Inhibition of lysyl oxidase-like 1 (LOXL1) expression arrests liver fibrosis progression in cirrhosis by reducing elastin crosslinking

Accepted Manuscript Inhibition of lysyl oxidase-like 1 (LOXL1) expression arrests liver fibrosis progression in cirrhosis by reducing elastin crosslinking

Wenshan Zhao, Aiting Yang, Wei Chen, Ping Wang, Tianhui Liu, Min Cong, Anjian Xu, Xuzhen Yan, Jidong Jia, Hong You PII: DOI: Reference:

S0925-4439(18)30030-9 https://doi.org/10.1016/j.bbadis.2018.01.019 BBADIS 65035

To appear in: Received date: Revised date: Accepted date:

13 July 2017 11 January 2018 19 January 2018

Please cite this article as: Wenshan Zhao, Aiting Yang, Wei Chen, Ping Wang, Tianhui Liu, Min Cong, Anjian Xu, Xuzhen Yan, Jidong Jia, Hong You , Inhibition of lysyl oxidaselike 1 (LOXL1) expression arrests liver fibrosis progression in cirrhosis by reducing elastin crosslinking. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Bbadis(2018), https://doi.org/10.1016/ j.bbadis.2018.01.019

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ACCEPTED MANUSCRIPT

Inhibition of lysyl oxidase-like 1 (LOXL1) expression arrests liver fibrosis progression in cirrhosis by reducing elastin crosslinking Wenshan Zhao1,3, Aiting Yang 2,3, Wei Chen 2, Ping Wang 1,3, Tianhui Liu 1,3, Min Cong 1,3, Anjian Xu 2, Xuzhen Yan 1,3, Jidong Jia 1,3, and Hong You 1,3* Liver Research Center, Beijing Key Laboratory of Translational Medicine in Liver Cirrhosis,

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Experimental and Translational Research Center, Beijing Friendship Hospital, Capital Medical

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Beijing Friendship Hospital, Capital Medical University, Beijing, China

University, Beijing, China.

National Clinical Research Center of Digestive Diseases, Beijing, China.

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Correspondence: Hong You, Liver Research Center, Beijing Key Laboratory of Translational Medicine in Liver Cirrhosis, Beijing Friendship Hospital, Capital Medical University; National

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Clinical Research Center of Digestive Diseases, Beijing, China.

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No. 95 Yong An Road, Xi Cheng District, Beijing, 100050, China. Tel.: +86-10-63139019; Fax: +86-10-83165944; E-mail: [email protected].

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Keywords: liver fibrosis; elastin crosslinking; LOXL1; ECM

Abbreviations: LOXs, lysyl oxidases; CCl4, carbon tetrachloride; LOXL1, lysyl oxidase-like 1; α-SMA, α-smooth muscle actin; HSCs, hepatic stellate cells; AAV, adeno-associated vector. 1

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Abstract Mature crosslinked-poly-elastin deposition has been found to be associated with liver fibrosis. However, the regulation of crosslinked/insoluble elastin in liver fibrosis remains largely unknown. Here, we investigated the contribution of lysyl oxidases (LOXs) family, mediated elastin

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crosslinking, to liver fibrogenesis. We established carbon tetrachloride (CCl4)-induced liver

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fibrotic and cirrhotic models and found that crosslinked/insoluble elastin levels spiked only in

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cirrhosis stage during disease progression, in comparison to collagen Ι levels which increased continuously though all stages. Among the LOXs family members, only LOX-like 1 (LOXL1)

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levels were coincident with the appearance of crosslinked/insoluble elastin. These coincidences

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included that LOXL1 expression increased (34 fold) in cirrhosis, localized with α-smooth muscle actin (SMA) and was absent in normal and fibrotic livers. In LX-2 cells, LOXL1 silencing

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arrested expression of α-SMA, elastin and collagen Ι.

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Our previously characterized adeno-associated vector (AAV) 2/8 shRNA was shown to effectively downregulate LOXL1 expression in CCl4 induced fibrosis mice models. These resulted

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in delicate and thinner septa and less crosslinked elastin, with a 58% loss of elastin area and 51%

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decrease of collagen area. Our findings strongly suggested that elastin crosslinking and LOXL1 were co-associated with liver cirrhosis, while selective inhibition of LOXL1 arrested disease progression by reducing crosslinking of elastin.

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Introduction Liver fibrosis is characterized by a sustained quantitative accumulation of extracellular matrix (ECM) [1, 2]. Most studies have put an emphasis on characterizing the non-homeostatic synthesis and degradation of ECM and attempt to develop therapeutic approaches [3-5]. As a

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major component of ECM, elastin plays an important role in liver fibrosis progression [6].

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Elastin is a stable protein formed by polymerization of tropoelastin monomer which is a

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soluble precursor in the biosynthesis, and is synthesized and secreted by hepatic stellate cells (HSCs) and portal fibroblasts [6, 7]. In normal liver, the content of elastin is much lower than

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collagen. However, elastin deposition appears to increase much faster during progression toward

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liver cirrhosis [8]. More importantly, it has been demonstrated that elastin deposition in ECM limits liver fibrosis improvement [9], perhaps leading to a failure of therapeutic response. Elastin

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crosslinking renders resistance to degradation by elastase [9] and may be responsible for a more

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permanent form of its deposition, which is consistent with therapeutic resistance. ECM including collagen and elastin crosslinking is mediated by lysyl oxidases (LOXs) [10].

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LOX family consists of five members, LOX and LOX-like 1 to 4 (LOXL1 to LOXL4). All these

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enzymes are copper-dependent amine oxidase with a conserved C-terminal domain, which could promote crosslinking formation via oxidation reactions [11]. LOX catalyzes the formation of collagen and elastin crosslinking [12, 13]. By contrast, LOXL1 is believed specially associated with elastin crosslinking [14]. Of the five members, LOXL2 has been mostly widely studied in liver fibrosis [15, 16] and carcinoma [17, 18]. LOXL2 monoclonal antibody (GS-6624) has been applied in nonalcoholic steatohepatitis patients in clinical trials [19]. It has been reported that the upregulation of LOX and LOXL2 expression are detected in Wilson disease and primary biliary

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ACCEPTED MANUSCRIPT cholangitis patients [20]. To date, there are few studies about LOXL3 or LOXL4 in liver fibrosis. Previous studies have focused on collagen crosslinking [15, 16, 21], while the involvement and regulation of LOXs in elastin crosslinking remains largely unknown. Therefore, we utilized both in vitro and in vivo studies to explore whether elastin crosslinking

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could impact liver fibrosis progression, as well as functional regulation of LOXs on elastin

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crosslinking in liver fibrogenesis.

Material & methods

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CCl4-induced liver fibrosis models

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Male C57BL/6 mice (8 weeks old, 20.0 g of body weight in average) were purchased from Beijing HFK Bioscience Company (China). The mice were group-housed under constant

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temperature (23 ± 2°C) and 40–60% humidity with humane care and maintained on a 12

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hour/12hour light/dark cycle. Food and water were accessed ad libitum. Study protocols about laboratory animal use were in accordance with the guidelines for the Beijing Friendship Hospital

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Animal Care and Ethics Committee. Liver fibrosis in C57BL/6 mice was induced by

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intraperitoneal injection of 12.5% CCl4 in olive oil (1/7, v/v), while control mice only received olive oil. Twice-a-week administration of CCl4 was performed at a dose of 0.01 mL/g body weight, which lasted for 4 weeks (fibrosis, n = 8) or 8 weeks (cirrhosis, n = 8). The degree of liver fibrosis was assessed in accordance with staging criterion which has been specially used for mouse models [22]. All animals were sacrificed and livers were harvested upon euthanization at the predetermined time points.

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Histological examination and hydroxyproline measurement Liver tissue specimens from mice models were fixed in 4% paraformaldehyde and embedded in paraffin wax. The liver samples were sliced in 4 μm thick. For fibrosis analysis, the sections were stained with hematoxylin and eosin (H&E) and sirius red staining. Elastin fibers

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were stained using EVG staining kit (BASO, China) as vendors’ protocol. To observe elastin

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fiber in liver tissues clearly, we just used basic fuchsin dye in the kit which could specifically

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bind to elastin fiber. Collagen and elastin area were quantified using Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, MD). All the staining was averaged from 10 high power fields

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(×100) per slide in all mice models [23] and positive vessel walls were excluded for an accurate

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quantification. Hydroxyproline contents, which could reflect the amount of collagen, were detected using the kits purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing,

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China) and performed as instructions of manufacturer.

Immunofluorescence and Immunohistochemistry

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Double immunofluorescent staining was performed according to standard protocols. Briefly,

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the slides of tissues were incubated with primary antibodies against with LOXL1 (dilution 1:50, sc-166632, Santa Cruz, USA) and α-SMA (dilution 1:100, ab5694, Abcam, USA), 4 °C overnight. After washing with phosphate-buffered saline (PBS) for three times, the tissues were incubated with a mixture of Alexa Fluor 488 anti-mouse Alexa (dilution 1:500, Invitrogen, USA) and Alexa Fluor 594 anti-Rabbit (dilution 1:500, Invitrogen, USA) conjugated secondary antibodies for 1 h at room temperature. Then the stained slides were viewed and photographed with a confocal microscope (Olympus, USA). In addition, immunohistochemistry were

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ACCEPTED MANUSCRIPT performed with paraffin-embedded liver sections for LOXL1 (dilution 1:100, NBPI-82827, Novus, USA) and α-SMA (dilution 1:100, 14395-1-AP, Proteintech, USA) analysis.

Determination of crosslinked elastin and collagen

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The contents of crosslinked/insoluble elastin and crosslinked/insoluble collagen by the

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Fastin ElastinTM Assay (Biocolor life science assays, UK) and SircolTM Insoluble Collagen Assay

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(Biocolor life science assays, UK) as published before [24-25]. In brief, crosslinked elastin, which was insoluble in liver samples, was treated by heat extraction with oxalic acid. To achieve

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complete dissolution of crosslinked elastin, heat extraction was repeated up to three times. After

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precipitation, centrifugation and bounding with dye reagent, the elastin-dye complex was collected, released into solution and measured at 513 nm. Similarly, the quantification of

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insoluble collagen was detected according to the instruction of manufacturers. All the samples

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were performed to analyze in duplicate.

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Relative quantitative determination of elastin and collagen crosslinks by ultra-performance

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liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) analysis In the study, we detected the content of pyridinoline and desmosine, which were common and unique enzymatic crosslinks in collagen and elastin [26, 27], respectively. Liver samples from mice models were washed and weighted. After weighting, tissues were hydrolyzed with 6N HCl for 24 hour as described before [26]. Then the hydrolysate was dried by a speed vacuum centrifuge and dissolved in distilled water. The solution was used for next UPLC-MS/MS analysis.

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ACCEPTED MANUSCRIPT The crosslinks were detected according to the method published before [28]. In brief, a total of 20 μL solution was injected into UPLC system (ACQUITY, Waters, USA) and ACQUITY UPLC BEH HILIC column (2.1 mm × 100 mm, 1.7 μm) was used for separation. The mobile phase was methanol and 0.1% aqueous formic acid. Firstly, the column was equilibrated with

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methanol/ 0.1% aqueous formic acid (98:2, v: v) for 5 minutes. Then, linear gradient elution was

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performed from 98% to 82% methanol in 0.1% aqueous formic acid in 8 minutes. The flow rate

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was 0.1 mL/min at room temperature. The MS experiments were carried out using a QTRAP 5500 hybrid triple quadrupole/linear ion trap mass spectrometer (AB SCIEX Foster City, USA)

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coupled with an electrospray ion (ESI) source. Quantification analyses were performed by The optimum MS parameters were as

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multiple reaction monitoring (MRM) in positive mode.

follow: source temperature, 450 ◦C; Ionspray Voltage, 4.5 kV; curtain gas (CUR), 20 psi;

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nebulizer gas (Gas1) and 50 psi and turbo gas (Gas2), 50 psi. Data were acquired and processed

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with Analyst software (version 1.5.1, USA). The mass transitions of the protonated precursor/product ion pairs that were used to record the selected ion mass chromatograms of

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desmosine and pyridinoline were m/z 526.3→436.3 (collision energy, 20 eV; declustering

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potential, 100 V) and 429.2→339.2 (collision energy, 20 eV; declustering potential, 100 V), respectively. In our study, the relative quantitative determination of pyridinoline and desmosine was carried out by MRM method.

Western blot analysis The extraction of protein from frozen liver tissues or cultured cells, samples preparation and western blotting protocol were carried out as previously described [29]. The primary antibodies

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ACCEPTED MANUSCRIPT were incubated as follows: LOXL1 (diluted 1:1000, ab81488, Abcam, USA), elastin (diluted 1:1000, MAB2503, Millipore, USA), α-SMA (diluted 1:1000, CBL171, Millipore, USA), collagen Ι (diluted 1:1000, 14695-1-AP, Proteintech, USA), GAPDH (diluted 1:8000, ab181603, Abcam, USA). At last, the membranes were visualized by the enhanced chemiluminescence light method.

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GAPDH was used as a loading control.

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Real-time PCR

Total mRNA extraction from liver tissue and LX-2 cells was acquired using Qiagen RNeasy

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Mini Kit (Qiagen, Germany). The Reverse Transcription, real-time polymerase-chain reaction

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system and data analysis were in accordance with Yang et al [29]. The primers were listed in

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Table 1.

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Cell culture

Human HSCs line, LX-2, was kindly provided by Professor Lieming Xu [30]. LX-2 cells

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were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA), which contained

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10% (v/v) fetal bovine serum (FBS), 10 U/ mL penicillin and 0.1 mg/mL streptomycin (complete medium).

Synthesis and transfection of siRNA targeted LOXL1 in vitro Small interfering RNA (siRNA) oligonucleotides were synthesized by Shanghai GenePharma Co.,Ltd. (Shanghai, China). The sequences used in the study were shown as following:

sense,

5’-GCACGUGAACCCAAAGUAUTT-3’;

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antisense,

ACCEPTED MANUSCRIPT 5’-AUACUUUGGGUUCACGUGCTT-3’. LX-2 cells at a total of 2×105 were cultured in 6-well plates the day before transfection. As the manufacturer recommended, transfection of 2 μg siRNA was performed by Lipofectamine 2000 (Invitrogen, USA). The ratio of Lipofectamine 2000 to siRNA was 1:2. After transfected for

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6 hours, the medium for LX-2 cells was changed into DMEM with 10% FBS. The efficiency of

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knockdown was corroborated by western blot analysis and transfection was independently

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repeated three times in triplicate plates.

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Construction and injection of shRNA against LOXL1 in vivo

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Adeno-associated viral vector (AAV) 2/8 encoding shRNA against LOXL1 (LOXL1-shRNA) and control vector were generated and packaged by Shanghai Taitool Bioscience Co.,Ltd. (China).

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The sequence was in accordance with siRNA which was used in HSCs. The titers of

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LOXL1-shRNA and negative control vectors were 2.03×1013 vector genomes per milliliter (v.g./mL) and 1.71×1011 v.g./mL, respectively. As recommended by protocol, the final dose of

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injection was 1×1010 v.g. /g which was diluted by PBS.

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According to our successfully established the methods of dilution and injection of AAV2/8-shRNA [23], C57BL/6 mice were injected with AAV2/8-shRNA through tail vein. After 24 hours of vectors injection, those mice (n = 6 in each group) were induced to liver fibrosis as methods mentioned above. The liver samples were harvested and frozen at - 80ºC for further analysis. Serum aminotransferase and albumin activities were measured by biochemical method using an automated biochemical analyzer (Olympus, Japan).

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Statistical analysis All results were shown as the means ± SD from three independent experiments. Student’s 𝑡-test was used to compare difference between two groups. Differences among mean values of multiple groups were analyzed using the nonparametric analysis of variance test (SPSS 16.0,

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USA). 𝑃 < 0.05 was considered to be significant.

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Results

Elastin accumulates strongly only in cirrhosis stage, while collagen accumulates at a

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continuous rate from onset of injury.

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C57BL/6 mice were treated with repeated CCl4 injection for up to 8 weeks to induce liver fibrosis. Repeated CCl4 injection led to continuous progressive accumulation of ECM during

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fibrogenesis, characterized as fibrosis pattern with septa extended from portal veins at week 4

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and evidence of cirrhosis with fibrotic septa divided hepatic lobule at week 8 (Figure 1A). Additionally, continuous elevation was detected in hepatic collagen levels along with

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fibrogenesis, which were determined via hydroxyproline, representing an increase of 1.9-fold in

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fibrosis and 2.8-fold in cirrhosis stage compared with normal group, respectively (P < 0.01, Figure 1B).

We analyzed the dynamic expression of elastin and collagen during the process of progression. Morphometrical quantification of liver sections stained for elastin revealed that elastin was notably deposited for 8 weeks of CCl4 administration (Figure 1A), with a 20-fold increase in elastin-stained area (P < 0.01, Figure 1C) compared with normal and fibrotic liver tissues. In line with the morphometrical quantification, elastin massively accumulated in

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ACCEPTED MANUSCRIPT cirrhosis stage both on mRNA and protein level (P < 0.05, Figure 1D and E). However, expression of collagen Ι was gradually elevated from fibrosis stage to cirrhosis stage, as shown in collagen-stained area and corresponding expression analysis of mRNA and protein (P < 0.05, Figure 1C, D and E).

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Next, crosslinked/insoluble elastin and collagen contents in livers were measured.

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Interestingly, as shown in Figure 1F, it was found that the crosslinked/insoluble elastin levels

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were remarkably and massively increased in cirrhosis stage (207.9 ± 34.7 μg in normal, 213.5 ± 26.7 μg in fibrosis and 352.0 ± 57.0 μg in cirrhosis, per 10 mg; P < 0.01), and was very unlike

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the continuously upregulated expression of crosslinked/insoluble collagen (56.4 ± 15.1 μg in

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normal, 186.1 ± 38.6 μg in fibrosis and 221.9 ± 28.0 μg in cirrhosis, per 10 mg; P < 0.05). In parallel with crosslinked/insoluble elastin content, the UPLC-MS/MS analysis revealed that the

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increased desmosine, uniquely reflected elastin crosslinks, was consistently observed in

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cirrhosis stage (P < 0.01, Figure 1G). This was different from the gradual increase expression of

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pyridinoline during fibrosis progression, represented collagen crosslinks.

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LOXL1 expression was strongly associated with liver fibrosis progression. As LOX family contributes to matrix crosslinking, we characterized the dynamic changes of LOX family during progressive period. Compared with other members in LOX family, LOXL1 was notably elevated after 8 weeks of CCl4 administration on mRNA level, up to 34-fold in cirrhosis stage (P < 0.001, Figure 2A), which was closely matched the dramatic increased expression of elastin. LOXL1 was seen in cirrhosis stage along with distribution of α-SMA by immunohistochemistry analysis, while was hardly detectable in normal and fibrosis stage (Figure

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ACCEPTED MANUSCRIPT 2B). Furthermore, the double immunofluorescence staining revealed that LOXL1 and α-SMA almost fully co-localized in the septa at cirrhosis stage (Figure 2C), which further confirmed the association between LOXL1 and HSCs.

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LOXL1 silencing suppressed activation of HSCs and expression of elastin in vitro

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As shown in Figure 2B & C, LOXL1 was strongly associated with HSCs and activation of

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HSCs is also a critical step during liver fibrogenesis. Thus we further evaluated whether LOXL1 had a direct effect on HSCs though LOXL1 suppression. After a decrease of 80.9% in LOXL1

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expression, activation of LX-2 cells was inhibited, which was demonstrated by a 53.2% reduction

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of α-SMA (P < 0.05, Figure 3A). In addition, both α-SMA and collagen Ι expression were significantly decreased after LOXL1 inhibition (P < 0.05, Figure 3A). These decreased

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pro-fibrotic markers were consistently seen on the protein level (Figure 3B).

LOXL1 inhibition suppressed elastin crosslinking and attenuated CCl4-induced liver fibrosis

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To determine whether LOXL1 affected liver fibrosis through elastin crosslinking during

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chronic liver injury, LOXL1 expression was suppressed in vivo. AAV2/8-LOXL1-shRNA was successfully established (Figure 4A) and its injection was performed to knockdown LOXL1 expression in CCl4-treated mice models, with negative vector as control group (Figure 4B). After repeated CCl4 treatment for 8 weeks, fibrosis was assessed by sirius red staining and elastin staining to observe the structure of collagen and elastin fibers, respectively. After LOXL1 inhibition, it revealed that elastin was significantly reduced in those mice treated with LOXL1-shRNA and was shown as being darkly stained and thinner fibers, with a 58% decrease of

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ACCEPTED MANUSCRIPT elastin-stained area during fibrosis progressive period (Figure 4C and D). With sirius red staining observation, fibrotic septa in LOXL1 inhibition mice became delicate and less extensive, accompanying with a 51% loss of collagen fibrils (Figure 4D). Similarly, the mRNA expressions of collagen Ι and elastin were remarkably suppressed in LOXL1-shRNA treatment mice compared

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with control group (Figure 4E), with a decrease of 60% (P < 0.05) and 80% (P < 0.05),

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reduction comparison with control group (P < 0.05, Figure 4F).

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respectively. Furthermore, by inhibition of LOXL1, hepatic hydroxyproline levels showed a 33%

The direct effect of LOXL1 activity on elastin crosslinking was tested by insoluble elastin

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extraction analysis. The average contents of insoluble elastin, considered as crosslinked elastin,

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represented 82.0 ± 40.7 μg per 10 mg liver samples in LOXL1 inhibition group, compared with control group which had 153.8 ± 24.0 μg per 10 mg liver samples (P < 0.05, Figure 4G). A

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reduction of 36.2% in desmosine expression was observed in LOXL1 inhibition mice compared

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with controls after CCl4 administration for 8 weeks (P < 0.05, Figure 4H).

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LOXL1 inhibition decreased myofibroblast activity in livers

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Next, we detected the expression of α-SMA in liver tissues with or without LOXL1-shRNA treatment. It has been demonstrated that the expression of LOXL1 was inhibited both on mRNA level (Figure 5A) and protein level (Figure 5B), with a decrease of 79% (Figure 5A, P < 0.05) in mRNA level. After LOXL1 was knockdown, the activation of HSCs, indicated by the expression of α-SMA, was remarkably inhibited by 61% compared with negative control group (Figure 5A, P < 0.05), according with the protein expression of α-SMA (Figure 5B). However, the downregulated HSCs activation mediated by LOXL1 inhibition did not significantly relieve liver

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ACCEPTED MANUSCRIPT function. There was a decreased trend of alanine aminotransferase (ALT) in mice treated with LOXL1-shRNA, while no improvement of aspartate transaminase (AST) and albumin (ALB) levels (not statistically significant, Figure 5C).

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Discussion

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In the current study, we directly analyzed the dynamic changes of crosslinked extracellular

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matrix (elastin and collagen) during liver fibrosis progression. Our studies demonstrated that both total elastin and crosslinked elastin accumulated only at cirrhosis stage, which was different

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from the continuously increasing trend of collagen from the onset of injury. Other studies

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represented failure of elastin degradation as an important factor for ECM accumulation in cirrhosis [31]. It was difficult to remodeling when elastin was crosslinked in fibrotic liver [32].

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Thus, in contrast to most studies focused on collagen stabilization, our data suggested that elastin

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crosslinking might play a key effect at cirrhosis stage and be a critical factor limited fibrosis improvement. To our knowledge, there are few studies directly focused on the regulation of

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elastin stabilization in liver fibrosis progression or regression.

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It has been demonstrated that LOX family could contribute to matrix crosslinking and LOXL1, one member of the family, most closely contributes to elastin crosslinking [12, 33]. It has been shown that loss of elastin, not collagen, is seen in LOXL1-deficient mice, suggesting LOXL1 was closely required for elastogenesis and elastin crosslinking rather than collagen [14]. Our study was fully consistent with this LOXL1/elastin crosslinking relationship and specifically demonstrated that LOXL1 expression significantly increased in cirrhosis stage compared with normal and fibrosis stage, which was similar with the overall trend of elastin deposition,

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ACCEPTED MANUSCRIPT indicating the underlying relevance between LOXL1 and elastin during liver fibrosis progression. Interestingly, our findings about LOXL1 immunoreactivity was differed from LOXL2 [16], which was only observed in cirrhosis stage. These data, taken together, suggested that LOXL1 might play its role mainly in cirrhosis stage and specifically on elastin biochemistry.

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Furthermore, we directly demonstrated that LOXL1 inhibition could lead to downregulation

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of insoluble/crosslinked elastin and the architectural structure of elastin in vivo. LOXL1

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inhibition, by AAV2/8 LOXL1-shRNA, retarded the progression of liver fibrosis during continuously chronic injury which was represented as decreased deposition of collagen. The

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most straightforward explanation is that elastin fibers in livers are shown as cobwebbed cords

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entangled with microfibrils [34]. With a reduction of elastin, collagen fibers were more exposed and readily available for degradation (Figure 6). Those results provided evidence that LOXL1

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mediated crosslinking directly contributes to elastin stabilization and progressive fibrosis in liver

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injury. Therefore, targets on architectural structure of elastin merits further investigation during liver fibrosis regression period.

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Currently, therapeutic targets focused on ECM stabilization are very attractive [35]. We

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believe this approach could retard progression and improve the reversion of fibrotic livers. Based on this view, there are a few reports to explore matrix stability. Recent reports suggested that antibody against to LOXL2, which inhibits collagen crosslinking, could significantly reduce collagen deposition and accelerated fibrosis regression [16]. It has been also demonstrated that the non-selective inhibitor of LOX family BAPN, which focused on ECM crosslinking, supported the evidence above, even without a reduction of collagen [21].Moreover, deficiency of ADAMTS which was responsible for catalyzing the N-propeptide excision of procollagens,

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ACCEPTED MANUSCRIPT could lead to improve liver fibrosis and promote reverse [36].Therefore, only if crosslinking is reduced, would collagen be more susceptible to be degradation. This viewpoint is parallel with our results. As one member of ECM, elastin plays an equally important role in liver diseases. Recently, elastin was proposed to be a target for molecular magnetic resonance (MR) to evaluate

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liver fibrosis [37]. A recent study revealed that elastin is not only correlated with advanced liver

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fibrosis, but also a predictor for hepatocellular carcinoma independently of collagen [38].

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However, in contrast to our results, downregulation of elastin fibers in liver injury was observed by scanning electron microscopy (SEM) analysis and gave rise to the increased liver stiffness

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[34].

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Importantly, LOXL1 activity is associated with the activation of HSCs, which is the critical step during fibrogenesis [39]. Our data revealed that LOXL1 inhibition in vitro induced a

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notably suppression of LX-2 cells activation, which was represented as a reduction of α-SMA

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expression and subsequent downregulated expression of elastin. Previous studies have demonstrated that changes of ECM and increased liver stiffness, due to crosslinking, may affect

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myofibroblast differentiation [40, 41]. These studies suggest that lysyl oxidases contribute in part

HSCs.

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to HSCs activation and our findings revealed the direct function of LOXL1 on the activation of

In conclusion, we reported that LOXL1 inhibition could retard liver fibrosis progression. Whether it has the similar anti-fibrotic effects during liver fibrosis regression should be further studied. Moreover, we found that insoluble/crosslinked elastin only accumulates in cirrhosis stage. Of all LOX family members, LOXL1 appears to be most associated with end stage fibrosis. Suppression of LOXL1 activity can reduce elastin crosslinking, limit liver fibrosis progression

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ACCEPTED MANUSCRIPT and inhibit HSCs activation. Therefore, our study specifically points to LOXL1 as a promising therapeutic target, which suggests further studies are warranted for LOXL1 inhibition in reversing cirrhosis.

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Conflict of interest

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No conflict of interest connected with the manuscript was declared.

Acknowledgments

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We thank Dr. Paul L Hermonat, Professor of Internal Medicine, from University of

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Arkansas for Medical Sciences (UAMS) and Dr. Leonard Kaps from University Mainz in critically revising the manuscript. This study was supported by the National Natural Science

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Foundation of China (81670539 and 81500456) and the Chinese Foundation for Hepatitis

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Prevention and Control of the WBN Research Foundation (CFHPC20151009).

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G1147-1154.

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Figure legends Figure 1 Elastin deposited strongly only in cirrhosis stage which induced by carbon tetrachloride (CCl4). (A) Representative section images of sirius red staining and elastin staining after CCl4-treated 0

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(normal), 4 (fibrosis) and 8 weeks (cirrhosis) (original magnification, ×100).

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(B) Hepatic collagen contents were determined via hydroxyproline assessment.

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(C) Quantification of collagen and elastin area were measured by sirius red staining and elastin staining, respectively.

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(D, E) Collagen I and elastin expressions on mRNA and protein levels were evaluated by real-time

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PCR and western blot, respectively. Elastin massively accumulated in cirrhosis stage, while expression of collagen I was gradually elevated from fibrosis stage to cirrhosis stage.

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(F) Insoluble/crosslinked collagen and elastin in liver tissue. Unlike insoluble/crosslinked collagen,

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the insoluble/crosslinked elastin content was remarkably and massively increased in cirrhosis stage.

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(G) Relative expression of pyridinoline and desmosine by UPLC-MS/MS analysis during

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fibrogenesis, the common and unique crosslinks in collagen and elastin, respectively. All data were expressed as means ± SD (*P < 0.05, **P < 0.01), n = 8 in each group.

Figure 2 Lysyl oxidase-like 1 (LOXL1) was strongly associated with cirrhosis stage. (A) The mRNA expressions of LOX family and α-SMA were assessed at different stages of fibrosis. LOXL1 expression notably increased at cirrhosis stage compared with other members of LOX family. All data were expressed as means ± SD (*P < 0.05, #P < 0.001), n = 8 in each group.

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ACCEPTED MANUSCRIPT (B) Immunohistochemistry analysis of LOXL1 and α-smooth muscle actin (α-SMA) during fibrosis progression (×100 and ×200, respectively). (C) Double immunofluorescence staining revealed the co-location of LOXL1 and α-SMA at

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cirrhosis stage (×100).

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Figure 3 LOXL1 silencing inhibited activation of HSCs and expression of ECM in vitro.

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(A, B) Profibrogenic markers expression (α-SMA, collagen I and elastin) were significantly

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data were expressed as means ± SD (*P < 0.05).

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decreased in LX-2 cells after LOXL1 silencing at levels of mRNA and protein expression. All

Figure 4 AAV2/8-LOXL1-shRNA treatment significantly inhibited matrix deposition and

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crosslinked elastin in liver fibrosis induced by CCl4 in vivo.

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(A) AAV2/8-LOXL1-shRNA was successfully established. (B) AAV2/8-LOXL1-shRNA (LOXL1-shRNA) were injected in C57BL/6 mice one day before

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repeated CCl4 administration (two times per week), and liver fibrosis were induced for 8 weeks;

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AAV2/8-control vector as control group. (C) Collagen and elastin deposition are determined by H&E, sirius red (magnification, ×100) and elastin staining (×100 and ×200, respectively). LOXL1-shRNA led to thinner elastin fibers and less extensive septa. (D) Quantification of collagen and elastin area in LOXL1-shRNA treated mice significantly decreased in LOXL1-shRNA group, which were measured based on sirius red staining and elastin staining, respectively.

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ACCEPTED MANUSCRIPT (E) Expression of collagen and elastin mRNA after suppressed LOXL1 expression. (F) Hepatic collagen levels in fibrotic livers after LOXL1 inhibition were decreased illustrated by hydroxyproline. (G, H) Both insoluble/crosslinked elastin content and desmosine expression were decreased after

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LOXL1 inhibition in fibrotic livers. All data were given as means ± SD (*P < 0.05), n = 6 in each

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group.

Figure 5 Downregulation of α-SMA in fibrosis models without yielding improvement of liver

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function after AAV2/8-LOXL1-shRNA treatment.

(A, B) It was demonstrated that LOXL1-shRNA attenuated HSCs activation by mRNA and

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western blot analysis of α-SMA.

(C) Serum levels of alanine aminotransferase (ALT)，aspartate transaminase (AST) and albumin

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(ALB) in AAV8-sh-LOXL1 group during liver injury compared with control. All data were

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given as means ± SD (*P < 0.05), n = 6 in each group.

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Figure 6 The schematic representation of LOXL1 and elastin crosslinking in ECM

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deposition during liver fibrosis.

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Table 1 Primers designed for real-time PCR Gene

Primers

mElastin

forward, 5’-AAGACCTGGCTTTGGACTTTCT-3’

mCollagen Ι

forward, 5’-GAGCGGAGAGTACTGGATCG-3’

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reverse, 5’-GCTTCTTTTCCTTGGGGTTC-3’

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reverse, 5’-CGGCCACAGGATTTCCCA-3’

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forward, 5’-GAGTGAAGAACCAAGGGACATCG-3’

mLOX

reverse, 5’-CATCAAGCAGGTCATAGTGGCTG-3’ forward, 5’-CCGCAGCAGTTCCCCTATC-3’

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mLOXL1

mLOXL2

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reverse, 5’-CGCGGGATCGTAGTTCTCA-3’ forward, 5’-TGTTTGGCTCTGCTTGTCTTGC-3’

forward, 5’-TTGAGGTGGAGCATCAGTTGC-3’

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mLOXL3

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reverse, 5’-CCTCATTGTGCTTCCTCTTCTGG-3’

reverse, 5’-AGCCTTTGTCACACACTTGCG-3’ forward, 5’-TGGTGACCTGTCGGCAACT-3’

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mLOXL4

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reverse, 5’-GAACTCCACTCATCACCACTTCTTT-3’

mα-SMA

forward, 5’-GATGAAGCCCAGAGCAAGAG-3’ reverse, 5’- GAACTCCACTCATCACCACTTCTTT-3’

mGAPDH

forward, 5’-TCCACTCACGGCAAATTCAAC-3’ reverse, 5’-CGCTCCTGGAAGATGGTGATG-3’

hLOXL1

forward, 5’-TGCCACCAGCATTACCACAG-3’ reverse, 5’-GAGGTTGCCGAAGTCACAGG-3’

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ACCEPTED MANUSCRIPT hα-SMA

forward, 5’-GACAATGGCTCTGGGCTCTGTAA-3’ reverse, 5’- CTGTGCTTCGTCACCCACGTA-3’ forward, 5’- GGCCATTCCTGGTGGAGTTCC -3’

hElastin

reverse, 5’- AACTGGCTTAAGAGGTTTGCCTCCA-3’ forward, 5’- TGATGGGATTCCCTGGACCT -3’

forward, 5’- TCCACTCACGGCAAATTCAAC-3’

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hGAPDH

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reverse, 5’- GGGCCTTGTTCACCTCTCTC-3’

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hCollagen Ι

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reverse, 5’- CGCTCCTGGAAGATGGTGATG-3’

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ACCEPTED MANUSCRIPT Highlights

1. Both crosslinked elastin and LOXL1 played important roles in cirrhosis. 2. Suppression of LOXL1 retarded liver fibrosis progression in vivo. 3. LOXL1 promoted hepatic stellated cells activation and elastin

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expression in vitro.

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Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6