DNA methylation predicts the outcome of COVID-19 patients with acute respiratory distress syndrome.

BACKGROUND: COVID-19 infections could be complicated by acute respiratory distress syndrome (ARDS), increasing mortality risk. We sought to assess the methylome of peripheral blood mononuclear cells in COVID-19 with ARDS. METHODS: We recruited 100 COVID-19 patients with ARDS under mechanical ventilation and 33 non-COVID-19 controls between April and July 2020. COVID-19 patients were followed at four time points for 60 days. DNA methylation and immune cell populations were measured at each time point. A multivariate cox proportional risk regression analysis was conducted to identify predictive signatures according to survival. RESULTS: The comparison of COVID-19 to controls at inclusion revealed the presence of a 14.4% difference in promoter-associated CpGs in genes that control immune-related pathways such as interferon-gamma and interferon-alpha responses. On day 60, 24% of patients died. The inter-comparison of baseline DNA methylation to the last recorded time point in both COVID-19 groups or the intra-comparison between inclusion and the end of follow-up in every group showed that most changes occurred as the disease progressed, mainly in the AIM gene, which is associated with an intensified immune response in those who recovered. The multivariate Cox proportional risk regression analysis showed that higher methylation of the "Apoptotic execution Pathway" genes (ROC1, ZNF789, and H1F0) at inclusion increases mortality risk by over twofold. CONCLUSION: We observed an epigenetic signature of immune-related genes in COVID-19 patients with ARDS. Further, Hypermethylation of the apoptotic execution pathway genes predicts the outcome. TRIAL REGISTRATION: IMRPOVIE study, NCT04473131.

Altered Circulating microRNAs in Patients with Diabetic Neuropathy and Corneal Nerve Loss: A Pilot Study.

An alteration in circulating miRNAs may have important diagnostic and therapeutic relevance in diabetic neuropathy. Patients with type 2 diabetes mellitus (T2DM) underwent an assessment of neuropathic symptoms using Douleur Neuropathique 4 (DN4), the vibration perception threshold (VPT) using a Neurothesiometer, sudomotor function using the Sudoscan, corneal nerve morphology using corneal confocal microscopy (CCM) and circulating miRNAs using high-throughput miRNA expression profiling. Patients with T2DM, with (n = 9) and without (n = 7) significant corneal nerve loss were comparable in age, gender, diabetes duration, BMI, HbA1c, eGFR, blood pressure, and lipid profile. The VPT was significantly higher (p < 0.05), and electrochemical skin conductance (p < 0.05), corneal nerve fiber density (p = 0.001), corneal nerve branch density (p = 0.013), and corneal nerve fiber length (p < 0.001) were significantly lower in T2DM patients with corneal nerve loss compared to those without corneal nerve loss. Following a q-PCR-based analysis of total plasma microRNAs, we found that miR-92b-3p (p = 0.008) was significantly downregulated, while miR-22-3p (p = 0.0001) was significantly upregulated in T2DM patients with corneal nerve loss. A network analysis revealed that these miRNAs regulate axonal guidance and neuroinflammation genes. These data support the need for more extensive studies to better understand the role of dysregulated miRNAs' in diabetic neuropathy.

Angiogenic content of microparticles in patients with diabetes and coronary artery disease predicts networks of endothelial dysfunction.

BACKGROUND: Elevated endothelial microparticles (EMPs) levels are surrogate markers of vascular dysfunction. We analyzed EMPs with apoptotic characteristics and assessed the angiogenic contents of microparticles in the blood of patients with type 2 diabetes (T2D) according to the presence of coronary artery disease (CAD). METHODS: A total of 80 participants were recruited and equally classified as (1) healthy without T2D, (2) T2D without cardiovascular complications, (3) T2D and chronic coronary artery disease (CAD), and (4) T2D and acute coronary syndrome (ACS). MPs were isolated from the peripheral circulation, and EMPs were characterized using flow cytometry of CD42 and CD31. CD62E was used to determine EMPs' apoptotic/activation state. MPs content was extracted and profiled using an angiogenesis array. RESULTS: Levels of CD42- CD31 + EMPs were significantly increased in T2D with ACS (257.5 ± 35.58) when compared to healthy subjects (105.7 ± 12.96, p < 0.01). There was no significant difference when comparing T2D with and without chronic CAD. The ratio of CD42-CD62 +/CD42-CD31 + EMPs was reduced in all T2D patients, with further reduction in ACS when compared to chronic CAD, reflecting a release by apoptotic endothelial cells. The angiogenic content of the full population of MPs was analyzed. It revealed a significant differential expression of 5 factors in patients with ACS and diabetes, including TGF-ß1, PD-ECGF, platelet factor 4, serpin E1, and thrombospondin 1. Ingenuity Pathway Analysis revealed that those five differentially expressed molecules, mainly TGF-ß1, inhibit key pathways involved in normal endothelial function. Further comparison of the three diabetes groups to healthy controls and diabetes without cardiovascular disease to diabetes with CAD identified networks that inhibit normal endothelial cell function. Interestingly, DDP-IV was the only differentially expressed protein between chronic CAD and ACS in patients with diabetes. CONCLUSION: Our data showed that the release of apoptosis-induced EMPs is increased in diabetes, irrespective of CAD, ACS patients having the highest levels. The protein contents of MPs interact in networks that indicate vascular dysfunction.

Whole-methylome analysis of circulating monocytes in acute diabetic Charcot foot reveals differentially methylated genes involved in the formation of osteoclasts.

AIM: To assess whether DNA methylation of monocytes play a role in the development of acute diabetic Charcot foot (CF). PATIENTS & METHODS: We studied the whole methylome (WM) of circulating monocytes in 18 patients with Type 2 diabetes (T2D) and acute CF, 18 T2D patients with equivalent neuropathy and 18 T2D patients without neuropathy, using the enhanced reduced representation bisulfite sequencing technique. RESULTS & CONCLUSION: WM analysis demonstrated that CF monocytes are differentially methylated compared with non-CF monocytes, in both CpG-site and gene-mapped analysis approaches. Among the methylated genes, several are involved in the migration process during monocyte differentiation into osteoclasts or are indirectly involved through the regulation of inflammatory pathways. Finally, we demonstrated an association between methylation and gene expression in cis- and trans-association.

Differentially expressed circulating microRNAs in the development of acute diabetic Charcot foot.

AIM: Charcot foot (CF) is a rare complication of Type 2 diabetes (T2D). MATERIALS & METHODS: We assessed circulating miRNAs in 17 patients with T2D and acute CF (G1), 17 patients with T2D (G2) and equivalent neuropathy and 17 patients with T2D without neuropathy (G3) using the high-throughput miRNA expression profiling. RESULTS: 51 significantly deregulated miRNAs were identified in G1 versus G2, 37 in G1 versus G3 and 64 in G2 versus G3. Furthermore, we demonstrated that 16 miRNAs differentially expressed between G1 versus G2 could be involved in osteoclastic differentiation. Among them, eight are key factors involved in CF pathophysiology. CONCLUSION: Our data reveal that CF patients exhibit an altered expression profile of circulating miRNAs.

Circulating microparticles in acute diabetic Charcot foot exhibit a high content of inflammatory cytokines, and support monocyte-to-osteoclast cell induction.

Circulating microparticles (MPs) are major mediators in cardiovascular complications of type 2 diabetes (T2D); however, their contribution to Charcot foot (CF) disease is not known. Here, we purified and assessed the origin, concentration and content of circulating MPs from 33 individuals: 11 with T2D and acute CF, 11 T2D patients with equivalent neuropathy and 11 non-diabetic controls. First, we demonstrated that there were no differences in the distribution of MPs of endothelial, platelet origin among the 3 groups. However, MPs from leukocytes and monocytes origin were increased in CF patients. Moreover, we demonstrated that monocytes-derived MPs originated more frequently from intermediate and non-classical monocytes in CF patients. Five cytokines (G-CSF, GM-CSF, IL-1-ra, IL-2 and IL-16) were significantly increased in MPs from acute CF patients. Applying ingenuity pathways analysis, we found that those cytokines interacted well and induced the activation of pathways that are involved in osteoclast formation. Further, we treated THP-1 monocytes and monocytes sorted from healthy patients with CF-derived MPs during their differentiation into osteoclasts, which increased their differentiation into multinucleated osteoclast-like cells. Altogether, our study suggests that circulating MPs in CF disease have a high content of inflammatory cytokines and could increase osteoclast differentiation in vitro.