Extracellular microRNAs and endothelial hyperglycaemic memory: a therapeutic opportunity?

Type 2 diabetes mellitus (T2DM) is a major cause of cardiovascular (CV) disease. Several large clinical trials have shown that the risk for patients with diabetes of developing CV complications is only partially reduced by early, intensive glycaemic control and lifestyle interventions, and that such complications result from changes in complex, not fully explored networks that contribute to the maintenance of endothelial function. The accumulation of senescent cells and the low-grade, systemic, inflammatory status that accompanies aging (inflammaging) are involved in the development of endothelial dysfunction. Such phenomena are modulated by epigenetic mechanisms, including microRNAs (miRNAs). MiRNAs can modulate virtually all gene transcripts. They can be secreted by living cells and taken up in active form by recipient cells, providing a new communication tool between tissues and organs. MiRNA deregulation has been associated with the development and progression of a number of age-related diseases, including the enduring gene expression changes seen in patients with diabetes. We review recent evidence on miRNA changes in T2DM, focusing on the ability of diabetes-associated miRNAs to modulate endothelial function, inflammaging and cellular senescence. We also discuss the hypothesis that miRNA-containing extracellular vesicles (i.e. exosomes and microvesicles) could be harnessed to restore a 'physiological' signature capable of preventing or delaying the harmful systemic effects of T2DM.

Potential of acetaminophen on the sublingual microcirculation and peripheral tissue perfusion of febrile septic patients: prospective observational study.

BACKGROUND: Acetaminophen (ACT) has been studied in septic patients with detectable plasmatic levels of cell-free hemoglobin (Hb), where it demonstrated to inhibit the hemoprotein-mediated lipid peroxidation and oxidative injury, with a potential of beneficial effect on the endothelium. On the basis of this background, the aim of this study was to evaluate the sublingual microcirculation and the peripheral tissue perfusion before-and-after administration of ACT on clinical judgment in a cohort of febrile septic and septic shock patients. METHODS: Prospective observational study. 50 adult septic and septic shocks treated with ACT for pyrexia, where the sublingual microcirculation and the peripheral tissue perfusion with Near Infrared Spectroscopy (NIRS) and vascular occlusion test (VOT) were evaluated before ACT (t0), after 30 min (t1) and after 2 h (t2). Cell-free Hb and the markers of oxidative stress and endothelial damage were measured at t0 and t2. RESULTS: The study showed a significant increase of the density of the perfused small and total vessels of the sublingual microcirculation 30 min after the infusion of ACT; it also showed an increase of the Microvascular Flow Index (MFI) and a decrease in the heterogeneity of the flow. At a peripheral muscular level, we found an acceleration in the reperfusion curve after VOT at t1, expression of a higher reactivity of the microvasculature. CONCLUSIONS: ACT infusion did not show a clear correlation with cell-free Hb; however, it exhibited protective effect toward the microcirculation that was evident in particular in septic patients. This correlation merits further exploration.

Leukocyte telomere length is associated with complications of type 2 diabetes mellitus.

OBJECTIVE: The key goal of diabetes management is to prevent complications. While the patho-physiological mechanisms responsible for diabetes complications have been extensively studied, at present it is impossible to predict which patient with diabetes could develop complications. In recent years, the role of leukocyte telomere length in the pathogenesis of cardiovascular disease and Type 2 diabetes has been investigated. However, studies aiming to investigate the role of telomeres in the development and progression of Type 2 diabetes, as well as diabetic complications, are still lacking. As a consequence, this study aimed to verify whether leukocyte telomere length is associated with the presence and the number of diabetic complications in a sample of patients with Type 2 diabetes. METHODS: This is a cross-sectional study. Nine hundred and one subjects were enrolled, including 501 patients with Type 2 diabetes, of whom 284 had at least one complication and 217 were without complications, and 400 control subjects. Leukocyte telomere length was measured by quantitative real-time PCR. RESULTS: Patients with diabetes complications had significantly shorter leukocyte telomere length than both patients without diabetes complications and healthy control subjects. Moreover, among patients with diabetes complications, leukocyte telomere length became significantly and gradually shorter with the increasing number of diabetes complications. The magnitude of the effect of the decrease of the abundance of telomeric template vs. a single-copy gene length (T/S ratio) on complications is described by the estimated odds ratio OR=5.44 (95%CI 3.52-8.42). CONCLUSIONS: The results of the study support the hypothesis that telomere attrition may be a marker associated with the presence and the number of diabetic complications.

SARS-CoV-2 identification in lungs, heart and kidney specimens by transmission and scanning electron microscopy.

From two COVID-19-related deaths, samples of lung, heart and kidney were collected and processed for Transmission and Scanning Electron Microscopy (TEM and SEM) with the aim of identifying the virus. Virions of SARS-CoV-2 were found in all tissues by TEM and SEM, corroborating the hypothesis that the virus enters the cells of different organs. This is the first report identifying SARS-CoV-2 in different human tissues by TEM and SEM.

Telomere/telomerase system impairment in circulating angiogenic cells of geriatric patients with heart failure.

BACKGROUND: The functional characteristics of circulating angiogenic cells (CACs) are impaired in congestive heart failure (CHF) patients, suggesting that CAC dysfunction could contribute to CHF pathogenesis. However, the underlying mechanisms are only partly unraveled. No data are currently available regarding telomere/telomerase system in CACs of CHF patients. METHODS: CACs were obtained from 80 subjects: 40 healthy control subjects (CTR) [median age (IQR), 80 (76-85 yrs)] and 40 patients affected by post-ischemic cardiomyopathy CHF [median age (IQR), 82 (77-89)]. CAC and leukocyte telomere length, assessed as T/S ratio, and telomerase (TERT) activity were determined in all the enrolled subjects. Specificity and sensitivity of CAC and leukocyte T/S in discriminating between CHF and CTR were evaluated using Receiver Operator Characteristic (ROC) curve analysis and reported as AUC values. CD34+/VEGFR2+ number and pro-inflammatory cytokines plasma levels, such as IL-6 and TNF-α, were also measured. RESULTS: CAC T/S and TERT activity were significantly reduced in CHF patients compared to CTR subjects. In leukocytes, only a significant T/S reduction was observed. AUC values were higher for CAC T/S with respect to leukocyte T/S (AUC=0.89, and AUC=0.73, P<0.01, respectively). In multivariate analysis, leukocyte T/S, CAC T/S, CAC TERT activity and NT-proBNP levels were confirmed as parameters significantly associated with CHF. CD34+/VEGFR2+ number, IL-6 and TNF-α plasma levels were significantly increased in CHF patients. CONCLUSIONS: CACs from CHF patients are characterized by telomere/telomerase system impairment, providing new insight into the clinical relevance of CACs in CHF pathogenesis.

Low FasL levels promote proliferation of human bone marrow-derived mesenchymal stem cells, higher levels inhibit their differentiation into adipocytes.

Mesenchymal stem cells (MSCs) are multipotent progenitor cells that can differentiate into several cell types. Bone marrow (BM)-MSCs mainly differentiate into osteoblasts or adipocytes. MSC interactions with their microenvironment directly affect their self-renewal/differentiation program. Here, we show for the first time that Fas ligand (FasL), a well-explored proapoptotic cytokine, can promote proliferation of BM-derived MSCs in vitro and inhibits their differentiation into adipocytes. BM-MSCs treated with a low FasL dose (0.5 ng/ml) proliferated more rapidly than untreated cells without undergoing spontaneous differentiation or apoptosis, whereas higher doses (25 ng/ml) induced significant though not massive BM-MSC death, with surviving cells maintaining a stem cell phenotype. At the molecular level, 0.5 ng/ml FasL induced ERK1/2 phosphorylation and survivin upregulation, whereas 25 ng/ml FasL induced caspase activation. Importantly, 25 ng/ml FasL reversibly prevented BM-MSC differentiation into adipocytes by modulating peroxisome proliferator-activated receptor gamma (PPARγ) and FABP4/aP2 expression induced by adipogenic medium. All such effects were inhibited by anti-Fas neutralizing antibody. The in vitro data regarding adipogenesis were confirmed using Fas(lpr) mutant mice, where higher PPARγ and FABP4/aP2 mRNA and protein levels were documented in whole tibia. These data show for the first time that the FasL/Fas system can have a role in BM-MSC biology via regulation of both proliferation and adipogenesis, and may have clinical relevance because circulating Fas/FasL levels decline with age and several age-related conditions, including osteoporosis, are characterized by adipocyte accumulation in BM.

Noncytotoxic functions of natural killer (NK) cells: large granular lymphocytes (LGL) produce a B cell growth factor (BCGF).

Highly purified human large granular (LGL), depleted of any detectable contaminant T and B cells or monocytes, were found to be potent producers in vitro of a soluble B cell growth factor (BCGF) able to sustain proliferation of B cells activated by anti-mu. Activation by lectins (phytohemagglutinin, PHA, concanavalin A, Con A; and pokeweed mitogen, PWM) was required to induce the production of high levels of this BCGF from cultured LGL. Production of BCGF was also detected after the binding of LGL with natural killer (NK)-sensitive (K562) but not with NK-resistant (RL male 1) target cells. In contrast to T cells, LGL did not need the additional presence of accessory cells to reach optimal production of BCGF by 72 hr of culture. The subpopulation of LGL responsible for the production of BCGF had phenotypic characteristics associated with NK cells (3G8+, HNK1+/OKT11+, DR-, OKT3-, Leu-M1-), and separated cells with these markers exerted high levels of NK activity. Selective production of BCGF also was obtained from cytotoxic clones derived from LGL. A partial characterization of the LGL-derived BCGF was performed by gel filtration. BCGF activity was detected in fractions with estimated m.w. of 20,000 and 45,000. The LGL-derived BCGF activity was resistant to reduction with 2-mercaptoethanol and was stable at -20 degrees C for months. Conversely, heating (56 degrees C for 1 hr) or digestion with trypsin greatly reduced the LGL-derived BCGF activity. These findings strongly suggest that LGL including those with NK activity can play an important positive role in the early events of the B cell-mediated immune response.

GTP-binding proteins transduce signals generated via human FC gamma receptor IIIA (CD16).

This study demonstrates that GTP-binding proteins regulate Fc gamma RIII-mediated signal transduction and inositol phosphate (IPn) generation in human NK cells. In addition the cross-linking of CD16 by mAb, guanosine 5'-o-3-thiophosphate induced 1,4,5 inositol trisphosphate (IP3) release in permeabilized NK cells and their membranes. By contrast, guanosine 5'-o-2-thiophosphate, almost completely inhibited IP3 generation induced by cross-linking with anti-CD16 mAb. Pretreatment of NK cells with 10 to 100 ng/ml Vibrio cholerae toxin (Ctx) almost completely inhibited the generation of IP3 and of other Ipn as well as Fc gamma RIII-operated cell functions such as antibody-dependent cell-mediated cytotoxicity against antibody-coated P815 mastocytoma cells. Isolated B subunit of Ctx was inactive. Bordetella pertussis toxin (0.1 to 1 microgram/ml) only marginally affected IP3 release and antibody-dependent cell-mediated cytotoxicity. Ctx increased cAMP levels in NK cells. However, inhibition of IP3 release preceded the rise of cAMP. Moreover, cAMP analogues (8-chlor-cAMP, 8-bromo-cAMP, dibutiryl-cAMP), as well as intracellular cAMP-enhancing agents (PGE1, PGE2, and forskolin) did not mimicked the effects of Ctx on IP3 generation, suggesting that the adenylate cyclase pathway is not responsible for the early effects of Ctx on Fc gamma RIII-mediated signalling. Overall these results demonstrate that signal transduction via Fc gamma RIII is mediated by Ctx-sensitive cellular membrane GTP-binding protein.

Effects of protein kinase C (PK-C) activators and inhibitors on human large granular lymphocytes (LGL): role of PK-C on natural killer (NK) activity.

The role of protein kinase C (PK-C) in the early metabolic events involved in human natural killer (NK) cell activation has been studied through the action of PK-C-specific activators and inhibitors. Highly purified human large granular lymphocytes (LGL) were treated for 1 hr with the diacylglycerol analog 1-oleoyl-2-acetyl glycerol (OAG) (10(-4)-10(-5) g/ml) or with 12-O-tetradecanoylphorbol-13-acetate (TPA) (10(-8)-10(-10) g/ml), both specific activators of PK-C. Both these agents consistently increased NK activity against K562 target cells. Suboptimal doses of either OAG or TPA also synergized with Ca2+ ionophores to augment spontaneous cytotoxic activity. Pretreatment of LGL with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrocloride (H7) (5-40 microM), a potent PK-C inhibitor, greatly reduced NK activity in a time- and dose-dependent fashion. By contrast, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride (HA 1004), a potent cAMP- and cGMP-dependent PK inhibitor with almost no effect on PK-C, marginally reduced NK activity. Moreover, almost complete NK activity inhibition was observed when H7 (10 microM), but not HA 1004 (50 microM), was present in the NK assay. Finally, 48 hr stimulation of LGL with TPA (10(-6) g/ml), a treatment able to inactivate most of the PK-C cellular pool, almost completely abrogated NK activity. This functional evidence was supported by phosphorylation of several endogenous substrates which occurs within 5 min in TPA-treated LGL. Two proteins of 70 and 56 kDa have been identified as major PK-C substrates, together with other phosphorylated proteins with MW ranging from 177 to 43 kDa. H7, but not HA 1004, almost completely inhibited the TPA-induced phosphorylation of all of these proteins in the NK cells. These data strongly suggest that selective activation of PK-C plays an essential role in the mechanisms of NK cell activation.

Production of multiple cytokines by clones of human large granular lymphocytes.

LGL in addition to mediating natural killer (NK) activity, can secrete a variety of lymphokines, depending on the stimulus used: interleukin-1 (IL-1), interleukin-2 (IL-2), interferon alpha and gamma (IFN), and B-cell growth factor (BCGF). To define more directly whether cells with NK activity can also secrete one or more cytokines, we obtained clones by limiting dilution assays from highly purified preparations of human LGL and cultured them in IL-2-containing medium for several weeks. All the clones tested spontaneously produced detectable levels of IFN-gamma and 35 of 40 clones (87%) produced higher levels when stimulated with PHA. A smaller proportion (16%) of clones (9 of 54) secreted IL-1 after stimulation with LPS, while 34% of the clones (17 of 49) produced IL-2 in response to PHA stimulation. Cytokine production was associated with both cytotoxic and noncytotoxic clones and did not correlate with their surface phenotype, as has been observed for fresh LGL. The ability to produce IL-1 or IL-2 was not usually found within the same clones following PHA and LPS stimulation, respectively; however two clones produced both IL-1 and IL-2 when stimulated in different experiments, but not at the same time. In addition, two of nine cloned LGL simultaneously produced IFN gamma and IL-1. These results indicate that LGL-derived clones have the ability to produce multiple cytokines, suggesting that the LGL population may play an important immunoregulatory role and may also be capable of self-regulation of cytolytic activity.

Comparative evaluation of multiple lymphoid and recombinant human interleukin-2 preparations.

Six lymphoid human interleukin-2s (nIL-2s) [four from peripheral blood mononuclear cells (PBMC) and two from JURKAT cells] and six recombinant IL-2s (rIL-2s) were obtained for comparative evaluation. The main issues addressed were possible differences among the preparations in potency in T cell growth assays and other functional assays, and the possible presence of other cytokine activities or contaminants. Each preparation was assigned a standardized IL-2 activity in reference units (RU) by comparing its T cell growth promoting activity against the Biological Response Modifiers Program IL-2 (JURKAT) reference reagent. Relative to the IL-2 unitage indicated by the suppliers, the RU varied from 110-fold less to 38.5-fold more for the various preparations. Two nIL-2s and two rIL-2s contained significant levels of endotoxin. One nIL-2 contained low levels of both alpha and gamma interferon (IFN), and one nIL-2 had a high level of gamma IFN. All other IL-2s were negative for IFN activity. All IL-2 preparations significantly augmented human natural killer (NK) activity, although the amount of RU required varied from 0.1 to 50 RU. Four nIL-2s and three rIL-2s induced human PBMC to produce gamma IFN, whereas two nIL-2s and one rIL2 did not. All nIL-2s had substantial amounts of B cell growth factor activity, whereas none of the rIL-2s consistently displayed this activity. All IL-2s stimulated the tritiated thymidine [3H]TdR incorporation of human PBMC in the absence of other stimuli, in addition to augmenting the response to mitogen or alloantigens. Some nILs and IL-2s had effects on human monocytes such as inhibiting migration, inducing cytotoxic or growth inhibitory activity against tumor cells, and causing changes in cell surface markers. The IL-2s were also tested for activity in vitro and in vivo in mice. Although there was a 12-fold variation in activity among the preparations, all but one of the IL-2s showed augmentation of the mixed lymphocyte reaction activity and all IL-2s tested stimulated macrophage cytotoxicity in vitro. All IL-2s tested enhanced the mixed lymphocyte-allogeneic tumor cell reaction resulting in greater production of cytotoxic T cells. However, significant quantitative differences in potency were evident among the various IL-2 preparations, especially the nIL-2s. Only very high doses of IL-2 (intraperitoneal injection of 100,000 RU/animal) induced in vivo augmentation of splenic or peritoneal NK cells, although all IL-2s tested increased NK activity against tumor target cells in vitro with substantially lower doses (10-100 RU/ml).(ABSTRACT TRUNCATED AT 400 WORDS)