Crenigacestat inhibits osteogenic differentiation of human valve interstitial cells and seems promising for treatment of cardiovascular calcification
ABSTRACT: Vascular calcification is common pathology various forms of which presented in the half of humans. Calcific aortic valve disease (CAVD) is one of the most dangerous forms of vascular calcification. Despite high mortality there is no target therapy against CAVD. Thus, we tested crenigacestat (LY3039478), inhibitor of Notch-signaling, and shRNA against RBPJk for inhibition of osteogenic differentiation of velve intersticial cells (VICs) in vitro. Both of them effectivelly enhibited osteogenic differentiation and we performed proteomics analysis do describe molecular mechanisms of their effect. Presented dataset contain results of shotgun proteomics analysis with ion mobility in TimsToF Pro instrument (in DDA PASEF mode) of VICs in classic osteogenic medium (OM) and OM supplemented with crenigacestat, DMSO or shCSL.
Project description:The treatment of bone defects caused by infection, trauma or neoplasms remains a clinical challenge. Autologous bone transplantation is limited by availability, donor site morbidity and surgical risk factors. This has given rise to stromal/stem-cell based therapy. Bone marrow derived stromal cells (BMSCs) have been studied to a large extent and show high regenerative potential but their use is limited by availability, donor site morbidity and the relatively low cell yield as they represent only <0.1% of cell harvested from bone marrow aspirate. At the same time, they are the closest mesenchymal stromal cells for bone tissue engineering given their tissue origin and, unlike other mesenchymal stromal cells, can support the formation of hematopoietic marrow. Adipose tissue derived stromal cells (ASCs) as part of the stromal vascular fraction of adipose tissue can as well undergo osteogenic differentiation but can be additionally isolated in a sufficient quantity from lipoaspirate after liposuction of abundant subcutaneous fat tissue. Here, it has been shown that there are no major differences in regard to proliferation or differentiation capacity of ASCs derived from subcutaneous fat of different anatomical regions. It has been shown that BMSCs are more prone to senescence during expansion and passage than ASCs and that ageing impacts proliferative capabilities of BMSCs more than that of ASCs while it has also been reported that osteogenic differentiation capacity is least impacted by age. Multiple studies have compared the characteristics of these two mesenchymal stromal cells in regard to bone tissue engineering in vitro. Most studies point to inferior extracellular matrix mineralization and lower expression of key osteogenic transcription markers like Runx2 in osteogenic differentiated ASCs compared to BMSCs. On the other hand, a study by Rath et al. found contrary results using particular culturing conditions like 3D bioglass scaffolds. An intraindividual comparison of human MSCs of three donors cultured on decellularized porcine bone confirmed superior osteogenic capacity of BMSCs compared to ASCs. In contrast to BMSCs, ASCs were not able to induce heterogenic ossification in a mouse model. In a sheep tibia defect model application of BMSCs resulted in a significantly higher amount of newly formed bone tissue. Importantly, Osteogenic differentiated ASCs do not support the formation of a hematopoietic marrow. Proteomics enables large-scale analysis of proteins present in a cell type and can be used to identify differentially regulated key proteins in a comparative approach. A comparative proteomic analysis of BMSCs and ASCs by Roche et al. in 2009 identified 556 proteins with 78% of these not being differentially regulated between these two cell populations, regarded as high similarity. Another comparative proteomic study of 2016 by Jeon et al. found 90 differentially regulated proteins out of 3000 total identified proteins. Both studies do not specify a number of different tissue donors and in part using cell lines. Looking for differences upon osteogenic differentiation, transcriptomic comparison of osteogenic differentiated porcine ASCs and BMSCs has been performed, resulting in 21 differentially expressed genes after 21 days of osteogenic culture conditions. Still, it remains unanswered, which are the key distinctive features of osteogenic differentiated ASCs and BMSCs at protein level that might help address the abovementioned weaknesses of ASCs in bone tissue engineering/regeneration for translational research. To overcome this need, an intraindividual comparative DIA based proteomic analysis of osteogenic differentiated human BMSC and ASCs was performed in this study.
Project description:we analyzed the expression level change of transcription factors in adipose derived stem cells during osteogenic differentiation and found a candidate target gene, Sox11. We defined that Sox11 suppresses osteogenic differentiation through overexpression and knock down of Sox11. total RNA obtained from adipose derived stem cells subjected to 1,3,6,10 or 14 days in osteogenic differentiation compared to undifferentiated control adipose derived stem cells.
Project description:We have previously reported that the deficiency of p53 alone or in combination with Rb (Rb-/- p53-/-) in adipose-derived MSCs (ASCs) promotes leiomyosarcoma-like tumors in vivo. Here, we hypothesized that the source of MSCs and/or the cell differentiation stage could determine the phenotype of sarcoma development. To investigate whether there is a link between the source of MSCs and sarcoma phenotype, we generated p53-/- and Rb-/-p53-/- MSCs from bone marrow (BM-MSCs). Both genotypes of BM-MSCs initiated leiomyosarcoma formation similar to p53-/- and Rb-/-p53-/- ASCs. In addition, gene expression profiling revealed a link between p53- or Rb-p53-deficient BM-MSCs and ASCs and muscle-associated sarcomagenesis. These data suggest that the tissue source of MSC does not seem a crucial factor in the development of a particular sarcoma phenotype. To analyze whether the differentiation stage defines the sarcoma phenotype, BM-MSCs and ASCs were induced to differentiate towards the osteogenic lineage, and both p53 and Rb were excised using Cre-expressing adenovectors at different stages along osteogenic differentiation. Regardless of the level of osteogenic commitment, the inactivation of Rb and p53 in BM-MSC-derived, but not in ASC-derived, osteogenic progenitors gave rise to osteosarcoma-like tumors which could be serially transplanted. This indicates that the osteogenic differentiation stage of BM-MSCs imposes the phenotype of in vivo sarcoma development, and that BM-MSC-derived osteogenic progenitors rather than undifferentiated BM-MSCs, undifferentiated ASCs or ASC-derived osteogenic progenitors, represent the cell of origin for osteosarcoma development. To analyse whether the BM-MSC and Fat-MSC (ASC) differentiation stage may define the sarcoma phenotype, RbloxP/loxPp53loxP/loxP BM-MSCs and ASCs were induced to differentiate towards the osteogenic lineage and both Rb and p53 were excised with adenoviral vectors expressing the Cre-recombinase gene (Ad-CMV-Cre) at different stages (