Rat Pulmonary Microvascular Endothelial Cells Research Articles

Inhibition of MRP4 alleviates sepsis-induced acute lung injury in rats

This study was undertaken to examine the regulatory role of multidrug resistance-associated protein 4 (MRP4) in an experimental model of sepsis-induced acute lung injury in rats. Sepsis was induced by cecal ligation and puncture in anesthetized rats. Animals were then randomly assigned to receive intravenous injection of vehicle or MRP4 inhibitor (MK571, 20 mg/kg). The pathological changes were observed by hematoxylin and eosin staining. Lung water content, lung vascular permeability and inflammatory cell count in bronchoalveolar lavage fluid (BALF) were quantified. Serum tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels were measured. In addition, lung tissue cyclic adenosine monophosphate (cAMP) levels were examined by enzyme-linked immunosorbent assay. Furthermore, the effects of MRP4 knockdown on lipopolysaccharide (LPS)-induced endothelial permeability and the cytoskeleton of rat pulmonary microvascular endothelial cells (PMVECs) were detected. The protein expression levels of MRP4, Rac1, VE-cadherin, β-catenin and ZO-1 were measured by Western blot analysis. MK571 significantly reduced lung tissue damage, lung water content and lung vascular permeability. Lung tissue cAMP levels were attenuated in MK571-treated animals compared with vehicle controls. MK571 also decreased sepsis-induced inflammatory cell accumulation in BALF. In addition, the MK571 group had significantly lower serum TNF-α and IL-6 levels compared with vehicle controls. Consistently, knockdown of MRP4 protected against LPS-induced increase in the endothelial permeability and the destruction of cytoskeleton in vitro. Furthermore, silencing MRP4 gene significantly reduced MRP4 protein expression and restored the protein expression of Rac1, VE-cadherin, β-catenin and ZO-1 in rat PMVECs in response to LPS stimulation. These data suggest that inhibition of MRP4 significantly alleviates sepsis-induced acute lung injury in rats.

Isolating pulmonary microvascular endothelial cells ex vivo: Implications for pulmonary arterial hypertension, and a caution on the use of commercial biomaterials.

Transcriptomic analysis of pulmonary microvascular endothelial cells from experimental models offers insight into pulmonary arterial hypertension (PAH) pathobiology. However, culturing may alter the molecular profile of endothelial cells prior to analysis, limiting the translational relevance of results. Here we present a novel and validated method for isolating RNA from pulmonary microvascular endothelial cells (PMVECs) ex vivo that does not require cell culturing. Initially, presumed rat PMVECs were isolated from rat peripheral lung tissue using tissue dissociation and enzymatic digestion, and cells were cultured until confluence to assess endothelial marker expression. Anti-CD31, anti-von Willebrand Factor, and anti-α-smooth muscle actin immunocytochemistry/immunofluorescence signal was detected in presumed rat PMVECs, but also in non-endothelial cell type controls. By contrast, flow cytometry using an anti-CD31 antibody and isolectin 1-B4 (from Griffonia simplicifolia) was highly specific for rat PMVECs. We next developed a strategy in which the addition of an immunomagnetic selection step for CD31+ cells permitted culture-free isolation of rat PMVECs ex vivo for RNA isolation and transcriptomic analysis using fluorescence-activated cell sorting. Heterogeneity in the validity and reproducibility of results using commercial antibodies against endothelial surface markers corresponded to a substantial burden on laboratory time, labor, and scientific budget. We demonstrate a novel protocol for the culture-free isolation and transcriptomic analysis of rat PMVECs with translational relevance to PAH. In doing so, we highlight wide variability in the quality of commonly used biological reagents, which emphasizes the importance of investigator-initiated validation of commercial biomaterials.

Hyperspectral imaging fluorescence excitation scanning (HIFEX) microscopy for live cell imaging.

In the past two decades, spectral imaging technologies have expanded the capacity of fluorescence microscopy for accurate detection of multiple labels, separation of labels from cellular and tissue autofluorescence, and analysis of autofluorescence signatures. These technologies have been implemented using a range of optical techniques, such as tunable filters, diffraction gratings, prisms, interferometry, and custom Bayer filters. Each of these techniques has associated strengths and weaknesses with regard to spectral resolution, spatial resolution, temporal resolution, and signal-to-noise characteristics. We have previously shown that spectral scanning of the fluorescence excitation spectrum can provide greatly increased signal strength compared to traditional emission-scanning approaches. Here, we present results from utilizing a Hyperspectral Imaging Fluorescence Excitation Scanning (HIFEX) microscope system for live cell imaging. Live cell signaling studies were performed using HEK 293 and rat pulmonary microvascular endothelial cells (PMVECs), transfected with either a cAMP FRET reporter or a Ca2+ reporter. Cells were further labeled to visualize subcellular structures (nuclei, membrane, mitochondria, etc.). Spectral images were acquired using a custom inverted microscope (TE2000, Nikon Instruments) equipped with a 300W Xe arc lamp and tunable excitation filter (VF-5, Sutter Instrument Co., equipped with VersaChrome filters, Semrock), and run through MicroManager. Timelapse spectral images were acquired from 350-550 nm, in 5 nm increments. Spectral image data were linearly unmixed using custom MATLAB scripts. Results indicate that the HIFEX microscope system can acquire live cell image data at acquisition speeds of 8 ms/wavelength band with minimal photobleaching, sufficient for studying moderate speed cAMP and Ca2+ events.

ACE-2/ANG1-7 ameliorates ER stress-induced apoptosis in seawater aspiration-induced acute lung injury.

Previous studies have shown that apoptosis of alveolar cells can be regulated by autocrine of angiotensin (ANG)II and its counter regulatory ACE-2/ANG1-7 axis. Our earlier study has shown that endoplasmic reticulum (ER) stress in response to seawater aspiration eventually led to apoptosis in lung tissue. In this study, we examined the hypothesis that ER stress-induced apoptosis in seawater aspiration-induced acute lung injury (ALI) might also be regulated by the ANGII/ANG1-7 system. ER stress was induced by seawater stimulation and proteasome inhibitor MG132 (an ER stress inductor). Moreover, ER stress in seawater-stimulated lung tissues and rat pulmonary microvascular endothelial cells (RPMVECs) promoted ANGII expression and decreased ACE-2/ANG1-7 expression. ER stress induced by seawater stimulation also led to apoptosis. Apoptosis induced by seawater stimulation and MG132 were inhibited by ANGII receptor blocker and abrogated by the addition of ANG1-7. These results suggest that apoptosis induced by ER stress in seawater aspiration-induced ALI is regulated by ANG II/ANG1-7 in lung tissues and RPMVECs. In addition, the active form of X-box binding protein 1 (XBP1), spliced XBP1 (XBP1s), a transcription factor that regulates ER-associated degradation genes during ER stress was significantly activated in seawater stimulated cells. Based on this phenomenon we designed a tandem gene, Wfs1 promoter (a target gene promoter of XBP1s)- ACE2 and ANG1-7 and transfected this tandem gene into seawater-stimulated cells. ACE-2/ANG1-7 expression were significantly promoted and apoptosis was inhibited in cells transfected with the tandem gene. These results suggest that stimulation of ACE-2/ANG1-7 may be a therapeutic target of ER stress-induced apoptosis in seawater aspiration-induced ALI.

Differential roles of p38 MAPK and ERK1/2 in angiopoietin-2-mediated rat pulmonary microvascular endothelial cell apoptosis induced by lipopolysaccharide.

Angiopoietin-2 (Ang-2) is a Tie-2 ligand that destabilizes vascular structures, enhances vascular permeability and induces vascular regression and endothelial cell apoptosis. Although there is evidence for the involvement of the Ang/Tie2 axis in acute lung injury (ALI), the underlying mechanisms involved in Ang-2-induced cell apoptosis are not well understood. In this study, whether Ang-2 contributes to microvascular endothelial cell injury and mediates lipopolysaccharide (LPS)-induced endothelial cell apoptosis and its associated signaling pathways was investigated. Exposure of rat pulmonary microvascular endothelial cells (RPMVECs) to LPS, Ang-2 and related inhibitors was performed to measure the expression levels of Ang-2, the activation of mitogen-activated protein kinases (MAPKs), the phosphorylation of extracellular signal-regulated kinase (ERK)1/2, and expression of the apoptosis-related proteins Bax and Bcl-2 using western blotting, reverse transcription-quantitative polymerase chain reaction, flow cytometry and fluorescence microscopy. The expression of Ang-2 in the RPMVECs was increased by LPS independent of time. The phosphorylation of p38 MAPK and ERK1/2 was significantly upregulated and the activation of apoptosis-related proteins Bax and Bcl was mediated by Ang-2. In addition, inhibition of the p38 pathway by SB203580 attenuated the Ang-2-mediated cell apoptosis, but inhibition of the ERK1/2 pathway by PD98059 exerted an anti-apoptotic effect against Ang-2. In conclusion, LPS-induced apoptosis is partly mediated via stimulation of p38 and ERK1/2 signaling pathways, where Ang-2 acts an inflammation-related factor to participate in the course of cell apoptosis in RPMVECs.

Luteolin suppresses inflammation through inhibiting cAMP-phosphodiesterases activity and expression of adhesion molecules in microvascular endothelial cells.

Luteolin, an anti-inflammatory ingredient found in the Chinese herb Folium perillae, can inhibit not only the cyclic adenosine monophosphate (cAMP)-phosphodiesterases (PDEs) activity of neutrophils, but also the expression of lymphocyte function-associated antigen-1 in neutrophils, both of which result in a decrease in the adhesion between neutrophils and microvascular endothelial cells. However, the effect of luteolin on the cAMP-PDEs activity and expression of adhesion molecules in endothelial cells are not clear. In the present study, primary rat pulmonary microvascular endothelial cells and a lipopolysaccharide-induced rat acute pneumonia model were used to explore the role of luteolin on cAMP-PDEs activity, expression of adhesion molecules, and leukocyte infiltration. We demonstrate that rat pulmonary microvascular endothelial cells expressed high levels of cAMP-PDEs, specifically PDE4, and further luteolin exhibited dose-dependent inhibition on the activity of cAMP-PDEs or PDE4 in endothelial cells. Luteolin also had a significant inhibitory effect on the expression of vascular cell adhesion molecule (VCAM)-1, but not intracellular cell adhesion molecule (ICAM)-1 in microvascular endothelial cells. Further, we show that luteolin decreased the levels of soluble ICAM-1 (sICAM-1), but not soluble E-selectin in the serum of rats subjected to acute pneumonia. We also show that luteolin treatment decreased the wet/dry weight ratio of lung tissue and reduced the total number of serum leukocytes in a dose-dependent manner in a rat acute pneumonia model. In conclusion, these results demonstrate that luteolin suppresses inflammation, at least in part, through inhibiting both cAMP-PDEs or PDE4 activity and the expression of VCAM-1 (in vitro) and sICAM-1 (in vivo) in endothelial cells.

Colquhounia Root Tablet Protects Rat Pulmonary Microvascular Endothelial Cells against TNF-α-Induced Injury by Upregulating the Expression of Tight Junction Proteins Claudin-5 and ZO-1.

Background There are currently limited effective pharmacotherapy agents for acute lung injury (ALI). Inflammatory response in the lungs is the main pathophysiological process of ALI. Our preliminary data have shown that colquhounia root tablet (CRT), a natural herbal medicine, alleviates the pulmonary inflammatory responses and edema in a rat model with oleic acid-induced ALI. However, the potential molecular action mechanisms underlining its protective effects against ALI are poorly understood. This study aimed to investigate the effects and mechanism of CRT in rat pulmonary microvascular endothelial cells (PMEC) with TNF-α-induced injury. Methods PMECs were divided into 6 groups: normal control, TNF-α (10 ng/mL TNF-α), Dex (1×10−6 M Dex + 10 ng/mL TNF-α), CRT high (1000 ng/mL CRT + 10 ng/mL TNF-α), CRT medium (500 ng/mL CRT + 10 ng/mL TNF-α), and CRT low group (250 ng/mL CRT + 10 ng/mL TNF-α). Cell proliferation and apoptosis were detected by MTT assay and flow cytometry. Cell micromorphology was observed under transmission electron microscope. The localization and expression of tight junction proteins Claudin-5 and ZO-1 were analyzed by immunofluorescence staining and Western blot, respectively. Results TNF-a had successfully induced an acute endothelial cell injury model. Dex and CRT treatments had significantly stimulated the growth and reduced the apoptosis of PMECs (all p < 0.05 or 0.01) and alleviated the TNF-α-induced cell injury. The expression of Claudin-5 and ZO-1 in Dex and all 3 CRT groups was markedly increased compared with TNF-a group (all p < 0.05 or 0.01). Conclusion CRT effectively protects PMECs from TNF-α-induced injury, which might be mediated via stabilizing the structure of tight junction. CRT might be a promising, effective, and safe therapeutic agent for the treatment of ALI.

Amelioration of lung ischemia‑reperfusion injury by JNK and p38 small interfering RNAs in rat pulmonary microvascular endothelial cells in an ischemia‑reperfusion injury lung transplantation model.

The inhibition of mitogen‑activated protein kinases (MAPKs), including c‑Jun NH2‑terminal protein kinase (JNK), p38 MAPK (p38) and extracellular signal‑regulated protein kinase 1/2 (ERK1/2), have an important effect on lung ischemia‑reperfusion injury (IRI) during lung transplantation (LT). However, the way in which combined MAPK inhibition exerts optimal protective effects on lung IRI remains to be elucidated. Therefore, the present study evaluated the therapeutic efficacy of the inhibition of MAPKs in rat pulmonary microvascular endothelial cells (PMVECs) in an IRI model of LT. The rat PMVECs were transfected with small interfering RNAs (siRNAs) against JNK, p38 or ERK1/2. Cotransfection was performed with siRNAs against JNK and p38 in the J+p group, JNK and ERK1/2 in the J+E group, p38 and ERK1/2 in the p+E group, or all three in the J+p+E group. Non‑targeting (NT) siRNA was used as a control. The PMVECs were then treated to induce IRI, and the levels of inflammation, apoptosis and oxidative stress were detected. Differences between compared groups were determined using Tukey's honest significant difference test. In all groups, silencing of the MAPKs was shown to attenuate inflammation, apoptosis and oxidative stress to differing extents, compared with the NT group. The J+p and J+p+E groups showed lower levels of interleukin (IL)‑1β, IL‑6 and malondialdehyde, a lower percentage of early‑apoptotic cells, and higher superoxide dismutase (SOD) activity, compared with the other groups. No significant differences were observed in the inflammatory response, SOD activity or early apoptosis between the J+p and J+p+E groups. These findings suggested that the dual inhibition of JNK and p38 led to maximal amelioration of lung IRI in the PMVECs of the IRI model of LT, which occurred through anti‑inflammatory, anti‑oxidative and anti‑apoptotic mechanisms.

Effects of MRP4 gene on lipopolysaccharide-induced endothelial permeability and the cytoskeleton of endothelial cells changes

Objective To investigate the effects of multidrug resistance-associated protein 4 (MRP4) on the cytoskeleton and cellular permeability of rat pulmonary microvascular endothelial cells (PMVECs) induced by lipopolysaccharide (LPS). Methods PMVECs were cultured for 3 to 6 generations were randomly divided into 4 groups: control group, LPS group, Ad-shMRP4 group(adenoviral expression of a short-hairpin RNA directed against MRP4), Ad-shRNA group. The infection rate of cells was detected by fluorescence microscope observation. The level of MRP4 was assayed by Western botting. Monolayer permeability was determined by the Transwell assay. The morphological characteristic and distribution of F-actin was measured by laser confocal fluorescence microscope. Results Compared with control group, the expression of MRP4 protein was up-regulated (P<0.05) and the significant increase in the permeability of endothelial cells (2 h, 6 h, 12 h and 24 h respectively: 0.28±0.02 vs.0.41±0.04, 0.32±0.02, 0.30±0.01 vs.0.53±0.04, 0.39±0.03, 0.33±0.04 vs.1.12±0.17, 0.70±0.07, 0.32±0.03 vs.0.79±0.02, 0.57±0.05, P<0.05), the F-actin was remodeled, and the stress fibers were formed in LPS group and Ad-shMRP4 group. However, compared with LPS group, the expression of MRP4 protein was down-regulated (P<0.05) and the markedly decrease in the permeability of endothelial cells (2 h, 6 h, 12 h and 24 h respectively: 0.41±0.04 vs.0.32±0.02, 0.53±0.04 vs.0.39±0.03, 1.12±0.17 vs.0.70±0.07, 0.79±0.02 vs.0.57±0.05, P<0.05) was found, and the remodeling of F-actin, and the formation of stress fibers were observed in Ad-shMRP4 group. Conclusions Silencing of MRP4 gene can effectively attenuates LPS-induced increase in the endothelial cell permeability and the destruction of cytoskeleton, thus playing an important role in the protection of endothelial cell barrier. Key words: Sepsis; Multidrug resistance-associated protein 4; Endothelial permeability; Cytoskeleton; Gene silencing

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.

Relationship between endothelin-1 and p38 MAPK and PI3K/Akt signaling pathways during mechanical stretch-induced enhancement of adhesion of rat pulmonary microvascular endothelial cells

Objective To evaluate the relationship between endothelin-1 (ET-1) and p38 mitogen-activated protein kinase (p38 MAPK) and phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathways during mechanical stretch-induced enhancement of adhesion of rat pulmonary microvascular endothelial cells (PMVECs). Methods Rat PMVECs were seeded in the culture plate at a density of 0.5×105 cells/ml (2 ml/well) and divided into 5 groups (n=24 each) using a random number table: control group (group C), mechanical stretch group (group MS), mechanical stretch plus specific PI3K inhibitor LY294002 group (LY group), mechanical stretch plus specific p38 MAPK inhibitor SB203580 group (SB group), and mechanical stretch plus selective ETA receptor blocker BQ123 group (BQ group). Cells were exposed to 20% cyclic stretch at 0.3 Hz for 4 h using a sine wave.In LY, SB and BQ groups, LY294002, SB203580 and BQ123 at the final concentration of 10 μmol/L were added, respectively, after mechanical stretch, cells were incubated for 10 min, and then extracted and purified rat polymorphonuclear neutrophil leukocytes (PMNs, 5×105 cells/well) were added and co-incubated with PMVECs for 30 min and then washed out.The concentrations of ET-1 and interleukin-6 (IL-6) in the culture medium were determined using enzyme-linked immunosorbent assay.The expression of phosphorylated p38 MAPK (p-p38 MAPK) and phosphorylated Akt (p-Akt) was detected by Western blot.Adhesion of PMNs was measured by immuno-histochemistry, and the adhesion rate was calculated.The expression of P-selectin mRNA was detected using real-time polymerase chain reaction. Results Compared with group C, the concentrations of IL-6 and ET-1 in the culture medium were significantly increased, the expression of p-p38 MAPK, p-Akt and P-selectin mRNA was up-regulated, and the adhesion rate of PMNs was increased in the other four groups (P<0.05). Compared with group MS, the concentration of IL-6 in the culture medium was significantly decreased, the expression of p-Akt and P-selectin mRNA was down-regulated, and the adhesion rate of PMNs was decreased in LY, SB and BQ groups, the concentration of ET-1 in the culture medium was significantly decreased in group BQ, and the expression of p-p38 MAPK was significantly down-regulated in SB and BQ groups (P<0.05). Conclusion The signaling mechanism underlying ET-1-mediated enhancement of rat PMVEC adhesion may be related to activating p38 MAPK and PI3K/Akt signaling pathways. Key words: Endothelin-1; p38 Mitogen-activated protein kinases; 1-Phosphatidylinositol 3-kinase; Protein-serine-threonine kinases; Lung; Endothelial cells; Cell adhesion

Endothelial semaphorin 7A promotes seawater aspiration-induced acute lung injury through plexin C1 and β1 integrin.

Inflammation and edema are two main characteristics in seawater aspiration‑induced acute lung injury (ALI). In a previous study of the authors, it was demonstrated that endothelial semaphorin 7A (SEMA7A) serves an important role in the development of seawater‑induced inflammation and edema. However, the mechanism of endothelial SEMA7A‑mediated ALI remains unclear. Therefore, the authors explored the effect of SEMA7A in rat pulmonary microvascular endothelial cells (RPMVECs) and the interaction between endothelial SEMA7A and alveolar macrophages during seawater aspiration‑induced ALI. The role of SEMA7A in endothelial permeability was detected using plexin C1 blocking antibody or SEMA7A small interfering (si)RNA. In addition, RPMVECs were co‑cultured with rat alveolar macrophage cell line‑NR8383 cells and pro‑inflammatory cytokine production was detected. Interaction between the β1 integrin and SEMA7A was detected using the β1 integrin blocking antibody or SEMA7A siRNA. Seawater stimulation induced endothelial cytoskeleton remodeling, endothelial permeability, phosphorylation of cofilin, and increased the vascular endothelial growth factor (VEGF) expression in RPMVECs. Moreover, seawater stimulation led to expression of proinflammatory cytokines and activated the nuclear factor‑κB pathway in co‑cultured cells. However, blockage with the plexin C1 antibody inhibited endothelial cytoskeleton remodeling, endothelial permeability, phosphorylation of cofilin, and treatment with SEMA7A siRNA inhibited expression of VEGF in RPMVECs. In addition, blockage with β1 integrin antibody reduced expression of proinflammatory cytokines and inhibited activation of NF‑κB in co‑culture cells. These results suggest that SEMA7A promotes seawater induced lung edema via plexin C1 and stimulates seawater induced lung inflammation via β1 integrin.

Effect of multidrug resistant associated protein 4 overexpression on lipopolysaccharide-induced vascular endothelial hyperpermeability and its mechanism

To investigate the effect of multidrug resistance protein 4 (MRP4) overexpression on lipopolysaccharide (LPS)-induced vascular endothelial hyperpermeability of rat pulmonary micro-vascular endothelial cells (PMVECs) and its molecule mechanism. Three to six passages of PMVECs were cultured in vitro, and they were divided into three groups: the cells in LPS group were only challenged by LPS 10 μg/mL after being cultured in serum-free medium for 24 hours; the cells in Ad-shRNA and Ad-MRP4 groups were infected with the empty virus control or recombinant adenovirus expressing MRP4 for 2 hours, and then were cultured in serum-free medium for 24 hours followed by stimulation of LPS 10 μg/mL. Endothelial permeability was assayed by the Transwell chamber models at 2, 6, 12, and 24 hours after LPS stimulation. Intracellular cyclic adenosine monophosphate (cAMP) levels were detected by enzyme-linked immunosorbent assay (ELISA). The morphological characteristics and distribution of F-actin was determined by laser confocal fluorescence microscope. The protein expressions of MRP4, β-catenin, vascular endothelium-cadherin (VE-cad) and ZO-1 were measured by Western Blot. (1) After LPS stimulation, endothelium permeability and intracellular cAMP levels in PMVECs were significantly increased, peaked at 12 hours, and then decreased after 24 hours. Compared with LPS group and Ad-shRNA group, PMVECs of Ad-MRP4 group were exhibited a significant increase in endothelial permeability [12-hour permeability (A value): 1.88±0.06 vs. 1.12±0.17, 1.10±0.18] and a significant decrease in intracellular cAMP level [12-hour cAMP (μg/L): 2.39±0.02 vs. 2.97±0.01, 3.00±0.02, all P < 0.05]. There was no significant difference in endothelium permeability and intracellular cAMP levels at all time points between the LPS group and the Ad-shRNA group (all P > 0.05). (2) Under laser confocal fluorescence microscope, after LPS stimulation, the stress fiber formation was induced in three groups. But there were pronounced irregular aggregation of fiber in PMVECs of Ad-MRP4 group. (3) Furthermore, compared with LPS group and Ad-shRNA group, protein expression of MRP4 in Ad-MRP4 group was dramatically increased (gray value: 0.76±0.03 vs. 0.44±0.02, 0.43±0.02, both P < 0.05), and the protein expressions of β-catenin, VE-cad, and ZO-1 were significantly decreased [β-catenin (gray value): 0.14±0.03 vs. 0.23±0.04, 0.23±0.03); VE-cad (gray value): 0.21±0.01 vs. 0.34±0.02, 0.35±0.04; ZO-1 (gray value): 0.14±0.02 vs. 0.37±0.06, 0.33±0.07, all P < 0.05]. There was no significant difference in all protein expressions between the LPS group and Ad-shRNA group (all P > 0.05). MRP4 overexpression can decrease intracellular cAMP levels, reduce intercellular junction protein expression, and then exaggerate LPS-induced vascular endothelial hyperpermeability.

Re-endothelialization of rat lung scaffolds through passive, gravity-driven seeding of segment-specific pulmonary endothelial cells.

Effective re-endothelialization is critical for the use of decellularized scaffolds for ex vivo lung engineering. Current approaches yield insufficiently re-endothelialized scaffolds that haemorrhage and become thrombogenic upon implantation. Herein, gravity-driven seeding coupled with bioreactor culture facilitated widespread distribution and engraftment of endothelial cells throughout rat lung scaffolds. Initially, human umbilical vein endothelial cells were seeded into the pulmonary artery by either gravity-driven, variable flow perfusion seeding or pump-driven, pulsatile flow perfusion seeding. Gravity seeding evenly distributed cells and supported cell survival and re-lining of the vascular walls while perfusion pump-driven seeding led to increased cell fragmentation and death. Using gravity seeding, rat pulmonary artery endothelial cells and rat pulmonary vein endothelial cells attached in intermediate and large vessels, while rat pulmonary microvascular endothelial cells deposited mostly in microvessels. Combination seeding of these cells led to positive vascular endothelial cadherin staining. In addition, combination seeding improved barrier function as assessed by serum albumin extravasation; however, leakage was observed in the distal portions of the re-endothelialized tissue suggesting that recellularization of the alveoli is necessary to complete barrier function of the capillary-alveolar network. Overall, these data indicate that vascular recellularization of rat lung scaffolds is achieved through gravity seeding. Copyright © 2016 John Wiley & Sons, Ltd.

Microparticle Uptake by Pulmonary Microvascular Endothelial Cells

Microparticles (MPs) are important for the exchange of information between cells. MPs are extracellular vesicles that range in size from 0.1 to 1 μm in diameter. They are constitutively released from many cell types and are thought to play a role in vascular homeostasis. Furthermore the number and content of MPs are altered in pathologies involving inflammation and vascular cell activation. MPs carry proteins and RNAs from parent cells and are extracellular packets of information that affect target cell function. However, the mechanisms of targeting and delivery of the message are unknown. Understanding the message delivery system between MPs and target cells is the focus of this project.We first tested whether MPs are internalized by rat pulmonary microvascular endothelial cells (PMVECs) in culture. Cells labeled with Hoechst nuclear stain were incubated with calcein green and PKH26 labeled MPs for four hours. The cells were imaged using an environmental chamber on a confocal microscope (Nikon A1R). An image stack (Z‐stack) was acquired every 15 minutes. MPs were observed within cells and the number of MPs within cells increased over the first 90 minutes of the experiment. Maximum MP‐cell association was observed at 1 hour and few MPs were observed inside cells after 3 hours. These results indicate that MPs are taken up by target PMVECs in a time‐dependent manner.We next examined MP localization with organelles within cells. Organelle markers included monoclonal antibodies against early endosomes (Rab5 and EEA1), lysosomes (LAMP1), cis‐golgi (GRASP65) and trans‐golgi (TGN65), as well as labels for mitochondria, microtubules and cell nuclei. MPs were isolated from cell culture media via serial centrifugation and stained with a lipophilic membrane stain (PKH26). PMVECs were treated with stained MPs for 1, 2, 4 and 6 hours before organelle labeling. At one hour MPs colocalized with early endosomes (EEA1) and a small percentage colocalized with lysosomes (LAMP1). At six hours MPs colocalized with trans‐golgi. No overlap between MP‐staining and cis‐golgi, mitochondria, microtubules, or nuclei was observed at any time point. Taken together, these data indicate that MPs are internalized by target PMVECs. After internalization by target cells, most MPs are trafficked to the trans‐golgi for possible recycling of MP‐contents. Future studies will investigate the mechanisms responsible for internalization, whether payloads of MPs are delivered to specific organelles, and whether MP‐delivered proteins alter target cell function.Support or Funding InformationThis work was supported by NIH T32HL076125, P01HL066299, S10RR027535, and AHA 11SDG7390037.

Reduced Membrane Cholesterol Following Chronic Hypoxia Limits Orai1 ‐Mediated Pulmonary Endothelial Ca2+ Entry

Diminished Ca2+‐dependent production of endothelium‐derived vasodilators like nitric oxide is observed in chronic hypoxia (CH)‐induced pulmonary hypertension. Our previous studies show that endothelial store‐operated calcium entry (SOCE) as well as membrane cholesterol (Chol) are reduced in pulmonary arteries from rats exposed to CH (4 wk, 380 Torr) compared to their normoxic controls. Interestingly, Chol supplementation restores both membrane Chol and endothelial SOCE in cells from CH rats. Substitution of membrane Chol with epicholesterol (EpiChol, epimeric form of Chol) in freshly isolated pulmonary artery endothelial cell (PAEC) sheets mimics the effect of CH to blunt endothelial SOCE. These data suggest that reduced membrane Chol following CH limits the activity of ion channels required for endothelial SOCE. However, the ion channels involved in Chol‐sensitive SOCE have not been identified. We hypothesized that membrane Chol facilitates Orai1‐mediated endothelial Ca2+ entry, and that reduced Ca2+ entry following CH is due to loss of membrane Chol. To test this hypothesis, we pretreated PAEC sheets isolated from control and CH rats with Chol (2 mM) or EpiChol (2 mM), and administered the Orai1 inhibitor AnCoA4 (20 μM), or vehicle. Ca2+ influx in response store depletion with cyclopiazonic acid (CPA, 10 μM) was assessed by Mn2+‐quenching of fura‐2 fluorescence. Whereas Chol supplementation restored diminished SOCE in PAEC sheets from CH rats, both EpiChol and AnCoA4 significantly reduced endothelial SOCE in normoxic controls, but not in cells from CH rats. However, the Orai1 inhibitor had no further effect in cells pretreated with EpiChol. Additional experiments examined SOCE following Orai1 knockdown using siRNA in cultured rat pulmonary microvascular endothelial cells (PMVEC). We found that both EpiChol and Orai1 siRNA significantly inhibited SOCE compared to vehicle or non‐targeting siRNA, respectively. EpiChol treatment had no additional effect in cells following knockdown of Orai1. Furthermore, using a proximity ligation assay, we observed that EpiChol treatment significantly reduced the STIM1‐Orai1 interaction required for SOCE when compared to either vehicle or Chol treated groups. We conclude that membrane Chol is required for STIM1‐Orai1 interaction necessary to elicit endothelial SOCE. Furthermore, reduced Ca2+ entry after CH is due to loss of membrane Chol thereby limiting Orai1 activity.Support or Funding InformationNIH R01 HL132883, NIH R01 HL111084, AHA 16GRNT27700010, AHA 15GRNT21080001,

RhoA/mDia1 pathway involved in the expression of p-ERM in the pulmonary micro-vascular endothelial cell induced by lipopolysaccharide

Objective To investigate the possibility of the involvement of RhoA/mDia1 pathway to cause the expression of phosphorylate ezrin-radixin-moesin (p-ERM) in rat pulmonary micro-vascular endothelial cell (PMVEC) after the stimulation of lipopolysaccharide (LPS). Methods The specimens of lung tissue were taken from healthy male SPF grade SD rat with 100-120 g body weight which was purchased from the laboratory animal center of Anhui province. After culture, the PMVECs were randomly divided into dose-dependent groups (0, 0.1, 1, 10 μg/mL LPS added in PMVECs and cultured for 30 min, n=8 in each), time-dependent groups (10 μg/mL LPS added to PMVECs cultured for 0, 15, 30, 60, 120 min, n=8 in each) and intervention group (n=8). In the intervention group, PMVECs were cultured with 1 μg/mL C3 transferase in serum free media for 240 min, followed by treatment with 10 μg/mL LPS for 30 min. Meanwhile, two control groups in serum-free DMEM medium were made by adding 10 μg/mL LPS to PMVECs and 1 μg/mL C3 transferase to PMVECs respectively cultured for 30 min (n=8 in each). Western blot was used to detect the level of p-ERM, ERM and mDia1. Data were analyzed with SPSS 16.0 software, while one way analysis of variance (ANOVA) was used to compare multiple sets of variables, the intergroup comparisons were analyzed by the least-significant-difference (LSD) tests, with P<0.05 for the statistically significant difference. Results ERM, p-ERM and mDia1 were presented in rat PMVEC. Stimulation with LPS up-regulated p-ERM, mDia1 in a dose-dependent manner: LPS [0 μg/mL LPS group: (0.520±0.101), 0.1 μg/mL LPS group: (0.657±0.092), 1 μg/mL LPS group: (0.891±0.167), 10 μg/mL LPS group: (1.227±0.106); 0 μg/mL group vs. 0.1 μg/mL group, P>0.05; the rest P 0.05; the rest P 0.05; the rest P<0.05); and the level of mDia1 increased at 15 min(0.779±0.035), peaked at 30 min(0.889±0.036)then decreased at 60 min (0.648±0.019), 90 min (0.582±0.068), but kept at higher level at 120 min (0.526±0.059) than that in control group(0.284±0.118, all P<0.01). C3 transferase caused a marked attenuation of LPS induced p-ERM expression [control group: (0.339±0.069); C3+ LPS group: (0.438±0.07); C3 control group: (0.352±0.071); LPS control group: (0.634±0.191), C3+ LPS group vs. LPS control group, P=0.01], as the same in mDia1[control group: (0.449±0.122); C3+ LPS group: (0.380±0.148); C3 control group: (0.404±0.164); LPS control group: (0.622±0.174), C3+ LPS group vs. LPS control group, P<0.01]. Conclusions LPS could up-regulated the expression of p-ERM protein, and inhibition of RhoA/mDia1 signal pathway by C3 transferase could down-regulated the p-ERM levels. Key words: Lipopolysaccharide; Pulmonary microvascular endothelial cell; Ezrin-Radixin-Moesin; Inflammation; Acute respiratory distress syndrome; RhoA/mDia1

Endothelial colony-forming cells ameliorate endothelial dysfunction via secreted factors following ischemia-reperfusion injury.

Damage to endothelial cells contributes to acute kidney injury (AKI) by leading to impaired perfusion. Endothelial colony-forming cells (ECFC) are endothelial precursor cells with high proliferative capacity, pro-angiogenic activity, and in vivo vessel forming potential. We hypothesized that ECFC may ameliorate the degree of AKI and/or promote repair of the renal vasculature following ischemia-reperfusion (I/R). Rat pulmonary microvascular endothelial cells (PMVEC) with high proliferative potential were compared with pulmonary artery endothelial cells (PAEC) with low proliferative potential in rats subjected to renal I/R. PMVEC administration reduced renal injury and hastened recovery as indicated by serum creatinine and tubular injury scores, while PAEC did not. Vehicle-treated control animals showed consistent reductions in renal medullary blood flow (MBF) within 2 h of reperfusion, while PMVEC protected against loss in MBF as measured by laser Doppler. Interestingly, PMVEC mediated protection occurred in the absence of homing to the kidney. Conditioned medium (CM) from human cultured cord blood ECFC also conveyed beneficial effects against I/R injury and loss of MBF. Moreover, ECFC-CM significantly reduced the expression of ICAM-1 and decreased the number of differentiated lymphocytes typically recruited into the kidney following renal ischemia. Taken together, these data suggest that ECFC secrete factors that preserve renal function post ischemia, in part, by preserving microvascular function.

1\u03b1,25-Dihydroxyvitamin D3 Ameliorates Seawater Aspiration-Induced Lung Injury By Inhibiting The Translocation Of NF-\u03baB and RhoA

Our previous study have reported that 1α,25-Dihydroxyvitamin D3 (calcitriol) suppresses seawater aspiration-induced ALI in vitro and in vivo. We also have confirmed that treatment with calcitriol ameliorates seawater aspiration-induced inflammation and pulmonary edema via the inhibition of NF-κB and RhoA/Rho kinase pathway activation. In our further work, we investigated the effect of calcitriol on nuclear translocation of NF-κB and membrane translocation of RhoA in vitro. A549 cells and rat pulmonary microvascular endothelial cells (RPMVECs) were cultured with calcitriol or not for 48 h and then stimulated with 25% seawater for 40 min. After these treatments, cells were collected and performed with immunofluorescent staining to observe the translocation of NF-κB and RhoA and the cytoskeleton remodeling. In vitro, seawater stimulation activates nuclear translocation of NF-κB and membrane translocation of RhoA in A549 cells. In addition, seawater administration also induced cytoskeleton remodeling in A549 cells and RPMVECs. However, pretreatment with calcitriol significantly inhibited the activation of NF-κB and RhoA/Rho kinase pathways, as demonstrated by the reduced nuclear translocation of NF-κB and membrane translocation of RhoA in A549 cells. Meanwhile, treatment of calcitriol also regulated the cytoskeleton remodeling in both A549 cells and RPMVECs. These results demonstrated that treatment with calcitriol ameliorates seawater aspiration-induced ALI via inhibition of nuclear translocation of NF-κB and membrane translocation of RhoA and protection of alveolar epithelial and pulmonary microvascular endothelial barrier.

MiR-126a-5p is involved in the hypoxia-induced endothelial-to-mesenchymal transition of neonatal pulmonary hypertension.

Persistent pulmonary hypertension of the newborn (PPHN) is a clinical syndrome characterized by increased medial and adventitial thickness of the lung vasculature. The underlying mechanisms that regulate the cell phenotype alteration during PPHN remodeling are largely unknown. We randomly selected newborn rats that were exposed to hypoxia (10-12%) or room air for 2 weeks and used a microarray to identify the lung tissue microRNAs (miRNAs) involved in PPHN progression. The role of a key miRNA that affects the endothelial-to-mesenchymal transition (EndMT) in primary cultured rat pulmonary microvascular endothelial cells (RPMECs) was investigated. The expression of miR-126a-5p was elevated in the PPHN model according to microarray analysis. The relative expression of miR-126a-5p in RPMECs increased when they were exposed to hypoxia (P<0.05), consistent with the microarray results. Pecam1 expression decreased, whereas alpha-smooth muscle actin (α-SMA) increased in the hypoxic RPMECs. Knockdown of miR-126a-5p in RPMECs followed by treatment with hypoxia for 48 h resulted in a significant increase in the expression of Pecam1 and a reduction in α-SMA expression, with a simultaneous increase in PI3K (p85β) and phosphorylation of AKT at serine 473 compared with the negative control. Finally, the circulating miR-126a-5p concentration was upregulated in the PPHN model compared with healthy neonates. We concluded that hypoxia changed the cell homeostasis and that miR-126a-5p was upregulated in PPHN, which is partly responsible for hypoxia-induced EndMT. The mechanism underlying the upregulation of miR-126a-5p by hypoxia probably acts through the p85-β/p-AKT pathway.