Purpose: Arteriogenesis is a process encompassing the growth of pre-existing collateral blood vessels to form functional arteries. Monocytes play a vital role in this process. Circulating monocytes are recruited to the sites of collateral growth where arteriogenesis is mediated through VEGFR1 signalling pathways, among others. However, diabetes mellitus causes monocyte dysfunction by interfering with the proper signal transduction downstream of VEGF–receptor-1 (VEGFR1). The impaired arteriogenesis seen in diabetic patients is thus linked to the reduced ability of monocytes to respond to VEGF stimulation. The underlying molecular mechanisms are incompletely understood. We focused on the role of 12/15-Lipoxygenase (LOX) in monocyte function.

Methods: Human monocytes from non-T2DM or T2DM patients were isolated from peripheral blood through gradient centrifugation and negative immunological magnetic isolation. Monocytic THP-1 cells and primary monocytes isolated from healthy donors were subjected to either normoglycaemia or hyperglycaemia for 7 days and 48 hours respectively.  Detection of lipid peroxidation end-product, malondialdehyde (MDA) was carried out using HPLC. The lipid hypdroperoxide accumulation was also directly detected using BODIPY-IAM using fluorescence microscopy. Protein tyrosine phosphatase 1B (PTP1B) activity was measured by dephosphorylation of phospho-peptide-based malachite green assay. Oxidation status of PTP1B was analysed through labelling the oxidized cysteine with biotin. Pharmacological inhibitors were used to inhibit LOX. To mimic hyper lipidperoxide conditions, lipid peroxidation by product 15-HPETE (15-S-hydroperoxy-5Z,8Z,11Z,13E-eicosatetraenoic acid)  or 4-Hydroxynonenal (HNE) was used. VEGF-A and PlGF-1-induced monocyte chemotaxis was assessed in the modified Boyden chamber assay.

Results: Hyperglycymia led to approximately 25% increase in malondialdehyde (MDA), which could be significantly blocked by a lipoxygenase inhibitor, AA861. Hyperglycemic monocytes and monocytes from T2DM patients revealed an upregulation of LOX genes. In hyperglycemia conditions, PTP1B tyrosine phosphatase was found to be oxidized in a lipid peroxide-dependent manner. The impaired catalytic activity of PTP1B could be partly rescued when the lipid peroxides are quenched, indicating a direct link to lipid peroxide-mediated PTP1B oxidation. Enhancing the accumulation of lipid hydroperoxides in normoglycemia by the exogenous addition of lipid peroxidation by-products 15-HPETE or HNE was sufficient to recapitulate the VEGF resistance phenotype. Inversely, the reduction of lipid peroxide accumulation in hyperglycemia using LOX inhibition improved.

Conclusions: Our results reveal that hyperglycemia and T2DM conditions induce the LOX gene expression resulting in enhanced lipid hydroperoxide formation in monocytes. The presented findings suggest that the hyperglycaemia-induced lipid peroxidate accumulation can impair the catalytic activity of PTP1B, which can influence the function of monocytes. Quenching of lipid peroxide generation can have beneficial effects on monocyte function in the diabetic environment.