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Loss of Smad4 drives vascular malformations via c-KIT-dependent high shear stress mimicry

ABSTRACT: Vascular endothelial cells (ECs) encode a homeostatic fluid shear stress (FSS) set point that is essential for vascular stability. Deviations above or below this threshold trigger adaptive remodeling to restore physiological shear levels. Disruption of this control mechanism leads to enlarged arterial-venous malformations (AVMs) in hereditary haemorrhagic telangiectasia (HHT), a vascular disorder caused by heterozygous loss-of-function (LOF) mutation in ALK1, ENG or SMAD4. Mechanistically, Smad4 deficient ECs are reset to a lower FSS set point value, resulting in AVMs that show characteristics of high FSS remodeling with elevated Klf4 and excessive activation of the downstream Akt. Here, we investigated the Klf4-Akt upstream mechanisms by which Smad4 sets the physiological FSS setpoint. We identified the receptor tyrosine kinase c-Kit as a novel component and regulator of the junctional mechanosensory receptor complex, which is highly upregulated in murine and human AVMs. Smad4 restrains flow signaling by limiting c-Kit-dependent Erk5 activation and Klf4 induction. Thus, Smad4 LOF leads to sustained c-Kit engagement in the sensory junctional apparatus, driving excessive and prolonged activation of the Erk5-Klf4-Akt signaling axis. These results show that Smad4 LOF mutations induce malformations by disabling a key homeostatic mechanism and identify c-Kit as a novel therapeutic target.

ORGANISM(S): Homo sapiens

PROVIDER: GSE330070 | GEO | 2026/05/11

REPOSITORIES: GEO

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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.

Project description:Tissue Factor Pathway Inhibitor-2 is Induced by Fluid Shear Stress in Vascular Smooth Muscle Cells and Affects Cell Proliferation and Survival Introduction: Vascular smooth muscle cells (SMCs) are exposed to fluid shear stress (FSS) after interventional procedures such as balloon-angioplasty. Whereas the effects of hemodynamic forces on endothelial cells are explored in detail, the influence of FSS on smooth muscle cell function is poorly characterized. Here, we investigated the effect of FSS on SMC gene expression and function. Methods: Laminar FSS of arterial level (14 dynes/cm2) was applied to SMC cultures for 24 h in a parallel-plate flow chamber. The effect of FSS on gene expression was first screened with microarray technology and the results further verified by real time (RT) PCR and immunoblotting. Protein expression was also studied in the rat carotid artery after balloon-injury and DNA synthesis and apoptosis was examined in SMCs in vitro. Results: Microarrays identified tissue-pathway inhibitor-2 (TFPI-2) as the most differentially expressed gene by FSS in cultured SMCs. The regulatory effect of FSS on the expression of TFPI-2 was confirmed by RT-PCR and immunobloting demonstrating a more than 400-fold increase in TFPI-2 expression in SMCs exposed to FSS compared to static controls and a consistent upregulation at the protein level. Functionally, SMC proliferation was decreased by FSS and recombinant TFPI-2 was found to inhibit SMC proliferation and induce SMC apoptosis as indicated by activation of caspase-3. In vivo, TFPI-2 expression was found to be up-regulated 5, 10 and 20 h after rat carotid balloon-injury and immunohistochemistry demonstrated TFPI-2 protein in luminal SMCs exposed to FSS in rat carotid intimal hyperplasia 10 days after balloon-injury. Conclusion: FSS strongly influence gene expression in cultured SMCs and induce TFPI-2 expression, which is also expressed after rat carotid balloon injury in luminal SMCs exposed to FSS. Functionally, TFPI-2 may play an important role in vessel wall repair by regulating SMC proliferation and survival. Further studies are needed to elucidate the mechanisms by which TFPI-2 control SMC function. 3 x 2 samples from a paired fluid shear stress experiment on smooth muscle cells.

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Loss of Smad4 drives vascular malformations via c-KIT-dependent high shear stress mimicry

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