1University Hospital Münster Vascular Signalling, Molecular Cardiology, Department of Cardiology I- Coronary and Peripheral Vascular Disease, Heart Failure Münster, Deutschland; 2German Institute of Human Nutrition Potsdam-Rehbruecke (DIfE) Department of Molecular Toxicology Nuthetal, Deutschland
Purpose: Diabetes mellitus, one of the major cardiovascular risk factors, induces monocytic dysfunction by inducing oxidative stress. This dysfunction accelerates the accumulation of monocytic cells in plaque regions, thereby driving atherosclerosis. The underlying molecular mechanisms of the diabetes-oxidative stress pathways are incompletely understood. In our study, we focused on the role of 12/15 lipoxygenase (LOX) in monocyte dysfunction in diabetes mellitus.
Methods: Human monocytes from non-T2DM or T2DM patients and healthy donors were isolated from peripheral blood using negative selection methods. Monocytes from healthy controls were preconditioned for 48 hours in either normoglycemia (NG) or hyperglycemia (HG). The lipid peroxidation end product malondialdehyde (MDA) was detected by HPLC.
Monocytic adhesion molecule expression was measured by FACS and RT-QPCR. In addition, pharmacological inhibition of LOX using the inhibitor AA861 and inhibition of PTP1B (protein tyrosine phosphatase 1B) in hyperglycemia were performed. Elevated lipid peroxide conditions were mimicked using the lipid peroxidation by product 15-HPETE (15-S-hydroperoxy-5Z,8Z,11Z,13E-eicosatetraenoic acid). Chemokine release was measured by ELISA and gene expression by 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.
Results: LOX genes are upregulated in T2DM and HG monocytes. T2DM or HG preconditioning increased MDA. Additional treatment with the lipoxygenase inhibitor AA861 significantly reduced HG-induced MDA increase. PTP1B tyrosine phosphatase appears in its oxidized activated form in HG in a lipid peroxide-dependent manner. The impaired catalytic activity of PTP1B could be partially rescued by lipid peroxide scavenging.
HG significantly induced the expression of adhesion molecules. We detected an upregulation of CD18, CD11a, CD11b and CD61 on the monocytic cell surface and also demonstrated an increase in adhesion molecule genes. Additional LOX inhibition by AA861 in the presence of HG quenched the elevated adhesion molecules. Mimicking enhanced lipid peroxide conditions with 15-HPETE increased adhesion molecules comparable to HG; pharmacological inhibition of PTP1B reversed 15-HPETE-induced adhesion molecule expression.
We also demonstrated a role for LOX in inflammation in monocytic cells. In the presence of 15-HPETE, IL6 and IL8 secretion and gene expression are increased, which can be reversed by the blockade of PTP1B function.
Conclusions: Our study provides novel evidence for LOX gene amplification in T2DM and HG conditions. This leads to increased lipid peroxide formation in monocytic cells, which directly affects the expression of adhesion molecules. Furthermore, the present results suggest that HG-induced lipid peroxide accumulation impairs the catalytic activity of PTP1B, which then accelerates an inflammatory phenotype and increases monocyte adhesion capacity. In conclusion, our study provides data on the role of LOX-mediated PTP1B oxidation for HG-induced adhesion molecule expression and inflammatory phenotype of monocytic cells. Rescue of lipid peroxide generation in HG may have beneficial effects on impaired monocytic function in diabetes. However, further studies are needed to understand the relationship between LOX and PTP1B signalling pathways.