Experimental study protocol of the project "MOtor function and VItamin D: Toolkit for motor performance and risk Assessment (MOVIDA)".

Musculoskeletal injuries, a public health priority also in the military context, are ascribed to several risk factors, including: increased reaction forces; low/reduced muscle strength, endurance, body mass, Vitamin D level, and bone density; inadequate lifestyles and environment. The MOVIDA Project-funded by the Italian Ministry of Defence-aims at developing a transportable toolkit (assessment instrumentation, assessment protocols and reference/risk thresholds) which integrates motor function assessment with biological, environmental and behavioural factors to help characterizing the risk of stress fracture, stress injury or muscle fatigue due to mechanical overload. The MOVIDA study has been designed following the STROBE guidelines for observational cross-sectional studies addressing healthy adults, both militaries and civilians, with varying levels of physical fitness (sedentary people, recreational athletes, and competitive athletes). The protocol of the study has been designed and validated and is hereby reported. It allows to collect and analyse anamnestic, diagnostic and lifestyle-related data, environmental parameters, and functional parameters measured through portable and wearable instrumentation during adapted 6 minutes walking test. The t-test, one and two-way ANOVA with post-hoc corrections, and ANCOVA tests will be used to investigate relevant differences among the groups with respect to biomechanical parameters; non-parametric statistics will be rather used for non-normal continuous variables and for quantitative discrete variables. Generalized linear models will be used to account for risk and confounding factors.

Aspirin-Dependent Effects on Purinergic P2Y1 Receptor Expression.

Chronic treatment with aspirin in healthy volunteers (HVs) is associated with recovery of adenosine diphosphate (ADP)-induced platelet activation. The purinergic P2Y1 receptor exerts its effects via a Gq-protein, which is the same biochemical pathway activated by thromboxane-A2 receptor. We hypothesized that recovery of ADP-induced platelet activation could be attributed to increased P2Y1 expression induced by chronic aspirin exposure. We performed a multi-phase investigation which embraced both in vitro and in vivo experiments conducted in (1) human megakaryoblastic DAMI cells, (2) human megakaryocytic progenitor cell cultures, (3) platelets obtained from HVs treated with aspirin and (4) platelets obtained from aspirin-treated patients. DAMI cells treated with aspirin or WY14643 (PPARα agonist) had a significant up-regulation of P2Y1 mRNA, which was shown to be a PPARα-dependent process. In human megakaryocytic progenitors, in the presence of aspirin or WY14643, P2Y1 mRNA expression was higher than in mock culture. P2Y1 expression increased in platelets obtained from HVs treated with aspirin for 8 weeks. Platelets obtained from patients who were on aspirin for more than 2 months had increased P2Y1 expression and ADP-induced aggregation compared with patients on aspirin treatment for less than a month. Overall, our results suggest that aspirin induces genomic changes in megakaryocytes leading to P2Y1 up-regulation and that PPARα is the nuclear receptor involved in this regulation. Since P2Y1 is coupled to the same Gq-protein of thromboxane-A2 receptor, platelet adaptation in response to pharmacological inhibition seems not to be receptor specific, but may involve other receptors with the same biochemical pathway.

MiR-21 role in aspirin-dependent PPARα and multidrug resistance protein 4 upregulation.

BACKGROUND: A mechanism involved in high on-aspirin treatment residual platelet reactivity is platelet multidrug resistance protein 4 (MRP4) overexpression. Aspirin enhances platelet MRP4 expression with a PPARα-dependent mechanism and reduces miR-21 expression that, in turn, downregulates PPARα expression. OBJECTIVE: The aim of our study was to verify the relationship between miR-21 and MRP4-PPARα levels induced by aspirin treatment. METHODS: We evaluated the changes in MRP4-PPARα, mRNA, MRP4 protein, and miR-21 expression induced by aspirin in: (i) in vitro-treated megakaryoblastic cell line (DAMI), (ii) primary megakaryocytes cultures and derived platelets, (iii) healthy volunteers' platelets treated with aspirin, and (iv) aspirinated patients (aspirin-treated patients) and in a control population (control). RESULTS: We observed an aspirin-induced reverse relationship between the expression of miR-21 and PPARα-MRP4. In DAMI cells the miR-21 mimic transfection reduces PPARα and MRP4 expression, even if cells were treated with aspirin after transfection. MiR-21 inhibitor transfection induces PPARα and MRP4 expression that are not enhanced by aspirin treatment. In human megakaryocytes, aspirin treatment lead to a miR-21 downregulation and a MRP4 upregulation and this trend is confirmed in derived platelets. In aspirin-treated volunteers, an inverse relationship between miR-21 and MRP4 platelet expression was found after aspirin treatment. A similar negative relationship was found in aspirin-treated patients vs the control population. CONCLUSION: The results reported in this study provide information that aspirin induces the modulation of platelet miR-21 expression levels and this modulation can be responsible for MRP4 enhancement in circulating platelets.

From Human Megakaryocytes to Platelets: Effects of Aspirin on High-Mobility Group Box 1/Receptor for Advanced Glycation End Products Axis.

Platelets (PLTs) are the major source of high-mobility group box 1 (HMGB1), a protein that is involved in sterile inflammation of blood vessels and thrombosis. Megakaryocytes (MKs) synthesize HMGB1 and transfer both protein and mRNA into PLTs and PLT-derived microvesicles (MV). Free HMGB1 found in supernatants of in vitro differentiated MKs and in a megakaryoblastic cell line (DAMI cells). Aspirin "in vivo" and "in vitro" not only reduces HMGB1 and receptor for advanced glycation end products expression on MKs and PLTs but also drives the movement of HMGB1 from MKs into PLTs and PLT-derived MV. These findings suggest that consumption of low doses of aspirin reduces the risk of atherosclerosis complications as well as reducing PLT aggregation by the inhibition of COX-1.

Aspirin influences megakaryocytic gene expression leading to up-regulation of multidrug resistance protein-4 in human platelets.

AIM: The aim of the study was to investigate whether human megakaryocytic cells have an adaptive response to aspirin treatment, leading to an enhancement of multidrug resistance protein-4 (MRP4) expression in circulating platelets responsible for a reduced aspirin action. We recently found that platelet MRP4 overexpression has a role in reducing aspirin action in patients after by-pass surgery. Aspirin enhances MRP4-mRNA levels in rat liver and drug administration transcriptionally regulates MRP4 gene expression through peroxisome proliferator-activated receptor-α (PPARα). METHODS: The effects induced by aspirin or PPARα agonist (WY14643) on MRP4 modulation were evaluated in vitro in a human megakaryoblastic DAMI cell line, in megakaryocytes (MKs) and in platelets obtained from human haematopoietic progenitor cell (HPC) cultures, and in vivo platelets obtained from aspirin treated healthy volunteers (HV). RESULTS: In DAMI cells, aspirin and WY14643 treatment induced a significant increase in MRP4 and PPARα expression. In human MKs grown in the presence of either aspirin or WY14643, MRP4 and PPARα-mRNA were higher than in control cultures and derived platelets showed an enhancement in MRP4 protein expression. The ability of aspirin to modulate MRP4 expression in MKs and to transfer it to platelets was also confirmed in vivo. In fact, we found the highest MRP4 mRNA and protein expression in platelets obtained from HV after 15 days' aspirin treatment. CONCLUSIONS: The present study provides evidence, for the first time, that aspirin treatment affects the platelet protein pattern through MK genomic modulation. This work represents an innovative and attractive approach, useful both to identify patients less sensitive to aspirin and to improve pharmacological treatment in cardiovascular high-risk patients.

Autocrine role of angiopoietins during megakaryocytic differentiation.

The tyrosine kinase Tie-2 and its ligands Angiopoietins (Angs) transduce critical signals for angiogenesis in endothelial cells. This receptor and Ang-1 are coexpressed in hematopoietic stem cells and in a subset of megakaryocytes, though a possible role of angiopoietins in megakaryocytic differentiation/proliferation remains to be demonstrated. To investigate a possible effect of Ang-1/Ang-2 on megakaryocytic proliferation/differentiation we have used both normal CD34(+) cells induced to megakaryocytic differentiation and the UT7 cells engineered to express the thrombopoietin receptor (TPOR, also known as c-mpl, UT7/mpl). Our results indicate that Ang-1/Ang-2 may have a role in megakaryopoiesis. Particularly, Ang-2 is predominantly produced and released by immature normal megakaryocytic cells and by undifferentiated UT7/mpl cells and slightly stimulated TPO-induced cell proliferation. Ang-1 production is markedly induced during megakaryocytic differentiation/maturation and potentiated TPO-driven megakaryocytic differentiation. Blocking endogenously released angiopoietins partially inhibited megakaryocytic differentiation, particularly for that concerns the process of polyploidization. According to these data it is suggested that an autocrine angiopoietin/Tie-2 loop controls megakaryocytic proliferation and differentiation.