Although glutamate is one of the most important excitatory neurotransmitters of the central nervous system, its excessive extracellular concentration leads to uncontrolled continuous depolarization of neurons, a toxic process called, excitotoxicity. In excitotoxicity glutamate triggers the rise of intracellular Ca2+ levels, followed by up regulation of nNOS, dysfunction of mitochondria, ROS production, ER stress, and release of lysosomal enzymes. Excessive calcium concentration is the key mediator of glutamate toxicity through over activation of ionotropic and metabotropic receptors. In addition, glutamate accumulation can also inhibit cystine (CySS) uptake by reversing the action of the CySS/glutamate antiporter. Reversal of the antiporter action reinforces the aforementioned events by depleting neurons of cysteine and eventually glutathione’s reducing potential. Various cell lines have been employed in the pursuit to understand the mechanism(s) by which excitotoxicity affects the cells leading them ultimately to their demise. In some cell lines glutamate toxicity is exerted mainly through over activation of NMDA, AMPA, or kainate receptors whereas in other cell lines lacking such receptors, the toxicity is due to glutamate induced oxidative stress. However, in the greatest majority of the cell lines ionotropic glutamate receptors are present, co-existing to CySS/glutamate antiporters and metabotropic glutamate receptors, supporting the assumption that excitotoxicity effect in these cells is accumulative. Different cell lines differ in their responses when exposed to glutamate. In this review article the responses of PC12, SH-SY5Y, HT-22, NT-2, OLCs, C6, primary rat cortical neurons, RGC-5, and SCN2.2 cell systems are systematically collected and analyzed.

The present review summarizes recent advances in the transcriptional regulation of the human apolipoprotein genes, focusing mostly, but not exclusively, on in-vivo studies and signaling mechanisms that affect apolipoprotein gene transcription. An attempt is made to explain how interactions of transcription factors that bind to proximal promoters and distal enhancers may bring about gene transcription. The experimental approaches used and the transcriptional regulatory mechanisms that emerge from these studies may also be applicable in other gene systems that are associated with human disease. Understanding extracellular stimuli and the specific mechanisms that underlie apolipoprotein gene transcription may in the long run allow us to selectively switch on antiatherogenic genes, and switch off proatherogenic genes. This may have beneficial effects and may confer protection from atherosclerosis to humans.

Stem cells from the apical papilla (SCAP) of human adult teeth are considered an accessible source of cells with angiogenic properties. The aims of this study were to investigate the endothelial transdifferentiation of SCAP, the secretion of pro-and antiangiogenic factors from SCAP, and the paracrine effects of SCAP when exposed to environmental stress to stimulate tissue damage. SCAP were exposed to serum deprivation (SD), glucose deprivation (GD), and oxygen deprivation/hypoxia (OD) conditions, individually or in combination. Endothelial transdifferentiation was evaluated by in vitro capillary-like formation assays, real-time polymerase chain reaction, western blot, and flow cytometric analyses of angiogenesis-related markers; secretome by antibody arrays and enzyme-linked immunosorbent assays (ELISA); and paracrine impact on human umbilical vein endothelial cells (HUVECs) by in vitro transwell migration and capillary-like formation assays. The short-term exposure of SCAP to glucose/oxygen deprivation (GOD) in the presence, but mainly in deprivation, of serum (SGOD) elicited a proangiogenesis effect indicated by expression of angiogenesis-related genes involved in vascular endothelial growth factor (VEGF)/VEGFR and angiopoietins/Tie pathways. This effect was unachievable under SD in normoxia, suggesting that the critical microenvironmental condition inducing rapid endothelial shift of SCAP is the combination of SGOD. Interestingly, SCAP showed high adaptability to these adverse conditions, retaining cell viability and acquiring a capillary-forming phenotype. SCAP secreted higher numbers and amounts of pro-(angiogenin, IGFBP-3, VEGF) and lower amounts of antiangiogenic factors (serpin-E1, TIMP-1, TSP-1) under SGOD compared with SOD or SD alone. Finally, secretome obtained under SGOD was most effective in inducing migration and capillary-like formation by HUVECs. These data provide new evidence on the microenvironmental factors favoring endothelial transdifferentiation of SCAP, uncovering the molecular mechanisms regulating their fate. They also validate the angiogenic properties of their secretome giving insights into preconditioning strategies enhancing their therapeutic potential.

BackgroundDevelopment of clinical-grade cell preparations is central to meeting the regulatory requirements for cellular therapies under good manufacturing practice-compliant (cGMP) conditions. Since addition of animal serum in culture media may compromise safe and efficient expansion of mesenchymal stem cells (MSCs) for clinical use, this study aimed to investigate the potential of two serum/xeno-free, cGMP culture systems to maintain long-term “stemness” of oral MSCs (dental pulp stem cells (DPSCs) and alveolar bone marrow MSCs (aBMMSCs)), compared to conventional serum-based expansion.MethodsDPSC and aBMMSC cultures (n = 6/cell type) were established from pulp and alveolar osseous biopsies respectively. Three culture systems were used: StemPro_MSC/SFM_XenoFree (Life Technologies); StemMacs_MSC/XF (Miltenyi Biotek); and α-MEM (Life Technologies) with 15% fetal bovine serum. Growth (population doublings (PDs)), immunophenotypic (flow cytometric analysis of MSC markers) and senescence (β-galactosidase (SA-β-gal) activity; telomere length) characteristics were determined during prolonged expansion. Gene expression patterns of osteogenic (ALP, BMP-2), adipogenic (LPL, PPAR-γ) and chondrogenic (ACAN, SOX-9) markers and maintenance of multilineage differentiation potential were determined by real-time PCR.ResultsSimilar isolation efficiency and stable growth dynamics up to passage 10 were observed for DPSCs under all expansion conditions. aBMMSCs showed lower cumulative PDs compared to DPSCs, and when StemMacs was used substantial delays in cell proliferation were noted after passages 6–7. Serum/xeno-free expansion produced cultures with homogeneous spindle-shaped phenotypes, while serum-based expansion preserved differential heterogeneous characteristics of each MSC population. Prolonged expansion of both MSC types but in particular the serum/xeno-free-expanded aBMMSCs was associated with downregulation of CD146, CD105, Stro-1, SSEA-1 and SSEA-4, but not CD90, CD73 and CD49f, in parallel with an increase of SA-gal-positive cells, cell size and granularity and a decrease in telomere length. Expansion under both serum-free systems resulted in “osteogenic pre-disposition”, evidenced by upregulation of osteogenic markers and elimination of chondrogenic and adipogenic markers, while serum-based expansion produced only minor changes. DPSCs retained a diminishing (CCM, StemPro) or increasing (StemMacs) mineralization potential with passaging, while aBMMSCs lost this potential after passages 6–7 under all expansion conditions.ConclusionsThese findings indicate there is still a vacant role for development of qualified protocols for clinical-grade expansion of oral MSCs; a key milestone achievement for translation of research from the bench to clinics.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-017-0705-0) contains supplementary material, which is available to authorized users.

Recent studies have revealed that hepatocyte nuclear factor 4 (HNF-4) is an essential positive regulator of another liver enriched transcription factor HNF-1, defining a transcriptional hierarchy between the two factors operating in hepatocytes. To assess the possible autoregulation of the HNF-1 gene we have examined the effect of HNF-1 on its own transcription. In transient transfection assays, HNF-1 strongly down-regulated transcription driven by its own promoter in HepG2 cells. In addition HNF-1 also repressed the activity of HNF-4 dependent ApoCIII and ApoAI promoters. The same effect was observed using vHNF-1, a distinct but highly related protein to HNF-1. Both HNF-1 and vHNF-1 downregulated HNF-4 activated transcription from intact and chimeric promoter constructs carrying various HNF-4 binding sites implying that they act by impeding HNF-4 binding or activity. DNA binding and cell free transcription experiments however failed to demonstrate any direct or indirect interaction of HNF-1 and vHNF-1 with the above regulatory regions. Both factors repressed HNF-4 induced transcription of the ApoCIII and HNF-1 genes in HeLa cells, arguing against the requirement of a hepatocyte specific function. These findings define an indirect negative autoregulatory mechanism involved in HNF-1 gene expression, which in turn may affect HNF-4 dependent transcription of other liver specific genes.

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Tissue engineering (TE) may provide effective alternative treatment for challenging temporomandibular joint (TMJ) pathologies associated with disc malpositioning or degeneration and leading to severe masticatory dysfunction. Aim of this study was to evaluate the potential of chitosan/alginate (Ch/Alg) scaffolds to promote fibro/chondrogenic differentiation of dental pulp stem cells (DPSCs) and production of fibrocartilage tissue, serving as a replacement of the natural TMJ disc. Ch/Alg scaffolds were fabricated by crosslinking with CaCl2 combined or not with glutaraldehyde, resulting in two scaffold types that were physicochemically characterized, seeded with DPSCs or human nucleus pulposus cells (hNPCs) used as control and evaluated for cell attachment, viability, and proliferation. The DPSCs/scaffold constructs were incubated for up to 8 weeks and assessed for extracellular matrix production by means of histology, immunofluorescence, and thermomechanical analysis. Both Ch/Alg scaffold types with a mass ratio of 1:1 presented a gel-like structure with interconnected pores. Scaffolds supported cell adhesion and long-term viability/proliferation of DPSCs and hNPCs. DPSCs cultured into Ch/Alg scaffolds demonstrated a significant increase of gene expression of fibrocartilaginous markers (COLI, COL X, SOX9, COM, ACAN) after up to 3 weeks in culture. Dynamic thermomechanical analysis revealed that scaffolds loaded with DPSCs significantly increased storage modulus and elastic response compared to cell-free scaffolds, obtaining values similar to those of native TMJ disc. Histological data and immunochemical staining for aggrecan after 4 to 8 weeks indicated that the scaffolds support abundant fibrocartilaginous tissue formation, thus providing a promising strategy for TMJ disc TE-based replacement.