Release of Pro-Inflammatory/Angiogenic Factors by Retinal Microvascular Cells Is Mediated by Extracellular Vesicles Derived from M1-Activated Microglia.

The interactions between the neuronal and vascular sides of the retina during diabetic retinopathy (DR) have gained increasing attention. Microglia is responsible for the immune response to inflammation inside the retina, which could be mediated by paracrine signals carried by extracellular vesicles (EVs). We aimed to characterize EVs released from immortalized human microglial cells in inflammation and investigate their effects on the retinal microvasculature and the anti-inflammatory potential of thiamine in this context. M1 pro-inflammatory polarization in microglia was induced through a cytokine cocktail. EVs were isolated from the supernatants, characterized, and used to stimulate human retinal endothelial cells (HRECs) and pericytes (HRPs). Microvascular cell functions and their release of pro-inflammatory/angiogenic factors were assessed. M1-derived EVs showed increased content of miR-21, miR-155, CCL2, MMP2, and MMP9, and enhanced apoptosis, proliferation, migration, and ROS production in HRPs and HRECs. IL-1ß, IL-6, MMP9, CCL2, and VEGF release increased in HRPs exposed to M1-derived EVs, while HRECs showed augmented IL-6, Ang2, VEGF, and PDFG-B. Addition of thiamine to M1-microglial cultures reverted most of these effects. In conclusion, M1-derived EVs stimulate functional changes and secretion of pro-inflammatory/angiogenic molecules in microvascular cells, exacerbating inflammatory damage and retinopathy features. Thiamine added to microglia exerts anti-inflammatory effects.

Multidrug-Resistant Bloodstream Infections in Internal Medicine: Results from a Single-Center Study.

OBJECTIVES: Infections due to multidrug-resistant organisms (MDROs) are expanding globally and are associated with higher mortality rates and hospital-related costs. The objectives of this study were to analyze the trends of MDRO bacteremia and antimicrobial resistance rates in Internal Medicine wards of our hospital and to identify the variables associated with these infections. METHODS: During a 6-year period (July 1, 2011-June 30, 2017), patients with positive blood culture isolates hospitalized in the Internal Medicine wards in the Santa Croce and Carle Hospital in Cuneo, Italy, were assessed. We performed an analysis taking into consideration the time trends and frequencies of MDRO infections, as well as a case-control study to identify clinical-demographic variables associated with MDRO bacteremias. RESULTS: During the study period a total of 596 blood cultures were performed in 577 patients. The most frequently identified organism was Escherichia coli (33.7%), followed by Staphylococcus aureus (15.6%) and S epidermidis (7.4%). The percentage of resistance to methicillin among S aureus isolates showed a decreasing trend, whereas rates of extended-spectrum ß-lactamase-producing Enterobacteriaceae and carbapenemase-producing Klebsiella pneumoniae increased during the study period. Multivariate analysis showed that the nosocomial origin of the infection, hospitalization during the previous 3 months, residence in long-term care facilities, presence of a device, antibiotic exposure during the previous 3 months, and cerebrovascular disease were independently associated with bacteremia by resistant microorganisms. CONCLUSIONS: Our analysis reveals a concerning microbiological situation in an Internal Medicine setting, in line with other national and regional data. The risk variables for infection by MDRO identified in our study correspond to those reported in the literature, although studies focused on Internal Medicine settings appear to be limited.

Characterization of an Immortalized Human Microglial Cell Line as a Tool for the Study of Diabetic Retinopathy.

The complexity of the retinal structure reflects on the difficulty to describe its composite cell interactions. Microglia is responsible for the immune reaction to inflammatory stimuli during diabetic retinopathy (DR), but most studies still use rodent cells. We characterized a commercially available immortalized human microglial line and tested its susceptibility to inflammation, to study the interactions between the neuro-vascular retinal portions in species-specific models. After checking the expression of microglial markers, we tried lipopolysaccharide (LPS) stimulation and several pro-inflammatory cocktails to select the best combination able to induce a significant M1 (inflammatory) response. We measured M1 induction through the expression of pro- and anti-inflammatory molecules and performed morphologic and functional assays. Marker expression confirmed the human microglial derivation of these cells. Differently from rodents, LPS did not induce a M1 profile. The best pro-inflammatory stimulus was an interleukin-1ß + tumor necrosis factor-α + interferon-γ cocktail, which induced morphology changes and increased proliferation, apoptosis, migration, reactive oxygen species, and the expression of inflammatory cytokines and miRNAs. In conclusion, this microglial line proved potentially useful to investigate the cascade of events leading to DR. In perspective, co-culture models involving microvascular cells will help in the understanding of multifaceted interactions of the neurovascular unit.

Peripheral neuropathy after viral eradication with direct-acting antivirals in chronic HCV hepatitis: A prospective study.

BACKGROUND: HCV-related extra-hepatic complications include peripheral neuropathies, with important prevalence and impact. A recent metanalysis of previous intervention trials concluded for insufficient data to support evidence-based treatments for this complication. In this longitudinal study, we assessed for the first time prevalence and outcome of neuropathy in a cohort of patients with chronic HCV, before and after direct-acting antiviral agent (DAA) treatment. METHOD: Ninety-four patients (mean age 58.5 ± 9.9, infection duration 22.2 ± 6.3 years) without systemic and metabolic diseases, underwent neurological examination and electroneurography studies before (T0) and 10.4 ± 1.7 months after the end of DAA therapy (T1), and cryoglobulins (CG) assessment. Muscle strength was evaluated by Medical Research Council (MRC) score; neuropathic pain, sensory function, disability, quality of life were assessed by validated questionnaires (DN4, NPSI, SSS, INCAT and Euro-QoL). RESULTS: At T0, sensory-motor neuropathy was detected in 22 patients (23%), reflexes were depressed in 32 (34%) with no association with infection duration, viral load, age, CG. Neuropathic pain (DN4 ≥4) was present in 37 patients (39%). At T1, out of the 22 patients with altered electroneurography, 3 had died or developed HCC, 4 showed normal electroneurography, and nerve amplitude parameters tended to improve in the whole group. Only 11 patients (12%) had depressed reflexes and 10 (11%) DN4 ≥4 (P < .05 compared to T0). Scores for MRC, questionnaires and Euro-QoL improved significantly (P < .05). CONCLUSION: Our study confirms the high prevalence of clinical and subclinical peripheral sensory-motor neuropathy in patients with HCV infection and indicates improvement after eradication by DAA. These results support the need for larger intervention studies.

Decreasing prevalence of retinopathy in childhood-onset type 1 diabetes over the last decade: A comparison of two cohorts diagnosed 10 years apart.

AIM: To ascertain whether the prevalence of retinopathy has declined over the last 2 decades in individuals with childhood-onset type 1 diabetes and whether this might be explained by changes in lifetime HbA1c. MATERIALS AND METHODS: A multicentre, retrospective, observational study, comparing 128 subjects with diabetes onset in 2000-2003 assessed for retinopathy in 2016-2019, with a previous cohort of 115 individuals diagnosed in 1990-1993 and assessed for retinopathy in 2007-2009, was conducted. The two cohorts had both a similar diabetes duration and age at diagnosis. Retinal photographs were centrally graded. Lifetime HbA1c and its variability, estimated as the ratio between intrapersonal mean and standard deviation of HbA1c, were evaluated. RESULTS: The prevalence of any retinopathy in the new and old cohort was 24.2% and 43.5% (P < .003), respectively, and that of severe retinopathy was 1.7% and 9.6% (P = .018). Lifetime HbA1c was lower in the new cohort (7.8% ± 0.8% vs. 8.1% ± 0.8%; P = .002) during all periods following the first 5 years after diagnosis. Patients without retinopathy in the two cohorts had similar levels of HbA1c. Compared with patients without retinopathy, those with retinopathy had higher lifetime HbA1c and long-term HbA1c variability. However, on multiple regression analysis, only lifetime HbA1c was independently associated with retinopathy (P = .0018). CONCLUSIONS: The risk of developing retinopathy was nearly halved in children who developed type 1 diabetes in the new millennium compared with previous cohorts. These results confirm that maintaining the lowest possible levels of HbA1c throughout lifetime protects from diabetic retinopathy.

Glargine insulin loaded lipid nanoparticles: Oral delivery of liquid and solid oral dosage forms.

BACKGROUND AND AIMS: The oral administration of insulin has so far been precluded by gastro-intestinal enzyme degradation and poor intestinal absorption. Preliminary evidence for peptide uptake by the gut has recently been obtained, by our research group, following the administration of nanostructured lipid-carrier suspensions loaded with glargine insulin in healthy animal models. METHODS AND RESULTS: In this experimental study, glargine insulin-loaded nanostructured lipid carriers have been converted into solid oral dosage forms (tablets, capsules), that are more suitable for administration in humans and have prolonged shelf-life. The liquid and solid oral dosage forms were tested for glargine insulin uptake and glucose responsivity in healthy and streptozotocin-induced diabetic rats (6 animals in each group). A suitable composition gave redispersible solid oral dosage forms from glargine insulin-loaded carriers, using both spray-drying and freeze-drying. It was observed that the liquid and solid formulations had relevant hypoglycaemic effects in healthy rats, while only capsules were efficacious in diabetic rats; probably because of gut alterations in these animal models. Detected glargine insulinaemia was consistent with a glycaemic profile. CONCLUSION: The formulations under study showed their potential as oral glucose-lowering agents, particularly when used as capsules. However, further study is needed to produce a useful orally-active insulin preparation.

Emerging drugs for the treatment of diabetic retinopathy.

INTRODUCTION: Diabetic retinopathy (DR) is one of the main pathological features of the diabetes mellitus spectrum. It is estimated that in 2020 about 4 million people worldwide suffered from blindness or visual impairment caused by DR. Many patients cannot access treatment, mostly because of high costs, while others discontinue it prematurely due to the high number of intravitreal administrations required, or the occurrence of ocular complications, or discomfort in quality of life. AREAS COVERED: The aims of this paper are to summarize the current understanding of the pathogenesis and treatment of diabetic retinopathy, focus on the most promising new approaches to treatment that are being evaluated in clinical trials, and outline the potential financial impact of new drugs in future markets. EXPERT OPINION: Slow-release systems with steroids, anti-VEGF or sunitinib are promising. Oral imatinib would avoid the ocular complications of intravitreal drugs. Brolucizumab and abicipar pegol may be superior to aflibercept and ranibizumab with the advantage of less frequent administrations. Faricimab, active on Tie-2 receptors, is being evaluated in two phase 3 clinical trials. Further knowledge of the efficacy and safety of these drugs is necessary before their final approval for the treatment of diabetic retinopathy.

Self-management education may improve blood pressure in people with type 2 diabetes. A randomized controlled clinical trial.

BACKGROUND AND AIMS: Diabetes is a suitable model to evaluate intervention programmes aimed at chronic diseases, because of its well-defined and measurable process and outcome indicators. In this study, we aimed at investigating the effects of group based self-management education on clinical and psychological variables in type 2 diabetes. METHODS AND RESULTS: Four-year randomized controlled clinical trial (ISRCTN14558376) comparing Group Care and traditional one-to-one care. Clinical and psychological variables were monitored at baseline, 2 and 4 years. Although differences between groups appear to be non-significant at univariate analysis, body weight, BMI and HbA1c, systolic and diastolic blood pressure improved in the patients followed by Group Care but not among Controls. Prescription of lipid-lowering and anti-hypertensive agents did not change among the patients on Group Care, whereas anti-hypertensives were stepped up among Controls without improving their blood pressure. Multivariable analysis suggests that blood pressure improvement among patients on Group Care was independent of BMI, duration of diabetes and antihypertensive medication, suggesting a direct effect of education, presumably by increasing adherence. The "Powerful Others" dimension of the Locus of Control worsened and fear of complications decreased among Controls. CONCLUSIONS: The results confirm that a multidisciplinary structured group educational approach improves blood pressure, presumably through better adherence to healthy lifestyle and medication, in people with type 2 diabetes. CLINICAL TRIAL REGISTRATION NUMBER: ISRCTN14558376.

Functional analysis of miR-21-3p, miR-30b-5p and miR-150-5p shuttled by extracellular vesicles from diabetic subjects reveals their association with diabetic retinopathy.

Microvascular dysfunctions due to altered interactions between endothelial cells (ECs) and pericytes are key-events in the pathogenesis of diabetic retinopathy. Extracellular vesicles (EVs) derived from mesenchymal stem cells cultured in diabetic-like conditions enter pericytes, cause their detachment and migration, and stimulate angiogenesis. We recently showed that EVs from diabetic patients with retinopathy have different miRNA profiling patterns from healthy controls, and determine features of retinopathy in in vitro models of retinal microvasculature. In particular, a role for intra-vesicle miR-150-5p, miR-21-3p and miR-30b-5p was hypothesized. In this work, we further characterized EVs from subjects with diabetic retinopathy and investigated miR-150-5p, miR-21-3p and miR-30b-5p functions inside microvascular cells. Human retinal pericytes and ECs were transfected with mimics or inhibitors, as appropriate, of miR-21-3p, miR-30b-5p and miR-150-5p, to evaluate their ability in promoting cell migration and tube formation. mRNA and protein profiling of EVs extracted from diabetic subjects with (DR group) or without retinopathy (noDR group), and healthy controls (CTR group) were also performed. Modulation of miR-150-5p, miR-21-3p and miR-30b-5p inside microvascular cells confirmed their involvement in abnormal angiogenesis. mRNA analysis revealed differing expression of 7 genes involved in angiogenesis, while subsequent protein analysis confirmed increased expression of HIF-1α in DR group. Since all these molecules are involved in the hypoxia-induced retinal damage characteristic of the disease, our data reinforce the hypothesis of a potential use of miR-150-5p, miR-21-3p and miR-30b-5p extracted from circulating EVs as prognostic biomarkers for diabetic retinopathy.

Molecular and functional characterization of circulating extracellular vesicles from diabetic patients with and without retinopathy and healthy subjects.

Diabetic retinopathy is a sight-threatening complication of diabetes, characterized by loss of retinal pericytes and abnormal angiogenesis. We previously demonstrated that extracellular vesicles (EVs) derived from mesenchymal stem cells cultured in diabetic-like conditions are able to enter the pericytes, causing their detachment and migration, and stimulating angiogenesis in vitro. The purpose of this work was the molecular and functional characterization of EVs derived from diabetic subjects with or without diabetic retinopathy, compared with healthy controls. Characterization of EVs extracted from serum/plasma of diabetic patients with or without retinopathy, and healthy controls, was performed by FACS and microarray analysis of microRNA (miRNA) content. Relevant miRNA expression was validated through qRT-PCR. EV influence on pericyte detachment, angiogenesis and permeability of the blood-retinal barrier was also investigated. Diabetic subjects had a 2.5 fold higher EV concentration than controls, while expression of surface molecules was unchanged. Microarray analysis revealed 11 differentially expressed miRNAs. Three of them (miR-150-5p, miR-21-3p and miR-30b-5p) were confirmed by qRT-PCR. Plasma EVs from subjects with diabetic retinopathy induced pericyte detachment and pericyte/endothelial cell migration, increased the permeability of pericyte/endothelial cell bilayers and the formation of vessel-like structures, when compared with EVs from controls. In conclusion, circulating EVs show differences between diabetic patients and healthy subjects. EVs extracted from plasma of diabetic retinopathy patients are able to induce features of retinopathy in in vitro models of retinal microvasculature. Our data suggest a role for miR-150-5p, miR-21-3p and miR-30b-5p as potential biomarkers of the onset of diabetic retinopathy.

Type 2 diabetes affects bone cells precursors and bone turnover.

BACKGROUND: Here we study the effect of type 2 diabetes (T2DM) on bone cell precursors, turnover and cytokines involved in the control of bone cell formation and activity. METHODS: We enrolled in the study 21 T2DM women and 21 non diabetic controls matched for age and body mass index (BMI). In each subject we measured bone cell precursors, Receptor Activator of Nuclear Factor κB (RANKL), Osteoprotegerin (OPG), Sclerostin (SCL) and Dickoppf-1 (DKK-1) as cytokines involved in the control of osteoblast and osteoclast formation and activity, bone density (BMD) and quality trough trabecular bone score (TBS) and bone turnover. T2DM patients and controls were compared for the analyzed variables by one way ANOVA for Gaussian ones and by Mann-Whitney or Kruskal-Wallis test for non-Gaussian variables. RESULTS: RANKL was decreased and DKK-1 increased in T2DM. Accordingly, patients with T2DM have lower bone turnover compared to controls. BMD and TBS were not significantly different from healthy controls. Bone precursor cells were more immature in T2DM. However the number of osteoclast precursors was increased and that of osteoblasts decreased. CONCLUSIONS: Patients with T2DM have more immature bone cells precursors, with increased number of osteoclasts and decreased osteoblasts, confirming low bone turnover and reduced cytokines such as RANKL and DKK-1. BMD and TBS are not significantly altered in T2DM although, in contrast with other studies, this may be due to the match of patients and controls for BMI rather than age.

Somatostatin protects human retinal pericytes from inflammation mediated by microglia.

Diabetic retinopathy (DR) is usually considered a microvascular disease. However, involvement of the neuroretina in the early stages of DR has recently gained major credit. Inflammatory processes, leading to glial activation and neuronal apoptosis, develop early in the retina of diabetic subjects. Pericytes constitute a link between the vascular and the neural retina, play a central role in blood-retinal barrier maintenance, and may influence neuroinflammation. Somatostatin (SST) is a potent neuroprotective factor, which is down-regulated during early DR. In this paper, we have investigated the effects of the inflammatory signals triggered by the activation of microglia on inflammation and apoptosis/survival pathways in pericytes. Microglia cells (Bv-2) were stimulated with lipopolysaccharide (LPS) and/or SST. Human retinal pericytes (HRP) were exposed to conditioned media (CM) collected from Bv-2 cells in physiological conditions and in the settings described above. A panel of inflammation, apoptosis and survival mediators was analyzed. HRP treated with LPS-CM showed a significant increase of pro-inflammatory (iNos and TNFα) and pro-apoptotic mediators (FasL, active caspase-8, tBid and Bax), and a concomitant decrease in pro-survival factors (BclxL and pAkt). SST added to LPS was able to counteract these effects in all conditions. In conclusion, SST is able to modulate apoptosis/survival pathways in HRP during microglia-mediated inflammation. These results demonstrate a crosstalk between microglia and retinal pericytes, evidencing a possible defensive role of microglia in the early phases of DR.

Urinary proteomics predict onset of microalbuminuria in normoalbuminuric type 2 diabetic patients, a sub-study of the DIRECT-Protect 2 study.

BACKGROUND: Early prevention of diabetic nephropathy is not successful as early interventions have shown conflicting results, partly because of a lack of early and precise indicators of disease development. Urinary proteomics has shown promise in this regard and could identify those at high risk who might benefit from treatment. In this study we investigate its utility in a large type 2 diabetic cohort with normoalbuminuria. METHODS: We performed a post hoc analysis in the Diabetic Retinopathy Candesartan Trials (DIRECT-Protect 2 study), a multi centric randomized clinical controlled trial. Patients were allocated to candesartan or placebo, with the aim of slowing the progression of retinopathy. The secondary endpoint was development of persistent microalbuminuria (three of four samples). We used a previously defined chronic kidney disease risk score based on proteomic measurement of 273 urinary peptides (CKD273-classifier). A Cox regression model for the progression of albuminuria was developed and evaluated with integrated discrimination improvement (IDI), continuous net reclassification index (cNRI) and receiver operating characteristic curve statistics. RESULTS: Seven hundred and thirty-seven patients were analysed and 89 developed persistent microalbuminuria (12%) with a mean follow-up of 4.1 years. At baseline the CKD273-classifier predicted development of microalbuminuria during follow-up, independent of treatment (candesartan/placebo), age, gender, systolic blood pressure, urine albumin excretion rate, estimated glomerular filtration rate, HbA1c and diabetes duration, with hazard ratio 2.5 [95% confidence interval (CI) 1.4-4.3; P = 0.002] and area under the curve 0.79 (95% CI 0.75-0.84; P < 0.0001). The CKD273-classifier improved the risk prediction (relative IDI 14%, P = 0.002; cNRI 0.10, P = 0.043). CONCLUSIONS: In this cohort of patients with type 2 diabetes and normoalbuminuria from a large intervention study, the CKD273-classifier was an independent predictor of microalbuminuria. This may help identify high-risk normoalbuminuric patients for preventive strategies for diabetic nephropathy.

Vena Cava Responsiveness to Controlled Isovolumetric Respiratory Efforts.

OBJECTIVES: Respirophasic variation of inferior vena cava (IVC) size is affected by large variability with spontaneous breathing. This study aims at characterizing the dependence of IVC size on controlled changes in intrathoracic pressure. METHODS: Ten healthy subjects, in supine position, performed controlled isovolumetric respiratory efforts at functional residual capacity, attaining positive (5, 10, and 15 mmHg) and negative (-5, -10, and -15 mmHg) alveolar pressure levels. The isovolumetric constraint implies that equivalent changes are exhibited by alveolar and intrathoracic pressures during respiratory tasks. RESULTS: The IVC cross-sectional area equal to 2.88 ± 0.43 cm2 at baseline (alveolar pressure = 0 mmHg) was progressively decreased by both expiratory and inspiratory efforts of increasing strength, with diaphragmatic efforts producing larger effects than thoracic ones: -55 ± 15% decrease, at +15 mmHg of alveolar pressure (P < .01), -80 ± 33 ± 12% at -15 mmHg diaphragmatic (P < .01), -33 ± 12% at -15 mmHg thoracic. Significant IVC changes in size (P < .01) and pulsatility (P < .05), along with non significant reduction in the response to respiratory efforts, were also observed during the first 30 minutes of supine rest, detecting an increase in vascular filling, and taking place after switching from the standing to the supine position. CONCLUSIONS: This study quantified the dependence of the IVC cross-sectional area on controlled intrathoracic pressure changes and evidenced the stronger influence of diaphragmatic over thoracic activity. Individual variability in thoracic/diaphragmatic respiratory pattern should be considered in the interpretation of the respirophasic modulations of IVC size.

Somatostatin protects photoreceptor cells against high glucose-induced apoptosis.

PURPOSE: Many cellular and molecular studies in experimental animals and early retinal function tests in patients with diabetic retinopathy (DR) have shown that retinal neurodegeneration is an early event in the pathogenesis of the disease. Somatostatin (SST) is one of the most important neuroprotective factors synthesized by the retina: SST levels are decreased in parallel to retinal neurodegeneration in early stages of DR. In this study, we characterized the induction of apoptosis (programmed cell death) in a 661W photoreceptor-like cell line cultured under high glucose (HG) conditions and the effect of SST. METHODS: A 661W photoreceptor-like cell line and retinal explants from 10-week-old male C57BL/6 mice were cultured under HG conditions and treated with SST. RESULTS: Hyperglycemia significantly reduced the cellular viability by increasing the percentage of apoptotic cells, and this effect was ameliorated by SST (p˂0.05). Activation of caspase-8 by hyperglycemia was found in the 661W cells and retinal explants and decreased in the presence of SST (p˂0.05). Moreover, we detected activation of calpain-2 associated with hyperglycemia-induced cell death, as well as increased protein tyrosine phosphatase 1B (PTP1B) protein levels; both had a pattern of cleavage that was absent in the presence of SST (p˂0.05). Treatment of the 661W cells and retinal explants with SST for 24 h increased the phosphorylation of type 1 insulin-like growth factor receptor (IGF-IR; tyrosine 1165/1166) and protein kinase B (Akt; serine 473), suggesting this survival signaling is activated in the neuroretina by SST (p˂0.05). CONCLUSIONS: This study has provided new mechanistic insights first into the involvement of calpain-2 and PTP1B in the loss of cell survival and increased caspase-8-dependent apoptosis induced by hyperglycemia in photoreceptor cells and second, on the protective effect of SST against apoptosis by the enhancement of IGF-IR-mediated Akt phosphorylation.

THE STORM (acute coronary Syndrome in paTients end Of life and Risk assesMent) study.

INTRODUCTION: Elderly patients with coexisting frailty and multiple comorbidities frequently present to the emergency department (ED). Because non-cardiovascular comorbidities and declining health status may affect their life expectancy, management of these patients should start in the ED. This study evaluated the role of Gold Standards Framework (GSF) criteria for identifying patients with acute coronary syndromes (ACS) approaching end of life. METHODS: All consecutive patients admitted to the ED and hospitalised with a diagnosis of ACS between May 2012 and July 2012 were included. According to GSF criteria, patients were labelled as positive GSF status when they met at least one general criterion and two heart disease criteria; furthermore, traditional cardiovascular risk scores (the Global Registry for Acute Coronary Events (GRACE) score and the Age, Creatinine and Ejection Fraction (ACEF) score) were calculated and WHOQOL-BREF was assessed. Mortality and repeat hospitalisation due to cardiovascular and non-cardiovascular causes were evaluated at 3-month and 12-month follow-up. RESULTS: From a total of 156 patients with ACS enrolled, 22 (14%) had a positive GSF. A positive GSF was associated with higher rate of non-cardiovascular events (22.7% vs 6.7%; p=0.03) at 3 months and higher rates of both cardiovascular and non-cardiovascular events (36% vs 16.4%; p=0.04 and 27.3% vs 6.7%; p=0.009, respectively) at 12 months. In multivariate analysis, an in-hospital GRACE score was a predictor of cardiovascular events, while a positive GSF independently predicted non-cardiovascular events. CONCLUSIONS: The GSF score independently predicts non-cardiovascular events in patients presenting with ACS and may be used along with traditional cardiovascular risk scores in choosing wisely the most appropriate treatment. The present results need to be externally validated on larger samples.

Human mesenchymal stem cell-derived microvesicles modulate T cell response to islet antigen glutamic acid decarboxylase in patients with type 1 diabetes.

AIMS/HYPOTHESIS: Mesenchymal stem cells (MSCs) have been shown to abrogate in vitro the proinflammatory response in type 1 diabetes. The mechanism involves paracrine factors, which may include microvesicles (MVs). We evaluated whether MVs derived from heterologous bone-marrow MSCs exert an immunomodulatory effect on T cell responses against GAD (glutamic acid decarboxylase) antigen in type 1 diabetes. METHODS: MVs were purified from heterologous human MSCs by differential centrifugation. Peripheral blood mononuclear cells (PBMCs) were obtained from patients with type 1 diabetes at disease onset, and responses to GAD65 stimulation were assessed by IFN-γ enzyme-linked immunosorbent spot analysis. Levels of cytokines and prostaglandin E2 (PGE2) were measured in the supernatant fraction, and T helper 17 (Th17) and regulatory T cell analysis was performed. RESULTS: MVs were internalised by PBMCs, as assessed by confocal microscopy and flow cytometry analyses. MVs significantly decreased IFN-γ spots and levels in GAD65-stimulated PBMCs, and significantly increased transforming growth factor-ß (TGF-ß), IL-10, IL-6 and PGE2 levels. Furthermore, MVs decreased the number of Th17 cells and the levels of IL-17, and increased FoxP3(+) regulatory T cells in GAD65-stimulated PBMCs. CONCLUSIONS/INTERPRETATION: These results provide evidence that MSC-derived MVs can inhibit in vitro a proinflammatory response to an islet antigenic stimulus in type 1 diabetes. The action of MVs involves PGE2 and TGF-ß signalling pathways and IL-10 secretion, suggesting a switch to an anti-inflammatory response of T cells.

Platelet-derived growth factor regulates the secretion of extracellular vesicles by adipose mesenchymal stem cells and enhances their angiogenic potential.

BACKGROUND: Several studies demonstrate the role of adipose mesenchymal stem cells (ASCs) in angiogenesis. The angiogenic mechanism has been ascribed to paracrine factors since these cells secrete a plenty of signal molecules and growth factors. Recently it has been suggested that besides soluble factors, extracellular vesicles (EVs) that include exosomes and microvesicles may play a major role in cell-to-cell communication. It has been shown that EVs are implicated in the angiogenic process. RESULTS: Herein we studied whether EVs released by ASCs may mediate the angiogenic activity of these cells. Our results demonstrated that ASC-derived EVs induced in vitro vessel-like structure formation by human microvascular endothelial cells (HMEC). EV-stimulated HMEC when injected subcutaneously within Matrigel in SCID mice formed vessels. Treatment of ASCs with platelet-derived growth factor (PDGF) stimulated the secretion of EVs, changed their protein composition and enhanced the angiogenic potential. At variance of EVs released in basal conditions, PDGF-EVs carried c-kit and SCF that played a role in angiogenesis as specific blocking antibodies inhibited in vitro vessel-like structure formation. The enhanced content of matrix metalloproteinases in PDGF-EVs may also account for their angiogenic activity. CONCLUSIONS: Our findings indicate that EVs released by ASCs may contribute to the ASC-induced angiogenesis and suggest that PDGF may trigger the release of EVs with an enhanced angiogenic potential.