Abstract
CCN-2 (connective tissue growth factor; CTGF) is a key factor in fibrosis. Plasma CCN-2 has biomarker potential in numerous fibrotic disorders, but it is unknown which pathophysiological factors determine plasma CCN-2 levels. The proteolytic amino-terminal fragment of CCN-2 is primarily eliminated by the kidney. Here, we investigated elimination and distribution profiles of full length CCN-2 by intravenous administration of recombinant CCN-2 to rodents. After bolus injection in mice, we observed a large initial distribution volume (454 mL/kg) and a fast initial clearance (120 mL/kg/min). Immunosorbent assay and immunostaining showed that CCN-2 distributed mainly to the liver and was taken up by hepatocytes. Steady state clearance in rats, determined by continuous infusion of CCN-2, was fast (45 mL/kg/min). Renal CCN-2 clearance, determined by arterial and renal vein sampling, accounted for only 12 % of total clearance. Co-infusion of CCN-2 with receptor-associated protein (RAP), an antagonist of LDL-receptor family proteins, showed that RAP prolonged CCN-2 half-life and completely prevented CCN-2 internalization by hepatocytes. This suggests that hepatic uptake of CCN-2 is mediated by a RAP-sensitive mechanism most likely involving LRP1, a member of the LDL-receptor family involved in hepatic clearance of various plasma proteins. Surface plasmon resonance binding studies confirmed that CCN-2 is an LRP1 ligand. Co-infusion of CCN-2 with an excess of the heparan sulphate-binding protamine lowered the large initial distribution volume of CCN-2 by 88 % and reduced interstitial staining of CCN-2, suggesting binding of CCN-2 to heparan sulphate proteoglycans (HSPGs). Protamine did not affect clearance rate, indicating that RAP-sensitive clearance of CCN-2 is HSPG independent. In conclusion, unlike its amino-terminal fragment which is cleared by the kidney, full length CCN-2 is primarily eliminated by the liver via a fast RAP-sensitive, probably LRP1-dependent pathway.
Highlights
CTGF/CYR61/NOV −2 (CCN-2, known as connective tissue growth factor or CTGF) is a key factor in the pathogenesis of organ fibrosis
The main finding of our studies is that circulating full length CCN-2 is primarily cleared by fast hepatic metabolism via a receptor-associated protein (RAP)-sensitive pathway, most likely involving lipoprotein receptor-related protein 1 (LRP1)
The half-life of CCN-2 was markedly prolonged and hepatic uptake substantially reduced after administration of RAP, which suggests that uptake in hepatocytes is mediated by an low density lipoprotein receptor (LDLR) family protein
Summary
CTGF/CYR61/NOV −2 (CCN-2, known as connective tissue growth factor or CTGF) is a key factor in the pathogenesis of organ fibrosis. Circulating CCN-2 correlates with fibrogenic activity in various chronic disorders and might be useful as a non-invasive marker for monitoring fibrosis, including liver fibrosis (Dendooven et al 2011; Gressner et al 2013; Guo-Qiu et al 2010; Bauer et al 2012; Honsawek et al 2013; Colak et al 2012). The N-fragment is the predominant CCN-2 molecule in plasma in healthy subjects and chronic kidney disease patients, while full length CCN-2 is hardly detectable by available techniques (Dendooven et al 2011). In chronic liver disease elevated circulating levels of the full length protein were reported (Gressner et al 2006; Guo-Qiu et al 2010). Sparse data are available on circulating C-fragment levels and reported levels were lower than those of the N-fragment and the full length molecule (Weitz and Usinger 2003; Dziadzio et al 2005)
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