Rapid hepatic clearance of full length CCN-2/CTGF: a putative role for LRP1-mediated endocytosis

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.

Keywords: Biomarker; CCN-2; CTGF; Hepatic clearance; LRP1.

Fig. 1

Pharmacokinetic profile after single intravenous dose of recombinant human CCN-2 in mice. a Plasma disappearance curve of CCN-2. Plasma concentration expressed as percentage of injected dose per mL plasma. Each dot represents one animal. b-c Immunofluorescence of liver b and kidney c, 15 min after CCN-2 administration, by using human anti- CCN-2 (green), visualized by fluorescence microscopy. d Estimated organ levels expressed as percentage of injected dose of recombinant human CCN-2 5–10 min after intravenous CCN-2 administration in mice (n = 8). e Immunofluorescence of liver by using human anti- CCN-2 (green) and TO-PRO 3 Iodide for nuclear staining (red), visualized by confocal laser scanning microscopy

Fig. 2

a-b Pharmacokinetic profile after single intravenous dose of recombinant human CCN-2 preceded by injection of an excess of GST-RAP in mice. a Plasma disappearance curve of CCN-2. GST-RAP prior to CCN-2 reduced the elimination rate but not initial distribution. Plasma concentration is expressed as percentage of injected dose per mL plasma. Each dot represents one animal. b Immunofluorescence of liver 15 min after CCN-2 administration, by using human anti- CCN-2 (green), visualized by confocal laser scanning microscopy. GST-RAP prior to CCN-2 (right) reduces uptake in hepatocytes. c-e Surface plasmon resonance analysis of human CCN-2 and its proteolytic fragments, run over immobilized LRP1. Response is depicted as pg/mm². c Dose-dependent response with concentrations of full length CCN-2 of 0, 6.25, 12.5, 25, 50, 100 and 200 nM. d Interaction of CCN-2 (1 uM) (I) with LRP1 is partially inhibited by GST-RAP (3 uM) (II). e Full length CCN-2 (200 nM) (I) and C-terminal fragment (200 nM) (II), but not N-terminal fragment (200 nM) (III), show interaction with LRP1

Fig. 3

a-b Pharmacokinetic profile after single intravenous dose of recombinant human CCN-2 preceded by injection of an excess of protamine in mice. a Plasma disappearance curve of CCN-2. Administration of protamine prior to CCN-2 reduced distribution volume but not elimination rate. Plasma concentration is expressed as percentage of injected dose per mL plasma. Each dot represents one animal. b Immunofluorescence of kidney and liver, 5 min after CCN-2 administration, by using human anti- CCN-2 (green), visualized by confocal laser scanning microscopy. Protamine prior to CCN-2 (right) reduces interstitial staining