Atrial natriuretic peptide (ANP) modulates stress-induced autophagy in endothelial cells.

Atrial natriuretic peptide (ANP), a cardiac hormone involved in the regulation of water/sodium balance and blood pressure, is also secreted by endothelial cells, where it exerts protective effects in response to stress. Autophagy is an intracellular self-renewal process involved in the degradation of dysfunctional cytoplasmic elements. ANP was recently reported to act as an extracellular regulator of cardiac autophagy. However, its role in the regulation of endothelial autophagy has never been investigated. Here, we tested the effects of ANP in the regulation of autophagy in human umbilical vein endothelial cells (HUVECs). We found that ANP rapidly increases autophagy and autophagic flux at physiological concentrations through its predominant pathway, mediated by natriuretic peptide receptor type A (NPR-A) and protein kinase G (PKG). We further observed that ANP is rapidly secreted by HUVEC under stress conditions, where it mediates stress-induced autophagy through autocrine and paracrine mechanisms. Finally, we found that the protective effects of ANP in response to high-salt loading or tumor necrosis factor (TNF)-α are blunted by concomitant inhibition of autophagy. Overall, our results suggest that ANP acts as an endogenous autophagy activator in endothelial cells. The autophagy mechanism mediates the protective endothelial effects exerted by ANP.

Seminological, Hormonal and Ultrasonographic Features of Male Factor Infertility Due to Genetic Causes: Results from a Large Monocentric Retrospective Study.

Objectives: Evaluate the prevalence of genetic factors in a large population of infertile subjects and define the seminological, hormonal, and ultrasonographic features for each alteration. Methods: This single-center retrospective study included male partners of infertile couples undergoing genetic investigations due to oligozoospermia or azoospermia evaluated from January 2012 to January 2022. The genetic investigations consist of karyotype, CFTR gene mutations plus variant of the IVS8-5T polymorphic trait, Y chromosome microdeletion, and Next Generation Sequencing panel to analyze genes implicated in congenital hypogonadotropic hypogonadism (CHH). Results: Overall, 15.4% (72/466) of patients received a diagnosis of genetic cause of infertility. Specifically, 23 patients (31.9%) harbor mutations in the CFTR gene, 22 (30.6%) have a 47, XXY karyotype, 14 (19.4%) patients show a Y chromosome microdeletion, 7 (9.7%) have structural chromosomal anomalies, and 6 (8.3%) have CHH. Overall, 80.6% of patients were azoospermic and 19.4% oligozoospermic (sperm concentration 3.5 ± 3.8 million/mL). Almost all patients presented hormonal alterations related to the specific genotype, while the main ultrasound alterations were testicular hypoplasia, calcifications/microcalcifications, and enlarged/hyperechoic epididymis. Conclusions: The prevalence of genetic abnormalities in males of infertile couples was 15.4% in our Center. CFTR gene disease-causing variants resulted in more frequent, with various clinical features, highlighting the complexity and heterogeneity of the presentation. Other investigations are needed to understand if conditions like ring chromosomes and other translocations are related to infertility or are incidental factors.

TRPA1 Contributes to FGFR2c Signaling and to Its Oncogenic Outcomes in Pancreatic Ductal Adenocarcinoma-Derived Cell Lines.

Fibroblast growth factor receptor (FGFR) signaling is a key modulator of cellular processes dysregulated in cancer. We recently found that the high expression of the mesenchymal FGFR2c variant in human pancreatic ductal adenocarcinoma (PDAC)-derived cells triggers the PKCε-mediated improvement of EMT and of MCL-1/SRC-dependent cell invasion. Since other membrane proteins can affect the receptor tyrosine kinase signaling, including transient receptor potential channels (TRPs), in this work, we investigated the role of TRPs in the FGFR2c/PKCε oncogenic axis. Our results highlighted that either the FGFR2c/PKCε axis shut-off obtained by shRNA or its sustained activation via ligand stimulation induces TRPA1 downregulation, suggesting a channel/receptor dependence. Indeed, biochemical molecular and immunofluorescence approaches demonstrated that the transient depletion of TRPA1 by siRNA was sufficient to attenuate FGFR2c downstream signaling pathways, as well as the consequent enhancement of EMT. Moreover, the biochemical check of MCL1/SRC signaling and the in vitro assay of cellular motility suggested that TRPA1 also contributes to the FGFR2c-induced enhancement of PDAC cell invasiveness. Finally, the use of a selective channel antagonist indicated that the contribution of TRPA1 to the FGFR2c oncogenic potential is independent of its pore function. Thus, TRPA1 could represent a putative candidate for future target therapies in PDAC.

The Impact of Non-Andrological Medications on Semen Characteristics, Oxidative Stress and Inflammatory Parameters.

Background and Objectives: The aim of this study was to evaluate the impact of medications on oxidative stress, inflammatory biomarkers and semen characteristics in males with idiopathic infertility. Materials and Methods: In this observational case-control clinical study, 50 men with idiopathic infertility were enrolled, of whom 38 (the study group) were on pharmacological treatment and 12 made up the control group. The study group was clustered according to the medications (Group A: anti-hypertensive, n = 10; Group B: thyroxine, n = 6; Group C: non-steroidal anti-inflammatory drugs, n = 13; Group D: miscellaneous, n = 6; Group E: lipid-lowering drugs, n = 4). Semen analyses were performed according to WHO 2010 guidelines. Interleukins (IL)-10, IL-1 beta, IL-4, IL-6, Tumor Necrosis Factor- alpha (TNF-alpha) and IL-1 alpha were determined using a solid-phase sandwich immunoassay. The diacron reactive oxygen metabolites, d-ROMs test, was performed by means of a colorimetric determination of reactive oxygen metabolites and measured with a spectrophotometer. Beta-2-microglobulin and cystatin-C were measured with an immunoturbidimetric analyzer. Results: No differences between the study and control groups for age and macroscopic and microscopic semen characteristics were found, nor were any differences found after clustering according to the drug categories. IL-1 alpha and IL-10 were significantly lower in the study group compared with the control group; IL-10 was significantly lower in groups A, B, C and D compared with the control group. Furthermore, a direct correlation between IL-1 alpha, IL-10 and TNF-alpha and leukocytes was found. Conclusions: Despite the sample size limitations, the data suggest a correlation between drug use and activation of the inflammatory response. This could clarify the pathogenic mechanism of action for several pharmacological classes on male infertility.

Atrial natriuretic peptide stimulates autophagy/mitophagy and improves mitochondrial function in chronic heart failure.

Mitochondrial dysfunction, causing increased reactive oxygen species (ROS) production, is a molecular feature of heart failure (HF). A defective antioxidant response and mitophagic flux were reported in circulating leucocytes of patients with chronic HF and reduced ejection fraction (HFrEF). Atrial natriuretic peptide (ANP) exerts many cardiac beneficial effects, including the ability to protect cardiomyocytes by promoting autophagy. We tested the impact of ANP on autophagy/mitophagy, altered mitochondrial structure and function and increased oxidative stress in HFrEF patients by both ex vivo and in vivo approaches. The ex vivo study included thirteen HFrEF patients whose peripheral blood mononuclear cells (PBMCs) were isolated and treated with αANP (10-11 M) for 4 h. The in vivo study included six HFrEF patients who received sacubitril/valsartan for two months. PBMCs were characterized before and after treatment. Both approaches analyzed mitochondrial structure and functionality. We found that levels of αANP increased upon sacubitril/valsartan, whereas levels of NT-proBNP decreased. Both the ex vivo direct exposure to αANP and the higher αANP level upon in vivo treatment with sacubitril/valsartan caused: (i) improvement of mitochondrial membrane potential; (ii) stimulation of the autophagic process; (iii) significant reduction of mitochondrial mass-index of mitophagy stimulation-and upregulation of mitophagy-related genes; (iv) reduction of mitochondrial damage with increased inner mitochondrial membrane (IMM)/outer mitochondrial membrane (OMM) index and reduced ROS generation. Herein we demonstrate that αANP stimulates both autophagy and mitophagy responses, counteracts mitochondrial dysfunction, and damages ultimately reducing mitochondrial oxidative stress generation in PBMCs from chronic HF patients. These properties were confirmed upon sacubitril/valsartan administration, a pivotal drug in HFrEF treatment.

FGFR2c Upregulation Contributes to Cancer-Associated Fibroblast Program Activation and to Enhanced Autophagy in Actinic Keratosis-Derived Dermal Fibroblasts: A Possible Role in Precancerous Cell/Stromal Cell Crosstalk.

Actinic keratosis (AK) is a preneoplastic skin disorder which can rapidly progress to cutaneous squamous cell carcinomas (SCCs). In light of our previous findings, indicating a possible oncogenic role of the mesenchymal isoform of FGFR2 (FGFR2c) aberrantly expressed in AK keratinocytes, we analyzed the possible tumor-promoting role of this receptor in the stromal AK counterpart in this work. Molecular analysis showed that, particularly in early AK lesions, FGFR2c dermal upregulation is accompanied by the downregulation of the cancer-associated fibroblasts (CAF) transcription repressor CSL, the upregulation of the CAF activator ULK3, and the consequent CAF gene induction. Immunofluorescence and molecular analysis, coupled with silencing approaches by siRNA, applied on primary cultures of KIN I-derived fibroblasts, indicated that FGFR2c upregulation contribute to CAF signature and the increased autophagy in response to FGF2. Magnetic bead-based multiplex assay, combined with FGFR2 signaling shut-off approaches, indicated that, especially in response to FGF2, IL-6 secretion could depend on FGFR2c high expression and signaling, suggesting the possible establishment of FGFR2c-dependent secretory autophagy, contributing to tumor-promoting factor release. Overall, our results identified FGFR2c as a signaling molecule involved in controlling precancerous/stromal cell oncogenic crosstalk, pointing to this receptor as a possible early molecular marker predictive for AK's rapid malignant progression.

Surfactant Protein B Plasma Levels: Reliability as a Biomarker in COPD Patients.

Background: The diagnosis of COPD is based on both clinical signs and functional tests. Although there are different functional tests used to assess COPD, no reliable biomarkers able to provide information on pathogenesis and severity are available. The aim of the present study is to explore the relationship between surfactant protein B (Sp-B) serum levels and clinical, radiological, and functional pulmonary parameters in COPD patients. Methods: Forty COPD patients and twenty smokers without airflow limitations or respiratory symptoms were enrolled. Each patient was given questionnaires (CAT and mMRC) and 6MWT, spirometry, DLCO, and computer tomography (CT) were performed. All participants underwent a venous blood sample drawing, and quantitative detection of their Sp-B plasma levels was performed by an enzyme-linked immunosorbent assay. The spirometry and Sp-B plasma levels were assessed after 12 months. Results: A statistically significant difference was found in the plasma Sp-B levels between COPD patients compared to the other group (4.72 + 3.2 ng/mL vs. 1.78 + 1.5 ng/mL; p < 0.001). The change in FEV1 after 12 months (Delta FEV1) showed a significantly negative correlation with respect to the change in Sp-B levels (Delta SpB) (r = −0.4; p < 0.05). This correlation indicates that increasing the plasma dosage of SpB is a foretoken of functional decline. Conclusions: SpB may be considered as a useful marker in COPD assessment and provides prognostic information on lung functional decline. Despite its usefulness, further studies are needed to define its reliability as a biomarker.

NPPA/atrial natriuretic peptide is an extracellular modulator of autophagy in the heart.

NPPA/atrial natriuretic peptide (natriuretic peptide type A) exerts critical pleiotropic effects in the cardiovascular system, limiting cardiomyocyte hypertrophy and death, reducing cardiac fibrosis and promoting vascular integrity. However, the molecular mechanisms underlying these beneficial effects still need to be clarified. We demonstrated for the first time that macroautophagy/autophagy is involved in the local protective effects of NPPA in cardiomyocytes (CMs), both in vitro and in vivo. Exogenous NPPA rapidly activates autophagy in CMs through NPR1/type A natriuretic peptide receptor and PRKG/protein kinase G signaling and also increases cardiac autophagy in mice. Remarkably, endogenous NPPA is secreted by CMs in response to glucose deprivation or hypoxia, thereby stimulating autophagy through autocrine/paracrine mechanisms. NPPA preserves cell viability and reduces hypertrophy in response to stress through autophagy activation. In vivo, we found that Nppa knockout mice undergoing ischemia-reperfusion (I/R) show increased infarct size and reduced autophagy. Reactivation of autophagy by Tat-Beclin D11 limits I/R injury. We also found that the protective effects of NPPA in reducing infarct size are abrogated in the presence of autophagy inhibition. Mechanistically, we found that NPPA stimulates autophagy through the activation of TFEB (transcription factor EB). Our data suggest that NPPA is a novel extracellular regulator of autophagy in the heart.

From Survey Results to a Decision-Making Matrix for Strategic Planning in Healthcare: The Case of Clinical Pathways.

BACKGROUND: It is a well-known fact that the information obtained from a survey can be used in a healthcare organizational analysis; however, it is very difficult to compare the different results found in the literature to each other, even through the use of metanalysis, as the methodology is often not consistent. METHODS: Data from a survey analyzing the organizational and managerial responses adopted in pathology-specific clinical pathways (CPs) during the first two waves of the COVID-19 pandemic were used for constructing a decisional matrix, a tool called SPRIS system, consisting of four different sheets. The first sheet reports the results of the survey and, using a streetlight color system, identifies strengths and weaknesses; the second one, by assigning a priority score, establishes the priority of intervention on each of the strengths and weaknesses identified; the third sheet reports the subjective items of the questionnaire in order to identify threats and opportunities and their probability of happening; in the last sheet, a SWOT Analysis is used to calculate the performance index of the whole organization. RESULTS: The SPRIS system, applied to data concerning the adaptation of four CPs to the COVID-19 pandemic, showed that, whereas all the CPs had a good performance index, some concerns remained unsolved and need be addressed. CONCLUSIONS: The SPRIS system showed to be an easily constructed tool that is able to give an overview of the organization analyzed by the survey and to produce an index that can be used in a direct quality comparison between different services or organizations.

Role of FGFR2c and Its PKCε Downstream Signaling in the Control of EMT and Autophagy in Pancreatic Ductal Adenocarcinoma Cells.

Pancreatic ductal adenocarcinoma (PDAC) is a treatment-resistant malignancy characterized by a high malignant phenotype including acquired EMT signature and deregulated autophagy. Since we have previously described that the aberrant expression of the mesenchymal FGFR2c and the triggering of the downstream PKCε signaling are involved in epidermal carcinogenesis, the aim of this work has been to assess the contribution of these oncogenic events also in the pancreatic context. Biochemical, molecular and immunofluorescence approaches showed that FGFR2c expression impacts on PDAC cell responsiveness to FGF2 in terms of intracellular signaling activation, upregulation of EMT-related transcription factors and modulation of epithelial and mesenchymal markers compatible with the pathological EMT. Moreover, shut-off via specific protein depletion of PKCε signaling, activated by high expression of FGFR2c resulted in a reversion of EMT profile, as well as in a recovery of the autophagic process. The detailed biochemical analysis of the intracellular signaling indicated that PKCε, bypassing AKT and directly converging on ERK1/2, could be a signaling molecule downstream FGFR2c whose inhibition could be considered as possible effective therapeutic approach in counteracting aggressive phenotype in cancer.

Trehalose, a natural disaccharide, reduces stroke occurrence in the stroke-prone spontaneously hypertensive rat.

Cerebrovascular disease, a frequent complication of hypertension, is a major public health issue for which novel therapeutic and preventive approaches are needed. Autophagy activation is emerging as a potential therapeutic and preventive strategy toward stroke. Among usual activators of autophagy, the natural disaccharide trehalose (TRE) has been reported to be beneficial in preclinical models of neurodegenerative diseases, atherosclerosis and myocardial infarction. In this study, we tested for the first time the effects of TRE in the stroke-prone spontaneously hypertensive rat (SHRSP) fed with a high-salt stroke permissive diet (JD). We found that TRE reduced stroke occurrence and renal damage in high salt-fed SHRSP. TRE was also able to decrease systolic blood pressure. Through ex-vivo studies, we assessed the beneficial effect of TRE on the vascular function of high salt-fed SHRSP. At the molecular level, TRE restored brain autophagy and reduced mitochondrial mass, along with the improvement of mitochondrial function. The beneficial effects of TRE were associated with increased nuclear translocation of TFEB, a transcriptional activator of autophagy. Our results suggest that TRE may be considered as a natural compound efficacious for the prevention of hypertension-related target organ damage, with particular regard to stroke and renal damage.

May Bacterial Infections Trigger Bullous Pemphigoid? Case Report and Review of Literature.

Bullous pemphigoid (BP) is an autoimmune blistering skin disease, mainly observed in the elderly. Infections have been suggested as possible disease triggers. However, infections may even heavily influence the disease clinical course and mortality. A 75-year-old woman was admitted to hospital for severe erythematosus blistering disease, accompanied by hyper-eosinophilia and hyper-IgE. The culture of bullous fluid was positive for Enterococcus faecalis, the blood culture was positive for Staphylococcus aureus, and the urine culture was positive for Proteus mirabilis and Escherichia coli. Moreover, circulating anti-BP180 IgG was present and the histopathological/ultrastructural examination of a lesional skin biopsy was compatible with BP. High eosinophil levels (up to 3170/µL) were found throughout the clinical course, while values below 1000/µL were associated with clinical improvement. The total IgE was 1273 IU/mL, and specific anti-G/V-penicillin/ampicillin IgE antibodies were positive. The patient had a complete clinical recovery in two months with methyl-prednisolone (40 then 20 mg/day) and low-dose azathioprine (50 mg/day) as a steroid-sparing agent. The steroid treatment was tapered until interruption during a one-year period and intravenous immunoglobulins have been administered for three years in order for azathioprine to also be interrupted. The patient stopped any treatment five years ago and, in this period, has always been in good health. In this case, the contemporaneous onset of different bacterial infections and BP is suggestive of bacterial infections acting as BP trigger(s), with allergic and autoimmune pathways contributing to the disease pathogenesis.

Expression of the E5 Oncoprotein of HPV16 Impacts on the Molecular Profiles of EMT-Related and Differentiation Genes in Ectocervical Low-Grade Lesions.

Infection with human papillomavirus type 16 (HPV16) is one of the major risk factors for the development of cervical cancer. Our previous studies have demonstrated the involvement of the early oncoprotein E5 of HPV16 (16E5) in the altered isoform switch of fibroblast growth factor receptor 2 (FGFR2) and the consequent expression in human keratinocytes of the mesenchymal FGFR2c isoform, whose aberrant signaling leads to EMT, invasiveness, and dysregulated differentiation. Here, we aimed to establish the possible direct link between these pathological features or the appearance of FGFR2c and the expression of 16E5 in low-grade squamous intraepithelial lesions (LSILs). Molecular analysis showed that the FGFR2c expression displayed a statistically significant positive correlation with that of the viral oncoprotein, whereas the expression values of the epithelial FGR2b variant, as well as those of the differentiation markers keratin 10 (K10), loricrin (LOR) and involucrin (INV), were inversely linked to the 16E5 expression. In contrast, the expression of EMT-related transcription factors Snail1 and ZEB1 overlapped with that of 16E5, becoming a statistically significant positive correlation in the case of Snail2. Parallel analysis performed in human cervical LSIL-derived W12 cells, containing episomal HPV16, revealed that the depletion of 16E5 by siRNA was able to counteract these molecular events, proving to represent an effective strategy to identify the specific role of this viral oncoprotein in determining LSIL oncogenic and more aggressive profiles. Overall, coupling in vitro approaches to the molecular transcript analysis in ectocervical early lesions could significantly contribute to the characterization of specific gene expression profiles prognostic for those LSILs with a greater probability of direct neoplastic progression.

Effects of Sorafenib, a Tyrosin Kinase Inhibitor, on Adrenocortical Cancer.

The lack of an effective medical treatment for adrenocortical carcinoma (ACC) has prompted the search for better treatment protocols for ACC neoplasms. Sorafenib, a tyrosine kinase inhibitor has exhibited effectiveness in the treatment of different human tumors. Therefore, the aim of this study was to understand the mechanism through which sorafenib acts on ACC, especially since treatment with sorafenib alone is sometimes unable to induce a long-lasting antiproliferative effect in this tumor type. The effects of sorafenib were tested on the ACC cell line H295R by evaluating cell viability, apoptosis and VEGF receptor signaling which was assessed by analyzing VE-cadherin and ß-catenin complex formation. We also tested sorafenib on an in vitro 3D cell culture model using the same cell line. Apoptosis was observed after sorafenib treatment, and coimmunoprecipitation data suggested that the drug prevents formation VEGFR-VE-cadherin and ß-catenin proteins complex. These results were confirmed both by ultrastructural analysis and by a 3D model where we observed a disaggregation of spheres into single cells, which is a crucial event that represents the first step of metastasis. Our findings suggest that although sorafenib induces apoptotic cell death a small portion of cells survive the treatment and have characteristics of a malignancy. Based on our data we recommend against the use of sorafenib in patients with ACC.

A protective role for autophagy in vitiligo.

A growing number of studies supports the existence of a dynamic interplay between energetic metabolism and autophagy, whose induction represents an adaptive response against several stress conditions. Autophagy is an evolutionarily conserved and a highly orchestrated catabolic recycling process that guarantees cellular homeostasis. To date, the exact role of autophagy in vitiligo pathogenesis is still not clear. Here, we provide the first evidence that autophagy occurs in melanocytes and fibroblasts from non-lesional skin of vitiligo patients, as a result of metabolic surveillance response. More precisely, this study is the first to reveal that induction of autophagy exerts a protective role against the intrinsic metabolic stress and attempts to antagonize degenerative processes in normal appearing vitiligo skin, where melanocytes and fibroblasts are already prone to premature senescence.

Novel ACTA1 mutation causes late-presenting nemaline myopathy with unusual dark cores.

ACTA1 gene encodes the skeletal muscle alpha-actin, the core of thin filaments of the sarcomere. ACTA1 mutations are responsible of several muscle disorders including nemaline, cores, actin aggregate myopathies and fiber-type disproportion. We report clinical, muscle imaging, histopatological and genetic data of an Italian family carrying a novel ACTA1 mutation. All affected members showed a late-presenting, diffuse muscle weakness with sternocleidomastoideus and temporalis atrophy. Mild dysmorphic features were also detected. The most affected muscles by muscle MRI were rectus abdominis, gluteus minimus, vastus intermedius and both gastrocnemii. Muscle biopsy showed the presence of nemaline bodies with several unusual dark areas at Gomori Trichrome, corresponding to unstructured cores with abundant electrodense material by electron microscopy. The molecular analysis revealed missense variant c.148G>A; p.(Gly50Ser) in the exon 3 of ACTA1, segregating with affected members in the family. We performed a functional essay of fibre contractility showing a higher pCa50 (a measure of the calcium sensitivity of force) of type 1 fibers compared to control subjects' type 1 muscle fibers. Our findings expand the clinico-pathological spectrum of ACTA1-related congenital myopathies and the genetic spectrum of core-rod myopathies.

Monoclonal Gammopathies of Renal Significance: Renal Biopsy and Beyond.

Monoclonal Gammopathies of Renal Significance (MGRS) are a rather heterogeneous group of renal disorders caused by a circulating monoclonal (MC) immunoglobulin (Ig) component, often in the absence of multiple myeloma (MM) or another clinically relevant lymphoproliferative disorder. Nevertheless, substantial kidney damage could occur, despite the "benign" features of the bone-marrow biopsy. One example is renal amyloidosis, often linked to a small clone of plasma cells, without the invasive features of MM. However, patients with amyloidosis may present with a nephrotic syndrome and renal failure, eventually leading to end-stage kidney disease. At the same time, other organs, such as the heart and the liver, may be severely damaged by Ig light chains or amyloid deposits, occasionally resulting in fatal arrhythmias and/or organ failure. Acute kidney injury (AKI) may as well result from massive excretion of MC proteins, with deposition disease in glomeruli or renal tubules, not rarely obstructed by luminal aggregates, or "casts". Proliferative glomerulonephritis with monoclonal Ig deposits is another, less frequent clinical presentation of an MGRS. The present review deals with the implications of MGRS for renal function and prognosis, and the potential of tools, such as the renal biopsy, for assessing clinical risk and guiding therapy of the underlying condition.

A peculiar adverse reaction to blue pigment tattoo.

The practice of tattooing dates back to 5000 years ago and is currently constantly growing. The most common pigment associated with tattoo complications is the red one. Another pigment, which could be associated with adverse reactions, even if less frequently than red, is blue. Traditionally, blue pigment contains cobalt, which causes allergic reactions. Here, we report a case of blue pigment granulomatous reaction in a 37-year-old male patient.

Atypical koebner phenomenon on a tattoo.

Tattooing is more and more popular in developed countries in recent years and many side effects are associated with this practice, including psoriatic lesions and Koebner phenomenon. We report the case of a lichenoid reaction to red pigment in a patient affected by psoriasis.