BMP pathway regulation of and by macrophages.

Pulmonary arterial hypertension (PAH) is a disease of progressively increasing pulmonary vascular resistance, associated with mutations of the type 2 receptor for the BMP pathway, BMPR2. The canonical signaling pathway for BMPR2 is through the SMAD family of transcription factors. BMPR2 is expressed in every cell type, but the impact of BMPR2 mutations affecting SMAD signaling, such as Bmpr2delx4+, had only previously been investigated in smooth muscle and endothelium. In the present study, we created a mouse with universal doxycycline-inducible expression of Bmpr2delx4+ in order to determine if broader expression had an impact relevant to the development of PAH. We found that the most obvious phenotype was a dramatic, but patchy, increase in pulmonary inflammation. We crossed these double transgenic mice onto an NF-κB reporter strain, and by luciferase assays on live mice, individual organs and isolated macrophages, we narrowed down the origin of the inflammatory phenotype to constitutive activation of tissue macrophages. Study of bone marrow-derived macrophages from mutant and wild-type mice suggested a baseline difference in differentiation state in Bmpr2 mutants. When activated with LPS, both mutant and wild-type macrophages secrete BMP pathway inhibitors sufficient to suppress BMP pathway activity in smooth muscle cells (SMC) treated with conditioned media. Functionally, co-culture with macrophages results in a BMP signaling-dependent increase in scratch closure in cultured SMC. We conclude that SMAD signaling through BMP is responsible, in part, for preventing macrophage activation in both live animals and in cells in culture, and that activated macrophages secrete BMP inhibitors in sufficient quantity to cause paracrine effect on vascular smooth muscle.

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.

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.

Idiopathic and heritable PAH perturb common molecular pathways, correlated with increased MSX1 expression.

The majority of pulmonary arterial hypertension (PAH) is not associated with BMPR2 mutation, and major risk factors for idiopathic PAH are not known. The objective of this study was to identify a gene expression signature for IPAH. To accomplish this, we used Affymetrix arrays to probe expression levels in 86 patient samples, including 22 healthy controls, 20 IPAH patients, 20 heritable PAH patients (HPAH), and 24 BMPR2 mutation carriers that were as yet unaffected (UMC). Culturing the patient cells removes the signatures of drug effects and inflammation which have made interpretation of results from freshly isolated lymphocytes problematic. We found that gene expression signatures from IPAH patients clustered either with HPAH patients or in a single distinct group. There were no groups of genes changed in IPAH that were not also changed in HPAH. HPAH, IPAH, and UMC had common changes in metabolism, actin dynamics, adhesion, cytokines, metabolism, channels, differentiation, and transcription factors. Common to IPAH and HPAH but not UMC were an upregulation of vesicle trafficking, oxidative/nitrosative stress, and cell cycle genes. The transcription factor MSX1, which is known to regulate BMP signaling, was the most upregulated gene (4×) in IPAH patients. These results suggest that IPAH cases have a shared molecular origin, which is closely related to, but distinct from, HPAH. HPAH and IPAH share the majority of altered signaling pathways, suggesting that treatments developed to target the molecular etiology of HPAH will also be effective against IPAH.

Oxidative injury is a common consequence of BMPR2 mutations.

BACKGROUND: Hereditary pulmonary arterial hypertension(PAH) is usually caused by mutations in BMPR2. Mutations are found throughout the gene, and common molecular consequences of different types of mutation are not known. Knowledge of common molecular consequences would provide insight into molecular etiology of disease. The objective of this study was to determine common molecular consequences across classes of BMPR2 mutation. METHODS #ENTITYSTARTX00026; RESULTS: Increased superoxide and peroxide production, and alterations in genes associated with oxidative stress were a common consequence of stable transfection of vascular smooth muscle cells with three distinct classes of BMPR2 mutation, in the ligand binding domain, the kinase domain, and the cytoplasmic tail domain. Measurement of oxidized lipids in whole lung from transgenic mice expressing a mutation in the BMPR2 cytoplasmic tail showed a 50% increase in isoprostanes and a twofold increase in isofurans, suggesting increased ROS of mitochondrial origin. Immunohistochemistry on BMPR2 transgenic mouse lung showed that oxidative stress was vascular-specific. Electron microscopy showed decreased mitochondrial size and variability in pulmonary vessels from BMPR2 mutant mice. Measurement of oxidized lipids in urine from humans with BMPR2 mutations demonstrated increased ROS, regardless of disease status. Immunohistochemistry on HPAH patient lung confirmed oxidative stress specific to the vasculature. CONCLUSIONS: Increased oxidative stress, likely of mitochondrial origin, is a common consequence of BMPR2 mutation across mutation types in cell culture, mice, and humans.

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.

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.

Comparison of pleural fluid N-terminal pro-brain natriuretic peptide and brain natriuretic-32 peptide levels.

BACKGROUND: Current evidence indicates that measurement of pleural fluid N-terminal pro-brain natriuretic peptide (NT-proBNP) levels can aid in distinguishing pleural effusions of cardiac origin from those of noncardiac origin. To date, only one study, to our knowledge, has described simultaneous measurement of pleural fluid brain natriuretic-32 peptide (BNP) and NT-proBNP. The purpose of the present study was to determine pleural fluid BNP and NT-proBNP levels and analyze the relationship between these two measurements. We hypothesized that there would be a positive correlation between pleural fluid NT-proBNP and BNP, whereas NT-proBNP levels would be higher than BNP levels. METHODS: Levels of pleural fluid NT-proBNP and BNP were measured by enzyme immunoassay in a total of 80 patients: 20 with congestive heart failure, 20 status post-coronary artery bypass graft, 20 with carcinoma, and 20 with pneumonia. RESULTS: Comparison of NT-proBNP and BNP concentrations using the Spearman method of statistical analysis revealed a correlation coefficient of 0.572, P < .001. Evaluation of the diagnostic accuracy of BNP and NT-proBNP in patients with pleural effusions of cardiac origin demonstrated an area under the receiver operating characteristic curve of 0.700 (95% CI, 0.569-0.831) and 0.835 (95% CI, 0.721-0.949), respectively. CONCLUSIONS: Although levels of pleural fluid BNP have a statistically significant correlation with those of NT-proBNP, this relationship only explains 32% of the variance in NT-proBNP levels. Furthermore, when compared with BNP, NT-proBNP is a more accurate diagnostic aid in the evaluation of pleural effusions of cardiac origin.

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.

Gene expression in BMPR2 mutation carriers with and without evidence of pulmonary arterial hypertension suggests pathways relevant to disease penetrance.

BACKGROUND: While BMPR2 mutation strongly predisposes to pulmonary arterial hypertension (PAH), only 20% of mutation carriers develop clinical disease. This finding suggests that modifier genes contribute to FPAH clinical expression. Since modifiers are likely to be common alleles, this problem is not tractable by traditional genetic approaches. Furthermore, examination of gene expression is complicated by confounding effects attributable to drugs and the disease process itself. METHODS: To resolve these problems, B-cells were isolated, EBV-immortalized, and cultured from familial PAH patients with BMPR2 mutations, mutation positive but disease-free family members, and family members without mutation. This allows examination of differences in gene expression without drug or disease-related effects. These differences were assayed by Affymetrix array, with follow-up by quantitative RT-PCR and additional statistical analyses. RESULTS: By gene array, we found consistent alterations in multiple pathways with known relationship to PAH, including actin organization, immune function, calcium balance, growth, and apoptosis. Selected genes were verified by quantitative RT-PCR using a larger sample set. One of these, CYP1B1, had tenfold lower expression than control groups in female but not male PAH patients. Analysis of overrepresented gene ontology groups suggests that risk of disease correlates with alterations in pathways more strongly than with any specific gene within those pathways. CONCLUSION: Disease status in BMPR2 mutation carriers was correlated with alterations in proliferation, GTP signaling, and stress response pathway expression. The estrogen metabolizing gene CYP1B1 is a strong candidate as a modifier gene in female PAH patients.

Mice expressing BMPR2R899X transgene in smooth muscle develop pulmonary vascular lesions.

Familial pulmonary arterial hypertension (PAH) is associated with mutations in bone morphogenetic protein type II receptor (BMPR2). Many of these mutations occur in the BMPR2 tail domain, leaving the SMAD functions intact. To determine the in vivo consequences of BMPR2 tail domain mutation, we created a smooth muscle-specific doxycycline-inducible BMPR2 mutation with an arginine to termination mutation at amino acid 899. When these SM22-rtTA x TetO(7)-BMPR2(R899X) mice had transgene induced for 9 wk, starting at 4 wk of age, they universally developed pulmonary vascular pruning as assessed by fluorescent microangiography. Approximately one-third of the time, the induced animals developed elevated right ventricular systolic pressures (RVSP), associated with extensive pruning, muscularization of small pulmonary vessels, and development of large structural pulmonary vascular changes. These lesions included large numbers of macrophages and T cells in their adventitial compartment as well as CD133-positive cells in the lumen. Small vessels filled with CD45-positive and sometimes CD3-positive cells were a common feature in all SM22-rtTA x TetO(7)-BMPR2(R899X) mice. Gene array experiments show changes in stress response, muscle organization and function, proliferation, and apoptosis and developmental pathways before RVSP increases. Our results show that the primary phenotypic result of BMPR2 tail domain mutation in smooth muscle is pulmonary vascular pruning leading to elevated RVSP, associated with early dysregulation in multiple pathways with clear relevance to PAH. This model should be useful to the research community in examining early molecular and physical events in the development of PAH and as a platform to validate potential treatments.

Imaging mass spectrometry of intact proteins from alcohol-preserved tissue specimens: bypassing formalin fixation.

Imaging mass spectrometry is becoming a key technology for the investigation of the molecular content of biological tissue sections in direct correlation with the underlying histology. Much of our work has been done with fresh-frozen tissue sections that has undergone minimal protein degradation between the time a tissue biopsy is sampled and the time it is snap-frozen so that no preserving or fixing agents need to be added to the frozen biopsy. However, in many sampling environments, immediate flash freezing may not be possible and so we have explored the use of ethanol-preserved, paraffin-embedded tissue specimens for proteomic analyses. Solvent-only preserved tissue specimens provide long-term preservation at room temperature, generation of high quality histological sections and little if any chemical alteration of the proteins. Using mouse organs, several key steps involved in the tissue dehydration process have been investigated to assess the potential of such preserved specimens for profiling and imaging mass spectrometry investigations.

Endoplasmic reticulum stress in alveolar epithelial cells is prominent in IPF: association with altered surfactant protein processing and herpesvirus infection.

Recent evidence suggests that dysfunctional type II alveolar epithelial cells (AECs) contribute to the pathogenesis of idiopathic pulmonary fibrosis (IPF). Based on the hypothesis that disease-causing mutations in surfactant protein C (SFTPC) provide an important paradigm for studying IPF, we investigated a potential mechanism of AEC dysfunction suggested to result from mutant SFTPC expression: induction of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). We evaluated biopsies from 23 IPF patients (including 3 family members with L188Q SFTPC mutations, 10 individuals with familial interstitial pneumonia without SFTPC mutations, and 10 individuals with sporadic IPF) and sections from 10 control lungs. After demonstrating UPR activation in cultured A549 cells expressing mutant SFTPC, we identified prominent expression of UPR markers in AECs in the lungs of patients with SFTPC mutation-associated fibrosis. In individuals with familial interstitial pneumonia without SFTPC mutations and patients with sporadic IPF, we also found UPR activation selectively in AECs lining areas of fibrotic remodeling. Because herpesviruses are found frequently in IPF lungs and can induce ER stress, we investigated expression of viral proteins in lung biopsies. Herpesvirus protein expression was found in AECs from 15/23 IPF patients and colocalized with UPR markers in AECs from these patients. ER stress and UPR activation are found in the alveolar epithelium in patients with IPF and could contribute to disease progression. Activation of these pathways may result from altered surfactant protein processing or chronic herpesvirus infection.

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.

Interaction of interleukin-6 and the BMP pathway in pulmonary smooth muscle.

The majority of familial pulmonary arterial hypertension (PAH) cases are caused by mutations in the type 2 bone morphogenetic protein receptor (BMPR2). However, less than one-half of BMPR2 mutation carriers develop PAH, suggesting that the most important function of BMPR2 mutation is to cause susceptibility to a "second hit." There is substantial evidence from the literature implicating dysregulated inflammation, in particular the cytokine IL-6, in the development of PAH. We thus hypothesized that the BMP pathway regulates IL-6 in pulmonary tissues and conversely that IL-6 regulates the BMP pathway. We tested this in vivo using transgenic mice expressing an inducible dominant negative BMPR2 in smooth muscle, using mice injected with an IL-6-expressing virus, and in vitro using small interfering RNA (siRNA) to BMPR2 in human pulmonary artery smooth muscle cells (PA SMC). Consistent with our hypothesis, we found upregulation of IL-6 in both the transgenic mice and in cultured PA SMC with siRNA to BMPR2; this could be abolished with p38(MAPK) inhibitors. We also found that IL-6 in vivo caused a twofold increase in expression of the BMP signaling target Id1 and caused increased BMP activity in a luciferase-reporter assay in PA SMC. Thus we have shown both in vitro and in vivo a complete negative feedback loop between IL-6 and BMP, suggesting that an important consequence of BMPR2 mutations may be poor regulation of cytokines and thus vulnerability to an inflammatory second hit.

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.

Differential expression of surfactant protein A in the nasal mucosa of patients with allergy symptoms.

OBJECTIVE: To characterize surfactant protein A (SP-A) expression in human nasal tissue and correlate differential expression of SP-A with symptoms suggestive of allergic rhinitis. DESIGN: Allergic rhinitis symptom data were prospectively collected in the form of the Rhinitis Symptom Utility Index, the Rhinoconjunctivitis Quality of Life Questionnaire, and a Visual Analog Scale. Immunohistochemical staining for SP-A was performed on resected nasal tissue. Quantitative polymerase chain reaction amplification of the SP-A gene referenced to beta-actin was performed on complementary DNA samples synthesized from total RNA isolates. SETTING: Academic tertiary referral center, department of otolaryngology laboratories. PATIENTS: Twenty-five consecutive patients undergoing nasal surgery. MAIN OUTCOME MEASURES: Immunohistochemical staining of SP-A in human nasal mucosa and submucosa, polymerase chain reaction amplification of SP-A messenger RNA, and rhinitis symptom scores. RESULTS: Immunostaining localized SP-A to the mucosa and submucosal glands in specimens. Quantitative polymerase chain reaction demonstrated correlation between SP-A messenger RNA concentration and the total Rhinitis Symptom Utility Index score (0.51, P = .009) as well as "sneezing over the previous week" (0.40, P = .049), "runny nose over the previous week" (0.55, P = .005), and "sneezing today" (0.47, P = .02). CONCLUSIONS: To our knowledge, this is the first report of SP-A expression in human nasal tissue. Furthermore, the degree of expression correlated with severity of disease as measured by the Rhinitis Symptom Utility Index in patients with allergic rhinitis symptoms.

Tenascin-C is induced by mutated BMP type II receptors in familial forms of pulmonary arterial hypertension.

Familial forms of human pulmonary arterial hypertension (FPAH) have been linked to mutations in bone morphogenetic protein (BMP) type II receptors (BMPR2s), yet the downstream targets of these receptors remain obscure. Here we show that pulmonary vascular lesions from patients harboring BMPR2 mutations express high levels of tenascin-C (TN-C), an extracellular matrix glycoprotein that promotes pulmonary artery (PA) smooth muscle cell (SMC) proliferation. To begin to define how TN-C is regulated, PA SMCs were cultured from normal subjects and from those with FPAH due to BMPR2 mutations. FPAH SMCs expressed higher levels of TN-C than normal SMCs. Similarly, expression of Prx1, a factor that drives TN-C transcription, was elevated in FPAH vascular lesions and SMCs derived thereof. Furthermore, Prx1 and TN-C promoter activities were significantly higher in FPAH vs. normal SMCs. To delineate how BMPR2s control TN-C, we focused on receptor (R)-Smads, downstream effectors activated by wild-type BMPR2s. Nuclear localization and phosphorylation of R-Smads was greater in normal vs. FPAH SMCs. As well, indirect blockade of R-Smad signaling with a kinase-deficient BMP receptor Ib upregulated TN-C in normal SMCs. Because ERK1/2 MAPKs inhibit the transcriptional activity of R-Smads, and because ERK1/2 promotes TN-C transcription, we determined whether ERK1/2 inhibits R-Smad signaling in FPAH SMCs and whether this activity is required for TN-C transcription. Indeed, ERK1/2 activity was greater in FPAH SMCs, and inhibition of ERK1/2 resulted in nuclear localization of R-Smads and inhibition of TN-C. These studies define a novel signaling network relevant to PAH underscored by BMPR2 mutations.

HIV-1 Tat interaction with cyclin T1 represses mannose receptor and the bone morphogenetic protein receptor-2 transcription.

Macrophage transcription is significantly altered by HIV-1 infection. HIV Tat, an immediate-early product of the viral lifecycle, interacts with host transcription factors to alter host gene expression. We have previously shown that Tat represses transcription from the mannose receptor (MR) and the bone morphogenetic protein receptor-2 (BMPR2) promoters. The current study shows that transcriptional repression of these receptors involves Tat interaction with cyclin T1. Assays using U937 human monocytic cells transiently expressing MR or BMPR2 promoter-luciferase constructs demonstrated equal repression by one- and two-exon Tat gene products. A mutant Tat expression vector encoding Tat protein lacking the cyclin T1 binding domain failed to inhibit MR and BMPR2 promoter activities. Over-expression of cyclin T1 in the presence of wild-type Tat resulted in recovered activity from both promoters. Finally, two inhibitors of cyclin-dependent kinase 9 (a dominant negative CDK9 and flavopiridol) repressed activity from the MR and BMPR2 promoters.