Quantitative evaluation of hepatic integrin α_vβ₃ expression by positron emission tomography imaging using ¹⁸F-FPP-RGD₂ in rats with non-alcoholic steatohepatitis

Non-alcoholic fatty liver disease (NAFLD) is one of the most common causes of chronic liver disease [1, 2], and the more severe form of this condition, non-alcoholic steatohepatitis (NASH), is a serious disease [3]. Approximately half of NASH patients develop liver fibrosis, which is the major predictor of subsequent liver cirrhosis, hepatocellular carcinoma and transplantation [4]. Consequently, early detection of fibrosis, before its progression, is important for the prevention and appropriate treatment of these pathologies. To date, liver biopsy has been regarded as the gold standard method of diagnosing and staging liver fibrosis [5, 6]. However, liver biopsy is invasive and is associated with several risks, including high sampling variability, because of the small size of the tissue samples obtained, inter-observer variability, pain and complications due to the procedure itself [7, 8]. A number of non-invasive methods of staging fibrosis and determining the treatment response have been reported in recent years [9‐13], but none of these have become well established as diagnostic techniques. The most successful non-invasive approaches in clinical practice are ultrasound- and magnetic resonance-based elastography, which assess liver stiffness [12, 13]. Consequently, they do not provide information regarding the molecular pathology of liver fibrosis [14, 15]. Therefore, the development and validation of a non-invasive biomarker for the diagnosis and staging of liver fibrosis would be valuable.

The pathology of NASH and the mechanism of progression of liver fibrosis have been well documented [16]. The activation of hepatic stellate cells (HSCs) is one of the most important events in liver fibrogenesis [17]. Activated HSCs play an important role in fibrogenesis by secreting proteins such as integrin α_vβ₃ that play a critical role in the transformation of HSCs to myofibroblasts, which express α-smooth muscle actin (α-SMA). This leads to excessive production of extracellular matrix (ECM) proteins, such as collagens type-1 and type-3 [18]. Therefore, the expression of integrin α_vβ₃ may have potential as a marker of fibrosis in the liver.

Integrin α_vβ₃ positron emission tomography (PET) probes, such as ¹⁸F-labelled RGD [19, 20], have previously been used to detect liver fibrosis in animal models. However, evaluation of the kinetic profile of the binding of ¹⁸F-FPP-RGD₂ to integrin α_vβ₃ in the livers of animal models of NASH is a critical step. There have been previous kinetic analyses of RGD peptide PET probes, such as ¹⁸F- and ⁶⁸Ga-labelled RGD [21, 22], in animal models of angiogenesis and neoplasia, but not in models of liver fibrosis and NASH. Therefore, we aimed to conduct a kinetic analysis to calculate the parameter V_T in an animal model of NASH.

In the present study, we have investigated the relationship between the hepatic V_T of ¹⁸F-FPP-RGD₂, calculated using a kinetic analysis, or the standard uptake value between 60 and 90 min (SUV_60–90 min) and integrin α_vβ₃ protein expression using PET imaging in a model of diet-induced NASH.

In the present study, we have evaluated the kinetics of ¹⁸F-FPP-RGD₂ in an animal model of NASH, as a means of quantifying hepatic integrin α_vβ₃ protein expression. We found strong correlations between hepatic V_{T (IDIF)}, evaluated using the 2T3C model or SUV_60–90 min, and hepatic integrin α_v or β₃ protein expression. The present findings indicate that higher hepatic V_{T (IDIF)} values for ¹⁸F-FPP-RGD₂ may be a predictor of fibrosis associated with NASH.

The CDHFD-fed rat is a suitable model of NASH because it demonstrates clinically relevant onset and progression of hepatic fibrosis [30, 31]. In the present study, histological and biochemical analysis confirmed the progression of NASH pathology, and the severity of the pathological changes increased alongside increases in hepatic integrin α_v and β₃ protein expression. These findings were in accordance with those of previous studies conducted in similar animal models [20, 30, 32]. Histopathological examination confirmed the development of NASH and fibrosis in CDHFD-fed rats. As shown by Kleiner et al. [33], CDHFD-feeding for 3–4 or 9–10 weeks caused borderline NASH and frank NASH, according to the NAFLD activity score. In addition, the area of fibrosis in the 9–10-week CDHFD-fed group was significantly larger than that in the normal diet-fed group, whereas the area of fibrosis in the 3–4-week CDHFD-fed group was not significantly higher than that in the 3–4-week normal diet-fed group. This indicates that 3–4 weeks of CDHFD feeding results in the development of NAFLD/NASH with minimal or no fibrosis and that 9–10 weeks of feeding results in NASH with moderate fibrosis.

HSC activation plays a pivotal role not only in the onset, but also in the progression of hepatic fibrosis [17]. The integrin α_vβ₃ expression level is low in quiescent HSCs, hepatocytes and non-parenchymal cells [34]. On the other hand, many several studies of liver fibrosis revealed that integrin α_vβ₃ expression level is upregulated on activated HSC [35‐38]. Therefore, integrin α_vβ₃ has been shown to be a biomarker of HSC activation [39]. In the present study, hepatic integrin α_v and β₃ protein expression in CDHFD-fed rats was higher from 3–4 weeks of feeding, prior to the development of significant fibrosis. Therefore, it seems that integrin α_vβ₃ protein expression increases, indicating HSC activation, before the development of fibrosis in rats with CDHFD-induced NASH.

The metabolite analysis confirmed that there is negligible metabolism of ¹⁸F-FPP-RGD₂ in the plasma and liver up to 90 min after intravenously administration. Hepatic TAC fitting revealed that the 2T3C model was more appropriate than the 1T2C model, and this was also quantitatively confirmed using the AIC values. Consistent with the results of a previous study [3], hepatic TACs for ¹⁸F-FPP-RGD₂ peaked rapidly after administration and then gradually decreased until the final time point, implying that ¹⁸F-FPP-RGD₂ kinetics are consistent with a reversible receptor-binding model. Therefore, in the present study, we did not evaluate an irreversible compartment model. These data indicate that ¹⁸F-FPP-RGD₂ binds to integrin α_vβ₃ proteins reversibly and that a 2T3C model is suitable for the analysis of ¹⁸F-FPP-RGD₂ kinetics. Moreover, we found that there is a strong correlation (Spearman's rank correlation r_s = 0.943, P < 0.05) between the V_{T (IDIF)} and V_{T (AIF)}. Therefore, the ¹⁸F-FPP-RGD₂ activity in left ventricular blood as IDIF was used for kinetic analysis, instead of AIF, with the assumption that there was no inter-individual variation in haematocrit.

¹⁸F-labelled RGD, which binds to integrin α_vβ₃, has previously been used to detect liver fibrosis in animal models [19, 20]. A previous study showed the colocalisation of integrin α_vβ₃ with α-SMA in activated HSCs by immunofluorescence staining and that fluorescently labelled RGD binds to activated HSCs [34]. In the present study, the hepatic SUV or V_{T (IDIF)} for ¹⁸F-FPP-RGD₂ positively correlated with hepatic integrin α_v and β₃ protein expression and the hepatic V_T of ¹⁸F-FPP-RGD₂ was reduced by the co-administration of unlabelled RGD. These findings indicate that ¹⁸F-FPP-RGD₂ specifically binds to hepatic integrin α_vβ₃ on HSCs in a rat model of NASH.

Next, we conducted 90-min dynamic PET scans using ¹⁸F-FPP-RGD₂ and determined the most suitable kinetic model for the evaluation of the liver in rats with CDHFD-induced NASH. In the 3–4-week CDHFD-fed groups, the image-derived input function was similar to that of the normal diet-fed groups. However, in the 9–10-week CDHFD-fed group, although there was no difference in the image-derived input function during the late phase, it was slightly lower during the early phase (0–1 min after administration). Moreover, the K₁, extravasation rate of the PET probe in the CDHFD-fed groups was lower than in the respective normal diet-fed groups. The hepatic SUVs of the CDHFD groups were higher than those of the respective normal diet-fed groups and significantly increased with the duration of the feeding period. These data indicate that hepatic integrin α_vβ₃ protein expression in CDHFD-fed rats is higher than that in normal diet-fed rats. The V_{T (IDIF)} for ¹⁸F-FPP-RGD₂, evaluated using the 2T3C model, was higher in the CDHFD-fed groups than in the normal diet-fed groups and strongly positively correlated with hepatic integrin α_v (r_s = 0.717) and β₃ (r_s = 0.644) protein expression. The strengths of these correlations were similar to those between SUV_60–90 min and hepatic integrin α_v (r_s = 0.680) or β₃ (r_s = 0.776) protein expression. Indeed, the clinical usefulness of V_T has recently been reported for pulmonary fibrosis related to other causes than cancer. Both idiopathic pulmonary fibrosis/healthy control V_T ratio and SUV ratio > 1. Thus, the V_T and SUV of [¹⁸F] FB-A20FMDV2 were quantify expression of integrin α_vβ₆ in the lungs of subjects with fibrotic interstitial lung disease.[40]. Our data suggest that hepatic V_T, obtained non-invasively using kinetic modelling, might be useful as a predictor of fibrosis and for determining the efficacy of new drugs in pre-clinical and clinical studies. Moreover, the correlation analyses (Fig. 7) suggest that hepatic SUV, which can be easily obtained using a static scan in the clinic, could be used as a predictor of fibrosis with similar efficacy to hepatic V_T obtained using a dynamic scan.

This study has two limitations. The first is delay or dispersion of input function. Several studies revealed that portal hypertension was induced in NASH patients and diet-induced animal models [41, 42], which lead to cardiovascular dysfunction, such as arterial blood pressure and blood volume decrease [43]. Moreover, the cardiac failure was developed in patients of cirrhosis, due to continuous of hyperdynamic state [44‐46]. Our NASH model also might develop cardiovascular dysfunction and have the decreased input function. But we have estimated the fitting without these points. The hepatic blood supply is derived from two vessels: the hepatic artery and the portal vein. However, in the present study, the left ventricular time-activity curves were used as the IDIF without considering the input function from the portal vein. Although the hepatic V_{T (IDIF)} of ¹⁸F-FPP-RGD₂ strongly positively correlated with hepatic integrin α_v and β₃ protein expression in the present study, further studies are required to evaluate the V_{T (IDIF)} using input functions from both the hepatic artery and portal vein.

Kinetic modelling studies using dynamic ¹⁸F-FPP-RGD₂ PET scans are feasible, on the basis of an image-derived input function, in a diet-induced animal model of NASH. Moreover, the kinetic modelling analysis performed in this study will be useful for the quantitative evaluation of ¹⁸F-FPP-RGD₂ binding to hepatic integrin α_vβ₃ proteins. The present findings indicate that higher hepatic V_T values for ¹⁸F-FPP-RGD₂ may offer novel strategies for the prediction of fibrosis associated with NASH.