The Expression Of Serum Non-phosphorylated Matrix Gla Protein And Its Correlation With Bone Remodeling In Ankylosing Spondylitis

Background Ankylosing spondylitis (AS) is a chronic inflammatory disease with enthesitis and syndesmophytosis as pathological hallmarks,which leads to progressive stiffening of the spine and functional impairment. In many patients, the pain is relieved by treatment with NSAIDs, and most NSAIDs-resistant patients respond to anti-tumor necrosis factor (TNF) therapy.Unfortunately, TNF blockade in NSAIDs-resistant patients does not seem to arrest bone erosion or syndesmophyte formation, even if there is symptomatic relief. It is hypothesised but not yet proven that early treatment of AS may prevent the development of ankylosis.Diagnosing AS is a clinically driven process based on the observation of clinical signs and structural changes on X-rays. The modified New York criteria for AS are widespread and have good sensitivity and specificity but It takes several years before these structural changes are visible on conventional X-rays. Recent evolution in the understanding of AS and SpA emphasise the need for biomarkers that facilitate understanding of the underlying pathophysiological processes, diagnostic ascertainment prediction of prognosis, assessment of burden of disease and, last but not least, prediction of treatment response Biomarkers for the stage of disease may also be of interest, especially since there is still a mean delay of 7 years between the first symptoms and diagnosis. A combination of markers for diagnosis and stage of disease may support the goals of early treatment.Matrix Gla-protein (MGP) is an 84-amino-acid (approximately 12 kDa) protein that contains five y-carboxyglutamic acid (Gla) residues. The function of Gla residues is to bind calcium ions or calcium crystals. In addition, it contains phosphorylated serines that may regulate its activity. MGP is mainly secreted by mature hypertrophic chondrocytes and vascular smooth muscle cells (VSMCs). it is unknown yet to which extent each tissue contributes to the serum levels of MGP. MGP has been proved to be an extracellular inhibitor of cardiovascular calcification and endochondral ossification. MGP may exist as various species according to their state of phosphorylation and/or carboxylation (phosphorylated [pMGP],non-phosphorylated [desphospho, dpMGP], carboxylated[cMGP] or uncarboxylated [ucMGP]).so, in the circulation different MGP species may be detected. the affinity of these species for calcium salts and their inhibitory activity may differ widely. These various circulating MGP species may reflect the degree of calcification or heterotopic ossification (inhibition) in lesion like the vascular wall or cartilage, and since MGP carboxylation is vitamin K-dependent,certain species may also reflect the availability of vitamin K present in lesion. It has been proved that ucMGP accumulate in sites of arterial calcification.So far,we have not encountered a previous study in which serum MGP levels were investigated for patients with AS on large samples.A research carried out in arthritis patients(five patients with AS) reported lower ucMGP levels,but without statistical significance. Because the form of syndesmophyte in way of endochondral ossification is the main hallmark of ossification for AS. Former researches about MGP had revealed its negative regulation of ectopic calcification and endochondral ossification. The mechanism of it’s role can be summed up as follows:1. Binding calcium ions to clear excess calcium into the circulation.2. Binding calcium crystals to inhibite crystal growth.3. Negative regulation of osteoinductive activity of bone morphogenetic protein-2 (BMP-2).4.Coordinated levels of MGP are required for chondrocyte differentiation and matrix mineralization during endochondral ossification. We hypothesize that MGP may be involved in the process of bone remodeling,in AS.Therefore,we aimed to investigate the serum dpMGP levels and its correlation with radiographic damage, bone remodelling and inflammation of AS.Objective To evaluate serum dpMGP level in patients with ankylosing spondylitis(AS) and its diagnostic utility, to investigate its relation to inflammation and bone remodelling process in AS.Methods1.SubjectsA total of 81 patiens with AS and 75 heathy individuals were enrolled in this cross-sectional study. Patients with AS were selected from Zhujiang Hospital,Guangzhoul,China. Controls were healthy individuals matched for age and sex.All patients had an established diagnosis of AS according to the modified New York criteria.All patients were selected independently of disease activity status.These patients and controls had no history of cardiovascular, chronic kidney, or rheumatic diseases and did not use VKS or VKA. The study was approved by the institutional ethics committee of Southern medical university,China. we obtained informed consent from all of the patients and healthy volunteers.2.Clinical assessmentAge, sex, medication history, BMI, duration of disease of AS were recorded.The disease activity of AS patients were evaluated by the Bath Ankylosing.Spondylitis Disease Activity Index (BASDAI).Radiographs obteined from AS patients included lateral views of the lumbar and cervical spine, were required for the scoring of structural damage using the modified Stoke AS Spine Score (SASSS).3.Laboratory assessmentSamples of peripheral blood were allowed to clot and then were centrifuged at 2,400×g for 10 min. The sera were frozen at-80℃ immediately after sample collection. Centrifuge the sample again after thawing before the assay. Avoid repeated freeze-thaw cycles.BALP and OC were measured by enzyme-linked immunosorbent assay (ELISA).The method for BALP and OC measurement was sandwich ELISA (NovaTeinBio, Massachusetts, USA)Acute-phase reactants,the erythrocytesedimentation rate (ESR, Westergren method) and the Creactive protein (CRP, nephelometer) level were recorded.We determined dpMGP by a competive-ELISA assay. The monoclonal antibody used (mAb-dpMGP;Vita K BV,Maastricht,The Netherlands) was directed against the nonphosphorylated peptide sequence SHESMESYELNPF. Biotinylated synthetic MGP homologous to the non-phosphorylated MGP sequence 3-15 (dpMGP3-15) was used as tracer. Test kits for MGP detection in serum were obtained from Biomedica (Vienna, Austria). The principle of the assay is that of a competitive enzyme-linked immunosorbent assay (ELISA) in which microwell plates are pre-coated with mouse monoclonal antibodies against human MGP3-15, mAb-dpMGP. The assay procedure is as follows:100ul Standards or samples diulate are added to the appropriate micro ELISA plate wells and Incubate for 90 minutes at 37℃. Remove the liquid of each wel,don’t wash. Immediately add 100μL of Biotinylated dpMGP3-15 to each well, Incubate for 1 hour at 37℃. Aspirate each well and wash, repeating the process three times with wash Buffer. Add 100μL of HRP Conjugate working solution to each well. Cover with the Plate sealer. Incubate for 30 minutes at 37℃. Add 90μL of Substrate Solution to each well. Incubate for about 15 minutes at 37℃. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but not more than 30minutes. When apparent gradient appeared in standard wells, user should terminate the reaction. Add 50μL of Stop Solution to each well. Then, the color turns to yellow immediately. Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters. The concentration of BALP in the samples is then determined by comparing the O.D. of the samples to the standard curve.4.Statistical analysisThe statistical analysis of the results were performed using the software Statistical Package Sciences (SPSS) for windows version 16.0. For the discrepancies among the groups, Student’s t test and the chi-square test,or Whitney U tests were employed. Spearman’s correlation tests were used to assess relations between quantitative parameters. The statistic results with p value below 0.01 were considered significant.We performed exploratory correlation analysis, using Spearman’s correlation, to identify any correlation between dpMGP and clinical parameters, biomarkers reflecting bone turnover and inflammation.We also calculated optimal cutoff levels of dpMGP for discriminating patients with AS from healthy people,using receiver operating characteristic (ROC) analysis.The value of this cutoff for predicting AS, was analyzed by calculating sensitivity, specificity, likelihood ratios.To determine whether the measure of dpMGP can be a good predictor for radiographic damage,we explored diagnostic properties of dpMGP for radiographic damage using Receiver operating characteristic analysis(ROC). Three arbitrary cut off levels for radiographic damage were investigated. And the ability of dpMGP to discriminate true positives and negatives, and false positives and negatives for the diagnosis of radiographic damage was described in detail with Cumulative probability plots and table.Results1. Patient characteristicseighty-two patients with AS were compared with controls. Thej-e was no significant difference about mean age or sex ratio, BMI between two groups. About 84 percent of patients is male.the mean age of patients is 39 years (mean±SD,38.8±8.9). SASSS scores(mean±SD,13.3±18.7) and BASDAI scores (mean±SD,3.9±2.2) were recorded according to score criterion.2. serum level of dpMGP and inflammational cytokines and bone remodelling markersSerum level of BALP and OC was measured by sandwich ELISA,according to the principle of kit.No significant difference was found between two groups in BALP or OC[mean (SD)),19.1 (5.3) VS 16.0 (6.0) IU/l; 12.1 (3.2) VS 8.9 (3.9) ng/ml].Serum level of ESR and CRP in patients with AS were more elevated relative to controls [mean (SD),33.5 (25.3) VS 9.7 (4.0) mm/h,P<0.01; 8.7 (1.2) VS 1.7 (0.1) mg/dl,P<0.01].3 Serum level of dpMGP for patients of AS with different agePatients with AS were divided into two groups,A group (20-40 years),B group(40-60 years),with segmentation of the range of age. Serum dpMGP was measured by competive ELISA.Serum level of dpMGP in AS patients[mean (SD), 9.2(2)nmol/l] was significantly lower than control[mean (SD) 13.7(4.5)nmol/l] (p<0.01),which were also seen for A (mean (SD),9.6 (2) nmol/1) and B (mean (SD),9.6 (2) nmol/l) groups than control.There is no difference between A and B group.Serum level of dpMGP for patients of AS with different age is analyzed.Patients with AS were divided into two groups,A group (20-40 years),B group(40-60 years),with segmentation of the range of age. Serum dpMGP was measured by sanwitch-ELISA.Serum level of dpMGP in AS patients[mean (SD), 9.2(2)nmol/l] was significantly lower than control[mean (SD) 13.7(4.5)nmol/l] (p<0.01),which were also seen for A (mean (SD),9.6 (2) nmol/1) and B (mean (SD),9.6 (2) nmol/1) groups than control.There is no difference between A and B group.4. Diagnostic value of dpMGPWe further investigated whether we could identify patients with AS by using the predictor of dpMGP. For this purpose, ROC analysis was used to identify the best cutoff levels of serum dpMGP (Picture 2).This analysis revealed that the best cutoff level of 11.5 nmol/l had the highest combination of sensitivity(88%) and specificity (69.7%) (ROC area under the curve(AUC),0.81).The positive and negative LRs using this cutoff were 2.75 and 0.18, respectively.5. characteristic of correlation between dpMGP and SASSSWe explored the association between serum level of dpMGP and serum biomarkers(ESR,CRP,OC,ALP),BASDAI,SASSS.Of all parameters analyzed, only SASSS correlated significantly with dpMGP (rs=-0.715, P<0.01). (Table 3) To investigate the characteristic of correlation between dpMGP and SASSS, we mapped the dpMGP-SASSS scatter plot. (Picture 3).We can see from this plot the correlation is not steady. The bigger the SASSS was, the stronger the correlation became (rs=-0.715,SASSS> 0; rs=-0.524, 0<SASSS<35; rs=-0.73,35≤SASSS≤70). The value of "P" were less than 0.01 for the interval analysis above.6.diagnostic propertiy of dpMGP for radiographic damageFor the gold standard defined at SASSS from 0 to 4,the AUC values range from 0.778 to 0.873.Obviously,the bigger the SASSS is,the more accurate dpMGP became for the diagnosis of radiographic damage.Three scatter plots of the cumulative SASSS versus dpMGP shows that if a higher cut off level for radiographic damage is chosen(from SASSS>0 to SASSS>4), the percentage of true positives decreases (from 52% to 37%), whereas the percentage of true negatives increases (from 20% to 43%). The false positive rate remains about the same (6%-7%), but the percentage of false negatives becomes smaller (from 22% to 13%) because of the increase of the true negatives. So in summary the percentage of patients accurately classified as having or not having radiographic damage increased (from 72% to 80%) along with the increase of cut off level for radiographic damage.Conclusions1.The serum dpMGP is a potential biomarker of AS for diagnosis,with sensitivity of 91.5%and specificity of 69.7%.2.Serum dpMGP may a potential biomarker indicating bone remodelling, especially in the late stage of AS.3.DpMGP protein might play negative role in the process of bone remodelling, rather than in inflammation,in AS.