Dataset Information

ECM characterization of aortas from Adamts5+/+ and Adamts5∆cat AngII-treated mice by LC-MS/MS

ABSTRACT: Objective - Thoracic aortic aneurysm (TAA), a degenerative disease of the aortic wall, is accompanied by changes in the structure and composition of the aortic extracellular matrix (ECM). The ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family has recently been implicated in TAA formation. This study aimed to investigate the contribution of ADAMTS-5 to TAA development. Approach and Results - A model of aortic dilatation by angiotensin II (AngII) infusion was adopted in mice lacking the catalytic domain of ADAMTS-5 (Adamts5Δcat). Adamts5Δcat mice showed an attenuated rise in blood pressure whilst displaying increased dilatation of the ascending aorta. Interestingly, a comparison of the aortic ECM from AngII-treated wildtype and Adamts5Δcat mice revealed versican as the most up-regulated ECM protein in Adamts5Δcat mice. This was accompanied by a marked reduction of ADAMTS-specific versican cleavage products (versikine) and a decrease of low-density lipoprotein-related protein 1 (LRP1). Silencing LRP1 expression in human aortic smooth muscle cells reduced the expression of ADAMTS5, attenuated the generation of versikine but increased soluble ADAMTS-1. A similar increase in ADAMTS-1 was observed in aortas of AngII-treated Adamts5Δcat mice, but was not sufficient to maintain versican processing and prevent aortic dilatation. Conclusions - Our results support the emerging role of ADAMTS proteases in TAA. ADAMTS-5 rather than ADAMTS-1 is the key protease for versican regulation in murine aortas. Further studies are needed to define the ECM substrates of the different ADAMTS proteases and their contribution to TAA formation.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Aorta

DISEASE(S): Dilatation Of The Ascending Aorta

SUBMITTER: Xiaoke Yin

LAB HEAD: Manuel Mayr

PROVIDER: PXD009410 | Pride | 2018-05-08

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	Marika_Mouse_ATS5_AngII_Top15_20161110_KO_19.raw	Raw
	Marika_Mouse_ATS5_AngII_Top15_20161110_KO_20.raw	Raw
	Marika_Mouse_ATS5_AngII_Top15_20161110_KO_21.raw	Raw
	Marika_Mouse_ATS5_AngII_Top15_20161110_KO_23.raw	Raw
	Marika_Mouse_ATS5_AngII_Top15_20161110_KO_24.raw	Raw

Items per page:

1 - 5 of 55

Publications

Role of ADAMTS-5 in Aortic Dilatation and Extracellular Matrix Remodeling.

Fava Marika M Barallobre-Barreiro Javier J Mayr Ursula U Lu Ruifang R Didangelos Athanasios A Baig Ferheen F Lynch Marc M Catibog Norman N Joshi Abhishek A Barwari Temo T Yin Xiaoke X Jahangiri Marjan M Mayr Manuel M

Arteriosclerosis, thrombosis, and vascular biology 20180405 7

<h4>Objective</h4>Thoracic aortic aneurysm (TAA), a degenerative disease of the aortic wall, is accompanied by changes in the structure and composition of the aortic ECM (extracellular matrix). The ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family of proteases has recently been implicated in TAA formation. This study aimed to investigate the contribution of ADAMTS-5 to TAA development.<h4>Approach and results</h4>A model of aortic dilatation by AngII (angiotensin II) ...[more]

PMID: 29622560

Similar Datasets

Project description:We identify a role for two evolutionarily related, secreted metalloproteases of the ADAMTS family (A disintegrin-like and metalloprotease domain with thrombospondin type-1 motif), ADAMTS20 and ADAMTS9, in palatogenesis. Adamts20 mutations cause the mouse white spotting mutant belted (bt), whereas Adamts9 is essential for survival beyond 7.5 days of gestation (E7.5). Functional overlap of Adamts9 with Adamts20 was established in bt/bt:Adamts9+/- mice, which have increased white spotting relative to bt mice, as previously reported, and a fully penetrant cleft palate. Palatal closure was delayed, although eventually completed, in both bt/+;Adamts9+/- and bt/bt mice, demonstrating a cooperative role of these related genes. Adamts9 and Adamts20 are both expressed in palatal mesenchyme, with Adamts9 expressed exclusively in microvascular endothelial cells. Palatal shelves from bt/bt:Adamts9+/- mice fused in culture, suggesting an intact TGF signaling pathway in palatal epithelium, and indicating a temporally specific delay in palatal shelf elevation and growth toward the midline. Palatal shelf mesenchymal cells showed a statistically significant decrease of cell proliferation at E13.5 and E14.5, as well as decreased processing of versican, an ADAMTS substrate, at these stages. Vcan haploinsufficiency led to a greater penetrance of cleft palate in bt mice, and impaired proliferation was also seen in palatal mesenchymal cells of these mice, suggesting a role for ADAMTS-mediated versican proteolysis in palatal closure. In a parallel with recent work identifying a role for a bioactive ADAMTS-generated versican fragment in regulating apoptosis during interdigital web regression, we propose that versican proteolysis may influence palatal mesenchymal cell proliferation. Palatal shelves were dissected from four E13.75 Adamts9+/-:bt/bt embyos (correspond to the 4 samples: Palate_Adamts9+/-:bt/bt_Rep1, Palate_Adamts9+/-:bt/bt_Rep2, Palate_Adamts9+/-:bt/bt_Rep3 and Palate_Adamts9+/-:bt/bt_Rep4) and age-matched 3 wild-type C57Bl/6 embryos (correspond to the 3 samples: Palate_WT_Rep1, Palate_WT_Rep2, and Palate_WT_Rep3) that were used as the controls

Project description:Vascular calcification and increased extracellular matrix (ECM) stiffness are hallmarks of vascular ageing. Sox9 (SRY-Box Transcription Factor 9) is a master regulator of chondrogenesis, also expressed in the vasculature, that has been implicated in vascular smooth muscle cell (VSMC) osteo-chondrogenic conversion. Here, we investigated the relationship between vascular ageing, calcification and Sox9-driven ECM regulation in VSMCs. Immunohistochemistry in human aortic samples showed that Sox9 was not spatially associated with vascular calcification but correlated with the senescence marker p16. Analysis of Sox9 expression in vitro showed it was mechanosensitive with increased expression and nuclear translocation in senescent cells and on stiff matrices. Manipulation of Sox9 via overexpression and depletion, combined with atomic force microscopy (AFM) and proteomics, revealed that Sox9 regulates ECM stiffness and organisation by orchestrating changes in collagen expression and reducing VSMC contractility, leading to the formation of an ECM that mirrored that of senescent cells. These ECM changes promoted phenotypic modulation of VSMCs whereby senescent cells plated onto ECM synthesized from cells depleted of Sox9 returned to a proliferative state, while proliferating cells on a matrix produced by Sox9 expressing cells showed reduced proliferation and increased DNA damage, reiterating features of senescent cells. Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3 (LH3) was identified as a Sox9 target, and key regulator of ECM stiffness. LH3 is packaged into extracellular vesicles (EVs) and Sox9 promoted EV secretion, leading to increased LH3 deposition within the ECM. These findings identify cellular senescence and Sox9 as a key regulators of ECM stiffness during VSMC ageing and highlight a crucial role for ECM structure and composition in regulating VSMC phenotype. We identify a positive feedback cycle whereby cellular senescence and increased ECM stiffening promote Sox9 expression which drives further ECM modifications that act to accelerate vascular stiffening and cellular senescence.

Project description:In mammals, the A Disintegrin-like And Metalloproteinase domain with Thrombospondin type 1 motifs (ADAMTS) family comprises 19 secreted proteases, including 6 members with the ability to cleave large aggregating proteoglycans: ADAMTS1, 4, 5, 9, 15, and 20. ADAMTS9 has an essential, non-redundant role during embryogenesis, as Adamts9 null embryos die prior to completing gastrulation and Adamts9 haploinsufficiency results in cardiovascular and ocular anomalies. ADAMTS9 proteolytic activity is required for proteostasis of versican, a widely distributed large aggregating proteoglycan/hyalectan in the provisional extracellular matrix present during early development. Despite its importance, ADAMTS9 proteoglycanase activity has not been directly compared to that of ADAMTS1, 4, and 5, due to difficulties in expressing and purifying the >200 kDa full-length form of ADAMTS9. Like ADAMTS1, 4, and 5, ADAMTS9 cleaves versican V1 isoform at the Glu441-Ala442 site, but unlike them, cleavages at other sites are unknown. Here, we expressed a truncated ADAMTS9 variant (ADAMTS9 MDTCS) consisting of all ADAMTS ‘core domains’ that render it more comparable in size and domain structure to ADAMTS1, 4, and 5 and compared its activity against versican, aggrecan and the small leucine-rich proteoglycan biglycan. A catalytically inactive ADAMTS9 MDTCS variant (ADAMTS9 EQ) was used as the control in all experiments. We identified cleavage sites in versican (V1 and V2 isoforms) and biglycan by all 4 ADAMTS family members using a quantitative label-free proteomics strategy. Moreover, using a quantitative substrate cleavage assay, we established that ADAMTS9 MDTCS versicanase activity at the Glu441-Ala442 site is 175-fold lower than ADAMTS5, 9-fold lower than ADAMTS4, and 5.5-fold higher lower than ADAMTS1. These provide systematic analysis of the proteoglycanase activity in the ADAMTS family and highlight differences and similarities in cleavage site specificities that could potentially be leveraged to design selective small molecule inhibitors for therapeutic applications.

Project description:BACKGROUND: A better understanding of the molecular mechanism of aortic valve development and bicuspid aortic valve (BAV) formation would significantly improve and optimize the therapeutic strategy for treating BAV patients. Over the past decade, the genes involved in aortic valve development and BAV formation has been increasingly recognized. On the other hand, ADAMTS gene family members have been reported to be able to modulate cardiovascular development and diseases. The present study aimed to further investigate the roles of ADAMTS family members in aortic valve development and BAV formation. METHODS: Morpholino-based ADAMTS family gene-targeted screening for zebrafish heart outflow tract phenotypes combined with DNA sequencing in a 304 cohort BAV patient registry study was initially carried out to identify potentially related genes. Both ADAMTS gene-specific fluorescence in situ hybridization assay and genetic tracing experiments were then performed to evaluate the expression pattern in the aortic valve Accordingly, related genetic mouse models (both knock-out and knock-in) were generated using the CRISPR/Cas9 method to further study the roles of ADAMTS family genes. The lineage tracing technique was used again to evaluate how the cellular activity of specific progenitor cells was regulated by ADAMTS genes. Bulk RNA sequencing was used to investigate the signaling pathways involved. Both inducible pluripotent stem cells (iPSC) derived from BAV patients and genetic mouse tissue were used to study the molecular mechanism of the gene. Immunohistochemistry using different antibodies was performed to describe the phenotype of cardiac valve anomalies, especially in the ECM components. RESULTS: ADAMTS genes targeted phenotype screening in zebrafish and targeted DNA sequencing on a cohort of BAV patients identified ADAMTS16 as a BAV-causing gene and found the ADAMTS16 p.H357Q variant in an inherited BAV family. In addition, both in situ hybridization and genetic tracing studies described a unique spatiotemporal pattern of ADAMTS16 expression during aortic valve development. Adamts16+/- and Adamts16+/H355Q mouse models both exhibited an R-NC-type BAV phenotype, with progressive aortic valve thickening associated with raphe formation (fusion of the commissure). Conditional Adamts16 mutant mouse models further demonstrated that Adamts16 deficiency mainly in endothelial lineage cells recapitulated the BAV phenotype. This was confirmed in lineage tracing mouse models in which Adamts16 deficiency affected endothelial and second heart field cells, not the neural crest cells. Accordingly, the changes were mainly detected in the noncoronary and right coronary leaflets. Bulk RNA sequencing using iPSC-induced endothelial cells (iPSC-ECs) and genetic mouse embryonic heart tissue unveiled enhanced FAK signaling, which was accompanied by elevated fibronectin levels. Both in vitro iPSC-ECs culture and ex vivo embryonic outflow tract explant studies validated the altered FAK signaling. CONCLUSIONS: Our present study identified a novel BAV-causing ADAMTS16 p. H357Q variant. ADAMTS16 deficiency led to BAV formation. We then performed gene expression profiling analysis using data obtained from RNA-seq of 15 different tissues of different genetic mice.