Association of significantly elevated plasma levels of NGAL and IGFBP4 in patients with diabetic nephropathy.

BACKGROUND: Diabetic nephropathy (DN) is a type of progressive kidney disease affecting approximately 40% of patients with diabetes. Current DN diagnostic criteria predominantly rely on albuminuria and serum creatinine (sCr) levels. However, the specificity and reliability of both markers are limited. Hence, reliable biomarkers are required for early diagnosis to effectively manage DN progression. METHODS: In this study, a cohort of 159 individuals were clinically evaluated and the plasma levels of NGAL, IGFBP-1, IGFBP-3, and IGFBP-4 were determined using Multiplexing Assays. Additionally, the association between the plasma levels of NGAL, IGFBP-1, IGFBP-3, and IGFBP-4 in patients with DN were compared to those in patients with T2D without kidney disease and control participants. RESULTS: Circulating level of NGAL were significantly higher in people with DN compared to people with T2D and non-diabetic groups (92.76 ± 7.5, 57.22 ± 8.7, and 52.47 ± 2.9 mg/L, respectively; p <  0.0001). IGFBP-4 showed a similar pattern, where it was highest in people with DN (795.61 ng/ml ±130.7) compared to T2D and non-diabetic people (374.56 ng/ml ±86.8, 273.06 ng/ml ±27.8 respectively, ANOVA p <  0.01). The data from this study shows a significant positive correlation between NGAL and IGFBP-4 in people with DN (ρ = .620, p <  0.005). IGFBP-4 also correlated positively with creatinine level and negatively with eGFR, in people with DN supporting its involvement in DN. CONCLUSION: The data from this study shows a parallel increase in the plasma levels of NGAL and IGFBP-4 in DN. This highlights the potential to use these markers for early diagnosis of DN.

The Rise of IGFBP4 in People with Obstructive Sleep Apnea and Multilevel Sleep Surgery Recovers Its Basal Levels.

IGFBP4 is the smallest member of the insulin-like growth factor binding protein family (IGFBP). It is a hepatic protein that plays a role in modulating the activity and bioavailability of IGF-I. The expression of IGFBP4 was found to increase under conditions of hypoxia. Obstructive sleep apnea (OSA) is a common disorder, characterized by cyclic episodes of intermittent hypoxia and fragmented sleep. Our aim was to quantify levels of circulating IGFBP1, IGFBP2, IGFBP3, IGFBP4, and IGFBP7 in fasting plasma samples of 69 Kuwaiti participants and explore its correlation with indices of OSA. The quantification was performed using multiplexing assay. The study involved 28 controls and 41 patients with OSA. Levels of circulating IGFBP4 were significantly higher in people with OSA (289.74 ± 23.30 ng/ml) compared to the control group (217.60 ± 21.74 ng/ml, p = 0.028). The increase in IGFBP4 correlated significantly and positively with AHI (r = .574, p = .01) and AI (r = .794, p = .001) in people with moderate and severe OSA. There was a significant decline in circulating IGFBP4 after 3 months of surgery (225.89 ± 18.16 ng/ml, p = 0.012). This was accompanied by a prominent improvement in OSA (AHI 8.97 ± 2.37 events/h, p = 0.001). In this study, our data showed a significant increase in circulating IGFBP4 in people with OSA. We also report a significant positive correlation between IGFBP4 and indices of OSA at baseline, which suggests IGFBP4 as a potential diagnostic biomarker for OSA. There was a significant improvement in OSA after 3 months of surgical intervention, which concurred with a significant decline in IGFBP4 levels. Altogether, the detected change suggests a potential link between IGFBP4 and OSA or an OSA-related factor, whereby OSA might play a role in triggering the induction of IGFBP4 expression.

Metabolic improvement after gastric bypass correlates with changes in IGF-regulatory proteins stanniocalcin-2 and IGFBP-4.

BACKGROUND: Pregnancy-associated plasma protein-A (PAPP-A) is an enzyme that increases IGF-activity through cleavage of IGF-binding proteins (IGFBPs), primarily IGFBP-4, whereby bound IGF-I becomes released as a free molecule. The enzymatic activity of PAPP-A is irreversibly suppressed by the glycoprotein stanniocalcin-2 (STC2). Pre-clinical and clinical studies suggest that the STC2 - PAPP-A - IGFBP-4 axis is important in controlling local IGF-action. STC2, PAPP-A and IGFBP-4 are expressed in adipose tissue, and as bariatric surgery markedly reduces the amount of fat, we found it relevant to study the impact of Roux-en-Y gastric bypass (RYGB) on circulating concentrations of this IGF-regulatory network. METHODS: Analysis of fasting blood samples from 20 obese subjects, hereof 10 with preoperative type 2 diabetes, investigated before RYGB, and 1 week, 3 months and 12 months post-surgery. Members of the IGF-system were analyzed by immunoassays, bioactive IGF by cell-based IGF-I receptor activation assay. We compared changes in IGF-system components with changes in fasting plasma insulin and glucose, and HbA1c. RESULTS: PAPP-A remained unchanged, but STC2 decreased following RYGB (p < 0.05). The PAPP-A substrate IGFBP-4 declined (p < 0.01), whereas levels of PAPP-A specific IGFBP-4 fragments increased (p < 0.05), indicating an increased PAPP-A enzymatic activity post-RYGB. Further, the reduction in intact IGFBP-4 correlated with increased levels of bioactive IGF (p < 0.05). In multivariable regression analyses, an improved glucose metabolism correlated with reductions in STC2 and IGFBP-4, and with increases in bioactive IGF and IGF-I (p < 0.05). CONCLUSION: After 12 months, RYGB caused reduced serum concentrations of intact IGFBP-4 and STC2, whereas serum PAPP-A remained at pre-operative levels. However, concentrations of PAPP-A generated IGFBP-4 fragments increased, pointing to an overall increased PAPP-A enzymatic activity following RYGB. Notably, reductions in intact IGFBP-4 and STC2 associated with improvements in glucose metabolism. Therefore, we propose that STC2 and IGFBP-4 are involved in the metabolic improvement that follows RYGB.

Cross-sectional study of plasma Axl, ferritin, IGFBP4 and sTNFR2 as biomarkers of disease activity in childhood-onset SLE: A study of the Pediatric Nephrology Research Consortium.

OBJECTIVE: To evaluate the performance of 4 plasma protein markers for detecting disease activity in childhood-onset systemic lupus erythematosus (SLE) patients. METHODS: Eighty-three consecutive pediatric patients fulfilling ≥4 ACR criteria for SLE and twenty-five healthy controls were prospectively recruited for serological testing of 4 protein markers identified by antibody-coated microarray screen, namely Axl, ferritin, IGFBP4 and sTNFR2. SLE disease activity was assessed using SLEDAI-2000 score. Fifty-seven patients had clinically active SLE (SLEDAI score ≥4, or having a flare). RESULTS: The plasma concentrations of Axl and ferritin were significantly higher in patients with active SLE than inactive SLE. Plasma Axl levels were significantly higher in active renal versus active non-renal SLE patients. Levels of Axl, ferritin and IGFBP4 correlated significantly with SLEDAI scores. Levels of Axl, IFGBP4 and sTNFR2 inversely correlated with plasma complement C3 levels. Only plasma Axl and ferritin levels correlated with degree of proteinuria. These markers were more specific, but less sensitive, in detecting concurrent SLE activity than elevated anti-dsDNA antibody titer or decreased C3. Ferritin and IGFBP4 levels were more specific for concurrent active lupus nephritis than anti-dsDNA or C3. Plasma ferritin was the best monitor of global SLE activity, followed by C3 then Axl, while both Axl and C3 were best monitors of clinical lupus nephritis activity. CONCLUSION: In childhood-onset SLE patients, plasma ferritin and Axl perform better than traditional yardsticks in identifying disease activity, either global or renal. The performance of these plasma markers should be explored further in longitudinal cohorts of SLE patients.

Novel Insights Into the Effects of Interleukin 6 Antagonism in Non-ST-Segment-Elevation Myocardial Infarction Employing the SOMAscan Proteomics Platform.

Background Interleukin 6 concentration is associated with myocardial injury, heart failure, and mortality after myocardial infarction. In the Norwegian tocilizumab non-ST-segment-elevation myocardial infarction trial, the first randomized trial of interleukin 6 blockade in myocardial infarction, concentration of both C-reactive protein and troponin T were reduced in the active treatment arm. In this follow-up study, an aptamer-based proteomic approach was employed to discover additional plasma proteins modulated by tocilizumab treatment to gain novel insights into the effects of this therapeutic approach. Methods and Results Plasma from percutaneous coronary intervention-treated patients, 24 in the active intervention and 24 in the placebo-control arm, drawn 48 hours postrandomization were randomly selected for analysis with the SOMAscan assay. Employing slow off-rate aptamers, the relative abundance of 1074 circulating proteins was measured. Proteins identified as being significantly different between groups were subsequently measured by enzyme immunoassay in the whole trial cohort (117 patients) at all time points (days 1-3 [7 time points] and 3 and 6 months). Five proteins identified by the SOMAscan assay, and subsequently confirmed by enzyme immunoassay, were significantly altered by tocilizumab administration. The acute-phase proteins lipopolysaccharide-binding protein, hepcidin, and insulin-like growth factor-binding protein 4 were all reduced during the hospitalization phase, as was the monocyte chemoattractant C-C motif chemokine ligand 23. Proteinase 3, released primarily from neutrophils, was significantly elevated. Conclusions Employing the SOMAscan aptamer-based proteomics platform, 5 proteins were newly identified that are modulated by interleukin 6 antagonism and may mediate the therapeutic effects of tocilizumab in non-ST-segment-elevation myocardial infarction.

Differential production of insulin-like growth factor-binding proteins in liver fibrosis progression.

Noninvasive methods for liver disease diagnoses offer great advantages over biopsy, but they cannot be utilized in all cases. Therefore, specific indicators for chronic liver disease management are necessary. The aim was to assess the production of insulin-like growth factor-binding proteins (IGFBPs) 1-7 and their correlation with the different stages of fibrosis in chronic hepatitis C (CHC). A prospective, cross-sectional, multicenter study was conducted. CHC patients were categorized by FibroTest® and/or FibroScan®. Serum concentrations of IGFBPs 1-7 were determined through multiple suspension arrangement array technology. Significant differences were validated by the Kruskal-Wallis and Mann-Whitney U tests. Logistic regression models were performed to assess the association between the IGFBPs and fibrosis stages. The association was determined utilizing odds ratios (ORs), and receiver operating characteristic (ROC) curves were constructed to distinguish the IGFBPs in relation to the diagnosis of fibrosis. IGFBP-1 and IGFBP-7 concentrations were higher in CHC than in the healthy individuals, whereas IGFBP-3, IGFBP-5, and IGFBP-6 were downregulated in the patients. An apparent increase of all the IGFBPs was found at fibrosis stage F4, but with different regulations. IGFBP-2, -4, -6, and -7 had the best OR, showing the relation to fibrosis progression. The ROC curves showed that IGFBP-7 was the only protein that distinguished F1 from F3 and F2 from F3. IGFBPs participate in liver fibrosis progression and could be employed as circulating novel protein panels for diagnosis and as possible therapeutic targets in liver fibrosis progression.

Increase of circulating IGFBP-4 following genotoxic stress and its implication for senescence.

Senescent cells secrete several molecules, collectively named senescence-associated secretory phenotype (SASP). In the SASP of cells that became senescent following several in vitro chemical and physical stress, we identified the IGFBP-4 protein that can be considered a general stress mediator. This factor appeared to play a key role in senescence-paracrine signaling. We provided evidences showing that genotoxic injury, such as low dose irradiation, may promote an IGFBP-4 release in bloodstream both in mice irradiated with 100 mGy X-ray and in human subjects that received Computer Tomography. Increased level of circulating IGFBP-4 may be responsible of pro-aging effect. We found a significant increase of senescent cells in the lungs, heart, and kidneys of mice that were intraperitoneally injected with IGFBP-4 twice a week for two months. We then analyzed how genotoxic stressors may promote the release of IGFBP-4 and the molecular pathways associated with the induction of senescence by this protein.

ANGPTL4: A Predictive Marker for Diabetic Nephropathy.

BACKGROUND: ANGPTL4 is a glycoprotein that is involved in regulating triglyceride metabolism by inhibiting LPL activity under fasting conditions. Additionally, ANGPTL4 has been suggested as a link between hypertriglyceridemia and albuminuria in the nephrotic syndrome. In this study, we examined levels of circulating ANGPTL4 in people with diabetic nephropathy (DN) and its association with established DN-associated proteins such as IGFBP1 and IGFBP4. METHODS: We quantified circulating ANGPTL4, IGFBP1, IGFBP3, and IGFBP4 in fasting plasma samples of 122 Kuwaiti participants using a multiplexing assay. The study involved 36 controls, as well as 86 people with type 2 diabetes (T2D) including 37 people with normal kidney function and 49 people with DN. RESULTS: ANGPTL4 level was increased in people with DN (241.56 ± 14.1 µg/ml) compared to the control group (178.43 ± 24.09 µg/ml). The increase in ANGPTL4 correlated with clinical parameters of DN including albumin-to-creatinine ratio (r = 0.271, P = 0.002), serum creatinine (r = 0.381, P = 0.0001), and eGFR (r = -0.349, P < 0.0001). Furthermore, ANGPTL4 correlated positively with both IGFBP1 (r = 0.202, P = 0.026) and IGFBP4 (r = 0.364, P < 0.0001). Multiple regression analysis showed increased IGFBP1 and TG as predictors of higher ANGPTL4 in people with DN. In people with T2D, only IGFBP1 acted as a positive predictor of a rise in ANGPTL4. CONCLUSION: In this study, our data showed a significant increase in circulating ANGPTL4, IGFBP1, and IGFBP4 in patients with DN. The elevation in ANGPTL4 correlated significantly with clinical markers of DN such as ACR, serum creatinine, and eGFR, as well as IGFBP1 and IGFBP4. Altogether, this suggests a potential role for ANGPTL4 in DN perhaps through its role in inhibiting LPL activity and promotes ANGPTL4 as a biochemical marker for the detection of a diabetic kidney disease in patients with T2D.

Quantitative iTRAQ-based proteomic analysis of differentially expressed proteins in aging in human and monkey.

BACKGROUND: The underlying physiological mechanisms associated with aging are still complex and unclear. As a very important tissue of human body, the circulatory system also plays a very important role in the process of aging. In this study, we use the isobaric tags for relative and absolute quantification (iTRAQ) method to identify differentially expressed proteins in plasma for humans and monkeys between young and aged. Western blotting and behavioral experiment in mice were performed to validate the expression of the candidate protein. RESULTS: Between the young / the old humans and the young / the old monkeys 74 and 69 proteins were found to be differently expressed, respectively. For the human samples, these included 38 up-regulated proteins and 36 down-regulated proteins (a fold change ≥1.3 or ≤ 0.667, p value ≤0.05).For the monkey samples, 51 up-regulated proteins and 18 down-regulated proteins (a fold change ≥1.3 or ≤ 0.667, p value ≤0.05). KEGG pathway analysis revealed that phagosome, focal adhesion, ECM-receptor interaction and PI3K/AKT signaling pathway were the most common pathways involved in aging. We found only IGFBP4 protein that existed in up-regulated proteins in aged both for human and monkey. In addition, the differential expression of IGFBP4 was validated by western blot analysis and IGFBP4 treatment mimicked aging-related cognitive dysfunction in mice. CONCLUSIONS: This first, the integrated proteomics for the plasma protein of human and monkey reveal one protein-IGFBP4, which was validated by western blotting and behavioral analysis can promote the process of aging. And, iTRAQ analysis showed that proteolytic systems, and inflammatory responses plays an important role in the process of aging. These findings provide a basis for better understanding of the underlying mechanisms involved in aging.

Free IGF-1, Intact IGFBP-4, and PicoPAPP-A are Altered in Acute Myocardial Infarction Compared to Stable Coronary Artery Disease and Healthy Controls.

Insulin-like growth factor-1 (IGF-1) and its binding proteins have been implicated in the pathophysiology of coronary artery disease (CAD) and myocardial infarction (MI). We investigated components of the IGF-1 system in circulation at the time of acute MI and following reperfusion in relation to levels of stable CAD patients and controls. Patients with MI (MI Group, n=31) treated with percutaneous coronary intervention (PCI) were compared to patients with stable CAD subjected to scheduled PCI (CAD Group, n=40) and controls with symptoms mimicking CAD without stenosis in angiography (Control Group, n=43). The number and extent of stenosis were recorded. Total and free IGF-1, total and intact IGF binding protein (IGFBP)-3 and -4, pico-Pregnancy Associated Plasma Protein-A (PAPP-A), and the known markers ALT, AST, CK and CK-MB were measured at baseline and 6 or 24 h after the intervention. Patients with MI had higher free IGF-1 (p=0.003) and PAPP-A (p=0.011), but lower intact IGFBP-4 (p=0.006) compared with patients with stable CAD or healthy controls. None of the investigated molecules changed following reperfusion or correlated with the extent of stenosis. AST (p<0.001), CK (p<0.001) and CK-MB (p<0.001), were also higher. Free IGF-1, intact IGFBP-4 and PAPP-A could predict MI, but with lower accuracy than CK-MB. In conclusion, free IGF-1 levels are higher in MI compared to CAD patients and controls and this could result from increased cleavage of its binding protein IGFBP-4 by the higher PAPP-A levels. Free IGF-1, intact IGFBP-4, and/or PAPP-A are inferior to CK-MB as predictors or markers of myocardial damage.

Serum Levels of Activins, Follistatins, and Growth Factors in Neoplasms of the Breast: A Case-Control Study.

Context: Breast cancer is the most common malignancy in women. Noninvasive biomarkers are needed for its early diagnosis and/or prognosis. Objective: The aim of this case-control study was the comparison of serum activins, follistatins, and members of the IGF family levels in women with benign vs malignant breast neoplasms vs apparently healthy controls. Design and Patients: Women with breast benign (n = 100) or malignant tumors (n = 145) and disease-free controls (n = 100) were recruited. Women with breast cancer were subsequently subdivided into recently diagnosed/treatment-naive (n = 112) and chemotherapy-treated (n = 33). Anthropometric, demographic, biochemical, and histological data were recorded. Setting: A breast cancer clinic in Thessaloniki, Greece. Main Outcome Measures: Serum levels of activin A, activin B, follistatin, follistatin-like (FSTL)-3, total IGF-1, total and intact insulin-like growth factor binding protein (IGFBP)-4 and pregnancy-associated plasma protein-A (PAPP-A) were measured with highly specific ELISA kits. Results: In adjusted comparisons, substantial differences in FSTL-3, total and intact IGFBP-4, PAPP-A, and total IGF-1 were observed between groups. In logistic regression analysis, primarily total IGFBP-4 levels were independently associated with the overall presence of breast malignancy. FSTL-3 was the only variable that could distinguish between a benign vs malignant breast mass. In linear regression analysis, FSTL-3 was independently associated with tumor size. Conclusions: We showed that members of the IGF-1/IGFBP-4/PAPP-A axis and FSTL-3 may serve as surrogate markers in breast cancer. Future mechanistic and longitudinal studies and/or clinical trials are needed to explore the efficacy of these molecules as noninvasive biomarkers and their possible therapeutic potential in breast cancer.

Prognostic value of the Stanniocalcin-2/PAPP-A/IGFBP-4 axis in ST-segment elevation myocardial infarction.

OBJECTIVE: The aim of the present study was to evaluate the prognostic value of the Stanniocalcin-2/PAPP-A/IGFBP-4 axis in patients with ST-segment elevation myocardial infarction (STEMI). METHODS: Observational cohort study performed in 1085 consecutive STEMI patients treated with early reperfusion between February 2011 and August 2014. Stanniocalcin-2, PAPP-A, and IGFBP-4 were measured using state-of-the art immunoassays. The primary outcome was the composite endpoint of all-cause mortality and readmission due to heart failure (HF). RESULTS: Median follow-up was 3.3 years (IQR 1.0-3.7), during which 176 patients (16.2%) presented a composite endpoint. Multivariable cox regression analysis revealed that Stanniocalcin-2 (HR 2.06; 95% CI 1.13-3.75; p = 0.018), IGFBP-4 (HR 1.73; 95% CI 1.14-2.64; p = 0.010), Killip-Kimball class III-IV (HR 1.40; 95% CI 1.13-1.74; p = 0.002), NT-ProBNP (HR 1.21; 95% CI 1.07-1.37; p = 0.002), age (HR 1.06; 95% CI 1.04-1.08; p < 0.001) and left ventricular ejection fraction (HR 0.97; 95% CI 0.95-0.98; p < 0.001) were independent predictors of the composite endpoint. A model containing Stanniocalcin-2 and IGFBP-4 on top of clinical variables significantly improved C-index discrimination (p = 0.036). Stanniocalcin-2 was also identified as independent predictor of all-cause mortality (HR 2.23; 95% CI 1.16-4.29; p = 0.017) and readmission due to HF (HR 3.42; 95% CI 1.22-9.60; p = 0.020). CONCLUSIONS: In STEMI patients, Stanniocalcin-2 and IGFBP-4 emerged as independent predictors of all-cause death and readmission due to HF. The Stanniocalcin-2/PAPP-A/IGFBP-4 axis exhibits a significant role in STEMI risk stratification.

Glycosylated and non-glycosylated NT-IGFBP-4 in circulation of acute coronary syndrome patients.

BACKGROUND: N-terminal and C-terminal proteolytic fragments of IGF binding protein 4 (NT-IGFBP-4 and CT-IGFBP-4) were recently shown to predict adverse cardiac events in acute coronary syndrome (ACS) patients. NT-IGFBP-4 and CT-IGFBP-4 are products of the pregnancy-associated plasma protein-A (PAPP-A)-mediated cleavage of IGFBP-4. It has been demonstrated that circulating IGFBP-4 is partially glycosylated in its N-terminal region, although the influence of this glycosylation on PAPP-A-mediated proteolysis and the ratio of glycosylated/non-glycosylated IGFBP-4 fragments in human blood remain unrevealed. The aims of this study were to investigate i) the presence of glycosylated NT-IGFBP-4 in the circulation, ii) the influence of the glycosylation of IGFBP-4 on its susceptibility to PAPP-A-mediated cleavage, and iii) the influence of glycosylation on NT-IGFBP-4 immunodetection. METHODS: Affinity purification was used for the extraction of IGFBP-4 and NT-IGFBP-4 from plasma samples. Purified proteins were quantified by Western blotting and specific sandwich immunoassays, while molecular masses were determined using mass spectrometry. RESULTS: Glycosylated NT-IGFBP-4 was identified in the blood of ACS patients. The fraction of glycosylated NT-IGFBP-4 in individual plasma samples was 9.8%-23.5% of the total levels of NT-IGFBP-4. PAPP-A-mediated proteolysis of glycosylated IGFBP-4 was 3-4 times less efficient (p < 0.001) than proteolysis of non-glycosylated protein. A sandwich fluoroimmunoassay that was designed for quantitative NT-IGFBP-4 measurements recognized both protein forms with the same efficiency. CONCLUSIONS: Although glycosylation suppresses PAPP-A-mediated IGFBP-4 cleavage, a considerable amount of glycosylated NT-IGFBP-4 is present in blood. Glycosylation does not influence NT-IGFBP-4 measurements using a specific sandwich immunoassay.

Physiological parameters regulating circulating levels of the IGFBP-4/Stanniocalcin-2/PAPP-A axis.

BACKGROUND: Insulin Growth Factor Binding Protein 4 (IGFBP-4), Stanniocalcin-2 (STC-2) and Pregnancy-Associated Plasma Protein-A (PAPP-A) have a well-documented involvement in several physiological functions in humans but predictors of their circulating levels remain largely unknown. We aimed to identify anthropometric and biochemical parameters associated with circulating levels of IGFBP-4/STC-2/PAPP-A axis (ISPa) cross-sectionally and to study their day-night variation and their regulation in response to mixed meal and exercise. METHODS: One hundred twenty two healthy individuals were evaluated cross-sectionally. Subgroups were subjected to standardized mixed meal ingestion in increasing quantities of 125mL or 250mL, or aerobic exercise for 30min, or day-night rhythm study. Main outcome measurements were circulating IGFBP-4 (total and intact), STC-2 and PAPP-A levels. RESULTS: In multivariate models, the main predictors of serum total IGFBP-4 were PAPP-A and female gender. Intact IGFBP-4 was positively associated with serum creatinine. Height was inversely and female gender and % of total body fat were positively correlated with STC-2. PAPP-A decreased after ingesting both the 125mL (p=0.03) and 250mL quantities (p=0.001), while total IGFBP-4 was reduced after the 250mL quantity (p=0.001). Exercise increased STC-2 and PAPP-A levels (p<0.001 for both). Intact, and to a lesser extent total, IGFBP-4 displayed a cortisol-like day/night variation. CONCLUSIONS: We report for the first time anthropometric and physiological modulators of ISPa serum levels in healthy humans.

Insulin-Like Growth Factor Bioactivity, Stanniocalcin-2, Pregnancy-Associated Plasma Protein-A, and IGF-Binding Protein-4 in Pleural Fluid and Serum From Patients With Pulmonary Disease.

Context: Members of the insulin-like growth factor (IGF) system are primarily produced in the liver and secreted into the circulation, but they are also produced, recruited, and activated locally in tissues. Objective: To compare activity and concentrations of IGF system components in pleural fluid and blood. Design: Pathological pleural fluid, secondary to lung cancer or nonmalignant disease, and matching blood samples were collected from 24 patients ages 66.7 to 81.9 years. Methods: IGF-related proteins and cytokine levels were measured by immunoassays or immunoblotting. Bioactive IGF was measured by an IGF-1 receptor phosphorylation assay. Results: Total IGF-1 concentration did not differ between the compartments, but concentrations of free IGF-1 and bioactive IGF were more than threefold higher in pleural fluid than in corresponding serum samples (P = 0.0004), regardless of etiology. Median pregnancy-associated plasma protein-A (PAPP-A) and interleukin (IL)-6 levels were increased 47-fold and 143-fold, respectively, in pleural fluid compared with plasma (P < 0.0001). PAPP-A and IL-6 concentrations correlated positively (r = 0.46; P = 0.02). In pleural fluid, levels of PAPP-A-generated IGF binding protein-4 fragments correlated inversely with that of stanniocalcin-2 (r ≤ -0.42; P ≤ 0.05), a PAPP-A inhibitor; such correlations were absent in plasma. Conclusion: Pathological pleural fluid is characterized by increased in vitro IGF bioactivity and elevated concentrations of PAPP-A, an IGF-activating proteinase. Thus, the tissue activity of the IGF system may differ substantially from that of the circulating IGF system. The correlation between IL-6 and PAPP-A indicates that inflammation plays a role in promoting local tissue IGF activity.

Insulin-Like Growth Factor Binding Protein 4 Fragments Provide Incremental Prognostic Information on Cardiovascular Events in Patients With ST-Segment Elevation Myocardial Infarction.

BACKGROUND: Fragments of insulin-like growth factor binding protein 4 (IGFBP-4) are potential new biomarkers for cardiac risk assessment. The fragments are generated on specific cleavage by pregnancy-associated plasma protein-A, which exerts proatherogenic activity. This study investigated the prognostic value of IGFBP-4 fragments in patients with ST-segment elevation myocardial infarction. METHODS AND RESULTS: We prospectively included 656 patients with ST-segment elevation myocardial infarction treated with percutaneous coronary intervention from September 2006 to December 2008. Blood samples were drawn before percutaneous coronary intervention, and levels of intact IGFBP-4 and N-terminal and C-terminal IGFBP-4 fragments were measured by specific assays. End points were 5-year all-cause and cardiovascular mortality and the combined end point of major adverse cardiac events. Prognostic potential was evaluated on top of a clinical model in terms of discrimination, calibration, and reclassification analysis. During follow-up, 166 patients experienced a major adverse cardiac event and 136 patients died, of whom 69 died from cardiovascular causes. Both IGFBP-4 fragments were associated with all end points (P<0.001). After multivariable adjustments, both N-terminal and C-terminal IGFBP-4 fragment levels remained associated with all end points, including cardiovascular mortality with hazard ratios per doubling in protein concentration of 2.54 (95% CI 1.59-4.07; P<0.001) and 2.07 (95% CI 1.41-3.04; P<0.001), respectively. Incorporation of IGFBP-4 fragments into a clinical model with 15 risk factors improved C-statistics and model calibration and provided incremental prognostic contribution, as assessed by net reclassification improvement and integrated discrimination improvement. CONCLUSIONS: IGFBP-4 fragments are associated with increased risk of all-cause mortality, cardiovascular mortality, and major adverse cardiac events in patients with ST-segment elevation myocardial infarction.

The IGF system in patients with type 2 diabetes: associations with markers of cardiovascular target organ damage.

OBJECTIVE: Perturbations in the insulin-like growth factor (IGF) system may contribute to the accelerated cardiovascular disease (CVD) that occurs in patients with type 2 diabetes (T2D). However, it remains unknown whether the IGF system is also involved in the development of early, subclinical CVD. We characterised the IGF system in T2D patients and matched controls and examined the associations with markers of subclinical target organ damage. METHODS: The study included 99 patients with recently diagnosed T2D and 99 age- and sex-matched controls. IGF-1 and IGFBP-1 to -4 were measured by immunoassays, as were pregnancy-associated plasma protein-A (PAPP-A) and the PAPP-A-generated N-terminal (NT) and C-terminal (CT) IGFBP-4 fragments, which are novel CVD risk markers. Arterial stiffness was evaluated by carotid-femoral pulse wave velocity (PWV). Cerebral white matter lesions (WMLs) and carotid artery remodelling were determined by MRI. RESULTS: After multivariate adjustments, patients with T2D had lower concentrations of IGFBP-2, IGFBP-4, NT- and CT-IGFBP-4, when compared with controls. IGFBP-2 was inversely correlated to PWV in all subjects in multivariate analysis (P < 0.05), and IGFBP-3 was inversely associated with severity of WMLs (P < 0.05). The NT-IGFBP-4 fragment was associated with the degree of carotid artery remodelling among all subjects (regression coefficient (95% CI): 2.95 (0.70, 5.16), P = 0.011). Levels of NT- and CT-IGFBP-4 were reduced in T2D patients receiving metformin compared to those in controls and patients not receiving metformin. CONCLUSIONS: Even in recently diagnosed and well-controlled T2D patients, IGF protein levels are altered and associated with CVD risk factors.

Insulin-Like Growth Factor Binding Protein-4 as a Marker of Chronic Lupus Nephritis.

Kidney biopsy remains the mainstay of Lupus Nephritis (LN) diagnosis and prognostication. The objective of this study is to identify non-invasive biomarkers that closely parallel renal pathology in LN. Previous reports have demonstrated that serum Insulin-like growth factor binding protein 4 (IGFBP-4) was increased in diabetic nephropathy in both animal models and patients. We proceeded to assess if IGFBP4 could be associated with LN. We performed ELISA using the serum of 86 patients with LN. Normal healthy adults (N = 23) and patients with other glomerular diseases (N = 20) served as controls. Compared to the healthy controls or other glomerular disease controls, serum IGFBP-4 levels were significantly higher in the patients with LN. Serum IGFBP-4 did not correlate well with systemic lupus erythematosus disease activity index (SLEDAI), renal SLEDAI or proteinuria, but it did correlate with estimated glomerular filtration rate (R = 0.609, P < 0.0001). Interestingly, in 18 patients with proliferative LN whose blood samples were obtained at the time of renal biopsy, serum IGFBP-4 levels correlated strongly with the chronicity index of renal pathology (R = 0.713, P < 0.001). IGFBP-4 emerges a potential marker of lupus nephritis, reflective of renal pathology chronicity changes.

Development and validation of a spontaneous preterm delivery predictor in asymptomatic women.

BACKGROUND: Preterm delivery remains the leading cause of perinatal mortality. Risk factors and biomarkers have traditionally failed to identify the majority of preterm deliveries. OBJECTIVE: To develop and validate a mass spectrometry-based serum test to predict spontaneous preterm delivery in asymptomatic pregnant women. STUDY DESIGN: A total of 5501 pregnant women were enrolled between 17(0/7) and 28(6/7) weeks gestational age in the prospective Proteomic Assessment of Preterm Risk study at 11 sites in the United States between 2011 and 2013. Maternal blood was collected at enrollment and outcomes collected following delivery. Maternal serum was processed by a proteomic workflow, and proteins were quantified by multiple reaction monitoring mass spectrometry. The discovery and verification process identified 2 serum proteins, insulin-like growth factor-binding protein 4 (IBP4) and sex hormone-binding globulin (SHBG), as predictors of spontaneous preterm delivery. We evaluated a predictor using the log ratio of the measures of IBP4 and SHBG (IBP4/SHBG) in a clinical validation study to classify spontaneous preterm delivery cases (<37(0/7) weeks gestational age) in a nested case-control cohort different from subjects used in discovery and verification. Strict blinding and independent statistical analyses were employed. RESULTS: The predictor had an area under the receiver operating characteristic curve value of 0.75 and sensitivity and specificity of 0.75 and 0.74, respectively. The IBP4/SHBG predictor at this sensitivity and specificity had an odds ratio of 5.04 for spontaneous preterm delivery. Accuracy of the IBP4/SHBG predictor increased using earlier case-vs-control gestational age cutoffs (eg, <35(0/7) vs ≥35(0/7) weeks gestational age). Importantly, higher-risk subjects defined by the IBP4/SHBG predictor score generally gave birth earlier than lower-risk subjects. CONCLUSION: A serum-based molecular predictor identifies asymptomatic pregnant women at risk of spontaneous preterm delivery, which may provide utility in identifying women at risk at an early stage of pregnancy to allow for clinical intervention. This early detection would guide enhanced levels of care and accelerate development of clinical strategies to prevent preterm delivery.

Serum IGFBP4 concentration decreased in dairy heifers towards day 18 of pregnancy.

This study was conducted to determine if the main components of the somatotropic axis change during the early phase of pregnancy in the maternal blood system and whether differences exist on day 18 after pregnancy recognition by the maternal organism. Blood samples of pregnant heifers (Holstein Friesian; n = 10 after embryo transfer) were obtained on the day of ovulation (day 0), as well as on days 7, 14, 16 and 18 and during pregnant, non-pregnant and negative control cycles. The concentrations of progesterone (P4), oestrogen, growth hormone (GH), insulin-like growth factor-1 and -2 (IGF1, -2) and IGF-binding protein-2, -3 and -4 (IGFBP2, -3, -4) were measured. The mRNA expressions of growth hormone receptor 1A, IGF1, IGF2, IGFBP2, IGFBP3 and IGFBP4 were detected using RT-qPCR in liver biopsy specimens (day 18). In all groups, total serum IGF1 decreased from day 0 to 16. Notably, IGFBP4 maternal blood concentrations were lower during pregnancy than during non-pregnant cycles and synchronized control cycles. It can be speculated that the lower IGFBP4 in maternal blood may result in an increase of free IGF1 for local action. Further studies regarding IGFBP4 concentration and healthy early pregnancy are warranted.