All-trans retinoic acid exhibits anti-proliferative and differentiating activity in Merkel cell carcinoma cells via retinoid pathway modulation.

BACKGROUND: The limited therapies available for treating Merkel cell carcinoma (MCC), a highly aggressive skin neoplasm, still pose clinical challenges, and novel treatments are required. Targeting retinoid signalling with retinoids, such as all-trans retinoic acid (ATRA), is a promising and clinically useful antitumor approach. ATRA drives tumour cell differentiation by modulating retinoid signalling, leading to anti-proliferative and pro-apoptotic effects. Although retinoid signalling is dysregulated in MCC, ATRA activity in this tumour is unknown. This study aimed to evaluate the impact of ATRA on the pathological phenotype of MCC cells. METHODS: The effect of ATRA was tested in various Merkel cell polyomavirus-positive and polyomavirus-negative MCC cell lines in terms of cell proliferation, viability, migration and clonogenic abilities. In addition, cell cycle, apoptosis/cell death and the retinoid gene signature were evaluated upon ATRA treatments. RESULTS: ATRA efficiently impaired MCC cell proliferation and viability in MCC cells. A strong effect in reducing cell migration and clonogenicity was determined in ATRA-treated cells. Moreover, ATRA resulted as strongly effective in arresting cell cycle and inducing apoptosis/cell death in all tested MCC cells. Enrichment analyses indicated that ATRA was effective in modulating the retinoid gene signature in MCC cells to promote cell differentiation pathways, which led to anti-proliferative and pro-apoptotic/cell death effects. CONCLUSIONS: These results underline the potential of retinoid-based therapy for MCC management and might open the way to novel experimental approaches with other retinoids and/or combinatorial treatments.

Spiropiperidine-Based Oligomycin-Analog Ligands To Counteract the Ischemia-Reperfusion Injury in a Renal Cell Model.

Finding a therapy for ischemia-reperfusion injury, which consists of cell death following restoration of blood flowing into the artery affected by ischemia, is a strong medical need. Nowadays, only the use of broad-spectrum molecular therapies has demonstrated a partial efficacy in protecting the organs following reperfusion, while randomized clinical trials focused on more specific drug targets have failed. In order to overcome this problem, we applied a combination of molecular modeling and chemical synthesis to identify novel spiropiperidine-based structures active in mitochondrial permeability transition pore opening inhibition as a key process to enhance cell survival after blood flow restoration. Our results were confirmed by biological assay on an in vitro cell model on HeLa and human renal proximal tubular epithelial cells and pave the way to further investigation on an in vivo model system.

The Complex Relationship between Hypoxia Signaling, Mitochondrial Dysfunction and Inflammation in Calcific Aortic Valve Disease: Insights from the Molecular Mechanisms to Therapeutic Approaches.

Calcific aortic valve stenosis (CAVS) is among the most common causes of cardiovascular mortality in an aging population worldwide. The pathomechanisms of CAVS are such a complex and multifactorial process that researchers are still making progress to understand its physiopathology as well as the complex players involved in CAVS pathogenesis. Currently, there is no successful and effective treatment to prevent or slow down the disease. Surgical and transcatheter valve replacement represents the only option available for treating CAVS. Insufficient oxygen availability (hypoxia) has a critical role in the pathogenesis of almost all CVDs. This process is orchestrated by the hallmark transcription factor, hypoxia-inducible factor 1 alpha subunit (HIF-1α), which plays a pivotal role in regulating various target hypoxic genes and metabolic adaptations. Recent studies have shown a great deal of interest in understanding the contribution of HIF-1α in the pathogenesis of CAVS. However, it is deeply intertwined with other major contributors, including sustained inflammation and mitochondrial impairments, which are attributed primarily to CAVS. The present review aims to cover the latest understanding of the complex interplay effect of hypoxia signaling pathways, mitochondrial dysfunction, and inflammation in CAVS. We propose further hypotheses and interconnections on the complexity of these impacts in a perspective of better understanding the pathophysiology. These interplays will be examined considering recent studies that shall help us better dissect the molecular mechanism to enable the design and development of potential future therapeutic approaches that can prevent or slow down CAVS processes.

Mitochondrial dysfunction and skeletal muscle atrophy: Causes, mechanisms, and treatment strategies.

Skeletal muscle, which accounts for approximately 40% of total body weight, is one of the most dynamic and plastic tissues in the human body and plays a vital role in movement, posture and force production. More than just a component of the locomotor system, skeletal muscle functions as an endocrine organ capable of producing and secreting hundreds of bioactive molecules. Therefore, maintaining healthy skeletal muscles is crucial for supporting overall body health. Various pathological conditions, such as prolonged immobilization, cachexia, aging, drug-induced toxicity, and cardiovascular diseases (CVDs), can disrupt the balance between muscle protein synthesis and degradation, leading to skeletal muscle atrophy. Mitochondrial dysfunction is a major contributing mechanism to skeletal muscle atrophy, as it plays crucial roles in various biological processes, including energy production, metabolic flexibility, maintenance of redox homeostasis, and regulation of apoptosis. In this review, we critically examine recent knowledge regarding the causes of muscle atrophy (disuse, cachexia, aging, etc.) and its contribution to CVDs. Additionally, we highlight the mitochondrial signaling pathways involvement to skeletal muscle atrophy, such as the ubiquitin-proteasome system, autophagy and mitophagy, mitochondrial fission-fusion, and mitochondrial biogenesis. Furthermore, we discuss current strategies, including exercise, mitochondria-targeted antioxidants, in vivo transfection of PGC-1α, and the potential use of mitochondrial transplantation as a possible therapeutic approach.

The Tricky Connection between Extracellular Vesicles and Mitochondria in Inflammatory-Related Diseases.

Mitochondria are organelles present in almost all eukaryotic cells, where they represent the main site of energy production. Mitochondria are involved in several important cell processes, such as calcium homeostasis, OXPHOS, autophagy, and apoptosis. Moreover, they play a pivotal role also in inflammation through the inter-organelle and inter-cellular communications, mediated by the release of mitochondrial damage-associated molecular patterns (mtDAMPs). It is currently well-documented that in addition to traditional endocrine and paracrine communication, the cells converse via extracellular vesicles (EVs). These small membrane-bound particles are released from cells in the extracellular milieu under physio-pathological conditions. Importantly, EVs have gained much attention for their crucial role in inter-cellular communication, translating inflammatory signals into recipient cells. EVs cargo includes plasma membrane and endosomal proteins, but EVs also contain material from other cellular compartments, including mitochondria. Studies have shown that EVs may transport mitochondrial portions, proteins, and/or mtDAMPs to modulate the metabolic and inflammatory responses of recipient cells. Overall, the relationship between EVs and mitochondria in inflammation is an active area of research, although further studies are needed to fully understand the mechanisms involved and how they may be targeted for therapeutic purposes. Here, we have reported and discussed the latest studies focused on this fascinating and recent area of research, discussing of tricky connection between mitochondria and EVs in inflammatory-related diseases.

1,3,8-Triazaspiro[4.5]decane Derivatives Inhibit Permeability Transition Pores through a FO-ATP Synthase c Subunit Glu119-Independent Mechanism That Prevents Oligomycin A-Related Side Effects.

Permeability transition pore (PTP) molecular composition and activity modulation have been a matter of research for several years, especially due to their importance in ischemia reperfusion injury (IRI). Notably, c subunit of ATP synthase (Csub) has been identified as one of the PTP-forming proteins and as a target for cardioprotection. Oligomycin A is a well-known Csub interactor that has been chemically modified in-depth for proposed new pharmacological approaches against cardiac reperfusion injury. Indeed, by taking advantage of its scaffold and through focused chemical improvements, innovative Csub-dependent PTP inhibitors (1,3,8-Triazaspiro[4.5]decane) have been synthetized in the past. Interestingly, four critical amino acids have been found to be involved in Oligomycin A-Csub binding in yeast. However, their position on the human sequence is unknown, as is their function in PTP inhibition. The aims of this study are to (i) identify for the first time the topologically equivalent residues in the human Csub sequence; (ii) provide their in vitro validation in Oligomycin A-mediated PTP inhibition and (iii) understand their relevance in the binding of 1,3,8-Triazaspiro[4.5]decane small molecules, as Oligomycin A derivatives, in order to provide insights into Csub interactions. Notably, in this study we demonstrated that 1,3,8-Triazaspiro[4.5]decane derivatives inhibit permeability transition pores through a FO-ATP synthase c subunit Glu119-independent mechanism that prevents Oligomycin A-related side effects.

Comprehensive Analysis of Mitochondrial Dynamics Alterations in Heart Diseases.

The most common alterations affecting mitochondria, and associated with cardiac pathological conditions, implicate a long list of defects. They include impairments of the mitochondrial electron transport chain activity, which is a crucial element for energy formation, and that determines the depletion of ATP generation and supply to metabolic switches, enhanced ROS generation, inflammation, as well as the dysregulation of the intracellular calcium homeostasis. All these signatures significantly concur in the impairment of cardiac electrical characteristics, loss of myocyte contractility and cardiomyocyte damage found in cardiac diseases. Mitochondrial dynamics, one of the quality control mechanisms at the basis of mitochondrial fitness, also result in being dysregulated, but the use of this knowledge for translational and therapeutic purposes is still in its infancy. In this review we tried to understand why this is, by summarizing methods, current opinions and molecular details underlying mitochondrial dynamics in cardiac diseases.

PML at mitochondria-associated membranes governs a trimeric complex with NLRP3 and P2X7R that modulates the tumor immune microenvironment.

Uncontrolled inflammatory response arising from the tumor microenvironment (TME) significantly contributes to cancer progression, prompting an investigation and careful evaluation of counter-regulatory mechanisms. We identified a trimeric complex at the mitochondria-associated membranes (MAMs), in which the purinergic P2X7 receptor - NLRP3 inflammasome liaison is fine-tuned by the tumor suppressor PML. PML downregulation drives an exacerbated immune response due to a loss of P2X7R-NLRP3 restraint that boosts tumor growth. PML mislocalization from MAMs elicits an uncontrolled NLRP3 activation, and consequent cytokines blast fueling cancer and worsening the tumor prognosis in different human cancers. New mechanistic insights are provided for the PML-P2X7R-NLRP3 axis to govern the TME in human carcinogenesis, fostering new targeted therapeutic approaches.

The Multifaceted Roles of Autophagy in Infectious, Obstructive, and Malignant Airway Diseases.

Autophagy is a highly conserved dynamic process by which cells deliver their contents to lysosomes for degradation, thus ensuring cell homeostasis. In response to environmental stress, the induction of autophagy is crucial for cell survival. The dysregulation of this degradative process has been implicated in a wide range of pathologies, including lung diseases, representing a relevant potential target with significant clinical outcomes. During lung disease progression and infections, autophagy may exert both protective and harmful effects on cells. In this review, we will explore the implications of autophagy and its selective forms in several lung infections, such as SARS-CoV-2, Respiratory Syncytial Virus (RSV) and Mycobacterium tuberculosis (Mtb) infections, and different lung diseases such as Cystic Fibrosis (CF), Chronic Obstructive Pulmonary Disease (COPD), and Malignant Mesothelioma (MM).

The "mitochondrial stress responses": the "Dr. Jekyll and Mr. Hyde" of neuronal disorders.

Neuronal disorders are associated with a profound loss of mitochondrial functions caused by various stress conditions, such as oxidative and metabolic stress, protein folding or import defects, and mitochondrial DNA alteration. Cells engage in different coordinated responses to safeguard mitochondrial homeostasis. In this review, we will explore the contribution of mitochondrial stress responses that are activated by the organelle to perceive these dangerous conditions, keep them under control and rescue the physiological condition of nervous cells. In the sections to come, particular attention will be dedicated to analyzing how compensatory mitochondrial hyperfusion, mitophagy, mitochondrial unfolding protein response, and apoptosis impact human neuronal diseases. Finally, we will discuss the relevance of the new concept: the "mito-inflammation", a mitochondria-mediated inflammatory response that is recently found to cover a relevant role in the pathogenesis of diverse inflammatory-related diseases, including neuronal disorders.

Increase of Parkin and ATG5 plasmatic levels following perinatal hypoxic-ischemic encephalopathy.

Brain injury at birth is an important cause of neurological and behavioral disorders. Hypoxic-ischemic encephalopathy (HIE) is a critical cerebral event occurring acutely or chronically at birth with high mortality and morbidity in newborns. Therapeutic strategies for the prevention of brain damage are still unknown, and the only medical intervention for newborns with moderate-to-severe HIE is therapeutic hypothermia (TH). Although the neurological outcome depends on the severity of the initial insult, emerging evidence suggests that infants with mild HIE who are not treated with TH have an increased risk for neurodevelopmental impairment; in the current clinical setting, there are no specific or validated biomarkers that can be used to both correlate the severity of the hypoxic insult at birth and monitor the trend in the insult over time. The aim of this work was to examine the presence of autophagic and mitophagic proteins in bodily fluids, to increase knowledge of what, early at birth, can inform therapeutic strategies in the first hours of life. This is a prospective multicentric study carried out from April 2019 to April 2020 in eight third-level neonatal intensive care units. All participants have been subjected to the plasma levels quantification of both Parkin (a protein involved in mitophagy) and ATG5 (involved in autophagy). These findings show that Parkin and ATG5 levels are related to hypoxic-ischemic insult and are reliable also at birth. These observations suggest a great potential diagnostic value for Parkin evaluation in the first 6 h of life.

Identification of small-molecule urea derivatives as PTPC modulators targeting the c subunit of F1/Fo-ATP synthase.

Maintaining a high percentage of living and functional cells in those pathologies in which excessive cell death occurs, such as neurodegenerative disorders and cardiovascular diseases, is one of the most intriguing challenges in the field of biochemical research for drug discovery. Here, mitochondrial permeability transition-driven regulated cell death is the main mechanism of mitochondrial impairment and cell fate; this pathway is still lacking of satisfying pharmacological treatments to counteract its becoming; for this reason, it needs continuous and intense research to find new compounds as modulator of the permeability transition pore complex (PTPC) activity. In this study, we report the identification of small-molecule urea derivatives able to inhibit PTPC opening following calcium overload and selected for future use in cytoprotection.

Some Insights into the Regulation of Cardiac Physiology and Pathology by the Hippo Pathway.

The heart is one of the most fascinating organs in living beings. It beats up to 100,000 times a day throughout the lifespan, without resting. The heart undergoes profound anatomical, biochemical, and functional changes during life, from hypoxemic fetal stages to a completely differentiated four-chambered cardiac muscle. In the middle, many biological events occur after and intersect with each other to regulate development, organ size, and, in some cases, regeneration. Several studies have defined the essential roles of the Hippo pathway in heart physiology through the regulation of apoptosis, autophagy, cell proliferation, and differentiation. This molecular route is composed of multiple components, some of which were recently discovered, and is highly interconnected with multiple known prosurvival pathways. The Hippo cascade is evolutionarily conserved among species, and in addition to its regulatory roles, it is involved in disease by drastically changing the heart phenotype and its function when its components are mutated, absent, or constitutively activated. In this review, we report some insights into the regulation of cardiac physiology and pathology by the Hippo pathway.

Calcium dysregulation in heart diseases: Targeting calcium channels to achieve a correct calcium homeostasis.

Intracellular calcium signaling is a universal language source shared by the most part of biological entities inside cells that, all together, give rise to physiological and functional anatomical units, the organ. Although preferentially recognized as signaling between cell life and death processes, in the heart it assumes additional relevance considered the importance of calcium cycling coupled to ATP consumption in excitation-contraction coupling. The concerted action of a plethora of exchangers, channels and pumps inward and outward calcium fluxes where needed, to convert energy and electric impulses in muscle contraction. All this without realizing it, thousands of times, every day. An improper function of those proteins (i.e., variation in expression, mutations onset, dysregulated channeling, differential protein-protein interactions) being part of this signaling network triggers a short circuit with severe acute and chronic pathological consequences reported as arrhythmias, cardiac remodeling, heart failure, reperfusion injury and cardiomyopathies. By acting with chemical, peptide-based and pharmacological modulators of these players, a correction of calcium homeostasis can be achieved accompanied by an amelioration of clinical symptoms. This review will focus on all those defects in calcium homeostasis which occur in the most common cardiac diseases, including myocardial infarction, arrhythmia, hypertrophy, heart failure and cardiomyopathies. This part will be introduced by the state of the art on the proteins involved in calcium homeostasis in cardiomyocytes and followed by the therapeutic treatments that to date, are able to target them and to revert the pathological phenotype.

The Interplay of Hypoxia Signaling on Mitochondrial Dysfunction and Inflammation in Cardiovascular Diseases and Cancer: From Molecular Mechanisms to Therapeutic Approaches.

Cardiovascular diseases (CVDs) and cancer continue to be the primary cause of mortality worldwide and their pathomechanisms are a complex and multifactorial process. Insufficient oxygen availability (hypoxia) plays critical roles in the pathogenesis of both CVDs and cancer diseases, and hypoxia-inducible factor 1 (HIF-1), the main sensor of hypoxia, acts as a central regulator of multiple target genes in the human body. Accumulating evidence demonstrates that mitochondria are the major target of hypoxic injury, the most common source of reactive oxygen species during hypoxia and key elements for inflammation regulation during the development of both CVDs and cancer. Taken together, observations propose that hypoxia, mitochondrial abnormality, oxidative stress, inflammation in CVDs, and cancer are closely linked. Based upon these facts, this review aims to deeply discuss these intimate relationships and to summarize current significant findings corroborating the molecular mechanisms and potential therapies involved in hypoxia and mitochondrial dysfunction in CVDs and cancer.

Impairment of mitophagy and autophagy accompanies calcific aortic valve stenosis favouring cell death and the severity of disease.

AIMS: In the last 15 years, some observations tried to shed light on the dysregulation of the cellular self-digestion process in calcific aortic valve stenosis (CAVS), but the results obtained remain still controversial. This work is aimed to definitively establish the trend of autophagy in patients affected by CAVS, to analyse the putative involvement of other determinants, which impact on the mitochondrial quality control mechanisms and to explore possible avenues for pharmacological interventions in the treatment of CAVS. METHODS AND RESULTS: This observational study, performed exclusively in ex vivo human samples (cells and serum), by using biochemical approaches and correlations with clinical data, describes new biological features of the calcified valve in terms of mitochondrial dysfunctions. In detail, we unveiled a significant deficiency in mitochondrial respiration and in ATP production coupled to increase production of lactates. In addition, mitochondrial population in the pathologic group is aged with significant alterations in biogenesis and mitophagy pathways. We are also reporting an updated view about autophagy accompanying the calcification process and advanced stages of the disease. We provided evidence for a rapamycin-based therapeutic strategy to revert the calcified phenotype to the wild type one. CONCLUSION: Our data suggest that the CAVS phenotype is featured by defects in mitochondrial quality control mechanisms and that autophagy is not activated enough to counteract cell death and sustain cell functions. Thus, boosting autophagy and mitophagy from short- to long-term reverts quite all pathological phenotypes.

Understanding the Role of Autophagy in Cancer Formation and Progression Is a Real Opportunity to Treat and Cure Human Cancers.

The malignant transformation of a cell produces the accumulation of several cellular adaptions. These changes determine variations in biological processes that are necessary for a cancerous cell to survive during stressful conditions. Autophagy is the main nutrient recycling and metabolic adaptor mechanism in eukaryotic cells, represents a continuous source of energy and biomolecules, and is fundamental to preserve the correct cellular homeostasis during unfavorable conditions. In recent decades, several findings demonstrate a close relationship between autophagy, malignant transformation, and cancer progression. The evidence suggests that autophagy in the cancer context has a bipolar role (it may act as a tumor suppressor and as a mechanism of cell survival for established tumors) and demonstrates that the targeting of autophagy may represent novel therapeutic opportunities. Accordingly, the modulation of autophagy has important clinical benefits in patients affected by diverse cancer types. Currently, about 30 clinical trials are actively investigating the efficacy of autophagy modulators to enhance the efficacy of cytotoxic chemotherapy treatments. A deeper understanding of the molecular pathways regulating autophagy in the cancer context will provide new ways to target autophagy for improving the therapeutic benefits. Herein, we describe how autophagy participates during malignant transformation and cancer progression, and we report the ultimate efforts to translate this knowledge into specific therapeutic approaches to treat and cure human cancers.

Biological Characterization of Human Autologous Pericardium Treated with the Ozaki Procedure for Aortic Valve Reconstruction.

BACKGROUND: The Ozaki procedure is an innovative surgical technique aiming at reconstructing aortic valves with human autologous pericardium. Even if this procedure is widely used, a comprehensive biological characterization of the glutaraldehyde (GA)-fixed pericardial tissue is still missing. METHODS: Morphological analysis was performed to assess the general organization of pericardium subjected to the Ozaki procedure (post-Ozaki) in comparison to native tissue (pre-Ozaki). The effect of GA treatment on cell viability and nuclear morphology was then investigated in whole biopsies and a cytotoxicity assay was executed to assess the biocompatibility of pericardium. Finally, human umbilical vein endothelial cells were seeded on post-Ozaki samples to evaluate the influence of GA in modulating the endothelialization ability in vitro and the production of pro-inflammatory mediators. RESULTS: The Ozaki procedure alters the arrangement of collagen and elastic fibers in the extracellular matrix and results in a significant reduction in cell viability compared to native tissue. GA treatment, however, is not cytotoxic to murine fibroblasts as compared to a commercially available bovine pericardium membrane. In addition, in in vitro experiments of endothelial cell adhesion, no difference in the inflammatory mediators with respect to the commercial patch was found. CONCLUSIONS: The Ozaki procedure, despite alteration of ECM organization and cell devitalization, allows for the establishment of a noncytotoxic environment in which endothelial cell repopulation occurs.