Procollagen type I N-terminal propeptide Karsdal, Morten Asser; Vassiliadis, Efstathios; Vainer, Ben ...
Fibrogenesis & tissue repair,
04/2010, Volume:
3
Journal Article
Peer reviewed
Background Fibrosis can be described as the excess deposition of extracellular matrix (ECM) components, such as collagens and proteoglycans. Fibrosis of the liver, which eventually leads to ...cirrhosis, is a major global health problem. Being able to measure fibrosis progression may enable timely preventative intervention. The aim of the current study was to investigate the utility of serum procollagen type I N-terminal propeptide (PINP) as a marker of hepatic fibrosis, as distinct from bone formation, during three different periods of fibrosis development following hepatic injury induced by bile duct ligation (BDL) in rats. Methods BDL was performed on 30 female Sprague-Dawley rats aged 6 months, and sham operations on 30 controls. Animals were killed after 14, 28, or 35 days. The extent of liver fibrosis was evaluated by quantitative histology after Sirus Red staining. Levels of serum PINP and osteocalcin (a marker solely for osteoblastic bone formation) were determined using ELISA at baseline and post termination. Results Collagen formation increased by 30% compared to 3% in sham-operated animals (P less than 0.0001). PINP levels increased significantly in all BDL groups compared with baseline (14 days: baseline 13.9 ng/ml, termination 17.7 ng/ml, P = 0.047; 28 days: baseline 17.9 ng/ml, termination 26.2 ng/ml, P = 0.005; 35 days: baseline 18.0 ng/ml, termination 27.4 ng/ml P = 0.015, an increase of 52%). PINP levels did not change from baseline in the sham-operated rats, indicating that the increased PINP levels were due to hepatic injury. The bone-specific marker, osteocalcin, did not increase in either BDL or sham-operated rats. PINP measured in serum correlated to the extent of liver fibrosis as evaluated by quantitative histology (R.sup.2 .sup.= 0.42, P less than 0.001). Conclusion PINP was associated with the development of liver fibrosis, but not bone formation, in mature rats subjected to BDL. Thus, PINP may be useful in studying the pathogenesis of liver fibrosis. However, caution should be applied when interpreting PINP levels in other disease states.
Salmon calcitonin (sCT) and human calcitonin (hCT) are pharmacologically distinct. However, the reason for the differences is unclear. Here we analyze the differences between sCT and hCT on the human ...calcitonin receptor (CT(a)R) with respect to activation of cAMP signaling, beta -arrestin recruitment, ligand binding kinetics and internalization. The study was conducted using mammalian cell lines heterologously expressing the human CT(a) receptor. CT(a)R downstream signaling was investigated with dose response profiles for cAMP production and beta -arrestin recruitment for sCT and hCT during short term (<2 hours) and prolonged (up to 72 hours) stimulation. CT(a)R kinetics and internalization was investigated with radio-labeled sCT and hCT ligands on cultured cells and isolated membrane preparations from the same cell line. We found that sCT and hCT are equipotent during short-term stimulations with differences manifesting themselves only during long-term stimulation with sCT inducing a prolonged activation up to 72 hours, while hCT loses activity markedly earlier. The prolonged sCT stimulation of both cAMP accumulation and beta -arrestin recruitment was attenuated, but not abrogated by acid wash, suggesting a role for sCT activated internalized receptors. We have demonstrated a novel phenomenon, namely that two distinct CT(a)R downstream signaling activation patterns are activated by two related ligands, thereby highlighting qualitatively different signaling responses in vitro that could have implications for sCT use in vivo.
Objective
Calcitonin has been suggested to have chondroprotective effects. One signaling pathway of calcitonin is via the second messenger cAMP. We undertook this study to investigate whether ...increased cAMP levels in chondrocytes would be chondroprotective.
Methods
Cartilage degradation was induced in bovine articular cartilage explants by 10 ng/ml oncostatin M (OSM) and 20 ng/ml tumor necrosis factor (TNF). In these cultures, cAMP levels were augmented by treatment with either forskolin (4, 16, or 64 μM) or 3‐isobutyl‐1‐methyl xanthine (IBMX; 4, 16, or 64 μM). Cartilage degradation was assessed by 1) quantification of C‐terminal crosslinking telopeptide of type II collagen fragments (CTX‐II), 2) matrix metalloproteinase (MMP)–mediated aggrecan degradation by 342FFGV‐ G2 assay, 3) aggrecanase‐mediated degradation by 374ARGS‐G2 assay, 4) release of sulfated glycosaminoglycans (sGAG) into culture medium, 5) immunohistochemistry with a monoclonal antibody recognizing the CTX‐II epitope, and 6) toluidine blue staining of proteoglycans. MMP expression and activity were assessed by gelatin zymography.
Results
OSM and TNF induced an 8,000% increase in CTX‐II compared with control (P < 0.001). Both forskolin and IBMX dose‐dependently inhibited release of CTX‐II (P < 0.001). OSM and TNF induced a 6‐fold increase in 342FFGV‐G2, which was abrogated by forskolin and IBMX (by >80%). OSM and TNF stimulated MMP expression as visualized by zymography, and MMP expression was dose‐dependently inhibited by forskolin and IBMX. The highest concentration of IBMX lowered cytokine‐induced release of sGAG by 72%.
Conclusion
Levels of cAMP in chondrocytes play a key role in controlling catabolic activity. Increased cAMP levels in chondrocytes inhibited MMP expression and activity and consequently strongly inhibited cartilage degradation. Specific cAMP modulators in chondrocytes may be potential treatments for cartilage degenerative diseases.
Endoscopy and the use of faecal calprotectin faecal CP are among the least-favoured methods for assessing disease activity by inflammatory bowel disease IBD patients; the handling/processing of ...faecal samples is also impractical. Therefore, we sought to develop a novel neo-epitope serum calprotectin enzyme-linked immunosorbent assay ELISA, CPa9-HNE, with the aim of quantifying neutrophil activity and neutrophil extracellular trap NET-osis and proposing a non-invasive method for monitoring disease activity in IBD patients.BACKGROUND AND AIMSEndoscopy and the use of faecal calprotectin faecal CP are among the least-favoured methods for assessing disease activity by inflammatory bowel disease IBD patients; the handling/processing of faecal samples is also impractical. Therefore, we sought to develop a novel neo-epitope serum calprotectin enzyme-linked immunosorbent assay ELISA, CPa9-HNE, with the aim of quantifying neutrophil activity and neutrophil extracellular trap NET-osis and proposing a non-invasive method for monitoring disease activity in IBD patients.In vitro cleavage was performed by mixing calprotectin S100A9/S100A8 with human neutrophil elastase HNE, and a novel HNE-derived calprotectin neo-epitope CPa9-HNE was identified by mass spectrometry for ELISA development. The CPa9-HNE ELISA was quantified in supernatants from ex vivo activated neutrophils and serum samples from patients with ulcerative colitis UC, n = 43, Crohn's disease CD, n = 93, and healthy subjects HS, n = 23. For comparison, faecal CP and MRP8/14 biomarkers were also measured.METHODSIn vitro cleavage was performed by mixing calprotectin S100A9/S100A8 with human neutrophil elastase HNE, and a novel HNE-derived calprotectin neo-epitope CPa9-HNE was identified by mass spectrometry for ELISA development. The CPa9-HNE ELISA was quantified in supernatants from ex vivo activated neutrophils and serum samples from patients with ulcerative colitis UC, n = 43, Crohn's disease CD, n = 93, and healthy subjects HS, n = 23. For comparison, faecal CP and MRP8/14 biomarkers were also measured.CPa9-HNE was specific for activated neutrophils ex vivo. Serum CPa9-HNE levels were 4-fold higher in CD p <0.0001 and UC p <0.0001 patients than in HS. CPa9-HNE correlated well with the Simple Endoscopic Score SES-CD score r = 0.61, p <0.0001, MES r = 0.46, p = 0.0141, and the full Mayo score r = 0.52, p = 0.0013. CPa9-HNE was able to differentiate between CD and UC patients in endoscopic remission and moderate/severe disease activity (CD: area under the curve AUC = 0.82 p = 0.0003, UC: AUC = 0.87 p = 0.0004). The performance of CPa9-HNE was equipotent or slightly better than that of faecal CP.RESULTSCPa9-HNE was specific for activated neutrophils ex vivo. Serum CPa9-HNE levels were 4-fold higher in CD p <0.0001 and UC p <0.0001 patients than in HS. CPa9-HNE correlated well with the Simple Endoscopic Score SES-CD score r = 0.61, p <0.0001, MES r = 0.46, p = 0.0141, and the full Mayo score r = 0.52, p = 0.0013. CPa9-HNE was able to differentiate between CD and UC patients in endoscopic remission and moderate/severe disease activity (CD: area under the curve AUC = 0.82 p = 0.0003, UC: AUC = 0.87 p = 0.0004). The performance of CPa9-HNE was equipotent or slightly better than that of faecal CP.Serum CPa9-HNE levels were highly associated with CD and UC patients. CPa9-HNE correlated with the SES-CD score and the full Mayo score, indicating a strong association with disease activity.CONCLUSIONSSerum CPa9-HNE levels were highly associated with CD and UC patients. CPa9-HNE correlated with the SES-CD score and the full Mayo score, indicating a strong association with disease activity.
Objective
To investigate whether oral calcitonin treatment influences the increases in type II collagen (CII) degradation and related surface erosions of articular cartilage in ovariectomized rats.
...Methods
Fifty rats were randomly allocated into 1 of the 5 following intervention groups: sham‐operated, ovariectomy, ovariectomy plus subcutaneously implanted 17β‐estradiol pellet, ovariectomy plus 2 mg/kg salmon calcitonin plus 50 mg/kg 5CNAC (carrier), or ovariectomy plus 50 mg/kg 5CNAC. Each treatment was administered for 9 weeks after the ovariectomy. Blood samples for biochemical marker analysis were collected from fasting animals at baseline, on day 3, and after 1, 2, 4, 6, and 9 weeks. CII degradation was quantified by specific immunoassay, and the changes in severity scores of articular cartilage erosions were visualized and scored in histologic sections of the knees.
Results
Ovariectomy resulted in a marked increase in serum levels of C‐telopeptide of type II collagen (CTX‐II) (P < 0.001), which could be effectively reversed by 17β‐estradiol supplementation. Oral administration of calcitonin elicited similar decreases in serum levels of CTX‐II (P < 0.001). Histologic scoring of cartilage erosion showed significantly less cartilage erosion in calcitonin‐treated ovariectomized rats versus control ovariectomized rats that were untreated or treated with 5CNAC alone. (P < 0.01).
Conclusion
The in vivo effects of calcitonin in rats suggest that calcitonin is able to counteract CII degradation and the accompanying structural disintegration of articular cartilage promoted by estrogen deficiency. Clinical assessment of the chondroprotective potential of calcitonin in postmenopausal women seems warranted.
PDF
