Jadranka Milosevic

University of Pittsburgh, Pittsburgh, PA, United States

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Publications (19)68 Total impact

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    ABSTRACT: Although asthma is recognized as a heterogeneous disease associated with clinical phenotypes, the molecular basis of these phenotypes remains poorly understood. While genomic studies have successfully broadened our understanding in diseases such as cancer, they have not been widely used in asthma studies. To link gene expression patterns to clinical asthma phenotypes, we utilized a microarray platform to analyze bronchial airway epithelial cell (BAEC) gene expression in relation to the asthma biomarker fractional exhaled nitric oxide (FeNO) in 155 asthmatic subjects and healthy controls from the Severe Asthma Research Program (SARP). We first identified a diverse set of 549 genes whose expression correlated with FeNO. We used K-means to cluster the patient samples according to the expression of these genes, identifying 5 asthma clusters/phenotypes with distinct clinical, physiological, cellular and gene transcription characteristics-termed 'subject clusters' (SCs). To then investigate differences in gene expression between SCs, a total of 1384 genes were identified which highly differentiated the SCs at an unadjusted p-value of <10-6. Hierarchical clustering of these 1384 genes identified 9 gene clusters (GCs) or 'biclusters', whose coexpression suggested biological characteristics unique to each SC. While genes related to Type-2 inflammation were present, novel pathways, including those related to neuronal function, WNT pathways and actin cytoskeleton, were also noted. These findings show that bronchial epithelial cell gene expression, as related to the asthma biomarker FeNO, can identify distinct asthma phenotypes, while also suggesting the presence of underlying novel gene pathways relevant to these phenotypes.
    American Journal of Respiratory and Critical Care Medicine 10/2014; · 11.04 Impact Factor
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    ABSTRACT: Background: Herpes simplex virus, type 1 (HSV-1) commonly produces lytic mucosal lesions. It invariably initiates latent infection in sensory ganglia enabling persistent, lifelong infection. Acute HSV-1 encephalitis is rare and definitive evidence of latent infection in the brain is lacking. However, exposure untraceable to encephalitis has been repeatedly associated with impaired working memory and executive functions, particularly among schizophrenia patients. Methods: Patterns of HSV-1 infection and gene expression changes were examined in human induced pluripotent stem cell (iPSC)-derived neurons. Separately, differences in blood oxygenation level-dependent (BOLD) responses to working memory challenges using letter n-back tests were investigated using functional magnetic resonance imaging (fMRI) among schizophrenia cases/controls. Results: HSV-1 induced lytic changes in iPSC-derived glutamatergic neurons and neuroprogenitor cells. In neurons, HSV-1 also entered a quiescent state following coincubation with antiviral drugs, with distinctive changes in gene expression related to functions such as glutamatergic signaling. In the fMRI studies, main effects of schizophrenia (P = .001) and HSV-1 exposure (1-back, P = 1.76 × 10(-) (4); 2-back, P = 1.39 × 10(-) (5)) on BOLD responses were observed. We also noted increased BOLD responses in the frontoparietal, thalamus, and midbrain regions among HSV-1 exposed schizophrenia cases and controls, compared with unexposed persons. Conclusions: The lytic/quiescent cycles in iPSC-derived neurons indicate that persistent neuronal infection can occur, altering cellular function. The fMRI studies affirm the associations between nonencephalitic HSV-1 infection and functional brain changes linked with working memory impairment. The fMRI and iPSC studies together provide putative mechanisms for the cognitive impairments linked to HSV-1 exposure.
    Schizophrenia Bulletin 03/2014; · 8.80 Impact Factor
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    ABSTRACT: Severe refractory asthma is associated with enhanced nitrative stress. To determine the mechanisms for high nitrative stress in human severe asthma (SA), 3-nitrotyrosine (3NT) was compared with Th1 and Th2 cytokine expression. In SA, high 3NT levels were associated with high interferon (IFN)-γ and low interleukin (IL)-13 expression, both of which have been reported to increase inducible nitric oxide synthase (iNOS) in human airway epithelial cells (HAECs). We found that IL-13 and IFN-γ synergistically enhanced iNOS, nitrite, and 3NT, corresponding with increased H2O2. Catalase inhibited whereas superoxide dismutase enhanced 3NT formation, supporting a critical role for H2O2, but not peroxynitrite, in 3NT generation. Dual oxidase-2 (DUOX2), central to H2O2 formation, was also synergistically induced by IL-13 and IFN-γ. The catalysis of nitrite and H2O2 to nitrogen dioxide radical (NO2(•)) requires an endogenous peroxidase in this epithelial cell system. Thyroid peroxidase (TPO) was identified by microarray analysis ex vivo as a gene distinguishing HAEC of SA from controls. IFN-γ induced TPO in HAEC and small interfering RNA knockdown decreased nitrated tyrosine residues. Ex vivo, DUOX2, TPO, and iNOS were higher in SA and correlated with 3NT. Thus, a novel iNOS-DUOX2-TPO-NO2(•) metabolome drives nitrative stress in HAEC and likely in SA.Mucosal Immunology advance online publication, 12 February 2014; doi:10.1038/mi.2014.6.
    Mucosal Immunology 02/2014; · 7.54 Impact Factor
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    ABSTRACT: MicroRNAs (microRNAs) are small non-coding RNAs that inhibit protein expression. We have previously shown that the inhibition of the microRNA let-7d in epithelial cells caused changes consistent with Epithelial to Mesenchymal Transition (EMT) both in vitro and in vivo. The aim of this study was to determine whether introduction of let-7d into fibroblasts will alter their mesenchymal properties. Transfection of primary fibroblasts with let-7d caused a decrease in expression of the mesenchymal markers αSMA, N-Cadherin and FSP-1, as well as an increase in the epithelial marker TJP-1. Phenotypic changes were also present, including a delay in wound healing, reduced motility, and proliferation of fibroblasts following transfection. In addition, we examined the effects of transfection on fibroblast responsiveness to TGFβ, an important factor in many fibrotic processes such as lung fibrosis, and found that let-7d transfection significantly attenuated HMGA2 level induction by TGFβ. Our results indicate administration of the epithelial microRNA let-7d can significantly alter the phenotype of primary fibroblasts.
    AJP Lung Cellular and Molecular Physiology 01/2014; · 3.52 Impact Factor
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    ABSTRACT: As miRNAs are associated with normal cellular processes, deregulation of miRNAs is thought to play a causative role in many complex diseases. Nevertheless, the precise contribution of miRNAs in fibrotic lung diseases, especially the idiopathic form (IPF), remains poorly understood. Given the poor response rate of IPF patients to current therapy, new insights into the pathogenic mechanisms controlling lung fibroblasts activation, the key cell type driving the fibrogenic process, are essential to develop new therapeutic strategies for this devastating disease. To identify miRNAs with potential roles in lung fibrogenesis, we performed a genome-wide assessment of miRNA expression in lungs from two different mouse strains known for their distinct susceptibility to develop lung fibrosis after bleomycin exposure. This led to the identification of miR-199a-5p as the best miRNA candidate associated with bleomycin response. Importantly, miR-199a-5p pulmonary expression was also significantly increased in IPF patients (94 IPF versus 83 controls). In particular, levels of miR-199a-5p were selectively increased in myofibroblasts from injured mouse lungs and fibroblastic foci, a histologic feature associated with IPF. Therefore, miR-199a-5p profibrotic effects were further investigated in cultured lung fibroblasts: miR-199a-5p expression was induced upon TGFβ exposure, and ectopic expression of miR-199a-5p was sufficient to promote the pathogenic activation of pulmonary fibroblasts including proliferation, migration, invasion, and differentiation into myofibroblasts. In addition, we demonstrated that miR-199a-5p is a key effector of TGFβ signaling in lung fibroblasts by regulating CAV1, a critical mediator of pulmonary fibrosis. Remarkably, aberrant expression of miR-199a-5p was also found in unilateral ureteral obstruction mouse model of kidney fibrosis, as well as in both bile duct ligation and CCl-induced mouse models of liver fibrosis, suggesting that dysregulation of miR-199a-5p represents a general mechanism contributing to the fibrotic process. MiR-199a-5p thus behaves as a major regulator of tissue fibrosis with therapeutic potency to treat fibroproliferative diseases.
    PLoS Genetics 02/2013; 9(2):e1003291. · 8.52 Impact Factor
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    ABSTRACT: Idiopathic pulmonary fibrosis (IPF) is a progressive and life threatening disease with median survival of 2.5-3 years. The IPF lung is characterized by abnormal lung remodeling, epithelial cell hyperplasia, myofibroblast foci formation, and extracellular matrix deposition. Analysis of gene expression microarray data revealed that cartilage oligomeric matrix protein (COMP), a non-collagenous extracellular matrix protein is among the most significantly up-regulated genes (Fold change 13, p-value <0.05) in IPF lungs. This finding was confirmed at the mRNA level by nCounter® expression analysis in additional 115 IPF lungs and 154 control lungs as well as at the protein level by western blot analysis. Immunohistochemical analysis revealed that COMP was expressed in dense fibrotic regions of IPF lungs and co-localized with vimentin and around pSMAD3 expressing cells. Stimulation of normal human lung fibroblasts with TGF-β1 induced an increase in COMP mRNA and protein expression. Silencing COMP in normal human lung fibroblasts significantly inhibited cell proliferation and negatively impacted the effects of TGF-β1 on COL1A1 and PAI1. COMP protein concentration measured by ELISA assay was significantly increased in serum of IPF patients compared to controls. Analysis of serum COMP concentrations in 23 patients who had prospective blood draws revealed that COMP levels increased in a time dependent fashion and correlated with declines in force vital capacity (FVC). Taken together, our results should encourage more research into the potential use of COMP as a biomarker for disease activity and TGF-β1 activity in patients with IPF. Hence, studies that explore modalities that affect COMP expression, alleviate extracellular matrix rigidity and lung restriction in IPF and interfere with the amplification of TGF-β1 signaling should be persuaded.
    PLoS ONE 01/2013; 8(12):e83120. · 3.53 Impact Factor
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    ABSTRACT: Rationale: In this study we explore the regulation and the role of upregulated microRNAs in Idiopathic pulmonary fibrosis (IPF), a progressive interstitial lung disease of unknown origin. Methods and Results: We analyzed the expression of microRNAs in IPF lungs and identified 43 significantly upregulated microRNAs. Impressively, 24 of the 43 increased microRNAs were localized to the chromosome 14q32 microRNA cluster. We validated the increased expression of miR-154, miR-134, miR-299-5p, miR-410, miR-382, miR-409-3p, miR-487b, miR-31 and miR-127 by qRT-PCR, and determined that they were similarly expressed in embryonic lungs. We did not find evidence for differential methylation in this region but analysis of transcription factor binding sites identified multiple SMAD3 binding elements in the 14q32 microRNA cluster. TGF-β1 stimulation of normal human lung fibroblast (NHLF) caused up-regulation of microRNAs on chr14q32 that were also increased in IPF lungs. Chromatin immunoprecipitation confirmed binding of SMAD3 to the putative promoter of miR-154. Mir-154 was increased in IPF fibroblasts and transfection of NHLF with miR-154 caused significant increases in cell proliferation and migration. The increase in proliferation induced by TGF-β was not observed when NHLF or IPF fibroblasts were transfected with a mir-154 inhibitor. Transfection with miR-154 caused activation of the WNT pathway in NHLF. ICG-001 and XAV939, inhibitors of the WNT/β-catenin pathway, reduced the proliferative effect of miR-154. Conclusion: The potential role of miR-154, one of multiple chr14q32 microRNA cluster members upregulated in IPF, and a regulator of fibroblast migration and proliferation, should be further explored in IPF.
    American Journal of Respiratory Cell and Molecular Biology 10/2012; · 4.15 Impact Factor
  • American Thoracic Society 2012 International Conference, May 18-23, 2012 • San Francisco, California; 05/2012
  • American Thoracic Society 2012 International Conference, May 18-23, 2012 • San Francisco, California; 05/2012
  • American Thoracic Society 2012 International Conference, May 18-23, 2012 • San Francisco, California; 05/2012
  • American Thoracic Society 2012 International Conference, May 18-23, 2012 • San Francisco, California; 05/2012
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    ABSTRACT: Human cytomegalovirus (HCMV) infection is one of the leading prenatal causes of congenital mental retardation and deformities world-wide. Access to cultured human neuronal lineages, necessary to understand the species specific pathogenic effects of HCMV, has been limited by difficulties in sustaining primary human neuronal cultures. Human induced pluripotent stem (iPS) cells now provide an opportunity for such research. We derived iPS cells from human adult fibroblasts and induced neural lineages to investigate their susceptibility to infection with HCMV strain Ad169. Analysis of iPS cells, iPS-derived neural stem cells (NSCs), neural progenitor cells (NPCs) and neurons suggests that (i) iPS cells are not permissive to HCMV infection, i.e., they do not permit a full viral replication cycle; (ii) Neural stem cells have impaired differentiation when infected by HCMV; (iii) NPCs are fully permissive for HCMV infection; altered expression of genes related to neural metabolism or neuronal differentiation is also observed; (iv) most iPS-derived neurons are not permissive to HCMV infection; and (v) infected neurons have impaired calcium influx in response to glutamate.
    PLoS ONE 01/2012; 7(11):e49700. · 3.53 Impact Factor
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    ABSTRACT: Primary human distal lung/parenchymal fibroblasts (DLFs) exhibit a different phenotype from airway fibroblasts (AFs), including the expression of high levels of α-smooth muscle actin (α-SMA). The scope of the differences between these anatomically differentiated fibroblasts, or the mechanisms driving them, has remained unknown. To determine whether the different characteristics of regional fibroblasts are predicted by distinct genomic differences in AFs versus DLFs, matched human fibroblast pairs were isolated from proximal and distal lung tissue and evaluated. Microarray analysis was performed on 12 matched fibroblast pairs (four normal and eight asthmatic samples) and validated by quantitative real-time PCR. The potential functional implications of these differences were analyzed using computational approaches. Four hundred seventy-four transcripts were up-regulated in AFs, and 611 were up-regulated in DLFs via microarray analysis. No differences in normal and asthmatic fibroblasts were evident, and the data were combined for subsequent analyses. Gene ontology and network analyses suggested distinct patterns of pathway activation between AFs and DLFs. The up-regulation of extracellular matrix-associated molecules in AFs was observed, whereas genes associated with actin binding and cytoskeletal organization were up-regulated in DLFs. The up-regulation of activated/total SMAD3 and c-Jun N-terminal kinase in DLFs may partly explain these myofibroblast-like characteristics in DLFs. Thus, marked genomic differences exist between these two populations of regional lung fibroblasts. These striking differences may help identify potential mechanisms by which AFs and DLFs differ in their responses to injury, regeneration, and remodeling in the lung.
    American Journal of Respiratory Cell and Molecular Biology 07/2011; 45(6):1256-62. · 4.15 Impact Factor
  • American Thoracic Society 2011 International Conference, May 13-18, 2011 • Denver Colorado; 05/2011
  • American Thoracic Society 2011 International Conference, May 13-18, 2011 • Denver Colorado; 05/2011
  • Kusum V Pandit, Jadranka Milosevic, Naftali Kaminski
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    ABSTRACT: In this review, we describe the recent advances in the understanding of the role of microRNAs in idiopathic pulmonary fibrosis (IPF), a chronic progressive and lethal fibrotic lung disease. Approximately 10% of the microRNAs are significantly changed in IPF lungs. Among the significantly downregulated microRNAs are members of let-7, mir-29, and mir-30 families as well as miR-17∼92 cluster among the upregulated mir-155 and mir-21. Downregulation of let-7 family members leads to changes consistent with epithelial mesenchymal transition in lung epithelial cells both in vitro and in vivo, whereas inhibition of mir-21 modulates fibrosis in the bleomycin model of lung fibrosis. Perturbations of mir-155 and mir-29 have profibrotic effects in vitro but have not yet been assessed in vivo in the context of lung fibrosis. A recurrent global theme is that many microRNAs studied in IPF are both regulated by transforming growth factor β1 (TGFβ1) and regulate TGFβ1 signaling pathway by their target genes. As a result, their aberrant expression leads to a release of inhibitions on the TGFβ1 pathway and to the creation of feed-forward loops. Coanalysis of published microRNA and gene expression microarray data in IPF reveals enrichment of the TGFβ1, Wnt, sonic hedgehog, p53, and vascular endothelial growth factor pathways and complex regulatory networks. The changes in microRNA expression in the IPF lung and the evidence for their role in the fibrosis suggest that microRNAs should be evaluated as therapeutic targets in IPF.
    Translational research : the journal of laboratory and clinical medicine. 04/2011; 157(4):191-9.
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    ABSTRACT: Uncontrolled extracellular matrix production by fibroblasts in response to tissue injury contributes to fibrotic diseases, such as idiopathic pulmonary fibrosis (IPF), a progressive and ultimately fatal process that currently has no cure. Although dysregulation of miRNAs is known to be involved in a variety of pathophysiologic processes, the role of miRNAs in fibrotic lung diseases is unclear. In this study, we found up-regulation of miR-21 in the lungs of mice with bleomycin-induced fibrosis and also in the lungs of patients with IPF. Increased miR-21 expression was primarily localized to myofibroblasts. Administration of miR-21 antisense probes diminished the severity of experimental lung fibrosis in mice, even when treatment was started 5-7 d after initiation of pulmonary injury. TGF-beta1, a central pathological mediator of fibrotic diseases, enhanced miR-21 expression in primary pulmonary fibroblasts. Increasing miR-21 levels promoted, whereas knocking down miR-21 attenuated, the pro-fibrogenic activity of TGF-beta1 in fibroblasts. A potential mechanism for the role of miR-21 in fibrosis is through regulating the expression of an inhibitory Smad, Smad7. These experiments demonstrate an important role for miR-21 in fibrotic lung diseases and also suggest a novel approach using miRNA therapeutics in treating clinically refractory fibrotic diseases, such as IPF.
    Journal of Experimental Medicine 08/2010; 207(8):1589-97. · 13.21 Impact Factor
  • American Thoracic Society 2009 International Conference, May 15-20, 2009 • San Diego, California; 04/2009
  • American Thoracic Society 2009 International Conference, May 15-20, 2009 • San Diego, California; 04/2009