Leucine-rich Α-2-glycoprotein 1 Research Articles

Diagnostic Utility of Serum Leucine-Rich α-2-Glycoprotein 1 for Acute Appendicitis in Children.

The aim of this study is to assess the diagnostic utility of serum leucine-rich α-2-glycoprotein 1 (LRG1) in pediatric patients with acute abdominal pain, admitted to the emergency surgical unit, in order to make a prompt and accurate diagnosis of acute appendicitis. Pediatric patients older than 5 years of age who presented to the emergency department from 15 October 2021 to 30 June 2022 with acute abdominal pain and suspected acute appendicitis were prospectively recruited in the study. Demographic and clinical data, as well as operative and postoperative data, were recorded. A total of 92 patients were equally distributed into two groups: children with acute appendicitis who underwent laparoscopic appendectomy and non-appendicitis patients, presenting with non-specific abdominal pain. LRG1 levels were determined using a commercially available LRG1 enzyme-linked immunosorbent assay (ELISA) kit. Serum LRG1 levels, as well as other inflammatory markers (white blood cell count (WBC), C-reactive protein (CRP) and absolute neutrophil count) were compared between groups. The median level of LRG1 in serum was significantly higher in the group of children with pathohistologically confirmed acute appendicitis than in the control group, at 350.3 µg/mL (interquartile range (IQR) 165.2-560.3) and 25.7 µg/mL (IQR 14.7-36.8) (p < 0.001), respectively. Receiver operating characteristic area under the curve for LRG1 from serum was 1.0 (95% CI 0.96-1.00; p < 0.001) and the value of >69.1 µg/mL was found to perfectly separate acute appendicitis cases from controls. Additionally, as expected, each of the examined laboratory inflammatory markers provided a significantly higher values in the acute appendicitis group compared to the control group: WBC 14.6 × 109/L (IQR 12.7, 18.7) vs. 7.0 × 109/L (IQR 5.4, 9.0) (p < 0.001), CRP 16.3 mg/dL (IQR 6.9, 50.4) vs. 2.2 mg/dL (IQR 2, 2) (p < 0.001) and absolute neutrophil count 84.6% (IQR 79.5, 89.0) vs. 59.5% (IQR 51.5, 68.6) (p < 0.001). LRG1 in the serum was found to be a promising novel biomarker, with excellent differentiation of acute appendicitis from non-appendicitis cases in children presenting with non-specific abdominal pain.

Diagnostic Accuracy of Leucine-Rich α-2-Glycoprotein 1 as a Non-Invasive Salivary Biomarker in Pediatric Appendicitis.

The aim of this study is to evaluate the diagnostic accuracy of leucine-rich α-2-glycoprotein 1 (LRG1) in saliva as a novel biomarker for acute appendicitis in the pediatric population. From October 2021 to June 2022, 92 children aged 5 to 17 years who presented with acute abdomen and suspected acute appendicitis were enrolled in this prospective study. The parameters documented included demographic and clinical information, as well as operative and postoperative data. Patients were divided into two groups: those with acute appendicitis who underwent laparoscopic appendectomy (n = 46) and those without appendicitis (n = 46). The total white blood cell (WBC) count, percent of neutrophils, C-reactive protein (CRP) level, and salivary LRG1 were compared between groups. A commercially available enzyme-linked immunosorbent assay (ELISA) LRG kit was used to measure the LRG levels. The median salivary LRG1 level was significantly higher in the group of children with pathohistologically confirmed acute appendicitis compared to the control group: 233.45 ng/mL (IQR 114.9, 531.2) vs. 55.95 ng/mL (IQR 51.5, 117.9), p < 0.001. LRG1 had an overall good receiver-operator characteristic area under the curve of 0.85 (95% CI 0.76-0.92; p < 0.001). The optimal LRG1 cutoff with best separation between acute appendicitis and the controls was >352.6 ng/mL (95% CI from >270.7 to >352.6). Although the specificity was 100% at this cutoff, the sensitivity for identifying appendicitis was 36%. In addition, a significant difference was found between groups in the laboratory values of all inflammatory markers tested: WBC, absolute neutrophil count, and CRP (p < 0.001 for all). Although LRG1 in saliva showed a good AUC parameter and significantly higher values in patients with acute appendicitis compared to the controls, its usefulness in the patient population who present at emergency departments with abdominal pain is debatable. Future studies should focus on investigating its diagnostic potential.

Associations of non-invasive indices of liver steatosis and fibrosis with progressive kidney impairment in adults with type 2 diabetes

Longitudinal data linking non-alcoholic fatty liver disease to kidney dysfunction in type 2 diabetes (T2D) are limited. This study evaluated the associations of non-invasive indices of liver steatosis and liver fibrosis with kidney impairment, and the mediatory role of the pro-angiogenic factor leucine-rich α-2 glycoprotein 1 (LRG1). T2D adults (n = 2057) were followed for a mean period of 6.1 ± 1.6years. Baseline liver steatosis [(hepatic steatosis index (HSI) and Zhejiang University index (ZJU)] and liver fibrosis [aspartate transaminase/alanine transaminase ratio (AAR) and BARD] indices derived from composite scoring systems were calculated. Plasma LRG1 levels were quantified using immunoassay. The study outcomes were progressive kidney function decline defined as estimated glomerular filtration rate (eGFR) decline of ≥ 40% and albuminuria progression defined as an increase in albuminuria category. Cross-sectionally, liver steatosis and liver fibrosis indices were associated with increased albuminuria (urinary albumin/creatinine ratio ≥ 30µg/mg) and reduced renal function (eGFR < 60mL/min/1.73m2) after covariate adjustment, respectively. Approximately 32% of the participants experienced progressive kidney function decline, while 38% had albuminuria worsening over time. Longitudinal analysis revealed that baseline AAR (hazard ratio: 1.56; 95% CI 1.15-2.11) and BARD (hazard ratio: 1.16, 95% CI 1.04-1.28) predicted progressive kidney function decline, partly mediated by LRG1. In contrast, liver steatosis (HSI and ZJU) but not liver fibrosis (AAR and BARD) indices were independently associated with albuminuria progression. Increased liver steatosis scores were associated with albuminuria deterioration. Conversely, liver fibrosis indices may be associated with progressive kidney function decline, potentially driven by increased inflammation and angiogenesis.

Hepatoprotective effects of moderate-intensity interval training along with ginger juice in an old male rat model.

Aging is a natural process coupled with oxidative stress and chronic inflammation, gradually associated with losing organ function over time. Therefore, the objective of the current work was to peruse the protective effects of 8-week moderate-intensity interval training (MIIT) and ginger extract supplementation on some biomarkers of oxidative stress, inflammation, and lipid metabolism in the liver of elderly males Wistar rats (animal study with ethical code IR.BMSU.REC.1401.015). A total of thirty-two 22-month-aged male Wistar rats were randomly assigned to four groups: (1) control, (2) MIIT, (3) ginger, and (4) MIIT + ginger. After 8weeks of treadmill training and ginger extract supplementation, the biochemical parameters (liver enzyme and lipid profile), inflammatory mediators (leucine-rich α-2 glycoprotein 1 (LRG1), tumor necrosis factor-alpha, and interleukin-6), pro-oxidant (malondialdehyde), antioxidant biomarkers (catalase, superoxide dismutase, total antioxidant capacity), some lipid metabolism regulators (carnitine palmitoyltransferase 1, adipose triglyceride lipase, acetyl-CoA carboxylase, CD36, and AMP-activated protein kinase), and liver histopathological changes were appraised. The acquired findings pointed out that MIIT combined with ginger extract appreciably diminished the serum levels of LRG1, liver enzymes, and lipid profile relative to the other groups after 8weeks of intervention. Furthermore, ginger + MIIT caused a great improvement in the liver levels of antioxidant biomarkers, pro-oxidant, pro-inflammatory biomarkers, lipid metabolism regulators, and liver tissue impairment compared to the other groups. The findings suggested that MIIT + ginger was more effective in improving examined indices relative to the other groups.

Urine Leucine-Rich α-2 Glycoprotein 1 (LRG1) Predicts the Risk of Progression to End-Stage Kidney Disease in Patients With Type 2 Diabetes.

Leucine-rich α-2 glycoprotein 1 (LRG1) was recently identified as an amplifier of transforming growth factor-β (TGF-β)-induced kidney fibrosis in animal models. We aimed to study whether urine LRG1 is associated with risk of progression to end-stage kidney disease (ESKD) in individuals with type 2 diabetes. A total of 1,837 participants with type 2 diabetes and estimated glomerular filtration rate (eGFR) >30 mL/min/1.73 m2 were recruited from a regional hospital and a primary care facility. Association of urine LRG1 with risk of ESKD (progression to sustained eGFR <15 mL/min/1.73 m2, dialysis, or death resulting from renal causes) was assessed by survival analyses. During a median follow-up of 8.6 (interquartile range 5.8-9.6) years, 134 incident ESKD events were identified. Compared with those in the lowest tertile, participants with baseline urine LRG1 in the highest tertile had a 1.91-fold (95% CI 1.04-3.50) increased risk of progression to ESKD, after adjustment for cardiorenal risk factors, including eGFR and albuminuria. As a continuous variable, 1 SD increment in urine LRG1 was associated with a 1.53-fold (95% CI 1.19-1.98) adjusted risk of ESKD. Of note, the association of urine LRG1 with ESKD was independent of plasma LRG1. Moreover, urine LRG1 was associated with rapid kidney function decline and progression to macroalbuminuria, two common pathways leading to ESKD. Urine LRG1, a TGF-β signaling modulator, predicts risk of progression to ESKD independently of clinical risk factors in patients with type 2 diabetes, suggesting that it may be a novel factor involved in the pathophysiological pathway leading to kidney disease progression.

Metformin suppresses LRG1 and TGFβ1/ALK1-induced angiogenesis and protects against ultrastructural changes in rat diabetic nephropathy

Diabetic nephropathy (DN) has high prevalence and poor prognosis which make it a research priority for scientists. Since metformin, a hypoglycaemic drug, has been found to prolong the survival of mice with DN. This study aims at investigating the molecular mechanisms leading to DN in rats and to explore the role of leucine-rich α-2-glycoprotein-1 (LRG1), activin-like kinase1 (ALK1), and transforming growth factor–β (TGFβ1) in the pathologic alterations seen in DN. The aim was also extended to explore the protective action of metformin against DN in rats and its influence on LRG1and ALK1/TGFβ1 induced renal angiogenesis. 24 male rats were used. Rats were assigned as, the vehicle group, the diabetic control group and diabetic + metformin (100 and 200 mg/kg) groups. Kidney samples were processed for histopathology, immunohistochemistry and biochemical analysis. Bioinformatic analysis of studied proteins was done to determine protein-protein interactions. Metformin reduced serum urea and creatinine significantly, decreased the inflammatory cytokine levels and reduced LRG1, TGFβ1, ALK1 and vascular endothelial growth factor (VEGF) proteins in rat kidneys. Bioinformatic analysis revealed interactions between the studied proteins. Metformin alleviated the histopathological changes observed in the diabetic rats such as the glomerular surface area and increased Bowman’s space diameter. Metformin groups showed decreased VEGF immunostaining compared to diabetic group. Metformin shows promising renoprotective effects in diabetic model that was at least partly mediated by downregulation of LRG1 and TGFβ1/ALK1-induced renal angiogenesis. These results further explain the molecular mechanism of metformin in DN management.

LRG1, a Novel Serum Biomarker for Idiopathic Multicentric Castleman Disease Activity

Objective: To investigate the relationship between serum Leucine-Rich α-2-Glycoprotein-1 (LRG1) and idiopathic multicentric Castleman disease (iMCD) activity, and to verify the effectiveness of LRG1 in monitoring treatment response. Methods: Clinical data and serum samples were collected from iMCD patients in flare and remission state diagnosed between August 2015 and April 2022 in Peking Union Medical College Hospital. Enzyme-linked Immunosorbent Assay was performed to quantify LRG1 concentration, which was further analyzed with patients' clinical data. Results: 81 patients were included, with 10 cases of iMCD-TAFRO and 40 cases of severe-disease, and 110 serum samples were acquired. The average LRG1 concentration was 113.5±14.27μg/mL in 76 samples of disease flare, 47.48±14.23μg/mL in 16 biochemical PR, and 14.4±1.18μg/mL in 18 biochemical CR samples. The difference is significant among different disease states (Flare vs PR, p = 0.0019; Flare vs CR, p < 0.0001; PR vs CR, p = 0.0349). Paired sample of 20 cases showed significant decrease of serum LRG1 from Flare to PR/CR (-35.36±5.94μg/mL, p < 0.0001) after treatment (figure 1A). Serum LRG1 level was also elevated in those iMCD-TAFRO cases with normal serum CRP level during disease flare. Baseline LRG1 level was significantly higher in patients with severe disease (142.6±23.2μg/mL vs 81.2±14.0μg/mL, p = 0.0271), which could be identified efficiently by baseline LRG1 over 57.75μg/mL (AUC = 0.655, p = 0.0203, sensitivity 70%, specificity 63.89%). Among 38 patients using thalidomide-cyclophosphamide-prednisone (TCP) regimen as first-line treatment, the median time to next treatment in patients with baseline LRG1 over 57.75μg/mL was significantly shorter than those under 57.75μg/mL (10 vs 30 months, HR: 2.254, 95% CI: 1.052-4.828, p = 0.0277) (figure 1B). Conclusion: Serum LRG1 level could mirror disease activity, and could be utilized to monitor the treatment response, especially in iMCD-TAFRO subtype. Serum LRG1 level over 57.75μg/mL could efficiently identify patients with severe iMCD, and had the potential to predict the efficacy of TCP regimen. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

LRG1 is an adipokine that promotes insulin sensitivity and suppresses inflammation.

While dysregulation of adipocyte endocrine function plays a central role in obesity and its complications, the vast majority of adipokines remain uncharacterized. We employed bio-orthogonal non-canonical amino acid tagging (BONCAT) and mass spectrometry to comprehensively characterize the secretome of murine visceral and subcutaneous white and interscapular brown adip ocytes. Over 600 proteins were identified, the majority of which showed cell type-specific enrichment. We here describe a metabolic role for leucine-rich α-2 glycoprotein 1 (LRG1) as an obesity-regulated adipokine secreted by mature adipocytes. LRG1 overexpression significantly improved glucose homeostasis in diet-induced and genetically obese mice. This was associated with markedly reduced white adipose tissue macrophage accumulation and systemic inflammation. Mechanistically, we found LRG1 binds cytochrome c in circulation to dampen its pro-inflammatory effect. These data support a new role for LRG1 as an insulin sensitizer with therapeutic potential given its immunomodulatory function at the nexus of obesity, inflammation, and associated pathology.

Circulatory cytokeratin 17, marginal zone B1 protein and leucine-rich α2-glycoprotein-1 as biomarkers for disease severity and fibrosis in systemic sclerosis patients.

IntroductionSystemic sclerosis (Ssc) is a multiorgan debilitating autoimmune disease that associates the triad: vascular involvement, tissue fibrosis and profound immune response alterations. Numerous previous studies focused on identification of candidate proteomic Ssc biomarkers using mass-spectrometry techniques and a large number of candidate Ssc biomarkers emerged. These biomarkers must firstly be confirmed in independent patient groups. The aim of the present study was to investigate the association of cytokeratin 17 (CK17), marginal zone B1 protein (MZB1) and leucine-rich α2-glycoprotein-1 (LRG1) with clinical and biological Ssc characteristics.Material and methodsSerum CK17, MZB1 and LRG1 were assessed in samples of the available Ssc biobank comprising of samples from 53 Ssc patients and 26 matched age and gender controls.ResultsCirculatory CK17, LRG1 and MZB1 concentrations were increased in Ssc patients. Cytokeratin 17 is independently associated with Ssc disease activity. Patients with pulmonary fibrosis expressed higher LRG1 and MZB1 concentrations. Serum MZB1 concentrations were also associated with extensive skin fibrosis.ConclusionsSerum CK17, MZB1 and LRG1 were confirmed biomarkers for Ssc. LRG1 seems a good biomarker for pulmonary fibrosis, while MZB1 is a good biomarker for extensive skin fibrosis. CK17 proved to be independently associated with Ssc disease severity, higher CK17 values being protective for a more active disease.

Increased LRG1 Levels in Overweight and Obese Adolescents and Its Association with Obesity Markers, Including Leptin, Chemerin, and High Sensitivity C-Reactive Protein.

Leucine-rich α-2 glycoprotein1 (LRG1) is a member of the leucine-rich repeat (LRR) family that is implicated in multiple diseases, including cancer, aging, and heart failure, as well as diabetes and obesity. LRG1 plays a key role in diet-induced hepatosteatosis and insulin resistance by mediating the crosstalk between adipocytes and hepatocytes. LRG1 also promotes hepatosteatosis by upregulating de novo lipogenesis in the liver and suppressing fatty acid β-oxidation. In this study, we investigated the association of LRG1 with obesity markers, including leptin and other adipokines in adolescents (11–14 years; n = 425). BMI-for-age classification based on WHO growth charts was used to define obesity. Plasma LRG1 was measured by ELISA, while other markers were measured by multiplexing assay. Median (IQR) of LRG1 levels was higher in obese (30 (25, 38) µg/mL) and overweight (30 (24, 39) µg/mL) adolescents, compared to normal-weight participants (27 (22, 35) µg/mL). The highest tertile of LRG1 had an OR [95% CI] of 2.55 [1.44, 4.53] for obesity. LRG1 was positively correlated to plasma levels of high sensitivity c-reactive protein (HsCRP) (ρ = 0.2), leptin (ρ = 0.2), and chemerin (ρ = 0.24) with p < 0.001. Additionally, it was positively associated with plasma level of IL6 (ρ = 0.17) and IL10 (ρ = 0.14) but not TNF-α. In conclusion, LRG1 levels are increased in obese adolescents and are associated with increased levels of adipogenic markers. These results suggest the usefulness of LRG1 as an early biomarker for obesity and its related pathologies in adolescents.

Association between Higher Circulating Leucine-Rich α-2 Glycoprotein 1 Concentrations and Specific Plasma Ceramides in Postmenopausal Women with Type 2 Diabetes.

Background: Although ceramides are involved in the pathophysiology of cardiovascular disease and other inflammation-associated disorders, there is a paucity of data on the association between plasma ceramides and inflammatory biomarkers in type 2 diabetes mellitus (T2DM). Therefore, we explored whether there was an association between plasma leucine-rich α-2 glycoprotein 1 (LRG1) concentrations (i.e., a novel proinflammatory signaling molecule) and specific plasma ceramides in postmenopausal women with T2DM. Methods: We measured six previously identified plasma ceramides, which have been associated with increased cardiovascular risk [plasma Cer(d18:1/16:0), Cer(d18:1/18:0), Cer(d18:1/20:0), Cer(d18:1/22:0), Cer(d18:1/24:0) and Cer(d18:1/24:1)], amongst 99 Caucasian postmenopausal women with non-insulin-treated T2DM (mean age 72 ± 8 years, mean hemoglobin A1c 6.9 ± 0.7%), who consecutively attended our diabetes outpatient service during a 3-month period. Plasma ceramide and LRG1 concentrations were measured with a targeted liquid chromatography-tandem mass spectrometry assay and a Milliplex® MAP human cardiovascular disease magnetic bead kit, respectively. Results: In linear regression analyses, higher plasma LRG1 levels (1st tertile vs. 2nd and 3rd tertiles combined) were associated with higher levels of plasma Cer(d18:1/16:0) (standardized β coefficient: 0.289, p = 0.004), Cer(d18:1/18:0) (standardized β coefficient: 0.307, p = 0.002), Cer(d18:1/20:0) (standardized β coefficient: 0.261, p = 0.009) or Cer(d18:1/24:1) (standardized β coefficient: 0.343, p < 0.001). These associations remained significant even after adjusting for age, body mass index, systolic blood pressure, total cholesterol level, hemoglobin A1c, insulin resistance and statin use. Conclusions: The results of our pilot exploratory study suggest that higher plasma LRG1 concentration was associated with higher levels of specific high-risk plasma ceramide molecules in elderly postmenopausal women with metabolically well-controlled T2DM, even after adjusting for known cardiovascular risk factors and other potential confounding variables.

The Effect of Aerobic Exercise on Angiogenesis Factors in Middle-aged Women

Background and Aim: Aging is a process, in which the physiological capacity of the body decreases. One of these basic changes is the reduction of blood supply caused by the inability of angiogenesis. This article was conducted to investigate the effect of aerobic exercise on angiogenic factors, including leucine-rich α-2-glycoprotein 1 (LRG-1) and thrombospondin-1 (TSP-1) in middle-aged women. Materials and Methods: This quasi-experimental study was conducted on 18 middle-aged women. The samples were randomly divided into two groups, including the aerobic (n=9) and control (n=9) groups. The experimental group performed aerobic exercise for eight weeks (three sessions per week). Then, 5 mL of blood samples was taken from each person 48 hours before and after training to measure LRG-1 and TSP-1 protein levels. The dependent and independent t-tests were used to analyze the data. Results: The levels of LRG-1 in the experimental group showed a significant increase in post-test compared to the control group. Also, in the experimental group, TSP-1 levels showed a significant decrease in post-test compared to the control group. Conclusion: The results of this study showed that aerobic exercise can increase the levels of LRG-1 protein and decrease the serum TSP-1 protein levels; therefore, by increasing LRG-1 and decreasing TSP-1 levels, the angiogenesis process in the body is enhanced. Accordingly, middle-aged women are advised to do aerobic exercise as a tool to improve angiogenesis and improve blood circulation.

An Immunological Axis Involving Interleukin 1β and Leucine-Rich-α2-Glycoprotein Reflects Therapeutic Response of Children with Kawasaki Disease: Implications from the KAWAKINRA Trial

PurposeA recent phase II open-label study of the interleukin 1 (IL-1) receptor antagonist (IL-1Ra) anakinra in treating IVIG-resistant Kawasaki disease (KD) patients reported promising results. Here, we aimed to characterize the immunological impact of IL-1 blockade in this unique study population.MethodsPatients’ and control sera and supernatants of cells (whole blood, neutrophils, coronary artery endothelial cells) stimulated with recombinant IL-1β were analyzed for single or multiple marker (n = 22) expression by ELISA or multiplexed bead array assay. Data were analyzed using unsupervised hierarchical clustering, multiple correlation, and multi-comparison statistics and were compared to retrospective analyses of KD transcriptomics.ResultsInflammation in IVIG-resistant KD (n = 16) is hallmarked by over-expression of innate immune mediators (particularly IL-6 > CXCL10 > S100A12 > IL-1Ra). Those as well as levels of immune or endothelial cell activation markers (sICAM-1, sVCAM-1) declined most significantly in course of anakinra treatment. Prior as well as following IL-1R blockade, over-expression of leucine-rich-α2-glycoprotein 1 (LRG1) associated best with remnant inflammatory activity and the necessity to escalate anakinra dosage and separated inflammatory KD patients from sJIA-MAS (n = 13) and MIS-C (n = 4). Protein as well as retrospective gene expression analyses indicated tight association of LRG1 with IL-1β signaling and neutrophilia, while particularly neutrophil stimulation with recombinant IL-1β resulted in concentration-dependent LRG1 release.ConclusionOur study identifies LRG1 as known trigger of endothelial activation and cardiac re-modeling to associate with IL-1β signaling in KD. Besides a potential patho-mechanistic implication of these findings, our data suggest blood leukocyte and neutrophil counts to best predict response to IL-1Ra treatment in IVIG-resistant KD.

Structural basis of human LRG1 recognition by Magacizumab, a humanized monoclonal antibody with therapeutic potential.

The formation of new dysfunctional blood vessels is a crucial stage in the development of various conditions such as macular degeneration, diabetes, cardiovascular disease, neurological disease and inflammatory disorders, as well as during tumor growth, eventually contributing to metastasis. An important factor involved in pathogenic angiogenesis is leucine-rich α-2-glycoprotein 1 (LRG1), the antibody blockade of which has been shown to lead to a reduction in both choroidal neovascularization and tumor growth in mouse models. In this work, the structural interactions between the LRG1 epitope and the Fab fragment of Magacizumab, a humanized function-blocking IgG4 against LRG1, are analysed, determining its specific binding mode and the key residues involved in LRG1 recognition. Based on these structural findings, a series of mutations are suggested that could be introduced into Magacizumab to increase its affinity for LRG1, as well as a model of the entire Fab-LRG1 complex that could enlighten new strategies to enhance affinity, consequently leading towards an even more efficient therapeutic.

LRG1 Accelerates Wound Healing in Diabetic Rats by Promoting Angiogenesis via the Wnt/β-Catenin Signaling Pathway.

Patients with diabetic wounds may end with lower extremity amputation or death. Leucine-rich α-2-glycoprotein 1 (LRG1) is an effective regulator of angiogenesis and essential for timely wound healing. However, its role in regulating angiogenesis in diabetic wounds remains unclear. This study aimed to investigate the pro-angiogenic function of exogenous LRG1 in diabetic wound healing and explore possible mechanisms. LRG1 expression patterns following injury in normal and diabetic wounds were determined by western blotting. Local injection of LRG1 was used to verify the effects on angiogenesis and wound healing in diabetic rats. Immunohistochemical staining for CD31 was used to analyze the vessel density. Human umbilical vein endothelial cells (HUVECs) cultured in hyperglycemia were used to explore how LRG1 promotes angiogenesis in diabetic wound healing. We found that the expression peak of LRG1 around the wounds was delayed in diabetic rats compared with that in normal rats. Exogenous administration of LRG1 significantly accelerated the wound closure rate and promoted angiogenesis in diabetic rats. In addition, exogenous LRG1 effectively restored the proliferation, migration, and tube formation ability of HUVECs under hyperglycemia. Mechanistically, LRG1 promoted angiogenesis and diabetic wound healing mainly by activating the Wnt/β-catenin pathway, which is inhibited in diabetic wounds. This research suggests that LRG1 promotes angiogenesis and wound closure in diabetic rats by improving angiogenesis via activation of the Wnt/β-catenin pathway. Hence, LRG1 may be a possible therapeutic strategy for diabetic foot treatment.

Tubular epithelial cell-to-macrophage communication forms a negative feedback loop via extracellular vesicle transfer to promote renal inflammation and apoptosis in diabetic nephropathy.

Background: Macrophage infiltration around lipotoxic tubular epithelial cells (TECs) is a hallmark of diabetic nephropathy (DN). However, how these two types of cells communicate remains obscure. We previously demonstrated that LRG1 was elevated in the process of kidney injury. Here, we demonstrated that macrophage-derived, LRG1-enriched extracellular vesicles (EVs) exacerbated DN.Methods: We induced an experimental T2DM mouse model with a HFD diet for four months. Renal primary epithelial cells and macrophage-derived EVs were isolated from T2D mice by differential ultracentrifugation. To investigate whether lipotoxic TEC-derived EV (EVe) activate macrophages, mouse bone marrow-derived macrophages (BMDMs) were incubated with EVe. To investigate whether activated macrophage-derived EVs (EVm) induce lipotoxic TEC apoptosis, EVm were cocultured with primary renal tubular epithelial cells. Subsequently, we evaluated the effect of LRG1 in EVe by investigating the apoptosis mechanism.Results: We demonstrated that incubation of primary TECs of DN or HK-2 mTECs with lysophosphatidyl choline (LPC) increased the release of EVe. Interestingly, TEC-derived EVe activated an inflammatory phenotype in macrophages and induced the release of macrophage-derived EVm. Furthermore, EVm could induce apoptosis in TECs injured by LPC. Importantly, we found that leucine-rich α-2-glycoprotein 1 (LRG1)-enriched EVe activated macrophages via a TGFβR1-dependent process and that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-enriched EVm induced apoptosis in injured TECs via a death receptor 5 (DR5)-dependent process.Conclusion: Our findings indicated a novel cell communication mechanism between tubular epithelial cells and macrophages in DN, which could be a potential therapeutic target.

Modulation of transforming growth factor-β-induced kidney fibrosis by leucine-rich ⍺-2 glycoprotein-1

Kidney fibrosis is considered the final convergent pathway for progressive chronic kidney diseases, but there is still a paucity of success in clinical application for effective therapy. We recently demonstrated that the expression of secreted leucine-rich α-2 glycoprotein-1 (LRG1) is associated with worsened kidney outcomes in patients with type 2 diabetes and that LRG1 enhances endothelial transforming growth factor-β signaling to promote diabetic kidney disease progression. While the increased expression of LRG1 was most prominent in the glomerular endothelial cells in diabetic kidneys, its increase was also observed in the tubulointerstitial compartment. Here, we explored the potential role of LRG1 in kidney epithelial cells and TGF-β-mediated tubulointerstitial fibrosis independent of diabetes. LRG1 expression was induced by tumor necrosis factor-α in cultured kidney epithelial cells and potentiated TGF-β/Smad3 signal transduction. Global Lrg1 loss in mice led to marked attenuation of tubulointerstitial fibrosis in models of unilateral ureteral obstruction and aristolochic acid fibrosis associated with concomitant decreases in Smad3 phosphorylation in tubule epithelial cells. In mice with kidney epithelial cell-specific LRG1 overexpression, while no significant phenotypes were observed at baseline, marked exacerbation of tubulointerstitial fibrosis was observed in the obstructed kidneys. This was associated with enhanced Smad3 phosphorylation in both kidney epithelial cells and α-smooth muscle actin-positive interstitial cells. Co-culture of kidney epithelial cells with primary kidney fibroblasts confirmed the potentiation of TGF-β-mediated Smad3 activation in kidney fibroblasts through epithelial-derived LRG1. Thus, our results indicate that enhanced LRG1 expression-induced epithelial injury is an amplifier of TGF-β signaling in autocrine and paracrine manners promoting tubulointerstitial fibrosis. Hence, therapeutic targeting of LRG1 may be an effective means to curtail kidney fibrosis progression in chronic kidney disease.

LRG1 destabilizes tumor vessels and restricts immunotherapeutic potency

A poorly functioning tumor vasculature is pro-oncogenic and may impede the delivery of therapeutics. Normalizing the vasculature, therefore, may be beneficial. We previously reported that the secreted glycoprotein leucine-rich α-2-glycoprotein 1 (LRG1) contributes to pathogenic neovascularization. Here, we investigate whether LRG1 in tumors is vasculopathic and whether its inhibition has therapeutic utility. Tumor growth and vascular structure were analyzed in subcutaneous and genetically engineered mouse models in wild-type and Lrg1 knockout mice. The effects of LRG1 antibody blockade as monotherapy, or in combination with co-therapies, on vascular function, tumor growth, and infiltrated lymphocytes were investigated. In mouse models of cancer, Lrg1 expression was induced in tumor endothelial cells, consistent with an increase in protein expression in human cancers. The expression of LRG1 affected tumor progression as Lrg1 gene deletion, or treatment with a LRG1 function-blocking antibody, inhibited tumor growth and improved survival. Inhibition of LRG1 increased endothelial cell pericyte coverage and improved vascular function, resulting in enhanced efficacy of cisplatin chemotherapy, adoptive Tcell therapy, and immune checkpoint inhibition (anti-PD1) therapy. With immunotherapy, LRG1 inhibition led to a significant shift in the tumor microenvironment from being predominantly immune silent to immune active. LRG1 drives vascular abnormalization, and its inhibition represents a novel and effective means of improving the efficacy of cancer therapeutics. Wellcome Trust (206413/B/17/Z), UKRI/MRC (G1000466, MR/N006410/1, MC/PC/14118, and MR/L008742/1), BHF (PG/16/50/32182), Health and Care Research Wales (CA05), CRUK (C42412/A24416 and A17196), ERC (ColonCan 311301 and AngioMature 787181), and DFG (CRC1366).

Macrophage-derived, LRG1-enriched extracellular vesicles exacerbate aristolochic acid nephropathy in a TGFβR1-dependent manner.

Aristolochic acid nephropathy (AAN) is a progressive kidney disease caused by some herbal medicines, but treatment remains ineffective. We previously found that leucine-rich α-2-glycoprotein 1 (LRG1), which regulates cellular processes, plays an important role in a kidney injury model. However, the underlying mechanism by which LRG1 regulates AAN is still unknown. In this study, we established an AAN model in vivo, a coculture system of macrophages and TECs, and a macrophage/TEC conditioned media culture model in vitro. We found that macrophage infiltration promoted injury, oxidative stress, and apoptosis in TECs. Furthermore, the role of macrophages in AAN was dependent on macrophage-derived extracellular vesicles (EVs). Importantly, we found that macrophage-derived, LRG1-enriched EVs induced TEC injury and apoptosis via a TGFβR1-dependent process. This study may help design a better therapeutic strategy to treat AAN patients.

Paradoxical Roles of Leucine-Rich α2-Glycoprotein-1 in Cell Death and Survival Modulated by Transforming Growth Factor-Beta 1 and Cytochrome c.

Leucine-rich α2-glycoprotein-1 (LRG1) has been shown to impact both apoptosis and cell survival, pleiotropic effects similar to one of its known ligands, transforming growth factor-beta 1 (TGF-β1). Recent studies have given insight into the TGF-β1 signaling pathways involved in LRG1-mediated death versus survival signaling, i.e., canonical or non-canonical. Interaction of LRG1 with another ligand, extracellular cytochrome c (Cyt c), promotes cell survival, at least for lymphocytes. LRG1 has been shown to bind Cyt c with high affinity, higher than it binds TGF-β1, making it sensitive to small changes in the level of extracellular Cyt c within a microenvironment that may arise from cell death. Evidence is presented here that LRG1 can bind TGF-β1 and Cyt c simultaneously, raising the possibility that the ternary complex may present a signaling module with the net effect of signaling, cell death versus survival, determined by the relative extent to which the LRG1 binding sites are occupied by these two ligands. A possible role for LRG1 should be considered in studies where extracellular effects of TGF-β1 and Cyt c have been observed in media supplemented with LRG1-containing serum.