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Anderson-Fabry disease (AFD) is a multisystem X-linked lysosomal storage disorder caused by a deficiency in the enzyme α-galactosidase A (α-Gal A). This deficiency results in the intracellular accumulation of glycosphingolipids, primarily uncleaved globotriaosylceramide (Gb3) and its deacylated form, lyso-globotriaosylceramide (Lyso-Gb3), leading to progressive organ damage and functional impairment. The diagnostic evaluation for AFD involves clinical assessment and family history, supported by biochemical testing (α-Gal A enzyme activity and Lyso-Gb3 levels) and genetic analysis of the GLA gene. In cases of unexplained renal impairment or when genetic analysis is inconclusive, kidney biopsy is often required to confirm the diagnosis and guide targeted treatments. However, histological findings in kidney biopsies may sometimes be nonspecific, complicating the diagnostic process. This article aims to provide an updated perspective on the role of kidney biopsy in AFD, illustrating two cases that exemplify its pivotal role in confirming or excluding the suspected disease, proving to be both decisive and confounding in this complex clinical setting., Competing Interests: Declarations. Ethics approval and consent to participate: The study was performed according to the Declaration of Helsinki. Consent for publication: Informed consent was obtained by all participants. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Background/objectives: Mummy studies allow to reconstruct the characteristic of a population in a specific spatiotemporal context, in terms of living conditions, pathologies and death. Radiology represents an efficient diagnostic technique able to establish the preservation state of mummified organs and to estimate the patient's pathological conditions. However, the radiological approach shows some limitations. Although bone structures are easy to differentiate, soft tissue components are much more challenging, especially when they overlap. For this reason, computed tomography, a well-established approach that achieves optimal image contrast and three-dimensional reconstruction, has been introduced. This original article focuses attention on the role of virtual dissection as a promising technology for exploring human mummy anatomy and considers the potential of environmental scanning electron microscopy and X-ray spectroscopy as complementary approaches useful to understand the state of preservation of mummified remains., Methods: Ancient mummy corps have been analyzed through Anatomage Table 10 and environmental scanning electron microscope equipped with X-ray spectrometer; Results: Anatomage Table 10 through various volumetric renderings allows us to describe spine alteration due to osteoarthritis, dental state, and other clinical-pathological characteristics of different mummies. Environmental scanning electron microscope, with the advantage of observing mummified samples without prior specimen preparation, details on the state of tissue fragments. Skin, tendon and muscle show a preserved morphology and keratinocytes, collagen fibers and tendon structures are easily recognizable. Furthermore, X-ray spectrometer reveals in our tissue remains, the presence of compounds related to soil contamination. This investigation identifies a plethora of organic and inorganic substances where the mummies were found, providing crucial information about the mummification environment., Conclusions: These morphological and analytical techniques make it possible to study mummified bodies and describe their anatomical details in real size, in a non-invasive and innovative way, demonstrating that these interdisciplinary approaches could have great potential for improving knowledge in the study of ancient corpses.

Due to the recent pandemic, new teaching methods have been trialed, even in traditional disciplines such as histopathology, which typically involve extensive hands-on laboratory work. In this study, we investigated the effects of three different learning modalities in histopathology teaching: optical microscopy, in-class virtual microscopy (VM), and online VM lectures. We examined relational aspects, didactic effectiveness, and satisfaction of these learning settings, both during class time and in private study, outside the dedicated spaces and hours of the university. The comparative design of the study, based on a set of surveys taken during the course and at its end, resulted in valuable methodological insights and a clear picture of the students' needs. These were characterized both by high technological expectations and by desire for a stimulating social environment. Our results show that the implementation of new learning tools has a positive impact on teaching, and not only at the didactic level. It also positively affects the way the university is experienced by the students, it facilitates their wanted socializing lifestyles, and it exposes them, as a new generation of professionals, to the latest technologies used by specialty physicians.

Background & Aims: The pathophysiology of irritable bowel syndrome (IBS) is multifactorial and includes epithelial barrier dysfunction, a key element at the interface between the gut lumen and the deeper intestinal layers. Beneath the epithelial barrier there is the vascular one representing the last barrier to avoid luminal antigen dissemination The aims of this study were to correlate morpho-functional aspects of epithelial and vascular barriers with symptom perception in IBS., Methods: Seventy-eight healthy subjects (controls) and 223 patients with IBS were enrolled in the study and phenotyped according to validated questionnaires. Sugar test was used to evaluate in vivo permeability. Immunohistochemistry, western blot, and electron microscopy were used to characterize the vascular barrier. Vascular permeability was evaluated by assessing the mucosal expression of plasmalemma vesicle-associated protein-1 and vascular endothelial cadherin. Caco-2 or human umbilical vein endothelial cell monolayers were incubated with soluble mediators released by mucosal biopsies to highlight the mechanisms involved in permeability alteration. Correlation analyses have been performed among experimental and clinical data., Results: The intestinal epithelial barrier was compromised in patients with IBS throughout the gastrointestinal tract. IBS-soluble mediators increased Caco-2 permeability via a downregulation of tight junction gene expression. Blood vessel density and vascular permeability were increased in the IBS colonic mucosa. IBS mucosal mediators increased permeability in human umbilical vein endothelial cell monolayers through the activation of protease-activated receptor-2 and histone deacetylase 11, resulting in vascular endothelial cadherin downregulation. Permeability changes correlated with intestinal and behavioral symptoms and health-related quality of life of patients with IBS., Conclusions: Epithelial and vascular barriers are compromised in patients with IBS and contribute to clinical manifestations., (Copyright © 2024 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background : Telocytes are interstitial stromal cells identified in various human organs, including the kidney. Their presence and role in human diabetic kidney disease remain unknown. Methods : To identify and localize telocytes in glomerular and tubule-interstitial compartments, both normal and diabetic human renal tissues were examined using immunohistochemistry, immunofluorescence, and transmission electron microscopy. Results : Renal telocytes are elongated interstitial cells with long, thin telopodes, showing alternating thin and thick segments. They expressed CD34, Nestin, α-SMA, and Vimentin markers. Occasionally, c-Kit expression was observed in some rounded and spindle cells, while no positivity was detected for PDGFR-β and NG2. Telocytes were identified around Bowman's capsule, tubules, and peritubular capillaries in both normal and diabetic conditions. In diabetic renal samples, there was a significant increase in α-SMA expressing telocytes, leading to periglomerular fibrosis. These telocytes also exhibited a synthetic phenotype with proteoglycan deposition in the extracellular matrix and, in some cases, showed pre-adipocytic differentiation. Conclusions : Telocytes were identified in normal and diabetic human kidneys. These cells form an elastic mechanical scaffold in the interstitium and are present in all renal cortical compartments. In diabetic samples, their increased α-SMA expression and synthetic phenotype suggest their potential role in the pathogenesis of diabetic nephropathy.

Background: Carotid endarterectomy (CEA) for the prevention of upcoming vascular and cerebral events is necessary in patients with high-grade stenosis (≥70%). In the framework of the Italian National project Age.It, a pilot study was proposed aiming at the discovery of a molecular signature with predictive potential of carotid stenosis comparing 65+ asymptomatic and symptomatic inpatients., Methods: A total of 42 inpatients have been enrolled, including 26 men and 16 women, with a mean age of 74 ± 6 years. Sixteen symptomatic and 26 asymptomatic inpatients with ≥70% carotid stenosis underwent CEA, according to the recommendations of the European Society for Vascular Surgery and the Society for Vascular Surgeons. Plaque biopsies and peripheral blood samples from the same individuals were obtained. Hematobiochemical analyses were conducted on all inpatients, and plasma cytokines/molecules, such as microRNAs (miRs), IL-6, sIL-6Ralpha, sgp130, myostatin (GDF8), follistatin, activin A, CXCL9, FGF21, and fibronectin, were measured using the ELISA standard technique. MiR profiles were obtained in the discovery phase including four symptomatic and four asymptomatic inpatients (both plasma and plaque samples), testing 734 miRs. MiRs emerging from the profiling comparison were validated through RT-qPCR analysis in the total cohort., Results and Conclusion: The two groups of inpatients differ in the expression levels of blood c-miRs-126-5p and -1271-5p (but not in their plaques), which are more expressed in symptomatic subjects. Three cytokines were significant between the two groups: IL-6, GDF8, and CXCL9. Using receiver operating characteristic (ROC) analysis with a machine learning-based approach, the most significant blood molecular signature encompasses albumin, C-reactive protein (CRP), the percentage of monocytes, and CXCL9, allowing for the distinction of the two groups (AUC = 0.83, 95% c.i. [0.85, 0.81], p = 0.0028). The potential of the molecular signature will be tested in a second cohort of monitored patients, allowing the application of a predictive model and the final evaluation of cost/benefit for an assessable screening test., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Capri, Fronterrè, Collura, Giampieri, Carrino, Feroldi, Ciurca, Conte, Olivieri, Ullo, Pini, Vacirca, Astolfi, Vasuri, La Manna, Pasquinelli and Gargiulo.)

IgA Nephropathy (IgAN) is the most prevalent glomerular disease worldwide. Complement system activation is crucial in its pathogenesis. Few studies correlated serum C3 and C4 with disease activity and prognosis. This retrospective study investigated the prognostic value of serum complement at the time of diagnosis in patients with IgAN. Specifically we evaluated whether adding serum C3 and C4 levels to established predictive models-one based on variables related to chronic kidney disease (CKD) progression and another incorporating variables from the International IgA Prediction Tool (IntIgAPT)-enhances the accuracy of outcome prediction. A composite renal outcome was defined as 50% decline in eGFR or onset of kidney failure. 101 patients were stratified according to baseline C3 levels in three groups (Low, Medium and High). During a median follow-up of 54 months, the Low group exhibited higher incidence of primary outcome (16.3 events vs 2.9 and 1.7 events × 100 pts/year, p = 0.0026). Model-1 (M1), consisting of CKD progression variables, and Model-3 (M3), comprising IntIgANPT variables, were implemented with baseline C3 and C4 to create Model-2 (M2) and Model-4 (M4), respectively. M2 demonstrated better predictive performance over M1, showing higher discrimination (lower AIC and BIC, higher C-index and NR2). Similarly, M4 outperformed M3, showing enhanced outcome prediction when C3 and C4 levels were added. Implementation of serum C3 and C4 can enhance prediction accuracy of already-validated prognostic models in IgAN. Lower C3 and higher C4 levels were associated with poorer prognosis, highlighting a more 'Complement-Pathic' subset of patients., (© 2024. The Author(s).)

Introduction: The image of a nurse is a source of concern due to its impact on recruitment into the profession, political decisions about the profession, and how the image affects nursing practice. For these reasons, one of the long-term challenges is to assess and maintain a favorable public image that respects the utility and value of the nursing profession., Aim: This study aims to validate an instrument for assessing the image of the nurse as perceived by Italian citizens., Methods: A non-probabilistic sample of 564 people participated in the study between 2022 and 2023. Sociodemographic information of the Italian citizens was collected, and the instrument used to evaluate the perceived public image was the Nursing Attitudes Questionnaire (NAQ). The psychometric properties of the Italian version of the NAQ (NAQ-IV) were calculated using Cronbach's alpha, item-total correlations, skewness, and kurtosis. Factor analysis was performed using principal axis factoring and the varimax rotation method., Results: Factor analysis revealed a four-factor model explaining more than 60.52% of the variance, with the largest variance explained by the "Role and Professionalism" factor (34.08%). The internal consistency calculation showed a Cronbach's alpha of 0.89 for the scale and between 0.88 and 0.89 among the factors; all the items verified the item-total correlation and response variability criteria., Conclusions: The NAQ-IV could be a valid tool for assessing the perception of Italian citizens. However, further studies are recommended to evaluate the reliability of the instrument, especially in the evolving professional profile and social health welfare.

Perilipin 2 (PLIN2) is a lipid droplet (LD)-coating protein playing important roles in lipid homeostasis and suppression of lipotoxicity in different tissues and cell types. Recently, a role for PLIN2 in supporting mitochondrial function has emerged. PLIN2 dysregulation is involved in many metabolic disorders and age-related diseases. However, the exact consequences of PLIN2 dysregulation are not yet completely understood. In this study, we knocked down (KD) PLIN2 in primary human dermal fibroblasts (hDFs) from young (mean age 29 years) and old (mean age 71 years) healthy donors. We have found that PLIN2 KD caused a decline of mitochondrial function only in hDFs from young donors, while mitochondria of hDFs from old donors (that are already partially impaired) did not significantly worsen upon PLIN2 KD. This mitochondrial impairment is associated with the increased expression of the stress-related mitokine growth differentiation factor 15 (GDF15) and the induction of cell senescence. Interestingly, the simultaneous KD of PLIN2 and GDF15 abrogated the induction of cell senescence, suggesting that the increase in GDF15 is the mediator of this phenomenon. Moreover, GDF15 KD caused a profound alteration of gene expression, as observed by RNA-Seq analysis. After a more stringent analysis, this alteration remained statistically significant only in hDFs from young subjects, further supporting the idea that cells from old and young donors react differently when undergoing manipulation of either PLIN2 or GDF15 genes, with the latter being likely a downstream mediator of the former., (© 2024 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

Background: Music evokes positive emotions and reduces stress and anxiety. Operating room (OR) staff face various challenges which can lead to high levels of stress. The aim of the study is to assess whether listening to music during intraoperative phases improves the work environment by reducing anxiety and stress in the entire surgical team., Methods: A prospective observational study was conducted from February to September 2023, involving medical personnel, nursing staff, and nursing students. They were divided into two groups: Group 1 with music during surgical procedures, and Group 2 without music. Participants were administered two validated instruments: the Zung Anxiety Self-Assessment Scale (SAS) to measure anxiety, and the Positive and Negative Affect Schedule to assess emotions generating stress. Additional items were included for demographics, job satisfaction, and the organization method., Results: Music did not impact anxiety, but increased positive emotions while reducing negative ones. Music had an ancillary effect, highlighting the need for significant organizational interventions aimed at increasing operator satisfaction, including offering voluntary instead of mandatory assignments to nursing staff., Conclusions: Music appears to reduce stress in the intraoperative team when supported by a positive work environment in which assigned operators have chosen to work in the OR.

Giacomo Torno was born in 1539 (or 1541) in Naples. At the age of 18 he joined the Clerics Regular Theatines in San Paolo Maggiore and was welcomed on 30 October 1558. He suffered a stroke on 4 December 1608 and died 45 days later. Contemporary sources report that he appeared to be tormented by the devil during his illness, suffering from constant spasms in his arm, which caused him great discomfort. During the analysis of his mummified body, a discontinuity of the skin surface at the level of the sacrum was discovered. All morphological features indicate a wound that developed during the subject's life. Based on historical sources, the fracture of the first coccygeal vertebra, the appearance of the lesion near death, and the shape of the lesion, suggest that this is the first recorded instance of the Kennedy terminal ulcer, identified through both direct and indirect sources., Competing Interests: Conflict of interest: the authors declare no conflict of interest., (Copyright © 2023, the Author(s).)

Cardiovascular disease (CVD), the leading cause of death globally, affects the heart and arteries with a variety of clinical manifestations, the most dramatic of which are myocardial infarction (MI), abdominal aortic aneurysm (AAA), and intracranial aneurysm (IA) rupture. In MI, necrosis of the myocardium, scar formation, and loss of cardiomyocytes result from insufficient blood supply due to coronary artery occlusion. Beyond stenosis, the arteries that are structurally and functionally connected to the cardiac tissue can undergo pathological dilation, i.e., aneurysmal dilation, with high risk of rupture. Aneurysms of the intracranial arteries (IAs) are more commonly seen in young adults, whereas those of the abdominal aorta (AAA) are predominantly seen in the elderly. IAs, unpredictably, can undergo rupture and cause life-threatening hemorrhage, while AAAs can result in rupture, internal bleeding and high mortality rate. In this clinical context, hydrogels, three-dimensional networks of water-seizing polymers, have emerged as promising biomaterials for cardiovascular tissue repair or protection due to their biocompatibility, tunable properties, and ability to encapsulate and release bioactive molecules. This review provides an overview of the current state of research on the use of hydrogels as an innovative platform to promote cardiovascular-specific tissue repair in MI and functional recovery or protection in aneurysmal dilation.

Human arteries show structural and functional peculiarities according to the nutrient and oxygen needs of a specific vascular district. This architectural heterogeneity is reflected in the pathological setting of cardiovascular diseases (CVDs). Indeed, the responsiveness to cardiovascular risk factors, and the morphological and molecular patterns are discriminating factors among CVDs affecting different vascular beds. MicroRNAs (miRNAs) are endogenous regulators of gene expression and fine-tuners of vascular cell differentiation; thus, these non-coding RNAs can modulate arterial heterogeneity. The identification of an artery-specific miRNA signature would be promising in the therapy of CVDs, especially in patients who are frail and elderly. In the present review, we will provide a concise description of the arterial tree heterogeneity on a structural and cellular basis, mainly in the pathological context. Secondly, we will address the miRNA potential as crucial mediators of arterial heterogeneity, focusing on the abdominal aorta and femoral artery, with the final goal of strengthening the search for more targeted therapies in CVDs and stratification approaches in patients who are frail and elderly.

Angiosarcomas (ASs) are rare malignant vascular entities that can affect several regions in our body, including the heart. Cardiac ASs comprise 25-40% of cardiac sarcomas and can cause death within months of diagnosis. Thus, our aim was to identify potential differences and/or similarities between cardiac and extra-cardiac ASs to enhance targeted therapies and, consequently, patients' prognosis. Whole-transcriptome analysis of three cardiac and eleven extra-cardiac non-cutaneous samples was performed to investigate differential gene expression and mutational events between the two groups. The gene signature of cardiac and extra-cardiac non-cutaneous ASs was also compared to that of cutaneous angiosarcomas (n = 9). H/N/K-RAS and TP53 alterations were more recurrent in extra-cardiac ASs, while POTE -gene family overexpression was peculiar to cardiac ASs. Additionally, in vitro functional analyses showed that POTEH upregulation conferred a growth advantage to recipient cells, partly supporting the cardiac AS aggressive phenotype and patients' scarce survival rate. These features should be considered when investigating alternative treatments.

Visceral sarcomas are a rare malignant subgroup of soft tissue sarcomas (STSs). STSs, accounting for 1% of all adult tumors, are derived from mesenchymal tissues and exhibit a wide heterogeneity. Their rarity and the high number of histotypes hinder the understanding of tumor development mechanisms and negatively influence clinical outcomes and treatment approaches. Although some STSs (~20%) have identifiable genetic markers, as specific mutations or translocations, most are characterized by complex genomic profiles. Thus, identification of new therapeutic targets and development of personalized therapies are urgent clinical needs. Although cell lines are useful for preclinical investigations, more reliable preclinical models are required to develop and test new potential therapies. Here, we provide an overview of the available in vitro and in vivo models of visceral sarcomas, whose gene signatures are still not well characterized, to highlight current challenges and provide insights for future studies.

Background: Thrombotic microangiopathy is a severe and potentially life-threatening condition inducing severe endothelial injury in many organs, particularly native and transplanted kidneys. Current pathological studies by our group have identified the use of Caveolin-1 immunohistochemistry as a potential marker of endothelial damage and progression degree of thrombotic microangiopathy. The aim of the present work was to evaluate Caveolin-1 as a marker of severity in thrombotic microangiopathy kidney disease, according to the ultrastructural progression of the disease evaluated by transmission electron microscopy., Materials and Methods: Twenty-nine patients (17 non-transplanted and 12 transplanted) were retrospectively selected, biopsied for suspected or histologically-confirmed thrombotic microangiopathy. Transmission electron microscopy was performed in all cases, and an ultrastructural score of thrombotic microangiopathy-related glomerular disease was assessed (from 0 to 3+). Immunohistochemistry for Caveolin-1 was automatically performed., Results: The mean percentage of Caveolin-1-positive glomerular capillaries was 53.2 ± 40.6% and 28.0 ± 42.8% in the active thrombotic microangiopathy versus previous thrombotic microangiopathy cases (p = 0.085), considering both native and transplanted kidneys. The presence of progressive disease correlated with diffuse Caveolin-1 immunoreactivity (p = 0.031), and ultrastructural score correlated with glomerular Caveolin-1 positivity, progressively increasing from 22.5% of the Score 0 group to 95.5% of the Score 3 group (p = 0.036)., Discussion: Caveolin-1 proved to be a very useful marker of early endothelial damage in the course of thrombotic microangiopathy for both native and transplanted kidneys, therefore worth considering in routine practice. Diffuse glomerular Caveolin-1 immunoreactivity correlates with the severity of the thrombotic disease and it can appear very early, even before ultrastructurally evident endothelial damage., (© 2023. The Author(s) under exclusive licence to Italian Society of Nephrology.)

Rationale & Objective: Fabry disease (FD) is an X-linked genetic disorder that causes lysosomal storage of glycosphingolipids, primarily globotriaosylceramide (Gb3) and its derivative globotriaosylsphingosine (lyso-Gb3), with multiorgan dysfunction including chronic kidney disease. Affected individuals may be carriers of gene variants that are of uncertain significance (GVUS). We describe kidney pathology at the early stages of FD-related kidney disease to gain insights into its association with GVUS and sex., Study Design: Single-center, case series., Setting & Participants: Thirty-five consecutively biopsied patients (aged 48.1±15.4 years, 22 females) from among 64 patients with genetically diagnosed FD. Biopsies were retrospectively screened using the International Study Group of Fabry Nephropathy Scoring System., Observations: Genetic mutation type, p.N215S and D313Y, sex, age, estimated glomerular filtration rate (eGFR), plasma lyso-Gb3 (pLyso-Gb3) levels, and histological parameters, including Gb3 deposits were recorded. Genetic analyses showed mostly missense mutations, p.N215S variant in 15, and the "benign polymorphism" D313Y in 4 of the biopsied patients. Morphological lesions were similar for men and women except for interstitial fibrosis and arteriolar hyalinosis being more common in men. Early in their clinical course, patients with normal/mild albuminuria had podocyte, tubular, and peritubular capillary vacuoles/inclusions, and evidence of chronicity, i.e., glomerulosclerosis, interstitial fibrosis, tubular atrophy. These findings appeared to be associated with pLyso-Gb3, eGFR, and age., Limitations: Retrospective design and inclusion of outpatients partially based on family pedigree., Conclusions: In early stages of kidney disease in the setting of FD, numerous histological abnormalities are present. These observations suggest that kidney biopsies early in FD may reveal activity of kidney involvement that may inform clinical management., (Copyright © 2023 National Kidney Foundation, Inc. Published by Elsevier Inc. All rights reserved.)

Little is known about the signaling network responsible for the organization of the perinuclear actin cap, a recently identified structure holding unique roles in the regulation of nuclear shape and cell directionality. In cancer cells expressing a constitutively active MET, we show a rearrangement of the actin cap filaments, which crash into perinuclear patches associated with spherical nuclei, meandering cell motility and inactivation of the mechano-transducer YAP1. MET ablation is sufficient to reactivate YAP1 and restore the cap, leading to enhanced directionality and flattened nuclei. Consistently, the introduction of a hyperactive MET in normal epithelial cells, enhances nuclear height and alters the cap organization, as also confirmed by TEM analysis. Finally, the constitutively active YAP1 mutant YAP5SA is able to overcome the effects of oncogenic MET. Overall, our work describes a signaling axis empowering MET-mediated YAP1 dampening and actin cap misalignment, with implications for nuclear shape and cell motility., (© 2023. Springer Nature Limited.)

Background: Diabetic donors are recognized as a reliable source of organs, although the discard rate of kidneys is still high. Few data are available on the histological evolution of these organs especially on kidneys transplanted into non-diabetic patients who remain euglycemic., Methods: We describe the histological evolution of ten kidney biopsies performed on non-diabetic recipients of diabetic donors., Results: Mean donor age was 69 ± 7 years, 60% were males. Two donors were treated with insulin, eight with oral antidiabetic drugs. Mean recipient age was 59.9 ± 7 years, 70% were males. The pre-existing diabetic lesions identified in the pre-implantation biopsies, encompassed all histological classes, and were associated with mild IF/TA and vascular damages. The median follow-up was 59.5 [IQR 32.5-99.0] months; at follow-up, 40% of cases did not change histologic classification, two patients with class IIb downgraded to IIa or I and one with class III downgraded to IIb. Conversely, three cases showed a worsening, from class 0 to I, I to IIb or from IIa to IIb. We also observed a moderate evolution of IF/TA and vascular damages. At follow-up visit, estimated GFR was stable (50.7 mL/min vs. 54.8 at baseline) and proteinuria was mild (51.1 ± 78.6 mg/day)., Conclusions: Kidneys from diabetic donors show variable evolution of the histologic features of diabetic nephropathy after transplant. This variability may be associated to recipients characteristics such as euglycemic milieu, in case of improvement, or obesity and hypertension, in case of worsening of histologic lesions., (© 2023. The Author(s).)

Introduction: Calcification of a vascular endograft and adjacent tissues (adventitia, media, and neointima) can result in graft failure. This report shows a rare case of intraluminal calcifications in the distal end of a thoracic endovascular aortic repair (TEVAR) endograft implanted 11 years previously for grade IV blunt traumatic aortic injury (BTAI) in a young patient., Report: A 24 year old man required TEVAR for a BTAI caused by a motorcycle accident. The procedure consisted of TEVAR and an emergency left carotid subclavian venous bypass. Eleven years after the procedure, he had severe hypertension. Intra-TEVAR calcifications appeared, gradually increasing on computed tomography angiography (CTA). Calcifications in the distal luminal end of the TEVAR were responsible for a 60% stenosis on CTA. An open approach was indicated after multidisciplinary discussion, based on the gradient value. The patient underwent explantation, with total replacement of the aortic arch and descending thoracic aorta with re-implantation of the supra-aortic vessels, under extracorporeal circulation. Macroscopic analysis showed no device degeneration but revealed a solid mass at the distal end of the TEVAR. Both microcomputed tomography and histopathology confirmed the calcific nature of the lesions., Conclusion: This case highlights a rare long term graft failure due to calcified neo-atherosclerosis in a TEVAR., Competing Interests: None., (© 2023 The Authors.)

Background: Arteriovenous fistula (AVF) for hemodialysis integrates outward remodeling with vessel wall thickening in response to drastic hemodynamic changes. Aim of this study is to determine the role of Ki67, a well-established proliferative marker, related to AVF, and its relationship with time-dependent histological morphologic changes., Materials and Methods: All patients were enrolled in 1 year and stratified in two groups: (A) pre-dialysis patients submitted to first AVF and (B) patients submitted to revision of AVF. Morphological changes: neo-angiogenesis (NAG), myointimal thickening (MIT), inflammatory infiltrate (IT), and aneurysmatic fistula degeneration (AD). The time of AVF creation was recorded. A biopsy of native vein in Group A and of arterialized vein in Group B was submitted to histological and immunohistochemical (IHC) analysis. IHC for Ki67 was automatically performed in all specimens. Ki67 immunoreactivity was assessed as the mean number of positive cells on several high-power fields, counted in the hot spots., Results: A total of 138 patients were enrolled, 69 (50.0%) Group A and 69 (50.0%) Group B. No NAG or MIT were found in Group A. Seven (10.1%) Group A veins showed a mild MIT. Analyzing the Group B, a moderate-to-severe MIT was present in 35 (50.7%), IT in 19 (27.5%), NAG in 37 (53.6%); AD was present in 10 (14.5%). All AVF of Group B with the exception of one (1.4%) showed a positivity for Ki67, with a mean of 12.31 ± 13.79 positive cells/hot spot (range 0-65). Ki67-immunoreactive cells had a subendothelial localization in 23 (33.3%) cases, a myointimal localization in SMC in 35 (50.7%) cases. The number of positive cells was significantly correlated with subendothelial localization of Ki67 ( p  = 0.001) and with NA ( p  = 0.001)., Conclusions: Native veins do not contain cycling cells. In contrast, vascular cell proliferation starts immediately after AVF creation and persists independently of the time the fistula is set up. The amount of proliferating cells is significantly associated with MIT and subendothelial localization of Ki67-immunoreactive cells, thus suggesting a role of Ki-67 index in predicting AVF failure.

The aim of this study was to optimize a coculture in vitro model established between the human Müller glial cells and human umbilical vein endothelial cells, mimicking the inner blood-retinal barrier, and to explore its resistance to damage induced by oxidative stress. A spontaneously immortalized human Müller cell line MIO-M1 and human umbilical vein endothelial cells (HUVEC) were plated together at a density ratio 1:1 and maintained up to the 8th passage (p8). The MIO-M1/HUVECs p1 through p8 were treated with increasing concentrations (range 200-800 μM) of H 2 O 2 to evaluate oxidative stress induced damage and comparing data with single cell cultures. The following features were assayed p1 through p8: doubling time maintenance, cell viability using MTS assay, ultrastructure of cell-cell contacts, immunofluorescence for Vimentin and GFAP, molecular biology (q-PCR) for GFAP and CD31 mRNA. MIO-M1/HUVECs cocultures maintained distinct cell cytotype up to p8 as shown by flow cytometry analysis, without evidence of cross activation, displaying cell-cell tight junctions mimicking those found in human retina, only acquiring a slight resistance to oxidative stress induction over the passages. This MIO-M1/HUVECs coculture represents a simple, reproducible and affordable model for in vitro studies on oxidative stress-induced retinal damages., (© 2022 The Authors. Microscopy Research and Technique published by Wiley Periodicals LLC.)

Background: Hypothermic oxygenated perfusion (HOPE) has become widespread for the preservation of liver grafts, making tangled the relationship among the use of extended criteria donors (ECD), graft histology and transplant outcome., Aims: To prospectively validate the impact of the graft histology on transplant outcome in recipient receiving liver grafts from ECD after HOPE., Methods: Ninety-three ECD grafts were prospectively enrolled; 49 (52.7 %) were perfused with HOPE according to our protocols. All clinical, histological and follow-up data were collected., Results: Grafts with portal fibrosis stage ≥ 3 according to Ishak's (evaluated with Reticulin stain) had a significantly higher incidence of early allograft dysfunction (EAD) and 6-month-dysfunction (p = 0.026 and p = 0.049), with more days in Intensive Care Unit (p = 0.050). Lobular fibrosis correlated with post-liver transplant kidney function (p = 0.019). Moderate-to-severe chronic portal inflammation was correlated with graft survival on both multivariate and univariate analyses (p < 0.001), but this risk factor is sensibly reduced by the execution of HOPE., Conclusions: The use of liver grafts with portal fibrosis stage ≥ 3 implies a higher risk of post-transplant complications. Portal inflammation represents an important prognostic factor as well, but the execution of HOPE represents a valid tool to improve graft survival., Competing Interests: Conflict of interest The authors have no conflicts of interest to disclose., (Copyright © 2023. Published by Elsevier GmbH.)

The failure of arteriovenous fistulas (AVFs) following intimal hyperplasia (IH) increases morbidity and mortality rates in patients undergoing hemodialysis for chronic kidney disease. The peroxisome-proliferator associated receptor (PPAR-γ) may be a therapeutic target in IH regulation. In the present study, we investigated PPAR-γ expression and tested the effect of pioglitazone, a PPAR-γ agonist, in different cell types involved in IH. As cell models, we used Human Endothelial Umbilical Vein Cells (HUVEC), Human Aortic Smooth Muscle Cells (HAOSMC), and AVF cells (AVFCs) isolated from (i) normal veins collected at the first AVF establishment (T0), and (ii) failed AVF with IH (T1). PPAR-γ was downregulated in AVF T1 tissues and cells, in comparison to T0 group. HUVEC, HAOSMC, and AVFC (T0 and T1) proliferation and migration were analyzed after pioglitazone administration, alone or in combination with the PPAR-γ inhibitor, GW9662. Pioglitazone negatively regulated HUVEC and HAOSMC proliferation and migration. The effect was antagonized by GW9662. These data were confirmed in AVFCs T1, where pioglitazone induced PPAR-γ expression and downregulated the invasive genes SLUG, MMP-9, and VIMENTIN. In summary, PPAR-γ modulation may represent a promising strategy to reduce the AVF failure risk by modulating cell proliferation and migration.

Clear cell sarcoma of the kidney (CCSK) is a rare pediatric renal tumor with a worse prognosis than Wilms' tumor. Although recently, BCOR internal tandem duplication (ITD) has been found as a driver mutation in more than 80% of cases, a deep molecular characterization of this tumor is still lacking, as well as its correlation with the clinical course. The aim of this study was to investigate the differential molecular signature between metastatic and localized BCOR-ITD-positive CCSK at diagnosis. Whole-exome sequencing (WES) and whole-transcriptome sequencing (WTS) were performed on six localized and three metastatic BCOR-ITD-positive CCSKs, confirming that this tumor carries a low mutational burden. No significant recurrences of somatic or germline mutations other than BCOR-ITD were identified among the evaluated samples. Supervised analysis of gene expression data showed enrichment of hundreds of genes, with a significant overrepresentation of the MAPK signaling pathway in metastatic cases ( p < 0.0001). Within the molecular signature of metastatic CCSK, five genes were highly and significantly over-expressed: FGF3, VEGFA, SPP1, ADM, and JUND. The role of FGF3 in the acquisition of a more aggressive phenotype was investigated in a cell model system obtained by introducing the ITD into the last exon of BCOR by Crispr/Cas9 gene editing of the HEK-293 cell line. Treatment with FGF3 of BCOR-ITD HEK-293 cell line induced a significant increase in cell migration versus both untreated and scramble cell clone. The identification of over-expressed genes in metastatic CCSKs, with a particular focus on FGF3, could offer new prognostic and therapeutic targets in more aggressive cases.

Introduction: Growth Differentiation Factor 15 (GDF15) is a mitochondrial-stress-responsive molecule whose expression strongly increases with aging and age-related diseases. However, its role in neurodegenerative diseases, including Alzheimer's disease (AD), is still debated., Methods: We have characterized the expression of GDF15 in brain samples from AD patients and non-demented subjects (controls) of different ages., Results: Although no difference in CSF levels of GDF15 was found between AD patients and controls, GDF15 was expressed in different brain areas and seems to be predominantly localized in neurons. The ratio between its mature and precursor form was higher in the frontal cortex of AD patients compared to age-matched controls ( p  < 0.05). Moreover, this ratio was even higher for centenarians ( p  < 0.01), indicating that aging also affects GDF15 expression and maturation. A lower expression of OXPHOS complexes I, III, and V in AD patients compared to controls was also noticed, and a positive correlation between GDF15 and IL-6 mRNA levels was observed. Finally, when GDF15 was silenced in vitro in dermal fibroblasts, a decrease in OXPHOS complexes transcript levels and an increase in IL-6 levels were observed., Discussion: Although GDF15 seems not to be a reliable CSF marker for AD, it is highly expressed in aging and AD brains, likely as a part of stress response aimed at counteracting mitochondrial dysfunction and neuroinflammation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Chiariello, Valente, Pasquinelli, Baracca, Sgarbi, Solaini, Medici, Fantini, Poloni, Tognocchi, Arcaro, Galimberti, Franceschi, Capri, Salvioli and Conte.)

Primary cardiac sarcomas are considered rare malignant entities associated with poor prognosis. In fact, knowledge regarding their gene signature and possible treatments is still limited. In our study, whole-transcriptome sequencing on formalin-fixed paraffin-embedded (FFPE) samples from one cardiac osteosarcoma and one cardiac leiomyosarcoma was performed, to investigate their mutational profiles and to highlight differences and/or similarities to other cardiac histotypes. Both cases have been deeply detailed from a pathological point of view. The osteosarcoma sample presented mutations involving ATRX , ERCC5 , and COL1A1 , while the leiomyosarcoma case showed EXT2 , DNM2 , and PSIP1 alterations. Altered genes, along with the most differentially expressed genes in the leiomyosarcoma or osteosarcoma sample versus the cardiac angiosarcomas and intimal sarcomas (e.g., YAF2 , PAK5 , and CRABP1 ), appeared to be associated with cell growth, proliferation, apoptosis, and the repair of DNA damage, which are key mechanisms involved in tumorigenesis. Moreover, a distinct gene expression profile was detected in the osteosarcoma sample when compared to other cardiac sarcomas. For instance, WIF1, a marker of osteoblastic differentiation, was upregulated in our bone tumor. These findings pave the way for further studies on these entities, in order to identify targeted therapies and, therefore, improve patients' prognoses.

Background: Mitochondrial tRNA (MTT) genes are hotspot for mitochondrial DNA mutation and are responsible of half mitochondrial disease. MTT mutations are associated with a broad spectrum of phenotype often with complex multisystem involvement and complex genotype-phenotype correlations. MT-TL1 mutations, among which the m.3243A>G mutation is the most frequent, are associated with myopathy, maternal inherited diabetes and deafness, MELAS, cardiomyopathy, and focal segmental glomerulosclerosis., Case Study: Here we report the case of an Italian 49-years old female presenting with encephalomyopathy, chronic proteinuric kidney disease and a new heteroplasmic m.3274&#95;3275delAC MT-TL1 gene mutation., Conclusions: Our case demonstrates a systemic mitochondrial disease caused by the heteroplasmic m.3274&#95;3275delAC MT-TL1 gene mutation, not yet described in the literature. A mitochondrial disease should be suspected in case of complex multisystem phenotypes, including steroid-resistant nephrotic syndrome with multisystemic involvement.

Mesenchymal stromal/stem cells (MSCs) have been identified in multiple human tissues, including the vascular wall. High proliferative potential, multilineage, and immunomodulatory properties make vascular MSCs promising candidates for regenerative medicine. Indeed, their location is strategic for controlling vascular and extra-vascular tissue homeostasis. However, the clinical application of MSCs, and in particular vascular MSCs, is still challenging. Current studies are focused on developing strategies to improve MSC therapeutic applications, like priming MSCs with stress conditions (hypoxia, nutrient deprivation) to achieve a higher therapeutic potential. The goal of the present study is to review the main findings regarding the MSCs isolated from the human vascular wall. Further, the main priming strategies tested on MSCs from different sources are reported, together with the experience on vascular MSCs isolated from healthy cryopreserved and pathological arteries. Stress induction can be a priming approach able to improve MSC effectiveness through several mechanisms that are discussed in this review. Nevertheless, these issues have not been completely explored in vascular MSCs and potential side effects need to be investigated., (© The Author(s) 2022. Published by Oxford University Press.)

The Serum Response Factor (SRF) is a transcription factor that regulates the expression of a wide set of genes involved in cell proliferation, migration, cytoskeletal organization and myogenesis. Accumulating evidence suggests that SRF may play a role in carcinogenesis and tumor progression in various neoplasms, where it is often involved in different fusion events. Here we investigated SRF rearrangements in soft tissue tumors, along with a gene expression profile analysis to gain insight into the oncogenic mechanism driven by SRF fusion. Whole transcriptome analysis of cell lines transiently overexpressing the SRF::E2F1 chimeric transcript uncovered the specific gene expression profile driven by the aberrant gene fusion, including overexpression of SRF-dependent target genes and of signatures related to myogenic commitment, inflammation and immune activation. This result was confirmed by the analysis of two cases of myoepitheliomas harboring SRF::E2F1 fusion with respect to EWSR1 -fusion positive tumors. The recognition of the specific gene signature driven by SRF rearrangement in soft tissue tumors could aid the molecular classification of this rare tumor entity and support therapeutic decisions.

The aim of the present study was to evaluate the homeostasis and trophism of liver sinusoidal endothelial cells (LSECs) in vivo in different stages of liver graft donation, in order to understand the effects of graft ischemia and perfusion on LSEC activity in liver grafts. Special attention was paid to grafts that underwent hypothermic oxygenated perfusion (HOPE). Forty-seven donors were prospectively enrolled, and two distinct biopsies were performed in each case: one allocation biopsy (at the stage of organ allocation) and one post-perfusion biopsy, performed after graft implant in the recipients. In all biopsies, immunohistochemistry and RT-PCR analyses were carried out for the endothelial markers CD34, ERG, Nestin, and VEGFR-2. We observed an increase in CD34 immunoreactivity in LSEC during the whole preservation/perfusion period (p < 0.001). Nestin and ERG expression was low in allocation biopsies, but increased in post-perfusion biopsies, in both immunohistochemistry and RT-PCR (p < 0.001). An inverse correlation was observed between ERG positivity and donor age. Our results indicate that LSEC trophism is severely depressed in liver grafts, but it is restored after reperfusion in standard conditions. The execution of HOPE seems to improve this recovery, confirming the effectiveness of this machine perfusion technique in restoring endothelial functions.

Background: Synthetic vascular graft calcification is a serious complication of graft placement. Here, we analysed migration and osteogenic genes of human umbilical vein endothelial cells (HUVEC) cultured with a poly-L-lactic acid (PLLA) electrospun mat. The role of epigallo-catechin-3-gallate (EGCG) in pathogenic processes involving HUVEC and peripheral blood mononuclear cells (PBMCs) was also tested., Methods: HUVEC were cultured in indirect contact with PLLA for 48 h, with or without EGCG, and processed for mRNA expression. HUVEC proliferation, migration and osteogenic differentiation were evaluated after EGCG treatment. EGCG was also administrated to human PBMCs, to analyse proliferation and migration toward HUVEC cultured with PLLA., Results: HUVEC cultured with PLLA exhibited increased expression of SLUG, VIMENTIN, MMP-9 (migration, vascular remodelling) and RUNX-2 (osteogenic transcription factor). EGCG at 25 μM significantly reduced HUVEC migration, osteogenic differentiation, without affecting cell viability, and mitigated PLLA influence on SLUG, MMP-9, VIMENTIN and RUNX-2 expression. EGCG affected PBMC proliferation and migration toward PLLA in a transwell co-culture system with HUVEC., Conclusion: Our study suggests the pro-calcific effect of PLLA, proposing EGCG as an anti-inflammatory modulatory approach. Research efforts need to deepen PLLA-vascular wall interactions for preventing vascular graft failure.

Extensive evidence indicates an association of air pollution exposure with an increased risk of cardiovascular disease (CVD) development. Fine particulate matter (PM) represents one of the main components of urban pollution, but the mechanisms by which it exerts adverse effects on cardiovascular system remain partially unknown and under investigation. The alteration of endothelial functions and inflammation are among the earliest pathophysiological impacts of environmental exposure on the cardiovascular system and represent critical mediators of PM-induced injury. In this context, endothelial stem/progenitor cells (EPCs) play an important role in vascular homeostasis, endothelial reparative capacity, and vasomotor functionality modulation. Several studies indicate the impairment of EPCs' vascular reparative capacity due to PM exposure. Since a central source of EPCs is bone marrow (BM), their number and function could be related to the population and functional status of stem cells (SCs) of this district. In this review, we provide an overview of the potential mechanisms by which PM exposure hinders vascular repair by the alteration of progenitor and stem cells' functionality., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Costa and Pasquinelli.)

Background: Neuroblastoma is a deadly childhood cancer, and MYCN-amplified neuroblastoma (MNA-NB) patients have the worst prognoses and are therapy-resistant. While retinoic acid (RA) is beneficial for some neuroblastoma patients, the cause of RA resistance is unknown. Thus, there remains a need for new therapies to treat neuroblastoma. Here we explored the possibility of combining a MYCN-specific antigene oligonucleotide BGA002 and RA as therapeutic approach to restore sensitivity to RA in NB., Methods: By molecular and cellular biology techniques, we assessed the combined effect of the two compounds in NB cell lines and in a xenograft mouse model MNA-NB., Results: We found that MYCN-specific inhibition by BGA002 in combination with RA (BGA002-RA) act synergistically and overcame resistance in NB cell lines. BGA002-RA also reactivated neuron differentiation (or led to apoptosis) and inhibited invasiveness capacity in MNA-NB. Moreover, we found that neuroblastoma had the highest level of mRNA expression of mTOR pathway genes, and that BGA002 led to mTOR pathway inhibition followed by autophagy reactivation in MNA-NB cells, which was strengthened by BGA002-RA. BGA002-RA in vivo treatment also eliminated tumor vascularization in a MNA-NB mouse model and significantly increased survival., Conclusion: Taken together, MYCN modulation mediates the therapeutic efficacy of RA and the development of RA resistance in MNA-NB. Furthermore, by targeting MYCN, a cancer-specific mTOR pathway inhibition occurs only in MNA-NB, thus avoiding the side effects of targeting mTOR in normal cells. These findings warrant clinical testing of BGA002-RA as a strategy for overcoming RA resistance in MNA-NB., (© 2022. The Author(s).)

BACKGROUND Rhabdomyolysis is a syndrome characterized by muscle necrosis and the subsequent release of intracellular muscle constituents into the bloodstream. Although the specific cause is frequently evident from the history or from the immediate events, such as a trauma, extraordinary physical exertion, or a recent infection, sometimes there are hidden risk factors that have to be identified. For instance, individuals with sickle cell trait (SCT) have been reported to be at increased risk for rare conditions, including rhabdomyolysis. Moreover, there have been a few case reports of SARS-CoV-2 infection-related rhabdomyolysis. CASE REPORT We present a case of a patient affected by unknown SCT and admitted with SARS-CoV-2 pneumonia, who suffered non-traumatic non-exertional rhabdomyolysis leading to acute kidney injury (AKI), requiring acute hemodialysis (HD). The patients underwent 13 dialysis session, of which 12 were carried out using an HFR-Supra H dialyzer. He underwent kidney biopsy, where rhabdomyolysis injury was ascertained. No viral traces were found on kidney biopsy samples. The muscle biopsy showed the presence of an "open nucleolus" in the muscle cell, which was consistent with virus-infected cells. After 40 days in the hospital, his serum creatinine was 1.62 mg/dL and CPK and Myoglobin were 188 U/L and 168 ng/mL, respectively; therefore, the patient was discharged. CONCLUSIONS SARS-CoV-2 infection resulted in severe rhabdomyolysis with AKI requiring acute HD. Since SARS-CoV-2 infection can trigger sickle-related complications like rhabdomyolysis, the presence of SCT needs to be ascertained in African patients.

The ability to form spheroids under non-adherent conditions is a well-known property of human mesenchymal stem cells (hMSCs), in addition to stemness and multilineage differentiation features. In the present study, we tested the ability of hMSCs isolated from the vascular wall (hVW-MSCs) to grow as spheres, and provide a characterization of this 3D model. hVW-MSCs were isolated from femoral arteries through enzymatic digestion. Spheres were obtained using ultra-low attachment and hanging drop methods. Immunophenotype and pluripotent genes (SOX-2, OCT-4, NANOG) were analyzed by immunocytochemistry and real-time PCR, respectively. Spheres histological and ultrastructural architecture were examined. Cell viability and proliferative capacity were measured using LIVE/DEATH assay and ki-67 proliferation marker. Metabolomic profile was obtained with liquid chromatography-mass spectrometry. In 2D, hVW-MSCs were spindle-shaped, expressed mesenchymal antigens, and displayed mesengenic potential. 3D cultures of hVW-MSCs were CD44 + , CD105 low , CD90 low , exhibited a low propensity to enter the cell cycle as indicated by low percentage of ki-67 expression and accumulated intermediate metabolites pointing to slowed metabolism. The 3D model of hVW-MSCs exhibits stemness, dormancy and slow metabolism, typically observed in stem cell niches. This culture strategy can represent an accurate model to investigate hMSCs features for future clinical applications in the vascular field., (© 2021 The Authors. Microscopy Research and Technique published by Wiley Periodicals LLC.)

Introduction: Different models have been proposed for the prediction of the risk/benefit ratio of surgery in patients with carotid atheromasic disease, mainly based on clinical patients' characteristics and risk factors, but no definite biological markers predictive of plaque instability and disease evolution have emerged so far, able to help the surgeon in the choice and timing of treatment. The main purpose of the present study was to assess the role of the polymorphism for genes commonly implicated in cell proliferation and neoangiogenesis in the clinical and histopathological carotid plaque vulnerability., Materials and Methods: We retrospectively studied 29 consecutive patients who underwent carotid endarterectomy in 6 months. All histological variables were collected, as well as patients' cardiovascular risk factors, clinical presentation, and brain computed tomography (CT) for the presence of ischemic lesions. Next-Generation Sequencing (NGS) was performed on 10-µm FFPE sections by means of a multi-gene panel used for sequencing 343 amplicons in 28 genes., Results: Among the gene variants observed, the polymorphism p.(Gln787=) in the EGFR gene was inversely correlated with intraplaque hemorrhage (p = 0.014), but also with the presence of ischemic brain lesions at CT (p = 0.001). Also p.(Gly105=) polymorphism in the IDH1 gene was inversely correlated with the presence of ischemic brain lesions (p = 0.038)., Conclusions: The variant p.(Gln787=) in the EGFR gene seems to play a role in plaque stability in patients with carotid atheromasic disease, on both histopathological and clinical grounds, probably acting on plaque matrix remodeling. This can open new scenarios on the pre-surgical management of these patients., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Innovative green, eco-friendly, and biologically derived hydrogels for non-load bearing bone sites were conceived and produced. Natural polysaccharides (copolymers of sodium D-mannuronate and L-guluronate) with natural polypeptides (gelatin) and bioactive mineral fillers (calcium silicates CaSi and dicalcium phosphate dihydrate DCPD) were used to obtain eco-sustainable biomaterials for oral bone defects. Three PP-x:y formulations were prepared (PP-16:16, PP-33:22, and PP-31:31), where PP represents the polysaccharide/polypeptide matrix and x and y represent the weight % of CaSi and DCPD, respectively. Hydrogels were tested for their chemical-physical properties (calcium release and alkalizing activity in deionized water, porosity, solubility, water sorption, radiopacity), surface microchemistry and micromorphology, apatite nucleation in HBSS by ESEM-EDX, FT-Raman, and micro-Raman spectroscopies. The expression of vascular ( CD31 ) and osteogenic (alkaline phosphatase ALP and osteocalcin OCN ) markers by mesenchymal stem cells (MSCs) derived from human vascular walls, cultured in direct contact with hydrogels or with 10% of extracts was analysed. All mineral-filled hydrogels, in particular PP-31:31 and PP-33:22, released Calcium ions and alkalized the soaking water for three days. Calcium ion leakage was high at all the endpoints (3 h-28 d), while pH values were high at 3 h-3 d and then significantly decreased after seven days ( p < 0.05). Porosity, solubility, and water sorption were higher for PP-31:31 ( p < 0.05). The ESEM of fresh samples showed a compact structure with a few pores containing small mineral granules agglomerated in some areas (size 5-20 microns). PP-CTRL degraded after 1-2 weeks in HBSS. EDX spectroscopy revealed constitutional compounds and elements of the hydrogel (C, O, N, and S) and of the mineral powders (Ca, Si and P). After 28 days in HBSS, the mineral-filled hydrogels revealed a more porous structure, partially covered with a thicker mineral layer on PP-31:31. EDX analyses of the mineral coating showed Ca and P, and Raman revealed the presence of B-type carbonated apatite and calcite. MSCs cultured in contact with mineral-filled hydrogels revealed the expression of genes related to vascular ( CD31 ) and osteogenic (mainly OCN ) differentiation. Lower gene expression was found when cells were cultured with extracts added to the culture medium. The incorporation of biointeractive mineral powders in a green bio-derived algae-based matrix allowed to produce bioactive porous hydrogels able to release biologically relevant ions and create a suitable micro-environment for stem cells, resulting in interesting materials for bone regeneration and healing in oral bone defects.

Introduction: Electron microscopy (EM) represents an indispensable technique for the diagnosis of kidney glomerular diseases. When dedicated tissue is not available, histological and cryostat sections can be reprocessed for EM using the pop-off technique. Here the practical value of this technique is analysed with emphasis on its accuracy in measuring basement membrane thickness and detecting immune deposits., Methods: Ninety-four histological sections of kidney tissues fixed in Serra's solution, stained with H&E, PAS, and Masson's Trichrome; for EM analysis, the sections were recovered from either treated or untreated microscope slides through the pop-off technique. Some sections were recovered from cryosections allocated for immunofluorescence., Results: The ultrastructural details were sufficiently maintained on tissues fixed with Serra's solution despite being considered disadvantageous for EM. The type of microscope slides and the time of biopsy storage did not affect the quality of section recovery. The histological stains had only moderate effects on the electron-density of the glomerular basement membrane (GBM). The pop-off technique reduced the GBM thickness when compared to the conventional EM processing but preserved the electron density of immune deposits., Conclusions: The application of the pop-off method to renal biopsy is a useful recovery method that produces limited but satisfactory results when there is no suitable material for EM. The ultrastructural morphology was retained even from tissues fixed with Serra's solution, and deposits maintained the expected electron density, however, we observed an overall thickness reduction of the GBM that could have a potential impact on thin membrane disease diagnosis., (© 2021. Italian Society of Nephrology.)

Atherosclerosis may starts early in life and each artery has peculiar characteristics likely affecting atherogenesis. The primary objective of the work was to underpin the microRNA (miR)-profiling differences in human normal femoral, abdominal aortic, and carotid arteries. The secondary aim was to investigate if those identified miRs, differently expressed in normal conditions, may also have a role in atherosclerotic arteries at adult ages. MiR-profiles were performed on normal tissues, revealing that aorta and carotid arteries are more similar than femoral arteries. MiRs emerging from profiling comparisons, i.e., miR-155-5p, -27a-5p, and -139-5p, were subjected to validation by RT-qPCR in normal arteries and also in pathological/atheroma counterparts, considering all the available 20 artery specimens. The three miRs were confirmed to be differentially expressed in normal femoral vs aorta/carotid arteries. Differential expression of those miRs was also observed in atherosclerotic arteries, together with some miR-target proteins, such as vimentin, CD44, E-cadherin and an additional marker SLUG. The different expression of miRs and targets/markers suggests that aorta/carotid and femoral arteries differently activate molecular drivers of pathological condition, thus conditioning the morphology of atheroma in adult life and likely suggesting the future use of artery-specific treatment to counteract atherosclerosis., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

We report the case of a 77-year-old woman affected by coronavirus disease-19 (COVID-19) who developed an occlusive arterial disease of the lower limb requiring a left leg amputation. We studied the mechanisms of vascular damage by SARS-CoV-2 by means of a comprehensive multi-technique in situ analysis on the diseased popliteal arterial district, including immunohistochemistry (IHC), transmission electron microscopy (TEM) and miRNA analysis. At histological analyses, we observed a lymphocytic inflammatory infiltrate, oedema and endothelialitis of adventitial vasa vasorum while the media was normal and the intima had only minor changes. The vasa vasorum expressed the ACE2 receptor and factor VIII; compared with the controls, VEGFR2 staining was reduced. TEM analyses showed endothelial injury and numerous Weibel-Palade bodies in the cytoplasm. No coronavirus particle was seen. IL-6 protein and mRNA, together with miR-155-5p and miRs-27a-5p, which can target IL-6, were significantly increased compared with that in the controls. Our case report suggests an involvement of adventitial artery microcirculation by inflammation in the course of COVID-19. Without evident signs of current infection by SARS-CoV-2, endothelial cells show a spectrum of structural and functional alterations that can fuel the cardiovascular complications observed in people infected with SARS-CoV-2.

Neisseria gonorrhoeae (GC) is the agent of one of the most common bacterial sexually transmitted infections worldwide. The possible development of 'untreatable' infections points out the need for antibiotic-sparing methods to reduce the number of gonococcal infections. In this context, fatty acids are interesting candidates as next-generation antibacterial agents. The aim of this study was to investigate the bactericidal effects of selected fatty acids on GC viability, as well as to observe their biological effects by means of transmission electron microscopy. The cytotoxicity of these compounds on human cervical cells (HeLa), chosen as a model of genital mucosa, was assessed as well. Lauric, myristic, and palmitic acid displayed high killing activity against GC in concentrations ranging between 100 μM and 25 μM, whereas the antimicrobial effect of oleic and butyric acids was present in concentrations between 1 mM and 0.25 mM. Modifications induced by fatty acids on the GC cell included the disorganization of the cytoplasmic structure, the distortion of pili/fimbriae, and the separation of the inner and outer membrane layers. For concentrations active against GC, fatty acids were not toxic for cervical cells. Our data can help in promoting innovative antibiotic-free compounds for the treatment of GC infections.

Bone development-related genes are enriched in healthy femoral arteries, which are more prone to calcification, as documented by the predominance of fibrocalcific plaques at the femoral location. We undertook a prospective histological study on the presence of calcifications in normal femoral arteries collected from donors. Since endothelial-to-mesenchymal transition (EndMT) participates in vascular remodeling, immunohistochemical (IHC) and molecular markers of EndMT and chondro-osteogenic differentiation were assessed. Transmission electron microscopy (TEM) was used to describe calcification at its inception. Two hundred and fourteen femoral arteries were enrolled. The mean age of the donors was 39.9 ± 12.9 years; male gender prevailed (M: 128). Histology showed a normal architecture; calcifications were found in 52 (24.3%) cases, without correlations with cardiovascular risk factors. Calcifications were seen on or just beneath the inner elastic lamina (IEL). At IHC, SLUG was increasingly expressed in the wall of focally calcified femoral arteries (FCFA). ETS-related gene (ERG), SLUG, CD44, and SOX-9 were positive in calcifications. RT-PCR showed increased levels of BPM-2, RUNX-2, alkaline phosphatase, and osteocalcin osteogenic transcripts and increased expression of the chondrogenic marker, SOX-9, in FCFA. TEM documented osteoblast-like cells adjacent to the IEL, releasing calcifying vesicles from the cell membrane. The vesicles were embedded in a proteoglycan-rich matrix and were entrapped in IEL fenestrations. In this study, ERG- and CD44-positive cell populations were found in the context of increased SLUG expression, thus supporting the participation of EndMT in FCFA; the increased transcript expression of osteochondrogenic markers, particularly SOX-9, reinforced the view that EndMT, osteochondrogenesis, and neoangiogenesis interact in the process of arterial calcification. Given its role as a transcription factor in the regulation of endothelial homeostasis, arterial ERG expression can be a clue of endothelial dysregulation and changes in IEL organization which can ultimately hinder calcifying vesicle diffusion through the IEL fenestrae. These results may have a broader implication for understanding arterial calcification within a disease context., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Vasuri, Valente, Motta, Degiovanni, Ciavarella and Pasquinelli.)

The endothelial to mesenchymal transition (End-MT) can be associated with vascular calcification, by providing mesengenic progenitors. In this study, we investigated a link between End-MT and the osteogenic process and explored the involvement of miR-30a-5p and miR-30d as potential regulators of these processes. End-MT was induced in Human Umbilical Vein Endothelial Cells (HUVEC) through transforming growth factor-β1 (TGF-β1), TGFβ-3 and tumor necrosis factor-α (TNF-α), for 24 h and 6 days. End-MT mediators, mesenchymal and osteo/chondrogenic markers were analyzed through Real-Time PCR, immunofluorescence, flow cytometry and Western Blot. miR-30a-5p and miR-30d over-expression was carried out in HUVEC to explore their effects on End-MT and osteogenic differentiation. HUVEC at 24 h and 6 days gained mesenchymal morphology markers, including matrix metalloproteinase 9 (MMP-9), SLUG, VIMENTIN and α-smooth muscle actin (α-SMA), and a significant migratory potential, notably with TNF-α. After 6 days, the osteo/chondrogenic markers runt-related transcription factor 2 (RUNX-2) and SRY box transcription factor 9 (SOX-9) were upregulated. At this time point, miR-30a-5p and miR-30d decreased. Over-expression of miR-30a-5p and miR-30d affected End-MT mediators and the osteogenic potency in HUVEC, by reducing SLUG, VIMENTIN and RUNX-2. Our data suggest that End-MT represents a key link between inflammation and vascular calcification. Further, miR-30a-5p and miR-30d can regulate both the End-MT and the osteogenic processes, prompting future studies for exploring their potential use as therapeutic targets or biomarkers in vascular diseases.

Background and Aim of the Study: COVID-19 is characterized by super spread events occurring in communities, e.g., hospitals. To limit virus diffusion among healthcare workers the use of personal protective equipment and screening tests are highly advised; also, isolation of virus positive professionals while monitoring their health condition is recommended. This study aims to assess, in a cohort of COVID-19 positive quarantined healthcare workers, the perceived source of infection and exposure risk as well as the clinical evolution of the disease through a surveillance interview., Methods: A retrospective observational study accounting 896 observations on 93 healthcare professionals tested positive for COVID-19. Data were collected from the Nursing and Technical Directorate of Romagna, Ravenna, Local Health Company, Italy., Results: 99.5% of the positive workers accepted phone interviews with management staff. 2.6% of workers were positive with increasing records in the specialist medical area. Nurses and social health professionals were mostly affected.  Patient exposure at a distance <1 m and a contact time > 2 hours was the first cause of positivity. In COVID-19 and territorial emergency departments, the first cause was the contact with colleagues. At the time of the infection, most of the staff wore a surgical mask. Cough, asthenia, fever, anosmia, dysgeusia, and rhinitis were common symptoms. Asymptomatic percentage was about 10%. The self-perceived physical condition was high (>7) and improved during the observation period., Conclusions: The diffusion rate of COVID-19 among healthcare workers is relatively low, probably due to the use of personal protective equipment. The distancing, also among colleagues, is a fundamental measure to reduce the possibility of infection. Symptoms are mild and can be controlled by surveillance measures. Constant contact with the organization is an essential strategy for promoting recovering of workers and reducing the spread of the virus within the healthcare organization.

We evaluated the long-term effects of sirolimus on three different cell in vitro models, cultured in physiological conditions mimicking sirolimus-eluted stent, in order to clarify the effectiveness of sirolimus in blocking cell proliferation and survival. Three cells lines (WPMY-1 myofibroblasts, HT-29 colorectal adenocarcinoma, and U2OS osteosarcoma) were selected and growth in 10 ml of Minimum Essential Medium for 5 weeks with serial dilutions of sirolimus. The number of colonies and the number of cells per colony were counted. As main result, the number of WPMY-1 surviving colonies increased in a dose-dependent manner when treated with sirolimus ( p = 0.0011), while the number of U2OS colonies progressively decreased ( p = 0.0011). The clonal capacity of HT-29 was not modified by the exposure to sirolimus ( p = 0.6679). In conclusion sirolimus showed the well-known cytostatic effect, but with an effect on clonogenic potential different among the different cell types. In the practice, the plaque typology and composition may influence the response to sirolimus and thus the effectiveness of eluted stent., Competing Interests: CONFLICTS OF INTEREST Authors declare no conflicts of interest.

Background: Abdominal aortic aneurysm (AAA) is a progressive dilation of the aortic wall, determined by the unbalanced activity of matrix metalloproteinase (MMPs). In vitro and in vivo studies support the pivotal role of MMP-9 to AAA pathogenesis. In our experience, we elucidated the expression of MMP-9 in an ex vivo model of human mesenchymal stem cells isolated from AAA specimen (AAA-MSCs). Thus, MMP-9 inhibition could be an attractive therapeutic strategy for inhibiting AAA degeneration and rupture. Our study was aimed at testing the effect of 3 different drugs (pioglitazone, doxycycline, simvastatin) on MMP-9 and peroxisome proliferator-activated receptor (PPAR)-γ expression in AAA-MSCs., Methods: Aneurysmal aortic wall segments were taken from AAA patients after the open surgical treatment. MSCs were isolated from AAA (n = 20) tissues through enzymatic digestion. AAA-MSCs were exposed to different doses of pioglitazone (5-10-25 μM), doxycycline (10-25 μM), and simvastatin (10 μM) for 24 h. The effect of each drug was evaluated in terms of cell survival, by crystal violet stain. MMP-9 and PPAR-γ mRNA were analyzed using real-time PCR., Results: AAA-MSCs were not affected by the exposure to the selected drugs, as shown by the analysis of cell viability. Interestingly, MMP-9 mRNA resulted significantly decreased after each treatment, recording a downregulation of 50% in presence of pioglitazone, 90% with doxycycline, and 40% with exposed to simvastatin, in comparison to untreated cells. We further analyzed the expression of PPAR-γ, target of pioglitazone, observing an upregulation in exposed AAA-MSCs to controls., Conclusions: Our data support the potential therapeutic effect of pioglitazone, doxycycline, and simvastatin on AAA by reducing the MMP-9 expression in a patient-specific model (AAA-MSCs). In addition, pioglitazone drives the increase of PPAR-G, another promising target for AAA therapy. Further studies are necessary to elucidate the mechanism driving this inhibitory pathway, which can reduces the mortality risk associated with AAA rupture., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Atherosclerosis is considered a chronic inflammatory disease of arteries associated with the aging process. Many risk factors have been identified and they are mainly related to life-styles, gene-environment interactions and socioeconomic status. Carotid and coronary artery diseases are the two major atherosclerotic conditions, being the primary cause of stroke and heart attack, respectively. Nevertheless, carotid plaque assumes particular aspects not only for the specific molecular mechanisms, but also for the types of atheroma which may be associated with a better or a worst prognosis. The identification of circulating blood biomarkers able to distinguish carotid plaque types (stable or vulnerable) is a crucial step for the improvement of adequate therapeutic approaches avoiding or delaying endarterectomy in the oldest old individuals (> 80 years), a population predicted to growth in the next years. The review highlights the most recent knowledge on carotid plaque molecular mechanisms, focusing on microRNAs (miRs), as a site-specific accelerated aging within the conceptual framework of Geroscience for new affordable therapies., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)