DNA methyltransferase isoforms regulate endothelial cell exosome proteome composition

Sampara Vasishta; Shruthi Ammankallu; Shashikiran Umakanth; Thottethodi Subrahmanya Keshava Prasad; Manjunath B Joshi

doi:10.1016/j.biochi.2024.05.010

DNA methyltransferase isoforms regulate endothelial cell exosome proteome composition

Biochimie. 2024 May 10:223:98-115. doi: 10.1016/j.biochi.2024.05.010. Online ahead of print.

Authors

Sampara Vasishta¹, Shruthi Ammankallu², Shashikiran Umakanth³, Thottethodi Subrahmanya Keshava Prasad², Manjunath B Joshi⁴

Affiliations

¹ Department of Ageing Research, Manipal School of Life Sciences, Manipal Academy of Higher Education, Manipal, 576104, Karnataka, India.
² Center for Systems Biology and Molecular Medicine, Yenepoya Research Centre, Yenepoya (Deemed to Be University), Mangalore, 575020, Karnataka, India.
³ Department of Medicine, Dr. TMA Pai Hospital, Udupi, 576101, Karnataka, India.
⁴ Department of Ageing Research, Manipal School of Life Sciences, Manipal Academy of Higher Education, Manipal, 576104, Karnataka, India. Electronic address: manjunath.joshi@manipal.edu.

PMID: 38735570
DOI: 10.1016/j.biochi.2024.05.010

Abstract

Extrinsic and intrinsic pathological stimuli in vascular disorders induce DNA methylation based epigenetic reprogramming in endothelial cells, which leads to perturbed gene expression and subsequently results in endothelial dysfunction (ED). ED is also characterized by release of exosomes with altered proteome leading to paracrine interactions in vasculature and subsequently contributing to manifestation, progression and severity of vascular complications. However, epigenetic regulation of exosome proteome is not known. Hence, our present study aimed to understand influence of DNA methylation on exosome proteome composition and their influence on endothelial cell (EC) function. DNMT isoforms (DNMT1, DNMT3A, and DNMT3B) were overexpressed using lentivirus in ECs. Exosomes were isolated and characterized from ECs overexpressing DNMT isoforms and C57BL/6 mice plasma treated with 5-aza-2'-deoxycytidine. 3D spheroid assay was performed to understand the influence of exosomes derived from cells overexpressing DNMTs on EC functions. Further, the exosomes were subjected to TMT labelled proteomics analysis followed by validation. 3D spheroid assay showed increase in the pro-angiogenic activity in response to exosomes derived from DNMT overexpressing cells which was impeded by inclusion of 5-aza-2'-deoxycytidine. Our results showed that exosome proteome and PTMs were significantly modulated and were associated with dysregulation of vascular homeostasis, metabolism, inflammation and endothelial cell functions. In vitro and in vivo validation showed elevated DNMT1 and TGF-β1 exosome proteins due to DNMT1 and DNMT3A overexpression, but not DNMT3B which was mitigated by 5-aza-2'-deoxycytidine indicating epigenetic regulation. Further, exosomes induced ED as evidenced by reduced expression of phospho-eNOS^ser1177. Our study unveils epigenetically regulated exosome proteins, aiding management of vascular complications.

Keywords: DNA methylation; Exosomes; Proteomics; Vascular complications.