cis-4-[18F]fluoro-L-proline Molecular Imaging Experimental Liver Fibrosis

Abstract

Introduction: Early-stage liver fibrosis is potentially reversible, but difficult to diagnose. Clinical management would be enhanced by the development of a non-invasive imaging technique able to identify hepatic injury early, before end-stage fibrosis ensues. The analog of the amino acid proline, cis-4-[¹⁸F]fluoro-L-proline ([¹⁸F]fluoro-proline), which targets collagenogenesis in hepatic stellate cells (HSC), was used to detect fibrosis. Methods: Acute steatohepatitis was induced in experimental animals by liquid ethanol diet for 8 weeks, intra-gastric binge feedings every 10th day along with lipopolysaccharide (LPS) injection. The control animals received control diet for 8 weeks and an equivalent volume of saline on the same schedule as the acute steatohepatitis model. First, in vitro cellular experiments were carried out to assess [³H]proline uptake by HSC, hepatocytes and Kupffer cells derived from rats with acute steatohepatitis (n = 14) and controls (n = 14). Next, ex vivo liver experiments were done to investigate unlabeled proline-mediated collagen synthesis and its associated proline transporter expression in acute steatohepatitis (n = 5) and controls (n = 5). Last, in vivo dynamic and static [¹⁸F]fluoro-proline micro-PET/CT imaging was performed in animal models of acute steatohepatitis (n = 7) and control (n = 7) mice. Results: [³H]proline uptake was 5-fold higher in the HSCs of steatohepatitis rats than controls after incubation of up to 60 min. There was an excellent correlation between [³H]proline uptake and liver collagen expression (r-value > 0.90, p < 0.05). Subsequent liver tissue studies demonstrated 2-3-fold higher proline transporter expression in acute steatohepatitis animals than in controls, and proline-related collagen synthesis was blocked by this transporter inhibitor. In vivo micro-PET/CT studies with [¹⁸F]fluoro-proline showed 2-3-fold higher uptake in the livers of acute steatohepatitis mice than in controls. There was an excellent correlation between [¹⁸F]fluoro-proline uptake and liver collagen expression in the livers of acute steatohepatitis mice (r-value = 0.97, p < 0.001). Conclusion: [¹⁸F]fluoro-proline localizes in the liver and correlates with collagenogenesis in acute steatohepatitis with a signal intensity that is sufficiently high to allow imaging with micro-PET/CT. Thus, [¹⁸F]fluoro-proline could serve as a PET imaging biomarker for detecting early-stage liver fibrosis.

Keywords: PET/CT; cis-4-[18F]fluoro-L-proline; early stage alcoholic liver fibrosis; hepatic stellate cells; molecular imaging; steatohepatitis.

FIGURE 1

[³H]proline uptake by HSCs isolated from healthy rats is incubation time-dependent and liver histology is normal. (A) HSC [³H]proline uptake (n = 5), isolated from 7 healthy rats, was dose-dependent 30 min after incubation with different radioactivity doses of [³H]proline. Counts were acquired using a liquid scintillation counter and calculated into counts per minute with one milligram of HSC protein representing activation of collagen synthesis. ***p < 0.001 or NS vs. [³H]proline uptake by HSC of normal rats at the dose of 37 MBq/L (A). (B) [³H]proline uptake is found to incubation time-dependent after HSCs (n = 5), isolated from 7 chow-fed rats, were incubated with 19 MBq/L of [³H]proline. ***p < 0.001 or NS vs. [³H]proline uptake by HSC at 60 min incubation. (C) representative photomicrographs of H&E staining (A) and reprehensive Trichrome staining (B) of liver tissue sections from healthy rats show no abnormal lipid, inflammatory, or collagen formation.

FIGURE 2

[³H]proline uptake and collagen type 1 expression by hepatic stellate cells (HSCs) isolated from rats with acute steatohepatitis vs. controls. (A) [³H]proline uptake by HSC isolated from 7 acute steatohepatitis rats; each red bar represents one acute steatohepatitis rat. (B) [³H]proline uptake by HSC isolated from 7 control rats; each gray bar indicates one control rat. Liquid scintillation radioactive counts were transformed into counts per minute in one-milligram HSC protein to represent activation of collagen synthesis. (C) collagen type 1 concentrations in culture media from HSCs of 7 individual acute steatohepatitis rats (red bars) using ELISA in the same order as in (A). (D) collagen type 1 concentration in HSC culture media from 7 individual control rats (gray bars) using ELISA with the same animal order as (B). Correlation r-values between [³H]proline uptake and collagen type 1 in acute steatohepatitis (r-value = 0.91) and controls (r-value = 0.93) were derived from the data in (A–D). (E–G) [³H]proline uptake using a liquid scintillation counter, α1(1) procollagen mRNA using RT-PCR and collagen type 1 protein using ELISA looking at HSCs from acute steatohepatitis mice (red straight line bar, n = 7) and controls (gray slanted square bar, n = 7); ***p < 0.001 vs. [³H]proline uptake by HSC isolated from the controls at baseline 0. (H,I), [³H]proline uptake by HSCs isolated from normal rats and treated with different doses (0–20 mg/L, n = 5) of LPS for 60 min and at different time points HSC incubated with 10 mg/L LPS for 0–120 min. NS, no significance, **P < 0.01 and ***p < 0.001 compared with HSCs not receiving [³H]proline (baseline 0 μg/L). (J,K) Collagen type 1 concentrations in control rat HSC culture media that were treated with different doses (0–20 mg/L, n = 5) of LPS and different time points (0–120 min). NS, no significance vs. control with no LPS added (0 μg/mL), **P < 0.01 and ***p < 0.001 vs. the values corresponding control at point of 0. Correlation r-values between [3H]proline uptake and collagen type 1 in relation to LPS dose (r-value = 0.93) and time course (r-value = 0.93) were derived from the data in (H–K). (L–N), [³H]proline uptake, α1(1) procollagen gene expression, and collagen type 1 concentration in HSCs isolated from acute steatohepatitis rats and treated and incubated with/without LPS, respectively; Red straight line bars represent HSC with LPS treatment (n = 5). Gray slanted square bars represent HSC with no LPS treatment (n = 5). ***p < 0.001 HSCs with LPS treatment (red straight bar) or no LPS treatment (gray slanted square bar) isolated from ASH animal livers vs. the corresponding controls.

FIGURE 3

[³H]proline uptake by hepatocytes, Kupffer cells, and HSC isolated from rat with acute steatohepatitis vs. control rats, with liver histology. Photomicrographs of histologic liver sections from rats with steatohepatitis (ASH) vs. control rats. (A,C) H&E stained liver sections from ASH (C) and control (A) rats, 60× magnification. (B,D) Trichrome staining of liver sections from ASH (D) and control (B) rats showing increased collagen staining in the ASH liver section compared with the control section, 60× magnification. (E) [³H]proline uptake by hepatic stellate cells (HSC) was significantly higher in the steatohepatitis rats (n = 7) compared with the control rats (n = 7). ***p < 0.001 representing [³H]proline uptake by HSC between these two groups. Gray square bar, control rats. Red bar, acute steatohepatitis model rats.

FIGURE 4

Collagen and proline transporter mRNA and protein expression in the livers of rats and mice with acute steatohepatitis and the assessment of proline transporter specificity of [³H]proline uptake by using unlabeled fluoro-proline and its transporter inhibitor. (A) Collagen levels in livers of rat (n = 7) and mouse (n = 5) with acute steatohepatitis (ASH) and controls. (B) Proline transporter mRNA expression at gene transcription level, and (C) proline transporter protein levels in livers of rats and mice with ASH. Investigate specificity of [³H]proline uptake in ex vivo liver tissue was investigated. (D) Liver collagen synthesis corresponding to different concentrations of unlabeled fluoro-proline in ASH mice (blue line) compared with control mice (red line), ex vivo. (E) Competitive transportation inhibition of liver [³H]proline uptake when unlabeled fluoro-proline (0–0.8 mM) and 1.85 MBq [³H]proline were incubated together with the liver tissues of mice with ASH (blue line, n = 5) compared with those of control mice (red line, n = 5), ex vivo. (F,G) Inhibition of unlabeled fluoro-proline-mediated liver collagen synthesis by the proline transporter inhibitor benztropine (0–2.0mM) in ASH mice (blue line, n = 5) compared with control mice (red line, n = 5), ex vivo. PT, proline transporter, ASH, acute steatohepatitis, Ctr, control. ***p < 0.001 vs. corresponding controls. NS, no significant difference between rats and mice with ASH.

FIGURE 5

Dynamic and static [¹⁸F]fluoro-proline PET/CT and [¹⁸F]fluoride PET imaging. (A–C) histograms show HPLC analysis of prep-purified tracer, and final purified tracer formulation, co-injected with commercial standard non-radiolabeled fluoro-proline. (D) A region of interest (ROI) of the liver on PET image. (E) ROI of the liver on whole body fused PET/CT image. (F) [¹⁸F]fluoro-proline distribution time course in different organs using dynamic and static PET/CT imaging of normal mice. The representative normal mouse body weight was 31 g and the injected dose was 9 MBq of [¹⁸F]fluoro-proline. (G,H) Comparison between [¹⁸F]fluoro-proline and [¹⁸F]fluoride distributions in the livers and other organs of healthy mice (MIP views are representative of the same normal mouse). The representative mouse body weight was 31 g and the injected dose was 9 MBq of [¹⁸F]fluoro-proline and 10 MBq of [¹⁸F]fluoride at the time of imaging experiments. (I,J) A comparison between the distributions of [¹⁸F]fluoro-proline (I) and [¹⁸F]fluoride (J) in the spinal region (yellow arrow) of the same normal mouse on two axial views. MIP, maximum intensity projection.

FIGURE 6

Histopathology, blood biochemistry, and body weight changes in acute steatohepatitis mouse model compared with control mice. (A,C) Control and acute steatohepatitis (ASH) mouse liver stained with H&E. (B,D), Control and ASH mouse liver stained with Mason’s trichrome stain. (E–H) Summary regarding grades of steatosis (E), inflammation (F), fibrosis (G), and ballooning (H) in livers of ASH mice (red straight line, n = 7) vs. control mice (gray slanted line, n = 7). (I,J) Two plasma hepatic enzymes, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), were measured in the serum of mice with ASH (red straight line, n = 7) vs. control mice (gray slanted line, n = 7). (K,L) Ethanol and LPS plasma levels in mice with ASH (red straight line, n = 7) vs. control mice (gray slanted line, n = 7). (M) Ratio of liver weights to body weights. (N) Body weight changes during the ethanol feeding periods. (O,P) hyaluronic acid (HA) and alpha-2 macroglobulin (A2M) concentrations in mice with ASH (red straight line, n = 7) vs. control mice (gray slanted line, n = 7). *p < 0.05, **p < 0.01 and ***p < 0.001 vs. corresponding controls. NS, no significant difference between mice with ASH and the control mice.

FIGURE 7

Static [¹⁸F]fluoro-proline fused PET/CT imaging in acute steatohepatitis mouse model compared with control mice. (A–C) Sagittal, coronal and transaxial views of [¹⁸F]fluoro-proline positron emission tomography/computed tomography (CT) (PET/CT) imaging in control mouse. (D–F) Sagittal, coronal and axial views of PET/CT imaging in mouse with acute steatohepatitis. Images were performed 60 min after [¹⁸F]fluoro-proline was administrated via the tail vein and total imaging length was acquired for 30 min. Green arrow indicates livers of the animals. The representative mouse body weights were 32 g of control mouse and 32 g of ASH mouse and the injected doses were 10 MBq, respectively, of [¹⁸F]fluoro-proline at the time of imaging experiments.

FIGURE 8

[¹⁸F]fluoro-proline PET imaging quantification of liver [¹⁸F]fluoro-proline uptake in acute steatohepatitis mice vs. control mice. (A) Voxel intensity of [¹⁸F]fluoro-proline activity in lung, pancreas, and liver and other 9 organs of acute steatohepatitis (ASH) mice (red column, n = 4) and control mice (gray column, n = 4). (B) Percentage of injected doses per gram body weight of [¹⁸F]fluoro-proline in livers of ASH mice model and control mice. (C) [¹⁸F]fluoro-proline activity in SUVmax in the livers of ASH mouse model and its controls. (B,C) Collagen type 1 concentrations in livers of these two groups (n = 7). ***p < 0.001 vs. corresponding controls.