Leucine-rich Alpha-2-glycoprotein 1 Research Articles

Graphene-based synthetic peptide electrochemical sensor for colorectal cancer diagnosis

This work reports the development of an electrochemical sensor using graphene-peptide conjugates for detecting colorectal cancer (CRC) biomarker leucine-rich alpha-2 glycoprotein-1 (LRG1). To enable LRG1 quantification, we rationally designed peptides with dual graphene anchoring motifs for optimal orientation and binding activity when immobilized on a reduced graphene oxide (rGO) electrochemical transducer surface. The graphene nanomaterial provides several advantages such as high conductivity, large surface area, and excellent stability that can enhance the sensor's analytical performance metrics. Furthermore, the synthetic peptides offer benefits like smaller size, specificity, ease of modification and cost-effective production compared to traditional antibody receptors. Under optimized conditions, the peptide sensor exhibited high sensitivity of 22.3 μA/(ng/mL.cm2), low limit of detection (75 pg/mL LRG1 in serum), accuracy of 101.1 % spiked recovery, and precision within 6 % RSD. Testing with colonoscopy-classified patient serum specimens discriminated normal, precancerous adenomatous polyps and malignant carcinoma stages based on LRG1 overexpression. A 24 % elevation for adenomas and 103 % higher levels in CRC were observed. Validation with spiked plasma samples indicated 97–104 % recovery and <7 % RSD, proving accurate detection capability. Comparison to antibody-based sensors showed superior linear range, sensitivity, reproducibility, and faster assay time. This demonstrates the promise of computational peptide designing combined with advanced nanomaterials for electrochemical detection of CRC progression through serum protein biomarkers.

Increased level of serum leucine-rich-alpha-2-glycoprotein 1 in patients with clear cell renal cell carcinoma

BackgroundCurrently, no useful serum markers exist for clear cell renal cell carcinoma (ccRCC), making early detection challenging as diagnosis relies solely on imaging tests. Radiation exposure is also a concern due to multiple required CT examinations during treatment. Renal cell carcinoma (RCC) histological types include ccRCC and non-clear cell RCC (non-ccRCC); however, treatment response to medications varies which necessitates accurate differentiation between the two. Therefore, we aimed to identify a novel serum marker of RCC. Increased LRG1 expression in the serum has been demonstrated in multiple cancer types. However, the expression of LRG1 expression in the serum and cancer tissues of patients with RCC has not been reported. Since ccRCC is a hypervascular tumor and LRG1 is capable of accelerating angiogenesis, we hypothesized that the LRG1 levels may be related to ccRCC. Therefore, we examined LRG1 expression in sera from patients with RCC.MethodsUsing an enzyme-linked immunosorbent assay, serum levels of leucine-rich-alpha-2-glycoprotein 1 (LRG1) were measured in 64 patients with ccRCC and 22 patients non-ccRCC who underwent radical or partial nephrectomy, as well as in 63 patients without cancer.ResultsMedian values of serum LRG1 and their inter-quartile ranges were 63.2 (42.8–94.2) µg/mL in ccRCC, 23.4 (17.7–29.6) µg/mL in non-ccRCC, and 36.0 (23.7–56.7) µg/mL in patients without cancer, respectively (ccRCC vs. non-ccRCC or patients without cancer: P < 0.001). C-reactive protein (CRP) levels (P = 0.002), anemia (P = 0.037), hypercalcemia (P = 0.023), and grade (P = 0.031) were independent predictors of serum LRG1 levels in ccRCC. To assess diagnostic performance, the area under the receiver operating characteristic curve of serum LRG1 was utilized to differentiate ccRCC from non-cancer and non-ccRCC, with values of 0.73 (95% CI, 0.64–0.82) and 0.91 (95% CI, 0.82–0.96), respectively.ConclusionsLRG1 served as a serum marker associated with inflammation, indicated by CRP, anemia, hypercalcemia, and malignant potential in ccRCC. Clinically, serum LRG1 levels may assist in differentiating ccRCC from non-ccRCC with excellent diagnostic accuracy.

Differences in the Synovial Fluid Proteome of Septic and Aseptic Implant Failure.

Implant loosening is a severe complication after total joint replacement. Here, differential diagnosis between septic and aseptic cases is crucial for further surgical treatment, but low-grade periprosthetic joint infections (PJIs) in particular remain a challenge. In this study, we analyzed the synovial fluid proteome of 21 patients undergoing revision surgery for septic (eight cases) or aseptic (thirteen cases) implant failure using LC-MS/MS to identify potential new biomarkers as future diagnostic tools. Staphylococci were found in four cases, Streptococci in two cases, Serratia marcescens and Cutibacterium acnes in one case. Proteomic analysis of the synovial fluid resulted in the identification of 515 different proteins based on at least two peptides. A statistical comparison revealed 37 differentially abundant proteins (p < 0.05), of which 17 proteins (46%) showed a higher abundance in the septic group. The proteins with the highest fold change included the known marker proteins c-reactive protein (7.57-fold) and the calprotectin components protein S100-A8 (4.41-fold) and protein S100-A9 (3.1-fold). However, the protein with the highest fold change was leucine-rich alpha-2-glycoprotein 1 (LRG1) (9.07-fold), a currently discussed new biomarker for inflammatory diseases. Elevated LRG1 levels could facilitate the diagnosis of PJI in the future, but their significance needs to be further investigated.

A proteomic classifier panel for early screening of colorectal cancer: a case control study

BackgroundDiagnosis of colorectal cancer (CRC) during early stages can greatly improve patient outcome. Although technical advances in the field of genomics and proteomics have identified a number of candidate biomarkers for non-invasive screening and diagnosis, developing more sensitive and specific methods with improved cost-effectiveness and patient compliance has tremendous potential to help combat the disease.MethodsWe enrolled three cohorts of 479 subjects, including 226 CRC cases, 197 healthy controls, and 56 advanced precancerous lesions (APC). In the discovery cohort, we used quantitative mass spectrometry to measure the expression profile of plasma proteins and applied machine-learning to select candidate proteins. We then developed a targeted mass spectrometry assay to measure plasma concentrations of seven proteins and a logistic regression classifier to distinguish CRC from healthy subjects. The classifier was further validated using two independent cohorts.ResultsThe seven-protein panel consisted of leucine rich alpha-2-glycoprotein 1 (LRG1), complement C9 (C9), insulin-like growth factor binding protein 2 (IGFBP2), carnosine dipeptidase 1 (CNDP1), inter-alpha-trypsin inhibitor heavy chain 3 (ITIH3), serpin family A member 1 (SERPINA1), and alpha-1-acid glycoprotein 1 (ORM1). The panel classified CRC and healthy subjects with high accuracy, since the area under curve (AUC) of the training and testing cohort reached 0.954 and 0.958. The AUC of the two independent validation cohorts was 0.905 and 0.909. In one validation cohort, the panel had an overall sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of 89.9%, 81.8%, 89.2%, and 82.9%, respectively. In another blinded validation cohort, the panel classified CRC from healthy subjects with a sensitivity of 81.5%, specificity of 97.9%, and overall accuracy of 92.0%. Finally, the panel was able to detect APC with a sensitivity of 49%.ConclusionsThis seven-protein classifier is a clear improvement compared to previously published blood-based protein biomarkers for detecting early-stage CRC, and is of translational potential to develop into a clinically useful assay.

LRG1 predicts the prognosis and is associated with immune infiltration in thyroid cancer: a bioinformatics study.

The diagnostic and prognostic value of the leucine-rich alpha-2-glycoprotein 1 (LRG1) gene in thyroid cancer remains unclear. Using the Cancer Genome Atlas (TCGA) database, we conducted a bioinformatics analysis to determine the role of LRG1 in thyroid cancer. Data from 512 patients with thyroid cancer and 59 normal individuals were collected from TCGA database. The Kruskal-Wallis test and logistic analysis were used to examine the relationship between LRG1 expression and clinicopathologic characteristics. Cox regression and Kaplan-Meier analysis were used to determine the predictive value of LRG1 on clinical outcomes. Single-sample gene set enrichment analysis (ssGSEA) was used to reveal associations between LRG1 expression and immune infiltration levels in thyroid cancer. LRG1 was highly expressed in thyroid cancer (P < 0.001) and could effectively distinguish tumor tissue (area under the curve = 0.875) from normal tissue. Moreover, LRG1 was significantly correlated with pathological N stage (odds ratio (OR) = 2.411 (1.659-3.505), P < 0.001). Kaplan-Meier survival analysis revealed that patients with high LRG1 expression had better overall survival (hazard ratio (HR) = 0.30, P = 0.038). Cox regression analysis indicated that pathological M stage was a risk factor for progression-free interval (HR = 5.964 (2.010-17.694), P < 0.001). Using ssGSEA, we found that LRG1 expression was positively correlated with the number of T helper 1 cells (R = 0.435, P < 0.001), dendritic cells (R = 0.442, P < 0.001), and macrophages (R = 0.459, P < 0.001). LRG1 may be an important biomarker for predicting the prognosis of thyroid cancer and represent a suitable target for immunotherapy associated with immune infiltration.

The Effect of High-intensity Functional Training on LRG1 Levels and Its Relationship with Cardiovascular Risk Factors and Atherogenic Indices in Overweight and Obese Women

Introduction: The present study aimed to investigate the effect of high-intensity functional training on leucine-rich alpha-2 glycoprotein 1(LRG1) levels and its relationship with cardiovascular risk factors and atherogenic indicators in overweight and obese women. Method: In this semi-experimental study, 19 overweight and obese women were randomly divided into two groups of high-intensity functional training (n=9) and control (n=10). The participants of the training group performed high-intensity functional exercises three times per week for eight weeks. Before and after the intervention, blood samples were taken to measure LRG1 concentration and lipid profile, in addition to the calculation of atherogenic indices. Analysis of covariance test was used to analyze the data. Results: After eight weeks of high-intensity functional training, LRG1 concentration (P=0.027), plasma atherogenic index (P=0.000), Castelli risk index-I (CRI-I) (P=0.001), and Castelli risk index-II (CRI-II) (P=0.002) were significantly decreased in the experimental group compared to the control group. A positive and significant correlation was observed between changes in LRG1 concentration and changes in body fat percentage (r=0.437, P=0.041), plasma atherogenic index (r=0.512, P=0.025), and CRI-I (r=0.479, P=0.038). A negative and significant correlation was observed between the changes of LRG1 and high-density lipoprotein cholesterol (HDL-C) (r =-0.569, P=0.011). Conclusion: Eight weeks of high-intensity functional training in overweight and obese women is effective in reducing LRG1 and improving cardiovascular risk factors and atherogenic indicators. Therefore, high-intensity functional exercises can be effective as auxiliary exercise interventions in controlling and reducing complications related to overweight and obesity

Correlation between LRG1 and Adipokines in Psoriasis

Background and Objective. Patients with psoriasis may exhibit abnormal changes in serum adipokine levels, which are often related to disease severity of the disease. Leucine-rich alpha-2-glycoprotein 1 (LRG1) is an acute-phase inflammatory protein that may be linked to adipokines in psoriasis. In this study, we evaluated the differences in the expression of adipokines and LRG1 between patients with psoriasis and healthy individuals, analyzed the correlation between the expression of LRG1 and adipokines, and explored their relationship with psoriatic lesions. Methods. In this cross-sectional study, patients with psoriasis (n = 54) and healthy controls (n = 26) were enrolled, and their clinical characteristics were recorded. Fasting venous blood samples were collected from each participant. The serum concentrations of leptin, resistin, adiponectin, and LRG1 in each sample were measured using the enzyme-linked immunosorbent assay. Results. The study included 54 patients with psoriasis vulgaris and 26 healthy controls. The serum levels of LRG1, leptin, and resistin were significantly higher in patients with psoriasis than in healthy controls. Conversely, adiponectin levels were significantly lower in patients with psoriasis. The study showed that LRG1 expression was positively correlated with leptin and resistin expression but negatively correlated with adiponectin expression. Interestingly, only leptin, resistin, and LRG1 expression showed a linear correlation with the Psoriasis Area and Severity Index (PASI). When we categorized patients with psoriasis based on their LRG1 levels, we observed that the group with high LRG1 levels showed a higher PASI. Conclusions. We observed a significant correlation between LRG1 and adipokine expression in patients with psoriasis. In addition, the expression levels of LRG1, leptin, and resistin were observed to be correlated with the severity of psoriasis. We believe that the occurrence and development of psoriasis are collectively influenced by LRG1 and leptin/resistin expression.

Exosome enriched leucine-rich alpha-2-glycoprotein-1 and extracellular matrix protein 1 proteins induce abnormal placental angiogenesis in pregnant mice

IntroductionGestational Diabetes Mellitus (GDM) is characterized by a high risk of fetal macrosomia and placenta hypervascularization. Exosomes has been known participating in various physiological and pathological processes, including pro-angiogenic function. However, the effects of umbilical cord blood derived exosomes from cases of GDM (GDM-exo) on placental vascular network formation remain unclear. MethodsIn the current study, we isolated and identified exosomes in umbilical cord blood from both normal (N-exo) and GDM pregnancies. Meanwhile, we investigated the effects of umbilical cord blood derived exosomes on placental angiogenesis both in vitro and in vivo. ResultsOur data indicated that in a mouse model, the placenta and fetus weight were significantly higher in the ones administrated with GDM-exo when compared with N-exo. Meanwhile, GDM-exo significantly enhanced placental endothelial cells functions in both HUVEC and HPMEC endothelial cell models. Importantly, we explored two up-regulated proteins in GDM-exo, namely leucine-rich alpha-2-glycoprotein-1 (LRG1) and extracellular matrix protein 1 (ECM1) by proteome analysis, which performed largely pro-angiogenic function and probably resulted in hypervascularization in GDM placenta. DiscussionThus, we proposed that abundant LRG1 and ECM1 enriched GDM-exo may take important roles in regulating pathological placental angiogenesis.

LRG1 Promotes ECM Integrity by Activating the TGF-β Signaling Pathway in Fibroblasts.

Leucine-rich alpha-2-glycoprotein 1 (LRG1) mediates skin repair and fibrosis by stimulating the transforming growth factor-beta (TGF-β) signaling pathway. In the present study, we investigated the effect of LRG1 on extracellular matrix (ECM) integrity in fibroblasts, as well as on skin aging. The treatment of dermal fibroblasts with purified recombinant human LRG1 increased type I collagen secretion and decreased matrix metalloproteinase-1 secretion. Additionally, LRG1 promoted SMAD2/SMAD3 phosphorylation in a pattern similar to that of TGF-β1 treatment. An inhibitor of TGF-β receptor 1 abolished LRG1-induced SMAD2 phosphorylation. RNA sequencing identified "extracellular region", "extracellular space", and "extracellular matrix" as the main Gene Ontology terms in the differentially expressed genes of fibroblasts treated with or without LRG1. LRG1 increased TGF-β1 mRNA levels, suggesting that LRG1 partially transactivates the expression of TGF-β1. Furthermore, an increased expression of type I collagen was also observed in fibroblasts grown in three-dimensional cultures on a collagen gel mimicking the dermis. LRG1 mRNA and protein levels were significantly reduced in elderly human skin tissues with weakened ECM integrity compared to in young human skin tissues. Taken together, our results suggest that LRG1 could retard skin aging by activating the TGF-β signaling pathway, increasing ECM deposition while decreasing its degradation.

Expression of LRG-1 in mice with hypertensive renal damage and its significance.

Long-term elevated blood pressure may lead to kidney damage, yet the pathogenesis of hypertensive kidney damage is still unclear. This study aims to explore the role and significance of leucine-rich alpha-2-glycoprotein-1 (LRG-1) in hypertensive renal damage through detecting the levels of LRG-1 in the serum and kidney of mice with hypertensive renal damage and its relationship with related indexes. C57BL/6 mice were used in this study and randomly divided into a control group, an angiotensin II (Ang II) group, and an Ang II+irbesartan group. The control group was gavaged with physiological saline. The Ang II group was pumped subcutaneously at a rate of 1.5 mg/(kg·d) for 28 days to establish the hypertensive renal damage model in mice, and then gavaged with equivalent physiological saline. The Ang II+irbesartan group used the same method to establish the hypertensive renal damage model, and then was gavaged with irbesartan. Immunohistochemistry and Western blotting were used to detect the expression of LRG-1 and fibrosis-related indicators (collagen I and fibronectin) in renal tissues. ELISA was used to evaluate the level of serum LRG-1 and inflammatory cytokines in mice. The urinary protein-creatinine ratio and renal function were determined, and correlation analysis was conducted. Compared with the control group, the levels of serum LRG-1, the expression of LRG-1 protein, collagen I, and fibronectin in kidney in the Ang II group were increased (all P<0.01). After treating with irbesartan, renal damage of hypertensive mice was alleviated, while the levels of LRG-1 in serum and kidney were decreased, and the expression of collagen I and fibronectin was down-regulated (all P<0.01). Correlation analysis showed that the level of serum LRG-1 was positively correlated with urinary protein-creatinine ratio, blood urea nitrogen, and blood creatinine level in hypertensive kidney damage mice. Serum level of LRG-1 was also positively correlated with serum inflammatory factors including TNF-α, IL-1β, and IL-6. Hypertensive renal damage mice display elevated expression of LRG-1 in serum and kidney, and irbesartan can reduce the expression of LRG-1 while alleviating renal damage. The level of serum LRG-1 is positively correlated with the degree of hypertensive renal damage, suggesting that it may participate in the occurrence and development of hypertensive renal damage.

Label-free quantitative proteomics reveals aberrant expression levels of LRG, C9, FN, A1AT and AGP1 in the plasma of patients with colorectal cancer

BackgroundColorectal cancer (CRC) is one of the major causes of cancer-related death worldwide. Although commercial biomarkers of CRC are currently available, they are still lacking in terms of sensitivity and specificity; thus, searching for reliable blood-based biomarkers are important for the primary screening of CRC.MethodsPlasma samples of patients with non-metastatic (NM) and metastatic (M) CRC and healthy controls were fractionated using MARS-14 immunoaffinity chromatography. The flow-through and elute fractions representing low- and high-abundant proteins, respectively, were analyzed by label-free quantitative proteomics mass spectrometry. The functional analysis of the proteins with greater than 1.5-fold differential expression level between the CRC and the healthy control groups were analyzed for their biological processes and molecular functions. In addition, the levels of plasma proteins showing large alterations in CRC patients were confirmed by immunoblotting using two independent cohorts. Moreover, receiver operating characteristic (ROC) curve analysis was performed for individual and combinations of biomarker candidates so as to evaluate the diagnostic performance of biomarker candidates.ResultsFrom 163 refined identifications, five proteins were up-regulated and two proteins were down-regulated in NM-CRC while eight proteins were up-regulated and three proteins were down-regulated in M-CRC, respectively. Altered plasma proteins in NM-CRC were mainly involved in complement activation, while those in M-CRC were clustered in acute-phase response, complement activation, and inflammatory response. Results from the study- and validation-cohorts indicate that the levels of leucine-rich alpha-2-glycoprotein-1(LRG), complement component C9 (C9), alpha-1-acid glycoprotein 1 (AGP1), and alpha-1-antitrypsin (A1AT) were statistically increased, while fibronectin (FN) level was statistically decreased in CRC patients compared to healthy controls, with most alterations found in a metastatic stage-dependent manner. ROC analysis revealed that FN exhibited the best diagnostic performance to discriminate CRC patients and healthy controls while AGP1 showed the best discrimination between the disease stages in both cohorts. The combined biomarker candidates, FN + A1AT + AGP1, exhibited perfect discriminatory power to discriminate between the CRC population and healthy controls whereas LRG + A1AT + AGP1 was likely to be the best panel to discriminate the metastatic stages in both cohorts.ConclusionsThis study identified and quantified distinct plasma proteome profiles of CRC patients. Selected CRC biomarker candidates including FN, LRG, C9, A1AT, and AGP1 may be further applied for screening larger cohorts including disease groups from other types of cancer or other diseases.

Alterations of plasma exosomal proteins and motabolies are associated with the progression of castration-resistant prostate cancer

BackgroundCurrent diagnosis tools for prostate cancer (PCa) such as serum PSA detection and prostate biopsy cannot distinguish dormant tumors from invasive malignancies, either be used as prognosis marker for castration resistant prostate cancer (CRPC), the lethal stage of PCa patients. Exosomes have been widely investigated as promising biomarkers for various diseases. We aim to characterize the proteomic and metabolomic profile of exosomes and to evaluate their potential value for the diagnosis of PCa, especially CRPC. We also investigate the functions of some specific exosome biomarkers in the progression of CRPC.MethodsIntegrated proteomics and metabolomics analysis were performed for plasma-derived exosomes collected from tumor-free controls (TFC), PCa and CRPC patients. Expression of specific exosomal proteins were further validated by targeted 4D-parallel reaction monitoring (PRM) mass spectrometry among the three cohorts. Tissue distribution and functional role of exosomal protein LRG1 was studied in clinical PCa tissue samples and cell line models.ResultsThree potential exosomal protein markers were identified. The apolipoprotein E level in PCa samples was 1.7-fold higher than that in TFC (receiver operating characteristic value, 0.74). Similarly, the levels of exosome-derived leucine-rich alpha2-glycoprotein 1 (LRG1) and inter-alpha-trypsin inhibitor heavy chain H3 (ITIH3) in the CRPC group were 1.7 and 2.04 times, respectively, higher than those in the PCa group (ROC values, 0.84 and 0.85, respectively), indicating that LRG1 and ITIH3 could serve as predictive markers for CRPC. For metabolomic evaluation of exosomes, a series of differentially expressed metabolites were identified, and a combined metabolite panel showed ROC value of 0.94 for distinguishing PCa from TFC and 0.97 for distinguishing CRPC from PCa. Immunohistochemistry of tissue microarray showed that LRG1 protein was significantly upregulated in advanced prostate cancer and functional assay revealed that ectopic expression of LRG1 can significantly enhance the malignant phenotype of prostate cancer cells. More importantly, PCa cell derived LRG1-overexpressed exosomes remarkably promoted angiogenesis.ConclusionIntegration of proteomics and metabolomics data generated proteomic and metabolic signatures of plasma exosomes that may facilitate discrimination of CRPC from PCa and TFC patients, suggesting the potential of exosomal proteins and metabolites as CRPC markers. The study also confirmed the important role of exosomal protein LRG1 in PCa malignant progression.

PS-B01-5: TISSUE-SPECIFIC REGULATION OF LRG1 EXPRESSION IN MULTIPLE HYPERTENSION MODELS OF MOUSE

Object: Leucine-rich alpha-2-glycoprotein-1 (LRG1), which is firstly isolated from human plasma, has been reported to be related to several diseases as a biomarker or a pathogenic factor. Serum LRG1 is associated with the prognosis of diabetic nephropathy and cardiovascular diseases, and we previously demonstrated that LRG1 especially plays a pivotal role in the initial development of diabetic nephropathy. In addition, a previous study showed that increased plasma LRG1 levels in patients with type 2 diabetes mellitus was associated with increased systolic blood pressure (BP), arterial stiffness estimated by pulse wave velocity, and decreased endothelium-dependent vasodilation estimated by laser doppler flowmetry. However, the relationship between LRG1 and BP have remained substantially unknown. With respect to the function of LRG1, LRG1 is expressed in endothelial cells and promotes pathological angiogenesis by modulating TGF-beta signaling. LRG1 switches TGF-beta signaling from the Smad2/3 pathway to the Smad1/5/8 pathway. In addition, the activation of Smad2/3 signaling enhanced endothelial nitric oxide synthase (eNOS) production and promoted endothelium-dependent vasodilation. Suppressing eNOS activity reduces NO levels and leads to endothelial dysfunction and hypertension. Moreover, LRG1 is increased in response to inflammation and oxidative stress in endothelial cells. However, little is known about the relationship between LRG1 in endothelial cells and eNOS. Based on the previous findings, we hypothesized that LRG1 may inhibit eNOS production by potentially blocking the Smad2/3 pathway, which will promote to form a vicious cycle that progresses vascular endothelial damage and hypertension. As the first step to elucidate this hypothesis, we investigated LRG1 expression in various tissues including vascular endothelium in some hypertension models with endothelial dysfunction. Design and method: Male 10–12 weeks-aged C57/BL mice were divided into control and hypertension with endothelial dysfunction. BP was measured using a tail-cuff method during the experiment, and mice were sacrificed and their organs were collected at the end of the experiment. We examined the expression of LRG1, Smad2/3, Smad1/5/8 and NOS in the vascular endothelium, kidney, heart, adipose tissue by PCR, Western blot and immunohistochemical analyses. Results: We are currently conducting the experiments and will present our findings at this conference. Conclusion: We plan to present the results of tissue-specific regulation of LRG1 expression in some hypertension models of mice at this conference.

Leucine-Rich Alpha-2-Glycoprotein 1 is a Systemic Biomarker of Early Brain Injury and Delayed Cerebral Ischemia After Subarachnoid Hemorrhage.

After subarachnoid hemorrhage (SAH), early brain injury (EBI) and delayed cerebral ischemia (DCI) lead to poor outcomes. Discovery of biomarkers indicative of disease severity and predictive of DCI is important. We tested whether leucine-rich alpha-2-glycoprotein 1 (LRG1) is a marker of severity, DCI, and functional outcomes after SAH. We performed untargeted proteomics using mass spectrometry in plasma samples collected at < 48h of SAH in two independent discovery cohorts (n = 27 and n = 45) and identified LRG1 as a biomarker for DCI. To validate our findings, we used enzyme-linked immunosorbent assay and confirmed this finding in an internal validation cohort of plasma from 72 study participants with SAH (22 DCI and 50 non-DCI). Further, we investigated the relationship between LRG1 and markers of EBI, DCI, and poor functional outcomes (quantified by the modified Rankin Scale). We also measured cerebrospinal fluid (CSF) levels of LRG1 and investigated its relationship to EBI, DCI, and clinical outcomes. Untargeted proteomics revealed higher plasma LRG1 levels across EBI severity and DCI in both discovery cohorts. In the validation cohort, the levels of LRG1 were higher in the DCI group compared with the non-DCI group (mean (SD):95 [44] vs. 72 [38]pg/ml, p < 0.05, Student's t-test) and in study participants who proceeded to have poor functional outcomes (84 [39.3] vs. 72 [43.2]pg/ml, p < 0.05). Elevated plasma LRG1 levels were also associated with markers of EBI. However, CSF levels of LRG1 were not associated with EBI severity or the occurrence of DCI. Plasma LRG1 isa biomarker for EBI, DCI, and functional outcomes after SAH. Further studies to elucidate the role of LRG1 in the pathophysiology of SAH are needed.

AGR2 expression as a predictive biomarker for therapy response in esophageal squamous cell carcinoma.

Despite multidisciplinary therapy, the prognosis is poor for esophageal squamous cell carcinoma (ESCC). In the locally advanced stage, neoadjuvant chemoradiotherapy (nCRT) followed by surgery could provide survival benefits to some patients. Here, we aimed to identify for tumor therapy response a biomarker based on RNA sequencing. We collected endoscopic biopsies of 32 ESCC patients, who were divided according to nCRT response, into two groups: the complete response group (n = 13) and the non-complete response group (n = 19). RNA-sequencing data showed that 464 genes were differentially expressed. Increased in non-complete response group, 4 genes increased expressions were AGR2 (anterior gradient 2), GADD45B (growth arrest and DNA damage inducible beta), PPP1R15A (protein phosphatase 1 regulatory subunit 15A) and LRG1 (leucine rich alpha-2-glycoprotein 1). The areas under the curve (AUC) of the AGR2 gene was 0.671 according to read counts of RNA-seq and therapy response of nCRT. In vitro study showed that apoptosis cell was significantly increased in the AGR2-knockdown TE-2 cell line treated with cisplatin and 5-Fluorouracil (5-FU), when compared with si-control. Results suggest that in ESCC, the AGR2 gene is a promising and predictive gene marker for the response to anti-tumor therapy.

Systemic Biomarkers and Unique Pathways in Different Phenotypes of Heart Failure with Preserved Ejection Fraction.

Heart failure with preserved ejection fraction (HFpEF) accounts for around 50% of all heart failure cases. It is a heterogeneous condition with poorly understood pathogenesis. Here, we aimed to identify unique pathogenic mechanisms in acute and chronic HFpEF and hypertrophic cardiomyopathy (HCM). We performed unbiased, comprehensive proteomic analyses of plasma samples from gender- and BMI-matched patients with acute HFpEF (n = 8), chronic HFpEF (n = 9) and HCM (n = 14) using liquid chromatography–mass spectrometry. Distinct molecular signatures were observed in different HFpEF forms. Clusters of biomarkers differentially abundant between HFpEF forms were predominantly associated with microvascular inflammation. New candidate protein markers were also identified, including leucine-rich alpha-2-glycoprotein 1 (LRG1), serum amyloid A1 (SAA1) and inter-alpha-trypsin inhibitor heavy chain 3 (ITIH3). Our study is the first to apply systematic, quantitative proteomic screening of plasma samples from patients with different subtypes of HFpEF and identify candidate biomarkers for improved management of acute and chronic HFpEF and HCM.

A new method for screening acute/chronic lymphocytic leukemia: dual-label time-resolved fluorescence immunoassay

BackgroundLymphocytic leukemia (LL) is a primary malignant tumor of hematopoietic tissue, which seriously affects the health of children and the elderly. The study aims to establish a new detection method for screening acute/chronic LL using time-resolved fluorescence immunoassay (TRFIA) via quantitative detection of S100 calcium binding protein A8 (S100A8) and leucine-rich alpha-2-glycoprotein 1 (LRG1) in serum.MethodsHere a sandwich TRFIA was optimized and established: Anti-S100A8/LRG1 caputre antibodies immobilized on 96-well plates captured S100A8/LRG1, and then banded together with the anti-S100A8/LRG1 detection antibodies labeled with Europium(III) (Eu3+)/samarium(III) (Sm3+) chelates. Finally time resolved fluorometry measured the fluorescence intensity.ResultsThe sensitivity of S100A8 was 1.15 ng/mL(LogY = 3.4027 + 0.4091 × LogX, R2 = 0.9828, P < 0.001, dynamic range: 2.1–10,000 ng/mL), and 3.2 ng/mL for LRG1 (LogY = 3.3009 + 0.4082 × LogX, R2 = 0.9748, P < 0.001, dynamic range: 4.0–10,000 ng/mL). The intra-assay and inter-assay CVs were low, ranging from 5.75% to 8.23% for S100A8 and 5.30% to 9.45% for LRG1 with high specificity and affinity in serum samples. Bland–Altman plots indicated TRFIA and ELISA kits have good agreement in clinical serum samples. Additionally, the cutoff values for S100A8 and LRG1 were 1849.18 ng/mL and 588.08 ng/mL, respectively.ConclusionThe present TRFIA method could be used for the quantitative detection of S100A8 and LRG1 in serum, and it has high sensitivity, accuracy and specificity. Clinically, this TRFIA method could be suitable for screening of LL via the quantitative detection of S100A8 and LRG1.

Abstract 3180: Targeting LRG1 to normalize tumor vasculature and enhance therapeutic efficacy

Abstract In recent years improving tumor vascular function, to render the tumour microenvironment less permissive and to improve delivery of therapeutics, has gained traction due to a growing body of supportive evidence. Identifying suitable targets that are tractable, ubiquitous and safe, however, has proven to be more challenging. Almost a decade ago we reported that a secreted glycoprotein, leucine-rich alpha-2-glycoprotein 1 (LRG1), was induced in ocular neovascular complications and contributed to the formation of dysfunctional neovessels1. More recently, we have reported that LRG1 is expressed in experimental and human tumors, and that its inhibition with a function-blocking antibody improves outcome in multiple primary2 and metastatic3 models of cancer. Crucially, we found that LRG1 blockade normalizes tumor vessels and enhances the efficacy of cisplatin, adoptive T cell and checkpoint inhibitor therapy, and that a humanized version of our blocking antibody named Magacizumab effectively inhibits tumour growth both alone and as an antibody-drug-conjugate4.Using animal models and in vitro assays we present here further data in support of LRG1 as a promising target for the treatment of solid cancers. We have further investigated the effects of LRG1 blockade on immune cell infiltration using flow cytometric and immunohistochemical analysis. In subcutaneous B16 melanoma-bearing mice treated with a PD1 checkpoint inhibitor, antibody blockade of LRG1 significantly enhanced the infiltration of CD3+ and CD8+ T cells, with the latter exhibiting a more activated phenotype as evidenced by higher GrzB, reduced PD1hi expression and increased proliferation. We also observed a reduction in the Tregs:Th ratio and a higher number of MHCII+ cells. These outcomes were also observed in LLC tumors alongside a reduction in the number of infiltrated neutrophils. In preliminary studies, where we investigated LRG1 blockade in the Rag1 mouse, we observed no effect suggesting that the downstream mode of action is mediated by impacting the immune system. Finally, to test the development of our humanized anti-LRG1 antibody Magacizumab, that only recognizes human LRG1, we demonstrated its efficacy in B16F0 tumors grown in a human LRG1 knock-in mouse. These studies provide compelling evidence that LRG1 is a novel, legitimate and potentially efficacious target for the treatment of various human solid cancers.

995-P: LRG1 Mediates Diet-Induced Hyperinsulinemia by Enhancing Glucose-Stimulated Insulin Secretion

Childhood obesity and metabolic syndrome sequelae associated with early-onset insulin resistance and type 2 diabetes are at epidemic levels globally with increasing evidence for worrisome young adult health sequelae trajectories. Pediatric obesity is often associated with dramatic rise in hyperinsulinemia and acanthosis during puberty, a risk for progression to type 2 diabetes and/or hepatic steatosis. Recently, we reported leucine-rich alpha-2-glycoprotein 1 (LRG1) as an adipokine, which levels in sera are positively associated with BMI in humans. LRG1 binds with multiple metabolic organs in vivo, including the liver and pancreas. Deletion of the Lrg1 gene in mice alleviates high-fat diet (HFD) -induced hyperinsulinemia, hepatosteatosis, insulin resistance, and obesity at a young age. Our current study indicates that treating the MIN6 beta-cells with LRG1 was sufficient to trigger insulin secretion both in the absence and presence of glucose in a dose-dependent manner, specifically enhancing the first phase of insulin secretion. We also identified an LRG1 monoclonal antibody that neutralizes LRG1-related stimulation of insulin secretion in MIN6 beta-cells. Serum levels of LRG1 in young mice fed with HFD led to enhanced levels of LRG1 in sera in these mice, which preceded hyperinsulinemia and subsequent increased weight gain. Together, our results reveal that up-regulated LRG1 in circulation could be a causal factor for persistent hyperinsulinemia by directly targeting pancreatic beta-cells to enhance insulin secretion in childhood obesity and a likely biomarker for early childhood hyperinsulinemia and treatment assessments. Understanding the impact of LRG1 on childhood obesity provides critical information to prevent early-stage insulin resistance and type 2 diabetes. Disclosure D.Morales: None. J.Ryu: None. J.L.Lynch: Consultant; Novo Nordisk, Novo Nordisk, Novo Nordisk, Research Support; Beta Bionics, Inc., Beta Bionics, Inc., Beta Bionics, Inc., Jaeb Center for Health Research, Jaeb Center for Health Research, Jaeb Center for Health Research. R.Duggirala: None. L.Q.Dong: None.

Dysregulation of Immune Response Mediators and Pain-Related Ion Channels Is Associated with Pain-like Behavior in the GLA KO Mouse Model of Fabry Disease.

Fabry disease (FD) is a rare life-threatening disorder caused by deficiency of the alpha-galactosidase A (GLA) enzyme with a characteristic pain phenotype. Impaired GLA production or function leads to the accumulation of the cell membrane compound globotriaosylceramide (Gb3) in the neurons of the dorsal root ganglia (DRG) of FD patients. Applying immunohistochemistry (IHC) and quantitative real-time polymerase chain reaction (qRT PCR) analysis on DRG tissue of the GLA knockout (KO) mouse model of FD, we address the question of how Gb3 accumulation may contribute to FD pain and focus on the immune system and pain-associated ion channel gene expression. We show a higher Gb3 load in the DRG of young (<6 months) (p < 0.01) and old (≥12 months) (p < 0.001) GLA KO mice compared to old wildtype (WT) littermates, and an overall suppressed immune response in the DRG of old GLA KO mice, represented by a reduced number of CD206+ macrophages (p < 0.01) and lower gene expression levels of the inflammation-associated targets interleukin(IL)1b (p < 0.05), IL10 (p < 0.001), glial fibrillary acidic protein (GFAP) (p < 0.05), and leucine rich alpha-2-glycoprotein 1 (LRG1) (p < 0.01) in the DRG of old GLA KO mice compared to old WT. Dysregulation of immune-related genes may be linked to lower gene expression levels of the pain-associated ion channels calcium-activated potassium channel 3.1 (KCa3.1) and transient receptor potential ankyrin 1 channel (TRPA1). Ion channel expression might further be disturbed by impaired sphingolipid recruitment mediated via the lipid raft marker flotillin-1 (FLOT1). This impairment is represented by an increased number of FLOT1+ DRG neurons with a membranous expression pattern in old GLA KO mice compared to young GLA KO, young WT, and old WT mice (p < 0.001 each). Further, we provide evidence for aberrant behavior of GLA KO mice, which might be linked to dysregulated ion channel gene expression levels and disturbed FLOT1 distribution patterns. Behavioral testing revealed mechanical hypersensitivity in young (p < 0.01) and old (p < 0.001) GLA KO mice compared to WT, heat hypersensitivity in young GLA KO mice (p < 0.001) compared to WT, age-dependent heat hyposensitivity in old GLA KO mice (p < 0.001) compared to young GLA KO mice, and cold hyposensitivity in young (p < 0.001) and old (p < 0.001) GLA KO mice compared to WT, which well reflects the clinical phenotype observed in FD patients.