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    • There are two major isoforms of LO in

    2023-08-22

    There are two major isoforms of 12/15-LO in mammalian, 12 and 15- LO, with different cellular distributions. To determine the relative contributions of these isoforms towards glucose-induced monocyte-endothelial interactions, the distribution of these isoforms in the two cell types involved in leukostasis; endothelial and monocytes, was determined. An intriguing and important finding described here is the demonstration that 12-LO and 15-LO both are found in retinal microvascular endothelial Sulindac while they were hardly detected in circulating human monocytes. These results are in agreement with prior studies showing a notable 15-LO expression in HRECs [43], but not in agreement with other studies which demonstrated undetectable 15-LO mRNA in basal or stimulated human aortic or umbilical endothelial cells [28,44]. The discrepancy between those results and our findings may be due to differences in endothelial properties in large vessels, such as aortic artery or umbilical vein, versus microvessels present in the retina [45]. This differential expression of 12/15-LO might explain contradictory findings regarding the contribution of 12/15-LO to vascular dysfunction found in various pathological conditions. For instance, it has been verified in three different mouse models (apoE, LDL-R, and apobec-1/LDL-R deficiency) by at least three research groups the role of 12/15-LO expressing bone marrow–derived cells (e.g. macrophages) in preference to macrovascular endothelial cells in initiating pro-atherogenic inflammatory response [46]. On the contrary, our results suggest a role for 12/15-LO expressing retinal microvascular endothelial cells, and not monocytic/macrophagic cells, in mediating inflammatory responses seen in early DR. Several potential mechanisms for the regulation of ICAM-1 expression by 12/15-LO can be considered. First, the 12/15-LO product, 12- or 15-HETE, which we have recently demonstrated to be increased in high glucose-treated HRECs [22], has shown to mediate the ICAM-1 induction through RhoA, protein kinase C (PKC), and NF-κB activation pathways [47]. Second, Bolick and colleagues postulated, in the same study [47], that the 12/15-LO product can activate a G protein–coupled receptor (GPCR) to initiate these events. Criteria for making this assumption were based on the activation of the small GTPase by 12/15-LO product as well as the reduction in ICAM-1 expression in response to blocking the G proteins G12 and G13 but not Gi in the endothelium [47]. The isolation of this receptor has proven later to be GPCR-31 for 12-HETE [48]. Nevertheless, no research group has “adopted” one of the orphan members as a 15-HETE receptor. Furthermore, whether 15-HETE can activate the same receptor as does 12-HETE has yet to be clarified. Others have demonstrated the ability of 12/15-LO products to bind an intracellular receptor that associates with other proteins, which is more reminiscent of nuclear receptor signaling [49,50]. Such observations leave an open question with regard to 12/15-LO products: whether their cellular effects are mediated by a secreted product binding to a receptor or whether they act as second messengers within the cell. Although our studies do not directly address this issue, previous studies from our group showed that exogenous addition of 12/15-LO products in nano-molar concentrations was sufficient to stimulate monocyte binding [22]. Yet, the possibility that 12/15-LO products may function intracellularly cannot be ruled out because it is generally recognized that lipophilic molecules can cross the cell membrane via passive diffusion. In any case, understanding the pathway of HREC activation under hyperglycemia from 12/15-LO induction to monocyte adhesion would be a substantial step forward in translating eicosanoid biology to therapeutics in DR. In summary, our data have shown that one mechanism by which glucose may mediate monocyte–endothelial cell interaction in the retinal endothelial cells is via the 12/15-LO pathway Furthermore, our current and previously published data [20,22] indicate a differential role of endothelial 12/15-LO versus the one in monocytic/macrophagic cells in mediating the inflammatory responses during DR. We, therefore, conclude that glucose promotes production of 12- or 15-HETE through retinal endothelial 12/15-LO rather than monocye/macrophage 12/15-LO, which results in autocrine activation of endothelial cells and induction of ICAM-1 on the surface of HRECs that would allow monocytes to firmly adhere to the endothelium (Fig. 6). Because the interaction between monocytes and the endothelium is a key early event in the development of DR, targeting this enzyme in endothelial cells may facilitate the development of more precisely targeted treatment strategies for DR.