Reelin immunoreactivity in dissociated cultures of the postnatal hippocampus

Reelin is an extracellular matrix glycoprotein expressed in different nerve cell populations in the developing, early postnatal and adult central nervous system. During histogenesis of the neocortex and hippocampus, reelin is present in Cajal-Retzius cells and other early neurons and contributes to correct layering of these regions. During early postnatal life, pioneer neurons disappear and reelin expression establishes in a subpopulation of cortical and hippocampal GABAergic interneurons, where it is maintained throughout adult life. We studied the developmental distribution pattern of reelin in dissociated cultures obtained from the early postnatal hippocampus to verify whether or not such a maturation phenomenon is maintained in vitro. Reelin is expressed both in Cajal-Retzius cells and multipolar and pyramidal neurons in younger cultures. The density of reelin-positive Cajal-Retzius cells dropped drastically by about 84% in 4-week-old cultures. Multipolar and pyramidal neurons containing reelin represented 12% of the total cell population in younger cultures and decreased by about 25% after 3 to 4 weeks of cultivation. Their density was significantly lower in cultures of the same age treated with glutamate receptor antagonists. These reelin-positive multipolar and pyramidal neurons were heterogeneous, including a larger amount of non-GABAergic, and 30-40% of GABAergic neurons. Cells double labeled for reelin and the GABA synthesizing enzyme glutamic acid decarboxylase represented about 4% of the total neuron population in culture and their density remained constant with age. It is thus possible that the decrease in the total reelin population may selectively be of importance to the larger non-GABAergic fraction of reelin cells. This study shows that reelin-expressing neurons are maintained in dissociated cultures of the neonatal hippocampus and their distribution and age-dependent changes in density resemble those of the early postnatal hippocampus in vivo.

NGS Nominated CELA1, HSPG2, and KCNK5 as Candidate Genes for Predisposition to Balkan Endemic Nephropathy

Balkan endemic nephropathy (BEN) is a familial chronic tubulointerstitial disease with insidious onset and slow progression leading to terminal renal failure. The results of molecular biological investigations propose that BEN is a multifactorial disease with genetic predisposition to environmental risk agents. Exome sequencing of 22 000 genes with Illumina Nextera Exome Enrichment Kit was performed on 22 DNA samples (11 Bulgarian patients and 11 Serbian patients). Software analysis was performed via NextGene, Provean, and PolyPhen. The frequency of all annotated genetic variants with deleterious/damaging effect was compared with those of European populations. Then we focused on nonannotated variants (with no data available about them and not found in healthy Bulgarian controls). There is no statistically significant difference between annotated variants in BEN patients and European populations. From nonannotated variants with more than 40% frequency in both patients' groups, we nominated 3 genes with possible deleterious/damaging variants-CELA1, HSPG2, and KCNK5. Mutant genes (CELA1, HSPG2, and KCNK5) in BEN patients encode proteins involved in basement membrane/extracellular matrix and vascular tone, tightly connected to process of angiogenesis. We suggest that an abnormal process of angiogenesis plays a key role in the molecular pathogenesis of BEN.

New insights into the pathobiology of Down syndrome--hyaluronan synthase-2 overexpression is regulated by collagen VI α2 chain.

Down syndrome (DS) is a common birth defect characterized by the trisomy of chromosome 21. DS-affected umbilical cords (UCs) of fetuses show altered architecture of the extracellular matrix. Overexpression of the chromosome 21 genes encoding the collagen type VI (COLVI) chains α1(VI) and α2(VI), COL6A1 and COL6A2, respectively, has also reported to occur in the nuchal skin of DS fetuses. The aim of this study was therefore to evaluate the COLVI content in euploid and DS-affected UCs and human skin fibroblasts, and to investigate the relationships between COLVI and hyaluronan (HA) and HA synthase-2 (HAS2). We found that the UCs of DS fetuses showed denser staining of COLVI and increased COL6A2 expression at both early and term gestational ages. In vitro expression studies in DS-derived fibroblasts showed similarly increased amounts of α1(VI) and α2(VI) chains at the protein and transcriptional level, supporting the hypothesis of the gene dosage effect. Furthermore, increased levels of HA and HAS2 were also found in DS-derived skin fibroblast cultures. Notably, silencing of COL6A2 in DS-derived cells resulted in downregulation of HAS2, with a simultaneous decrease in secreted HA. Exogenous addition of COLVI to normal fibroblasts did not have any effect on HAS2 expression. In conclusion, UCs and skin fibroblasts in DS show significant increases in COLVI and HA; the overexpression of COL6A2 in DS tissue and cells is closely related to the increased expression of HAS2. These data may explain the DS phenotypes and their effects in organ tissue maturation.

Tenascins in stem cell niches.

Tenascins are extracellular matrix proteins with distinct spatial and temporal expression during development, tissue homeostasis and disease. Based on their expression patterns and knockout phenotypes an important role of tenascins in tissue formation, cell adhesion modulation, regulation of proliferation and differentiation has been demonstrated. All of these features are of importance in stem cell niches where a precise regulation of growth versus differentiation has to be guaranteed. In this review we summarize the expression and possible functions of tenascins in neural, epithelial and osteogenic stem cell niches during normal development and organ turnover, in the hematopoietic and pro-inflammatory niche as well as in the metastatic niche during cancer progression.

Proteomic analysis of porcine bone-conditioned medium.

PURPOSE Autografts are considered to support bone regeneration. Paracrine factors released from cortical bone might contribute to the overall process of graft consolidation. The aim of this study was to characterize the paracrine factors by means of proteomic analysis. MATERIALS AND METHODS Bone-conditioned medium (BCM) was prepared from fresh bone chips of porcine mandibles and subjected to proteomic analysis. Proteins were categorized and clustered using the bioinformatic tools UNIPROT and PANTHER, respectively. RESULTS Proteomic analysis showed that BCM contains more than 150 proteins, of which 43 were categorized into "secreted" and "extracellular matrix." Growth factors that are not only detectable in BCM, but potentially also target cellular processes involved in bone regeneration, eg, pleiotrophin, galectin-1, transforming growth factor beta (TGF-β)-induced gene (TGFBI), lactotransferrin, insulin-like growth factor (IGF)-binding protein 5, latency-associated peptide forming a complex with TGF-β1, and TGF-β2, were discovered. CONCLUSION The present results demonstrate that cortical bone chips release a large spectrum of proteins with the possibility of modulating cellular aspects of bone regeneration. The data provide the basis for future studies to understand how these paracrine factors may contribute to the complex process of graft consolidation.

Beneficial effects of adiponectin on periodontal ligament cells under normal and regenerative conditions

Type 2 diabetes and obesity are increasing worldwide and linked to periodontitis, a chronic disease which is characterized by the irreversible destruction of the tooth-supporting tissues, that is, periodontium. The mechanisms underlying the association of diabetes mellitus and obesity with periodontal destruction and compromised periodontal healing are not well understood, but decreased plasma levels of adiponectin, as found in diabetic and obese individuals, might be a critical mechanistic link. The aim of this in vitro study was to examine the effects of adiponectin on periodontal ligament (PDL) cells under normal and regenerative conditions, and to study the regulation of adiponectin and its receptors in these cells. Adiponectin stimulated significantly the expression of growth factors and extracellular matrix, proliferation, and in vitro wound healing, reduced significantly the constitutive tumor necrosis factor-α expression, and caused a significant upregulation of its own expression. The beneficial actions of enamel matrix derivative on a number of PDL cell functions critical for periodontal regeneration were partially enhanced by adiponectin. The periodontopathogen Porphyromonas gingivalis inhibited the adiponectin expression and stimulated the expression of its receptors. In conclusion, reduced levels of adiponectin, as found in type 2 diabetes and obesity, may compromise periodontal health and healing.

Endogenous and synthetic MMP inhibitors in CNS physiopathology.

Matrix metalloproteinases (MMPs, including the membrane-type MMPs (MT-MMPs)), a disintegrin and metalloproteinase (ADAM), and ADAM with thrombospondin motifs belong to the metzincins, a subclass of metalloproteinases that contain a Met residue and a Zn(2+) ion at the catalytic site necessary for enzymatic reaction. MMP proteolytic activity is mainly controlled by their natural tissue inhibitors of metalloproteinase (TIMP). A number of synthetic inhibitors have been developed to control deleterious MMP activity. The roles of MMPs and some of their ECM substrates in CNS physiology and pathology are covered by other chapters of the present volume and will thus not be addressed in depth. This chapter will focus (i) on the endogenous MMP inhibitors in the CNS, (ii) on MMP and TIMP regulations in three large classes of neuropathologic processes (inflammatory, neurodegenerative, and infectious), and (iii) on synthetic inhibitors of MMPs and the perspective of their use in different brain diseases.

Tenascin-C is required for normal Wnt/β-catenin signaling in the whisker follicle stem cell niche.

Whisker follicles have multiple stem cell niches, including epidermal stem cells in the bulge as well as neural crest-derived stem cells and mast cell progenitors in the trabecular region. The neural crest-derived stem cells are a pool of melanocyte precursors. Previously, we found that the extracellular matrix glycoproteins tenascin-C and tenascin-W are expressed near CD34-positive cells in the trabecular stem cell niche of mouse whisker follicles. Here, we analyzed whiskers from tenascin-C knockout mice and found intrafollicular adipocytes and supernumerary mast cells. As Wnt/β-catenin signaling promotes melanogenesis and suppresses the differentiation of adipocytes and mast cells, we analyzed β-catenin subcellular localization in the trabecular niche. We found cytoplasmic and nuclear β-catenin in wild-type mice reflecting active Wnt/β-catenin signaling, whereas β-catenin in tenascin-C knockout mice was mostly cell membrane-associated and thus transcriptionally inactive. Furthermore, cells expressing the Wnt/β-catenin target gene cyclin D1 were enriched in the CD34-positive niches of wild-type compared to tenascin-C knockout mice. We then tested the effects of tenascins on this signaling pathway. We found that tenascin-C and tenascin-W can be co-precipitated with Wnt3a. In vitro, substrate bound tenascins promoted β-catenin-mediated transcription in the presence of Wnt3a, presumably due to the sequestration and concentration of Wnt3a near the cell surface. We conclude that the presence of tenascin-C in whiskers assures active Wnt/β-catenin signaling in the niche thereby maintaining the stem cell pool and suppressing aberrant differentiation, while in the knockout mice with reduced Wnt/β-catenin signaling, stem cells from the trabecular niche can differentiate into ectopic adipocytes and mast cells.

How best to preserve and reveal the structural intricacies of cartilaginous tissue.

No single processing technique is capable of optimally preserving each and all of the structural entities of cartilaginous tissue. Hence, the choice of methodology must necessarily be governed by the nature of the component that is targeted for analysis, for example, fibrillar collagens or proteoglycans within the extracellular matrix, or the chondrocytes themselves. This article affords an insight into the pitfalls that are to be encountered when implementing the available techniques and how best to circumvent them. Adult articular cartilage is taken as a representative pars pro toto of the different bodily types. In mammals, this layer of tissue is a component of the synovial joints, wherein it fulfills crucial and diverse biomechanical functions. The biomechanical functions of articular cartilage have their structural and molecular correlates. During the natural course of postnatal development and after the onset of pathological disease processes, such as osteoarthritis, the tissue undergoes structural changes which are intimately reflected in biomechanical modulations. The fine structural intricacies that subserve the changes in tissue function can be accurately assessed only if they are faithfully preserved at the molecular level. For this reason, a careful consideration of the tissue-processing technique is indispensable. Since, as aforementioned, no single methodological tool is capable of optimally preserving all constituents, the approach must be pre-selected with a targeted structure in view. Guidance in this choice is offered.

Mechanical Loading Promoted Discogenic Differentiation of Human Mesenchymal Stem Cells Incorporated in 3D-PEG Scaffolds with RhGDF5 and RGD

Hydrogels have been described as ideal scaffolds for cells of 3D tissue constructs and hold strong promises with respect to in vitro 3D-cell-culture, where cells are isolated from native extracellular matrix (ECM). Synthesized polyethyleneglycol (PEG) hydrogels are appealing with regard to potential for cell therapy or as vehicles for drug delivery or even to regenerate tissue with similar hydrogel-like properties such as the nucleus pulposus of the intervertebral disc (IVD). Here, we tested whether incorporation of RGD motive would hinder discogenic differentiation of primary bone marrow-derived human mesenchymal stem cells (hMSCs) but favor proliferation of undifferentiated hMSCs. HMSCs were embedded in +RGD containing or without RGD PEG hydrogel and pre-conditioned with or without growth and differentiation factor-5 (rhGDF-5) for 13 days. Afterwards, all hMSCs-PEG gels were subsequently cyclically loaded (15% strain, 1Hz) for 5 consecutive days in a bioreactor to generate an IVD-like phenotype. Higher metabolic activity (resazurin assay) was found in groups with rhGDF5 in both gel types with and without RGD. Cell viability and morphology measured by confocal laser microscopy and DNA content showed decreased values (~60%) after 18 days of culture. Real-time RT-PCR of an array of 15 key genes suspected to be distinctive for IVD cells revealed moderate response to rhGDF5 and mechanical loading as also shown by histology staining. Preconditioning and mechanical loading showed relatively moderate responses revealed from both RT-PCR and histology although hMSCs were demonstrated to be potent to differentiate into chondrocyte-progenitor cells in micro- mass and 3D alginate bead culture.

Alpha1a-adrenoceptor genetic variant induces cardiomyoblast-to-fibroblast-like cell transition via distinct signaling pathways.

The role of naturally occurring human α1a-Adrenergic Receptor (α1aAR) genetic variants associated with cardiovascular disorders is poorly understood. Here, we present the novel findings that expression of human α1aAR-247R (247R) genetic variant in cardiomyoblasts leads to transition of cardiomyoblasts into a fibroblast-like phenotype, evidenced by morphology and distinct de novo expression of characteristic genes. These fibroblast-like cells exhibit constitutive, high proliferative capacity and agonist-induced hypertrophy compared with cells prior to transition. We demonstrate that constitutive, synergistic activation of EGFR, Src and ERK kinases is the potential molecular mechanism of this transition. We also demonstrate that 247R triggers two distinct EGFR transactivation-dependent signaling pathways: 1) constitutive Gq-independent β-arrestin-1/Src/MMP/EGFR/ERK-dependent hyperproliferation and 2) agonist-induced Gq- and EGFR/STAT-dependent hypertrophy. Interestingly, in cardiomyoblasts agonist-independent hyperproliferation is MMP-dependent, but in fibroblast-like cells it is MMP-independent, suggesting that expression of α1aAR genetic variant in cardiomyocytes may trigger extracellular matrix remodeling. Thus, these novel findings demonstrate that EGFR transactivation by α1aAR-247R leads to hyperproliferation, hypertrophy and alterations in cardiomyoblasts, suggesting that these unique genetically-mediated alterations in signaling pathways and cellular function may lead to myocardial fibrosis. Such extracellular matrix remodeling may contribute to the genesis of arrhythmias in certain types of heart failure.

Differential inflammatory response of dental pulp explants and fibroblasts to saliva

Abstract AIM: To investigate the inflammatory response of dental pulp fibroblasts and the respective explants to whole saliva. METHODOLOGY: Explants from human and porcine dental pulp tissue and isolated dental pulp fibroblasts were used to investigate the inflammatory response to sterile saliva. Cytokine and chemokine expression was assessed by RT-PCR. Western blot analysis and pharmacologic inhibitors were used to determine the involvement of signalling pathways. RESULTS: Dental pulp explants of human and porcine origin exposed to human saliva exhibited no major changes of IL-6 and IL-8 mRNA expression (P > 0.05). In contrast, isolated porcine and human dental pulp fibroblasts, when stimulated with human saliva, exhibited a vastly increased expression of IL-6 and IL-8 mRNA (P < 0.05). In pulp fibroblasts, saliva also increased the expression of other cytokines and chemokines via activation of NFkappaB, ERK and p38 signalling. Notably, a significantly reduced inflammatory response was elicited when pulp fibroblasts were transiently exposed to saliva. CONCLUSIONS: Saliva has a potential impact on inflammation of dental pulp fibroblasts in vitro but not when cells are embedded in the intrinsic extracellular matrix of the explant tissue.

Profiling invasiveness in head and neck cancer: recent contributions of genomic and transcriptomic approaches.

High-throughput molecular profiling approaches have emerged as precious research tools in the field of head and neck translational oncology. Such approaches have identified and/or confirmed the role of several genes or pathways in the acquisition/maintenance of an invasive phenotype and the execution of cellular programs related to cell invasion. Recently published new-generation sequencing studies in head and neck squamous cell carcinoma (HNSCC) have unveiled prominent roles in carcinogenesis and cell invasion of mutations involving NOTCH1 and PI3K-patwhay components. Gene-expression profiling studies combined with systems biology approaches have allowed identifying and gaining further mechanistic understanding into pathways commonly enriched in invasive HNSCC. These pathways include antigen-presenting and leucocyte adhesion molecules, as well as genes involved in cell-extracellular matrix interactions. Here we review the major insights into invasiveness in head and neck cancer provided by high-throughput molecular profiling approaches.

NonViral Gene Delivery of Growth and Differentiation Factor 6 (GDF6) to whole bovine Intervertebral Disc

Question: Low back pain is an increasing global health problem, which is associated with intervertebral disc (IVD) damage and de- generation. Major changes occur in the nucleus pulposus (NP), with the degradation of the extracellular matrix (ECM) [1]. Further studies showed that growth factors from the transforming growth factor (TGF) and bone morphogenic proteins (BMP) family may induce chondrogenic differentiation of mesenchymal stem cells (MSC) [2]. Focusing on non-viral gene therapies and their possible translation into the clinics, we investigated if GDF6 (syn. BMP13 or CDMP2) can induce regeneration of degraded NP. We hypothesized that IVD transfected with plasmid over-expressing GDF6 also up-regulates other NP- and chondrogenic cell markers and enhances ECM deposition. Methods: Bovine IVD cells were isolated by pronase/collagenase II overnight digestion. After monolayer expansion up to passage 3, cells were transfected with the plasmid pGDF6 (RG211366, Origene, SF) or with green fluorescence protein (GFP) control using the NeonÒ transfection system (Invitrogen, Basel), both equipped with a Cy- tomegalovirus (CMV) promotor to induce over-expression. We tested a range of yet unpublished parameters for each of the primary disc cells to optimize efficiency. To test a non-viral gene therapy applied directly to 3D whole organ culture, bovine IVDs were harvested from fresh tails obtained from the abattoir within 5 h post-mortem [3]. Discs were then pre-incubated for 24 h in high glucose Dulbecco’s Modified Eagle Medium and 5 % fetal calf serum. Each disc was transfected by injection of 5 lg of plasmid GDF6 (Origene, RG211366) into the center by 25G needle and using Hamilton sy- ringe. Electroporation was performed using 2-needle array electrode or tweezertrodes; 8 pulses at 200mv/cm with an interval of 10 ms were applied using ECM830 Square Wave Electroporation System (Harvard Apparatus, MA) (Fig. 1). After transfection discs were cultured for 72 h to allow expression of GFP or GDF6. Discs were then fixed, cryosectioned and analysed by immunofluorescence against GDF6. Results: We successfully transfected bovine NP and AF cells in monolayer culture with the two plasmids using a 1,400 V, 20 ms and 2 pulses with a *25 % efficiency using 0.15 M cells and 3 lg DNA (Fig. 1). Organ IVD culture transfection revealed GFP6 positive staining in the centre of the disc using 2-needle array electrode. Results from tweezertrodes did not show any GFP posi- tive cells. Conclusions: We identified novel parameters to successfully transfect primary bovine IVD cells. For transfection of whole IVD explants electroporation parameters need to be further optimized. Acknowledgments: This study was supported by the Lindenhof Foundation ‘‘Forschung und Lehre’’ (Project no. 13-02-F). References 1. Roughly PJ (2004) Spine (Phila) 29:2691–2699 2. 3. Clarke LE, McConell JC, Sherratt MJ, Derby B, Richardson SM, Hoyland JA (2014) Arthritis Res Ther 16:R67 Chan SC, Gantenbein-Ritter B (2012) J Vis Exp 60(60):e3490

Transcriptional regulation of tenascin genes

Extracellular matrix proteins of the tenascin family resemble each other in their domain structure, and also share functions in modulating cell adhesion and cellular responses to growth factors. Despite these common features, the 4 vertebrate tenascins exhibit vastly different expression patterns. Tenascin-R is specific to the central nervous system. Tenascin-C is an "oncofetal" protein controlled by many stimuli (growth factors, cytokines, mechanical stress), but with restricted occurrence in space and time. In contrast, tenascin-X is a constituitive component of connective tissues, and its level is barely affected by external factors. Finally, the expression of tenascin-W is similar to that of tenascin-C but even more limited. In accordance with their highly regulated expression, the promoters of the tenascin-C and -W genes contain TATA boxes, whereas those of the other 2 tenascins do not. This article summarizes what is currently known about the complex transcriptional regulation of the 4 tenascin genes in development and disease.