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Activation and proliferation of HSCs during the progression of CCl4-induced liver fibrosis in mice. a Schedule for injection of CCl4 in mice (n = 5). Female C57BL/6 mice (6-week-old) were intraperitoneally injected with CCl4 twice a week. b Progression of liver fibrosis in mice injected with CCl4. Liver tissues were collected for analysis by histochemistry (H&E staining), macrophages (CD68⁺), collagen (Sirius Red staining), and aHSCs (αSMA⁺). c Quantitative evaluation on liver fibrosis according to the Ishak score, collagen (Sirius Red and hydroxyproline), aHSCs (αSMA⁺ cells), and fibrotic markers including TGFβ1, BMP7, MMP2, and TIMP1. d ALT and AST in the blood
Source publication
Liver fibrosis usually progresses to liver cirrhosis and even hepatocellular carcinoma. Since activated hepatic stellate cells (aHSCs) are responsible for liver fibrosis, reducing the quantity of aHSCs was considered the essential strategy for clinical antihepatofibrotic therapy. Due to the overexpression of TRAIL receptor 2 (DR5) in aHSCs, human T...
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Citations
... All experiments were performed using C57BL/6 mice (6 weeks old) under the guidance of the Animal Care and Use Committee of the University. Liver fibrosis in mice were induced by treatment with CCl 4 according to our previous work [22] or by bile duct ligation (BDL) according to the descriptions by Miyoshi et al. [23]. To construct the liver fibrosis spontaneous recovery models in CCl 4 -treated mice, administration of CCl 4 was ceased at 4 weeks after the initial injection of CCl 4 . ...
... To construct the liver fibrosis spontaneous recovery models in CCl 4 -treated mice, administration of CCl 4 was ceased at 4 weeks after the initial injection of CCl 4 . To induce the regression of liver fibrosis in CCl 4 -treated mice, aHSC-cytotoxic ABD-TRAIL [22,24] was intravenously injected into the mice at 10 mg/kg three times per week from 4 to 6 weeks after initial CCl 4 injection. ...
... To determine liver fibrosis, hematoxylin and eosin (H&E) staining, immunohistochemistry, immunofluorescence, and Sirius red staining were performed according to our previous descriptions [22]. At least three images of the tissues were captured and used for measuring the immunofluorescence of biomarkers with ImageJ software. ...
Purpose
Noninvasive quantifying activated hepatic stellate cells (aHSCs) by molecular imaging is helpful for assessing disease progression and therapeutic responses of liver fibrosis. Our purpose is to develop platelet-derived growth factor receptor β (PDGFRβ)-targeted radioactive tracer for assessing liver fibrosis by positron emission tomography (PET) imaging of aHSCs.
Methods
Comparative transcriptomics, immunofluorescence staining and flow cytometry were used to evaluate PDGFRβ as biomarker for human aHSCs and determine the correlation of PDGFRβ with the severity of liver fibrosis. The high affinity affibody for PDGFRβ (ZPDGFRβ) was labeled with gallium-68 (⁶⁸Ga) for PET imaging of mice with carbon tetrachloride (CCl4)-induced liver fibrosis. Binding of the [⁶⁸Ga]Ga-labeled ZPDGFRβ ([⁶⁸Ga]Ga-DOTA-ZPDGFRβ) for aHSCs in human liver tissues was measured by autoradiography.
Results
PDGFRβ overexpressed in aHSCs was highly correlated with the severity of liver fibrosis in patients and CCl4-treated mice. The ⁶⁸Ga-labeled ZPDGFRβ affibody ([⁶⁸Ga]Ga-DOTA-ZPDGFRβ) showed PDGFRβ-dependent binding to aHSCs. According to the PET imaging, hepatic uptake of [⁶⁸Ga]Ga-DOTA-ZPDGFRβ increased with the accumulation of aHSCs and collagens in the fibrotic livers of mice. In contrast, hepatic uptake of [⁶⁸Ga]Ga-DOTA-ZPDGFRβ decreased with spontaneous recovery or treatment of liver fibrosis, indicating that the progression and therapeutic responses of liver fibrosis in mice could be visualized by PDGFRβ-targeted PET imaging. [⁶⁸Ga]Ga-DOTA-ZPDGFRβ also bound human aHSCs and visualized fibrosis in patient-derived liver tissues.
Conclusions
PDGFRβ is a reliable biomarker for both human and mouse aHSCs. PDGFRβ-targeted PET imaging could be used for noninvasive monitoring of liver fibrosis in mice and has great potential for clinical translation.
... Peptides based on the Affibody molecule scaffold (58-amino-acid length in a triple-α-helix conformation) with nanomolar affinity towards PDGFRβ have been described [69]. Peptides based on this scaffold have been labeled by both fluorescence [70] and fluorine-18 [40] and successfully evaluated in preclinical liver fibrosis models for detection of fibrogenic processes and activation of HSCs. Clinical translation of a gallium-68-labeled version of the PDGFRβ-targeting Affibody molecule, named [ 68 Ga]Ga-DOTA-Cys-ATH001, is ongoing. ...
Fibrosis accompanies various pathologies, and there is thus an unmet medical need for non-invasive, sensitive, and quantitative methods for the assessment of fibrotic processes. Currently, needle biopsy with subsequent histological analysis is routinely used for the diagnosis along with morphological imaging techniques, such as computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound (US). However, none of these imaging techniques are sufficiently sensitive and accurate to detect minor changes in fibrosis. More importantly, they do not provide information on fibrotic activity on the molecular level, which is critical for fundamental understanding of the underlying biology and disease course. Molecular imaging technology using positron emission tomography (PET) offers the possibility of imaging not only physiological real-time activity, but also high-sensitivity and accurate quantification. This diagnostic tool is well established in oncology and has exhibited exponential development during the last two decades. However, PET diagnostics has only recently been widely applied in the area of fibrosis. This review presents the progress of development of radiopharmaceuticals for non-invasive detection of fibrotic processes, including the fibrotic scar itself, the deposition of new fibrotic components (fibrogenesis), or the degradation of existing fibrosis (fibrolysis).
... Nevertheless, the tracer was rapidly cleared from liver within 0.5 h, which would not interfere with tumor imaging. Interestingly, although PDGFRβ is an exclusive biomarker of pericytes with low expression in normal tissues, activated hepatic stellate cell would also express PDGFRβ during inflammatory repair, which should be noted to differentiate from malignancies (37). To evaluate the acute liver and kidney toxicity, mice were intravenously injected with 50 MBq of [ 68 Ga]Ga-DOTA-Z TRI or equal moles of Z TRI (50 µg), and mice treated with PBS were considered as the control group (n = 5). ...
Purpose
Hepatocellular carcinoma (HCC) is a highly vascularized solid carcinoma and tumor vessel–targeted molecular imaging might be effective for early diagnosis of HCC. Herein, we developed a novel trimeric affibody (Z TRI ) with highly specific binding to the platelet-derived growth factor receptor beta (PDGFRβ). The aim of this study is to evaluate the feasibility of ⁶⁸ Ga-radiolabeled Z TRI ([ ⁶⁸ Ga]Ga-DOTA-Z TRI ) as PET tracer for diagnosis of HCC.
Methods
The bioinformatics analysis of clinical database and immunoblotting of clinical specimens were performed to validate the potential of PDGFRβ as HCC biomarker. The trimeric affibody Z TRI was conjugated with DOTA-NHS-ester and radiolabeled with ⁶⁸ Ga to produce [ ⁶⁸ Ga]Ga-DOTA-Z TRI conjugate. Immunoreactivity and specific uptake of [ ⁶⁸ Ga]Ga-DOTA-Z TRI were assessed by dose-dependent cell binding, autoradiography, and biodistribution analysis. [ ⁶⁸ Ga]Ga-DOTA-Z TRI PET/CT scanning of diethylnitrosamine (DEN)-induced primary HCC rats and a rare case of idiopathical HCC rhesus monkey was performed to evaluate the imaging capability and radiation dosimetry of [ ⁶⁸ Ga]Ga-DOTA-Z TRI in vivo.
Results
Excessive PDGFRβ was validated as a representative biomarker of HCC neovascularization. The radiolabeling of [ ⁶⁸ Ga]Ga-DOTA-Z TRI was achieved at more than 95% radiochemical yield. In vitro assays showed specific uptake of [ ⁶⁸ Ga]Ga-DOTA-Z TRI in HCC tumor vessels by autoradiography. Animal PET/CT imaging with [ ⁶⁸ Ga]Ga-DOTA-Z TRI successfully visualized the tumor lesions in primary HCC rats and rhesus monkey, and indicated radiation absorbed dose of 2.03E-02 mSv/MBq for each scanning.
Conclusions
Our results demonstrated that [ ⁶⁸ Ga]Ga-DOTA-Z TRI conjugate could be applied as a promising PET tracer for early diagnosis of hepatocellular carcinoma.
... Recently, it was found that resistance of myofibroblasts to apoptosis played a critical role in fibrotic diseases, and the induction of apoptosis in myofibroblasts could be an effective strategy to alleviate liver fibrosis (Hinz and Lagares 2020;Li et al. 2019Li et al. , 2020Oh et al. 2016). In addition, Aghajanian et al. (2019) reported that cardiac fibrosis could be alleviated through specific killing of activated cardiac fibroblasts by FAP-specific CAR T cells, indicating that specific killing of activated fibroblasts could also be an effective way to inhibit tissue fibrosis. ...
Background
Chronic liver injury induces pathological repair, resulting in fibrosis, during which hepatic stellate cells (HSCs) are activated and transform into myofibroblasts. CD248 is mainly expressed on myofibroblasts and was considered as a promising target to treat fibrosis. The primary aim of this study was to generate a CD248 specific antibody-drug conjugate (ADC) and evaluate its therapeutic efficacy for liver fibrosis and its safety in vivo.
Methods
CD248 expression was examined in patients with liver cirrhosis and in mice with CCl 4 -induced liver fibrosis. The ADC IgG78-DM1, which targets CD248, was prepared and its bioactivity on activated primary HSCs was studied. The anti-fibrotic effects of IgG78-DM1 on liver fibrosis were evaluated in CCl 4 -induced mice. The reproductive safety and biosafety of IgG78-DM1 were also evaluated in vivo.
Results
CD248 expression was upregulated in patients with liver cirrhosis and in CCl 4 -induced mice, and was mainly expressed on alpha smooth muscle actin (α-SMA) ⁺ myofibroblasts. IgG78-DM1 was successfully generated, which could effectively bind with and kill CD248 ⁺ activated HSCs in vitro and inhibit liver fibrosis in vivo. In addition, IgG78-DM1 was demonstrated to have qualified biosafety and reproductive safety in vivo.
Conclusions
Our study demonstrated that CD248 could be an ideal target for myofibroblasts in liver fibrosis, and CD248-targeting IgG78-DM1 had excellent anti-fibrotic effects in mice with liver fibrosis. Our study provided a novel strategy to treat liver fibrosis and expanded the application of ADCs beyond tumors.
Graphic Abstract
... 10 For example, in liver fibrosis, Li et al. designed a fusion protein Z-hTRAIL, which is composed of the Z PDGFRβ , an affibody with high affinity for platelet-derived growth factor receptor beta (PDGFRβ) and human TNF-related apoptosis-inducing ligand (hTRAIL), and demonstrated that this fusion protein could specifically target PDGFRβpositive activated liver fibroblasts (hepatic stellate cells, HSCs) and induce their apoptosis, thus alleviating liver fibrosis. 11 In pulmonary fibrosis, Ashley et al. reported that blocking the inhibitor of apoptosis protein (IAP) family proteins, which had an antiapoptotic function, could enhance myofibroblast apoptosis and attenuates pulmonary fibrosis. 12 Recently, Aghajanian et al demonstrated that specific killing of the activated cardiac fibroblasts by fibroblast activation protein (FAP)-specific chimeric antigen receptor (CAR)-T cells could reduce cardiac fibrosis and restore cardiac function in mice. ...
Myofibroblasts, or activated fibroblasts, play a critical role in the process of renal fibrosis. Targeting myofibroblasts to inhibit their activation or induce specific cell death has been considered to be an effective strategy to attenuate renal fibrosis. However, specific biomarkers for myofibroblasts are needed to ensure the efficacy of these strategies. Here, we verified that CD248 was mainly expressed in myofibroblasts in patients with chronic kidney disease, which was inversely correlated with renal function. The same result was also confirmed in renal fibrotic mice induced by unilateral ureteral obstruction and aristolochic acid nephropathy. By using an antibody‐drug conjugate (ADC) named IgG78‐DM1, in which maytansinoid (DM1) was linked to a fully human antibody IgG78 through an uncleavable SMCC linker, we demonstrated that it could effectively bind with and kill CD248⁺ fibroblasts in vitro and alleviate renal fibrosis in mice models. Besides, we confirmed that IgG78‐DM1 had qualified biosafety in vivo. Our results confirmed that CD248 can be used as a specific marker for myofibroblasts, and specific killing of CD248⁺ myofibroblasts by IgG78‐DM1 has excellent anti‐fibrotic effect in renal fibrotic mice. Our study expanded the application of ADC and provided a novel strategy for the treatment of renal fibrosis.
Background and Aim
Hydronidone (HDD) is a novel pirfenidone derivative developed initially to reduce hepatotoxicity. Our previous studies in animals and humans have demonstrated that HDD treatment effectively attenuates liver fibrosis, yet the underlying mechanism remains unclear. This study aimed to investigate whether HDD exerts its anti‐fibrotic effect by inducing apoptosis in activated hepatic stellate cells (aHSCs) through the endoplasmic reticulum stress (ERS)‐associated mitochondrial apoptotic pathway.
Methods
The carbon tetrachloride (CCl 4 )‐ and 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine (DDC)‐induced liver fibrosis models were used for in vivo studies. In vitro studies were conducted using the human hepatic stellate cell line LX‐2. The apoptotic effect of HDD on aHSCs was examined using TUNEL and flow cytometry assays. The small interfering RNA (siRNA) technique was employed to downregulate the expression of interest genes.
Results
HDD treatment significantly promoted apoptosis in aHSCs in both the CCl 4 ‐ and DDC‐induced liver fibrosis in mice and LX‐2 cells. Mechanistic studies revealed that HDD triggered ERS and subsequently activated the IRE1α‐ASK1‐JNK pathway. Furthermore, the influx of cytochrome c from the mitochondria into the cytoplasm was increased, leading to mitochondrial dysfunction and ultimately triggering apoptosis in aHSCs. Notably, inhibition of IRE1α or ASK1 by siRNA partially abrogated the pro‐apoptotic effect of HDD in aHSCs.
Conclusions
The findings of both in vivo and in vitro studies suggest that HDD induces apoptosis in aHSCs via the ERS‐associated mitochondrial apoptotic pathway, potentially contributing to the amelioration of liver fibrosis.
Purpose
Human umbilical cord mesenchymal stem cell (hucMSC)-derived small extracellular vesicles (sEVs) are natural nanocarriers with promising potential in treating liver fibrosis and have widespread applications in the fields of nanomedicine and regenerative medicine. However, the therapeutic efficacy of natural hucMSC-sEVs is currently limited owing to their non-specific distribution in vivo and partial removal by mononuclear macrophages following systemic delivery. Thus, the therapeutic efficacy can be improved through the development of engineered hucMSC-sEVs capable to overcome these limitations.
Patients and Methods
To improve the anti-liver fibrosis efficacy of hucMSC-sEVs, we genetically engineered hucMSC-sEVs to overexpress the anti-fibrotic gene bone morphogenic protein 7 (BMP7) in parental cells. This was achieved using lentiviral transfection, following which BMP7-loaded hucMSC-sEVs were isolated through ultracentrifugation. First, the liver fibrosis was induced in C57BL/6J mice by intraperitoneal injection of 50% carbon tetrachloride (CCL4) twice a week for 8 weeks. These mice were subsequently treated with BMP7+sEVs via tail vein injection, and the anti-liver fibrosis effect of BMP7+sEVs was validated using small animal in vivo imaging, immunohistochemistry (IHC), tissue immunofluorescence, and enzyme-linked immunosorbent assay (ELISA). Finally, cell function studies were performed to confirm the in vivo results.
Results
Liver imaging and liver histopathology confirmed that the engineered hucMSC-sEVs could reach the liver of mice and aggregate around activated hepatic stellate cells (aHSCs) with a significantly stronger anti-liver fibrosis effect of BMP7-loaded hucMSC-sEVs compared to those of blank or negative control-transfected hucMSC-sEVs. In vitro, BMP7-loaded hucMSC-sEVs promoted the phenotypic reversal of aHSCs and inhibited their proliferation to enhance the anti-fibrotic effects.
Conclusion
These engineered BMP7-loaded hucMSC-sEVs offer a novel and promising strategy for the clinical treatment of liver fibrosis.
Liver fibrosis and hepatic carcinoma (HCC) pose a huge challenge worldwide due to the lack of effective treatment options for end-stage liver diseases. According to their functions and roles, hepatic myofibroblasts mainly include nontumoral fibroblasts (mainly activated hepatic stellate cells (HSCs)), which are involved in the wound-healing process of liver fibrosis, and cancer-associated fibroblasts (CAFs) in hepatic HCC. HSCs play a significant role in regulating extracellular matrix (ECM) deposition in progressive liver fibrosis. CAFs can be derived from activated HSCs and differentiate into ECM-producing myofibroblasts. Moreover, growing evidence shows that CAFs are the primary regulators of the HCC microenvironment, releasing growth factors and cytokines and suppressing the antitumor immune response. Combined therapeutic strategies show reduced drug resistance and side effects. Nanotechnology-based combined strategies aim to improve the delivery efficiency of various therapeutic agents with reduced toxicity via multiple mechanisms. In this review, we will discuss recent developments in combinational strategies based on nanotechnology that regulate myofibroblasts and the diseased microenvironment for liver fibrosis and HCC treatment. We will also identify the major challenges that the field is facing and offer some insights for future drug discovery.
Truncated transforming growth factor β receptor type II (tTβRII) is a promising anti-liver fibrotic candidate because it serves as a trap for binding excessive TGF-β1 by means of competing with wild type TβRII (wtTβRII). However, the widespread application of tTβRII for the treatment of liver fibrosis has been limited by its poor fibrotic liver-homing capacity. Herein, we designed a novel tTβRII variant Z-tTβRII by fusing the platelet-derived growth factor β receptor (PDGFβR)-specific affibody ZPDGFβR to the N-terminus of tTβRII. The target protein Z-tTβRII was produced using Escherichia coli expression system. In vitro and in vivo studies showed that Z-tTβRII has a superior specific fibrotic liver-targeting potential via the engagement of PDGFβR-overexpressing activated hepatic stellate cells (aHSCs) in liver fibrosis. Moreover, Z-tTβRII significantly inhibited cell migration and invasion, and downregulated fibrosis- and TGF-β1/Smad pathway-related protein levels in TGF-β1-stimiluated HSC-T6 cells. Furthermore, Z-tTβRII remarkably ameliorated liver histopathology, mitigated the fibrosis responses and blocked TGF-β1/Smad signaling pathway in CCl4-induced liver fibrotic mice. More importantly, Z-tTβRII exhibits a higher fibrotic liver-targeting potential and stronger anti-fibrotic effects than either its parent tTβRII or former variant BiPPB-tTβRII (PDGFβR-binding peptide BiPPB modified tTβRII). In addition, Z-tTβRII shows no significant sign of potential side effects in other vital organs in liver fibrotic mice. Taken together, we conclude that Z-tTβRII with its a high fibrotic liver-homing potential, holds a superior anti-fibrotic activity in liver fibrosis in vitro and in vivo, which may be a potential candidate for targeted therapy for liver fibrosis.
