Original Article
Biomedical
Effect of Hydroxytyrosol on Human Mesenchymal Stromal/Stem Cell Differentiation into Adipocytes and Osteoblasts

https://doi.org/10.1016/j.arcmed.2016.06.006Get rights and content

Background and Aims

Natural phenolic compounds are known for their antioxidant capacity, showing biological activity in numerous physiological processes. Such chemicals have been proposed for prevention or treatment of pathologies like osteoporosis and diabetes. One of these is hydroxytyrosol (HT), which may be involved in the differentiation of human mesenchymal stromal/stem cells (MSCs), which are precursors of osteoblasts and adipocytes. Yet, little information is available. Therefore, our objective was to study the possible effect of HT on MSC differentiation.

Methods

Differentiation markers were analyzed while human bone marrow MSCs were differentiated into osteoblasts or adipocytes in the presence of 1 or 100 μmol HT.

Results

High HT concentrations repressed the expression of osteoblastic markers in MSCs differentiating into osteoblasts, whereas they increased the expression of adipogenic genes and the formation of fat vesicles in MSCs differentiating into adipocytes.

Conclusions

High HT concentrations may inhibit osteoblastogenesis and promote adipogenesis, which can lead to bone loss. Therefore, the possible pharmacological use of extracts rich in HT should take into account this undesirable effect.

Introduction

Hydroxytyrosol (HT) or 4-(2-hydroxyethyl)-1,2-benzenediol is a phenolic compound present in olive trees (Olea europaea), mainly in their leaves and fruits. It is also present in olive tree products like olive oil (1). It has been obtained from olive tree leaf extract (2) and two-phase pomace called “alperujo” (paste-like waste), generated by the two-phase method of olive oil extraction mainly used in Spain 3, 4.

The antioxidant activity of HT is even higher than that of vitamin E and butylated hydroxytoluene (BHT) as demonstrated by both in vitro and in vivo studies 1, 5. Indeed, HT has a high biological activity (6) with potent antigenotoxic effects against hydrogen peroxide-induced damage in Drosophila melanogaster. HT inhibits the oxidation of low-density lipoproteins (LDL) (7), inducing caspase-3-dependent apoptosis in HL60 cells (8). Therefore, it has been considered a potential molecule for the treatment of cardiovascular diseases, cancer and human immunodeficiency virus (HIV) infection or acquired immune deficiency syndrome (AIDS) (9).

Osteoporosis is a disorder representing a serious public health issue, being characterized by loss of both bone mass and strength, leading to fragility fractures. The fundamental pathogenic mechanisms underlying this disease include excessive osteoclastic bone resorption and defects in osteoblastic bone formation (10). Increased levels of osteoporosis have been observed in aging bone. Some authors have suggested that it could be due, at least in part, to a disorder in mesenchymal cell differentiation into adipocytes instead of osteoblast-osteocyte cells (11). This was also supported by the progressive accumulation of adipose tissue within the bone marrow of osteoporotic aged bones (12).

All these bone metabolic processes could be modulated by several phytochemicals as we have demonstrated (13), probably through a higher scavenging activity of free radicals involved in osteoclastogenesis and bone resorption (14). In the same way, phytochemicals may promote a particular mitochondrial pro-adipocyte function. Thus, they may stimulate adipocyte differentiation while decreasing free fatty acids within the adipocytes (15).

Mesenchymal stromal/stem stem cells (MSCs) are defined as a type of multipotent cells that can differentiate into a variety of cell types including adipocytes, myocytes, chondrocytes or osteoblasts. They can be isolated from different sources such as bone marrow and adipose tissue and can be cryopreserved to be used for research applications as we have described (16).

The most commonly used medications for treatment of osteoporosis are antiresorptive agents, which act by inhibiting osteoclast-mediated bone resorption, whereas anabolic agents can directly stimulate osteoblastic formation of new bone. Teriparatide (TPT), the only anabolic agent approved to treat osteoporosis, corresponds to the recombinant 34 N-terminal amino acids of the 84 amino acid human parathyroid hormone (hPTH) (17). The intermittent TPT administration increases the number and activity of osteoblasts by inducing the differentiation of bone marrow MSCs and preventing osteoblast apoptosis (18). Nevertheless, TPT treatments are approved by the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) only for a limited duration, ranging from 18–24 months (17). This circumstance makes it necessary to find other alternatives that may allow longer bone anabolic treatments. In this study, MSCs have been used as an in vitro human model to assess the osteoblastogenic and adipogenic potential effects of HT in humans.

Section snippets

Donor Selection and Bone Marrow Processing

Volunteer donors were recruited by the Bone Marrow Transplant Program of the Hematology Service at the Reina Sofía University Hospital (Córdoba, Spain). All bone marrow donors gave written informed consent. Human MSCs were isolated from bone marrow, cryopreserved and characterized as positive for mesenchymal (CD73, CD90 and CD105) and negative for hematopoietic (CD14, CD34 and CD45) markers following a methodology that we previously optimized (16). Bone MSCs were thawed and cultured in flasks

Cell Number

Cells in culture induced into osteoblast decreased in the presence of 100 μmol HT at days 7 and 14 and with 1 μmol HT at day 14 (Figure 1A). On the contrary, HT produced no change in cell number of cultures induced to differentiate into adipocytes (Figure 1B).

HT Effect on Osteoblastic Markers

ALPL activity, mineralization and expression of osteoblastic gene markers were analyzed in MSC induced to differentiate into osteoblast. Such activity decreased with both 1 and 100 μmol HT at day 7 (Figure 2A). Additionally, 100 μmol HT

Discussion

Medicinal uses of naturally derived phenolic substances are being increasingly considered due to their beneficial effects in the prevention of several diseases like osteoporosis, diabetes and cancer 13, 20, 21. Our results show that a high concentration of HT (100 μmol) decreased the expression of some osteoblastic markers during MSC differentiation. These results partially agree with those previously reported (22), showing that olive tree polyphenols prevent bone mass loss at similar

Acknowledgments

Supported by grants P06-FQM-01515, PI-00200/2009 and “Ayuda a la Intensificación de la Investigación” (Junta de Andalucía, Spain). We thank Sebastian Demyda for his helpful comments.

Conflicts of interest: The authors declare no conflicts of interest in this manuscript.

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