Biology subjects

Aldred, M. A.

Publications and source records attributed to Aldred, M. A..

6 recordsLinked to original sources

bioRxiv2024

Single-cell transcriptomics reveal diverging pathobiology and opportunities for precision targeting in scleroderma-associated versus idiopathic pulmonary arterial hypertension

IntroductionPulmonary arterial hypertension (PAH) involves progressive cellular and molecular change within the pulmonary vasculature, leading to increased vascular resistance. Current therapies targeting nitric oxide (NO), endothelin, and prostacyclin pathways yield variable treatment responses. Patients with systemic sclerosis-associated PAH (SSc-PAH) often experience worse outcomes than those with idiopathic PAH (IPAH). MethodsLung tissue samples from four SSc-PAH, four IPAH, and four failed donor specimens were obtained from the Pulmonary Hypertension Breakthrough Initiative (PHBI) lung tissue bank. Single-cell RNA sequencing (scRNAseq) was performed using the 10X Genomics Chromium Flex platform. Data normalization, clustering, and differential expression analysis were conducted using Seurat. Additional analyses included gene set enrichment analysis (GSEA), transcription factor activity analysis, and ligand-receptor signaling. Pharmacotranscriptomic screening was performed using the Connectivity Map. ResultsSSc-PAH samples showed a higher proportion of fibroblasts and dendritic cells/macrophages compared to IPAH and donor samples. GSEA revealed enriched pathways related to epithelial-to-mesenchymal transition (EMT), apoptosis, and vascular remodeling in SSc-PAH samples. There was pronounced differential gene expression across diverse pulmonary vascular cell types and in various epithelial cell types in both IPAH and SSc-PAH, with epithelial to endothelial cell signaling observed. Macrophage to endothelial cell signaling was particularly pronounced in SSc-PAH. Pharmacotranscriptomic screening identified TIE2, GSK-3, and PKC inhibitors, among other compounds, as potential drug candidates for reversing SSc-PAH gene expression signatures. DiscussionOverlapping and distinct gene expression patterns exist in SSc-PAH versus IPAH, with significant molecular differences suggesting unique pathogenic mechanisms in SSc-PAH. These findings highlight the potential for precision-targeted therapies to improve SSc-PAH patient outcomes. Future studies should validate these targets clinically and explore their therapeutic efficacy.

bioinformatics↗

bioRxiv2024

Role of Forkhead box F1 in the Pathobiology of Pulmonary Arterial Hypertension

RationaleApproximately 80% of patients with non-familial pulmonary arterial hypertension (PAH) lack identifiable pathogenic genetic variants. While most genetic studies of PAH have focused on predicted loss-of-function variants, recent approaches have identified ultra-rare missense variants associated with the disease. FOXF1 encodes a highly conserved transcription factor, essential for angiogenesis and vasculogenesis in human and mouse lungs. ObjectivesWe identified a rare FOXF1 missense coding variant in two unrelated probands with PAH. FOXF1 is an evolutionarily conserved transcription factor required for lung vascular development and vascular integrity. Our aims were to determine the frequency of FOXF1 variants in larger PAH cohorts compared to the general population, study FOXF1 expression in explanted lung tissue from PAH patients versus control (failed-donor) lungs, and define potential downstream targets linked to PAH development. MethodsThree independent, international, multicenter cohorts were analyzed to evaluate the frequency of FOXF1 rare variants. Various composite prediction models assessed the deleteriousness of individual variants. Bulk RNA sequencing datasets from human explanted lung tissues were compared to failed-donor controls to determine FOXF1 expression. Bioinformatic tools identified putative FOXF1 binding targets, which were orthogonally validated using mouse ChIP-seq datasets. Measurements and Main ResultsSeven novel or ultra-rare missense coding variants were identified across three patient cohorts in different regions of the FOXF1 gene, including the DNA binding domain. FOXF1 expression was dysregulated in PAH lungs, correlating with disease severity. Histological analysis showed heterogeneous FOXF1 expression, with the lowest levels in phenotypically abnormal endothelial cells within complex vascular lesions in PAH samples. A hybrid bioinformatic approach identified FOXF1 downstream targets potentially involved in PAH pathogenesis, including BMPR2. ConclusionsLarge genomic and transcriptomic datasets suggest that decreased FOXF1 expression or predicted dysfunction is associated with PAH.

genomics↗

bioRxiv2024

Loss of Tbx4 Affects Postnatal Lung Development and Predisposes to Pulmonary Hypertension.

Pulmonary arterial hypertension (PAH) is a progressive vascular disease characterized by remodeling of the precapillary pulmonary arteries. Genomic variation within the T-box 4 (TBX4) transcription factor is the second most common genetic cause of PAH, and can also cause severe lung developmental disorders with neonatal PH. Currently, the effect of TBX4 loss-of-function on later stages of lung development and predisposition to lung disease, including PH, is not well understood. Therefore, we have generated Tbx4 conditional knockout (Tbx4-CKO) mice in which Cre recombinase deletes exon 5 of Tbx4 within the embryonic lung mesenchyme to create a null allele. We harvested lungs from these mice at various timepoints to examine alveologenesis, vascularization, vascular remodeling, lung cellular composition, and disruption of transcriptional activity compared with control lungs. Right ventricular systolic pressure (RVSP) was measured in six-month-old mice to evaluate for PH. Tbx4-CKO lungs show enlargement of airspaces, as confirmed by an increase in mean linear intercept at P14 (24.9%), P36 (31.5%), and P180 (49.6%). These lungs also show a 39.3% decrease in von Willebrand Factor-positive vessels and a 14.2% increase in vessel wall thickness. Consistent with these results, Tbx4-CKO mice show a statistically significant increase of 15.7% in RVSP and 16.3% in the Fulton index. Bulk-RNA sequencing analysis revealed enrichment of pathways and genes relevant to lung alveologenesis, angiogenesis, and PH. Our results show that disruption of Tbx4 expression during early lung development is sufficient to disrupt postnatal lung development and circulation.

genetics↗

bioRxiv2023

Transcriptome-wide analysis of primary human endothelial cell responses to 1 hour of protein translation inhibition identify nonsense mediated decay targets and a non-coding SLC11A2 exon as an acute biomarker

Nonsense mediated decay (NMD) lowers the cellular concentration of spliced RNAs harboring premature termination codons (PTC), and inhibition has been proposed as a potential therapeutic method. Conversely, NMD plays regulatory roles throughout the eukaryotic kingdom, including when protein translation is inhibited acutely as part of the integrated stress response. To define tools for endothelial evaluations of therapeutic NMD inhibition, and quantification of subtle cellular stress states, natural endothelial-expressed targets were examined via whole transcriptome RNA sequencing of primary human microvascular endothelial cells (HMECs) treated for 1h with cycloheximide, a protein translation and NMD inhibitor. Genes differentially expressed after 1h cycloheximide overlapped with genes differentially expressed many days after NMD-specific knockdown in other cell types. For endothelial cells, customized novel scripts used 255,500 exons in media-treated HMEC and 261,725 exons in cycloheximide-treated HMEC to predict 1h cycloheximide-stabilized exons. RT-PCR and RNASeq validations in other endothelial cells highlighted exon 3B of the iron transporter SLC11A2 (also known as NRAMP2/DMT1) as a novel exon in a transcript most consistently stabilized. Exact junctional alignments to SLC11A2 exon 3B were confirmed in blood outgrowth endothelial cells (BOECs) from 3 donors at mean 5.9% (standard deviation 2.0%) of adjacent constitutive exon expression, increasing 3.7-fold following 1h treatment with cycloheximide. Relevance beyond endothelial cells is supported by SLC11A2s wide expression profiles, genome-wide associations with microcytic anemia, biomarker status for poor prognosis ovarian cancer, and exon 3B sequence in RefSeq non-coding transcript NR_183176.1. The studies contribute understanding to functions affected acutely by NMD/translation inhibition and provide a stimulus for further studies in experimental, stress, and therapeutic settings.

molecular biology↗

bioRxiv2023

Integrative Multiomics to Dissect the Lung Transcriptional Landscape of Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) remains an incurable and often fatal disease despite currently available therapies. Multiomics systems biology analysis can shed new light on PAH pathobiology and inform translational research efforts. Using RNA sequencing on the largest PAH lung biobank to date (96 disease and 52 control), we aim to identify gene co-expression network modules associated with PAH and potential therapeutic targets. Co-expression network analysis was performed to identify modules of co-expressed genes which were then assessed for and prioritized by importance in PAH, regulatory role, and therapeutic potential via integration with clinicopathologic data, human genome-wide association studies (GWAS) of PAH, lung Bayesian regulatory networks, single-cell RNA-sequencing data, and pharmacotranscriptomic profiles. We identified a co-expression module of 266 genes, called the pink module, which may be a response to the underlying disease process to counteract disease progression in PAH. This module was associated not only with PAH severity such as increased PVR and intimal thickness, but also with compensated PAH such as lower number of hospitalizations, WHO functional class and NT-proBNP. GWAS integration demonstrated the pink module is enriched for PAH-associated genetic variation in multiple cohorts. Regulatory network analysis revealed that BMPR2 regulates the main target of FDA-approved riociguat, GUCY1A2, in the pink module. Analysis of pathway enrichment and pink hub genes (i.e. ANTXR1 and SFRP4) suggests the pink module inhibits Wnt signaling and epithelial-mesenchymal transition. Cell type deconvolution showed the pink module correlates with higher vascular cell fractions (i.e. myofibroblasts). A pharmacotranscriptomic screen discovered ubiquitin-specific peptidases (USPs) as potential therapeutic targets to mimic the pink module signature. Our multiomics integrative study uncovered a novel gene subnetwork associated with clinicopathologic severity, genetic risk, specific vascular cell types, and new therapeutic targets in PAH. Future studies are warranted to investigate the role and therapeutic potential of the pink module and targeting USPs in PAH.

genomics↗

bioRxiv2021

Heterozygous transcriptional and nonsense decay signatures in blood outgrowth endothelial cells from patients with hereditary haemorrhagic telangiectasia

Frameshift and nonsense DNA variants represent the commonest causes of monogenic inherited diseases. They usually generate premature termination codon (PTC)-containing RNA transcripts that produce truncated proteins in recombinant systems, but endogenously are subject to nonsense mediated decay. To examine native consequences of these variants, we derived cells from pre-genotyped patients. Blood outgrowth endothelial cells (BOECs) were established from individuals with hereditary hemorrhagic telangiectasia (HHT) due to a heterozygous nonsense variant in ACVRL1, ENG or SMAD4 that each encode an endothelial cell-expressed protein mediating bone morphogenetic protein (BMP)/ transforming growth factor (TGF)-{beta} signalling. RNA sequencing alignments to PTC alleles varied from 8-23% of expected, and differed between same-donor replicates. Differential gene expression analyses were validated by single cell qRT-PCR, and identification of changes in wider, disease-specific BMP/TGF-{beta} pathway components. However, the most differentially expressed genes clustered to disease-independent terms for post translational protein modification (isopeptide bond; ubiquitin-like conjugation). They were the only terms meeting Benjamini significance after clustering Bonferroni-ranked, differentially expressed genes from the 5,013 meeting 10% intraassay coefficients of variation, and significance was robust to normalisation methods. Optimised pulse chase experiments supported perturbed wildtype protein maturation, but no PTC-truncated protein was identified. Unexpectedly, BOEC cultures with highest PTC persistence were discriminated in unsupervised hierarchical clustering of low GINI coefficient invariant housekeeper genes, and patterns were compatible with higher cellular stress. The findings support a model whereby PTCs are more of a burden in stressed cells, and lead us to conclude that overlooked and varying PTC burdens contribute to biological variability.

cell biology↗