Procollagen Peptide Research Articles

Abstract 2210: Importance of fibrillar collagen quality, not quantity, for patients with solid tumors

Abstract Background: Abnormal architectures of collagen fibers in the extracellular matrix (ECM) is directly related to cancer associated fibroblast (CAF) activity and subtype and are hallmarks of tumor fibrosis and key for determining prognosis and response to treatment in patients with solid tumors. Quantifiable tumor fibrosis biomarkers are needed for patient stratification and a non-invasive approach (liquid biopsy) is preferred in particular in the advanced setting. Type I collagen (COL1) and type V collagen (COL5) are two collagens responsible for the densely packed fibrils in the fibrotic tumor microenvironment. While COL1 is the most abundant collagen, COL5 is known for controlling collagen fibril size and orientation, and therefore fibrillar collagen quality. Increased fibrotic activity results in release of collagen pro-peptides into the bloodstream of patients with cancer. In this study, we first evaluated the clinical utility of non-invasive collagen based liquid biomarkers of COL1 and COL5 in patients with various solid tumors and subsequently evaluated prognostic utility in patients with pancreatic cancer (PC). Methods: In a discovery cohort, pro-peptides of COL1 (PRO-C1) and COL5 (PRO-C5) were measured in serum from patients with various stages of bladder-, breast-, colorectal-, head and neck-, kidney-, lung-, melanoma-, pancreatic-, prostate-, and stomach cancer (n=220) and compared to healthy controls (n=33). Based on these results, the biomarker potential of PRO-C5 was validated further in 814 patients with PC from the BIOPAC cohort (n=15, 201, 164 and 434 for stage 1-4, respectively) including assessment for associations with overall survival (OS) analyzed by Kaplan-Meyer curves and Cox regression analysis, alone, and after adjusting for age, gender, BMI, diabetes, smoking, CA19-9, stage, no. of metastatic sites, performance status, and cachexia. Results: PRO-C5, not PRO-C1, was significantly elevated in serum from all types of cancer patients compared to healthy controls (p<0.05 - p<0.0001). Levels of PRO-C5 were similar in patients from the BIOPAC cohort to those found in the discovery cohort. Furthermore, when dividing patients from the BIOPAC cohort into quartiles based on PRO-C5 levels (Q1-Q4) a stepwise decrease was detected in median OS time (log rank, p<0.0001). Moreover, patients in the upper quartile of PRO-C5 (Q4) had 65% increased risk of dying compared to patients in Q1 (HR=1.65, 1.35-2.02, p<0.0001) and this remained significant after adjusting for other common risk factors by multivariate analysis (HR=1.69, 1.30-2.20, p=0.0001). Conclusion: Type V collagen (COL5) pro-peptides measured in serum (PRO-C5) is a promising pan-tumor fibrosis liquid biopsy with indications of independent prognostic value in patients with pancreatic cancer. In the future, this may provide means for serological subtyping of CAF subtypes which will aid in treating cancer patients. Citation Format: Neel Ingemann Nissen, Astrid Z. Johansen, Inna M. Chen, Christina Jensen, Carsten Palnæs Hansen, Jeppe Thorlacius-Ussing, Morten Karsdal, Julia S. Johansen, Nicholas Willumsen. Importance of fibrillar collagen quality, not quantity, for patients with solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2210.

Changes in bone quality after switching from a TDF to a TAF based ART: A pilot randomized study.

The impact of tenofovir disoproxil fumarate (TDF) antiretroviral (ART) regimens on bone health has been characterized mostly by bone mineral density (BMD), but recently also by bone quality (BQ). The aim of this pilot study is to assess the changes in BMD and BQ after switch from TDF to tenofovir alafenamide (TAF) ART. HIV individuals receiving TDF-based ART were randomized to switch to Bictegravir-TAF-Emtricitabine or to remain in the same regimen. At baseline and 24-weeks after randomization, participants underwent bone mineral density (BMD) by DXA and BQ assessment using bone microindentation, a validated technique that measures bone tissue quality expressed as bone material strength index (BMSi). A panel of plasma bone turnover biomarkers were measured by ELISA at the same time-points. Values are expressed as median [interquartile range] and non-parametric tests were used where appropriate. A total of 24 HIV individuals were included in the study, 19 of which were men (80%). Median age at baseline was 43 years (IQR 38-54). Half of individuals were allocated in the TDF group while the other half changed to TAF treatment. No differences at baseline between both groups were detected in any parameter. Non-significant changes nor in lumbar or femoral BMD at week 24 was found in any regimen. In contrast, there was an increase in BMSi in the TAF arm at 24 weeks, and thus an improvement in BQ[81.6 (79-83) to 86 (80-88) (+5.1%);p=0.041], whereas the TDF arm remained stable from 82 (76-85) at baseline to 82 (73-83);p=0.812. Hence, at week 24 there were significant differences in BQ between arms (p=0.049). A reduction in bone formation markers was found at week 24 in both regimens: N-terminal propeptide of type-1 collagen decreased a 20% (-35 - -0.6); p=0.031 with TAF and -16% (-25 - -5); p=0.032 with TDF. Also a decrease in bone resorption marker C-telopeptide with TAF was detected [-10% (-19 - -5);p=0.028] but not with TDF (p=0.232), suggesting a less metabolically active bone after switching to TAF. A bone quality improvement was found after switching from a TDF to a TAF based ART independently of BMD, suggesting that the bone health benefits of TAF may extend beyond BMD. Future research should be directed to confirm these findings and to identify the underlying mechanisms of ART related bone toxicity.

High serum levels of the C-propetide of type V collagen (PRO-C5) are prognostic for short overall survival in patients with pancreatic ductal adenocarcinoma.

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is characterized by a pronounced fibrotic tumor microenvironment, which impairs treatment response. Type I and V collagens are responsible for the densely packed fibrils in the tumor fibrosis environment. While the role of the major type I collagen in cancer is well described, less is known about the minor type V collagen. Quantifying collagen propeptides in serum has been shown to have prognostic and predictive value. In this study, we evaluated the clinical utility of measuring the propeptide of type V collagen (PRO-C5) in serum from a discovery cohort and a validation cohort of patients with PDAC as well as in non-pancreatic solid tumor types to explore the relevance of the PRO-C5 biomarker in cancer. Methods: Serum PRO-C5 was measured in three cohorts: a discovery cohort (19 healthy controls, 12 patients with chronic pancreatitis and 33 patients with PDAC (stage I-IV)), a validation cohort (800 patients with PDAC (stage I-IV)), and a non-pancreatic solid tumor type cohort of 33 healthy controls and 200 patients with 10 different non-pancreatic solid tumor types. The levels of serum PRO-C5 in patients with cancer were compared to levels in healthy controls. The association between PRO-C5 levels and overall survival (OS) was evaluated in patients with PDAC after adjusting for established prognostic factors. Results: PRO-C5 was significantly increased in serum from patients with PDAC compared to healthy controls (p < 0.001). High PRO-C5 levels were significantly associated with short OS in both the discovery- and the validation cohort, especially in early stages of PDAC (validation cohort stage II, HR = 2.0, 95%CI1.2-3.4). The association was independent of other prognostic parameters including stage, performance status and CA19-9. Furthermore, serum levels of PRO-C5 were significantly increased in serum from patients with other non-pancreatic solid tumor types compared to healthy controls. Conclusion: High levels of serum PRO-C5 is prognostic for short OS in patients with PDAC and may provide clinical value in many other tumor types beyond PDAC. This underlines the importance of type V collagen in tumor fibrosis. PRO-C5 could have the potential to be used in several aspects within drug discovery, patient stratification and drug efficacy.

Extracorporeal Shock Wave Therapy Improves Nontraumatic Knee Contracture in a Rat Model.

Joint contractures occur frequently after trauma or immobilization, but few reliable treatments are available. Extracorporeal shock wave therapy (ESWT) is often used for various musculoskeletal conditions, but whether it is effective for treating joint contractures and the mechanisms through which it might work for that condition remain unclear. Using a rat model, we asked, does ESWT (1) inhibit the progression of knee contracture, (2) ameliorate histopathologic joint changes, and (3) improve serum and myofascial fibrosis-related factors? We also asked, (4) what is the possible mechanism by which ESWT inhibits knee contracture? Thirty-two male Sprague-Dawley rats (12 weeks old and weighing 300 to 400 g) were randomly separated into two groups: control group (eight rats) and noncontrol group (24) in the first week. Rats in the control group were kept free in cages for 4 weeks, and the right lower limbs of the rats in the noncontrol group were immobilized in plaster for 4 weeks. ROM was then measured for each rat with or without 4 weeks of immobilization. After ROM measurement, rats in the noncontrol group were randomly separated into three groups: immobilization group (eight rats), remobilization group (eight rats), and remobilization with ESWT group (eight rats) at Week 4. Knee contracture was induced in rats by fixing the right knee with a plaster cast as in a previous study. The plaster cast was removed after 4 weeks; knee contracture was established when passive ROM was decreased and dysfunction such as abnormal gait occurred. Subsequently, rats with a remobilized joint contracture were treated with or without ESWT for 15 days (on Days 5, 10, and 15). The therapeutic effect was examined using ROM, joint diameter (as an indication of swelling), histopathologic changes, and the levels of fibrosis-related extracellular matrix component factors (hyaluronic acid, serum procollagen peptide, and laminin). The effect of ESWT on fibrosis protein was also evaluated using immunohistochemistry, quantitative polymerase chain reaction (qPCR), and Western blot. The expressions of factors in the TGF-β/SMADs pathway were also determined using Western blot and qPCR. ESWT mitigated immobilization-induced knee contracture in rats by improving ROM (immobilization versus remobilization with ESWT: 53° ± 8° versus 32° ± 8° [95% confidence interval 13° to 30°]; p < 0.001) and joint swelling (immobilization versus remobilization with ESWT: 8 ± 0.8 cm versus 6 ± 0.3 cm [95% CI 0.4 to 2.2 cm]; p = 0.01). Histopathologic features of remission were alleviated after ESWT (immobilization versus remobilization with ESWT: thickness of the knee space: 0.2 ± 0.03 mm versus 0.6 ± 0.01 mm [95% CI -0.49 to -0.33 mm]; p < 0.001. On Masson staining, the positive expression area, which indicates collagen fiber deposition, was 24% ± 5% versus 9% ± 2% ([95% CI 10% to 21%]; p < 0.001). ESWT improved the serum fibrosis factors of hyaluronic acid, procollagen peptide, and laminin (immobilization versus remobilization with ESWT: hyaluronic acid: 412 ± 32 versus 326 ±15 ng/mL [95% CI 29 to 144 ng/mL]; p = 0.003; serum procollagen peptide: 19 ± 1 versus 12 ±1 ng/mL [95% CI 3 to 11 ng/mL]; p < 0.001; laminin: 624 ± 78 versus 468 ±9 ng/mL [95% CI 81 to 231 ng/mL]; p = 0.006) and myofascial factors of α-SMA and Type I collagen associated with immobilization-induced contractures. The findings suggest that ESWT improved joint contracture by inhibiting the TGF-β1/SMADs signaling pathway in rats. This work suggests ESWT may be worth exploring in preliminary research in humans to determine whether it may be a treatment option for patients with nontraumatic knee contractures. If the mechanism of ESWT can be confirmed in humans, ESWT might be a therapy for diseases involved in the TGF-β1/SMADs signaling pathway, such as hypertroic scarring and scleroderma.

Cross-Sectional and Longitudinal Performance of Non-Invasive Tests of Liver Fibrosis in Patients with Non-Alcoholic Fatty Liver Disease.

Background and aims: Non-invasive tests (NITs) are needed in clinical practice to replace histology for the identification of liver fibrosis and prognostication in Non-Alcoholic Fatty Liver Disease (NAFLD). Novel collagen-derived fibrogenesis markers including N-terminal type III collagen pro-peptide (PRO-C3) are among the most promising tools in this field. The aim of this study was to assess the diagnostic accuracy of PRO-C3, the derivative ADAPT score, and other NITs for the identification of advanced fibrosis (stages 3-4) and changes over 12 months of follow-up. Methods: In this longitudinal study, 96 patients with biopsy-proven NAFLD were evaluated at baseline, of which 50 underwent a follow-up visit after 12 months. Clinical-biochemical parameters, liver stiffness (LS) by transient elastography, PRO-C3, and other NITs (ADAPT, FIB-4, NFS, APRI) were collected at baseline and follow-up. Results: LS showed the best accuracy for the identification of advanced fibrosis, with Area under the Receiving Operator Curve (AUROC) 0.82 (0.73-0.89) for a cut-off value of 9.4 kPa. Among the other NITs, the ADAPT score showed the best accuracy, with AUROC 0.80 (0.71-0.88) for a cut-off of 5.02 (Se 62%, Sp 89%, PPV 74%, NPV 83%). The comparison between the AUROC of LS with that of ADAPT was not statistically different (DeLong test p value 0.348). At follow-up, LS was slightly reduced, whilst PRO-C3 displayed a significant increase from baseline median 11.2 ng/mL to 13.9 ng/mL at follow-up (p = 0.017). Accordingly, ADAPT score increased from median 5.3 to 6.1 (p = 0.019). The other NITs did not significantly change over 12 months. Conclusions: The ADAPT score shows the best performance among non-invasive scores for the identification of advanced fibrosis, not different from LS. Collagen-derived biomarker PRO-C3 and the derivative score ADAPT display significant changes over time, and may be useful tools for monitoring the progression of liver disease or assessing responses to treatments.

Fetuin A functionalisation of biodegradable PLLA-co-PEG nonwovens towards enhanced biomineralisation and osteoblastic growth behaviour.

Therapy for large-scale bone defects remains a major challenge in regenerative medicine. In this context, biodegradable electrospun nonwovens are a promising material to be applied as a temporary implantable scaffold as their fibre diameters are in the micro- and nanometre range and possess a high surface-to-volume ratio paired with high porosity. In this work, in vitro assessment of biodegradable PLLA-co-PEG nonwovens with fetuin A covalently anchored to the surface has been performed in terms of biomineralisation and the influence on MG-63 osteoblast cell metabolic activity, biosynthesis of type I collagen propeptide and inflammatory potential. Our finding was that covalent fetuin A funtionalisation of the nonwoven material leads to a distinct increase in calcium affinity, thus enhancing biomineralisation while maintaining the distinct fibre morphology of the nonwoven. The cell seeding experiments showed that the fetuin A functionalised and subsequently in vitro biomineralised PLLA-co-PEG nonwovens did not show negative effects on MG-63 growth. Fetuin A funtionalisation and enhanced biomineralisation supported cell attachment, leading to improved cell morphology, spreading and infiltration into the material. Furthermore, no signs of increase in the inflammatory potential of the material have been detected by flow cytometry experiments. Overall, this study provides a contribution towards the development of artificial scaffolds for guided bone regeneration with the potential to enhance osteoinduction and osteogenesis.

703: IMMUNOCOMPROMISED CHILDREN WITH ARDS POSSESS A DISTINCT CIRCULATING INFLAMMATORY PROFILE

Introduction: Immunocompromised status, with and without stem cell transplant (SCT), confers a worse prognosis in pediatric acute respiratory distress syndrome (ARDS). An improved understanding of the biochemistry of immunocompromised children with ARDS would inform whether specific pathways are targetable, or merely bystanders, in order to improve outcomes in this high-risk subgroup. Therefore, we aimed to identify a biomarker profile of immunocompromised children, with and without SCT, independent of illness severity. Methods: We performed a secondary analysis of a prospective cohort of intubated children with Berlin-defined ARDS with existing biomarker measurements from the Children’s Hospital of Philadelphia between 2014 and 2019. In order to identify biomarker levels independent of illness severity, we computed predicted (log-transformed) biomarker levels after adjusting for confounders (age, ARDS etiology, oxygenation index, vasopressor score, and absolute neutrophil count [ANC]) using linear regression, and then compared these severity-adjusted biomarker levels between immunocompetent and immunocompromised (with and without SCT) subjects using analyses of variance and post-hoc Bonferroni. Results: In 333 children with ARDS, 84 were immunocompromised, of whom 39 had SCT. ANC strongly correlated with biomarkers, with 14 of 18 proteins differentially expressed in patients with versus without neutropenia. After multivariable adjustment, 11 biomarkers were higher in immunocompromised subjects without SCT, relative to immunocompetent, reflecting differences in inflammation (interleukin [IL]-6 and IL-8), endotheliopathy (angiopoietin 2), and tissue damage (type III procollagen peptide). Immunocompromised subjects with SCT had adjusted biomarker levels averaging between immunocompetent subjects and immunocompromised subjects without SCT. A single biomarker, CCL22, was lower in immunocompromised subjects with and without SCT, consistent with dysregulated regulatory T cell activity. Conclusions: Immunocompromised children with ARDS were characterized by elevated pro-inflammatory biomarkers and endotheliopathy. Our study provides insight into mechanisms underlying the molecular heterogeneity of pediatric ARDS, and potentially identifies targetable pathways to mitigate their increased mortality risk.

Blood-based tumor fibrosis markers as diagnostic and prognostic biomarkers in patients with biliary tract cancer.

Biliary tract cancer (BTC) is characterized by a desmoplastic extracellular matrix (ECM). We tested the diagnostic and prognostic use of seven circulating biomarkers of ECM remodeling: pro-peptides of type III collagen (PRO-C3), VI (PRO-C6) and XI (PRO-C11), matrix metalloprotease (MMP) degraded type III collagen (C3M) and type IV collagen (C4M) fragments, granzyme B degraded type IV collagen fragments (C4G) and MMP degraded and citrullinated vimentin (VICM) a marker of macrophage activation. The study included 269 patients with all stages of BTC and 49 patients with benign biliary tract diseases. Serum samples from BTC patients were collected before surgery, or before first- or second-line chemotherapy. C3M, C4M, PRO-C3, PRO-C6, PRO-C11 and VICM levels were elevated in patients with BTC compared to patients with benign disease. Receiver operating characteristics curve analyses identified PRO-C3 (area under curve [AUC]=0.87) as the ECM marker with the best diagnostic performance. The ECM biomarkers correlated with inflammation biomarkers (C-reactive protein [CRP], interleukin-6 [IL-6] and YKL-40) but not with CA19-9. To investigate prognostic performance, patients were split into three cohorts (first-line, second-line and surgery). Elevated ECM biomarker levels were associated with short overall survival (OS), but only pretreatment PRO-C3 and PRO-C6 were associated with OS in both the first-line and second-line settings when adjusting for CA19-9, performance status and stage in a multivariate Cox-regression analyses. Our results indicate that collagen remodeling is increased in patients with BTC and associated with survival. The collagen pro-peptides (PRO-C3 and PRO-C6) could be used as novel biomarkers in these patients.

Association between active cytomegalovirus infection and lung fibroproliferation in adult patients with acute respiratory distress syndrome: a retrospective study

BackgroundCytomegalovirus (CMV) has high seroprevalence, and its active infection is associated with several adverse prognoses in adult patients with acute respiratory distress syndrome (ARDS). However, the role of active CMV infection in ARDS-associated fibroproliferation is unknown. This study aimed at determining the association between active CMV infection and lung fibroproliferation in adult patients with ARDS.MethodsWe retrospectively reviewed the medical records of all adult patients with ARDS who were admitted to the intensive care unit (ICU) from January 2018 to December 2020 at a national university-affiliated hospital in China. Study subjects were divided into active and non-active CMV infection groups based on CMV DNAemia within a 28-day ICU hospitalization. Lung fibroproliferation was measured using chest high-resolution computed tomography (HRCT) and N-terminal peptide of serum procollagen III (NT-PCP-III) within the first 28 days of ICU admission. Pulmonary fibrosis, clinical features, laboratory findings, treatment measures, and clinical outcomes were compared between the two groups.ResultsAmong the 87 ARDS patients included in this study, the incidence of active CMV infection was 16.1% within the 28-day ICU admission period. In logistic regression analyze, active CMV infection was found to be associated with higher pulmonary fibrogenesis, pulmonary fibrosis score, and NT-PCP-III level (P < 0.05). The duration of ICU stay in ARDS patients with active CMV infection was significantly higher than in those without active CMV infection (P < 0.05).ConclusionsAmong adult patients with ARDS, active CMV infection was related to poor clinical outcomes. Active CMV infection was associated with ARDS-associated fibroproliferation. Prophylactic and preemptive use of anti-CMV agents on pulmonary fibrosis should be assessed to determine a consensus therapeutic strategy.

Plasma Kallikrein-Activated TGF-β Is Prognostic for Poor Overall Survival in Patients with Pancreatic Ductal Adenocarcinoma and Associates with Increased Fibrogenesis.

Pancreatic ductal adenocarcinoma (PDAC) is a hard-to-treat cancer due to the collagen-rich (fibrotic) and immune-suppressed microenvironment. A major driver of this phenomenon is transforming growth factor beta (TGF-β). TGF-β is produced in an inactive complex with a latency-associated protein (LAP) that can be cleaved by plasma kallikrein (PLK), hereby releasing active TGF-β. The aim of this study was to evaluate LAP cleaved by PLK as a non-invasive biomarker for PDAC and tumor fibrosis. An ELISA was developed for the quantification of PLK-cleaved LAP-TGF-β in the serum of 34 patients with PDAC (stage 1–4) and 20 healthy individuals. Biomarker levels were correlated with overall survival (OS) and compared to serum type III collagen (PRO-C3) and type VI collagen (PRO-C6) pro-peptides. PLK-cleaved LAP-TGF-β was higher in patients with PDAC compared to healthy individuals (p < 0.0001). High levels (>median) of PLK-cleaved LAP-TGF-β were associated with poor OS in patients with PDAC independent of age and stage (HR 2.57, 95% CI: 1.22–5.44, p = 0.0135). High levels of PLK-cleaved LAP-TGF-β were associated with high PRO-C3 and PRO-C6, indicating a relationship between the PLK-cleaved LAP-TGF-β fragment, TGF-β activity, and tumor fibrosis. If these preliminary results are validated, circulating PLK-cleaved LAP-TGF-β may be a biomarker for future clinical trials.

Type III procollagen peptide level can indicate liver dysfunction associated with volume overload in acute heart failure.

AimThe role of serum type III procollagen peptide (P3P) level in the acute phase of acute heart failure (AHF) requires clarification. We hypothesized that serum P3P level is temporarily higher during the acute phase, reflecting liver dysfunction due to congestion.Methods and resultsA total of 800 AHF patients were screened, and data from 643 patients were analysed. Heart failure was diagnosed by the treating physician according to the European Society of Cardiology (ESC) guidelines, and included patients being treated with high‐concentration oxygen inhalation (including mechanical support) for orthopnea, inotrope administration, or mechanical support for low blood pressure, and various types of diuretics for peripheral or pulmonary oedema. In all cases, diuretics or vasodilators were administered to treat AHF. The patients were divided into three groups according to their quartile (Q) serum P3P level: low‐P3P (Q1, P3P ≤ 0.6 U/mL), mid‐P3P (Q2/Q3, 0.6 < P3P <1.2 U/mL), and high‐P3P (Q4, P3P ≥ 1.2 U/mL). The plasma volume status (PVS) was calculated using the following formula: ([actual PV − ideal PV]/ideal PV) × 100 (%). The primary endpoint was 365 day mortality. A Kaplan–Meier curve analysis showed that prognoses, including all‐cause mortality and heart failure events within 365 days, were significantly (P < 0.001) worse in the high‐P3P group when compared with the mid‐P3P and low‐P3P groups. A multivariate logistic regression analysis showed that high PVS (Q4, odds ratio [OR]: 4.702, 95% CI: 2.012–20.989, P < 0.001), high fibrosis‐4 index (Q4, OR: 2.627, 95% CI: 1.311–5.261, P = 0.006), and low estimated glomerular filtration rate per 10 mL/min/1.73 m2 decrease (OR: 1.996, 95% CI: 1.718–2.326, P < 0.001) were associated with high P3P values. The Kaplan–Meier curve analysis demonstrated a significantly lower survival rate, as well as a higher rate of heart failure events, in the high‐P3P and high‐PVS groups when compared with the other groups. A multivariate Cox regression model identified high P3P level and high PVS as an independent predictor of 365 day all‐cause mortality (hazard ratio [HR]: 2.249; 95% CI: 1.081–3.356; P = 0.026) and heart failure events (HR: 1.586, 95% CI: 1.005–2.503, P = 0.048).ConclusionA high P3P level during the acute phase of AHF served as a comprehensive biomarker of liver dysfunction with volume overload (i.e. liver congestion) and renal dysfunction. A high P3P level at admission may be able to predict adverse outcomes in AHF patients.

Crosstalk between Irisin Levels, Liver Fibrogenesis and Liver Damage in Non-Obese, Non-Diabetic Individuals with Non-Alcoholic Fatty Liver Disease.

Background: Insulin resistance plays a relevant role in the onset of non-alcoholic fatty liver disease (NAFLD) and its progression to non-alcoholic steatohepatitis (NASH) and fibrosis. Irisin is an exercise-induced myokine involved in the regulation of energy homeostasis and glucose metabolism. Additionally, pre-clinical models have shown a potential role of irisin in the pathogenesis of NAFLD. The aim of this study is to explore the association between irisin, histological features and biomarkers of liver fibrogenesis in non-diabetic, non-obese, biopsy-proven NAFLD individuals. Methods: Forty-one patients with histological evidence of NAFLD were included. Circulating irisin and direct markers of fibrogenesis N-terminal type III collagen propeptide (PRO-C3) and type VI collagen cleavage product (PRO-C6) were measured by ELISA. Results: Median age of the cohort was 45 years (41–51) and 80.4% were male. Significant fibrosis (stage ≥ 2) was present in 36.6% of cases. Circulating irisin, PRO-C3 and PRO-C6 levels were significantly higher in subjects with fibrosis stage ≥ 2 when compared to those with fibrosis stage < 2 (5.96 ng/mL (95% CI = 4.42–9.19) vs. 2.42 ng/mL (95% CI = 1.73–5.95), p = 0.033; 9.5 ng/mL (95% CI = 7.7–13.6) vs. 6.2 ng/mL (95% CI = 4.9–8.9), p = 0.016; 6.6 ng/mL (95% CI = 5.6–7.9) vs. 5.1 ng/mL (95% CI = 4.2–5.4), p = 0.013, respectively). Irisin levels were similarly distributed between the features of NASH. Circulating irisin positively correlated with both PRO-C3 and PRO-C6 levels (r = 0.47, p = 0.008 and r = 0.46, p = 0.002). Conclusions: Increased circulating irisin levels may identify a more aggressive phenotype of liver disease with increased fibrogenesis and more severe liver damage.

Are Bone Metabolism and Bone Material Strength Evaluations Worthwhile?: Commentary on an article by Tamara D. Rozental, MD, et al.: "Longitudinal Changes in Serum Markers of Bone Metabolism and Bone Material Strength in Premenopausal Women with Distal Radial Fracture".

Commentary The article by Dr. Tamara D. Rozental and colleagues introduces us to 2 innovative methods to improve our understanding of bone strength and fracture-healing biochemistry that maybe relevant to clinical practice. Bone mineral density (BMD) is the standard for determining bone quality and fracture risk but does not define the structural and mechanical properties of bone1. Impact microindentation provides information on the material properties of cortical bone that we as orthopaedic surgeons need in order to improve our choice of fixation strategy2. Markers of bone metabolism (MBM) have been used to assess the results of osteoporotic treatment but have not been applied to the non-osteoporotic patient to assess fracture healing or to predict potential osteoporosis3,4. Impact microindentation is a minimally invasive technique that measures the ability of the tibial cortex to resist a standardized impact force. This measures the bone’s material quality, which is the interaction of the bone’s mineral and organic elements to resist failure. This technique might allow for a better determination of the ability of bone to hold implants that are used for fracture fixation (for example, the ability to achieve screw purchase). A greater understanding of such variables would facilitate fixation decisions, including the potential need to augment fixation with use of locking screws or bone substitutes. In this study, the microindentation technique did not prove valuable in assessing bone quality, but the investigators demonstrated that the test was possible to administer in a clinical setting. MBM have demonstrated promise as markers of the healing response following the treatment of a fracture5. Sclerostin (a glycoprotein produced by osteocytes) and Dickkopf 1 (DKK1) both decrease bone formation were initially increased but then both dropped to low levels allowing the healing response to occur. Tartrate-resistant acid phosphatase (TRAP) and C-terminal telopeptide (both of which are indicators of osteoclastic activity) peaked early, indicating the need for bone removal secondary to fracture site necrosis. Bone-formation markers such as N-terminal extension propeptide of type-I collagen (P1NP) and bone-specific alkaline phosphatase (BSAP) increased throughout the healing phase. These longitudinal results are evidence that, in premenopausal women, the biochemistry of fracture healing can be evaluated. This study provides the impetus to evaluate whether MBM can determine problems in fracture healing earlier than radiographic evidence and also whether MBM can serve as an indication of early osteopenia and osteoporosis in premenopausal women6. This article had a number of strengths. Patient selection was appropriate (although it was limited to a specific group of premenopausal women with a distal radial fracture). The fractures were classified with use of an acceptable system that can be used to determine severity on the basis of the mechanism of injury (i.e., high or low energy). Finally, the study included both operatively and nonoperatively managed patients, with the former demonstrating an elevated BSAP. This article also had also several areas of concern. MBM vary depending on patient activity and the time of day when the samples are taken. The authors acknowledge this limitation, but it is important for the reader to understand that the controls were only sampled once, so any assumption regarding longitudinal MBM levels may not hold. Another issue is that there is no consensus on what constitutes a meaningful clinical difference in these results. As this study is the first to measure MBM during fracture healing, one is unsure of what these results mean. It is obvious that the results appear to coincide with our understanding of fracture healing, but this relationship has not been proven. Although this study has limitations, including a small sample size in a very specific patient population, it opens the door to a better understanding of fracture healing. Orthopaedic surgeons, particularly those specializing in fracture care, should consider this study as an impetus to further investigate the physiology and biochemistry of fracture healing.

PLC, PTH and NF-κB increased during orthodontic bone remodeling in chronic stress rats

This study was designed to investigate the potential effects of chronic stress on periodontal bone remodeling and its mechanism during orthodontic tooth movement in rats. Forty-eight male SD rats aged 8 weeks were randomly divided into control group and chronic stress group. Chronic unpredictable mild stress model (CUMS) was established in the stress group, which was validated by behavioral experiment as well as cortisol (CORT) and adrenocorticotropic hormone (ATCH) levels. Then, the two groups were further divided into three distinct groups, namely group with no orthodontic force, group with 30 g orthodontic force and group with 50 g orthodontic force respectively to construct orthodontic tooth movement model. The rats were sacrificed after 14 days and maxilla on the loading side was obtained to measure tooth movement distance. It was found that compared with the control group, the chronic stress group displayed increased parathyroid hormone (PTH), amino terminal peptide of type I procollagen (PINP) and c-terminal peptide of type I collagen branch (CTX) levels as measured by enzyme-linked immunosorbent assay (ELISA). Hematoxylin and Eosin (HE) and TRAP staining showed fewer osteoblasts and more number of osteoclasts. The results of western blot showed no significant change in expression of Adenylate cyclase (AC) but increased phospholipase C (PLC) levels were noted. In addition, increased NF-κB expression was observed by immunohistochemistry. Overall, chronic stress can affect bone remodeling during orthodontic tooth movement by increasing the content of PTH in the blood and increasing PLC and NF-κB.

Study of risk factors for the osteoporosis development in rheumatoid arthritis in real clinical practice

Introduction. Osteoporosis is a common complication of rheumatoid arthritis. Its development is associated with the mechanisms underlying in the progression of autoimmune inflammatory diseases and therapeutic approaches used for them. The study of risk factors for osteoporosis can contribute to the clarification of its pathogenesis components, as well as the development of new methods for prevention, diagnosis and treatment of this condition.Aim. To study the role of anamnestic, clinical and laboratory factors for secondary osteoporosis in women with rheumatoid arthritis.Materials and methods. 102 women with rheumatoid arthritis were enrolled in our study. Exclusion criterias were type 2 diabetes mellitus, hepatic cirrhosis, hepatocarcinoma and level of alanine aminotransferase ≥ than 3 upper limit ofnormal. The cumulative dose, duration and daily dose of glucocorticoids (GC) were determined by patient intake. All patients undergone standard clinical and immunological examination. Serum fetuin-A, 25-hydroxycalciferol, C-telopeptide of collagen I type, N-terminal propeptide of collagen I type levels were determined using ELISA. X-ray of afflicted joints and dual-energy x-ray absorptiometry were performed. Statistical analysis was performed using conventional methods. Forced data entry was used to perform multiple logistic regression. Hereinafter data is presented as odds ratio (OR) and 95% confirmation intervals (CI).Results. OR for osteoporosis were higher in women of age ≥ 58.5 years (OR 1,07 (1.02–1.12)), body mass index (BMI) ≤ 27 kg/m2 (OR 1.1 (1.01–1.2)), cumulative dose of GC ≥ 7.6 g (OR 1.09 (1.02–1.17), serum fetuin-A levels ≤ 660 μg/ml (OR 1,05 (1,01–1,09) and if the duration of GC intake is more than 3 months (hereinafter if dose of glucocorticoids is ≥ 5 mg for prednisolone daily) (OR 12.3 (4.12–36.5). Adjusted OR for osteoporosis were higher in women of age ≥ 58.5 years old (adjOR 1.08 (1.01–1.16), serum fetuin-A levels ≤ 660 μg/ml (adjOR 1.08 (1.01–1.15) andif the duration of GC intake is ≥ than 3 months (adjOR 12.1 (1.44–102.3).Conclusions. Women with RA of ≥ than 58.5 years old, duration of GCs intake more than 3 months and serum fetuin-A levels ≤ than 660 μg/ml had higher odds for osteoporosis.These are independent factors for osteoporosis in women with rheumatoid arthritis, whichshould be used in patient’s management.

Abstract 9416: Monocyte Subsets and Exercise Capacity in Patients With Atrial Fibrillation With Preserved Left Ventricular Function

Introduction: Monocytes play important roles in inflammation, angiogenesis and tissue repair in cardiovascular disease. This study examined differences in monocyte subset numbers, subsequent markers of inflammation and cardiac fibrosis in patients with permanent atrial fibrillation and preserved left ventricular function with regards to peak exercise capacity (cardiopulmonary exercise testing and 6 minute walk test), quality of life and hospitalisation. Furthermore, the effects of spironolactone vs placebo on the quantitative measurement of these potential biomarkers were assessed in a double blind randomised study over a 2 year period. Methods: Three monocyte subsets [CD14++CD16-CCR2+ (Mon1), CD14++CD16+CCR2+ (Mon2) and CD14+CD16++CCR2- (Mon3)] were analysed by flow cytometry and ELISA to quantify makers of fibrosis in the form of Galectin 3, Type III Procollagen Peptide and Type I Procollagen Peptide. SPSS software was used to carry out statistical analysis of all 250 patients in the study cohort using stepwise multivariant linear regression analysis. Independent samples T testing between spironolactone and placebo groups were carried out. Results: Monocyte subsets CD16 Mon3 (p=0.001) and CD42 MPA3 (p=0.030) were found to be predictors of peak VO 2 . Comparison of monocyte subsets at end of study showed no differences between Mon1 (p=0.86), Mon2 (p=0.66) or Mon3 (p=0.81) subsets and their subsequent makers of inflammation and fibrosis between the treatment and placebo control group. This held true for further markers of cardiac fibrosis with regards to 6 minute walk test , quality of life assessment and hospitalisation. Conclusions: This study shows that monocyte subsets can be potential biomarkers of exercise capacity which in the form of cardiopulmonary testing is known to have morbidity and mortality implications. CD16 Mon3 were significantly related to beneficial outcomes with regards to a peak exercise capacity in respect to VO 2 max (higher CD16 Mon3 count associated with higher peak VO 2 performance). Spironolactone has no impact on primary or secondary outcome in this study.

Patients With Cirrhosis Have Elevated Bone Turnover but Normal Hepatic Production of Osteoprotegerin.

Severe osteodystrophy is common in patients with liver dysfunction. Markers of bone metabolism may help in early diagnosis of osteodystrophy and in understanding underlying pathophysiological mechanisms. To elucidate changes in bone metabolism associated with cirrhosis and to determine the route of elimination for the markers. Case-control study at a public university hospital. Fifty-nine patients with cirrhosis (47 alcoholic and 12 nonalcoholic cirrhosis) and 20 controls were included. Participants underwent catheterization of the femoral artery, and the hepatic, renal, and femoral veins with collection of blood from all 4 sites. Regional arteriovenous differences in concentrations of bone metabolism markers were determined: procollagen of type I collagen propeptide (PINP), C-terminal cross-linking telopeptide of type I collagen (CTX), osteocalcin, tartrate-resistant acid phosphatase isoform 5b (TRAcP5b), osteoprotegerin (OPG), and sclerostin and correlated with degree of disease (Child-Pugh classification). PINP concentration was higher (median: 87.9 µg/L) in patients with cirrhosis than in controls (52.6 µg/L) (P = .001), while hepatic extraction was lower (4.3% vs 14.5%) (P < .001). Both CTX and TRAcP5b were higher in patients with cirrhosis (340 ng/L and 3.20 U/L) than in controls (215 ng/L and 1.60 U/L) (P < .001 and P < .0001). Hepatic sclerostin extraction was lower in patients with cirrhosis (14.6%) than in controls (28.7%) (P < .0001). In both groups OPG showed a hepatic release rate (production) of 6%. Patients with cirrhosis have increased bone resorption, but unaltered bone formation. Sclerostin is eliminated through the liver while OPG is produced in the liver. Bone markers may prove useful in evaluating bone turnover in patients with cirrhosis.

O13 A case of chronic recurrent multifocal osteomyelitis

O13 A case of chronic recurrent multifocal osteomyelitis

Enhanced liver fibrosis score as a surrogate of liver-related complications and mortality in primary biliary cholangitis.

The presence of bridging fibrosis predicts survival of primary biliary cholangitis (PBC). This study aimed to compare serum parameters for the estimation of liver fibrosis and prediction of clinical outcomes in PBC.Out of 392 patients with PBC, 102 who underwent liver biopsy and in whom fibrosis indices, platelet count, hyaluronic acid, type IV collagen 7 second domain, procollagen type III amino-terminal peptide, tissue inhibitor of metalloproteinases 1, Mac-2 binding protein glycosylation isomer, N-terminal type III collagen propeptide levels; fibrosis index based on 4 factors, aspartate aminotransferase-to-platelet ratio index, and enhanced liver fibrosis (ELF) score were determined, were included. The correlation of histological stages based on both Scheuer and Nakanuma classifications with fibrosis indices was investigated. The Nakanuma system comprises grading for liver fibrosis and bile duct loss. Diagnostic performances of 10 fibrosis indices were evaluated to identify patients with poor prognosis. Moreover, correlations of those with PBC clinical manifestation and survival were also investigated.Enhances liver fibrosis (ELF) score had the highest correlation coefficient for liver fibrosis evaluated according to either the Scheuer or Nakanuma classification among 10 serum fibrosis indices. It also had the highest diagnostic performance in estimating Scheuer stage III and Nakanuma fibrosis score 2, both of which represent portal-bridging fibrosis. Patients with an ELF score of ≥10.0 had shorter survival and presented more frequently clinical complications than those with an ELF score of <10.0.ELF score determines the severity of liver fibrosis and predicts the occurrence of complications and survival in patients with PBC.

Andrographolide sulfonate attenuates alveolar hypercoagulation and fibrinolytic inhibition partly via NF-κB pathway in LPS-induced acute respiratory distress syndrome in mice

BackgroundAlveolar hypercoagulation and fibrinolytic inhibition are important characteristics during acute respiratory distress syndrome (ARDS), and NF-κB p65 signaling pathway is involved to regulate these pathophysiologies. We hypothesize that targeting NF-κB signal pathway could ameliorate alveolar hypercoagulation and fibrinolyitc inhibition, thus attenuating lung injury in ARDS. PurposeWe explore the efficacy and the potential mechanism of andrographolide sulfonate (Andro-S) on alveolar hypercoagulation and fibrinolytic inhibition in LPS-induced ARDS in mice. MethodsARDS was made by lipopolysaccharide (LPS) inhalation in C57BLmice. Andrographolide sulfonate (2.5, 5 and 10 mg/kg) was intraperitoneally given to the mice (once a day for three consecutive days) before LPS administration. NEMO binding domain peptide (NBD), an inhibitor of NF-κB, was used as the positive control and it replaced Andro-S in mice of NBD group. Mice in normal control received saline instead of LPS. Lung tissues and bronchoalveolar lavage fluid (BALF) were collected for analysis of alveolar coagulation, fibrinolytic inhibition as well as of pulmonary inflammatory response after 8 h of LPS inhalation. NF-κB signal pathway in lung tissue was simultaneously determined. ResultsAndro-S dose-dependently inhibited tissue factor (TF) and plasminogen activator inhibitor (PAI)-1 expressions either in mRNA or in protein in lung tissue of ARDS mice, and it also decreased the concentrations of TF, PAI-1, thrombin-antithrombin complex (TAT), procollagen peptide type Ⅲ (PⅢP) while promoting the production of activated protein C (APC) in BALF. Meanwhile, Andro-S effectively inhibited inflammatory response (interleukin 1β and myeloperoxidase) induced by LPS. LPS stimulation dramatically activated NF-κB signal pathway, indicated by increased expressions of phosphorylation of p65 (p-p65), p-IKKα/β and p-IκBα and the higher p65-DNA binding activity, which were all dose-dependently reversed by Andro-S. Andro-S and NBD presented similar efficacies. ConclusionsAndro-S treatment improves alveolar hypercoagulation and fibrinolytic inhibition and attenuates pulmonary inflammation in LPS-induced ARDS in mice partly through NF-κB pathway inactivation. The drug is expected to be an effective choice for ARDS.