Biology subjects

Oshins, R.

Publications and source records attributed to Oshins, R..

6 recordsLinked to original sources

bioRxiv2024

Novel Transgenic Humanized Alpha-1 Antitrypsin Deficiency Mouse Model on Murine SERPINA1 Null Background

Alpha-1 antitrypsin deficiency (AATD) is a rare genetic disorder caused by accumulation of misfolded -1 antitrypsin within hepatocytes. AATD patients are prone to develop liver disease that remains undiagnosed until the late stages of the disease. Due to challenges in manipulating the -1 antitrypsin genes in mice, determining a true loss of function of -1 antitrypsin in previous AATD mouse models has been challenging. Here, we report generation and liver characterization of a new humanized transgenic mouse model for AATD with a background of a CRISPR-Cas9 generated SERPINA1-null mouse. Male and female transgenic mice for normal (Pi*M) and mutant (Pi*Z) variants of human -1 antitrypsin at 4-6 months of age were subjected to this study. The accumulation of human -1 antitrypsin in the hepatocytes and fibrotic features of the liver were monitored by performing an in vivo study. We demonstrate a strong liver phenotype satisfying clinically relevant manifestations of liver pathology associated with AATD, including hepatic accumulation of human -1 antitrypsin globules, liver deposition of extracellular matrix proteins, hepatic ER stress, and liver fibrosis in Pi*Z mice, in addition to mild systemic inflammation. In addition to major phenotypic criteria of AATD-associated liver fibrosis, accompanying single-nucleus RNA-seq data demonstrate activation of pathways associated with liver metabolic changes, inflammation, and regeneration. Data from this study suggest our humanized transgenic AATD mouse model could provide a suitable model to study -1 antitrypsin loss of function, replicate the pathophysiology of AATD associated liver disease, and evaluate therapeutic reagents against this disease. NEW & NOTEWORTHYWe have characterized a new humanized transgenic mouse model of -1 antitrypsin deficiency with a SERPINA1-null background that shows strong manifestations of liver disease. Our data explores the altered phenotype of -1 antitrypsin deficient hepatocytes and suggests a relationship between liver cell types during disease progression. This model may become a useful tool for investigating -1 antitrypsin loss of function, pathogenic mechanisms, and for drug discovery aimed at both prevention and treatment of the disease.

molecular biology↗

bioRxiv2024

Extracellular Vesicle-Associated Neutrophil Elastase Activates Hepatic Stellate Cells and Promotes Liver Fibrogenesis via ERK1/2 Pathway

Liver fibrosis associated with increased mortality is caused by activation of hepatic stellate cells and excessive production and accumulation of extracellular matrix in response to fibrotic insults. It has been shown that in addition to liver inflammation, systemic inflammation also contributes to liver fibrogenesis. A deeper understanding of mechanisms that control liver fibrotic response to intra-and extra-hepatic inflammation is essential to develop novel clinical strategies against this disease. Extracellular vesicles (EV) have been recognized as immune mediators that facilitate activation of hepatic stellate cells. In inflammatory diseases, activated neutrophils release neutrophil elastase (NE) bound to EV, which has been identified as a significant contributor to inflammation by promoting immune cell activation. Here, we aimed to explore the role of inflammation derived plasma EV-associated NE in liver fibrogenesis and its potential mechanisms. We show EV-associated NE induces activation, proliferation and migration of hepatic stellate cells by promoting activation of the ERK1/2 signaling pathway. This effect did not occur through EV without surface NE, and Sivelestat, a NE inhibitor, inhibited activation of the ERK1/2 signaling pathway mediated by EV-associated NE. Moreover, we found plasma EV-associated NE increases deposition of collagen1 and -smooth muscle actin in the liver of a mouse model of liver fibrosis (Mdr2-/-). Notably, this effect does not occur in control mice without preexisting liver disease. These data suggest that EV-associated NE is a pro-fibrogenic factor for hepatic stellate cell activation via the ERK1/2 signaling pathway in pre-existing liver injuries. Inhibition of the plasma EV-associated NE in inflammatory conditions may be a therapeutic target for liver fibrosis in patients with inflammatory diseases.

cell biology↗

bioRxiv2023

Profiling plasma extracellular vesicle-derived microRNAs for noninvasive diagnosis of alpha-1 antitrypsin deficiency associated liver disease

Alpha-1 antitrypsin deficiency (AATD) is a rare genetic disease characterized by reduced circulating levels of alpha-1 antitrypsin (AAT) due to the retention of misfolded AAT in the hepatocytes. The toxic AAT aggregates in hepatocytes cause liver fibrosis, cirrhosis, and hepatocellular carcinoma. Most patients remain asymptomatic until the final stage in which liver transplantation becomes the only treatment. Timely diagnosis of AATD liver disease plays a critical role in intervention and finding curative solutions. Assessing the prevalence and severity of AATD liver disease remains challenging due to limitations in current methods. Liver biopsy, the gold standard for evaluating the hepatic AAT accumulation, the initiating stage for AATD liver disease, is hindered by invasiveness and sampling errors. To address these limitations, we conducted a study using unique and precious clinical samples. We analyzed plasma extracellular vesicle (EV) derived miRNAs and liver transcriptomes from AATD individuals to develop a sensitive and noninvasive diagnostic approach for AATD liver disease. In the testing stage of our study, we enrolled 17 AATD individuals with different stages of liver disease, as determined by liver biopsy, and 20 controls. We identified differential expression of 178 miRNAs within the AATD group compared to controls by miRNA sequencing. Among those miRNAs, we selected miR-223-3p, miR-23a-3p, miR-15b-5p, let-7a-5p, let-7f-5p, and miR-374a-5p for further validation in an independent cohort of 45 AATD individuals. Using a logistic model that combined three miRNAs, we achieved an AUC of 0.737 for detecting AATD liver disease. Adding a fourth miRNA into this model increased the AUC to 0.751. The changes in EV miRNAs are correlated to dysregulated expression of liver mRNAs in AATD individuals with different stages of liver disease. We propose that plasma-circulating EV exhibit distinct miRNAs in AATD individuals and could serve as clinically significant biomarkers for the early detection of AATD liver disease.

molecular biology↗

bioRxiv2022

Correlation of Alpha-1 Antitrypsin Levels and Exosome Associated Neutrophil Elastase Endothelial Injury in Subjects with SARS-CoV2 Infection

BackgroundSevere acute respiratory syndrome caused by a novel coronavirus 2 (SARS-CoV-2) has infected more than 18 million people worldwide. The activation of endothelial cells is a hallmark of signs of SARS-CoV-2 infection that includes altered integrity of vessel barrier and endothelial inflammation. ObjectivesPulmonary endothelial activation is suggested to be related to the profound neutrophil elastase (NE) activity, which is necessary for sterilization of phagocytosed bacterial pathogens. However, unopposed activity of NE increases alveolocapillary permeability and extracellular matrix degradation. The uncontrolled protease activity of NE during the inflammatory phase of lung diseases might be due to the resistance of exosome associated NE to inhibition by alpha-1 antitrypsin. Method31 subjects with a diagnosis of SARS-CoV2 infection were recruited in the disease group and samples from 30 voluntaries matched for age and sex were also collected for control. ResultsWe measured the plasma levels of exosome-associated NE in SARS-CoV-2 patients which, was positively correlated with the endothelial damage in those patients. Notably, we also found strong correlation with plasma levels of alpha-1 antitrypsin and exosome-associated NE in SARS-CoV-2 patients. Using macrovascular endothelial cells, we also observed that purified NE activity is inhibited by purified alpha-1 antitrypsin while, NE associated with exosomes are resistant to inhibition and show less sensitivity to alpha-1 antitrypsin inhibitory activity, in vitro. ConclusionsOur results point out the role of exosome-associated NE in exacerbation of endothelial injury in SARS-CoV-2 infection. We have demonstrated that exosome-associated NE could be served as a new potential therapeutic target of severe systemic manifestations of SARS-CoV-2 infection.

cell biology↗

bioRxiv2022

Cigarette smoke exposed airway epithelial cell-derived EVs promote pro-inflammatory macrophage activation in alpha-1 antitrypsin deficiency

BackgroundAlpha-1 antitrypsin deficiency (AATD) is a genetic disorder most commonly secondary to a single mutation in the SERPINA1 gene (PI*Z) that causes misfolding and accumulation of alpha-1 antitrypsin (AAT) in hepatocytes and mononuclear phagocytes which reduces plasma AAT and creates a toxic gain of function. This toxic gain of function promotes a pro-inflammatory phenotype in macrophages that contributes to lung inflammation and early-onset COPD, especially in individuals who smoke cigarettes. The aim of this study is to determine the role of cigarette exposed AATD macrophages and bronchial epithelial cells in AATD-mediated lung inflammation. MethodsPeripheral blood mononuclear cells from AATD and healthy individuals were differentiated into alveolar-like macrophages and exposed to air or cigarette smoke while in culture. Macrophage endoplasmic reticulum stress was quantified and secreted cytokines were measured using qPCR and cytokine ELISAs. To determine whether there is "cross talk" between epithelial cells and macrophages, macrophages were exposed to extracellular vesicles released by airway epithelial cells exposed to cigarette smoke and their inflammatory response was determined. ResultsAATD macrophages spontaneously produce several-fold more pro-inflammatory cytokines as compared to normal macrophages. AATD macrophages have an enhanced inflammatory response when exposed to cigarette smoke-induced extracellular vesicles (EVs) released from airway epithelial cells. Cigarette smoke-induced EVs induce expression of GM-CSF and IL-8 in AATD macrophages but have no effect on normal macrophages. Release of AAT polymers, potent neutrophil chemo attractants, were also increased from AATD macrophages after exposure to cigarette smoke-induced EVs. ConclusionsThe expression of mutated AAT confers an inflammatory phenotype in AATD macrophages which disposes them to an exaggerated inflammatory response to cigarette smoke-induced EVs, and thus could contribute to progressive lung inflammation and damage in AATD individuals.

cell biology↗

bioRxiv2018

Neutrophil Elastase Activates Macrophage MMPs, Promotes Cell Adhesion And Cytokine Production Via Integrin-Src Kinases Pathway

There are a number of diseases characterized by the presence of neutrophil elastase (NE) activity in tissues including cystic fibrosis and alpha-1-antitrypsin deficiency induced lung destruction. It is generally accepted that NE actively contributes to this pathological process, but the precise mechanisms has yet to be determined. We hypothesized that NE activates the macrophages (M{square}) pro-inflammatory program. We demonstrate that following NE exposure, monocyte-derived M{square} release proteolytic activity composed of several matrix metalloproteinases (MMPs) which could contribute to extracellular matrix (ECM) degradation. NE upregulates expression of M{square} derived pro-inflammatory cytokines including TNF, IL-1{beta}, and IL-8. Thus, NE-activated M{square} can contribute to tissue destruction through the proteolytic activity of metalloproteinases and by supporting chronic inflammation through expression of pro-inflammatory cytokines. We also demonstrate that NE increases M{square} adhesion that is attenuated by antibodies specific to integrin subunits. We show that the effects of NE on M{square} can be mediated through an activation of integrin pathways. In support of integrin involvement, we demonstrate that NE activates the Src kinase family, a hallmark of integrin signaling activation. Moreover, pretreatment of macrophages with a specific Src kinase inhibitor, PP2, completely prevents NE-induced inflammatory cytokine production. Taken together these findings indicate that NE has effect on lung destruction that extends beyond direct proteolytic degradation of matrix proteins.

cell biology↗