Clin Res Cardiol (2022). https://doi.org/10.1007/s00392-022-02002-5

Hyperhomocysteinemia potentiates monocyte activation through the methylation-dependent dysregulation of PTEN phosphatase function

M. Dorenkamp1, D. Semo1, L. Miao1, S. Koch1, H. Reinecke1, R. Godfrey1

1Klinik für Kardiologie I: Koronare Herzkrankheit, Herzinsuffizienz und Angiologie, Universitätsklinikum Münster, Münster;

Purpose: Activation of monocytes imparts a pro-migratory and pro-adhesive phenotype which contributes to the initiation and progression of atherosclerotic plaques. Therefore, uncovering mechanisms of how monocyte migration and adhesion phenotype get enhanced is essential to find strategies to reduce atherosclerosis. Although elevated levels of homocysteine (HCY) are known as an independent risk factor for the development of atherosclerosis, the specific impact of HCY on monocyte function is not characterized yet and was addressed in the current study. Methods: Primary human monocytes were isolated from peripheral blood through the immuno-magnetic method. Monocytes were incubated with a clinically relevant dose of 400 µM HCY for 24 hours. Transwell migration assays were used to study monocyte migration. Adhesion to inflamed endothelium was studied under static and physiological flow conditions. Western Blot, RT-qPCR and FACS were performed to quantify the expression of relevant molecules. To decrypt the epigenetic regulation of HCY-induced PTEN function, 5-azacytidine was used to inhibit DNA methyltransferase 1 and SF1670 to inhibit PTEN. Results: Incubation of monocytes with HCY induced enhanced chemokinesis (1.5-fold, p=0.006) and accelerated chemotaxis to pro-inflammatory chemokine MCP-1 (1.32-fold, p=0.02) and fMLP (1.61-fold, p=0.003), indicating a pro-migratory phenotype. Moreover, HCY-treated monocytes revealed significantly augmented adhesion to inflamed endothelial cells (HUVEC). This pro-adhesive switch could be observed under static (1.68-fold, p=0.01) and physiological flow conditions (4.1-fold, p=0.001). Complementing these functional data, we detected elevated expression of CD11a on HCY-treated monocytes (1.24-fold, p=0.03). Mechanistically, we found downregulation of the expression and activity of the PTEN phosphatase after HCY-treatment (-36%, p<0.001) which lead to enhanced AKT activation at serine 473 (2.1-fold, p=0.02). In line with that, the application of PTEN inhibitor alone was sufficient to recapitulate aggravated monocyte activation (1.9-fold, p=0.002). Based on the possibility that HCY influences the methylation status of PTEN and thereby regulates its expression, we inhibited the DNA methyltransferase 1 (DNMT1) with 5-azacytidine (AZA). Coherently, co-treatment of monocytes with AZA could reverse HCY-induced impairment of PTEN expression (p=0.003). In line with that, DNMT1 inhibition was sufficient to reverse the aberrant migration (p=0.02) and adhesion (p=0.02) of HCY-treated monocytes completely. Finally, HCY-induced pro-migratory and pro-adhesive phenotype was also rescued by co-treatment with the cofactors (30µM VitaminB12 and 3µM folic acid) responsible for homocysteine to methionine catabolism. Aptly, normalized PTEN expression despite HCY-treatment was detected in these samples (p=0.002). Conclusions: Our results reveal a basis for a hitherto unknown pro-atherosclerotic activation of monocytes under hyperhomocysteinemic conditions. Furthermore, we decrypt the associated signalling pathway aberration induced by HCY, which is centred on the methylation-dependent inactivation of PTEN phosphatase. In addition, we identified the counteracting strategies to reset monocyte activation by targeting the PTEN methylator, DNMT1 or aiding HCY catabolism through VitaminB12 and folic acid. These data provide a basis for potential new approaches to attenuate monocyte activation in hyperhomocysteinemic conditions.

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