200,000+ products from a single source!
sales@angenechem.com
Home > PDGFRβ‑targeted TRAIL specifically induces apoptosis of activated hepatic stellate cells and ameliorates liver fibrosis
PDGFRβ‑targeted TRAIL specifically induces apoptosis of activated hepatic stellate cells and ameliorates liver fibrosis
Rui Li1 · Zhao Li1 · Yanru Feng1 · Hao Yang1 · Qiuxiao Shi1 · Ze Tao1 · Jingqiu Cheng1 · Xiaofeng Lu
Introduction
Liver fibrosis, which is characterized by the excessive deposition of extracellular matrix (ECM) in the liver, is a grue- some consequence of chronic liver diseases (CLD) such as virus hepatitis, schistosomiasis, nonalcoholic fatty liver disease (NAFLD), and alcoholic liver disease (ALD) [1]. Sustained liver fibrosis eventually progresses to liver cir- rhosis and even hepatocellular carcinoma, which requires liver transplantation. However, due to a serious shortage of donors, numerous patients die while on the waiting list [2].Global deaths induced by liver cirrhosis have increased steadily in past decades. Since 2010, liver cirrhosis has caused more than one million deaths worldwide (approxi-mately 2% global deaths) each year [3]. In addition, a recent investigation indicated that the global burden of CLD has exceeded 1.3 billion [4]. Once these liver diseases progress to liver cirrhosis, the global healthcare burden will be drasti- cally increased. However, the Food and Drug Administration (FDA) of the United States has not approved any targeted antihepatofibrotic therapy.
Although long-term antivirus therapy leads to cirrhosis reversion in some hepatitis B virus (HBV)-infected patients, the other CLD-associated cirrhosis might not be reverted by removing the causative agent [5]. Generally, the regression of CLD at the stage of cirrhosis is a clinical challenge. Fibro- sis is an early step in the progression of cirrhosis. If a patient is treated at the stage of liver fibrosis, it might not be difficult to regress and even prevent cirrhosis. Excessive accumula- tion of ECM in the liver is a hallmark of liver fibrosis. Since activated hepatic stellate cells (aHSCs) are responsible for most of the ECM deposition, reducing the quantity of aHSCs was considered the essential strategy for the targeted therapy of liver fibrosis. Numerous studies revealed that the fate of aHSCs during liver fibrosis recovery include phenotype reversion (being reverted into quiescent HSCs (qHSCs)), senescence, and apoptosis [6, 7]. However, spontaneous res- olution of liver fibrosis predominantly relies on the apoptosis of aHSCs. Inducing the apoptosis of aHSCs is more feasi- ble. It was found that adiponectin, cannabinoids, hepatocyte growth factor (HGF), nerve growth factor (NGF), Fas ligand (Fas L), tumor necrosis factor α (TNFα), and TNF-related apoptosis-inducing ligand (TRAIL) could directly induce the apoptosis of aHSCs [8]. Other cytokines such as interferon γ (IFNγ) and interferon α (IFNα) could directly or indirectly kill aHSCs [9].
Considering biosafety, TRAIL was the most appealing apoptosis inducer. In previous studies, TRAIL receptor 1 (TRAIL R1, DR4) and TRAIL receptor 2 (TRAIL R2, DR5) were found to be overexpressed in tumor cells, whereas the normal cells ubiquitously express the decoy receptors (DcRs) of TRAIL. The ligation of TRAIL to DR4 and/or DR5, but not DcRs, triggers intracellular proapoptotic sig- nals. Consequently, recombinant human TRAIL (hTRAIL) effectively induces apoptosis in tumor cells and not in nor- mal cells, thus showing it is sufficiently safe for cancer patients and promoting the development of hTRAIL and its variants as anticancer drugs [10, 11]. Recently, it was found that DR5 is overexpressed in aHSCs [12]. TRAIL is used by NK cells and Kupffer cells to control liver fibrosis by inducing apoptosis of aHSCs [13–16].These results indicate the important roles of TRAIL in the resolution of liver fibro- sis, which triggered our interest in developing recombinant hTRAIL as a novel antihepatofibrotic agent. Nevertheless, according to the lessons from hTRAIL as an anticancer drug, the antihepotofibrotic effect of hTRAIL in patients is limited by its short half-life and poor aHSCs-targeting. Although the half-life of hTRAIL can be significantly extended by chemical modifications [17–19], there are no efficient tools available for the targeted delivery of hTRAIL to aHSCs.
Theoretically, biomarkers on the surface of aHSCs might serve as mediators for aHSCs-targeted delivery. It is known that the expression of integrin αvβ3, vimentin and desmin, mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF-IIR), collagen type VI recep- tor (CVIR), and platelet-derived growth factor receptor β (PDGFRβ) is upregulated during HSCs activation. As the most potent mediator of aHSCs proliferation, PDGFRβ was considered as an ideal target for aHSCs-targeted drug delivery [20, 21]. By using PDGFRβ-binding peptides as carriers, IFNγ and its mimics have been selectively deliv- ered to aHSCs [22]. In addition, a versatile aHSCs-tar- geted drug delivery system was built by modifying human serum albumin with a PDGFRβ-binding peptide [21]. However, these small peptides usually show low affinity (KD ~ μM) for their ligands, which might limit their effi- cacy as drug carriers [23]. Recently, numerous affibodies against PDGFRβ have been identified by Lindborg et al. [24]. Of these affibodies, ZPDGFRβ showed high affinity (KD ~ nM) for PDGFRβ. In previous studies, we success- fully delivered hTRAIL to PDGFRβ-positive pericytes of tumor vessels, thus significantly improving its antitumor effect and suggesting the enhancement of the antihepatofi- brotic effect of hTRAIL by aHSCs-targeted delivery of hTRAIL with ZPDGFRβ as a carrier [25]. As the aHSCs- targeted delivery might be hindered by the progressive alteration in the structure and biochemistry of the fibrotic liver [26], investigating whether ZPDGFRβ could deliver hTRAIL to aHSCs and enhance the ability to ameliorate liver fibrosis is needed.
In this study, we first determined the coexpression of PDGFRβ and DR5 in aHSCs and evaluated the potential of ZPDGFRβ as an aHSC-targeting carrier for antihepatofi- brotic therapy. Subsequently, we produced the fusion protein Z-hTRAIL by genetically conjugating ZPDGFRβ to hTRAIL and evaluated the roles of the fused ZPDGFRβ in aHSCs-targeted delivery, as well as enhancing the anti- hepatofibrotic effects of hTRAIL. Moreover, we prepared PEGylated Z-hTRAIL and investigated the impact of PEGylation on half-life extension and antihepatofibrotic effect improvement. Finally, we evaluated the acute tox- icities of Z-hTRAIL with or without PEGylation. All the results suggested the importance of further investigating PEGylated Z-hTRAIL as an aHSCs-targeting and long- acting antifibrotic agent for liver fibrosis.
Materials and methods
Preparation of recombinant proteins
Recombinant expression, purification and identification of ZPDGFRβ, hTRAIL, and the fusion protein Z-hTRAIL con- taining ZPDGFRβ and hTRAIL were performed as previously published [25]. The PEGylated Z-hTRAIL (Z-hTRAIL- 10K) was prepared by conjugating 10 kDa PEG to the N-terminus of Z-hTRAIL. The purity and pharmacokinet- ics of Z-hTRAIL-10K were examined according to our previous study [27].
Cell culture and cytotoxicity assays
The HSCs used here included primary human HSCs (hHSCs, ScienCell, CA, USA) and the LX2 cell line (BeNa Culture Collection, Beijing, China). HSCs were activated under hypoxic conditions [28]. Endothelial cells (ECs), smooth muscle cells (SMCs) and normal hepatocytes (NCTC 1469) are obtained from the American Type Culture Collection (ATCC, VA, USA). To examine the cytotoxicity, 4 × 104 cells in 400 μL medium were inoculated into 24-well plates and treated with proteins at different concentrations for 24 h followed by measuring the surviving cells using a cell counting kit-8 (CCK-8, Dojindo, Japan). The viability of protein-treated cells was expressed as a percentage of that of PBS-treated cells. The half maximal inhibitory con- centration values of proteins (IC50) were calculated using the SPSS 21.0 software according to their respective cell viability curves.
Apoptosis assays
Apoptosis of cultured cells was illustrated by flow cytom- etry after FITC-Annexin V and propidium iodide (PI) dual staining. The involvement of the caspase pathway in protein- induced apoptosis was revealed by using caspase-specific substrates and a pan-caspase inhibitor Z-VAD-FKM. The terminal deoxynucleotidyl transferase UTP nick end labeling (TUNEL) assay (Promega, WI, USA) was used to visualize apoptotic cells in the liver [25].
Expression of biomarkers
Expression of PDGFRβ, αSMA, DR5, CD31, CD68 was detected by immunofluorescence, immunohistochemistry, or flow cytometry with corresponding primary and second- ary antibodies. The primary antibodies used here include anti-PDGFRβ (AF385, R&D, MN, USA; ab32570, Abcam, MN, USA), anti-CD31 (102401, Biolegend, CA, USA), anti- DR5 (ab8416, Abcam, MN, USA), anti-CD68 (ab125212,
Abcam, MN, USA), and anti-αSMA (ab32575, Abcam, MN, USA). All the secondary antibodies obtained from Abcam (MN, USA) include donkey anti-goat IgG, (ab150133, ab96932), goat anti-rat IgG (ab96888), donkey anti-rabbit IgG (ab150073, ab96892). These secondary antibodies were labeled with Alexa Fluor® 488 or DyLight® 550. The goat anti-mouse IgG labeled with horseradish peroxidase (HRP) was purchased from ZSGB-Bio Corporation (Beijing, China). The primary antibodies were incubated with tissues at 37 °C or with cultured cells at 4 °C for 1.5 h followed by incubation with secondary antibodies for 30–45 min. The slides were washed three times with phosphate buffer (PBS, 137 mM NaCl, 10 mM Na2HPO4, 2.68 mM KCl, 2 mM KH2PO4, pH 7.4). At least five images were used to meas- ure the surface area of biomarkers with Image J software.
Cell binding and location of protein
Proteins were labeled with 5(6)-carboxyfluorescein (FAM) (Sigma, MA, USA) or DyLight800 (Thermo Scientific, MA, USA) according to our previous work [25]. To deter- mine the cell binding capability of the protein, cells were incubated with FAM- or DyLight800-labeled proteins prior to flow cytometry analysis and observation under fluores- cence microscope or optical imaging system. To localize the recombinant proteins in PDGFRβ-expressing cells in the fibrotic liver, mice exposed to CCl4 were intravenously injected with a single dose of FAM-labeled protein. Subse- quently, the livers were collected and sectioned under fro- zen conditions 4 h post-injection. The PDGFRβ-expressing cells were identified by immunofluorescence with antibodies against PDGFRβ prior to observation under the fluorescence microscope. To monitor the accumulation of proteins in the fibrotic liver, a single dose of DyLight800-labeled proteins were intravenously injected into mice (n = 3). Subsequently, dynamic optical imaging of the mice and their livers was performed according to the methods published by Luli et al. [29].
CCl4‑induced liver fibrosis in mice and treatment
All the applicable institutional guidelines for the care and use of animals were followed. Female C57BL/6 mice (16–18 g, 6-week-old) were purchased from Dossy Experi- mental Animal Corporation (Chengdu, China) and housed in experimental animal center (SPF grade) of the hospital. To induce liver fibrosis, mice were intraperitoneally injected with 25% (v/v) CCl4 dissolved in olive oil at 5 ml/kg (2.5 ml/ kg for the first injection) twice a week. The liver tissues and blood samples of mice (n = 5–7) were collected at different time (0, 2, 4, or 6 weeks) post-CCl4 injection. The histopa- thology of the liver tissues was examined by hematoxylin and eosin (H&E) staining. The aHSCs in the fibrotic region were identified by immunohistochemistry with an antibody against αSMA or PDGFRβ. Macrophages were indicated by immunofluorescence with antibody against CD68. Blood vessels were illustrated by immunofluorescence with anti- CD31. The fibrotic degree of liver was evaluated using the Ishak scoring system according to the collagen deposition visualized by Sirius Red staining [30]. The hydroxyproline in the liver tissues was quantified using a kit (Jiancheng, Nanjing, China). Fibrotic markers including TGFβ1, BMP7, MMP2, and TIMP1 in liver tissues were determined by enzyme-linked immunosorbent assay (ELISA) kits (Boster, Wuhan, China). The glutamic-pyruvic transaminase (AST) and glutamic-oxaloacetic transaminase (ALT) in the blood were measured using specific kits (JianCheng, Nanjing, China).
To determine their antifibrotic ability, proteins (10 mg/kg for ZPDGFRβ, hTRAIL, and Z-hTRAIL, 3.5 mg/kg ZPDGFRβ plus 10 mg/kg hTRAIL for ZPDGFRβ + hTRAIL) were intrave- nously injected into mice (three times per week for 2 weeks) starting the 4th week post-initial CCl4 injection. Compared to the mice injected with PBS or olive oil, reduction of collagen deposition and fibrotic markers in liver of mice treated with protein reflect the antihepatofibrotic effect of these proteins.
The spontaneous recovery model of liver fibrosis was constructed by withdrawal of CCl4. Briefly, to induce liver fibrosis, mice were intraperitoneally injected with CCl4 twice a week for 2 weeks. Subsequently, these mice were allowed to recover for an additional 2 weeks without injec- tion of CCl4.
Over 100,000 products now available from Angene:
© 2019 Angene International Limited. All rights Reserved.