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.     

Protein targets of 1,4-benzoquinone and 1,4-naphthoquinone in human bronchial epithelial cells

Many aspects of the toxicity of xenobiotic compounds have been attributed to the consequences of covalent modification of specific proteins, but the nature and specificity of protein targets for classes of electrophilic toxins remain largely uncharacterized. For inhaled toxicants, the point of exposure or absorption lies with epithelial cells lining the pulmonary tree. In this study, abundant proteins in human bronchial epithelial cells that are arylated in vitro by two quinonoid compounds, 1,4-benzoquinone (BQ) and 1,4-naphthoquinone (NQ) have been detected using (14)C-labeled quinones and two-dimensional gel electrophoresis. These proteins were identified using matrix assisted laser desorption/ionization mass spectrometry for tryptic mass mapping followed by sequence database searching. Corroborative identification of protein targets was obtained from the apparent isoelectric points, molecular weights, and the use of antibody probes. There were subtle differences in the protein targets of BQ and NQ, but both associated with the following abundant proteins, nucleophosmin, galectin-1, probable protein disulfide isomerase, protein disulfide isomerase, 60 kDa heat shock protein, mitochondrial stress-70 protein, epithelial cell marker protein, and S100-type calcium binding protein A14. We further delineate the properties of these proteins that make them preferred targets and the evidence these adducts present for delivery of these quinones to subcellular compartments.     

Discriminating pulmonary hypertension caused by monocrotaline toxicity from chronic hypoxia by near-infrared spectroscopy and multivariate methods of analysis

A new method has been developed for the determination of tissue pathology caused by chronic hypoxia and monocrotaline toxicity. The method is based on the use of near-infrared (NIR) spectrophotometry to measure spectra of lung tissue from normal chronic hypoxia (CH) and monocrotaline (MCT) models of pulmonary hypertension (PH), followed by analysis using multivariate methods, that is, principal component analysis (PCA) and partial least squares (PLS). Synergistic use of NIR with the PCA/PLS method makes it possible, for the first time, not only to divide different lung tissue samples into their respective groups (normal, CH, and MCT) but also to gain insight into mechanisms of PH caused by CH and MCT toxicity. Specifically, MCT metabolites and other hypertensive conditions are known to produce subtle and minor chemical changes in the compositions of tissue (e.g., proteins, carbohydrates, lipids). Although these changes were detected by the NIR technique, they were too small to be discerned through visual inspection of the spectra. However, they can be accurately classified and properly assigned by the PCA/PLS method. The fact that different tissue types can be accurately divided into their corresponding groups by the NIR and PCA/PLS methods suggests that chemical alterations and mechanisms of pulmonary vascular remodeling and PH induced by MCT are different from those induced by CH.     

Analysis of cysteine residues in peptides and proteins alkylated with volatile reagents

Summary Conditions are described for the reduction and alkylation of cysteines in peptides and proteins with volatile reagents by use of triethylphosphine as reductant, bromopropane as alkylating reagent and triethylamine as base. Alkylated samples need only be vacuum dried prior to subsequent analysis steps. Alkylated samples have been acid hydrolyzed and analyzed on an amino acid analyzer with recoveries of cysteine within 10% of the expected value. Alkylated samples have been directly applied to a sequencer membrane, dried on the surface and cysteines identified by sequence analysis without additional wash steps. In addition proteins blotted onto PVDF have been alkylatedin situ and sequenced with identification of cysteines. On the analyzer and sequencer the S-propylcysteine derivative elutes at a unique position allowing for the unambiguous identification of cysteine. Cysteine residues are quantitativly alkylated under the conditions developed. The ease of this procedure allows the routine analysis of cysteine in peptides and proteins without additional, time consuming repurification or dialysis steps.Abbreviations dptu diphenylthiourea - dmptu dimethylphenylthiourea - prop-cys S-propylcysteine     

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.     

Induction of apoptosis and endothelin-1 secretion in primary human lung endothelial cells by HIV-1 gp120 proteins

Pulmonary hypertension associated with human immunodeficiency virus (HIV) infection also involves injury to the lung endothelium. However, the pathogenesis of HIV-induced pulmonary hypertension is not known; we hypothesized that HIV or secreted viral proteins could play a role in vascular injury and the increased frequency of pulmonary hypertension observed in HIV-infected patients. Here, we report that exposure of HIV-1 gp120 proteins to primary human lung microvascular endothelial cells causes apoptosis, as assessed by TUNEL assay, Annexin-V staining, and DNA laddering. Using ribonuclease protection assay and Western blotting we find that gp120-induced apoptosis of lung endothelial cells involves a down-regulation in Bcl-xl mRNA and proteins. In addition, gp120 significantly increases secretion of the potent vasoconstrictor endothelin-1 by human lung endothelial cells. These data suggest that secreted HIV gp120 proteins induce lung endothelial cell injury and could contribute to the development of HIV-associated pulmonary hypertension.     

Depletion of the ATPase NSF from Golgi membranes with hypo-S-nitrosylation of vasorelevant proteins in endothelial cells exposed to monocrotaline pyrrole

Investigations of regulated S-nitrosylation and denitrosylation of vasorelevant proteins are a newly emergent area in vascular biology. We previously showed that monocrotaline pyrrole (MCTP)-induced megalocytosis of pulmonary arterial endothelial cells (PAECs), which underlies the development of pulmonary arterial hypertension, was associated with a Golgi blockade characterized by the trapping of diverse vesicle tethers, soluble N-ethylmaleimide-sensitive factor (NSF)-attachment protein receptors (SNAREs), and soluble NSF-attachment proteins (SNAPs) in the Golgi; reduced trafficking of caveolin-1 (cav-1) and endotheial nitric oxide (NO) synthase (eNOS) from the Golgi to the plasma membrane; and decreased caveolar NO. We have investigated whether NSF, the ATPase involved in all SNARE disassembly, might be the upstream target of MCTP and whether MCTP might regulate NSF by S-nitrosylation. Immunofluorescence microscopy and Golgi purification techniques revealed the discordant decrease of NSF by approximately 50% in Golgi membranes after MCTP despite increases in alpha-SNAP, cav-1, eNOS, and syntaxin-6. The NO scavenger (4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide failed to affect the initiation or progression of MCTP megalocytosis despite a reduction of 4,5-diaminofluorescein diacetate fluorescence and inhibition of S-nitrosylation of eNOS as assayed using the biotin-switch method. Moreover, the latter assay not only revealed constitutive S-nitrosylation of NSF, eNOS, cav-1, and clathrin heavy chain (CHC) in PAECs but also a dramatic 70-95% decrease in the S-nitrosylation of NSF, eNOS, cav-1, and CHC after MCTP. These data point to depletion of NSF from Golgi membranes as a mechanism for Golgi blockade after MCTP and to denitrosylation of vasorelevant proteins as critical to the development of endothelial cell megalocytosis.     

Oxidant and angiotensin II-induced subcellular translocation of protein kinase C in pulmonary artery endothelial cells

We recently reported that nitrogen dioxide (NO₂), an environmental oxidant, alters the dynamics of the plasma membrane lipid bilayer structure, resulting in increased phosphatidylserine content and angiotensin II (Ang II) receptor binding. Angiotensin II is known to elicit receptor-mediated stimulation of diacylglycerol (DAG) production in pulmonary artery endothelial cells. Because protein kinase C (PKC) is a phosphatidylserine-dependent enzyme and is activated by DAG, we examined whether NO₂ resulted in activation and/or translocation of PKC from predominantly cytosolic to membrane fractions of these cells. We also evaluated whether NO₂ exposure resulted in increased production of DAG in pulmonary artery endothelial cells. Exposure to 5 ppm NO₂ for 1–24 hr resulted in significant increases in PKC activity in the cytosolic and membrane fractions (p < 0.05 for both fractions) compared to activities in control fractions. Exposure to Ang II resulted in translocation of PKC activity from cytosol to membrane fractions of both control and NO₂-exposed cells. This translocation of PKC from cytosolic to membrane fraction was prevented by the specific receptor antagonist [Sar¹ Ile⁸] Ang II. Exposure of 5 ppm NO₂ for 1–24 hr provoked rapid increases in [³H]glycerol labeling of DAG in pulmonary artery endothelial cells. These results demonstrate that exposure to NO₂ increases the production of second messenger DAG and activates PKC in both the cytosolic and membrane fractions, whereas Ang II stimulates the redistribution of PKC from cytosolic to membrane fractions of pulmonary artery endothelial cells.     

Featured Article: microRNA-125a in pulmonary hypertension: Regulator of a proliferative phenotype of endothelial cells

Vascular remodeling due to excessive proliferation of endothelial and smooth muscle cells is a hallmark feature of pulmonary hypertension. microRNAs (miRNAs) are a class of small, non-coding RNA fragments that have recently been associated with remodeling of pulmonary arteries, in particular by silencing the bone morphogenetic protein receptor type II (BMPR2). Here we identified a novel pathway involving the concerted action of miR-125a, BMPR2 and cyclin-dependent kinase inhibitors (CDKN) that controls a proliferative phenotype of endothelial cells. An in silico approach predicted miR-125a to target BMPR2. Functional inhibition of miR-125a resulted in increased proliferation of these cells, an effect that was found accompanied by upregulation of BMPR2 and reduced expression of the tumor suppressors CDKN1A (p21) and CDKN2A (p16). These data were confirmed in experimental pulmonary hypertension in vivo. Levels of miR-125a were elevated in lung tissue of hypoxic animals that develop pulmonary hypertension. In contrast, circulating levels of miR-125a were found to be lower in mice with pulmonary hypertension as compared to control mice. Similar findings were observed in a small cohort of patients with precapillary pulmonary hypertension. These translational data emphasize the pathogenetic role of miR-125a in pulmonary vascular remodeling.     

On the cysteine and cystine content of proteins

Summary Analysis of published data on the cysteine and half-cystine content of proteins indicates that most intracellular proteins may be classified as sulfhydryl proteins (those containing cysteine but little or no half-cystine) and that such sulf-hydryl proteins have a low cysteine content. The mean cysteine content found for 32 intracellular mammalian proteins was 1.6 % and intracellular proteins of many bacteria have similar or lower values. Extracellular mammalian proteins are primarily disulfide proteins (those containing half-cystine but little or no cysteine) and have a high half-cystine content, the mean value found for some 34 extracellular mammalian proteins being 4.1 %. This is contrasted with many of the extracellular proteins from facultative bacteria which are cyst(e)ine-free proteins, being lacking in both cysteine and half-cystine. These and related observations are interpreted in terms of the evolution of life in a reducing atmosphere and the subsequent transition to an oxidizing environment. It is suggested that disulfide proteins evolved primarily after the accumulation of oxygen in the atmosphere.     

Determinants of an elevated pulmonary arterial pressure in patients with pulmonary arterial hypertension

Given the difficulty of diagnosing early-stage pulmonary arterial hypertension (PAH) due to the lack of signs and symptoms, and the risk of an open lung biopsy, the precise pathological features of presymptomatic stage lung tissue remain unknown. It has been suggested that the maximum elevation of the mean pulmonary arterial pressure (P_pa) is achieved during the early symptomatic stage, indicating that the elevation of the mean P_pa is primarily driven by the pulmonary vascular tone and/or some degree of pulmonary vascular remodeling completed during this stage. Recently, the examination of a rat model of severe PAH suggested that the severe PAH may be primarily determined by the presence of intimal lesions and/or the vascular tone in the early stage. Human data seem to indicate that intimal lesions are essential for the severely increased pulmonary arterial blood pressure in the late stage of the disease.However, many questions remain. For instance, how does the pulmonary hemodynamics change during the course of the disease, and what drives the development of severe PAH? Although it is generally acknowledged that both pulmonary vascular remodeling and the vascular tone are important determinants of an elevated pulmonary arterial pressure, which is the root cause of the time-dependent progression of the disease? Here we review the recent histopathological concepts of PAH with respect to the progression of the lung vascular disease.     

Distribution of the pulmonary artery and vein in the chimpanzee lung

Lungs of two chimpanzees (Pan troglodytes) were examined. The right pulmonary artery runs across the ventral side of the right upper lobe bronchiole and, then across the dorsal side of the right middle lobe bronchiole. Thereafter, it runs between the dorsal bronchiole system and the lateral bronchiole system, along the right bronchus. During its course, it gives off arterial branches which run along each bronchiole. The left pulmonary artery runs across the dorsal side of the left middle lobe bronchiole and then between the dorsal bronchiole system and the lateral bronchiole system. The branches of the pulmonary artery run mainly along the dorsal or lateral side of the bronchiole. The pulmonary veins run mainly along the ventral or medial side of the bronchioles, and between them. Finally, they enter the left atrium with four large veins, i.e. the common trunk of the right upper lobe vein and the right middle lobe vein, right lower lobe pulmonary venous trunk, left middle lobe vein, and left lower lobe pulmonary venous trunk.     

增殖细胞核抗原在低氧大鼠肺动脉平滑肌细胞中表达

目的通过建立低氧性肺动脉高压大鼠模型,探讨增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)在大鼠低氧性肺血管平滑肌细胞中的表达。方法将SPF级SD大鼠随机分为正常对照组(n=10)、模型组(n=10),通过间断常压低氧法建立大鼠低氧性肺动脉高压模型,肺组织切片经HE染色后图像分析技术定量检测大鼠肺小动脉的形态改变;免疫组织化学染色法测定肺血管平滑肌细胞内PCNA蛋白表达,并经图像分析半定量检测其表达强度。结果 4周后,模型组SD大鼠MT%、MA%与对照组比较,差异具有显著性(P<0.05);模型组SD大鼠肺血管平滑肌细胞内PCNA核蛋白表达(积分面积、累积光密度)与对照组比较,差异具有显著性(P<0.05)。结论常压低氧4周可成功建立肺动脉高压大鼠模型,PCNA在肺血管平滑肌细胞中的表达量具有差异性提示其可能在肺动脉高压形成过程中起重要作用。     

Anxiety and depression disorders in patients with pulmonary arterial hypertension and chronic thromboembolic pulmonary hypertension

Background

The objective of this prospective study was to assess the prevalence of anxiety and depression disorders and their association with quality of life (QoL), clinical parameters and survival in patients with pulmonary hypertension (PH).

Methods

We prospectively assessed 158 patients invasively diagnosed with pulmonary arterial hypertension (n = 138) and inoperable chronic thromboembolic PH (n = 20) by clinical measures including quality of life (QoL, SF-36 questionnaire), cardiopulmonary exercise testing and six minute walking distance and by questionnaires for depression (PHQ-9) and anxiety (GAD-7). According to the results of the clinical examination and the questionnaires for mental disorders (MD) patients were classified into two groups, 1) with moderate to severe MD (n = 36, 22,8%), and 2) with mild or no MD (n = 122). Patients were followed for a median of 2.7 years. Investigators of QoL, SF-36 were blinded to the clinical data.

Results

At baseline the 2 groups did not differ in their severity of PH or exercise capacity. Patients with moderate to severe MD (group 1) had a significantly lower QoL shown in all subscales of SF-36 (p < 0.002). QoL impairment significantly correlated with the severity of depression (p < 0.001) and anxiety (p < 0.05). During follow-up period 32 patients died and 3 were lost to follow-up. There was no significant difference between groups regarding survival. Only 8% of the patients with MD received psychopharmacological treatment.

Conclusion

Anxiety and depression were frequently diagnosed in our patients and significantly correlated with quality of life, but not with long term survival. Further prospective studies are needed to confirm the results.     

Sepiapterin improves angiogenesis of pulmonary artery endothelial cells with in utero pulmonary hypertension by recoupling endothelial nitric oxide synthase

Persistent pulmonary hypertension of the newborn (PPHN) is associated with decreased blood vessel density that contributes to increased pulmonary vascular resistance. Previous studies showed that uncoupled endothelial nitric oxide (NO) synthase (eNOS) activity and increased NADPH oxidase activity resulted in marked decreases in NO bioavailability and impaired angiogenesis in PPHN. In the present study, we hypothesize that loss of tetrahydrobiopterin (BH4), a critical cofactor for eNOS, induces uncoupled eNOS activity and impairs angiogenesis in PPHN. Pulmonary artery endothelial cells (PAEC) isolated from fetal lambs with PPHN (HTFL-PAEC) or control lambs (NFL-PAEC) were used to investigate the cellular mechanisms impairing angiogenesis in PPHN. Cellular mechanisms were examined with respect to BH4 levels, GTP-cyclohydrolase-1 (GCH-1) expression, eNOS dimer formation, and eNOS-heat shock protein 90 (hsp90) interactions under basal conditions and after sepiapterin (Sep) supplementation. Cellular levels of BH4, GCH-1 expression, and eNOS dimer formation were decreased in HTFL-PAEC compared with NFL-PAEC. Sep supplementation decreased apoptosis and increased in vitro angiogenesis in HTFL-PAEC and ex vivo pulmonary artery sprouting angiogenesis. Sep also increased cellular BH4 content, NO production, eNOS dimer formation, and eNOS-hsp90 association and decreased the superoxide formation in HTFL-PAEC. These data demonstrate that Sep improves NO production and angiogenic potential of HTFL-PAEC by recoupling eNOS activity. Increasing BH4 levels via Sep supplementation may be an important therapy for improving eNOS function and restoring angiogenesis in PPHN.     

Distribution of the pulmonary artery and vein of the orangutan lung

The distribution of the pulmonary artery and vein of the orangutan lung was examined. The right pulmonary artery runs obliquely across the ventral side of the right bronchus at the caudally to the right upper lobe bronchiole. It then runs across the dorsal side of the right middle lobe bronchiole. Thereafter it runs obliquely across the dorsal side of the right bronchus, and then along the dorso-medial side of the right bronchus. This course is different from that in other mammals. During its course, it gives off branches which run mainly along the dorsal or lateral side of each bronchiole. The left pulmonary artery runs across the dorsal side of the left middle lobe bronchiole, then along the dorso-lateral side of the left bronchus, giving off branches which run along each bronchiole. The pulmonary veins run mainly the ventral or medial side of, along or between the bronchioles. In the left lung, the left middle lobe vein has two trunks; one enters the left atrium, and the other enters the left lower lobe pulmonary venous trunk. This is also different from that found in most mammals. Finally, the pulmonary veins enter the left atrium with four large veins.     

15-HETE suppresses K+ channel activity and inhibits apoptosis in pulmonary artery smooth muscle cells

15-Hydroxyeicosatetraenoic acid (15-HETE) is an important hypoxic product from arachidonic acid (AA) in the wall of pulmonary vessels. Although its effects on pulmonary artery constriction are well known, it remains unclear whether 15-HETE acts on the apoptotic responses in pulmonary artery smooth muscle cells (PASMCs) and whether K⁺ channels participate in this process. These hypothesises were validated by cell viability assay, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling, mitochondrial potentials assay, caspase activity assay and western blot. We found that 15-HETE enhanced cell survival, suppressed the expression and activity of caspase-3, upregulated bcl-2 and attenuated mitochondrial depolarization, prevented chromatin condensation and partly reversed K⁺ channel opener-induced apoptosis in PASMCs under serum-deprived conditions. Our data indicated that 15-HETE inhibits the apoptosis in PASMCs through, at least in part, inactivating K⁺ channels. Yumei Li and Qian Li contributed equally to this work.     

Mitochondrial ROS-K+ channel signaling pathway regulated secretion of human pulmonary artery endothelial cells

The objective was to investigate the molecular mechanism of mitochondrial reactive oxygen species (ROS) signaling regulation of pulmonary artery endothelial cell (HPAEC) secretion in the condition of oxidative stress. Acrolein (40 μM) induced HPAEC mitochondrial generation of ROS, rotenone (2 μmol/L) blocked mitochondrial respiratory chain complex I, cesium chloride (CsCl, 40 mmol/L)blocked K⁺channels, and saline (0.9 g/dl) were used as control. The generations of NOS, ET-1 and VEGF were determined with ELISA in the condition of different treatment reagents namely acrolein, acrolein plus rotenone, acrolein plus CsCl and saline. In the different reagent treatment of HPAECs, acrolein increased mitochondrial ROS, membrane potential, Kv1.5 mRNA and protein expression, intracellular calcium and the generation of NOS (determining NO production), ET-1 and VEGF, and those were reduced by rotenone. CsCl decreased the increment of membrane potential, the elevation of intracellular calcium and the upregulation of NOS, E-1 and VEGF expressions, which were induced by acrolein. The present study demonstrated that mitochondrial ROS-K⁺channel regulated HPAEC secretion of NO, ET-1 and VEGF in the condition of oxidative stress. Kv1.5 channel may be an important component of ROS-K+ channel signaling pathway, and intracellular calcium contributed to mitochondrial ROS-K⁺ channel signaling modulation of HPAEC secretion.