Patients with inflammatory rheumatic diseases die prematurely, largely due to cardiovascular (CV) disease. Atherosclerosis, the main determinant of CV morbidity and mortality, occurs prematurely in Rheumatoid Arthritis (RA) patients [1]. These patients have almost a two-fold increase in cardiovascular events in comparison with the general population, contributing to about 40% of the reasons of death in these patients [2, 3]. This seems to be an effect of the disease per se, reinforced by an increased prevalence of some CV risk factors [3-5].

Atherosclerosis is an inflammatory disease characterized by the accumulation of lipid in infiltrated macrophages within the arterial wall causing it to thicken [6]. Endothelial dysfunction, intimal edema, foam cell formation and migration of leukocytes and macrophages compose the pathogenesis of atherosclerosis, culminating in the rupture of a plaque with subsequent thrombus formation [7]. Vascular calcification, a cell-controlled process in which mineral is ectopically deposited in blood vessels, is a surrogate marker for atherosclerosis and increased CV risk. RA patients have an increased prevalence of vascular calcification [8].

There are several common features between bone metabolism and atherosclerosis, such as the RANK/RANKL/OPG system, the Wnt signaling pathway or pro-inflammatory cytokines, such as interleukin (IL)-6 [9-11].

Given the current evidence we hypothesize that in RA, bone and vascular disturbances share common pathophysiological mechanisms and that a connection between the progression of both conditions exists.

Thus, the main goal of this study is to test the association between atherosclerosis progression and bone disturbances in the context of RA and the effects of cytokines blockage on subclinical atherosclerosis progression and bone disturbances.

To address our objectives we will test the association between subclinical atherosclerosis progression and bone metabolical, structural and mechanical disturbances in an animal model of arthritis and atherosclerosis. Further we will test the effect of cytokines blockage on the progression of both conditions. Additionally, we intend to evaluate the expression of inflammation related genes and of bone related genes expressed in human atherosclerotic plaques and its correlation with serological markers of inflammation and of bone turnover.

A mouse model of arthritis and atherosclerosis will be produced by giving a fat diet to the K/BxAg7 mice, as described by Rose S. and colleagues [12]. The K/BxAg7 mice model is a model of arthritis that develop a severe inflammatory arthritis, and express the transgenic T cell receptor (TCR) KRN and the MHC class II allele Ag7. ApoE-/- (a mouse model of spontaneous atherosclerosis) feed with fat diet, and K/Bx Ag7 mice feed with chaw diet and C57BL/6J mice fedd with fat diet will be used as control groups. We will use 10 mice (two groups of 5) from each strain. Mice will start the fat diet at 10 weeks of age and will be euthanized at several time-points (2, 4, 5, 6 and 7 months) in order to follow the development of both diseases. A blood sample will be immediately collected for seric biomarkers analysis. Aortas will be removed for atherosclerotic lesion quantification and also for conventional histology. We will also use the aortas for RNA extraction and gene expression analysis. Bones will be removed for mechanical and structural testing and RNA extraction.

A convenient sample constituted by all consecutive patients that will be submitted to endarterectomy at the vascular surgery departments of Hospital de Santa Maria (HSM) during two years will be studied and we expect to recruit 80 patients. All surgeries will be performed with standard surgical techniques and minimal manipulation of the specimen. The study will be conducted in accordance with regulations concerning clinical trials such as the Declaration of Helsinki as amended in Edinburgh (2000) and approved by HSM and HGO Ethics Committees. All patients will sign an informed consent. We will obtain the carotid atherosclerotic plaques to perform gene expression analysis, histology and immunohistochemistry analysis. We will also obtain a blood sample from each patient for serum biomarkers analysis. Patients will also perform a dual x-ray absorptiometry (DXA) before the surgery.
The identification of common mechanisms between atherosclerosis progression and bone disturbances adds to the knowledge of the pathophysiology of these diseases. Thus, the present study will contribute to the identification of potential targets for the development of new preventive and/or therapeutical strategies aiming at a better control of these two, often associated, pathologies.

References

1. Santos, M.J. and J.E. Fonseca, Metabolic syndrome, inflammation and atherosclerosis – the role of adipokines in health and in systemic inflammatory rheumatic diseases. Acta Reumatol Port, 2009. 34(4): 590-8.

2. Sidiropoulos, P.I., S.A. Karvounaris, and D.T. Boumpas, Metabolic syndrome in rheumatic diseases: epidemiology, pathophysiology, and clinical implications. Arthritis Res Ther, 2008. 10(3): 207.

3. Santos, M.J., et al., Cardiovascular risk profile in systemic lupus erythematosus and rheumatoid arthritis: a comparative study of female patients. Acta Reumatol Port, 2010. 35(3): 325-32.

4. Santos, M.J., et al., Body composition phenotypes in systemic lupus erythematosus and rheumatoid arthritis: a comparative study of Caucasian female patients. Clin Exp Rheumatol, 2011. 29.

5. Symmons, D.P. and S.E. Gabriel, Epidemiology of CVD in rheumatic disease, with a focus on RA and SLE. Nat Rev Rheumatol, 2011.

6. Hoekstra, M., et al., The peripheral blood mononuclear cell microRNA signature of coronary artery disease. Biochem Biophys Res Commun, 2010. 394(3): 792-7.

7. Hofbauer, L.C., et al., Vascular calcification and osteoporosis–from clinical observation towards molecular understanding. Osteoporos Int, 2007. 18(3): 251-9.

8. Wang, S., et al., Prevalence and extent of calcification over aorta, coronary and carotid arteries in patients with rheumatoid arthritis. J Intern Med, 2009. 266(5): 445-52.

9. Fonseca, J.E., et al., Interleukin-6 as a key player in systemic inflammation and joint destruction. Autoimmun Rev, 2009. 8(7): 538-42.

10. Persy, V. and P. D’Haese, Vascular calcification and bone disease: the calcification paradox. Trends Mol Med, 2009. 15(9): 405-16.

11. Cannata-Andia, J.B., P. Roman-Garcia, and K. Hruska, The connections between vascular calcification and bone health. Nephrol Dial Transplant, 2011. 26(11): 3429-36.

12. Rose, S., et al., A novel mouse model that develops spontaneous arthritis and is predisposed towards atherosclerosis. Ann Rheum Dis, 2013. 72(1): 89-95.

Diana Fernandes
Estudante de Doutoramento