Article

Analysis of Disease-Associated Protein Expression Using Quantitative Proteomics—Fibulin-5 Is Expressed in Association with Hepatic Fibrosis

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Abstract

Hepatic fibrosis and cirrhosis are major health problems worldwide. Up to now, highly invasive biopsy remains the diagnostic gold standard despite many disadvantages. In order to develop non-invasive diagnostic assays for the assessment of liver fibrosis it is urgently necessary to identify molecules which are robustly expressed in association with the disease. We analyzed biopsied tissue samples from 95 patients with HBV/HCV-associated hepatic fibrosis using three different quantification methods. We performed a label-free proteomics discovery study to identify novel disease-associated proteins using a subset of the cohort (n = 27). Subsequently, gene expression data from all available clinical samples were analyzed (n= 77). Finally, we performed a targeted proteomics approach (MRM) to verify the disease-associated expression in samples independent from the discovery approach (n = 68). We identified Fibulin-5 (FBLN5) as a novel protein expressed in relation to hepatic fibrosis. Furthermore, we confirmed the altered expression of microfibril-associated glycoprotein 4 (MFAP4), lumican (LUM) and collagen alpha-1(XIV) chain (COL14A1) in association to hepatic fibrosis. To our knowledge, no tissue-based quantitative proteomics study for hepatic fibrosis has been performed using a cohort of comparable size. By this means, we add substantial evidence for the disease-related expression of the proteins examined in this study.

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... These fibrillar collagens form arrays incorporating fibril-associated collagens with interrupted triple helices (FACIT), such as collagens XII and XIV [114]. Hepatic collagen XII amounts decreased with increasing stages of HCV-related fibrosis, whereas collagen XIV levels increased [59,84]. Collagen XVI, another FACIT not associated with collagens I, III, or V fibrils [115], was found underexpressed, especially at the cirrhotic stage of HCV-related fibrosis [59]. ...
... During chronic hepatitis C, the upregulation of gelatinases will lead to a major reshuffling of the basement membrane, with the destruction of the structural support of cells, and the downregulation of collagenases will amplify the deposition of insoluble collagen fibers and aggravate the fibrotic phenotype. As mentioned earlier, hepatic collagen XIV is increased during HCV-related fibrosis [59,84]; as this collagen is collagenase-sensitive, this observation is in line with the downregulation of collagenases [57]. ...
... Mechanistic analyses revealed that the overproduction of fibromodulin was under the control of oxidative stress, locally induced by the liver injury [60] (and by HCV-induced liver injury in particular [163]). Lumican was also found overexpressed in liver fibrosis [102], and in HCV-induced fibrosis in particular, on liver biopsies [84] and specifically in the ECM [59]. Lumican expression correlated with the severity of the (HCV-related) fibrosis [81,84,102], and high levels of lumican were reported in the plasma of HCV-positive patients with liver disease [81,84]. ...
Article
Full-text available
Chronic infection by the hepatitis C virus (HCV) is a major cause of liver diseases, predisposing to fibrosis and hepatocellular carcinoma. Liver fibrosis is characterized by an overly abundant accumulation of components of the hepatic extracellular matrix, such as collagen and elastin, with consequences on the properties of this microenvironment and cancer initiation and growth. This review will provide an update on mechanistic concepts of HCV-related liver fibrosis/cirrhosis and early stages of carcinogenesis, with a dissection of the molecular details of the crosstalk during disease progression between hepatocytes, the extracellular matrix, and hepatic stellate cells.
... In the current phase 2 studies (PROVE1, PROVE2, and PROVE3) of the direct-acting antiviral drug telaprevir, serum samples from responders and nonresponders to HCV treatment were analyzed by proteomic profiling and 15 differentially expressed proteins, with seven of them belonging to focal adhesion proteins or other macromolecular assemblies that constitute structural links between integrins and the actin cytoskeleton, were observed[84]. The ultimate goal of performing pretreatment serum proteome profiling prior to treatment is to predict sustained virological response (SVR) and nonresponse (NR) to antiviral drugs in chronic HCV infection and design suitable treatments for each patient.Ceruloplasmin 2D-DIGE Serum HCV +[78]Clusterin 2D-DIGE Serum HBV[74]Collagen 2D-DIGE Serum HBV +[74]LC-MS Liver biopsy HCV +[80]Complement component 4a 2D-DIGE Serum HCV +[78]Cystathione beta synthase LC-MS Liver biopsy HCV[77]Cysteine and glycine rich protein 2LC-MS Liver biopsy HCV +[80]Cytochrome b-245 beta LC-MS Liver biopsy HCV +[77]Cytochrome c LC-MS Liver biopsy HCV +[76]Fetuin A 2D-PAGE Serum HCV[73]Fibrinogen 2D-DIGE Serum HCV +[78]2D-DIGE Serum HBV +[74]Fibulin-5 LC-MS Liver biopsy HCV +[80]FK506 binding protein 14 LC-MS Liver biopsy HCV[77]Gelsolin 2D-PAGE Serum HBV[81]Glutamate dehydrogenase 1 2D-DIGE Mouse liver tissue HBV[79]Gluthatione-S-transferases LC-MS Liver biopsy HCV[77]Haptoglobin 2D-PAGE Serum HCV[73]2D-DIGE Serum HBV[74]Hemopexin 2D-PAGE Serum HCV +[73]2D-DIGE Serum HBV +[74]High mobility group 1 2D-DIGE Mouse liver tissue HBV[79]ProteinPre-angiotensionogen LC-MS Liver biopsy HCV +/[77]Prolyl 4-hydroxylase 2D-PAGE liver tissue HCV +[72]Protein disulfide isomerase precursor 2D-DIGE Mouse liver tissue HBV +[79]Proteosome beta subunit type 4 LC-MS Liver biopsy HCV +[77]Retinal dehydrogenase LC-MS Liver biopsy HCV +/[76]Serotransferrin 2D-DIGE Serum HBV[74]Serum amyloid A1 LC-MS Liver biopsy HCV +/[77]Superoxide dismutase 1 LC-MS Liver biopsy HCV[77]Thioredoxin reductase 1 LC-MS Liver biopsy HCV +[77]Transgelin LC-MS Liver biopsy HCV +[80]2D-PAGE liver tissue HCV +[72]Tropomyosin 2D-PAGE liver tissue HCV +[72]Tumor rejection antigen, GRP94 2D-DIGE Mouse liver tissue HBV +[79]VitaminCan Proteomic Profiling Identify Biomarkers and/or Therapeutic Targets for Liver Fibrosis? http://dx.doi.org/10.5772/65608 ...
... In the current phase 2 studies (PROVE1, PROVE2, and PROVE3) of the direct-acting antiviral drug telaprevir, serum samples from responders and nonresponders to HCV treatment were analyzed by proteomic profiling and 15 differentially expressed proteins, with seven of them belonging to focal adhesion proteins or other macromolecular assemblies that constitute structural links between integrins and the actin cytoskeleton, were observed[84]. The ultimate goal of performing pretreatment serum proteome profiling prior to treatment is to predict sustained virological response (SVR) and nonresponse (NR) to antiviral drugs in chronic HCV infection and design suitable treatments for each patient.Ceruloplasmin 2D-DIGE Serum HCV +[78]Clusterin 2D-DIGE Serum HBV[74]Collagen 2D-DIGE Serum HBV +[74]LC-MS Liver biopsy HCV +[80]Complement component 4a 2D-DIGE Serum HCV +[78]Cystathione beta synthase LC-MS Liver biopsy HCV[77]Cysteine and glycine rich protein 2LC-MS Liver biopsy HCV +[80]Cytochrome b-245 beta LC-MS Liver biopsy HCV +[77]Cytochrome c LC-MS Liver biopsy HCV +[76]Fetuin A 2D-PAGE Serum HCV[73]Fibrinogen 2D-DIGE Serum HCV +[78]2D-DIGE Serum HBV +[74]Fibulin-5 LC-MS Liver biopsy HCV +[80]FK506 binding protein 14 LC-MS Liver biopsy HCV[77]Gelsolin 2D-PAGE Serum HBV[81]Glutamate dehydrogenase 1 2D-DIGE Mouse liver tissue HBV[79]Gluthatione-S-transferases LC-MS Liver biopsy HCV[77]Haptoglobin 2D-PAGE Serum HCV[73]2D-DIGE Serum HBV[74]Hemopexin 2D-PAGE Serum HCV +[73]2D-DIGE Serum HBV +[74]High mobility group 1 2D-DIGE Mouse liver tissue HBV[79]ProteinPre-angiotensionogen LC-MS Liver biopsy HCV +/[77]Prolyl 4-hydroxylase 2D-PAGE liver tissue HCV +[72]Protein disulfide isomerase precursor 2D-DIGE Mouse liver tissue HBV +[79]Proteosome beta subunit type 4 LC-MS Liver biopsy HCV +[77]Retinal dehydrogenase LC-MS Liver biopsy HCV +/[76]Serotransferrin 2D-DIGE Serum HBV[74]Serum amyloid A1 LC-MS Liver biopsy HCV +/[77]Superoxide dismutase 1 LC-MS Liver biopsy HCV[77]Thioredoxin reductase 1 LC-MS Liver biopsy HCV +[77]Transgelin LC-MS Liver biopsy HCV +[80]2D-PAGE liver tissue HCV +[72]Tropomyosin 2D-PAGE liver tissue HCV +[72]Tumor rejection antigen, GRP94 2D-DIGE Mouse liver tissue HBV +[79]VitaminCan Proteomic Profiling Identify Biomarkers and/or Therapeutic Targets for Liver Fibrosis? http://dx.doi.org/10.5772/65608 ...
... In the current phase 2 studies (PROVE1, PROVE2, and PROVE3) of the direct-acting antiviral drug telaprevir, serum samples from responders and nonresponders to HCV treatment were analyzed by proteomic profiling and 15 differentially expressed proteins, with seven of them belonging to focal adhesion proteins or other macromolecular assemblies that constitute structural links between integrins and the actin cytoskeleton, were observed[84]. The ultimate goal of performing pretreatment serum proteome profiling prior to treatment is to predict sustained virological response (SVR) and nonresponse (NR) to antiviral drugs in chronic HCV infection and design suitable treatments for each patient.Ceruloplasmin 2D-DIGE Serum HCV +[78]Clusterin 2D-DIGE Serum HBV[74]Collagen 2D-DIGE Serum HBV +[74]LC-MS Liver biopsy HCV +[80]Complement component 4a 2D-DIGE Serum HCV +[78]Cystathione beta synthase LC-MS Liver biopsy HCV[77]Cysteine and glycine rich protein 2LC-MS Liver biopsy HCV +[80]Cytochrome b-245 beta LC-MS Liver biopsy HCV +[77]Cytochrome c LC-MS Liver biopsy HCV +[76]Fetuin A 2D-PAGE Serum HCV[73]Fibrinogen 2D-DIGE Serum HCV +[78]2D-DIGE Serum HBV +[74]Fibulin-5 LC-MS Liver biopsy HCV +[80]FK506 binding protein 14 LC-MS Liver biopsy HCV[77]Gelsolin 2D-PAGE Serum HBV[81]Glutamate dehydrogenase 1 2D-DIGE Mouse liver tissue HBV[79]Gluthatione-S-transferases LC-MS Liver biopsy HCV[77]Haptoglobin 2D-PAGE Serum HCV[73]2D-DIGE Serum HBV[74]Hemopexin 2D-PAGE Serum HCV +[73]2D-DIGE Serum HBV +[74]High mobility group 1 2D-DIGE Mouse liver tissue HBV[79]ProteinPre-angiotensionogen LC-MS Liver biopsy HCV +/[77]Prolyl 4-hydroxylase 2D-PAGE liver tissue HCV +[72]Protein disulfide isomerase precursor 2D-DIGE Mouse liver tissue HBV +[79]Proteosome beta subunit type 4 LC-MS Liver biopsy HCV +[77]Retinal dehydrogenase LC-MS Liver biopsy HCV +/[76]Serotransferrin 2D-DIGE Serum HBV[74]Serum amyloid A1 LC-MS Liver biopsy HCV +/[77]Superoxide dismutase 1 LC-MS Liver biopsy HCV[77]Thioredoxin reductase 1 LC-MS Liver biopsy HCV +[77]Transgelin LC-MS Liver biopsy HCV +[80]2D-PAGE liver tissue HCV +[72]Tropomyosin 2D-PAGE liver tissue HCV +[72]Tumor rejection antigen, GRP94 2D-DIGE Mouse liver tissue HBV +[79]VitaminCan Proteomic Profiling Identify Biomarkers and/or Therapeutic Targets for Liver Fibrosis? http://dx.doi.org/10.5772/65608 ...
Chapter
Full-text available
Liver fibrosis is a serious disease that affects around 350–400 million people worldwide. The main approach for fibrosis staging is liver biopsy, which is an invasive procedure that is not endured pretty well by patients. Currently, some serum-based biomarker panels are available for diagnosis and staging of liver fibrosis. Recent high-throughput proteomic studies are also very promising for identification of novel biomarkers for diagnosis and/or treatment of liver fibrosis. We hereby review the application of proteomic profiling studies for identification of fibrosis biomarkers with their advantages and drawbacks.
... WNT1-inducible signaling pathway protein 2 was more highly expressed in gastric cancer tissues compared to adjacent normal tissues and might be a favorable biomarker for the early stage prediction and prognosis of gastric cancer (Li et al., 2017). Fibulin-5 was reported to be a potential biomarker for hepatic fibrosis, early-stage hepatocellular carcinoma and calcified thoracic aortic aneurysms (Bracht et al., 2015;Matsumoto et al., 2012;Naboulsi et al., 2016). Plastin-2 may have potential as a biomarker for the prediction of type 2 diabetes mellitus (Chan et al., 2012). ...
... Previous studies have demonstrated that self-factors and environmental factors can cause differences in the urine proteome (Guo et Ethnic group differences may be an influencing factor in urine proteome studies and should be considered when human urine samples are used for biomarker discovery. (Reszka, 2012) Pro-epidermal growth factor P01133 Type 2 diabetic nephropathy (Guo et al., 2015a) WNT1-inducible-signaling pathway protein 2 O76076 Gastric cancer (Li et al., 2017) Fibulin-5 Q9UBX5 Hepatic fibrosis (Bracht et al., 2015) Hepatocellular carcinoma (Naboulsi et al., 2016) Calcified thoracic aortic aneurysms (Matsumoto et al., 2012) Plastin-2 P13796 Type 2 diabetes mellitus (Chan et al., 2012) Neutrophil gelatinase-associated lipocalin Thrombospondin-4 P35443 S-Adenosylhomocysteine hydrolase (AHCY) deficiency (Sedic et al., 2011) Colorectal cancer (Greco et al., 2010) Coactosin-like protein Q14019 Small cell lung cancer (Jeong et al., 2011) Peptidoglycan recognition protein 1 O75594 Oral Inflammation in Kidney Disease (Nylund et al., 2017) Atherosclerosis (Rohatgi et al., 2009) Putative elongation factor 1-alpha-like 3 Q5VTE0 Hepatocellular carcinoma (Chen et al., 2018) Schistosomiasis (Onile et al., 2017) Pancreatic cancer (Buanes, 2016) Dipeptidase 1 P16444 Colorectal cancer (Toiyama et al., 2011;Eisenach et al., 2013;Tachibana et al., 2017) Gastrointestinal cancer (Arner et al., 2015) Neurodegenerative diseases (Yin et al., 2009) Lamin A/C Q5TCI8 Cervical cancer (Capo-chichi et al., 2016) Gastric carcinoma (Wu et al., 2009) Colorectal cancer (Willis et al., 2008) Carbonic anhydrase 1 P00915 Schistosoma mansoni infection (Ward et al., 2017) Type 2 diabetic (Bellei et al., 2008) Non-small cell lung cancer L-xylulose reductase Q7Z4W1 Prostate adenocarcinoma (Cho-Vega et al., 2007) ...
Article
Biomarkers indicate changes associated with disease. Blood is relatively stable due to the homeostatic mechanisms of the body; however, urine accumulates metabolites from changes in the body, making it a better source for early biomarker discovery. The Li ethnic group is a unique minority ethnic group that has only lived on Hainan Island for approximately 5,000 years. Studies have shown that various specific genetic variations are different between the Li and Han ethnic groups. However, whether the urinary proteome between these two ethnic groups is significantly different remains unknown. In this study, differential urinary proteins were identified in the Li and Han ethnic groups using liquid chromatography tandem mass spectrometry (LC-MS/MS). In total, 1,555 urinary proteins were identified. Twenty-five of the urinary proteins were statistically significantly different, 16 of which have been previously reported to be biomarkers of many diseases, and that these significantly different proteins were caused by ethnic differences rather than random differences. Ethnic group differences may be an influencing factor in urine proteome studies and should be considered when human urine samples are used for biomarker discovery.
... MFAP4 is a ubiquitous protein that plays an increasingly noteworthy part in elastin fiber formation and ECM remodeling processes during vascular injury and multitudinous fibrotic diseases, including myocardium, liver, joint and renal fibrosis [6][7][8][9][10][11][12]. In viral hepatitis and cirrhosis patients, transcription and protein levels experiment and histochemical analysis showed that MFAP4 levels increased significantly while progressing from non-fibrosis to severe stages [13,14]. Moreover, extensive research has shown that serum MFAP4 levels can be used as a diagnostic predictor of varying degrees of liver cirrhosis and liver fibrosis [15]. ...
... Current research has shown that MFAP4 expression is correlated with a number of functions, including coagulation, angiogenesis, tissue growth and remodeling and innate immunity [29][30][31]. Furthermore, increased MFAP4 protein levels play an extremely important role in the development of fibrotic diseases, including heart, liver, joint and kidney fibrosis [10,13,14,16,25,32,33]. However, whether MFAP4 is related to the OSF procedure has not been intensively investigated. ...
Article
Full-text available
Background Oral submucous fibrosis (OSF), distinguished by abnormal collagen deposition, is a potentially malignant disorder with 4.2% (95% CI 2.7–5.6%) of malignant transformation and rising global prevalence. However, the precise pathogenesis and effective treatment remain elusive and controversial despite the abundance of literature on this topic. Therefore, it is crucial to explore the clinicopathological characteristics and potential markers for the diagnosis and prognosis of OSF. The objective of this study was to evaluate the influence and correlation of Microfibrillar-associated protein 4 (MFAP4) and tropoelastin (TE) in the development of OSF patients. Material and methods Clinicopathological factors, hematoxylin–eosin (HE) and Masson trichome staining, immunohistochemical characteristics and the correlation between MFAP4 and TE were recorded and compared among different stages of OSF progression among cases (n = 60) and controls (n = 10). Student's t test, ANOVA analysis, and the chi-square test were performed to compare the categorical variables for clinicopathological characteristics and the expression level of MFAP4 and TE between the fibrotic and normal tissues. Correlation analysis of MFAP4 and TE was performed using Pearson's correlation test and linear regression. Results MFAP4 and TE proteins are upregulated and increased gradually in patients with varying stages of OSF, relative to the control group. Furthermore, statistical analyses revealed that the expression level of MFAP4 was positively associated with TE, with a Pearson correlation coefficient of 0.3781 ( p = 0.0048). Clinically, we found that OSF affected more males than females, with a ratio of 29:1. The age range was 16–60 years, and the mean age was 36.25 ± 10.25 years. In patients younger than 40 years, the positive expression rate of MFAP4 and TE was higher than in those over 40 years. All OSF cases had chewed areca nut, with 51.67% smoking tobacco. Conclusions Our study elucidates that the accumulation of MFAP4 and TE proteins may play a vital role in the occurrence and development of OSF and may be promising candidate moleculars for prevention, diagnosis, and treatment strategies for OSF in the future.
... MFAP4 is a ubiquitous protein playing an increasingly noteworthy part in elastin fiber formation and ECM remodeling processes during vascular injury and multitudinous fibrotic diseases, including myocardium, liver, joint and renal fibrosis [6][7][8][9][10][11][12]. In viral hepatitis and cirrhosis patients, MFAP4 levels increased significantly from non-fibrosis stage to the severe stage via transcription and protein levels experiment and histochemical analysis [13,14]. Meanwhile, extensive research has shown that serum MFAP4 level can be used as a diagnosis predictor of varying degrees of liver cirrhosis and liver fibrosis [15]. ...
... Current research has shown that MFAP4 expression is correlated with numbers of functions, including coagulation, angiogenesis, tissue growth and remodeling and innate immunity [28][29][30]. Furthermore, increased MFAP4 protein play an extremely significant role in the development of fibrotic diseases, including heart, liver, joint and kidney fibrosis [10,13,14,16,25,31,32]. However, whether MFAP4 is related to OSF procedure has not been intensively investigated. ...
Preprint
Full-text available
Background: Oral submucous fibrosis (OSF), distinguished by abnormal collagen deposition, is a precancerous disorder with 7%-30% of malignant transformation and rising global prevalence. However, the precise pathogenesis and effective treatment still remains elusive and controversial despite superfluity of literature. Therefore, it is extremely necessary and significant to explore the clinicopathological characteristics and potential markers for diagnosis and prognosis of OSF. Here, the objective of this research is to evaluate the influence and correlation of Microfibrillar-associated protein 4 [MFAP4] and tropoelastin [TE] on the development of OSF patients. Material and Methods: Classic clinicopathological factors, HE and Masson trichome staining, immunohistochemical characteristics and the correlation (MFAP4 and TE) were recorded and compared among different stages of OSF cases (n = 60) and among those normal individuals (n = 10). Then, the comparison using Student's t test, ANOVA analysis, the chi-square test for categorical variables was conducted in clinicopathological characteristics and the expression level of MFAP4 and TE between the patients' and normal tissue. The correlation analysis of MFAP4 and TE were assessed via means of Pearson's correlation test and linear regression. Results: MFAP4 and TE proteins are upregulated and even increasing gradually in varying grades of OSF patients relative to the normal cases. Furthermore, statistical analyses yielded that the expression level of MFAP4 was positively associated with TE, and the Pearson correlation coefficient was 0.3781 (p = 0.0048). Clinically, we found that OSF affected more male than female with a ratio of 29: 1. The age range was 16-60 years, and the mean age was 36.25 ± 10.25 years old. Moreover, the positive expression rate of MFAP4 and TE in patients less than 40 years old is higher than that of those over 40 years old. Meanwhile, all OSF cases had chewed areca nut, with 51.67% smoking tobacco. Conclusions: Our study elucidates that the accumulation of MFAP4 and TE proteins may play a vitally important effect in the occurrence and development of OSF and has a hope to become a promising candidate molecular for prevention, diagnosis, and treatment strategies of OSF in the future.
... At present, proteomics technologies facilitate the discovery of novel disease/syndrome-associated proteins in an unbiased and non-hypothesis driven manner. 9 Label-free quantitative (LFQ) approaches are increasingly employed as an alternative or complement to labeling methods because of the ease of the experimental procedures and lack of limitations regarding the source and number of samples. 10 However, altered expression of proteins quantified using label-free proteomics techniques should be further verified on a larger scale. ...
... 11 In this study, differentially expressed proteins (DEPs) were analyzed using enzyme-linked immunosorbent assay (ELISA), which is the gold standard for quantifying low-abundance proteins in blood samples. 9 In the present study, we screened characteristic proteins in the plasma of patients with HCC and spleen deficiency syndrome (SDS) and identified a potential indicator of the efficacy of treatment with the Jianpi Liqi Fang (健 脾理气方, JPLQF) combined with TACE. These findings should provide new ideas for the treatment of HCC. ...
Article
Objective: To investigate the therapeutic effect of the Jianpi Liqi Fang ( ,JPLQF) combined with transcatheter arterial chemoembolization (TACE) in patients with hepatocellular carcinoma (HCC) and spleen deficiency syndrome (SDS) and identify a potential indicator of efficacy. Methods: Ninety-nine patients with HCC who were diagnosed with SDS, non-spleen deficiency syndrome (NSDS), or no syndrome (NS) were treated with JPLQF combined with TACE for three periods. Therapeutic efficacy was compared among the groups. Plasma proteins were screened using label-free discovery analysis and verified via enzyme-linked immunosorbent assay (ELISA). Furthermore, receiver operating characteristic (ROC) curves were analyzed to evaluate therapeutic indicators. Results: After treatment, the Karnofsky Performance Status was significantly improved in the SDS group and significantly better than that in the NS group. The Traditional Chinese Medicine (TCM) syndrome scores were lower in the SDS group after treatment and lower than those in the NSDS group. However, alanine aminotransferase, carbohydrate antigen 19-9, alpha-fetoprotein, and carcinoembryonic antigen levels and white blood cell and platelet counts did not differ among the groups. Serum aspartate aminotransferase levels in the SDS group were significantly lower after treatment than before treatment, and total bilirubin levels were significantly lower in the SDS group than in the NSDS group. Label-free analysis identified 24 differentially expressed proteins (DEPs) between the SDS and NS groups, including 17 and 7 upregulated and downregulated proteins, respectively. Fibulin-5 (FBLN5) displayed the largest difference in expression between the groups. ELISA confirmed that FBLN5 levels were significantly lower in the NSDS and NS groups than in the SDS group. Following treatment with JPLQF and TACE, FBLN5 expression was upregulated only in the SDS group. Furthermore, ROC curve analysis indicated that FBLN5 may serve as a potential indicator of the efficacy of JPLQF combined with TACE in patients with HCC and SDS. Conclusion: JPLQF combined with TACE improved quality of life, clinical TCM symptoms, and liver function in patients with HCC and SDS. FBLN5 expression was significantly altered by treatment with JPLQF and TACE in patients with HCC and SDS.
... However, the level of serum MFAP4 also varies with sex, age and smoking, 18 and is moderately affected by the presence of stable atherosclerosis, 1 peripheral artery disease, 19 presence of abdominal aorta aneurysms, 20 asthma, 21 and chronic obstructive lung disease 22 in addition to more markedly induced levels in liver fibrosis/cirrhosis. 11,[23][24][25][26] Considering sMFAP4 as a biomarker for chronic fibrosing disease warrants detailed evaluation of both technical and normal physiological variation. In this regard, we have previously shown that MFAP4 has a remarkable pre-analytical stability. ...
... Previously, we identified sMFAP4 as a biomarker candidate for liver fibrosis and cirrhosis in hepatitis C patients possibly reflecting the underlying ECM metabolism. sMFAP4 was able to reliably identify patients with severe fibrosis stages 11,25,26,28 in these studies. The accepted best available standard for determining the presence and degree of liver fibrosis is liver biopsy, despite well-documented limitations regarding sampling and interpretation variation as well as procedure related complications. ...
Article
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Serum microfibrillar-associated protein 4 (sMFAP4) has been investigated as a biomarker for various diseases and is demonstrated to show significant gradual increase with severity of liver fibrosis. Ideal biomarkers used for disease diagnosis or prognosis should display deviating levels in affected individuals only and be robust to factors unrelated to the disease. Here we show the impact of normal physiological variation of sMFAP4 by characterizing the circadian variation, week-to-week variation, and physical exercise-induced levels. Serum samples from 3 groups of healthy volunteers were drawn: 7 times during a 24-hour period, 5 times during a 3-week period, and before and after a standardized physical exercise challenge. sMFAP4 was determined by AlphaLISA. Statistical analysis was performed using mixed effects modeling of repeated measurements. Circadian variation of sMFAP4 was demonstrated, with time of peak and nadir values depending on age and gender. For males, the peak values were observed during nighttime whereas for females, peak values were observed in the morning. Individual sMFAP4 levels remained stable over a period of 3 weeks and physical exercise inferred a mild negative influence. In conclusion, the circadian sMFAP4 variation was significant, and the levels could be influenced by physical activity. However, these variations were of limited magnitude relative to previously observed disease-induced levels in support of the biomarker potential of sMFAP4.
... Type I collagen [169,175,178,179] ↑↑ ↑↑↑ Type III collagen [7,169,175,[177][178][179] ↑↑ ↑↑ Type IV collagen [169,[175][176][177][178] ↑ ↑ ↑ Type V collagen [151,169,175,177] (↑) ↑↑↑ Type VI collagen [7,169,175,177,178,180,181] ↑-↑↑ Type X collagen [169] ↓ ↑↑↑ Type XII collagen [169] (↑) ↓↓ Type XIV collagen [169,182] ↓ -Type XVI collagen [169] ↓ ↑ Type XVIII collagen [165,169] ↑ ↑↑↑ Elastin [169,183,184] (↑) ↑↑ Fibulin-5 [169,185] (↑) ↑↑↑ MFAP-4 [169,186,187] (↑) ↑↑↑ Vitronectin [169] (↑) ↑↑ Lumican [169] ↑ ↑ ↑ Biglycan [169] ↓ ↓ ↓ Fibronectin [167,169] ↑ ↑ ↑ Laminin [176] (↑) ↑ relative loss of some types of collagens and the overexpression of others, or with collagens deposited "at the wrong place". Advanced fibrosis/cisshosis is associated with an up to 10-fold increase in fibrillar (type I, III and V) collagens relative to liver dry weight, and an up to 6-fold increase of collagen type IV, as well as of other noncollagenous ECM components, which results in n nincrease of liver stiffness [4,175]. ...
Article
Usually the dense extracellular structure in fibrotic tissues is described as extracellular matrix (ECM) or simply as collagen. However, fibrosis is not just fibrosis, which is already exemplified by the variant morphological characteristics of fibrosis due to viral versus cholestatic, autoimmune or toxic liver injury, with reticular, chicken wire and bridging fibrosis. Importantly, the overall composition of the ECM, especially the relative amounts of the many types of collagens, which represent the most abundant ECM molecules and which centrally modulate cellular functions and physiological processes, changes dramatically during fibrosis progression.
... Among the genes that increase in accessibility, we focused on those that exemplify the largest gain in accessibility by selecting the top 25th percentile of change in accessibility and lower 25th percentile of read counts in the control sample (Fig. 6A). Among those were homologs of known human liver fibrosis markers such as Apolipoprotein A-IV [54] or Fibulin-5 [55] (Fig. 6C, D). In clusters G and H we observe a decrease in promoter accessibility (Fig. 4D, Supp. ...
Article
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Background Liver fibrosis is a wound-healing response to tissue injury and inflammation hallmarked by the extracellular matrix (ECM) protein deposition in the liver parenchyma and tissue remodelling. Different cell types of the liver are known to play distinct roles in liver injury response. Hepatocytes and liver endothelial cells receive molecular signals indicating tissue injury and activate hepatic stellate cells which produce ECM proteins upon their activation. Despite the growing knowledge on the molecular mechanism underlying hepatic fibrosis in general, the cell-type-specific gene regulatory network associated with the initial response to hepatotoxic injury is still poorly characterized. Results In this study, we used thioacetamide (TAA) to induce hepatic injury in adult zebrafish. We isolated three major liver cell types - hepatocytes, endothelial cells and hepatic stellate cells - and identified cell-type-specific chromatin accessibility and transcriptional changes in an early stage of liver injury. We found that TAA induced transcriptional shifts in all three cell types hallmarked by significant alterations in the expression of genes related to fatty acid and carbohydrate metabolism, as well as immune response-associated and vascular-specific genes. Interestingly, liver endothelial cells exhibit the most pronounced response to liver injury at the transcriptome and chromatin level, hallmarked by the loss of their angiogenic phenotype. Conclusion Our results uncovered cell-type-specific transcriptome and epigenome responses to early stage liver injury, which provide valuable insights into understanding the molecular mechanism implicated in the early response of the liver to pro-fibrotic signals.
... Overexpression of thioredoxin has been reported to prevent TAA-induced hepatic fibrosis in mice (Okuyama et al. 2005). Lumican is a prerequisite for liver fibrosis, and the altered expression of lumican has been associated with liver fibrosis (Bracht et al. 2015;Krishnan et al. 2012). There were also some differential proteins discovered that have never been reported to relate to liver fibrosis, such as torsin-1A-interacting protein 2, putative lysozyme C-2, and cathepsin L1. ...
Book
This book demonstrates the potential of urine as a biomarker resource for early disease detection, covering the related theory, strategies, tools and findings. Biomarkers are measurable changes associated with diseases. Blood, as a critical part of its internal environment, is closely monitored and controlled by the body to maintain homeostasis, especially in the early stages of diseases. In contrast, urine, as a form of waste excreted by the body, collects a variety of substance changes. Accordingly, urine can offer an ideal resource for early biomarker discovery. In addition, urine is more stable than blood in vitro, and is easy to store and analyze. The book discusses exciting preliminary applications of urine biomarkers for diseases affecting major biological systems. Its main goal is to make scientists, clinicians and medical companies aware of this important, exciting, undeveloped, and profitable field.
... Both qPSCs and aPSCs express COL4A1 and COL4A2 (Fig. 2b), but aPSCs showed higher levels of other collagen genes such as COL5A2, COL6A3 and components of the basement membrane such as laminin proteins LAMA2 and LAMB1 (Fig. 2b). Furthermore, we detected higher levels of SLIT2, FBLN5 and LUM, known mediators of fibrogenesis and migration in hepatic stellate cells 26,27 (Fig. 2c). The smallest cluster of cells identified in this study is represented by Schwann cells (22 nuclei, constituting 0.02% of the total) expressing specific markers such as CDH19, S100B, CRYAB, PMP22 and SCN7A (Fig. 2b). ...
Preprint
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The cellular heterogeneity of the human pancreas has not been previously characterized due to the presence of extreme digestive enzymatic activities, causing rapid degradation of cells and RNA upon resection. Therefore, previous cellular mapping studies based on gene expression were focused on pancreatic islets, leading to a vast underrepresentation of the exocrine compartment. By profiling the transcriptome of more than 110,000 cells from human donors, we created the first comprehensive pancreas cell atlas including all the tissue components. We unveiled the existence of four different acinar cell states and suggest a division of labor for enzyme production within the healthy exocrine pancreas, which has so far been considered a homogeneous tissue. This work provides a novel and rich resource for future investigations of the healthy and diseased pancreas.
... ECM remodeling is associated with the progression of hepatic fibrosis [24]. Sitek found that FBLN5 expression increases significantly on both transcript level and protein level as the fibrosis stage of the patient advances [25]. The current results found significantly higher FBLN5 expression in NAFLD subjects than in MHO subjects, and the ECM organization also differed significantly between the two groups in GO analysis. ...
Article
Obese subjects with non-alcoholic fatty liver disease (NAFLD) and considered metabolically healthy have not been well differentiated. In this study, obese subjects were divided into metabolic healthy obesity (MHO) and NAFLD groups. Liver tissues were sampled from these two types of subjects undergoing bariatric surgery, and proteins in the liver tissues that expressed differently between the two groups of subjects were identified by Tandem Mass Tags (TMT) assay. Compared with the MHO group, 132 proteins were found to be upregulated and 84 proteins were found to be downregulated (mainly localized in mitochondria) in NAFLD group. The KEGG pathway analysis showed that significantly upregulated metabolic pathways include PPAR signaling, ECM-receptor interaction and oxidative phosphorylation was significantly downregulated. The GO analysis revealed that upregulated proteins were involved in extracellular structure organization, extracellular matrix organization and downregulated proteins took part in the oxidation–reduction process and so on. FBLN5 and DHRS2 were further validated by Western blot, immunohistochemistry and ELISA. All results demonstrate that FBLN5 expression was significantly upregulated but DHRS2 was significantly downregulated. Significance The variation between MHO and NAFLD was studied by mass spectroscopy to evaluate the mechanism with which MHO subjects resist the harmful effects induced by obesity.
... Bioinformatics analyses of DEGs identified fibulin-5 as a novel candidate factor in the crosstalk between the ERK5 and TGF-β1 signaling pathways that was induced by TGF-β1 treatment. Fibulin-5 -an elastin-binding protein-was upregulated in hepatic fibrosis and in the fibrotic skin of patients with systemic scleroderma, which is considered a biomarker for elastogenesis in lung tissues with COPD [29][30][31]. Fibulin-5, which is induced via canonical TGF-β1/Smad signaling in human lung fibroblasts, was shown to exert profibrotic effects [32] and induce skin fibrosis in part by activating dermal fibroblasts and stimulating the proliferation and migration of smooth muscle cells in the airways [31] [33][34]. Additionally, blockade of ERK5 did not only suppressed Smad3, α-SMA, and fibronectin -detected in the ERK5 (MAPK7) networks by VaProS analysis; but also gel contraction and migration as well as fibulin-5 expression. ...
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Background/aims: Lung fibrosis is associated with lung tissue contraction due to abnormal accumulation of myofibroblasts, which aggressively promote the fibrotic process. Transforming growth factor (TGF)-β signaling in fibroblasts promotes extracellular matrix (ECM) synthesis and fibroblast migration and differentiation into myofibroblasts. Inhibition of extracellular signal-regulated kinase (ERK)5 blocks lung fibroblast activation by suppressing TGF-β signaling. Here, we examined the effects of an ERK5 inhibitor on TGF-β1-induced fibrosis in lung fibroblasts. Methods: The effects of ERK5 inhibition following TGF-β1 exposure were evaluated in lung fibroblasts isolated from fibrotic human lung tissues. Fibroblast-mediated collagen gel contraction and fibroblast migration towards fibronectin were assessed. Phenotypic differences in fibrotic fibroblasts were examined using the cap analysis gene expression method for genome-wide quantification of promoter activity. Results: TGF-β1stimulated contraction of collagen gels, fibroblast migration, and α-smooth muscle actin and fibronectin expression, and Smad3 phosphorylation were increased in fibrotic fibroblasts as compared to normal lung fibroblasts. Treatment with the ERK5 inhibitor blocked these responses to a greater extent in fibroblasts from patients with usual interstitial pneumonia as compared to nonspecific interstitial pneumonia, independent of bone morphogenetic protein/Smad1 regulation. Moreover, 223 genes including fibulin-5 -which is involved in the TGF-β1-ERK5 signaling network- were upregulated in fibrotic fibroblasts, and ECM regulation was found to be enriched in the Reactome analysis. Conclusion: ERK5 inhibition attenuated the high sensitivity of fibrotic fibroblasts to TGF-β1/Smad3 signaling. Thus, the ERK5 pathway components and fibulin-5 are potential therapeutic targets to prevent lung fibrosis progression.
... In one of these research, HCV-infected human liver tissue from 15 patients at different stages of fibrosis were analyzed by 16 O/ 18 O stable isotope labeling in combination with the accurate mass and time (AMT) tag approach and demonstrated association of oxidative stress and hepatic mitochondrial dysfunction with HCV pathogenesis [18]. A recent study performed differential label-free proteomics approach using 27 biopsies from patients with HCV-associated hepatic fibrosis and reported alterations in lumican (LUM), fibulin 5 (FBLN5), cysteine and glycine-rich protein 2 (CSRP2), calponin 2 (CNN2), transgelin (TAGLN), collagen alpha-1(XIV) chain (COL14A1), and MFAP-4, then verify the expression of these proteins on a transcriptional level and with targeted proteomic approach in different cohorts composed by a total of 77 and 68 HBV or HCV infected patients with liver fibrosis, respectively [19]. These mentioned studies focuses on proteomics of HCV associated liver fibrosis, only the latter offered a partial targeted proteomic information about limited group corresponding to different fibrotic stages in HBV infection. ...
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Background Hepatitis B virus (HBV) is a global health problem, and infected patients if left untreated may develop cirrhosis and eventually hepatocellular carcinoma. This study aims to enlighten pathways associated with HBV related liver fibrosis for delineation of potential new therapeutic targets and biomarkers. Methods Tissue samples from 47 HBV infected patients with different fibrotic stages (F1 to F6) were enrolled for 2D-DIGE proteomic screening. Differentially expressed proteins were identified by mass spectrometry and verified by western blotting. Functional proteomic associations were analyzed by EnrichNet application. ResultsFibrotic stage variations were observed for apolipoprotein A1 (APOA1), pyruvate kinase PKM (KPYM), glyceraldehyde 3-phospahate dehydrogenase (GAPDH), glutamate dehydrogenase (DHE3), aldehyde dehydrogenase (ALDH2), alcohol dehydrogenase (ALDH1A1), transferrin (TRFE), peroxiredoxin 3 (PRDX3), phenazine biosynthesis-like domain-containing protein (PBLD), immuglobulin kappa chain C region (IGKC), annexin A4 (ANXA4), keratin 5 (KRT5). Enrichment analysis with Reactome and Kegg databases highlighted the possible involvement of platelet release, glycolysis and HDL mediated lipid transport pathways. Moreover, string analysis revealed that HIF-1α (Hypoxia-inducible factor 1-alpha), one of the interacting partners of HBx (Hepatitis B X protein), may play a role in the altered glycolytic response and oxidative stress observed in liver fibrosis. Conclusions To our knowledge, this is the first protomic research that studies HBV infected fibrotic human liver tissues to investigate alterations in protein levels and affected pathways among different fibrotic stages. Observed changes in the glycolytic pathway caused by HBx presence and therefore its interactions with HIF-1α can be a target pathway for novel therapeutic purposes.
... Elite instrument (Thermo Fisher Scientific) coupled online to an upstream-connected Ultimate 3000 RSLCnano highperformance liquid chromatography system (Dionex, Idstein, Germany), as previously described. 39 Briefly, preconcentration of peptides was performed on a C18 trap column (Acclaim PepMap 100; 100 mm  2 cm, 5 mm, 100 Å; Thermo Fisher Scientific) within 7 minutes at a flow rate of 30 mL/minute with 0.1% trifluoroacetic acid. The peptides were then transferred to a Nano Viper C18 analytical column (Acclaim PepMap RSLC; 75 mm  50 cm, 2 mm, 100 Å; Thermo Fisher Scientific). ...
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Histopathological differentiation between severe urocystitis with reactive urothelial atypia and carcinoma in situ (CIS) can be difficult, particularly after a treatment that deliberately induces an inflammatory reaction, such as intravesical instillation of Bacillus Calmette-Guèrin. However, precise grading in bladder cancer is critical for therapeutic decision making and thus requires reliable immunohistochemical biomarkers. Herein, an exemplary potential biomarker in bladder cancer was identified by the novel approach of Fourier transform infrared imaging for label-free tissue annotation of tissue thin sections. Identified regions of interest are collected by laser microdissection to provide homogeneous samples for liquid chromatography–tandem mass spectrometry–based proteomic analysis. This approach afforded label-free spatial classification with a high accuracy and without interobserver variability, along with the molecular resolution of the proteomic analysis. Cystitis and invasive high-grade urothelial carcinoma samples were analyzed. Three candidate biomarkers were identified and verified by immunohistochemistry in a small cohort, including low-grade urothelial carcinoma samples. The best-performing candidate AHNAK2 was further evaluated in a much larger independent verification cohort that also included CIS samples. Reactive urothelial atypia and CIS were distinguishable on the basis of the expression of this newly identified and verified immunohistochemical biomarker, with a sensitivity of 97% and a specificity of 69%. AHNAK2 can differentiate between reactive urothelial atypia in the setting of an acute or chronic cystitis and nonmuscle invasive-type CIS.
... Overexpression of the E2A isoforms resulted in altered proximal tubule cell morphology, F-actic cytoskeletal rearrangement, decreased E-cadherin expression, and increased ACTA2 expression, thus inducing EMT in HK-2 cells (Slattery, McMorrow, and Ryan 2006). COL14A1 encodes the alpha chain of type XIV collagen, a member of the FACIT (fibril-associated collagens with interrupted triple helices) collagen family which has been implicated in pulmonary and hepatic fibroses (Bracht et al. 2015;Tzortzaki et al. 2003;Bolarin and Azinge 2007). ACTA2 encodes alpha-smooth muscle actin, an actin-isoform typical of smooth muscle differentiation and prototypical marker of EMT used to define myofibroblasts (Nagamoto, Eguchi, and Beebe 2000). ...
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The use of calcineurin inhibitors has revolutionized solid organ transplantation increasing survival rates dramatically. However, calcineurin inhibitor induced nephrotoxicity severely limits the use of this class of drugs in transplantation and other diseases. Here we set out to define a microRNA (miRNA)-messenger RNA (mRNA) interaction map to identify the role of miRNAs in cyclosporine-induced nephrotoxicity and the gene pathways they regulate. By integrating miRNA and mRNA expression profiling with photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PARCLIP) against endogenous Argonaute 2 (AGO2) protein in human proximal tubule cells, we identified miRNAs and mRNAs undergoing active targeting in cyclosporine A (CsA) treated-cells and vehicle-treated controls. First, expression profiling of miRNAs and mRNAs in CsA-treated versus vehicle-treated cells identified the key canonical pathways and cellular processes which are dysregulated in proximal tubule epithelial cells (PTECs) by CsA. Our data support a model whereby CsA induces and epithelial-to-mesenchymallike (EMT-like) re-programming of PTECs by down-regulation of key apical surface, cell junction, and adherens junction genes as well as up-regulation of major pro-EMT cell signaling pathways such as PI3K/AKT, ERK, and TGF-β. Our data indicate that CsA causes specific changes in miRNAs and mRNAs associated with the RNA-Induced Silencing Complex (RISC) complex. Pathway enrichment analysis identified canonical pathways specifically under regulation by miRNAs following CsA treatment. Analysis of active miRNA-mRNA targeting interactions revealed that CsA suppresses an upstream regulator of JNK and p38 MAPKs by inducing targeting of MAP3K1 by miR-101-3p thereby uncovering a previously undefined mechanism by which CsA affects calcineurinindependent molecular pathways. These insights into the molecular pathways governing expression of genes involved in cyclosporine-induced nephrotoxicity may provide novel therapeutic approaches to preventing chronic renal injury in transplant recipients.
... MFAP4 was initially identified as a gene generally deleted in patients with Smith-Magenis syndrome [35]. One study showed that MFAP4 has the potential to serve as a promising biomarker for noninvasive assessment of various conditions, such as chronic obstructive pulmonary disease [36,37], hepatic cirrhosis [38,39], diabetic neuropathy [40], and cardiovascular complications [41]. However, the biological functions of MFAP4 in tumors remain largely unknown. ...
Article
Primary platinum-based chemoresistance occurs in approximately one-third of patients with serous ovarian cancer (SOC); however, traditional clinical indicators are poor predictors of chemoresistance. So we aimed to identify novel genes as predictors of primary platinum-based chemoresistance. Gene expression microarray analyses were performed to identify the genes related to primary platinum resistance in SOC on two discovery datasets (GSE51373, GSE63885) and one validation dataset (TCGA). Univariate and multivariate analyses with logistic regression were performed to evaluate the predictive values of the genes for platinum resistance. Machine learning algorithms (linear kernel support vector machine and artificial neural network) were applied to build prediction models. Univariate and multivariate analyses with Cox proportional hazards regression and log-rank tests were used to assess the effects of these gene signatures for platinum resistance on prognosis in two independent datasets (GSE9891, GSE32062). AGGF1 and MFAP4 were found highly expressed in patients with platinum-resistant SOC and independently predicted platinum resistance. Platinum resistance prediction models based on these targets had robust predictive power (highest AUC: 0.8056, 95% CI: 0.6338-0.9773; lowest AUC: 0.7245, 95% CI: 0.6052-0.8438). An AGGF1- and MFAP4-centered protein interaction network was built, and hypothetical regulatory pathways were identified. Enrichment analysis indicated that aberrations of extracellular matrix may play important roles in platinum resistance in SOC. High AGGF1 and MFAP4 expression levels were also related to shorter recurrence-free and overall survival in patients with SOC after adjustment for other clinical variables. Therefore, AGGF1 and MFAP4 are potential predictive biomarkers for response to platinum-based chemotherapy and survival outcomes in SOC.
... Thermo Fisher Scientific, Rockford, IL). 13 The mass spectra were searched against the UniProtKB/Swiss-Prot database (2017_11; 556 196 entries) restricted to Homo sapiens (20 243 sequences) using Mascot (version 2.3.0.2, Matrix Science Ltd., London, U.K.). ...
Article
Exosomes are extracellular vesicles that function in intercellular communication. We have previously reported that exosomes play an important role in the transmission of antiviral molecules during interferon-α (IFN-α) treatment. In this study, the protein profiles of THP-1-derived macrophages with or without interferon-α treatment and the exosomes secreted from these cells were analyzed by label-free LC-MS/MS quantitation technologies. A total of 1845 and 1550 protein groups were identified in the THP-1 macrophages and the corresponding exosomes, respectively. Treating the cells with IFN-α resulted in the differential abundance of 94 proteins in cells and 67 proteins in exosomes (greater than 2.0-fold), among which 23 proteins were up-regulated in both the IFN-α treated cells and corresponding exosomes while 14 proteins were specifically up-regulated in exosomes but not in the donor cells. GO and KEGG analysis of the identified proteins suggested that IFN-α promoted the abundance of proteins involved in the “defense response to virus” and “Type Ⅰ interferon signaling pathway” in both exosomes and cells. Functional analysis further indicated that exosomes from IFN-α-treated cells exhibited potent antiviral activity that restored the impaired antiviral response of IFN-α in hepatitis B virus-replicating hepatocytes. These results have deepened the understanding of the exosome-mediated transfer of IFN-α-induced antiviral molecules and may provide a new basis for therapeutic strategies to control viral infection.
... Recently, we confirmed the elevated abundance of MFAP4 in hepatic fibrosis in tissue samples from a large patient cohort on transcript and protein level. 19 MFAP4 is an oligomeric ECM protein that is expressed in association with elastic fibers in the entire body, especially in elastin rich tissues like the heart or the lung. 20 It has been demonstrated to interact with Tropoelastin and Fibrillin during formation of elastic fibers 21 and was found to be involved in smooth muscle cell activation in vascular and pulmonary remodeling. ...
Article
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Background/aims: An estimated 80 million people worldwide are infected with viremic hepatitis C virus (HCV). Even after eradication of HCV with direct acting antivirals (DAAs), hepatic fibrosis remains a risk factor for hepatocarcinogenesis. Recently, we confirmed the applicability of microfibrillar-associated protein 4 (MFAP4) as a serum biomarker for the assessment of hepatic fibrosis. The aim of the present study was to assess the usefulness of MFAP4 as a biomarker of liver fibrosis after HCV eliminating therapy with DAAs. Methods: MFAP4 was measured using an immunoassay in 50 hepatitis C patients at baseline (BL), the end-of-therapy (EoT), and the 12-week follow-up visit (FU). Changes in MFAP4 from BL to FU and their association with laboratory parameters including alanine aminotransferase (ALT), aspartate aminotransferase (AST), platelets, the AST to platelet ratio index (APRI), fibrosis-4 score (FIB-4), and albumin were analyzed. Results: MFAP4 serum levels were representative of the severity of hepatic fibrosis at BL and correlated well with laboratory parameters, especially APRI (Spearman correlation, R²=0.80). Laboratory parameters decreased significantly from BL to EoT. MFAP4 serum levels were found to decrease from BL and EoT to FU with high statistical significance (Wilcoxon P<0.001 for both). Conclusions: Our findings indicate that viral eradication resulted in reduced MFAP4 serum levels, presumably representing a decrease in hepatic fibrogenesis or fibrosis. Hence, MFAP4 may be a useful tool for risk assessment in hepatitis C patients with advanced fibrosis after eradication of the virus.
... However, the tissue response to injury and the activation and trans-differentiation of HSCs lead to a complete remodeling of the ECM, from both a qualitative and a quantitative point of view [156,157]. In particular, the deposition of collagens increases, with a relevant proportion of fibrillar collagens, and ECM proteins such as fibulin-5, vitronectin and lumican [149,[157][158][159][160][161]. This becomes even more apparent as disease progresses, as demonstrated by an interesting study of liver transcriptome of NAFLD patients which has revealed the upregulation of genes related to ECM organization in NASH compared to NAFL patients, mediated by the activation of Hedgehog pathway [162]. ...
Article
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Non-alcoholic fatty liver disease (NAFLD) is a chronic liver disease characterized by lipid accumulation in hepatocytes in the absence of excessive alcohol consumption. The global prevalence of NAFLD is constantly increasing. NAFLD is a disease spectrum comprising distinct stages with different prognoses. Non-alcoholic steatohepatitis (NASH) is a progressive condition, characterized by liver inflammation and hepatocyte ballooning, with or without fibrosis. The natural history of NAFLD is negatively influenced by NASH onset and by the progression towards advanced fibrosis. Pathogenetic mechanisms and cellular interactions leading to NASH and fibrosis involve hepatocytes, liver macrophages, myofibroblast cell subpopulations, and the resident progenitor cell niche. These cells are implied in the regenerative trajectories following liver injury, and impairment or perturbation of these mechanisms could lead to NASH and fibrosis. Recent evidence underlines the contribution of extra-hepatic organs/tissues (e.g., gut, adipose tissue) in influencing NASH development by interacting with hepatic cells through various molecular pathways. The present review aims to summarize the role of hepatic parenchymal and non-parenchymal cells, their mutual influence, and the possible interactions with extra-hepatic tissues and organs in the pathogenesis of NAFLD.
... Binding kinetics to 50 nM fluorescently labelled dexamethasone (F-DEX, Thermo Fischer Scientific, Bremen, Germany) were recorded at 20 °C NanoLC-ESI-MS/MS. 200 ng tryptically digested samples were measured by nanoLC-ESI-MS/MS as described previously 79 . An UliMate 3,000 RSLC nano LC system (Thermo Fischer Scientific, Bremen, Germany) was utilized for nano HPLC analysis using the following solvent system: (A) 0.1% FA; (B) 84% ACN, 0.1% FA. ...
Article
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The function of steroid receptors in the cell depends on the chaperone machinery of Hsp90, as Hsp90 primes steroid receptors for hormone binding and transcriptional activation. Several conserved proteins are known to additionally participate in receptor chaperone assemblies, but the regulation of the process is not understood in detail. Also, it is unknown to what extent the contribution of these cofactors is conserved in other eukaryotes. We here examine the reconstituted C. elegans and human chaperone assemblies. We find that the nematode phosphatase PPH-5 and the prolyl isomerase FKB-6 facilitate the formation of glucocorticoid receptor (GR) complexes with Hsp90. Within these complexes, Hsp90 can perform its closing reaction more efficiently. By combining chemical crosslinking and mass spectrometry, we define contact sites within these assemblies. Compared to the nematode Hsp90 system, the human system shows less cooperative client interaction and a stricter requirement for the co-chaperone p23 to complete the closing reaction of GR·Hsp90·Pp5/Fkbp51/Fkbp52 complexes. In both systems, hormone binding to GR is accelerated by Hsp90 alone and in the presence of its cofactors. Our results show that cooperative complex formation and hormone binding patterns are, in many aspects, conserved between the nematode and human systems.
... Lumican is a prerequisite for liver fibrosis [65] and the altered expression of lumican has 350 been associated with liver fibrosis [66] . Alpha-1-antiproteinase (A1AT) is the most 351 abundant liver-derived glycoprotein in plasma. ...
... TNS1 is also localized to focal adhesions and acts as a bridge linking ECM and the actin cytoskeleton [40]. EFEMP1 and FBLN5 are ECM proteins and highly express in portal fibroblasts, they have been proved to play a role in progressive liver fibrosis [41,42]. SORBS1 involves in cytoskeleton organization and insulin signaling pathway in human hepatoma cell line [43]. ...
Article
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Background Portal hypertension induced esophageal and gastric variceal bleeding is the main cause of death among patients of decompensated liver cirrhosis. Therefore, a standardized, biomarker-based test, to make an early-stage non-invasive risk assessment of portal hypertension, is highly desirable. However, no fit-for-purpose biomarkers have yet been identified. Methods We conducted a pilot study consisting of 5 portal hypertensive gastropathy (PHG) patients and 5 normal controls, sampling the gastric mucosa of normal controls and PHG patients before and after endoscopic cyanoacrylate injection, using label-free quantitative (LFQ) mass spectrometry, to identify potential biomarker candidates in gastric mucosa from PHG patients and normal controls. Then we further used parallel reaction monitoring (PRM) to verify the abundance of the targeted protein. Results LFQ analyses identified 423 significantly differentially expressed proteins. 17 proteins that significantly elevated in the gastric mucosa of PHG patients were further validated using PRM. Conclusions This is the first application of an LFQ-PRM workflow to identify and validate PHG–specific biomarkers in patient gastric mucosa samples. Our findings lay the foundation for comprehending the molecular mechanisms of PHG pathogenesis, and provide potential applications for useful biomarkers in early diagnosis and treatment. Trial registration and ethics approval : Trial registration was completed (ChiCTR2000029840) on February 25, 2020. Ethics Approvals were completed on July 17, 2017 (NYSZYYEC20180003) and February 15, 2020 (NYSZYYEC20200005).
... These genes were enriched for extracellular matrix (ECM) organization genes. Included in this cluster of genes are multiple genes that encode collagens including COL14A1 and COL15A1, which have well-defined roles in ECM creation and can be overexpressed during fibrosis [21][22][23]. Among the most strongly downregulated genes in the quiescent state log 2 ...
Article
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Background Quiescence, reversible exit from the cell division cycle, is characterized by large-scale changes in steady-state gene expression, yet mechanisms controlling these changes are in need of further elucidation. In order to characterize the effects of post-transcriptional control on the quiescent transcriptome in human fibroblasts, we determined mRNA decay rates for over 10,000 genes using a transcription shut-off time-course. ResultsWe found that ~500 of the genes monitored exhibited significant changes in decay rate upon quiescence induction. Genes involved in RNA processing and ribosome biogenesis were destabilized with quiescence, while genes involved in the developmental process were stabilized with quiescence. Moreover, extracellular matrix genes demonstrated an upregulation of gene expression that corresponded with a stabilization of these transcripts. Additionally, targets of a quiescence-associated microRNA (miR-29) were significantly enriched in the fraction of transcripts that were stabilized during quiescence. Conclusion Coordinated stability changes in clusters of genes with important functions in fibroblast quiescence maintenance are highly correlated with quiescence gene expression patterns. Analysis of miR-29 target decay rates suggests that microRNA-induced changes in RNA stability are important contributors to the quiescence gene expression program in fibroblasts. The identification of multiple stability-related gene clusters suggests that other posttranscriptional regulators of transcript stability may contribute to the coordination of quiescence gene expression. Such regulators may ultimately prove to be valuable targets for therapeutics that target proliferative cells, for instance, in cancer or fibrosis.
... Both, EFEMP2 and FBLN5 are essential for elastic fiber formation in connective tissues 25,26 . Proteomics studies have also shown increased fibulin-5 protein levels with hepatic fibrosis 27 and recent functional studies show that fibulin-4 is essential for elastin and collagen fiber crosslinking and extracellular matrix assembly via lysyloxidase (LOX) 28 . THBS2 (thrombospondin-2) also encodes a secreted ECM glycoprotein, which modestly correlates with histologic severity of NASH and fibrosis in a recent study 29 . ...
Article
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Non-alcoholic fatty liver disease (NAFLD) is the most common cause of liver disease worldwide. In adults with NAFLD, fibrosis can develop and progress to liver cirrhosis and liver failure. However, the underlying molecular mechanisms of fibrosis progression are not fully understood. Using total RNA-Seq, we investigated the molecular mechanisms of NAFLD and fibrosis. We sequenced liver tissue from 143 adults across the full spectrum of fibrosis stage including those with stage 4 fibrosis (cirrhosis). We identified gene expression clusters that strongly correlate with fibrosis stage including four genes that have been found consistently across previously published transcriptomic studies on NASH i.e. COL1A2 , EFEMP2 , FBLN5 and THBS2 . Using cell type deconvolution, we estimated the loss of hepatocytes versus gain of hepatic stellate cells, macrophages and cholangiocytes with advancing fibrosis stage. Hepatocyte-specific functional analysis indicated increase of pro-apoptotic pathways and markers of bipotent hepatocyte/cholangiocyte precursors. Regression modelling was used to derive predictors of fibrosis stage. This study elucidated molecular and cell composition changes associated with increasing fibrosis stage in NAFLD and defined informative gene signatures for the disease.
... Among the genes that increase in accessibility, we focused on those that exemplify the largest gain in accessibility by selecting the top 25th percentile of change in accessibility and lower 25th percentile of read counts in the control sample (Fig. 6A). Among those were homologs of known human liver brosis markers such as Apolipoprotein A-IV [52] or Fibulin-5 [53] (Fig. 6C, Fig. 6D). In clusters G and H we observe a decrease in promoter accessibility (Fig. 4D, Supp. Figure 3C, Supp. Figure 3D) accompanied by reduced gene expression (Fig. 3C). ...
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Background Liver fibrosis is a wound-healing response to tissue injury and inflammation hallmarked by the extracellular matrix (ECM) protein deposition in the liver parenchyma and tissue remodelling. Different cell types of the liver are known to play distinct roles in liver injury response. Hepatocytes and liver endothelial cells receive molecular signals indicating tissue injury and activate hepatic stellate cells which produce ECM proteins upon their activation. Despite the growing knowledge on the molecular mechanism underlying hepatic fibrosis in general, the cell-type-specific gene regulatory network associated with the initial response to hepatotoxic injury is still poorly characterized. Results In this study, we used thioacetamide (TAA) to induce hepatic injury in adult zebrafish. We isolated three major liver cell types - hepatocytes, endothelial cells and hepatic stellate cells - and identified cell-type-specific chromatin accessibility and transcriptional changes in an early stage of liver injury. We found that TAA induced transcriptional shifts in all three cell types hallmarked by significant alterations in the expression of genes related to fatty acid and carbohydrate metabolism, as well as immune response-associated and vascular-specific genes. Interestingly, liver endothelial cells exhibit the most pronounced response to liver injury at the transcriptome and chromatin level, hallmarked by the loss of their angiogenic phenotype. Conclusion Our results uncovered cell-type-specific transcriptome and epigenome responses to early stage liver injury, which provide valuable insights into understanding the molecular mechanism implicated in the early response of the liver to pro-fibrotic signals.
... Of interest, we measured increased abundance of several proteins in the Achilles IFM with ageing that have been associated with fibrosis in connective tissues, including collagen VI and XIV, periostin and tenascin. Collagen VI and XIV are overexpressed in several fibrotic diseases, including pulmonary fibrosis, hepatic fibrosis and adhesive capsulitis, and collagen VI-null mice exhibit improved cardiac structure and function post-myocardial infarction [49][50][51][52][53] . Indeed, recent studies have implicated collagen VI as a major determinant of fibrosis [54] . ...
Article
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Tendon consists of highly aligned collagen-rich fascicles surrounded by interfascicular matrix (IFM). Some tendons act as energy stores to improve locomotion efficiency, but such tendons commonly obtain debilitating injuries. In equine tendons, energy storing is achieved primarily through specialisation of the IFM. However, no studies have investigated IFM structure-function specialisation in human tendons. Here, we compare the human positional anterior tibial tendon and energy storing Achilles tendons, testing the hypothesis that the Achilles tendon IFM has specialised composition and mechanical properties, which are lost with ageing. Data demonstrate IFM specialisation in the energy storing Achilles, with greater elasticity and fatigue resistance than in the positional anterior tibial tendon. With ageing, alterations occur predominantly to the proteome of the Achilles IFM, which are likely responsible for the observed trends towards decreased fatigue resistance. Knowledge of these key energy storing specialisations and their changes with ageing offers crucial insight towards developing treatments for tendinopathy. Statement of Significance Developing effective therapeutics or preventative measures for tendon injury necessitates the understanding of healthy tendon function and mechanics. By establishing structure-function relationships in human tendon and determining how these are affected by ageing, potential targets for therapeutics can be identified. In this study, we have used a combination of mechanical testing, immunolabelling and proteomics analysis to study structure-function specialisations in human tendon. We demonstrate that the interfascicular matrix is specialised for energy storing in the Achilles tendon, and that its proteome altered with ageing, which is likely responsible for the observed trends towards decreased fatigue resistance. Knowledge of these key energy storing specialisations and their changes with ageing offers crucial insight towards developing treatments and preventative approaches for tendinopathy.
... ECM remodeling is related to liver fibrosis. Bracht has found that the expression of FBLN5 at the transcription and protein level increased significantly with the progression of the fibrosis stage in patients [39]. It was also noted that the expression of FBLN5 in a NAFLD group is significantly higher than in other groups. ...
Article
Objective: To analyze differentially expressed genes (DEGs) related to liver fibrosis, and clarify the key genes and the possible targets in the progression of liver fibrosis. Methods: Using microarray datasets, GSE38199 was extracted from Gene Expression Omnibus (GEO), and a bioinformatics method was performed to find DEGs and transcription factors related to liver fibrosis. Results: A total of 58 DEGs were screened out according to GEO2R online analysis tool, which included 49 up-regulated and 9 down-regulated genes. These DEGs were mainly involved in formation with the extracellular region and extracellular exosome through gene ontology (GO) enrichment analysis. Kyoto Encyclopedia of Gene and Genome (KEGG) pathway enrichment analysis showed that DEGs mainly participated in the PI3K-Akt signaling pathway, focal adhesion, ECM-receptor interaction, and metabolic pathways. Based on the results of the Protein-Protein Interaction (PPI) network and Molecular Complex Detection (MCODE) analysis, 9 key genes (COL1A1, FBN1, BGN, COL6A3, MMP2, FBLN5, LUM, PDGFRB, LOXL1) were screened out. A total of 30 transcription factors were found according to these 9 key genes, of which 4 transcription factors (Stat3, Trp53, NF-κB1, Sp1) were enriched. Conclusion: Stat3, Trp53, NF-κB1, and Sp1 were all related to the development of liver fibrosis, and FBLN5 might be a target for liver fibrosis.
... MFAP4 was initially identified as a gene that is commonly missing in patients with Smith-Magenis syndrome [10]. Studies have shown that MFAP4 can be useful for non-invasive assessment of various diseases, such as chronic obstructive pulmonary disease [11,12], liver cirrhosis [13,14], diabetic neuropathy [15], and cardiovascular complications [16]. However, the biological function of MFAP4 in tumors is still unknown. ...
Article
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BACKGROUND Oral squamous cell carcinoma (OSCC) is a common tumor of the head and neck. Its treatment usually requires multiple modalities. Currently, there are no molecular biomarkers to guide these treatment strategies. Studies have shown that microfibril-associated protein 4 (MFAP4) is potentially useful for non-invasive assessment of various diseases; however, its biological function in tumors is still unknown. In this study, we propose that MFAP4 is a new prognostic target for OSCC. MATERIAL AND METHODS First, we collected OSCC data (GSE25099 and GSE30784 datasets) from the Gene Expression Omnibus (GEO) database and compared the differential expression of MFAP4 gene between the patients (tumor) and normal (control) groups. The comparison was done with University of California Santa Cruz Xena (https://xenabrowser.net/Datapages/), and we calculated the difference in MFAP4 gene expression between normal and tumor tissues in a pan-cancer analysis. Then, we compared the 2 groups with high and low expression of MFAP4 gene in terms of tumor mutation burden (TMB), miRNA regulation, and immune cell infiltration. RESULTS We found that the expression of MFAP4 gene was significantly decreased in tumors. Our research also showed that high expression of MFAP4 was related to better prognosis of patients and may be related to tumor gene mutation, miRNA regulation, and infiltration of different immune cells. CONCLUSIONS Our work provides evidence that expression of MFAP4 can be used as a prognostic biomarker for risk stratification of OSCC patients and elaborates on its relation with the regulation of TMB, miRNAs, and immune cell infiltration.
... TNS1 is also localized to focal adhesions and acts as a bridge linking ECM and the actin cytoskeleton (39). EFEMP1 and FBLN5 are ECM proteins and highly express in portal broblasts, they have been proved to play a role in progressive liver brosis (40,41). ...
Preprint
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Background: Portal hypertension induced esophageal and gastric varix bleeding is the main cause of death among patients of decompensated liver cirrhosis. Therefore, a standardized, biomarker-based test, to make an early-stage non-invasive risk assessment of portal hypertension, is highly desirable. However, no fit-for-purpose biomarkers have yet been identified. Methods: We conducted a pilot study consisting of 5 portal hypertensive gastropathy (PHG) patients and 5 normal controls, sampling the gastric mucosa of normal controls and PHG patients before and after endoscopic cyanoacrylate injection, using label-free quantitative (LFQ) mass spectrometry, to identify potential biomarker candidates in gastric mucosa from PHG patients and normal controls. Then we further used parallel reaction monitoring (PRM) to verify the abundance of the targeted protein. Results: LFQ analyses identified 423 significantly differentially expressed proteins. 17 protein that significantly elevated in the gastric mucosa of PHG patients were further validated using PRM. Conclusions: This is the first application of an LFQ-PRM workflow to identify and validate PHG–specific biomarkers in patient gastric mucosa samples. Our findings lay the foundation for comprehending the molecular mechanisms of PHG pathogenesis, and provide potential applications for useful biomarkers in early diagnosis and treatment. Trial Registration: Trial registration was completed (ChiCTR2000029840) on February 25, 2020.
... Both qPSCs and aPSCs express COL4A1 and COL4A2, but aPSCs showed higher levels of mRNAs encoding other collagens such as COL5A2, COL6A3 and components of the basement membrane such as laminin proteins LAMA2 and LAMB1 (Figure 3Band SupplementaryFigure 3). Furthermore, in aPSCs we detected higher levels of SLIT2 and LUM, known mediators of fibrogenesis and migration in hepatic stellate cells16,17 (Figure 3Band Supplementary ...
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Background & aims: Molecular evidence of cellular heterogeneity in the human exocrine pancreas has not been yet established because of the local concentration and cascade of hydrolytic enzymes that can rapidly degrade cells and RNA upon pancreatic resection. We sought to better understand the heterogeneity and cellular composition of the pancreas in neonates and adults in healthy and diseased conditions using single-cell-sequencing approaches. Methods: We innovated single-nucleus RNA-sequencing protocols and profiled more than 120,000 cells from pancreata of adult and neonatal human donors. We validated the single-nucleus findings using RNA fluorescence in situ hybridization, in situ sequencing, and computational approaches. Results: We created the first comprehensive atlas of human pancreas cells, to our knowledge, including epithelial and nonepithelial constituents, and uncovered 3 distinct acinar cell types, with possible implications for homeostatic and inflammatory processes of the pancreas. The comparison with neonatal single-nucleus-sequencing data showed a different cellular composition of the endocrine tissue, highlighting the tissue dynamics occurring during development. By applying spatial cartography, involving cell proximity mapping through in situ sequencing, we found evidence of specific cell type neighborhoods, dynamic topographies in the endocrine and exocrine pancreas, and principles of morphologic organization of the organ. Furthermore, similar analyses in chronic pancreatitis biopsy samples showed the presence of acinar-REG+ cells, a reciprocal association between macrophages and activated stellate cells, and a new potential role of tuft cells in this disease. Conclusions: Our human pancreas cell atlas can be interrogated to understand pancreatic cell biology and provides a crucial reference set for comparisons with diseased tissue samples to map the cellular foundations of pancreatic diseases.
... In the liver, lumican expression level increases during fibrosis in rat models of hepatic fibrosis (160) and in patients with NAFLD, NASH, HCV-, and HBV-associated fibrosis (161,162,163,164), where it correlates with progression of fibrosis stage. In liver biopsies from patients with mild and severe NASH, lumican was found increased in sinusoids compared to tissue from obese patients with normal liver or with liver steatosis (162). ...
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Chronic liver disease when accompanied by underlying fibrosis, is characterized by an accumulation of extracellular matrix (ECM) proteins and chronic inflammation. Although traditionally considered as a passive and largely architectural structure, the ECM is now being recognized as a source of potent damage-associated molecular pattern (DAMP)s with immune-active peptides and domains. In parallel, the ECM anchors a range of cytokines, chemokines and growth factors, all of which are capable of modulating immune responses. A growing body of evidence shows that ECM proteins themselves are capable of modulating immunity either directly via ligation with immune cell receptors including integrins and TLRs, or indirectly through release of immunoactive molecules such as cytokines which are stored within the ECM structure. Notably, ECM deposition and remodeling during injury and fibrosis can result in release or formation of ECM-DAMPs within the tissue, which can promote local inflammatory immune response and chemotactic immune cell recruitment and inflammation. It is well described that the ECM and immune response are interlinked and mutually participate in driving fibrosis, although their precise interactions in the context of chronic liver disease are poorly understood. This review aims to describe the known pro-/anti-inflammatory and fibrogenic properties of ECM proteins and DAMPs, with particular reference to the immunomodulatory properties of the ECM in the context of chronic liver disease. Finally, we discuss the importance of developing novel biotechnological platforms based on decellularized ECM-scaffolds, which provide opportunities to directly explore liver ECM-immune cell interactions in greater detail.
... Lumican is a prerequisite for liver fibrosis [65] and the altered expression of lumican has 350 been associated with liver fibrosis [66] . Alpha-1-antiproteinase (A1AT) is the most 351 abundant liver-derived glycoprotein in plasma. ...
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Biomarker is the change associated with the disease. Blood is relatively stable because of the homeostatic mechanisms of the body. However, urine accumulates changes of the body, which makes it a better early biomarker source. Liver fibrosis, which results from the deposition of extracellular matrix (ECM) components, is a reversible pathological condition, whereas cirrhosis, the end-stage of liver fibrosis, is irreversible. Consequently, noninvasive early biomarkers for fibrosis are desperately needed. In this study, differential urinary proteins were identified in the thioacetamide (TAA) liver fibrosis rat model using tandem mass tagging and two-dimensional liquid chromatography tandem mass spectrometry (2DLC-MS/MS). A total of 766 urinary proteins were identified, 143 and 118 of which were significantly changed in the TAA 1-week and 3-week groups, respectively. Multiple reaction monitoring (MRM)-targeted proteomics was used to further validate the abundant differentially expressed proteins in the TAA 1-week, 3-week, 6-week and 8-week groups. A total of 40 urinary proteins were statistically significant (fold change >2 and p<0.05), 15 of which had been previously reported as biomarkers of liver fibrosis, cirrhosis or other related diseases and 10 of which had been reported to be associated with the pathology and mechanism of liver fibrosis. These differential proteins were detected in urine before the alanine aminotransferase (ALT) and aspartate transaminase (AST) changes in the serum and before fibrosis was observed upon hematoxylin and eosin (HE) and Masson staining.
... Overexpression of thioredoxin has been reported to prevent TAA-induced hepatic fibrosis in mice (Okuyama et al., 2005). Lumican is a prerequisite for liver fibrosis (Krishnan et al., 2012) and the altered expression of lumican has been associated with liver fibrosis (Bracht et al., 2015). Alpha-1antiproteinase (A1AT) is the most abundant liver-derived glycoprotein in plasma. ...
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Biomarker is the change associated with the disease. Blood is relatively stable because of the homeostatic mechanisms of the body. However, urine accumulates changes of the body, which makes it a better early biomarker source. Liver fibrosis is a reversible pathological condition, whereas cirrhosis, the end-stage of liver fibrosis, is irreversible. Consequently, noninvasive early biomarkers for fibrosis are desperately needed. In this study, differential urinary proteins were identified in the thioacetamide liver fibrosis rat model using tandem mass tagging and two-dimensional liquid chromatography tandem mass spectrometry. A total of 766 urinary proteins were identified, 143 and 118 of which were significantly changed in the TAA 1-week and 3-week groups, respectively. Multiple reaction monitoring (MRM)-targeted proteomics was used to further validate the abundant differentially expressed proteins. A total of 40 urinary proteins were statistically significant, 15 of which had been previously reported as biomarkers of liver fibrosis, cirrhosis or other related diseases and 10 of which had been reported to be associated with the pathology and mechanism of liver fibrosis. These differential proteins were detected in urine before the alanine aminotransferase and aspartate transaminase changes in the serum and before fibrosis was observed upon hematoxylin and eosin (HE) and Masson’s staining.
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Hepatitis B virus (HBV) infection is a major health problem worldwide. Recent evidence suggests that some viruses can manipulate the infection process by packing specific viral and cellular components into exosomes, small nanometer-sized (30-150 nm) vesicles secreted from various cells. However, the impact of HBV replication on the content of exosomes produced by hepatocytes has not been fully delineated. In this work, an HBV-inducible cell line HepAD38 was used to directly compare changes in the protein content of exosomes secreted from HepAD38 cells with or without HBV replication. Exosomes were isolated from supernantants of HepAD38 cells cultured with or without doxycycline (dox) and their purity was confirmed by transmission electron microscopy (TEM) and Western immunoblotting assays. Ion-intensity based label-free LC-MS/MS quantitation technologies were applied to analyze protein content of exosomes from HBV replicating cells(referred as HepAD38 (dox- )-exo) and from HBV non-replicating cells (referred as HepAD38 (dox+ )-exo). A total of 1412 exosomal protein groups were identified, among which the abundance of 35 proteins was significantly changed following HBV replication. Strikingly, 5 subunit proteins from the 26S proteasome complex, including PSMC1, PSMC2, PSMD1, PSMD7 and PSMD14 were consistently enhanced in HepAD38 (dox- )-exo. Bioinformatic analysis of differential exosomal proteins confirmed the significant enrichment of components involved in the proteasomal catabolic process. Proteasome activity assays further suggested that HepAD38 (dox- )-exo had enhanced proteolytic activity compared to HepAD38 (dox+ )-exo. Furthermore, human peripheral monocytes incubated with HepAD38 (dox- )-exo induced a significantly lower level of IL-6 secretion compared to IL-6 levels from HepAD38 (dox+ )-exo. Irreversible inhibition of proteasomal activity within exosomes restored higher production of IL-6 by monocytes, suggesting that transmission of proteasome subunit proteins by HepAD38 (dox- )-exo might modulate the production of pro-inflammatory molecules in the recipient monocytes. These results revealed the composition and potential function of exosomes produced during HBV replication, thus providing a new perspective on the role of exosomes in HBV-host interaction.
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Nonalcoholic steatohepatitis (NASH) is strongly associated with obesity and T2D. The molecular factors underlying the development of inflammation and severe fibrosis in NASH remain largely unknown. The purpose of this study was to identify gene expression patterns related to obesity-related NASH inflammation and fibrosis. We performed sequencing-based mRNA profiling analysis of liver samples from individuals with normal histology (N=24), lobular inflammation (N=53), or bridging fibrosis, incomplete cirrhosis, or cirrhosis (N=65). Hepatic expression of a subset of mRNAs was validated using an orthogonal method, analyzed in a hepatic stellate cell line, and used to identify transcriptional patterns shared by other forms of cirrhosis. We observed evidence for differential levels of 3820 and 2980 transcripts in lobular inflammation and advanced fibrosis, respectively, compared with normal histology (FDR ≤ 0.05), including 176 genes specific to fibrosis. Functional enrichment analysis of these genes revealed participation in pathways involving cytokine-cytokine receptor interaction, PI3K-Akt signaling pathway, focal adhesion, ECM-receptor interaction. We identified 34 differentially expressed transcripts in comparisons of lobular inflammation and fibrosis, a proportion of which were also upregulated during activation of hepatic stellate cells. A set of 16 genes from a previous independent study of NASH bridging fibrosis/cirrhosis were replicated, several of which have also been associated with advanced fibrosis/cirrhosis due to hepatitis viruses or alcohol in human patients. Dysregulated mRNA expression is associated with inflammation and fibrosis in NASH. Advanced NASH fibrosis is characterized by distinct set of molecular changes that are shared with other causes of cirrhosis.
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Microfibrillar-associated protein 4 (MFAP4) is an extracellular matrix protein belonging to the fibrinogen-related domain family. It has been localized to elastic fiber-rich regions in several tissues including the arteries, lungs, heart and skin. MFAP4 binds collagen, fibrillins and tropoelastin and contributes to the process of microfibrillar assembly and maturation of elastic fibers. MFAP4 can also bind RGD-dependent integrins, predominantly αVβ3 and αVβ5 through its N-terminal RGD sequence, modulating cellular behavior. Circulating MFAP4 was suggested as a robust biomarker for hepatitis C virus- and alcoholic liver disease-related liver fibrosis, cardiovascular disorders and chronic obstructive pulmonary disease. In mice, MFAP4 seems to have a widely redundant role under homeostatic conditions, as global MFAP4 deficiency results in a mild pulmonary phenotype, causing emphysema-like airspace enlargement that progresses with age. However, emerging in vivo and in vitro data suggest that MFAP4 is actively involved in the pathogenesis of remodeling-associated diseases, including fibrosis, cardiovascular disorders, aging, asthma and cancer through activation of integrin-mediated signaling as well as by modulating TGF-β pathway, thus supporting maladaptive matrix remodeling. This review summarizes the current knowledge about MFAP4 structure and localization, its mechanisms of action in disease-induced tissue remodeling as well as its potential role as a clinical biomarker.
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Cerebrospinal fluid (CSF) is in direct contact with the brain and serves as a valuable specimen to examine diseases of the central nervous system through analyzing its components. These include the analysis of metabolites, cells as well as proteins. For identifying new suitable diagnostic protein biomarkers bottom-up data-dependent acquisition (DDA) mass spectrometry-based approaches are most popular. Drawbacks of this method are stochastic and irreproducible precursor ion selection. Recently, data-independent acquisition (DIA) emerged as an alternative method. It overcomes several limitations of DDA, since it combines the benefits of DDA and targeted methods like selected reaction monitoring (SRM). We established a DIA method for in-depth proteome analysis of CSF. For this, four spectral libraries were generated with samples from native CSF (n = 5), CSF fractionation (15 in total) and substantia nigra fractionation (54 in total) and applied to three CSF DIA replicates. The DDA and DIA methods for CSF were conducted with the same nanoLC parameters using a 180 min gradient. Compared to a conventional DDA method, our DIA approach increased the number of identified protein groups from 648 identifications in DDA to 1574 in DIA using a comprehensive spectral library generated with DDA measurements from five native CSF and 54 substantia nigra fractions. We also could show that a sample specific spectral library generated from native CSF only increased the identification reproducibility from three DIA replicates to 90% (77% with a DDA method). Moreover, by utilizing a substantia nigra specific spectral library for CSF DIA, over 60 brain-originated proteins could be identified compared to only 11 with DDA. In conclusion, the here presented optimized DIA method substantially outperforms DDA and could develop into a powerful tool for biomarker discovery in CSF. Data are available via ProteomeXchange with the identifiers PXD010698, PXD010708, PXD010690, PXD010705, and PXD009624.
Chapter
Liver fibrosis is the common factor of liver diseases regardless of the underlying cause. Continuous accumulation of extracellular matrix (ECM) proteins will eventually lead to organ failure. The extracellular matrix is composed of fibrous and non-fibrous collagens, elastin, and proteoglycans. In advanced stages of fibrosis the balance of the ECM remodeling is disturbed resulting in alterations of the quality and constitution of ECM liver proteins. As a result, the extracellular matrix density maximizes and a superfluous build-up of fibrous tissue as well as a general change in the protein profile and liver structure occurs. In fact, compared with a healthy liver, a cirrhotic liver may comprise nearly six times as much collagen; where type I and III collagens are the most plentiful. Formation and disease relevant degradation products of extracellular and intracellular proteins released into the circulation are the result of the extracellular matrix remodeling. Serving as serological biomarker targets these fragments can hold valuable clues about the disease pathogenesis. Being able to identify early signs of liver fibrosis with the aim of stopping further progression is a clinical need. Unlike in histology, serological markers can potentially mirror both the activity of the fibrotic processes and the total extracellular matrix mass going through a modification. The most prevalent ECM remodeling markers such as hyaluronic acid, N-terminal procollagen type III, and type IV collagen will be discussed in the following chapter. A novel alternative: The Protein Fingerprint Technology will also be introduced. This technology has become widely recognized for its ability to identify and quantify disease-specific protein fragments in body liquids, demonstrating a sound potential as a serological biomarker of extracellular matrix remodeling in fatty liver diseases with fibrosis.
Chapter
Digestive diseases are disorders of the digestive organs, which may range from benign to serious. There is an inherent difficulty in diagnosis and treatment of various chronic digestive diseases; thus noninvasive early biomarkers are desperately needed. Without homeostasis mechanisms, urine is an ideal biomarker source that can reflect the early changes of disease theoretically. In this section, we will introduce the new studies of the urine proteome in rat models of three digestive diseases including liver fibrosis, chronic pancreatitis and inflammatory bowel disease. Many potential biomarkers were found earlier than clinical symptom and significant pathological changes that may provide important clues for the early detection of these diseases. We think urine proteome has a broad application prospect in the early diagnosis, treatment, monitoring, and prognosis of digestive diseases.
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Targeted proteomics techniques allow accurate quantitative measurements of analytes in complex matrices with dynamic linear ranges that span up to 4–5 orders of magnitude. Hence, targeted methods are promising for the development of robust protein assays in several sensitive areas, e.g. in health care. However, exploiting the full method potential requires reliable determination of the dynamic range along with related quantification limits for each analyte. Here, we present a software named CalibraCurve that enables an automated batch‐mode determination of dynamic linear ranges and quantification limits for both targeted proteomics and similar assays. The software uses a variety of measures to assess the accuracy of the calibration, namely precision and trueness. Two different kinds of customizable graphs are created (calibration curves and response factor plots). The accuracy measures and the graphs offer an intuitive, detailed and reliable opportunity to assess the quality of the model fit. Thus, we deem CalibraCurve a highly useful and flexible tool to facilitate the development and control of reliable SRM/MRM‐MS‐based proteomics assays. This article is protected by copyright. All rights reserved
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Introduction: Chronic kidney disease (CKD) is a widespread health problem, in which mortality is most frequently due to cardiovascular diseases. Microfibrillar-associated protein 4 (MFAP4) is an extracellular matrix protein. MFAP4 is involved in several biological processes, particularly the maintenance of vascular integrity and extracellular matrix remodeling. Our review of the literature revealed no data concerning MFAP4 levels in CKD and its relationship with myocardial functions. Objective: The purpose of this study was therefore to investigate MFAP4 levels in CKD, parameters affecting these, and the relationship with myocardial functions. Materials and methods: Seventy-nine CKD patients and 30 healthy controls were included in the study. Routine biochemical tests and echocardiography were performed once demographic data had been recorded. Blood specimens were collected for MFAP4 analysis, and the results were subjected to statistical analysis. Results: MFAP4 levels were significantly higher in the patient group than in the control group (p< 0.001). Doppler parameters revealed more frequent LV diastolic impairment in the patient group. Tissue Doppler systolic velocity and global longitudinal strain were significantly impaired, revealing the subclinical LV systolic dysfunction in CKD patients. MFAP4 elevation in the patient group was positively correlated with aortic root (AR), global circumferential strain (GCS), and GCS rate. Conclusion: Our results showed MFAP4 elevation in CKD for the first time in the literature, and that this elevation may be related to GCS and AR dilation. We think that, once supported by further studies, MFAP4 may constitute a marker in the evaluation of myocardial functions in CKD.
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Microfibrillar‐associated proteins (MFAPs) are extracellular matrix glycoproteins, which play a role in microfibril assembly, elastinogenesis, and tissue homeostasis. MFAPs consist of five subfamily members, including MFAP1, MFAP2, MFAP3, MFAP4, and MFAP5. Among these, MFAP2 and MFAP5 are most closely related, and exhibit very limited amino acid sequence homology with MFAP1, MFAP3, and MFAP4. Gene expression profiling analysis reveals that MFAP2, MFAP5, and MFAP4 are specifically expressed in osteoblastic like cells, whereas MFAP1 and MFAP3 are more ubiquitously expressed, indicative of their diverse role in the tropism of tissues. Molecular structural analysis shows that each MFAP family member has distinct features, and functional evidence reveals discrete purposes of individual MFAPs. Animal studies indicate that MFAP2‐deficient mice exhibit progressive osteopenia with elevated receptor activator of NF‐κB ligand (RANKL) expression, whereas MFAP5‐deficient mice are neutropenic, and MFAP4‐deficient mice displayed emphysema‐like pathology and the impaired formation of neointimal hyperplasia. Emerging data also suggest that MFAPs are involved in cancer progression and fat metabolism. Further understanding of tissue‐specific pathophysiology of MFAPs might offer potential novel therapeutic targets for related diseases, such as skeletal and metabolic disorders, and cancers.
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In shotgun proteomics, the analysis of label-free quantification experiments is typically limited by the identification rate and the noise level in the quantitative data. This generally causes a low sensitivity in differential expression analysis. Here, we propose a quantification-first approach for peptides that reverses the classical identification-first workflow, thereby preventing valuable information from being discarded in the identification stage. Specifically, we introduce a method, Quandenser, that applies unsupervised clustering on both MS1 and MS2 level to summarize all analytes of interest without assigning identities. This reduces search time due to the data reduction. We can now employ open modification and de novo searches to identify analytes of interest that would have gone unnoticed in traditional pipelines. Quandenser+Triqler outperforms the state-of-the-art method MaxQuant+Perseus, consistently reporting more differentially abundant proteins for all tested datasets. Software is available for all major operating systems at https://github.com/statisticalbiotechnology/quandenser, under Apache 2.0 license.
Chapter
Targeted proteomics represents an efficient method to quantify proteins of interest with high sensitivity and accuracy. Targeted approaches were first established for triple quadrupole instruments, but the emergence of hybrid instruments allowing for high-resolution and accurate-mass measurements of MS/MS fragment ions enabled the development of parallel reaction monitoring (PRM). In PRM analysis, specific peptides are measured as representatives of proteins in complex samples, with the full product ion spectra being acquired, allowing for identification and quantification of the peptides. Ideally, corresponding stable isotope-labeled peptides are spiked into the analyzed samples to account for technical variation and enhance the precision. Here, we describe the development of a PRM assay including the selection of appropriate peptides that fulfill the criteria to serve as unique surrogates of the targeted proteins. We depict the sequential steps of method development and the generation of calibration curves. Furthermore, we present the open-access tool CalibraCurve for the determination of the linear concentration ranges and limits of quantification (LOQ).
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ProteomicsDB (https://www.ProteomicsDB.org) is a multi-omics and multi-organism resource for life science research. In this update, we present our efforts to continuously develop and expand ProteomicsDB. The major focus over the last two years was improving the findability, accessibility, interoperability and reusability (FAIR) of the data as well as its implementation. For this purpose, we release a new application programming interface (API) that provides systematic access to essentially all data in ProteomicsDB. Second, we release a new open-source user interface (UI) and show the advantages the scientific community gains from such software. With the new interface, two new visualizations of protein primary, secondary and tertiary structure as well an updated spectrum viewer were added. Furthermore, we integrated ProteomicsDB with our deep-neural-network Prosit that can predict the fragmentation characteristics and retention time of peptides. The result is an automatic processing pipeline that can be used to reevaluate database search engine results stored in ProteomicsDB. In addition, we extended the data content with experiments investigating different human biology as well as a newly supported organism.
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Corona is a layer of macromolecules formed on nanoparticle surface in vivo. It can substantially change the biological identity of nanomaterials and possibly trigger adverse responses from the body tissue. Dissecting the role of corona in the development of a particular disease may provide profound insights for understanding toxicity of nanomaterials in general. In our present study, we explored the capability of different silica nanoparticles (SiNP) in inducing silicosis in the mice lung, and analyzed the composition of coronas formed on these particles. We found that SiNP of certain size and surface chemistry could specifically recruit transforming growth factor beta 1 (TGF-β1) into their corona, which subsequently induce the development of lung fibrosis. Once embedded into corona on SiNP, TGF-β1 was remarkably more stable than in its free form; and its fibrosis-triggering activity was significantly prolonged. Our study meaningfully demonstrates that a specific corona component on a certain nanoparticle could initiate a particular pathogenic process in a clinically-relevant disease model. Our findings may shed lights on the understanding of molecular mechanisms of human health risks correlated with exposure to small-scale substances.
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Liver fibrosis is the excessive accumulation of extracellular matrix proteins including collagen that occurs in most types of chronic liver diseases. Advanced liver fibrosis results in cirrhosis, liver failure, and portal hypertension and often requires liver transplantation. Our knowledge of the cellular and molecular mechanisms of liver fibrosis has greatly advanced. Activated hepatic stellate cells, portal fibroblasts, and myofibroblasts of bone marrow origin have been identified as major collagen-producing cells in the injured liver. These cells are activated by fibrogenic cytokines such as TGF-β1, angiotensin II, and leptin. Reversibility of advanced liver fibrosis in patients has been recently documented, which has stimulated researchers to develop antifibrotic drugs. Emerging antifibrotic therapies are aimed at inhibiting the accumulation of fibrogenic cells and/or preventing the deposition of extracellular matrix proteins. Although many therapeutic interventions are effective in experimental models of liver fibrosis, their efficacy and safety in humans is unknown. This review summarizes recent progress in the study of the pathogenesis and diagnosis of liver fibrosis and discusses current antifibrotic strategies.
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Background: Chronic obstructive pulmonary disease (COPD) is a progressive, incurable lung disease characterised by abnormal tissue repair causing emphysema and small airways fibrosis. Since current therapy cannot modify this abnormal repair, it is crucial to unravel its underlying molecular mechanisms. Unbiased analysis of genome-wide gene expression profiles in lung tissue provides a powerful tool to investigate this. Methods: We performed genome-wide gene expression profiling in 581 lung tissue samples from current and ex-smokers with (n=311) and without COPD (n=270). Subsequently, quantitative PCR, western blot and immunohistochemical analyses were performed to validate our main findings. Results: 112 genes were found to be upregulated in patients with COPD compared with controls, whereas 61 genes were downregulated. Among the most upregulated genes were fibulin-5 (FBLN5), elastin (ELN), latent transforming growth factor β binding protein 2 (LTBP2) and microfibrillar associated protein 4 (MFAP4), all implicated in elastogenesis. Our gene expression findings were validated at mRNA and protein level. We demonstrated higher ELN gene expression in COPD lung tissue and similar trends for FBLN5 and MFAP4, and negative correlations with lung function. FBLN5 protein levels were increased in COPD lung tissue and cleaved, possibly non-functional FBLN5 protein was present. Strong coexpression of FBLN5, ELN, LTBP2 and MFAP4 in lung tissue and in silico analysis indicated cofunctionality of these genes. Finally, colocalisation of FBLN5, MFAP4 and LTBP2 with elastic fibres was demonstrated in lung tissue. Conclusions: We identified a clear gene signature for elastogenesis in COPD and propose FBLN5 as a novel player in tissue repair in COPD.
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Unlabelled: The cost-effectiveness of noninvasive tests (NITs) as alternatives to liver biopsy is unknown. We compared the cost-effectiveness of using NITs to inform treatment decisions in adult patients with chronic hepatitis C (CHC). We conducted a systematic review and meta-analysis to calculate the diagnostic accuracy of various NITs using a bivariate random-effects model. We constructed a probabilistic decision analytical model to estimate health care costs and outcomes (quality-adjusted life-years; QALYs) using data from the meta-analysis, literature, and national UK data. We compared the cost-effectiveness of four treatment strategies: testing with NITs and treating patients with fibrosis stage≥F2; testing with liver biopsy and treating patients with ≥F2; treat none; and treat all irrespective of fibrosis. We compared all NITs and tested the cost-effectiveness using current triple therapy with boceprevir or telaprevir, but also modeled new, more-potent antivirals. Treating all patients without any previous NIT was the most effective strategy and had an incremental cost-effectiveness ratio (ICER) of £9,204 per additional QALY gained. The exploratory analysis of currently licensed sofosbuvir treatment regimens found that treat all was cost-effective, compared to using an NIT to decide on treatment, with an ICER of £16,028 per QALY gained. The exploratory analysis to assess the possible effect on results of new treatments, found that if SVR rates increased to >90% for genotypes 1-4, the incremental treatment cost threshold for the "treat all" strategy to remain the most cost-effective strategy would be £37,500. Above this threshold, the most cost-effective option would be noninvasive testing with magnetic resonance elastography (ICER=£9,189). Conclusions: Treating all adult patients with CHC, irrespective of fibrosis stage, is the most cost-effective strategy with currently available drugs in developed countries.
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Microfibrillar-associated protein 4 (MFAP4) is located in the extracellular matrix (ECM). We sought to identify tissues with high levels of MFAP4 mRNA and MFAP4 protein expression. Moreover, we aimed to evaluate the significance of MFAP4 as a marker of cardiovascular disease (CVD) and to correlate MFAP4 with other known ECM markers, such as fibulin-1, osteoprotegerin (OPG), and osteopontin (OPN). Quantitative real-time PCR demonstrated that MFAP4 mRNA was more highly expressed in the heart, lung, and intestine than in other elastic tissues. Immunohistochemical studies demonstrated high levels of MFAP4 protein mainly at sites rich in elastic fibers and within blood vessels in all tissues investigated. The AlphaLISA technique was used to determine serum MFAP4 levels in a clinical cohort of 172 patients consisting of 5 matched groups with varying degrees of CVD: 1: patients with ST elevation myocardial infarction (STEMI), 2: patients with non-STEMI, 3: patients destined for vascular surgery because of various atherosclerotic diseases (stable atherosclerotic disease), 4: apparently healthy individuals with documented coronary artery calcification (CAC-positive), and 5: apparently healthy individuals without signs of coronary artery calcification (CAC-negative). Serum MFAP4 levels were significantly lower in patients with stable atherosclerotic disease than CAC-negative individuals (p
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The essential role of mechanical signals in regulating the function of living cells is universally observed. However, how mechanical signals are transduced in cells to regulate gene expression is largely unknown. We previously demonstrated that the gene encoding h2-calponin (Cnn2) is sensitively regulated by mechanical tension. In the present study, mouse genomic DNA containing the Cnn2 promoter was cloned, and a nested set of 5′ truncations was studied. Transcriptional activity of the Cnn2 promoter-reporter constructs was examined in transfected NIH/3T3, HEK293, and C2C12 cells for their responses to the stiffness of culture substrate. The results showed significant transcriptional activities of the −1.00- and −1.24-kb promoter constructs, whereas the −0.61-kb construct was inactive. The −1.38-, −1.57-, and −2.12-kb constructs showed higher transcriptional activity, whereas only the −1.57- and −2.12-kb constructs exhibited repression of expression when the host cells were cultured on low stiffness substrate. Internal deletion of the segment between −1.57 and −1.38 kb in the −2.12-kb promoter construct abolished the low substrate stiffness-induced repression. Site-specific deletion or mutation of an HES-1 transcription factor binding site in this region also abolished this repression effect. The level of HES-1 increased in cells cultured under a low tension condition, corresponding to the down-regulation of h2-calponin. h2-Calponin gene expression is further affected by the treatment of cells with Notch inhibitor and activator, suggesting an upstream signaling mechanism.
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Multi-OMICS approaches aim on the integration of quantitative data obtained for different biological molecules in order to understand their interrelation and the functioning of larger systems. This paper deals with several data integration and data processing issues that frequently occur within this context. To this end, the data processing workflow within the PROFILE project is presented, a multi-OMICS project that aims on identification of novel biomarkers and the development of new therapeutic targets for seven important liver diseases. Furthermore, a software called CrossPlatformCommander is sketched, which facilitates several steps of the proposed workflow in a semi-automatic manner.
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Background Microfibrillar-associated protein 4 (MFAP4) is a matricellular glycoprotein that co-localises with elastic fibres and is highly expressed in the lungs. The aim of this study was to test the hypothesis that plasma MFAP4 (pMFAP4) reflects clinical outcomes in chronic obstructive pulmonary disease (COPD). Methods pMFAP4 was measured by an AlphaLISA immunoassay in stable COPD (n=69) at baseline and at follow-up until 24 months after inclusion and in acute exacerbations of COPD (AECOPD) (n=14) at baseline and until 6 months after inclusion. Results The majority of patients (89%) were in GOLD II and III. Multiple linear regressions showed positive associations between pMFAP4 and the Global initiative for Obstructive Lung Disease (GOLD) grade (p=0.01), modified Medical Research Council score (p<0.0001) and BODE index (p=0.04). Negative associations were found with 6-minute walking distance (p=0.04) and bronchodilator-induced reversibility (p=0.02). The pMFAP4 levels varied less than 25% between the baseline and a 3-month follow-up in 83% of the patients. The pMFAP4 levels appeared unaffected in the acute phase of severe AECOPD but rose to an increased stable level within one month after hospitalization. Conclusion Increased pMFAP4 was associated to the severity in COPD and has the potential to serve as a stable disease biomarker. This observation warrants confirmation in a larger longitudinal COPD population.
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Proteomic information about the hepatitis C virus (HCV) infection leads to a better understanding of the protein function and interaction in liver cirrhosis. HCV is a causative agent of chronic liver disease leading to cirrhosis, liver failure and hepatocellular carcinoma with anticipated prevalence of 3% of the world population. In order to examine the nuclear membrane proteins of infected liver tissues, liver biopsy sample were subjected to SDS-PAGE followed by 2-DE analysis with narrow pI ranges. The expressed proteins revealed interesting results which contributes to the evaluation and understanding of HCV infection leading to cirrhosis. To support our results and to provide a conclusive data, a comparative study is carried out to elucidate the differentially expressed proteins in serum of HCV infected individuals. These results might be useful for rapid translation of findings from basic research to practical means of anticipation, control and therapeutic advancement of liver diseases.
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An emerging approach for multiplexed targeted proteomics involves bottom-up LC-multiple-reaction monitoring (MRM)-MS, with stable isotope-labeled internal standard peptides, to accurately quantitate panels of putative disease biomarkers in biofluids. In this paper, we used this approach to quantitate 27 candidate cancer-biomarker proteins in human plasma that had not been treated by immunoaffinity depletion or enrichment techniques. These proteins have been reported as biomarkers for a variety of human cancers, from laryngeal to ovarian, with breast cancer having the highest correlation. We implemented measures to minimize the analytical variability, improve the quantitative accuracy, and increase the feasibility and applicability of this MRM-based method. We have demonstrated excellent retention time reproducibility (median inter-day CV: 0.08%) and signal stability (median inter-day CV: 4.5% for the analytical platform and 6.1% for the bottom-up workflow) for the 27 biomarker proteins (represented by 57 interference-free peptides). The linear dynamic range for the MRM assays spanned four orders-of-magnitude, with 25 assays covering a 10(3) -10(4) range in protein concentration. The lowest-abundance quantifiable protein in our biomarker panel was insulin-like growth factor 1 (calculated concentration: 127 ng/mL). Overall, the analytical performance of this assay demonstrates high robustness and sensitivity, and provides the necessary throughput and multiplexing capabilities required to verify and validate cancer-associated protein biomarker panels in human plasma, prior to clinical use.
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Cirrhosis of the liver is the end stage of chronic liver disease. Among the many liver disorders that can lead to cirrhosis, some progress rapidly (years) and others more slowly (decades). In Germany, cirrhosis is often a consequence of fatty liver disease due to alcoholism or other causes, but can also be caused by hepatitis B and hepatitis C. Cirrhosis is more common in overweight persons and smokers. The underlying causes of cirrhosis determine its rate of progression and are the focus of preventive efforts and treatment. The prevalence of cirrhosis in Germany is rising; it now ranks among the top 20 causes of death in the country. This article is based on a selective review of pertinent literature, including reviews and current guidelines. Strictly speaking, cirrhosis is a pathological diagnosis; it is, nevertheless, usually diagnosed clinically, by history, physical examination (e.g., cutaneous signs of liver disease), ancillary testing (e.g., ultrasonography, transient elastography) and laboratory analyses (e,g., APRI, which is the quotient of the GOT concentration and the platelet count). There are no laboratory cutoff values for the diagnosis of cirrhosis. Early detection of chronic liver disease, followed by individually tailored, risk-adapted treatment, is the best way to prevent it. Esophagogastroduodenoscopy can be performed early on to assess the risk of variceal bleeding. In most patients, the progression of fibrosis can be averted by early detection and appropriate treatment. Screening for chronic liver disease should include history and physical examination, serum transaminase me