Lipid peroxidative products impact a list of human disorders, such as diabetes, cancer, cardiovascular disease, neurological disorders, and some inflammatory diseases. Lipid peroxidative products, such as aldehydes, act as biomarkers to indicate some disorder processes such as Parkinson’s and Alzheimer’s. Deeper study on lipid peroxidative products would be more important to identify novel signaling pathways in various human disorders, possible abnormal biomarkers, and to find novel therapeutic approaches. IsoF and IsoP are synthesized under comparable conditions. Higher oxygen intensity favors IsoF production, however, it counts against the IsoP formation. Thus, oxygen tension decides the ratio of IsoP/IsoF , which could be applied as an indicator of brain peroxidation  and cellular oxidative stress status . However, other pathological properties of the IsoF are still uncovered. Human disorders with abnormal angiogenesis, including atherosclerosis , cancer [7, 8], diabetes , retinopathy  is closely associated with oxidative stress and lipid peroxidation. In those conditions IsoF is advantageous to be produced. To investigate the pathological property of the novel found lipid peroxidative product, IsoF, we performed this project. In this study, the pro-angiogenic activity of the IsoF was verified by endothelial cellular proliferation and migration assays in the RBECs. Experiments to rescue the staurosporine-induced cellular apoptosis revealed the endothelial cellular protective potential of the IsoF. Induction of calcium release and ERK1/2 activation revealed the signaling manner of IsoF. Finally, IsoF clearly induced TNFα, Tie2, and VEGF-A gene up-regulation. Those gene alterations may be involved in the pathological process of IsoF-induced abnormal angiogenesis. These data firstly reveal the pro-angiogenic potential of IsoF. The findings of this study may open a door to investigate the pathogenic activity of the IsoF in other abnormal angiogenic disorders.
IsoP, as another kind of lipid product which was formed through a very similar pathway to IsoF, reveal an activity on vascular regulation. For example, PGE2 and PGI2 act as arterial vasodilation factors [22, 23]. Different PGs isoforms present different regulatory effects on angiogenesis as well. High expression of aldo-keto reductase 1C3 promotes the growth of skin squamous cell carcinoma via inhibition of PGD2, indicating that PGD2 has an anti-angiogenic potential . However,PGF2a is increased and acts as a pro-angiogenic factor in sarcoma . Decrease of PGE2 is associated with angiogenesis and tumor spread in fibroblast tumor . Here, we showed that IsoF present a pro-angiogenic potential. Other activities concerning on vascular regulation by IsoF need to be addressed further.
In this study, we found that 1µM IsoF created maximal biological response to induce RBEC’s proliferation. Fessel JP et al (1) have measured the concentration of IsoF in rat plasma and indicated that the IsoF concentration of rat plasma is 334 ng/ml, that is equal to 0.571µM (based on the calculation with 585 as the molecular weight of IsoF). Thus, it is reasonable that doubling of this level (1µM) of the IsoF could be enough to induce a pathological response. However, this data only shows the response dose for RBEC (rat brain vascular endothelium). This dose may not be optimal for other endothelium because endothelium has a well-known district specificity as well as different tissues having different physiological levels of IsoF. For example, the IsoF’s level in human plasma is 71ng/ml (0.121µM)(1). We recommend doing a dose response assay of the IsoF for different endothelium when performing experiments in different endothelium.
Although the sequence homology is not the same between classes, all GPCRs obtain a universal structure and similar signal transduction mechanism. Particularly, the C-tail of GPCR usually contains either serine or threonine residues. Once they are phosphorylated, the intracellular structure will bind scaffolding proteins called β-arrestins  to interrupt G-protein coupling and enlist other proteins, resulting in signaling complexes activation, including ERK pathway activation, or receptor endocytosis. Thus, IsoF-induced cellular ERK1/2 phosphorylation (Fig. 5) hints that the receptor of the IsoF might be a GPCR.
In phosphatidylinositol signal pathway of GPCR, ligand binds with Gq to activate phospholipase Cs located on the plasma membrane. The lipase hydrolyzes phosphatidylinositol 4,5-bisphosphate into either diacylglycerol or inositol 1,4,5-trisphosphate (IP3). The IP3 associates with the IP3 receptor in the membrane of the smooth endoplasmic reticulum and mitochondria to open Ca2+ channels. On the other hand, ligand-binding with Gβγ trigger various ion channels, including N-type voltage-gated Ca2+ channels to induce calcium release. . In this study, the Ca2+ release by the IsoF (Fig. 4) suggests that the receptor of IsoF could be either a Gq or a Gβγ type GPCR. However, whether a GPCR is the receptor of IsoF need further detailed investigation.
In our previous study, TNFα and Tie2 play a crucial role in the protease-activated receptor 2-induced endothelial angiogenesis via ERK1/2 signaling. Interestingly, here we showed that IsoF not only up-regulates both Tie2 and TNFα, but also activates ERK1/2 signaling, suggesting that the ERK1/2-TNFα-Tie2 axis participates in the pro-angiogenic mechanism of the IsoF. Further, VEGF-A up-regulation by IsoF may be one of the pro-angiogenic mechanism of the IsoF as well.
In this study, we performed Coverslip-removing migration assay(15) to address the pro-angiogenic property of the IsoF. This method is created based on the Wound and Healing migration assay, but it overcomes a weak point of Wound and Healing migration assay where the migration borders are manually defined. This novel method applies a 10mm diameter coverslip that has a clear, constant, and standard borders. In addition, it employs MTT stain method to figure out the migrated cells. This way, the assay is getting easy and mechanical operation without any infection from subjective awareness of operators. In the contrary, manually drawing the migrated area is performed in the Wound and Healing migration assay, which could be impacted by subjective awareness of operators. The operator can easily get bored and fatigued because of the repeated manual works. Due to the 48–72 hours assay processes in Coverslip-removing migration assay, to overcome the impact of results caused by cellular proliferation, mitomicine D is employed to inhibit the cellular proliferation. Indeed, Wound and Healing migration assay has this problem as well.
The limitation of this study is that it did not define whether interfering ERK1/2 affects Tie2, TNFα, or VEGF-A gene/protein expression, to get the direct evidence that the ERK1/2-TNFα-Tie2-VEGF-A axis participate in the IsoF-induced angiogenesis. In addition, lipid peroxidation is closely associated with inflammation. Whether the pro-angiogenic activity of the IsoF is caused due to the promotion of inflammatory reaction needs to be further addressed, because inflammation response is always companied with angiogenic process. Indeed, addressing those questions is already scheduled in our next research plan.