Biology subjects

Altemeier, W. A.

Publications and source records attributed to Altemeier, W. A..

7 recordsLinked to original sources

bioRxiv2026

Dynamics of bleomycin-induced lung injury at single-nucleus resolution

Single-cell RNA sequencing (scRNA-seq) has emerged as a powerful tool for studying individual cell populations in complex tissues during injury and repair. However, it involves extensive sample manipulation, limiting its application in rare and difficult-to-dissociate cell populations. Single-nucleus RNA sequencing (snRNA-seq) bypasses several of these challenges, but its performance in lung disease models is not well characterized. In a longitudinal murine model of bleomycin-induced lung injury, we performed whole lung snRNA-seq to systematically map 46 cell populations and states with minimal tissue processing. Our approach captured the emergence of Krt8+/Cdkn1a+ transitional alveolar cells and an expansion of macrophage populations during injury. Additionally, we identified sizeable populations of rare cell types, such as pericytes, and cells sensitive to digestion protocols, including a population of activated fibroblasts. These results indicate that snRNA-seq recapitulates the dynamic changes associated with injury as previously reported using single-cell methods and outperforms these approaches in the representation of cell types that are sensitive to processing, thereby highlighting its utility for high resolution analysis of heterogenous tissues such as lung.

bioinformatics↗

bioRxiv2025

Pressure Points: Endothelial Responses to Shear Stress and Pressure in Health and Pulmonary Arterial Hypertension

BackgroundHemodynamic forces exert a profound influence on endothelial signaling and, when abnormal, contribute centrally to human vascular disease. Pulmonary arterial hypertension (PAH) is characterized by both hemodynamic derangement and pulmonary arterial endothelial cell (PAEC) dysfunction. Despite importance in disease initiation and progression, the combined effects of shear and pressure forces on PAEC biology remain incompletely understood, particularly in the context of PAH. MethodsPAECs obtained at explant from controls and patients with idiopathic PAH or congenital heart disease-associated PAH (CHD-PAH) were cultured in a custom resistor-coupled microfluidic platform and exposed to static, low (3 dyne/cm{superscript 2}), or high (20 dyne/cm{superscript 2}) shear stress under either low or elevated (60 mmHg) pressure. After 24 hours, we assessed cellular morphology and performed transcriptomic analysis via bulk RNA sequencing, incorporating analyses of PAH subtype and donor sex. ResultsMorphologically, PAECs (n=18 donors) aligned with flow under high, but not low, shear, and alignment was not significantly altered by disease state or pressure. As expected, shear stress fundamentally reorganized the PAEC transcriptome. The "dose-response" to increasing shear differed across biological pathways in six statistically significant patterns. Increasing shear led to divergence in transcription between control and PAH cells, particularly in pathways involved in immune activation, stress signaling, and vascular remodeling, with subtype differences also observed. Pressure had modest effects on transcription, with CHD-PAH PAECs notably displaying pressure-induced stress and inflammatory signaling. We identified sexual dimorphism in the endothelial shear response, including that male cells under shear enriched for proliferative and angiogenic pathways and female cells for fatty acid metabolism and stress responses. ConclusionsWe provide a systems-level overview of how shear and pressure shape PAEC transcription, revealing divergent responses across disease state, PAH subtype, and donor sex. These findings highlight the need for further investigation into mechanosensitive pathways in PAH as potential novel therapeutic targets.

bioengineering↗

bioRxiv2025

Immunomodulation of the Innate Host Response by Mesenchymal-Derived Versican during Influenza A Virus Infection

Viral and bacterial lung infections place a significant burden on public health. Versican, an extracellular matrix (ECM) chondroitin sulfate proteoglycan, coordinates the innate immune response in multiple experimental models. Versicans potential as an immunomodulatory molecule makes it a promising therapeutic target for controlling the hosts immune response to lung infection. However, versicans contribution to lung inflammation, injury, and immune cell activity during influenza A virus (IAV) infection represents a critical knowledge gap. To address our central hypothesis that mesenchymal-derived versican is pro-inflammatory and enhances the innate immune response to IAV infection, we generated a tamoxifen-inducible mouse deficient in mesenchymal-derived versican (B6. Col1a2-CreERT+/-/Vcantm1.1Cwf, Col1a2/Vcan-/-). We report that mesenchymal-derived versican plays a critical role in neutrophil, monocyte, and dendritic cell migration into the lungs and airways early in IAV infection. Intriguingly, mesenchymal-derived versican deficiency had the most substantial negative impact on neutrophil emigration into the lungs. We found that neutrophils were less adhesive to the ECM of Col1a2/Vcan-/- mouse lung fibroblasts (mLFs), which had a significant decrease in versican compared to wild-type mLFs. Additionally, Col1a2/Vcan-/- mLFs treated with poly(I:C) in vitro have reduced cell-associated hyaluronan. These findings suggest that fibroblast-derived versican is necessary for adhesion to lung fibroblasts by neutrophils as they transit into the lung interstitium and airways from the pulmonary vasculature. Our findings demonstrate that mesenchymal-derived versican is a key regulator of the early host immune responses to IAV. NEW & NOTEWORTHYWe report the novel finding that mesenchymal-derived versican is critical for neutrophil, monocyte, and dendritic cell migration into the lungs and airways early in influenza A virus infection. Additionally, a differentiated neutrophil-cell line is less adherent to versican-deficient fibroblasts, and versican-deficient fibroblasts have significantly reduced cell-associated hyaluronan (HA) content in vitro. These findings suggest that mesenchymal-derived versican and cell-associated HA are necessary for the adhesion of neutrophils and monocytes to lung fibroblasts.

molecular biology↗

bioRxiv2024

Regulation of Versican Expression in Macrophages is Mediated byCanonical Type I Interferon Signaling via ISGF3

Growing evidence supports a role for versican as an important component of the inflammatory response, with both pro- and anti-inflammatory roles depending on the specific context of the system or disease under investigation. Our goal is to understand the regulation of macrophage-derived versican and the role it plays in innate immunity. In previous work, we showed that LPS triggers a signaling cascade involving TLR4, the Trif adaptor, type I interferons, and the type I interferon receptor, leading to increased versican expression by macrophages. In the present study, we used a combination of chromatin immunoprecipitation, siRNA, chemical inhibitors, and mouse model approaches to investigate the regulatory events downstream of the type I interferon receptor to better define the mechanism controlling versican expression. Results indicate that transcriptional regulation by canonical type I interferon signaling via the heterotrimeric transcription factor, ISGF3, controls versican expression in macrophages exposed to LPS. This pathway is not dependent on MAPK signaling, which has been shown to regulate versican expression in other cell types. The stability of versican mRNA may also contribute to prolonged versican expression in macrophages. These findings strongly support a role for macrophage-derived versican as a type I interferon-stimulated gene and further our understanding of versicans role in regulating inflammation.

cell biology↗

bioRxiv2022

CryoGrid-PIXUL-RNA: High throughput RNA isolation platform for tissue transcript analysis

Disease molecular complexity requires high throughput workflows to map disease pathways through analysis of vast tissue repositories. Great progress has been made in life sciences analytical technologies. To match the high throughput of these advanced analytical platforms, we have previously developed a multipurpose microplate sonicator, PIXUL, that can be used in multiple workflows to extract analytes from cultured cells and tissue fragments for various downstream molecular assays. And yet, the sample preparation devices, such as PIXUL, along with the downstream analytical capabilities have not been fully exploited to interrogate tissues because storing and sampling of such specimens remain, in comparison, inefficient. To mitigate this bottleneck, we have developed a low-cost user-friendly system, the CryoGrid, that consists of CryoBlock, thermometer/thermocouple, and QR coded CryoTrays to freeze and store frozen tissue fragments, and hand-held CryoCore tool for tissue sampling supported by iPad and Google apps to display tissues, direct coring and share metadata. RNA is one of the most studied analytes. There is a decades-long history of developing methods to isolate and analyze RNA. Still, the throughput of sampling and RNA extraction from tissues has not matched that of the high throughput transcriptome analytical platforms. To address this need, we have integrated the CryoGrid system with PIXUL-based methods to isolate RNA for gene-specific qPCR and genome-wide transcript analyses. TRIzol is commonly used to isolate RNA but it is labor-intensive, hazardous, requires fume-hoods, and is an expensive reagent. We developed a PIXUL-based TRIzol-free RNA isolation fast protocol that uses a buffer containing proteinase K (PK). Virtually every disease (and often therapeutic agents toxicity) is a systemic syndrome but often only one organ is examined. CryoGrid-PIXUL, integrated with either TRIzol or PK buffer RNA isolation protocols, yielded similar RNA profiles in a multiorgan (brain, heart, kidney and liver) mouse model of sepsis. Thus, RNA isolation using the CryoGrid-PIXUL system combined with the PK buffer offers an inexpensive user-friendly workflow to study transcriptional responses in tissues in health and disease as well as in therapeutic interventions.

molecular biology↗

bioRxiv2019

PERICYTE-LIKE CELLS UNDERGO TRANSCRIPTIONAL REPROGRAMMING AND DISTINCT FUNCTIONAL ADAPTATIONS IN ACUTE LUNG INJURY

BackgroundWe previously reported on the role of pericyte-like cells as functional sentinel immune cells in lung injury. However, much about the biological role of pericytes in lung injury remains unknown. Lung pericyte-like cells are well-positioned to sense disruption to the epithelial barrier and coordinate local inflammatory responses due to their anatomic niche within the alveoli. In this report, we characterized transcriptional responses and functional changes in pericyte-like cells following activation by alveolar components from injured and uninjured lungs in a mouse model of acute lung injury (ALI). MethodsWe purified pericyte-like cells from lung digests using PDGFR{beta} as a selection marker and expanded them in culture as previously described (1). We induced sterile acute lung injury in mice with recombinant human Fas ligand (rhFasL) instillation followed by mechanical ventilation (1). We then collected bronchoalveolar lavage fluid (BALF) from injured and uninjured mice. Purified pericyte-like cells in culture were exposed to growth media only (control), BALF from uninjured mice, and BALF from injured mice for 6 and 24 h. RNA collected from these treatment conditions were processed for RNAseq. Targets of interest identified by pathway analysis were validated using in vitro and in vivo assays. ResultsWe observed robust global transcriptional changes in pericyte-like cells following treatment with uninjured and injured BALF at 6 h, but this response persisted for 24 h only after exposure to injured BALF. Functional enrichment analysis of pericytes treated with injured BALF revealed activation of immuno-inflammatory, cell migration and angiogenesis-related pathways, whereas processes associated with tissue development and remodeling were down-regulated. We validated select targets in the inflammatory, angiogenesis-related, and cell migratory pathways using functional biological assays in vitro and in vivo. ConclusionLung pericyte-like cells are highly responsive to alveolar compartment content from both uninjured and injured lungs, but injured BALF elicits a more sustained response. The inflammatory, angiogenic, and migratory changes exhibited by activated pericyte-like cells underscore the phenotypic plasticity of these specialized stromal cells in the setting of acute lung injury.

cell biology↗

bioRxiv2019

The Effects of Genotype x Phenotype Interactions on Silver Nanoparticle Toxicity in Organotypic Cultures of Murine Tracheal Epithelial Cells

Silver nanoparticles (AgNP) are used in multiple applications but primarily in the manufacturing of antimicrobial products. Previous studies have identified AgNP toxicity in airway epithelial cells, but no in vitro studies to date have used organotypic cultures as a high-content in vitro model of the conducting airway to characterize the effects of interactions between host genetic and acquired factors, or gene x phenotype interactions (GxP), on AgNP toxicity. In the present study, we derived organotypic cultures from primary murine tracheal epithelial cells (MTEC) to characterize nominal and dosimetric dose-response relationships for AgNP-induced barrier dysfunction, glutathione (GSH) depletion, reactive oxygen species (ROS) production, lipid peroxidation, and cytotoxicity across two genotypes (A/J and C57BL/6J mice), two phenotypes (\"Normal\" and \"Type 2 [T2]-Skewed\"), and two exposures (an acute exposure of 24 h and a subacute exposure of 4 hours, every other day, over 5 days [5x4 h]). We characterized the \"T2-Skewed\" phenotype as an in vitro model of chronic respiratory diseases, which was marked by increased sensitivity to AgNP-induced barrier dysfunction, GSH depletion, ROS production, lipid peroxidation, and cytotoxicity, suggesting that asthmatics are a sensitive population to AgNP exposures in occupational settings. This also suggests that exposure limits, which should be based upon the most sensitive population, should be derived using in vitro and in vivo models of chronic respiratory diseases. This study highlights the importance of considering dosimetry as well as GxP effects when screening and prioritizing potential respiratory toxicants. Such in vitro studies can be used to inform regulatory policy aimed at special protections for all populations.

pharmacology and toxicology↗