Abstract
Vascular endothelial cells (ECs) play a key role in physiology by controlling arterial contraction and relaxation, and molecular transport. EC dysfunction is associated with multiple pathologies. Here, we characterize the cellular and extracellular matrix (ECM) proteomes of primary human coronary artery ECs, from multiple donors, and oxidation/nitration products formed on these during cell culture, using liquid chromatography-mass spectrometry. In total ∼9900 proteins were identified in cells from 3 donors, with ∼7000 proteins per donor. Of these ∼5300 were consistently identified, indicating some heterogeneity across the donors, with age a possible cause. Multiple endogenous oxidation products were detected on both ECM and cellular proteins (and particularly endoplasmic reticulum species). In contrast, nitration was mostly detected on cell proteins and particularly cytoskeletal proteins, consistent with intracellular generation of nitrating agents, possibly from endothelial nitric oxide synthase (eNOS) or peroxidase enzymes. The modifications are ascribed to both physiological enzymatic activity (hydroxylation at proline/lysine; predominantly on ECM proteins and especially collagens) and the formation of reactive species (oxidation at tryptophan/tyrosine/histidine; nitration at tryptophan/tyrosine). The identified sites are present on a limited number of peptides (104 oxidized; 23 nitrated) from a modest number of proteins. A small number of proteins were detected with multiple modifications, consistent with these being selective and specific targets. Several nitrated peptides were consistently detected across all donors, and also in human smooth muscle cells suggesting that these are major targets in the vascular proteome. These data provide a ‘background’ proteome dataset for studies of endothelial dysfunction in disease.
| Originalsprog | Engelsk |
|---|---|
| Artikelnummer | 103524 |
| Tidsskrift | Redox Biology |
| Vol/bind | 81 |
| Antal sider | 14 |
| ISSN | 2213-2317 |
| DOI | |
| Status | Udgivet - 2025 |
Bibliografisk note
Funding Information:Multiple different processes are likely to contribute to the modifications detected in the current study. The oxidation of Pro and Lys is likely to arise from the enzymatic activity of multiple enzymes and is an intentional PTM required for biological function (e.g. at Pro to stabilize collagen triple helices, or at Lys for protein cross-linking and sugar attachment [38,40,41]). In contrast, the oxidative modifications at Trp, Tyr and His, and the nitration of Trp and Tyr, probably arise from unintended processes. Whilst multiple processes are known to generate oxidative modifications (reviewed [31,33]), the nitration is likely to arise from nitrating species (e.g. peroxynitrous acid/peroxynitrite, NO2., N2O3 [34,57]) arising from the enzymatic activity of eNOS, a constitutive enzyme of HCAEC, and critical for the biological vasorelaxation activity of these cells. Peroxynitrous acid/peroxynitrite is also a well-established oxidant, and may contribute to the oxidations detected at Trp, Tyr and His [34,57]. NO2. can also be formed from the reaction of nitrite (NO2\u2212) with activated peroxidase enzymes (e.g. myeloperoxidase (MPO) and peroxidasin (PXDN) [58\u201360]), and this radical can generate nitrated products from Tyr and Trp residues [34,57]. However, there are few data supporting the presence of MPO in primary endothelial cells ex vivo, although MPO can be detected in arterial wall ECs in vivo at sites of inflammation [61]. Thus, MPO is unlikely to be a significant source of nitration in the current study. In contrast, PXDN is expressed in ECs, and has been reported to be essential for EC survival [62]. This enzyme is therefore a potential candidate for the source of the observed nitration, in addition to peroxynitrous acid/peroxynitrite or N2O3. Further studies are required to examine these possibilities.This work was supported by the Novo Nordisk Foundation (Laureate Continuation Grant: NNF20SA0064214 to MJD) and the China Scholarship Council (PhD scholarship 201806270237 to Shuqi Xu). We thank Dr. Lasse G. Lorentzen for valuable scientific discussions.
Funding Information:
This work was supported by the Novo Nordisk Foundation (Laureate Continuation Grant: NNF20SA0064214 to MJD) and the China Scholarship Council (PhD scholarship 201806270237 to Shuqi Xu). We thank Dr. Lasse G. Lorentzen for valuable scientific discussions.
Publisher Copyright:
© 2025 The Authors