Biology subjects

Wojciak-Stothard, B.

Publications and source records attributed to Wojciak-Stothard, B..

5 recordsLinked to original sources

bioRxiv2026

Organ on chip model of respiratory vascular interactions under COPD relevant oxidative stress

Oxidative stress-induced airway injury contributes to chronic obstructive pulmonary disease (COPD). Cardiovascular complications increase COPD morbidity and mortality, but mechanistic links between airway injury and vascular dysfunction remain unclear, largely due to limitations of in vitro models that fail to replicate the multicellular lung environment. We developed REVAS, a modular organ-on-chip platform to study human respiratory-vascular cell-cell interactions at baseline and under oxidative stress conditions. REVAS consists of two respiratory chips hosting airway epithelium and microvascular endothelium, and a vascular chip hosting pulmonary artery endothelial cells co-cultured with vascular support cells, including smooth muscle cells, pericytes and fibroblasts. We studied effects of vascular support and respiratory cells on vascular endothelial phenotype at baseline and under H2O2-induced epithelial oxidative stress using functional assays, proteomic and transcriptomic analyses. Multicellular environment enhanced vascular endothelial barrier function and promoted respiratory and vascular cell differentiation at baseline. Mural cells altered endothelial cell-matrix interactions, metabolism and cytoskeletal remodelling, while respiratory cells promoted endothelial aerobic respiration and quiescent phenotype. Epithelial oxidative stress triggered inflammatory gene expression across all respiratory and vascular cells alongside apoptotic, reparative and pro-angiogenic signalling in endothelial and mural cells, accompanied by increased release of COPD-relevant cytokines and chemokines, including IL-6, TNF-/{beta}, IL-8, CCL5, CXCL9, PDGF, TGF-{beta}. Comparative analyses with COPD endothelial datasets confirmed that REVAS recapitulates key features of disease-associated endothelial dysfunction. These findings demonstrate that airway epithelial injury drives downstream vascular responses linked to inflammation and vascular remodelling, establishing REVAS as a human-relevant platform for mechanistic and therapeutic evaluation of cell-cell interactions in COPD and related lung diseases. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=164 HEIGHT=200 SRC="FIGDIR/small/730087v1_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@e29fd8org.highwire.dtl.DTLVardef@6c3d22org.highwire.dtl.DTLVardef@21a53forg.highwire.dtl.DTLVardef@e7f432_HPS_FORMAT_FIGEXP M_FIG C_FIG REVAS: a microfluidic platform developed to model multicellular interactions between airway epithelium and pulmonary vasculature under basal and oxidative stress. COPD: Chronic Obstructive Pulmonary Disease; EMT: endothelial-to-mesenchymal transition; HsEpCs: human small airway epithelial cells; HPMVECs: human pulmonary microvascular endothelial cells; HPAECs: human pulmonary artery endothelial cells; HPASMCs: human pulmonary artery smooth mucle cells; HPFs: human pulmonary fibcroblasts; HPCs: human pericytes.

bioengineering↗

bioRxiv2024

KLF6 in Pulmonary Hypertension: The Dual Role of Friend and Foe

BackgroundPulmonary arterial hypertension (PAH) is a severe lung condition with unmet clinical needs, marked by endothelial damage, excessive repair, and arterial narrowing, though mechanisms remain unclear. MethodsThis study investigates Kruppel-like transcription factor 6 (KLF6), known for its role in tissue injury response and cancer onset, in PAH through functional and expression analyses in human pulmonary artery endothelial cells (HPAECs) and human and rodent PAH lung tissues. FindingsKLF6 expression increased in early experimental PAH in response to hypoxia and inflammation, while the expression of endothelial homeostasis regulators KLF2 and KLF4, previously linked to PAH, decreased. KLF6 overexpression enhanced pulmonary endothelial survival and angiogenesis through broad transcriptomic remodelling, including changes in genes governing endothelial homeostasis and arterial identity (e.g., SOX17, ERG, BMPR2) and promoted human pulmonary artery smooth muscle cells (HPASMCs) proliferation, which was inhibited by bosentan and imatinib. KLF6 functional and transcriptomic responses differed from those of KLF2 and KLF4. Comparative analysis of RNA-seq PAH databases and spatial transcriptomic analysis of human idiopathic PAH (IPAH) tissues highlighted strong association of KLF6 with vascular remodelling, especially with the formation of angioproliferative (plexiform) lesions. High KLF6 expression was observed in IPAH vascular endothelium and IPAH blood-derived endothelial progenitor cells. Single nucleus RNA-seq in PAH associated with Alveolar Capillary Dysplasia confirmed disease-related elevated KLF6 expression in arterial endothelial cells. InterpretationAccumulation and reorganization of KLF6+ endothelial cells characterize human PAH. KLF6 drives endothelial repair and an apoptosis-resistant, angioproliferative endothelial phenotype. Targeting KLF6 could be a novel therapeutic approach for PAH. RESEARCH IN CONTEXTO_ST_ABSEvidence before this studyC_ST_ABSPulmonary arterial hypertension (PAH) is a progressive and life-shortening lung disease with no cure. In PAH development, endothelial damage is believed to initiate an abnormal repair process, leading to extensive vascular remodelling and the formation of complex angio-proliferative (plexiform) lesions. We conducted a systematic search of the PubMed database to identify transcription factors potentially involved in driving endothelial repair and promoting an apoptosis-resistant, angio-proliferative vascular phenotype. Previous research has linked the loss of endothelial homeostasis in PAH to the inhibition of transcription factors KLF2 and KLF4. While KLF6 is known to play a vital role in vascular development and supports endothelial repair, its specific role in PAH remains unexplored. Added value of this studyThis study is the first to establish a connection between KLF6 and PAH pathogenesis. Our findings reveal that KLF6 activation is a key feature of an apoptosis-resistant, angio-proliferative endothelial phenotype characteristic of human PAH-associated plexogenic arteriopathy. Furthermore, we identify both overlapping and unique activation patterns and transcriptional programs regulated by KLF2, KLF4, and KLF6 in lung endothelial cells, highlighting KLF6s unique role in driving endothelial dysfunction in PAH. Implications of all the available evidenceTargeting KLF6 offers a promising therapeutic strategy to counteract excessive vascular repair and prevent the vascular remodelling in PAH. FUNDINGPhD studentship from the University of Hafr Al Batin, KSA, and the Saudi Cultural Bureau in London (UKSACB) (Rehab Alharbi). Spatial transcriptomic reagents were funded by the British Heart Foundation Centre of Research Excellence Award and Senior BHF Fellowship FS/18/52/33808 (Allan Lawrie). Human samples used in this research project were obtained from the Imperial College Healthcare Tissue Bank (ICHTB) supported by the National Institute for Health Research (NIHR) Biomedical Research Centre based at Imperial College Healthcare NHS Trust and Imperial College London.

cell biology↗

bioRxiv2023

ARF6 as a novel activator of HIF-2alpha in pulmonary arterial hypertension

ADP-ribosylation factor 6 (ARF6), a GTPase associated with cancer metastasis, is activated in the lung endothelium in pulmonary arterial hypertension (PAH). To identify ARF6-regulated pathways relevant to PAH, we performed a state-of-the-art proteomic analysis of human pulmonary artery endothelial cells (HPAECs) overexpressing the wildtype, constitutively active, fast-cycling and dominant negative mutants of ARF6. The analysis revealed a novel link of ARF6 with hypoxia-inducible factor (HIF), in addition to endocytotic vesicle trafficking, cell proliferation, angiogenesis, oxidative stress and lipid metabolism. Active ARF6 markedly increased expression and activity of HIF-2, critical in PAH, with HIF-1 relatively unaffected. Hypoxic ARF6 activation was a prerequisite for HIF-2 activation and HIF-dependent gene expression in HPAECs, PAH blood-derived late outgrowth endothelial colony forming cells (ECFCs) and hypoxic mouse lungs in vivo. A novel ARF6 inhibitor, chlortetracycline (CTC), reduced hypoxia-induced HIF-2 activation, proliferation and angiogenesis in HPAECs and reduced HIF-2 expression in lung and heart tissues of hypoxic mice. PAH ECFCs showed elevated expression and activity of ARF6 and HIF2, which was attenuated by CTC. Oral CTC attenuated development of PH in chronically hypoxic mice. In conclusion, we are first to demonstrate a key role of ARF6 in the regulation of HIF-2 activation in vitro and in vivo and show that HIF-2, a master-regulator of vascular remodelling in PAH, can be targeted by a clinically approved antibiotic chlortetracycline.

cell biology↗

bioRxiv2020

Role of endothelial microRNA-150 in pulmonary arterial hypertension

Endothelial dysfunction contributes to the vascular pathology in pulmonary arterial hypertension (PAH). Circulating levels of endothelial miR-150 are reduced in PAH and act as an independent predictor of patient survival. The role of endothelial miR-150 in vascular dysfunction in PAH is not well understood. Endothelium-targeted miR-150 delivery prevented the disease in Sugen/hypoxia mice, while endothelial knockdown of miR-150 had adverse effects. miR-150 target genes revealed significant associations with PAH pathways, including proliferation, inflammation and phospholipid signaling, with PTEN-like mitochondrial phosphatase (PTPMT1) most markedly altered. PTPMT1 reduced inflammation, apoptosis and improved mitochondrial function in human pulmonary endothelial cells and blood-derived endothelial colony forming cells (ECFCs) from idiopathic PAH. Beneficial effects of miR-150 in vitro and in vivo were linked with PTPMT1-dependent biosynthesis of mitochondrial phospholipid cardiolipin and reduced expression of pro-apoptotic, pro-inflammatory and pro-fibrotic genes, including c-MYB, NOTCH3, TGF-{beta} and Col1a1. In conclusion, we are first to show that miR-150-PTPMT1-cardiolipin pathway attenuates pulmonary endothelial damage induced by vascular stresses and may be considered as a potential therapeutic strategy in PAH.

physiology↗

bioRxiv2019

Krüppel-like factor 2- induced microRNAs: implications for treatment of pulmonary hypertension

Flow-activated transcription factor Kruppel-like factor 2 (KLF2) signaling is compromised in pulmonary arterial hypertension (PAH). We aimed to identify KLF2-induced endothelium-protective exosomal microRNAs of potential therapeutic significance.\n\nEight exosomal microRNAs elevated by KLF2 but reduced in PAH were transfected into human pulmonary artery endothelial cells. Of these, only miR-181a-5p and miR-324-5p had anti-apoptotic, anti-inflammatory and anti-proliferative effect on endothelial cells and reduced proliferation of vascular smooth muscle cells in vitro. RNA sequencing of miRNA-transfected HPAECs revealed reduced expression of multiple genes implicated in vascular remodelling, including ETS-1, NOTCH4, ACTA2, TNF-, IL-1, MMP10, MAPK and NFATC2. KLF2, miR-181a-5p and miR-324-5p were reduced, while their target genes were elevated in blood-derived endothelial colony forming cells and lung tissues from idiopathic and heritable PAH patients with disabling KLF2 mutation and Sugen/hypoxia mice. Supplementation of miR-181a-5p and miR-324-5p or silencing of their target genes attenuated proliferative and angiogenic responses in endothelial cells from idiopathic PAH and prevented development of pulmonary hypertension in Sugen/hypoxia mice.\n\nThis study highlights potential therapeutic role of KLF2-induced exosomal microRNAs in PAH.

cell biology↗