The Appearance and Modulation of Osteocyte Marker Expression during Calcification of Vascular Smooth Muscle Cells

Vascular calcification is an indicator of elevated cardiovascular risk. Vascular smooth muscle cells (VSMCs), the predominant cell type involved in medial vascular calcification, can undergo phenotypic transition to both osteoblastic and chondrocytic cells within a calcifying environment.In the present study, using in vitro VSMC calcification studies in conjunction with ex vivo analyses of a mouse model of medial calcification, we show that vascular calcification is also associated with the expression of osteocyte phenotype markers. As controls, the terminal differentiation of murine calvarial osteoblasts into osteocytes was induced in vitro in the presence of calcifying medium (containing ß-glycerophosphate and ascorbic acid), as determined by increased expression of the osteocyte markers DMP-1, E11 and sclerostin. Culture of murine aortic VSMCs under identical conditions confirmed that the calcification of these cells can also be induced in similar calcifying medium. Calcified VSMCs had increased alkaline phosphatase activity and PiT-1 expression, which are recognized markers of vascular calcification. Expression of DMP-1, E11 and sclerostin was up-regulated during VSMC calcification in vitro. Increased protein expression of E11, an early osteocyte marker, and sclerostin, expressed by more mature osteocytes was also observed in the calcified media of Enpp1(-/-) mouse aortic tissue.This study has demonstrated the up-regulation of key osteocytic molecules during the vascular calcification process. A fuller understanding of the functional role of osteocyte formation and specifically sclerostin and E11 expression in the vascular calcification process may identify novel potential therapeutic strategies for clinical intervention

Cross-recognition between histones and La/SSB may account for anti-DNA reactivity in SLE patients

Antibodies to La/SSB are detected in sera of patients with primary Sjogren's syndrome (pSS) and systemic lupus erythematosus (SLE). The vast majority of anti-La/SSB positive sera contain antibodies directed towards a linear B-cell epitope of La/SSB spanning the sequence 349–364aa (pep349–364). The aim of this study was to evaluate the fluctuation of antibody levels to major B-cell epitopes of La/SSB over time and investigate for their possible crossreactions. Sequential sera from 15 SLE and 15 pSS patients, followed from 3 to 10 years were obtained. All patients with SLE were positive for anti-Ro/SSA, anti-La/SSB and anti-dsDNA antibodies and patients with pSS were positive for anti-Ro/SSA and anti-La/SSB antibodies. Sera from 30 patients with SLE without anti-La/SSB antibodies and 30 healthy individuals served as disease and negative control respectivelly. All sera tested for the presence of anti-pep349–364 antibodies, using a specific ELISA. Specific anti-pep349–364 IgG was purified from sera of SLE patients and evaluated for cross reactivity against dsDNA and histones. In all SLE sera the levels of anti-pep349–364 antibodies varied in time and fluctuated in parallel with anti dsDNA antibodies. Anti-pep349–364 IgG purified from 7 SLE patients. Five out of 7 were found to react with calf thymus DNA in ELISA. All purified (7/7) anti-pep349–364 IgG preparations reacted with histone H1 and failed to produce a positive immunofluorescence pattern in Crithidia luciliae anti-dsDNA assay which lacks histones. Competative inhibition experiments demonstrated that histone H1 could inhibit completely the binding of anti-pep349–364 IgG to pep349–364 while pep349–364 inhibited by 70% the binding of anti-pep349–364 IgG to histone H1. These findings indicate that a subgroup of SLE patients possess cross-reacting anti-histone H1 antibodies and anti-pep349–364 antibodies, which can be faulty considered as anti-dsDNA reactivity in regular ELISA techniques