Angiotensin-converting-enzyme insertion/deletion polymorphism, ACE activity, and COVID-19: A rather controversial hypothesis. A case-control study.

Accumulating data has shown a contribution of the renin-angiotensin system in COVID-19 pathogenesis. The role of angiotensin-converting enzyme (ACE) insertion (I)/deletion (D) polymorphism as a risk factor in developing COVID-19 disease comes from epidemiological data and is controversially discussed. We conducted a retrospective case-control study and assessed the impact of ACE I/D genotype in COVID-19 disease prevalence and severity. In 81 COVID-19 patients explicitly characterized and 316 controls, recruited during the first wave of COVID-19 pandemic, ACE I/D genotype, and ACE activity were determined. A generalized linear model was used and Poisson regression analysis estimated the risk ratios (RRs) of alleles and genotypes for disease severity. DD patients had almost 2.0-fold increased risk (RR: 1.886, confidence limit [CL] 95%: 1.266-2.810, p = 0.0018) of developing a more severe disease when contrasted to ID and II individuals, as did D allele carriers compared to I carriers (RR: 1.372; CL 95%: 1.051-1.791; p = 0.0201). ACE activity (expressed as arbitrary units, AU/L) was lower in patients (3.62 ± 0.26) than in controls (4.65 ± 0.13) (p < 0.0001), and this reduction was observed mainly among DD patients compared to DD controls (3.97 ± 0.29 vs. 5.38 ± 0.21; p = 0.0014). Our results demonstrate that ACE DD genotype may predispose to COVID-19 increased disease severity via a mechanism associated, at least in part, with the significant fall in their ACE activity. Our findings suggest a more complex pattern of synergy between this polymorphism and ACE activity in COVID-19 patients compared to healthy individuals and set the grounds for large-scale studies assessing ACE genotype-based optimized therapies with ACE inhibitors and angiotensin receptor blockers.

Glutamine synthetase regulation by dexamethasone, RU486, and compound A in astrocytes derived from aged mouse cerebral hemispheres is mediated via glucocorticoid receptor.

Glucocorticoids (GCs) regulate astrocyte function, while glutamine synthetase (GS), an enzyme highly expressed in astrocytes, is one of the most remarkable GCs-induced genes. GCs mediate their effects through their cognate glucocorticoid receptor (GRα and GRß isoforms); however, the mechanism via which these isoforms regulate GS activity in astrocytes remains unknown. We used dexamethasone (DEX), a classical GRα/GRß agonist, RU486, which is a specific GRß ligand, and Compound A, a known "dissociated" ligand, to delineate the mechanism via which GR modulates GS activity. Aged Mouse Cerebral Hemisphere astrocytes were treated with DEX (1 µM), RU486 (1 nM-1 µM) or compound A (10 µM), alone or in combination with DEX. GS activity and expression, GR isoforms (mRNA and protein levels), and GRα subcellular trafficking were measured. DEX increased GS activity in parallel with GRα nuclear translocation. RU486 increased GS activity in absence of GRα nuclear translocation implicating thus a role of GRß-mediated mechanism compound A had no effect on GS activity implicating a GRα-GRE-mediated mechanism. None of the compounds affected whole-cell GRα protein content. DEX reduced GRα and GRß mRNA levels, while RU486 increased GRß gene expression. We provide evidence that GS activity, in astrocytes, is regulated via GRα- and GRß-mediated pathways with important implications in pathological conditions in which astrocytes are involved.

Mitochondrial Glucocorticoid Receptors and Their Actions.

Mitochondria are membrane organelles present in almost all eukaryotic cells. In addition to their well-known role in energy production, mitochondria regulate central cellular processes, including calcium homeostasis, Reactive Oxygen Species (ROS) generation, cell death, thermogenesis, and biosynthesis of lipids, nucleic acids, and steroid hormones. Glucocorticoids (GCs) regulate the mitochondrially encoded oxidative phosphorylation gene expression and mitochondrial energy metabolism. The identification of Glucocorticoid Response Elements (GREs) in mitochondrial sequences and the detection of Glucocorticoid Receptor (GR) in mitochondria of different cell types gave support to hypothesis that mitochondrial GR directly regulates mitochondrial gene expression. Numerous studies have revealed changes in mitochondrial gene expression alongside with GR import/export in mitochondria, confirming the direct effects of GCs on mitochondrial genome. Further evidence has made clear that mitochondrial GR is involved in mitochondrial function and apoptosis-mediated processes, through interacting or altering the distribution of Bcl2 family members. Even though its exact translocation mechanisms remain unknown, data have shown that GR chaperones (Hsp70/90, Bag-1, FKBP51), the anti-apoptotic protein Bcl-2, the HDAC6- mediated deacetylation and the outer mitochondrial translocation complexes (Tom complexes) co-ordinate GR mitochondrial trafficking. A role of mitochondrial GR in stress and depression as well as in lung and hepatic inflammation has also been demonstrated.

Glucocorticoid Receptor Signaling in Diabetes.

Stress and depression increase the risk of Type 2 Diabetes (T2D) development. Evidence demonstrates that the Glucocorticoid (GC) negative feedback is impaired (GC resistance) in T2D patients resulting in Hypothalamic-Pituitary-Adrenal (HPA) axis hyperactivity and hypercortisolism. High GCs, in turn, activate multiple aspects of glucose homeostasis in peripheral tissues leading to hyperglycemia. Elucidation of the underlying molecular mechanisms revealed that Glucocorticoid Receptor (GR) mediates the GC-induced dysregulation of glucose production, uptake and insulin signaling in GC-sensitive peripheral tissues, such as liver, skeletal muscle, adipose tissue, and pancreas. In contrast to increased GR peripheral sensitivity, an impaired GR signaling in Peripheral Blood Mononuclear Cells (PBMCs) of T2D patients, associated with hyperglycemia, hyperlipidemia, and increased inflammation, has been shown. Given that GR changes in immune cells parallel those in brain, the above data implicate that a reduced brain GR function may be the biological link among stress, HPA hyperactivity, hypercortisolism and hyperglycemia. GR polymorphisms have also been associated with metabolic disturbances in T2D while dysregulation of micro-RNAs-known to target GR mRNA-has been described. Collectively, GR has a crucial role in T2D, acting in a cell-type and context-specific manner, leading to either GC sensitivity or GC resistance. Selective modulation of GR signaling in T2D therapy warrants further investigation.

Blood-based analysis of 84 microRNAs identifies molecules deregulated in individuals with type-2 diabetes, risk factors for the disease or metabolic syndrome.

AIM: Micro-RNAs (miRNAs) are implicated in insulin-signaling and the development of type-2 diabetes (T2D). Their deregulated expression is mostly described in the pancreas, liver, skeletal muscle, or adipose tissue of diabetic animals. Relevant studies in humans are limited due to difficulties in accessing tissue-biopsies. Though, circulating miRNAs are indicators of organ-specific pathophysiological events and could potentially serve as disease biomarkers. We explored the profile of 84 T2D-related miRNAs in peripheral blood of subjects with or without the disease. METHODS: An RT-qPCR array screening 84 T2D-related miRNAs was applied in samples of T2D (n = 6) versus non-T2D (n = 6) subjects. The deregulated miRNAs were thereafter analyzed in peripheral blood samples of a validation cohort of 40 T2D and 37 non-T2D individuals [16 controls and 21 subjects with metabolic syndrome (Met-S) and/or T2D risk factors (T2D-RF)], using specific RT-qPCR assays. Correlations with clinicopathological parameters and risk factors were evaluated. RESULTS: Subjects with the disease displayed decreased levels of miR-214-3p, miR-24-3p and let-7f-5p, compared to those without. MiRNA levels correlated with serum insulin and HbA1c levels in individuals with T2D or Met-S/T2D-RF, and with higher BMI, dyslipidemia and family history in controls. CONCLUSIONS: Blood levels of miR-214-3p, miR-24-3p and let-7f-5p are down-regulated in T2D- and Met-S/T2D-RF subjects. Future studies are needed to evaluate their potential as disease biomarkers and elucidate the associated tissue-specific pathogenetic mechanisms.

Decreased expression of microRNAs targeting type-2 diabetes susceptibility genes in peripheral blood of patients and predisposed individuals.

AIM: Certain microRNA molecules (miRNAs) that target genes involved in beta-cell growth and insulin resistance are found deregulated in patients with type-2 diabetes mellitus (T2D) and correlate with its complications. However, the expression profile of miRNAs that regulate genes bearing T2D-related single-nucleotide polymorphisms has been hardly studied. We recently reported that the mRNA patterns of specific T2D-susceptibility genes are impaired in patients, and associate with disease parameters and risk factors. The aim of this study was to explore the levels of miRNAs that target those genes, in peripheral blood of patients versus controls. METHODS: A panel of 14 miRNAs validated to target the CDKN2A, CDK5, IGF2BP2, KCNQ1, and TSPAN8 genes, was developed upon combined search throughout the DIANNA TarBase v7.0, miRTarBase, miRSearch v3.0-Exiqon, miRGator v3.0, and miRTarget Link Human algorithms. Specifically developed poly(A)polyadenylation(PAP)-reverse transcription(RT)-qPCR protocols were applied in peripheral blood RNA samples from patients and controls. Possible correlations with the disease, clinicopathological parameters and/or risk factors were evaluated. RESULTS: T2D patients expressed decreased levels of let-7b-5p, miR-1-3p, miR-24-3p, miR-34a-5p, miR-98-5p, and miR-133a-3p, compared with controls. Moreover, these levels correlated with certain disease features including insulin and % HbA1c levels in patients, as well as BMI, triglycerides' levels and family history in controls. CONCLUSIONS: A T2D-specific expression profile of miRNAs that target disease-susceptibility genes is for the first time described. Future studies are needed to elucidate the associated transcription-regulatory mechanisms, perchance involved in T2D pathogenesis, and to evaluate the potential of these molecules as possible biomarkers for this disorder.

Blood-based analysis of type-2 diabetes mellitus susceptibility genes identifies specific transcript variants with deregulated expression and association with disease risk.

Despite significant progress by genome-wide association studies, the ability of genetic variants to conduce to the prediction or prognosis of type-2 diabetes (T2D) is weak. Expression analysis of the corresponding genes may suggest possible links between single-nucleotide polymorphisms and T2D phenotype and/or risk. Herein, we investigated the expression patterns of 24 T2D-susceptibility genes, and their individual transcript variants (tv), in peripheral blood of T2D patients and controls (CTs), applying RNA-seq and real-time qPCR methodologies, and explore possible associations with disease features. Our data revealed the deregulation of certain transcripts in T2D patients. Among them, the down-regulation of CAPN10 tv3 was confirmed as an independent predictor for T2D. In patients, increased expression of CDK5 tv2, CDKN2A tv3 or THADA tv5 correlated positively with serum insulin levels, of CDK5 tv1 positively with % HbA1c levels, while in controls, elevated levels of TSPAN8 were associated positively with the presence of T2D family history. Herein, a T2D-specific expression profile of specific transcripts of disease-susceptibility genes is for the first time described in human peripheral blood. Large-scale studies are needed to evaluate the potential of these molecules to serve as disease biomarkers.

Effect of Vinca Alkaloids on the Expression Levels of microRNAs Targeting Apoptosis-related Genes in Breast Cancer Cell Lines.

BACKGROUND: Treatment of cancer with natural chemotherapeutic agents induces apoptosis along with remarkable alterations in the expression of apoptosis-related genes. Deregulation of microRNA (miRNA) expression is implicated in several human malignancies. Vinca alkaloids compose a class of antimitotic drugs preventing cancer cells from dividing, leading to apoptosis. They are commonly used in clinical practice for breast cancer treatment. OBJECTIVE: The present study focused on the effects of vinca alkaloids (vincristine, vinblastine, and vinorelbine) on miRNA expression of treated breast cancer cells. METHODS: We investigated the effect of vincristine, vinblastine, and vinorelbine on the expression of oncogenic and tumor-suppressive miRNAs (miR-15a-5p, miR-16-5p, miR-21-5p, miR-25-3p, miR- 29b-3p, miR-125b-5p, miR-148a-3p, miR-214-3p, miR-221-3p, miR-222-3p, and miR-421), as well as on the expression of the apoptosis-related genes BAX, BCL2, TP53, and CDKN1B in BT-20 and SKBR- 3 breast adenocarcinoma cells. RESULTS: Treatment of BT-20 cells with vincristine, vinblastine, and/or vinorelbine resulted in upregulation of TP53 expression. However, no alterations in the mRNA levels of the pivotal BCL2 family members BAX and BCL2 were observed. On the other hand, treatment of SK-BR-3 cells with any of these vinca alkaloids led to an increase in the BAX/BCL2 mRNA ratio, implying the activation of the intrinsic apoptotic pathway. No concomitant alteration in TP53 expression was observed in treated SKBR- 3 cells. Regarding the miRNAs examined in this study, miR-222-3p expression exhibited the most remarkable modulations in both treated cell lines. CONCLUSION: This study suggests the possible involvement of miR-222-3p expression in breast cancer cell apoptosis, triggered by vincristine, vinblastine, and vinorelbine.