The redox-active defensive Selenoprotein T as a novel stress sensor protein playing a key role in the pathophysiology of heart failure.

Maladaptive cardiac hypertrophy contributes to the development of heart failure (HF). The oxidoreductase Selenoprotein T (SELENOT) emerged as a key regulator during rat cardiogenesis and acute cardiac protection. However, its action in chronic settings of cardiac dysfunction is not understood. Here, we investigated the role of SELENOT in the pathophysiology of HF: (i) by designing a small peptide (PSELT), recapitulating SELENOT activity via the redox site, and assessed its beneficial action in a preclinical model of HF [aged spontaneously hypertensive heart failure (SHHF) rats] and against isoproterenol (ISO)-induced hypertrophy in rat ventricular H9c2 and adult human AC16 cardiomyocytes; (ii) by evaluating the SELENOT intra-cardiomyocyte production and secretion under hypertrophied stimulation. Results showed that PSELT attenuated systemic inflammation, lipopolysaccharide (LPS)-induced macrophage M1 polarization, myocardial injury, and the severe ultrastructural alterations, while counteracting key mediators of cardiac fibrosis, aging, and DNA damage and restoring desmin downregulation and SELENOT upregulation in the failing hearts. In the hemodynamic assessment, PSELT improved the contractile impairment at baseline and following ischemia/reperfusion injury, and reduced infarct size in normal and failing hearts. At cellular level, PSELT counteracted ISO-mediated hypertrophy and ultrastructural alterations through its redox motif, while mitigating ISO-triggered SELENOT intracellular production and secretion, a phenomenon that presumably reflects the extent of cell damage. Altogether, these results indicate that SELENOT could represent a novel sensor of hypertrophied cardiomyocytes and a potential PSELT-based new therapeutic approach in myocardial hypertrophy and HF.

Targeting of mitochondrial fission through natural flavanones elicits anti-myeloma activity.

BACKGROUND: Mitochondrial alterations, often dependent on unbalanced mitochondrial dynamics, feature in the pathobiology of human cancers, including multiple myeloma (MM). Flavanones are natural flavonoids endowed with mitochondrial targeting activities. Herein, we investigated the capability of Hesperetin (Hes) and Naringenin (Nar), two aglycones of Hesperidin and Naringin flavanone glycosides, to selectively target Drp1, a pivotal regulator of mitochondrial dynamics, prompting anti-MM activity. METHODS: Molecular docking analyses were performed on the crystallographic structure of Dynamin-1-like protein (Drp1), using Hes and Nar molecular structures. Cell viability and apoptosis were assessed in MM cell lines, or in co-culture systems with primary bone marrow stromal cells, using Cell Titer Glo and Annexin V-7AAD staining, respectively; clonogenicity was determined using methylcellulose colony assays. Transcriptomic analyses were carried out using the Ion AmpliSeq™ platform; mRNA and protein expression levels were determined by quantitative RT-PCR and western blotting, respectively. Mitochondrial architecture was assessed by transmission electron microscopy. Real time measurement of oxygen consumption was performed by high resolution respirometry in living cells. In vivo anti-tumor activity was evaluated in NOD-SCID mice subcutaneously engrafted with MM cells. RESULTS: Hes and Nar were found to accommodate within the GTPase binding site of Drp1, and to inhibit Drp1 expression and activity, leading to hyperfused mitochondria with reduced OXPHOS. In vitro, Hes and Nar reduced MM clonogenicity and viability, even in the presence of patient-derived bone marrow stromal cells, triggering ER stress and apoptosis. Interestingly, Hes and Nar rewired MM cell metabolism through the down-regulation of master transcriptional activators (SREBF-1, c-MYC) of lipogenesis genes. An extract of Tacle, a Citrus variety rich in Hesperidin and Naringin, was capable to recapitulate the phenotypic and molecular perturbations of each flavanone, triggering anti-MM activity in vivo. CONCLUSION: Hes and Nar inhibit proliferation, rewire the metabolism and induce apoptosis of MM cells via antagonism of the mitochondrial fission driver Drp1. These results provide a framework for the development of natural anti-MM therapeutics targeting aberrant mitochondrial dependencies.

Atherosclerosis: From Molecular Biology to Therapeutic Perspective 3.0.

Atherosclerosis is a multifactorial chronic disease triggered and sustained by different risk factors such as dyslipidemia, hypertension, diabetes mellitus (DM), smoke, elevated homocysteine, and hormones [...].

Atherosclerosis: From Molecular Biology to Therapeutic Perspective.

Atherosclerosis is a chronic and progressive inflammatory disease of the arteries initiated by the functional and structural alteration of the endothelial layer responsible for promoting the subendothelial retention of modified low-density lipoproteins (LDL), which in turn generate an active proinflammatory state in which environmental factors, such as oxidizing agents, growth factors, cytokines, monocyte-macrophages and smooth muscle cells (SMCs), work in cooperation to promote the formation of plaque [...].

Atherosclerosis: From Molecular Biology to Therapeutic Perspective 2.0.

Atherosclerosis is a chronic inflammatory disease of large- and medium-sized arteries involving aberrant immune-inflammatory responses, dysfunctional molecular pathways, and impaired tissue repair mechanisms [...].

The microscopic anatomy of endothelial cells in human atherosclerosis: Focus on ER and mitochondria.

Once regarded merely as a bland lipid storage disease consequence of aging, atherosclerosis is currently considered a slow and continuous inflammatory process (partially controllable by treatment) with complex etiology involving a multitude of genetic and environmental risk factors which ultimately result in the formation of the plaque. The vascular endothelium, a monolayer of endothelial cells (ECs), is an important regulatory "organ" critical for cardiovascular homeostasis in health which also contributes significantly to the pathomechanisms of several disease states, including atherosclerosis. Over the years, there has been evidence highlighting the central role of endoplasmic reticulum (ER) in the maintenance of endothelial function and perturbations in ER biology have been proposed to adversely affect a diverse range of endothelial functions. Of particular interest is the evidence that under certain pathophysiological circumstances, abnormal ER ultrastructure correlates with altered ER function and signaling and can contribute to cell injury and apoptosis. Therefore, the ultrastructural traits of ER membranes can have important implications not only for their functional bearings but also for the etiology and pathophysiology of diverse human disorders. With regard to atherosclerosis, the focus of ER research has been centered on the molecular signals originated from the ER to manage conditions of stress, leaving the fine structure of this organelle an almost unexplored (but promising) area of studies. There is, also, increasing evidence that mitochondrial dysfunction plays a critical role in promoting cell apoptosis, inflammation, and oxidative stress, thereby contributing to atheroma growth. It is within this context that the present study has been undertaken to investigate the microscopic architecture of ECs in human atherosclerosis and to determine whether the potential structural abnormalities of ER and mitochondria may play a central pathogenic role in atherogenesis or may merely reflect the condition of a tissue whose integrity has already been disturbed or destroyed. For this purpose, transmission electron microscopy (TEM) remains a powerful technique that can not only provide information about the ultrastructural state of cell organelles but also allow the correlation between different subcellular alterations indicative of a certain pathophysiological condition and cellular response. The present study expands the spectrum of ultrastructural defects known to exist in human atherosclerosis and suggests that ER alterations may be of great importance in the pathogenesis of the disease. The architectural changes of ER may be considered early pathological events that precede any overt histologic abnormalities in the vascular endothelium and its subcellular organelles, primarily the mitochondrial pool.

ER-phagy in human atherosclerosis: an exploratory ultrastructural study.

Autophagy is a vacuolar self-digesting mechanism responsible for the removal of damaged organelles, indigestible aggregates, and nonfunctional long-lived proteins by lysosome. Autophagy is dynamically connected to the endoplasmic reticulum (ER) in several ways. It is capable to counteract the possible harmful effects linked to the impairment of protein folding in the ER; the ER has been proposed as the source for autophagosomal membranes. Also, the ER itself can undergo a selective form of autophagy (called ER-phagy) which ensures the maintenance of ER's morphology and function. Autophagy has been widely investigated in the cardiovascular system however there is no evidence to date regarding the occurrence of ER-phagy into the blood vessel wall. This study has been undertaken to explore the existence of this selective control mechanism in the cells of human atherosclerotic plaques. Transmission Electron Microscopy (TEM) analysis revealed that in the plaque cells the smooth ER profiles reorganized into concentric whorls and closely packed membranes arranged in curved and parallel arrays. Circular, often ring-shaped, ER membranes studded with ribosomes and enclosed in a sequestering vesicle have been also frequently observed. This preliminary study demonstrates the existence of a distinct machinery for the specific turnover of ER membranes in human atherosclerosis and provides the first ultrastructural description of ER-phagy in the diseased vascular tissue. These results may open new perspectives for future investigation in the cardiovascular field.

Occurrence and characterization of lipofuscin and ceroid in human atherosclerotic plaque.

Atherosclerotic plaque formation starts early in life, develops silently over decades, and often displays clear evidence of accelerated biological aging. Lipofuscin has been classically defined as "the most consistent and phylogenetically conserved cellular morphologic change of aging," however, despite this traditional view different lines of evidence have recently demonstrated that, besides aging, various noxious influences can engeder its accumulation in cells and also that specific experimental conditions can revert this effect. Lipofuscin has been also proven to interact with disease-related factors to enhance cell loss. Along with lipofuscin, ceroid, another autofluorescent lipopigment usually produced under various pathological conditions unrelated to aging, has been suggested to jeopardize cell performance and viability by inducing membrane fragility, mitochondrial dysfunction, DNA damage, and oxidative stress-induced apoptosis. With regard to atherosclerosis, very few investigations have been conducted to assess whether a link could exist between lipofuscin/ceroid accumulation and the progression of the disease and no information still exist regarding the anatomy and the ultrastructural diversification of lipofuscin and ceroid in the lesional vascular tissue. At the same time, data concerning their potential toxicity at the cellular level are fragmentary, dated, and scarce. The present study investigates the occurrence and distribution of lipofuscin and ceroid in human atherosclerotic plaque and adjacent healthy tissues and analyzes the ultrastructural changes associated with their accumulation within the cell.

Ultrastructural, Elemental and Mineralogical Analysis of Vascular Calcification in Atherosclerosis.

Over the past few decades, remarkable progress has been achieved in terms of understanding the molecular and cellular mechanisms of atherosclerotic vascular calcification and the important role of matrix vesicles in initiating and propagating pathologic tissue mineralization has been widely recognized. Despite these recent advances, however, no definitive data are currently available regarding the texture and composition of the minerals that grow in the vessel wall during the course of the disease. Using different electron microscopy imaging and analysis, we demonstrate that vascular cells can produce and secrete more than one type of matrix vesicles which act as sites for initial mineral deposition independently of their structural features. Our results reveal that apatite formation in the atherosclerotic lesions of the human aorta occur through the deposition of amorphous calcium phosphate that matures over time, transforms into crystalline hydroxyapatite, and radiates towards the lumen of the vesicles, finally forming the calcified spherules. Elemental and mineralogical analyses also demonstrate that the presence of mature and stable amorphous calcium phosphate deposits in the affected tissues is linked to the incorporation of magnesium, which probably delay the conversion to the crystalline phase. Though more rarely, the presence of calcium oxalate crystals has been also documented.

The origin of the autophagosomal membrane in human atherosclerotic plaque: a preliminary ultrastructural study.

Autophagy is an evolutionarily conserved process that occurs ubiquitously and functions as a primary route for the degradation of damaged organelles and proteins in response to starvation, oxidative stress, and other harmful conditions. The initial event upon autophagy induction is the formation of a membranous cistern called the phagophore or isolation membrane, a cup-shaped structure that elongates, engulfs cytoplasmic "cargo", and fuses at its rims to give rise to the autophagosome within which cytoplasmic material is enclosed. Although thoroughly studied in diverse cell culture systems, few attempts have been made to analyze the membrane dynamics during phagophore biogenesis in tissues. With respect to the cardiovascular system, no structural information is currently available regarding the sources that may contribute to the nucleation and growth of the phagophore membrane. The results presented here demonstrate that in the cells of human atherosclerotic plaque the phagophores are in contact with the endoplasmic reticulum (ER) membranes. Initially, the phagophore appears as a membrane sac that enwraps injured organelles and dysfunctional proteins and then matures into a double-membrane, closed structure often containing portions of the ER. These structural data indicate that the membrane source that elongates the phagophore might probably come from the ER. The topographical relationship between the ER tubules and the phagophore might also favor an efficient mechanism to transfer lipids from their site of synthesis to the nascent membrane, thus promoting its elongation and, ultimately, the formation of the autophagosome.

Interaction between lipid droplets and endoplasmic reticulum in human atherosclerotic plaques.

Lipid droplets (LDs) are intracellular organelles that are found in nearly all cell types, where they serve fundamental roles in lipid metabolism and energy homeostasis. Traditionally considered as simple lipid storage deposits, these dynamic and remarkable organelles have recently been implicated in a number of other cellular functions, ranging from protein storage and degradation to virus replication. Intracellular accumulation of LDs is also a hallmark feature of diverse human diseases including diabetes type II, obesity, hepatosteatosis, cancer, and atherosclerosis. LDs are highly motile and possess the capability to interact with a variety of cell organelles, such as the endoplasmic reticulum membranes, mitochondria, endosomes, and peroxisomes. To date, however, much remains to be learned regarding the existence and structure of these interactions in the cardiovascular system. The results presented herein demonstrate that in the foam cells of human atherosclerotic plaques, LDs are preferentially associated with ER membranes probably to form a lipid-buffer system that allows the storage of free fatty acids and reduces the risk of lipotoxicity.

Exosomes in human atherosclerosis: An ultrastructural analysis study.

Cell-to-cell communication, or signaling, is absolutely essential in orchestrating the activities of cells in multicellular organisms, to grow, develop, detect environmental changes and compensate for them in an internal, coordinated fashion. In the last few years, a considerable amount of new data have demonstrated the occurrence of a sophisticated intercellular signaling pathway based on the release of specialized vesicular structures, called exosomes, whose secretion appears to be regulated by various natural and experimental stimuli, physiological states, and disease processes. In the cardiovascular system, the study of exosomes is still in its infancy. Here, we aim to provide the first ultrastructural evidence for the presence of exosomes in human atherosclerotic plaque. We demonstrate by means of transmission electron microscopy that both lesional smooth muscle cells and endothelial cells are able to generate these membraneous microvesicles within specific compartments of the cell, called multivesicular bodies. Notably, in our series no signs of apoptosis have been detected in vascular cells secreting exosomes and no evidence of calcification has been observed associated with these structures in the extracellular space. Our results suggest the possible existence of a new mechanism of intercellular communication in the plaque milieu.

Bergapten drives autophagy through the up-regulation of PTEN expression in breast cancer cells.

BACKGROUND: Bergapten (5-methoxypsoralen), a natural psoralen derivative present in many fruits and vegetables, has shown antitumoral effects in a variety of cell types. In this study, it has been addressed how Bergapten in breast cancer cells induces autophagic process. RESULTS: In MCF7 and ZR-75 breast cancer cells Bergapten exhibited anti-survival response by inducing the autophagic process increasing Beclin1, PI3KIII, UVRAG, AMBRA expression and conversion of LC3-I to LC3-II. LC3-GFP, Acridine orange assay and transmission electron microscopy even confirmed the increased autophagosome formations in treated cells. Bergapten-induced autophagy is dependent by PTEN up-regulation, since silencing this gene, the induction of Beclin1 and the p-AKT/p-mTOR signal down-regulation were reversed. PTEN is transcriptionally regulated by Bergapten through the involvement of p38MAPK/NF-Y, as evidenced by the use of p38MAPK inhibitor SB203580, site-direct mutagenesis of NF-Y element and NF-Y siRNA. Furthermore NF-Y knockdown prevented Bergapten-induced acid vesicular organelle accumulations (AVOs), strengthening the role of this element in mediating autophagy. CONCLUSIONS: Our data indicate PTEN as a key target of Bergapten action in breast cancer cells for the induction of autophagy. These findings add further details on the mechanism of action of Bergapten, therefore suggesting that phytochemical compounds may be implemented in the novel strategies for breast cancer treatment.

Human kidney podocyte cell population as a novel biological target of nerve growth factor.

Human podocytes are highly specialized cells with a key role in kidney physiology. Alteration of their structure as a consequence of injury or developmental failure leads to severe renal diseases. Although several studies have tried to elucidate the molecular framework of this cellular system, the functional bases for the maintenance of podocytes in their specialized state to sustain kidney barrier filtration are not completely understood. In this study, the capability of podocytes to produce and secrete the nerve growth factor (NGF) has been demonstrated via a validated in vitro model. During the process of cell differentiation, NGF and its receptors are modulated in human podocytes just as NGF-responsive neurons. Blockade of NGF biological activity results in severe changes of cell morphology. Collectively, our results outline a novel function of the neurotrophin and add a new cellular target in the complex biological framework of NGF.

Human sperm molecular anatomy: the enzyme 5α-reductase (SRD5A) is present in the sperm and may be involved in the varicocele-related infertility.

The most common cause of male infertility is the testicular varicocele, a condition that impairs production and decreases quality of sperm. Male fertility also strictly depends on androgens acting through their own receptor. The enzyme 5α-reductase (SRD5A) is involved in the conversion of testosterone to 5α-dihydrotestosterone, both required for the development and maintenance of male reproductive function. Here, we evaluated, by western blotting analysis, the presence of SRD5A in human ejaculated spermatozoa and evidenced differences in sperm SRD5A content between healthy donors and varicocele-affected patients. Additionally, SRD5A sperm ultrastructural localization was also assessed by transmission electron microscopy and immunogold assay. We evidenced that SRD5A enzyme is present in the human spermatozoa and that its cellular content is lowered in sperm samples from varicocele patients compared to healthy subjects. The presence of SRD5A in human ejaculated spermatozoa highlights the potential role of this enzyme in sperm physiopathology suggesting that the decrease in its content, by affecting the conversion of testosterone into 5α-dihydrotestosterone, may be an important additional mechanism involved in the harmful effect of varicocele in male fertility.

Human sperm liver receptor homolog-1 (LRH-1) acts as a downstream target of the estrogen signaling pathway.

In the last decade, the study of human sperm anatomy, at molecular level, has revealed the presence of several nuclear protein receptors. In this work, we examined the expression profile and the ultrastructural localization of liver receptor homolog-1 (LRH-1) in human spermatozoa. We evidenced the presence of the receptor by Western blotting and real time-RT-PCR. Furthermore, we used immunogold electron microscopy to investigate the sperm anatomical regions containing LRH-1. The receptor was mainly located in the sperm head, whereas its expression was reduced in the neck and across the tail. Interestingly, we observed the presence of LRH-1 in different stages of testicular germ cell development by immunohistochemistry. In somatic cells, it has been suggested that the LRH-1 pathway is tightly linked with estrogen signaling and the important role of estradiol has been widely studied in sperm cells. To assess the significance of LRH-1 in male gametes and to deepen understanding of the role of estrogens in these cells, we investigated important sperm features such as motility, survival and capacitation. Spermatozoa were treated with 10 nm estradiol and the inhibition of LRH-1 reversed the estradiol stimulatory action. From our data, we discovered that human spermatozoa can be considered a new site of expression for LRH-1, evidencing its role in sperm motility, survival and cholesterol efflux. Furthermore, we may presume that in spermatozoa the LRH-1 effects are closely integrated with the estrogen signaling, supporting LRH-1 as a downstream effector of the estradiol pathway on some sperm functions.

HIF-1α and VEGF: Immunohistochemical Profile and Possible Function in Human Aortic Valve Stenosis.

Calcific aortic stenosis (CAS) is the most common valvular disease in Western countries. Histological findings in patients with CAS extremely resemble those of atherosclerosis and include accumulation and modification of lipoproteins, inflammation, extracellular matrix remodeling, and calcification. Angiogenesis is another prominent feature of CAS; however, there is only a limited amount of data available regarding the mechanisms behind the pathological neovascularization of a structure that is originally avascular. The present study aims to identify the molecular basis that regulates blood vessel growth in stenotic aortic valves, focusing on the role of HIF-1α and VEGF pathway. A total of 19 native degenerating aortic valves obtained at valve replacement surgery have been processed for Western blot, immunohistochemical, morphometric, and ultrastructural analyses. First, we have demonstrated the adverse ECM remodeling and the significant thickening of the leaflet also showing that HIF-1α and VEGF are significantly upregulated in the stenotic valves, are locally produced and colocalize with angiogenesis and areas of calcification. Next, we have characterized, for the first time to the best of our knowledge, the morphological features of the neovasculature evidencing the presence of intact blood vessels in close proximity to the mineralized zones. These results suggest that the complex structural remodeling of the matrix might reduce oxygen availability in the valve cusp contributing to the stabilization of HIF-1α that in turn induces a metabolic adaptation through the upregulation of VEGF and the formation of new blood vessels not only to overcome the hypoxic state but also to sustain the calcification process.

A novel functional interplay between Progesterone Receptor-B and PTEN, via AKT, modulates autophagy in breast cancer cells.

The tumour suppressor activity of the phosphatase and tensin homologue on chromosome 10 (PTEN) is subject of intense investigative efforts, although limited information on its regulation in breast cancer is available. Herein, we report that, in breast cancer cells, progesterone (OHPg), through its cognate receptor PR-B, positively modulates PTEN expression by inducing its mRNA and protein levels, and increasing PTEN-promoter activity. The OHPg-dependent up-regulation of PTEN gene activity requires binding of the PR-B to an Sp1-rich region within the PTEN gene promoter. Indeed, ChIP and EMSA analyses showed that OHPg treatment induced the occupancy of PTEN promoter by PR and Sp1 together with transcriptional coactivators such as SRC1 and CBP. PR-B isoform knockdown abolished the complex formation indicating its specific involvement. The OHPg/PR-B dependent induction of PTEN causes the down-regulation of PI3K/AKT signal, switching on the autophagy process through an enhanced expression of UVRAG and leading to a reduced cell survival. Altogether these findings highlight a novel functional connection between OHPg/PR-B and tumour suppressor pathways in breast cancer.

Human sperm anatomy and endocrinology in varicocele: role of androgen receptor.

The study of androgens involved in male reproduction has been object of intense efforts, while their reported action on human male gametes is limited. We previously described the presence of androgen receptor (AR) in sperm with a role related to the modulation of the PI3K pathway. In the present study, we investigated the expression of AR and its ultrastructural location in normal sperm as well as in spermatozoa obtained from varicocele patients. We observed a reduced AR content in varicocele sperm with respect to healthy sperm by western blot analysis and transmission electron microscopy (TEM). The ultrastructural location of AR was detected mainly on the head membrane as well as in the nucleus, neck, and mitochondria. Influence of dihydrotestosterone (DHT) treatment on cholesterol efflux was increased in normal sperm, while it was reduced or absent in varicocele sperm. To better understand DHT/AR significance in human male gametes, we evaluated triglyceride content and lipase, acyl-CoA dehydrogenase, and glucose-6-phosphate dehydrogenase activities upon DHT treatment. The metabolic outcome glimpsed in normal sperm was an increased metabolic rate, while 'varicocele' sperm economized energy. Taken together, our results reveal DHT and AR as new players in sperm endocrinology, indicating that varicocele sperm may have difficulty in switching to the capacitated status. A decreased AR expression and a consequent reduced responsiveness to DHT in sperm may represent molecular mechanisms involved in the pathophysiology of varicocele leading to male infertility. This study revealed new detrimental effects of varicocele on sperm at the molecular level.

Expression profile and subcellular localization of GAPDH in the smooth muscle cells of human atherosclerotic plaque: an immunohistochemical and ultrastructural study with biological therapeutic perspectives.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has long been considered a classical glycolytic enzyme involved exclusively in cytosolic energy production. Several recent studies, however, have demonstrated that GAPDH is a multifunctional protein whose presence and activity can be regulated by disease states and/or experimental manipulation. Expression levels of GAPDH have been shown to be altered in certain tumors as well as in proliferating and differentiating cells. Since dedifferentiation and proliferation of smooth muscle cells (SMCs) are important features of human atherosclerosis, we have characterized the expression profile of GAPDH in the SMCs of atherosclerotic plaques and its putative interrelationship with the synthetic/proliferative status of these cells utilizing the proliferating cell nuclear antigen (PCNA) antibody, a valuable marker of cell proliferation. Western blot data revealed that GAPDH was significantly upregulated in atherosclerotic plaque specimens. Immunohistochemical stains demonstrated that GAPDH accumulated in the nucleus of dedifferentiated SMCs that also showed positive immunoreactivity for PCNA, but remained cytoplasmatic in the contractile SMCs (PCNA-negative), thus reflecting the proliferative, structural and synthetic differences between them. We suggest that, in human atherosclerotic plaque, GAPDH might exert additional functions that are independent of its well-documented glycolytic activity and might play key roles in development of the disease.