Dottorato in INNOVATION IN IMMUNO-MEDIATED AND HEMATOLOGICAL DISORDERS

Titolo della tesi: CAFFEINE IMPROVES SYSTEMIC LUPUS ERYTHEMATOSUS ENDOTHELIAL DYSFUNCTION BY PROMOTING ENDOTHELIAL PROGENITOR CELLS SURVIVAL

Introduction: circulating endothelial progenitor cells (EPCs) are widely demonstrated biomarkers of endothelial function. Their frequency and function varied in systemic lupus erythematosus (SLE) patients, with a significant association with subclinical atherosclerosis. Caffeine, one of the most widely consumed products in the world, seems to improve endothelial cells number and EPCs migration in coronary artery disease both in mouse models and in patients. We recently demonstrated the impact of caffeine on SLE disease activity status, in terms of SLE Disease Activity Index 2000 (SLEDAI-2K) values and serum cytokine levels. Aim: the aim of this study was to evaluate the possible role of caffeine intake on endothelial function in SLE patients, by assessing its effect on number and function of EPCs both ex vivo in SLE patients and in vitro in healthy donors (HD) treated with SLE sera. Methods: we performed a cross-sectional study enrolling SLE patients, excluding patients with history of traditional cardiovascular risks factors. Caffeine intake was evaluated using a 7-day food frequency questionnaire. At the end of questionnaire filling blood samples were collected from each patient to assess circulating EPC percentage. EPCs were detected by using a flow cytometry analysis defined as KDR CD34 double positive cells. Subsequently, EPCs pooled from 6 HD were co-cultured with caffeine at 0.5 mM and 1 mM with and without SLE sera. After 7 days, we evaluated the cells morphology and the ability to form colonies; moreover, we analyzed for the percentage of annexin V-positive (AV) apoptotic cells by flow cytometry analysis and for levels of autophagy and apoptotic markers LC3-II, p62 and Bcl2 by western blot, alone or in the presence of protease lysosomal inhibitors E64d and Pepstatin A. Finally, we performed a WB analysis to assess the A2AR/SIRT3/AMPK pathway. Results: we enrolled 31 SLE patients (F:M 30:1, median age 43 years, IQR 18; median disease duration 144 months, IQR 180). We found a EPCs median percentage of 0.03% (IQR 0.04) observing a positive correlation between caffeine intake and circulating EPCs percentage (p=0.03, r=0.4). Moving on in vitro experiments, HD EPCs treated with SLE sera and caffeine showed an improvement in morphology and in number of EPCs-CFU in comparison with those incubated without caffeine (p=0.0003). The colonies treated with SLE sera were poorly organized in comparison with HD; the addition of caffeine restored the colony structure. After treating HD-EPCs with SLE sera we observed an increase in AV positive cells and p62 values and a reduction of LC3-II and Bcl2 values; the addition of caffeine was able to significantly reduce AV positive cells and p62 values and to significantly increased LC3-II and Bcl2 values, without any significant differences between caffeine 0.5 mM and 1 mM. After E64d and pepstatin A treatment, both LC3IIB and p62 trend didn’t change, compared to untreated cells. Finally, we observed after caffeine treatment, in comparison with SLE sera alone, a significantly reduction in A2AR levels leading to an increase in protein levels of SIRT3 and subsequently AMPK phosphorylation. Conclusions: we demonstrated, for the first time, a protective role of caffeine on endothelial function in SLE patients. Caffeine intake positively correlated with the percentage of circulating EPCs in SLE patients; moreover, caffeine in vitro treatment was able to improve EPCs survival and vitality through the inhibition of apoptosis and the promotion of autophagy via A2AR/SIRT3/AMPK pathway.