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Advances in functional and molecular MRI technologies in chronic liver diseases

  • Iris Y. Zhou
    Affiliations
    Athinoula A. Martinos Center for Biomedical Imaging, Charlestown, MA, United States

    Harvard Medical School, Boston, MA, USA

    Institute for Innovation in Imaging (i3), Department of Radiology, Massachusetts General Hospital, Charlestown, MA, USA
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  • Onofrio A. Catalano
    Affiliations
    Athinoula A. Martinos Center for Biomedical Imaging, Charlestown, MA, United States

    Harvard Medical School, Boston, MA, USA

    Division of Abdominal Imaging, Department of Radiology, Massachusetts General Hospital, Boston, MA, United States
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  • Peter Caravan
    Correspondence
    Corresponding author. Address: 149 13th St, Boston, MA 02129. Tel.: 617-643-0193.
    Affiliations
    Athinoula A. Martinos Center for Biomedical Imaging, Charlestown, MA, United States

    Harvard Medical School, Boston, MA, USA

    Institute for Innovation in Imaging (i3), Department of Radiology, Massachusetts General Hospital, Charlestown, MA, USA
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      Summary

      MRI has emerged as the most comprehensive non-invasive diagnostic tool for liver diseases. In recent years, the value of MRI in hepatology has been significantly enhanced by a wide range of contrast agents, both clinically available and under development, that add functional information to anatomically detailed morphological images, or increase the distinction between normal and pathological tissues by targeting molecular and cellular events. Several classes of contrast agents are available for contrast-enhanced hepatic MRI, including i) conventional non-specific extracellular fluid contrast agents for assessing tissue perfusion; ii) hepatobiliary-specific contrast agents that are taken up by functioning hepatocytes and excreted through the biliary system for evaluating hepatobiliary function; iii) superparamagnetic iron oxide particles that accumulate in Kupffer cells; and iv) novel molecular contrast agents that are biochemically targeted to specific molecular/cellular processes for staging liver diseases or detecting treatment responses. The use of different functional and molecular MRI methods enables the non-invasive assessment of disease burden, progression, and treatment response in a variety of liver diseases. A high diagnostic performance can be achieved with MRI by combining imaging biomarkers.

      Keywords

      Introduction

      Chronic liver disease (CLD) is a major public health problem worldwide. The major causes of CLD include hepatic viral infections, chronic exposure to toxins or drugs (e.g., alcohol abuse), chronic metabolic alterations, and persistent autoimmune reaction. CLD may induce steatosis, iron dysregulation, inflammation and/or fibrogenesis. Liver fibrosis, a common feature of almost all causes of progressive CLD, involves the accumulation of collagen, proteoglycans, and other macromolecules within the extracellular matrix. Without removal of exposure to the specific aetiology, fibrosis tends to progress, leading to cirrhosis, hepatic dysfunction, and portal hypertension. Therefore, it is crucial to be able to differentiate between the stages of liver fibrosis, since treatment decisions and monitoring algorithms are related to the degree of fibrosis.
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      The value of MRI in the liver is significantly enhanced by a wide range of contrast agents, both clinically available and under development, that increase the distinction between normal and pathological tissues and can be used to assess liver function.
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      and MR elastography
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      MR elastography of the liver: preliminary results.
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      have been applied to assess liver fibrosis. Besides these endogenous contrast mechanisms, a wide range of contrast agents, both clinically available and under development, add functional information to the anatomically detailed morphological images, or increase the distinction between normal and pathological tissues by targeting molecular and cellular events. The diagnostic value of contrast-enhanced MRI in liver lesion detection and characterisation has been extensively reviewed
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      Magnetic resonance imaging of liver tumors.
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      Focal liver lesions: practical magnetic resonance imaging approach.
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      Hepatobiliary-specific MR contrast agents: role in imaging the liver and biliary tree.
      and will not be discussed here. In this review, we will provide an overview of the MRI contrast agents available for functional and molecular liver MRI in the context of diffuse liver diseases, as well as discussing their use in clinical and preclinical studies of CLDs and looking at their future clinical applications.
      Dynamic contrast-enhanced MRI with conventional gadolinium-based extracellular fluid intravenous contrast agents is used to detect changes in perfusion, blood flow, and vascularity associated with liver lesions, cirrhosis or chronic liver disease.

      Classification of MRI contrast agents for liver imaging

      Contrast agents used in hepatobiliary imaging can be classified into 4 major categories depending on their mechanism of biodistribution: i) conventional non-specific extracellular fluid (ECF) contrast agents that accumulate in the vascular space and extracellular extravascular space by a passive distribution mechanism; ii) hepatobiliary contrast agents that are taken up specifically by hepatocytes and partially excreted through the biliary system; iii) superparamagnetic iron oxide (SPIO) particles that are taken up by the reticuloendothelial system (RES), particularly by resident hepatic macrophages and Kupffer cells; iv) molecular contrast agents that are biochemically targeted to specific molecular processes. Table 1 lists examples of contrast agents from each category. The majority of these agents are gadolinium (Gd) chelates.
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      Gd3+ is highly paramagnetic resulting in a shortening of the T1 and T2 relaxation times of nearby water protons. The T1 shortening effect of Gd chelates predominates under most conditions, which increases the signal intensity of T1-weighted MRI scan in regions where the agents are distributed.
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      ,
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      Contrast agents for MR imaging of the liver.
      Therefore, the Gd-based contrast agents are also called positive contrast agents. SPIO particles, on the other hand, are referred to as negative contrast agents owing to their susceptibility effect, which causes local magnetic field inhomogeneities and a resultant signal reduction on T2- or T2∗-weighted images.
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      Table 1Functional and molecular MRI contrast agents for imaging liver diseases.
      Agent (trade name)Distribution type/targetDevelopment stageIndication
      Gd-DTPA (Magnevist)ECFApproved (1988); suspended (EU 2017)Liver lesions
      Gd-DOTA (Dotarem, Clariscan)ECFApproved (EU 1989; US 2013)Liver lesions
      Gd-HPDO3A (ProHance)ECFApproved (1992)Liver lesions
      Gd-DO3A-butrol (EU: Gadovist; US: Gadavist)ECFApproved (EU 1998; US 2011)Liver lesions
      Gd-BOPTA (Multihance)ECF/hepatobiliaryApproved (EU 2000; US 2004)Liver lesions; hepatocyte function
      Gd-EOB-DTPA (EU: Primovist; US: Eovist)HepatobiliaryApproved (EU 2004; US 2008)Liver lesions; hepatocyte function; liver fibrosis/cirrhosis (animal studies)
      EP-3533Type I collagenAnimal studies (CCl4, BDL, DEN, CDAHFD); treatment response (rapamycin, farnesoid X receptor agonist, PPAR-α/δ agonist)Liver fibrosis
      CM-101Type I collagenAnimal studies (BDL, CCl4)Liver fibrosis
      ProCA32.collagen1Type I collagenAnimal studies (TAA/alcohol, DEN, NASH diet)Liver fibrosis
      Gd-ESMAElastinAnimal studies (CCl4)Liver fibrosis
      Gd-HydAllysine/oxidised collagenAnimal studies (CCl4, CDAHFD); treatment response (farnesoid X receptor agonist, PPAR-α/δ agonist)Liver fibrogenesis, NASH
      c(RGDyC)-USPIOIntegrin αvβ3 (HSCs)Animal studies (CCl4)Liver fibrogenesis
      Gd-PFibrin-fibronectinAnimal studies (CCl4)Liver fibrosis
      EP-2104RFibrinAnimal studies (DEN)Liver inflammation
      MPO-GdMyeloperoxidaseAnimal studies (MCD diet); human biopsy samplesLiver inflammation, NASH
      SPIO (Endorem, Feridex; Resovist)RES/Kupffer cellsApproved but withdrawn or discontinuedLiver lesions, fibrosis
      USPIO ferumoxytol (Feraheme)RES/Kupffer cellsApproved (EU 2013; US 2009) only for use in iron replacement therapyLiver lesions
      BDL, bile duct ligation; CCl4, carbon tetrachloride; CDAHFD, choline-deficient L-amino acid-defined high-fat diet; DEN, diethylnitrosamine; ECF, extracellular fluid; HSC, hepatic stellate cell; MCD, methionine and choline-deficient; NASH, non-alcoholic steatohepatitis; RES, reticuloendothelial system; TAA, thioacetamide.

      Extracellular fluid agents

      ECF agents are small, hydrophilic chelates of Gd3+ that exhibit no protein binding and are used to measure the perfusion, blood flow, and vascularity of liver tissue. ECF agents enter the liver via the hepatic artery and portal vein, rapidly distribute throughout the extracellular space, and are excreted almost entirely by glomerular filtration.
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      Focal liver lesions: practical magnetic resonance imaging approach.
      ,
      • Gandhi S.N.
      • Brown M.A.
      • Wong J.G.
      • Aguirre D.A.
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      MR contrast agents for liver imaging: what, when, how.
      ,
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      • Press W.R.
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      Characteristics of gadolinium-DTPA complex: a potential NMR contrast agent.
      Compared to iodinated CT contrast agents, Gd-based ECF agents are detected with orders of magnitude better sensitivity,
      • Balci N.C.
      • Semelka R.C.
      Contrast agents for MR imaging of the liver.
      which allows for better enhancement of the blood pool at equilibrium phase and better delineation of subtle areas of contrast agent accumulation.
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      CT and MR imaging of benign hepatic and biliary tumors.
      ,
      • Nelson R.C.
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      Delayed magnetic resonance hepatic imaging with gadolinium-DTPA.
      ECF agents are the least expensive and the most commonly used contrast agents, and are generally considered safe when administered at low dosage.
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      Risk of nephrogenic systemic fibrosis: evaluation of gadolinium chelate contrast agents at four American universities.
      ,
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      • Almen T.
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      • Bertolotto M.
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      Nephrogenic systemic fibrosis and gadolinium-based contrast media: updated ESUR Contrast Medium Safety Committee guidelines.
      The ECF agents can be categorised as linear or macrocyclic based on their chemical structure. Macrocyclic agents are considered safer than linear agents with respect to any release of Gd ion into the body and are now more widely used.

      Hepatobiliary agents

      Hepatobiliary agents are paramagnetic compounds taken up by functioning hepatocytes and excreted in bile. Hepatobiliary agents have been available for over 20 years, starting from the now discontinued mangafodipir trisodium (Mn-DPDP). Mn-DPDP is the only manganese-based contrast agent ever approved for clinical use, but it was withdrawn from all markets owing to a lack of commercial success. It was administered by slow infusion and provided a strong positive liver signal but was not suitable for dynamic imaging. The 2 hepatobiliary agents currently available, gadoxetic acid (Gd-EOB-DTPA) and gadobenate dimeglumine (Gd-BOPTA), are both Gd based and exhibit higher T1 relaxivity (the ability to change T1 relaxation) than Mn-DPDP or Gd-based ECF agents. Gd-EOB-DTPA is an amphiphilic Gd chelate first described in 1991.
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      • Gries H.
      A new lipophilic gadolinium chelate as a tissue-specific contrast medium for MRI.
      Unlike ECF agents, it displays liver-specific properties based on organic anion-transporting polypeptide (OATP1B1 and B3)-mediated hepatic uptake and multidrug resistance protein (MRP2)-mediated biliary excretion by hepatocytes
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      Primovist, Eovist: what to expect?.
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      Visualization of hepatic uptake transporter function in healthy subjects by using gadoxetic acid-enhanced MR imaging.
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      Characterization of the intestinal and hepatic uptake/efflux transport of the magnetic resonance imaging contrast agent gadolinium-ethoxylbenzyl-diethylenetriamine-pentaacetic acid.
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      • et al.
      Expression of OATP1B3 determines uptake of Gd-EOB-DTPA in hepatocellular carcinoma.
      (Fig. 1A). In healthy individuals, the elimination is 50% hepatic and 50% by glomerular filtration.
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      Phase II clinical evaluation of Gd-EOB-DTPA: dose, safety aspects, and pulse sequence.
      ,
      • Hamm B.
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      Phase I clinical evaluation of Gd-EOB-DTPA as a hepatobiliary MR contrast agent: safety, pharmacokinetics, and MR imaging.
      Under pathological conditions, there are 2 main factors that determine the cellular uptake and excretion of Gd-EOB-DTPA. Alterations in expression of OATPs will cause altered uptake of the contrast agent, while the intracellular concentration of Gd-EOB-DTPA after it enters the hepatocyte is strongly influenced by the ability of MRPs to excrete the contrast agent into the bile ducts or back into the sinusoids. Gd-BOPTA shares several properties with Gd-EOB-DTPA, including initial distribution in the ECF compartment and being taken up via OATPs on hepatocytes. Therefore, both can be used as ECF agents for hepatic arterial and portal venous phase imaging and as a hepatobiliary agents for delayed hepatocellular phase imaging. While Gd-BOPTA is administered at a 4-fold higher dose than Gd-EOB-DTPA, only 3–5% of Gd-BOPTA is cleared via biliary excretion, significantly lower than the 50% for Gd-EOB-DTPA. Because of its greater hepatic uptake, Gd-EOB-DTPA results in greater hepatocellular phase enhancement
      In contrast to global liver function tests such as ICG-R15, dynamic contrast-enhanced MRI with hepatocyte-specific contrast agents such as Gd-EOB-DTPA offers regional information about hepatocyte transport function during the hepatobiliary phase, improving the assessment of diseased hepatobiliary systems.
      than Gd-BOPTA and is thus a preferred hepatobiliary agent. In addition, Gd-EOB-DTPA is cleared from the body more rapidly, according to the elimination half-lives (1 hour for Gd-EOB-DTPA and 1–2 hours for Gd-BOPTA).
      Figure thumbnail gr1
      Fig. 1Uptake and excretion mechanism of hepatobiliary agent Gd-EOB-DTPA and Gd-EOB-DTPA-enhanced MRI for assessment of hepatocyte transport function.
      (A) Diagram shows the hepatocyte uptake and biliary excretion mechanism of Gd-EOB-DTPA. Axial T1-weighted fat saturated 3D gradient echo images show hepatobiliary phase of enhancement imaged at 20 minutes post administration for Gd-EOB-DTPA for (B) a healthy individual and (C) a patient with NASH-induced cirrhosis. The biliary ducts filled by Gd-EOB-DTPA are indicated by white arrowheads. The morphologic stigmata of cirrhosis such as hypertrophy of the caudate lobe and left lobe of the liver are indicated by yellow arrows. (D) Hepatocyte uptake ratio map in a 57-year-old man with hepatitis B cirrhosis and Child-Pugh class A disease reveals a hepatocyte uptake ratio value of 3.64; indocyanine green retention test is 14.6%. (E) Hepatocyte uptake ratio map in a 55-year-old man with hepatitis B cirrhosis and Child-Pugh class A disease reveals a hepatocyte uptake ratio value of 1.58; indocyanine green retention test is 22.9%. (A created with BioRender.com; D and E adapted from.
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      Quantitative assessment of liver function by using gadoxetic acid-enhanced MRI: hepatocyte uptake ratio.
      ) Gd-EOB-DTPA, gadoxetic acid; NASH, non-alcoholic steatohepatitis.

      Superparamagnetic iron oxide particles

      SPIO particles are composed of small crystalline cores of iron oxides coated with a biocompatible material, e.g. dextran or citrate, to improve solubility and tolerability. Typically given as a slow infusion, SPIOs are phagocytosed by macrophages throughout the body, and especially by Kupffer cells which line the hepatic sinusoids.
      • Sjogren C.E.
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      • Sontum P.C.
      • Briley-Saebo K.
      • Fahlvik A.K.
      Crystal size and properties of superparamagnetic iron oxide (SPIO) particles.
      SPIO contrast agents can be classified in 2 groups, superparamagnetic iron oxide particles (SPIO, <150 nm in diameter) and ultra-small superparamagnetic iron oxide particles (USPIO, <30 nm in diameter).
      • Ferrucci J.T.
      • Stark D.D.
      Iron oxide-enhanced MR imaging of the liver and spleen: review of the first 5 years.
      ,
      • Fretz C.J.
      • Elizondo G.
      • Weissleder R.
      • Hahn P.F.
      • Stark D.D.
      • Ferrucci Jr., J.T.
      Superparamagnetic iron oxide-enhanced MR imaging: pulse sequence optimization for detection of liver cancer.
      ,
      • Weissleder R.
      • Elizondo G.
      • Wittenberg J.
      • Rabito C.A.
      • Bengele H.H.
      • Josephson L.
      Ultrasmall superparamagnetic iron oxide: characterization of a new class of contrast agents for MR imaging.
      Typically, SPIOs of >80 nm diameter are rapidly taken up by the RES, while smaller USPIOs can evade macrophage capture and exhibit a longer blood circulation time of hours to days. While several SPIOs have been approved as liver-specific contrast agents, they have since been withdrawn from one or all major markets due to poor sales. The USPIO ferumoxytol is currently available as an iron replacement therapy, but it sees some off-label use as a contrast agent. Compared to liver-specific SPIOs used previously, uptake of ferumoxytol into Kupffer cells is slow.

      Molecularly targeted agents

      Molecularly targeted probes are small molecules, peptides, or antibodies that recognise a specific protein, receptor, or biological process. They are tagged with a contrast generating moiety, such as chelated Gd for MRI visualisation. There has been considerable effort to develop targeted MRI probes for non-invasive imaging of fibrotic or inflammatory molecular pathways, common features shared by almost all causes of progressive CLDs. While still under preclinical development, targeted probes provide unique means to characterise and quantify dysregulated molecular events, playing an important role in optimising the drug discovery and validation processes. Their clinical translation, if successful, can profoundly impact the detection, staging, prognosis, and treatment monitoring of disease, as well as elucidating complex biology.

      Functional MRI of diffuse liver diseases

      Perfusion imaging

      Dynamic contrast-enhanced (DCE) MRI with ECF agents has become an integral part of clinical liver MRI, providing information on changes in tissue perfusion. A volumetric T1-weighted fat-suppressed gradient echo technique is commonly used to achieve high spatial resolution and sufficient temporal resolution in DCE MRI. To ensure alignment of the liver in serial images, motion control is achieved either prospectively using respiratory control techniques, e.g. multiple breath holds, navigated acquisition, and respiratory triggering, or retrospectively by image registration.
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      Fundamentals of tracer kinetics for dynamic contrast-enhanced MRI.
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      • Koh D.M.
      Functional imaging of the liver.
      Fat suppression is necessary to reduce the high T1-weighted signal from subcutaneous and juxtadiaphragmatic fat stores that is responsible for chemical shift artifact and is often visible as motion artifact.
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      Magnetic resonance imaging of the liver: sequence optimization and artifacts.
      Perfusion MR parameters can be derived using model-free or model-based techniques.
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      Perfusion magnetic resonance imaging of the liver.
      Model-free analysis refers to the parameters extracted from time-signal intensity curves, including area under the curve (AUC), time to peak, peak enhancement, wash-in slope, etc. The AUC over 60–120 seconds from the onset of contrast enhancement is a widely used parameter, which represents the leakage of contrast into the extracellular space and therefore, indirectly reflects tissue perfusion. Model-based approaches involve curve fitting of a dual-input single-compartmental model.
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      ,
      • Materne R.
      • Smith A.M.
      • Peeters F.
      • Dehoux J.P.
      • Keyeux A.
      • Horsmans Y.
      • et al.
      Assessment of hepatic perfusion parameters with dynamic MRI.
      The model-derived parameters quantify the pharmacokinetic distribution of the agent in physiological terms like arterial and portal venous blood flow, distribution volume, and mean transit time, although it should be noted that several assumptions underlie the model-based approaches, which can affect the accuracy of the extracted parameters.
      Quantitative DCE MRI was used in research trials to assess the microcirculatory changes that occur in liver fibrosis and cirrhosis. The deposition of collagen in the space of Disse and sinusoidal capillarisation results in an increased resistance to incoming sinusoidal blood flow,
      • Pandharipande P.V.
      • Krinsky G.A.
      • Rusinek H.
      • Lee V.S.
      Perfusion imaging of the liver: current challenges and future goals.
      which leads to a decrease in portal venous flow to the liver, an increase in hepatic arterial flow, and the subsequent formation of intrahepatic and portosystemic shunts.
      • Faria S.C.
      • Ganesan K.
      • Mwangi I.
      • Shiehmorteza M.
      • Viamonte B.
      • Mazhar S.
      • et al.
      MR imaging of liver fibrosis: current state of the art.
      Transfer of the ECF agent from the vascular sinusoids into the interstitial space is thus increasingly impeded by liver fibrosis.
      • Thng C.H.
      • Koh T.S.
      • Collins D.J.
      • Koh D.M.
      Perfusion magnetic resonance imaging of the liver.
      In patients with cirrhosis, decreased portal and total hepatic perfusion is observed, as well as increased arterial perfusion and mean transit time, with preserved or increased distribution volume. These perfusion changes already occur at intermediate stages of liver fibrosis, but are more marked in cirrhosis, where they correlate with the degree of liver dysfunction and portal hypertension.
      • Annet L.
      • Materne R.
      • Danse E.
      • Jamart J.
      • Horsmans Y.
      • Van Beers B.E.
      Hepatic flow parameters measured with MR imaging and Doppler US: correlations with degree of cirrhosis and portal hypertension.
      A dual-input, single-compartment, model-based study reported an increase in absolute arterial blood flow, arterial fraction, distribution volume, and mean transit time and a decrease in portal venous fraction, in patients with advanced liver fibrosis compared to patients with early-stage liver fibrosis.
      • Hagiwara M.
      • Rusinek H.
      • Lee V.S.
      • Losada M.
      • Bannan M.A.
      • Krinsky G.A.
      • et al.
      Advanced liver fibrosis: diagnosis with 3D whole-liver perfusion MR imaging--initial experience.
      Among all the estimated perfusion parameters, the distribution volume had the best performance in predicting advanced liver fibrosis, with a sensitivity of 77% and a specificity of 79%.

      Imaging hepatocyte function

      Active transport of a hepatobiliary contrast agent into the hepatocytes offers a unique means for functional assessment of the liver. In addition to the initial dynamic phase, a hepatobiliary phase (HBP) of enhancement is imaged at about 20 minutes post contrast administration for Gd-EOB-DTPA and at 1–2 hours post Gd-BOPTA. During the HBP, only the liver parenchyma (not the vessels) is enhanced, while impaired hepatocytes commonly show hypointensity.
      • Van Beers B.E.
      • Pastor C.M.
      • Hussain H.K.
      Primovist, Eovist: what to expect?.
      Signal enhancement in HBP images reflects the balance between uptake and excretory transporters. It also reflects the total number of hepatocytes; for instance, in fibrosis and cirrhosis decreased relative liver enhancement (RLE) results from the loss of some normal hepatocytes due to fibrotic replacement. Based on a variety of pharmacokinetic properties, including liver perfusion, vascular permeability, extracellular diffusion, and hepatocyte transporter expression, the hepatobiliary contrast agents (particularly Gd-EOB-DTPA) simultaneously provide a combination of morphologic and functional information in hepatobiliary systems.
      • Van Beers B.E.
      • Pastor C.M.
      • Hussain H.K.
      Primovist, Eovist: what to expect?.
      ,
      • Choi Y.
      • Huh J.
      • Woo D.C.
      • Kim K.W.
      Use of gadoxetate disodium for functional MRI based on its unique molecular mechanism.
      ,
      • Geier A.
      • Dietrich C.G.
      • Voigt S.
      • Kim S.K.
      • Gerloff T.
      • Kullak-Ublick G.A.
      • et al.
      Effects of proinflammatory cytokines on rat organic anion transporters during toxic liver injury and cholestasis.
      Typically, liver function assessment is performed using the signal enhancement on the HBP images acquired at 20 minutes post Gd-EOB-DTPA, with some studies using the indocyanine green retention test (ICG-R15) or clinical liver function for comparison.
      • Seale M.K.
      • Catalano O.A.
      • Saini S.
      • Hahn P.F.
      • Sahani D.V.
      Hepatobiliary-specific MR contrast agents: role in imaging the liver and biliary tree.
      ,
      • Van Beers B.E.
      • Pastor C.M.
      • Hussain H.K.
      Primovist, Eovist: what to expect?.
      ,
      • Haimerl M.
      • Schlabeck M.
      • Verloh N.
      • Zeman F.
      • Fellner C.
      • Nickel D.
      • et al.
      Volume-assisted estimation of liver function based on Gd-EOB-DTPA-enhanced MR relaxometry.
      • Kukuk G.M.
      • Schaefer S.G.
      • Fimmers R.
      • Hadizadeh D.R.
      • Ezziddin S.
      • Spengler U.
      • et al.
      Hepatobiliary magnetic resonance imaging in patients with liver disease: correlation of liver enhancement with biochemical liver function tests.
      • Yamada A.
      • Hara T.
      • Li F.
      • Fujinaga Y.
      • Ueda K.
      • Kadoya M.
      • et al.
      Quantitative evaluation of liver function with use of gadoxetate disodium-enhanced MR imaging.
      However, a simple signal intensity measurement at a single timepoint may be subject to technical variations between scanners as well as physiological variations between healthy individuals and patients. Moreover, strong functional heterogeneity exists, particularly in diseased livers, which cannot be captured by region-of-interest based signal intensity.
      • Nilsson H.
      • Karlgren S.
      • Blomqvist L.
      • Jonas E.
      The inhomogeneous distribution of liver function: possible impact on the prediction of post-operative remnant liver function.
      Therefore, dynamic image acquisition is increasingly employed to enable pharmacokinetic analysis, extracting both temporal and spatial information. The feasibility of deriving hepatic extraction fraction (HEF) from DCE MRI (with Gd-EOB-DTPA) by deconvolutional analysis was demonstrated early on in healthy individuals.
      • Clement O.
      • Muhler A.
      • Vexler V.
      • Berthezene Y.
      • Brasch R.C.
      Gadolinium-ethoxybenzyl-DTPA, a new liver-specific magnetic resonance contrast agent. Kinetic and enhancement patterns in normal and cholestatic rats.
      In a rabbit model of carbon tetrachloride (CCl4)–induced liver fibrosis, the HEF was calculated by deconvoluting aortic and hepatic parenchymal time-intensity curves from DCE MRI of Gd-EOB-DTPA.
      • Ryeom H.K.
      • Kim S.H.
      • Kim J.Y.
      • Kim H.J.
      • Lee J.M.
      • Chang Y.M.
      • et al.
      Quantitative evaluation of liver function with MRI Using Gd-EOB-DTPA.
      A negative correlation was found between the HEF and changes in ICG-R15. A multicentre study determined liver function using DCE MRI of Gd-EOB-DTPA in patients scheduled to undergo hepatectomy or radiofrequency ablation for hepatocellular carcinoma.
      • Yoon J.H.
      • Choi J.I.
      • Jeong Y.Y.
      • Schenk A.
      • Chen L.
      • Laue H.
      • et al.
      Pre-treatment estimation of future remnant liver function using gadoxetic acid MRI in patients with HCC.
      The predicted remnant liver function, calculated as the pre-treatment HEF multiplied by the remnant liver volume, showed a significant negative correlation with post-treatment ICG-R15. In contrast with global liver function tests such as ICG-R15, DCE MRI with Gd-EOB-DTPA demonstrated the potential to include regional variations of liver function into planning a personalised preoperative strategy. With pharmacokinetic analysis of DCE MRI data, liver perfusion and hepatocyte transport function can be assessed separately using deconvolutional or compartmental analysis.
      • Nilsson H.
      • Nordell A.
      • Vargas R.
      • Douglas L.
      • Jonas E.
      • Blomqvist L.
      Assessment of hepatic extraction fraction and input relative blood flow using dynamic hepatocyte-specific contrast-enhanced MRI.
      ,
      • Sourbron S.
      • Sommer W.H.
      • Reiser M.F.
      • Zech C.J.
      Combined quantification of liver perfusion and function with dynamic gadoxetic acid-enhanced MR imaging.
      While these quantitative methods are promising, standardisation of processing and analysis will be needed to obtain clinically useful metrics.
      Liver perfusion and hepatocyte transport function can be assessed separately using pharmacokinetic analysis of dynamic Gd-EOB-DTPA-enhanced MRI data.
      Gd-EOB-DTPA-enhanced MRI has been used to assess hepatobiliary function in liver fibrosis, cirrhosis, and non-alcoholic steatohepatitis (NASH).
      • Lagadec M.
      • Doblas S.
      • Giraudeau C.
      • Ronot M.
      • Lambert S.A.
      • Fasseu M.
      • et al.
      Advanced fibrosis: correlation between pharmacokinetic parameters at dynamic gadoxetate-enhanced MR imaging and hepatocyte organic anion transporter expression in rat liver.
      • Bastati N.
      • Feier D.
      • Wibmer A.
      • Traussnigg S.
      • Balassy C.
      • Tamandl D.
      • et al.
      Noninvasive differentiation of simple steatosis and steatohepatitis by using gadoxetic acid-enhanced MR imaging in patients with nonalcoholic fatty liver disease: a proof-of-concept study.
      • Nilsson H.
      • Blomqvist L.
      • Douglas L.
      • Nordell A.
      • Janczewska I.
      • Naslund E.
      • et al.
      Gd-EOB-DTPA-enhanced MRI for the assessment of liver function and volume in liver cirrhosis.
      • Feier D.
      • Balassy C.
      • Bastati N.
      • Stift J.
      • Badea R.
      • Ba-Ssalamah A.
      Liver fibrosis: histopathologic and biochemical influences on diagnostic efficacy of hepatobiliary contrast-enhanced MR imaging in staging.
      Axial T1-weighted fat saturated 3D gradient echo images obtained 20 minutes after injection of Gd-EOB-DTPA are shown in a volunteer with normal liver function (Fig. 1B) and in a patient suffering from NASH-induced cirrhosis (Fig. 1C). The biliary ducts filled by Gd-EOB-DTPA are indicated by arrowheads. The liver parenchyma demonstrates marked and homogenously increased signal intensity in the healthy liver and only mild signal enhancement in the cirrhotic liver. The morphologic stigmata of cirrhosis such as hypertrophy of the caudate lobe and left lobe of the liver are also seen (yellow arrows). In an animal model of advanced liver fibrosis, the enhancement parameters (maximum enhancement, and time to peak) and the pharmacokinetic parameters (HEF and mean residence time) measured from DCE MRI significantly decreased in diseased rats relative to those in control rats, which correlated with the expression of the hepatic organic anion transporters.
      • Lagadec M.
      • Doblas S.
      • Giraudeau C.
      • Ronot M.
      • Lambert S.A.
      • Fasseu M.
      • et al.
      Advanced fibrosis: correlation between pharmacokinetic parameters at dynamic gadoxetate-enhanced MR imaging and hepatocyte organic anion transporter expression in rat liver.
      In a clinical study in patients with CLD or cirrhosis, Gd-EOB-DTPA signal enhancement in the liver was normalised to that in the spleen to quantify the hepatocyte uptake ratio.
      • Yoon J.H.
      • Lee J.M.
      • Kang H.J.
      • Ahn S.J.
      • Yang H.
      • Kim E.
      • et al.
      Quantitative assessment of liver function by using gadoxetic acid-enhanced MRI: hepatocyte uptake ratio.
      Hepatocyte uptake ratios are negatively correlated with liver function measured by ICG-R15. In patients with CLD or Child-Pugh class A, significantly higher hepatocyte uptake ratio was found for those with ICG-R15 ≤20% (Fig. 1D) than those with ICG-R15 ≥20% (Fig. 1E). Hepatocyte uptake ratios demonstrated better performance for detecting ICG-R15 ≥20% with the highest AUROC (0.96; 95% CI 0.89–0.99), followed by postcontrast liver T1 value (0.89; 95% CI 0.76–0.97) and liver volume (0.70; 95% CI 0.55–0.83). On the other hand, impaired hepatic uptake of Gd-EOB-DTPA was found to correlate with fibrosis stage in patients with liver fibrosis/cirrhosis.
      • Watanabe H.
      • Kanematsu M.
      • Goshima S.
      • Kondo H.
      • Onozuka M.
      • Moriyama N.
      • et al.
      Staging hepatic fibrosis: comparison of gadoxetate disodium-enhanced and diffusion-weighted MR imaging--preliminary observations.
      ,
      • Feier D.
      • Balassy C.
      • Bastati N.
      • Stift J.
      • Badea R.
      • Ba-Ssalamah A.
      Liver fibrosis: histopathologic and biochemical influences on diagnostic efficacy of hepatobiliary contrast-enhanced MR imaging in staging.
      ,
      • Goshima S.
      • Kanematsu M.
      • Watanabe H.
      • Kondo H.
      • Kawada H.
      • Moriyama N.
      • et al.
      Gd-EOB-DTPA-enhanced MR imaging: prediction of hepatic fibrosis stages using liver contrast enhancement index and liver-to-spleen volumetric ratio.
      ,
      • Verloh N.
      • Utpatel K.
      • Haimerl M.
      • Zeman F.
      • Fellner C.
      • Fichtner-Feigl S.
      • et al.
      Liver fibrosis and Gd-EOB-DTPA-enhanced MRI: a histopathologic correlation.
      Recently, a semi-quantitative functional liver imaging score (FLIS) derived from the HBP image of Gd-EOB-DTPA was used to predict hepatic decompensation and transplant-free survival in patients with CLD.
      • Bastati N.
      • Beer L.
      • Mandorfer M.
      • Poetter-Lang S.
      • Tamandl D.
      • Bican Y.
      • et al.
      Does the functional liver imaging score derived from gadoxetic acid-enhanced MRI predict outcomes in chronic liver disease?.
      Because FLIS requires no dynamic acquisition or signal modelling and is independent of MRI field-strength and vendor, it may be easier to implement in routine clinical practice.
      In the past decade, Gd-EOB-DTPA-enhanced MRI has increasingly been used to assess the risk of post-hepatectomy liver failure and graft liver dysfunction. An accurate estimate of hepatic functional reserve before resection of diseased liver is crucial to avoid post-hepatectomy liver failure.
      • van den Broek M.A.
      • Olde Damink S.W.
      • Dejong C.H.
      • Lang H.
      • Malago M.
      • Jalan R.
      • et al.
      Liver failure after partial hepatic resection: definition, pathophysiology, risk factors and treatment.
      Several clinical studies showed that lower preoperative Gd-EOB-DTPA uptake in the liver was associated with a higher probability of post-hepatectomy liver failure.
      • Costa A.F.
      • Tremblay St-Germain A.
      • Abdolell M.
      • Smoot R.L.
      • Cleary S.
      • Jhaveri K.S.
      Can contrast-enhanced MRI with gadoxetic acid predict liver failure and other complications after major hepatic resection?.
      • Wibmer A.
      • Prusa A.M.
      • Nolz R.
      • Gruenberger T.
      • Schindl M.
      • Ba-Ssalamah A.
      Liver failure after major liver resection: risk assessment by using preoperative Gadoxetic acid-enhanced 3-T MR imaging.
      • Cho S.H.
      • Kang U.R.
      • Kim J.D.
      • Han Y.S.
      • Choi D.L.
      The value of gadoxetate disodium-enhanced MR imaging for predicting posthepatectomy liver failure after major hepatic resection: a preliminary study.
      Orthotopic liver transplantation is the only curative therapeutic option for end-stage liver diseases, and prompt diagnosis and aggressive management of possible complications are critically needed to give each graft the best chance of survival. Gd-EOB-DTPA–enhanced MRI was used to evaluate global and regional liver function in liver transplant recipients with regard to serum biomarkers and mortality at 1 year from imaging.
      • Wibmer A.
      • Aliya Q.
      • Steininger R.
      • Trauner M.
      • Maresch J.
      • Muhlbacher F.
      • et al.
      Liver transplantation: impaired biliary excretion of gadoxate is associated with an inferior 1-year retransplantation-free survival.
      Decreased hepatic uptake and delayed or absent excretion of Gd-EOB-DTPA, in the absence of biliary obstruction, was found to correlate with inferior short-term graft survival, whereas normal hepatobiliary excretion was associated with 1-year retransplantation-free survival.
      • Wibmer A.
      • Aliya Q.
      • Steininger R.
      • Trauner M.
      • Maresch J.
      • Muhlbacher F.
      • et al.
      Liver transplantation: impaired biliary excretion of gadoxate is associated with an inferior 1-year retransplantation-free survival.
      The same group described a qualitative scoring system based on 3 simple features derived from Gd-EOB-DTPA-enhanced MRI to predict graft survival for 3 years.
      • Bastati N.
      • Wibmer A.
      • Tamandl D.
      • Einspieler H.
      • Hodge J.C.
      • Poetter-Lang S.
      • et al.
      Assessment of orthotopic liver transplant graft survival on gadoxetic acid-enhanced magnetic resonance imaging using qualitative and quantitative parameters.
      Liver fibrosis is a common feature of almost all causes of progressive chronic liver disease, which is associated with a cascade of cellular and molecular processes such as immune system activation, vascular leak, extravascular coagulation, collagen deposition coagulation, inflammation, excessive deposition of extracellular matrix and cross-linking.
      In patients with end-stage renal failure, liver enhancement in HBP was markedly reduced, attributed to elevated serum ferritin levels. Increased hepatic accumulation of iron, given its superparamagnetic properties, reduces the signal enhancement induced by Gd chelates. Therefore, Gd-EOB-DTPA is not administrated in patients with elevated serum ferritin (>200 ng/ml in men and >150 ng/ml in women recommended by the World Health Organisation) or total serum bilirubin >3 mg/dl, per drug's prescribing information, due to inadequate liver enhancement.
      Compared to Gd-EOB-DTPA, a much lower biliary excretion and a higher typical dose of Gd-BOPTA result in higher enhancement of the hepatic arterial, portal and hepatic venous branches during dynamic liver imaging, but also in a delayed onset of the HBP (1–2 hours vs. 20 minutes for Gd-EOB-DTPA) and in a relatively weaker HBP signal enhancement. Like Gd-EOB-DTPA, the RLE after Gd-BOPTA might provide insights into liver function. RLE was inversely correlated with prothrombin time and total bilirubin levels and directly correlated with serum albumin levels, helping distinguish Child-Pugh A from Child-Pugh B patients.
      • Zhao X.
      • Huang M.
      • Zhu Q.
      • Wang T.
      • Liu Q.
      The relationship between liver function and liver parenchymal contrast enhancement on Gd-BOPTA-enhanced MR imaging in the hepatocyte phase.
      However, the late HBP and varied excretion time of Gd-BOPTA make it less suitable for assessing liver function.

      Double contrast-enhanced MRI of liver fibrosis

      Fibrotic tissue has reduced Kupffer cell density and less accumulation of iron oxides. Therefore, SPIO-enhanced MRI shows liver fibrosis as hyperintense reticulations with the background liver darkened preferentially.
      • Lucidarme O.
      • Baleston F.
      • Cadi M.
      • Bellin M.F.
      • Charlotte F.
      • Ratziu V.
      • et al.
      Non-invasive detection of liver fibrosis: is superparamagnetic iron oxide particle-enhanced MR imaging a contributive technique?.
      SPIOs can be combined with ECF agents for double contrast-enhanced MRI.
      • Guo D.M.
      • Qiu T.S.
      • Bian J.
      • Liu S.F.
      • Wang C.Z.
      Detection and characterization of hepatocellular carcinoma in rats with liver cirrhosis: diagnostic value of combined use of MR positive and negative contrast agents.
      ,
      • Ward J.
      • Robinson P.J.
      Combined use of MR contrast agents for evaluating liver disease.
      The SPIO produces a dark liver background, while the Gd-based ECF agent provides a delayed enhancement of septal and bridging fibrosis. The 2 agents synergistically improve fibrosis-to-normal liver contrast with higher clarity than can be achieved with either agent alone.
      • Aguirre D.A.
      • Behling C.A.
      • Alpert E.
      • Hassanein T.I.
      • Sirlin C.B.
      Liver fibrosis: noninvasive diagnosis with double contrast material-enhanced MR imaging.
      ,
      • Hughes-Cassidy F.
      • Chavez A.D.
      • Schlang A.
      • Hassanein T.
      • Gamst A.
      • Wolfson T.
      • et al.
      Superparamagnetic iron oxides and low molecular weight gadolinium chelates are synergistic for direct visualization of advanced liver fibrosis.
      Aguirre et al. showed that the hepatic texture alterations seen on double contrast-enhanced MRI differentiated advanced hepatic fibrosis from mild or absent fibrosis with an accuracy of 93%.
      • Aguirre D.A.
      • Behling C.A.
      • Alpert E.
      • Hassanein T.I.
      • Sirlin C.B.
      Liver fibrosis: noninvasive diagnosis with double contrast material-enhanced MR imaging.
      The authors suggested that combining texture, surface, and secondary imaging features would probably further improve diagnostic performance. The main limitations of double contrast-enhanced MRI are the higher cost and inconvenience associated with dual contrast administration, as well as the limited availability of SPIOs.

      Imaging molecular processes involved in liver fibrosis

      Liver fibrosis results from repeated hepatic injury which causes the chronic activation of tissue repair mechanisms that replace necrotic tissue with extracellular matrix (ECM) proteins.
      • Hernandez-Gea V.
      • Friedman S.L.
      Pathogenesis of liver fibrosis.
      Fig. 2 illustrates the cellular and molecular processes involved in the pathogenesis of liver fibrosis. First, inflammatory responses to the injury occur, which involve the recruitment and stimulation of immune cells to the site of injury, as well as the stimulation of Kupffer cells. Loss of endothelial fenestrations allows inflammatory immune cells to infiltrate into the hepatic parenchyma along with plasma proteins like fibrinogen. Thrombin-mediated extravascular coagulation results in fibrin deposition. Soluble inflammatory mediators including chemokines and cytokines released by the inflammatory cells activate the quiescent hepatic stellate cells (HSCs). Activated HSCs transdifferentiate into myofibroblasts and produce and deposit collagen. Activated HSCs and other matrix-producing cells including bone marrow-derived fibrocytes and portal fibroblasts, drive enhanced expression, secretion, and deposition of ECM.
      Figure thumbnail gr2
      Fig. 2Cellular and molecular mechanisms in the pathogenesis of liver fibrosis.
      Injury to hepatocytes results in the recruitment and stimulation of inflammatory immune cells to the site of injury, as well as the stimulation of resident macrophages, i.e. Kupffer cells. The loss of endothelial fenestrations allows inflammatory cells and plasma proteins like fibrinogen to infiltrate in the hepatic parenchyma. Thrombin-driven hepatic fibrin deposition is also frequently observed in the parenchyma. Soluble mediators including chemokines and cytokines released by the inflammatory cells activate the quiescent HSCs. The activated HSCs transdifferentiate to myofibroblasts, which are the predominant source of collagen synthesis and deposition. Activated HSCs, complemented by other matrix-producing cells including bone marrow-derived fibrocytes and portal fibroblasts, lead to enhanced expression and secretion of extracellular matrix and matrix deposition. (Illustration created with BioRender.com.) ECM, extracellular matrix; HSCs, hepatic stellate cells.

      Imaging matrix deposition and cross-linking

      Excess deposition of type I collagen is the hallmark of liver fibrosis. EP-3533 is a molecular probe that contains a cyclic peptide specific to type I collagen and 3 Gd-DTPA moieties to boost the MR signal.
      • Helm P.A.
      • Caravan P.
      • French B.A.
      • Jacques V.
      • Shen L.
      • Xu Y.
      • et al.
      Postinfarction myocardial scarring in mice: molecular MR imaging with use of a collagen-targeting contrast agent.
      ,
      • Caravan P.
      • Das B.
      • Dumas S.
      • Epstein F.H.
      • Helm P.A.
      • Jacques V.
      • et al.
      Collagen-targeted MRI contrast agent for molecular imaging of fibrosis.
      Compared to the ECF agent Gd-DTPA, EP-3533-enhanced MRI could distinguish fibrotic liver from controls in a rat model of diethylnitrosamine (DEN)-induced fibrosis and a mouse model of CCl4-induced liver fibrosis.
      • Polasek M.
      • Fuchs B.C.
      • Uppal R.
      • Schuhle D.T.
      • Alford J.K.
      • Loving G.S.
      • et al.
      Molecular MR imaging of liver fibrosis: a feasibility study using rat and mouse models.
      The change in MRI liver-to-muscle contrast correlated linearly to collagen content determined biochemically by hydroxyproline analysis and to the Ishak score of fibrosis. In CCl4-injured mice, rats with bile duct ligation (BDL), and DEN-injured rats, EP-3533 was shown to accurately stage liver fibrosis.
      • Fuchs B.C.
      • Wang H.
      • Yang Y.
      • Wei L.
      • Polasek M.
      • Schuhle D.T.
      • et al.
      Molecular MRI of collagen to diagnose and stage liver fibrosis.
      • Farrar C.T.
      • DePeralta D.K.
      • Day H.
      • Rietz T.A.
      • Wei L.
      • Lauwers G.Y.
      • et al.
      3D molecular MR imaging of liver fibrosis and response to rapamycin therapy in a bile duct ligation rat model.
      • Zhu B.
      • Wei L.
      • Rotile N.
      • Day H.
      • Rietz T.
      • Farrar C.T.
      • et al.
      Combined magnetic resonance elastography and collagen molecular magnetic resonance imaging accurately stage liver fibrosis in a rat model.
      EP-3533-enhanced MRI could also be used to assess treatment response, which has historically relied on histologic analysis.
      • Neuschwander-Tetri B.A.
      • Loomba R.
      • Sanyal A.J.
      • Lavine J.E.
      • Van Natta M.L.
      • Abdelmalek M.F.
      • et al.
      Farnesoid X nuclear receptor ligand obeticholic acid for non-cirrhotic, non-alcoholic steatohepatitis (FLINT): a multicentre, randomised, placebo-controlled trial.
      ,
      • Sanyal A.J.
      • Brunt E.M.
      • Kleiner D.E.
      • Kowdley K.V.
      • Chalasani N.
      • Lavine J.E.
      • et al.
      Endpoints and clinical trial design for nonalcoholic steatohepatitis.
      In a rat BDL model, EP-3533-enhanced MRI detected significant changes in fibrosis in response to rapamycin therapy and could prospectively identify treatment responders from non-responders.
      • Farrar C.T.
      • DePeralta D.K.
      • Day H.
      • Rietz T.A.
      • Wei L.
      • Lauwers G.Y.
      • et al.
      3D molecular MR imaging of liver fibrosis and response to rapamycin therapy in a bile duct ligation rat model.
      Moreover, 3D molecular MRI enabled characterisation of intrahepatic heterogeneity throughout the whole liver in a rapamycin non-responder rat, in which the fibrosis heterogeneity was confirmed by histology (Fig. 3A–C). EP-3533-enhanced MRI also detected decreased fibrosis in BDL rats in response to the farnesoid X receptor agonist EDP-305.
      • Erstad D.J.
      • Farrar C.T.
      • Ghoshal S.
      • Masia R.
      • Ferreira D.S.
      • Chen Y.I.
      • et al.
      Molecular magnetic resonance imaging accurately measures the antifibrotic effect of EDP-305, a novel farnesoid X receptor agonist.
      To improve the translational potential of EP-3533, a new version, called CM-101, employing the highly stable macrocyclic Gd-DOTA chelate was developed, and was shown to detect fibrosis in rat BDL and mouse CCl4 models with faster rapid blood clearance and minimal accumulation of Gd in bone or tissue compared to EP-3533.
      • Farrar C.T.
      • Gale E.M.
      • Kennan R.
      • Ramsay I.
      • Masia R.
      • Arora G.
      • et al.
      CM-101: type I collagen-targeted MR imaging probe for detection of liver fibrosis.
      Figure thumbnail gr3
      Fig. 3Type I collagen imaging in murine models of liver fibrosis.
      (Fig. 3A–C and captions adapted from,
      • Farrar C.T.
      • DePeralta D.K.
      • Day H.
      • Rietz T.A.
      • Wei L.
      • Lauwers G.Y.
      • et al.
      3D molecular MR imaging of liver fibrosis and response to rapamycin therapy in a bile duct ligation rat model.
      Fig. 3D–E and captions adapted from.
      • Salarian M.
      • Turaga R.C.
      • Xue S.
      • Nezafati M.
      • Hekmatyar K.
      • Qiao J.
      • et al.
      Early detection and staging of chronic liver diseases with a protein MRI contrast agent.
      )
      (A) Pre- and post-EP-3533 longitudinal relaxation rate (R1) maps of a bile duct ligated rat treated with rapamycin. Striking heterogeneity in liver fibrosis with greater R1 in the right liver lobe compared to the left liver lobe. (B) While the pre-EP-3533 histogram (blue) of liver R1 values was homogeneous the post EP-3533 R1 histogram (red) demonstrated a bimodal R1 distribution. (C) Corresponding Sirius Red images from the left and right liver lobes confirmed the fibrosis heterogeneity. (D) R1 maps of normal (Ishak stage), early-stage (Ishak stage 3), and late-stage (Ishak stage 5) liver fibrosis before and 24 h after injection of ProCA32.collagen1. (E) αSMA, H&E, and Sirius red staining of early- and late-stage fibrotic liver compared to normal liver confirms the stage of liver fibrosis.
      ProCA32 is a small protein that was engineered to bind tightly to Gd and to have a very high relaxivity. Grafting the same type I collagen targeting peptide used in EP-3533 to ProCA32 created ProCA32.collagen1 – this reagent was evaluated in alcohol/chemical-induced mouse models of liver fibrosis and a diet-induced mouse model of NASH.
      • Salarian M.
      • Turaga R.C.
      • Xue S.
      • Nezafati M.
      • Hekmatyar K.
      • Qiao J.
      • et al.
      Early detection and staging of chronic liver diseases with a protein MRI contrast agent.
      ProCA32.collagen1-enhanced MRI could detect early-stage alcohol-induced liver fibrosis (Ishak stage 3) and early-stage NASH (Ishak stage 1) using both longitudinal and transverse relaxation (R1 and R2) maps at 24 hours post-injection. R1 quantification of ProCA32.collagen1 could distinguish early-stage (Ishak stage 3) from late-stage fibrosis (Ishak stage 5), which correlated with histologic quantification of fibrosis (Fig. 3D–E). Because of its much larger size, ProCA32.collagen1 has a longer blood residency time than either EP-3533 or CM-101. Therefore, the delayed imaging took place hours after injection or the next day compared to within 10–40 minutes with EP-3533 or CM-101.
      Novel molecular contrast agents that are biochemically targeted to specific cellular/molecular processes are undergoing preclinical development.
      Elastin accumulates at advanced stages of liver fibrosis due to both increased synthesis and decreased macrophage metalloelastase (MMP12)-mediated degradation.
      • Blaauboer M.E.
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      • Verschuren L.
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      • et al.
      Novel combination of collagen dynamics analysis and transcriptional profiling reveals fibrosis-relevant genes and pathways.
      ,
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      • et al.
      Elastin accumulation is regulated at the level of degradation by macrophage metalloelastase (MMP-12) during experimental liver fibrosis.
      The Gd-based elastin-specific MR probe, Gd-ESMA, was used to monitor ECM-remodelling during liver fibrosis.
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      • et al.
      Elastin-based molecular MRI of liver fibrosis.
      Gd-ESMA administration resulted in significant perivascular signal enhancement in the livers of CCl4-treated mice, but not in control mice. The periportal elastin deposition detected by Gd-ESMA-enhanced MRI was further confirmed by elastin-specific Elastica-Van-Gieson staining.
      Fibrin-fibronectin complexes are deposited in the fibrotic liver as a result of cross-linking between fibrin/fibrinogen and fibronectin,
      • Neubauer K.
      • Knittel T.
      • Armbrust T.
      • Ramadori G.
      Accumulation and cellular localization of fibrinogen/fibrin during short-term and long-term rat liver injury.
      and can serve as a molecular target for liver fibrosis. Fibronectin deposition in the liver was evaluated using Gd-P, a CGLIIQKNEC (CLT1) peptide-targeted nanoglobular probe that binds to fibrin-fibronectin complexes. In a mouse model of CCl4-induced liver injury, an increased amount of fibronectin in the extracellular space in insulted livers was confirmed by histology, and Gd-P induced signal enhancement was significantly higher in injured mice than controls.
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      • Tan M.
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      • Fan S.J.
      • Cheung J.S.
      • Man K.
      • et al.
      Molecular MRI of liver fibrosis by a peptide-targeted contrast agent in an experimental mouse model.

      Imaging fibrogenesis

      Fibrogenesis, or the active deposition of ECM, is characterised by high levels of allysine, a reactive aldehyde generated as part of a lysyl oxidase (LOX)-mediated collagen cross-linking process. Allysine-targeting imaging probes can be used to monitor fibrogenesis.
      • Waghorn P.A.
      • Jones C.M.
      • Rotile N.J.
      • Koerner S.K.
      • Ferreira D.S.
      • Chen H.H.
      • et al.
      Molecular magnetic resonance imaging of lung fibrogenesis with an oxyamine-based probe.
      ,
      • Akam E.A.
      • Abston E.
      • Rotile N.J.
      • Slattery H.R.
      • Zhou I.Y.
      • Lanuti M.
      • et al.
      Improving the reactivity of hydrazine-bearing MRI probes for in vivo imaging of lung fibrogenesis.
      The hydrazide-containing, aldehyde-targeting agent Gd-Hyd detected and staged disease progression caused by CCl4 injury in mice and detected reduced fibrogenesis in mice after withdrawal of CCl4, as confirmed by histology and Lox gene expression.
      • Chen H.H.
      • Waghorn P.A.
      • Wei L.
      • Tapias L.F.
      • Schu Hle D.T.
      • Rotile N.J.
      • et al.
      Molecular imaging of oxidized collagen quantifies pulmonary and hepatic fibrogenesis.
      In mice fed a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) – a model of NASH – Gd-Hyd detected reduced fibrogenesis after treatment with the farnesoid X receptor agonist EDP-305, corroborated by picrosirius red collagen staining, hydroxyproline biochemical quantification, and Lox gene expression analyses.
      • Erstad D.J.
      • Farrar C.T.
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      • Masia R.
      • Ferreira D.S.
      • Chen Y.I.
      • et al.
      Molecular magnetic resonance imaging accurately measures the antifibrotic effect of EDP-305, a novel farnesoid X receptor agonist.
      These data suggest that Gd-Hyd could be used to monitor disease progression and regression. This probe could also be used to predict treatment responses; in this regard, its performance compared to serum-based markers warrants further evaluation.
      Activated HSCs express integrin αvβ3.
      • Tsuchida T.
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      Mechanisms of hepatic stellate cell activation.
      ,
      • Higashi T.
      • Friedman S.L.
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      Hepatic stellate cells as key target in liver fibrosis.
      ECM proteins bind to integrins like αvβ3 by means of the tri-amino acid sequence arginine-glycine-aspartate (RGD).
      • Giancotti F.G.
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      Integrin signaling.
      ,
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      • et al.
      Targeting of alphav integrin identifies a core molecular pathway that regulates fibrosis in several organs.
      Numerous RGD-based integrin-binding agents have been developed for imaging αvβ3 in activated HSCs.
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      Molecular imaging of hepatic stellate cell activity by visualization of hepatic integrin alphavbeta3 expression with SPECT in rat.
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      Small-animal SPECT/CT of the progression and recovery of rat liver fibrosis by using an integrin alphavbeta3-targeting radiotracer.
      • Zhang X.
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      Assessing activation of hepatic stellate cells by (99m)Tc-3PRGD2 scintigraphy targeting integrin alphavbeta3: a feasibility study.
      • Wang Q.B.
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      • Chen K.M.
      • Liu B.Y.
      • et al.
      MR imaging of activated hepatic stellate cells in liver injured by CCl4 of rats with integrin-targeted ultrasmall superparamagnetic iron oxide.
      • Rokugawa T.
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      • et al.
      Evaluation of hepatic integrin alphavbeta3 expression in non-alcoholic steatohepatitis (NASH) model mouse by (18)F-FPP-RGD2 PET.
      For example, an RGD peptide-based USPIO, a T2 MRI contrast agent, was developed to image the activated HSCs in CCl4-injured rats.
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      • Jiang T.T.
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      • Chen K.M.
      • Liu B.Y.
      • et al.
      MR imaging of activated hepatic stellate cells in liver injured by CCl4 of rats with integrin-targeted ultrasmall superparamagnetic iron oxide.
      The integrin-targeted iron particles were engulfed by activated HSCs, resulting in reduction of T2 relaxation times. However, the USPIOs were also taken up by Kupffer cells in both normal and injured livers resulting in a non-specific background.

      Imaging the inflammatory response

      Tissue injury triggers extravascular coagulation as a part of inflammatory response and fibroproliferative processes of tissue repair,
      • Mercer P.F.
      • Chambers R.C.
      Coagulation and coagulation signalling in fibrosis.
      and results in fibrin deposition.
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      • et al.
      Increased local expression of coagulation factor X contributes to the fibrotic response in human and murine lung injury.
      Abnormal extravascular deposition of fibrin was detected in rodent models of liver injury.
      • Neubauer K.
      • Knittel T.
      • Armbrust T.
      • Ramadori G.
      Accumulation and cellular localization of fibrinogen/fibrin during short-term and long-term rat liver injury.
      ,
      • Joshi N.
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      • Towery K.L.
      • Cline-Fedewa H.
      • Williams K.J.
      • et al.
      Coagulation-driven platelet activation reduces cholestatic liver injury and fibrosis in mice.
      EP-2104R is a fibrin-specific, peptide-based, Gd probe that has been used for molecular imaging of extravascular fibrin in mouse models of cancer and pulmonary fibrosis.
      • Shea B.S.
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      Uncoupling of the profibrotic and hemostatic effects of thrombin in lung fibrosis.
      • Uppal R.
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      Molecular imaging of fibrin in a breast cancer xenograft mouse model.
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      • et al.
      EP-2104R: a fibrin-specific gadolinium-Based MRI contrast agent for detection of thrombus.
      In DEN-injured rats, signal enhancement with EP-2104R was significantly higher in animals imaged at 1 day post-DEN dosing when liver inflammation was high compared with 7 days post-DEN when inflammation had subsided, as confirmed by histology (Fig. 4A–B).
      • Atanasova I.
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      Molecular magnetic resonance imaging of fibrin deposition in the liver as an indicator of tissue injury and inflammation.
      Collagen imaging with EP-3533 showed a similar T1 change when imaging rats 1 day or 7 days post-DEN, consistent with equivalent fibrosis assessed by Ishak score, demonstrating that fibrin/inflammation could be imaged on a background of liver fibrosis.
      Figure thumbnail gr4
      Fig. 4Molecular MRI of inflammatory responses.
      (A) Molecular MRI using the fibrin-specific MRI probe EP-2104R for the non-invasive imaging of fibrin as a marker of liver inflammation in DEN-injured liver. Representative T1-weighted axial images of rats dosed weekly for 4 weeks with PBS (top), or with 100 mg/kg of DEN and imaged 1 day after the last DEN dose (bottom). False colour overlay represents subtraction image of precontrast image from the 1-minute post EP-2104R image. (B) Histological analysis of liver specimens from rats received PBS (top) or DEN (bottom). H&E staining of liver tissue showed inflammatory regions labelled with pointed black arrows. Sirius Red staining of liver tissue showed fibrosis by intense red staining of collagen fibres. Immunohistochemistry staining of fibrin in liver tissue. (C) Molecular MPO-Gd–enhanced MRI depicting inflammatory activity in liver biopsy samples from NASH (top) and control patients (bottom). Pseudo-coloured MPO-Gd–enhanced MR images are shown with the biopsy sample outlined by dashed line. Increased signal after incubation with MPO-Gd is seen on samples from patients with NASH, but not control patients. (D) Immunohistochemistry for MPO in liver biopsy samples from NASH (top) and control patients (bottom) (bar = 100 μm) demonstrated clusters of MPO-expressing cells in samples from patients with NASH but not in samples from control patients. (A–B and captions adapted from
      • Atanasova I.
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      • Geraldes C.
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      Molecular magnetic resonance imaging of fibrin deposition in the liver as an indicator of tissue injury and inflammation.
      ; C–D and captions adapted from.
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      Multiple sclerosis: myeloperoxidase immunoradiology improves detection of acute and chronic disease in experimental model.
      ) DEN, diethylnitrosamine; Gd, gadolinium; MPO, myeloperoxidase; NASH, non-alcoholic steatohepatitis.
      The inflammatory response also results in recruitment of immune cells including monocytes and neutrophils to the site of injury.
      • Bataller R.
      • Brenner D.A.
      Liver fibrosis.
      ,
      • Browning J.D.
      • Horton J.D.
      Molecular mediators of hepatic steatosis and liver injury.
      Myeloperoxidase (MPO), a heme-containing enzyme capable of generating reactive oxygen and nitrogen species (ROS and RNS) for host defence, is abundant in these pro-inflammatory immune cells.
      • Klebanoff S.J.
      Myeloperoxidase: friend and foe.
      Increased MPO is found in human livers with NASH compared with steatotic specimens.
      • Rensen S.S.
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      • Nijhuis J.
      • Jans A.
      • Bieghs V.
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      • et al.
      Increased hepatic myeloperoxidase activity in obese subjects with nonalcoholic steatohepatitis.
      Inflammation and fibrosis were also decreased in MPO-deficient mice fed a high-fat diet,
      • Rensen S.S.
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      • Bakker J.A.
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      • et al.
      Neutrophil-derived myeloperoxidase aggravates non-alcoholic steatohepatitis in low-density lipoprotein receptor-deficient mice.
      indicating the possible role of MPO in the pathogenesis of NASH. Moreover, MPO may be important in the development of liver fibrosis through activation of HSCs.
      • Pulli B.
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      • et al.
      Myeloperoxidase-hepatocyte-stellate cell cross talk promotes hepatocyte injury and fibrosis in experimental nonalcoholic steatohepatitis.
      An activatable probe, MPO-Gd, consists of a Gd-chelate bearing two 5-hydroxytryptamide moieties that are readily oxidised by MPO.
      • Querol M.
      • Chen J.W.
      • Weissleder R.
      • Bogdanov A.
      DTPA-bisamide-based MR sensor agents for peroxidase imaging.
      ,
      • Querol M.
      • Chen J.W.
      • Bogdanov Jr., A.A.
      A paramagnetic contrast agent with myeloperoxidase-sensing properties.
      Oxidation of MPO-Gd leads to its oligomerisation and/or covalent addition to proteins; both processes increase relaxivity and help retain the probe at the site of injury, thereby allowing for non-invasive assessment of MPO activity.
      • Nahrendorf M.
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      • Chen J.W.
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      • et al.
      Activatable magnetic resonance imaging agent reports myeloperoxidase activity in healing infarcts and noninvasively detects the antiinflammatory effects of atorvastatin on ischemia-reperfusion injury.
      ,
      • Rodriguez E.
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      • Weissleder R.
      • Chen J.W.
      Activatable magnetic resonance imaging agents for myeloperoxidase sensing: mechanism of activation, stability, and toxicity.
      MPO-Gd was used for in vivo detection of MPO activity in animal models of myocardial infarction, multiple sclerosis, stroke, and vasculitis.
      • Nahrendorf M.
      • Sosnovik D.
      • Chen J.W.
      • Panizzi P.
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      • et al.
      Activatable magnetic resonance imaging agent reports myeloperoxidase activity in healing infarcts and noninvasively detects the antiinflammatory effects of atorvastatin on ischemia-reperfusion injury.
      ,
      • Chen J.W.
      • Querol Sans M.
      • Bogdanov Jr., A.
      • Weissleder R.
      Imaging of myeloperoxidase in mice by using novel amplifiable paramagnetic substrates.
      • Ronald J.A.
      • Chen J.W.
      • Chen Y.
      • Hamilton A.M.
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      • Reynolds F.
      • et al.
      Enzyme-sensitive magnetic resonance imaging targeting myeloperoxidase identifies active inflammation in experimental rabbit atherosclerotic plaques.
      • Breckwoldt M.O.
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      • Qiu S.
      • et al.
      Tracking the inflammatory response in stroke in vivo by sensing the enzyme myeloperoxidase.
      • Pulli B.
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      • Wojtkiewicz G.R.
      • Iwamoto Y.
      • Ali M.
      • Li D.
      • et al.
      Multiple sclerosis: myeloperoxidase immunoradiology improves detection of acute and chronic disease in experimental model.
      • Su H.S.
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      • Rodriguez E.
      • Iwamoto Y.
      • et al.
      Vasculitis: molecular imaging by targeting the inflammatory enzyme myeloperoxidase.
      In a study using 2 mouse models of steatohepatitis, elevated MPO activity could be detected with MPO-Gd, allowing steatohepatitis to be differentiated from steatosis.
      • Pulli B.
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      • Ali M.
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      • et al.
      Molecular MR imaging of myeloperoxidase distinguishes steatosis from steatohepatitis in nonalcoholic fatty liver disease.
      Ex vivo molecular MRI of liver biopsy samples from patients with NASH and controls confirmed the results from animal studies (Fig. 4C–D). Improved MPO-targeted probes using the more stable and translatable macrocyclic Gd-DOTA have been reported.
      • Wadghiri Y.Z.
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      High-resolution imaging of myeloperoxidase activity sensors in human cerebrovascular disease.
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      • et al.
      Peroxidase sensitive amplifiable probe for molecular magnetic resonance imaging of pulmonary inflammation.
      Recently a redox-active iron complex, Fe-PyC3A, was shown to detect tissue inflammation and in vivo Fe-PyC3A-induced signal enhancement correlated strongly with ex vivo quantitation of MPO activity.
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      Molecular magnetic resonance imaging using a redox-active iron complex.

      Multiparametric imaging of diffuse liver diseases

      In addition to functional and molecular liver MRI, state-of-the-art MRI techniques – including MR elastography, diffusion-weighted imaging and MRI quantification of liver fat or iron content – are
      Given the multiparametric capabilities of MRI, functional and molecular MRI can be readily performed in conjunction with advanced MRI techniques such as MR elastography, diffusion-weighted imaging, MRI quantification of liver fat or iron content to provide a more comprehensive characterisation of liver diseases and to assess their response to treatment.
      currently being used in clinical practice, or are under development, for the non-invasive assessment of CLD. Molecular and/or functional imaging can be readily added to these advanced MRI protocols to provide a more comprehensive assessment of CLD. For instance Gd-Hyd-enhanced MRI of fibrogenesis and Dixon MRI-based quantitative fat imaging were used in a mouse model of NASH.
      • Erstad D.J.
      • Farrar C.T.
      • Ghoshal S.
      • Masia R.
      • Ferreira D.S.
      • Chen Y.I.
      • et al.
      Molecular magnetic resonance imaging accurately measures the antifibrotic effect of EDP-305, a novel farnesoid X receptor agonist.
      In a DEN-induced rat model of fibrosis, collagen imaging with EP-3533 was shown to be most sensitive to early fibrosis, while tissue stiffness assessed by MR elastography was more sensitive to advanced fibrosis.
      • Zhu B.
      • Wei L.
      • Rotile N.
      • Day H.
      • Rietz T.
      • Farrar C.T.
      • et al.
      Combined magnetic resonance elastography and collagen molecular magnetic resonance imaging accurately stage liver fibrosis in a rat model.
      A composite score formulated from these 2 measurements resulted in increased diagnostic accuracy for staging fibrosis.
      • Zhu B.
      • Wei L.
      • Rotile N.
      • Day H.
      • Rietz T.
      • Farrar C.T.
      • et al.
      Combined magnetic resonance elastography and collagen molecular magnetic resonance imaging accurately stage liver fibrosis in a rat model.
      Another multiparametric study with diffusion-weighted imaging, susceptibility-weighted imaging, and Gd-EOB-DTPA-enhanced MRI showed improved performance in staging fibrosis.
      • Feier D.
      • Balassy C.
      • Bastati N.
      • Fragner R.
      • Wrba F.
      • Ba-Ssalamah A.
      The diagnostic efficacy of quantitative liver MR imaging with diffusion-weighted, SWI, and hepato-specific contrast-enhanced sequences in staging liver fibrosis--a multiparametric approach.
      Multiparametric approaches combining native T1 mapping for fibrosis/inflammation staging, T2∗ mapping for liver iron quantification, and proton MR spectroscopy for liver fat quantification have been trialled to detect liver fibrosis, steatosis, and hemosiderosis, respectively.
      • Eddowes P.J.
      • McDonald N.
      • Davies N.
      • Semple S.I.K.
      • Kendall T.J.
      • Hodson J.
      • et al.
      Utility and cost evaluation of multiparametric magnetic resonance imaging for the assessment of non-alcoholic fatty liver disease.
      ,
      • Pavlides M.
      • Banerjee R.
      • Tunnicliffe E.M.
      • Kelly C.
      • Collier J.
      • Wang L.M.
      • et al.
      Multiparametric magnetic resonance imaging for the assessment of non-alcoholic fatty liver disease severity.
      Recently a multiparametric MRI study, using a type-1-collagen-specific probe (EP-3533), an allysine-targeted fibrogenesis probe (Gd-Hyd), MR elastography, and native T1, was used to characterise fibrosis and to assess treatment response in rats fed with CDAHFD as a model of NASH.
      • Zhou I.Y.
      • Jordan V.C.
      • Rotile N.
      • Akam E.A.
      • Krishnan S.
      • Arora G.
      • et al.
      Advanced MRI of liver fibrosis and treatment response in a rat model of nonalcoholic steatohepatitis.
      The collagen-targeted molecular probe most accurately detected the early onset of liver fibrosis with an AUROC of 0.95, superior to other MRI measures. The fibrogenesis imaging probe Gd-Hyd had the highest accuracy for detecting treatment responders. These advanced MRI techniques are complementary in their capture of the fibrotic process. All techniques were performed in a single protocol, highlighting the strength of MRI for characterising liver disease in the context of NASH. Despite higher cost, the rich information provided by such imaging protocols raises new possibilities for clinical imaging and for assessing treatment response for new therapies in development.

      Challenges and future directions

      Several challenges exist in developing and translating molecular MRI probes for the clinic. First, newly developed contrast agents are considered novel drugs and therefore require extensive preclinical safety evaluations, and manufacturing must be performed to meet good manufacturing practice standards, both of which create a large cost barrier for first-in-human evaluation. Second, animal models may not reflect target concentrations in human disease, and probe pharmacokinetics and metabolism may also differ. Furthermore, quantification of sensitivity and specificity will require an effective truth standard like histology, which may not be readily available. Quantification also requires rigorous assessment of reproducibility among individuals and different vendors' scanners. Ultimately outcome data will be required to establish the clinical utility of the probes.
      However, in the era of precision medicine and targeted therapy, the need for non-invasive quantification of liver function and visualisation of cellular/molecular processes underlying liver diseases is continuously growing. With the development of expensive new anti-inflammatory/antifibrotic therapeutics, imaging could be used to stratify patients and to monitor treatment response. The functional and molecular imaging approaches described herein represent the first steps towards these goals. While much of the work was performed in preclinical models or a small cohort of patients with a single disease condition, the successful translation into clinical practice will hinge upon improved and standardised dynamic MRI and post-processing methods, rational design and optimisation of the molecular probes, and further prospective validation studies.

      Conclusion

      Advances in MRI, particularly with the wide deployment of hepatobiliary-specific contrast agents and the development of molecularly targeted probes, have provided the opportunity to revolutionise how we evaluate various liver diseases. Despite the challenges involved in clinical application, we anticipate that the coalescence of major advances in engineering, molecular biology, chemistry, immunology, and genetics will continue to fuel multidisciplinary innovations and drive the field of clinical non-invasive imaging – this will ultimately lead to improved disease identification, risk stratification, and treatment response monitoring, with unparalleled sensitivity and specificity. Moreover, techniques that enable imaging of molecular and cellular events go hand in hand with the development of molecular therapies.

      Abbreviations

      AUC, area under the curve; BDL, bile duct ligation; CCl4, carbon tetrachloride; DCE, dynamic contrast-enhanced; DEN, diethylnitrosamine; CDAHFD, choline-deficient, L-amino acid-defined, high-fat diet; CLD, chronic liver disease; ECF, extracellular fluid; ECM, extracellular matrix; FLIS, functional liver imaging score; Gd, gadolinium; Gd-EOB-DTPA, gadoxetic acid; Gd-BOPTA, gadobenate dimeglumine; HBP, hepatobiliary phase; HEF, hepatic extraction fraction; HSC, hepatic stellate cell; ICG-R15, indocyanine green retention test; LOX, lysyl oxidase; Mn-DPDP, mangafodipir trisodium; MPO, myeloperoxidase; NASH, non-alcoholic steatohepatitis; RES, reticuloendothelial system; RGD, arginine-glycine-aspartate; RLE, relative liver enhancement; RNS, reactive nitrogen species; ROS, reactive oxygen species; SPIO, superparamagnetic iron oxide; USPIO, ultra-small superparamagnetic iron oxide.

      Financial support

      We acknowledge support from the National Institute of Diabetes and Digestive and Kidney Diseases with grants DK104956 , DK104302 , DK121789 .

      Authors' contributions

      All authors contributed to the draft of the text. IYZ and PC created the figures and table. OAC and PC made critical revisions.

      Conflict of interest

      P.C. has equity in and is a consultant to Collagen Medical LLC which owns the patent rights to EP-3533 and CM-101, has equity in Reveal Pharmaceuticals Inc, and has research support from Pliant Therapeutics, Celgene, Takeda, and Indalo Therapeutics.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Supplementary data

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