Proteomics analysis on aortic smooth muscle cells reveals new potential targets for the treatment of Marfan syndrome-associated thoracic aortic aneurysm
Authors: Rega S , Bouhuis S , Vescio M , Gaspardo A , Munno M , Pattini L , Banfi C , Pompilio G , Perrucci G L 
Published: May 2024

Abstract

Funding Acknowledgements
Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Ministry of Health

Background
Thoracic aortic aneurysm (TAA) is characterized by the abnormal enlargement of the initial segment of the aorta. Patients with genetic Marfan syndrome (MFS) develop TAA in young age, since mutations of fibrillin-1 contribute to thoracic aortic wall detrimental weakening. Presently, effective treatment options for this life-threatening condition are lacking, and existing pharmacological approaches are merely palliative, by postponing the need for surgical aortic replacement. To identify new treatments or MFS-related TAA (MFS-TAA), current research is concentrated on pinpointing specific biological targets and mechanisms underlying this aortic disorder.

Purpose
This study is aimed to unveil potential therapeutic targets that could be exploited for treating MFS-TAA by adopting an advanced proteomics technique.

Material and Methods
A label-free quantitative mass spectrometry technique was employed, utilizing a hybrid quadrupole-time of flight mass spectrometer coupled with a UPLC Mclass system and a nano-source, to analyze and compare the protein expression levels in vascular smooth muscle cells (VSMC) isolated from the aortas of MFS patients (MFS-VSMC) and healthy subjects (HC-VSMC). The proteomics findings were subsequently validated through qRT-PCR and Western blot assays.

Results
The proteomics investigation yielded 16 proteins displaying statistically significant differences. Among these, five proteins were notably more abundant in MFS-VSMC compared to HC-VSMC: nestin (NES, p&lt;0.01), filamin B (FLNB, p&lt;0.05), A-kinase anchor protein 12 (AKAP12, p&lt;0.05), catenin delta-1 (CTNND1, p&lt;0.05), and heparan sulfate proteoglycan core protein (HSPG2, p&lt;0.05). The Gene Ontology (GO) analysis performed on cellular components of these 5 items using the clusterProfiler R package highlighted the enrichment of GO terms generally related to transmembrane proteins and anchoring junctions. Specifically, enriched GO terms included cell cortex (p&lt;0.002), focal adhesion (p&lt;0.002), cell-substrate junction (p&lt;0.002), and intermediate filament binding (p&lt;0.03). To verify these findings, two out of the five proteins with increased expression in MFS-VSMC (i.e., nestin and filamin B) were subjected to validation experiments. Results confirmed the elevated gene expression and protein levels of both nestin (p&lt;0.05) and filamin B (p&lt;0.05) in MFS-VSMC samples when compared with HC-VSMC.

Conclusions
Taken together, these results suggest that proteins with increased expression in MFS-VSMC may represent valuable biological target of MFS-TAA. This may encompass their potential both as diagnostic tools and/or mechanistic mediators of TAA in MFS subjects. In details, the pharmacological targeting of nestin and filamin B may represent a novel and exploitable therapeutic option for limiting/inhibiting TAA onset or progression in MFS pathological scenario.