Regenerative Medicine and Eye Diseases

The chapter examines the use of stem cells in ophthalmological pathologies affecting both the anterior and posterior segments. The authors review the clinical trials that have most contributed to defining the role and potential of stem cell regenerative therapy in corneal and retinal pathology. The results described in the scientific literature are analyzed and commented, without neglecting the possible side effects related to the use of this therapy. Within the anterior segment, the greatest efforts were made to study the possible uses of limbal epithelial stem cells (LESCs). They were the first stem cells to be discovered at the level of the anterior segment and currently the only ones involved in clinical practice with satisfactory results. At this juncture there have been significant successes in the treatment of corneal stem cell deficiency and of corneal scars. The chapter later investigates the possible applications of stem cell therapy in degenerative retinal diseases, with particular reference to retinitis pigmentosa, Stargardt’s disease, and age-related macular degeneration. It then describes how the use of cell therapies, in particular those that use ADSC, can contribute, through various methods, to the containment of the evolution of retinal degenerative diseases. These mechanisms cover various biological aspects and can be summarized as follows: neurotrophism, oxidation, vascular changes, apoptosis, inflammation, or immunology. The ophthalmological modalities of the cell graft and what is the ideal approach for an ophthalmological cellular surgery are later on described. Finally, the technique used by the author and the possible outcomes in the course of degenerative retinopathy are described

Selection of reference genes is critical for miRNA expression analysis in human cardiac tissue. A focus on atrial fibrillation

MicroRNAs (miRNAs) are emerging as key regulators of complex biological processes in several cardiovascular diseases, including atrial fibrillation (AF). Reverse transcription-quantitative polymerase chain reaction is a powerful technique to quantitatively assess miRNA expression profile, but reliable results depend on proper data normalization by suitable reference genes. Despite the increasing number of studies assessing miRNAs in cardiac disease, no consensus on the best reference genes has been reached. This work aims to assess reference genes stability in human cardiac tissue with a focus on AF investigation. We evaluated the stability of five reference genes (U6, SNORD48, SNORD44, miR-16, and 5S) in atrial tissue samples from eighteen cardiac-surgery patients in sinus rhythm and AF. Stability was quantified by combining BestKeeper, delta-Cq, GeNorm, and NormFinder statistical tools. All methods assessed SNORD48 as the best and U6 as the worst reference gene. Applications of different normalization strategies significantly impacted miRNA expression profiles in the study population. Our results point out the necessity of a consensus on data normalization in AF studies to avoid the emergence of divergent biological conclusions

Albiglutide and cardiovascular outcomes in patients with type 2 diabetes and cardiovascular disease (Harmony Outcomes): a double-blind, randomised placebo-controlled trial

Background: Glucagon-like peptide 1 receptor agonists differ in chemical structure, duration of action, and in their effects on clinical outcomes. The cardiovascular effects of once-weekly albiglutide in type 2 diabetes are unknown. We aimed to determine the safety and efficacy of albiglutide in preventing cardiovascular death, myocardial infarction, or stroke. Methods: We did a double-blind, randomised, placebo-controlled trial in 610 sites across 28 countries. We randomly assigned patients aged 40 years and older with type 2 diabetes and cardiovascular disease (at a 1:1 ratio) to groups that either received a subcutaneous injection of albiglutide (30–50 mg, based on glycaemic response and tolerability) or of a matched volume of placebo once a week, in addition to their standard care. Investigators used an interactive voice or web response system to obtain treatment assignment, and patients and all study investigators were masked to their treatment allocation. We hypothesised that albiglutide would be non-inferior to placebo for the primary outcome of the first occurrence of cardiovascular death, myocardial infarction, or stroke, which was assessed in the intention-to-treat population. If non-inferiority was confirmed by an upper limit of the 95% CI for a hazard ratio of less than 1·30, closed testing for superiority was prespecified. This study is registered with ClinicalTrials.gov, number NCT02465515. Findings: Patients were screened between July 1, 2015, and Nov 24, 2016. 10 793 patients were screened and 9463 participants were enrolled and randomly assigned to groups: 4731 patients were assigned to receive albiglutide and 4732 patients to receive placebo. On Nov 8, 2017, it was determined that 611 primary endpoints and a median follow-up of at least 1·5 years had accrued, and participants returned for a final visit and discontinuation from study treatment; the last patient visit was on March 12, 2018. These 9463 patients, the intention-to-treat population, were evaluated for a median duration of 1·6 years and were assessed for the primary outcome. The primary composite outcome occurred in 338 (7%) of 4731 patients at an incidence rate of 4·6 events per 100 person-years in the albiglutide group and in 428 (9%) of 4732 patients at an incidence rate of 5·9 events per 100 person-years in the placebo group (hazard ratio 0·78, 95% CI 0·68–0·90), which indicated that albiglutide was superior to placebo (p<0·0001 for non-inferiority; p=0·0006 for superiority). The incidence of acute pancreatitis (ten patients in the albiglutide group and seven patients in the placebo group), pancreatic cancer (six patients in the albiglutide group and five patients in the placebo group), medullary thyroid carcinoma (zero patients in both groups), and other serious adverse events did not differ between the two groups. There were three (<1%) deaths in the placebo group that were assessed by investigators, who were masked to study drug assignment, to be treatment-related and two (<1%) deaths in the albiglutide group. Interpretation: In patients with type 2 diabetes and cardiovascular disease, albiglutide was superior to placebo with respect to major adverse cardiovascular events. Evidence-based glucagon-like peptide 1 receptor agonists should therefore be considered as part of a comprehensive strategy to reduce the risk of cardiovascular events in patients with type 2 diabetes. Funding: GlaxoSmithKline

Investigation of the structural and molecular substrate of atrial fibrillation

Atrial fibrillation (AF) is the most common sustained arrhythmia worldwide and a frequent cause of hospitalization. Moreover, it represents one of the most frequent complication following cardiac surgery with an incidence of around 30% and an important predictor of patient morbidity. The exact pathophysiological mechanisms responsible for the onset and perpetuation of AF are not completely understood. However, clinical and experimental insights on the factors causing AF have suggested that atrial fibrillation is a multi-factorial phenomenon. Atrial fibrillation is characterized by a highly complex and irregular electrical activation of the atrial tissue, which is the manifestation of diverse abnormalities (electrical, structural, metabolic, neurohormonal, and/or molecular alterations) in diverse pathological conditions. In particular, it has been shown that fibrosis, a phenomenon in which extracellular matrix (ECM) components, mainly fibrillar collagen, accumulate between cardiomyocytes, leads to the inhomogeneous atrial electrical conduction typical of fibrillation. Recent studies have suggested that the deregulation of gene expression may act as a molecular mechanism of arrhythmogenesis. In particular, miRNAs, a new class of non-coding RNAs have rapidly emerged as one of the key players in the gene expression regulatory network, so variations in their expression levels may constitute a pathway for the arrhythmia-induced atrial remodeling. The present study aims to investigate the structural and molecular features of atrial tissue, with particular attention to fibrosis, which may be involved in the formation of a pro-arrhythmic substrate. By using both histological and advanced microscopy techniques, intramural fibrotic content and 3D collagen network properties were determined in atrial samples, collected during cardiac surgery in patients who developed or not AF. The quantitative analysis indicated a general decrease of collagen content from the outer (the epicardium) to the inner (the endocardium) myocardial wall, in the overall patient population. However, AF patients presented higher fibrotic values compared to sinus rhythm (SR) patients in the deeper myocardial layers, thus supporting the hypothesis that an accumulation of fibrotic tissue within the myocardial wall may represent an important structural contributor in the pathophysiology of AF. In addition to a quantitative assessment, collagen properties such as fibers orientation (degree and anisotropy) and scale dimension, were determined by non-linear optical microscopy techniques. The analysis revealed that in SR patients collagen network showed a fine architecture characterized by thin fibrils with changing angles and directions compared to AF, where fibers tended to pack-up in larger bundles of defined directions. A quantitative analysis of the 3D collagen network features, throughout the atrial wall, revealed that fibers orientation and scale dimension changed along tissue depth in both SR and AF patients, with larger values of orientation and fiber changes in AF tissues. These results highlight the spatial rearrangement and thickening of the 3D collagen network in AF patients, suggesting its possible role in the maintenance of the arrhythmia. Numerous evidence indicated that also an altered regulation of gene expression may play an important role in the mechanisms of atrial remodeling which underlie AF. In this perspective, the expression pattern of some miRNAs known to target different genes involved in diverse mechanisms that underlie AF was evaluated. A panel of miRNAs (miR-1, miR-133a/b, miR-30c, miR-29a/b, miR-208a/b, miR-328, miR-499, miR-590 and miR-21), principally involved in the formation of a pro-arrhythmic substrate, was selected after an accurate review of the literature and analyzed by RT-qPCR, in AF patients versus SR individuals. To accurately determine the levels of analyzed miRNAs, their expression data are usually normalized relatively to endogenous and/or exogenous reference genes. To date, no general agreement between different normalization strategies has been found, in particular in cardiac tissue, for the study of AF. For these reasons, a preliminary study aiming to establish the best endogenous reference genes for miRNAs data normalization was performed. Specifically, different well-established analysis tools such as NormFinder, GeNorm, BestKeeper and ∆Ct method, were applied on five commonly used endogenous reference transcripts such as 5S, U6, SNORD48, SNORD44 and miR-16. The suitable reference gene obtained, SNORD48, was applied for miRNAs data normalization. Our findings revealed that miRNAs expression levels were different in AF compared to SR patients. MiR-208a and miR-208b displayed statistically significant differences between the two populations. To investigate possible relationships between miRNAs expression levels and the fibrotic content a correlation measurement was also performed. Our analysis revealed that miR-21 and miR-208b were close to a significant correlation with fibrosis. In conclusion, this work introduced new techniques and implemented new methods of analysis for the study of the substrate of AF. In particular, the results obtained with this multiscale approach, from structural to molecular level, exacerbated the role of fibrosis as a critical contributor in the formation of a pro-arrhythmic substrate. Nonetheless, further studies are needed for a better understanding of the ways in which structural, molecular and also cellular remodeling may alter the impulse propagation in the myocardium

Effectiveness of dapagliflozin versus comparators on renal endpoints in the real world. A multicentre retrospective study

SGLT-2 inhibitors improve plasma glucose, blood pressure, and body weight. In randomized controlled trials, SGLT-2 inhibitors also reduced albumin excretion rate (AER) and improved renal outcomes in patients with type 2 diabetes (T2D). We herein report the changes in renal endpoints in T2D patients treated with dapagliflozin versus other glucose lowering medications in routine clinical practice

Endocrine-metabolic assessment checklist for cancer patients treated with immunotherapy: A proposal by the Italian Association of Medical Oncology (AIOM), Italian Association of Medical Diabetologists (AMD), Italian Society of Diabetology (SID), Italian Society of Endocrinology (SIE) and Italian Society of Pharmacology (SIF) multidisciplinary group

Immunotherapy with immune checkpoint inhibitors (ICI) is increasingly employed in oncology. National and international endocrine and oncologic scientific societies have provided guidelines for the management of endocrine immune-related adverse events. However, guidelines recommendations differ according to the specific filed, particularly pertaining to recommendations for the timing of endocrine testing. In this position paper, a panel of experts of the Italian Association of Medical Oncology (AIOM), Italian Association of Medical Diabetologists (AMD), Italian Society of Diabetology (SID), Italian Society of Endocrinology (SIE), and Italian Society of Pharmacology (SIF) offers a critical multidisciplinary consensus for a clear, simple, useful, and easily applicable endocrine-metabolic assessment checklist for cancer patients on immunotherapy

Polybrominated Diphenyl Ethers (PBDEs) and Human Health: Effects on Metabolism, Diabetes and Cancer

There is increasing evidence of the role of endocrine disruptors (EDs) derived from commonly employed compounds for manufacturing and processing in altering hormonal signaling and function. Due to their prolonged half-life and persistence, EDs can usually be found not only in industrial products but also in households and in the environment, creating the premises for long-lasting exposure. Polybrominated diphenyl ethers (PBDEs) are common EDs used in industrial products such as flame retardants, and recent studies are increasingly showing that they may interfere with both metabolic and oncogenic pathways. In this article, a multidisciplinary panel of experts of the Italian Association of Medical Diabetologists (AMD), the Italian Society of Diabetology (SID), the Italian Association of Medical Oncology (AIOM), the Italian Society of Endocrinology (SIE) and the Italian Society of Pharmacology (SIF) provides a review on the potential role of PBDEs in human health and disease, exploring both molecular and clinical aspects and focusing on metabolic and oncogenic pathways

Generation in Human Plasma of Misfolded, Aggregation-Prone Electronegative Low Density Lipoprotein

Human plasma contains small amounts of a low density lipoprotein in which apoprotein is misfolded. Originally identified and isolated by means of anion-exchange chromatography, this component was subsequently described as electronegative low density lipoprotein (LDL)(−), with increased concentrations associated with elevated cardiovascular disease risk. It has been recognized recently as the trigger of LDL amyloidogenesis, which produces aggregates similar to subendothelial droplets observed in vivo in early atherogenesis. Although LDL(−) has been produced in vitro through various manipulations, the mechanisms involved in its generation in vivo remain obscure. By using a more physiological model, we demonstrate spontaneous, sustained and noticeable production of LDL(−) during incubation of unprocessed human plasma at 37°C. In addition to a higher fraction of amyloidogenic LDL(−), LDL purified from incubated plasma contains an increased level of lysophospholipids and free fatty acids; analysis of LDL lipids packing shows their loosening. As a result, during plasma incubation, lipid destabilization and protein misfolding take place, and aggregation-prone particles are generated. All these phenomena can be prevented by inhibiting calcium-dependent secretory phospholipases A2. Our plasma incubation model, without removal of reaction products, effectively shows a lipid-protein interplay in LDL, where lipid destabilization after lipolysis threatens the apoprotein's structure, which misfolds and becomes aggregation-prone