context of 18% cyclic stretch. In summary, these data indicate an important regulatory role for hnRNPA1 in MYLK splicing, and they increase understanding of MYLK splicing in the regulation of lung vascular integrity during acute lung inflammation and excessive mechanical stress, such as that observed in ARDS and VILI.

ITE inhibits growth of human pulmonary artery endothelial cells

ABSTRACTAim: Pulmonary arterial hypertension (PAH), a deadly disorder is associated with excessive growth of human pulmonary artery endothelial (HPAECs) and smooth muscle (HPASMCs) cells. Current therapies primarily aim at promoting vasodilation, which only ameliorates clinical symptoms without a cure. 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) is an endogenous aryl hydrocarbon receptor (AhR) ligand, and mediates many cellular function including cell growth. However, the roles of ITE in human lung endothelial cells remain elusive. Herein, we tested a hypothesis that ITE inhibits growth of human pulmonary artery endothelial cells via AhR. Materials and Methods: Immunohistochemistry was performed to localize AhR expression in human lung tissues. The crystal violet method and MTT assay were used to determine ITE's effects on growth of HPAECs. The AhR activation in HPAECs was confirmed using Western blotting and RT-qPCR. The role of AhR in ITE-affected proliferation of HPAECs was assessed using siRNA knockdown method followed by the crystal violet method. Results: Immunohistochemistry revealed that AhR was present in human lung tissues, primarily in endothelial and smooth muscle cells of pulmonary veins and arteries, as well as in bronchial and alveolar sac epithelia. We also found that ITE dose- and time-dependently inhibited proliferation of HPAECs with a maximum inhibition of 83% at 20 µM after 6 days of treatment. ITE rapidly decreased AhR protein levels, while it increased mRNA levels of cytochrome P450 (CYP), family 1, member A1 (CYP1A1) and B1 (CYP1B1), indicating activation of the AhR/CYP1A1 and AhR/CYP1B1 pathways in HPAECs. The AhR siRNA significantly suppressed AhR protein expression, whereas it did not significantly alter ITE-inhibited growth of HPAECs. Conclusions: ITE suppresses growth of HPAECs independent of AhR, suggesting that ITE may play an important role in preventing excessive growth of lung endothelial cells.

Loss of Syndecan-1 Abrogates the Pulmonary Protective Phenotype Induced by Plasma After Hemorrhagic Shock.

Syndecan-1 (Sdc1) is considered a biomarker of injury to the endothelial glycocalyx following hemorrhagic shock, with shedding of Sdc1 deleterious. Resuscitation with fresh frozen plasma (FFP) has been correlated with restitution of pulmonary Sdc1 and reduction of lung injury, but the precise contribution of Sdc1 to FFPs protection in the lung remains unclear. Human lung endothelial cells were used to assess the time and dose-dependent effect of FFP on Sdc1 expression and the effect of Sdc1 silencing on in vitro endothelial cell permeability and actin stress fiber formation. Wild-type and Sdc1 mice were subjected to hemorrhagic shock followed by resuscitation with lactated Ringers (LR) or FFP and compared with shock alone and shams. Lungs were harvested after 3 h for analysis of permeability, histology, and inflammation and for measurement of syndecan- 2 and 4 expression. In vitro, FFP enhanced pulmonary endothelial Sdc1 expression in time- and dose-dependent manners and loss of Sdc1 in pulmonary endothelial cells worsened permeability and stress fiber formation by FFP. Loss of Sdc1 in vivo led to equivalency between LR and FFP in restoring pulmonary injury, inflammation, and permeability after shock. Lastly, Sdc1 mice demonstrated a significant increase in pulmonary syndecan 4 expression after hemorrhagic shock and FFP-based resuscitation. Taken together, our findings support a key role for Sdc1 in modulating pulmonary protection by FFP after hemorrhagic shock. Our results also suggest that other members of the syndecan family may at least be contributing to FFP's effects on the endothelium, an area that warrants further investigation.

A microengineered model of RBC transfusion-induced pulmonary vascular injury

Red blood cell (RBC) transfusion poses significant risks to critically ill patients by increasing their susceptibility to acute respiratory distress syndrome. While the underlying mechanisms of this life-threatening syndrome remain elusive, studies suggest that RBC-induced microvascular injury in the distal lung plays a central role in the development of lung injury following blood transfusion. Here we present a novel microengineering strategy to model and investigate this key disease process. Specifically, we created a microdevice for culturing primary human lung endothelial cells under physiological flow conditions to recapitulate the morphology and hemodynamic environment of the pulmonary microvascular endothelium in vivo. Perfusion of the microengineered vessel with human RBCs resulted in abnormal cytoskeletal rearrangement and release of intracellular molecules associated with regulated necrotic cell death, replicating the characteristics of acute endothelial injury in transfused lungs in vivo. Our data also revealed the significant effect of hemodynamic shear stress on RBC-induced microvascular injury. Furthermore, we integrated the microfluidic endothelium with a computer-controlled mechanical stretching system to show that breathing-induced physiological deformation of the pulmonary microvasculature may exacerbate vascular injury during RBC transfusion. Our biomimetic microsystem provides an enabling platform to mechanistically study transfusion-associated pulmonary vascular complications in susceptible patient populations.

PEGylation of the GALA Peptide Enhances the Lung-Targeting Activity of Nanocarriers That Contain Encapsulated siRNA

A α-helical GALA peptide (WEAALAEALAEALAEHLAEALAEALEALAA) has been found to possess dual functions: a pH-dependent inducer of endosomal escape, and a ligand that targets lung endothelium. In the present study, the flexibility of GALA was improved by modifying the edge with polyethylene glycol linker, to increase lung-targeting activity. We first investigated the uptake of the GALA-modified liposomes in which GALA was directly conjugated to the lipid (Cholesterol: GALA/Chol) or the phospholipid-PEG (GALA/PEG2000). The liposomes that were modified with GALA/PEG2000 (GALA/PEG2000-LPs) were taken up at a higher level by human lung endothelial cells (HMVEC-L), in comparison with particles that were modified with GALA/Chol (GALA/Chol-LPs). Small-interfering RNA–encapsulating liposomal-based nanocarriers (multifunctional envelope-type nano device: MEND) that were formulated with a vitamin E-scaffold SS-cleavable pH-activated lipid-like material, namely GALA/PEG2000-MENDssPalmE were also modified with GALA/PEG2000. Gene silencing activity in the lung endothelium was then evaluated against an endothelial marker; CD31. In comparison with the unmodified MENDssPalmE, GALA/PEG2000-MENDssPalmE exhibited a higher silencing activity in the lung. Optimization of GALA/PEG2000-MENDssPalmE resulted in silencing activity in the lung with an ED50 value of 0.21 mg/kg, while non-specific gene silencing in liver was marginal. Collectively, PEGylated GALA is a promising device for use in targeting the lung endothelium.

Memantine, the dual α7‐nAChR/NMDAR antagonist displays potent anti‐angiogenic activity in lung cancer

Non‐small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) bear a strong etiological association with smoking habits. Nicotine, the addictive component of cigarettes, promotes angiogenesis in lung cancer via the α7‐nicotinic acetylcholine receptor (α7‐nAChR) subunit in human lung endothelial cells. Therefore, we conjectured that α7‐nAChR‐antagonists should display potent anti‐angiogenic and antitumor activity in lung cancers. Memantine is a dual α7‐nAChR/NMDAR antagonist, which is used in the clinic for the treatment of patients suffering from mild‐to‐moderate Alzheimer's disease. Receptor binding assays have shown that the affinity of Memantine for α7‐nAChR is greater than NMDAR. Here we show that memantine attenuates nicotine‐induced angiogenesis in human microvascular endothelial cells of the lung (HMEC‐Ls). Most interestingly, the levels of α7‐nAChR in tumor‐associated endothelial cells was greater than normal lung endothelial cells. The anti‐angiogenetic activity of memantine was mediated by the α7‐nAChR (and not by NMDARs) on lung endothelial cells. Furthermore, the α7‐nAChR antagonist memantine displayed potent anti‐angiogenic activity in the chicken chorioallantoic membrane (CAM) model. Our studies show that α7‐nAChR antagonists may be useful anti‐tumor agents relevant for the treatment of human lung cancer.Support or Funding InformationFunding for our study was supported by the an NIH R15‐AREA Grant (2R15CA161491‐02), WVU‐MU Health Partnership Grant and by ACTSI Grant from Marshall University

Simvastatin-induced sphingosine 1-phosphate receptor 1 expression is KLF2-dependent in human lung endothelial cells.

We have demonstrated that simvastatin and sphingosine 1−phosphate (S1P) both attenuate increased vascular permeability in preclinical models of acute respiratory distress syndrome. However, the underlying mechanisms remain unclear. As Krüppel-like factor 2 (KLF2) serves as a critical regulator for cellular stress response in endothelial cells (EC), we hypothesized that simvastatin enhances endothelial barrier function via increasing expression of the barrier-promoting S1P receptor, S1PR1, via a KLF2-dependent mechanism. S1PR1 luciferase reporter promoter activity in human lung artery EC (HPAEC) was tested after simvastatin (5 μM), and S1PR1 and KLF2 protein expression detected by immunoblotting. In vivo, transcription and expression of S1PR1 and KLF2 in mice lungs were detected by microarray profiling and immunoblotting after exposure to simvastatin (10 mg/kg). Endothelial barrier function was measured by trans-endothelial electrical resistance with the S1PR1 agonist FTY720-(S)-phosphonate. Both S1PR1 and KLF2 gene expression (mRNA, protein) were significantly increased by simvastatin in vitro and in vivo. S1PR1 promoter activity was significantly increased by simvastatin (P < 0.05), which was significantly attenuated by KLF2 silencing (siRNA). Simvastatin induced KLF2 recruitment to the S1PR1 promoter, and consequently, significantly augmented the effects of the S1PR1 agonist on EC barrier enhancement (P < 0.05), which was significantly attenuated by KLF2 silencing (P < 0.05). These results suggest that simvastatin upregulates S1PR1 transcription and expression via the transcription factor KLF2, and consequently augments the effects of S1PR1 agonists on preserving vascular barrier integrity. These results may lead to novel combinatorial therapeutic strategies for lung inflammatory syndromes.

Mechanical Stress and Single Nucleotide Variants Regulate Alternative Splicing of the MYLK Gene.

The nonmuscle (nm) myosin light-chain kinase isoform (MLCK), encoded by the MYLK gene, is a vital participant in regulating vascular barrier responses to mechanical and inflammatory stimuli. We determined that MYLK is alternatively spliced, yielding functionally distinct nmMLCK splice variants including nmMLCK2, a splice variant highly expressed in vascular endothelial cells (EC) and associated with reduced EC barrier integrity. We demonstrated previously that the nmMLCK2 variant lacks exon 11, which encodes a key regulatory region containing two differentially phosphorylated tyrosine residues (Y464 and Y471) that influence vascular barrier function during inflammation. In this study, we used minigene constructs and RT-PCR to interrogate biophysical factors (mechanical stress) and genetic variants (MYLK single-nucleotide polymorphisms [SNPs]) that are potentially involved in regulating MYLK alternative splicing and nmMLCK2 generation. Human lung EC exposed to pathologic mechanical stress (18% cyclic stretch) produced increased nmMLCK2 expression relative to levels of nmMLCK1 with alternative splicing significantly influenced by MYLK SNPs rs77323602 and rs147245669. In silico analyses predicted that these variants would alter exon 11 donor and acceptor sites for alternative splicing, computational predictions that were confirmed by minigene studies. The introduction of rs77323602 favored wild-type nmMLCK expression, whereas rs147245669 favored alternative splicing and deletion of exon 11, yielding increased nmMLCK2 expression. Finally, lymphoblastoid cell lines selectively harboring these MYLK SNPs (rs77323602 and rs147245669) directly validated SNP-specific effects on MYLK alternative splicing and nmMLCK2 generation. Together, these studies demonstrate that mechanical stress and MYLK SNPs regulate MYLK alternative splicing and generation of a splice variant, nmMLCK2, that contributes to the severity of inflammatory injury.

Endocytic Pathways Used by Andes Virus to Enter Primary Human Lung Endothelial Cells.

Andes virus (ANDV) is the major cause of hantavirus pulmonary syndrome (HPS) in South America. Despite a high fatality rate (up to 40%), no vaccines or antiviral therapies are approved to treat ANDV infection. To understand the role of endocytic pathways in ANDV infection, we used 3 complementary approaches to identify cellular factors required for ANDV entry into human lung microvascular endothelial cells. We screened an siRNA library targeting 140 genes involved in membrane trafficking, and identified 55 genes required for ANDV infection. These genes control the major endocytic pathways, endosomal transport, cell signaling, and cytoskeleton rearrangement. We then used infectious ANDV and retroviral pseudovirions to further characterize the possible involvement of 9 of these genes in the early steps of ANDV entry. In addition, we used markers of cellular endocytosis along with chemical inhibitors of known endocytic pathways to show that ANDV uses multiple routes of entry to infect target cells. These entry mechanisms are mainly clathrin-, dynamin-, and cholesterol-dependent, but can also occur via a clathrin-independent manner.

Parkin regulates lipopolysaccharide-induced proinflammatory responses in acute lung injury

The acute respiratory distress syndrome (ARDS) is a serious condition resulting from direct or indirect lung injury that is associated with high mortality and morbidity. A key biological event in the pathogenesis of the acute lung injury (ALI) that causes acute respiratory distress syndrome is activation of the lung endothelium cells (ECs), which is triggered by a variety of inflammatory insults leading to barrier disruption and excessive accumulation of neutrophils. Recently, we demonstrated that imatinib protects against lipopolysaccharide (LPS)-induced EC activation by inhibiting c-Abl kinase. In the present study, we explored the role of parkin, a novel c-Abl substrate, in ALI. Parkin is an E3 ubiquitin ligase originally characterized in the pathogenesis of Parkinson disease; however, its potential role in acute inflammatory processes and lung EC function remains largely unknown. Using parkin deficient (PARK2-/-) mice, we now demonstrate that parkin mediates LPS-induced ALI. After LPS, PARK2-/- mice have reduced total protein and cell levels in bronchoalveolar lavage (BAL) compared to wild type. Moreover, in LPS-treated PARK2-/- lungs, the sequestration and activation of neutrophils and release of inflammatory cytokines (interleukin 6 [IL-6], tumor necrosis factor alpha [TNF-α]) are significantly reduced. The BAL levels of soluble VCAM-1 and ICAM-1 are also decreased in LPS-treated PARK2-/- mice compared to wild type. In cultured human lung endothelial cells, downregulation of parkin by small interfering RNA decreases LPS-induced VCAM-1 expression, IL-8 and IL-6 secretion, and NF-kB phosphorylation. These results suggest a previously unidentified role of parkin in mediating endotoxin-induced endothelial proinflammatory signaling and indicate that it may play a critical role in acute inflammation.

Selective HDAC6 inhibition prevents TNF-α-induced lung endothelial cell barrier disruption and endotoxin-induced pulmonary edema.

Lung endothelial damage contributes to the pathogenesis of acute lung injury. New strategies against lung endothelial barrier dysfunction may provide therapeutic benefits against lung vascular injury. Cell-cell junctions and microtubule cytoskeleton are basic components in maintaining endothelial barrier integrity. HDAC6, a deacetylase primarily localized in the cytoplasm, has been reported to modulate nonnuclear protein function through deacetylation. Both α-tubulin and β-catenin are substrates for HDAC6. Here, we examined the effects of tubastatin A, a highly selective HDAC6 inhibitor, on TNF-α induced lung endothelial cell barrier disruption and endotoxin-induced pulmonary edema. Selective HDAC6 inhibition by tubastatin A blocked TNF-α-induced lung endothelial cell hyperpermeability, which was associated with increased α-tubulin acetylation and microtubule stability. Tubastatin A pretreatment inhibited TNF-α-induced endothelial cell contraction and actin stress fiber formation with reduced myosin light chain phosphorylation. Selective HDAC6 inhibition by tubastatin A also induced β-catenin acetylation in human lung endothelial cells, which was associated with increased membrane localization of β-catenin and stabilization of adherens junctions. HDAC6 knockdown by small interfering RNA also prevented TNF-α-induced barrier dysfunction and increased α-tubulin and β-catenin acetylation in endothelial cells. Furthermore, in a mouse model of endotoxemia, tubastatin A was able to prevent endotoxin-induced deacetylation of α-tubulin and β-catenin in lung tissues, which was associated with reduced pulmonary edema. Collectively, our data indicate that selective HDAC6 inhibition by tubastatin A is a potent approach against lung endothelial barrier dysfunction.