Under oxidative stress, phospholipids (PLs) containing polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA), arachidonic acid (AA) and linoleic acid (LA), undergo oxidation and truncation to generate a multitude of reactive aldehydes including ¿-hydroxy-a,ß-unsaturated aldehyde-PLs such as 4-hydroxy-7-oxo-hept-5-enoyl phospholipids (HOHA-PLs), 5-hydroxy-8-oxooct-6-enoyl phospholipids (HOOA-PLs) and 9-hydroxy-12-oxododec-10-enoyl phospholipids (HODA-PLs), and the corresponding ¿-oxo-a,ß- unsaturated aldehyde-PLs such as 4-keto-7-oxohept-5-enoyl phospholipids (KOHA-PLs), 5-keto-8-oxooct-6-enoyl phospholipids (KOOA-PLs) and 9-keto-12-oxododec-10-enoyl phospholipids (KODA-PLs) from DHA-PLs, AA-PLs and LA-PLs, respectively. Recently, HOHA-PLs and HOOA-PLs were found to undergo spontaneous deacylation via an intramolecular transesterification mechanism to generate the corresponding HOHA-lactone and HOOA-lactone under physiological conditions. More recently, HOHA-lactone was found to be a precursor for the generation of 2-(¿-carboxyethyl)pyrrole (CEP) derivatives of proteins and ethanolamine phospholipids. The present thesis documents that CEP derivatives accumulate in RPE cells either by exogenous addition of HOHA-lactone or by HOHA-lactone generated endogenously in RPE cells exposed to oxidative or inflammatory insults. In addition, HOHA-lactone was found to exhibit hermetic effects on RPE cellular activity: low levels of HOHA-lactone induce proliferation and cell growth of RPEs while high levels of HOHA-lactone induce apoptosis. At high concentrations, HOHA-lactone induces RPE cell death by activating caspase-3 apoptotic signaling, where p53 may be involved in this apoptotic process, indicated by the induction and phosphorylation of p53, nuclear accumulation of p53, and the degradation of MDM2. Moreover, it has also been confirmed that HOHA-lactone can readily diffuse into RPE cells where it is detoxified by conjugation with GSH in the cytosol, forming an aldehyde adduct HOHA-lactone-GSH (=O) and an alcohol HOHA-lactone-GSH (-OH), which are then transported from cytosol to extracellular medium. At low levels, HOHA-lactone induces the secretion of VEGF in ARPE-19 cells, which correlates well with an increase in intracellular ROS and a decrease in intracellular GSH. VEGF secreted into the media showed angiogenic properties as indicated by increased migration and tube formation of HUVECs in matrigel when grown in media from ARPE-19 cells treated with HOHA-lactone. Wound healing and tube formation assays showed that HOHA-lactone-GSH conjugates have pro-angiogenic effects. The results of these studies show for the first time, that HOHA-lactone causes angiogenesis in HUVECs by more than one molecular pathway. In an indirect mechanism, HOHA-lactone induces the secretion of VEGF by RPE cells and VEGF can promote angiogenesis. In two other molecular mechanisms HOHA-lactone reacts with GSH or with the primary amino groups of biomolecules to form the corresponding GSH conjugates or CEPs that, in turn, also
promote angiogenesis. A fourth angiogenesis pathway induced by HOHA-lactone may involve the formation of HOHA-lactone-GSH (=O), which then produces CEP derivatives by reaction with primary amino groups of biomolecules and CEP then promotes angiogenesis.
In analogy with the generation of CEP from HOHA-lactone, HOOA-lactone was found to serve as a precursor of CPP modifications both in vitro and in vivo. In analogy with the biological activity of HOHA-lactone, HOOA-lactone was demonstrated to be capable of inducing intracellular oxidative stress and of causing apoptosis of ARPE-19 cells at high concentrations via activation of caspase-3. In view of the toxic potential of HOOA-lactone to ARPE-19 cells, the detoxification of HOOA-lactone in this cell type was also studied and the results showed that HOOA-lactone can easily diffuse through the cell membrane into ARPE-19 cells where it is detoxified by conjugation with GSH to form HOOA-lactone-GSH (=O) and HOOA-lactone-GSH (-OH).
Recently, KODA-PLs, one of the LPO products from LA-PLs, were found to form the 4-ketoamide derivatives of the Lys residues of proteins. KOHA-PLs were expected to modify biomolecules to give similar stable 4-ketoamide adducts: 4-oxo-heptanedioic amide (OHdiA) derivatives. Using LC-MS/MS, KOHA-PL was found not only modify the primary e-amino group of protein Lys residues but also those of the ethanolamine headgroup of ethanolamine phospholipids (EPs) to produce OHdiA derivatives in vitro. In addition, OHdiA derivatives were also detected in vivo, evident by the presence of OHdiA derivatives in blood from both SCD patients and healthy individuals. Moreover, OHdiA adducts were shown to have pro-angiogenic effects and this OHdiA-driven angiogenesis was shown to be TLR2 dependent similar to angiogenesis promoted by CEP but different from VEGF promoted angiogenesis. Furthermore, anti-OHdiA antibody was found to exhibit significant cross-reactivity with CEP-HSA while anti-CEP antibody showed high structural specificity and did not show cross-reactivity with OHdiA-HSA.
While ¿-hydroxy-a,ß-unsaturated aldehyde-PLs like HOHA-PLs, HOOA-PLs and HODA-PLs were previously shown to react with primary amino groups of proteins to produce the corresponding carboxyethylpyrroles (CEPs), carboxypropylpyrroles (CPPs) and carboxyheptylpyrroles (CHPs), respectively, the extent of the formation of analogous derivatives of the primary amino groups of EPs in vivo and the biological activities of those modified EPs remain poorly characterized. In the current study, an LC-MS/MS assay that allows simultaneous quantification of global CAP- and PP-modified EPs was developed by measuring levels of CAP-and PP-ETN released through hydrolysis of lipid extracts under catalysis by PLD from Strdptomyces chromofuscus. The presence of CAP-EPs and PP-EPs in vivo was established. A small pilot study revealed that levels of CAP-EPs and PP-EPs, except CHP-EPs, are significantly elevated in plasma samples from clinical SCD patients compared to those of hospitalized SCD patients.