Leucine-rich Α2-glycoprotein Research Articles

Abstract 1039: Leucine-rich α2 glycoprotein modulates the effect of TGFβ1 on the growth of lewis lung carcinoma cells

Abstract Background: Leucine-rich α2 glycoprotein (LRG) is an approximately 50 kDa serum glycoprotein. It has been reported that the expression levels of LRG were elevated in the sera and tissues in several cancers. While it has been demonstrated that LRG modulated TGFβ1 signaling in endothelial cells and resulted in the promotion of pathogenic angiogenesis, the precise function of LRG in cancer remains unclear. The aim of this study was to investigate the role of LRG on the cellular response by TGFβ1 in Lewis lung carcinoma (LLC) cells. Methods: Parental LLC, mLRG-overexpressing LLC or control vector transfected LLC cells were subcutaneously transplanted into LRG knockout (KO) or Wild-type (WT) C57BL/6J mice and tumor growth was monitored. Cell proliferation treated with TGFβ1 was evaluated by WST-8 assay. Caspase activity was measured with Caspase-GloTM assay kit, protein expression levels were analyzed by western blotting and gene expression was analyzed with quantitative real-time PCR. LLC tumor growth were monitored in LRG KO or WT mice treated with TGFβRI inhibitor (SB431542) or vehicle. Apoptosis of LLC tumor was evaluated by TUNEL staining. Result: In LRG KO mice, LLC tumor growth was significantly increased compared with that in WT mice. In contrast, overexpression of mLRG significantly inhibited the growth of LLC tumors in WT mice. Treatment of TGFβ1 inhibited the proliferation of LLC cells in vitro. Addition of TGFβ1 strongly inhibited the proliferation of mLRG-overexpressing LLC cells than control vector LLC cells by induction of apoptosis. By TGFβ1 stimulation, induction of apoptosis via activation of smad2 and downstream signaling pathway was significantly increased in mLRG overexpressing LLC compared with control LLC cells in vitro. Treatment of TGFβRI inhibitor significantly enhanced growth and inhibited apoptosis of LLC tumor in WT mice compared with LRG KO mice. Conclusion: We found that LRG promoted TGFβ1-induced apoptosis in LLC, in vitro and in vivo. LRG is involved in the enhancement of TGFβ1-smad signal transduction. Citation Format: Norihiko Takemoto, Satoshi Serada, Minoru Fujimoto, Hidenori Inohara, Tetsuji Naka. Leucine-rich α2 glycoprotein modulates the effect of TGFβ1 on the growth of lewis lung carcinoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1039. doi:10.1158/1538-7445.AM2015-1039

Serum leucine-rich α2-glycoprotein is a useful biomarker for monitoring disease activity in patients with adult-onset Still’s disease

Objective: To investigate whether serum leucine-rich α2-glycoprotein (LRG) levels are elevated in patients with adult-onset Still’s disease (AOSD) and determine their correlation with disease activity parameters.Method: We enrolled 39 patients with AOSD, 47 patients with rheumatoid arthritis (RA), and 39 controls. Forty-five serum samples from the patients with AOSD were assayed for LRG using an enzyme-linked immunosorbent assay (ELISA). Comprehensive AOSD activity was determined by a modified Pouchot score.Results: Serum LRG levels were significantly elevated in patients with AOSD (128.8 ± 40.8 ng/mL) compared to those in patients with RA and in controls (33.9 ± 15.2 ng/mL, p < 0.001 and 22.4 ± 6.1 ng/mL, p < 0.001, respectively). Patients with active AOSD had significantly higher LRG levels than those with inactive disease (141.4 ± 31.3 ng/mL vs. 79.8 ± 37.1 ng/mL, p = 0.002). Serum LRG levels were positively correlated with C-reactive protein (CRP; γ = 0.387, p = 0.015), lactate dehydrogenase (LDH; γ = 0.370, p = 0.026), ferritin (γ = 0.687, p < 0.001) levels, and the modified Pouchot score (γ = 0.756, p < 0.001). Serum LRG levels decreased significantly after treatment in all six patients with active AOSD who had follow-up evaluations (p = 0.007). The best cut-off value for LRG to distinguish AOSD from RA was 67.9 ng/mL, with a sensitivity of 92.3% and a specificity of 97.9%.Conclusions: Serum LRG levels were increased in patients with AOSD and correlated well with disease activity measures. LRG may be a useful biomarker for distinguishing AOSD from RA and for monitoring the disease activity of AOSD.

LRG1 modulates invasion and migration of glioma cell lines through TGF-β signaling pathway

Studies have shown that the abnormal expression of leucine-rich α2 glycoprotein 1 (LRG1) is associated with multiple malignancies, yet its role in glioma pathology remains to be elucidated. In this study, we investigated the role of LRG1 in regulating proliferation, migration and invasion of glioma cells by establishing glioma cell strains with constitutively silenced or elevated LRG1 expression. LRG1 overexpression and silenced cell lines demonstrated modulation of glioma cellular proliferation, migration and invasion through MTT, cell scratching and Transwell assays. Furthermore, overexpression of LRG1 led to augmented activation of transforming growth factor-β (TGF-β) signaling pathway as well as downregulation of E-cadherin and resultant enhanced invasiveness, which was reversed by TGF-β signaling pathway inhibitor SB431542. In summary, our findings suggest that LRG1 promotes invasion and migration of glioma cells through TGF-β signaling pathway.

Leucine-rich α2 -glycoprotein as a potential biomarker for joint inflammation during anti-interleukin-6 biologic therapy in rheumatoid arthritis.

To investigate whether leucine-rich α2 -glycoprotein (LRG) could be a biomarker for disease activity during interleukin-6 (IL-6) blockade treatment of rheumatoid arthritis (RA). In 59 RA patients who were treated with tocilizumab for 24 weeks, serum LRG levels were determined by enzyme-linked immunosorbent assay. RA disease activity was evaluated by the Clinical Disease Activity Index (CDAI). Receiver operating characteristic (ROC) curve analysis was used to examine the diagnostic performance of LRG and other biomarkers. In monkeys with experimental autoimmune arthritis, swollen joint counts, joint pathologic changes, and blood levels of C-reactive protein (CRP) and LRG were evaluated after treatment with anti-IL-6 receptor antibody. Among tocilizumab-treated RA patients, those with active disease (CDAI >2.8) had significantly higher serum LRG levels compared to those whose disease was in remission. ROC curve analysis suggested that the LRG level was more useful than the CRP or matrix metalloproteinase 3 level or the erythrocyte sedimentation rate in discriminating between remission and active disease during therapy with tocilizumab. In monkeys treated with IL-6 blockade, joint scores were more closely correlated with LRG levels than with CRP levels. Histologic analysis of joints revealed that LRG levels correlated significantly with granulomatous tissue formation, cartilage degeneration, and bone destruction in IL-6 blockade-treated monkeys with low levels of CRP. Under conditions of IL-6 inhibition, LRG was more useful than other biomarkers in discriminating between active and inactive disease in human RA and in detecting joint inflammation in experimental arthritis. LRG may serve as a convenient biomarker for RA disease activity during IL-6 blockade treatment.

Idiopathic normal pressure hydrocephalus has a different cerebrospinal fluid biomarker profile from Alzheimer's disease.

The diagnosis of idiopathic normal pressure hydrocephalus (iNPH) is sometimes complicated by concomitant Alzheimer's disease (AD) pathology. The purpose of the present study is to identify an iNPH-specific cerebrospinal fluid (CSF) biomarker dynamics and to assess its ability to differentiate iNPH from AD. Total tau (t-tau), tau phosphorylated at threonine 181 (p-tau), amyloid-β (Aβ) 42 and 40, and leucine-rich α-2-glycoprotein (LRG) were measured in 93 consecutive CSF samples consisting of 55 iNPH (46 tap test responders), 20 AD, 11 corticobasal syndrome, and 7 spinocerebeller disease. Levels of t-tau and p-tau were significantly decreased in iNPH patients especially in tap test responders compared to AD. Correlation was observed between Mini-Mental State Examination scores and Aβ42 in AD (R = 0.44) and mildly in iNPH (R = 0.28). Although Aβ42/40 ratio showed no significant difference between iNPH and AD (p = 0.08), the levels of Aβ40 and Aβ42 correlated positively with each other in iNPH (R = 0.73) but much less in AD (R = 0.26), suggesting that they have discrete amyloid clearance and pathology. LRG levels did not differ between the two. Thus, our study shows that although CSF biomarkers of iNPH patients can be affected by concomitant tau and/or amyloid pathology, CSF t-tau and p-tau are highly useful for differentiation of iNPH and AD.

Stable knockdown of LRG1 by RNA interference inhibits growth and promotes apoptosis of glioblastoma cells in vitro and in vivo.

Leucine-rich α2 glycoprotein 1 (LRG1) has been shown to be aberrantly expressed in multiple human malignancies. However, the biological functions of LRG1 in human glioblastoma remain unknown. Here, we report for the first time the role of LRG1 in glioblastoma development based on the preliminary in vitro and in vivo data. We first confirmed the expression of LRG1 in human glioblastoma cell lines. Next, to investigate the role of LRG1 in the tumorigenesis and development of glioblastoma, a short hairpin RNA (shRNA) construct targeting LRG1 mRNA was transfected into U251 glioblastoma cells to generate a cell line with stably silenced LRG1 expression. The results showed that silencing of LRG1 significantly inhibited cell proliferation, induced cell cycle arrest at G0/G1 phase, and enhanced apoptosis in U251 cells in vitro. Consistently, LRG1 silencing resulted in the downregulation of key cell cycle factors including cyclin D1, B, and E and apoptotic gene Bcl-2 while elevated the levels of pro-apoptotic Bax and cleaved caspase-3, as determined by Western blot analysis. We further demonstrate that the silencing of LRG1 expression effectively reduced the tumorigenicity of U251 cells, delayed tumor formation, and promoted apoptosis in a xenograft tumor model in vivo. In conclusion, silencing the expression of LRG1 suppresses the growth of glioblastoma U251 cells in vitro and in vivo, suggesting that LRG1 may play a critical role in glioblastoma development, and it may have potential clinical implications in glioblastoma therapy.

Neutrophils Promote Hematopoiesis Via a Novel Mechanism Involving Leucine Rich α-2 Glycoprotein (LRG)

Leucine-rich α2 glycoprotein (LRG), the founding member of the leucine-rich repeat superfamily of proteins, was initially identified in serum more than 30 years ago, but its biologic function has remained elusive. A role for LRG has been implicated in inflammation and angiogenesis. Our laboratory previously identified cDNA and genomic clones for human and murine LRG, and showed that ectopically expressed LRG localizes to the granule compartment in transfected myeloid cell lines and promotes their granulocytic differentiation. We also demonstrated that expression of LRG is transcriptionally regulated during neutrophil granulocyte differentiation in a manner similar to that reported for genes encoding the different subsets of neutrophil granule proteins. The presence of LRG in primary neutrophils and a role for LRG in hematopoiesis, however, have not been previously described. Based on our prior studies in transfected myeloid cell lines, we considered the tantalizing possibility that LRG is a novel neutrophil granule protein that is secreted extracellularly upon neutrophil activation to modulate hematopoiesis. To investigate this, we examined LRG in primary human neutrophils isolated from healthy volunteers. Immunoblot analysis of whole cell lysates from neutrophils (97% purity) identified a higher molecular weight LRG species in neutrophils (62 kDa) compared to serum (50 kDa); our data demonstrate the difference in apparent molecular weight is due to differential glycosylation. Immunofluorescence microscopy using antibodies to human LRG and antibodies to the neutrophil granule proteins myeloperoxidase (MPO), lactoferrin (LF), and matrix metalloproteinase 9 (MMP-9, also known as gelatinase), along with fluorescently-labeled secondary antibodies, demonstrated the presence of LRG in the cytoplasm of neutrophils in a compartment corresponding to LF. ELISA and immunoblot analyses of subcellular fractions from isolated neutrophils prepared by nitrogen cavitation demonstrated the presence of LRG in LF-containing fractions as well as some MMP-9-containing fractions, consistent with localization of LRG to the secondary/tertiary granule compartment. Neutrophil exocytosis assays using ionomycin, phorbol-12-myristate 13-acetate, and f-Met-Leu-Phe as stimulants also indicated that LRG is co-released with LF and MMP9, but not with MPO. Notably, LRG secreted from activated neutrophils could bind cytochrome c as reported for LRG purified from serum. Recent reports that LRG can also bind to the TGFβR1 receptor on endothelial cells prompted us to investigate the effects of LRG on TGFβ signaling in hematopoietic cells. LRG significantly antagonized the inhibitory effect of TGFβ on HL-60 cell proliferation (n=3; p<0.05) and also on colony growth of human hematopoietic progenitor cells. When LRG was added to hematopoietic progenitor cells cultured in TGFβ-containing Methocult (SF H4436, serum free), a 50% increase in CFU-GMs was observed. Collectively, these data suggest a novel mechanism whereby neutrophils modulate hematopoiesis in the microenvironment via extracellular release of LRG, and invoke an additional role for neutrophils in innate immunity that has not previously been reported DisclosuresNo relevant conflicts of interest to declare.

Identification of host-immune response protein candidates in the sera of human oral squamous cell carcinoma patients.

One of the most common cancers worldwide is oral squamous cell carcinoma (OSCC), which is associated with a significant death rate and has been linked to several risk factors. Notably, failure to detect these neoplasms at an early stage represents a fundamental barrier to improving the survival and quality of life of OSCC patients. In the present study, serum samples from OSCC patients (n = 25) and healthy controls (n = 25) were subjected to two-dimensional gel electrophoresis (2-DE) and silver staining in order to identify biomarkers that might allow early diagnosis. In this regard, 2-DE spots corresponding to various up- and down-regulated proteins were sequenced via high-resolution MALDI-TOF mass spectrometry and analyzed using the MASCOT database. We identified the following differentially expressed host-specific proteins within sera from OSCC patients: leucine-rich α2-glycoprotein (LRG), alpha-1-B-glycoprotein (ABG), clusterin (CLU), PRO2044, haptoglobin (HAP), complement C3c (C3), proapolipoprotein A1 (proapo-A1), and retinol-binding protein 4 precursor (RBP4). Moreover, five non-host factors were detected, including bacterial antigens from Acinetobacter lwoffii, Burkholderia multivorans, Myxococcus xanthus, Laribacter hongkongensis, and Streptococcus salivarius. Subsequently, we analyzed the immunogenicity of these proteins using pooled sera from OSCC patients. In this regard, five of these candidate biomarkers were found to be immunoreactive: CLU, HAP, C3, proapo-A1 and RBP4. Taken together, our immunoproteomics approach has identified various serum biomarkers that could facilitate the development of early diagnostic tools for OSCC.

178: Leucine-rich α2 glycoprotein promotes TGFβ1-induced apoptosis in the lewis lung carcinoma cell lines

Aims Leucine-rich α2 glycoprotein (LRG) is an approximately 50 kDa glycoprotein originally found in the blood. It has been reported that the expression levels of LRG were elevated in the sera of several cancers. While it has been demonstrated that LRG modulated TGFβ1 signaling in endothelial cells and promoted pathogenic angiogenesis, the precise function of LRG in cancer remains unclear. In this study, we investigated the role of LRG on the cellular response by TGFβ1 in Lewis lung carcinoma (LLC) cell line. Methods Parental LLC, mLRG-overexpressing LLC or control vector transfeced LLC cells were subcutaneously transplanted into LRG Knockout(KO) or Wild-type(WT) C57BL/6 J mice and tumor growth was monitored. Cell proliferation treated with TGFβ1 was evaluated by WST-8 assay. Caspase activity was measured with Casapase-Glo™ assay kit, protein expression levels were analyzed by western blotting and gene expression was analyzed with Quantitative real-time PCR. LLC tumor growth were monitored in LRG KO or WT mice treated with TGFβRI inhibitor(SB431542) or vehicle. Apoptosis of LLC tumor was evaluated by TUNEL staining. Result LLC tumor growth in LRG KO mice was significantly enhanced compared with that in WT mice. Conversely, overexpression of mLRG significantly inhibited the growth of LLC tumors in WT mice. TGFβ1 inhibited the proliferation of LLC cells in vitro. Addition of TGFβ1 strongly inhibited the proliferation of mLRG-overexpressing LLC cells than control vector LLC cells by induction of apoptosis. By TGFβ1 stimulation, induction of apoptosis via activation of smad2 and downstream signaling pathway was significantly increased in mLRG overexpressing LLC compared with control LLC cells in vitro. TGFβRI inhibitor significantly enhanced growth and inhibited apoptosis of LLC tumor in WT mice compared with LRG KO. Conclusion Our data showed that LRG promotes TGFβ1-induced apoptosis in LLC, in vitro and in vivo. LRG might be classified as a novel protein that modulates the effects of cytokines.

THU0409 Leucine-Rich Alpha2-Glycoprotein is A Useful Biomarker for Monitoring of Disease Activity in Patients with Adult-Onset Still's Disease

BackgroundLeucine-rich alpha2-glycoprotein (LRG) is a protein which contains leucine-rich repeats. Though physiologic functions of LRG have not been clearly defined, its expression was up-regulated during neutrophil differentiation and its levels...

Leucine-Rich α2-Glycoprotein Is a Novel Biomarker of Neurodegenerative Disease in Human Cerebrospinal Fluid and Causes Neurodegeneration in Mouse Cerebral Cortex

Leucine-rich α2-glycoprotein (LRG) is a protein induced by inflammation. It contains a leucine-rich repeat (LRR) structure and easily binds with other molecules. However, the function of LRG in the brain during aging and neurodegenerative diseases has not been investigated. Here, we measured human LRG (hLRG) concentration in the cerebrospinal fluid (CSF) and observed hLRG expression in post-mortem human cerebral cortex. We then generated transgenic (Tg) mice that over-expressed mouse LRG (mLRG) in the brain to examine the effects of mLRG accumulation. Finally, we examined protein-protein interactions using a protein microarray method to screen proteins with a high affinity for hLRG. The CSF concentration of hLRG increases with age and is significantly higher in patients with Parkinson’s disease with dementia (PDD) and progressive supranuclear palsy (PSP) than in healthy elderly people, idiopathic normal pressure hydrocephalus (iNPH) patients, and individuals with Alzheimer’s disease (AD). Tg mice exhibited neuronal degeneration and neuronal decline. Accumulation of LRG in the brains of PDD and PSP patients is not a primary etiological factor, but it is thought to be one of the causes of neurodegeneration. It is anticipated that hLRG CSF levels will be a useful biomarker for the early diagnosis of PDD and PSP.

Differentially Expressed Proteins in Chronic Active Hepatitis, Cirrhosis, and HCC Related to HCV Infection in Comparison With HBV Infection: A proteomics study

BackgroundHepatocellular carcinoma is a highly progressive cancer in the case of late diagnosis which is frequently associated with HBV and HCV viral infections.ObjectivesTo identify differentially expressed serum proteins among three main stages of HCV infection and healthy individuals, and their comparisons with sera from patients with the same stage of HBV infection.Patients and MethodsTwo-dimensional polyacrylamide gel electrophoresis combined with liquid chromatography-tandem mass spectrometry was performed on 47 sera from healthy volunteers, those with chronic active hepatitis, cirrhosis and HCC patients associated with HBV and HCV infections.ResultsAmong these, 62 spots were differentially expressed (≥ 1.5 fold; P < 0.05), of which 42 spots that corresponded to 15 proteins were identified by liquid chromatography-tandem mass spectrometry. CD5-like antigen (CD5L) was differentially expressed between cirrhosis and HCC patients with HCV infection. Leucine-rich α2-glycoprotein (LRG) and haptoglobin (HP) α2 isoforms differed in the HCC that was associated with either HCV or HBV infections.ConclusionsCD5L might be a useful biomarker for early diagnosis of HCC in HCV cirrhotic patients. LRG and HP α2 isoforms could be potential markers for distinguishing viral HCC. Our results also further support the presence of varying molecules involved in hepatocarcinogenesis in HBV when compared with HCV infection.

Tumour expression of bladder cancer‐associated urinary proteins

WHAT'S KNOWN ON THE SUBJECT? AND WHAT DOES THE STUDY ADD?: The current basis for diagnosis and prognosis in urinary bladder cancer is based on the pathologists' assessment of a biopsy of the tumour. Urinary biomarkers are preferable as they can be non-invasively sampled. Urinary cytology is the only test with widespread use but is hampered by poor reproducibility and low sensitivity. By studying the protein expression in bladder tumour tissue samples of proteins previously found in elevated levels in the urine of patients with bladder cancer, we have been able to show that these proteins originate from the tumour. The immunoreactivity of three of the investigated proteins increased with higher stage. Also a serine peptidase inhibitor was found to be predictive of progression from non-muscle-invasive to muscle-invasive tumours. To analyse the expression of five bladder cancer-associated urinary proteins and investigate if expression is related to the malignant phenotype of the tumour. To explore the possible prognostic value of these proteins. Urine samples, 16 from patients with bladder cancer and 26 from controls, were used in Western Blotting experiments. Tissue microarrays with bladder tissue from 344 patients diagnosed with bladder cancer between 1984 and 2005 was used in immunohistochemistry experiments. The proteins apolipoprotein E (APOE), fibrinogen β chain precursor (FGB), leucine-rich α2-glycoprotein (LRG1), polymerase (RNA) I polypeptide E (POLR1E), α1-antitrypsin (SERPINA1) and topoisomerase 2A (TOP2A) were probed with antibodies validated by the Human Protein Atlas. Increased expressions of APOE, FGB and POLR1E were correlated with increased tumour stage (P < 0.001). Expression of SERPINA1 in Ta and T1 tumours was found to increase the risk of tumour progression (hazard ratio 2.57, 95% confidence interval 1.13-5.87; P = 0.025) CONCLUSIONS: All proteins previously detected in urine from patients with bladder cancer were also expressed in bladder cancer tissue. The expression of APOE, FGB and POLR1E increased with stage and they are potential diagnostic markers. SERPINA1 was identified as a prognostic marker candidate.

Detection and Diagnostic Value of Urine Leucine-Rich α-2-Glycoprotein in Children With Suspected Acute Appendicitis

Previously, we used a proteomics approach for the discovery of new diagnostic markers of acute appendicitis and identified leucine-rich α-2-glycoprotein (LRG) that was elevated in the urine of children with acute appendicitis and enriched in diseased appendices. Here, we sought to evaluate the diagnostic utility of enzyme-linked immunosorbent assay (ELISA) of urine LRG in a blinded, prospective, cohort study of children being evaluated for acute abdominal pain. Urine LRG concentration was measured with a commercially available LRG ELISA and selected ion monitoring mass spectrometry. Urine LRG test performance was evaluated blindly against the pathologic diagnosis and histologic grade of appendicitis. Urine LRG was measured in 49 patients. Mean urine LRG concentration measured with commercial LRG ELISA was significantly elevated in patients with acute appendicitis but exhibited an interference effect. Direct measurements using selected ion monitoring mass spectrometry demonstrated that LRG was elevated more than 100-fold in patients with acute appendicitis compared with those without, with the receiver operating characteristic area under the curve of 0.98 (95% confidence interval 0.96 to 1.0). Among patients with acute appendicitis, elevations of urine LRG measured with ELISA and selected ion monitoring mass spectrometry correlated with the histologic severity of appendicitis. Urine LRG ELISA allows for discrimination between patients with and without acute appendicitis but exhibits limited accuracy because of immunoassay interference. Direct measurements of urine LRG with selected ion monitoring mass spectrometry demonstrate superior diagnostic performance. Development of a clinical-grade urine LRG assay is needed to advance the diagnostic accuracy of clinical evaluations of appendicitis.

Proteomic Analysis of Coronary Sinus Serum Reveals Leucine-Rich α2-Glycoprotein as a Novel Biomarker of Ventricular Dysfunction and Heart Failure

Background— Heart failure (HF) prevention strategies require biomarkers that identify disease manifestation. Increases in B-type natriuretic peptide (BNP) correlate with increased risk of cardiovascular events and HF development. We hypothesize that coronary sinus serum from a high BNP hypertensive population reflects an active pathological process and can be used for biomarker exploration. Our aim was to discover differentially expressed disease-associated proteins that identify patients with ventricular dysfunction and HF. Methods and Results— Coronary sinus serum from 11 asymptomatic, hypertensive patients underwent quantitative differential protein expression analysis by 2-dimensional difference gel electrophoresis. Proteins were identified using mass spectrometry and then studied by enzyme-linked immunosorbent assay in sera from 40 asymptomatic, hypertensive patients and 105 patients across the spectrum of ventricular dysfunction (32 asymptomatic left ventricular diastolic dysfunction, 26 diastolic HF, and 47 systolic HF patients). Leucine-rich α2-glycoprotein (LRG) was consistently overexpressed in high BNP serum. LRG levels correlate significantly with BNP in hypertensive, asymptomatic left ventricular diastolic dysfunction, diastolic HF, and systolic HF patient groups ( P ≤0.05). LRG levels were able to identify HF independent of BNP. LRG correlates with coronary sinus serum levels of tumor necrosis factor-α ( P =0.009) and interleukin-6 ( P =0.021). LRG is expressed in myocardial tissue and correlates with transforming growth factor-βR1 ( P &lt;0.001) and α-smooth muscle actin ( P =0.025) expression. Conclusions— LRG was identified as a serum biomarker that accurately identifies patients with HF. Multivariable modeling confirmed that LRG is a stronger identifier of HF than BNP and this is independent of age, sex, creatinine, ischemia, β-blocker therapy, and BNP.

ITRAQ-based proteomic identification of leucine-rich α-2 glycoprotein as a novel inflammatory biomarker in autoimmune diseases

ObjectiveTo identify a novel serum biomarker of disease activity in inflammatory autoimmune disorders.MethodsSera obtained from rheumatoid arthritis (RA) patients before and after anti-TNF therapy were analysed by iTRAQ (isobaric tags...

Potential diagnostic biomarkers in serum of idiopathic pulmonary arterial hypertension

The pathogenesis of idiopathic pulmonary arterial hypertension (IPAH) is unknown, and the syndrome of IPAH remains a diagnostic and therapeutic challenge. The present study investigated the disease-specific proteins that aid in the diagnosis of IPAH and thus to study their role in the disease process. A comparative proteomic analysis was used for clinical screening of serum proteins in 10 patients with IPAH and compared with 10 normal subjects. Furthermore, enzyme linked immunosorbent assay (ELISA) was performed for comparison with serum proteins between individual IPAH patients and controls. Nine proteins and their isoforms, including leucine-rich alpha-2-glycoprotein (LRG), haptoglobin precursor, albumin isoform 2, transferrin variant, C3 complement, hydroxypyruvate reductase isoform 1, RAF1, fibrinogen isoformgamma-A and fibrinogen isoformgamma-B showed significant changes in serum of IPAH patients compared with controls by proteomic analysis. And significant higher serum levels of LRG in IPAH patients compared with controls were found by ELISA. Correlation analysis disclosed a significant association between serum LRG concentrations and New York Heart Association (NYHA) functional class (r=0.71, P<0.01) and cardiac output (CO) (r=-0.65, P<0.01). These results indicate that there are significant differences in the expression of proteins in the serum of patients with IPAH and normal subjects. And the measurement of LRG, RAF1 and C3 complement levels in the serum may be helpful for the diagnosis of IPAH. In particular, LRG may be a specific prognostical biomarker of IPAH.

Posttransplant Proteinuria Associated With Everolimus

Anti-mTOR may induce proteinuria when utilized after renal transplantation. Little is known about the pathogenesis and composition of proteinuria. To clarify this unresolved aspect, we analyzed urinary protein composition utilizing an integrated proteomics approach, including quantitative assays, 2-dimensional electrophoresis, MALDI-TOF, and Western blots among 48 renal transplant recipients treated with everolimus (EVL; n = 31) or enteric-coated mycophenolic acid (EC-MPA; n = 17). High (>3 g/d) or intermediate levels of proteinuria (1–3 g) developed in 12 EVL patients (39%) compared with 4 subjects (23%) in the EC-MPA group. Proteinuria, which started during the first 2 days after EVL, tended to reduce during the follow-up. Quantitative proteomics showed an increase in low molecular proteins β2 microglobulin ( P < .001) and α1 microglobulin ( P < .025). Qualitative proteomics showed a marked increase among all urinary components in EVL and EC-MPA patients. Major changes involved typical components of glomerular damage: albumin, Zn-α1 glycoprotein, α2HS glycoprotein, and leucine-rich α2 glycoprotein. In addition, we observed specific biomarkers for EVL: clusters of α1-antitrypsin fragments and monoclonal λ chains. In conclusion, EVL induced proteinuria of a mixed glomerular and tubular origin that correlated with the start of treatment and reached nephrotic ranges in few cases. The specific urinary markers may reflect renal alterations related to the transplant or specific alterations associated with the drug.

Up-regulation of the expression of leucine-rich α 2-glycoprotein in hepatocytes by the mediators of acute-phase response

Leucine-rich α 2-glycoprotein (LRG) is a plasma protein in which leucine-rich repeats (LRRs) were first discovered. Although the physiological function of LRG is not known, increases in the serum level of LRG have been reported in various diseases. In this study, we found that LRG was induced by recombinant human IL-6 in human hepatoma HepG2 cells. The induction of LRG by IL-6 was up-regulated synergistically with either IL-1β or TNFα in a pattern similar to those for type 1 acute-phase proteins. We also found that lipopolysaccharide (LPS) administered intraperitoneally to mice enhanced dose-dependently the expression of LRG mRNA in the liver as well as those for mouse major acute-phase proteins. These results strongly suggest that LRG was a secretory type 1 acute-phase protein whose expression was up-regulated by the mediator of acute-phase response.

LRG-accelerated differentiation defines unique G-CSFR signaling pathways downstream of PU.1 and C/EBPε that modulate neutrophil activation

Expression of leucine-rich alpha2 glycoprotein (LRG), a member of the leucine-rich repeat family of proteins, was recently shown to be up-regulated during neutrophil differentiation. Its precise role in granulopoiesis, however, remains unknown. In this paper, we show that the transcription factors PU.1 and C/EBPepsilon that regulate the expression of multiple myeloid-specific genes also bind to the LRG promoter. We also demonstrate that LRG localizes to the same cytoplasmic compartment as myeloperoxidase and that G-CSF treatment of the 32Dcl3 myeloid cell line induces nuclear translocation of LRG. Stable transfection of LRG into 32Dcl3 cells resulted in accelerated, G-CSF-mediated neutrophil differentiation and induction of CD11b expression. In contrast, constitutive expression of LRG in 32Dwt18 cells, expressing a chimeric erythropoietin (Epo)/G-CSFR consisting of the EpoR extracellular domain fused to the G-CSFR transmembrane and cytoplasmic domains, failed to induce accelerated neutrophil differentiation and CD11b expression in response to Epo stimulation. LRG-mediated accelerated differentiation and CD11b expression were found to correlate with an increased level of phospho-Stat3 but not with PU.1 or p27(kip1) levels. Hence, similar to other genes involved in neutrophil differentiation, the expression of LRG also appears to be regulated by PU.1 and C/EBPepsilon. Collectively, these findings suggest a role for LRG in modulating neutrophil differentiation and expression of CD11b via nonredundant G-CSFR signals.