RNA-Seq: Smad4-iECKO vs Smad4f/f P7 isolated retinal endothelial cells (iECKO=inducible, endothelial cell specific knockout)
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE116230 | GEO | 2018/12/18
REPOSITORIES: GEO
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Project description:Hereditary hemorrhagic telangiectasia (HHT) is an inherited autosomal dominant disorder associated with mutations in the bone morphogenetic protein (BMP) pathway. Inherited heterozygous loss of function mutations and acquired loss of heterozygosity in either Alk1, Eng, or Smad4 lead to the development of arteriovenous malformations (AVMs), which trigger local vessel instability, hypoxia and vessel leakage or rupture. Current models assume common cellular pathomechanisms culminating in altered endothelial cell (EC) shape regulation, directional migration and proliferation control as a consequence of deficient BMP pathway signaling in ECs under the influence of blood-flow mediated shear stress. Here we report that loss of Alk1 or Smad4 surprisingly triggers very distinct endothelial phenotypes, signaling dynamics and transcriptional changes in ECs, both in vitro and in vivo. EC behavior in both in vivo and mosaic flow cultures illustrate that cells deficient in Smad4 effectively migrate against the direction of fluid shear, from veins to arteries, whereas cells lacking Alk1 fail to polarize and migrate against flow. Our data suggest that AVMs triggered by Smad4 mutations occur through hyperpruning of capillaries, thus precipitating flow in a single shunt, whereas AVMs caused by Alk1 mutation grow by cell accumulations close to the vein, as well as the persistence of a hyperdense plexus that drives nidus formation. We propose that the cellular pathomechanisms leading to AVM formation are not the same if the upstream BMP receptor Alk1, or the downstream common transcription factor Smad4 are mutated, raising the prospect for urgently needed, mechanism-based, therapeutic avenues that need to be tailored to correcting the specific pathomechanism.
2025-02-18 | GSE282952 | GEO
Project description:To investigate transcriptomic changes in the endothelial cells of a mouse model of JP/HHT (Smad4), endothelial cells were isolated from the lungs of P5 Smad4 control (Smad4 conditional floxed mice; Smad4 f/f) and Smad4 HHT mice (Smad4 f/f;Cdh5-CreERT2). Data sets were compared to reveal differentially expressed genes that may provide clues into the molecular changes driving HHT phenotypes.
2025-12-04 | GSE312375 | GEO
Project description:Purpose: Hereditary Hemorrhagic Telangiectasia (HHT) is an autosomal dominant vascular disorder characterized by arteriovenous malformations (AVMs). Over 90% of HHT patients carry heterozygous loss-of-function mutations in Transforming Growth Factor-beta (TGFb) signaling components activin receptor-like kinase 1 (ACVRL1/ALK1), endoglin (ENG), or mothers against decapentaplegic homolog 4 (SMAD4). Brain AVMs can be lethal for HHT patients. Here, we use an inducible endothelial (EC)-specific mouse line to investigate brain AVMs characteristics in Alk1-, Eng-, and Smad4-HHT mouse models. Angiopientin-2 (Ang2) inhibition diminishes HHT associated cerebral vascular defects. We aim to identify the shared transcriptional changes in brain ECs between these three HHT types, and provide insights for transcriptional changes upon Ang2 inhibition. Methods: ECs were isolated from brains of both WT and HHT mutant mice at postnatal day 7 (P7) for Smad4 and Eng models, P6 for Alk1 model. Total RNA was extracted from isolated brain ECs and quantified using Qubit RNA High Sensitivity Assay Kit. RNA integrity was determined using the Bioanalyzer RNA 6000 Nano assay kit. RNA library construction was performed with the TruSeq RNA Library Prep Kit v2 from Illumina. The resulting mRNA library was quantified using Qubit dsDNA High Sensitivity Assay Kit and verified using the Bioanalyzer DNA1000 assay kit. Verified samples were sequenced using the NextSeq 500/550 High Output Kit v2.5 (150 Cycles) on a Nextseq 550 system. Sequenced reads were aligned to the mouse (mm10) reference genome with RNA-seq alignment tool (version 2.0.1). The aligned reads were used to quantify mRNA expression and determine differentially expressed genes using the RNA-seq Differential Expression tool (version 1.0.1). Both alignment and differential expression analysis were performed using the tools in the BaseSpace Sequence Hub. Results: We identified 5509, 5381, and 2663 differentially expressed genes in Smad4, Alk1, and Eng HHT models respectively. Also, Ang2 inhibition mainly funcioned through angiogenesis and cell migration processes to normalize cerebrovascular defects in Smad4-HHT model. Conclusions: A common but unique pro-angiogenic program among all HHT models included 14 consistent upregulated and 5 consistent downregulated angiogic genes. Also, Ang2 inhibition mainly funcioned through angiogenesis and cell migration processes to normalize cerebrovascular defects in Smad4-HHT model.
2023-05-19 | GSE197105 | GEO
Project description:Vascular networks form, remodel and mature under the influence of both fluid shear stress (FSS) and soluble factors. Physiological FSS promotes and maintains vascular stability via synergy with Bone Morphogenic Protein 9 (BMP9) and BMP10. Conversely, mutation of the BMP receptors ALK1, Endoglin or the downstream effector SMAD4 leads to Hereditary Hemorrhagic Telangiectasia (HHT), characterized by fragile and leaky arterial-venous malformations (AVMs). But how endothelial cells (EC) integrate FSS and BMP signals in vascular development and homeostasis, and how mutations give rise to vascular malformations is not well understood. Here, we aimed to elucidate the mechanism of synergy between fluid shear stress and SMAD signaling in vascular stability and its failure in HHT. We have now found that loss of Smad4 increases ECs’ sensitivity to flow by lowering the FSS set point with resulting AVMs exhibiting features of excessive flow-mediated morphological responses. Mechanistically, loss of Smad4 disinhibits flow-mediated Klf4-Tie2-PI3K/Akt signaling leading to increased EC proliferation and loss of arterial identity due to Klf4-mediated repression of cyclin dependent Kinase (CDK) inhibitors, CDKN2A and CDKN2B. Thus, AVMs caused by Smad4 deletion are characterized by chronic high flow remodeling with excessive EC proliferation and loss of arterial identity as triggering events.
2023-07-03 | GSE232443 | GEO
Project description:We previously identified somatic activating KRAS mutations in a majority of human arteriovenous malformations (AVMs), using whole exome sequencing, which were enhanced in AVM endothelial cell fractions. We have now performed whole genome sequencing on AVM endothelial and non-endothelial cell fractions, as well as paired blood samples, in order to identify further somatic mutations.
| EGAS00001006719 | EGA
Project description:Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy characterized by a median survival rate of approximately six months. While genetic profiling has uncovered common mutations in PDAC, developing targeted therapeutic strategies remains challenging. SMAD4 is frequently mutated or deleted in 30-55% of PDAC patients and correlates with poor survival rates. Such mutations frequently result in loss-of-function, thereby disrupting normal cell cycle regulation and contributing to tumorigenesis. Therefore, translating SMAD4 genotype into actionable targets are highly desired for therapeutic innovation in PDAC. In this study, we performed a SMAD4-focused oncogenic protein-protein interaction (oncoPPI) network mapping and revealed a direct physical interaction between SMAD4 and NFATc1. We found that SMAD4 interacts with NFATc1 in a TGF-independent and NFATc1 phosphorylation-dependent manner. Further, SMAD4 sequesters NFATc1 in cytoplasm and inhibits NFATc1 transcriptional activity. In PDAC cells, SMAD4-loss releases its inhibitory activity on NFATc1, activates NFATc1 transcriptional activity which drives STAT3 mRNA and protein upregulation. Pharmacological profiling identified multiple STAT3 inhibitors selectively inhibit the growth of SMAD4-loss PDAC cells. These results suggested a rewired SMAD4-NFATc1-STAT3 axis and targeting STAT3 as a potential therapeutic strategy in SMAD4-loss PDAC.
2026-02-02 | PXD067749 | Pride
Project description:To identify potential biological targets of the TGFβ pathway involved in AVM formation, we performed ChIP-sequencing experiments on BMP9 stimulated Ms1 endothelial cells (ECs). This data provides a comprehensive list of all SMAD4 binding sites in endothelial cells.
2018-12-18 | GSE115921 | GEO
Project description:Background: Hereditary hemorrhagic telangiectasia (HHT) is an inherited vascular disorder characterized by arteriovenous malformations (AVMs). Loss-of-function mutations in Activin receptor-like kinase 1 (ALK1) cause type 2 HHT and Alk1 knockout (KO) mice develop AVMs along with overactivation of VEGFR2/PI3K/AKT signaling. The full spectrum of signaling alterations resulting from ALK1 mutations remains unknown, and more effective and specific inhibitors to combat AVM formation in patients are needed. Methods: Single-cell RNA sequencing of endothelial-specific Alk1 KO mouse retinas and controls was performed. Overexpression of fluid shear stress signaling signatures including the mechanosensitive ion channel PIEZO1 was confirmed in mouse and human HHT2 lesions. Genetic and pharmacological PIEZO1 inhibition was tested in Alk1 KO mice, along with downstream PIEZO1 signaling. Results: A cluster of Alk1 mutant endothelial cells with altered arterio-venous identity overexpressed pathways related to fluid shear stress, hypoxia, inflammation, cell cycle and VEGFR2/PI3K/AKT signaling. Piezo1 deletion and pharmacological inhibition in Alk1-deficient mice mitigated AVM formation, whereas Piezo1 overexpression enhanced AVM formation induced by ALK1 ligand blockade. Mechanistically, PIEZO1 inhibition reduced elevated VEGFR2/AKT, ERK5-p62-KLF4, eNOS, hypoxia, proliferation and inflammation in ALK1 deficient endothelium. Conclusions: PIEZO1 expression and signaling are elevated in HHT2. PIEZO1 blockade reduces AVM formation and alleviates cellular and molecular hallmarks of ALK1-deficient cells. This finding provides new insights into the mechanistic underpinnings of ALK1-related vascular diseases and identifies potential therapeutic targets to prevent AVMs.
2025-07-18 | GSE298122 | GEO
Project description:RNA-Seq: Smad4-iECKO vs Smad4f/f P7 isolated retinal endothelial cells (iECKO=inducible, endothelial cell specific knockout)
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Project description:RNA sequencing of mouse lung endothelial cells from male wildtype (TET2fl/fl Cre negative, tamoxifen-injected) or endothelial-specific TET2 knockout mice (TET2fl/fl, Cdh5-CreERT2 positive, tamoxifen-injected) fed either a standard chow diet or a high fat diet for 20 weeks.
2025-07-23 | GSE232888 | GEO