Vitamin MK-7 enhances vitamin D3-induced osteogenesis in hMSCs: modulation of key effectors in mineralization and vascularization.

The osteoblast is the bone-forming cell and is derived from mesenchymal stem cells (MSCs). Osteo-inductive substances could represent a useful therapeutic approach during the fracture repair process. The aim of this work was to evaluate the effects of vitamin MK-7, alone or in association with vitamin D3, in differentiating human MSCs (hMSCs) in vitro along the osteoblastic lineage. In particular, primary endpoints of the study include gene and protein markers of osteoblast differentiation. Considering genes involved in bone formation and mineralization, our data show that vitamin MK-7 enhances vitamin D3 gene induction of osteocalcin (OC). Among genes related to cell growth and differentiation, a specific effect of vitamin MK-7 was observed for growth differentiation factor-10 (GDF10) and insulin-like growth factor 1 (IGF1), the latter being also involved in the induction of vascular endothelial growth factors (VEGFA). Accordingly, vitamin co-supplementation greatly affected VEGFA and its receptor fms-related tyrosine kinase 1 (FLT1), a key factor in both angiogenic and osteogenic processes. These results stress the relevance of MK-7 and D3 co-supplementation in the bone-healing process as able to modulate the expression of genes involved in both mineralization and angiogenesis. Moreover, at the protein level co-association of vitamins might provide an optimal balance between induction and carboxylation of osteocalcin, essential for its functionality in the extracellular matrix (ECM). Our results may provide hints for therapeutic application of hMSCs in bone disease, clarifying mechanisms involved in stem cell-mediated bone development, and they also highlight the relevance of co-supplementation strategies, since single supplementations might result in a suboptimal effect.

Reference gene validation for qPCR on normoxia- and hypoxia-cultured human dermal fibroblasts exposed to UVA: is ß-actin a reliable normalizer for photoaging studies?

Data normalization of gene expression on human dermal fibroblasts (HDF) exposed to UVA has commonly been done using either GAPDH or ß-actin as reference genes without any validation of their expression stability. Since this aspect, important for accurate normalization, has been overlooked, we aimed to establish a suitable set of reference genes for studies on UVA-treated HDF cultured under both standard atmospheric oxygen tension (normoxia, 21%) and under a physiological, low oxygen tension for these cells (hypoxia, 5%). The stability of six commonly used reference genes was assessed using the geNorm and NormFinder softwares subsequent to reverse-transcription quantitative real-time PCR (RT-qPCR). GAPDH/SDHA were found to be the most stable genes under normoxia, while SDHA/TBP or HPRT1/ß2M were the most stable ones under hypoxia in HDF exposed to 18 J/cm(2) UVA. ß-Actin was always the most unstable reference gene. To emphasize the importance of selecting the most stably expressed reference genes for obtaining reliable results, mRNA expression levels of MMP-1 and COL1A1 were analyzed vs the best reference genes and the worst one. These reference genes are hence recommended for future qPCR analyses in studies concerning photo-damage on UVA-treated HDF.

Effect of Coenzyme Q10 in mitigating oxidative DNA damage in Down syndrome patients, a double blind randomized controlled trial.

Down syndrome (DS) is a chromosomal abnormality (trisomy 21) associated with a complex phenotype. Oxidative stress is known to play a major role in this pathology both due to genetic and epigenetic factors, suggesting that oxidative imbalance contributes to the clinical manifestation of DS. In particular, the implications of oxidative DNA damage in Down syndrome has been linked with neurodegeneration. Here we report the results of a double blind controlled trial aimed at investigating the protective effect of Coenzyme Q(10) on DNA oxidation in this clinical setting using the single cell gel electrophoresis technique.

Coenzyme Q10 and oxidative imbalance in Down syndrome: biochemical and clinical aspects.

Down syndrome (DS) is a chromosomal abnormality (trisomy 21) associated with mental retardation and Alzheimer-like dementia, characteristic change of the individual's phenotype and premature ageing. Oxidative stress is known to play a major role in this pathology since a gene dose effect leads to elevated ratio of superoxide dismutase to catalase/glutathione peroxidase compared to controls in all age categories suggesting that oxidative imbalance contributes to the clinical manifestation of DS. Hyperuricemia is another feature of DS that has an interesting relationship with oxidative stress since uric acid represents an important free radical scavenger. However its formation is connected to the conversion of Xanthine dehydrogenase (XDH) to Xanthine oxidase (XO) which leads to concomitant production of free radicals. Here we report that plasma samples from DS patients in pediatric age, despite an increased total antioxidant capacity, largely due to elevated Uric acid content (UA), present significantly elevated markers of oxidative damage such as increased allantoin levels. Moreover DS plasma samples do not differ from healthy control ones in terms of Coenzyme Q10 and susceptibility to peroxidative stimuli. On the contrary, lymphocyte and platelet CoQ10 content was significantly lower in DS patients, a fact that might underlie oxidative imbalance at a cellular level.

Effect of UV-C mediated oxidative stress in leukemia cell lines and its relation to ubiquinone content.

UV-C radiation is able to impair cellular functions by directly damaging DNA, and by inducing an increased formation of reactive oxygen species that leads to a condition of oxidative stress. In this study we evaluated different responses to UV insult of two leukemia cell lines, HL-60 and Raji, and the relationship with their CoQ10 content. DNA damage was monitored by means of the alkaline single cell gel electrophoresis (Comet assay); intracellular levels of ROS, mitochondrial depolarization and cell viability was measured by flow cytometry. Raji cells appeared more resistant to the UV insult; moreover, they did not show any increase in ROS content and the extent of mitochondrial depolarisation was much lower than in HL 60 cell line. Raji cells also contained significantly higher levels of CoQ10 and their ability to incorporate and to reduce exogenous CoQ10 added to the culture medium was remarkably elevated compared with HL 60.