[Show abstract][Hide abstract] ABSTRACT: Rationale: Pulmonary Arterial Hypertension (PAH) is a progressive disease characterized by elevated pulmonary artery pressure, vascular remodeling, and ultimately right ventricular heart failure. PAH can have a genetic component (Heritable PAH), most often through mutations of bone morphogenetic protein receptor 2, as well as idiopathic and associated forms. Heritable PAH is not completely penetrant within families, with ~20% concurrence of inactivating bone morphogenetic protein receptor 2 mutations and delayed onset of PAH disease. Since one of the treatment options is using prostacyclin analogs, we hypothesized that prostacyclin synthase promoter sequence variants associated with increased mRNA expression may play a protective role in the bone morphogenetic protein receptor 2 unaffected carriers. Objectives: To characterize the range of prostacyclin synthase promoter variants and assess their transcriptional activities in PAH relevant cell types. Determine the distribution of prostacyclin synthase promoter variants in PAH, unaffected carriers in Heritable PAH families, and control populations. Methods: Polymerase chain reaction approaches were used to genotype prostacyclin synthase promoter variants in over 300 individuals. Prostacyclin synthase promoter haplotypes' transcriptional activities were determined with luciferase reporter assays. Measurements and Main Results: We identified a comprehensive set of prostacyclin synthase promoter variants and tested their transcriptional activities in PAH relevant cell types. We demonstrated differences of prostacyclin synthase promoter activities dependent on their haplotype. Conclusions: Prostacyclin synthase promoter sequence variants exhibit a range of transcriptional activities. We discovered a significant bias for more active prostacyclin synthase promoter variants in unaffected carriers as compared to affected PAH patients.
American Journal of Respiratory and Critical Care Medicine 03/2014; · 11.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chromosomal instability is central to the process of carcinogenesis. The genome-wide detection of somatic chromosomal alterations (SCAs) in small premalignant lesions remains challenging since sample heterogeneity dilutes the aberrant cell information. To overcome this hurdle, we focused on the B allele frequency data from single nucleotide polymorphism microarrays (SNP arrays). The difference of allelic fractions between paired tumor and normal samples from the same patient (delta-θ) provides a simple but sensitive detection of SCA in affected tissue. We applied the delta-θ approach to small, heterogeneous clinical specimens including endobronchial biopsies and brushings. Regions identified by delta-θ were validated by FISH and qPCR in heterogeneous samples. Distinctive genomic variations were successfully detected across the whole genome in all invasive cancer cases (6/6), carcinoma in situ (3/3), and high grade dysplasia (severe or moderate) (3/11). Not only well-described SCAs in lung squamous cell carcinoma, but also several novel chromosomal alterations were frequently found across the pre-invasive dysplastic cases. Within these novel regions, losses of putative tumor suppressors (RNF20 and SSBP2) and an amplification of RASGRP3 gene with oncogenic activity were observed. Widespread sampling of the airway during bronchoscopy demonstrated that field cancerization reflected by SCAs at multiple sites was detectable. SNP arrays combined with delta-θ analysis can detect SCAs in heterogeneous clinical sample and expand our ability to assess genomic instability in the airway epithelium as a biomarker of lung cancer risk.
Cancer Prevention Research 12/2013; · 4.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Genomics and proteomics have emerged as key technologies in biomedical research, resulting in a surge of interest in training by investigators keen to incorporate these technologies into their research. At least two types of training can be envisioned in order to produce meaningful results, quality publications and successful grant applications: (1) immediate short-term training workshops and (2) long-term graduate education or visiting scientist programs. We aimed to fill the former need by providing a comprehensive hands-on training course in genomics, proteomics and informatics in a coherent, experimentally-based framework. This was accomplished through a National Heart, Lung, and Blood Institute (NHLBI)-sponsored 10-day Genomics and Proteomics Hands-on Workshop held at National Jewish Health (NJH) and the University of Colorado School of Medicine (UCD). The course content included comprehensive lectures and laboratories in mass spectrometry and genomics technologies, extensive hands-on experience with instrumentation and software, video demonstrations, optional workshops, online sessions, invited keynote speakers, and local and national guest faculty. Here we describe the detailed curriculum and present the results of short- and long-term evaluations from course attendees. Our educational program consistently received positive reviews from participants and had a substantial impact on grant writing and review, manuscript submissions and publications.
[Show abstract][Hide abstract] ABSTRACT: Rationale: Schistosomiasis is one of the most common causes of pulmonary arterial hypertension worldwide, but the pathogenic mechanism by which the host inflammatory response contributes to vascular remodeling is unknown. We sought to identify signaling pathways that play protective or pathogenic roles in experimental Schistosoma-induced pulmonary vascular disease by whole-lung transcriptome analysis. Methods: Wildtype mice were experimentally exposed to S. mansoni ova by intraperitoneal sensitization followed by tail vein augmentation, and the phenotype assessed by right ventricular catheterization and tissue histology, RNA and protein analysis. Whole-lung transcriptome analysis by microarray and RNA sequencing was performed, the latter analyzed using 2 bioinformatic methods. Functional testing of the candidate IL-6 pathway was determined using IL6-knockout mice and the STAT3 inhibitor STI-201. Results: Wild-type mice exposed to S. mansoni had increased right ventricular systolic pressure and thickness of the pulmonary vascular media. Whole lung transcriptome analysis identified the IL6-STAT3-NFATc2 pathway as being upregulated, which was confirmed by PCR and immunostaining of lung tissue from S. mansoni-exposed mice and patients who died of the disease. Mice lacking IL6 or treated with STI-201 developed pulmonary hypertension associated with significant intima remodeling after exposure to S. mansoni. Conclusions: Whole lung transcriptome analysis identified upregulation of the IL6-STAT3-NFATc2 pathway, and IL6 signaling was found to be protective against Schistosoma-induced intimal remodeling.
American Journal of Respiratory Cell and Molecular Biology 07/2013; · 4.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: There is incredible potential to advance our understanding of disease pathogenesis, enhance our diagnostic capability, and revolutionize our treatment modalities with the advent of advanced systems approaches to genetic, genomic, and epigenetic discoveries. Investigation using these technologies is beginning to impact our understanding of pulmonary arterial hypertension (PAH). The following review details work to date on single gene mutations in PAH, and expression array analysis in the disease. The wider use of DNA-based arrays for genome wide association studies (GWAS) and copy number alterations is examined. The impact of epigenomic modulation in the pathobiology of PAH and its therapeutic implications is investigated. Finally, a summary of the capabilities and promises for next-generation sequencing is discussed. A framework for studies of the future is proposed.
[Show abstract][Hide abstract] ABSTRACT: Sepsis, a systemic inflammatory response to infection, commonly progresses to acute lung injury (ALI), an inflammatory lung disease with high morbidity. We postulated that sepsis-associated ALI is initiated by degradation of the pulmonary endothelial glycocalyx, leading to neutrophil adherence and inflammation. Using intravital microscopy, we found that endotoxemia in mice rapidly induced pulmonary microvascular glycocalyx degradation via tumor necrosis factor-α (TNF-α)-dependent mechanisms. Glycocalyx degradation involved the specific loss of heparan sulfate and coincided with activation of endothelial heparanase, a TNF-α-responsive, heparan sulfate-specific glucuronidase. Glycocalyx degradation increased the availability of endothelial surface adhesion molecules to circulating microspheres and contributed to neutrophil adhesion. Heparanase inhibition prevented endotoxemia-associated glycocalyx loss and neutrophil adhesion and, accordingly, attenuated sepsis-induced ALI and mortality in mice. These findings are potentially relevant to human disease, as sepsis-associated respiratory failure in humans was associated with higher plasma heparan sulfate degradation activity; moreover, heparanase content was higher in human lung biopsies showing diffuse alveolar damage than in normal human lung tissue.
[Show abstract][Hide abstract] ABSTRACT: ABSTRACT BACKGROUND: Lymphangiogenesis responds to tissue injury as a key component of normal wound healing. The development of fibrosis in the idiopathic interstitial pneumonias (IIPs) may result from abnormal wound healing in response to injury. We hypothesize that increased lymphatic vessel length, a marker of lymphangiogenesis, is associated with parenchymal components of the fibroblast reticulum (organizing collagen, fibrotic collagen and fibroblast foci), and its extent correlates with disease severity. METHODS: We assessed stereologically the parenchymal structure of fibrotic lungs and its associated lymphatic network, which was highlighted immunohistochemically in age matched samples of usual interstitial pneumonia (UIP), non-specific interstitial pneumonia (NSIP) with FVC <80%, chronic obstructive pulmonary disease (COPD) with a GOLD (Global Initiative for Obstructive Lung Disease) stage 0, and normal control lungs. RESULTS: Lymphatic vessel length density, as opposed to vessel volume density, was found to be associated with organizing and fibrotic collagen density (p<0.0001). Length density of lymphatic vessels and the volume density of organizing and fibrotic collagen were significantly associated with severity of both FVC% (p<0.001) and DLco% (p<0.001). CONCLUSIONS: Severity of disease in UIP and NSIP is associated with increased lymphatic vessel length and is strongly associated with components of the fibroblast reticulum, namely organizing and fibrotic collagen, which supports a pathogenic role of lymphatic vessels in these two diseases. Furthermore, the absence of definable differences between UIP and NSIP, suggests that lymphatic vessels is a unifying mechanism for the development of fibrosis in these fibrotic lung diseases.
[Show abstract][Hide abstract] ABSTRACT: The G-quadruplex, a non-B DNA motif that forms in certain G-rich sequences, is often located near transcription start sites in growth regulatory genes. Multiple lines of evidence show that reactive oxygen species generated as second messengers during physiologic signaling target specific DNA sequences for oxidative base modifications. Because guanine repeats are uniquely sensitive to oxidative damage, and G4 sequences are known "hot spots" for genetic mutation and DNA translocation, we hypothesized that G4 sequences are targeted for oxidative base modifications in hypoxic signaling. Approximately 25% of hypoxia-regulated genes in pulmonary artery endothelial cells harbored G4 sequences within their promoters. Chromatin immunoprecipitation showed that common base oxidation product 8-oxoguanine was selectively introduced into G4s, in promoters of hypoxia up-, down-, and nonregulated genes. Additionally, base excision DNA repair (BER) enzymes were recruited, and transient strand breaks formed in these sequences. Transcription factor Sp1, constitutively bound to G4 sequences in normoxia, was evicted as 8-oxoguanine accumulated during hypoxic exposure. Blocking hypoxia-induced oxidant production prevented both base modifications and decreased Sp1 binding. These findings suggest that oxidant stress in hypoxia causes oxidative base modifications, recruitment of BER enzymes, and transient strand breaks in G4 promoter sequences potentially altering G4 integrity and function.
[Show abstract][Hide abstract] ABSTRACT: Gonadotrope and null cell pituitary tumors cause significant morbidity, often presenting with signs of hypogonadism together with visual disturbances due to mass effects. Surgery and radiation are the only therapeutic options to date. To identify dysregulated genes and pathways that may play a role in tumorigenesis and/or progression, molecular profiling was performed on 14 gonadotrope tumors, with nine normal human pituitaries obtained at autopsy serving as controls. Bioinformatic analysis identified putative downstream effectors of tumor protein 53 (p53) that were consistently repressed in gonadotrope pituitary tumors, including RPRM, P21, and PMAIP1, with concomitant inhibition of the upstream p53 regulator, PLAGL1(Zac1). Further analysis of the growth arrest and DNA damage-inducible (GADD45) family revealed no change in the p53 target, GADD45α, but identified repression of GADD45β in pituitary tumors in addition to the previously reported inhibition of GADD45γ. Overexpression of GADD45β in LβT2 mouse gonadotrope cells blocked tumor cell proliferation and increased rates of apoptosis in response to growth factor withdrawal. Stable gonadotrope cell transfectants expressing increased GADD45β showed decreased colony formation in soft agar, confirming its normal role as a tumor suppressor. Unlike previous studies of GADD45γ in pituitary tumors and α and β in other tumors, bisulfite sequencing showed no evidence of hypermethylation of the GADD45β promoter in human pituitary tumor samples to explain the repression of its expression. Thus, GADD45β is a novel pituitary tumor suppressor whose reexpression blocks proliferation, survival, and tumorigenesis. Together these studies identify new targets and mechanisms to explore in pituitary tumor initiation and progression.
[Show abstract][Hide abstract] ABSTRACT: The mortality rate for acute lung injury (ALI) is reported to be between 35-40%, and there are very few treatment strategies that improve the death rate from this condition. Previous studies have suggested that signaling through the prostaglandin (PG) I(2) receptor may protect against bleomycin-induced ALI in mice. We found that mice that overexpress PGI synthase (PGIS) in the airway epithelium were significantly protected against bleomycin-induced mortality and had reduced parenchymal consolidation, apoptosis of lung tissue, and generation of F(2)-isoprostanes compared with littermate wild-type controls. In addition, we show for the first time in both in vivo and in vitro experiments that PGI(2) induced the expression of NADP (H): quinoneoxidoreductase 1 (Nqo 1), an enzyme that prevents the generation of reactive oxygen species. PGI(2) induction of Nqo 1 provides a possible novel mechanism by which this prostanoid protects against bleomycin-induced mortality and identifies a potential therapeutic target for human ALI.
[Show abstract][Hide abstract] ABSTRACT: There are no established chemopreventive agents for lung cancer, the leading cause of cancer death in the United States. Prostacyclin levels are low in lung cancer and supplementation prevents lung cancer in preclinical models. We carried out a multicenter double-blind, randomized, phase II placebo-controlled trial of oral iloprost in current or former smokers with sputum cytologic atypia or endobronchial dysplasia. Bronchoscopy was performed at study entry and after completion of six months of therapy. Within each subject, the results were calculated by using the average score of all biopsies (Avg), the worst biopsy score (Max), and the dysplasia index (DI). Change in Avg was the primary end point, evaluated in all subjects, as well as in current and former smokers. The accrual goal of 152 subjects was reached and 125 completed both bronchoscopies (60/75 iloprost, 65/77 placebo). Treatment groups were well matched for age, tobacco exposure, and baseline histology. Baseline histology was significantly worse for current smokers (Avg 3.0) than former smokers (Avg 2.1). When compared with placebo, former smokers receiving oral iloprost exhibited a significantly greater improvement in Avg (0.41 units better, P = 0.010), in Max (1.10 units better, P = 0.002), and in DI (12.45%, P = 0.006). No histologic improvement occurred in current smokers. Oral iloprost significantly improves endobronchial histology in former smokers and deserves further study to determine if it can prevent the development of lung cancer.
Cancer Prevention Research 06/2011; 4(6):793-802. · 4.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Growth differentiation factor (GDF)-15 is a secreted member of the transforming growth factor-β cytokine superfamily. GDF-15 levels are elevated in the serum of patients with cardiovascular diseases. We hypothesized that GDF-15 levels would also be increased in the plasma and lung tissue of patients with systemic sclerosis-associated pulmonary arterial hypertension (SSc-PAH).
GDF-15 levels were measured in plasma in subjects with SSc-PAH (n = 30) and compared with subjects with systemic sclerosis (SSc) without pulmonary arterial hypertension (PAH) (n = 24). Patients with idiopathic PAH (IPAH) (n = 44) and normal individuals (n = 13) served as control subjects. Immunohistochemistry and immunofluorescence assay identified GDF-15 protein in lung tissue from patients with SSc-PAH and IPAH.
Patients with SSc-PAH had significantly higher mean circulating levels of GDF-15 in plasma compared with patients with SSc without PAH (422.3 ± 369.5 pg/mL vs 108.1 ± 192.8 pg/mL, P = .004). GDF-15 levels correlated positively with estimated right ventricular systolic pressure on echocardiogram and plasma levels of the amino terminal propeptide form of brain natriuretic peptide. There was an inverse correlation between circulating GDF-15 and diffusing capacity of the lung for carbon monoxide (Dlco) and a positive correlation with the FVC to Dlco ratio on pulmonary function test. GDF-15 levels > 125 pg/mL were associated with reduced survival. GDF-15 protein expression was increased in lung tissue from patients with SSc-PAH.
GDF-15 may be a useful biomarker in PAH associated with SSc. Its presence in lung tissue may suggest a role in the pathology of the disease.
[Show abstract][Hide abstract] ABSTRACT: The Division of Lung Diseases of the National Heart, Lung, and Blood Institute, with the Office of Rare Diseases Research, held a workshop to identify priority areas and strategic goals to enhance and accelerate research that will result in improved understanding of the lung vasculature, translational research needs, and ultimately the care of patients with pulmonary vascular diseases. Multidisciplinary experts with diverse experience in laboratory, translational, and clinical studies identified seven priority areas and discussed limitations in our current knowledge, technologies, and approaches. The focus for future research efforts include the following: (1) better characterizing vascular genotype-phenotype relationships and incorporating systems biology approaches when appropriate; (2) advancing our understanding of pulmonary vascular metabolic regulatory signaling in health and disease; (3) expanding our knowledge of the biologic relationships between the lung circulation and circulating elements, systemic vascular function, and right heart function and disease; (4) improving translational research for identifying disease-modifying therapies for the pulmonary hypertensive diseases; (5) establishing an appropriate and effective platform for advancing translational findings into clinical studies testing; and (6) developing the specific technologies and tools that will be enabling for these goals, such as question-guided imaging techniques and lung vascular investigator training programs. Recommendations from this workshop will be used within the Lung Vascular Biology and Disease Extramural Research Program for planning and strategic implementation purposes.
American Journal of Respiratory and Critical Care Medicine 12/2010; 182(12):1554-62. · 11.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Vascular remodeling in pulmonary arterial hypertension (PAH) involves proliferation and migration of endothelial and smooth muscle cells, leading to obliterative vascular lesions. Previous studies have indicated that the endothelial cell proliferation is quasineoplastic, with evidence of monoclonality and instability of short DNA microsatellite sequences.
To assess whether there is larger-scale genomic instability.
We performed genome-wide microarray copy number analysis on pulmonary artery endothelial cells and smooth muscle cells isolated from the lungs of patients with PAH.
Mosaic chromosomal abnormalities were detected in PAEC cultures from five of nine PAH lungs but not in normal (n = 8) or disease control subjects (n = 5). Fluorescent in situ hybridization analysis confirmed the presence of these abnormalities in vivo in two of three cases. One patient harbored a germline mutation of BMPR2, the primary genetic cause of PAH, and somatic loss of chromosome-13, which constitutes a second hit in the same pathway by deleting Smad-8. In two female subjects with mosaic loss of the X chromosome, methylation analysis showed that the active X was deleted. One subject also showed completely skewed X-inactivation in the nondeleted cells, suggesting the pulmonary artery endothelial cell population was clonal before the acquisition of the chromosome abnormality.
Our data indicate a high frequency of genetically abnormal subclones within PAH lung vessels and provide the first definitive evidence of a second genetic hit in a patient with a germline BMPR2 mutation. We propose that these chromosome abnormalities may confer a growth advantage and thus contribute to the progression of PAH.
American Journal of Respiratory and Critical Care Medicine 11/2010; 182(9):1153-60. · 11.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Pulmonary arterial hypertension is a common and fatal complication of scleroderma that may involve inflammatory and autoimmune mechanisms. Alterations in the gene expression of peripheral blood mononuclear cells have been previously described in patients with pulmonary arterial hypertension. Our goal is to identify differentially expressed genes in peripheral blood mononuclear cells in scleroderma patients with and without pulmonary hypertension as biomarkers of disease. Gene expression analysis was performed on a Microarray Cohort of scleroderma patients with (n = 10) and without (n = 10) pulmonary hypertension. Differentially expressed genes were confirmed in the Microarray Cohort and validated in a Validation Cohort of scleroderma patients with (n = 15) and without (n = 19) pulmonary hypertension by RT-qPCR. We identified inflammatory and immune-related genes including interleukin-7 receptor (IL-7R) and chemokine receptor 7 as differentially expressed in patients with scleroderma-associated pulmonary hypertension. Flow cytometry confirmed decreased expression of IL-7R on circulating CD4+ T-cells from scleroderma patients with pulmonary hypertension. Differences exist in the expression of inflammatory and immune-related genes in peripheral blood cells from patients with scleroderma-related pulmonary hypertension compared to those with normal pulmonary artery pressures. These findings may have implications as biomarkers to screen at-risk populations for early diagnosis and provide insight into mechanisms of scleroderma-related pulmonary hypertension.
Clinical and Translational Science 10/2010; 3(5):210-8. · 2.33 Impact Factor