Single cell analysis of docosahexaenoic acid suppression of sequential LPS-induced proinflammatory and interferon-regulated gene expression in the macrophage.

Preclinical and clinical studies suggest that consumption of long chain omega-3 polyunsaturated fatty acids (PUFAs) reduces severity of chronic inflammatory and autoimmune diseases. While these ameliorative effects are conventionally associated with downregulated expression of proinflammatory cytokine and chemokine genes, our laboratory has recently identified Type 1 interferon (IFN1)-regulated gene expression to be another key target of omega-3 PUFAs. Here we used single cell RNA sequencing (scRNAseq) to gain new mechanistic perspectives on how the omega-3 PUFA docosahexaenoic acid (DHA) influences TLR4-driven proinflammatory and IFN1-regulated gene expression in a novel self-renewing murine fetal liver-derived macrophage (FLM) model. FLMs were cultured with 25 µM DHA or vehicle for 24 h, treated with modest concentration of LPS (20 ng/ml) for 1 and 4 h, and then subjected to scRNAseq using the 10X Chromium System. At 0 h (i.e., in the absence of LPS), DHA increased expression of genes associated with the NRF2 antioxidant response (e.g. Sqstm1, Hmox1, Chchd10) and metal homeostasis (e.g.Mt1, Mt2, Ftl1, Fth1), both of which are consistent with DHA-induced polarization of FLMs to a more anti-inflammatory phenotype. At 1 h post-LPS treatment, DHA inhibited LPS-induced cholesterol synthesis genes (e.g. Scd1, Scd2, Pmvk, Cyp51, Hmgcs1, and Fdps) which potentially could contribute to interference with TLR4-mediated inflammatory signaling. At 4 h post-LPS treatment, LPS-treated FLMs reflected a more robust inflammatory response including upregulation of proinflammatory cytokine (e.g. Il1a, Il1b, Tnf) and chemokine (e.g.Ccl2, Ccl3, Ccl4, Ccl7) genes as well as IFN1-regulated genes (e.g. Irf7, Mx1, Oasl1, Ifit1), many of which were suppressed by DHA. Using single-cell regulatory network inference and clustering (SCENIC) to identify gene expression networks, we found DHA modestly downregulated LPS-induced expression of NF-κB-target genes. Importantly, LPS induced a subset of FLMs simultaneously expressing NF-κB- and IRF7/STAT1/STAT2-target genes that were conspicuously absent in DHA-pretreated FLMs. Thus, DHA potently targeted both the NF-κB and the IFN1 responses. Altogether, scRNAseq generated a valuable dataset that provides new insights into multiple overlapping mechanisms by which DHA may transcriptionally or post-transcriptionally regulate LPS-induced proinflammatory and IFN1-driven responses in macrophages.

Oncogenic RAS sensitizes cells to drug-induced replication stress via transcriptional silencing of P53.

Cancer cells often experience high basal levels of DNA replication stress (RS), for example due to hyperactivation of oncoproteins like MYC or RAS. Therefore, cancer cells are considered to be sensitive to drugs that exacerbate the level of RS or block the intra S-phase checkpoint. Consequently, RS-inducing drugs including ATR and CHK1 inhibitors are used or evaluated as anti-cancer therapies. However, drug resistance and lack of biomarkers predicting therapeutic efficacy limit efficient use. This raises the question what determines sensitivity of individual cancer cells to RS. Here, we report that oncogenic RAS does not only enhance the sensitivity to ATR/CHK1 inhibitors by directly causing RS. Instead, we observed that HRASG12V dampens the activation of the P53-dependent transcriptional response to drug-induced RS, which in turn confers sensitivity to RS. We demonstrate that inducible expression of HRASG12V sensitized cells to ATR and CHK1 inhibitors. Using RNA-sequencing of FACS-sorted cells we discovered that P53 signaling is the sole transcriptional response to RS. However, oncogenic RAS attenuates the transcription of P53 and TGF-ß pathway components which consequently dampens P53 target gene expression. Accordingly, live cell imaging showed that HRASG12V exacerbates RS in S/G2-phase, which could be rescued by stabilization of P53. Thus, our results demonstrate that transcriptional control of P53 target genes is the prime determinant in the response to ATR/CHK1 inhibitors and show that hyperactivation of the MAPK pathway impedes this response. Our findings suggest that the level of oncogenic MAPK signaling could predict sensitivity to intra-S-phase checkpoint inhibition in cancers with intact P53.

Folate Receptor Expression by Human Monocyte-Derived Macrophage Subtypes and Effects of Corticosteroids.

OBJECTIVE: Folate receptor beta (FR-ß) has been used as a clinical marker and target in multiple inflammatory diseases, including osteoarthritis (OA) and rheumatoid arthritis (RA). However, the conditions under which FR-ß+ macrophages arise remain unclear and could be affected by corticosteroids. Therefore, we studied FR-ß expression in vitro in macrophage subtypes and determined their response to triamcinolone acetonide (TA), a clinically often-used corticosteroid. DESIGN: Human monocyte-derived macrophages were differentiated to the known M0, M1, or M2 macrophage phenotypes. The phenotype and FR-ß expression and plasticity of the macrophage subtypes were determined using flow cytometry, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and enzyme-linked immunosorbent assay (ELISA). RESULTS: FR-ß expression was low in granulocyte-macrophage colony-stimulating factor (GM-CSF)-generated (M1-like) macrophages and high in macrophage colony-stimulating factor (M-CSF)-generated (M0 and M2-like) macrophages. FR-ß expression remained high once the M0 or M2 macrophages were stimulated with pro-inflammatory stimuli (interferon-γ plus lipopolysaccharide) to induce M1-like macrophages. On the contrary, anti-inflammatory TA treatment skewed GM-CSF macrophage differentiation toward an M2 and FR-ß+ phenotype. CONCLUSIONS: As corticosteroids skewed monocytes toward an FR-ß-expressing, anti-inflammatory phenotype, even in an M1 priming GM-CSF environment, FR-ß has potential as a biomarker to monitor success of treatment with corticosteroids. Without corticosteroid treatment, M-CSF alone induces high FR-ß expression which remains high under pro-inflammatory conditions. This explains why pro-inflammatory FR-ß+ macrophages (exposed to M-CSF) are observed in arthritis patients and correlate with disease severity.

Circulating MicroRNAs as Non-invasive Biomarkers for Canine Cushing's Syndrome.

Canine Cushing's syndrome (hypercortisolism) can be caused by a pituitary tumor (pituitary-dependent hypercortisolism; PDH) or a cortisol-secreting adrenocortical tumor (csACT). For both cases, non-invasive biomarkers that could pre-operatively predict the risk of recurrence after surgery would greatly impact clinical decision making. The aim of this study was to determine whether circulating microRNAs (miRNAs) can be used as diagnostic (presence of PDH or csACT) and/or prognostic (disease recurrence, histological grade) non-invasive biomarkers for canine Cushing's syndrome. After a pilot study with 40 miRNAs in blood samples of healthy dogs (n = 3), dogs with PDH (n = 3) and dogs with a csACT (n = 4), we selected a total of 20 miRNAs for the definitive study. In the definitive study, these 20 miRNAs were analyzed in blood samples of healthy dogs (n = 6), dogs with PDH (n = 19, pre- and post-operative samples) and dogs with a csACT (n = 26, pre-operative samples). In dogs with PDH, six miRNAs (miR-122-5p, miR-126-5p, miR-141-3p, miR-222-3p, miR-375-3p and miR-483-3p) were differentially expressed compared to healthy dogs. Of one miRNA, miR-122-5p, the expression levels did not overlap between healthy dogs and dogs with PDH (p = 2.9x10-4), significantly decreased after hypophysectomy (p = 0.013), and were significantly higher (p = 0.017) in dogs with recurrence (n = 3) than in dogs without recurrence for at least one year after hypophysectomy (n = 7). In dogs with csACTs, two miRNAs (miR-483-3p and miR-223-3p) were differentially expressed compared to healthy dogs. Additionally, miR-141-3p was expressed significantly lower (p = 0.009) in dogs with csACTs that had a histopathological Utrecht score of ≥ 11 compared to those with a score of <11. These results indicate that circulating miRNAs have the potential to be non-invasive biomarkers in dogs with Cushing's syndrome that may contribute to clinical decision making.

Enhanced Extracellular Matrix Breakdown Characterizes the Early Distraction Phase of Canine Knee Joint Distraction.

OBJECTIVE: Joint distraction triggers intrinsic cartilage repair in animal models of osteoarthritis (OA), corroborating observations in human OA patients treated with joint distraction. The present study explores the still largely elusive mechanism initiating this repair process. DESIGN: Unilateral OA was induced in the knee joint of 8 dogs using the groove model; the contralateral joint served as a control. After 10 weeks, 4 animals received joint distraction, the other 4 serving as OA controls. Halfway the distraction period (after 4 weeks of a standard 8-week distraction treatment), all animals were euthanized, and joint tissues were collected. A targeted quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis was performed of commonly involved processes including matrix catabolism/anabolism, inflammation, and known signaling pathways in OA. In addition, cartilage changes were determined on tissue sections using the canine OARSI (Osteoarthritis Research Society International) histopathology score and collagen type II (COL2A1) immunostaining. RESULTS: Midway distraction, the distracted OA joint showed an upregulation of proteolytic genes, for example, ADAMTS5, MMP9, MMP13, compared to OA alone and the healthy joints, which correlated with an increased OARSI score. Additionally, genes of the transforming growth factor (TGF)-ß and Notch pathway, and markers associated with progenitor cells were increased. CONCLUSIONS: Joint distraction initiates both catabolic and anabolic transcriptional responses. The enhanced turnover, and thereby renewal of the matrix, could be the key to the cartilage repair observed in the months after joint distraction.

Notochordal Cell-Based Treatment Strategies and Their Potential in Intervertebral Disc Regeneration.

Chronic low back pain is the number one cause of years lived with disability. In about 40% of patients, chronic lower back pain is related to intervertebral disc (IVD) degeneration. The standard-of-care focuses on symptomatic relief, while surgery is the last resort. Emerging therapeutic strategies target the underlying cause of IVD degeneration and increasingly focus on the relatively overlooked notochordal cells (NCs). NCs are derived from the notochord and once the notochord regresses they remain in the core of the developing IVD, the nucleus pulposus. The large vacuolated NCs rapidly decline after birth and are replaced by the smaller nucleus pulposus cells with maturation, ageing, and degeneration. Here, we provide an update on the journey of NCs and discuss the cell markers and tools that can be used to study their fate and regenerative capacity. We review the therapeutic potential of NCs for the treatment of IVD-related lower back pain and outline important future directions in this area. Promising studies indicate that NCs and their secretome exerts regenerative effects, via increased proliferation, extracellular matrix production, and anti-inflammatory effects. Reports on NC-like cells derived from embryonic- or induced pluripotent-stem cells claim to have successfully generated NC-like cells but did not compare them with native NCs for phenotypic markers or in terms of their regenerative capacity. Altogether, this is an emerging and active field of research with exciting possibilities. NC-based studies demonstrate that cues from developmental biology can pave the path for future clinical therapies focused on regenerating the diseased IVD.

Excessive E2F Transcription in Single Cancer Cells Precludes Transient Cell-Cycle Exit after DNA Damage.

E2F transcription factors control the expression of cell-cycle genes. Cancers often demonstrate enhanced E2F target gene expression, which can be explained by increased percentages of replicating cells. However, we demonstrate in human cancer biopsy specimens that individual neoplastic cells display abnormally high levels of E2F-dependent transcription. To mimic this situation, we delete the atypical E2F repressors (E2F7/8) or overexpress the E2F3 activator in untransformed cells. Cells with elevated E2F activity during S/G2 phase fail to exit the cell cycle after DNA damage and undergo mitosis. In contrast, wild-type cells complete S phase and then exit the cell cycle by activating the APC/CCdh1 via repression of the E2F target Emi1. Many arrested wild-type cells eventually inactivate APC/CCdh1 to execute a second round of DNA replication and mitosis, thereby becoming tetraploid. Cells with elevated E2F transcription fail to exit the cell cycle after DNA damage, which potentially causes genomic instability, promotes malignant progression, and reduces drug sensitivity.

Dog as a Model for Osteoarthritis: The FGF4 Retrogene Insertion May Matter.

Osteoarthritis (OA) is a degenerative joint disease associated with chronic pain and disability in humans and companion animals. The canine species can be subdivided into non-chondrodystrophic (NCD) and chondrodystrophic (CD) dogs, the latter having disproportionally short limbs due to disturbance in endochondral ossification of long bones. This phenotype is associated with retrogene insertions of the fibroblast growth factor 4 (FGF4) gene, resulting in enhanced fibroblast growth factor receptor 3 (FGFR3) signaling. The effect on cartilage is unknown and in experimental studies with dogs, breeds are seemingly employed randomly. The aim of this study was to determine whether CD- and NCD-derived cartilage differs on a structural and biochemical level, and to explore the relationship between FGF4 associated chondrodystrophy and OA. Cartilage explants from CD and NCD dogs were cultured for 21 days. Activation of canonical Wnt signaling was assessed in primary canine chondrocytes. OA and synovitis severity from an experimental OA model were compared between healthy and OA samples from CD and NCD dogs. Release of glycosaminoglycans, DNA content, and cyclooxygenase 2 (COX-2) expression were higher in NCD cartilage explants. Healthy cartilage from NCD dogs displayed higher cartilage degeneration and synovitis scores, which was aggravated by the induction of OA. Dikkopf-3 gene expression was higher in NCD cartilage. No differences in other Wnt pathway read outs were found. To conclude, chondrodystrophy associated with the FGF4 retrogene seems to render CD dogs less susceptible to the development of OA when compared with NCD dogs. These differences should be considered when choosing a canine model to study the pathobiology and new treatment strategies of OA. © 2019 The Authors. Journal of Orthopaedic Research® Published by Wiley Periodicals, Inc. J Orthop Res 37:2550-2560, 2019.

Hedgehog proteins and parathyroid hormone-related protein are involved in intervertebral disc maturation, degeneration, and calcification.

Parathyroid hormone-related protein (PTHrP) and hedgehog signaling play an important role in chondrocyte development, (hypertrophic) differentiation, and/or calcification, but their role in intervertebral disc (IVD) degeneration is unknown. Better understanding their involvement may provide therapeutic clues for low back pain due to IVD degeneration. Therefore, this study aimed to explore the role of PTHrP and hedgehog proteins in postnatal canine and human IVDs during the aging/degenerative process. The expression of PTHrP, hedgehog proteins and related receptors was studied during the natural loss of the notochordal cell (NC) phenotype during IVD maturation using tissue samples and de-differentiation in vitro and degeneration by real-time quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry. Correlations between their expression and calcification levels (Alizarin Red S staining) were determined. In addition, the effect of PTHrP and hedgehog proteins on canine and human chondrocyte-like cells (CLCs) was determined in vitro focusing on the propensity to induce calcification. The expression of PTHrP, its receptor (PTHR1) and hedgehog receptors decreased during loss of the NC phenotype. N-terminal (active) hedgehog (Indian hedgehog/Sonic hedgehog) protein expression did not change during maturation or degeneration, whereas expression of PTHrP, PTHR1 and hedgehog receptors increased during IVD degeneration. Hedgehog and PTHR1 immunopositivity were increased in nucleus pulposus tissue with abundant vs no/low calcification. In vitro, hedgehog proteins facilitated calcification in CLCs, whereas PTHrP did not affect calcification levels. In conclusion, hedgehog and PTHrP expression is present in healthy and degenerated IVDs. Hedgehog proteins had the propensity to induce calcification in CLCs from degenerated IVDs, indicating that in the future, inhibiting hedgehog signaling could be an approach to inhibit calcification during IVD degeneration.

Growth plate expression profiling: Large and small breed dogs provide new insights in endochondral bone formation.

The difference in the adult height of mammals, and hence in endochondral bone formation, is not yet fully understood and may serve to identify targets for bone and cartilage regeneration. In line with this hypothesis, the intra-species disparity between the adult height of Great Danes and Miniature Poodles was investigated at a transcriptional level. Microarray analysis of the growth plate of five Great Danes and five Miniature Poodles revealed 2,981 unique genes that were differentially expressed, including many genes with an unknown role in skeletal development. A signaling pathway impact analysis indicated activation of the cell cycle, extracellular matrix receptor interaction and the tight junction pathway, and inhibition of pathways associated with inflammation and the complement cascade. In additional validation steps, the gene expression profile of the separate growth plate zones for both dog breeds were determined. Given that the BMP signaling is known for its crucial role in skeletal development and fracture healing, and BMP-2 is used in orthopaedic and spine procedures for bone augmentation, further investigations concentrated on the BMP pathway.The canonical BMP-2 and BMP-6 signaling pathway was activated in the Great Danes compared to Miniature Poodles. In conclusion, investigating the differential expression of genes involved in endochondral bone formation in small and large breed dogs, could be a game changing strategy to provide new insights in growth plate development and identify new targets for bone and cartilage regeneration. © 2017 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals, Inc. on behalf of the Orthopaedic Research Society. J Orthop Res 36:138-148, 2018.

Bone Morphogenetic Protein-2, But Not Mesenchymal Stromal Cells, Exert Regenerative Effects on Canine and Human Nucleus Pulposus Cells.

Chronic back pain is related to intervertebral disc (IVD) degeneration and dogs are employed as animal models to develop growth factor- and cell-based regenerative treatments. In this respect, the differential effects of transforming growth factor beta-1 (TGF-ß1) and bone morphogenetic protein-2 (BMP2) on canine and human chondrocyte-like cells (CLCs) derived from the nucleus pulposus of degenerated IVDs were studied. Human and canine CLCs were cultured in 3D microaggregates in basal culture medium supplemented with/without TGF-ß1 (10 ng/mL) or BMP2 (100 or 250 ng/mL). Both TGF-ß1 and BMP2 increased proliferation and glycosaminoglycan (GAG) deposition of human and canine CLCs. TGF-ß1 induced collagen type I deposition and fibrotic (re)differentiation, whereas BMP2 induced more collagen type II deposition. In dogs, TGF-ß1 induced Smad1 and Smad2 signaling, whereas in humans, it only tended to induce Smad2 signaling. BMP2 supplementation increased Smad1 signaling in both species. This altogether indicates that Smad1 signaling was associated with collagen type II production, whereas Smad2 signaling was associated with fibrotic CLC (re)differentiation. As a step toward preclinical translation, treatment with BMP2 alone and combined with mesenchymal stromal cells (MSCs) was further investigated. Canine male CLCs were seeded in albumin-based hydrogels with/without female bone marrow-derived MSCs (50:50) in basal or 250 ng/mL BMP2-supplemented culture medium. Although the results indicate that a sufficient amount of MSCs survived the culture period, total GAG production was not increased and GAG production per cell was even decreased by the addition of MSCs, implying that MSCs did not exert additive regenerative effects on the CLCs.

Reference genes for reverse transcription quantitative PCR in canine brain tissue.

BACKGROUND: In the last decade canine models have been used extensively to study genetic causes of neurological disorders such as epilepsy and Alzheimer's disease and unravel their pathophysiological pathways. Reverse transcription quantitative polymerase chain reaction is a sensitive and inexpensive method to study expression levels of genes involved in disease processes. Accurate normalisation with stably expressed so-called reference genes is crucial for reliable expression analysis. RESULTS: Following the minimum information for publication of quantitative real-time PCR experiments precise guidelines, the expression of ten frequently used reference genes, namely YWHAZ, HMBS, B2M, SDHA, GAPDH, HPRT, RPL13A, RPS5, RPS19 and GUSB was evaluated in seven brain regions (frontal lobe, parietal lobe, occipital lobe, temporal lobe, thalamus, hippocampus and cerebellum) and whole brain of healthy dogs. The stability of expression varied between different brain areas. Using the GeNorm and Normfinder software HMBS, GAPDH and HPRT were the most reliable reference genes for whole brain. Furthermore based on GeNorm calculations it was concluded that as little as two to three reference genes are sufficient to obtain reliable normalisation, irrespective the brain area. CONCLUSIONS: Our results amend/extend the limited previously published data on canine brain reference genes. Despite the excellent expression stability of HMBS, GAPDH and HRPT, the evaluation of expression stability of reference genes must be a standard and integral part of experimental design and subsequent data analysis.

Biocompatibility and intradiscal application of a thermoreversible celecoxib-loaded poly-N-isopropylacrylamide MgFe-layered double hydroxide hydrogel in a canine model.

INTRODUCTION: Chronic low back pain due to intervertebral disc (IVD) degeneration is associated with increased levels of inflammatory mediators. Current medical treatment consists of oral anti-inflammatory drugs to alleviate pain. In this study, the efficacy and safety of a novel thermoreversible poly-N-isopropylacrylamide MgFe-layered double hydroxide (pNIPAAM MgFe-LDH) hydrogel was evaluated for intradiscal controlled delivery of the selective cyclooxygenase (COX) 2 inhibitor and anti-inflammatory drug celecoxib (CXB). METHODS: Degradation, release behavior, and the ability of a CXB-loaded pNIPAAM MgFe-LDH hydrogel to suppress prostaglandin E2 (PGE2) levels in a controlled manner in the presence of a proinflammatory stimulus (TNF-α) were evaluated in vitro. Biocompatibility was evaluated histologically after subcutaneous injection in mice. Safety of intradiscal application of the loaded and unloaded hydrogels was studied in a canine model of spontaneous mild IVD degeneration by histological, biomolecular, and biochemical evaluation. After the hydrogel was shown to be biocompatible and safe, an in vivo dose-response study was performed in order to determine safety and efficacy of the pNIPAAM MgFe-LDH hydrogel for intradiscal controlled delivery of CXB. RESULTS: CXB release correlated to hydrogel degradation in vitro. Furthermore, controlled release from CXB-loaded hydrogels was demonstrated to suppress PGE2 levels in the presence of TNF-α. The hydrogel was shown to exhibit a good biocompatibility upon subcutaneous injection in mice. Upon intradiscal injection in a canine model, the hydrogel exhibited excellent biocompatibility based on histological evaluation of the treated IVDs. Gene expression and biochemical analyses supported the finding that no substantial negative effects of the hydrogel were observed. Safety of application was further confirmed by the absence of clinical symptoms, IVD herniation or progression of degeneration. Controlled release of CXB resulted in a nonsignificant maximal inhibition (approximately 35 %) of PGE2 levels in the mildly degenerated canine IVDs. CONCLUSIONS: In conclusion, this study showed biocompatibility and safe intradiscal application of an MgFe LDH-pNIPAAM hydrogel. Controlled release of CXB resulted in only limited inhibition of PGE2 in this model with mild IVD degeneration, and further studies should concentrate on application of controlled release from this type of hydrogel in animal models with more severe IVD degeneration.

Effect of coculturing canine notochordal, nucleus pulposus and mesenchymal stromal cells for intervertebral disc regeneration.

INTRODUCTION: Early degenerative changes in the nucleus pulposus (NP) are observed after the disappearance of notochordal cells (NCs). Thus, it has been suggested that NCs play an important role in maintaining the NP and may have a regenerative potential on other cells of the NP. As the number of resident NP cells (NPCs) decreases in a degenerating disc, mesenchymal stromal (stem) cells (MSCs) may be used for cell supplementation. In this study, using cells of one species, the regenerative potential of canine NCs was assessed in long-term three-dimensional coculture with canine NPCs or MSCs. METHODS: Canine NCs and canine NPCs or MSCs were cocultured in alginate beads for 28 days under hypoxic and high-osmolarity conditions. Cell viability, cell morphology and DNA content, extracellular matrix production and expression of genes related to NC markers (Brachyury, KRT18) and NP matrix production (ACAN, COL2A1, COL1A1) were assessed after 1, 15 and 28 days of culture. RESULTS: NCs did not completely maintain their phenotype (morphology, matrix production, gene expression) during 28 days of culture. In cocultures of NPCs and NCs, both extracellular matrix content and anabolic gene expression remained unchanged compared with monoculture groups, whereas cocultures of MSCs and NCs showed increased glycosaminoglycan/DNA. However, the deposition of these proteoglycans was observed near the NCs and not the MSCs. Brachyury expression in the MSC and NC coculture group increased in time. The latter two findings indicate a trophic effect of MSCs on NCs rather than vice versa. CONCLUSIONS: No regenerative potential of canine NCs on canine NPCs or MSCs was observed in this study. However, significant changes in NC phenotype in long-term culture may have resulted in a suboptimal regenerative potential of these NCs. In this respect, NC-conditioned medium may be better than coculture for future studies of the regenerative potential of NCs.

A mRNA landscape of bovine embryos after standard and MAPK-inhibited culture conditions: a comparative analysis.

BACKGROUND: Genes and signalling pathways involved in pluripotency have been studied extensively in mouse and human pre-implantation embryos and embryonic stem (ES) cells. The unsuccessful attempts to generate ES cell lines from other species including cattle suggests that other genes and pathways are involved in maintaining pluripotency in these species. To investigate which genes are involved in bovine pluripotency, expression profiles were generated from morula, blastocyst, trophectoderm and inner cell mass (ICM) samples using microarray analysis. As MAPK inhibition can increase the NANOG/GATA6 ratio in the inner cell mass, additionally blastocysts were cultured in the presence of a MAPK inhibitor and changes in gene expression in the inner cell mass were analysed. RESULTS: Between morula and blastocyst 3,774 genes were differentially expressed and the largest differences were found in blastocyst up-regulated genes. Gene ontology (GO) analysis shows lipid metabolic process as the term most enriched with genes expressed at higher levels in blastocysts. Genes with higher expression levels in morulae were enriched in the RNA processing GO term. Of the 497 differentially expressed genes comparing ICM and TE, the expression of NANOG, SOX2 and POU5F1 was increased in the ICM confirming their evolutionary preserved role in pluripotency. Several genes implicated to be involved in differentiation or fate determination were also expressed at higher levels in the ICM. Genes expressed at higher levels in the ICM were enriched in the RNA splicing and regulation of gene expression GO term. Although NANOG expression was elevated upon MAPK inhibition, SOX2 and POU5F1 expression showed little increase. Expression of other genes in the MAPK pathway including DUSP4 and SPRY4, or influenced by MAPK inhibition such as IFNT, was down-regulated. CONCLUSION: The data obtained from the microarray studies provide further insight in gene expression during bovine embryonic development. They show an expression profile in pluripotent cells that indicates a pluripotent, epiblast-like state. The inability to culture ICM cells as stem cells in the presence of an inhibitor of MAPK activity together with the reported data indicates that MAPK inhibition alone is not sufficient to maintain a pluripotent character in bovine cells.

The paracrine feedback loop between vitamin D3 (1,25(OH)2D3) and PTHrP in prehypertrophic chondrocytes.

The endocrine feedback loop between vitamin D3(1,25(OH)2D3) and parathyroid hormone (PTH) plays a central role in skeletal development. PTH-related protein (PTHrP) shares homology and its receptor (PTHR1) with PTH. The aim of this study was to investigate whether there is a functional paracrine feedback loop between 1,25(OH)2D3 and PTHrP in the growth plate, in parallel with the endocrine feedback loop between 1,25(OH)2D3 and PTH. This was investigated in ATDC5 cells treated with 10(-8) M 1,25(OH)2D3 or PTHrP, Col2-pd2EGFP transgenic mice, and primary Col2-pd2EGFP growth plate chondrocytes isolated by FACS, using RT-qPCR, Western blot, PTHrP ELISA, chromatin immunoprecipitation (ChIP) assay, silencing of the 1,25(OH)2D3 receptor (VDR), immunofluorescent staining, immunohistochemistry, and histomorphometric analysis of the growth plate. The ChIP assay confirmed functional binding of the VDR to the PTHrP promoter, but not to the PTHR1 promoter. Treatment with 1,25(OH)2D3 decreased PTHrP protein production, an effect which was prevented by silencing of the VDR. Treatment with PTHrP significantly induced VDR production, but did not affect 1α- and 24-hydroxylase expression. Hypertrophic differentiation was inhibited by PTHrP and 1,25(OH)2D3 treatment. Taken together, these findings indicate that there is a functional paracrine feedback loop between 1,25(OH)2D3 and PTHrP in the growth plate. 1,25(OH)2D3 decreases PTHrP production, while PTHrP increases chondrocyte sensitivity to 1,25(OH)2D3 by increasing VDR production. In light of the role of 1,25(OH)2D3 and PTHrP in modulating chondrocyte differentiation, 1,25(OH)2D3 in addition to PTHrP could potentially be used to prevent undesirable hypertrophic chondrocyte differentiation during cartilage repair or regeneration.

Expression stability of reference genes for quantitative RT-PCR of healthy and diseased pituitary tissue samples varies between humans, mice, and dogs.

Pituitary surgery generates pituitary tissue for histology, immunohistochemistry, and molecular biological research. In the last decade, the pathogenesis of pituitary adenomas has been extensively studied in humans, and to a lesser degree in dogs, and tumor oncogenesis has been studied in knock-out mice, often by means of quantitative reversed-transcriptase PCR (RT-qPCR). A precondition of such analyses is that so-called reference genes are stably expressed regardless of changes in disease status or treatment. In this study, the expression of six frequently used reference genes, namely, tata box binding protein (tbp), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (ywhaz), hydroxymethylbilane synthase (hmbs), beta-2-microglobulin (b2m), succinate dehydrogenase complex subunit A (sdha), and glyceraldehyde 3 phosphate dehydrogenase 1 (gapdh), was studied in pituitary tissue (normal and adenoma) from three species (humans, mice, and dogs). The stability of expression of these reference genes differed between species and between healthy and diseased tissue within one species. Quantitative analysis based on a single reference gene that is assumed to be stably expressed might lead to wrong conclusions. This cross-species analysis clearly emphasizes the need to evaluate the expression stability of reference genes as a standard and integral aspect of study design and data analysis, in order to improve the validity of the conclusions drawn on the basis of quantitative molecular analyses.

Gene expression profiling of early intervertebral disc degeneration reveals a down-regulation of canonical Wnt signaling and caveolin-1 expression: implications for development of regenerative strategies.

INTRODUCTION: Early degeneration of the intervertebral disc (IVD) involves a change in cellular differentiation from notochordal cells (NCs) in the nucleus pulposus (NP) to chondrocyte-like cells (CLCs). The purpose of this study was to investigate the gene expression profiles involved in this process using NP tissue from non-chondrodystrophic and chondrodystrophic dogs, a species with naturally occurring IVD degeneration. METHODS: Dual channel DNA microarrays were used to compare 1) healthy NP tissue containing only NCs (NC-rich), 2) NP tissue with a mixed population of NCs and CLCs (Mixed), and 3) NP tissue containing solely CLCs (CLC-rich) in both non-chondrodystrophic and chondrodystrophic dogs. Based on previous reports and the findings of the microarray analyses, canonical Wnt signaling was further evaluated using qPCR of relevant Wnt target genes. We hypothesized that caveolin-1, a regulator of Wnt signaling that showed significant changes in gene expression in the microarray analyses, played a significant role in early IVD degeneration. Caveolin-1 expression was investigated in IVD tissue sections and in cultured NCs. To investigate the significance of Caveolin-1 in IVD health and degeneration, the NP of 3-month-old Caveolin-1 knock-out mice was histopathologically evaluated and compared with the NP of wild-type mice of the same age. RESULTS: Early IVD degeneration involved significant changes in numerous pathways, including Wnt/ß-catenin signaling. With regard to Wnt/ß-catenin signaling, axin2 gene expression was significantly higher in chondrodystrophic dogs compared with non-chondrodystrophic dogs. IVD degeneration involved significant down-regulation of axin2 gene expression. IVD degeneration involved significant down-regulation in Caveolin-1 gene and protein expression. NCs showed abundant caveolin-1 expression in vivo and in vitro, whereas CLCs did not. The NP of wild-type mice was rich in viable NCs, whereas the NP of Caveolin-1 knock-out mice contained chondroid-like matrix with mainly apoptotic, small, rounded cells. CONCLUSIONS: Early IVD degeneration involves down-regulation of canonical Wnt signaling and Caveolin-1 expression, which appears to be essential to the physiology and preservation of NCs. Therefore, Caveolin-1 may be regarded an exciting target for developing strategies for IVD regeneration.

Canonical Wnt signaling in the notochordal cell is upregulated in early intervertebral disk degeneration.

The notochordal cell (NC) of the nucleus pulposus (NP) is considered a potential NP progenitor cell, and early intervertebral disk (IVD) degeneration involves replacement of NCs by chondrocyte-like cells (CLCs). Wnt/ß-catenin signaling plays a crucial role in maintaining the notochordal fate during embryogenesis, but is also involved in tissue degeneration and regeneration. The canine species, which can be subdivided into non-chondrodystrophic and chondrodystrophic breeds, is characterized by differential maintenance of the NC: in non-chondrodystrophic dogs, the NC remains the predominant cell type during the majority of life, with IVD degeneration only occurring at old age; conversely, in chondrodystrophic dogs the NC is lost early in life, with concurrent degeneration of all IVDs. This study investigated Wnt/ß-catenin signaling in the healthy, NC-rich NP and early degenerated, CLC-rich NP of both breed types by immunohistochemistry of ß-catenin and relative gene expression of brachyury and cytokeratin 8 (notochordal markers) and Wnt targets axin2, cyclin D1, and c-myc. Both NCs and CLCs showed nuclear and cytoplasmic ß-catenin protein expression and axin2 gene expression, but ß-catenin signal intensity and Wnt target gene expression were higher in the CLC-rich NP. Primary NCs in monolayer culture (normoxic conditions) showed Wnt/ß-catenin signaling comparable to the in vivo situation, with increased cyclin D1 and c-myc gene expression. In conclusion, Wnt/ß-catenin signaling activity in the NC within the NC-rich NP and in culture supports the role of this cell as a potential progenitor cell; increased Wnt/ß-catenin signaling activity in early IVD degeneration may be a reflection of its dual role.

Identification and characterisation of side population cells in the canine pituitary gland.

To date, stem/progenitor cells have not been identified in the canine pituitary gland. Cells that efficiently exclude the vital dye Hoechst 33342 can be visualised and identified using fluorescence activated cell sorting (FACS) as a 'side population' (SP), distinct from the main population (MP). Such SPs have been identified in several tissues and display stem/progenitor cell characteristics. In this study, a small SP (1.3%, n=6) was detected in the anterior pituitary glands of healthy dogs. Quantitative PCR indicated significantly higher expression of CD34 and Thy1 in this SP, but no differences in the expression of CD133, Bmi-1, Axin2 or Shh. Pro-opiomelanocortin (POMC) and Lhx3 expression were significantly higher in the MP than in the SP, but no differences in the expression of Tpit, GH or PRL were found. The study demonstrated the existence of an SP of cells in the normal canine pituitary gland, encompassing cells with stem cell characteristics and without POMC expression.