https://orcid.org/0000-0001-5026-8783
Abstract
We studied the role of caffeine intake on endothelial function in SLE by assessing its effect on circulating endothelial progenitor cells (EPCs) both ex vivo in SLE patients and in vitro in healthy donors (HD) treated with SLE sera.
We enrolled SLE patients without traditional cardiovascular risks factors. Caffeine intake was evaluated with a 7-day food frequency questionnaire. EPCs percentage was assessed by flow cytometry analysis and, subsequently, EPCs pooled from six HD were co-cultured with caffeine with and without SLE sera. After 7 days, we evaluated cells’ morphology and ability to form colonies, the percentage of apoptotic cells by flow cytometry analysis and the levels of autophagy and apoptotic markers by western blot. Finally, we performed a western blot analysis to assess the A2AR/SIRT3/AMPK pathway.
We enrolled 31 SLE patients, and observed a positive correlation between caffeine intake and circulating EPCs percentage. HD EPCs treated with SLE sera and caffeine showed an improvement in morphology and in number of EPCs colony-forming units in comparison with those incubated without caffeine. Caffeine was able to restore autophagy and apoptotic markers in HD EPCs as before SLE sera treatment. Finally, caffeine treatment was able to significantly reduce A2AR levels, leading to an increase in protein levels of SIRT3 and subsequently AMPK phosphorylation.
Caffeine intake positively correlated with the percentage of circulating EPCs in SLE patients; moreover, caffeine in vitro treatment was able to improve EPC survival and vitality through the inhibition of apoptosis and the promotion of autophagy via A2AR/SIRT3/AMPK pathway.
Caffeine intake positively correlated with the percentage of circulating EPCs in lupus patients.
In vitro caffeine treatment improves EPC survival and vitality through apoptosis inhibition and autophagy promotion
Introduction
SLE is a multifactorial systemic autoimmune disease with a broad spectrum of clinical presentations. Its pathogenesis is the result of complex interactions between genetic, epigenetic, immunoregulatory, ethnic, hormonal and environmental factors [1].
Among the environmental factors, there has been an emerging interest in dietary factors: a diet rich in vitamin D and A, polyunsaturated fatty acid and phenols, and poor in sodium seems to play a role in decreasing the inflammatory burden [2, 3].
The most common cause of late mortality in SLE patients is represented by cardiovascular disease (CVD), linked both to disease-related and traditional risk factors [4]. The increased vascular damage associated with an inadequate repair identified in SLE patients seems to be linked to endothelial dysfunction, crucial alteration for the development of atherosclerosis. In particular, an impairment in number and function of circulating endothelial progenitor cells (EPCs) has been demonstrated in these patients [5–9]. EPCs are bone marrow-derived cells essential for vascular damage repair and a decrease in their number is associated with subclinical atherosclerosis [10, 11].
Caffeine is an alkaloid (1,3,7-trimethylxanthine), found in various plant constituents, and it is one of the most widely consumed products in the world. Besides the well-known psychostimulant effect, caffeine exerts an anti-inflammatory dose-dependent action, binding with high affinity the adenosine receptors A2A expressed on the surface of immune cells [12]. The effect of caffeine consumption on CVD has been widely investigated, with conflicting results: recent studies tend to report an inverse relationship between habitual caffeine consumption and CVD morbidity and mortality [13, 14]. Furthermore, coffee consumption seems to be able to increase endothelial function in healthy subjects and to improve endothelial function in patients with CVD [15–21]. To date, only one study has focused on the effect of caffeine on EPC, demonstrating a significant improvement in EPC migration in relation to coffee consumption in coronary artery disease both in mouse models and in patients, via an AMPK-dependent mechanism [22].
Finally, we recently analysed the impact of caffeine intake on SLE disease activity and phenotype, showing an inverse correlation between caffeine consumption and disease activity—as assessed by SLEDAI-2K—and serum cytokine levels. Moreover, a low caffeine intake was associated with more frequent major organ involvement and anti-dsDNA positivity [23].
At the best of our knowledge, there are no data in the literature about the effect of caffeine on cardiovascular risk in SLE patients. Therefore, the aim of this study was to evaluate the role of caffeine intake in 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
Ex vivo analysis
We performed a cross-sectional study enrolling SLE patients, fulfilling the 2019 EULAR/ACR criteria [24], referring to the Lupus Clinic of the Rheumatology Unit, Sapienza University of Rome (Sapienza lupus cohort). Patients with history of smoking, CVD, chronic kidney failure, dyslipidaemia and/or diabetes were excluded. For each patient, the clinical and laboratory data, including demographics, past medical history with the date of diagnosis and treatments, were collected in a standardized, computerized and electronically filled form.
The study protocol included the determination of autoantibodies and the evaluation of C3 and C4 serum levels. ANAs were determined by indirect immunofluorescence (IIF) on HEp-2 (titre ≥1:160 or ++ on a scale from + to ++++); anti-dsDNA was determined with IIF on Crithidia luciliae (titre ≥1:10); ENAs (including anti-Ro/SSA, anti-La/SSB, anti-Sm and anti-RNP) were analysed by ELISA considering titres above the cut-off of the reference laboratory; aCL (IgG/IgM isotype) was analysed by ELISA, in serum or plasma, at medium or high titres (e.g. >40 GPL or MPL or above the 99th percentile); anti-β2 glycoprotein I (IgG/IgM isotype) was analysed by ELISA, in serum (above the 99th percentile); LA was analysed according to the guidelines of the International Society on Thrombosis and Haemostasis. Finally, C3 and C4 serum levels were determined by radial immunodiffusion. Disease activity was assessed using the SLEDAI-2K [25] and chronic damage by the SLICC Damage Index [26].
Caffeine intake was evaluated using a 7-day food frequency questionnaire reporting all the main sources of caffeine, previously applied in SLE patients [23].
At the end of questionnaire filling, blood samples were collected from each patient to assess circulating EPC percentage.
Peripheral blood mononuclear cells (PBMCs) were obtained by density gradient centrifugation (Lympholyte-H; Cedarlane Laboratories, Hornby, Ontario, Canada); phenotypic characterization was performed as previously described [27]. In brief, after incubation with FcR-blocking reagent (Miltenyi Biotec, Bergisch-Gladbach, Germany), 1 × 106 PBMCs were incubated for 30 min on ice with fluorescein isothiocyanate (FITC)-labelled mAb anti-CD34 (BD Immunocytometry Systems, San Jose, CA, USA) and phycoerythrin (PE)-labelled mAb anti-VEGF R2/KDR (BD Immunocytometry Systems, San Jose, CA, USA). Appropriate isotype controls were used. Acquisition was performed on a FACS Calibur (BD Immunocytometry Systems, San Jose, CA, USA) and included 100 000–400 000 events per sample. Data were analysed using the CellQuest Pro software (BD Immunocytometry Systems, San Jose, CA, USA). EPCs were defined as CD34/KDR double-positive cells, and their number was expressed as a percentage of cells within the lymphocyte gate.
In vitro analysis
We used 20 ml of venous blood from HD for EPCs isolation. Samples were processed within 4 h of collection, and PBMC were isolated by Ficoll density-gradient centrifugation; 5 × 106 PBMCs were plated on human fibronectin-precoated (10 μg/ml Sigma-Aldrich, six-well plates), cultivated in growth medium 199 (Gibco, Fisher Scientific, Italia) containing 20% fetal bovine serum (FBS) and penicillin (100 U/ml)/streptomycin (100 μg/ml, Gibco, Fisher Scientific, Italia), and then incubated (37°C and 5% CO2). After 3 days, non-adherent cells were removed and fresh culture medium was supplied and changed every other day. After 7 days of culture, EPCs colony-forming units (CFU) were enumerated by light microscopy (Scpe.A1, ZEISS). The ability to form colonies was evaluated after in vitro treatment of EPCs from HD with SLE sera with and without caffeine at two different dosages, 0.5 mM and 1 mM. In details, all the colonies present in each single 6-well plates were counted at least in triplicate for each condition. Two independent operators contributed to the count.
For this in vitro treatment we used sera from SLE patients who did not consume caffeine and have SLEDAI ≥4. Similarly, we enrolled HD who did not consume caffeine.
By day 7, most of the adherent cells had acquired a spindle shape, typical of early EPCs; flow cytometry analysis with CD34 FITC/KDR PE was performed.
Autophagy and apoptosis analysis
Subsequently, we analysed autophagy and apoptosis in EPCs cultures treated with caffeine with and without SLE sera.
Autophagy was assessed by immunoblotting for LC3-IIB (anti-LC3B; Cell Signalling) in combination with p62 (anti–sequestosome 1/p62; Cell Signalling). Interpretation of autophagy was conducted according to published guidelines [28]. Specifically, LC3-II reflects autophagosome formation following activation of autophagy and conjugation of the microtubule-associated protein 1A/1B LC3-I with phosphatidylethanolamine [29], and p62 is a substrate whose degradation has been observed during activation of autophagy [30]. Dynamic activation of autophagy may emerge as either an increase in LC3-II (autophagosome formation) or a decrease in LC3-II (autophagosome degradation upon fusion with lysosomes), both in the presence of a concomitant reduction of p62 [30]. Finally, we assessed autophagy also with lysosomal inhibitors E64d and pepstatin A.
Apoptosis was evaluated by flow cytometry analysis for the percentage of annexin V (AV)/propidium iodide (PI) and by western blotting (WB) evaluating the antiapoptotic molecule Bcl-2 (anti-Bcl-2; no. GTX100064; GeneTex). Beta-actin was used as a loading control (no. SAB5600204; Sigma-Aldrich).
A2AR/SIRT3/AMPK pathway
Finally, to assess the A2AR/SIRT3/AMPK pathway and its role in EPC autophagy and apoptosis, we performed a WB analysis with the following primary antibodies: SIRT3 (no. PA528402, Life Technologies), ADORA2A (A2AR) (no. MA5-31611, Life Technologies), AMPK alpha-1 (no. BSM-33236M, Sial) and phospho-AMPK alpha-1 (Ser485, no. PA5-104982, Life Technologies).
Statistical analysis
Categorical variables were expressed as frequencies and percentages, while continuous variables were presented as means and s.d. or median (range) and interquartile range (IQR), if normally or non-normally distributed, respectively. The comparisons for continuous variables were made with the Mann–Whitney test. Univariate comparisons between nominal variables were calculated using χ2 test or Fisher’s exact test where appropriate. The significance of any correlation was determined by Spearman’s rank correlation coefficient. Any P-values <0.05 were considered significant. Statistical analysis was performed using GraphPadPrism 6 software (GraphPad Software, San Diego, CA, USA).
Results
Ex vivo analysis
We enrolled 31 SLE patients (F/M 30/1, median age 43 years, IQR 18; median disease duration 144 months, IQR 180). Demographic, clinical and laboratory features and treatment during the disease course are reported in Supplementary Table S1, available at Rheumatology online. Supplementary Table S2, available at Rheumatology online describes the patients’ treatment at the time of enrolment.
The median intake of caffeine in our cohort was 166 mg/day (IQR 194) and coffee was the most frequent source referred by patients [median intake 160 mg/day (IQR 183)].
Median percentage of EPCs was 0.03 (IQR 0.04). Patients with history of neuropsychiatric lupus and those under treatment with glucocorticoids showed a significantly lower percentage of EPCs (0.01 vs 0.04, P = 0.0003; 0.03 vs 0.04, P = 0.04, respectively).
Interestingly, we observed a positive correlation between daily caffeine intake and EPC percentage (P = 0.03, r = 0.4) (Fig. 1).
Correlation between KDR+/CD34+ cells and daily caffeine intake
In vitro analysis
In the in vitro experiments, EPCs pooled from six 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. HD EPCs treated with SLE sera and caffeine showed an improvement in morphology and in number of EPCs CFU in comparison with those incubated with SLE sera without caffeine (number ± s.d.; 15 ± 1 vs 5 ± 1, P = 0.0003; Fig. 2A). Moreover, the colonies treated with SLE sera were poorly organized in comparison with HD and the addition of caffeine was able to restore the colony structure (Fig. 2B). We did not observe any significant difference between the two dosages of caffeine.
In vitro effect of caffeine treatment and SLE sera on EPC CFU number and morphology represented as histogram (A) showing the mean number of EPC CFU, and images showing the change of morphology and organization at two different microscopic magnifications (B and C). EPC: endothelial progenitor cell; HD: healthy donor; CFU: colony-forming units
Autophagy and apoptosis analysis
Subsequently, autophagy was evaluated in PBMCs from six HD co-cultured with SLE sera with and without caffeine at 0.5 mM and 1 mM. We observed a decrease in LC3-II levels in HD patients PBMCs treated with SLE sera (20% FBS vs SLE sera, P = 0.001) and an increase after caffeine treatment, restoring LC3-II levels to those before SLE sera treatment (SLE sera vs SLE sera + caffeine 0.5 mM, P = 0.01; SLE sera vs SLE sera + caffeine 1 mM, P = 0.01). A concomitant significant increase in p62 levels was observed in HD patients’ PBMCs treated with SLE sera (20% FBS vs SLE-sera, P < 0.0001) and a decrease after caffeine treatment, restoring p62 levels to those before SLE sera treatment (SLE sera vs SLE sera + caffeine 0.5 mM, P = 0.0002; SLE sera vs SLE sera + caffeine 1 mM, P = 0.0003) (Fig. 3).
LC3IIB (A) and p62 (B) levels in healthy donor EPCs treated with SLE sera and caffeine at 0.5 mM and 1 mM. EPC: endothelial progenitor cell
According to these results, we hypothesized an autophagy dysfunction and/or blockade. Thus, we assessed in vitro experiments with lysosomal inhibitors E64d and pepstatin A. Both LC3IIB and p62 trend did not change, compared with untreated cells. Indeed, as before, in the presence of lysosomal inhibitors, we observed a decrease in LC3-II levels in HD patients PBMCs treated with SLE sera (20% FBS vs SLE-sera, P = 0.0004) and an increase after caffeine treatment (SLE sera vs SLE sera + caffeine 1 mM, P = 0.007). A concomitant significant increase in p62 levels was observed in HD patients PBMCs treated with SLE sera (20% FBS vs SLE sera, P = 0.001) and a decrease after caffeine treatment (SLE sera vs SLE sera + caffeine, P = 0.001) (Fig. 4).
LC3IIB (A) and p62 (B) levels in healthy donor EPCs treated with SLE sera and caffeine after lysosomal inhibitors E64d and pepstatin A treatment. EPC: endothelial progenitor cell; FBS: fetal bovine serum
Similarly, apoptosis was evaluated in PBMCs from six HD co-cultured with caffeine at 0.5 mM and 1 mM with and without SLE sera, by flow cytometry analysis for the percentage of AV/PI and by WB evaluating the antiapoptotic molecule Bcl-2.
We observed an increase in AV-positive cells percentage in HD PBMCs treated with SLE sera (20% FBS vs SLE sera, P = 0.001) and a decrease after caffeine treatment, restoring the AV-positive cell percentage to that before SLE sera treatment (SLE sera vs SLE sera + caffeine 0.5 mM, P = 0.001; SLE sera vs SLE sera + caffeine 1 mM, P = 0.001) (Fig. 5A).
(A) Annexin V (AV)-positive cells percentage in healthy donors EPCs treated with SLE sera and caffeine at 0.5 mM and 1 mM. (B) Bcl2 levels in healthy donors EPCs treated with SLE sera and caffeine at 0.5 mM and 1 mM. EPC: endothelial progenitor cell; FBS: fetal bovine serum
In WB analysis, a concomitant significant decrease in Bcl2 levels was observed in HD PBMCs treated with SLE sera (20% FBS vs SLE sera, P = 0.04) and an increase after caffeine treatment, restoring Bcl2 levels to those before SLE sera treatment (SLE sera vs SLE sera + caffeine 0.5 mM, P = 0.03; SLE sera vs SLE sera + caffeine 1 mM, P = 0.005) (Fig. 5B).
A2AR/SIRT3/AMPK pathway
Finally, the signalling pathway that could lead to the activation of autophagy by caffeine in EPCs was investigated. Caffeine treatment, in comparison with SLE sera, significantly decreased A2AR levels leading to an increase in protein levels of SIRT3 and subsequently AMPK phosphorylation.
In detail, we observed an increase in A2AR levels in HD patients PBMCs treated with SLE sera (20% FBS vs SLE sera, P = 0.02) and a subsequent decrease after caffeine treatment (SLE sera vs SLE sera + caffeine 0.5 mM, P = ns; SLE sera vs SLE sera + caffeine 1 mM, P = 0.02) (Fig. 6A). A concomitant significant decrease in SIRT3 and p-AMPK/AMPK levels was observed in HD patients PBMCs treated with SLE sera (SIRT3: 20% FBS vs SLE sera, P = 0.001; p-AMPK/AMPK: 20% FBS vs SLE-sera, P = 0.01) and an increase after caffeine treatment (SIRT3: SLE sera vs SLE sera + caffeine 0.5 mM, P = 0.001, SLE sera vs SLE sera + caffeine 1 mM, P = 0.0004; p-AMPK/AMPK: SLE sera vs SLE sera + caffeine 0.5 mM, P = 0.0003, SLE sera vs SLE sera + caffeine 1 mM, P = 0.0008) (Fig. 6B and C).
A2AR (A), SIRT3 (B) and p-AMPK/AMPK (C) levels in healthy donors EPCs treated with SLE sera and caffeine at 0.5 mM and 1 mM. EPC: endothelial progenitor cell; FBS: fetal bovine serum
Discussion
In the present study 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, in vitro caffeine treatment was able to improve EPC survival and vitality through the inhibition of apoptosis and the promotion of autophagy via the A2AR/SIRT3/AMPK pathway.
CVD represents one of the most frequent causes of late mortality in SLE patients. Endothelial dysfunction, the earliest and reversible stage of atherosclerotic process, is widely documented in SLE patients and provides diagnostic and prognostic data on atherosclerotic risk [9]. Seeking a deeper understanding of the upstream mechanisms leading to endothelial dysfunction, various investigators have intensively studied the cellular mechanisms that lead to endothelial function impairment in SLE patients. In particular, the study of EPCs has attracted much interest in the past 20 years. EPCs are immature precursors of endothelial cells, recruited from bone marrow, mirroring the reparative capacity of healthy endothelium; therefore, alterations of number and functions of EPCs reflect endothelial dysfunction [31]. In this context, observational studies and a meta-analysis revealed that circulating EPCs were significantly reduced in SLE patients compared with healthy people, even in patients who have not experienced a cardiovascular event [5–8, 32]. A variety of markers have been used to identify and characterize EPCs within SLE [33]. Altered morphology and reduced expression of VEGF and hence reduced VEGF-driven mobilization of EPCs have been demonstrated in patients with SLE [5, 7]. In addition to reduced quantity, the crucial functions of EPCs, including migration and tube- and colony-forming abilities, were found to be reduced in patients with SLE compared with healthy individuals [5]. Furthermore, studies in SLE patients showed a negative effect of type I IFN on EPCs, which was already demonstrated in animal models [6]. Upregulation of the genes encoding IFN-α was observed in EPCs in patients with SLE, which suppressed VEGF and hepatocyte growth factor expression and subsequent angiogenic activity [5]. IFN-α induces EPC apoptosis, reduces proliferation of EPCs and inhibits pro-angiogenic transcriptomes induced by IL-1β, leading to reduction of VEGF expression and increase of IL-18-induced inflammasome activation, widely demonstrated as a crucial downstream pathway promoting aberrant vasculogenesis [5, 6, 34–36]. Furthermore, BAFF plays a part in reducing EPC quantity and function: indeed, BAFF receptors are expressed on EPCs and mediate EPC apoptosis when engaged with BAFF [37]. EPC apoptosis and impairment of colony structure were shown to be reversible by treating EPCs from SLE patients with belimumab, suggesting for BAFF a pro-apoptotic effect on EPCs [37].
In order to prevent endothelial dysfunction morbidity and mortality, it is crucial to identify modifiable factors such as lifestyle and diet. In this regard, the central role of environmental factors in SLE has been widely demonstrated—among these, there has been an emerging interest in dietary factors. In this context, caffeine represents one of the most widely consumed products in the world. We recently provided information about the role of caffeine in SLE immune and clinical phenotype. Our results suggested a possible immunoregulatory dose-dependent effect of caffeine through the modulation of several serum cytokine levels; similarly, caffeine could influence disease clinical phenotype favouring a less severe disease picture [23]. Adenosine receptors, in particular A2A receptors, seem to have a protective role in atherosclerosis as well [38]. Recent studies suggest an inverse relationship between habitual caffeine consumption and CVD morbidity and mortality [13, 14]. On the other hand, very few data are available about the effect of caffeine on endothelial function, with conflicting results [15–21, 39–41].
To the best of our knowledge there are no data in the literature about the effect of caffeine on cardiovascular risk in SLE patients. Our ex vivo results demonstrated a positive correlation between daily caffeine intake and circulating EPCs in SLE patients. To date, only one study evaluating the effect of caffeine on EPCs has been published, showing similar results. Indeed, the authors demonstrated in patients with coronary artery disease that caffeinated coffee significantly increased the migratory activity of patient EPCs, in comparison with decaffeinated coffee. Furthermore, treatment with caffeine in a mouse model was able to improve endothelial repair, stimulating re-endothelialization through an AMPK-dependent mechanism [22].
In the in vitro experiments, we demonstrated for the first time an improvement in EPCs morphology and in number of EPCs CFU, after caffeine treatment in HD treated with SLE sera in comparison with those incubated without caffeine. Moreover, the colonies treated with SLE sera were poorly organized in comparison with HD and the addition of caffeine was able to restore the colony structure. To better understand the caffeine effect on EPCs, we analysed autophagy and apoptosis markers. SLE sera increased apoptosis and decreased autophagy of EPCs and, interestingly, caffeine treatment was able to restore apoptotic and autophagy markers levels to those before SLE sera treatment.
We could hypothesize that the positive caffeine effect, observed in SLE patients and in in vitro culture, is linked to a direct action on EPC survival and vitality through the inhibition of apoptosis and the promotion of autophagy. Indeed, autophagy was shown to be extremely important for cardiovascular health, and the dysfunction of autophagy contributes to cardiovascular disorders, especially atherosclerosis; it has been well-documented that deficiency of autophagy may be a major mechanism and risk factor that elicits endothelial dysfunction [42]. It has been demonstrated that the promotion of autophagy was able to accelerate EPC maturation, highlighting the critical role of autophagy on angiogenesis [43]. Moreover, Wang and colleagues demonstrated that autophagy through AMPK signalling in EPCs led to an increased angiogenic differentiation and accelerated wound healing rate [39].
Finally, we demonstrated that EPC caffeine treatment, in comparison with SLE sera alone, significantly decreased A2AR levels leading to an increase in SIRT3 levels and subsequently AMPK phosphorylation. SIRT3, the mitochondrial nicotinamide adenine dinucleotide(+)-dependent deacetylase, has emerged as an important player in CVD, influencing endothelial dysfunction, atherosclerosis, oxidative stress and inflammation [44]. AMPK, a key energy sensor, is a downstream target of SIRT3 and a known positive regulator of autophagy [45]. Data from the literature showed that the SIRT3-AMPK signalling pathway plays an important role in inhibiting vascular inflammation and endothelial dysfunction [46, 47]. In this context, Gong and colleagues demonstrated that serum SIRT3 levels were significantly lower in diabetic patients with CAD in comparison with normal coronary arteries [44]. Moreover, in mouse atherosclerotic models, SIRT3 levels were closely related with apoptotic markers and SIRT3 expression was shown to increase in early sepsis, protecting against endothelial dysfunction and reducing mortality [48, 49].
Taken together, these data supported our results demonstrating that the positive effect of caffeine observed in vitro and in vivo could be mediated by SIRT3/AMPK stimulating autophagy.
To date, the effect of caffeine on the A2AR/SIRT3/AMPK pathway has been investigated only in skin cells by Li and colleagues. They demonstrated, both in vivo and in vitro, that caffeine protects skin from oxidative stress–induced senescence through activating the A2AR/SIRT3/AMPK-mediated autophagy, similar to ours results in EPCs [50]. Moreover, our data are in line with previous results highlighting the positive AMPK-inducing effect of caffeine on EPCs [22].
Certainly, the present study has some limitations; in particular, it would be helpful to measure caffeine levels in the serum of the participants. This would allow a better comparison of the caffeine serum levels with the caffeine concentrations used in the in vitro studies.
In conclusion, we demonstrated a protective role of caffeine on endothelial function in vivo in SLE patients and in vitro in HD treated with SLE sera. In our SLE cohort, caffeine intake positively correlated with the percentage of circulating EPCs. In addition, caffeine in vitro treatment was able to restore autophagy and apoptosis to the levels seen before SLE sera addition via the A2AR/SIRT3/AMPK pathway.
Supplementary material
Supplementary material is available at Rheumatology online.
Data availability
The main data have been reported in the text.
Funding
No specific funding was received for this work.
Disclosure statement: The authors have not conflict of interest to declare.
Ethics: The study complies with the Declaration of Helsinki. The ethic committee of Policlinico Umberto I—Roma has approved the research protocol and a written informed consent has been obtained from the subjects involved.
Comments