Evaluation of hepatic integrin αvβ3 expression in non-alcoholic steatohepatitis (NASH) model mouse by 18F-FPP-RGD2 PET

Evaluation of hepatic integrin αvβ3 expression in non-alcoholic steatohepatitis (NASH) model... Background: Activated hepatic stellate cells (HSCs), which express integrin αvβ3, are a major fibrogenic factor in 18 18 NASH pathophysiology. F-labeled cyclic arginine-glycine-aspartic acid penta-peptide ( F-FPP-RGD ) has been used as a PET probe for tumors expressing integrin αvβ3. The aim of this study was to assess the potential of PET with F-FPP-RGD to detect hepatic integrin αvβ3 expression in non-alcoholic steatohepatitis (NASH) model mice. Results: Thirty-two male C57BL/6 mice aged 6 weeks were fed a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) for 3 and 8 weeks. F-FPP-RGD PET imaging of the liver was performed at 3 and 8 weeks after CDAHFD feeding. After PET scanning, levels of hepatic integrin αvβ,3α-smooth muscle actin (α-SMA), and collagen type 1 alpha 1(col1a1) were measured. Histopathological analysis of hepatic steatosis, inflammation, and fibrosis, as well as blood biochemistry analysis, was also performed. CDAHFD for 3 and 8 weeks produced a moderate-to-severe steatosis and inflammation of the liver in mice. NAFLD activity score (NAS) in mice fed the CDAHFD for 3 and 8 weeks were more than 4 indicating NASH or borderline NASH pathology. Fibrosis was observed only in mice fed the CDAHFD for 8 weeks. PET imaging showed that the hepatic standardized uptake value, SUV , was increased with prolonged 80–90 min CDAHFD feeding compared with the respective controls (CDAHFD 3 weeks 0.32 ± 0.06 vs 0.48 ± 0.05, p < 0.01; CDAHFD 8 weeks 0.35 ± 0.04 vs 0.75 ± 0.07, p < 0.01, respectively). Prolonged CDAHFD feeding increased hepatic mRNA and protein levels of integrin αvand β3 at 3 and 8 weeks. Hepatic F-FPP-RGD uptake and amount of integrin αvand β3 protein were well correlated (r =0.593, p <0.05 and r =0.835, p < 0.001, respectively). Hepatic F-FPP-RGD uptake also showed a positive correlation with Sirius red-positive area. Conclusions: The hepatic uptake of F-FPP-RGD correlated well with integrin αvand β3 expression and histological fibrosis in a mouse model of NASH, suggesting the predictability of fibrosis in NASH pathology. Keywords: Non-alcoholic fatty liver disease, Non-alcoholic steatohepatitis, Fibrosis, Positron emission tomography, F-FPP-RGD , Modified methionine choline-deficient, High-fat diet, Integrin αvβ3 Background progress to fibrosis and about 10% progress to cir- Non-alcoholic fatty liver disease (NAFLD), one of the rhosis [2]. The prognosis of NAFLD depends on the most common forms of chronic liver disease in pa- histological severity, particularly of liver fibrosis, tients without a history of alcoholic abuse, encom- which is the strongest predictor of liver morbidity passes a wide spectrum of conditions from simple and mortality [3]. To prevent liver-related mortality, steatosis to non-alcoholic steatohepatitis (NASH) [1]. it is important to reverse advanced fibrosis or prevent It was reported that 30–40% of NASH patients the progression to fibrosis in NASH patients. From a clinical view point, liver biopsy is the gold standard for the diagnosis of NASH and staging liver * Correspondence: takemi.rokugawa@shionogi.co.jp fibrosis [4]. However, liver biopsy has limitations in- Translational Research Unit, Biomarker R&D Department, Shionogi & Co., cluding sampling error and it is invasive, painful and Ltd., 3-1-1, Futaba-cho, Toyonaka, Osaka 561-0825, Japan Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Rokugawa et al. EJNMMI Research (2018) 8:40 Page 2 of 9 results in poor patient compliance [5]. Therefore, the [21]. Therefore, RGD imaging might be a useful pre- development of a non-invasive strategy to evaluate dictor for the onset of fibrosis in NASH. liver fibrosis is required. Magnetic resonance elasto- In the present study, we investigated the relationship be- graphy and ultrasound-based transient elastography tween the hepatic uptake of F-FPP-RGD and integrin have been developed to assess liver fibrosis [6, 7]. αvβ3 expression using PET imaging in a NASH mouse These methods can discriminate moderate and ad- model induced by feeding with a choline-deficient, vanced liver fibrosis from early-stage liver injury or L-amino acid-defined, high-fat diet (CDAHFD) [22]. the normal patient population. However, these ap- proaches have reported a lower accuracy for the de- Methods tection of early stage liver fibrosis [8]. Furthermore, Animals and experimental design especially in NASH patients, steatosis may produce a Male C57BL/6J mice, aged 6 weeks old, were purchased softer liver because of fat deposition in the liver par- from CLEA Japan (Shizuoka, Japan). Mice were given enchyma [9, 10]. These non-invasive tools are not free access to water and either a normal diet or likely to be sensitive enough to identify mild changes CDAHFD, which contained 0.1% methionine, no choline or early stages of fibrosis. Preclinical studies recently and 60 kcal% fat, prepared by Research Diets (New reported the imaging of collagen or elastin probes for Brunswick, NJ, USA), for 3 and 8 weeks. They were thereliableassessmentof fibrosis [11, 12]. Thus, the housed in a temperature-controlled room maintained on development of sensitive imaging markers of fibrogen- a 12 h light/dark cycle with lights on at 7:00 am. The ex- esis is important to predict the prognosis and deter- perimental protocols were reviewed and approved by the mine the precise therapeutic intervention required. Institutional Animal Care and Use Committee of Osaka Activated hepatic stellate cells, myofibroblasts, cholan- University Graduate School of Medicine. giocytes, macrophages, and components of the patho- logical extracellular matrix are major fibrogenic factors. Activation and proliferation of hepatic stellate cells (HSCs) are considered key factors of hepatic fibrosis Biochemical and histopathological analysis [13]. Following HSC activation, they transform to Mice were euthanized by exsanguination under isoflur- myofibroblast-like cells characterized by α-smooth ane anesthesia. Plasma (200 μL) was collected and muscle actin (α-SMA) expression and produce excessive assayed for the content of alanine aminotransferase amounts of extra cellular matrix proteins such as type 1 (ALT), aspartate aminotransferase (AST), triglyceride and type 3 collagen [13]. With the activation of HSCs, (TG), total cholesterol (TC), and high-density lipopro- integrin αvβ3, which plays an important role in cell sig- tein cholesterol (HDLC). The left hepatic lobes were naling such as cell-to-cell adhesion, apoptosis, and fixed in 10% formalin and sectioned, and 4-μm sections cell-matrix interactions, is expressed on HSCs [14]. were stained with hematoxylin and eosin (H&E) and Sir- Thus, monitoring the expression of integrin αvβ3in ius red. Steatosis, inflammation, ballooning, and fibrosis NASH liver might be used as a marker to predict the in the liver were assessed based on the severity and size onset of fibrosis. of the lesion. Steatosis and inflammation scores ranged The three-amino-acid sequence of arginine-gly from 0 to 3: normal = 0; minimal = 1; moderate = 2; cine-aspartic acid (RGD) has a high binding affinity to marked = 3. Ballooning score ranged from 0 to 2: nor- integrin αvβ3[15, 16]. Many studies reported that mal = 0; minimal = 1; marked = 2. NAFLD activity score radiolabeled cyclic RGD peptides (cRGD) imaged by (NAS) was calculated by using the sum of each histo- positron emission tomography (PET) and single pho- logical score. To assess hepatic fibrosis, five different ton emission computed tomography (SPECT) have areas (× 200 magnification) were selected per mouse, been developed as a new radio tracer for selective in- and Sirius red-positive areas were measured using Win- tegrin αvβ3 positive tumors [17, 18]. In a recent fluor- ROOF software (Mitsutani, Tokyo, Japan). escence trace study, cRGD was accumulated in activated but not quiescent HSCs [19]. Furthermore, Radiopharmaceutical preparation hepatic integrin αvβ3 imaging by SPECT using I 99m F-FPP-RGD was radio synthesized using a two-step and Tc-labeled cRGD and magnetic resonance im- method as reported previously [23]. Conjugation be- aging using cRGD labeled by contrast agent USPIO 18 18 tween F-4-nitrophenyl-2-fluoropropionate ( F-NFP) detected rodent hepatic fibrosis induced by thioaceta- and the RGD dimeric peptide (PEG -c[RGDyK] )was mide or CCl treatment [19, 20]. These hepatic fibrosis 3 2 performed. Radiochemical purity and specific activity models develop fibrosis more quickly than common were > 99% and 445.6 ± 107.6 GBq/μmol, respectively, NASH models. In NASH pathology, HSCs are acti- with a radiosynthesis and purification time of 90 min. vated before or at the early stage of hepatic fibrosis Rokugawa et al. EJNMMI Research (2018) 8:40 Page 3 of 9 Table 1 Body, liver weight, and blood parameters in mice fed a CDAHFD Parameter Control 3 weeks CDAHFD 3 weeks Control 8 weeks CDAHFD 8 weeks Body weight (g) 24.44 ± 0.91 20.81 ± 0.96** 26.41 ± 1.56 21.66 ± 2.23** Liver weight (g) 1.11 ± 0.11 1.47 ± 0.17** 1.12 ± 0.13 1.69 ± 0.24** AST (IU/L) 32.80 ± 3.83 436.6 ± 76.35** 41.20 ± 12.69 402.70 ± 242.78** ALT (IU/L) 19.20 ± 3.45 772.10 ± 128.63** 31.90 ± 16.52 584.6 ± 475.74** TG (mg/dL) 155.7 ± 48.57 41.50 ± 17.69** 146.80 ± 65.67 21.50 ± 3.54** TC (mg/dL) 80.40 ± 7.91 49.00 ± 6.95** 79.70 ± 9.50 36.3 ± 4.14** Statistical differences were assessed using Steel-Dwass test AST aspartate transaminase, ALT alanine aminotransferase, TC total cholesterol, TG, triglyceride **p < 0.01 compared with respective control mice PET imaging PET images were automatically fused and analyzed by PET scan and X-ray CT imaging were performed with a PMOD v3.6 (PMOD Technologies Ltd., Zürich, Pre-Clinical Imaging System Triumph LbPET12/CT Switzerland). To calculate hepatic SUV 100 mm elliptic (TriFoil Imaging Inc., Chatsworth, CA, USA). Mice fed a two regions-of-interest (ROI) were chosen excluding the CDAHFD or control diet for 3 or 8 weeks were anesthe- aorta on the liver tissue were analyzed. Time 10–20 s tized with 2% isoflurane. Eight mice per group were used PET image and CT image were used to set ROI in order for PET imaging. In each group, five mice were used for to avoid the aorta on the liver. In addition, to clarify the non-blockade study and three mice were used for block- influence of CDAHFD diet on systemic exposure, left ade study. Under isoflurane anesthesia, a venous catheter ventricle of the heart instead of analysis of blood radio- was introduced through the tail vein and used for the activity was set as ROI for input analysis. TACs of liver administration of F-FPP-RGD . After mice placed in and left ventricle were decay-corrected to the injection an abdominal position on the PET scanner gantry, ap- time and expressed as the standardized uptake value proximately 7–12 MBq F-FPP-RGD were continu- (SUV), where SUV = tissue radioactivity concentration ously injected (0.2 mL/30 s) into the tail vein. PET scans (MBq/cm )/injected radioactivity (MBq) × body weight were started immediately after F-FPP-RGD injection (g). After the PET/CT scan, each mouse was euthanized was started. To confirm the F-FPP-RGD binding to and the plasma and liver were collected. Plasma and the integrin αvβ3, blockade experiments were performed liver were immediately frozen in liquid nitrogen and by the co-injection of 60 μg c(RGDfK) with stored at − 80 °C until protein assay. Liver was also col- F-FPP-RGD in three mice. Dynamic data acquisition lected and fixed by 10% formalin for histology. For was performed for 90 min. After the PET scans, CT quantitative RT-PCR, parts of livers were collected in scans were performed to acquire anatomical information RNAlater stabilization solution and stored at − 80 °C and to obtain data for the attenuation collection of PET after 24 h stored at 4 °C. images. The CT images were reconstructed using the fil- tered back-projection method (512 slices) and acquired Western blotting PET images were reconstructed by the 3D-MLEM The hepatic amount of integrin αv and β3 subunit s pro- method with CT-based attenuation correction. Dynamic tein was determined by western blot analysis, as de- images (6 × 10 s, 4 × 1 min, 11 × 5 min, 3 × 10 min) for a scribed previously [24]. Briefly, liver homogenates were time activity curve (TAC) were reconstructed. CT and prepared, and 15 μg of protein was separated by Table 2 Histological analysis of the liver in mice fed a CDAHFD Parameter Control 3 weeks CDAHFD 3 weeks Control 8 weeks CDAHFD 8 weeks Steatosis score 0.00 ± 0.00 1.75 ± 0.71** 0.00 ± 0.00 1.88 ± 0.35** Inflammation score 0.00 ± 0.00 2.88 ± 0.35** 0.00 ± 0.00 3.00 ± 0.00** Ballooning score 0.00 ± 0.00 0.38 ± 0.52** 0.00 ± 0.00 0.88 ± 0.64** NAFLD activity score 0.00 ± 0.00 5.00 ± 1.07** 0.00 ± 0.00 5.75 ± 0.71** ## Fibrosis area (%) 0.61 ± 0.29 0.47 ± 0.19 0.65 ± 0.18 3.89 ± 1.81** Representative photomicrographs of hepatic histology stained with hematoxylin and eosin (H&E) and Sirius red. Steatosis and inflammation scores ranged from 0 to 3 (normal = 0; minimal = 1; moderate = 2; marked = 3). Ballooning score ranged from 0 to 2 (normal = 0; minimal = 1; marked = 2). NAFLD activity score (NAS) was calculated by using the sum of each histological score. Data are expressed as the mean ± SD (n = 8 mice per group). Statistical differences were assessed using Steel-Dwass test. Hematoxylin and eosin (H&E) and Sirius red, × 200 magnification ## *p < 0.05, **p < 0.01 compared with respective control mice. p < 0.01 compared with CDAHFD 3 weeks Rokugawa et al. EJNMMI Research (2018) 8:40 Page 4 of 9 Fig. 1 Hepatic histopathology in mice fed a control or choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) for 3 or 8 weeks. Representative photomicrographs of hepatic histology stained with hematoxylin and eosin (H&E) (left) and Sirius red (right) (× 200 magnification) electrophoresis on 4–12% gradient polyacrylamide gels. GTACTGGATCG-3′, Col1a1-R: 5′-TACTCGAACGGGA After transfer to a polyvinylidene fluoride membrane, ATCCATC-3′.18S-F:5′-CGGCTACCACATCCAAGGA they were blocked by 5% BSA buffer and incubated over- A-3′,18S-R: 5′-GCTGGAATTACCGCGCCT-3′. night at 4 °C with antibodies to mouse anti-integrin αv (1:100; BD Biosciences), rabbit anti-integrin β3 (1:1000; Statistical analysis Abcam) and GAPDH (1:5000; Cell Signaling). After Quantitative data were expressed as the mean ± SD. washing, the membrane was incubated with horseradish Means were compared using Steel-Dwass test or Wil- peroxidase-conjugated secondary antibodies and de- coxon test. Spearman’s ranked correlation test was per- tected by LAS-3000. formed for evaluation of the correlation between protein expression of integrin αvβ3 and 80–90 min liver SUV of Quantitative RT-PCR analysis F-FPP-RGD . p values < 0.05 were considered statisti- The hepatic messenger RNA (mRNA) of integrin αvand cally significant. β3 subunits, α-smooth muscle actin (α-SMA), and colla- gen type 1 alpha 1 (col1a1) was analyzed by RT-PCR as Results described previously [25]. Briefly, gene expression was Blood biochemistry and liver histopathology in measured using the 7500 Real-Time PCR System and CDAHFD-fed mice Power SYBR™ Green PCR Master Mix (Applied Biosys- Plasma ALT and AST levels were significantly higher in 3- tems, CA, USA). The amplification method used 50 cycles and 8-week CDAHFD-fed mice compared with respective of 95 °C for 5 min, 95 °C for 10 s, and 65 °C for 30 s. The control mice (ALT 19.20 ± 3.45 vs 772.10 ± 128.63, p < 2-ΔΔCT method was used to calculate the relative mRNA 0.01; 31.90 ± 16.52 vs 584.6 ± 475.74, p < 0.01, respectively. expression normalized to 18S ribosomal RNA. The PCR AST 32.80 ± 3.83 vs 436.6 ± 76.35, p < 0.01; 41.20 ± 12.69 primer sequences were as follows: Integrin αv-F: 5′-TCGT vs 402.70 ± 242.78, p < 0.01, respectively) (Table 1). Histo- TTCTATCCCACCGCAG-3′.Integrin αv-R: 5′-TCGT logical analysis revealed that mice fed a CDAHFD for 3 TTCTATCCCACCGCAG-3′.Integrin β3-F: 5′-AGTG and 8 weeks showed moderate-to-marked steatosis and GCCGGGACAACTCT-3′,Integrin β3-R: 5′-AGACAAA inflammation was observed (Table 2, Fig. 1). No fibrotic GTCTCATCTGAGCACCA-3′. α-SMA-F: 5′-GAGCATC areas were observed in mice fed the CDAHFD for 3 weeks CGACACTGCTGACA-3′, α-SMA-R: 5′-AGCACAGCC but were observed in those with CDAHFD for 8 weeks TGAATAGCCACATAC-3′. Col1a1-F: 5′-GAGCGGAGA (0.47 ± 0.19 vs 3.89 ± 1.81, p < 0.01) (Table 2, Fig. 1). Fig. 2 Representative PET/CT fusion images in the livers of mice fed a control or choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) at 80–90 min Rokugawa et al. EJNMMI Research (2018) 8:40 Page 5 of 9 Fig. 3 Hepatic time activity curves after F-FPP-RGD injection in mice fed a control (a) or methionine choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) (b)(n = 5 per group). Sixty micrograms of c(RGDfK) was co-injected with F-FPP-RGD into each group for the blockade study (n = 3 per group). white circle, control 3 weeks; black circle, CDAHFD 3 weeks; white triangle, control 3 weeks + cRGDfK; black triangle, CDAHFD 3 weeks + cRGDfK; white square, control 8 weeks; black square, CDAHFD 8 weeks; white diamond, control 8 weeks + cRGDfK; and black diamond, CDAHFD 8 weeks + cRGDfK F-RPP-RGD PET imaging in CDAHFD-fed mice SUV of mice fed the CDAHFD at 3 and 8 weeks was sig- PET images of F-FPP-RGD at 80–90 min and time nificantly higher than that of respective control mice activity curves (TACs) of the liver and heart, mainly the (0.32 ± 0.06 vs 0.48 ± 0.05, p < 0.05, 0.35 ± 0.04 vs 0.75 ± covered left ventricle, are shown in Figs. 2, 3, and 4. 0.07, p < 0.05) (Fig. 5). In the blockade study, all groups 18 18 Higher uptake of F-FPP-RGD was observed in mice had accelerated liver clearance of F-FPP-RGD and de- 2 2 fed the CDAHFD for 3 and 8 weeks compared with con- creased SUV at 80–90 min compared with the respective trol mice. Hepatic TACs revealed that the clearance of control groups. F-FPP-RGD in CDAHFD-fed mice, which was calcu- lated using the following equation: ((SUV − 0–5min SUV )/SUV ), was slower than that of re- Hepatic expression of integrin αv and β3 in CDAHFD-fed 80–90 min 0–5min spective control mice (control 3 weeks vs CDAHFD mice 3 weeks = 0.69 vs 0.56, control 8 weeks vs CDAHFD Hepatic mRNA and protein levels of integrin αv and β3 8 weeks = 0.66 vs 0.45). F-FPP-RGD uptake in the were increased by prolonged CDAHFD (Fig. 6). Mice fed heart was highest at 20 s and was eliminated rapidly the CDAHFD for 8 weeks had the highest protein and from all groups (Fig. 4a, b). Hepatic radioactivity of ex- mRNA expression of all groups. Hepatic α-SMA and cess cold-c(RGDfK) co-injection groups were rapidly Col1a1 mRNA expressions were also markedly increased cleared from the liver (Fig. 3a, b). At 80–90 min, the in 3- and 8-week CDAHFD-fed mice (Fig. 7). Fig. 4 Left ventricle time activity curves after F-FPP-RGD injection in mice fed a control (a) or methionine choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) (b)(n = 5 per group). Sixty micrograms of c(RGDfK) was co-injected with F-FPP-RGD into each group for the blockade study (n = 3 per group). white circle, control 3 weeks; black circle, CDAHFD 3 weeks; white triangle, control 3 weeks + cRGDfK; black triangle, CDAHFD 3 weeks + cRGDfK; white square, control 8 weeks; black square, CDAHFD 8 weeks; white diamond, control 8 weeks + cRGDfK; and black diamond, CDAHFD 8 weeks + cRGDfK Rokugawa et al. EJNMMI Research (2018) 8:40 Page 6 of 9 Fig. 5 Hepatic SUV at 80–90 min. Data are expressed as the mean ±SD (n = 5 per group or n = 3 per group (+cRGDfK)). Statistical differences were assessed using Steel-Dwass test; *p < 0.05, compared with respective control mice, p < 0.05 compared with mice fed the CDAHFD for 3 weeks and Wilcoxon test, p < 0.05 compared with respective blockade group. White bar, control group; black bar, CDAHFD group Correlation of hepatic uptake of F-FPP-RGD and protein expression of integrin αvor β3, or Sirius red- positive area in CDAHFD-fed mice We evaluated the correlation between the hepatic uptake of F-FPP-RGD SUV at 80–90 min and protein expres- sion of integrin αvor β3. Liver SUV at 80–90 min showed Fig. 6 Hepatic mRNA (a) and protein levels (b) of integrin αv and β3 a positive correlation with integrin αvor β3(r =0.593, p < subunits in the livers of control and choline-deficient, L-amino 0.05 and r =0.835, p <0.001) (Fig. 8a, b). We also evalu- acid-defined, high-fat diet (CDAHFD)-fed mice. Data are expressed as ated correlation between the hepatic of F-FPP-RGD the mean ± SD (n = 8 mice per group). Statistical differences were SUV at 80–90 min and Sirius red-positive area. Liver SUV assessed using Steel-Dwass test. *p <0.05, **p < 0.01 compared with ## at 80–90 min showed a positive correlation with Sirius respective control mice. p < 0.01, compared with the CDAHFD 3 weeks group. White bar, integrin αv; black bar, integrin β3 red-positive area (r =0.593, p < 0.05) (Fig. 8c). Discussion only observed in CDAHFD mice fed for 8 weeks. These In this study, we clearly showed that the hepatic uptake results indicate that CDAHFD mice fed for 3 weeks de- of F-FPP-RGD was correlated with the hepatic ex- veloped NAFLD/NASH with minimal or no fibrosis and pression of integrin αvβ3 in CDAHFD-fed NASH model that CDAHFD mice fed for 8 weeks developed moderate mice. The CDAHFD-fed model was developed as a new fibrosis. A previous study reported that the mRNA and NAFLD/NASH mouse model and has a rapid onset and protein expressions of integrin αvβ3 were increased dur- progression of hepatic fibrosis compared with the ing the development and progression of liver fibrosis in methionine-choline-deficient diet-fed mouse model [20]. CCl and thioacetamide models, which are used as hep- In the present study, high ALT and ASL levels, steatosis, atic fibrosis models commonly [19, 20, 27]. In this study, inflammation, and ballooning were observed in 3- and CDAHFD mice fed for 3 weeks had increased hepatic in- 8-week CDAHFD-fed mice. To assess the NASH path- tegrin αv and β3 and α-SMA mRNA expressions, which ology, the sum of steatosis score, inflammation score, are indicators of HSCs. Mouse fed a methionine and ballooning score called NAS, were used. According choline-deficient diet, another commonly used NASH to the criteria, a NAS of 5 or more is diagnosed as “de- model, revealed an increase in α-SMA before fibrosis finitive NASH” and NAS of 3 or 4 is diagnosed as ‘bor- [21]. Therefore, it was considered that integrin αvβ3 derline NASH’ [26]. Therefore, in this study, CDAHFD 3 might be increased with HSC activation rather than fi- and 8 weeks fed mice were considered as NASH-like brosis in the livers of NASH model mice induced by a pathology. However, Sirius red stain-positive areas were methionine choline-deficient diet. Rokugawa et al. EJNMMI Research (2018) 8:40 Page 7 of 9 18 18 F-FPP-RGD is a commonly used F labeled RGD PET probe in clinical and non-clinical studies of tumors that express integrin αvβ3[17, 28]. To our knowledge, this is the first study to evaluate the relationship be- tween the hepatic uptake of F-FPP-RGD and integrin αvβ3 expression using PET in CDAHFD-fed NASH/ NAFLD model mice. In a previous study, Li et al. re- ported that integrin αvβ3 was co-localized with α-SMA positive areas by immunofluorescence and that fluores- cent labeled RGD bound to activate HSCs [19]. Caiyuan et al. also reported that integrin β3 was co-localized with α-SMA and that MRI contrast agent labeled RGD accu- mulated in HSCs [20] indicating F-FPP-RGD also spe- cifically bound to integrin αvβ3 on HSCs. In a PET imaging study, the hepatic uptake of F-FPP-RGD was increased with CDAHFD feeding and excess unlabeled cRGD co-injection reduced F-FPP-RGD accumulation in all groups. These results indicated that F-FPP-RGD bound to integrin αvβ3 in vivo. The hepatic uptake (SUV) of F-FPP-RGD remained constant from 30 to 90 min in both control and model mice. On the other hand, the liver ratio to the heart of F-FPP-RGD up- take was maximum at 90 min, suggesting the minimum effects of blood radioactivity to tissue radioactivity. Therefore, we used this time point for the SUV values in all analyses. At 80–90 min, the hepatic SUV of Fig. 7 Hepatic mRNA levels of a α-smooth muscle actin (α-SMA) F-FPP-RGD in mice fed the CDAHFD for 3 and and b collagen type 1a1 (Col1a1) in the livers of mice fed a control 8 weeks was increased compared with the respective or choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD). control mice and the highest uptake was in mice fed the Data are expressed as the mean ± SD (n = 8 mice per group). CDAHFD for 8 weeks. A previous SPECT scintigraphy Statistical differences were assessed using Steel-Dwass test. 99m ## study using Tc-labeled cRGD in a thioacetamide **p < 0.01 compared with respective control mice. p < 0.01, compared with the CDAHFD 3 weeks group treated rat model reported that the radioactivity of liver-to-heart ratio was increased with integrin αvβ3 ex- pression and fibrosis stage [19]. On the other hand, there was no correlation study between SPECT and in- tegrin αvβ3 expression or fibrosis stage using the same Fig. 8 Correlation between hepatic F-FPP-RGD uptake and integrin αv(a), β3(b) subunits, and Sirius red-positive area (c). Correlations were analyzed using Spearman’s ranked correlation test. white triangle, control; black circle, CDAHFD 3 weeks; black square,CDAHFD 8 weeks Rokugawa et al. EJNMMI Research (2018) 8:40 Page 8 of 9 animals. In the present study, the hepatic SUV of for assessing the quantitative analysis of the hepatic up- 18 18 F-FPP-RGD well correlated with integrin αv and β3 take of F-FPP-RGD in small animal. 2 2 protein expression. The hepatic SUV of F-FPP-RGD also showed correlation with Sirius red-positive area. On Conclusions the other hand, in CDAHFD for 3 weeks fed mice, hep- PET imaging showed F-FPP-RGD uptake was in- atic SUV of F-FPP-RGD was increased before fibrosis. creased before the onset of fibrosis and correlated with While integrin αv is expressed by parenchymal cells and integrin αvβ3 expression, especially β3 expression. HSCs, most integrin β3 was expressed on HSCs [29]. F-FPP-RGD PET imaging might be useful to Furthermore, while integrin αv forms complexes with non-invasively predict the fibrosis risk in NASH several β subunits (β1, β3, β5, β6, β8), integrin β3 forms patients. complexes with only two type α subunits, αv and αIIb Acknowledgements [30]. It might be thought that amount of integrin αvβ3is We thank Radio Isotope Laboratory, Osaka University, for preparing and feeding similar to integrin β3 rather than integrin αv. Therefore, the animals. hepatic SUV of F-FPP-RGD might be correlated with Availability of data and materials integrin β3rather than integrin αv and Sirius red-positive All data generated or analyzed during this study are included in this area. These results indicate that F-FPP-RGD PET has published article. the potential to evaluate the expression of integrin αvβ3 Authors’ contributions on hepatic cells including activated HSCs and might TR participated in the design of the study, performed and analyzed the PET have potential to predict fibrosis. Furthermore, experiments, statistics analyses, and drafted the manuscript. MI performed F-FPP-RGD PET might be helpful for development of the PET experiments. HI performed the radiosynthesis. HK performed the histological study and assessment, western blotting, and qRT-PCR. RN anti-fibrotic agent and decision about therapeutic inter- performed the histological assessment and drafted the manuscript. ES vention at early fibrosis stage in NASH patient. Steatosis and JH were the supervisors of the study. KA participated in the study and inflammation score are used to evaluate NAFLD ac- coordination and the design of the study and drafted the manuscript. All authors read and approved the final manuscript. tivity score, which discriminates NASH from simple steatosis. However, steatosis and inflammation were se- Ethics approval vere without any significant difference between mice fed The experimental protocols were reviewed and approved by the Institutional the CDAHFD for 3 and 8 weeks. Therefore, further Animal Care and Use Committee of Osaka University Graduate School of Medicine. studies are needed to prove that F-FPP-RGD PET could evaluate NASH pathology. Competing interests In quantitative PET imaging study, it is important to The authors declare that they have no competing interests. evaluate input function. Because of the small size of mice, arterial input function through blood sampling is Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in published technically challenging and we did not collect the arter- maps and institutional affiliations. ial blood. A previous study used radioactivity of the left ventricle from a PET image as the image-derived input Author details Translational Research Unit, Biomarker R&D Department, Shionogi & Co., function [31]. In the present study, the radioactivity of Ltd., 3-1-1, Futaba-cho, Toyonaka, Osaka 561-0825, Japan. Obesity and the heart, mainly including the left ventricle increased Metabolic Diseases, Drug Discovery and Disease Research Laboratory, immediately after injection and then decreased rapidly. Shionogi & Co., Ltd., Osaka, Japan. Department of Applied Chemistry and Analysis, Research Laboratory for Development, Shionogi & Co., Ltd., Osaka, Therefore, most of the radioactivity in the heart would Japan. Department of Molecular Imaging in Medicine, Osaka University be thought as image-derived input function. In mice fed Graduate School of Medicine, Osaka, Japan. Department of Nuclear the CDAHFD for 3 weeks, image-derived input function Medicine and Tracer Kinetics, Osaka University Graduate School of Medicine, Osaka, Japan. PET Molecular Imaging Center, Osaka University Graduate was not changed significantly. On the other hand, in School of Medicine, Osaka, Japan. mice fed the CDAHFD for 8 weeks, although there was no change of image-derived input function at early phase Received: 8 March 2018 Accepted: 1 May 2018 (about 0–5 min), a slight increase was observed at late phase. These data indicate that increased liver uptake of References F-FPP-RGD partly might be due to increased input 1. Bhala N, Angulo P, van der Poorten D, Lee E, Hui JM, Saracco G, et al. The function in mice given CDAHFD for 8 weeks. Because natural history of nonalcoholic fatty liver disease with advanced fibrosis or cirrhosis: an international collaborative study. Hepatology. 2011;54(4):1208–16. mouse ventricle was small, it is difficult to exclude the 2. Angulo P. Medical progress: nonalcoholic fatty liver disease. N Engl J Med. cardiac muscle completely in setting the region of inter- 2002;346:1221–31. est. Then, although left ventricle ROI set carefully, 3. Angulo P, Kleiner DE, Dam-Larsen S, et al. Liver fibrosis, but no other histologic features, is associated with long-term outcomes of patients with image-derived input function might include the spillover nonalcoholic fatty liver disease. Gastroenterology. 2015;149:389–97.e10. of radioactivity in cardiac muscle. Further studies, espe- 4. Talwalkar JA. Motion—all patients with NASH need to have a liver biopsy cially correct input function evaluation, will be needed arguments for the motion. Can J Gastroenterol. 2002;16(10):718–21. Rokugawa et al. EJNMMI Research (2018) 8:40 Page 9 of 9 5. Bedossa P, Darger D, Paradis V. Sampling variability of liver fibrosis in 29. Patsenker E, Popov Y, Wiesner M, Goodman SL, Schuppan D. chronic hepatitis C. Hepatology. 2003;38(6):1449–57. Pharmacological inhibition of the vitronectin receptor abrogates PDGF-BB- 6. Takeuchi H, Sugimoto K, Oshiro H, Iwatsuka K, Kono S, Yoshimasu Y, et al. induced hepatic stellate cell migration and activation in vitro. J Hepatol. Liver fibrosis: noninvasive assessment using supersonic shear imaging and 2007;46(5):878–87. FIB4 index in patients with non-alcoholic fatty liver disease. J Med Ultrason. 30. Hudson SV, Dolin CE, Poole LG, Massey VL, Wilkey D, Beier JI, et al. Modeling 2017; https://doi.org/10.1007/s10396-017-0840-3. the kinetics of integrin receptor binding to hepatic extracellular matrix 7. Chen J, Talwalkar JA, Yin M, Glaser KJ, Sanderson SO, Ehman RL. Early proteins. 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Pharmacological inhibition of integrin alphavbeta3 aggravates experimental liver fibrosis and suppresses hepatic angiogenesis. Hepatology. 2009;50:501–1511. 25. Itoh M, Suganami T, Nakagawa N, Tanaka M, Yamamoto Y, Kmei Y, et al. Melanocortin 4 receptor-deficient mice as a novel mouse model of nonalcoholic steatohepatitis. Am J Pathol. 2011;179(5):2545–63. 26. Kleiner DE, Brunt EM, Van Natta M, et al. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology. 2005;41:1313–21. 27. Li D, He L, Guo H, Chen H, Shan H. Targeting activated hepatic stellate cells (aHSCs) for liver fibrosis imaging. EJNMMI Res. 2015;5(1):71. 28. Chin FT, She B, Liu S, Berganos RA, Chang E, Mittra E, et al. First experience with clinical-grade ([18F]FPP(RGD )): an automated multi-step radiosynthesis for clinical PET studies. Mol Imaging Biol. 2012;14(1):99–5. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png EJNMMI Research Springer Journals

Evaluation of hepatic integrin αvβ3 expression in non-alcoholic steatohepatitis (NASH) model mouse by 18F-FPP-RGD2 PET

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Medicine & Public Health; Nuclear Medicine; Imaging / Radiology; Orthopedics; Cardiology; Oncology
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Abstract

Background: Activated hepatic stellate cells (HSCs), which express integrin αvβ3, are a major fibrogenic factor in 18 18 NASH pathophysiology. F-labeled cyclic arginine-glycine-aspartic acid penta-peptide ( F-FPP-RGD ) has been used as a PET probe for tumors expressing integrin αvβ3. The aim of this study was to assess the potential of PET with F-FPP-RGD to detect hepatic integrin αvβ3 expression in non-alcoholic steatohepatitis (NASH) model mice. Results: Thirty-two male C57BL/6 mice aged 6 weeks were fed a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) for 3 and 8 weeks. F-FPP-RGD PET imaging of the liver was performed at 3 and 8 weeks after CDAHFD feeding. After PET scanning, levels of hepatic integrin αvβ,3α-smooth muscle actin (α-SMA), and collagen type 1 alpha 1(col1a1) were measured. Histopathological analysis of hepatic steatosis, inflammation, and fibrosis, as well as blood biochemistry analysis, was also performed. CDAHFD for 3 and 8 weeks produced a moderate-to-severe steatosis and inflammation of the liver in mice. NAFLD activity score (NAS) in mice fed the CDAHFD for 3 and 8 weeks were more than 4 indicating NASH or borderline NASH pathology. Fibrosis was observed only in mice fed the CDAHFD for 8 weeks. PET imaging showed that the hepatic standardized uptake value, SUV , was increased with prolonged 80–90 min CDAHFD feeding compared with the respective controls (CDAHFD 3 weeks 0.32 ± 0.06 vs 0.48 ± 0.05, p < 0.01; CDAHFD 8 weeks 0.35 ± 0.04 vs 0.75 ± 0.07, p < 0.01, respectively). Prolonged CDAHFD feeding increased hepatic mRNA and protein levels of integrin αvand β3 at 3 and 8 weeks. Hepatic F-FPP-RGD uptake and amount of integrin αvand β3 protein were well correlated (r =0.593, p <0.05 and r =0.835, p < 0.001, respectively). Hepatic F-FPP-RGD uptake also showed a positive correlation with Sirius red-positive area. Conclusions: The hepatic uptake of F-FPP-RGD correlated well with integrin αvand β3 expression and histological fibrosis in a mouse model of NASH, suggesting the predictability of fibrosis in NASH pathology. Keywords: Non-alcoholic fatty liver disease, Non-alcoholic steatohepatitis, Fibrosis, Positron emission tomography, F-FPP-RGD , Modified methionine choline-deficient, High-fat diet, Integrin αvβ3 Background progress to fibrosis and about 10% progress to cir- Non-alcoholic fatty liver disease (NAFLD), one of the rhosis [2]. The prognosis of NAFLD depends on the most common forms of chronic liver disease in pa- histological severity, particularly of liver fibrosis, tients without a history of alcoholic abuse, encom- which is the strongest predictor of liver morbidity passes a wide spectrum of conditions from simple and mortality [3]. To prevent liver-related mortality, steatosis to non-alcoholic steatohepatitis (NASH) [1]. it is important to reverse advanced fibrosis or prevent It was reported that 30–40% of NASH patients the progression to fibrosis in NASH patients. From a clinical view point, liver biopsy is the gold standard for the diagnosis of NASH and staging liver * Correspondence: takemi.rokugawa@shionogi.co.jp fibrosis [4]. However, liver biopsy has limitations in- Translational Research Unit, Biomarker R&D Department, Shionogi & Co., cluding sampling error and it is invasive, painful and Ltd., 3-1-1, Futaba-cho, Toyonaka, Osaka 561-0825, Japan Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Rokugawa et al. EJNMMI Research (2018) 8:40 Page 2 of 9 results in poor patient compliance [5]. Therefore, the [21]. Therefore, RGD imaging might be a useful pre- development of a non-invasive strategy to evaluate dictor for the onset of fibrosis in NASH. liver fibrosis is required. Magnetic resonance elasto- In the present study, we investigated the relationship be- graphy and ultrasound-based transient elastography tween the hepatic uptake of F-FPP-RGD and integrin have been developed to assess liver fibrosis [6, 7]. αvβ3 expression using PET imaging in a NASH mouse These methods can discriminate moderate and ad- model induced by feeding with a choline-deficient, vanced liver fibrosis from early-stage liver injury or L-amino acid-defined, high-fat diet (CDAHFD) [22]. the normal patient population. However, these ap- proaches have reported a lower accuracy for the de- Methods tection of early stage liver fibrosis [8]. Furthermore, Animals and experimental design especially in NASH patients, steatosis may produce a Male C57BL/6J mice, aged 6 weeks old, were purchased softer liver because of fat deposition in the liver par- from CLEA Japan (Shizuoka, Japan). Mice were given enchyma [9, 10]. These non-invasive tools are not free access to water and either a normal diet or likely to be sensitive enough to identify mild changes CDAHFD, which contained 0.1% methionine, no choline or early stages of fibrosis. Preclinical studies recently and 60 kcal% fat, prepared by Research Diets (New reported the imaging of collagen or elastin probes for Brunswick, NJ, USA), for 3 and 8 weeks. They were thereliableassessmentof fibrosis [11, 12]. Thus, the housed in a temperature-controlled room maintained on development of sensitive imaging markers of fibrogen- a 12 h light/dark cycle with lights on at 7:00 am. The ex- esis is important to predict the prognosis and deter- perimental protocols were reviewed and approved by the mine the precise therapeutic intervention required. Institutional Animal Care and Use Committee of Osaka Activated hepatic stellate cells, myofibroblasts, cholan- University Graduate School of Medicine. giocytes, macrophages, and components of the patho- logical extracellular matrix are major fibrogenic factors. Activation and proliferation of hepatic stellate cells (HSCs) are considered key factors of hepatic fibrosis Biochemical and histopathological analysis [13]. Following HSC activation, they transform to Mice were euthanized by exsanguination under isoflur- myofibroblast-like cells characterized by α-smooth ane anesthesia. Plasma (200 μL) was collected and muscle actin (α-SMA) expression and produce excessive assayed for the content of alanine aminotransferase amounts of extra cellular matrix proteins such as type 1 (ALT), aspartate aminotransferase (AST), triglyceride and type 3 collagen [13]. With the activation of HSCs, (TG), total cholesterol (TC), and high-density lipopro- integrin αvβ3, which plays an important role in cell sig- tein cholesterol (HDLC). The left hepatic lobes were naling such as cell-to-cell adhesion, apoptosis, and fixed in 10% formalin and sectioned, and 4-μm sections cell-matrix interactions, is expressed on HSCs [14]. were stained with hematoxylin and eosin (H&E) and Sir- Thus, monitoring the expression of integrin αvβ3in ius red. Steatosis, inflammation, ballooning, and fibrosis NASH liver might be used as a marker to predict the in the liver were assessed based on the severity and size onset of fibrosis. of the lesion. Steatosis and inflammation scores ranged The three-amino-acid sequence of arginine-gly from 0 to 3: normal = 0; minimal = 1; moderate = 2; cine-aspartic acid (RGD) has a high binding affinity to marked = 3. Ballooning score ranged from 0 to 2: nor- integrin αvβ3[15, 16]. Many studies reported that mal = 0; minimal = 1; marked = 2. NAFLD activity score radiolabeled cyclic RGD peptides (cRGD) imaged by (NAS) was calculated by using the sum of each histo- positron emission tomography (PET) and single pho- logical score. To assess hepatic fibrosis, five different ton emission computed tomography (SPECT) have areas (× 200 magnification) were selected per mouse, been developed as a new radio tracer for selective in- and Sirius red-positive areas were measured using Win- tegrin αvβ3 positive tumors [17, 18]. In a recent fluor- ROOF software (Mitsutani, Tokyo, Japan). escence trace study, cRGD was accumulated in activated but not quiescent HSCs [19]. Furthermore, Radiopharmaceutical preparation hepatic integrin αvβ3 imaging by SPECT using I 99m F-FPP-RGD was radio synthesized using a two-step and Tc-labeled cRGD and magnetic resonance im- method as reported previously [23]. Conjugation be- aging using cRGD labeled by contrast agent USPIO 18 18 tween F-4-nitrophenyl-2-fluoropropionate ( F-NFP) detected rodent hepatic fibrosis induced by thioaceta- and the RGD dimeric peptide (PEG -c[RGDyK] )was mide or CCl treatment [19, 20]. These hepatic fibrosis 3 2 performed. Radiochemical purity and specific activity models develop fibrosis more quickly than common were > 99% and 445.6 ± 107.6 GBq/μmol, respectively, NASH models. In NASH pathology, HSCs are acti- with a radiosynthesis and purification time of 90 min. vated before or at the early stage of hepatic fibrosis Rokugawa et al. EJNMMI Research (2018) 8:40 Page 3 of 9 Table 1 Body, liver weight, and blood parameters in mice fed a CDAHFD Parameter Control 3 weeks CDAHFD 3 weeks Control 8 weeks CDAHFD 8 weeks Body weight (g) 24.44 ± 0.91 20.81 ± 0.96** 26.41 ± 1.56 21.66 ± 2.23** Liver weight (g) 1.11 ± 0.11 1.47 ± 0.17** 1.12 ± 0.13 1.69 ± 0.24** AST (IU/L) 32.80 ± 3.83 436.6 ± 76.35** 41.20 ± 12.69 402.70 ± 242.78** ALT (IU/L) 19.20 ± 3.45 772.10 ± 128.63** 31.90 ± 16.52 584.6 ± 475.74** TG (mg/dL) 155.7 ± 48.57 41.50 ± 17.69** 146.80 ± 65.67 21.50 ± 3.54** TC (mg/dL) 80.40 ± 7.91 49.00 ± 6.95** 79.70 ± 9.50 36.3 ± 4.14** Statistical differences were assessed using Steel-Dwass test AST aspartate transaminase, ALT alanine aminotransferase, TC total cholesterol, TG, triglyceride **p < 0.01 compared with respective control mice PET imaging PET images were automatically fused and analyzed by PET scan and X-ray CT imaging were performed with a PMOD v3.6 (PMOD Technologies Ltd., Zürich, Pre-Clinical Imaging System Triumph LbPET12/CT Switzerland). To calculate hepatic SUV 100 mm elliptic (TriFoil Imaging Inc., Chatsworth, CA, USA). Mice fed a two regions-of-interest (ROI) were chosen excluding the CDAHFD or control diet for 3 or 8 weeks were anesthe- aorta on the liver tissue were analyzed. Time 10–20 s tized with 2% isoflurane. Eight mice per group were used PET image and CT image were used to set ROI in order for PET imaging. In each group, five mice were used for to avoid the aorta on the liver. In addition, to clarify the non-blockade study and three mice were used for block- influence of CDAHFD diet on systemic exposure, left ade study. Under isoflurane anesthesia, a venous catheter ventricle of the heart instead of analysis of blood radio- was introduced through the tail vein and used for the activity was set as ROI for input analysis. TACs of liver administration of F-FPP-RGD . After mice placed in and left ventricle were decay-corrected to the injection an abdominal position on the PET scanner gantry, ap- time and expressed as the standardized uptake value proximately 7–12 MBq F-FPP-RGD were continu- (SUV), where SUV = tissue radioactivity concentration ously injected (0.2 mL/30 s) into the tail vein. PET scans (MBq/cm )/injected radioactivity (MBq) × body weight were started immediately after F-FPP-RGD injection (g). After the PET/CT scan, each mouse was euthanized was started. To confirm the F-FPP-RGD binding to and the plasma and liver were collected. Plasma and the integrin αvβ3, blockade experiments were performed liver were immediately frozen in liquid nitrogen and by the co-injection of 60 μg c(RGDfK) with stored at − 80 °C until protein assay. Liver was also col- F-FPP-RGD in three mice. Dynamic data acquisition lected and fixed by 10% formalin for histology. For was performed for 90 min. After the PET scans, CT quantitative RT-PCR, parts of livers were collected in scans were performed to acquire anatomical information RNAlater stabilization solution and stored at − 80 °C and to obtain data for the attenuation collection of PET after 24 h stored at 4 °C. images. The CT images were reconstructed using the fil- tered back-projection method (512 slices) and acquired Western blotting PET images were reconstructed by the 3D-MLEM The hepatic amount of integrin αv and β3 subunit s pro- method with CT-based attenuation correction. Dynamic tein was determined by western blot analysis, as de- images (6 × 10 s, 4 × 1 min, 11 × 5 min, 3 × 10 min) for a scribed previously [24]. Briefly, liver homogenates were time activity curve (TAC) were reconstructed. CT and prepared, and 15 μg of protein was separated by Table 2 Histological analysis of the liver in mice fed a CDAHFD Parameter Control 3 weeks CDAHFD 3 weeks Control 8 weeks CDAHFD 8 weeks Steatosis score 0.00 ± 0.00 1.75 ± 0.71** 0.00 ± 0.00 1.88 ± 0.35** Inflammation score 0.00 ± 0.00 2.88 ± 0.35** 0.00 ± 0.00 3.00 ± 0.00** Ballooning score 0.00 ± 0.00 0.38 ± 0.52** 0.00 ± 0.00 0.88 ± 0.64** NAFLD activity score 0.00 ± 0.00 5.00 ± 1.07** 0.00 ± 0.00 5.75 ± 0.71** ## Fibrosis area (%) 0.61 ± 0.29 0.47 ± 0.19 0.65 ± 0.18 3.89 ± 1.81** Representative photomicrographs of hepatic histology stained with hematoxylin and eosin (H&E) and Sirius red. Steatosis and inflammation scores ranged from 0 to 3 (normal = 0; minimal = 1; moderate = 2; marked = 3). Ballooning score ranged from 0 to 2 (normal = 0; minimal = 1; marked = 2). NAFLD activity score (NAS) was calculated by using the sum of each histological score. Data are expressed as the mean ± SD (n = 8 mice per group). Statistical differences were assessed using Steel-Dwass test. Hematoxylin and eosin (H&E) and Sirius red, × 200 magnification ## *p < 0.05, **p < 0.01 compared with respective control mice. p < 0.01 compared with CDAHFD 3 weeks Rokugawa et al. EJNMMI Research (2018) 8:40 Page 4 of 9 Fig. 1 Hepatic histopathology in mice fed a control or choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) for 3 or 8 weeks. Representative photomicrographs of hepatic histology stained with hematoxylin and eosin (H&E) (left) and Sirius red (right) (× 200 magnification) electrophoresis on 4–12% gradient polyacrylamide gels. GTACTGGATCG-3′, Col1a1-R: 5′-TACTCGAACGGGA After transfer to a polyvinylidene fluoride membrane, ATCCATC-3′.18S-F:5′-CGGCTACCACATCCAAGGA they were blocked by 5% BSA buffer and incubated over- A-3′,18S-R: 5′-GCTGGAATTACCGCGCCT-3′. night at 4 °C with antibodies to mouse anti-integrin αv (1:100; BD Biosciences), rabbit anti-integrin β3 (1:1000; Statistical analysis Abcam) and GAPDH (1:5000; Cell Signaling). After Quantitative data were expressed as the mean ± SD. washing, the membrane was incubated with horseradish Means were compared using Steel-Dwass test or Wil- peroxidase-conjugated secondary antibodies and de- coxon test. Spearman’s ranked correlation test was per- tected by LAS-3000. formed for evaluation of the correlation between protein expression of integrin αvβ3 and 80–90 min liver SUV of Quantitative RT-PCR analysis F-FPP-RGD . p values < 0.05 were considered statisti- The hepatic messenger RNA (mRNA) of integrin αvand cally significant. β3 subunits, α-smooth muscle actin (α-SMA), and colla- gen type 1 alpha 1 (col1a1) was analyzed by RT-PCR as Results described previously [25]. Briefly, gene expression was Blood biochemistry and liver histopathology in measured using the 7500 Real-Time PCR System and CDAHFD-fed mice Power SYBR™ Green PCR Master Mix (Applied Biosys- Plasma ALT and AST levels were significantly higher in 3- tems, CA, USA). The amplification method used 50 cycles and 8-week CDAHFD-fed mice compared with respective of 95 °C for 5 min, 95 °C for 10 s, and 65 °C for 30 s. The control mice (ALT 19.20 ± 3.45 vs 772.10 ± 128.63, p < 2-ΔΔCT method was used to calculate the relative mRNA 0.01; 31.90 ± 16.52 vs 584.6 ± 475.74, p < 0.01, respectively. expression normalized to 18S ribosomal RNA. The PCR AST 32.80 ± 3.83 vs 436.6 ± 76.35, p < 0.01; 41.20 ± 12.69 primer sequences were as follows: Integrin αv-F: 5′-TCGT vs 402.70 ± 242.78, p < 0.01, respectively) (Table 1). Histo- TTCTATCCCACCGCAG-3′.Integrin αv-R: 5′-TCGT logical analysis revealed that mice fed a CDAHFD for 3 TTCTATCCCACCGCAG-3′.Integrin β3-F: 5′-AGTG and 8 weeks showed moderate-to-marked steatosis and GCCGGGACAACTCT-3′,Integrin β3-R: 5′-AGACAAA inflammation was observed (Table 2, Fig. 1). No fibrotic GTCTCATCTGAGCACCA-3′. α-SMA-F: 5′-GAGCATC areas were observed in mice fed the CDAHFD for 3 weeks CGACACTGCTGACA-3′, α-SMA-R: 5′-AGCACAGCC but were observed in those with CDAHFD for 8 weeks TGAATAGCCACATAC-3′. Col1a1-F: 5′-GAGCGGAGA (0.47 ± 0.19 vs 3.89 ± 1.81, p < 0.01) (Table 2, Fig. 1). Fig. 2 Representative PET/CT fusion images in the livers of mice fed a control or choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) at 80–90 min Rokugawa et al. EJNMMI Research (2018) 8:40 Page 5 of 9 Fig. 3 Hepatic time activity curves after F-FPP-RGD injection in mice fed a control (a) or methionine choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) (b)(n = 5 per group). Sixty micrograms of c(RGDfK) was co-injected with F-FPP-RGD into each group for the blockade study (n = 3 per group). white circle, control 3 weeks; black circle, CDAHFD 3 weeks; white triangle, control 3 weeks + cRGDfK; black triangle, CDAHFD 3 weeks + cRGDfK; white square, control 8 weeks; black square, CDAHFD 8 weeks; white diamond, control 8 weeks + cRGDfK; and black diamond, CDAHFD 8 weeks + cRGDfK F-RPP-RGD PET imaging in CDAHFD-fed mice SUV of mice fed the CDAHFD at 3 and 8 weeks was sig- PET images of F-FPP-RGD at 80–90 min and time nificantly higher than that of respective control mice activity curves (TACs) of the liver and heart, mainly the (0.32 ± 0.06 vs 0.48 ± 0.05, p < 0.05, 0.35 ± 0.04 vs 0.75 ± covered left ventricle, are shown in Figs. 2, 3, and 4. 0.07, p < 0.05) (Fig. 5). In the blockade study, all groups 18 18 Higher uptake of F-FPP-RGD was observed in mice had accelerated liver clearance of F-FPP-RGD and de- 2 2 fed the CDAHFD for 3 and 8 weeks compared with con- creased SUV at 80–90 min compared with the respective trol mice. Hepatic TACs revealed that the clearance of control groups. F-FPP-RGD in CDAHFD-fed mice, which was calcu- lated using the following equation: ((SUV − 0–5min SUV )/SUV ), was slower than that of re- Hepatic expression of integrin αv and β3 in CDAHFD-fed 80–90 min 0–5min spective control mice (control 3 weeks vs CDAHFD mice 3 weeks = 0.69 vs 0.56, control 8 weeks vs CDAHFD Hepatic mRNA and protein levels of integrin αv and β3 8 weeks = 0.66 vs 0.45). F-FPP-RGD uptake in the were increased by prolonged CDAHFD (Fig. 6). Mice fed heart was highest at 20 s and was eliminated rapidly the CDAHFD for 8 weeks had the highest protein and from all groups (Fig. 4a, b). Hepatic radioactivity of ex- mRNA expression of all groups. Hepatic α-SMA and cess cold-c(RGDfK) co-injection groups were rapidly Col1a1 mRNA expressions were also markedly increased cleared from the liver (Fig. 3a, b). At 80–90 min, the in 3- and 8-week CDAHFD-fed mice (Fig. 7). Fig. 4 Left ventricle time activity curves after F-FPP-RGD injection in mice fed a control (a) or methionine choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) (b)(n = 5 per group). Sixty micrograms of c(RGDfK) was co-injected with F-FPP-RGD into each group for the blockade study (n = 3 per group). white circle, control 3 weeks; black circle, CDAHFD 3 weeks; white triangle, control 3 weeks + cRGDfK; black triangle, CDAHFD 3 weeks + cRGDfK; white square, control 8 weeks; black square, CDAHFD 8 weeks; white diamond, control 8 weeks + cRGDfK; and black diamond, CDAHFD 8 weeks + cRGDfK Rokugawa et al. EJNMMI Research (2018) 8:40 Page 6 of 9 Fig. 5 Hepatic SUV at 80–90 min. Data are expressed as the mean ±SD (n = 5 per group or n = 3 per group (+cRGDfK)). Statistical differences were assessed using Steel-Dwass test; *p < 0.05, compared with respective control mice, p < 0.05 compared with mice fed the CDAHFD for 3 weeks and Wilcoxon test, p < 0.05 compared with respective blockade group. White bar, control group; black bar, CDAHFD group Correlation of hepatic uptake of F-FPP-RGD and protein expression of integrin αvor β3, or Sirius red- positive area in CDAHFD-fed mice We evaluated the correlation between the hepatic uptake of F-FPP-RGD SUV at 80–90 min and protein expres- sion of integrin αvor β3. Liver SUV at 80–90 min showed Fig. 6 Hepatic mRNA (a) and protein levels (b) of integrin αv and β3 a positive correlation with integrin αvor β3(r =0.593, p < subunits in the livers of control and choline-deficient, L-amino 0.05 and r =0.835, p <0.001) (Fig. 8a, b). We also evalu- acid-defined, high-fat diet (CDAHFD)-fed mice. Data are expressed as ated correlation between the hepatic of F-FPP-RGD the mean ± SD (n = 8 mice per group). Statistical differences were SUV at 80–90 min and Sirius red-positive area. Liver SUV assessed using Steel-Dwass test. *p <0.05, **p < 0.01 compared with ## at 80–90 min showed a positive correlation with Sirius respective control mice. p < 0.01, compared with the CDAHFD 3 weeks group. White bar, integrin αv; black bar, integrin β3 red-positive area (r =0.593, p < 0.05) (Fig. 8c). Discussion only observed in CDAHFD mice fed for 8 weeks. These In this study, we clearly showed that the hepatic uptake results indicate that CDAHFD mice fed for 3 weeks de- of F-FPP-RGD was correlated with the hepatic ex- veloped NAFLD/NASH with minimal or no fibrosis and pression of integrin αvβ3 in CDAHFD-fed NASH model that CDAHFD mice fed for 8 weeks developed moderate mice. The CDAHFD-fed model was developed as a new fibrosis. A previous study reported that the mRNA and NAFLD/NASH mouse model and has a rapid onset and protein expressions of integrin αvβ3 were increased dur- progression of hepatic fibrosis compared with the ing the development and progression of liver fibrosis in methionine-choline-deficient diet-fed mouse model [20]. CCl and thioacetamide models, which are used as hep- In the present study, high ALT and ASL levels, steatosis, atic fibrosis models commonly [19, 20, 27]. In this study, inflammation, and ballooning were observed in 3- and CDAHFD mice fed for 3 weeks had increased hepatic in- 8-week CDAHFD-fed mice. To assess the NASH path- tegrin αv and β3 and α-SMA mRNA expressions, which ology, the sum of steatosis score, inflammation score, are indicators of HSCs. Mouse fed a methionine and ballooning score called NAS, were used. According choline-deficient diet, another commonly used NASH to the criteria, a NAS of 5 or more is diagnosed as “de- model, revealed an increase in α-SMA before fibrosis finitive NASH” and NAS of 3 or 4 is diagnosed as ‘bor- [21]. Therefore, it was considered that integrin αvβ3 derline NASH’ [26]. Therefore, in this study, CDAHFD 3 might be increased with HSC activation rather than fi- and 8 weeks fed mice were considered as NASH-like brosis in the livers of NASH model mice induced by a pathology. However, Sirius red stain-positive areas were methionine choline-deficient diet. Rokugawa et al. EJNMMI Research (2018) 8:40 Page 7 of 9 18 18 F-FPP-RGD is a commonly used F labeled RGD PET probe in clinical and non-clinical studies of tumors that express integrin αvβ3[17, 28]. To our knowledge, this is the first study to evaluate the relationship be- tween the hepatic uptake of F-FPP-RGD and integrin αvβ3 expression using PET in CDAHFD-fed NASH/ NAFLD model mice. In a previous study, Li et al. re- ported that integrin αvβ3 was co-localized with α-SMA positive areas by immunofluorescence and that fluores- cent labeled RGD bound to activate HSCs [19]. Caiyuan et al. also reported that integrin β3 was co-localized with α-SMA and that MRI contrast agent labeled RGD accu- mulated in HSCs [20] indicating F-FPP-RGD also spe- cifically bound to integrin αvβ3 on HSCs. In a PET imaging study, the hepatic uptake of F-FPP-RGD was increased with CDAHFD feeding and excess unlabeled cRGD co-injection reduced F-FPP-RGD accumulation in all groups. These results indicated that F-FPP-RGD bound to integrin αvβ3 in vivo. The hepatic uptake (SUV) of F-FPP-RGD remained constant from 30 to 90 min in both control and model mice. On the other hand, the liver ratio to the heart of F-FPP-RGD up- take was maximum at 90 min, suggesting the minimum effects of blood radioactivity to tissue radioactivity. Therefore, we used this time point for the SUV values in all analyses. At 80–90 min, the hepatic SUV of Fig. 7 Hepatic mRNA levels of a α-smooth muscle actin (α-SMA) F-FPP-RGD in mice fed the CDAHFD for 3 and and b collagen type 1a1 (Col1a1) in the livers of mice fed a control 8 weeks was increased compared with the respective or choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD). control mice and the highest uptake was in mice fed the Data are expressed as the mean ± SD (n = 8 mice per group). CDAHFD for 8 weeks. A previous SPECT scintigraphy Statistical differences were assessed using Steel-Dwass test. 99m ## study using Tc-labeled cRGD in a thioacetamide **p < 0.01 compared with respective control mice. p < 0.01, compared with the CDAHFD 3 weeks group treated rat model reported that the radioactivity of liver-to-heart ratio was increased with integrin αvβ3 ex- pression and fibrosis stage [19]. On the other hand, there was no correlation study between SPECT and in- tegrin αvβ3 expression or fibrosis stage using the same Fig. 8 Correlation between hepatic F-FPP-RGD uptake and integrin αv(a), β3(b) subunits, and Sirius red-positive area (c). Correlations were analyzed using Spearman’s ranked correlation test. white triangle, control; black circle, CDAHFD 3 weeks; black square,CDAHFD 8 weeks Rokugawa et al. EJNMMI Research (2018) 8:40 Page 8 of 9 animals. In the present study, the hepatic SUV of for assessing the quantitative analysis of the hepatic up- 18 18 F-FPP-RGD well correlated with integrin αv and β3 take of F-FPP-RGD in small animal. 2 2 protein expression. The hepatic SUV of F-FPP-RGD also showed correlation with Sirius red-positive area. On Conclusions the other hand, in CDAHFD for 3 weeks fed mice, hep- PET imaging showed F-FPP-RGD uptake was in- atic SUV of F-FPP-RGD was increased before fibrosis. creased before the onset of fibrosis and correlated with While integrin αv is expressed by parenchymal cells and integrin αvβ3 expression, especially β3 expression. HSCs, most integrin β3 was expressed on HSCs [29]. F-FPP-RGD PET imaging might be useful to Furthermore, while integrin αv forms complexes with non-invasively predict the fibrosis risk in NASH several β subunits (β1, β3, β5, β6, β8), integrin β3 forms patients. complexes with only two type α subunits, αv and αIIb Acknowledgements [30]. It might be thought that amount of integrin αvβ3is We thank Radio Isotope Laboratory, Osaka University, for preparing and feeding similar to integrin β3 rather than integrin αv. Therefore, the animals. hepatic SUV of F-FPP-RGD might be correlated with Availability of data and materials integrin β3rather than integrin αv and Sirius red-positive All data generated or analyzed during this study are included in this area. These results indicate that F-FPP-RGD PET has published article. the potential to evaluate the expression of integrin αvβ3 Authors’ contributions on hepatic cells including activated HSCs and might TR participated in the design of the study, performed and analyzed the PET have potential to predict fibrosis. Furthermore, experiments, statistics analyses, and drafted the manuscript. MI performed F-FPP-RGD PET might be helpful for development of the PET experiments. HI performed the radiosynthesis. HK performed the histological study and assessment, western blotting, and qRT-PCR. RN anti-fibrotic agent and decision about therapeutic inter- performed the histological assessment and drafted the manuscript. ES vention at early fibrosis stage in NASH patient. Steatosis and JH were the supervisors of the study. KA participated in the study and inflammation score are used to evaluate NAFLD ac- coordination and the design of the study and drafted the manuscript. All authors read and approved the final manuscript. tivity score, which discriminates NASH from simple steatosis. However, steatosis and inflammation were se- Ethics approval vere without any significant difference between mice fed The experimental protocols were reviewed and approved by the Institutional the CDAHFD for 3 and 8 weeks. Therefore, further Animal Care and Use Committee of Osaka University Graduate School of Medicine. studies are needed to prove that F-FPP-RGD PET could evaluate NASH pathology. Competing interests In quantitative PET imaging study, it is important to The authors declare that they have no competing interests. evaluate input function. Because of the small size of mice, arterial input function through blood sampling is Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in published technically challenging and we did not collect the arter- maps and institutional affiliations. ial blood. A previous study used radioactivity of the left ventricle from a PET image as the image-derived input Author details Translational Research Unit, Biomarker R&D Department, Shionogi & Co., function [31]. In the present study, the radioactivity of Ltd., 3-1-1, Futaba-cho, Toyonaka, Osaka 561-0825, Japan. Obesity and the heart, mainly including the left ventricle increased Metabolic Diseases, Drug Discovery and Disease Research Laboratory, immediately after injection and then decreased rapidly. Shionogi & Co., Ltd., Osaka, Japan. Department of Applied Chemistry and Analysis, Research Laboratory for Development, Shionogi & Co., Ltd., Osaka, Therefore, most of the radioactivity in the heart would Japan. Department of Molecular Imaging in Medicine, Osaka University be thought as image-derived input function. In mice fed Graduate School of Medicine, Osaka, Japan. Department of Nuclear the CDAHFD for 3 weeks, image-derived input function Medicine and Tracer Kinetics, Osaka University Graduate School of Medicine, Osaka, Japan. PET Molecular Imaging Center, Osaka University Graduate was not changed significantly. On the other hand, in School of Medicine, Osaka, Japan. mice fed the CDAHFD for 8 weeks, although there was no change of image-derived input function at early phase Received: 8 March 2018 Accepted: 1 May 2018 (about 0–5 min), a slight increase was observed at late phase. These data indicate that increased liver uptake of References F-FPP-RGD partly might be due to increased input 1. Bhala N, Angulo P, van der Poorten D, Lee E, Hui JM, Saracco G, et al. The function in mice given CDAHFD for 8 weeks. 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EJNMMI ResearchSpringer Journals

Published: May 31, 2018

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