Liver vitronectin release into the bloodstream increases due to reduced vagal muscarinic signaling after cerebral stroke in female mice.

Vitronectin (VTN) is a glycoprotein enriched in the blood and activates integrin receptors. VTN blood levels increase only in female mice 24 h after an ischemic stroke and exacerbate brain injury through IL-6-driven inflammation, but the VTN induction mechanism is unknown. Here, a 30 min middle cerebral artery occlusion (MCAO) in female mice induced VTN protein in the liver (normally the main source) in concert with plasma VTN. Male mice were excluded as VTN is not induced after stroke. MCAO also increased plasma VTN levels after de novo expression of VTN in the liver of VTN-/- female mice, using a hepatocyte-specific (SERPINA1) promoter. MCAO did not affect SERPINA1 or VTN mRNA in the liver, brain, or several peripheral organs, or platelet VTN, compared to sham mice. Thus, hepatocytes are the source of stroke-induced increases in plasma VTN, which is independent of transcription. The cholinergic innervation by the parasympathetic vagus nerve is a potential source of brain-liver signaling after stroke. Right-sided vagotomy at the cervical level led to increased plasma VTN levels, suggesting that VTN release is inhibited by vagal tone. Co-culture of hepatocytes with cholinergic neurons or treatment with acetylcholine, but not noradrenaline (sympathetic transmitter), suppressed VTN expression. Hepatocytes have muscarinic receptors and the M1/M3 agonist bethanechol decreased VTN mRNA and protein release in vitro via M1 receptors. Finally, systemic bethanechol treatment blocked stroke-induced plasma VTN. Thus, VTN translation and release are inhibited by muscarinic signaling from the vagus nerve and presents a novel target for lessening detrimental VTN expression.

Over-expression of vitronectin correlates with impaired survival in gastric cancers.

ABSTRACT: Over-expression of vitronectin (VN) is associated with tumorigenesis. The present study aimed to evaluate the prognostic value of VN expression in gastric cancer.The least absolute shrinkage and selection operator analysis was performed to screen the hub gene from The Cancer Genome Atlas gastric cancer patients with complete follow-up data, and 347 patients were finally included. Moreover, 102 patients were enrolled from the Affiliated Fuzhou First Hospital of Fujian Medical University. VN expression in paired gastric cancer and adjacent gastric normal tissues was detected using immunohistochemistry, and the clinicopathological significance of VN expression was evaluated. The prognostic significance of VN expression in gastric cancer patients was evaluated using by Kaplan-Meier method and Cox regression analysis and confirmed using Oncomine.VN was the prognosis relative gene which screened by The Cancer Genome Atlas dataset. Moreover, we identified the VN expression in an external dataset by immunohistochemistry. The result demonstrated that VN expression was remarkedly elevated in gastric cancer tissues (P < .001). High VN expression correlated with higher pathological Tumor-Node-Metastasis stage, and poorer survival outcomes. Cox regression analysis showed that VN expression was independently predictive of overall survival (OS) and disease-free survival (P = .004, P < .001, respectively). A prognostic risk score for OS was built based on VN expression. A meta-analysis from Oncomine datasets revealed that significantly lower VN mRNA levels in gastric cancer correlated with poorer OS.VN expression could be a prognostic marker of gastric cancer.

Serum biomarker discovery related to pathogenesis in acute coronary syndrome by proteomic approach.

Acute coronary syndrome (ACS) results from inadequate supply of blood flow from the coronary arteries to the heart or ischemia. ACS has an extremely high morbidity and mortality. The levels of biomarkers currently used for detection of ACS also increase in response to myocardial necrosis and other diseases and are not elevated immediately after symptoms appear, thus limiting their diagnostic capacity. Therefore, we aimed to discover new ACS diagnostic biomarkers with high sensitivity and specificity that are specifically related to ACS pathogenesis. Sera from 50 patients with ACS and healthy controls (discovery cohort) each were analyzed using mass spectrometry (MS) to identify differentially expressed proteins, and protein candidates were evaluated as ACS biomarkers in 120 people in each group (validation cohort). α-1-acid glycoprotein 1 (AGP1), complement C5 (C5), leucine-rich α-2-glycoprotein (LRG), and vitronectin (VN) were identified as biomarkers whose levels increase and gelsolin (GSN) as a biomarker whose levels decrease in patients with ACS. We concluded that these biomarkers are associated with the pathogenesis of ACS and can predict the onset of ACS prior to the appearance of necrotic biomarkers.

Biomarker Discovery of Acute Coronary Syndrome Using Proteomic Approach.

Acute coronary syndrome (ACS) is a condition in which the coronary artery supplying blood to the heart is infarcted via formation of a plaque and thrombus, resulting in abnormal blood supply and high mortality and morbidity. Therefore, the prompt and efficient diagnosis of ACS and the need for new ACS diagnostic biomarkers are important. In this study, we aimed to identify new ACS diagnostic biomarkers with high sensitivity and specificity using a proteomic approach. A discovery set with samples from 20 patients with ACS and 20 healthy controls was analyzed using mass spectrometry. Among the proteins identified, those showing a significant difference between each group were selected. Functional analysis of these proteins was conducted to confirm their association with functions in the diseased state. To determine ACS diagnostic biomarkers, standard peptides of the selected protein candidates from the discovery set were quantified, and these protein candidates were validated in a validation set consisting of the sera of 50 patients with ACS and 50 healthy controls. We showed that hemopexin, leucine-rich α-2-glycoprotein, and vitronectin levels were upregulated, whereas fibronectin level was downregulated, in patients with ACS. Thus, the use of these biomarkers may increase the accuracy of ACS diagnosis.

The Plasma Levels and Polymorphisms of Vitronectin Predict Radiation Pneumonitis in Patients With Lung Cancer Receiving Thoracic Radiation Therapy.

PURPOSE: Our previous findings have identified vitronectin (VTN) as a potential biomarker for radiation pneumonitis (RP) through proteomics and molecular mechanism studies. In a recent study, we further explored associations of plasma level and single nucleotide polymorphisms of VTN with the risk of RP in patients with lung cancer receiving radiation therapy. METHODS AND MATERIALS: A total of 165 patients with lung cancer were prospectively enrolled with detection of VTN concentration before radiation therapy. VTN reference single nucleotide polymorphisms, rs704 and rs2227721, were genotyped by Taqman probe method. Cox proportional hazard models were performed to identify clinical variables and genotypes associated with the risk of RP on univariate and multivariate analyses, and t tests and analysis of variance were conducted to evaluate the expression level of VTN. RESULTS: The baseline secretion level of VTN in patients with grade ≥3 RP was significantly higher than that in grade <3 RP patients (P < .0001), and elevated levels were observed in patients having the AA genotype compared with GA/GG genotypes of rs704. The VTN rs704 GA/GG and rs2227721 AA/AC genotypes had a significantly lower risk of RP (hazard ratio [HR], 0.448, P = .005; HR, 0.419, P = .008, respectively). In addition, combining cut-off values of mean lung dose (MLD) and VTN plasma level, grade ≥3 RP risk groupings were as follows: high risk: MLD ≥12 Gy and VTN level ≥132 µg/mL (RP rate, 10 of 16 patients, 62.5%); intermediate risk: MLD ≥12 Gy and VTN level <132 µg/mL or MLD <12 Gy and VTN level ≥132 µg/mL (8 of 70 patients, 11.4%); and low risk: MLD <12 Gy and VTN level <132 µg/mL (1 of 79 patients, 1.3%) (P < .0001). CONCLUSIONS: Among patients receiving radiation therapy, relatively high plasma levels of VTN before radiation therapy were associated with the higher incidence of RP, and VTN rs704 and rs2227721 each had a significant effect on predicting RP risk. Combining VTN concentration with MLD appeared to facilitate stratification of patients with lung cancer who received radiation therapy into low-, intermediate-, and high-risk RP groups. This study indicated that VTN may serve as a blood biomarker for susceptibility to RP in patients with lung cancer.

Blood Vitronectin Induces Detrimental Brain Interleukin-6 and Correlates With Outcomes After Stroke Only in Female Mice.

Background and Purpose- Women have worse stroke outcomes than men, especially after menopause. Few studies have focused on female-specific mechanisms, other than hormones. We investigated the role of the blood protein VTN (vitronectin) after ischemic stroke in mice. Methods- Adult male and female VTN knockout and wild-type littermates and C57BL/6 mice received a middle cerebral artery occlusion and the injured brain tissue analyzed 24 hours to 3 weeks later for cell loss and inflammation, as well as neurological function. Blood VTN levels were measured before and after stroke. Results- Intravenously injected VTN leaked extensively from bloodstream into brain infarct and penumbra by 24 hours after stroke. Strikingly, VTN was detrimental in female, but not male, mice, as shown by reduced brain injury (26.2±2.6% versus 13.4±3.8%; P=0.018; n=6 and 5) and forelimb dysfunction in female VTN knockout mice. Stroke increased plasma VTN 2- to 8-fold at 24 hours in females (36±4 versus 145±24 µg/mL; P<0.0001; n=10 and 7), but not males (62±8 versus 68±6; P>0.99; n=10 and 7), and returned to control levels by 7 days. Individually variable VTN levels at 24 hours correlated with stroke-induced brain injury at 7 days only in females. VTN promoted stroke-induced microglia/macrophage activation and leukocyte infiltration in females. Proinflammatory IL (interleukin)-6 greatly increased in the striatum at 24 hours in wild-type mice but was increased ≈60% less in female (739±159 versus 268±111; P=0.02; n=7 and 6), but not male (889±178 versus 1179±295; P=0.73; n=10 and 11), knockout mice. In individual wild-type females, plasma VTN levels correlated with striatal IL-6 expression at 24 hours. The female-specific effect of VTN-induced IL-6 expression following stroke was not due to gonadal hormones, as shown by ovariectomy and castration. Lastly, intrastriatal injection of IL-6 in female mice immediately before stroke reversed the VTN knockout phenotypes of reduced brain injury and microglia/macrophage activation. Conclusions- VTN plays a novel sexually dimorphic detrimental pathophysiological role in females and might ultimately be a therapeutic target to improve stroke outcomes in women.

Evaluation of Serum Levels of Inflammation, Fibrinolysis and Oxidative Stress Markers in Coronary Artery Disease Prediction: A Cross-Sectional Study.

BACKGROUND: Coronary Artery Disease (CAD) has long been recognized as a global health issue. Inflammation, Fibrinolysis and Oxidative Stress play an important role in the disruption of plaques leading to CAD. Markers that reflect this pathophysiologic mechanism may have prognostic value. OBJECTIVE: To estimate the serum concentrations of high-sensitivity C-reactive protein (hs-CRP), sialic acid (SA), vitronectin (VN), plasminogen activator inhibitor-1 (PAI-1), oxidized low density lipoprotein (OX-LDL) and malondialdehyde (MDA) with significant prognostic value in patients with CAD. METHODS: The markers included, hs-CRP, SA, VN, PAI-1, OX-LDL and MDA, were compared between 160 angiographically diagnosed CAD patients and 20 age- and sex-matched healthy individuals. The subjects were divided into 4 groups according to angiography results, and association between all risk factors of CAD was studied. Serum levels of SA, VN, PAI-1, and OX-LDL were measured by enzyme-linked immunosorbent assay (ELISA); MDA was measured based on reaction with thiobarbituric acid (TBA); and hs-CRP level was estimated by immunoturbidimetry using a commercial kit. The diagnostic value of these variables was further assessed by ROC curve analysis. Multiple logistic regression was used to evaluate the diagnostic power of the combination. Furthermore, p < 0.05 was considered as significant. RESULTS: Serum levels of hs-CRP, SA, VN, PAI-1, and OX-LDL were significantly higher in patient groups compared to control group (p < 0.001). Using both normal and CAD patients as subjects, ROC analysis was performed. The cutoff for OX-LDL, MDA, PAI-1, VN, hs-CRP and SA was 2.67 (ug/mL), 5.49 (mmol/mL), 67 (ng/mL), 254 (ng/mL), 3.4 (mg/dL), 7/89 (mg/dL), respectively. Eventually, the complete diagnostic efficacy was classified as: SA, hs-CRP, PAI-1, OX-LDL, MDA and VN. CONCLUSION: Serum levels SA, hs-CRP, VN, PAI-1, OX-LDL and MDA may be predictive of adverse cardiovascular outcomes. Interestingly, these analyses can help as diagnostic and monitoring markers in CAD patients.

Plasma levels of soluble membrane attack complex are elevated despite viral suppression in HIV patients with poor immune reconstitution.

The complement system is now a therapeutic target for the management of serious and life-threatening conditions such as paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, glomerulonephritis and other diseases caused by complement deficiencies or genetic variants. As complement therapeutics expand into more clinical conditions, monitoring complement activation is increasingly important, as is the baseline levels of complement activation fragments in blood or other body fluid levels. Although baseline complement levels have been reported in the literature, the majority of these data were generated using non-standard assays and with variable sample handling, potentially skewing results. In this study, we examined the plasma and serum levels of the soluble membrane attack complex of complement (sMAC). sMAC is formed in the fluid phase when complement is activated through the terminal pathway. It binds the regulatory proteins vitronectin and/or clusterin and cannot insert into cell membranes, and can serve as a soluble diagnostic marker in infectious disease settings, as previously shown for intraventricular shunt infections. Here we show that in healthy adults, serum sMAC levels were significantly higher than those in plasma, that plasma sMAC levels were similar between in African Americans and Caucasians and that plasma sMAC levels increase with age. Plasma sMAC levels were significantly higher in virally suppressed people living with HIV (PLWH) compared to non-HIV infected healthy donors. More specifically, PLWH with CD4+ T cell counts below 200 had even greater sMAC levels, suggesting diagnostic value in monitoring sMAC levels in this group.

Evaluation of Serum Integrin αvß3 & Vitronectin in the Early Diagnosis of Breast Cancer.

BACKGROUND: This study aimed to evaluate the relationship of serum vitronectin and integrin alpha V beta 3 (αvß3) levels with various clinicopathological parameters of breast cancer and to assess the diagnostic value of these markers alone or in combination with the conventional breast cancer biomarker CA15.3. METHODS: This study included 50 early diagnosed stage I - II primary breast cancer patients, 20 patients with fibroadenoma benign lesions, and 20 apparently normal healthy controls. Integrin αVß3, vitronectin, and CA15.3 levels were measured using ELISA technique. RESULTS: Serum levels of integrin αVß3 and vitronectin were significantly higher in the malignant group than those in the benign group and the control group with (p < 0.001). Significant positive correlation between integrin αvß3 and vitronectin concentrations was found. Both markers showed significant statistically difference with lymph node, histological grade, tumor stage, and tumor size (p < 0.05). Integrin αvß3 exhibited the highest sensitivity (70%) and specificity (68%), then vitronectin with 67% and 68%, respectively, followed by CA15.3 showing the least sensitivity and specificity (65% and 62%, respectively). All assessed parameters revealed comparable area under the receiver-operating characteristic curve (AUC) 95% confidence interval (CI) range of 0.581 - 0.822. CONCLUSIONS: Integrin αvß3 is a promising biomarker alone or in combination with vitronectin and CA15.3 for diagnosis and prognosis of early stage breast cancer.

Parallel reaction monitoring with multiplex immunoprecipitation of N-glycoproteins in human serum for detection of hepatocellular carcinoma.

The N-glycosylation of proteins is one of the most important post-translational modifications relevant to various biological functions. The identification and quantification of N-glycoproteins in liquid chromatography-mass spectrometry (LC-MS) is challenging because of their low analytical sensitivity and selectivity. This is due to their microheterogeneity and the difficulty of synthesizing N-glycopeptides as an internal standard. Parallel reaction monitoring (PRM) is widely used in targeted LC-MS. The key advantage of LC-PRM is that it can identify N-glycopeptides using tandem mass spectrometry (MS/MS) fragmentation, even without an internal standard. We investigated the feasibility of analyzing N-glycoproteins using multiplex immunoprecipitation to improve sensitivity and selectivity. We targeted N-glycoproteins [α-fetoprotein (AFP), vitronectin (VTN), and α-1-antichymotrypsin (AACT)] that are abnormally glycosylated in hepatocellular carcinoma (HCC). Their tryptic N-glycopeptides were selected to determine the percentages of fucosylated N-glycopeptides using Y ions, which include glycopeptide fragments with amino acid sequences. Finally, we confirmed that the area under the receiver operating characteristic curve (AUC = 0.944) for the combination of AFP and VTN increased more so than for a single glycopeptide (AUC = 0.889 for AFP and 0.792 for VTN) with respect to discriminating between HCC and cirrhosis serum. This study shows that an LC-PRM method using multiplex N-glycoproteins immunoprecipitated from serum could be applied to develop and verify cancer biomarkers. Graphical abstract.

Exosomal miR-99a-5p is elevated in sera of ovarian cancer patients and promotes cancer cell invasion by increasing fibronectin and vitronectin expression in neighboring peritoneal mesothelial cells.

BACKGROUND: microRNAs (miRNAs) stably exist in circulating blood and are encapsulated in extracellular vesicles such as exosomes. The aims of this study were to identify which exosomal miRNAs are highly produced from epithelial ovarian cancer (EOC) cells, to analyze whether serum miRNA can be used to discriminate patients with EOC from healthy volunteers, and to investigate the functional role of exosomal miRNAs in ovarian cancer progression. METHODS: Exosomes were collected from the culture media of serous ovarian cancer cell lines, namely TYK-nu and HeyA8 cells. An exosomal miRNA microarray revealed that several miRNAs including miR-99a-5p were specifically elevated in EOC-derived exosomes. Expression levels of serum miR-99a-5p in 62 patients with EOC, 26 patients with benign ovarian tumors, and 20 healthy volunteers were determined by miRNA quantitative reverse transcription-polymerase chain reaction. To investigate the role of exosomal miR-99a-5p in peritoneal dissemination, neighboring human peritoneal mesothelial cells (HPMCs) were treated with EOC-derived exosomes and then expression levels of miR-99a-5p were examined. Furthermore, mimics of miR-99a-5p were transfected into HPMCs and the effect of miR-99a-5p on cancer invasion was analyzed using a 3D culture model. Proteomic analysis with the tandem mass tag method was performed on HPMCs transfected with miR-99a-5p and then potential target genes of miR-99a-5p were examined. RESULTS: The serum miR-99a-5p levels were significantly increased in patients with EOC, compared with those in benign tumor patients and healthy volunteers (1.7-fold and 2.8-fold, respectively). A receiver operating characteristic curve analysis showed with a cut-off of 1.41 showed sensitivity and specificity of 0.85 and 0.75, respectively, for detecting EOC (area under the curve = 0.88). Serum miR-99a-5p expression levels were significantly decreased after EOC surgeries (1.8 to 1.3, p = 0.002), indicating that miR-99a-5p reflects tumor burden. Treatment with EOC-derived exosomes significantly increased miR-99a-5p expression in HPMCs. HPMCs transfected with miR-99a-5p promoted ovarian cancer invasion and exhibited increased expression levels of fibronectin and vitronectin. CONCLUSIONS: Serum miR-99a-5p is significantly elevated in ovarian cancer patients. Exosomal miR-99a-5p from EOC cells promotes cell invasion by affecting HPMCs through fibronectin and vitronectin upregulation and may serve as a target for inhibiting ovarian cancer progression.

The relationship between vitronectin and hepatic insulin resistance in type 2 diabetes mellitus.

The World Health Organization (WHO) estimates that approximately 300 million people will suffer from diabetes mellitus by 2025. Type 2 diabetes mellitus (T2DM) is much more prevalent. T2DM comprises approximately 90% of diabetes mellitus cases, and it is caused by a combination of insulin resistance and inadequate compensatory insulin secretory response. In this study, we aimed to compare the plasma vitronectin (VN) levels between patients with T2DM and insulin resistance (IR) and healthy controls. Seventy patients with IR and 70 age- and body mass index (BMI)-matched healthy controls were included in the study. The insulin, Waist-to-Hip Ratio (WHR), C-peptide (CP) and VN levels of all participants were examined. The homeostasis model of assessment for insulin resistence index (HOMA-IR (CP)) formula was used to calculate insulin resistance. The levels of BMI, fasting plasma gluose (FPG), 2-hour postprandial glucose (2hPG), glycated hemoglobins (HbA1c), and HOMA-IR (CP) were significantly elevated in case group compared with controls. VN was found to be significantly decreased in case group. (VN Mean (Std): 8.55 (2.92) versus 12.88 (1.26) ng/mL p < 0.001). Multiple linear regression analysis was performed. This model explained 43.42% of the total variability of VN. Multiple linear regression analysis showed that HOMA-IR (CP) and age independently predicted VN levels. The VN may be a candidate target for the appraisal of hepatic insulin resistance in patients with T2DM.

Overexpression of the Vitronectin V10 Subunit in Patients with Nonalcoholic Steatohepatitis: Implications for Noninvasive Diagnosis of NASH.

Nonalcoholic steatohepatitis (NASH) is the critical stage of nonalcoholic fatty liver disease (NAFLD). The persistence of necroinflammatory lesions and fibrogenesis in NASH is the leading cause of liver cirrhosis and, ultimately, hepatocellular carcinoma. To date, the histological examination of liver biopsies, albeit invasive, remains the means to distinguish NASH from simple steatosis (NAFL). Therefore, a noninvasive diagnosis by serum biomarkers is eagerly needed. Here, by a proteomic approach, we analysed the soluble low-molecular-weight protein fragments flushed out from the liver tissue of NAFL and NASH patients. On the basis of the assumption that steatohepatitis leads to the remodelling of the liver extracellular matrix (ECM), NASH-specific fragments were in silico analysed for their involvement in the ECM molecular composition. The 10 kDa C-terminal fragment of the ECM protein vitronectin (VTN) was then selected as a promising circulating biomarker in discriminating NASH. The analysis of sera of patients provided these major findings: the circulating VTN fragment (i) is overexpressed in NASH patients and positively correlates with the NASH activity score (NAS); (ii) originates from the disulfide bond reduction between the V10 and the V65 subunits. In conclusion, V10 determination in the serum could represent a reliable tool for the noninvasive discrimination of NASH from simple steatosis.

Blood vitronectin is a major activator of LIF and IL-6 in the brain through integrin-FAK and uPAR signaling.

We defined how blood-derived vitronectin (VTN) rapidly and potently activates leukemia inhibitory factor (LIF) and pro-inflammatory interleukin 6 (IL-6) in vitro and after vascular injury in the brain. Treatment with VTN (but not fibrinogen, fibronectin, laminin-111 or collagen-I) substantially increased LIF and IL-6 within 4 h in C6-astroglioma cells, while VTN-/- mouse plasma was less effective than that from wild-type mice. LIF and IL-6 were induced by intracerebral injection of recombinant human (rh)VTN in mice, but induction seen upon intracerebral hemorrhage was less in VTN-/- mice than in wild-type littermates. In vitro, VTN effects were inhibited by RGD, αvß3 and αvß5 integrin-blocking peptides and antibodies. VTN activated focal adhesion kinase (FAK; also known as PTK2), whereas pharmacological- or siRNA-mediated inhibition of FAK, but not PYK2, reduced the expression of LIF and IL-6 in C6 and endothelial cells and after traumatic cell injury. Dominant-negative FAK (Y397F) reduced the amount of injury-induced LIF and IL-6. Pharmacological inhibition or knockdown of uPAR (also known as PLAUR), which binds VTN, also reduced cytokine expression, possibly through a common target of uPAR and integrins. We propose that VTN leakage into tissues promotes inflammation. Integrin-FAK signaling is therefore a novel IL-6 and LIF regulation mechanism relevant to the inflammation and stem cell fields.

Vitronectin (Vn) glycosylation patterned by lectin affinity assays-A potent glycoproteomic tool to discriminate plasma Vn from cancer ascites Vn.

Changes in glycosylation have been associated with human cancer, but their complexity poses an analytical challenge. Ovarian cancer is a major cause of death in women because of an often late diagnosis. At least one-third of patients presents ascites fluid at diagnosis, and almost all have ascites at recurrence. Vitronectin (Vn) is a multifunctional glycoprotein that is suggested to be implicated in ovarian cancer metastasis and is found within ascites. The present study evaluated the potential of using lectin affinity for characterizing the glycosylation pattern of Vn. Human Vn was purified from 1 sample of ovarian cancer ascites or a pool of plasma samples. Consistent findings were observed with both dot blot and lectin array assays. Based on a panel of 40 lectins, the lectin array revealed discriminant patterns of lectin binding to Vn glycans. Interestingly, almost all the highlighted interactions were found to be higher with Vn from ascites relative to the plasma counterpart. Also, the lectin array was able to discriminate profiles of lectin interactions (ConA, SNA-I, PHA-E, PHA-L) between Vn samples that were not evident using dot blot, indicating its high sensitivity. The model of ConA binding during thermal unfolding of Vn confirmed the higher accessibility of mannosylated glycans in Vn from ascites as monitored by turbidimetry. Thus, this study demonstrated the usefulness of lectins and the lectin array as a glycoproteomic tool for high throughput and sensitive analysis of glycosylation patterns. Our data provide novel insights concerning Vn glycosylation patterns in clinical specimens, paving the way for further investigations regarding their functional impact and clinical interest.

Serum osteopontin and vitronectin levels in systemic sclerosis.

BACKGROUND: Osteopontin a matricellular protein has pro-fibrotic effects and binds integrin such as αvß1 and αvß3. Vitronectin is one of the integrin αvß3 ligands and is a multifunctional glycoprotein. OBJECTIVES: The aim of the present study was to evaluate serum osteopontin and vitronectin levels in a cohort of patients with systemic sclerosis (SSc). MATERIAL AND METHODS: Eighty-six patients with SSc, 46 patients with systemic lupus erythematosus (SLE), and 38 healthy controls (HC) were enrolled in the study. Serum osteopontin, vitronectin, IL-6, and TGF-ß levels were analyzed. RESULTS: Serum osteopontin levels were higher in the SSc and SLE groups compared to the HC group (p < 0.01 and p < 0.001, respectively). However, it was not correlated with disease activity and severity scores in the SSc group. On the other hand, serum vitronectin levels were lower in the SSc group than in the SLE and HC groups (p < 0.001 for both). CONCLUSIONS: These results may suggest that osteopontin levels may be increased due to the inflammatory process and osteopontin has not a specific role on fibrosis in SSc. On the other hand, serum vitronectin levels decrease in SSc in contrast to SLE. It may be concluded that the one cause of decreased serum vitronectin levels in SSc may be its accumulation in fibrotic area.

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.

Diagnostic and prognostic roles of serum vitronectin in hepatitis B-related hepatocellular carcinoma.

BACKGROUND: Vitronectin (VN) might be involved in the progression of hepatocellular carcinoma (HCC). OBJECTIVE: This study was designed to evaluate the diagnostic and prognostic value of serum vitronectin among HCC patients. METHODS: A total of 105 patients with HCC, 91 with liver cirrhosis, 102 with chronic hepatitis, and 100 healthy subjects were recruited. Serum VN and alpha-fetoprotein (AFP) levels were measured. RESULTS: Serum VN levels were significantly higher in HCC patients than in the other groups. Based on area under receiver operating characteristic curve, serum VN had similar diagnostic value, compared with serum AFP, in distinguishing HCC from the groups, and also improved the diagnostic value of AFP alone. Serum VN levels were associated with the degree of histological differentiation, multiple foci, vascular tumor thrombosis and tumor node metastasis stage. Serum VN was an independent predictor for early recurrence and disease-free survival. Moreover, serum VN possessed similar prognostic predictive performance as compared to serum AFP and also significantly enhanced the prognostic value of AFP alone. CONCLUSIONS: Elevated serum VN levels represented high diagnostic value and had close relation to clinicopathological factors and early recurrence, suggesting that serum VN might be a useful diagnostic and prognostic marker for HCC.

Diagnostic and prognostic value of serum vitronectin levels in human glioma.

OBJECTIVE: Vitronectin is an extracellular matrix protein, the synthesis of which by glioma cells correlates with tumor grade. The current study was designed to investigate the relationship between serum vitronectin levels and clinicopathological characteristics, diagnosis and prognosis in glioma patients. METHODS: In a prospective observatory study, a total of 98 glioma patients, 98 healthy controls, 98 other non-glioma brain tumors, and 98 other non-tumor neurological diseases were recruited. Following univariate analyses, multivariate analyses were performed to explore the associations of serum vitronectin levels with survival and clinicopathological parameters. Receiver operating characteristic curve analysis was done to assess its diagnostic and prognostic predictive value. RESULTS: Serum vitronectin levels were significantly elevated in glioma patients as compared with other groups. High Wealth Health Organization grade was independently associated with high vitronectin levels. Serum vitronectin levels could significantly distinguish glioma patients from other groups and discriminate high-grade glioma from low-grade glioma. Vitronectin levels markedly predicted 5-year progression and 5-year mortality. Moreover, serum vitronectin was identified as an independent predictor for 5-year overall survival and 5-year progression-free survival as well as 5-year mortality and 5-year progression. CONCLUSION: Serum vitronectin may be a promising diagnostic and prognostic biomarker that can be detected in the peripheral blood of patients with glioma.