Whole-transcriptome analyses of ovine lung microvascular endothelial cells infected with bluetongue virus.

Bluetongue virus (BTV) infection induces profound and intricate changes in the transcriptional profile of the host to facilitate its survival and replication. However, there have been no whole-transcriptome studies on ovine lung microvascular endothelial cells (OLMECs) infected with BTV. In this study, we comprehensively analysed the whole-transcriptome sequences of BTV-1 serotype-infected and mock-infected OLMECs and subsequently performed bioinformatics differential analysis. Our analysis revealed 1215 differentially expressed mRNA transcripts, 82 differentially expressed long noncoding RNAs (lncRNAs) transcripts, 63 differentially expressed microRNAs (miRNAs) transcripts, and 42 differentially expressed circular RNAs (circRNAs) transcripts. Annotation from Gene Ontology, enrichment from the Kyoto Encyclopedia of Genes and Genomes, and construction of endogenous competing RNA network analysis revealed that the differentially expressed RNAs primarily participated in viral sensing and signal transduction pathways, antiviral and immune responses, inflammation, and extracellular matrix (ECM)-related pathways. Furthermore, protein‒protein interaction network analysis revealed that BTV may regulate the conformation of ECM receptor proteins and change their biological activity through a series of complex mechanisms. Finally, on the basis of real-time fluorescence quantitative polymerase chain reaction results, the expression trends of the differentially expressed RNA were consistent with the whole-transcriptome sequencing data, such as downregulation of the expression of COL4A1, ITGA8, ITGB5, and TNC and upregulation of the expression of CXCL10, RNASEL, IRF3, IRF7, and IFIHI. This study provides a novel perspective for further investigations of the mechanism of the ECM in the BTV-host interactome and the pathogenesis of lung microvascular endothelial cells.

Local Complement Contributes to Pathogenic Activation of Lung Endothelial Cells in SARS-CoV-2 Infection.

Endothelial dysfunction and inflammation contribute to the vascular pathology of coronavirus disease (COVID-19). However, emerging evidence does not support direct infection of endothelial or other vascular wall cells, and thus inflammation may be better explained as a secondary response to epithelial cell infection. In this study, we sought to determine whether lung endothelial or other resident vascular cells are susceptible to productive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and how local complement activation contributes to endothelial dysfunction and inflammation in response to hypoxia and SARS-CoV-2-infected lung alveolar epithelial cells. We found that ACE2 (angiotensin-converting enzyme 2) and TMPRSS2 (transmembrane serine protease 2) mRNA expression in lung vascular cells, including primary human lung microvascular endothelial cells (HLMVECs), pericytes, smooth muscle cells, and fibroblasts, was 20- to 90-fold lower compared with primary human alveolar epithelial type II cells. Consistently, we found that HLMVECs and other resident vascular cells were not susceptible to productive SARS-CoV-2 infection under either normoxic or hypoxic conditions. However, viral uptake without replication (abortive infection) was observed in HLMVECs when exposed to conditioned medium from SARS-CoV-2-infected human ACE2 stably transfected A549 epithelial cells. Furthermore, we demonstrated that exposure of HLMVECs to conditioned medium from SARS-CoV-2-infected human ACE2 stably transfected A549 epithelial cells and hypoxia resulted in upregulation of inflammatory factors such as ICAM-1 (intercellular adhesion molecule 1), VCAM-1 (vascular cell adhesion molecule 1), and IL-6 (interleukin 6) as well as complement components such as C3 (complement C3), C3AR1 (complement C3a receptor 1), C1QA (complement C1q A chain), and CFB (complement factor B). Taken together, our data support a model in which lung endothelial and vascular dysfunction during COVID-19 involves the activation of complement and inflammatory signaling and does not involve productive viral infection of endothelial cells.

c-Jun-mediated miR-19b expression induces endothelial barrier dysfunction in an in vitro model of hemorrhagic shock.

BACKGROUND: Our previous data demonstrated that miR-19b expression was increased in human lung microvascular endothelial cells in-vitro-, in-vivo and in patients with hemorrhagic shock, leading to a decrease in syndecan-1 mRNA and protein and resulting in loss of endothelial barrier function. However, the mechanism underlying increased miR-19b expression remains unclear. The objective of the current study was to determine if c-Jun mediates the early responsive microRNA, miR-19b, to cause endothelial barrier dysfunction. METHOD: Human lung microvascular endothelial cells (HLMEC) or HEK293T cells were transfected with c-Jun overexpressing vector, c-Jun siRNA, miR-19b promoter vector, miR-19b mutated promoter vector, miR-19b oligo inhibitor, then subjected to hypoxia/reoxygenation as in-vitro model of hemorrhagic shock. Levels of protein, miRNA, and luciferase activity were measured. Transwell permeability of endothelial monolayers were also determined. Plasma levels of c-Jun were measured in injured patients with hemorrhagic shock. RESULT: Hypoxia/reoxygenation induced primary (pri-)miR-19b, mature miR-19b, and c-Jun expression over time in a comparable timeframe. c-Jun silencing by transfection with its specific siRNA reduced both pri-miR-19b and mature miR-19b levels. Conversely, c-Jun overexpression enhanced H/R-induced pri-miR-19b. Studies using a luciferase reporter assay revealed that in cells transfected with vectors containing the wild-type miR-19b promoter and luciferase reporter, c-Jun overexpression or hypoxia/ reoxygenation significantly increased luciferase activity. c-Jun knockdown reduced the luciferase activity in these cells, suggesting that the miR-19b promoter is directly activated by c-Jun. Further, chromatin immunoprecipitation assay confirmed that c-Jun directly bound to the promoter DNA of miR-19b and hypoxia/reoxygenation significantly increased this interaction. Additionally, c-Jun silencing prevented cell surface syndecan-1 loss and endothelial barrier dysfunction in HLMECs after hypoxia/reoxygenation. Lastly, c-Jun was significantly elevated in patients with hemorrhagic shock compared to healthy controls. CONCLUSION: Transcription factor c-Jun is inducible by hypoxia/reoxygenation, binds to and activates the miR-19b promoter. Using an in-vitro model of hemorrhagic shock, our findings identified a novel cellular mechanism whereby hypoxia/ reoxygenation increases miR-19b transcription by inducing c-Jun and leads to syndecan-1 decrease and endothelial cell barrier dysfunction. This finding supports that miR-19b could be a potential therapeutic target for hemorrhage shock.

A nonapoptotic endothelial barrier-protective role for caspase-3.

Noncanonical roles for caspase-3 are emerging in the fields of cancer and developmental biology. However, little is known of nonapoptotic functions of caspase-3 in most cell types. We have recently demonstrated a disassociation between caspase-3 activation and execution of apoptosis with accompanying cytoplasmic caspase-3 sequestration and preserved endothelial barrier function. Therefore, we tested the hypothesis that nonapoptotic caspase-3 activation promotes endothelial barrier integrity. Human lung microvascular endothelial cells were exposed to thrombin, a nonapoptotic stimulus, and endothelial barrier function was assessed using electric cell-substrate impedance sensing. Actin cytoskeletal rearrangement and paracellular gap formation were assessed using phalloidin staining. Cell stiffness was evaluated using magnetic twisting cytometry. In addition, cell lysates were harvested for protein analyses. Caspase-3 was inhibited pharmacologically with pan-caspase and a caspase-3-specific inhibitor. Molecular inhibition of caspase-3 was achieved using RNA interference. Cells exposed to thrombin exhibited a cytoplasmic activation of caspase-3 with transient and nonapoptotic decrease in endothelial barrier function as measured by a drop in electrical resistance followed by a rapid recovery. Inhibition of caspases led to a more pronounced and rapid drop in thrombin-induced endothelial barrier function, accompanied by increased endothelial cell stiffness and paracellular gaps. Caspase-3-specific inhibition and caspase-3 knockdown both resulted in more pronounced thrombin-induced endothelial barrier disruption. Taken together, our results suggest cytoplasmic caspase-3 has nonapoptotic functions in human endothelium and can promote endothelial barrier integrity.

Unfractionated Heparin Alleviates Sepsis-Induced Acute Lung Injury by Protecting Tight Junctions.

BACKGROUND: Unfractionated heparin (UFH) has been shown to ameliorate lung edema and lung vascular leakage in lipopolysaccharide-induced lung injury. Impaired tight junction (TJ) function is a sign of sepsis-induced acute respiratory distress syndrome (ARDS) and acute lung injury (ALI), which is closely related to the downregulated expression of TJ-specific proteins or the upregulated expression of inflammatory cytokines. Because UFH has been intensively studied in modulating inflammation, we hypothesize that UFH may play a positive role in treating sepsis-induced ARDS/ALI by protecting TJs. MATERIAL AND METHODS: Rat sepsis-induced lung injury was induced by cecal ligation and puncture and treated with UFH. Hematoxylin and eosin staining, lung wet/dry weight (W/D) ratio, and pulmonary microvascular leakage were evaluated to assess lung injury. Cytokines in bronchoalveolar lavage fluid were detected to determine lung inflammation. A transendothelial electrical resistance assay, a Transwell permeability assay, and transmission electron microscopy were used to study endothelial TJs in human lung microvascular endothelial cells. TJ protein expression was measured by western blotting or immunohistochemistry. RESULTS: UFH treatment alleviated lung injury in vivo by reducing IL-6 in bronchoalveolar lavage fluid and protecting TJs in LMVECs. UFH also protected TJs against lipopolysaccharide-stimulated damage and functioned upstream by inhibiting the ERK1/2 MAPK pathway to attenuate endothelial hyperpermeability and downregulating the expression of TJ proteins such as claudin-5, occludin, and ZO-1. CONCLUSIONS: These findings suggested that UFH has therapeutic potential for sepsis-induced ARDS or ALI through protecting TJs in LMVECs.

Sevoflurane prevents lipopolysaccharide-induced barrier dysfunction in human lung microvascular endothelial cells: Rho-mediated alterations of VE-cadherin.

Acute lung injury (ALI) mainly occurs as increased permeability of lung tissue and pleural effusion. Inhaled anesthetic sevoflurane has been demonstrated to alleviate lung permeability by upregulating junction proteins after ischemia-reperfusion. However, the exact mechanisms of its protective effect on reperfusion injury remain elusive. The aim of this study was to assess possible preconditioning with sevoflurane in an in vitro model of lipopolysaccharide (LPS)-induced barrier dysfunction in human lung microvascular endothelial cells (HMVEC-Ls). In this study, HMVEC-Ls were exposed to minimum alveolar concentration of sevoflurane for 2 h. LPS significantly increased the permeability of HMVEC-L. Moreover, the distribution of junction protein, vascular endothelial (VE)-cadherin, in cell-cell junction area and the total expression in HMVEC-Ls were significantly decreased by LPS treatment. However, the abnormal distribution and decreased expression of VE-cadherin and hyperpermeability of HMVEC-Ls were significantly reversed by pretreatment with sevoflurane. Furthermore, LPS-induced activation of the RhoA/ROCK signaling pathway was significantly inhibited with sevoflurane. Such activation, abnormal distribution and decreased expression of VE-cadherin and hyperpermeability of HMVEC-Ls were significantly inhibited with sevoflurane pretreatment or knockdown of RhoA or ROCK-2. In conclusion, sevoflurane prevented LPS-induced rupture of HMVEC-L monolayers by suppressing the RhoA/ROCK-mediated VE-cadherin signaling pathway. Our results may explain, at least in part, some beneficial effects of sevoflurane on pulmonary dysfunction such as ischemia-reperfusion injury.

LPA2 Alleviates Septic Acute Lung Injury via Protective Endothelial Barrier Function Through Activation of PLC-PKC-FAK.

Background: Increased endothelial permeability of pulmonary vessels is a primary pathological characteristic of septic acute lung injury (ALI). Previously, elevated lysophosphatidic acid (LPA) levels and LPA2 (an LPA receptor) expression have been found in the peripheral blood and lungs of septic mice, respectively. However, the specific role of LPA2 in septic ALI remains unclear. Methods: A lipopolysaccharide (LPS)-induced model of sepsis was established in wild-type (WT) and global LPA2 knockout (Lpar2-/-) mice. We examined mortality, lung injury, assessed endothelial permeability through Evans blue dye (EBD) assay in vivo, and transendothelial electrical resistance (TEER) of mouse lung microvascular endothelial cells (MLMECs) in vitro. Enzyme-linked immunosorbent assay (ELISA), histopathological, immunofluorescence, immunohistochemistry, and Western blot were employed to investigate the role of LPA2 in septic ALI. Results: Lpar2 deficiency increased vascular endothelial permeability, impaired lung injury, and increased mortality. Histological examination revealed aggravated inflammation, edema, hemorrhage and alveolar septal thickening in the lungs of septic Lpar2-/- mice. In vitro, loss of Lpar2 resulted in increased permeability of MLMECs. Pharmacological activation of LPA2 by the agonist DBIBB led to significantly reduced inflammation, edema and hemorrhage, as well as increased expression of the vascular endothelial tight junction (TJ) protein zonula occludens-1 (ZO-1) and claudin-5, as well as the adheren junction (AJ) protein VE-cadherin. Moreover, DBIBB treatment was found to alleviate mortality by protecting against vascular endothelial permeability. Mechanistically, we demonstrated that vascular endothelial permeability was alleviated through LPA-LPA2 signaling via the PLC-PKC-FAK pathway. Conclusion: These data provide a novel mechanism of endothelial barrier protection via PLC-PKC-FAK pathway and suggest that LPA2 may contribute to the therapeutic effects of septic ALI.

Modeling the Immune Response for Pathogenic and Nonpathogenic Orthohantavirus Infections in Human Lung Microvasculature Endothelial Cells.

Hantaviruses, genus Orthohantavirus, family Hantaviridae, order Bunyavirales, are negative-sense, single-stranded, tri-segmented RNA viruses that persistently infect rodents, shrews, and moles. Of these, only certain virus species harbored by rodents are pathogenic to humans. Infection begins with inhalation of virus particles into the lung and trafficking to the lung microvascular endothelial cells (LMVEC). The reason why certain rodent-borne hantavirus species are pathogenic has long been hypothesized to be related to their ability to downregulate and dysregulate the immune response as well as increase vascular permeability of infected endothelial cells. We set out to study the temporal dynamics of host immune response modulation in primary human LMVECs following infection by Prospect Hill (nonpathogenic), Andes (pathogenic), and Hantaan (pathogenic) viruses. We measured the level of RNA transcripts for genes representing antiviral, proinflammatory, anti-inflammatory, and metabolic pathways from 12 to 72 h with time points every 12 h. Gene expression analysis in conjunction with mathematical modeling revealed a similar profile for all three viruses in terms of upregulated genes that partake in interferon signaling (TLR3, IRF7, IFNB1), host immune cell recruitment (CXCL10, CXCL11, and CCL5), and host immune response modulation (IDO1). We examined secreted protein levels of IFN-ß, CXCL10, CXCL11, CCL5, and IDO in two male and two female primary HLMVEC donors at 48 and 60 h post infection. All three viruses induced similar levels of CCL5, CXCL10, and CXCL11 within a particular donor, and the levels were similar in three of the four donors. All three viruses induced different protein secretion levels for both IFN-ß and IDO and secretion levels differed between donors. In conclusion, we show that there was no difference in the transcriptional profiles of key genes in primary HLMVECs following infection by pathogenic and nonpathogenic hantaviruses, with protein secretion levels being more donor-specific than virus-specific.

Human PBMCs Form Lipid Droplets in Response to Spike Proteins.

Intracellular lipid droplets (LDs) can accumulate in response to inflammation, metabolic stresses, and other physiological/pathological processes. Herein, we investigated whether spike proteins of SARS-CoV-2 induce LDs in human peripheral blood mononuclear cells (PBMCs) and in pulmonary microvascular endothelial cells (HPMECs). PBMCs or HPMECs were incubated alone or with endotoxin-free recombinant variants of trimeric spike glycoproteins (Alpha, Beta, Delta, and Omicron, 12 µg/mL). Afterward, cells were stained with Oil Red O for LDs, cytokine release was determined through ELISA, and the gene expression was analyzed through real-time PCR using TaqMan assays. Our data show that spikes induce LDs in PBMCs but not in HPMECs. In line with this, in PBMCs, spike proteins lower the expression of genes involving lipid metabolism and LD formation, such as SREBF1, HMGCS1, LDLR, and CD36. On the other hand, PBMCs exposed to spikes for 6 or 18 h did not increase in IL-1ß, IL-6, IL-8, MCP-1, and TNFα release or expression as compared to non-treated controls. Thus, spike-induced LD formation in PBMCs seems to not be related to cell inflammatory activation. Further detailed studies are warranted to investigate in which specific immune cells spikes induce LDs, and what are the pathophysiological mechanisms and consequences of this induction in vivo.

Effects of Hyperoxia and Hyperoxic Oscillations on the Proteome of Murine Lung Microvascular Endothelium.

Patients presenting with insufficient tissue oxygenation and impaired lung function as in acute respiratory distress syndrome (ARDS) frequently require mechanical ventilation with supplemental oxygen. Despite the lung being used to experiencing the highest partial pressure of oxygen during healthy breathing, the organ is susceptible to oxygen-induced injury at supraphysiological concentrations. Hyperoxia-induced lung injury (HALI) has been regarded as a second hit to pre-existing lung injury and ventilator-induced lung injury (VILI) attributed to oxidative stress. The injured lung has a tendency to form atelectasis, a cyclic collapse and reopening of alveoli. The affected lung areas experience oxygen conditions that oscillate between hyperoxia and hypoxia rather than remaining in a constant hyperoxic state. Mechanisms of HALI have been investigated in many animal models previously. These studies provided insights into the effects of hyperoxia on the whole organism. However, cell type-specific responses have not been dissected in detail, but are necessary for a complete mechanistic understanding of ongoing pathological processes. In our study, we investigated the effects of constant and intermittent hyperoxia on the lung endothelium from a mouse by an in vitro proteomic approach. We demonstrate that these oxygen conditions have characteristic effects on the pulmonary endothelial proteome that underlie the physiological (patho)mechanisms.

miRNA expression in COVID-19.

Coronavirus disease 2019 (COVID-19) is regarded as a challenge in health system. Several studies have assessed the immune-related aspect of this disorder to identify the host-related factors that affect the course of COVID-19. microRNAs (miRNAs) as potent regulators of immune responses have gained much attention in this regard. Recent studies have shown aberrant expression of miRNAs in COVID-19 in association with disease course. Differentially expressed miRNAs have been enriched in pathways related with inflammation and antiviral immune response. miRNAs have also been regarded as potential therapeutic targets in COVID-19, particularly for management of pathological consequences of COVID-19. In the current review, we summarize the data about dysregulation of miRNAs in COVID-19.

Monocytes promote pyroptosis of endothelial cells during lung ischemia-reperfusion via IL-1R/NF-κB/NLRP3 signaling.

In our previous study, we observed that donor pulmonary intravascular nonclassical monocytes play a major role in early PGF, but the specific mechanism remained unclear. In this study, we investigated the mechanistic role of monocytes in inducing pyroptosis of human pulmonary microvascular endothelial cells (HPMECs) during IRI. A murine hilar ligation model of IRI was utilized whereby left lungs underwent 1 h of ischemia and 23 h of reperfusion. Monocyte depletion by intraperitoneal clodronate-liposome treatment on pulmonary edema and pyroptosis activation were determined. In vitro experiments, we performed the co-culture experiments under hypoxia-reoxygenation (H/R) conditions to mimic the IRI environment. We monitored the expression of NLRP3, caspase-1 and IL-1ß in co-cultures of monocytes (U937 cells) and HPMECs under H/R conditions. NLRP3, IL-1ß and IL-1R siRNA knockdown, caspase-1 and NF-κB pathway inhibitors were employed to elucidate the mechanism modulating HPMEC pyroptosis during H/R. Treatment of mice with clodronate-liposome attenuated IR-induced pulmonary edema, cytokine production and pyroptosis activation. In vitro, NLRP3 knockdown in monocytes reduced caspase-1 and IL-1ß secretion in co-cultures of monocytes and HPMECs. Reduced HPMEC pyroptosis was also observed either containing HPMECs with genetically engineered IL-1R knockdown or in co-culture treated with a Triplotide inhibitor that disrupts NF-κB signaling. Monocytes play a vital role in the development of transplant-associated ischemia-reperfusion injury. A potential role is that monocytes secrete IL-1ß to induce HPMEC pyroptosis via the IL-1R/NF-κB/NLRP3 pathway.

Sphingosine 1 Phosphate (S1P) Receptor 1 Is Decreased in Human Lung Microvascular Endothelial Cells of Smokers and Mediates S1P Effect on Autophagy.

Destruction of alveoli by apoptosis induced by cigarette smoke (CS) is a major driver of emphysema pathogenesis. However, when compared to cells isolated from non-smokers, primary human lung microvascular endothelial cells (HLMVECs) isolated from chronic smokers are more resilient when exposed to apoptosis-inducing ceramide. Whether this adaptation restores homeostasis is unknown. To better understand the phenotype of HLMVEC in smokers, we interrogated a major pro-survival pathway supported by sphingosine-1-phosphate (S1P) signaling via S1P receptor 1 (S1P1). Primary HLMVECs from lungs of non-smoker or smoker donors were isolated and studied in culture for up to five passages. S1P1 mRNA and protein abundance were significantly decreased in HLMVECs from smokers compared to non-smokers. S1P1 was also decreased in situ in lungs of mice chronically exposed to CS. Levels of S1P1 expression tended to correlate with those of autophagy markers, and increasing S1P (via S1P lyase knockdown with siRNA) stimulated baseline macroautophagy with lysosomal degradation. In turn, loss of S1P1 (siRNA) inhibited these effects of S1P on HLMVECs autophagy. These findings suggest that the anti-apoptotic phenotype of HLMVECs from smokers may be maladaptive, since it is associated with decreased S1P1 expression that may impair their autophagic response to S1P.

Oxygen conditions oscillating between hypoxia and hyperoxia induce different effects in the pulmonary endothelium compared to constant oxygen conditions.

The pulmonary endothelium is an immediate recipient of high oxygen concentrations upon oxygen therapy and mediates down-stream responses. Cyclic collapse and reopening of atelectatic lung areas during mechanical ventilation with high fractions of inspired oxygen result in the propagation of oxygen oscillations in the hypoxic/hyperoxic range. We used primary murine lung endothelial cell cultures to investigate cell responses to constant and oscillating oxygen conditions in the hypoxic to hyperoxic range. Severe constant hyperoxia had pro-inflammatory and cytotoxic effects including an increase in expression of ICAM1, E-selectin, and RAGE at 24 hr exposure. The coagulative/fibrinolytic system responded by upregulation of uPA, tPA, and vWF and PAI1 under constant severe hyperoxia. Among antioxidant enzymes, the upregulation of SOD2, TXN1, TXNRD3, GPX1, and Gstp1 at 24 hr, but downregulation of SOD3 at 72 hr constant hyperoxia was evident. Hypoxic/hyperoxic oscillating oxygen conditions induced pro-inflammatory cytokine release to a lesser extent and later than constant hyperoxia. Gene expression analyses showed upregulation of NFKB p65 mRNA at 72 hr. More evident was a biphasic response of NOS3 and ACE1 gene expression (downregulation until 24 hr and upregulation at 72 hr). ACE2 mRNA was upregulated until 72 hr, but shedding of the mature protein from the cell surface favored ACE1. Oscillations resulted in severe production of peroxynitrite, but apart from upregulation of Gstp1 at 24 hr responses of antioxidative proteins were less pronounced than under constant hyperoxia. Oscillating oxygen in the hypoxic/hyperoxic range has a characteristical impact on vasoactive mediators like NOS3 and on the activation of the renin-angiotensin system in the lung endothelium.

A high-yield isolation and enrichment strategy for human lung microvascular endothelial cells.

Vasculopathies, characterized by the formation of fragile and abnormal microvessels, are associated with the severity of many chronic lung diseases, including pulmonary fibrosis, emphysema/chronic obstructive pulmonary disease, systemic sclerosis, and hypertension. However, the study of human lung vasculature has been limited by the ability to isolate generous quantities of microvascular endothelial cells (MVEC) free from mesenchymal contamination. Expansion and passaging of primary human MVEC in vitro typically results in loss of a traditional phenotype in favor of an intermediate mesenchymal one, as early as passage five. Here we provide a detailed protocol for the selection of large quantities of enriched primary human lung MVEC based upon differential adherence from mesenchyme and simple magnetic separation, which decreases the need for excessive passaging, in order to obtain sufficient cell numbers to successfully freeze stock cultures. Additional protocols are provided for Ac-di-LDL selection, characterization, and a sandwich angiogenesis method of functional tube formation. The complete protocol including cell isolation and characterization takes approximately six weeks to complete.

Ability of plasmid DNA complexed with histidinylated lPEI and lPEI to cross in vitro lung and muscle vascular endothelial barriers.

DNA complexes made with cationic polymers (polyplexes) developed as nonviral vectors for gene therapy must be enabled to cross through vascular endothelium to transfect underlying tissues upon their administration in the blood circulation. Here, we evaluated the transendothelial passage (TEP) of DNA complexes made with histidinylated linear polyethylenimine (His-lPEI) or linear polyethylenimine (lPEI). In vitro studies were performed by using established transwell lung and skeletal muscle vascular endothelial barriers. The models were composed of a monolayer of human lung microvascular endothelial (HMVEC-L) cells and mouse cardiac endothelial (MCEC) cells formed on a PET insert and immortalized human tracheal epithelial (ΣCFTE29o-) cells and mouse myoblasts (C2C12) as target cells cultured in the lower chamber, respectively. When the vascular endothelium monolayer was established and characterized, the transfection efficiency of target (ΣCFTE29o- and C2C12) cells with plasmid DNA encoding luciferase was used to evaluate TEP of polyplexes. The luciferase activities with His-lPEI and lPEI polyplexes compared to those obtained in the absence of endothelial cell monolayer were 6.5% and 4.3% into ΣCFTE29o- cells, and 18.5% and 0.23% into C2C12 cells, respectively. The estimated rate for His-lPEI polyplexes was 0.135 µg/cm(2).h and 0.385 µg/cm(2).h through the HMVEC-L and MCEC monolayers, respectively. These results indicate that His-lPEI polyplexes can pass through the lung and skeletal muscle vascular endothelium and can transfect underlying cells.

A cellular model to mimic exhaled cigarette smokeinduced lung microvascular endothelial cell injury and death.

Tobacco smoke exhaled from smokers is a key component of secondhand smoke, contributing to lung alveolar wall destruction seen in chronic lung diseases. Although mainstream and sidestream tobacco smoke are cyto-toxic to lung cells, it is unclear whether exhaled smoke induces lung cell injury or even death. We sought to establish an in vitro model to examine the effects of exhaled smoke on lung cells. Phosphate-buffered saline-conditioned cigarette smoke (CCS) derived from a blow-by system was used to mimic exhaled tobacco smoke exposure. Exposure of medium to CCS leads to dose-dependent increases in nicotine/cotinine levels. Scanning spectrophotometric analysis of the CCS-exposed medium reveals an absorption peak at 290 nm wavelength. The OD values at 290 nm are correlated with nicotine levels in the exposed medium, indicating that a simple measurement of OD at 290 nm can be used to monitor CCS exposure. Tobacco smoke contacts the microvascular endothelium located at lung alveoli, before it enters the blood stream. Hence, human lung microvascular endothelial cells (hMVEC) were exposed to CCS and assessed for cell injury and death. Exposure of hMVEC to CCS equivalent to burning 12-16 cigarettes leads to increased LDH release from the cells into the medium. This suggests that CCS can induce lung cell injury. CCS at a low level increases cell growth, whereas the high level of CCS decreases cell viability. In addition, CCS exposure induces cell detachment and morphological changes. Our results demonstrate that exposure of buffer-conditioned mainstream cigarette smoke leads to increased nicotine/cotinine levels and cell injury/death, which may contribute to the pathophysiology of passive smoking-associated lung diseases.