OBJECTIVE: This study aimed to elucidate the polarization of M1 and M2 macrophage from CAD patients as well as to investigate the expression of MerTK in these macrophage phenotypes.
METHODS: A total of 14 (n) CAD patients were recruited and subsequently grouped into "no apparent CAD", "non-obstructive CAD" and "obstructive CAD" according to the degree of stenosis. Thirty ml of venous blood was withdrawn to obtain monocyte from the patients. The M1 macrophage was generated by treating the monocyte with GMCSF, LPS and IFN-γ while MCSF, IL-4 and IL-13 were employed to differentiate monocyte into M2 macrophage. After 7 days of polarization, analysis of cell surface differentiation markers (CD86+/CD80+ for M1 and CD206+/CD200R+ for M2) and measurement of MerTK expression were performed using flow cytometry.
RESULTS: Both M1 and M2 macrophage expressed similar level of CD86, CD80 and CD206 in all groups of CAD patients. MerTK expression in no apparent CAD patients was significantly higher in M2 macrophage compared to M1 macrophage [12.58 ± 4.40 vs. 6.58 ± 1.37, p = 0.040].
CONCLUSION: Differential polarization of macrophage into M1 and M2 was highly dynamic and can be varied due to the microenvironment stimuli in atherosclerotic plaque. Besides, higher expression of MerTK in patients with the least coronary obstructive suggest its vital involvement in efferocytosis.
METHODS: Transfection of ANXA1 siRNA was conducted to downregulate ANXA1 expression in Jurkat, K562 and U937 cells. Apoptosis and cell cycle assays were conducted using flow cytometry. Western blot was performed to evaluate ANXA1, caspases and Bcl-2 proteins expression. Phagocytosis was determined using hematoxylin and eosin staining.
RESULTS: The expression of ANXA1 after the knockdown was significantly downregulated in all cell lines. Genistein significantly induced apoptosis associated with an upregulation of procaspase-3, -9, and - 1 in Jurkat cells. The Bcl-2 expression showed no significant difference in Jurkat, K562 and U937 cells. Treatment with phytoestrogens increased procaspase-1 expression in Jurkat and U937 cells while no changes were detected in K562 cells. Flow cytometry analysis demonstrated that after ANXA1 knockdown, coumestrol and genistein caused cell cycle arrest at G2/M phase in selected type of cells. The percentage of phagocytosis and phagocytosis index increased after the treatment with phytoestrogens in all cell lines.
CONCLUSION: Phytoestrogens induced cell death in ANXA1-knockdown leukemia cells, mediated by Annexin A1 proteins. Graphical abstract.