Oxidative dysregulation of PTP1B via 12/15-Lipoxygenase drives monocyte dysfunction in hyperglycemia

Abstract Monocytes play a critical role in atherosclerosis by adhering to activated endothelial cells, a process driven by inflammatory and oxidative stress signals in diabetes mellitus(DM). Once adhered, monocytes transmigrate into the subendothelial space and contribute to plaque formation by perpetuating inflammation. The underlying molecular mechanisms of the diabetes-oxidative stress pathways are incompletely understood; therefore, we investigated the role of 12/15 lipoxygenase(LOX) in monocyte dysfunction in DM. The lipid peroxidation end product malondialdehyde(MDA) was detected by HPLC in primary human monocytes either obtained from peripheral blood of T2DM(Type 2 Diabetes mellitus) or non-T2DM patients using negative selection methods. Moreover, MDA was measured in monocytes from healthy controls, which were preconditioned in normoglycemia(NG) or hyperglycemia(HG). The lipid hypdroperoxide accumulation was measured using BODIPY-IAM fluorescence microscopy. LOX and PTP1B was inhibited using AA861 or TCS 401. Enhanced lipid peroxide environment was mimicked using the lipid peroxidation byproduct 15-HPETE. The adhesion molecule expression was evaluated by FACS and RT-qPCR. PTP1B activity was analyzed by a phospho-peptide-based malachite green dephosphorylation assay and oxidation status was measured by biotin labelling of the catalytic cysteine. Chemokine release was measured by ELISA and gene expression by RT-qPCR. In this study, an approximate 25% increase in malondialdehyde (MDA) in HG monocytes was demonstrated. The increase was significantly reversed by LOX inhibition. We detected enhanced expression of LOX transcripts in monocytes, which were preconditioned in HG or isolated from patients with T2DM. We also identified PTP1B oxidation and the subsequent increase in activation of monocytes in HG is regulated in a lipid-peroxide-dependent manner. The impairment of PTP1B catalytic activity was partially diminished by scavenging lipid peroxide. HG has been demonstrated to induce the upregulation of monocytic surface CD18 (70%, p-value=0.01), CD11b (twofold, p-value=0.0008) and CD61 (60%, p-value=0.02), as well as adhesion molecule genes. This could be reversed by both LOX and PTP1B inhibition. Moremore, monocytes treated with 15-HPETE displayed activation characteristics similar to that of HG monocytes. The presented findings demonstrate an induction of LOX gene expression in conditions of HG and T2DM resulting in an enhanced lipid hydroperoxide formation in monocytes. Consequently, this results in elevated levels of adhesion molecules. Furthermore, we suggest that the HG-induced lipid peroxidate accumulation impedes the catalytic activity of PTP1B. The latter plays a central role in monocytic function. These findings underscore the pivotal role of LOX-mediated and PTP1B-dependent lipid peroxidation in HG-driven monocyte dysfunction and suggest that targeting this pathway may offer a therapeutic strategy to mitigate vascular inflammation in T2DM.