18 In the present study we further evaluated hepatic integrin αvβ3 expression and identified the cell type that expressed majority of integrin αvβ3 at different stages of liver fibrosis in rats, then used radiolabeled cRGD as a SPECT radiotracer to image hepatic integrin αvβ3 expression in order to develop a noninvasive approach to monitor HSC activity during fibrotic progression. aHSCs, activated hepatic stellate cells; α-SMA, α-smooth muscle actin; BDL, bile duct ligation; CCl4, carbon tetrachloride; cRGD, cyclic arginine-glycine-aspartic acid peptides; ECM, extracellular matrix; FAM, carboxyfluorescein; HSCs, hepatic stellate cells;
125I, iodine-125; 125I-cRGD, 125I-labeled cRGD; LB, liver biopsy; MRAR, the mean radioactivity Daporinad mw ratio of liver to heart; qHSCs, quiescent hepatic stellate cells; TAA, thioacetamide; 99mTc, technetium-99m; 99mTc-cRGD, 99mTc-labeled cRGD; SPECT, single photon emission computed tomography. Eight-week-old DNA/RNA Synthesis inhibitor inbred male Sprague-Dawley rats (body weight 200 ± 20 g) were obtained from the Laboratory Animal Research Center of Fudan University (Shanghai, China) and fed standard laboratory rat chow on a 12-hour light/dark cycle with free access to water and food.
The study was approved by the Institutional Ethical Committee of Animal Experimentation and all experiments were performed strictly according to governmental and international guidelines on animal experimentation. cRGD (cyclo[Arg-Gly-Asp-D-Phe-Lys], cRGDfK) was synthesized at the Fudan-Pharmaco Targeting Drug Research Center, Fudan University (Shanghai, China) as described.19 The synthesis of carboxyfluorescein-labeled cRGD (FAM-cRGD) was carried out by mixing the resin containing the cyclic peptides with preactivated 5-FAM (Sigma-Aldrich, Hong Kong, China) for 3 hours before cleaving. cRGD was labeled with iodine-125 (125I) by dissolving
the peptide (20 μg) into [125I]NaI (37 MBq) (PerkinElmer, Hong Kong, China) in a 1.5-mL polypropylene vial coated with 100 μg of iodogen as described.20 To label cRGD with technetium-99m (99mTc), 20 μg of cRGD was added to 1.0 mL of Na[99mTcO4] solution (10 Methane monooxygenase μCi) (Shenke Medicinal, Beijing, China) as described.14 Analytical reverse-phase high-performance liquid chromatography (RP-HPLC) was performed to examine the purity of cRGD and its derivatives. Electrospray ionization mass spectrometry (ESI-MS) analysis was conducted to examine the molecular weight of final products. HSCs and hepatocytes (HCs) were isolated from rats (450-550 g) by two steps of collagenase digestion.21 Primary rat HSCs cultured for 3 or 7 days after isolation (referred to as day-3 or day-7 HSCs) and primary HC cultured for 24 hours after isolation were used for further experiments. Human umbilical vein endothelial cells (provided by Chinese Academy of Sciences Shanghai Branch) were used as a control.