Opportunities and challenges for the discovery and validation of 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. </jats:p

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) &lt;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.</p

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

Abstract 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

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-Aa and VEGF-Ab, 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-rtTATetoCreLoxP-VEGF-Ato 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-Aa and VEGF-Ab in terms of proliferation and matrix expression. Increased VEGF-Ab 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-Ab inhibited the development of pulmonary fibrosis, as did treatment with intraperitoneal delivery of VEGF-Ab 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-Aa to VEGF-Ab, are critical in development of pulmonary fibrosis and may be a paradigm for the regulation of tissue repair

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

peer reviewedMicrofluidic 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

C3f and V65 ELISA development and validation.

<p>(A) A typical standard curve for C3f sandwich ELISA. Graph represent the log of C3f peptide concentration (from 0–1 μg/ml) against reading of optical density (OD) values fitted with nonlinear regression curve (Four Parameters Logistic Regression(4PL)). (B) Recovery of C3f peptide from spiked normal human serum diluted 1:50 to 1:400. C3f peptide was added at 1 μg/ml into assay buffer as positive control (C3f control) and to human serum diluted at 1:50, 1:100, 1:200 and 1:400 to test recovery. Diluted serum without spiked C3f was tested as negative control. Recovery of spiked C3f in serum was calculated in comparison to C3f control and showed 65%, 86%, 88% and 96% recovery at 1:50, 1:100, 1:200 and 1:400 dilutions respectively. Data plotted as means ± SEM. (C) A typical standard curve for V65 competitive ELISA. Graph represent the log of V65 peptide concentration (from 0–4 μg/ml) fitted with nonlinear regression curve (4PL). (D) Graph represent the concentration of V65 peptide against OD values. Spiking experiment was carried out with 2-fold serial dilution starting at 1μg/ml of V65 peptide using normal human serum (at 1:100 or 1:400). Data plotted as means ± SEM.</p

C3f in human serum samples.

<p>(A) Measurements of C3f in μg/ml in serum samples from OA patients (n = 13), RA patients (n = 13) and NC (n = 13). All samples were analysed at 1 in 50 dilution. Concentration of C3f was significantly increased in the RA group in comparison to the OA and NC group (***p>0.0001) and no increase was observed for the OA group. Data were analysed using Kruskal-Wallis with post hoc Dunn’s analysis and plotted as means ± SEM. (B) Schematic representation of human C3 complement cleavage to generated C3a, C3b, C3f and iC3b fragments. (C) C3f sandwich ELISA performed on filtered serum samples from the 3 study groups. RA patient’s sample (n = 5) and 1 NC sample were filtered using 3kDa and 10kDa cut-off filter and assessed by C3f sandwich ELISA. Unfiltered serum were tested as control. Data plotted as means ± SEM.</p

Additional file 4: Figure S3. of Effects of hypoxia and hyperoxia on the differential expression of VEGF-A isoforms and receptors in Idiopathic Pulmonary Fibrosis (IPF)

Expression of VEGF-A receptor and co-receptor mRNA and proteins in response to hypoxia and hyperoxia in normal (NF) and fibrotic (FF) fibroblasts. a) Quantitative RT-PCR of VEGFR1, neuropilin (NP) 1, and NP2 mRNA expression in total RNA cell lysates in NF and FF. VEGFR1 (***p < 0.001) and NP2 (*p < 0.05) mRNA levels were significantly up-regulated in NF in response to exposure to hypoxia, whilst NF NP1 mRNA levels were significantly downregulated (*p < 0.05). Similarly, FF VEGFR1 (*p < 0.05) and NP2 (****p < 0.0001) mRNA levels were significantly upregulated in response to hypoxia, but NP1 mRNA levels were unaffected. Hyperoxia had no significant effect on VEGFR1 or NP2 mRNA levels in neither NF or FF, whilst hyperoxia significantly upregulated (*p < 0.05) FF NP1 mRNA levels. Data are presented as mean fold change in expression (2-△△CT) with SEM, data analysis performed on △△CT values (NF and FF n = 6). b) The effect of hypoxia and hyperoxia on VEGFR1 protein expression in NF and FF as measured by western blotting (above) and densitometric analysis (below). VEGFR1 protein expression was significantly upregulated (***p < 0.001) in response to hypoxia in NF but not in FF. Hyperoxia had no statistically significant effect on VEGFR1 expression in NF or FF. c) Hypoxia resulted in the significant down-regulation of NP1 protein expression in NF cell lysates (*p < 0.05), but had no significant effect on FF. Hyperoxia up-regulated NP1 protein expression in FF (p* < 0.05), but had no significant effect on NF. d) Hypoxia and hyperoxia had no significant effect in the expression of NP2 protein. Data presented as means with SEM (n = 4, n = 2 shown in each western blot image). Normal: Normal fibroblasts, Fibrotic: Fibrotic fibroblasts, N: Normoxia, HO: Hypoxia, HE: Hyperoxia. Tubulin: loading control. Analysis of variance with post hoc Dunnett’s multiple comparisons analysis used throughout. (JPEG 114 kb