Transcriptome Profile of the Rat Adrenal Gland: Parenchymal and Interstitial Cells.

The homeostasis of the adrenal gland plays a decisive role in its proper functioning, both in non-stressful conditions and under the influence of various types of stress. This consists of interactions between all types of cells that make up the organ, including parenchymal and interstitial cells. The amount of available information on this subject in the rat adrenal glands under non-stressful conditions is insufficient; the aim of the research was to determine the expression of marker genes for rat adrenal cells depending on their location. The material for the study consisted of adrenal glands taken from intact adult male rats that were separated into appropriate zones. Transcriptome analysis by means of Affymetrix® Rat Gene 2.1 ST Array was used in the study, followed by real-time PCR validation. Expression analysis of interstitial cell marker genes revealed both the amount of expression of these genes and the zone in which they were expressed. The expression of marker genes for fibroblasts was particularly high in the cells of the ZG zone, while the highest expression of specific macrophage genes was observed in the adrenal medulla. The results of this study, especially with regard to interstitial cells, provide a so far undescribed model of marker gene expression of various cells, both in the cortex and medulla of the sexually mature rat adrenal gland. The interdependence between parenchymal and interstitial cells creates a specific microenvironment that is highly heterogeneous within the gland with respect to some of the interstitial cells. This phenomenon most likely depends on the interaction with the differentiated parenchymal cells of the cortex, as well as the medulla of the gland.

New Gene Markers Expressed in Porcine Oviductal Epithelial Cells Cultured Primary In Vitro Are Involved in Ontological Groups Representing Physiological Processes of Porcine Oocytes.

Changes that occur within oviducts after fertilization are dependent on post-ovulation events, including oocyte-oviduct interactions. Although general processes are well-defined, the molecular basis are poorly understood. Recently, new marker genes involved in 'cell development', 'cell growth', 'cell differentiation' and 'cell maturation' processes have been identified in porcine oocytes. The aim of the study was to assess the expression profile of genes in primary in vitro cultured oviductal epithelial cells (OECs), clustered in Gene Ontology groups which enveloped markers also identified in porcine oocytes. OECs (from 45 gilts) were surgically removed and cultured in vitro for ≤ 30 days, and then subjected to molecular analyses. The transcriptomic and proteomic profiles of cells cultured during 7, 15 and 30 days were investigated. Additionally, morphological/histochemical analyzes were performed. The results of genes expression profiles were validated after using RT-qPCR. The results showed a significant upregulation of UNC45B, NOX4, VLDLR, ITGB3, FMOD, SGCE, COL1A2, LOX, LIPG, THY1 and downregulation of SERPINB2, CD274, TXNIP, CELA1, DDX60, CRABP2, SLC5A1, IDO1, ANPEP, FST. Detailed knowledge of the molecular pathways occurring in the OECs and the gametes that contact them may contribute both to developments of basic science of physiology, and new possibilities in advanced biotechnology of assisted reproduction.

Transcriptomic analysis of expression of genes regulating cell cycle progression in porcine ovarian granulosa cells during short-term in vitro primary culture.

The primary function of ovarian granulosa cells (GCs) is the support of oocytes during maturation and development. Molecular analyses of granulosa cell-associated processes, leading to improvement of understanding of the cell cycle events during the formation of ovarian follicles (folliculogenesis), may be key to improve the in vitro fertilization procedures. Primary in vitro culture of porcine GCs was employed to examine the changes in the transcriptomic profile of genes belonging to "cell cycle", "cell division", "cell cycle process", "cell cycle phase transition", "cell cycle G1/S phase transition", "cell cycle G2/M phase transition" and "cell cycle checkpoint" ontology groups. During the analysis, microarrays were employed to study the transcriptome of GCs, analyzing the total RNA of cells from specific periods of in vitro cultures. This research was based on material obtained from 40 landrace gilts of similar weight, age and the same living conditions. RNA was isolated at specific timeframes: before the culture was established (0 h) and after 48 h, 96 h and 144 h in vitro. Out of 133 differentially expressed genes, we chose the 10 most up-regulated (SFRP2, PDPN, PDE3A, FGFR2, PLK2, THBS1, ETS1, LIF, ANXA1, TGFB1) and the 10 most downregulated (IGF1, NCAPD2, CABLES1, H1FOO, NEK2, PPAT, TXNIP, NUP210, RGS2 and CCNE2). Some of these genes known to play key roles in the regulation of correct cell cycle passage (up-regulated SFRP2, PDE3A, PLK2, LIF and down-regulated CCNE2, TXNIP, NEK2). The data obtained provide a potential reference for studies on the process of mammalian folliculogenesis, as well as suggests possible new genetic markers for cell cycle progress in in vitro cultured porcine granulosa cells.

Genes regulating hormone stimulus and response to protein signaling revealed differential expression pattern during porcine oocyte in vitro maturation, confirmed by lipid concentration.

Genes influencing oocyte maturation may be valuable for predicting their developmental potential, as well as discerning the mechanistic pathways regulating oocyte development. In the presented research microarray gene expression analysis of immature and in vitro matured porcine oocytes was performed. Two groups of oocytes were compared in the study: before (3 × n = 50) and after in vitro maturation (3 × n = 50). The selection of viable oocytes was performed using the brilliant cresyl blue (BCB) test. Furthermore, microarrays and RT-qPCR was used to analyze the transcriptome of the oocytes before and after IVM. The study focused on the genes undergoing differential expression in two gene-ontology groups: "Cellular response to hormone stimulus" and "Cellular response to unfolded protein", which contain genes that may directly or indirectly be involved in signal transduction during oocyte maturation. Examination of all the genes of interest showed a lower level of their expression after IVM. From the total number of genes in these gene ontologies ten of the highest change in expression were identified: FOS, ID2, BTG2, CYR61, ESR1, AR, TACR3, CCND2, EGR2 and TGFBR3. The successful maturation of the oocytes was additionally confirmed with the use of lipid droplet assay. The genes were briefly described and related to the literature sources, to investigate their potential roles in the process of oocyte maturation. The results of the study may serve as a basic molecular reference for further research aimed at improving the methods of oocyte in vitro maturation, which plays an important role in the procedures of assisted reproduction.

New Molecular Markers Involved in Regulation of Ovarian Granulosa Cell Morphogenesis, Development and Differentiation during Short-Term Primary In Vitro Culture-Transcriptomic and Histochemical Study Based on Ovaries and Individual Separated Follicles.

Nowadays, science has a lot of knowledge about the physiology of ovarian processes, especially folliculogenesis, hormone production and ovulation. However, the molecular basis for these processes remains largely undiscovered. The cell layer surrounding the growing oocyte-granulosa cells-are characterized by high physiological capabilities (e.g., proliferation, differentiation) and potential for growth in primary cultures, which predisposes them for analysis in the context of possible application of their cultures in advanced methods of assisted reproduction. In this study, we have used standard molecular approaches to analyze markers of these processes in primarily in vitro cultured porcine granulosa, subjected to conditions usually applied to cultures of similar cells. The material for our research came from commercially slaughtered pigs. The cells were obtained by enzymatic digestion of tissues and in vitro culture in appropriate conditions. The obtained genetic material (RNA) was collected at specific time intervals (0 h-before culture; reference, 48, 98, 144 h) and then analyzed using expression microarrays. Genes that showed a fold change greater than |2| and an adjusted p value lower than 0.05 were described as differentially expressed. Three groups of genes: "Cell morphogenesis", "cell differentiation" and "cell development" were analyzed. From 265 differently expressed genes that belong to chosen ontology groups we have selected DAPL1, CXCL10, NEBL, IHH, TGFBR3, SCUBE1, DAB1, ITM2A, MCOLN3, IGF1 which are most downregulated and PDPN, CAV1, TMOD1, TAGLN, IGFBP5, ITGB3, LAMB1, FN1, ITGA2, POSTN genes whose expression is upregulated through the time of culture, on which we focused in downstream analysis. The results were also validated using RT-qPCR. The aim of our work was to conduct primary in vitro culture of granulosa cells, as well as to analyze the expression of gene groups in relation to the proliferation of follicular granulosa cells in the model of primary culture in real time. This knowledge should provide us with a molecular insight into the processes occurring during the in vitro cultures of porcine granulosa cells, serving as a basic molecular entry on the extent of the loss of their physiological properties, as well as gain of new, culture-specific traits.

Differences in Expression of Genes Involved in Bone Development and Morphogenesis in the Walls of Internal Thoracic Artery and Saphenous Vein Conduits May Provide Markers Useful for Evaluation Graft Patency.

Coronary artery bypass grafting (CABG) is one of the most efficient procedures for patients with advanced coronary artery disease. From all the blood vessels with the potential to be used in this procedure, the internal thoracic artery (ITA) and the saphenous vein (SV) are the most commonly applied as aortocoronary conduits. Nevertheless, in order to evaluate the graft patency and efficiency effectively, basic knowledge should be constantly expanding at the molecular level as well, as the understanding of predictive factors is still limited. In this study, we have employed the expressive microarray approach, validated with Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR), to analyze the transcriptome of both venous and arterial grafts. Searching for potential molecular factors, we analyzed differentially expressed gene ontologies involved in bone development and morphogenesis, for the possibility of discovery of new markers for the evaluation of ITA and SV segment quality. Among three ontological groups of interest-"endochondral bone morphogenesis", "ossification", and "skeletal system development"-we found six genes common to all of them. BMP6, SHOX2, COL13A1, CSGALNACT1, RUNX2, and STC1 showed differential expression patterns in both analyzed vessels. STC1 and COL13A1 were upregulated in ITA samples, whereas others were upregulated in SV. With regard to the Runx2 protein function in osteogenic phenotype regulation, the RUNX2 gene seems to be of paramount importance in assessing the potential of ITA, SV, and other vessels used in the CABG procedure. Overall, the presented study provided valuable insight into the molecular background of conduit characterization, and thus indicated genes that may be the target of subsequent studies, also at the protein level. Moreover, it has been suggested that RUNX2 may be recognized as a molecular marker of osteogenic changes in human blood vessels.

"Biological Adhesion" is a Significantly Regulated Molecular Process during Long-Term Primary In Vitro Culture of Oviductal Epithelial Cells (Oecs): A Transcriptomic and Proteomic Study.

Oviductal epithelial cells (OECs) actively produce stimulating and protecting factors, favoring survival and viability of gametes and early embryos. The oviduct participates in the initial reproductive events, which strongly depends on adhesion. The analysis of differential gene expression in OECs, during long-term in vitro culture, enables recognition of new molecular markers regulating several processes, including "biological adhesion". Porcine oviducts were stained with hematoxylin and eosin, as well as with antibodies against epithelial markers. Then, OECs were long-term in vitro cultured and after 24 h, 7, 15, and 30 days of culture were subjected to transcriptomic and proteomic assays. Microarrays were employed to evaluate gene expression, with Matrix-assisted laser desorption/ionization-time of light (MALDI-TOF) mass spectrometry applied to determine the proteome. The results revealed proper morphology of the oviducts and typical epithelial structure of OECs during the culture. From the set of differentially expressed genes (DEGs), we have selected the 130 that encoded proteins detected by MALDI-TOF MS analysis. From this gene pool, 18 significantly enriched gene ontology biological processes (GO BP) terms were extracted. Among them we focused on genes belonging to "biological adhesion" GO BP. It is suggested that increased expression of studied genes can be attributed to the process of intensive secretion of substances that exhibit favorable influence on oviductal environment, which prime gametes adhesion and viability, fertilization, and early embryo journey.

Transcriptomic Pattern of Genes Regulating Protein Response and Status of Mitochondrial Activity Are Related to Oocyte Maturational Competence-A Transcriptomic Study.

This paper aims to identify and describe new genetic markers involved in the processes of protein expression and modification reflected in the change of mitochondrial activity before and after in vitro maturation of the oocyte. Porcine oocytes collected from the ovaries of slaughtered landrace gilts were subjected to the process of in vitro maturation. Transcriptomic changes in the expression profile of oocyte genes involved in response to hypoxia, the transmembrane protein receptor serine threonine kinase signaling pathway, the "transforming growth factor ß receptor signaling pathway", "response to protein stimulus", and "response to organic substance" were investigated using microarrays. The expression values of these genes in oocytes was analyzed before (immature) and after (mature) in vitro maturation, with significant differences found. All the significantly altered genes showed downregulation after the maturation process. The most changed genes from these gene ontologies, FOS, ID2, VEGFA, BTG2, CYR61, ESR1, AR, TACR3, CCND2, CHRDL1, were chosen to be further validated, described and related to the literature. Additionally, the mitochondrial activity of the analyzed oocytes was measured using specific dyes. We found that the mitochondrial activity was higher before the maturation process. The analysis of these results and the available literature provides a novel insight on the processes that occur during in vitro oocyte maturation. While this knowledge may prove to be useful in further research of the procedures commonly associated with in vitro fertilization procedures, it serves mostly as a basic reference for further proteomic, in vivo, and clinical studies that are necessary to translate it into practical applications.

Vascular restenosis in coronary artery bypass grafting might be associated with VEGF-C/VEGFR-3 signaling pathway.

The vascular endothelial growth factor (VEGF) family of peptides and caveolins (CAVs) are reported to contribute, in early graft failure in patients, a coronary artery bypass grafting (CABG). To investigate the possible association of ultimate luminal occlusion to VEGFs and CAVs expression, a functional analysis (based on the molecular biology, bioinformatics, histology, and clinical studies) was performed. Twenty-four hundred and sixty-eight CABG patients diagnosed with multivessel stable coronary artery disease (CAD) were enrolled into prospective study and assigned to two subgroups: double- and triple-vessel CAD subjects. Distal parts of all the harvested saphenous vein (SV) and internal thoracic artery (ITA) segments were used for further tests. ITA graft failure did not differ between double-vessel and triple-vessel CAD patients. The number of SV occlusions was significantly higher in triple-vessel CAD subjects. The microarray analysis performed on SV and ITA samples obtained exclusively from triple-vessel CAD patients who developed early graft occlusion revealed 383 genes with increased and 301 genes with decreased expression in ITA samples as compared to SV grafts. This was followed by functional analysis of 'blood vessel development' group of genes. Average VEGF-C expression in ITA grafts was higher than in corresponding SV grafts; FLT4 expression was significantly higher in SV than in ITA transplants. VEGFR-3 and CAV3 expression demonstrated immunohistochemically in SMCs of the tunica media of SV grafts predicted their early restenosis in triple-vessel CAD patients. CAV2 protein expression in SMCs of ITA grafts indicated the risk of early graft failure both in double-vessel and triple-vessel CAD subjects.

The Unique Mechanisms of Cellular Proliferation, Migration and Apoptosis are Regulated through Oocyte Maturational Development-A Complete Transcriptomic and Histochemical Study.

The growth and development of oocyte affect the functional activities of the surrounding somatic cells. These cells are regulated by various types of hormones, proteins, metabolites, and regulatory molecules through gap communication, ultimately leading to the development and maturation of oocytes. The close association between somatic cells and oocytes, which together form the cumulus-oocyte complexes (COCs), and their bi-directional communication are crucial for the acquisition of developmental competences by the oocyte. In this study, oocytes were extracted from the ovaries obtained from crossbred landrace gilts and subjected to in vitro maturation. RNA isolated from those oocytes was used for the subsequent microarray analysis. The data obtained shows, for the first time, variable levels of gene expression (fold changes higher than |2| and adjusted p-value < 0.05) belonging to four ontological groups: regulation of cell proliferation (GO:0042127), regulation of cell migration (GO:0030334), and regulation of programmed cell death (GO:0043067) that can be used together as proliferation, migration or apoptosis markers. We have identified several genes of porcine oocytes (ID2, VEGFA, BTG2, ESR1, CCND2, EDNRA, ANGPTL4, TGFBR3, GJA1, LAMA2, KIT, TPM1, VCP, GRID2, MEF2C, RPS3A, PLD1, BTG3, CD47, MITF), whose expression after in vitro maturation (IVM) is downregulated with different degrees. Our results may be helpful in further elucidating the molecular basis and functional significance of a number of gene markers associated with the processes of migration, proliferation and angiogenesis occurring in COCs.

Preoperative factors predicting saphenous vein graft occlusion in coronary artery bypass grafting: a multivariate analysis.

Saphenous vein segments are frequently used as aortocoronary bypass grafts, particularly in patients over 65 years of age. In the majority of patients, venous grafts maintain their patency for 5-6 years; however, some become occluded within 12 months after surgery. There are some defined predictive biological factors used to assess saphenous vein graft long-term patency rates, but little is known about molecular parameters for estimating the risk of early vein occlusion. The pathogenesis of this process involves the proliferation of stem cells, as well as progenitor cells, in the graft wall. Histologically, this is reflected by CD34 and CD133 expression in endothelial and smooth muscle cells. Thus, the aim of present work was to perform a multivariate analysis of stem cell and progenitor cell markers in saphenous vein graft walls before transplantation to arterial circulation and correlate these results with early graft occlusion. A total of 718 patients, who underwent coronary artery bypass grafting using a saphenous vein graft, were enrolled in this prospective study. CD34, CD133 and von Willebrand factor expression was evaluated via immunohistochemistry. A multivariate analysis revealed that strong CD133 expression in smooth muscle cells can be considered a risk factor for early graft failure. Our findings suggest that CD133 expression in smooth muscle cells of the tunica media in saphenous vein grafts obtained from coronary artery bypass graft patients before graft transplantation to coronary circulation might predict the possibility of early graft occlusion.

The processes of cellular growth, aging, and programmed cell death are involved in lifespan of ovarian granulosa cells during short-term IVC - Study based on animal model.

The oogenesis and folliculogenesis are closely linked and occur simultaneously in the growing ovarian follicles. Biochemical and morphological changes in oocytes (OC) and surrounding granulosa cells (GCs) are highly complex and depend on many factors, including intercellular communication. GCs are cells with many functions, often crucial for the proper viability of the oocyte and subsequent positive fertilization. The purpose of this study was to analyze gene expression in porcine GCs, to define differentially expressed genes belongs to the "cell growth", "aging", "positive regulation of cell death", "apoptotic process", "regulation of cell death", "cell death" and "negative regulation of cell death" ontology groups during the short - term primary in vitro culture. Microarrays were employed to study the transcriptome contained in the total RNA of the cultured GCs. Ovaries were obtained after slaughter, from 40 gilts of swine aged 170 days. The cells were obtained through puncture of the ovaries, collection of follicular fluid, removal of the cumulus - oocyte complexes and centrifugation. The cells were then cultured in vitro. The RNA material was obtained before the culture was established (0h) and then after 48h, 96h and 144h of its course. From 182 differently expressed genes belonging to the these ontology groups, we have selected POSTN, FN1, FMOD, ITGB3, DCN, SERPINB2, SFRP2, IGFBP5, EMP1, and CCL2 which were upregulated, as well as DAPL1, ESR1, IHH, TGFBR3, PPARD, PDK4, TXNIP, IFIT3, CSRNP3, and TNFSF10 genes whose expression was downregulated during the time of in vitro culture of the GCs. The significance of the differential gene expression is to provide new information on the molecular aspects of in vitro granulosa cell culture.

New markers for regulation of transcription and macromolecule metabolic process in porcine oocytes during in vitro maturation.

Oocyte maturation is essential for proper fertilization, embryo implantation and early development. While the physiological conditions of these processes are relatively well­known, its exact molecular mechanisms remain widely undiscovered. Oocyte growth, differentiation and maturation are therefore the subject of scientific debate. Precious literature has indicated that the oocyte itself serves a regulatory role in the mechanisms underlying these processes. Hence, the present study performed expression microarrays to analyze the complete transcriptome of porcine oocytes during their in vitro maturation (IVM). Pig material was used for experimentation, as it possesses similarities to the reproductive processes and general genetic proximities of Sus scrofa to human. Oocytes, isolated from the ovaries of slaughtered animals were assessed via the Brilliant Cresyl Blue test and directed to IVM. A number of oocytes were left to be analyzed as the 'before IVM' group. Oocyte mRNA was isolated and used for microarray analysis, which was subsequently validated via RT­qPCR. The current study particularly focused on genes belonging to 'positive regulation of transcription, DNA­dependent', 'positive regulation of gene expression', 'positive regulation of macromolecule metabolic process' and 'positive regulation of transcription from RNA polymerase II promoter' ontologies. FOS, VEGFA, ESR1, AR, CCND2, EGR2, ENDRA, GJA1, INHBA, IHH, INSR, APP, WWTR1, SMARCA1, NFAT5, SMAD4, MAP3K1, EGR1, RORA, ECE1, NR5A1, KIT, IKZF2, MEF2C, SH3D19, MITF and PSMB4 were all determined to be significantly altered (fold change, >|2|; P<0.05) among these groups, with their downregulation being observed after IVM. Genes with the most altered expressions were analyzed and considered to be potential markers of maturation associated with transcription regulation and macromolecule metabolism process.

New Gene Markers Involved in Molecular Processes of Tissue Repair, Response to Wounding and Regeneration Are Differently Expressed in Fibroblasts from Porcine Oral Mucosa during Long-Term Primary Culture.

The mechanisms of wound healing and vascularization are crucial steps of the complex morphological process of tissue reconstruction. In addition to epithelial cells, fibroblasts play an important role in this process. They are characterized by dynamic proliferation and they form the stroma for epithelial cells. In this study, we have used primary cultures of oral fibroblasts, obtained from porcine buccal mucosa. Cells were maintained long-term in in vitro conditions, in order to investigate the expression profile of the molecular markers involved in wound healing and vascularization. Based on the Affymetrix assays, we have observed three ontological groups of markers as wound healing group, response to wounding group and vascularization group, represented by different genes characterized by their expression profile during long-term primary in vitro culture (IVC) of porcine oral fibroblasts. Following the analysis of gene expression in three previously identified groups of genes, we have identified that transforming growth factor beta 1 (TGFB1), ITGB3, PDPN, and ETS1 are involved in all three processes, suggesting that these genes could be recognized as markers of repair specific for oral fibroblasts within the porcine mucosal tissue.

Cortical Granule Distribution and Expression Pattern of Genes Regulating Cellular Component Size, Morphogenesis, and Potential to Differentiation are Related to Oocyte Developmental Competence and Maturational Capacity In Vivo and In Vitro.

Polyspermia is an adverse phenomenon during mammalian fertilization when more than one sperm fuses with a single oocyte. The egg cell is prepared to prevent polyspermia by, among other ways, producing cortical granules (CGs), which are specialized intracellular structures containing enzymes that aim to harden the zona pellucida and block the fusion of subsequent sperm. This work focused on exploring the expression profile of genes that may be associated with cortical reactions, and evaluated the distribution of CGs in immature oocytes and the peripheral density of CGs in mature oocytes. Oocytes were isolated and then processed for in vitro maturation (IVM). Transcriptomic analysis of genes belonging to five ontological groups has been conducted. Six genes showed increased expression after IVM (ARHGEF2, MAP1B, CXCL12, FN1, DAB2, and SOX9), while the majority of genes decreased expression after IVM. Using CG distribution analysis in immature oocytes, movement towards the cortical zone of the oocyte during meiotic competence acquisition was observed. CGs peripheral density decreased with the rise in meiotic competence during the IVM process. The current results reveal important new insights into the in vitro maturation of oocytes. Our results may serve as a basis for further studies to investigate the cortical reaction of oocytes.

An innovative panel to assess endothelial integrity of pedicled and skeletonized internal thoracic artery used as aortocoronary bypass graft: a randomized comparative histologic and immunohistochemical study.

BACKGROUND: Optimal preservation of endothelial integrity of the vessels used as aortocoronary grafts is a crucial determinant of long-term clinical success of coronary artery bypass grafting (CABG). The purpose of this study was to evaluate an impact of two common techniques to harvest left internal thoracic artery (LITA) on endothelial integrity. METHODS: One hundred twenty consecutive patients (84 males and 36 females) with a mean age of 64.9±8.8 years undergoing CABG were randomized to receive pedicled (group P; n=60) or skeletonized (group S; n=60) LITA grafts. During surgery LITA was harvested by the same experienced cardiac surgeon. The most peripheral surplus segments of LITA were obtained and then analysed histologically under light microscope. Additionally, endothelial expression of CD31, CD34, CD133 and nitric oxide synthase (eNOS) were evaluated by means of immunohistochemistry. RESULTS: In both groups, no cases of major arterial wall damage such as disruption, dissection, thrombosis or subadventitial hematoma were noted on LITA cross sections. Immunohistochemical assessment of protein expression revealed no differences in endothelial expression of CD133, CD34 antigens (markers of regeneration potential) and eNOS (indicating preserved functional integrity) between studied groups. Contrary to them, endothelial immunoreactivity of CD31, a marker of the morphological integrity of the endothelium, was revealed to be stronger in group P. CONCLUSIONS: The skeletonized method of LITA harvesting may be associated with worse preservation of morphological integrity of endothelium but without compromising functional integrity and potential for tissue regeneration.

Caveolin 2: a facultative marker of unfavourable prognosis in long-term patency rate of internal thoracic artery grafts used in coronary artery bypass grafting. Preliminary report.

OBJECTIVES: Intimal hyperplasia leading to graft failure in patients undergoing coronary artery bypass grafting (CABG) is related to vascular smooth muscle cells (SMCs) proliferation. SMCs respond to a variety of mediators, the most important of which is platelet-derived growth factor (PDGF). The platelet-derived growth factor-induced cellular response has been shown to be mediated by caveolins. The aim of this study was to analyze CAV1-3 expression in internal thoracic artery (ITA) grafts used in CABG and correlate their expression with graft occlusion. METHODS: Six hundred patients undergoing CABG with the use of ITA grafts between 2008 and 2014 were enrolled into this prospective study. CAV1-3 expression in the ITA grafts was analyzed prior to graft transplantation into the coronary circulation via immunohistochemistry. Estimated caveolins expression pattern was then correlated with the occurrence of ITA graft failure observed within 24-months of surgery. RESULTS: Thirty-four patients developed ITA graft failure (subgroup A) and 566 study participants presented no adverse events (subgroup B). CAV1 and CAV3 expression levels in SMCs of the tunica media of the ITA grafts did not differ between the study subgroups and were not associated with the risk of graft failure. CAV2 was expressed within SMCs of the ITA grafts in 94.1% of the patients from subgroup A and 2.5% from subgroup B, and its expression was associated with ITA graft occlusion observed within 24-months after CABG. CONCLUSIONS: CAV2 expression in SMCs of the tunica media in autologous ITA transplants might indicate the risk of graft failure.

Quantification of the asymmetric migration of the lipophilic dyes, DiO and DiD, in homotypic co-cultures of chondrosarcoma SW-1353 cells.

DiO and DiD are lipophilic cell labelling dyes used in the staining of cells in vivo and in vitro. The aim of the present study was to quantify the asymmetrical distribution of dyes in co­cultured cells and to measure the intercellular transfer of DiO and DiD. DiO and DiD were applied separately to stain two identical populations of SW­1353 human chondrosarcoma cells that were subsequently co­cultured (homotypic co­culture). The intercellular migration of dyes in the co­cultured cells was measured by flow cytometry and recorded under a fluorescent microscope. DiD and DiO caused no effect on the proliferation of cells, the degradation rate of the two dyes was comparable and crossover effects between dyes were negligible. The results of the present study suggested that asymmetrical intercellular migration of DiD and DiO was responsible for the asymmetrical distribution of these dyes in co­cultured cells. To take advantage of the lipophilic dyes migration in the double-stained co-cultured cells we suggest to apply mixed-dyes controls prior to the flow cytometric analysis. These controls are performed by staining cells with a 1:1 mix of the two dyes and would enable the estimation of the intensity of intercellular contact in co­culture systems. A 1:1 premix of DiO and DiD was applied to estimate cellular effect on intercellular exchange of lipid dyes in co­cultures incubated with cycloheximide and cytochalasin B. The cellular effect contributed 6­7% of intercellular migration of the lipophilic dyes, DiO and DiD. The majority of the observed intercellular transfer of these dyes was due to non­cellular, passive transfer.

Co­culture of human nucleus pulposus cells with multipotent mesenchymal stromal cells from human bone marrow reveals formation of tunnelling nanotubes.

Degeneration of the intervertebral disc (IVD) is the main cause of age-related damage of spinal tissues. Using multipotent mesenchymal stromal cells (MSCs) regenerative medicine intends to restore the IVD components of annulus fibrosus (AF) and nucleus pulposus (NP). In the present study NP cells (NPCs) and MSCs obtained from adolescent patients suffering from scoliosis were used. IVDs and vertebrae were obtained during surgery and subsequently processed in order to establish cultures of NPCs and MSCs. The two cell types were co-cultured in 1-µm pore size insert system (indirect co-culture) or on one surface (direct co-culture). Prior to co-culture in these systems one of the cell types was stained by lipophilic fluorescent dye DiD (red). The results demonstrated that regardless of the cell type, the flow of DiD from stained to non-stained cells was more efficient in the direct co-culture in comparison with the insert system. Moreover, in the direct system the DiD flow was more efficient from MSCs towards NPCs compared with that in the opposite direction. These data indicated that the membrane interchange between the two cell types was asymmetric. To discriminate the subpopulation of cells that underwent membrane interchange, cells were double stained with DiD and DiO (green). In the first part of the experiment NPCs were stained by DiO and MSCs by DiD. In the second, NPCs were stained by DiD and MSCs by DiO. The cells were co-cultured in the direct system for 8 days and subsequently analyzed by flow cytometry and confocal microscopy. This analysis revealed that >50% of cells were stained by the DiO and DiD dyes. NPCs and MSCs formed structures similar to tunnelling nanotubes (TnT). In conclusion, the formation of TnT-like structures is able to promote, phenotypic changes during the direct co-culture of NPCs with MSCs.

Nuclear localization of P-glycoprotein is responsible for protection of the nucleus from doxorubicin in the resistant LoVo cell line.

The high expression of P-glycoprotein (P-gp) belongs to one of the most important factors causing multidrug-resistant (MDR) of cancer cells. P-gp is primarily associated with plasma membrane; however, small fraction of that protein is present in the nuclear envelope. Such phenomenon is observed in cancer cells and may result in the selection of MDR cells as the secondary tumor and/or resistant metastasis that significantly shorten patient survival rate. Here, we confirmed nuclear localization of P-gp in resistant LoVo cells and demonstrated its impact on doxorubicin efflux from the nucleus to cytoplasm. Furthermore, we showed that P-gp located at the nuclear envelope might have a different glycoside chain when compared to the form located in the cytoplasm. It suggests that the glycoside chain plays a role in the intracellular trafficking of P-gp and may decide about the destination place in the cell.