Elsevier

Journal of Hepatology

Volume 73, Issue 1, July 2020, Pages 161-169
Journal of Hepatology

Research Article
[18F]-Alfatide PET imaging of integrin αvβ3 for the non-invasive quantification of liver fibrosis

https://doi.org/10.1016/j.jhep.2020.02.018Get rights and content

Highlights

  • [18F]-Alfatide was demonstrated to bind specifically with integrin αvβ3 (mainly expressed on activated HSCs).

  • [18F]-Alfatide can detect fibrosis progression both in animal liver fibrotic models and human liver tissues.

  • Imaging integrin αvβ3 with PET/[18F]-Alfatide offers a potential non-invasive method for monitoring fibrosis progression.

Background & Aims

The vitronectin receptor integrin αvβ3 drives fibrogenic activation of hepatic stellate cells (HSCs). Molecular imaging targeting the integrin αvβ3 could provide a non-invasive method for evaluating the expression and the function of the integrin αvβ3 on activated HSCs (aHSCs) in the injured liver. In this study, we sought to compare differences in the uptake of [18F]-Alfatide between normal and injured liver to evaluate its utility for assessment of hepatic fibrogenesis.

Methods

PET with [18F]-Alfatide, non-enhanced CT, histopathology, immunofluorescence staining, immunoblotting and gene analysis were performed to evaluate and quantify hepatic integrin αvβ3 levels and liver fibrosis progression in mouse models of fibrosis (carbon tetrachloride [CCl4] and bile duct ligation [BDL]). The liver AUC divided by the blood AUC over 30 min was used as an integrin αvβ3–PET index to quantify fibrosis progression. Ex vivo analysis of frozen liver tissue from patients with fibrosis and cirrhosis verified the animal findings.

Results

Fibrotic mouse livers showed enhanced [18F]-Alfatide uptake and retention compared to control livers. The radiotracer was demonstrated to bind specifically with integrin αvβ3, which is mainly expressed on aHSCs. Autoradiography and histopathology confirmed the PET imaging results. Further, the mRNA and protein level of integrin αvβ3 and its signaling complex were higher in CCl4 and BDL models than controls. The results obtained from analyses on human fibrotic liver sections supported the animal findings.

Conclusions

Imaging hepatic integrin αvβ3 with PET and [18F]-Alfatide offers a potential non-invasive method for monitoring the progression of liver fibrosis.

Lay summary

Integrin αvβ3 expression on activated hepatic stellate cells (aHSCs) is associated with HSC proliferation during hepatic fibrogenesis. Herein, we show that a radioactive tracer, [18F]-Alfatide, binds to integrin αvβ3 with high affinity and specificity. [18F]-Alfatide could thus be used as a non-invasive imaging biomarker to track hepatic fibrosis progression.

Introduction

Liver fibrosis is a wound-healing process characterized by the accumulation of extracellular matrix (ECM) proteins in response to chronic injury.1 The accumulation of ECM proteins (mainly collagen type I and III) distorts the normal hepatic architecture and forms fibrous scars. As collagen deposition increases, nodules of regenerating hepatocytes result in cirrhosis with its high morbidity and mortality.2 Current methods for evaluating liver fibrosis often provide an incomplete picture of disease. Procedures and biomarkers for determining fibrosis include ultrasonography, elastography, age, platelet count, albumin, and serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) values.3,4 Even when these techniques are incorporated into multifactorial scoring systems, they still suffer from low sensitivity and specificity, and often do not correlate with the degree of fibrosis.5,6 Liver biopsy is the gold standard for assessing fibrosis, but unfortunately this technique samples a limited hepatic volume and can only be performed at a limited frequency due to concerns regarding complication rates.7

There is reason to believe that hepatic integrin αvβ3 may be a useful surrogate diagnostic biomarker for the extent of fibrosis. Hepatic stellate cells (HSCs) are important collagen-producing cells and αvβ3 expression on activated HSCs is associated with HSC proliferation during fibrogenesis.8,9 In addition, imaging hepatic integrin αvβ3 may provide information about therapeutic strategies designed to reduce collagen-producing activated HSCs (aHSCs), either by disrupting αvβ3-ECM interactions or through other approaches.10

The PET tracer used here to image hepatic integrin αvβ3, [18F]-Alfatide, has several advantages. Developed by Chen and coworkers at the NIH,11,12 [18F]-Alfatide is a dimer of RGD peptides, utilizing multivalent affinity enhancement to increase its interactions with its integrin target.[13], [14], [15] Second, [18F]-Alfatide features a radiosynthesis whereby the fluoride ion (18F-) tenaciously binds the aluminum of an Al3+: NOTA complex rather than undergoing formation of covalent 18F-C bond.12,16,17 [18F]-Alfatide's relatively simple radiosynthesis is an advantage when multisite clinical trials are considered. Finally, [18F]-Alfatide has been used in several clinical studies.[18], [19], [20]

Thus, a series of factors, the shortcomings of current liver fibrosis diagnostic methods, the importance of αvβ3 expression on aHSCs for collagen synthesis, the simplicity of [18F]-Alfatide radiochemistry and the prior clinical uses of [18F]-Alfatide, all suggest an [18F]-Alfatide/PET combination might be useful for imaging hepatic integrin αvβ3. To further this possibility, we employed [18F]-Alfatide/PET with the carbon tetrachloride (CCl4) and bile duct ligation (BDL) mouse models of hepatic disease. With both models, the 18F-Alfatide/PET technique imaged hepatic integrin αvβ3, and integrin αvβ3 expression correlated with disease severity and the degree of fibrosis. These results suggest [18F]-Alfatide/PET may offer a non-invasive imaging modality for quantifying hepatic αvβ3, which serves as a surrogate PET biomarker for liver fibrosis in clinical settings.

Section snippets

Animal models of liver fibrosis

We studied liver fibrosis in 2 experimental models. In the first model, 10-week-old CD1 male mice were treated with CCl4 (Sigma, St. Louis, MO) at a dose of 0.2 ml/kg in a 1:5 CCl4: olive oil mixture, administered by means of intraperitoneal (i.p.) injection twice a week for 12 weeks. Control animals were treated with vehicle (olive oil). In the second, BDL-induced biliary fibrosis model, mice were transected at the common bile duct after midline laparotomy. Sham-treated mice underwent midline

Designation of mild and severe levels of liver fibrosis in animal models

Following 6 weeks of CCl4 administration, a mild infiltration of inflammatory cells along with aggregated lymphocytes was observed in the portal areas (indicated by arrows, Fig. 1A,b) by H&E staining. With Sirius red, fibrotic lesions stained weakly in the portal area (mild fibrosis, Fig. 1A,e), and inflammatory cells and formation of regenerative nodules of liver parenchyma separated by fibrotic septa were observed (severe fibrosis, Fig. 1A,c,f). For the BDL model, at 10-day post operation,

Discussion

To our knowledge, this is the first study that systematically validates [18F]-Alfatide imaging in liver fibrosis by performing a spatially precise correlative analysis of PET/CT imaging, histopathology, integrin αvβ3 expression and autoradiography examination in both preclinical rodent models and clinical fibrotic liver specimens. We employed 2 commonly used fibrosis models: CCl4 intoxication and surgical BDL. CCl4 toxicity results from the generation of trichloromethyl (CCl3) radicals by

Financial support

The work presented in this study was supported by NIH grants R01AI136715, R01DK108370.

Authors' contributions

T.S. designed the study, performed experiments and drafted manuscript, Z.C., V.B., H.F., X.D., J.R., Q.Y., L.L., S.R., W.L., and L.J. performed experiments, X.W., V.K. and A.S. performed shear wave elastography experiments, S.R. provided some samples. X.C., R.C. and S.L. conceived project, designed the study and wrote the manuscript.

Conflict of interest

The authors declare no conflicts of interest that pertain to this work.

Please refer to the accompanying ICMJE disclosure forms for further details.

References (39)

  • D.C. Rockey et al.

    Noninvasive measures of liver fibrosis

    Hepatology

    (2006)
  • D.C. Rockey et al.

    Liver biopsy

    Hepatology

    (2009)
  • K. Patel et al.

    Clinical utility of biomarkers of liver fibrosis

    Gastroenterol Hepatol

    (2006)
  • H.T. Schon et al.

    Pharmacological Intervention in hepatic stellate cell activation and hepatic fibrosis

    Front Pharmacol

    (2016)
  • N. Guo et al.

    Quantitative analysis and comparison study of [18F]AlF-NOTA-PRGD2, [18F]FPPRGD2 and [68Ga]Ga-NOTA-PRGD2 using a reference tissue model

    PLoS One

    (2012)
  • L. Lang et al.

    Comparison study of [18F]FAl-NOTA-PRGD2, [18F]FPPRGD2, and [68Ga]Ga-NOTA-PRGD2 for PET imaging of U87MG tumors in mice

    Bioconjug Chem

    (2011)
  • M. Mammen et al.

    Polyvalent interactions in biological systems: implications for design and use of multivalent ligands and inhibitors

    Angew Chem Int Ed Engl

    (1998)
  • X. Montet et al.

    Multivalent effects of RGD peptides obtained by nanoparticle display

    J Med Chem

    (2006)
  • S. Liu

    Radiolabeled multimeric cyclic RGD peptides as integrin alphavbeta3 targeted radiotracers for tumor imaging

    Mol Pharm

    (2006)
  • Cited by (15)

    • Liver fibrosis alters the molecular structures of hepatic glycogen

      2022, Carbohydrate Polymers
      Citation Excerpt :

      TP and ALB did not show a significant difference between LF and control rats (Fig. 1C and Fig. 1D), while a higher GLB content was observed in the serum of LF rats (Fig. 1E). However, higher concentrations of ALT, AST and TBIL (Fig. 1F–H) were seen in the serum samples of LF rats, which indicates hepatocellular damage (Ruart et al., 2019; Shao et al., 2020). Results showed that liver from LF rats displayed an abnormal liver architecture compared with the controls that did not show liver damage.

    • [<sup>18</sup>F]MAGL-4-11 positron emission tomography molecular imaging of monoacylglycerol lipase changes in preclinical liver fibrosis models

      2022, Acta Pharmaceutica Sinica B
      Citation Excerpt :

      Until now, liver biopsy has been the gold standard for identifying liver fibrosis but has well-known limitations. Other procedures include ultrasonography and elastography still suffer from low sensitivity and specificity4. Accordingly, the development of novel PET imaging agents with alternative binding mechanisms and/or differential signaling transduction pathways is of particular interest for the physiologic processes and treatment of liver fibrosis.

    View all citing articles on Scopus

    Author names in bold designate shared co-first authorship

    View full text