Background: Atherosclerosis is a chronic inflammatory disease in which oxidative stress and macrophages play fundamental roles. Macrophages are heterogeneous cells in term of morphology and function and the prevalence of a specific morpho-phenotype may influence the progression or the regression of the plaque. Human coronary plaque macrophages are not easily obtainable and monocyte-derived macrophages (MDMs) are widely accepted as a good surrogate. Our previous study has reported that healthy subjects show two dominant MDM morphotypes, spindle and round, present in the same percentage. Aim: The aims of the study were to assess the biochemical and functional profile of MDMs obtained from CAD patients and to investigate the relationship between these characteristics and plaque features detected in vivo. Material and methods: ninety CAD patients and twenty-five healthy volunteers were enrolled. MDMs were obtained by culturing monocytes for 7 days in medium supplemented with 10% autologous serum. Transglutaminase2 (TG2), tissue factor (TF), nuclear factor erythroid 2–related factor 2 (Nrf2) and heme-oxygenase (HO-1) were determined by western blotting and immunofluorescence. The uptake of apoptotic Jurkat T cells was detected by flow cytometry and thrombin formation was assessed by thrombinoscope. Fatty acids composition and oxidative stress status, determined as the ratio between the reduced and oxidized forms of glutathione (GSH and GSSG, respectively) were analyzed by liquid-chromatography tandem mass spectrometry (LC-MS/MS). Plaque features were detected in vivo by optical coherence tomography (OCT). Results: MDMs obtained from CAD patients were characterized by the predominance of round cells. These cells exhibited a lower efferocytic capacity and higher capacity to generate thrombin in respect to those of healthy subjects, reflecting the low expression of TG2 and the enhance of TF levels. Moreover, MDMs of CAD patients showed a higher oxidative stress status, evidenced by the lower GSH/GSSG ratio, in respect to those of healthy subjects. Finally, we found a positive correlation between oxidative stress status and membrane fluidity (MFI) as evidenced by the ratio of C18:1 (oleic acid)/C18:2 (linoleic acid), as well as MFI and capacity to engulf apoptotic cells. Regarding OCT analysis, patients with a higher round MDMs prevalence exhibited more frequently a lipid rich plaque, a TCFA (thin cap fibroatheroma), a greater intra-plaque macrophage content, and a ruptured plaque, characteristics of vulnerable plaque. Furthermore, patients with high TF levels more frequently presented a intra-plaque macrophages accumulation, a ruptured plaque and a presence of thrombus. In addition, vulnerable plaque characteristics were associated with high HO-1 levels. Conclusions: MDMs of CAD patients present a pro-inflammatory and high thrombogenic profile in respect to those of healthy volunteers. The high oxidative stress present in these cells influences the membrane fluidity that in turn influences the efferocytic capability. This specific cellular profile detected in in vitro obtained MDMs is associated with the detection of high-risk and rupture-prone coronary plaques in vivo, at OCT investigation. Different MDM phenotypes might be novel diagnostic and therapeutic target able to counteract the progression of atherosclerosis.

BIOCHEMICAL AND FUNCTIONAL PROFILE OF SPONTANEOUSLY DIFFERENTIATED HUMAN MONOCYTE-DERIVED MACROPHAGES IN PATIENTS WITH ISCHEMIC HEART DISEASE / S. Fiorelli ; tutor: D. Caruso ; coordinatore: S. Sonnino. - : . DIPARTIMENTO DI SCIENZE FARMACOLOGICHE E BIOMOLECOLARI, 2018 Jan 25. ((30. ciclo, Anno Accademico 2017. [10.13130/s-fiorelli_phd2018-01-25].

BIOCHEMICAL AND FUNCTIONAL PROFILE OF SPONTANEOUSLY DIFFERENTIATED HUMAN MONOCYTE-DERIVED MACROPHAGES IN PATIENTS WITH ISCHEMIC HEART DISEASE

S. Fiorelli
2018-01-25

Abstract

Background: Atherosclerosis is a chronic inflammatory disease in which oxidative stress and macrophages play fundamental roles. Macrophages are heterogeneous cells in term of morphology and function and the prevalence of a specific morpho-phenotype may influence the progression or the regression of the plaque. Human coronary plaque macrophages are not easily obtainable and monocyte-derived macrophages (MDMs) are widely accepted as a good surrogate. Our previous study has reported that healthy subjects show two dominant MDM morphotypes, spindle and round, present in the same percentage. Aim: The aims of the study were to assess the biochemical and functional profile of MDMs obtained from CAD patients and to investigate the relationship between these characteristics and plaque features detected in vivo. Material and methods: ninety CAD patients and twenty-five healthy volunteers were enrolled. MDMs were obtained by culturing monocytes for 7 days in medium supplemented with 10% autologous serum. Transglutaminase2 (TG2), tissue factor (TF), nuclear factor erythroid 2–related factor 2 (Nrf2) and heme-oxygenase (HO-1) were determined by western blotting and immunofluorescence. The uptake of apoptotic Jurkat T cells was detected by flow cytometry and thrombin formation was assessed by thrombinoscope. Fatty acids composition and oxidative stress status, determined as the ratio between the reduced and oxidized forms of glutathione (GSH and GSSG, respectively) were analyzed by liquid-chromatography tandem mass spectrometry (LC-MS/MS). Plaque features were detected in vivo by optical coherence tomography (OCT). Results: MDMs obtained from CAD patients were characterized by the predominance of round cells. These cells exhibited a lower efferocytic capacity and higher capacity to generate thrombin in respect to those of healthy subjects, reflecting the low expression of TG2 and the enhance of TF levels. Moreover, MDMs of CAD patients showed a higher oxidative stress status, evidenced by the lower GSH/GSSG ratio, in respect to those of healthy subjects. Finally, we found a positive correlation between oxidative stress status and membrane fluidity (MFI) as evidenced by the ratio of C18:1 (oleic acid)/C18:2 (linoleic acid), as well as MFI and capacity to engulf apoptotic cells. Regarding OCT analysis, patients with a higher round MDMs prevalence exhibited more frequently a lipid rich plaque, a TCFA (thin cap fibroatheroma), a greater intra-plaque macrophage content, and a ruptured plaque, characteristics of vulnerable plaque. Furthermore, patients with high TF levels more frequently presented a intra-plaque macrophages accumulation, a ruptured plaque and a presence of thrombus. In addition, vulnerable plaque characteristics were associated with high HO-1 levels. Conclusions: MDMs of CAD patients present a pro-inflammatory and high thrombogenic profile in respect to those of healthy volunteers. The high oxidative stress present in these cells influences the membrane fluidity that in turn influences the efferocytic capability. This specific cellular profile detected in in vitro obtained MDMs is associated with the detection of high-risk and rupture-prone coronary plaques in vivo, at OCT investigation. Different MDM phenotypes might be novel diagnostic and therapeutic target able to counteract the progression of atherosclerosis.
CARUSO, DONATELLA
SONNINO, SANDRO
Settore BIO/10 - Biochimica
BIOCHEMICAL AND FUNCTIONAL PROFILE OF SPONTANEOUSLY DIFFERENTIATED HUMAN MONOCYTE-DERIVED MACROPHAGES IN PATIENTS WITH ISCHEMIC HEART DISEASE / S. Fiorelli ; tutor: D. Caruso ; coordinatore: S. Sonnino. - : . DIPARTIMENTO DI SCIENZE FARMACOLOGICHE E BIOMOLECOLARI, 2018 Jan 25. ((30. ciclo, Anno Accademico 2017. [10.13130/s-fiorelli_phd2018-01-25].
Doctoral Thesis
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/544434
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