Phospholipid oxidation and its products are increasingly believed to be associated with diseases. Oxidative cleavage of phospholipids containing docosahexaenoic acid produces reactive electrophilic 4-hydroxy-7-oxohept-5-enoates and 4-keto-7-oxohept-5-enoates. The former one can convert the primary amino group of protein lysyl residues into 2-(ω-carboxyethyl)pyrrole (CEP) derivatives, while the latter one can generate 4-oxo
-heptanedioic amide (OHdiA) derivatives. Arachidonyl acid containing phospholipids and Linoleic acid containing phospholipids undergo the same transformations to generate
2-(ω-carboxypropyl)pyrrole derivatives (CPP), 2-(ω-carboxyheptyl)pyrrole (CHP) and 2-pentylpyrrole derivatives (PP). For the first time, OHdiA derivatives of proteins, a dipeptide, and an ethanolamine phospholipid was synthesized, characterized and found to be angiogenic. Anti-OHdiA polyclonal antibodies are generated and purified. ELISA experiments confirm that those antibodies are specific to the OHdiA epitopes with little cross-reaction from CEPs, CPPs, and CHPs. CEPs and OHdiAs are generated during inflammation and wound healing and accumulate at high levels in aging tissues. The CEP-driven and OHdiA-driven angiogenesis may be different from other proangiogenic growth factors, including VEGF, providing an attractive new therapeutic target, especially in cancers resistant to anti-VEGF therapy and as an adjunct to anti-VEGF therapy for AMD.
To further investigate the influence of carboxyalkyl chain length in carboxyalkyl pyrrole compounds, three different compounds other than CEP, CPP and CHP were also synthesized and characterized. A new and effective synthesis of CAP is described which facilitates scale up because intermediates are crystalline. This improved synthesis facilitates their use as agonists to test their receptor-mediated responses, and as standards to measure levels of them in vivo.
The CEP levels of both Melanoma patients and healthy controls were measured by the ELISA assay using CEP-HSA as standard. It was found that there was no significant difference between the two groups. Similarly, five melanoma tumor-injected WT-WT mice, five melanoma tumor-injected WT-TLR2 mice, seven melanoma tumor-injected TLR2-WT mice, three CEP mAb-injected plus wound assay mice and two nonspecific IgG2a-injected plus wound assay mice were measured for their CEP protein adducts levels by competitive ELISA, and no significant difference was found in CEP levels among different mice.