Biology subjects

Kopras, E. J.

Publications and source records attributed to Kopras, E. J..

6 recordsLinked to original sources

bioRxiv2023

Heterogeneity in Neutrophil Extracellular Traps from Healthy Human Subjects

Neutrophil Extracellular Traps (NETs), a key component of early defense against microbial infection, are also associated with tissue injury. NET composition has been reported to vary with some disease states, but the composition and variability of NETs across many healthy subjects provides a critical comparison that has not been well investigated. We evaluated NETs from twelve healthy subjects of varying ages isolated from multiple blood draws over a three and one half-year period to delineate the variability in extracellular DNA, protein, enzymatic activities, and susceptibility to protease inhibitors. We calculated correlations for NET constituents and loss of human bronchial epithelial barrier integrity, measured by transepithelial electrical resistance, after NET exposure. We found that although there was some variability within the same subject over time, the mean numbers of neutrophils, protein, LDH, serine protease activities, and cytokines IL-8, IL-1RA, and G-CSF in isolated NETs were consistent across subjects. Total DNA and double stranded DNA content in NETs were different across donors. NETs had little or no TNF, IL-17A, or GM-CSF. NET DNA concentration correlated with increased NET neutrophil elastase activity and higher NET IL-1RA concentrations. NET serine protease activity varied considerably within the same donor from day-to-day. Mean response to protease inhibitors was significantly different across donors. NET DNA concentration correlated best with reductions in barrier integrity of human bronchial epithelia. Defining NET concentration by DNA content correlates with other NET components and reductions in NET-driven epithelial barrier dysfunction, suggesting DNA is a reasonable surrogate measurement for these complex structures in healthy subjects.

cell biology↗

bioRxiv2023

Pulmonary osteoclast-like cells in silica induced pulmonary fibrosis

The pathophysiology of silicosis is poorly understood, limiting development of therapies for those who have been exposed to the respirable particle. We explored the mechanisms of silica-induced pulmonary fibrosis in a mouse model using multiple modalities including wholelung single-nucleus RNA sequencing. These analyses revealed that in addition to pulmonary inflammation and fibrosis, intratracheal silica challenge induced osteoclast-like differentiation of alveolar macrophages and recruited monocytes, driven by induction of the osteoclastogenic cytokine, receptor activator of nuclear factor-{kappa}B ligand (RANKL) in pulmonary lymphocytes and alveolar type II cells. Furthermore, anti-RANKL monoclonal antibody treatment suppressed silica-induced osteoclast-like differentiation in the lung and attenuated silica-induced pulmonary fibrosis. We conclude that silica induces osteoclast-like differentiation of distinct recruited and tissue resident monocyte populations, leading to progressive lung injury, likely due to sustained elaboration of bone resorbing proteases and hydrochloric acid. Interrupting osteoclast-like differentiation may therefore constitute a promising avenue for moderating lung damage in silicosis. One Sentence SummarySilica induces the alveolar epithelium to reprogram recruited and resident pulmonary myeloid cells to become osteoclasts that contribute to pulmonary fibrosis.

pathology↗

bioRxiv2022

Integrative Single Cell Multiomic Profiling Analysis Reveals HOX-PBX Gene Regulatory Network Contributing to the Survival of mTOR Hyperactive Cells

Lymphangioleiomyomatosis (LAM) is a rare, debilitating lung disease that predominantly affects women of reproductive age. LAM is characterized by the infiltration of the lungs by abnormally proliferating smooth muscle-like cells of unknown origin via an estrogen-dependent metastatic mechanism. LAM cells carry deleterious mutations of tuberous sclerosis complex (TSC1/TSC2) genes, resulting in hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1) and ultimately dysregulated cell growth. Sirolimus, an FDA approved mTORC1 inhibitor and current best-choice medication for LAM stabilizes lung function in most LAM patients. However, it requires sustained application and remains inefficacious in some patients. The greatest barriers to finding a cure for LAM include its undetermined origin and unclear underlying pathogenesis. Our study aims to advance knowledge on the origin of LAM, and ultimately serve as a premise for the development of novel therapeutic targets for LAM. Single cell RNA sequencing (scRNA-seq) is a powerful tool in biomedical research that informs gene expression differences at the cellular level and may provide insights into the most fundamental origin of LAM cells. Our scRNA-seq analysis of LAM cells revealed a unique population of cells (LAMCORE), expressing uterine-similar homeobox transcription factors (HOX) and Pre-B-cell leukemia homeobox 1 (PBX1), which are absent in normal lung, suggesting that the uterus is the primary origin of LAM. PBX1 is a transcription factor critical for female reproductive tract development and maintenance, and its overexpression is implicated in some female reproductive cancers. In this study we hypothesize that PBX1 promotes survival and lung colonization of LAM (TSC2-null) cells. Using LAM patient-derived cells, we validated the transcriptional profile, gene expression and protein levels of PBX1. We have the first functional evidence that PBX1 and its downstream targets are upregulated in LAM cells. In a mouse model of LAM, we monitored the effect of suppression of PBX1 by short hairpin RNA-mediated gene silencing on lung colonization and tumor growth. We also found that pharmacological suppression of PBX1 attenuates LAM lung colonization and promotes death of LAM cells in vivo and vitro. Our data collectively suggests that PBX1 is a critical regulator of LAM progression.

cancer biology↗

bioRxiv2022

Dysregulation of Acid Ceramidase-mediated Sphingolipid Metabolism Contributes to Tumor Progression in Tuberous Sclerosis Complex

Tuberous Sclerosis Complex (TSC) is disorder of multi-system benign neoplasia in the brain, heart, kidneys and lungs. Lymphangioleiomyomatosis (LAM) is a progressive pulmonary disease affecting exclusively women. Both are caused by mutations in TSC1 and TSC2, resulting in mTORC1 hyperactivation. Single cell RNA sequencing of LAM lungs identified activation of genes in the sphingolipid pathway. Independent validation studies showed that acid ceramidase (ASAH1) and dihydroceramide desaturase (DEGS1), key enzyme for regulating sphingolipid and ceramide metabolism, were significantly increased in TSC2-null cells, and their expression and activity were rapamycin-insensitive. TSC2 negatively regulated the biosynthesis of tumorigenic sphingolipids. Suppression of ASAH1 by shRNA or the inhibitor ARN14976 (17a) markedly decreased the viability of TSC2-null cells. In vivo, 17a significantly decreased the growth of Tsc2-null cell derived mouse xenografts. When combined with rapamycin, 17a more strongly inhibited the progression of renal cystadenomas in Tsc2+/- mice than either agent alone, evaluated by pathology and MRI. Collectively, our studies identify a rapamycin-insensitive disorder of sphingolipid metabolism in TSC2-null cells and tumors and validate the novel hypothesis that TSC2 regulates sphingolipid production and action via ASAH1. Targeting aberrant sphingolipid metabolism pathways may have therapeutic value in TSC and LAM, and possibly in mTORC1-hyperactive neoplasms.

cell biology↗

bioRxiv2021

Novel insights into pulmonary phosphate homeostasis and osteoclastogenesis emerge from the study of pulmonary alveolar microlithiasis.

Pulmonary alveolar microlithiasis (PAM) is an autosomal recessive lung disease caused by a deficiency in the pulmonary epithelial Npt2b sodium-phosphate co-transporter that results in accumulation of phosphate and formation of hydroxyapatite microliths in the alveolar space. The single cell transcriptomic analysis of a PAM lung explant showing a robust osteoclast gene signature in alveolar monocytes and the finding that calcium phosphate microliths contain a rich protein and lipid matrix that includes bone resorbing osteoclast enzymes suggested a role for osteoclast-like cells in the defense against microliths. While investigating the mechanisms of microlith clearance, we found that Npt2b modulates pulmonary phosphate homeostasis through effects on alternative phosphate transporter activity and alveolar osteoprotegerin, and that microliths induce osteoclast formation and activity in a receptor activator of nuclear factor-{kappa}B ligand (RANKL) and dietary phosphate dependent manner. This work reveals that Npt2b and pulmonary osteoclast-like cells play key roles in pulmonary homeostasis and suggest potential new therapeutic targets for the treatment of lung disease.

molecular biology↗

bioRxiv2019

Identification of the lymphangioleiomyomatosis cell and its uterine origin

Lymphangioleiomyomatosis (LAM) is a metastasizing neoplasm of reproductive age women that causes cystic lung remodeling and progressive respiratory failure. The source of LAM cells that invade the lung and the reasons that LAM targets women have remained elusive. We employed single cell and single nuclei RNA sequencing on LAM lesions within explanted LAM lungs, known to contain smooth muscle like cells bearing mTOR activating mutations in TSC1 or TSC2, and identified a unique population of cells that were readily distinguished from those of endogenous lung cells. LAMCORE cells shared closest transcriptomic similarity to normal uterus and neural crest. Immunofluorescence microscopy demonstrated the expression of LAMCORE cell signature genes within LAM lesions in both lung and uterus. Serum aptamer proteomics and ELISA identified biomarkers predicted to be secreted by LAMCORE cells. Single cell transcriptomics strongly supports a uterine neural crest origin of LAMCORE cells; providing insights into disease pathogenesis and informing future treatment strategies for LAM.\n\nSIGNIFICANCEPresent study identified a novel population of LAMCORE cells, which is likely originated from uterine neural crest; identified novel LAM cell-specific secretome proteins that hold promise as potential biomarkers and therapeutic targets. Advancing the understanding of LAM pathogenesis and metastasis model may yield broader insights into the biology of cancer.

cancer biology↗