TUNEL-positive cells per high-power field (200×) were counted. All measurements were performed blindly. Results are expressed as the mean ± standard error of the

mean. Significance was established using Student t test, two-way analysis of variance PD-1 inhibitor with Bonferroni’s post hoc test and Mann-Whitney assay. Differences were considered significant if P < 0.05. Other methods are shown in Supporting Materials and Methods. Losartan was conjugated to manose-6-phosphate coupled to human serum albumin (M6PHSA) (Fig. 1A). After its reaction to the linker at a stoichiometric ratio (Fig. 1B), the losartan-ULS adduct was conjugated to M6PHSA. An average of seven losartan-ULS molecules were coupled to M6PHSA, as assessed by HPLC and confirmed

by inductive coupled plasma-atomic emission spectroscopy (ICP-AES) (data not shown). Conjugation of losartan to M6PHSA did not change the charge or size features of M6PHSA, as assessed by anion-exchange chromatography and size exclusion chromatography, respectively (Fig. 1C,D). Because ULS is a derivative of cisplatin, an antitumor agent that may cause cell toxicity, we studied the effects of losartan-M6PHSA on cultured HSCs. Losartan-M6PHSA did not cause cell toxicity, this website while cisplatin induced cell death, suggesting that occupation of the coordinative sites of platinum with drug and carrier prevents its disruptive reactivity with cellular components (Fig. 1E). To test whether losartan-M6PHSA is biologically active in cultured HSCs, cells were stimulated with angiotensin II in the presence or absence of either free losartan or losartan-M6PHSA. We found that both treatments equally blunted angiotensin II–induced intracellular calcium increase (Fig. 1F). Also, we detected intracellular staining for HSA after incubating HSCs with losartan-M6PHSA for 10 minutes. Thiamine-diphosphate kinase This staining was strongly blunted by excess of M6P sugars and an antibody against the M6P/IGF II receptor. We found 25.2 ± 2.4, 0.2 ± 0.1, and 5.3

± 0.6 positive cells in cultures incubated with isotype-matched antibody, excess of M6P, and anti-IGFRII antibody, respectively (P < 0.001 of isotype-matched antibody respect to the other two conditions) (Fig. 2A). These results indicate that losartan-M6PHSA directly interacts with IGF II receptors present in HSCs, and is internalized to inhibit angiotensin II–induced biological actions. M6PHSA binds to M6P/IGFII-R, which is expressed in activated HSCs in the fibrotic liver.16 In the bile duct ligation model, we administered losartan-M6PHSA (3.3 mg/kg, corresponding to 125 μg losartan/kg) daily from day 12-14 and animals were sacrificed at day 15. For pharmacokinetic purposes, a subgroup of the animals received an additional dose of the conjugate at 10 minutes before sacrifice. Control groups were treated with equivalent doses of M6PHSA (3.