The lower in vitro chondrogenic potential of canine adipose tissue-derived mesenchymal stromal cells (MSC) compared to bone marrow-derived MSC is not improved by BMP-2 or BMP-6.

Mesenchymal stromal cells (MSC) are used for cell-based treatment for canine osteoarthritis (OA). Compared with human MSCs, detailed information on the functional characterisation of canine MSCs is limited. In particular, the chondrogenic differentiation of canine adipose tissue-derived MSCs (cAT-MSCs) is challenging. In this study, we aimed to compare cAT-MSCs with bone marrow-derived MSCs (cBM-MSCs), focusing specifically on their in vitro chondrogenic potential, with or without bone morphogenetic proteins (BMP). cBM-MSCs and cAT-MSCs were characterised using flow cytometry and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The chondrogenic differentiation potential of all cMSC preparations in the presence of TGF-ß1 alone or when supplemented with 10, 100, or 250 ng/mL BMP-2 or BMP-6 was investigated using RT-qPCR, and biochemical, histochemical and immunohistological analyses. Both cBM-MSCs and cAT-MSCs expressed the surface markers CD90, CD73, and CD29, and were negative for CD45 and CD34, although the expression of CD73 and CD271 varied with donor and tissue origin. Interestingly, expression of ACAN and SOX9 was higher in cBM-MSCs than cAT-MSCs. In contrast with cBM-MSCs, cAT-MSCs could not differentiate toward the chondrogenic lineage without BMP-2/-6, and their in vitro chondrogenesis was inferior to cBM-MSCs with BMP-2/-6. Thus, cAT-MSCs have lower in vitro chondrogenic capacity than cBM-MSC under the studied culture conditions with 10, 100, or 250 ng/mL BMP-2 or BMP-6. Therefore, further characterisation is necessary to explore the potential of cAT-MSCs for cell-based OA treatments.

The mRNA expression of PTTG1 is a strong prognostic indicator for recurrence after hypophysectomy in dogs with corticotroph pituitary adenomas.

Pituitary-dependent hypercortisolism (PDH) is a common endocrinopathy in dogs, but the promotors and initiators of the tumourigenesis of corticotroph pituitary adenomas remain unknown. Based on human data, we investigated mRNA expression of pituitary tumour transforming gene 1 (PTTG1) with quantitative RT-PCR in canine corticotroph pituitary adenomas. PTTG1 was overexpressed in adenomas approximately 3-fold. A strong association was observed between PTTG1 expression and disease-free interval; dogs with high PTTG1 expression had a significantly (4 times; P=0.02) shorter disease-free interval than dogs with low PTTG1 expression. This paper shows that PTTG1 expression is a negative prognosticator in relation to disease-free interval and recurrence in dogs undergoing transsphenoidal hypophysectomy as treatment for PDH.

Soluble and pelletable factors in porcine, canine and human notochordal cell-conditioned medium: implications for IVD regeneration.

During intervertebral disc (IVD) maturation, notochordal cells (NCs) are replaced by chondrocyte-like cells (CLCs) in the nucleus pulposus, suggesting that NCs play a role in maintaining tissue health. Affirmatively, NC-conditioned medium (NCCM) exerts regenerative effects on CLC proliferation and extracellular matrix (ECM) production. The aim of this study was to identify NC-secreted substances that stimulate IVD regeneration. By mass spectrometry of porcine, canine and human NCCM, 149, 170 and 217 proteins were identified, respectively, with 66 proteins in common. Mainly ECM-related proteins were identified, but also organelle-derived and membrane-bound vesicle proteins. To determine whether the effect of NCCM was mediated by soluble and/or pelletable factors, porcine and canine NCCM were separated into a soluble (NCCM-S; peptides and proteins) and pelletable (NCCM-P; protein aggregates and extracellular vesicles) fraction by ultracentrifugation, and tested on bovine and canine CLCs in vitro, respectively. In each model, NCCM-S exerted a more pronounced anabolic effect than NCCM-P. However, glycosaminoglycan (GAG) uptake from the medium into the carrier gel prevented more definite conclusions. While the effect of porcine NCCM-P on bovine CLCs was negligible, canine NCCM-P appeared to enhance GAG and collagen type II deposition by canine CLCs. In conclusion, porcine and canine NCCM exerted their anabolic effects mainly through soluble factors, but also the pelletable NCCM factors showed moderate regenerative potential. Although the regenerative potential of NCCM-P should not be overlooked, future studies should focus on unraveling the protein-based regenerative mechanism from NCCM produced from isolated NCs, e.g. by NCCM fractionation and pathway blocking studies.

Inflammation and wound healing in cats with chronic gingivitis/stomatitis after extraction of all premolars and molars were not affected by feeding of two diets with different omega-6/omega-3 polyunsaturated fatty acid ratios.

Feline chronic gingivitis/stomatitis (FCGS) is a painful inflammatory disease in cats. Extraction of teeth, including all premolars and molars, has been shown to be the therapy of choice in cats not responding sufficiently to home care (e.g. tooth brushing) and/or medical treatment (corticosteroids and/or antibiotics). In this study, we hypothesize that a cat food with an omega-6 polyunsaturated fatty acid (ω6 PUFA) to ω3 PUFA ratio of 10:1 reduces inflammation of the FCGS and accelerates soft tissue wound healing of the gingiva after dental extractions, compared to a cat food with a ω6:ω3 PUFA ratio of 40:1. The cats were fed diets with chicken fat and fish oil as sources of fatty acids. In one diet, part of the fish oil was replaced by safflower oil, resulting in two diets with ω6:ω3 PUFA ratios of 10:1 and 40:1. This double-blinded study in two groups of seven cats revealed that dietary fatty acids influence the composition of plasma cholesteryl esters and plasma levels of inflammatory cytokines. The diet with the 10:1 ratio lowered PGD(2) , PGE(2) and LTB(4) plasma levels significantly, compared to the diet with the 40:1 ratio (p = 0.05, p = 0.04, and p = 0.02 respectively). However, feeding diets with dietary ω6:ω3 PUFA ratios of 10:1 and 40:1, given to cats with FCGS for 4 weeks after extraction of all premolars and molars, did not alter the degree of inflammation or wound healing.

Intraspecies disparity in growth rate is associated with differences in expression of local growth plate regulators.

Disparities in longitudinal growth within a species can be partly explained by endocrinological differences. We hypothesized that regulatory networks acting locally in the growth plate may also be important. We tested this hypothesis by evaluating the IGF/IGFBP expression, the vitamin D pathway, and the PTHrP-Indian hedgehog (IHH) feedback loop in rib growth plates from 10- and 21-wk-old small- (Miniature Poodles, MP) and large-breed dogs (Great Danes, GD) using immunohistochemistry and quantitative (q)PCR. The rib growth plates of GD were 1.7 times thicker compared with those of MP, with larger proliferative (in absolute terms) and larger hypertrophic (in absolute and relative terms) zones. IGF/IGFBP gene expression profiling of the growth plates revealed decreased gene expression of igfbp2, -4, and -6 and an unaltered expression of igf-I and igf-II and their respective receptors in GD vs. MP. Immunohistochemistry and qPCR findings showed that the vitamin D pathway was more active in GD than in MP. Staining for 1α- and 24-hydroxylase was more abundant and intense in GD and the gene expressions of 1α-hydroxylase and the vitamin D receptor-driven 24-hydroxylase were six- and eightfold higher in GD vs. MP, respectively. Consistent with the immunohistochemistry findings, the expression of mRNA for components of the parathyroid hormone-related peptide (PTHrP)-IHH loop was different in GD compared with MP, with there being a relative threefold downregulation of Pthrp and a tenfold upregulation of Ihh in GD vs MP. These differences suggest that the effects of IHH in the regulation of chondrocyte proliferation and hypertrophy, both independently of PTHrP, can become more dominant during rapid growth rates. In conclusion, our data suggest that, in addition to modest endocrine differences, more pronounced changes in the expression of locally acting regulatory networks, such as the IGF system, vitamin D pathway, and PTHrP-IHH feedback loop are important contributors to within-species disparities in growth rates.

L-Dopa stimulates expression of the antioxidant enzyme NAD(P)H:quinone oxidoreductase (NQO) in cultured astroglial cells.

The autooxidation of L-Dopa, a catecholamine used in the symptomatic treatment of Parkinson's disease, generally yields reactive oxygen species and neurotoxic quinones. NAD(P)H:quinone oxidoreductase (NQO) is a flavoenzyme that is implicated in the detoxication of quinones, including those formed during L-Dopa autooxidation. Through the action of this enzyme, deleterious redox-labile quinones are turned into less toxic and more stable hydroquinones that are amenable to further detoxication and/or cellular excretion. In the present study, using primary rat astrocytes and C6 astroglioma as a model to evaluate the neuroprotective response of astroglial cells upon exposure to L-Dopa, we demonstrate that this compound, or more correctly its quinone (auto)oxidation products, up-regulates astroglial NQO in a time- and concentration-dependent way as assessed at the level of mRNA expression, protein level, and enzymatic activity. Moreover, under similar conditions cellular glutathione content was enhanced. It is concluded that, similar to glutathione, the oxidative stress limiting NQO is likely to contribute to the capacity of astroglial cells to protect dopaminergic neurons against L-Dopa, and, hence, may be considered as a potential target for the development of neuroprotective strategies for Parkinson's disease.