Essential Role of Adhesion GPCR, GPR123, for Human Pluripotent Stem Cells and Reprogramming towards Pluripotency.

G-protein-coupled receptors (GPCRs) are the largest family of cell surface receptors. They modulate key physiological functions and are required in diverse developmental processes including embryogenesis, but their role in pluripotency maintenance and acquisition during the reprogramming towards hiPSCs draws little attention. Meanwhile, it is known that more than 106 GPCRs are overexpressed in human pluripotent stem cells (hPSCs). Previously, to identify novel effectors of reprogramming, we performed a high-throughput RNA interference (RNAi) screening assay and identified adhesion GPCR, GPR123, as a potential reprogramming effector. Its role has not been explored before. Herein, by employing GPR123 RNAi we addressed the role of GPR123 for hPSCs. The suppression of GPR123 in hPSCs leads to the loss of pluripotency and differentiation, impacted colony morphology, accumulation of cells at the G2 phase of the cell cycle, and absence of the scratch closure. Application of the GPR123 RNAi at the initiation stage of reprogramming leads to a decrease in the percentage of the "true" hiPSC colonies, a drop in E-cadherin expression, a decrease in the percentage of NANOG+ nuclei, and the absence of actin cytoskeleton remodeling. Together this leads to the absence of the alkaline-phosphatase-positive hiPSCs colonies on the 18th day of the reprogramming process. Overall, these data indicate for the first time the essential role of GPR123 in the maintenance and acquisition of pluripotency.

Hsp70-containing extracellular vesicles are capable of activating of adaptive immunity in models of mouse melanoma and colon carcinoma.

The release of Hsp70 chaperone from tumor cells is found to trigger the full-scale anti-cancer immune response. Such release and the proper immune reaction can be induced by the delivery of recombinant Hsp70 to a tumor and we sought to explore how the endogenous Hsp70 can be transported to extracellular space leading to the burst of anti-cancer activity. Hsp70 transport mechanisms were studied by analyzing its intracellular tracks with Rab proteins as well as by using specific inhibitors of membrane domains. To study Hsp70 forms released from cells we employed the assay consisting of two affinity chromatography methods. Hsp70 content in culture medium and extracellular vesicles (EVs) was measured with the aid of ELISA. The properties and composition of EVs were assessed using nanoparticle tracking analysis and immunoblotting. The activity of immune cells was studied using an assay of cytotoxic lymphocytes, and for in vivo studies we employed methods of affinity separation of lymphocyte fractions. Analyzing B16 melanoma cells treated with recombinant Hsp70 we found that the chaperone triggered extracellular transport of its endogenous analog in soluble and enclosed in EVs forms; both species efficiently penetrated adjacent cells and this secondary transport was corroborated with the strong increase of Natural Killer (NK) cell toxicity towards melanoma. When B16 and CT-26 colon cancer cells before their injection in animals were treated with Hsp70-enriched EVs, a powerful anti-cancer effect was observed as shown by a two-fold reduction in tumor growth rate and elevation of life span. We found that the immunomodulatory effect was due to the enhancement of the CD8-positive response and anti-tumor cytokine accumulation; supporting this there was no delay in CT-26 tumor growth when Hsp70-enriched EVs were grafted in nude mice. Importantly, pre-treatment of B16 cells with Hsp70-bearing EVs resulted in a decline of arginase-1-positive macrophages, showing no generation of tumor-associated macrophages. In conclusion, Hsp70-containing EVs generated by specifically treated cancer cells give a full-scale and effective pattern of anti-tumor immune responses.

Suppression of mTORC1 activity in senescent Ras-transformed cells neither restores autophagy nor abrogates apoptotic death caused by inhibition of MEK/ERK kinases.

Autophagy is conservative catabolic process that degrades organelles, in particular, mitochondria, and misfolded proteins within the lysosomes, thus maintaining cellular viability. Despite the close relationship between mitochondrial dysfunction and cellular senescence, it is unclear how mitochondria damage can induce autophagy in senescent cells. We show that MEK/ERK suppression induces mitochondria damage followed by apoptosis of senescent Ras-expressing cells. To understand the role of persistent mTORC1 signaling in breaking the cAMPK-induced autophagy caused by mitochondrial damage, we inhibited mTORС1 with low concentrations of pp242. mTORC1 suppression neither restores the AMPK-induced autophagy nor decreases the level of apoptosis upon MEK/ERK inhibition. We discovered the existence of an alternative autophagy-like way that partially increases the viability of senescent cells under suppressed mTORC1. The pp242-treated cells survive due to formation of the non-autophagous LC3-negative vacuoles, which contain the damaged mitochondria and lysosomes with the following excretion the content from the cell. MEK/ERK activity is required to implement this process in senescent cells. Senescent cells exhibit distinctive spatial distribution of organelles and proteins that provides uncoupling of final participants of autophagy. We show that this feature stops the process of cytoprotective autophagy in response to MEK/ERK suppression, thus allowing selective elimination of senescent Ras-expressing cells.

Targeted elimination of senescent Ras-transformed cells by suppression of MEK/ERK pathway.

The Ras-Raf-MEK-ERK pathway plays a central role in tumorigenesis and is a target for anticancer therapy. The successful strategy based on the activation of cell death in Ras-expressing cells is associated with the suppression of kinases involved in Ras pathway. However, activation of cytoprotective autophagy overcomes antiproliferative effect of the inhibitors and develops drug resistance. We studied whether cellular senescence induced by HDAC inhibitor sodium butyrate in E1a+cHa-Ras-transformed rat embryo fibroblasts (ERas) and A549 human Ki-Ras mutated lung adenocarcinoma cells would enhance the tumor suppressor effect of MEK/ERK inhibition. Treatment of control ERas cells with PD0325901 for 24 h results in mitochondria damage and apoptotic death of a part of cellular population. However, the activation of AMPK-dependent autophagy overcomes pro-apoptotic effects of MEK/ERK inhibitor and results in restoration of the mitochondria and rescue of viability. Senescent ERas cells do not develop cytoprotective autophagy upon inhibition of MEK/ERK pathway due to spatial dissociation of lysosomes and autophagosomes in the senescent cells. Senescent cells are unable to form the autophagolysosomes and to remove the damaged mitochondria resulting in apoptotic death. Our data show that suppression of MEK/ERK pathway in senescent cells provides a new strategy for elimination of Ras-expressing cells.

mTOR kinase inhibitor pp242 causes mitophagy terminated by apoptotic cell death in E1A-Ras transformed cells.

mTOR is a critical target for controlling cell cycle progression, senescence and cell death in mammalian cancer cells. Here we studied the role of mTOR-dependent autophagy in implementating the antiprolifrative effect of mTORC1-specific inhibitor rapamycin and ATP-competitive mTOR kinase inhibitor pp242. We carried out a comprehensive analysis of pp242- and rapamycin-induced autophagy in ERas tumor cells. Rapamycin exerts cytostatic effect on ERas tumor cells, thus causing a temporary and reversible cell cycle arrest, activation of non-selective autophagy not accompanied by cell death. The rapamycin-treated cells are able to continue proliferation after drug removal. The ATP-competitive mTORC1/mTORC2 kinase inhibitor pp242 is highly cytotoxic by suppressing the function of mTORC1-4EBP1 axis and mTORC1-dependent phosphorylation of mTORC1 target--ULK1-Ser757 (Atg1). In contrast to rapamycin, pp242 activates the selective autophagy targeting mitochondria (mitophagy). The pp242-induced mitophagy is accompanied by accumulation of LC3 and conversion of LC3-I form to LC3-II. However reduced degradation of p62/SQSTM indicates abnormal flux of autophagic process. According to transmission electron microscopy data, short-term pp242-treated ERas cells exhibit numerous heavily damaged mitochondria, which are included in single membrane-bound autophagic/autolysophagic vacuoles (mitophagy). Despite the lack of typical for apoptosis features, ERas-treated cells with induced mitophagy revealed the activation of caspase 3, 9 and nucleosomal DNA fragmentation. Thus, pp242 activates autophagy with suppressed later stages, leading to impaired recycling and accumulation of dysfunctional mitochondria and cell death. Better understanding of how autophagy determines the fate of a cell--survival or cell death, can help to development of new strategy for cancer therapy.

Changes of fractional anisotropy (FA) and apparent diffusion coefficient (ADC) in the model of experimental acute hydrocephalus in rabbits.

BACKGROUND: To study the integrity of white matter, we investigated the correlation between the changes in neuroradiological and morphological parameters in an animal model of acute obstructive hydrocephalus. METHODS: Hydrocephalus was induced in New Zealand rabbits (n = 10) by stereotactic injection of kaolin into the lateral ventricles. Control animals received saline in place of kaolin (n = 10). The progression of hydrocephalus was assessed using magnetic resonance imaging. Regional fractional anisotropy (FA) and the apparent diffusion coefficient (ADC) were measured in several white matter regions before and after the infusion of kaolin. Morphology of myelinated nerve fibers as well as of the blood-brain barrier were studied with the help of transmission electron microscopy (TEM) and light microscopy. RESULTS: Compared with control animals, kaolin injection into the ventricles resulted in a dramatic increase in ventricular volume with compression of basal cisterns, brain shift and periventricular edema (as observed on magnetic resonance imaging [MRI]). The values of ADC in the periventricular and periaqueductal areas significantly increased in the experimental group (P < 0.05). FA decreased by a factor of 2 in the zones of periventricular, periaqueductal white matter and corpus collosum. Histological analysis demonstrated the impairment of the white matter and necrobiotic changes in the cortex. Microsctructural alterations of the myelin fibers were further proved with the help of TEM. Blood-brain barrier ultrastructure assessment showed the loss of its integrity. CONCLUSIONS: The study demonstrated the correlation of the neuroradiological parameters with morphological changes. The abnormality of the FA and ADC parameters in the obstructive hydrocephalus represents a significant implication for the diagnostics and management of hydrocephalus in patients.

Superparamagnetic iron oxide nanoparticles conjugated with epidermal growth factor (SPION-EGF) for targeting brain tumors.

Superparamagnetic iron oxide nanoparticles (SPIONs) conjugated with recombinant human epidermal growth factor (SPION-EGF) were studied as a potential agent for magnetic resonance imaging contrast enhancement of malignant brain tumors. Synthesized conjugates were characterized by transmission electron microscopy, dynamic light scattering, and nuclear magnetic resonance relaxometry. The interaction of SPION-EGF conjugates with cells was analyzed in a C6 glioma cell culture. The distribution of the nanoparticles and their accumulation in tumors were assessed by magnetic resonance imaging in an orthotopic model of C6 gliomas. SPION-EGF nanosuspensions had the properties of a negative contrast agent with high coefficients of relaxation efficiency. In vitro studies of SPION-EGF nanoparticles showed high intracellular incorporation and the absence of a toxic influence on C6 cell viability and proliferation. Intravenous administration of SPION-EGF conjugates in animals provided receptor-mediated targeted delivery across the blood-brain barrier and tumor retention of the nanoparticles; this was more efficient than with unconjugated SPIONs. The accumulation of conjugates in the glioma was revealed as hypotensive zones on T2-weighted images with a twofold reduction in T2 relaxation time in comparison to unconjugated SPIONs (P<0.001). SPION-EGF conjugates provide targeted delivery and efficient magnetic resonance contrast enhancement of EGFR-overexpressing C6 gliomas.

The process of granule exocytosis in non-stimulated atrial granular cells of the snail, Achatina achatina: an ultrastructural, histochemical and immunocytochemical study.

Abundant secretory granular cells (GCs) in the Giant African land snail atrium harbor a range of bioactive substances and undergo rapid total degranulation in response to stimulation of the cardiac nerve or stressful influences. Here we have analyzed exocytotic events in the non-stimulated GCs. It was shown that the GCs contain three major distinct types of granules that differ histochemically, immunocytochemically and ultrastructurally, each performing specific functions. The type I granules characteristically filled with electron-lucent homogeneous materials exhibit intense immunoreactivity for bioactive proteins and therefore are considered to be storage granules. Histochemistry using vital staining with Acridine Orange and Gomori acid phosphatase technique has revealed lysosomal-related nature of the electron-dense type II granules. Digestion remnants appearing as fine filamentous materials fill the type III granules. Only the type III granules fuse together and with the plasma membrane form degranulation channels and surface pores, through which the debris is removed from the cell. The finding of granules exhibiting intermediate ultrastructural, histochemical and immunocytochemical features suggests that the major granule types represent most stable states along a granule empting continuum. Thus, under physiological conditions, the GCs continuously produce secretory proteins and so maintain readiness for stress-response, but use protein degradation machinery to prevent massive release of these bioactive substances into hemolymph.

Tumor targeting using magnetic nanoparticle Hsp70 conjugate in a model of C6 glioma.

BACKGROUND: Superparamagnetic iron oxide nanoparticles (SPIONs), due to their unique magnetic properties, have the ability to function both as magnetic resonance (MR) contrast agents, and can be used for thermotherapy. SPIONs conjugated to the heat shock protein Hsp70 that selectively binds to the CD40 receptor present on glioma cells, could be used for MR contrast enhancement of experimental C6 glioma. METHODS: The magnetic properties of the Hsp70-SPIONs were measured by NMR relaxometry method. The uptake of nanoparticles was assessed on the C6 glioma cells by confocal and electron microscopes. The tumor selectivity of Hsp70-SPIONs being intravenously administered was analyzed in the experimental model of C6 glioma in the MRI scanner. RESULTS: Hsp70-SPIONs relaxivity corresponded to the properties of negative contrast agents with a hypointensive change of resonance signal in MR imaging. A significant accumulation of the Hsp70-SPIONs but not the non-conjugated nanoparticles was observed by confocal microscopy within C6 cells. Negative contrast tumor enhancement in the T2-weighted MR images was higher in the case of Hsp70-SPIONs in comparison to non-modified SPIONs. Histological analysis of the brain sections confirmed the retention of the Hsp70-SPIONs in the glioma tumor but not in the adjacent normal brain tissues. CONCLUSION: The study demonstrated that Hsp70-SPION conjugate intravenously administered in C6 glioma model accumulated in the tumors and enhanced the contrast of their MR images.

Early incorporation of obscurin into nascent sarcomeres: implication for myofibril assembly during cardiac myogenesis.

Obscurin is a recently identified giant multidomain muscle protein whose functions remain poorly understood. The goal of this study was to investigate the process of assembly of obscurin into nascent sarcomeres during the transition from non-striated myofibril precursors to striated structure of differentiating myofibrils in cell cultures of neonatal rat cardiac myocytes. Double immunofluorescent labeling and high resolution confocal microscopy demonstrated intense incorporation of obscurin in the areas of transition from non-striated to striated regions on the tips of developing myofibrils and at the sites of lateral fusion of nascent sarcomere bundles. We found that obscurin rapidly and precisely accumulated in the middle of the A-band regions of the terminal newly assembled half-sarcomeres in the zones of transition from the continuous, non-striated pattern of sarcomeric alpha-actinin distribution to cross-striated structure of laterally expanding nascent Z-discs. The striated pattern of obscurin typically ended at these points. This occurred before the assembly of morphologically differentiated terminal Z-discs of the assembling sarcomeres on the tips of growing myofibrils. The presence of obscurin in the areas of the terminal Z-discs of each new sarcomere was detected at the same time or shortly after complete assembly of sarcomeric structure. Many non-striated fibers with very low concentration of obscurin were already immunopositive for sarcomeric actin and myosin. This suggests that obscurin may serve for organization and alignment of myofilaments into the striated pattern. The comparison of obscurin and titin localization in these areas showed that obscurin assembly into the A-bands occurred soon after or concomitantly with incorporation of titin. Electron microscopy of growing myofibrils demonstrated intense formation and integration of myosin filaments into the "open" half-assembled sarcomeres in the areas of the terminal Z-I structures and at the lateral surfaces of newly formed, terminally located nascent sarcomeres. This process progressed before the assembly of the second-formed, terminal Z-discs of new sarcomeres and before the development of ultrastructurally detectable mature M-lines that define the completion of myofibril assembly, which supports the data of immunocytochemical study. Abundant non-aligned sarcomeres in immature myofibrils located on the growing tips were spatially separated and underwent the transition to the registered, aligned pattern. The sarcoplasmic reticulum, the organelle known to interact with obscurin, assembled around each new sarcomere. These results suggest that obscurin is directly involved in the proper positioning and alignment of myofilaments within nascent sarcomeres and in the establishment of the registered pattern of newly assembled myofibrils and the sarcoplasmic reticulum at advanced stages of myofibrillogenesis.

The release of Hsp70 from A431 carcinoma cells is mediated by secretory-like granules.

In our earlier work we have demonstrated that the treatment of squamous carcinoma cell line A431 with a pharmacological inhibitor of phospholipase C activity, U73122, resulted in fast release of stress-inducible heat shock protein 70 (Hsp70) into the extracellular medium (Evdonin et al., Cancer Cell Int., 4, 2, 2004). The purpose of the present study was to identify cellular organelles involved in the release of Hsp70 from A431 cells. We determined that Hsp70 is present in granules located at the periphery of cells, which had been treated with U73122 or subjected to heat shock. An inhibitor of the classical protein export pathway, brefeldin A was found to prevent the U73122-induced appearance of Hsp70 in the extracellular medium and in the peripheral granules. These findings suggest that vesicular transport is involved in Hsp70 release. The Hsp70-containing granules did not carry markers specific for lipid bodies, endosomes, or lysosomes. However, they were positive for a marker of secretory granules, i.e. chromogranin A. The levels of extracellular Hsp70 and chromogranin A were found to increase simultaneously. The secretory-like granule-dependent transport of Hsp70 was also studied in minimally transformed human HaCaT keratinocytes. We found that after U73122 and heat stress treatment, HaCaT cells secreted Hsp70 in a manner similar to A431 cells. Collectively our results suggest that human keratinocyte-derived cells release Hsp70 in the extracellular medium through a pathway involving secretory-like granules.

The presence of ANP in rat peritoneal mast cells.

Atrial natriuretic peptide (ANP) is an important component of the natriuretic peptide system. A great role in many regulatory systems is played by mast cells. Meanwhile involvement of these cells in ANP activity is poorly studied. In this work, we have shown the presence of ANP in rat peritoneal mast cells. Pure fraction of mast cells was obtained by separation of rat peritoneal cells on a Percoll density gradient. By Western blotting, two ANP-immunoreactive proteins of molecular masses of 2.5 kDa and 16.9 kDa were detected in lysates from these mast cells. Electron microscope immunogold labeling has revealed the presence of ANP-immunoreactive material in storage, secreting and released granules of mast cells. Our findings indicate the rat peritoneal mast cells to contain both ANP prohormone and ANP. These both peptides are located in mast cell secretory granules and released by mechanism of degranulation. It is discussed that many mast cell functions might be due to production of natriuretic peptides by these cells.

Proliferation and differentiation processes in the heart muscle elements in different phylogenetic groups.

This article reviews, discusses, and summarizes data about the generative behavior of muscle tissue cells, the mechanisms of its regulation, and the organization of the endocrine function of the heart in the main phylogenetic groups. With respect to the ratio of processes of proliferation and differentiation, cell organization, and growth mechanism, muscle tissues of propulsive organs can be divided into three types, each revealed in one of three main groups of animals, lophotrochozoans, ecdysozoans, and chordates. Ecdysterone is likely to play the key role in the regulation of proliferation and differentiation processes in the heart muscle of crustaceans, and, most probably, also of molluscs. In each of the three main phylogenetic groups the endocrine function of the heart consisting of secretion of natriuretic peptides has a peculiar organization. Vertebrate cardiomyocytes are known to combine contractile and endocrine differentiation. Such functional dualism is absent in heart muscle elements of Lophotrochozoa and Ecdysozoa; in the heart of lopfotrochozoans, secretion of natriuretic peptides is performed by endothelial cells and their derivatives. Homology of the heart muscle in the animal kingdom as well as possible mechanisms of genomic and epigenomic regulation of different types of cardiomyogenesis are discussed.

Heart and the peritoneal cover of the gut sinus in the polychaete Arenicola marina: an ultrastructural and autoradiographic study.

To elucidate the cellular mechanism underlying the growth of the peritoneal cover of the gut sinus and the heart in the polychaete Arenicola marina, cellular organization of these structures and proliferative potential of their cells were investigated using electron microscopy and electron microscopic autoradiography. Arenicola has a pair of dorsolaterally situated hearts connected to the gut sinus via a short duct and composed of two muscular layers separated by a layer of the extracellular matrix (ECM). The peritoneal cover of the gut sinus and the outer muscular layer of the heart present a myoepithelial layer resting on the ECM. The inner muscular layer of the heart is composed of myofibril-containing cells lacking well-defined polarity in arrangement of organelles. However, their persistent connection to branches of the ECM and the adherens-like intercellular junctions allow for considering the inner layer a modified myoepithelium. In the peritoneal cover of the gut sinus and in both myoepithelial layers of the heart, noncontractile epithelial cells have been observed. As determined by thymidine labeling, these epithelial cells are capable of DNA synthesis, while myoepithelial cells are not. Some suggestions are made about the myogenic nature of the epithelial cells in the investigated structures of A. marina.

Undifferentiated cells in the snail myocardium are capable of DNA synthesis and myodifferentiation.

Cellular mechanisms of heart-muscle growth in the snail Achatina fulica have been studied using cytophotometry and electron microscopic autoradiography. Cytophotometric DNA measurements showed that the snail cardiomyocytes are mononucleated cells with diploid nuclei. Ultrastructural analysis of the snail myocardium revealed that, in addition to mature myocytes, it contains small roundish undifferentiated cells (UCs) and poorly differentiated muscle cells. EM autoradiography detected silver grains over the nuclei of UCs 2 h after injection of tritiated thymidine ([(3)H]Tdr), while the nuclei of both mature and poorly differentiated myocytes remained unlabeled. In EM autographs of the myocardial tissue fixed 14 days after [(3)H]Tdr administration, labeled myonuclei were evident, which may suggest some myodifferentiation of prelabeled UCs. Many labeled UCs persist for 14 days after a single [(3)H]Tdr injection, suggesting that not all UCs undergo myodifferentiation after passing through the cell cycle, and that those that do not can enter the next cycle. UCs in the snail myocardium presumably provide not only reserve but also stem cells for myocytes. Thus, the heart muscle of the adult snail consists of mononucleated diploid myocytes with blocked proliferative activity and a renewable population of precursor myogenic cells. The results obtained suggest that the growth of this muscle involves a myoblastic mechanism of myogenesis; this mechanism differs from that of vertebrate cardiac muscle growth, which is non-myoblastic-that is, based on proliferation or polyploidization of cardiomyocytes. Evolutionary aspects of cellular mechanisms of the heart-muscle growth are discussed.

Ultrastructural study of the heart and its relationship with the connective tissue sheath of the stomach in Rhynchonella psittacea (Tentaculata, Brachiopoda).

The fine structure of the heart and connective tissue sheath surrounding the stomach of the brachiopod Rhynchonella psittacea has been studied. The stomach wall is lined externally with peritoneal epithelium. Between the bases of the peritoneal epithelial cells and those of the stomach epithelial cells is an extracellular amorphous matrix. The exterior part of the matrix is occupied by smooth muscle cells and the interior part by fibroblasts. The heart wall shows continuity with the peritoneal epithelium covering the stomach wall and consists of three layers: an outer layer of smooth myoepithelial and epithelial cells, an intermediate thick layer of extracellular matrix, and an inner discontinuous layer of fibroblasts. In myoepithelial cells, nucleated heads protruding freely into the coelom and contractile parts embedded in the extracellular matrix can easily be distinguished. These cells contain no sarcoplasmic reticulum or any elements of a T system. The epithelial cells are non-muscular mononucleated cells scattered among the myoepithelial cells and closely associated with these basally. They possess a well-developed rough endoplasmic reticulum. In rare cases, a small amount of myofibrils occurs basally in the epithelial cells. Morphologically the epithelial cells in the myocardium are very similar to the peritoneal epithelial cells covering the stomach wall. Both epithelial and myoepithelial cells are ciliated. No nerve elements have been found in the brachiopod heart. The structure of the brachiopod heart is compared with that of other invertebrates; similarity of cellular composition of the brachiopod heart and stomach cover is considered evidence of origin of the heart cells from the cells of the connective tissue sheath of the stomach. The myogenic role of the peritoneal cells and epithelial cells of the myocardium is suggested. J. Morphol. 234:69-77, 1997. © 1997 Wiley-Liss, Inc.