Oleic acid supplementation reduces oxidant-mediated dysfunction of cultured porcine pulmonary artery endothelial cells

We have previously shown that supplementing cultured porcine pulmonary artery endothelial cells (PAEC) with exogenous oleic acid (18:1ω9) alters the fatty acid composition of the cells and reduces oxidant-mediated cytotoxicity. Because the mechanisms by which lipid alterations modulate oxidant susceptibility have not been defined, the ability of 18:1 to reduce hydrogen peroxide (H₂O₂)-mediated PAEC dysfunction was evaluated. PAEC monolayers on polycarbonate filters were incubated for 3 h in maintenance medium supplemented with either 0.1 mM 18.1 in ethanol vehicle (ETOH) or with an equivalent volume of vehicle alone. Twenty-four hours later monolayers were treated for 30 min with 50 or 100 μM H₂O₂ in Hanks' balanced salt solution (HBSS) or with HBSS alone (nonoxidant control). As a functional index of PAEC monolayer integrity, the permeability of monolayers to albumin was then measured for 3 h. Treatment with 100 μM H₂O₂ caused cytotoxicity and progressive increases in PAEC monolayer permeability that were attenuated by 18:1 supplementation, whereas 50 μM H₂O₂ caused only a transient increase in permeability without cytotoxicity. Supplementation with 18:1 also attenuated H₂O₂-induced reductions in PAEC adenosine triphosphate (ATP) content and disruption of PAEC microfilament architecture. The ATP content of PAEC monolayers was reversibly reduced in the absence of oxidant stress by incubation with glucose-depleted medium containing deoxyglucose and antimycin A. Metabolic inhibitor-induced ATP depletion increased monolayer permeability and altered cytoskeletal architecture, alterations that resolved during recovery of PAEC ATP content. These results demonstrate that ATP depletion plays a critical role in barrier dysfunction and suggests that the ability of 18:1 to reduce oxidant-mediated PAEC dysfunction and injury may relate directly to its ability to preserve PAEC ATP content. © 1993 Wiley-Liss, Inc.     

Epidermal growth factor-like domain 7 protects endothelial cells from hyperoxia-induced cell death

Hyperoxia is one of the major contributors to the development of bronchopulmonary dysplasia (BPD), a chronic lung disease in premature infants. Emerging evidence suggests that the arrested lung development of BPD is associated with pulmonary endothelial cell death and vascular dysfunction resulting from hyperoxia-induced lung injury. A better understanding of the mechanism of hyperoxia-induced endothelial cell death will provide critical information for the pathogenesis and therapeutic development of BPD. Epidermal growth factor-like domain 7 (EGFL7) is a protein secreted from endothelial cells. It plays an important role in vascular tubulogenesis. In the present study, we found that Egfl7 gene expression was significantly decreased in the neonatal rat lungs after hyperoxic exposure. The Egfl7 expression was returned to near normal level 2 wk after discounting oxygen exposure during recovery period. In cultured human endothelial cells, hyperoxia also significantly reduced Egfl7 expression. These observations suggest that diminished levels of Egfl7 expression might be associated with hyperoxia-induced endothelial cell death and lung injury. When we overexpressed human Egfl7 (hEgfl7) in EA.hy926 human endothelial cell line, we found that hEgfl7 overexpression could partially block cytochrome c release from mitochondria and decrease caspase-3 activation. Further Western blotting analyses showed that hEgfl7 overexpression could reduce expression of a proapoptotic protein, Bax, and increase expression of an antiapoptotic protein, Bcl-xL. Theses findings indicate that hEGFL7 may protect endothelial cell from hyperoxia-induced apoptosis by inhibition of mitochondria-dependent apoptosis pathway.     

The DNA glycosylase Ogg1 defends against oxidant-induced mtDNA damage and apoptosis in pulmonary artery endothelial cells

Emerging evidence suggests that mitochondrial (mt) DNA damage may be a trigger for apoptosis in oxidant-challenged pulmonary artery endothelial cells (PAECs). Understanding the rate-limiting determinants of mtDNA repair may point to new targets for intervention in acute lung injury. The base excision repair (BER) pathway is the only pathway for oxidative damage repair in mtDNA. One of the key BER enzymes is Ogg1, which excises the base oxidation product 8-oxoguanine. Previously we demonstrated that overexpression of mitochondrially targeted Ogg1 in PAECs attenuated apoptosis induced by xanthine oxidase (XO) treatment. To test the idea that Ogg1 is a potentially rate-limiting BER determinant protecting cells from oxidant-mediated death, PAECs transfected with siRNA to Ogg1 were challenged with XO and the extent of mitochondrial and nuclear DNA damage was determined along with indices of apoptosis. Transfected cells demonstrated significantly reduced Ogg1 activity, which was accompanied by delayed repair of XO-induced mtDNA damage and linked to increased XO-mediated apoptosis. The nuclear genome was undamaged by XO in either control PAECs or cells depleted of Ogg1. These observations suggest that Ogg1 plays a critical and possibly rate-limiting role in defending PAECs from oxidant-induced apoptosis by limiting the persistence of oxidative damage in the mitochondrial genome.     

Ghrelin protects human pulmonary artery endothelial cells against hypoxia-induced injury via PI3-kinase/Akt

Endothelial injury and diminished NO release induced by hypoxia is thought to be a critical factor in the development of pulmonary artery hypertension (PAH). Ghrelin (Ghr) is a well-characterized hormone and has protective effects on the cardiovascular system, specifically by promoting the vascular endothelial cell function. The aim of this study was to investigate the effect of the Ghr on the hypoxia-induced injury in human pulmonary artery endothelial cells (HPAECs) and on the involved transduction pathway. Effects were investigated by treating cells with varying concentrations of Ghr in the absence or presence of inhibitors that target phosphoinositide 3-kinase (PI3K), in normoxic or hypoxic conditions for 24 h. Our results indicated that the treatment with 10⁻⁷ mol/l Ghr significantly enhanced cell viability (P < 0.05, n = 5) and upregulated the ratio of Bcl-2/Bax under hypoxic condition (P < 0.05, n = 4), as compared with the hypoxic condition alone. However, an addition of the PI3K/Akt inhibitor LY294002 inhibited these Ghr-mediated effects. Moreover, the Ghr (10⁻⁷ mol/l) significantly increased NO secretion and eNOS phosphorylation in comparison with the hypoxia or normoxia alone group (P < 0.05, n = 4). Nevertheless, the treatment with LY294002 (20 μ mol/l) decreased the Ghr-induced NO release as well as the eNOS activity. In conclusion, the Ghr could inhibit hypoxia-mediated HPAECs dysfunction via the PI3K/Akt pathway, and the bcl-2/bax ratio was also involved in the protective action of the Ghr in HPAECs. As such, the Ghr demonstrates a significant potential to prevent and treat PAH affected by the endothelial dysfunction.     

Pigment epithelium-derived factor protects retinal pigment epithelium from oxidant-mediated barrier dysfunction

Retinal pigment epithelium (RPE) cells form a monolayer at the blood-retina barrier between the retina and choriocapillaries. The barrier function may be damaged by multiple stresses to the cell, including the repeated exposure to oxidants that are generated by photoreceptor cell turnover. The purpose of our study was to document the protective effect of pigment epithelium-derived factor (PEDF), a tropic factor produced by the RPE, on H(2)O(2)-induced RPE barrier dysfunction. When assayed by a FITC-labeled dextran transepithelial flux, the increased permeability of the RPE barrier (induced by H(2)O(2)) was prevented by PEDF pretreatment. To further explore the mechanism leading to this permeability change, we investigated the distribution of cytoskeleton and junctional proteins. The redistribution of the two junctional proteins occludin, and N-cadherin and actin reorganization in RPE, induced by H(2)O(2), can be prevented by PEDF pretreatment. PEDF can also prevent H(2)O(2)-induced stress kinase p38/27-kDa heat shock protein signaling which is known to mediate actin rearrangement. These findings indicated that PEDF can stabilize actin, maintain normal membrane occludin and N-cadherin structure, and preserve the barrier function of RPE cells against oxidative stress.     

Collagen synthesis by cloned pulmonary artery endothelial cells

Pulmonary artery endothelial cells were isolated from bovine fetal blood vessels and used for biosynthetic studies. At confluence, cultures were incubated in minimal essential medium (MEM) without serum containing [U-14C]proline. After 24 hours, medium was removed and labeled proteins were precipitated by the addition of ammonium sulfate and fractionated by diethylaminoethyl (DEAE)-cellulose chromatography. The elution profile showed four major peaks and one minor peak. Fractions within each peak were pooled, subjected to digestion by chymotrypsin and/or collagenase, and analyzed by polyacrylamide gel electrophoresis. Peak l contained a collagen which contained approximately 6% of the 3-hydroxyproline isomer while total hydroxyproline content was approximately 45%. This material was digested by purified bacterial collagenase and had a mobility slightly slower than that of alpha 1(III) which did not change under conditions that reduce disulfide bonds. Upon digestion with chymotrypsin under conditions where native procollagens are converted to alpha-chains, this material was digested. These properties suggest that this material is type VIII or EC (endothelial cell) collagen. Peak 2 contained substantial fibronectin while peak 3 contained primarily type III procollagen. The last major peak contained a mixture of collagenous and noncollagenous material. Upon digestion with chymotrypsin, several peptides were generated which were sensitive to bacterial collagenases. The two major chymotrypsin-resistant components had mobilities slower than that of alpha(III) and were not disulfide-bonded.     

Role of the Na/H antiport in pH-dependent cell death in pulmonary artery endothelial cells

We investigated the role of intracellular pH (pH(i)) and Na/H exchange in cell death in human pulmonary artery endothelial cells (HPAEC) following a metabolic insult (inhibition-oxidative phosphorylation, glycolysis). Metabolic inhibition in medium at pH 7. 4 decreased viability (0-15% live cells) over 6 h. Cell death was attenuated by maneuvers that decreased pH(i) and inhibited Na/H exchange (acidosis, Na/H antiport inhibitors). In contrast, cell death was potentiated by maneuvers that elevated pH(i) or increased Na/H exchange (monensin, phorbol ester treatment) before the insult. HPAEC demonstrated a biphasic pH(i) response following a metabolic insult. An initial decrease in pH(i) was followed by a return to baseline over 60 min. Maneuvers that protected HPAEC and inhibited Na/H exchange (acidosis, Na(+)-free medium, antiport inhibitors) altered this pattern. pH(i) decreased, but no recovery was observed, suggesting that the return of pH(i) to normal was mediated by antiport activation. Although Na/H antiport activity was reduced (55-60% of control) following a metabolic insult, the cells still demonstrated active Na/H exchange despite significant ATP depletion. Phorbol ester pretreatment, which potentiated cell death, increased Na/H antiport activity above the level observed in monolayers subjected to a metabolic insult alone. These results demonstrate that HPAEC undergo a pH-dependent loss of viability linked to active Na/H exchange following a metabolic insult. Potentiation of cell death with phorbol ester treatment suggests that this cell death pathway involves protein kinase C-mediated phosphorylation events.     

Elastin production by cultured calf pulmonary artery endothelial cells

Calf pulmonary artery (CPA) endothelial cells synthesize and secrete soluble elastin when incubated in medium conditioned by arterial smooth muscle cells. Endothelial cell tropoelastin cross-reacts with antiserum to bovine ligamentum nuchae elastin and comigrates on SDS-PAGE with tropoelastins from fetal bovine ligamentum nuchae fibroblasts, aortic smooth muscle cells, and ear chondroblasts at an apparent molecular weight of 70,000. Endothelial cells synthesize only one-third as much elastin as these other cell types, however. Approximately 80% of the elastin synthesized by endothelial cells in confluent culture is released into the culture medium. The remaining 20% remains associated with the cell layer and is readily extractable with dilute acetic acid as un-cross-linked, 70,000-dalton tropoelastin. The addition of beta-aminopropionitrile to culture medium did not alter the ratio of tropoelastin in the medium and cell layer, suggesting that cross-linking of tropoelastin does not occur in culture. Immunofluorescent staining of confluent endothelial cell cultures with antielastin serum demonstrated elastin occurring as a web-like network of fine filaments extending throughout the extracellular space. The fibrous elastin was different in organization and distribution from fibers stained with antifibronectin serum, which were localized primarily beneath the cell layer and in regions of cell-cell contact. Extracellular matrix remaining after solubilization of cellular material with Triton X-100 stained positive for fibronectin, but not for elastin.     

Effects of biaxial deformation on pulmonary artery endothelial cells

An apparatus has been designed to subject vascular cells grown on a compliant substrate in vitro to uniform, quantifiable levels of biaxial deformation. The system described can be controlled with respect to strain level, rate, and frequency to mimic the pulsatile force to which vascular cells are exposed in vivo under both physiologic and pathologic conditions. In the experiments presented here, bovine pulmonary artery endothelial cells were grown on a substrate of segmented polyurethane urea (Mitrathane). Cell growth and morphology on this substrate were compared with those of cells grown on standard tissue culture polystyrene with no difference noted between the two substrates. Primary cultures of pulmonary artery endothelial cells were seeded onto Mitrathane, which was then subjected to cyclic biaxial deformation-producing strains of 0.78%, 1.76%, 4.9%, or 12.5% at a frequency of 1 sec-1 and a duty cycle of 0.5 sec-1 for 7 h. Cells subjected to deformations generating strains of either 4.9% or 12.5% secreted significantly less fibronectin than nondeformed cells. Similar results were obtained in experiments using cloned pulmonary artery endothelial cells on Mitrathane subjected to the 4.9% strain; however, total protein synthesis was increased. Cell viability and DNA synthesis were not affected by cyclic biaxial deformation in these experiments.     

Isolation and culture of pulmonary artery endothelial cells.

It has become increasingly evident that endothelial cells function as far more than a mechanical barrier between blood and parenchyma. Endothelial cells from one vesicular bed are known to differ structurally from those of another, and it has been suggested that they may differ functionally. Further to test the hypothesis that endothelial cells from one site may differ in terms of function from those of another site, it is necessary to test endothelium from various source after having obtained these cells in pure, well-characterized cultures. To facilitate such studies, we herein describe in detail means for the isolation, culture and characterization of endothelial cells from calf pulmonary artery. These cells may be of major interest in terms of specific metabolic activities as it has become evident that the lungs play a prominent role in determining the hormonal composition of systemic arterial blood.     

Alkaline stress-induced apoptosis in human pulmonary artery endothelial cells

The effect of alkaline stress, or an increase in extracellular pH (pHext), on cell viability is poorly defined. Human pulmonary artery endothelial cells (HPAEC) were subjected to alkaline stress using different methods of increasing pHext. Viability and mode of cell death following alkaline stress were determined by assessing nuclear morphology, ultrastructural features, and caspase-3 activity. Incubation of monolayers in media set to different pHext values (7.4–8.4) for 24-h induced morphological changes suggesting apoptosis (35–45% apoptotic cells) following severe alkaline stress. The magnitude of apoptosis was related to the severity of alkaline stress. These findings were confirmed with an assessment of ultrastructural changes and caspase-3 activation. While there was no difference in the intracellular calcium level ([Ca²⁺]_i) in monolayers set to pHext 7.4 versus 8.4 following the first hour of alkaline stress, blockade of calcium uptake with the chelator, EGTA, potentiated the magnitude of apoptosis under these conditions. Potentiation of apoptosis was reduced by calcium supplementation of the media. Finally, alkaline stress was associated with an increase in intracellular pH. This is the first report of apoptosis following alkaline stress in endothelial cells in the absence of other cell death stimuli.     

Zinc-related metallothionein metabolism in bovine pulmonary artery endothelial cells

Bovine pulmonary artery endothelial cells (BPAEC) were cultured in vitro under a variety of conditions to investigate how metallothionein (MT) might participate in zinc homeostasis. Experimental conditions included 10% serum to ensure that the in vitro environment would be a better reflection of the in vivo situation than with protein-free medium. MT was increased by acutely high zinc concentrations (100-200 micromol/L) in the extracellular environment. MT was relatively insensitive to moderate changes in zinc concentration (2-50 micromol/L), even after prolonged exposure for 7 to 12 days. BPAEC had reduced MT content when grown in medium containing serum that had been dialyzed to remove components with a molecular mass of less than 1,000, including zinc. Because the principal source of the major minerals in the experimental medium was not the serum, their concentrations in the final medium were not significantly influenced by serum dialysis. Restoring the zinc concentration in the medium containing the dialyzed serum did not restore MT content in BPAEC, suggesting that some small molecular weight molecule other than zinc established their basal MT content. This study did not identify these putative factors in serum, but hormones are likely candidates. Forty-eight-hour incubations of BPAEC with interleukin (IL-6) or dexamethasone increased cellular MT; however, 17beta-estradiol decreased MT, and IL-1 and adenosine 3',5'-cyclic phosphate (cAMP) had no discernible effect. We conclude that extracellular zinc concentrations have relatively little impact on the cellular concentrations of MT and zinc of BPAEC in vitro. Zinc homeostasis by BPAEC is not maintained by changing the MT concentration in response to changes in the extracellular zinc environment. (J. Nutr. Biochem. 10:00-00, 1999).     

8,9-Epoxyeicosatrienoic acid analog protects pulmonary artery smooth muscle cells from apoptosis via ROCK pathway

Epoxyeicosatrienoic acids (EETs), metabolites of arachidonic acid (AA) catalyzed by cytochrome P450 (CYP), have many essential biologic roles in the cardiovascular system including inhibition of apoptosis in cardiomyocytes. In the present study, we tested the potential of 8,9-EET and derivatives to protect pulmonary artery smooth muscle cells (PASMCs) from starvation induced apoptosis. We found 8,9-epoxy-eicos-11(Z)-enoic acid (8,9-EET analog (214)), but not 8,9-EET, increased cell viability, decreased activation of caspase-3 and caspase-9, and decreased TUNEL-positive cells or nuclear condensation induced by serum deprivation (SD) in PASMCs. These effects were reversed after blocking the Rho-kinase (ROCK) pathway with Y-27632 or HA-1077. Therefore, 8,9-EET analog (214) protects PASMC from serum deprivation-induced apoptosis, mediated at least in part via the ROCK pathway. Serum deprivation of PASMCs resulted in mitochondrial membrane depolarization, decreased expression of Bcl-2 and enhanced expression of Bax, all effects were reversed by 8,9-EET analog (214) in a ROCK dependent manner. Because 8,9-EET and not the 8,9-EET analog (214) protects pulmonary artery endothelial cells (PAECs), these observations suggest the potential to differentially promote apoptosis or survival with 8,9-EET or analogs in pulmonary arteries.     

Rad9 protects cells from topoisomerase poison-induced cell death

Previous studies have suggested two possible roles for Rad9 in mammalian cells subjected to replication stress or DNA damage. One model suggests that a Rad9-containing clamp is loaded onto damaged DNA, where it participates in Chk1 activation and subsequent events that contribute to cell survival. The other model suggests that Rad9 translocates to mitochondria, where it triggers apoptosis by binding to and inhibiting Bcl-2 and Bcl-x(L). To further study the role of Rad9, parental and Rad9(-/-) murine embryonic stem (ES) cells were treated with camptothecin, etoposide, or cytarabine, all prototypic examples of three classes of widely used anticancer agents. All three agents induced Rad9 chromatin binding. Each of these agents also triggered S-phase checkpoint activation in parental ES cells, as indicated by a caffeine-inhibitable decrease in [3H]thymidine incorporation into DNA and Cdc25A down-regulation. Interestingly, the ability of cytarabine to activate the S-phase checkpoint was severely compromised in Rad9(-/-) cells, whereas activation of this checkpoint by camptothecin and etoposide was unaltered, suggesting that the action of cytarabine is readily distinguished from that of classical topoisomerase poisons. Nonetheless, Rad9 deletion sensitized ES cells to the cytotoxic effects of all three agents, as evidenced by enhanced apoptosis and diminished colony formation. Collectively, these results suggest that the predominant role of Rad9 in ES cells is to promote survival after replicative stress and topoisomerase-mediated DNA damage.     

Transforming growth factor-beta1 protects against pulmonary artery endothelial cell apoptosis via ALK5

Transforming growth factor (TGF)-beta1 has been reported to cause endothelial cell apoptosis. However, conflicting data have also demonstrated that TGF-beta1 promotes endothelial cell survival. In this study, the effect of TGF-beta1 on apoptosis of cultured bovine pulmonary artery endothelial cells (PAEC) induced by multiple stimuli was investigated. TGF-beta1 protected against apoptosis of bovine PAEC induced by serum deprivation or the VEGF receptor inhibitor SU-5416, but not by UV light exposure or TNFalpha. Neither caspase-8 nor caspase-12 was activated by serum deprivation or the VEGF receptor blocker. However, blockade of VEGF receptors activated caspase-9, an effect that was abolished by TGF-beta1. Furthermore, serum deprivation and inhibition of VEGF receptors significantly decreased the protein level of Bcl-2, an effect that was also abrogated by TGF-beta1. In addition, the baseline level of Bcl-2 was enhanced by TGF-beta1 and reduced by inhibition of activin receptor-like kinase 5 (ALK5), a TGF-beta1 type I receptor. Furthermore, inhibition of ALK5 caused apoptosis of bovine PAEC. These results suggest that TGF-beta1 signaling is critical for maintenance of bovine PAEC survival. Finally, the protective effects of TGF-beta1 on bovine PAEC apoptosis and Bcl-2 reduction were abolished by ALK5 inhibition, but not by inhibition of non-SMAD signaling pathways. Also, TGF-beta1 activated SMAD2 and SMAD1/5, an effect that was abolished by ALK5 inhibition. The results of this study suggest that TGF-beta1 protects against bovine PAEC apoptosis, possibly through ALK5-mediated Bcl-2 induction and subsequent inhibition of the mitochondria-mediated intrinsic pathway of apoptosis. Understanding the mechanism by which TGF-beta1 promotes endothelial cell survival may provide a better treatment for apoptosis-dependent vascular diseases, such as emphysema.     

Protein targets of monocrotaline pyrrole in pulmonary artery endothelial cells

A single administration of monocrotaline to rats results in pathologic alterations in the lung and heart similar to human pulmonary hypertension. In order to produce these lesions, monocrotaline is oxidized to monocrotaline pyrrole in the liver followed by hematogenous transport to the lung where it injures pulmonary endothelium. In this study, we determined specific endothelial targets for (14)C-monocrotaline pyrrole using two-dimensional gel electrophoresis and autoradiographic detection of protein metabolite adducts. Selective labeling of specific proteins was observed. Labeled proteins were digested with trypsin, and the resulting peptides were analyzed using matrix-assisted laser desorption ionization mass spectrometry. The results were searched against sequence data bases to identify the adducted proteins. Five abundant adducted proteins were identified as galectin-1, protein-disulfide isomerase, probable protein-disulfide isomerase (ER60), beta- or gamma-cytoplasmic actin, and cytoskeletal tropomyosin (TM30-NM). With the exception of actin, the proteins identified in this study have never been identified as potential targets for pyrroles, and the majority of these proteins have either received no or minimal attention as targets for other electrophilic compounds. The known functions of these proteins are discussed in terms of their potential for explaining the pulmonary toxicity of monocrotaline.     

Osteoprotegerin protects endothelial cells against apoptotic cell death induced by Porphyromonas gingivalis cysteine proteinases

Osteoprotegerin (OPG) is a key regulator of osteoclastogenesis during the progression of periodontitis. Recent reports suggest that osteoprotegerin may also prevent arterial calcification and contribute to endothelial cell survival. To determine whether the vascular functions of osteoprotegerin are involved in periodontitis, we examined whether osteoprotegerin contributed to the survival of endothelial cells damaged by Porphyromonas gingivalis cysteine proteinases (gingipains). Gingipain proteinases cleave a broad range of host proteins, and are important virulence factors of P. gingivalis, a major causative bacterium of adult periodontitis. Human microvascular endothelial cells (HMVEC) were exposed to activated gingipain extracts from P. gingivalis 381, with and without pretreatment with osteoprotegerin. Cell viability was quantified by the tetrazolium (WST-8) reduction assay, and apoptosis was examined using Hoechst 33342 nuclear staining. After 16 h of treatment with activated gingipain extracts, HMVEC showed near-complete detachment from the tissue culture dish, and apoptosis was evident by 24 h. Pretreatment of HMVEC with osteoprotegerin reduced the extent of both cellular detachment and apoptotic cell death. Our results indicated that osteoprotegerin pretreatment protected HMVEC against detachment and apoptotic cell death induced by gingipain-active bacterial cell extracts. These results also suggest that osteoprotegerin may function as a survival factor for endothelial cells during periodontitis.