Sinusoidal dedifferentiation is a complicated process induced by several factors, and exists in early stage of diverse liver diseases. The mechanism of sinusoidal dedifferentiation is poorly unknown. In this study, we established a NaAsO2-induced sinusoidal dedifferentiation mice model. Liver sinusoidal endothelial cells were isolated and isobaric tag for relative and absolute quantitation (iTRAQ) based proteomic approach was adopted to globally examine the effects of arsenic on liver sinusoidal endothelial cells (LSECs) during the progression of sinusoidal dedifferentiation. In all, 4205 proteins were identified and quantified by iTRAQ combined with LC-MS/MS analysis, of which 310 proteins were significantly changed in NaAsO2 group, compared with the normal control. Validation by western blot showed increased level of clathrin-associated sorting protein Disabled 2 (Dab2) in NaAsO2 group, indicating that it may regulate receptor endocytosis, which served as a mechanism to augment intracellular VEGF signaling. Moreover, we found that knockdown of Dab2 reduced the uptake of VEGF in LSECs, furthermore blocking VEGF-mediated LSEC dedifferentiation and angiogenesis.

Liver sinusoidal endothelial cell (LSEC) is a type of liver specific microvascular cell, which characterizes unique phenotype and function. In normal liver, differentiated LSECs form capillaries of microvasculature and facilitate filtration by fenestrae as a selectively permeable barrier between liver parenchyma and sinusoid1. Upon liver injury (e.g., fibrosis2,3,4, hepatitis5,6, alcoholic liver injury7 and arsenic exposing8), LSECs loss their highly specialized fenestration and gain an organized basement membrane, which calls LSEC dedifferentiation or capillarization. Although the mechanism of LSEC dedifferentiation has been comprehensively studied, the molecular mechanisms driving dedifferentiation have not been fully elucidated. So far, there are few ideal models to study the molecular mechanisms of LSEC dedifferentiation in vivo, as most models cause cirrhotic fibrosis simultaneously, obfuscating the real issues of LSEC dedifferentiation. However, Straub et al.9 tested the effects of sodium arsenite (NaAsO2) on dedifferentiated LSECs and proved that NaAsO2 induced LSEC dedifferentiation without fibrosis initiation. Therefore, NaAsO2-induced LSEC dedifferentiation mice model could be applied to study LSEC dedifferentiation mechanisms.

In this study, we used NaAsO2 in drinking water of mice for 5 weeks as the early injury phase to induce LSEC dedifferentiation, comparing with normal mice. Then LSECs from these two groups were isolated and lysed. Isobaric tags for relative and absolute quantification (iTRAQ) coupled with LC-MS/MS was used for relative quantification of proteins in vivo, based on a more powerful and sensitive proteomic method than traditional approaches, especially quantifying low-abundance proteins10,11,12. Protein identification and quantification was accurately performed using Protein Pilot Software with specifically developed algorithms.

For our experiments, iTRAQ-labeled LSECs in NaAsO2-induced LSEC dedifferentiation mice model were first used for differentially expressed proteome analysis through Protein Pilot software, and 4205 proteins were identified. Among these, there were 207 up-regulated proteins and 103 down-regulated proteins, respectively. For functional analysis in depth, we found that two significantly increased proteins, disabled homolog 2 (Dab2) and clathrin heavy chain (CLTC), were involved in VEGF receptor endocytosis, serving as a mechanism to induce intracellular receptor signaling upon the stimulation of VEGF signal, which is referred as a regulator leading to angiogenesis13.

0.25 μg/mL NaAsO2 in drinking water was administered for 5 weeks in mice to induced LSEC dedifferentiation as described previously9. The open fenestrae of liver sinusoids were decreased in arsenic exposed group, compared with the normal counterparts (7.46 ± 0.41% vs 3.56 ± 0.76%) (Fig. 1A). The permeability of isolated LSECs from arsenic exposed group, identified by porosity of cell surface, was also reduced (22.64 ± 5.38% vs 13.35 ± 2.78%) (Fig. 1B). Meanwhile, the expression of LSEC dedifferentiation markers, such as CD31, Caveolin-1 and Rac1, was increased in arsenic exposed group (Fig. 1C). But LSEC differentiation marker, the uptake of acetylated low density lipoprotein (Ac-LDL), was instead reduced in arsenic exposed group (Fig. 1D). These results suggested that 5-week NaAsO2 administration successfully induced LSEC dedifferentiation in mice.

Figure 1
Figure 1

NaAsO2-induced LSEC dedifferentiation. (A) Representative SEM images of liver sinusoids (n = 5) and quantitative porosity of sinusoidal fenestrae in normal and NaAsO2-induced groups. Bar = 1μm. **P < 0.01. (B) Representative TEM images of primary LSECs isolated from normal (n = 5) and NaAsO2-induced group (n = 5) and quantitative porosity of sinusoidal fenestrae. Bar = 1μm. **P < 0.01. (C) The expressions of CD31, Caveoloin-1 and Rac1 in normal and NaAsO2-induced groups were analyzed by western blot. All western blot experiments were repeated at least three times. Full-length blots are included in Supplemental Information. (D) Uptake of Ac-LDL was analyzed by fluorescence microscopy in primary LSECs isolated from normal and NaAsO2-induced groups and cultured overnight. All experiments in (C) and (D) were repeated at least three times.

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To generate LSEC proteome, we isolated LSECs from normal and arsenic exposed mice respectively using a modified protocol including two-step collagenase perfusion, centrifugation and magnetic beads sorting, as described previously14,15,16. Purity and viability of LSECs were up to 93.6 ± 1.7% and 88.4 ± 0.5% confirmed by CD146 + F4/80- and 7-AAD + flow cytometry analysis separately, and yield of LSECs was approximately (2.1 ± 0.2) × 106 per mouse, (Supplemental Fig. 1A–C). Nextly, we assessed the quality of primary LSECs to exclude the false positive analysis of flow cytometry. After 8 h culture and extensive washing, LSEC monolayer showed a typical cobblestone, sheet-like appearance (Supplemental Fig. 1D). In liver, Ac-LDL was mainly taken up by LSECs17. Therefore, fluorescently labeled Ac-LDL was used to confirm LSEC quality18. After overnight culture, above 98% LSECs were labeled by Ac-LDL, according to Ac-LDL endocytosis assay (Supplemental Fig. 1E). These results demonstrated that primary LSECs with high purity and viability were obtained.

To elucidate the molecular mechanisms of LSEC dedifferentiation, quantitative proteomic analysis based on iTRAQ labeling was executed between NaAsO2 induced LSEC dedifferentiation mice model and the counterparts. Total 7763 proteins were identified in two independent biological replicates (FDR < 1%). Among these, 54.16% (4205/7763) proteins were shared by these two experiments (Supplemental Fig. 2A and Supplemental Table 1). In addition, linear regression analysis was performed with ln [115/116 ratio] and ln [116/115 ratio] in these two independent experiments to examine the biological reproducibility, and the Pearson correlation coefficient was 0.7181 (P < 0.0001), indicating high biological reproducibility of our experiments. To identify significant up- or down-regulated proteins during LSEC dedifferentiation, the threshold values of 115/116 or 116/115 ratios were ≥1.50 or ≤0.67 (≥1.5-fold) in both two iTRAQ analyses. Accordingly, 207 and 103 proteins were significantly up- or down-regulated, respectively, in dedifferentiated LSECs (Supplemental Tables 2 and 3), suggesting dramatic alterations during LSEC dedifferentiation.

Figure 2
Figure 2

Subcellular localization and biological functions of differential proteins during LSEC dedifferentiation (A) Subcellular localization of altered proteins during LSEC dedifferentiation. (B) Biological functions of differential proteins during LSEC dedifferentiation. The top biological functions of up-regulated and down-regulated proteins were determined by Gene Ontology and DAVID annotation analysis.

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The 310 differentially expressed proteins were categorized by their cellular component and biological function using Gene Ontology analysis (GO) or DAVID functional annotation. Most of up-regulated proteins were localized in plasma membrane and cytosol, while down-regulated proteins in endoplasmic reticulum and mitochondrion (Fig. 2A), indicating that differentially expressed proteins are significantly devided at the subcellular level. The enriched biological functions of up-regulated proteins were mainly associated with multiple component metabolism (such as nucleotide, single-organism, organic acid, small molecular, lipid, et al.), oxidative stress, cell survival and endocytosis (Fig. 2B), showing oxidative stress and energy metabolism are involved in LSEC dedifferentiation process, in accordance with the findings described previously8,9. Interestingly, endocytosis was the unique function with top enrichment score, suggesting endocytosis may mediate LSEC dedifferentiation. Meanwhile, proteins associated with transcription regulation, immune system process, ribosome biogenesis, apoptotic process, et al., were all down-regulated during LSEC dedifferentiation (Fig. 2B), among which the reduced immune system process might disable defense line against arsenic insult. These findings discovered that endocytosis induced LSEC dedifferentiation for the first time. In addition, LSEC were also found to lose the defense ability against injury insults.

Differential expressions of 5 selected proteins were further validated by western blot, focusing on those involved in receptor endocytosis and innate immune response. Compared with the normal LSECs, proteins involved in receptor endocytosis (CLTC) and clathrin coat assembly (Dab2) were significantly up-regulated in arsenic induced dedifferentiated LSECs, whereas three proteins (Galectin-3, SAMHD1, Rab10) related to innate immune response and antigen presentation showed significant down-regulation in dedifferentiated LSECs. The western blot results were in keeping with the iTRAQ data (Fig. 3A).

Figure 3
Figure 3

Validation of selected proteins in LSECs from NaAsO2-induced LSEC dedifferentiation and CCl4-induced liver injury mice model. (A) Differential expressions of 2 up-regulated proteins (CLTC, Dab2) and 3 down-regulated proteins (Galectin-3, SAMHD1, Rab-10) were validated by Western blot. The relative ratios of proteins by iTRAQ analysis were shown right. All western blot experiments were repeated at least three times. Full-length blots are included in Supplemental Information. (B) Representative SEM images of liver sinusoids (n = 5) and quantitative porosity of sinusoidal fenestrae at 0 and 6th week with CCl4 administration. Bar = 1μm. (C) Masson’s trichrome staining and αSMA expression in liver at 6th week with CCl4 administration (n = 5) were analyzed by immunohistochemistry analysis, Bar = 100μm. (D) Western blot analysis of primary LSECs isolated from 0 and 6th week for the levels of CLTC, Dab2, Galectin-3, SAMHD1 and Rab-10 proteins, and GAPDH was used as loading control. All western blot experiments were repeated at least three times. Full-length blots are included in Supplemental Information. (E) CLTC expression in liver at normal control mice and mice at 6th week with CCl4 administration (n = 5) were analyzed by immunohistochemistry analysis, Bar = 100μm.

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For further validation, the chronic liver injury mice model induced by carbon tetrachloride (CCl4) was established as described previously19, generating LSEC dedifferentiation at 6th week after CCl4 administration (Fig. 3B). And this model was further verified by Masson trichrome staining and αSMA immunohistochemistry, showing obvious chronic liver injury (Fig. 3C). Differential expression of selected proteins was further evaluated by Western blot in LSECs isolated from in vivo model, and confirmed CLTC and Dab 2 were increased at the 6th week after CCl4 administration, when LSECs were dedifferentiated and fibrotic septa formed. Galectin-3, SAMHD1, Rab10 showed down-regulation as displayed in Fig. 3D. The expression of CLTC, which is marker of clathrin-coat associated receptor endocytosis, was increased in liver of CCl4-treated mice at the 6th week, comparing with normal counterparts (olive given only) (Fig. 3E). These results further proved the expression levels of these proteins in chronic liver injury.

Expression of clathrin heavy chain…