Endoplasmic reticulum stress enhances fibrotic remodeling in the lungs.

Evidence of endoplasmic reticulum (ER) stress has been found in lungs of patients with familial and sporadic idiopathic pulmonary fibrosis. We tested whether ER stress causes or exacerbates lung fibrosis by (i) conditional expression of a mutant form of surfactant protein C (L188Q SFTPC) found in familial interstitial pneumonia and (ii) intratracheal treatment with the protein misfolding agent tunicamycin. We developed transgenic mice expressing L188Q SFTPC exclusively in type II alveolar epithelium by using the Tet-On system. Expression of L188Q SFTPC induced ER stress, as determined by increased expression of heavy-chain Ig binding protein (BiP) and splicing of X-box binding protein 1 (XBP1) mRNA, but no lung fibrosis was identified in the absence of a second profibrotic stimulus. After intratracheal bleomycin, L188Q SFTPC-expressing mice developed exaggerated lung fibrosis and reduced static lung compliance compared with controls. Bleomycin-treated L188Q SFTPC mice also demonstrated increased apoptosis of alveolar epithelial cells and greater numbers of fibroblasts in the lungs. With a complementary model, intratracheal tunicamycin treatment failed to induce lung remodeling yet resulted in augmentation of bleomycin-induced fibrosis. These data support the concept that ER stress produces a dysfunctional epithelial cell phenotype that facilitates fibrotic remodeling. ER stress pathways may serve as important therapeutic targets in idiopathic pulmonary fibrosis.

Cytoskeletal defects in Bmpr2-associated pulmonary arterial hypertension.

The heritable form of pulmonary arterial hypertension (PAH) is typically caused by a mutation in bone morphogenic protein receptor type 2 (BMPR2), and mice expressing Bmpr2 mutations develop PAH with features similar to human disease. BMPR2 is known to interact with the cytoskeleton, and human array studies in PAH patients confirm alterations in cytoskeletal pathways. The goal of this study was to evaluate cytoskeletal defects in BMPR2-associated PAH. Expression arrays on our Bmpr2 mutant mouse lungs revealed cytoskeletal defects as a prominent molecular consequence of universal expression of a Bmpr2 mutation (Rosa26-Bmpr2(R899X)). Pulmonary microvascular endothelial cells cultured from these mice have histological and functional cytoskeletal defects. Stable transfection of different BMPR2 mutations into pulmonary microvascular endothelial cells revealed that cytoskeletal defects are common to multiple BMPR2 mutations and are associated with activation of the Rho GTPase, Rac1. Rac1 defects are corrected in cell culture and in vivo through administration of exogenous recombinant human angiotensin-converting enzyme 2 (rhACE2). rhACE2 reverses 77% of gene expression changes in Rosa26-Bmpr2(R899X) transgenic mice, in particular, correcting defects in cytoskeletal function. Administration of rhACE2 to Rosa26-Bmpr2(R899X) mice with established PAH normalizes pulmonary pressures. Together, these findings suggest that cytoskeletal function is central to the development of BMPR2-associated PAH and that intervention against cytoskeletal defects may reverse established disease.

Iodopovidone is as effective as doxycycline in producing pleurodesis in rabbits.

BACKGROUND AND OBJECTIVE: The mechanism by which iodopovidone achieves pleurodesis is unknown. This study investigated whether iodopovidone is as effective as doxycycline in producing pleurodesis and whether systemic corticosteroids diminish its efficacy. METHODS: Four groups of seven New Zealand rabbits were assigned to the following intrapleural treatment groups: 2 mL of 2% iodopovidone, 2 mL of 4% iodopovidone, 2 mL of 4% iodopovidone plus 0.8 mg/kg triamcinolone intramuscularly weekly and 10 mL/kg doxycycline in 2 mL. Pleural fluid was collected 24, 48 and 72 h after intrapleural injections and analysed for WCC, protein and LDH levels. The rabbits were killed 2 weeks after the injections. Pleurodesis was graded macroscopically on a scale from 1 to 8. The degree of microscopic pleural fibrosis and pleural inflammation was graded from the HE stain slides. RESULTS: The mean volume of pleural fluid as well as the mean total WCC was significantly lower in the steroid-treated group than in the other groups. The degree of the resulting pleurodesis was similar in the 2% iodopovidone (7.00 +/- 1.29), 4% iodopovidone (7.71 +/- 0.76) and doxycycline (7.14 +/- 0.90) groups (P > 0.05) whereas the pleurodesis score of the steroid group (3.71 +/- 1.98) was significantly lower than all other groups (P < 0.05). The degree of microscopic pleural fibrosis and pleural inflammation was significantly lower in the steroid group than in the 2% iodopovidone or 4% iodopovidone group. CONCLUSIONS: Both 2% and 4% iodopovidone can induce pleurodesis as efficaciously as doxycycline in rabbits. Systemic corticosteroids significantly decrease the efficacy of iodopovidone in producing pleurodesis.

Single-chain urokinase in empyema induced by Pasturella multocida.

Intrapleural fibrin deposition and subsequent fibrosis characterize evolving empyema and contribute to the morbidity associated with this condition. Single-chain urokinase (scuPA) is proenzyme form of the urokinase plasminogen activator, which has recently been shown to effectively clear intrapleural loculation in tetracycline-induced pleurodesis in rabbits. The authors therefore hypothesized that scuPA could likewise improve intrapleural injury associated with empyema. The authors used a rabbit model of empyema induced by intrapleural administration of Pasturella multocida to test this hypothesis and determined the effects of intrapleural scuPA on pleural fluids indices of inflammation and intrapleural fibrosis. The authors found that intrapleural administration of scuPA was well tolerated, generated readily detectable fibrinolytic activity in the empyema fluids and did not induce intrapleural or systemic bleeding. Pleural fluid volume, intrapleural protein, and D-dimer concentrations were increased at 24 and 48 hours (P < .01, respectively) after induction of empyema. Intrapleural loculation did not occur in the scuPA- or vehicle control-treated animals and there was no significant change in the pleural empyema or thickening scores. These findings confirm that intrapleural scuPA generates fibrinolysis in empyema fluids but does not alter fibrotic repair at the pleural surface or the intensity of intrapleural inflammation in this empyema model.

Diagnostic value of pleural fluid N-terminal pro-brain natriuretic peptide levels in patients with cardiovascular diseases.

BACKGROUND AND OBJECTIVE: The diagnosis of the cause of pleural effusions caused by cardiovascular diseases such as congestive heart failure (CHF) and acute pulmonary embolism is sometimes difficult. The purpose of the present study was to evaluate the utility of pleural fluid levels of N-terminal pro-brain natriuretic peptide (NT-proBNP) in differentiating pleural effusions due to CHF, pulmonary embolism and post-coronary artery bypass graft (CABG) surgery. METHODS: The levels of pleural fluid NT-proBNP were measured by ELISA in a total of 40 patients: 10 with CHF, 10 with pulmonary embolism, 10 post-CABG and 10 with carcinoma. RESULTS: The median level of NT-proBNP in the pleural fluid of patients with CHF was 5390 pg/mL (25th to 75th percentiles, 4566 to 8158 pg/mL), which was significantly higher than that in patients with post-CABG effusions (424 pg/mL, 352 to 873), with pulmonary embolism (311 pg/mL, 212 to 1159), or with carcinoma (302 pg/mL, 208 to 626) (P < 0.001, CHF group vs all other groups). In receiver-operating curve analysis, an NT-proBNP level of >or=2220 pg/mL demonstrated a sensitivity of 100% and a specificity of 96.7% for the identification of CHF. CONCLUSIONS: Measurement of the NT-proBNP level in pleural fluid is accurate in diagnosing the etiology of the effusion as CHF. Pleural fluid levels above 2220 pg/mL are essentially diagnostic that the pleural effusion is due to CHF.

The short-term administration of Ketoprofen does not decrease the effect of Pleurodesis induced by talc or Doxycycline in rabbits.

OBJECTIVE: To determine whether the concomitant administration of ketoprofen, a non-steroidal anti-inflammatory drug (NSAID) has any effect on the pleurodesis induced by talc or doxycycline in rabbits. METHODS: Four groups of seven New Zealand rabbits were assigned to receive the following treatments: 400mg/kg of talc intrapleurally only (group 1), 400mg/kg of talc plus 1mg/kg of ketoprofen intramuscularly (group 2), 10mg/kg of doxycycline intrapleurally only (group 3) and 10mg/kg of doxycycline plus 1mg/kg of ketoprofen intramuscularly (group 4). Intramuscular administration of ketoprofen began 4h before the intrapleural administration of the sclerosing agents, followed by twice daily administrations for 1 week. Pleural fluid was collected 24, 48 and 72h after intrapleural injections. Pleurodesis was evaluated macroscopically and microscopically after 14 days. RESULTS: The concomitant use of ketoprofen at 1mg/kg does not decrease the WBC, LDH, and protein in pleural fluid at 24h following intrapleural injection of talc or doxycycline. There were no significant differences in the macroscopic pleurodesis scores, the degree of microscopic pleural fibrosis, the thickness of the pleura or the percent of the pleura occupied with angiogenesis. CONCLUSIONS: The study shows that the short-term systemic administration of NSAIDs does not affect the efficacy of pleurodesis induced by talc or doxycycline in rabbits.

HIV-1 TAT represses transcription of the bone morphogenic protein receptor-2 in U937 monocytic cells.

The bone morphogenetic protein receptor-2 (BMPR2) is a member of the transforming growth factor-beta receptor family and is expressed on the surface of several cell types including endothelial cells and macrophages. Recently, a cause for familial primary pulmonary hypertension (FPPH) has been identified as mutations in the gene encoding BMPR2. Three forms of pulmonary hypertension (PH) exist, including PPH, FPPH, and PH secondary to other etiologies (sporadic PH) such as drug abuse and human immunodeficiency virus (HIV) infection. It is interesting that these subtypes are histologically indistinguishable. The macrophage is a key target cell for HIV-1, significantly altering macrophage cell function upon infection. HIV-1 trans-activator of transcription (Tat), an immediate-early product of the HIV-1 lifecycle, plays an important role in mediating HIV-induced modulation of host cell function. Our laboratory has previously shown that Tat represses mannose receptor transcription in macrophages. In the current study, we examined activity from the BMPR2 promoter in the macrophage cell line U937 and potential regulation by Tat. Transfection of U937 cells with BMPR2 promoter-reporter constructs revealed dose-dependent repression of BMPR2 promoter activity in the presence of Tat. Experiments using truncations of the BMPR2 promoter localized Tat-mediated repression to the first 208 bases of the promoter. Decreased BMPR2 transcription resulted in altered downstream signaling. Similar to mothers against decapentaplegics (SMAD) phosphorylation and SMAD6 expression, in response to BMP2 treatment, were down-regulated after Tat treatment. Finally, HIV-1 infection and treatment with Tat protein of the U937 human monocytic cell line resulted in a decreased, endogenous BMPR2 transcript copy number.

Combination therapy with intrapleural doxycycline and talc in reduced doses is effective in producing pleurodesis in rabbits.

BACKGROUND: It has been suggested that talc and doxycycline might be acting through different pathways in creating pleurodesis. We hypothesized that combining doxycycline and talc in half the usual doses would be synergistic in inducing pleurodesis. METHODS: Thirty-two rabbits were equally allocated into four groups: group 1, half-dose combination (5 mg/kg of doxycycline and 200 mg/kg of talc slurry); group 2, quarter-dose combination (2.5 mg/kg of doxycycline and 100 mg/kg of talc slurry); group 3, half-dose doxycycline (5 mg/kg of doxycycline); and group 4, half-dose talc (100 mg/kg of talc slurry). The pleurodesis scores from historical groups that received a full dose of talc (400 mg/kg) or doxycycline (10 mg/kg) were also compared to those obtained in the current study. Pleural fluid lactate dehydrogenase and protein levels were measured 24 h after the injection. Pleurodesis was graded from 1 (none) to 8 (> 50% symphysis) by two observers blinded to treatment groups. All rabbits underwent an ultrasonic examination on each side of their chest for the evaluation of pleurodesis. RESULTS: The mean pleurodesis score in the half-dose combination group was significantly higher than that in the half-dose talc group, half-dose doxycycline group, and the historical full-dose talc group (p = 0.009, p = 0.01, and p < 0.05, respectively). The quarter-dose combination group also had a significantly higher mean pleurodesis score compared to the half-dose talc group (p = 0.013). The difference between the historical full-dose doxycycline and the half-dose combination or quarter-dose combination groups was not significant (p > 0.05). A significantly positive correlation existed between the pleurodesis score and the ultrasound scores (r = 0.876, p = 0.000000005). CONCLUSIONS: This study demonstrates that the combination of half doses of talc and doxycycline is more effective than the half dose of either drug alone or the full dose of talc in producing pleurodesis in rabbits. In addition, ultrasound is an accurate imaging modality for the evaluation of pleurodesis, in that the absence of pleural gliding on ultrasound correlates well with the presence of a pleurodesis in rabbits.

Transforming growth factor-beta3 induces pleurodesis in rabbits and collagen production of human mesothelial cells.

STUDY OBJECTIVE: The aim of this study was to examined whether transforming growth factor (TGF)-beta(3) induces pleurodesis in rabbits and collagen production by human pleural mesothelial cells in vitro. DESIGN: A combined animal and in vitro study. METHODS: TGF-beta(3) was injected intrapleurally in rabbits at the following doses: 0.167 mug, 0.5 mug, 1.67 mug, and 5 mug (five rabbits per group). The rabbits were killed 14 days following injection, and pleurodesis was graded from 1 (none) to 8 (complete symphysis). Pleural mesothelial cell cultures were established from benign pleural effusions and treated with TGF-beta(3) for 48 h at the following doses: 0 (control), 5 ng/mL, 1.5 ng/mL, 5 ng/mL, and 15 ng/mL and 50 ng/mL (two independent experiments). Collagen I messenger RNA (mRNA) expression was quantified using real-time polymerase chain reaction. RESULTS: The median pleurodesis score was 5 (interquartile range [IQR], 4.5) in the 0.167-mug group, 6 (IQR, 5) in the 0.5-mug group, 8 (IQR, 1) in the 1.67-mug group, and 8 (IQR, 0.5) in the 5-mug group (p = 0.012). Higher TGF-beta(3) doses induced the production of significantly more pleural fluid than the lower doses (p = 0.005). The pleural fluid induced by higher doses contained significantly lower numbers of nucleated cells (p = 0.014) and lactate dehydrogenase levels (p = 0.013) than the pleural fluid induced by lower doses of the agent. TGF-beta(3) markedly enhanced collagen I mRNA expression by the human pleural mesothelial cells. This effect peaked at 5 ng/mL TGF-beta(3). With this dose of TGF-beta(3), the collagen I mRNA expression was increased 16-fold over control levels. CONCLUSION: TGF-beta(3) causes a dose-dependent pleurodesis when administered intrapleurally in rabbits, and induces collagen messenger RNA synthesis from human pleural mesothelial cells.

Efficacy of ultrasound in the diagnosis of pleurodesis in rabbits.

INTRODUCTION: The treatment of recurrent pleural effusion or recurrent pneumothorax frequently involves the creation of a pleurodesis. Ultrasound is being used more frequently to assess the presence of pleural fluid or pneumothorax. With ultrasound, the gliding sign displays the gliding of the visceral pleura over the parietal pleura during respiration. The absence of a gliding sign is indicative of a pneumothorax. HYPOTHESIS: We hypothesized that the presence of pleurodesis would be indicated by the absence of a gliding sign on ultrasound. METHODS: To create a pleurodesis, a single intrapleural injection of transforming growth factor-beta2 at a dosage of 1.70 microg/kg or doxycycline at a dosage of 10 mg/kg in a volume of 2.0 mL was administered randomly to one side of a New Zealand White rabbit. Prior to death on day 14 after intrapleural injection, all rabbits underwent an ultrasonic examination at three marked sites on each side of the chest. At each site, three ultrasonic features (gliding sign, pleural thickening, and pleural effusion) were evaluated and graded. The gliding sign was graded as follows: 0 = gliding sign definitely present, 1 = gliding sign questionable, 2 = gliding sign absent. RESULTS: In a preliminary study for developing skill in assessing the gliding sign, the correlation between the gliding sign and the pleurodesis score in 16 rabbits was highly significant (r = 0.568, p = 0.02). In the subsequent main study with 18 additional rabbits, the correlation between the gliding sign score and the pleurodesis score was even better (r = 0.806, p = 0.00009). The gliding sign was definitely present on the noninjected side in all rabbits. CONCLUSIONS: The presence of a pleurodesis is indicated by the absence of a pleural guiding sign on ultrasound.

Pleurodesis is inhibited by anti-vascular endothelial growth factor antibody.

STUDY OBJECTIVES: The intrapleural injection of transforming growth factor (TGF)-beta2 produces pleurodesis in rabbits associated with large pleural effusions. This study investigated whether anti-vascular endothelial growth factor (VEGF) antibody has any effect on the fluid production or the pleurodesis induced by TGF-beta2. INTERVENTIONS AND MEASUREMENTS: Three groups of seven New Zealand white rabbits were administered TGF-beta2 5.0 microg intrapleurally. Two groups received anti-VEGF antibody (10 mg/kg and 25 mg/kg) IV 24 h before TGF-beta2 injection, and the third group received no antibody. The rabbits were killed at 2 weeks, and the macroscopic pleurodesis score was determined. The degree of pleural angiogenesis was assessed by immunohistochemical staining for factor VIII. RESULTS: The administration of anti-VEGF antibodies had no significant effect on the pleural fluid volume or the characteristics of the fluid. The mean pleurodesis score of the seven rabbits in the control group (7.71 +/- 0.76) was significantly (p < 0.05) higher than that for seven rabbits in the low-dose treatment group (4.43 +/- 2.37) and the seven rabbits in the high-dose treatment group (4.57 +/- 2.36) [+/- ]. The percentage of pleural tissue demonstrating angiogenesis in the control group (4.87 +/- 0.43%) was significantly (p < 0.05) higher than that for the low-dose (2.94 +/- 0.68%) or high-dose (2.67 +/- 0.64%) antibody groups. When all rabbits were considered, there was a highly significant correlation between the pleural vascular density scores and the pleurodesis scores (r = 0.84, p < 0.01). CONCLUSION: VEGF and angiogenesis appear to play a pivotal role in the production of a pleurodesis.

Pneumothorax-associated pleural eosinophilia in mice is interleukin-5 but not interleukin-13 dependent.

STUDY OBJECTIVES: To establish a murine model of pneumothorax-associated pleural eosinophilia and to examine the role of interleukin (IL)-5 and IL-13 in the pathogenesis of this reaction. DESIGN: An animal study. INTERVENTIONS: One hundred thirty-seven C57/Bl-6 mice were used in this study. Wild-type animals were injected intrapleurally with 0.4 mL of air and were killed at different time points (30 min to 7 days) after the injection. IL-5 knockout and IL-13 knockout animals were killed 24 h and 48 h after the injection. Pleural inflammation was assessed by pleural lavage (PL). MEASUREMENTS AND RESULTS: PL cells were significantly increased following the induction of pneumothorax. The peak number of neutrophils, observed at 12 h, was 900 times higher than the control. The peak number of eosinophils, observed at 48 h, was 700 times higher than the control. Lymphocytes and mononuclear cells increased threefold and fourfold, respectively. IL-5 knockout mice had significantly less PL eosinophils than that the wild-type or the IL-13 knockout mice at 24 h (150 +/- 46/microL, 903 +/- 244/microL, and 912 +/- 168/microL, respectively; p = 0.013) and 48 h (181 +/- 45/microL, 1,587 +/- 212/microL, and 1,379 +/- 364/microL, respectively; p = 0.003). CONCLUSION: Pneumothorax induces an inflammatory reaction of the mouse pleura, mainly characterized by increased neutrophils and eosinophils. IL-5 but not IL-13 is required for pneumothorax-associated pleural eosinophilia.

Oral forms of tetracycline and doxycycline are effective in producing pleurodesis.

PURPOSES: We investigated whether oral tetracyclines could produce an efficient and safe pleurodesis as does parenteral doxycycline, which is currently unavailable in many countries. METHODS: Parenteral doxycycline (10 mg/kg), oral tetracycline (35 mg/kg), or doxycycline (10 mg/kg) was injected intrapleurally through a right chest tube in rabbits. The oral forms were dissolved in saline solution and passed through a sterile membrane filter. When daily aspirated pleural fluid was < 5 mL/24 h, the chest tube was removed. Fluid WBC, lactate dehydrogenase (LDH), and protein levels were measured 24 h after the injection. After the death of the animals on day 14, pleurodesis was graded from 1 (none) to 8 (> 50% symphysis) by two observers blinded to treatment groups. RESULTS: The right pleurodesis score of the combined oral groups (median, 7.0; interquartile range [IQR], 4.0; n = 26) did not differ significantly (p = 0.349) from that of the parenteral group (median, 7.5; IQR, 6.0; n = 10). Oral tetracycline (capsule or tablet, n = 6 in each group) and doxycycline (capsule or tablet, n = 7 in each group) were as effective as parenteral doxycycline in producing pleurodesis: tetracycline capsule (median, 7.50; IQR, 6.00); tetracycline tablet (median, 6.50; IQR, 6.00); doxycycline capsule (median, 4.00; IQR, 1.00); doxycycline tablet (median, 8.00; IQR, 5.00), and parenteral doxycycline (median, 7.50; IQR, 6.00) [p = 0.235]. The left pleurodesis scores were 1.00 in all 36 rabbits. Fluid total volume, WBC, LDH, and protein levels were comparable between each oral and parenteral group, excluding WBCs in the tetracycline tablet group (p = 0.047). The complications were nonfatal (right hemothorax: tetracycline capsule [n = 3]/tetracycline tablet [n = 2], doxycycline tablet [n = 2], parenteral doxycycline [n = 2]; left hemothorax: tetracycline capsule [n = 1]; ascites: parenteral doxycycline [n = 1]). There was no growth on all filtrate cultures. Oral forms cost less than parenteral doxycycline (<1 US dollar vs 4.72 US dollars per rabbit). Filtering costs were 1.12 US dollars per rabbit. CONCLUSION: Oral tetracycline or doxycycline is as effective and safe as parenteral doxycycline in producing pleurodesis in rabbits; thus, they may also be used in humans.

Pleural space as a site of ectopic gene delivery: transfection of pleural mesothelial cells with systemic distribution of gene product.

STUDY OBJECTIVES: Successful ectopic gene therapy requires the transfection of the cells at the ectopic site, with local and systemic delivery of the gene product. This study aimed to evaluate the pleural mesothelial surface as a potential site for ectopic gene therapy. DESIGN: A secreted placental alkaline phosphatase (PALP) plasmid was injected bilaterally into the pleural spaces of seven rabbits via a chest tube, while an irrelevant reporter plasmid was injected into seven control rabbits. Blood was collected at baseline and at 24, 48, and 72 h after the injections. Pleural fluid was collected by lavage at 24, 48, and 72 h after the injections. The PALP level was measured by chemiluminesence. MEASUREMENTS AND RESULTS: Significant expressions of PALP proteins were observed in the serum of the treatment rabbits, with a threefold increase over baseline at 24 h, a ninefold increase at 48 h, and a twofold increase at 72 h. The serum PALP levels in the control rabbits remained at baseline levels at all time points. The pleural fluid PALP levels peaked at 24 h and decreased over the next 72 h. Mimicking the in vivo pattern, pleural mesothelial cells transfected in vitro demonstrated a similar increase in PALP levels. CONCLUSIONS: The results of the present short-term pilot study suggest that pleural mesothelial cells can be successfully transfected with plasmids, with increases in both the local and systemic levels of the gene product. The pleural space should be further evaluated for ectopic gene therapy.

Pleural fluid levels of vascular cell adhesion molecule-1 are elevated in eosinophilic pleural effusions.

STUDY OBJECTIVES: The mechanisms responsible for the accumulation of eosinophils in pleural fluid are not fully understood. The objective of the present study was to examine the relationship between pleural fluid eosinophilia and the levels of vascular cell adhesion molecule (VCAM)-1, eotaxin, RANTES (regulated upon activation, normal T-cell expressed and secreted), and interleukin (IL)-4 in pleural effusions. PATIENTS AND METHODS: Thirty-one patients with eosinophilic pleural effusion (EPE) [eosinophil percentage > 10% of the pleural fluid nucleated cells] and 10 patients without EPE were evaluated. VCAM-1, eotaxin, RANTES, and IL-4 in all pleural fluids were measured using enzyme-linked immunosorbent assay kits. IL-5 levels of the same fluids were measured in a previous study. RESULTS: VCAM-1, eotaxin, and RANTES but not IL-4 were detectable in the pleural fluids. The mean level of VCAM-1 in EPE (336 +/- 85 ng/mL) was significantly higher (p = 0.011) than that in the noneosinophilic effusions (260 +/- 34 ng/mL) [mean +/- SD]. VCAM-1 levels were significantly correlated with the eosinophil count and percentage in all pleural fluids (r = 0.43, p = 0.005, and r = 0.37, p = 0.019, respectively). Multiple linear regression analysis disclosed that both IL-5 (beta, 0.63; p < 0.001) and VCAM-1 (beta, 0.27, p = 0.025) are independent predictors of the number of eosinophils in all pleural fluids. RANTES and eotaxin did not differ significantly between EPEs and non-EPEs, and were not correlated with the number of pleural fluid eosinophils. CONCLUSION: The levels of VCAM-1 are increased in EPE, suggesting that VCAM-1 is important in the pathogenesis of EPE. Neither eotaxin nor RANTES is associated with pleural fluid eosinophilia.

Pleural fluid levels of interleukin-5 and eosinophils are closely correlated.

BACKGROUND: The mechanisms responsible for the accumulation of eosinophils in pleural fluid are not fully understood. The purpose of this study was to evaluate the relationship between eosinophil accumulation and the levels of interleukin (IL)-5, IL-3, and granulocyte/macrophage colony-simulating factor (GM-CSF) in pleural effusions. METHODS: We evaluated 30 patients with eosinophilic pleural effusions (eosinophil count > 10% nucleated cells in pleural fluid) and 10 patients with noneosinophilic pleural effusions. The patients with eosinophilic pleural effusions included 22 patients with post-coronary artery bypass graft surgery pleural effusions and 8 patients with eosinophilic pleural effusions caused by other causes. IL-5, IL-3, and GM-CSF in all pleural fluids were measured using enzyme-linked immunosorbent assay kits. RESULTS: The mean level of IL-5 in eosinophilic pleural effusions (283.1 +/- 341.6 pg/mL) was significantly (p < 0.025) higher than that in the noneosinophilic effusions (28.2 +/- 19.0 pg/mL). The absolute eosinophil count and percentage correlated significantly with the level of IL-5 in all patients (r = 0.55, p < 0.001, and r = 0.54, p < 0.001, respectively). There was no significant correlation between IL-5 levels and RBC counts in all patients (r = 0.24, p > 0.05). GM-CSF and IL-3 levels were below the detectable range in all pleural fluids. CONCLUSION: There is a significant relationship between the levels of IL-5 in pleural fluid and the total number and percentage of eosinophils in the pleural fluid. IL-5 seems to be related to the eosinophil accumulation associated with blood or air in the pleural space and other eosinophilic pleural effusions.

Altered prostanoid production by fibroblasts cultured from the lungs of human subjects with idiopathic pulmonary fibrosis.

BACKGROUND: Prostanoids are known to participate in the process of fibrogenesis. Because lung fibroblasts produce prostanoids and are believed to play a central role in the pathogenesis of idiopathic pulmonary fibrosis (IPF), we hypothesized that fibroblasts (HF) cultured from the lungs of patients with IPF (HF-IPF) have an altered balance between profibrotic (thromboxane [TX]A2) and antifibrotic (prostacyclin [PGI2]) prostaglandins (PGs) when compared with normal human lung fibroblasts (HF-NL). METHODS: We measured inducible cyclooxygenase (COX)-2 gene and protein expression, and a profile of prostanoids at baseline and after IL-1beta stimulation. RESULTS: In both HF-IPF and HF-NL COX-2 expression was undetectable at baseline, but was significantly upregulated by IL-1beta. PGE2 was the predominant COX product in IL-1beta-stimulated cells with no significant difference between HF-IPF and HF-NL (28.35 [9.09-89.09] vs. 17.12 [8.58-29.33] ng/10(6) cells/30 min, respectively; P = 0.25). TXB2 (the stable metabolite of TXA2) production was significantly higher in IL-1beta-stimulated HF-IPF compared to HF-NL (1.92 [1.27-2.57] vs. 0.61 [0.21-1.64] ng/10(6) cells/30 min, respectively; P = 0.007) and the ratio of PGI2 (as measured by its stable metabolite 6-keto-PGF1alpha) to TXB2 was significantly lower at baseline in HF-IPF (0.08 [0.04-0.52] vs. 0.12 [0.11-0.89] in HF-NL; P = 0.028) and with IL-1beta stimulation (0.24 [0.05-1.53] vs. 1.08 [0.51-3.79] in HF-NL; P = 0.09). CONCLUSION: An alteration in the balance of profibrotic and antifibrotic PGs in HF-IPF may play a role in the pathogeneses of IPF.

Subcellular mechanisms in pulmonary arterial hypertension: combinatorial modalities that inhibit anterograde trafficking and cause bone morphogenetic protein receptor type 2 mislocalization.

Abstract The natural history of familial pulmonary arterial hypertension (PAH) typically involves mutations in and/or haploinsuffciency of BMPR2 (gene for bone morphogenetic protein receptor type 2) but with low penetrance (10%-15%), delayed onset (in the third or fourth decade), and a gender bias (two- to fourfold more prevalent in postpubertal women). Thus, investigators have sought an understanding of "second-hit" modalities that might affect BMPR2 anterograde trafficking and/or function. Indeed, vascular lung lesions in PAH have been reported to contain enlarged "vacuolated" endothelial and smooth muscle cells with dilated endoplasmic reticulum (ER) cisternae, increased ER structural protein reticulon 4 (also called Nogo-B), and enlarged and fragmented Golgi apparatus. We recently replicated this cellular phenotype in primary human pulmonary arterial endothelial cells and human pulmonary arterial smooth muscle cells in culture by acute knockdown of the estradiol 17ß (E2)-responsive proteins signal transducer and activator of transcription 5a (STAT5a) and STAT5b using small interfering RNAs (siRNAs). We have now investigated whether functional haploinsufficiences of these molecules, alone or in combination with other modalities, might interfere with anterograde membrane trafficking using (a) the quantitative tsO45VSV-G-GFP trafficking assay and (b) assays for cell-surface localization of Flag-tagged BMPR2 molecules. The G glycoprotein of the vesicular stomatitis virus (VSV-G) trafficking assay was validated in EA.hy926 endothelial cells by showing that cells exposed to monocrotaline pyrrole displayed reduced anterograde trafficking. Thereafter, the combinatorial knockdowns of STAT5a, STAT5b, BMPR2, and/or endothelial nitric oxide synthase as well as exposure to E2 or 2-methoxyestradiol were observed to significantly inhibit VSV-G trafficking. These combinations also led to intracellular trapping of wild-type Flag-tagged BMPR2. Overexpression of the PAH disease-derived F14 and KDF mutants of BMPR2, which were trapped in the ER/Golgi, also inhibited VSV-G trafficking in trans. Moreover, probenecid, a chemical chaperone in clinical use today, partially restored cell-surface localization of the KDF but not the F14 mutant. These data identify several combinatorial modalities that inhibit VSV-G anterograde trafficking and cause mislocalization of BMPR2. These modalities merit consideration in defining aspects of the late-developing and gender-biased natural history of human PAH.

BMPR2 expression is suppressed by signaling through the estrogen receptor.

BACKGROUND: Studies in multiple organ systems have shown cross-talk between signaling through the bone morphogenetic protein receptor type 2 (BMPR2) and estrogen pathways. In humans, pulmonary arterial hypertension (PAH) has a female predominance, and is associated with decreased BMPR2 expression. The goal of this study was to determine if estrogens suppress BMPR2 expression. METHODS: A variety of techniques were utilized across several model platforms to evaluate the relationship between estrogens and BMPR2 gene expression. We used quantitative RT-PCR, gel mobility shift, and luciferase activity assays in human samples, live mice, and cell culture. RESULTS: BMPR2 expression is reduced in lymphocytes from female patients compared with male patients, and in whole lungs from female mice compared with male mice. There is an evolutionarily conserved estrogen receptor binding site in the BMPR2 promoter, which binds estrogen receptor by gel-shift assay. Increased exogenous estrogen decreases BMPR2 expression in cell culture, particularly when induced to proliferate. Transfection of increasing quantities of estrogen receptor alpha correlates strongly with decreasing expression of BMPR2. CONCLUSIONS: BMPR2 gene expression is reduced in females compared to males in live humans and in mice, likely through direct estrogen receptor alpha binding to the BMPR2 promoter. This reduced BMPR2 expression may contribute to the increased prevalence of PAH in females.