Angiogenesis / Oxidized phospholipids stimulate production of stem cell factor via NRF2dependent mechanisms

Receptor tyrosine kinase c-Kit and its ligand stem cell factor (SCF) regulate resident vascular wall cells and recruit circulating progenitors. We tested whether SCF may be induced by oxidized palmitoyl-arachidonoyl-phosphatidylcholine (OxPAPC) known to accumulate in atherosclerotic vessels. Gene expression analysis demonstrated OxPAPC-induced upregulation of SCF mRNA and protein in different types of endothelial cells (ECs). Elevated levels of SCF mRNA were observed in aortas of ApoE/ knockout mice. ECs produced biologically active SCF because conditioned medium from OxPAPC-treated cells stimulated activation (phosphorylation) of c-Kit in naïve ECs. Induction of SCF by OxPAPC was inhibited by knocking down transcription factor NRF2. Inhibition or stimulation of NRF2 by pharmacological or molecular tools induced corresponding changes in SCF expression. Finally, we observed decreased levels of SCF mRNA in aortas of NRF2 knockout mice. We characterize OxPLs as a novel pathology-associated stimulus inducing expression of SCF in endothelial cells. Furthermore, our data point to transcription factor NRF2 as a major mediator of OxPL-induced upregulation of SCF. This mechanism may represent one of the facets of pleiotropic action of NRF2 in vascular wall.(VLID)473560

Unbiased Identification of Proteins Covalently Modified by Complex Mixtures of Peroxidized Lipids Using a Combination of Electrophoretic Mobility Band Shift with Mass Spectrometry

Covalent modification of functionally important cell proteins by lipid oxidation products (LOPs) is a known mechanism initiating pathological consequences of oxidative stress. Identification of new proteins covalently modified by electrophilic lipids can be performed by a combination of chemical, immunological, and mass spectrometry-based methods, but requires prior knowledge either on the exact molecular structure of LOPs (e.g., 4-hydroxynonenal) or candidate protein targets. However, under the conditions of oxidative stress in vivo, a complex mixture of proteins (e.g., cytosolic proteome) reacts with a complex mixture of LOPs. Here we describe a method for detection of lipid-modified proteins that does not require an a priori knowledge on the chemical structure of LOPs or identity of target proteins. The method is based on the change of electrophoretic mobility of lipid-modified proteins, which is induced by conformational changes and cross-linking with other proteins. Abnormally migrating proteins are detected by mass spectrometry-based protein peptide sequencing. We applied this method to study effects of oxidized palmitoyl-arachidonoyl-phosphatidylcholine (OxPAPC) on endothelial cells. Several known, but also many new, OxPAPC-binding proteins were identified. We expect that this technically relatively simple method can be widely applied for label-free analysis of lipid-protein interactions in complex protein samples treated with different LOPs

Oxidized phospholipids regulate expression of ATF4 and VEGF in endothelial cells via NRF2-dependent mechanism : novel point of convergence between electrophilic and unfolded protein stress pathways

The ATF4 arm of the unfolded protein response is increasingly recognized for its relevance to pathology, and in particular to angiogenic reactions. Oxidized phospholipids (OxPLs), known to accumulate in atherosclerotic vessels, were shown to upregulate vascular endothelial growth factor (VEGF) and induce angiogenesis via an ATF4-dependent mechanism. In this study, we analyzed the mechanism of ATF4 upregulation by OxPLs and more specifically the involvement of NRF2, the major transcriptional mediator of electrophilic stress response

Analysis of fragmented oxidized phosphatidylcholines in human plasma using mass spectrometry: Comparison with immune assays.

Circulating oxidized phospholipids are increasingly recognized as biomarkers of atherosclerosis. Clinical association studies have been mainly performed using an immune assay based on monoclonal antibody E06, which recognizes a variety of molecular species of oxidized phosphatidylcholine (OxPC) in lipoproteins, cell membranes or covalently bound to plasma proteins. Accumulating evidence shows that individual molecular species of OxPC demonstrate different biological activities and have different half-life times. Therefore, it is likely that certain molecular species can be associated with pathology more strongly than others. This hypothesis can only be tested using LC-MS/MS allowing quantification of individual molecular species of OxPCs. In order to ensure that laborious LC-MS/MS methods do not simply replicate the results of a technically simpler E06-OxPCs assay, we have performed relative quantification of 8 truncated molecular species of OxPCs in plasma of 132 probands and compared the data with the results of the E06-OxPCs and OxLDL assays. We have found a strong correlation between individual molecular species of OxPCs but only a weak correlation of LC-MS/MS-OxPCs data with the E06-OxPCs assay and no correlation with the OxLDL assay. Furthermore, in contrast to the results of E06-OxPCs or OxLDL assays, 7 out of 8 OxPC species were associated with hypertension. The data suggest that the results of the LC-MS/MS-OxPCs assay do not replicate the results of two ELISA-based lipid oxidation tests and therefore may produce additional diagnostic information. These findings necessitate development of simplified mass spectrometric procedures for high-throughput and affordable analysis of selected molecular species of OxPCs