Biology subjects

Stewart, D. J.

Publications and source records attributed to Stewart, D. J..

10 recordsLinked to original sources

bioRxiv2025

Artificial Intelligence-enabled Histological Analysis in Preclinical Respiratory Disease Models: A Scoping Review

Histological analysis is a cornerstone of preclinical respiratory disease research, enabling assessment of pathology, therapeutic effects, and mechanisms. However, conventional approaches rely on manual scoring, which is subjective, time-consuming, and difficult to scale due to low throughput and inter-observer variability. Artificial intelligence (AI), particularly deep learning, offers potential to automate histology workflows, but its use and evaluation in preclinical respiratory models have not been synthesized. We conducted a scoping review following Joanna Briggs Institute guidelines, searching MEDLINE and Embase (inception-January 2025) for preclinical studies using AI to analyze histology in respiratory disease models. Screening, full-text review, and data extraction were performed in duplicate. Of 6271 studies screened, 29 met inclusion criteria. Most used murine models (76%) and investigated lung cancer (28%), pulmonary fibrosis (24%), or tuberculosis (17%). Hematoxylin and eosin was the most common stain (48%), with others targeting collagen or immune markers. AI tasks included image classification (n=20), segmentation (n=10), and object detection (n=4), predominantly using convolutional neural networks (69%). Preprocessing methods (e.g., stain normalization) were common, but annotation and training practices were inconsistently reported. Performance was generally high (accuracy [≥]90%; 7 studies) though validation metrics varied, and external validation was absent. Most studies used "black box" models, with minimal application of explainability techniques. Reproducibility measures, such as sharing datasets or code were rarely reported. AI tools are poised to transform histological analysis in preclinical respiratory research. By addressing gaps in validation, transparency, and standardization, the field can harness these technologies to deliver robust, efficient, and scalable workflows. Registration: Open Science Framework https://doi.org/10.17605/OSF.IO/NM94E

cell biology↗

bioRxiv2025

Natural killer cell TGF- signaling regulates senolytic activity and vascular patterning in the postnatal lung

BackgroundBronchopulmonary dysplasia (BPD) is a disease of neonatal lung development that is linked to impaired pulmonary vascularization, dysregulated transforming growth factor-{beta} (TGF-{beta}) signaling and the accumulation of senescent cells. Despite the established role for TGF-{beta} signaling in promoting vascular remodeling and suppressing the senolytic activity of natural killer (NK) cells, the contribution of NK cell TGF-{beta} signaling to postnatal lung patterning and the pathogenesis of BPD remains unclear. MethodsMice bearing an NK cell-selective deletion of the type-II TGF-{beta} receptor (Tgfbr2NK-/-) were analyzed for vascular and alveolar structure, lung NK cell infiltration, senescence markers and lung function testing across neonatal and adult timepoints. Single-cell RNA sequencing of lung tissue from both neonatal mice and human infants with BPD was performed. The effect of enhanced NK cell activity in a hyperoxia-induced model of BPD was assessed in Tgfbr2NK-/-neonates, as well as pharmacologically, using the TGF-{beta} ligand trap/IL-15 superagonist, HCW9218. ResultsNeonatal Tgfbr2NK-/- mice exhibited a baseline reduction in distal arteriolar density, impaired alveolarization, and sex-specific deficits in long-term lung function. Single-cell RNA sequencing identified the excessive clearance of senescent endothelial cells by TGF-{beta} insensitive NK cells in the lungs of Tgfbr2NK-/- neonates, which served as a contributor of the BPD-like phenotype observed in naive animals. Tgfbr2NK-/- mice were protected from impaired lung development in the hyperoxia model. Sequencing from lung tissue from infants with BPD confirmed excessive TGF-{beta} signaling and cytotoxic impairment in NK cells. Treatment with HCW9218 prevented senescent cell accumulation and rescued lung development in the hyperoxia mouse model. ConclusionsThese findings identify TGF-{beta} as a tunable regulator of NK cell senolytic activity that is essential to normal postnatal lung development. Excessive NK cell TGF-{beta} signaling contributes to impaired lung development following exposure to neonatal hyperoxia and may serve as a viable therapeutic target for human BPD.

developmental biology↗

bioRxiv2023

Vicious cycle of hemodynamic perturbation and endothelial injury in development and progression of pulmonary arterial hypertension

BackgroundPulmonary arterial hypertension (PAH) is a devastating disease caused by loss of effective lung microvasculature for which there is no curative treatment. Evidence from preclinical models and human disease-causing genetic mutations point to endothelial cell (EC) injury and apoptosis as a central trigger for the initiation of PAH. However, how EC apoptosis leads to pulmonary hypertension (PH) and complex arteriolar remodeling is uncertain. MethodsRats were subjected to SU5416-hypoxia (SUHx) and EC apoptosis, pulmonary vascular remodeling and arterial volume was assessed by immunohistochemistry, histology and microCT, respectively. Left pulmonary artery banding (LPAB) was performed, either 1 week before (prevention) or 5 weeks after SU injection (reversal), to study the effect of hemodynamic offloading. ResultsIn the SUHx model, EC apoptosis was markedly increased as early as 3 days post-SU, persisting through PAH development, and this was associated with a profound arterial pruning with reduction in lung arterial volume ([~]80%). LPAB abrogated lung EC apoptosis in the banded left lung and prevented as well as reversed arteriolar pruning. Moreover, in the reversal protocol, removal of the band at 10 weeks resulted in improvement in pulmonary hemodynamics and RV function at 13 weeks. ConclusionThese data demonstrate that perturbed hemodynamic factors triggered by lung microvascular arteriolar loss play a requisite role in perpetuating endothelial injury in experimental PAH, leading to persistent arterial EC injury and disease progression. Importantly, vascular loss, arterial remodeling and PH are reversible once the cycle of perturbed hemodynamics and EC injury is broken by unilateral lung banding.

physiology↗

bioRxiv2023

Emergence of disease specific endothelial and stromal cell populations involved in arterial remodeling during development of pulmonary arterial hypertension

AbstractPulmonary arterial hypertension (PAH) is a severe and lethal pulmonary vascular disease characterized by arteriolar pruning and occlusive vascular remodeling leading to increased pulmonary vascular resistance and eventually right heart failure. While endothelial cell (EC) injury and apoptosis are known triggers for this disease, the mechanisms by which they lead to complex arterial remodeling remain obscure. We employed multiplexed single-cell RNA sequencing (scRNA-seq) at multiple timepoints during the onset and progression of disease in a model of severe PAH to identify mechanisms involved in the development of occlusive arterial lesions. There was significant loss of arterial volume as early as 1-week by microCT, preceding any evidence of occlusive arteriopathy, consistent with early arteriolar dropout. Maximal arterial pruning was seen by 5 to 8 weeks, with signs of progressive occlusive remodeling. Analysis of the scRNA-seq data resolved 44 lung cell populations, with widespread early transcriptomic changes at 1 week affecting endothelial, stromal and immune cell populations. Notably, this included emergence of a relatively dedifferentiated (dD) EC population that was enriched for Cd74 expression compared to general capillary (gCap) ECs which were primed to undergo endothelial-mesenchymal transition, as evidenced by RNA velocity analysis. However, at late timepoints (5 and 8 weeks), activated arterial ECs (aAECs) were the only cell population exhibiting persistent differential gene expression. This was characterized by a growth regulated state, including high expression of Tm4sf1, a gene implicated in cancer cell growth, which was also expressed by a smooth muscle (SM)-like pericyte cluster. Both these populations were localized to regions of arterial remodeling in the rat model and PAH patients, with aAECs contributing to intimal occlusive lesions and SM-like pericytes forming bands of medial muscularization. Together these findings implicate disease-specific vascular cells in PAH progression and suggest that TM4SF1 may be a novel therapeutic target for arterial remodeling.

molecular biology↗

bioRxiv2022

Single-cell microencapsulation improves lung retention of endothelial colony forming cells after intravascular delivery and unmasks therapeutic benefit in severe pulmonary arterial hypertension

BackgroundPulmonary arterial hypertension (PAH) is triggered by pulmonary vascular endothelial cell apoptosis and microvascular loss; therefore, therapies that can regenerate lost vasculature may offer therapeutic benefit. Endothelial colony forming cells (ECFCs) can directly repair damaged blood vessels and may have therapeutic potential for the treatment of PAH. However, poor retention of ECFCs in the lungs following intravenous delivery greatly limits their therapeutic application. Therefore, we studied whether cellular microencapsulation could enhance ECFCs viability and retention in the lung after systemic delivery and improve therapeutic efficacy of ECFCs in a rat monocrotaline (MCT) PAH model. MethodsECFCs were encapsulated by vortex-emulsion using various concentrations of agarose, and capsule size and initial cell viability were assessed. Encapsulated and free ECFCs were transduced with luciferase and administered to Sprague-Dawley rats three days after injection of MCT. ECFCs were tracked in vivo by bioluminescence imaging (BLI) to assess cell persistence and bio-distribution. At end-study, right ventricular systolic pressure (RVSP) and right ventricular hypertrophy were assessed for therapeutic efficacy. ResultsMicrogel encapsulation using 3.5% agarose improved cells survival and supported cell migration from capsules. At 15 minutes after delivery, BLI radiance were similar for free and microencapsulated ECFCs; however, only encapsulated cells could be detected by BLI at 4 and 24 hours. Transplantation of microencapsulated ECFCs led to significant improvement in RVSP three weeks after delivery compared to non-encapsulated ECFCs. ConclusionTogether, microencapsulation increased retention of ECFCs within the lungs. Furthermore, even a modest increase in ECFCs persistence over 24 hours can provide an important therapeutic benefit in the rat MCT model of PAH.

bioengineering↗

bioRxiv2022

Targeting extracellular vesicle delivery to the lungs by microgel encapsulation

Extracellular vesicles (EVs) secreted by stem and progenitor cells have significant potential as cell-free cellular therapeutics. Yet, small EVs (<200 nm) are rapidly cleared after systemic administration, mainly by the liver, presenting challenges targeting EVs to a specific organ or tissue. Microencapsulation using natural nano-porous hydrogels (microgels) has been shown to enhance engraftment and increase the survival of transplanted cells. We sought to encapsulate EVs within microgels to target their delivery to the lung by virtue of their size-based retention within the pulmonary microcirculation. Mesenchymal stromal cell (MSC) derived EVs were labelled with the lipophilic dye (DiR) and encapsulated within agarose-gelatin microgels. Endothelial cells and bone marrow derived macrophages were able to take up EVs encapsulated in microgels in vitro, but less efficiently than the uptake of free EVs. Following intrajugular administration, microgel encapsulated EVs were selectively retained within the lungs for 72 hours, while free EVs were rapidly cleared by the liver. Furthermore, microgel loaded EVs demonstrated greater uptake by lung cells, in particular CD45+ immune cells, as assessed by flow cytometry compared to free EVs. Microencapsulation of EVs may be a novel tool for enhancing targeted delivery of EVs for future therapeutic applications.

bioengineering↗

bioRxiv2021

Newly Emergent Apelin Expressing Endothelial Stem-like Cells Orchestrate Lung Microvascular Repair

QuestionWe sought to define the mechanism underlying lung microvascular regeneration in a severe acute lung injury (ALI) model induced by selective lung endothelial cell ablation. MethodsChanges in lung cell populations and gene expression profiles were determined in transgenic mice expressing human diphtheria toxin (DT) receptor targeted to ECs using single-cell RNA sequencing at baseline (day 0) and days 3, 5 and 7 after lung EC ablation. ResultsEight distinct endothelial clusters were resolved, including alveolar aerocytes (aCap) ECs expressing apelin at baseline, and general capillary (gCap) ECs expressing the apelin receptor. Intratracheal instillation of DT resulted in ablation of >70% of lung ECs, producing severe ALI with near complete resolution by 7 days. At 3 days post injury, a novel gCap population emerged characterized by de novo expression of apelin, together with the stem cell marker, protein C receptor. These stem-like cells transitioned to proliferative ECs, expressing apelin receptor together with the pro-proliferative transcription factor, FoxM1. This progenitor-like cell population was responsible for the rapid replenishment of all depleted EC populations by 7 days post injury, including aerocytes which play a critical role in re-establishment of the air-blood barrier. Treatment with an apelin receptor antagonist prevented recovery and resulted in excessive mortality, consistent with a central role for apelin signaling in EC regeneration and microvascular repair. ConclusionThe lung has a remarkable capacity for microvasculature EC regeneration which is orchestrated by signaling between newly emergent apelin-expressing gCap endothelial stem-like cells and highly proliferative, apelin receptor positive endothelial progenitors. Take-Home messageUsing sublethal lung endothelial cell (EC) ablation, we show for the first that EC regeneration and resolution of acute lung injury is orchestrated by novel apelin-expressing, gCap endothelial stem-like cells by a mechanism requiring apelin signaling. Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY A schematic representation of EC populations contributing to microvascular repair. At baseline (Day 0), there are two main alveolar groups of capillary ECs: larger apelin positive aCap ECs, termed aerocytes, that play a key structural role in forming the air-blood barrier; and smaller apelin receptor (Aplnr) expressing gCap ECs, which are found in the thicker regions at the corners of the alveoli. After DT-induced EC ablation, there is a marked depletion of both EC populations and the appearance of novel transitional and transient populations. At Day 3, there is the appearance of stem-like gCap ECs that paradoxically express apelin, but not its receptor, and are characterized by various stem and progenitor cell markers but show no evidence of proliferation. By Day 5, these transition to ECs expressing Aplnr which have a strong proliferative phenotype, as evidenced by FoxM1 and Ki67 expression, and then rapidly replenish depleted EC pools, including aCap ECs, by Day 7. This transition is orchestrated by the interaction of apelin with its receptor as a critical mechanism in lung microvascular regeneration after EC injury. AT1 = alveolar type -1 epithelial cell; AT2 = alveolar type-2 epithelial cell; APLNR = apelin receptor; ANGPT2 = angiopoietin 2; EPCR = Endothelial protein C receptor.

cell biology↗

bioRxiv2019

Accuracy and Reproducibility of Somatic Point Mutation Calling in Clinical-Type Targeted Sequencing Data

BackgroundTreating cancer depends in part on identifying the mutations driving each patients disease. Many clinical laboratories are adopting high-throughput sequencing for assaying patients tumours, applying targeted panels to formalin-fixed paraffin-embedded tumour tissues to detect clinically-relevant mutations. While there have been some benchmarking and best practices studies of this scenario, much variant-calling work focuses on whole-genome or whole-exome studies, with fresh or fresh-frozen tissue. Thus, definitive guidance on best choices for sequencing platforms, sequencing strategies, and variant calling for clinical variant detection is still being developed. ResultsBecause ground truth for clinical specimens is rarely known, we used the well-characterized Coriell cell lines GM12878 and GM12877 to generate data. We prepared samples to mimic as closely as possible clinical biopsies, including formalin fixation and paraffin embedding. We evaluated two well-known targeted sequencing panels, Illuminas TruSight 170 panel and the Oncomine Focus panel. Sequencing was performed on an Illumina NextSeq500 and an Ion Torrent PGM respectively. We performed multiple biological replicates of each assay, to test reproducibility. Finally, we applied five different public and freely-available somatic single-nucleotide variant (SNV) callers to the data, MuTect2, SAMtools, VarScan2, Pisces and VarDict. Although the TruSight 170 and Oncomine Focus panels cover different amounts of the genome, we did not observe major differences in variant calling success within the regions that each covers. We observed substantial discrepancies between the five variant callers. All had high sensitivity, detecting known SNVs, but highly varying and non-overlapping false positive detections. Harmonizing variant caller parameters or intersecting the results of multiple variant callers reduced disagreements. However, intersecting results from biological replicates was even better at eliminating false positives. ConclusionsReproducibility and accuracy of targeted clinical sequencing results depends less on sequencing platform and panel than on downstream bioinformatics and biological variability. Differences in variant callers default parameters are a greater influence on algorithm disagreement than other differences between the algorithms. Contrary to typical clinical practice, we recommend analyzing replicate samples, as this greatly decreases false positive calls.

bioinformatics↗

bioRxiv2019

COMMD1 and PtdIns(4,5)P2 interaction maintain ATP7B copper transporter trafficking fidelity in HepG2 cells

Copper-responsive intracellular ATP7B trafficking is critical to maintain copper balance in mammalian hepatocytes and thus organismal copper levels. The COMMD1 protein binds both the ATP7B copper transporter and phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P2), while COMMD1 loss causes hepatocyte copper accumulation. Although it is clear that COMMD1 is included in endocytic trafficking complexes, a direct function for COMMD1 in ATP7B trafficking has not been defined. In this study, experiments using quantitative reveal that COMMD1 modulates the copper-responsive ATP7B trafficking through recruitment to PtdIns(4,5)P2. Decreased COMMD1 abundance results in loss of ATP7B from lysosomes and the trans-Golgi network (TGN) in high copper conditions, while excess expression of COMMD1 also disrupts ATP7B trafficking and TGN structure. Overexpression of COMMD1 mutated to inhibit PtdIns(4,5)P2 binding has little impact on ATP7B trafficking. A mechanistic PtdIns(4,5)P2-mediated function for COMMD1 is proposed that is consistent with decreased cellular copper export due to disruption of the ATP7B trafficking itinerary and accumulation in the early endosome when COMMD1 is depleted. PtdIns(4,5)P2 interaction with COMMD1 as well as COMMD1 abundance may both be important in maintenance of specific membrane protein trafficking pathways.\n\nSUMMARYQuantitative analysis of 3D protein colocalization defines the cellular function of COMMD1 in maintenance of ATP7B copper transporter trafficking fidelity and the importance of PtdIns(4,5)P2 in this action.

cell biology↗

bioRxiv2019

Transcriptomically-guided mesendoderm induction of human pluripotent stem cells using a systematically defined culture scheme

Human pluripotent stem cells (hPSCs) are an essential cell source in tissue engineering, studies of development, and disease modeling. Efficient, broadly amenable protocols for rapid lineage induction of hPSCs are of great interest in the stem cell biology field. We describe a simple, robust method for differentiation of hPSCs into mesendoderm in defined conditions utilizing single-cell seeding (SCS) and BMP4 and Activin A (BA) treatment. Gene sets and gene ontology terms related to mesoderm and endoderm differentiation were enriched after 48 hours of BA treatment. BA treatment was readily incorporated into existing protocols for chondrogenic and endothelial progenitor cell differentiation. After prolonged differentiation in vitro or in vivo, BA pre-treatment resulted in higher mesoderm and endoderm levels at the expense of ectoderm formation. These data demonstrate that SCS with BA treatment is a powerful method for induction of mesendoderm that can be integrated into protocols for mesoderm and endoderm differentiation.

bioengineering↗