3D-Printed SERS Chips for Highly Specific Detection of Denatured Type I and IV Collagens in Blood for Early Hepatic Fibrosis Diagnosis.

Hepatic fibrosis, the insidious progression of chronic liver scarring leading to life-threatening cirrhosis and hepatocellular carcinoma, necessitates the urgent development of noninvasive and precise diagnostic methodologies. Denatured collagen emerges as a critical biomarker in the pathogenesis of hepatic fibrosis. Herein, we have for the first time developed 3D-printed collagen capture chips for highly specific surface-enhanced Raman scattering (SERS) detection of denatured type I and type IV collagen in blood, facilitating the early diagnosis of hepatic fibrosis. Employing a novel blend of denatured collagen-targeting peptide-modified silver nanoparticle probes (Ag@DCTP) and polyethylene glycol diacrylate (PEGDA), we engineered a robust ink for the 3D fabrication of these collagen capture chips. The chips are further equipped with specialized SERS peptide probes, Ag@ICTP@R1 (S-I) and Ag@IVCTP@R2 (S-IV), tailored for the targeted detection of type I and IV collagen, respectively. The SERS chip platform demonstrated exceptional specificity and sensitivity in capturing and detecting denatured type I and IV collagen, achieving detection limits of 3.5 ng/mL for type I and 3.2 ng/mL for type IV collagen within a 10-400 ng/mL range. When tested on serum samples from hepatic fibrosis mouse models across a spectrum of fibrosis stages (S0-S4), the chips consistently measured denatured type I collagen and detected a progressive increase in type IV collagen concentration, which correlated with the severity of fibrosis. This novel strategy establishes a benchmark for the multiplexed detection of collagen biomarkers, enhancing our capacity to assess the stages of hepatic fibrosis.

Predictive scoring system for advanced liver fibrosis in Japanese patients with severe obesity.

PURPOSE: The aim of this study was to examine the predictive scoring system of advanced liver fibrosis in severely obese Japanese patients. METHODS: Seventy-two patients underwent laparoscopic sleeve gastrectomy and intraoperative liver biopsies. We classified these patients into two groups: Brunt stage ≥ 2 (advanced fibrosis) and 0/1 (none/mild fibrosis). A logistic regression analysis was performed to identify the predictors of advanced fibrosis. RESULTS: Sixteen patients had advanced fibrosis, while 56 had no/mild fibrosis. The prevalence of type 2 diabetes mellitus (T2DM) in advanced fibrosis group was significantly higher than in none/mild fibrosis. An univariate analysis of the factors predicting advanced fibrosis showed significant differences in AST/ALT ratio, serum insulin levels, HOMA-IR, and type IV collagen 7S in the T2DM group. According to a multivariate analysis, type IV collagen 7S was an independent predictor and the cutoff value was 5.6 ng/mL. We created a flow chart; high risk (T2DM and type IV collagen 7S ≥ 5.6 ng/mL), moderate risk (T2DM and type IV collagen 7S < 5.6 ng/mL), and low risk (non-DM). For those at high risk, the sensitivity, specificity, positive predictive value, and negative predictive value were 56.2%, 94.4%, 75.0%, and 87.9%, respectively. CONCLUSION: This classification system has the potential to accurately categorize the risk of liver fibrosis.

Type IV Collagen 7s Reflects Central Venous Pressure and Right Ventricular End-Diastolic Pressure in Patients with Congenital Heart Disease After Biventricular Repair.

After congenital heart disease repair, right heart dysfunction facilitates venous stasis and elevated central venous pressure; however, methods to evaluate right heart dysfunction are limited. We aimed to evaluate right heart function using liver biomarkers. We investigated 62 patients more than 5 years after congenital heart surgery. The patients underwent cardiac catheterization in our hospital between January 2015 and December 2019. To evaluate liver status, type IV collagen 7s, procollagen type III peptide, and hyaluronic acid levels were measured. The mean age of the 62 patients was 14.0 ± 7.2 years. The mean central venous pressure was 6.8 ± 3.5 mmHg and mean right ventricular end-diastolic pressure was 7.9 ± 3.5 mmHg. The mean levels of serum type IV collagen 7s, procollagen type III peptide, and hyaluronic acid were 5.9 ± 1.6 ng/mL, 24.3 ± 15.5 ng/mL, and 18.5 ± 13.6 ng/mL, respectively. There was a good correlation between central venous pressure, right ventricular end-diastolic pressure and type IV collagen 7s (r = 0.67 and r = 0.64). There was no correlation between central venous pressure and the procollagen type III peptide (r = 0.003), and slight correlation between central venous pressure and hyaluronic acid (r = 0.31). There was no correlation between right ventricular end-diastolic pressure and the procollagen type III peptide (r = 0.003), and slight correlation between right ventricular end-diastolic pressure and hyaluronic acid (r = 0.31). We found that changes in the hemodynamics of the right heart system can be evaluated using liver fibrosis markers. Type IV collagen 7s reflects central venous pressure and right ventricular end-diastolic pressure in postoperative patients with congenital heart disease.

Usefulness of collagen type IV in the detection of significant liver fibrosis in nonalcoholic fatty liver disease.

INTRODUCTION/AIMS: Liver fibrosis assessment is a key issue in the evaluation of nonalcoholic fatty liver disease (NAFLD) patients. In the present study, we aimed to validate a noninvasive marker panel to assess significant and advanced fibrosis in these patients. METHOD: 126 biopsy-proven NAFLD patients were included. NAFLD diagnosis was based on histological criteria. Fibrosis stages were determined according to NASH-Clinical Research Network criteria. Clinical and laboratorial data were collected during the interval of three months before or after liver biopsy. Histological fibrosis stages were classified as significant fibrosis (F2-F4) and advanced fibrosis (F3-F4). Five serum biomarkers [hyaluronic acid (HA), collagen type IV (cIV), procollagen type III (PC III), laminin (LN) and cholylglycine (CG)] were assessed by chemiluminescence immunoassays. RESULTS: Most patients were female (61.61%), mean age: 55.7 ±â€¯9.13 years old and mean BMI was 32.1 ±â€¯5.9 kg/m2. Prevalence of diabetes, dyslipidemia, arterial hypertension, and metabolic syndrome was 68.75%, 82.29%, 63.54% and 81.05%, respectively. Patients with cIV above 30 ng/mL had a 5.57-times (IC: 1.86-16.69) the chance of having significant fibrosis and 7.61-times (IC: 2.27-25.54) the chance of having advanced fibrosis versus patients with values below 30 ng/mL. HA, PC III, LN and CG did not detect the presence of significant and advanced fibrosis. The AUROC of clV for detection of significant (0.718) and advanced fibrosis (0.791) was better than that of other serum biomarkers. CONCLUSION: Type 4 collagen could predict the presence of significant and advanced fibrosis in NAFLD patients and it would be a useful tool in routine clinical practice.

Serological Biomarkers of Tissue Turnover Identify Responders to Anti-TNF Therapy in Crohn's Disease: A Pilot Study.

INTRODUCTION: Anti-tumor necrosis factor (TNF) therapy is effective in inducing remission in Crohn's disease in 60% of patients. No serological biomarkers are available, which can predict response to anti-TNF. We aimed to investigate serological markers of collagen turnover reflecting tissue inflammation as predictors of response to anti-TNF. METHODS: In 2 retrospective observational cohorts, markers for matrix metalloproteinase-degraded type III and IV collagens (C3M and C4M, respectively) and for formation of type III and IV collagens (PRO-C3 and PRO-C4, respectively) were measured in serum and compared with standard C-reactive protein in patients with active Crohn's disease who started infliximab (IFX, n = 21) or adalimumab (ADA, n = 21). Disease activity was classified by the Harvey-Bradshaw index (active disease ≥5); response was defined as clinical remission. RESULTS: Seventeen patients (81%) treated with IFX were in remission at week 14; 15 patients (71%) treated with ADA were in remission at week 8. Serum C4M at baseline was increased in nonresponders compared with responders (IFX: 35.0 ± 2.4 vs 23.2 ± 2.6, P = 0.04, ADA: 53.0 ± 3.2 vs 34.1 ± 2.8, P = 0.006). C4M levels at baseline predicted response in both cohorts (IFX: odds ratio 39 [95% confidence interval, 2.4-523.9] P = 0.02, cutoff 35.2 nmol/L; ADA: odds ratio 26 [95% confidence interval, 1.8-332.5], P = 0.01, cutoff 46.9 nmol/L). C-reactive protein was not able to predict response to anti-TNF. DISCUSSION: Response to anti-TNF therapy within the first 14 weeks of treatment can be predicted based on baseline levels of basement membrane marker C4M. This marker could be used as biomarker for response to anti-TNF and could aid in early therapy decision making. Validation in larger well-defined cohorts is needed.

Pre-hepatectomy type IV collagen 7S predicts post-hepatectomy liver failure and recovery.

BACKGROUND: Liver resection is an effective treatment for benign and malignant liver tumors. However, a method for preoperative evaluation of hepatic reserve has not yet been established. Previously reported assessments of preoperative hepatic reserve focused only on liver failure in the early postoperative period and did not consider the long-term recovery of hepatic reserve. When determining eligibility for hepatectomy, the underlying pathophysiology needs to be considered to determine if the functional hepatic reserve can withstand both surgery and any postoperative therapy. AIM: To identify pre-hepatectomy factors associated with both early postoperative liver failure and long-term postoperative liver function recovery. METHODS: This study was a retrospective cohort study. We retrospectively investigated 215 patients who underwent hepatectomy at our hospital between May 2013 and December 2016. Early post-hepatectomy liver failure (PHLF) was defined using the International Study Group of Liver Surgery's definition of PHLF. Long-term postoperative recovery of liver function was defined as the time taken for serum total bilirubin and albumin levels to return to levels of < 2 mg/dL and > 2.8 g/dL, respectively, and the time taken for Child-Pugh score to return to Child-Pugh class A. RESULTS: Preoperative type IV collagen 7S was identified as a significant independent factor associated with both PHLF and postoperative long-term recovery of liver function. Further analysis revealed that the time taken for the recovery of Child-Pugh scores and serum total bilirubin and albumin levels was significantly shorter in patients with type IV collagen 7S ≤ 6 ng/mL than in those with type IV collagen 7S > 6 ng/mL. In additional analyses, similar results were observed in patients without chronic viral hepatitis associated with fibrosis. CONCLUSION: Preoperative type IV collagen 7S is a preoperative predictor of PHLF and long-term postoperative liver function recovery. It can also be used in patients without chronic hepatitis virus.

Collagen IV, a promising serum biomarker for evaluating the prognosis of revascularization in a 2-kidney, 1-clip hypertensive rat model.

OBJECTIVES: The goal of this study was to investigate the expression of serum collagen IV and its value for evaluating the prognosis of revascularization in a 2-kidney, 1-clip hypertensive rat model. METHODS: A total of 40 Sprague-Dawley rats were randomly and evenly divided into a control group and 3-, 10- and 20-day (D) groups (namely, the ischaemic time for 3, 10 and 20 days, respectively). The systolic blood pressure and laboratory values such as serum creatinine and collagen IV levels were measured before and after clipping the renal artery. Histological Masson staining and immunohistochemical staining of collagen IV were conducted in a kidney specimen from each group to assess the severity of renal fibrosis and the level of collagen IV expression. RESULTS: After clipping, systolic blood pressure in the 3D, 10D and 20D groups increased significantly from 108 ± 8 to 126 ± 7 and from 153 ± 8 to 157 ± 6 mmHg, respectively (10D vs 20D group, P = 0.224; between other groups, P < 0.001). The expression of serum creatinine in the 3D, 10D and 20D groups increased significantly from 35.39 ± 5.64 to 57.53 ± 7.05, 101.86 ± 8.94 and 119.76 ± 9.37 mmol/l, respectively (between each group: P < 0.001). Serum collagen IV levels in the 10D and 20D groups increased significantly from 38.5 ± 10.4 to 60.8 ± 15.0 and 87.3 ± 11.5 ng/ml, respectively (control vs 3D group, P = 0.718; between other groups, P < 0.001). The Masson staining indicated that sclerotic changes in the glomeruli of the 10D and 20D groups significantly increased from 2.20 ± 1.03 to 15.20 ± 5.03 and 28.20 ± 7.07%, respectively (control vs 3D group, P = 0.175; between other groups, P < 0.001). The grade of tubulointerstitial damage in the 3D, 10D and 20D groups increased significantly from 0.30 ± 0.48 to 1.90 ± 0.74, 1.80 ± 0.79 and 3.20 ± 0.79, respectively (3D vs 10D group, P = 0.755; between other groups, P < 0.001). The semi-quantification from immunohistochemical staining indicated that the percentage of collagen IV positive areas in the 3D, 10D and 20D groups increased significantly from 3.50 ± 1.58 to 8.60 ± 2.11, 16.60 ± 8.55 and 23.10 ± 6.15, respectively (control vs 3D group, P = 0.043; 3D vs 10D group, P = 0.002; 10D vs 20D group, P = 0.011; between other groups, P < 0.001). The area under the curve of the receiver operating characteristic curve was 0.783 (P = 0.008; 95% confidence interval 0.634-0.932). There were positive associations of serum collagen IV levels with systolic blood pressure, serum creatinine and collagen IV quantification in kidney with correlation coefficients of 0.665, 0.775 and 0.628, respectively (P < 0.001). CONCLUSIONS: As the clear ischaemia time-response relationship identified in our study indicates, the increase in serum collagen IV levels may be a satisfactory biomarker to indicate a poor prognosis of renal artery revascularization in a 2-kidney, 1-clip hypertensive rat model. However, it is perhaps not a good early biomarker for the early detection of renovascular hypertension.

Circulating levels of PRO-C3 reflect liver fibrosis and liver function in HIV positive patients receiving modern cART.

BACKGROUND AND AIMS: Although combined antiretroviral treatment (cART) has improved overall survival of HIV infected patients, liver fibrosis and liver related-mortality still constitute major challenges in HIV positive patients. Collagen accumulates in the liver during fibrogenesis. Recent studies showed that circulating levels of extracellular matrix (ECM) fragments might reflect degree of portal hypertension and fibrosis stage in liver disease. In this study, we analyzed the correlation between liver fibrosis assessed by Fibroscan and levels of the formation and degradation markers of type III and IV collagen in HIV positive patients receiving cART. METHODS: 116 HIV positive patients (82.7% male, median age 47 years) were enrolled into the study. Liver stiffness and liver fat content were determined using a Fibroscan with integrated CAP function. We quantified ECM formation and degradation fragments of collagen III and IV: PRO-C3, PRO-C4, C3M and C4M. These fragments were measured in peripheral serum by using specific ELISAs. RESULTS: Fifteen (12.9%) out of the 116 HIV positive patients had relevant fibrosis with a liver stiffness ≥ 7.1 kPa, and 79 patients had relevant steatosis with a CAP value > 248 dB/m. Circulating PRO-C3 levels significantly correlated with increasing degree of liver fibrosis assessed by Fibroscan (p = 0.0005), as well as with APRI score (p = 0.015). Interestingly, circulating PRO-C3 levels were significantly correlated with bilirubin (p = 0.022), reduced platelet count (p = 0.0008) and low albumin levels (p = 0.001), suggesting the association of type III collagen deposition with impaired liver function. None of the other measured ECM components significantly correlated with fibrosis or steatosis. CONCLUSION: The formation marker of type III collagen, PRO-C3 not only reflects liver fibrosis, but might also mirror liver dysfunction in HIV positive patients receiving cART. Therefore, the circulating levels of PRO-C3 might be suitable to monitor progression of liver fibrosis and deterioration of liver function in HIV positive patients receiving cART.

Angiotensin blockade attenuates diabetic nephropathy in hypogonadal adult male rats.

This study examined the effect of the aromatase inhibitor letrozole (0.5 mg/kg) alone or in combination with the angiotensin-receptor blocker valsartan (30 mg/kg) against streptozocin-induced diabetic nephropathy (DN) in hypogonadal (HG) rats for 12 weeks. First, we tested the HG effect on hormone levels, inflammatory cytokines, and oxidative stress in nondiabetic (ND) and diabetic (D) rats. HG was induced with the luteinizing hormone-releasing hormone antagonist cetrorelix (0.71 mg/kg). Diabetes enhanced hormonal hypogonadism and increased inflammation and oxidative stress. Next, experiments examined the effect of early letrozole and valsartan intervention on DN in HG rats. HG-ND and HG-D rats were treated with letrozole alone or in combination with valsartan. HG-D rats developed proteinuria and had increased blood urea nitrogen and creatinine, and histopathological evidence of renal injury, including glomerular hypertrophy and mesangial expansion. Valsartan alone or in combination with letrozole reduced proteinuria, improved renal functions, and reduced diabetes-induced renal angiotensin II. Both agents ameliorated nuclear factor kappa light chain enhancer of activated B cells, interleukin 1ß, interleukin 6, and tumor necrosis factor alpha levels. The combination decreased superoxide dismutase, malondialdehyde, and glutathione peroxidase levels, and prevented glomerular hypertrophy. In HG-D rats, valsartan reduced renal collagen IV and transforming growth factor-beta 1, especially when the testosterone level was corrected by letrozole. Thus, normalizing testosterone and inhibiting renal angiotensin II have a renoprotective effect against DN in HG male rats.

Elevated non-invasive liver fibrosis markers and risk of liver carcinoma in adult patients after repair of tetralogy of Fallot.

BACKGROUND: Congestive hepatopathy and hepatocellular carcinoma is a serious complication after Fontan procedure. Liver fibrosis due to hepatic congestion could occur also in adult patients after repair of tetralogy of Fallot (rTOF). However, the incidence and severity remain unclear. METHODS: A total of 111 patients with adult congenital heart disease between 2009 and 2016 were enrolled. Liver fibrosis markers and hemodynamic parameters assessed by cardiac magnetic resonance imaging and catheterization were analyzed in 50 rTOF patients having significant pulmonary regurgitation and/or stenosis, 50 Fontan patients and 11 controls. RESULTS: Liver fibrosis markers in patients with rTOF were significantly higher than controls, and tended to be lower than Fontan patients (median, hyaluronic acid: 25.8 vs. 15.9 vs. 40.8, type IV collagen: 129 vs. 113 vs. 166, ng/mL, p < 0.05, respectively). Patients with rTOF showed abnormal hyaluronic acid levels more frequently than controls, and less frequently than Fontan patients (22% vs. 0% vs. 38%, respectively, p < 0.05). Multivariate analyses indicated a positive association of right atrial pressure with type IV-collagen or hyaluronic acid levels (each, p < 0.001, p = 0.003). Abdominal ultrasonography revealed hepatic congestion in 50% of rTOF patients tested. Liver biopsy of the two rTOF patients with highest hyaluronic acid levels showed pathological evidence of moderate and severe (F2 and F3) liver fibrosis and one had combined hepatocellular and cholangiocarcinoma. CONCLUSIONS: We first demonstrated elevated liver fibrosis markers in adult patients with rTOF. These levels may help to predict the progressive liver disease as well as consider the timing of pulmonary valve replacement.

Type IV collagen turnover is predictive of mortality in COPD: a comparison to fibrinogen in a prospective analysis of the ECLIPSE cohort.

BACKGROUND: Identifying subjects with chronic obstructive pulmonary disease (COPD) at high risk of exacerbation and mortality is key to aid individual management of COPD. The only FDA approved blood-based drug development biomarker for patients at high risk of mortality, is plasma fibrinogen. In this study, we benchmarked two biomarkers of basement membrane remodeling, a characteristic of COPD, against plasma fibrinogen alone and as a combination. The biomarkers of basement membrane remodeling are two neoepitopes from of the alpha 3 chain of type IV collagen (COL4A3). MATERIALS AND METHODS: COL4A3 degradation was assessed by the biomarkers C4Ma3 and tumstatin (TUM) in year 1 plasma samples in 984 COPD subjects, 95 non-smoking controls and 95 smoking controls from the Evaluation of COPD Longitudinally to Identify Predictive Surrogate End-points (ECLIPSE) cohort. They were measured by competitive ELISA using monoclonal antibodies recognizing two specific MMP-generated cleavage site within COL4A3. The level of fibrinogen was previously assessed in year 1 plasma. RESULTS: In COPD subjects, plasma C4Ma3 levels were significantly correlated with plasma fibrinogen levels (0.389 (P < 0.0001)). Cox proportional-hazards regression adjusted for relevant confounders showed that high levels of plasma C4Ma3, but not TUM, were related to a higher risk of mortality (hazard ratio 5.12 (95% CI 2.28-11.50), P < 0.0001). High levels of plasma fibrinogen were not associated with all-cause mortality in this subpopulation, contradictory to published results. Whereas plasma C4Ma3 multiplied by fibrinogen showed to be related to a higher risk of mortality (hazard ratio 5.74 (95% CI 2.65-12.41), P < 0.0001). Plasma C4Ma3 levels were related to the number of hospitalizations due to COPD exacerbations in the year before study start (P = 0.0375). Fibrinogen levels were related to hospitalized exacerbations prior to study start (P = 0.0058) and were also related to future exacerbations (P < 0.0001). CONCLUSION: We compared herein fibrinogen, C4Ma3 and TUM as biomarkers for COPD prognosis. Fibrinogen was related to future exacerbation, whereas C4Ma3 and the combination of C4Ma3 with fibrinogen were superior to fibrinogen alone in predicting mortality. This pilot study suggests that the assessment of plasma C4Ma3 could be important for identifying COPD patients with a poor prognosis. TRIAL REGISTRATION: NCT00292552 , GSK Study No. SCO104960.

Exosomes increased angiogenesis in papillary thyroid cancer microenvironment.

Tumour-derived exosomes under hypoxic conditions contain informative miRNAs involved in the interaction of cancer and para-carcinoma cells, thus contributing to tissue remodelling of the tumour microenvironment (TME). Exosomes isolated from hypoxic papillary thyroid cancer cells, BCPAP cells and KTC-1 cells enhanced the angiogenesis of human umbilical vein endothelial cells (HUVECs) compared with exosomes isolated from normal thyroid follicular cell line (Nthy-ori-3-1), normoxic BCPAP or KTC-1 cells both in vitro and in vivo. miR-21-5p was significantly upregulated in exosomes from papillary thyroid cancer BCPAP cells under hypoxic conditions, while the exosomes isolated from hypoxic BCPAP cells with knockdown of miR-21-5p attenuated the promoting effect of angiogenesis. In addition, our experiment revealed that miR-21-5p directly targeted and suppressed TGFBI and COL4A1, thereby increasing endothelial tube formation. Furthermore, elevated levels of exosomal miR-21-5p are found in the sera of papillary thyroid cancer patients, which promote the angiogenesis of HUVECs. Taken together, our study reveals the cell interaction between hypoxic papillary thyroid cancer cells and endothelial cells, elucidating a new mechanism by which hypoxic papillary thyroid cancer cells increase angiogenesis via exosomal miR-21-5p/TGFBI and miR-21-5p/COL4A1 regulatory pathway.

Combination leflunomide and methotrexate impedes the recovery of liver fibrosis, partly through inhibition of myeloid cell admittance.

The process of liver fibrosis is reversible and involves a recovery phase. In the present study, the potential side effects of combination leflunomide and methotrexate (LEF+MTX), a conventional rheumatoid arthritis therapy used in the resolution of liver fibrosis, was investigated. In a carbon tetrachloride­induced liver fibrosis model, the results of hepatic pathology demonstrated that the LEF+MTX combination delayed the recovery of fibrosis, although the activation of hepatic stellate cells in vitro was inhibited. A total of four liver fibrosis­associated indicators, hyaluronic acid, laminin, procollagen type III and collagen IV, maintained high levels in the serum of LEF+MTX­treated mice, while detection of bone marrow­driven monocytes in the blood by flow cytometry indicated that they were significantly decreased. Notably, the results of immunofluorescence staining of hepatic myeloid cells and detection of vascular growth factor A (VEGF­A) in blood and liver suggested that the reduced degeneration of collagen in liver sinusoids was associated with decreased myeloid cell adhesion and the downregulation of VEGF­A in the liver. The present results suggested that in certain cases, treatment with LEF+MTX may impede the recovery of hepatic fibrosis­associated diseases in mice.

Circulating markers of collagen types I, III, and IV in patients with dilated cardiomyopathy: relationships with myocardial collagen expression.

AIMS: Collagen-derived peptides such as collagen I C-terminal telopeptide (CITP) and procollagen III N-terminal propeptide (PIIINP) have been conventionally used as markers of cardiac fibrosis. Collagen IV 7S domain (P4NP 7S) has been recently reported to be correlated with haemodynamics in patients with acute heart failure. We investigated whether these markers reflect cardiac remodelling and myocardial collagen expression. METHODS AND RESULTS: In 80 patients with dilated cardiomyopathy, relationships of CITP, PIIINP, and P4NP 7S to clinical and echocardiographic variables were analysed. CITP and PIIINP were inversely correlated with estimated glomerular filtration rate (r = -0.41, P < 0.001 and r = -0.32, P = 0.004, respectively); P4NP 7S was positively correlated with B-type natriuretic peptide (r = 0.32, P = 0.003) and γ-glutamyltransferase (r = 0.38, P < 0.001). These correlations were significant even after adjustment by potential confounders, whereas all three collagen markers were not independently correlated with ejection fraction nor with left ventricular (LV) diastolic diameter. In 33 patients undergoing endomyocardial biopsy, myocardial collagen I and III mRNA expressions were correlated with LV end-diastolic volume index (r = 0.42, P = 0.02 and r = 0.54, P = 0.002, respectively), whereas myocardial collagen IV mRNA expression was not correlated with LV end-diastolic volume index nor with ejection fraction. Each collagen-derived peptide was not significantly correlated with the myocardial expression of their corresponding collagen mRNA. CONCLUSIONS: Our study shows that CITP, PIIINP, and P4NP 7S do not reflect myocardial collagen mRNA expression but presumably reflect extra-cardiac organ injury in heart failure.

Evaluation of the Impact of Preoperative Values of Hyaluronic Acid and Type IV Collagen on the Outcome of Patients with Hepatocellular Carcinoma After Hepatectomy.

BACKGROUND: Recently, some reports have revealed a relationship between post-hepatectomy prognosis in hepatocellular carcinoma (HCC) and hepatic fibrosis markers. We evaluated the relationship between these markers of hepatic fibrosis, clinicopathological findings, and prognosis. METHODS: Three hundred and sixty patients underwent hepatectomy for HCC in the Nippon Medical School Hospital between 1993 and 2013. We divided these patients into two groups: normal serum hyaluronic acid (HA) levels and abnormal levels. We also divided patients into groups with normal serum type IV collagen levels and abnormal levels. RESULTS: The overall survival rate and recurrence-free survival rate of the normal group were significantly higher than those of the abnormal group. In the normal hyaluronic acid group, serum albumin and prothrombin time were significantly higher than in the abnormal group, and age, hepatitis C virus antibody (HCV)-Ab positivity, Child-Pugh grade B, liver cirrhosis, indocyanine green retention rate at 15 min (ICGR15), type IV collagen level, and type IV collagen 7s level were significantly lower than those in the abnormal group. In the normal type IV collagen group, HCV-Ab positivity, liver cirrhosis, ICGR15, HA level, and type IV collagen 7s level were significantly lower than those in the abnormal group, and the serum albumin level was significantly higher than that in the abnormal group. Multivariate analysis independently revealed the significant effect of serum type IV collagen on the overall survival rate as well as the significant effect of serum HA on the recurrence-free survival rate in patients who underwent hepatectomy for HCC. CONCLUSIONS: Preoperative examinations of serum hyaluronic acid levels and type IV collagen levels are imperative for hepatic resection for HCC because these markers are significantly associated with liver function and prognosis.

Beneficial effects of cilostazol on liver injury induced by common bile duct ligation in rats: Role of SIRT1 signaling pathway.

Liver fibrosis is a health challenge requiring alternative therapeutic approaches. Cilostazol is a selective phosphodiesterase-3 inhibitor and possesses antioxidant, anti-inflammatory and antifibrotic properties. Sirtuin 1 (SIRT1) is a member of the silent information regulator 2 family. Cilostazol upregulates SIRT1 expression. Cilostazol protects against the cholestatic liver insults caused by bile duct obstruction. Involvement of SIRT1 pathway in this protective effect has not been studied yet. So, we hypothesized that SIRT1 signaling may have a role in cilostazol protective effects against bile duct ligation-induced liver damages. Rats were subjected to common bile duct ligation then treated with cilostazol (9 mg/kg or 27 mg/kg) in the presence or absence of specific SIRT1 inhibitor EX527. Cilostazol improved liver function, reduced inflammation, enhanced antioxidant status, ameliorated cholestatic liver injury and upregulated hepatic SIRT1. However, these protective effects were abrogated by EX527, suggesting that SIRT1 signaling may have a role in these effects. In conclusion, cilostazol in a dose-dependent way produced hepatoprotective effects via anti-inflammatory, antioxidant, antifibrotic effects which were mediated, in part, through SIRT1 upregulation.

Assessment of extracellular matrix-related biomarkers in patients with lower extremity artery disease.

BACKGROUND: The prevalence of lower extremity artery disease (LEAD) is high (20%-25%) in the population older than 65 years, but patients are seldom identified until the disease is advanced. Circulating markers of disease activity might provide patients with a key opportunity for timely treatment. We tested the hypothesis that measuring blood-specific fragments generated during degradation of the extracellular matrix (ECM) could provide further insight into the pathophysiologic mechanism of arterial remodeling. METHODS: The protein profile of diseased arteries from patients undergoing infrainguinal limb revascularization was assessed by a liquid chromatography and tandem mass spectrometry, nontargeted proteomic approach. The information retrieved was the basis for measurement of neoepitope fragments of ECM proteins in the blood of 195 consecutive patients with LEAD by specific enzyme-linked immunosorbent assays. RESULTS: Histologic and proteomic analyses confirmed the structural disorganization of affected arteries. Fourteen of 81 proteins were identified as differentially expressed in diseased arteries with respect to healthy tissues. Most of them were related to ECM components, and the difference in expression was used in multivariate analyses to establish that severe arterial lesions in LEAD patients have a specific proteome. Analysis of neoepitope fragments in blood revealed that fragments of versican and collagen type IV, alone or in combination, segregated patients with mild to moderate symptoms (intermittent claudication, Fontaine I-II) from those with severe LEAD (critical limb ischemia, Fontaine III-IV). CONCLUSIONS: We propose noninvasive candidate biomarkers with the ability to be clinically useful across the LEAD spectrum.

Circulating Vascular Basement Membrane Fragments are Associated with the Diameter of the Abdominal Aorta and Their Expression Pattern is Altered in AAA Tissue.

OBJECTIVE: Abdominal aortic aneurysm (AAA) is characterised by enhanced proteolytic activity, and extracellular matrix (ECM) remodelling in the vascular wall. Type IV and XVIII collagen/endostatin are structural proteins in vascular basement membrane (VBM), a specialised ECM structure. Here the association between plasma levels of these collagens with the aortic diameter and expansion rate is studied, and their expression in aortic tissue characterised. METHODS: This was a retrospective population based cohort study. Type IV and XVIII collagen/endostatin were analysed in plasma by ELISA assay in 615 men, divided into three groups based on the aortic diameter: 1) normal aorta ≤ 25 mm, 2) sub-aneurysmal aorta (SAA) 26-29 mm, and 3) AAA ≥ 30 mm. Follow up data were available for 159 men. The association between collagen levels and aortic diameter at baseline, and with the expansion rate at follow up were analysed in ordinal logistic regression and linear regression models, controlling for common confounding factors. Tissue expression of the collagens was analysed in normal aorta (n = 6) and AAA (n = 6) by immunofluorescence. RESULTS: Plasma levels of type XVIII collagen/endostatin (136 ng/mL [SD 29] in individuals with a normal aorta diameter, 154 ng/ml [SD 45] in SAA, and 162 ng/ml [SD 46] in AAA; p = .001) and type IV collagen (105 ng/mL [SD 42] normal aorta, 124 ng/ml [SD 46] SAA, and 127 ng/ml [SD 47] AAA; p = .037) were associated with a larger aortic diameter. A significant association was found between the baseline levels of type XVIII/endostatin and the aortic expansion rate (p = .035), but in the multivariable model, only the initial aortic diameter remained significantly associated with expansion (p = .005). Altered expression patterns of both collagens were observed in AAA tissue. CONCLUSION: Plasma levels of circulating type IV and XVIII collagen/endostatin increase with AAA diameter. The expression pattern of VBM proteins is altered in the aneurysm wall.

Wisteria floribunda agglutinin-positive Mac-2-binding protein and type 4 collagen 7S: useful markers for the diagnosis of significant fibrosis in patients with non-alcoholic fatty liver disease.

BACKGROUND AND AIM: The fibrosis stage of liver is associated with the long-term outcomes in patients with non-alcoholic fatty liver disease (NAFLD). However, significant fibrosis, defined as fibrosis stages 2-4, is associated with an elevated risk of progression to severe liver disease; there have been scant reports about diagnosing significant fibrosis. We compare the noninvasive method and aim to identify appropriate liver fibrosis markers for detecting significant fibrosis in NAFLD patients. METHODS: We compared the usefulness of liver fibrosis markers (Wisteria floribunda agglutinin-positive Mac-2-binding protein [WFA+ -M2BP], type 4 collagen 7S, etc.), clinical scoring systems, and liver stiffness measurement obtained using vibration-controlled transient elastography and magnetic resonance imaging-based magnetic resonance elastography in the same individuals and identified the most appropriate noninvasive method for detecting significant fibrosis in 165 patients with liver biopsy-diagnosed NAFLD. RESULTS: The area under the receiver operating characteristic curve based on the serum cutoff index values of WFA+ -M2BP/the serum levels of type IV collagen 7S for the diagnosis of significant fibrosis was 0.832 (95% confidence interval: 0.771-0.894)/0.837 (95% confidence interval: 0.778-0.898). "WFA+ -M2BP (cutoff index) ≥ 0.83 or type IV collagen 7S ≥ 5.2 ng/mL" showed a high sensitivity (91.4%) and negative predictive value (87.9%) for the diagnosis of significant fibrosis. CONCLUSIONS: We showed that serum WFA+ -M2BP or type IV collagen 7S levels serve as useful independent markers for detecting significant fibrosis and that use of both WFA+ -M2BP and type IV collagen 7S together increased the sensitivity and negative predictive value for the diagnosis of liver fibrosis. These results need to be validated in larger populations from multiple clinical centers.

Thioacetamide-induced hepatic fibrosis in the common marmoset.

The common marmoset (Callithrix jacchus) is a nonhuman primate that is used for preclinical research on stem cell transplantation therapies due to its similarity to human beings as well as its small size, enabling researchers to perform experiments without preparing a large number of cells. In this study, we developed a marmoset hepatic fibrosis model for regenerative medicine research. Six female marmosets aged 4-6 years were administered thioacetamide (TAA) at a dose of 2.5-40 mg/kg two or three times a week. Hepatic fibrosis was assessed by liver biopsy when blood chemistry indicated liver damage. Administration of TAA increased total bile acid, aspartate aminotransferase, and total bilirubin and decreased serum albumin levels. Following more than 11 weeks of continuous injection of TAA, histological analyses detected hepatic fibrosis in all animals. Type IV collagen 7S serum levels in animals with hepatic fibrosis were significantly higher than in normal animals as a possible marker of hepatic fibrosis in marmosets. Serial liver biopsies following the last administration of TAA revealed that induced fibrosis remained up to 11 weeks. The results suggest that continuous TAA administration induces persistent hepatic fibrosis in the common marmoset and this nonhuman primate hepatic fibrosis model have the possibility to evaluate the therapeutic effects of test samples to ameliorate hepatic fibrosis.