Effect of Atorvastatin on OPG/RANK/RANKL Expression in Immune Cells and Circulating Levels of Osteoprogenitor Cells

Background: A considerable number of clinical studies have shown that osteoporosis carries a significant increase in cardiovascular risk. Although the pathophysiological substrate of the so-called bone-vascular axis is still elusive, an important role is attributed to the OPG/RANK/RANKL triad, a master regulator of bone remodeling. In addition, circulating osteoprogenitors have been recently described as a new subset of immature cells harbouring pro-calcific potential in the vasculature and heart valves. Nevertheless, a number of studies indicated that the accumulation of lipid oxidation products within the skeleton may contribute to pathological bone resorption, mainly through the inhibition of osteoblast differentiation and the induction of osteoclast maturation/activation. Starting from these notions, we sought to investigate whether treatment with Atorvastatin can affect circulating levels of osteo-progenitor cells and OPG/RANK/RANKL expression in T cells and monocytes in hypercholesterolemic postmenopausal osteoporotic women.

Methods: We will enrol 20 consecutive hypercholesterolemic (LDL-C ≥130 mg/dL) women with newly diagnosed osteoporosis who refer to the Osteoporosis and Bone Metabolism Unit of the Cà Foncello Hospital in Treviso. Diagnosis of osteoporosis was based on T-score ≤2.5 SD at either the lumbar spine or femoral neck. All patients will receive Atorvastatin 40 mg/day for 3 months. Blood samplings will be performed at the time of enrolment and at the end of the treatment period. During the study, the patients will not be treated with calcium, vitamin D, and bisphosphonates. We will exclude women with clinical judgment of high risk of bone fracture. At the beginning of the study and after 3 months, serum samples will be collected to assess the lipid profile (total cholesterol [TC], LDL-C, HDL-C, triglycerides [TG]), level of hs-CRP, osteocalcin (OCN), bone alkaline phosphatase (BAP), cross-linked carboxy-terminal telopeptide of type I collagen (CTX-I), and OPG. Blood samples at enrolment and after three months will be also obtained to quantify circulating osteoprogenitor cells (flow cytometry) and gene expression of OPG/RANK/RANKL in mononuclear cells (RT-PCR).

Flow cytometry analysis (FACS): identification and quantification of circulating osteoprogenitor cells will be performed using polychromatic flow cytometry. Briefly, after red blood cell lysis, peripheral blood progenitor cells will be analysed for the surface expression of CD34, OCN and BAP using Fitc-conjugated anti-human CD34, PE-conjugated anti-human OCN, and APC-conjugated anti-human BAP.

Gene expression analysis: pure and viable monocytes and T cells will be obtained from blood samples by negative isolation using Dynabeads Untouched Human Monocytes and Dynabeads Untouched Human T cells respectively. Total RNA from both cell types will be collected using and stored at -80°Cuntil analysis. The levels of RANK, RANKL, and OPG transcripts will be then quantified by real-time PCR .