Highly multiplexed quantitative proteomic and phosphoproteomic in vascular smooth muscle cell dedifferentiation

Restenosis, re-narrowing of arterial lumen following intervention for cardiovascular disease, remains a major issue limiting the long-term therapeutic efficacy of treatment. The signaling molecules, TGF beta (transforming growth factor-beta) and Smad3, play important roles in vascular restenosis, but very little is yet known about the down-stream dynamics in global protein expression and phosphorylation. Here, we develop a highly multiplexed quantitative proteomic and phosphoproteomic strategy employing 12-plex N,N-dimethyl leucine (DiLeu) isobaric tags and The DiLeu Tool software to globally assess protein expression and phosphorylation changes in smooth muscle cells (SMCs) treated with TGF beta/Smad3 and/or SDF-1 alpha (stromal cell-derived factor). A total of 4086 proteins were quantified in the combined dataset of proteome and phosphoproteome across 12-plex DiLeu-labeled SMC samples. 2317 localized phosphorylation sites were quantified, corresponding to 1193 phosphoproteins. TGFb/Smad3 induced upregulation of 40 phosphosites and down-regulation of 50 phosphosites, and TGF beta/Smad3-specific SDF-1 alpha exclusively facilitated up-regulation of 27 phosphosites and down-regulation of 47 phosphosites. TGF beta/Smad3 inhibited the expression of contractile-associated proteins including smooth muscle myosin heavy chain, calponin, cardiac muscle alpha-actin, and smooth muscle protein 22 alpha. Gene ontology and pathway enrichment analysis revealed that elevated TGFb/Smad3 activated cell proliferation and TGF beta signaling pathway, sequentially stimulating phosphorylation of CXCR4 (C-X-C chemokine receptor 4). SDF-1 alpha/CXCR4 activated extracellular signal-regulating kinase signaling pathway and facilitated the expression of synthetic marker, osteopontin, which was validated through targeted analysis. These findings provide new insights into the mechanisms of TGF beta regulated SMC dedifferentiation, as well as new avenues for designing effective therapeutics for vascular disease. (C) 2020 Elsevier B.V. All rights reserved.