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Next Generation Sequencing to analyse regulators of constitutive VWF secretion

ABSTRACT: Next Generation Sequencing to analyse regulators of constitutive VWF secretion

PROVIDER: PRJNA637494 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR11931203_1.fastq.gz	Fastqsanger.gz
	SRR11931203_2.fastq.gz	Fastqsanger.gz
	SRR11931204_1.fastq.gz	Fastqsanger.gz
	SRR11931204_2.fastq.gz	Fastqsanger.gz
	SRR11931205_1.fastq.gz	Fastqsanger.gz

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Next Generation Sequencing to analyse regulators of constitutive VWF secretion

Project description:Constitutive VWF secretion can be increased by a range of factors; changes in VWF expression, levels of TNF-alpha or other environmental cues. An RNAseq analysis revealed that expression of RGS4 (Regulator of G protein signalling 4) was reduced in endothelial cells (HUVECs) grown under these conditions. si-RGS4 treatment of HUVECs increased constitutive basolateral secretion of VWF, probably by affecting the anterograde secretory pathway. In a simple model of endothelial damage we show that RGS4-silenced cells increased platelet recruitment onto the subendothelial matrix under flow. These results show that changes in RGS4 expression alter levels of subendothelial VWF, affecting platelet recruitment. This introduces a novel control over VWF function.

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Proximity proteomics of endothelial Weibel-Palade bodies identifies novel regulator of VWF secretion

Project description:Weibel-Palade bodies (WPB) are unique secretory organelles of endothelial cells that store factors regulating vascular haemostasis and local inflammation. Endothelial activation triggers the acute exocytosis of WPB, leading to the surface presentation of adhesion molecules relevant for leukocyte rolling (P-selectin) and platelet plug formation (von-Willebrand factor, VWF). Despite its role as an important secretory organelle, a comprehensive compilation of WPB-associated factors has not been carried out. We addressed this by a proximity proteomics approach employing the peroxidase APEX2 coupled to two known WPB-associated proteins, the RabGTPases Rab3b and Rab27a. We show that APEX2-Rab3b/27a fusion constructs are correctly targeted to WPB and that proteins in their close proximity can be biotinylated through the WPB-recruited APEX2. Mass spectrometry analysis of the biotinylated proteins identified 183 WPB-associated proteins. While some of the factors identified have been reported before to localize to WPB, the majority comprises proteins not previously associated with WPB biology. These include the SNARE-interacting protein Munc13-2, which specifically localizes to WPB and serves as a novel factor promoting histamine-evoked WPB exocytosis and VWF secretion.

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Multifaceted Pathomolecular Mechanism of a VWF Large Deletion Involved in the Pathogenesis of Severe VWD

Project description:An in-frame heterozygous large deletion of exons 4-34 of the von Willebrand factor (VWF) gene was identified in an index patient (IP) with type 3 von Willebrand disease (VWD), as the only mutation. The IP exhibited severe bleeding episodes despite prophylaxis treatment, with a short VWF half-life after infusion of VWF/FVIII concentrates. This study intends to elucidate the causal molecular mechanism of this large deletion. Transcript analysis confirmed transcription of normal VWF mRNA besides an aberrant deleted transcript. The amount of secreted VWF from blood outgrowth endothelial cells (BOECs) isolated from the IP was not significantly different from that of controls. However, IP-BOECs exhibited a deficiency in the assembly of VWF multimers and biogenesis of the Weibel-Palade bodies (WPBs). Furthermore, immunostaining of IP-BOECs demonstrated subcellular mislocalization of WPBs pro-inflammatory cargos angiopoietin-2 (Ang2) and P-selectin. Additionally, whole-transcriptome RNA-sequencing of the BOECs and subsequent Ingenuity Pathway Analysis indicated the significant alterations of canonical pathways in IP-BOECs related to inflammatory responses, cell adhesion, extracellular organization, and angiogenesis (e.g. granulocyte adhesion and Rho-related signaling pathways), which are known downstream signaling pathways induced by Ang2. Accordingly, the IP-BOECs exhibited an increased adhesiveness to leukocytes which may contribute to accelerated VWF clearance. In conclusion, deleted VWF has a dominant-negative impact on the elongation of multimers and biogenesis of WPBs. Aberrant WPBs lead to the alternative trafficking of its cargos in a specific way, which, in turn, may cause distinctive perturbations in cellular signaling pathways, resulting in the exceptional phenotypes in the current patient.

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Project description:A type 3 von Willebrand disease (VWD) index patient (IP) remains mutation-negative after completion of conventional diagnostic analysis, including multiplex ligation-dependent probe amplification and sequencing of the promotor, exons, and flanking intronic regions of VWF gene (VWF). In this study, we intended to elucidate causitive genetic defect through screening of the whole VWF (including complete intronic region), mRNA analysis, and study of the patient-derived endothelial colony-forming cells (ECFCs). The entire VWF was analyzed by next-generation sequencing (NGS) on an Illumina platform. The NGS revealed a novel variant in VWF intron 8 (997+118 T>G). The subsequent assessments using bioinformatics tools (e.g. SpliceAl) predicted this variant creates a new donor splice site (ss) in intron 8, which could outcompete the consensus 5’ donor ss at exon/intron 8 junction. This leads to an aberrant mRNA which contains a premature stop codon, targeting it to nonsense-mediated mRNA decay. The VWF mRNA from whole blood and isolated ECFCs were quantified using the TaqMan assay on an ABI 7500 real-time PCR system. The quantitative analysis confirmed the virtual absence of VWF mRNA. Additionally, the level of secreted VWF from IP ECFCs was considerably reduced (~6% of healthy donors).

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Syntaxin 5 determines Weibel-Palade body size and Von Willebrand factor secretion by controlling Golgi architecture

Project description:Abstract Von Willebrand factor (VWF) is a multimeric hemostatic protein primarily synthesized in endothelial cells (ECs). VWF is stored in endothelial storage organelles, the Weibel-Palade bodies (WPBs), whose biogenesis strongly depends on VWF anterograde trafficking and Golgi architecture. Elongated WPB morphology is correlated to longer VWF strings with better adhesive properties. We previously identified the SNARE SEC22B, which is involved in anterograde ER-to-Golgi transport, as a novel regulator of WPB elongation. To elucidate novel determinants of WPB morphology we explored endothelial SEC22B interaction partners in a mass spectrometry-based approach, identifying the Golgi SNARE Syntaxin 5 (STX5). We established STX5 knockdown in ECs using shRNA-dependent silencing and analyzed WPB and Golgi morphology, using confocal and electron microscopy. STX5-depleted ECs exhibited extensive Golgi fragmentation and decreased WPB length, which was associated with reduced intracellular VWF levels, and impaired stimulated VWF secretion. However, the secretion incompetent organelles in shSTX5 cells maintained WPB markers such as Angiopoietin 2, P-selectin, Rab27A, and CD63. Taken together, our study has identified SNARE protein STX5 as a novel regulator of WPB biogenesis

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