Multivariable logistic and linear regression models for identification of clinically useful biomarkers for osteoarthritis.

Osteoarthritis (OA) is the most common chronic degenerative joint disease which causes substantial joint pain, deformity and loss of activities of daily living. Currently, there are over 500 million OA cases worldwide, and there is an urgent need to identify biomarkers for early detection, and monitoring disease progression in patients without obvious radiographic damage to the joint. We have used regression modelling to describe the association of 19 of the currently available biomarkers (predictors) with key radiographic and clinical features of OA (outcomes) in one of the largest and best characterised OA cohort (NIH Osteoarthritis Initiative). We demonstrate that of the 19 currently available biomarkers only 4 (serum Coll2-1 NO2, CS846, COMP and urinary CTXII) were consistently associated with established radiographic and/or clinical features of OA. These biomarkers are independent of one another and provide additional predictive power over, and above established predictors of OA such as age, gender, BMI and race. We also show that that urinary CTXII had the strongest and consistent associations with clinical symptoms of OA as well as radiographic evidence of joint damage. Accordingly, urinary CTXII may aid in early diagnosis of OA in symptomatic patients without radiographic evidence of OA.

Effects of hypoxia and hyperoxia on the differential expression of VEGF-A isoforms and receptors in Idiopathic Pulmonary Fibrosis (IPF).

Dysregulation of VEGF-A bioavailability has been implicated in the development of lung injury/fibrosis, exemplified by Idiopathic Pulmonary Fibrosis (IPF). VEGF-A is a target of the hypoxic response via its translational regulation by HIF-1α. The role of hypoxia and hyperoxia in the development and progression of IPF has not been explored. In normal lung (NF) and IPF-derived fibroblasts (FF) VEGF-Axxxa protein expression was upregulated by hypoxia, mediated through activation of VEGF-Axxxa gene transcription. VEGF-A receptors and co-receptors were differentially expressed by hypoxia and hyperoxia. Our data supports a potential role for hypoxia, hyperoxia and VEGF-Axxxa isoforms as drivers of fibrogenesis.

Opportunities and challenges for the discovery and validation of proteomic biomarkers for common arthritic diseases.

Osteoarthritis (OA) and rheumatoid arthritis (RA) are most prevalent among all the rheumatic diseases, and currently, there are no reliable biochemical measures for early diagnosis or for predicting who is likely to progress. Early diagnosis is important for making decisions on treatment options and for better management of patients. This narrative review highlights the first-generation biomarkers identified over the last two decades and focuses on the discovery and validation of candidate OA biomarkers from recent mass-spectrometry-based proteomic studies for diagnosis and monitoring disease outcomes in human. It discusses the challenges and opportunities for discovery of novel biomarkers and progress in the development of techniques for measuring biomarkers, and provides directions for future discovery and validation of biomarkers for OA and rheumatoid arthritis.

Development and validation of novel biomarker assays for osteoarthritis.

BACKGROUND: Osteoarthritis (OA) is the most common chronic joint disease usually diagnosed at relatively advanced stages when there is irreparable damage to the joint(s). Recently, we have identified two novel biomarkers C3f and V65 which appear to be OA-specific and therefore potential markers of early disease. We report the development of immunoassays for quantitative measure of these two novel biomarkers. METHOD: Monoclonal and polyclonal antibodies were generated by immunising mouse and rabbits respectively with peptide-carrier conjugates of C3f and V65. Affinity purified antibodies were used for immunoassays development and assays validated using serum from OA patients and controls. RESULTS: The ELISAs developed showed spiked recovery of up to 96% for C3f and V65 peptides depending on serum dilutions with a coefficient of variation (CV) <10%. The intra- and inter-assay CVs for C3f and V65 were 1.3-10.8% and 4.2-10.3% respectively. Both assays were insensitive for measurements of the peptides in patients and the use of different signal amplification systems did not increase assay sensitivity. CONCLUSION: We have developed two immunoassays for measurements of C3f and V65 peptides biomarkers discovered by our earlier proteomic study. These assays could detect the endogenous peptides in serum samples from patients and controls but lacked sensitivity for accurate measurements of the peptides in patients. Our study highlights the difficulties and challenges of validating biomarker from proteomic studies and demonstrates how to overcome some of the technical challenges associated with developing immunoassays for small peptides.

Differential Expression of VEGF-Axxx Isoforms Is Critical for Development of Pulmonary Fibrosis.

RATIONALE: Fibrosis after lung injury is related to poor outcome, and idiopathic pulmonary fibrosis (IPF) can be regarded as an exemplar. Vascular endothelial growth factor (VEGF)-A has been implicated in this context, but there are conflicting reports as to whether it is a contributory or protective factor. Differential splicing of the VEGF-A gene produces multiple functional isoforms including VEGF-A165a and VEGF-A165b, a member of the inhibitory family. To date there is no clear information on the role of VEGF-A in IPF. OBJECTIVES: To establish VEGF-A isoform expression and functional effects in IPF. METHODS: We used tissue sections, plasma, and lung fibroblasts from patients with IPF and control subjects. In a bleomycin-induced lung fibrosis model we used wild-type MMTV mice and a triple transgenic mouse SPC-rtTA+/-TetoCre+/-LoxP-VEGF-A+/+ to conditionally induce VEGF-A isoform deletion specifically in the alveolar type II (ATII) cells of adult mice. MEASUREMENTS AND MAIN RESULTS: IPF and normal lung fibroblasts differentially expressed and responded to VEGF-A165a and VEGF-A165b in terms of proliferation and matrix expression. Increased VEGF-A165b was detected in plasma of progressing patients with IPF. In a mouse model of pulmonary fibrosis, ATII-specific deficiency of VEGF-A or constitutive overexpression of VEGF-A165b inhibited the development of pulmonary fibrosis, as did treatment with intraperitoneal delivery of VEGF-A165b to wild-type mice. CONCLUSIONS: These results indicate that changes in the bioavailability of VEGF-A sourced from ATII cells, namely the ratio of VEGF-Axxxa to VEGF-Axxxb, are critical in development of pulmonary fibrosis and may be a paradigm for the regulation of tissue repair.

VEGF isoforms have differential effects on permeability of human pulmonary microvascular endothelial cells.

BACKGROUND: Alternative splicing of Vascular endothelial growth factor-A mRNA transcripts (commonly referred as VEGF) leads to the generation of functionally differing isoforms, the relative amounts of which have potentially significant physiological outcomes in conditions such as acute respiratory distress syndrome (ARDS). The effect of such isoforms on pulmonary vascular permeability is unknown. We hypothesised that VEGF165a and VEGF165b isoforms would have differing effects on pulmonary vascular permeability caused by differential activation of intercellular signal transduction pathways. METHOD: To test this hypothesis we investigated the physiological effect of VEGF165a and VEGF165b on Human Pulmonary Microvascular Endothelial Cell (HPMEC) permeability using three different methods: trans-endothelial electrical resistance (TEER), Electric cell-substrate impedance sensing (ECIS) and FITC-BSA passage. In addition, potential downstream signalling pathways of the VEGF isoforms were investigated by Western blotting and the use of specific signalling inhibitors. RESULTS: VEGF165a increased HPMEC permeability using all three methods (paracellular and transcellular) and led to associated VE-cadherin and actin stress fibre changes. In contrast, VEGF165b decreased paracellular permeability and did not induce changes in VE-cadherin cell distribution. Furthermore, VEGF165a and VEGF165b had differing effects on both the phosphorylation of VEGF receptors and downstream signalling proteins pMEK, p42/44MAPK, p38 MAPK, pAKT and peNOS. Interestingly specific inhibition of the pMEK, p38 MAPK, PI3 kinase and eNOS pathways blocked the effects of both VEGF165a and VEGF165b on paracellular permeability and the effect of VEGF165a on proliferation/migration, suggesting that this difference in cellular response is mediated by an as yet unidentified signalling pathway(s). CONCLUSION: This study demonstrates that the novel isoform VEGF165a and VEGF165b induce differing effects on permeability in pulmonary microvascular endothelial cells.

Validation of a new method by nano-liquid chromatography on chip tandem mass spectrometry for combined quantitation of C3f and the V65 vitronectin fragment as biomarkers of diagnosis and severity of osteoarthritis.

Microfluidic liquid chromatography coupled to a nanoelectrospray source ion trap mass spectrometry was used for the absolute and simultaneous quantitation of C3f and the V65 vitronectin fragment in serum. The method was first carefully optimized and then validated in serum biological matrix. Stable isotopes for the two biomarkers of interest were used as stable isotope labeled peptide standards. A weighted 1/x2 quadratic regression for C3f and a weighted 1/x quadratic regression for the V65 vitronectin peptide were selected for calibration curves. Trueness (with a relative bias <10%), precision (repeatability and intermediate precision <15%) and accuracy (risk <15%) of the method were successfully demonstrated. The linearity of results was validated in the concentration range of 2.5-200ng/mL for C3f and 2.5-100ng/mL for the V65 vitronectin fragment. Serum samples (n=147) classified in 7 groups [(healthy volunteers, OA with 5 grades of severity and rheumatoid arthritis (RA) patients] were analyzed with our new quantitative method. Our data confirm that C3f and the V65 vitronectin fragment are biomarkers of OA severity, but also that C3f fragment is further related to OA severity whereas the V65 vitronectin fragment is more related to early OA detection.