Biomedical subjects

Gregory M Dolganov

Publications and source records attributed to Gregory M Dolganov.

12 recordsLinked to original sources

PubMed2006

Epithelial mucin stores are increased in the large airways of smokers with airflow obstruction.

BACKGROUND: Habitual cigarette smoking is associated with chronic mucus hypersecretion, but the relationship between mucus abnormalities and airflow obstruction in smokers is uncertain. METHODS: We collected bronchial biopsy samples and epithelial brushings from 24 smokers with and without airflow obstruction and 19 nonsmoking healthy control subjects. Epithelial mucin stores, mucin immunostains, and goblet cell morphology were quantified in bronchial biopsy samples using stereology, and mucin gene expression was quantified in epithelial brushings using real-time reverse transcriptase-polymerase chain reaction. RESULTS: Goblet cell size and number were higher than normal in smokers (both p < 0.05), leading to a 2.2-fold increase in the volume of stored mucin in the epithelium per surface area of basal lamina (1.94 +/- 0.31 microm(3)/microm(2) vs 4.32 +/- 0.55 microm(3)/microm(2) in control subjects vs smokers, p = 0.001). The increase in stored mucin occurred because of an increase in MUC5AC (p = 0.018) and despite a decrease in MUC5B (p < 0.0001). Stored mucin was significantly higher in the subgroup of smokers with airflow obstruction (p = 0.029) and correlated with FEV(1)/FVC even when controlling for diffusing capacity as a measure of emphysema (p = 0.034). CONCLUSIONS: Epithelial mucin stores are increased in habitual smokers because of goblet cell hypertrophy and hyperplasia, and the pattern of mucin gene expression is abnormal. The highest epithelial mucin stores are found in smokers with airflow obstruction, suggesting a mechanistic link between epithelial mucin dysregulation and airflow obstruction.

Adult↗

PubMed2005

Comprehensive transcriptome of proteases and protease inhibitors in vascular cells.

BACKGROUND AND PURPOSE: Smooth muscle cells, endothelial cells, and macrophages are essential components of the vasculature, of which the homeostatic gene expression participate importantly in the maintenance of vascular wall integrity. The pathogenesis of vascular diseases, such as cerebral ischemia, atherosclerosis, and abdominal aortic aneurysms, often associates with inflammation and altered gene expression, including proteolytic enzymes that play multiple and important roles in extracellular matrix degradation, cell proliferation and migration, and latent enzyme or growth factor activation. METHODS: Human saphenous vein smooth muscle cells, endothelial cells, and monocyte-derived macrophages from 3 independent donors were stimulated with interleukin 1beta, interferon gamma, tumor necrosis factor alpha, basic fibroblast growth factor, and vascular endothelial growth factor, 5 common proinflammatory mediators often found in diseased human microvessels and macrovessels. Quantitative real-time PCR was used to examine the mRNA levels of 49 proteolytic enzymes and their inhibitors, selected from 4 protease families, in these vascular cells. CONCLUSIONS: Although primary cultured cells from different donors may behave differently in response to these proinflammatory cytokines, data from this study revealed a broad view of vascular cell protease expression profiles under inflammatory conditions, critical to studies of inflammation-associated vascular tissue remodeling.

Aortic Aneurysm, Abdominal↗

PubMed2005

Immune complex-dependent remodeling of the airway vasculature in response to a chronic bacterial infection.

Chronic inflammation in the airways is associated with dramatic architectural changes in the walls of the airways and in the vasculature they contain. In this study, we show that the adaptive immune system is essential for airway remodeling that occurs in mice that are chronically infected with the respiratory pathogen Mycoplasma pulmonis. Angiogenesis, lymphangiogenesis, and epithelial remodeling were greatly reduced in mice that lacked B cells. Substantiating a role for Ab and airway immune complexes, we found that the transfer of immune serum to B cell-deficient mice could reconstitute pathogen-induced angiogenesis. Inflammatory cells recruited to the infected airways were activated by the humoral response, and this activation correlated with the induction of genes for remodeling factors such as vascular endothelial growth factor-D. The results reveal a novel pathway whereby T cell-dependent humoral immunity to a persistent airway infection can induce inflammation-dependent angiogenesis, lymphangiogenesis, and chronic airway pathology.

Animals↗

PubMed2005

A distinctive alveolar macrophage activation state induced by cigarette smoking.

RATIONALE: Macrophages are believed to play a central role in emphysema based largely on data from mouse models. However, the relevance of these models to smoking-related lung disease in humans is uncertain. OBJECTIVES: We sought to comprehensively characterize the effects of smoking on gene expression in human alveolar macrophages and to compare these with effects seen in transgenic mouse models of emphysema. METHODS: We used DNA microarrays with genomewide coverage to analyze alveolar macrophages from 15 smokers, 15 nonsmokers, and 15 subjects with asthma (disease control). Selected gene expression changes were validated by polymerase chain reaction and ELISA. Expression changes were compared with those identified by microarray analysis of interleukin-13-overexpressing and integrin-beta6-deficient mice, which both develop emphysema. MEASUREMENTS AND MAIN RESULTS: All 15 smokers shared a common pattern of macrophage gene expression that distinguished them from nonsmokers, a finding not observed in subjects with asthma. We identified 110 genes as differentially expressed in smokers despite using conservative statistical methods. Matrix metalloproteinase 12, a proteinase that plays a critical role in mouse models, was the third most highly induced gene in smokers (ninefold, p < 0.0001). However, most changes in smokers were not reflected in mouse models. One such finding was increased osteopontin expression in smokers (fourfold, p = 0.006), which was confirmed at the protein level and correlated with the degree of airway obstruction. CONCLUSIONS: Smoking induces a remarkably consistent and distinctive pattern of alveolar macrophage activation. These studies identify aspects of mouse models that are directly relevant to human smokers and also reveal novel potential mediators of smoking-related diseases.

Adult↗

PubMed2005

Cathepsin L expression and regulation in human abdominal aortic aneurysm, atherosclerosis, and vascular cells.

The cysteine protease cathepsin L is one of the most potent mammalian elastases and collagenases, widely expressed at basal levels in most tested tissues and cell types, and regulated by pro-inflammatory stimuli. The inflammatory arterial diseases abdominal aortic aneurysm (AAA) and atherosclerosis involve extensive vascular remodeling that requires elastolysis and collagenolysis. This study examined the hypothesis that cathepsin L is over-expressed in human AAA and atherosclerotic lesions and its expression in vascular cell types found in these lesions is regulated by pro-inflammatory cytokines. Immunohistochemical and tissue extract immunoblot analysis demonstrated increased expression of cathepsin L in human AAA and atheromata and localized its expression to lesional smooth muscle cells (SMC), endothelial cells (EC), and macrophages. In primary cultured human SMC, EC, and monocyte-derived macrophages, pro-inflammatory cytokines or growth factors induced the expression of cathepsin L and its activity against extracellular collagen and elastin. Patients with coronary artery stenosis (n=65) had higher serum cathepsin L levels than those without lesions detectable by quantitative coronary angiography (n=30) (1.47+/-0.33 ng/ml versus 0.60+/-0.06 ng/ml, p<0.02). A strong correlation between the percent of stenosis of left anterior descending coronary artery and serum cathepsin L levels in patients with stenosis (R=0.542, p<0.0001), also suggests involvement of cathepsin L in these vascular diseases.

Animals↗

PubMed2005

Increased DNA microarray hybridization specificity using sscDNA targets.

BACKGROUND: The most widely used amplification method for microarray analysis of gene expression uses T7 RNA polymerase-driven in vitro transcription (IVT) to produce complementary RNA (cRNA) that can be hybridized to arrays. However, multiple rounds of amplification are required when assaying very small amounts of starting RNA. Moreover, certain cRNA-DNA mismatches are more stable than the analogous cDNA-DNA mismatches and this might increase non-specific hybridization. We sought to determine whether a recently developed linear isothermal amplification method (ribo-SPIA) that produces single stranded cDNA would offer advantages over traditional IVT-based methods for microarray-based analyses of transcript expression. RESULTS: A single round of ribo-SPIA amplification produced sufficient sscDNA for hybridizations when as little as 5 ng of starting total RNA was used. Comparisons of probe set signal intensities obtained from replicate amplifications showed consistently high correlations (r = 0.99). We compared gene expression in two different human RNA samples using ribo-SPIA. Compared with one round IVT, ribo-SPIA had a larger dynamic range and correlated better with quantitative PCR results even though we used 1000-fold less starting RNA. The improved dynamic range was associated with decreases in hybridization to mismatch control probes. CONCLUSION: The use of amplified sscDNA may offer substantial advantages over IVT-based amplification methods, especially when very limited amounts of starting RNA are available. The use of sscDNA targets instead of cRNA targets appears to improve hybridization specificity.

Animals↗

PubMed2005

CXCR4 expression reflects tumor progression and regulates motility of bladder cancer cells.

Transitional cell carcinoma of the urinary bladder remains life threatening due to the high occurrence of metastases. Emerging evidence suggests that chemokines and their receptors play a critical role in tumor metastases. In our study, we performed a systematic analysis of the mRNA and protein expression levels of all 18 chemokine receptors in normal urothelium and bladder cancer. CXCR4 was the only chemokine receptor whose mRNA expression level was upregulated in bladder cancer cell lines as well as in invasive and locally advanced bladder cancer tissue samples (pT2-pT4). In contrast, superficial bladder tumors (pTa and pT1) displayed low CXCR4 expression levels and normal urothelial cells were negative for CXCR4. Immunohistochemistry of a bladder cancer tissue microarray (TMA) confirmed that a subgroup of invasive bladder cancers revealed a high CXCR4 protein expression, while superficial bladder tumors showed low immunoreactivity. To investigate the functional significance of CXCR4 expression, we performed migration and invasion assays. Exposure of CXCR4-positive bladder cancer cells to CXCL12 in a Boyden chamber type assay provoked a significant increase in migration as well as invasion across a Matrigel barrier. Enhanced migration and invasion were inhibited by a CXCR4-specific blocking antibody. In contrast, normal urothelial cells did not respond to CXCL12 and lacked chemotactic migration. In conclusion, bladder cancer cells express CXCR4 progressively with advanced tumorigenesis and this receptor interacts with CXCL12 to mediate tumor chemotaxis and invasion through connective tissue. These properties identify CXCR4 as a potential target for the attenuation of bladder cancer metastases.

Breast Neoplasms↗

PubMed2005

c-Kit immunophenotyping and metalloproteinase expression profiles of mast cells in interstitial lung diseases.

Diverse interstitial lung diseases (ILD) demonstrate mesenchymal infiltration by an abundance of activated mast cells whose role in parenchymal fibrogenesis remains unclear. Since mast cells differentiate in a dynamic, tissue-specific manner via signals transduced by c-Kit receptor, we examined the effect of ILD microenvironments on c-Kit expression and metalloproteinase phenotypes of mesenchymal mast cell populations. Immunohistochemical and flow cytometric analyses characterized surface expression of c-Kit on mast cells in tissues obtained from patients with idiopathic pulmonary fibrosis, systemic sclerosis, sarcoidosis, and lymphangioleiomyomatosis, thus identifying a unique immunophenotype not shared by normal lung mast cells. Isolation of c-Kit+/FcepsilonRI+/CD34- mast cells via immunocytometric sorting of heterogeneous cell populations from mechanically disaggregated lung tissues permitted analysis of gene expression patterns by two-step real-time polymerase chain reaction. Transcriptional profiling identified expression of c-Kit and the neutral serine proteases, tryptase and chymase, establishing the identity of sorted populations as mature mast cells. Mast cells harvested from ILD tissues demonstrated characteristic metalloproteinase phenotypes which included expression of matrix metalloproteinase (MMP)-1 and a disintegrin and metalloproteinase (ADAM)-9, -10, and -17. Immunohistochemical co-localization guided by gene profiling data confirmed expression of chymase, MMP-1, and ADAM-17 protein in subpopulations of mast cells in remodelling interstitium. Gene profiling of harvested mast cells also showed increased transcript copy numbers for TNFalpha and CC chemokine receptor 2, which play critical roles in lung injury. We conclude that ILD microenvironments induce unique c-Kit receptor and metalloproteinase mast cell phenotypes.

Cell Differentiation↗

PubMed2005

Dissecting asthma using focused transgenic modeling and functional genomics.

BACKGROUND: Asthma functional genomics studies are challenging because it is difficult to relate gene expression changes to specific disease mechanisms or pathophysiologic features. Use of simplified model systems might help to address this problem. One such model is the IL-13/Epi (IL-13-overexpressing transgenic mice with STAT6 expression limited to epithelial cells) focused transgenic mouse, which isolates the effects of a single mediator, IL-13, on a single cell type, the airway epithelial cell. These mice develop airway hyperreactivity and mucus overproduction but not airway inflammation. OBJECTIVE: To identify how effects of IL-13 on airway epithelial cells contribute to gene expression changes in murine asthma models and determine whether similar changes are seen in people with asthma. METHODS: We analyzed gene expression in ovalbumin allergic mice, IL-13-overexpressing mice, and IL-13/Epi mice with microarrays. We analyzed the expression of human orthologues of genes identified in the mouse studies in airway epithelial cells from subjects with asthma and control subjects. RESULTS: In comparison with the other 2 models, IL-13/Epi mice had a remarkably small subset of gene expression changes. Human orthologues of some genes identified as increased in the mouse models were more highly expressed in airway epithelial cells from subjects with asthma than in controls. These included calcium-activated chloride channel 1, 15-lipoxygenase, trefoil factor 2, and intelectin. CONCLUSION: The combination of focused transgenic models, DNA microarray analyses, and translational studies provides a powerful approach for analyzing the contributions of specific mediators and cell types and for focusing attention on a limited number of genes associated with specific pathophysiologic aspects of asthma.

Animals↗

PubMed2004

Hyperplasia of smooth muscle in mild to moderate asthma without changes in cell size or gene expression.

Bronchial hyperresponsiveness in mild to moderate asthma may result from airway smooth muscle cell proliferation or acquisition of a hypercontractile phenotype. Because these cells have not been well characterized in mild to moderate asthma, we examined the morphometric and gene expression characteristics of smooth muscle cells in this subgroup of patients with asthma. Using bronchial biopsies from 14 subjects with mild to moderate asthma and 15 control subjects, we quantified smooth muscle cell morphology by stereology and the expression of a panel of genes related to a hypercontractile phenotype of airway smooth muscle, using laser microdissection and two-step real-time polymerase chain reaction. We found that airway smooth muscle cell size was similar in both groups, but cell number was nearly twofold higher in subjects with asthma (p = 0.03), and the amount of smooth muscle in the submucosa was increased 50-83% (p < 0.005). Gene expression profiling in smooth muscle cells showed no difference in the expression of genes encoding phenotypic markers in cells from healthy subjects and subjects with asthma (all p > 0.1). We conclude that airway smooth muscle proliferation is a pathologic characteristic of subjects with mild to moderate asthma. However, smooth muscle cells in mild to moderate asthma do not show hypertrophy or gene expression changes of a hypercontractile phenotype observed in vitro.

Adult↗

PubMed2003

Inhibition of respiration by nitric oxide induces a Mycobacterium tuberculosis dormancy program.

An estimated two billion persons are latently infected with Mycobacterium tuberculosis. The host factors that initiate and maintain this latent state and the mechanisms by which M. tuberculosis survives within latent lesions are compelling but unanswered questions. One such host factor may be nitric oxide (NO), a product of activated macrophages that exhibits antimycobacterial properties. Evidence for the possible significance of NO comes from murine models of tuberculosis showing progressive infection in animals unable to produce the inducible isoform of NO synthase and in animals treated with a NO synthase inhibitor. Here, we show that O2 and low, nontoxic concentrations of NO competitively modulate the expression of a 48-gene regulon, which is expressed in vivo and prepares bacilli for survival during long periods of in vitro dormancy. NO was found to reversibly inhibit aerobic respiration and growth. A heme-containing enzyme, possibly the terminal oxidase in the respiratory pathway, likely senses and integrates NO and O2 levels and signals the regulon. These data lead to a model postulating that, within granulomas, inhibition of respiration by NO production and O2 limitation constrains M. tuberculosis replication rates in persons with latent tuberculosis.

Mycobacterium tuberculosis↗

PubMed2002

Direct effects of interleukin-13 on epithelial cells cause airway hyperreactivity and mucus overproduction in asthma.

Asthma is an increasingly common disease that remains poorly understood and difficult to manage. This disease is characterized by airway hyperreactivity (AHR, defined by exaggerated airflow obstruction in response to bronchoconstrictors), mucus overproduction and chronic eosinophilic inflammation. AHR and mucus overproduction are consistently linked to asthma symptoms and morbidity. Asthma is mediated by Th2 lymphocytes, which produce a limited repertoire of cytokines, including interleukin-4 (IL-4), IL-5, IL-9 and IL-13. Although each of these cytokines has been implicated in asthma, IL-13 is now thought to be especially critical. In animal models of allergic asthma, blockade of IL-13 markedly inhibits allergen-induced AHR, mucus production and eosinophilia. Furthermore, IL-13 delivery to the airway causes all of these effects. IL-13 is thus both necessary and sufficient for experimental models of asthma. However, the IL-13-responsive cells causing these effects have not been identified. Here we show that mice lacking signal transducer and activator of transcription 6 (STAT6) were protected from all pulmonary effects of IL-13. Reconstitution of STAT6 only in epithelial cells was sufficient for IL-13-induced AHR and mucus production in the absence of inflammation, fibrosis or other lung pathology. These results demonstrate the importance of direct effects of IL-13 on epithelial cells in causing two central features of asthma.

Animals↗