Rat Pulmonary Endothelial Cells Research Articles

Carbonic Anhydrase IX and Hypoxia Promote Rat Pulmonary Endothelial Cell Survival during Infection.

Low tidal volume ventilation protects the lung in mechanically ventilated patients. The impact of the accompanying permissive hypoxemia and hypercapnia on endothelial cell recovery from injury is poorly understood. CA (carbonic anhydrase) IX is expressed in pulmonary microvascular endothelial cells (PMVECs), where it contributes to CO2 and pH homeostasis, bioenergetics, and angiogenesis. We hypothesized that CA IX is important for PMVEC survival and that CA IX expression and release from PMVECs are increased during infection. Although the plasma concentration of CA IX was unchanged in human and rat pneumonia, there was a trend toward increasing CA IX in the bronchoalveolar fluid of mechanically ventilated critically ill patients with pneumonia and a significant increase in CA IX in the lung tissue lysates of pneumonia rats. To investigate the functional implications of the lung CA IX increase, we generated PMVEC cell lines harboring domain-specific CA IX mutations. By using these cells, we found that infection promotes intracellular (IC) expression, release, and MMP (metalloproteinase)-mediated extracellular cleavage of CA IX in PMVECs. IC domain deletion uniquely impaired CA IX membrane localization. Loss of the CA IX IC domain promoted cell death after infection, suggesting that the IC domain has an important role in PMVEC survival. We also found that hypoxia improves survival, whereas hypercapnia reverses the protective effect of hypoxia, during infection. Thus, we report 1) that CA IX increases in the lungs of pneumonia rats and 2) that the CA IX IC domain and hypoxia promote PMVEC survival during infection.

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa.

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary tissue during pneumonia results in the production of long-lived cytotoxins that can lead to subsequent end organ failure. We have developed in vitro assays to test the hypothesis that cytotoxins are produced during pulmonary infection. Isolated rat pulmonary endothelial cells and the bacterium Pseudomonas aeruginosa are used as model systems, and the production of cytoxins following infection of the endothelial cells by the bacteria is demonstrated using cell culture followed by direct quantitation using lactate dehydrogenase assays and a novel microscopic method utilizing ImageJ technology. The amyloid nature of these cytotoxins was demonstrated by thioflavin T binding assays and by immunoblotting and immunodepletion using A11 anti-amyloid antibody. Further analyses using immunoblotting demonstrated that oligomeric tau and Aβ were produced and released by endothelial cells following infection by P. aeruginosa. These methods should be readily adaptable to analyses of human clinical samples.

Intrauterine Malnutrition Reduced Long Leptin Receptor Isoform Expression and Proinflammatory Cytokine Production in Male Rat Pulmonary Endothelial Cells Stimulated by Lipopolysaccharide.

Background/Aims We have previously shown that low birth weight (LBW) rats exposed to intrauterine malnutrition have an impaired lung inflammatory response and reduced levels of inflammatory mediators; however, circulating leptin levels were not increased. We evaluated long leptin receptor isoform (ObRb) expression in lung endothelial cells from low birth weight rats and examined its role in the production of lipid mediators and cytokines. Methods Lung endothelial cells were obtained from normal birth weight (NBW) rats or LBW rats subjected to intrauterine malnutrition. These cells were stimulated with leptin (10 ng/mL), LPS (lipopolysaccharide, 1 μg/mL), or leptin plus LPS. Six hours after stimulation, the production of inflammatory mediators (PGE2, LTB4, IL-1β, and IL-6) was evaluated using commercial ELISA kits, and Western blotting was performed to investigate p38MAPK, NF-κB, and ObRb expression. Results Leptin increased IL-1β levels in only cells from the NBW group, whereas LPS increased PGE2 and LTB4 levels in cells from both groups; leptin addition potentiated lipid mediator production induced by LPS in the NBW group. LPS enhanced the production of IL-1β and IL-6 in only endothelial cells from NBW rats. Leptin receptor expression was decreased (63%) in endothelial cells from LBW rats. None of the stimuli increased NF-κB or p38 signaling pathway expression in cells from LBW rats. Conclusion These results suggest that intrauterine malnutrition compromises leptin receptor expression and cytokine production in pulmonary endothelial cells stimulated by LPS; these effects seem to involve the NF-κB and p38MAPK signaling pathways.

Tetrahydropalmatine protects rat pulmonary endothelial cells from irradiation-induced apoptosis by inhibiting oxidative stress and the calcium sensing receptor/phospholipase C-γ1 pathway

The aim of this study was to confirm the protective effect of tetrahydropalmatine (THP) against irradiation-induced rat pulmonary endothelial cell apoptosis and to explore the underlying mechanism, with a focus on the calcium-sensing receptor (CaSR)/phospholipase C-γ1 (PLC-γ1) pathway. We established a model of irradiation-induced primary rat pulmonary endothelial cell injury. Cell apoptosis and mitochondrial membrane potential (Δψm) were measured by flow cytometry. The expression of CaSR, cytochrome c, PLC-γ1, reactive oxygen species (ROS) and [Ca2+]i was also determined. Caspase-3 and caspase-9 activities were measured using commercial kits. Inositol triphosphate (IP3) and the production of inflammatory cytokines were detected by enzyme-linked immunosorbent assay. The results showed that THP significantly inhibited irradiation-induced cell apoptosis and intracellular accumulation of ROS. Pretreatment with THP significantly decreased the expression of CaSR, inhibited the CaSR/PLC-γ1 pathway and subsequent [Ca2+]i overload stimulated by irradiation. THP, NPS2390 (inhibitor of CaSR), U73122 (inhibitor of PLC-γ1) and 2-APB (inhibitor of IP3) further decreased cell apoptosis, along with down-regulation of cytochrome c, caspase-3 and caspase-9 activation, disruption of Δψm and the production of inflammatory cytokines. These findings suggest that THP protects primary rat pulmonary endothelial cells against irradiation-induced apoptosis by inhibiting oxidative stress and the CaSR/PLC-γ1 pathway.

Active trafficking of alpha 1 antitrypsin across the lung endothelium.

The homeostatic lung protective effects of alpha-1 antitrypsin (A1AT) may require the transport of circulating proteinase inhibitor across an intact lung endothelial barrier. We hypothesized that uninjured pulmonary endothelial cells transport A1AT to lung epithelial cells. Purified human A1AT was rapidly taken up by confluent primary rat pulmonary endothelial cell monolayers, was secreted extracellularly, both apically and basolaterally, and was taken up by adjacent rat lung epithelial cells co-cultured on polarized transwells. Similarly, polarized primary human lung epithelial cells took up basolaterally-, but not apically-supplied A1AT, followed by apical secretion. Evidence of A1AT transcytosis across lung microcirculation was confirmed in vivo by two-photon intravital microscopy in mice. Time-lapse confocal microscopy indicated that A1AT co-localized with Golgi in the endothelium whilst inhibition of the classical secretory pathway with tunicamycin significantly increased intracellular retention of A1AT. However, inhibition of Golgi secretion promoted non-classical A1AT secretion, associated with microparticle release. Polymerized A1AT or A1AT supplied to endothelial cells exposed to soluble cigarette smoke extract had decreased transcytosis. These results suggest previously unappreciated pathways of A1AT bidirectional uptake and secretion from lung endothelial cells towards the alveolar epithelium and airspaces. A1AT trafficking may determine its functional bioavailablity in the lung, which could be impaired in individuals exposed to smoking or in those with A1AT deficiency.

Efficient combinatorial approach to isolating rat pulmonary endothelial cell phenotypes

Endothelium from discrete vascular segments within the lung displays phenotypic and functional heterogeneity. However, isolation of segment specific endothelial cells (ECs) is a time and cost intensive process. Thus the aim of this current work was to develop an efficient method for obtaining ECs from rat pulmonary artery (PAECs), pulmonary vein (PVECs) and pulmonary microvasculature (PMVECs). Following surgical removal, vessels were digested using type II collagenase and trypsin. The enzymatically obtained cells were plated in D‐valine/heparin endothelial selective growth media on standard culture dishes. Non‐ECs resistant to the D‐valine media were removed by scraping. Lectin panning and differential cell binding were utilized to remove any remaining contaminating cells. Cell cultures were characterized using an EC marker panel: platelet endothelial cell adhesion molecule 1 (PECAM1), endothelial nitric oxide synthase (eNOS), von Willebrand factor (vWF), vascular endothelial cadherin (VE‐cadherin), and acetylated LDL uptake. Specific lectin binding was used to confirm the segmental origin of the ECs. PAECs bound Helix pomatia and Sambucus nigra, while PMVECs bound Griffonia simplicifolia. This combinatorial approach to isolating rat lung endothelial cells consistently yields multiple cell lines from individual animals. Supported by 5PO1HL066299.

Hypoxia upregulates GLUT‐1 expression in rat pulmonary endothelial cells even in hyperglycemia

Hyperglycemia is common in critical illness and is associated with worse outcome. Why this occurs is not clear. Glucose entry into endothelial cells occurs mainly through the sodium‐independent, non‐insulin regulated, glucose transporter 1 (GLUT‐1). The level of GLUT‐1 expression is a key determinant of how much glucose enters endothelial cells. Hyperglycemia decreases GLUT‐1 expression, whereas hypoxia and inflammation increase expression. In critical illness, where hyperglycemia, hypoxia and inflammation are all present, the net effect on GLUT‐1 expression in pulmonary artery endothelial cells is unknown. Cultured Rat Pulmonary Artery Endothelial Cells (RPAECs) were exposed to hypoxia (5% O2) and either high (20 mmol) or low (5 mmol) glucose, and GLUT‐1 protein expression was determined. Hypoxia (5% oxygen) increased GLUT‐1 protein expression in RPAECs in both high (20 mmol) and low (5 mmol) glucose based on Western blot analysis and immunocytochemistry. Additionally, the hypoxia‐induced upregulation of GLUT‐1 correlated with increased glucose uptake. In conclusion, hypoxia upregulates GLUT‐1 even in hyperglycemia and increases glucose uptake in RPAECs. This may be important in understanding the detrimental effects of hyperglycemia in critical illness. [This work is supported by funding from the NIH/NHLBI grant HL073244 and the NIH training grant HL076125.]

Depletion of the DNA repair enzyme, Ogg1, increases rat pulmonary endothelial cell (PAEC) vulnerability to xanthine oxidase (XO)–induced cytotoxicity

8-oxoguanine (8-oxoG) is prominent among reactive oxygen species (ROS)-induced DNA lesions because it is the predominant oxidized purine base product and exhibits a high mutagenic potential. The DNA glycosylase, Ogg1, is one of several important components of the base excision pathway repairing oxidative DNA damage. We previously showed that over-expression of mitochondrially-targeted Ogg1 in PAECs attenuated apoptotic cell death induced by XO treatment (Am. J. Physiol: Lung Cell Molec. Physiol. 288: L530, 2005), thus suggesting that Ogg1 is potentially a rate-limiting determinant in the mechanism protecting PAECs from ROS-mediated death. To further explore the critical role of Ogg1, the present study tested the hypothesis that depletion of Ogg1 in PAECs increases sensitivity to oxidant-induced death. PAECs transfected with siRNA to Ogg1 or a scrambled siRNA sequence were challenged with ascending doses of XO plus hypoxanthine. In comparison to treatment with the scrambled siRNA sequence, the specific siRNA afforded a significant reduction of Ogg1 activity detected in total cell lysate. Xanthine oxidase-induced PAEC cytotoxicity as well as caspase 3 activation were augmented in cells with reduced Ogg1 activity. These observations suggest that Ogg1 plays a critical, and possibly rate-limiting role in defending PAECs from oxidant-induced apoptosis. Supported by NIH.

Antisense intercellular adhesion molecule-1 (ICAM-1) oligodeoxyribonucleotide delivered during organ preservation inhibits posttransplant ICAM-1 expression and reduces primary lung isograft failure.

Transiently increased expression of leukocyte adhesion receptors after lung preservation contributes to early graft demise by recruiting leukocytes, activating complement, and causing microcirculatory stasis. We hypothesized that inhibiting intercellular adhesion molecule-1 (ICAM-1) expression even briefly may significantly improve lung graft function and that the preservation period might provide a unique window to deliver a therapeutic pulse of antisense oligonucleotide ICAM-1 to inhibit ICAM-1 expression after transplantation. Interleukin-1beta-treated rat pulmonary endothelial cells given a 20-mer phosphorothioate oligonucleotide comprising an antisense span targeted to the 3'-untranslated region of rat ICAM-1 demonstrated an oligonucleotide dose-dependent reduction in ICAM-1 expression. Using a cationic liposomal carrier, this same antisense oligonucleotide (but not the sense control) instilled into the pulmonary vasculature at the time of preservation reduced subsequent graft ICAM-1 expression and graft leukostasis and markedly improved oxygenation, pulmonary blood flow, and graft survival. These experiments demonstrate that the preservation period presents a window during which to target an anti-ICAM-1 expression strategy to inhibit early adhesion receptor expression and improve functional outcome after lung transplantation.

Endothelin-1 inhibits endothelin-converting enzyme-1 expression in cultured rat pulmonary endothelial cells.

The lung expresses large amounts of endothelin-converting enzyme-1 (ECE-1), which catalyzes a step in the biosynthesis of potent vasoactive endothelin-1 (ET-1) from the inactive intermediate big ET-1. Because there has been no report concerning a possible relationship between ET-1 and ECE-1, we investigated the effects of ET-1 on ECE-1 expression in cultured rat pulmonary endothelial cells. ECE-1 messenger RNA (mRNA) and protein expression in cultured endothelial cells were assayed by Northern and Western blotting, respectively. Incubation with ET-1 for 6 hours caused a significant decrease in ECE-1 mRNA expression. The action of ET-1 on ECE-1 mRNA expression was antagonized by pretreatment with BQ788, a specific ETB receptor antagonist, but not by pretreatment with BQ123, a specific ETA receptor antagonist. The expression of ECE-1 protein was also inhibited at 6 hours after incubation with ET-1. The effects of ET-1 on ECE-1 mRNA and protein expression were shown to be mimicked by ionomycin, a calcium ionophore, but not by 12-O-tetradecanoylphorbol 13-acetate, a protein kinase C activator. The present results demonstrate that ET-1 suppressed ECE-1 protein levels by inhibiting ECE-1 mRNA expression through the ETB receptor, suggesting the existence of a feedback action of ET-1 on ECE-1 in pulmonary endothelial cells.

Oxidant stress regulates basal endothelin-1 production by cultured rat pulmonary endothelial cells.

Endothelin-1 (ET-1) is a pluripotent mediator that modulates vascular tone and influences the inflammatory response. Patients with inflammatory lung disorders frequently have elevated circulating ET-1 levels. Because these pathophysiological conditions generate reactive oxygen species that can regulate gene expression, we investigated whether the level of oxidant stress influences ET-1 production in cultured rat pulmonary arterial endothelial cells (RPAEC). Treatment with the antioxidant 1,3-dimethyl-2-thiourea (10 mM) or the iron chelator deferoxamine (1.8 microM) doubles basal ET-1 release. Conversely, exposing cells to H2O2 generated by glucose and glucose oxidase (0.1-10 mU/ml) for 4 h causes a concentration-dependent decrease in ET-1 release. This effect occurs at concentrations of glucose oxidase that do not affect [3H]leucine incorporation or specific 51Cr release from RPAEC. Catalase prevents the decrease in ET-1 synthesis caused by glucose and glucose oxidase. Glucose and glucose oxidase decrease not only ET-1 generation but also ET-1 mRNA as assessed by semiquantitative polymerase chain reaction. Our results indicate that changes in oxidative stress can either up- or downregulate basal ET-1 generation by cultured pulmonary endothelial cells.

Effects of vasoactive and inflammatory mediators on endothelin-1 expression in pulmonary endothelial cells.

Endothelin-1 (ET-1) is known to be involved in a variety of pathophysiologic conditions, especially of the pulmonary vasculature. The aim of this study was to investigate physiologic mediators potentially involved in the pathogenesis of pulmonary hypertension, for their effects on ET-1 gene expression at both the transcriptional and translational level. Rat microvascular and pulmonary artery endothelial cells grown in culture were exposed to vasoactive mediators (thrombin or an anoxic gas mixture) and inflammatory mediators (lipopolysaccharide, interleukin 1 alpha, interleukin 1 beta, or tumor necrosis factor alpha) for various time periods. The change in prepro-ET-1 (ppET-1) mRNA levels in these cells in response to stimuli was a time-dependent phenomenon. The inflammatory mediators caused an acute rise in ppET-1 mRNA levels whereby peak induction occurred after 1 h with a rapid decline to control levels by 4 h. The vasoactive mediators elicited a more sustained response whereby a significant elevation in ppET-1 mRNA expression occurred quickly and remained elevated through 4 h. The pattern of induction was more rapid for thrombin than for anoxic gas exposure. Radioimmunoassay analysis demonstrated a similar response for thrombin and the inflammatory mediators in ET-1 mature peptide release, whereas the effect of anoxic gas exposure was divergent. Significant elevations were noted after 6 h for thrombin as well as each of the inflammatory mediators except IL-1 alpha. In response to the anoxic gas exposure, however, a significant rise in ET-1 peptide release was not evident until after 24 h. To determine the level at which ppET-1 mRNA induction is regulated, cells were cotreated with each of the stimuli and actinomycin D or cycloheximide. Results indicate that the induction of ppET-1 mRNA levels is likely due to de novo transcription, as well as mRNA stabilization. In summary, inflammatory and vasoactive agents are important regulators of ET-1 gene expression in rat pulmonary endothelial cells; most important, we observed a differential response at the mRNA or peptide level depending on the mediator involved.

The hsp56 immunophilin component of untransformed steroid receptor complexes is localized both to microtubules in the cytoplasm and to the same nonrandom regions within the nucleus as the steroid receptor.

In their unliganded state, mouse glucocorticoid receptors (GR) that are overexpressed in the WCL2 line of Chinese hamster ovary cells are distributed in a nonrandom manner throughout all planes of the nucleus. These untransformed nuclear receptors exist in a heterocomplex containing three heat shock proteins, hsp90, hsp70, and hsp56, the latter being an immunophilin of the FK506 binding type whose cellular function is unknown. Because a knowledge of the cellular distribution of hsp56 could provide important clues to its function in steroid-receptor heterocomplexes, we have examined hsp56 localization in intact cells by indirect immunofluorescence using the UPJ56 antibody. The majority of hsp56 is located in the nucleus, with substantial amounts also visualized in the cytoplasm of intact cells. The cytoplasmic hsp56 was examined in rat pulmonary endothelial cells where the protein was found to colocalize with microtubules. The nuclear hsp56 was examined in the WCL2 cells, where the protein was found by confocal imaging to colocalize throughout all planes of the nucleus in the same mottled pattern as the overexpressed GR. Like the GR, the nuclear hsp56 is recovered largely in the cytosolic fraction after hypotonic rupture of WCL2 cells. An observation potentially related to the microtubule-associated fraction of hsp56 is that immunoadsorption of hsp56 from WCL2 cytosol is accompanied by coadsorption of the microtubule-associated protein-1C complex. These observations are discussed with respect to the possible biological functions of hsp56 in the folding and/or cytoplasmic-nuclear trafficking of the receptor.

Regulation of rat pulmonary endothelial cell interleukin-6 production by bleomycin: effects of cellular fatty acid composition.

Previous studies have shown upregulation of lung cell interleukin-6 (IL-6) production in bleomycin-induced pulmonary fibrosis. To further elucidate the regulatory mechanisms governing this disease, the effects of bleomycin on the production of the pleiotropic cytokine, IL-6, were investigated in lung endothelial cells. Rat pulmonary artery endothelial cells were treated with bleomycin at doses previously shown to be effective in upregulating cytokine production in these cells, and the conditioned media was collected and assayed for IL-6 activity. The results show that these endothelial cells constitutively produced IL-6 and that bleomycin increased the production in a time- and dose-dependent manner. Feeding rats diets deficient in n-6 fatty acids is known to ameliorate bleomycin-induced lung fibrosis. In order to examine if fatty acids could modulate IL-6 production in vitro, cells were lipid depleted and then supplemented with 18:1n-9, 18:2n-6, or 18:3n-3 fatty acids, and the effects of bleomycin on IL-6 production reexamined. This regimen resulted in significant depletion of arachidonate in the 18:1n-9 and 18:3n-3 supplemented cells, which was associated with significantly reduced IL-6 production relative to the 18:2n-6-supplemented cells, both constitutively and when stimulated with bleomycin. Preincubation with indomethacin did not significantly inhibit the production of IL-6 by all three groups of cells, nor did supplementation with a stable prostacyclin analog increase IL-6 production. These results suggest that endothelial cell IL-6 production is not directly dependent on prostacyclin or other cyclooxygenase metabolites but may require or be upregulated by 18:2n-6 and/or metabolites derived from it.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of manganese superoxide dismutase: IL-1 and TNF induction in pulmonary artery and microvascular endothelial cells

IL-1 and TNF are important mediators in the inflammatory response, and have been associated with endothelial cell damage in the lung. TNF and IL-1 cell-mediated injury has been proposed to occur through an increase in intracellular oxygen free radical production. However, these cytokines have also been shown to protect the lung from hyperoxia-mediated oxidant injury. In this paper we evaluated the response of the antioxidant enzymes, MnSOD and Cu/ZnSOD to IL-1, TNF, and LPS in both rat pulmonary artery and microvascular endothelial cells. These mediators produced an increase in MnSOD but not Cu/ZnSOD expression in both rat pulmonary endothelial cells. An additive effect was observed with co-treatment by the cytokines with LPS. The MnSOD mRNA induction is dependent upon a transcriptional event, but did not require de novo protein synthesis.

Regulation of xanthine dehydrogenase and xanthine oxidase activity and gene expression in cultured rat pulmonary endothelial cells.

The central importance of xanthine dehydrogenase (XDH) and xanthine oxidase (XO) in the pathobiochemistry of a number of clinical disorders underscores the need for a comprehensive understanding of the regulation of their expression. This study was undertaken to examine the effects of cytokines on XDH/XO activity and gene expression in pulmonary endothelial cells. The results indicate that IFN-gamma is a potent inducer of XDH/XO activity in rat lung endothelial cells derived from both the microvasculature (LMVC) and the pulmonary artery. In contrast, interferon-alpha/beta, tumor necrosis factor-alpha, interleukin-1 or -6, lipopolysaccharide and phorbol myristate acetate have no demonstrable effect. The increase in XDH/XO activity requires new protein synthesis. By Northern analysis, IFN-gamma markedly increases the level of the 5.0-kb XDH/XO mRNA in LMVC. The increase is due, in part, to increased transcription rate of the XDH/XO gene. Transcriptional activation does not require new protein synthesis. The physiologic relevance of these observations was evaluated by administering IFN-gamma to rats. Intraperitoneal administration leads to an increased XDH/XO activity and XDH/XO mRNA level in rat lungs. In sum, IFN-gamma is a potent and biologically relevant inducer of XDH/XO expression; the major site of upregulation occurs at the transcriptional level.

Recombinant gene expression in pulmonary vascular endothelial cells: polarized secretion in vivo

A unique, spontaneously immortalized rat pulmonary endothelial cell line was transfected with a human growth hormone (hGH) fusion gene generating a line of stably transfected cells that expresses high levels of hGH (Ec/XGH-1). These cells produced significant serum levels of hGH when implanted intraperitoneally, subcutaneously, or intravenously into nude mice. Implantation of Ec/XGH-1 cells under the renal capsule resulted in the formation of large cysts that contained concentrations of hGH that were several thousand times greater than those in serum assayed simultaneously from the same animals. This study presents a new technique for in vivo gene expression using a convenient line of pulmonary vascular endothelial cells as gene carriers. In addition, this system demonstrates the special physiological features of transfected endothelial cells in forming large cysts.

Regulation of Manganese superoxide dismutase (MnSOD) in rat pulmonary endothelial cells by inflammatory mediators

Regulation of Manganese superoxide dismutase (MnSOD) in rat pulmonary endothelial cells by inflammatory mediators