An integrated proteomics analysis of bone tissues in response to mechanical stimulation

Bone cells can sense physical forces and convert mechanical stimulation conditions into biochemical signals that lead to expression of mechanically sensitive genes and proteins. However, it is still poorly understood how genes and proteins in bone cells are orchestrated to respond to mechanical stimulations. In this research, we applied integrated proteomics, statistical, and network biology techniques to study proteome-level changes to bone tissue cells in response to two different conditions, normal loading and fatigue loading. We harvested ulna midshafts and isolated proteins from the control, loaded, and fatigue loaded Rats. Using a label-free liquid chromatography tandem mass spectrometry (LC-MS/MS) experimental proteomics technique, we derived a comprehensive list of 1,058 proteins that are differentially expressed among normal loading, fatigue loading, and controls. By carefully developing protein selection filters and statistical models, we were able to identify 42 proteins representing 21 Rat genes that were significantly associated with bone cells' response to quantitative changes between normal loading and fatigue loading conditions. We further applied network biology techniques by building a fatigue loading activated protein-protein interaction subnetwork involving 9 of the human-homolog counterpart of the 21 rat genes in a large connected network component. Our study shows that the combination of decreased anti-apoptotic factor, Raf1, and increased pro-apoptotic factor, PDCD8, results in significant increase in the number of apoptotic osteocytes following fatigue loading. We believe controlling osteoblast differentiation/proliferation and osteocyte apoptosis could be promising directions for developing future therapeutic solutions for related bone diseases

Proteomic differences between native and tissue-engineered tendon and ligament

Tendons and ligaments (T/Ls) play key roles in the musculoskeletal system, but they are susceptible to traumatic or age‐related rupture, leading to severe morbidity as well as increased susceptibility to degenerative joint diseases such as osteoarthritis. Tissue engineering represents an attractive therapeutic approach to treating T/L injury but it is hampered by our poor understanding of the defining characteristics of the two tissues. The present study aimed to determine differences in the proteomic profile between native T/Ls and tissue engineered (TE) T/L constructs. The canine long digital extensor tendon and anterior cruciate ligament were analyzed along with 3D TE fibrin‐based constructs created from their cells. Native tendon and ligament differed in their content of key structural proteins, with the ligament being more abundant in fibrocartilaginous proteins. 3D T/L TE constructs contained less extracellular matrix (ECM) proteins and had a greater proportion of cellular‐associated proteins than native tissue, corresponding to their low collagen and high DNA content. Constructs were able to recapitulate native T/L tissue characteristics particularly with regard to ECM proteins. However, 3D T/L TE constructs had similar ECM and cellular protein compositions indicating that cell source may not be an important factor for T/L tissue engineering

Cellular response to physical exercise: analysis of serum proteins modulation and expression profiles in circulating progenitor cells

Physical activity plays an important role against pathological degenerative conditions and metabolic diseases. In particular, it works as a modulator of the mutually exclusive osteogenic or adipogenic fates of mesenchymal stem cells through a direct action on differentiation-related gene expression. On the other hand, it has also been reported that oxidative stress generated by strenous physical efforts (e.g. marathon running) can affect cell functions. The purpose of this study was to investigate the effects induced by a half marathon in male amateur runners. In particular the investigation focused on: i) serum proteins modulation in response to the oxidative environment, ii) the modulation of circulating progenitor cells commitment, monitored in terms of gene expression; iii) progenitor cells proliferation and homeostasis, monitored through the expression levels of genes related to telomerase activity and autophagic induction, respectively; iv) the effects of soluble factors present in runners\u2019 sera on differentiation process in an in vitro cellular model. The shotgun proteomic approach applied to runners\u2019 sera confirmed the production of reactive oxygen species, counteracted by an increased production of detoxifying and scavenger proteins. Overall, the proteome modulation profile suggests a consequent positive effect of the trained condition. Gene expression analyses showed an upregulation of osteogenesis related genes in Circulating Progenitor cells (CPs) after training, in particular RUNX2 and BMPs. In addition, chondrogenesis related genes such as SOX9, COMP and COL2A1 were upregulated after the run. At the same time, the higher expression of BMP3 suggests a stimulation of CPs proliferation which justifies as well the increased expression of telomerase-related genes, TERT and TERF1. The enhanced expression of autophagyrelated genes (ATG3 and ULK1) correlates positively with the induction of MSCs differentition. Data based on an in vitro model (i.e. Bone Marrow-derived MSCs supplemented with pre- and post-run sera), suggest that intense physical exercise enhances BM-MSC potential for osteo-chondrogenic commitment at the expense of the mutually exclusive adipogenesis. The in vitro deposition of calcium salts demonstrates mineralization, i.e. complete maturation of osteoblasts promoted by soluble factors in runners\u2019 sera. In conclusion, changes induced by physical activity may be considered positive in terms of: i) oxidative stress management during oxigen reactive species production; ii) progenitor cells proliferation, under autophagy-mediated positive selection; iii) osteochondrogenic induction of CPs; iv) production of circulating soluble factors which support complete maturation of committed osteoblasts. All data seem to suggest that physical activity has positive effects on overall health

Deficiency of the bone mineralization inhibitor NPP1 protects against obesity and diabetes

The emergence of bone as an endocrine regulator has prompted a re-evaluation of the role of bone mineralization factors in the development of metabolic disease. Ectonucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) controls bone mineralization through the generation of pyrophosphate, and levels of NPP1 are elevated both in dermal fibroblast cultures and muscle of individuals with insulin resistance. We investigated the metabolic phenotype associated with impaired bone metabolism in mice lacking the gene that encodes NPP1 (Enpp1−/− mice). Enpp1−/− mice exhibited mildly improved glucose homeostasis on a normal diet but showed a pronounced resistance to obesity and insulin resistance in response to chronic high-fat feeding. Enpp1−/− mice had increased levels of the insulin-sensitizing bone-derived hormone osteocalcin but unchanged insulin signalling within osteoblasts. A fuller understanding of the pathways of NPP1 could inform the development of novel therapeutic strategies for treating insulin resistance

NOTCH Activation Promotes Valve Formation by Regulating the Endocardial Secretome.

The endocardium is a specialized endothelium that lines the inner surface of the heart. Functional studies in mice and zebrafish have established that the endocardium is a source of instructive signals for the development of cardiac structures, including the heart valves and chambers. Here, we characterized the NOTCH-dependent endocardial secretome by manipulating NOTCH activity in mouse embryonic endocardial cells (MEEC) followed by mass spectrometry-based proteomics. We profiled different sets of soluble factors whose secretion not only responds to NOTCH activation but also shows differential ligand specificity, suggesting that ligand-specific inputs may regulate the expression of secreted proteins involved in different cardiac development processes. NOTCH signaling activation correlates with a transforming growth factor-β2 (TGFβ2)-rich secretome and the delivery of paracrine signals involved in focal adhesion and extracellular matrix (ECM) deposition and remodeling. In contrast, NOTCH inhibition is accompanied by the up-regulation of specific semaphorins that may modulate cell migration. The secretome protein expression data showed a good correlation with gene profiling of RNA expression in embryonic endocardial cells. Additional characterization by in situ hybridization in mouse embryos revealed expression of various NOTCH candidate effector genes (Tgfβ2, Loxl2, Ptx3, Timp3, Fbln2, and Dcn) in heart valve endocardium and/or mesenchyme. Validating these results, mice with conditional Dll4 or Jag1 loss-of-function mutations showed gene expression alterations similar to those observed at the protein level in vitro These results provide the first description of the NOTCH-dependent endocardial secretome and validate MEEC as a tool for assaying the endocardial secretome response to a variety of stimuli and the potential use of this system for drug screening.We thank C. Martí Gómez-Aldaraví for help with graphic representation and critical reading of the manuscript, and S. Bartlett for English editing. RTC is supported by a Foundation La Caixa PhD fellowship (Ref LCF/BQ/ES15/10360023). LLZ is supported by a Ramón y Cajal postdoctoral contract (Ref: RYC-2016-20917). JLdlP is funded by grants SAF2016-78370-R, CB16/11/00399 (CIBER CV), and RD16/0011/0021 (TERCEL) from the Ministerio de Ciencia, Innovación y Universidades, and grants from the Fundación BBVA (Ref.: BIO14_298) and Fundación La Marató TV3 (Ref.: 20153431). JV is supported by grants BIO2015-67580-P and CB16/11/00277 (CIBER CV) from the Ministerio de Ciencia, Innovación y Universidades, and Carlos III Institute of Health-Fondo de Investigación Sanitaria (Grant ProteoRed-PRB3-IPT17/0019-ISCIII-SGEFI/ERDF), the Fundación La Marató TV3 (Ref. 122/C/2015) and “La Caixa” Banking Foundation (project code HR17-00247). The cost of this publication was supported in part with funds from the ERDF. The CNIC is supported by the Ministerio de Ciencia, Innovación y Universidades and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505).S

3D gelatin-chitosan hybrid hydrogels combined with human platelet lysate highly support human mesenchymal stem cell proliferation and osteogenic differentiation

Bone marrow and adipose tissue human mesenchymal stem cells were seeded in highly performing 3D gelatin–chitosan hybrid hydrogels of varying chitosan content in the presence of human platelet lysate and evaluated for their proliferation and osteogenic differentiation. Both bone marrow and adipose tissue human mesenchymal stem cells in gelatin–chitosan hybrid hydrogel 1 (chitosan content 8.1%) or gelatin–chitosan hybrid hydrogel 2 (chitosan 14.9%) showed high levels of viability (80%–90%), and their proliferation and osteogenic differentiation was significantly higher with human platelet lysate compared to fetal bovine serum, particularly in gelatin–chitosan hybrid hydrogel 1. Mineralization was detected early, after 21 days of culture, when human platelet lysate was used in the presence of osteogenic stimuli. Proteomic characterization of human platelet lysate highlighted 59 proteins mainly involved in functions related to cell adhesion, cellular repairing mechanisms, and regulation of cell differentiation. In conclusion, the combination of our gelatin–chitosan hybrid hydrogels with hPL represents a promising strategy for bone regenerative medicine using human mesenchymal stem cells

Mechanobiology of Stem Cells: Implications for Vascular Tissue Engineering

Current challenges in vascular medicine (e.g., bypass grafting, stenting, and angioplasty.) have driven the field of vascular regenerative medicine. Bone marrow-derived mesenchymal stem cells (BMMSCs) are adult stem cells which may be a suitable cell source for vascular regenerative medicine applications. While it is well known that BMMSCs readily differentiate into musculoskeletal cells, recent studies have provided evidence for their differentiation into smooth muscle cells (SMCs) and endothelial cells (ECs). We and others have demonstrated the ability of the mechanical stimulus of cyclic stretch to drive BMMSC differentiation towards SMCs in vitro, but a rigorous, systematic analysis of other relevant forces is lacking. The working hypothesis that this work addressed is that mechanical stimuli relevant to the vasculature will guide BMMSC differentiation towards SMCs and ECs. To test this hypothesis, rat BMMSCs were exposed to physiologically relevant magnitudes and frequencies of a Mechanical Panel, which consisted of cyclic stretch, cyclic pressure, and shear stress, each applied in parallel to subcultures of BMMSCs. Quantitative changes in morphology, proliferation, and gene and protein expression were assessed to determine the differential effect of each stimulus in a dose- and frequency-dependant manner. Next, we investigated the importance of the duration of applied stimulation to BMMSC differentiation as well as tissue commitment (i.e., cell plasticity) following mechanical stimulation.Our results demonstrate that mechanical stimulation differentially altered BMMSC morphology, proliferation, and gene and protein expression towards the cardiovascular lineage while limiting expression for other lineages including bone, fat, and chondrocyte. This was particularly evident for cyclic stretch, which caused an elongated, spindle-shape and expression of the SMC proteins alpha-actin, calponin, and myosin heavy chain. Furthermore, we found that cyclic pressure and shear stress tended to increase endothelial gene expression when these stimuli are applied to confluent BMMSCs. While our findings as a whole tended to support our hypothesis, our data indicate that SMC protein expression is more readily increased by mechanical stimulation, and is highly variable, even without associated changes in gene expression. Future work employing systems biology approaches that take into consideration the resulting transcriptional and proteomic changes in BMMSCs from these mechanical stimuli will be necessary to more accurately identify how mechanical stimulation can be used as a tool for regenerative medicine