Unbiased proteomics identifies plasminogen activator inhibitor-1 as a negative regulator of endothelial nitric oxide synthase

Abstract

Nitric oxide (NO) produced by endothelial nitric oxide synthase (eNOS) is a critical mediator of vascular function. eNOS is tightly regulated at various levels, including transcription, co- and posttranslational modifications, and by various protein–protein interactions. Using stable isotope labeling with amino acids in cell culture (SILAC) and mass spectrometry (MS), we identified several eNOS interactors, including the protein plasminogen activator inhibitor-1 (PAI-1). In cultured human umbilical vein endothelial cells (HUVECs), PAI-1 and eNOS colocalize and proximity ligation assays demonstrate a protein–protein interaction between PAI-1 and eNOS. Knockdown of PAI-1 or eNOS eliminates the proximity ligation assay (PLA) signal in endothelial cells. Overexpression of eNOS and HA-tagged PAI-1 in COS7 cells confirmed the colocalization observations in HUVECs. Furthermore, the source of intracellular PAI-1 interacting with eNOS was shown to be endocytosis derived. The interaction between PAI-1 and eNOS is a direct interaction as supported in experiments with purified proteins. Moreover, PAI-1 directly inhibits eNOS activity, reducing NO synthesis, and the knockdown or antagonism of PAI-1 increases NO bioavailability. Taken together, these findings place PAI-1 as a negative regulator of eNOS and disruptions in eNOS–PAI-1 binding promote increases in NO production and enhance vasodilation in vivo.

Deferoxamine accelerates endothelial progenitor cell senescence and compromises angiogenesis

“…More importantly, plasminogen activator inhibitor-1 (PAI-1) and p53 were significantly upregulated, while endothelial nitric oxide synthase (eNOS) was downregulated ( Figure 3A , 3B ). The DFO-altered protein expression patterns in the current study are highly mimicking the phenotypes induced by repeat replication as described [ 14 – 16 ].…”

“…We previously demonstrated that angiogenic activity was impaired in senescent EPCs [ 14 ]. Therefore, we examined the effects of DFO on EPC migration.…”

“…Accordingly, we examined the effects of DFO on mitochondrial respiratory efficiency in EPCs. The oxygen consumption rate (OCR) curve moved markedly downward in the DFO-treated group ( Figure 4A ), in a pattern highly resembling that of EPCs isolated from old animals [ 14 ]. Among the quantified respiratory parameters, the mitochondrial reserve capacity and the maximal respiratory capacity of the EPCs were significantly reduced upon DFO treatment ( Figure 4B ), indicating that DFO reduced the respiratory efficiency.…”

Polarized Proteins in Endothelium and Their Contribution to Function

“…This is likely a result of eNOS-lipid interactions at the MEJ microdomain and another example of how protein localization to microdomains impacts its function [56]. Regulators of eNOS, including hemoglobin alpha, CYB5R3, plasminogen activator inhibitor 1, and caveolin-1, also localize to the MEJ [124, 125, 181-183]. Hemoglobin alpha, a protein specifically expressed in the resistance artery endothelium [125], is also enriched within the MEJ, where it regulates eNOS [124, 125] and may influence the dominance of EDH-based vasodilation in these vessels [46].…”

Regulation of NOS expression in vascular diseases

“…In addition to CaM, several proteins like caveolin-1, cell division cycle 37 (Cdc37), C-terminal hsp70-interacting protein (CHIP), connexin 37 and 40 (Cx37/40), G-protein-coupled receptor (GPCR) kinase interactor-1 (GIT1), hemoglobin alpha (Hbα), heat shock protein 90 (Hsp90), integrin-linked kinase (ILK), NOS3 interacting protein (NOSIP) and NOS3 traffic inducer (NOSTRIN), proviral integration site for Moloney murine leukemia virus 1 (Pim1), prolyl isomerase (Pin) 1, and stromal cell-derived factor 2 (SDF2), have been shown to interact and regulate NOS3 (see [66] for a recent review). In addition, the plasminogen activator inhibitor-1 (PAI-1) interacts with NOS3 and inhibits its activity [67].…”