Sirtuin 1 ameliorates defenestration in hepatic sinusoidal endothelial cells during liver fibrosis via inhibiting stress‐induced premature senescence

Abstract Objective Premature senescence is related to progerin and involves in endothelial dysfunction and liver diseases. Activating sirtuin 1 (SIRT1) ameliorates liver fibrosis. However, the mechanisms of premature senescence in defenestration of hepatic sinusoidal endothelial cells (HSECs) and how SIRT1 affects HSECs fenestrae remain elusive. Methods We employed the CCl4‐induced liver fibrogenesis rat models and cultured primary HSECs in vitro, administered with the SIRT1‐adenovirus vector, the activator of SIRT1 and knockdown NOX2. We measured the activity of senescence‐associated β‐galactosidase (SA‐β‐gal) in HSECs. Meanwhile, the protein expression of SIRT1, NOX2, progerin, Lamin A/C, Ac p53 K381 and total p53 was detected by Western blot, co‐immunoprecipitation and immunofluorescence. Results In vivo, premature senescence was triggered by oxidative stress during CCl4‐induced HSECs defenestration and liver fibrogenesis, whereas overexpressing SIRT1 with adenovirus vector lessened premature senescence to relieve CCl4‐induced HSECs defenestration and liver fibrosis. In vitro, HSECs fenestrae disappeared, with emerging progerin‐associated premature senescence; these effects were aggravated by H2O2. Nevertheless, knockdown of NOX2, activation of SIRT1 with resveratrol and SIRT1‐adenovirus vector inhibited progerin‐associated premature senescence to maintain fenestrae through deacetylating p53. Furthermore, more Ac p53 K381 and progerin co‐localized with the abnormal accumulation of actin filament (F‐actin) in the nuclear envelope of H2O2‐treated HSECs; in contrast, these effects were rescued by overexpressing SIRT1. Conclusion SIRT1‐mediated deacetylation maintains HSECs fenestrae and attenuates liver fibrogenesis through inhibiting oxidative stress‐induced premature senescence.


| INTRODUC TI ON
Premature senescence involves in cellular dysfunction and various chronic diseases, and its character is the inhibition of cell proliferation in advance when suffering from noxious stimuli. 1 Emerging evidence confirms that due to old age, the phenotypes of all hepatic cells have changed, such as the loss of fenestration in hepatic sinusoidal endothelial cells (HSECs). Furthermore, the defenestration and capillarization of HSECs are observed in some premature senescence-related disease paradigms. 2,3 The research indicates that premature senescence in HSECs may be closely related to HSECs defenestration and liver fibrogenesis. Hence, elucidation of the underlying mechanisms for premature senescence may be a key to our understanding of defenestration in HSECs and liver fibrosis pathogenesis.
The contraction and dilatation of fenestrae in HSECs are regulated by actin cytoskeleton (including F-actin). 4 Our previous studies reveal that oxidative damage facilitates HSECs defenestration during liver fibrogenesis via F-actin remodelling. 5,6 Novel findings show that lamins and their associated proteins, which regulate nucleoskeleton and cytoskeleton, affect cellular differentiation and senescence. 7,8 Especially, progerin is a mutant Lamin A protein, and the accumulation of progerin brings about abnormal nucleoskeleton and cellular premature senescence, so as to promote the occurrence and development of chronic liver diseases. [7][8][9] Thus, we speculate that progerin may contribute to premature senescence-associated HSECs defenestration via abnormal cytoskeleton remodelling.
Sirtuin 1 (SIRT1) is an essential protector against oxidative stress and senescence to reverse the progression of chronic liver diseases. 10 Recent studies emphasize that overexpressing or activating SIRT1 inhibits hepatic senescence and activation of HSCs to ameliorate liver fibrosis. 11,12 Besides, a significant finding demonstrates that the activation of SIRT1 prevents the endothelial cells from oxidative stress-induced senescence and dysfunction. 13,14 Nevertheless, the effects of SIRT1 on premature senescence and defenestration in HSECs during liver fibrogenesis remain elusive.
Herein, our present study investigates the underlying mechanisms and the intervening target linking premature senescence and HSECs defenestration, and the role of SIRT1 in HSECs defenestration in vitro and in vivo. We specifically focus on the SIRT1-mediated deacetylation, which may influence premature senescence-associated defenestration of HSECs in liver fibrogenesis.

| Animal experimental design
The animal experiments were approved by the Committee on the Rats were housed under a 12:12 h light/dark cycle at 22-24°C.

| The treatment of SIRT1 adenovirus vector
To investigate the role of SIRT1 in the fenestrae of primary HSECs and liver fibrogenesis, the GFP-SIRT1-adenovirus vector and the GFP-blank vector were produced by Hanbio AdenoVector Institute (Shanghai, China), and the dose of 10 11 viral particles was injected through a caudal vein to rats 1 week before the intraperitoneal injection of CCl 4 -olive oil solution. We employed the CCl 4 -induced liver fibrosis rat models (n = 6 per group for 6 days and n = 6 per group for 28 days). The vehicle group (n = 6 per group for 6 days and n = 6 per group for 28 days) was subjected to intraperitoneal injection of the same volume of olive oil, twice a week for 28 days. The AV-CTR + CCl 4 group and the AV-SIRT1 + CCl 4 group (n = 6 per group for 6 days and n = 6 per group for 28 days) were subjected to intraperitoneal injection of CCl 4 -olive oil solution F I G U R E 1 CCl 4 induces progerin-associated premature senescence in defenestrated HSECs during liver fibrogenesis. (A) Magnification of scanning electron micrograph (SEM) of hepatic sinusoidal endothelium in CCl 4 -induced rat models (Day 0, Day 3, Day 6 and Day 28), revealing fenestrae structures in hepatic sinusoidal endothelium (Scale bar: 5 μm). The white triangles indicated fenestrae in hepatic sinusoidal endothelium. The porosity of hepatic sinusoidal endothelium was quantified in the graph, right. * P < .05 vs Day 0. (B) The SAβ-Gal activity on primary HSECs, isolated from CCl 4 -induced rat models (Day 0, Day 3, Day 6 and Day 28) was observed by SAβ-Gal staining (Scale bar: 25 μm). The black triangles indicated the SAβ-Gal-positive cells. The SAβ-Gal-positive cells were quantified in the graph, right. * P < .05 vs Day 0. (C) Representative immunoblots of progerin, SIRT1, Ac p53 K381 and total p53 of primary HSECs, isolated from CCl 4induced rat models (Day 0, Day 3, Day 6, Day 14 and Day 28). The relative protein expression of progerin and SIRT1, as well as the ratio of Ac p53 K381 and total p53 protein levels were quantified in the graph, right. * P < .05 vs progerin relative protein level on Day 0; # P < .05 vs SIRT1 relative protein level on Day 0; $ P < .05 vs the ratio of Ac p53 K381 and total p53 protein levels on Day 0. (D) The immunofluorescent co-localization of vWF (red) with progerin (green) of liver biopsy specimens in CCl 4 -induced rat models (Day 0, Day 3, Day 6 and Day 28), visualized by confocal microscopy (Scale bar: 10 μm, 50 μm). Nuclear was showed by DAPI (blue). (E) The immunofluorescent co-localization of vWF (red) with SIRT1 (green) of liver biopsy specimens in CCl 4 -induced rat models (Day 0, Day 3, Day 6 and Day 28), visualized by confocal microscopy (Scale bar: 10 μm, 50 μm). Nuclear was showed by DAPI (blue). (F) The immunohistochemical (IHC) staining for Ac p53 K381 of liver biopsy specimens in CCl 4 -induced rat models (Day 0, Day 3, Day 6 and Day 28; Scale bar: 50 μm). The semi-quantitative score of IHC staining for Ac p53 K381 was in the graph, right. * P < .05 vs Day 0. n = 6 per group twice a week after administering vectors. On Day 6 and 28, the rat models were randomly sacrificed. The SIRT1 sequences were used: sense (5-CGGGCCCTCTAGACTCGAGCGGCCGCATG ATTGGCACCGATCCTC-3).

| Histological analysis and immunohistochemistry
Paraffin sections (4 μm) of liver tissue of the model rats were prepared with haematoxylin and eosin (H&E) staining. The rat liver histological inflammation and fibrosis stage are assessed with the Ishak inflammation and fibrosis score (from the supplementary method). Immunohistochemical detection of α-SMA, vWF and Ac p53 K381 was performed on paraffin sections (3 μm) of liver tissue, and subsequent sections were exposed to HRP-antibody coloured with DAB, and visualized by microscopy (BX51, Olympus, Japan). The degree of liver fibrosis and the number of α-SMA-, vWF-or Ac p53 K381-positive cells were quantified with Image J software.

| Cell isolation, identification, culture and treatment
Primary HSECs were isolated from normal male SD rats and identified by SEM, based on modified method. 5

| Scanning electron microscopy (SEM)
The liver tissue of the model rats and primary HSECs was fixed with 2.5% glutaraldehyde and subsequently dehydrated, and then coated with gold using the coating apparatus, based on the modified method. 5 Eventually, fenestrae in primary HSECs of samples were observed with SEM at 15-kV acceleration voltage.

| SIRT1 adenovirus transfection
The recombinant adenovirus was produced by Hanbio AdenoVector Institute (Shanghai, China). To construct Flag protein-tagged SIRT1, full-length SIRT1 cDNA was amplified from a human cDNA library and fused at its C-terminus with sequences encoding the mono-
The following progerin siRNA sequences were used: sense

| Extraction of nuclear and cytoplasmic protein of primary HSECs
Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, P0028) was used to extract nuclear and cytoplasmic protein of primary HSECs (10 7 cells per group). Nuclear and cytoplasmic protein was processed and detected for Western blotting. F I G U R E 2 Overexpression of SIRT1 relieves progerin-associated premature senescence to attenuate CCl 4 -induced defenestration in HSECs. (A) Representative immunoblots of SIRT1, vWF, NOX2, Ac p53 K381, total p53 and progerin of primary HSECs, isolated from CCl 4induced rat models on Day 6 and Day 28. The relative protein expression of SIRT1, vWF, NOX2 and progerin, as well as the ratio of Ac p53 K381 and total p53 protein levels were quantified in the graph, down. * P < .05 vs the vehicle group on Day 6; # P < .05 vs the CCl 4 group and the CCl 4 + AV-CTR group on Day 6; $ P < .05 vs the vehicle group on Day 28; & P < .05 vs the CCl 4 group and the CCl 4 + AV-CTR group on Day 28. (B) Magnification of SEM of hepatic sinusoidal endothelium in CCl 4 -induced rat models on Day 6, revealing fenestrae structures in hepatic sinusoidal endothelium (Scale bar: 5 μm). The white triangles indicated fenestrae in hepatic sinusoidal endothelium. The porosity of hepatic sinusoidal endothelium was quantified in the graph, right. * P < .05 vs the vehicle group; # P < .05 vs the CCl 4

| Immunocytochemistry
Paraformaldehyde-fixed primary HSECs were incubated with primary antibodies, followed by the secondary antibodies, and subsequently mounted with DAPI. The primary antibodies included anti-NOX2 (1:200), anti-progerin (1:50) and anti-Ac p53 K381 (1:200). After incubation with primary antibodies and the secondary antibodies, HSECs were stained with the phallotoxin to detect F I G U R E 4 Oxidative stress inhibits SIRT1-mediated deacetylation and aggravates progerin-associated premature senescence to facilitate defenestration in HSECs. Freshly primary HSECs, isolated from normal rats and cultured in vitro, were treated with H 2 O 2 (10 μM) from 12 hours to 48 hours. (A) Representative immunoblots of NOX2, SIRT1, Ac p53 K381, total p53 and vWF of HSECs in 12 hours, 24 hours and 48 hours. The relative protein expression of NOX2, SIRT1 and vWF, as well as the ratio of Ac p53 K381 and total p53 protein levels were quantified in the graph, right. * P < .05 vs NOX2 protein level in the concurrent control group; # P < .05 vs SIRT1 protein level in the concurrent control group; $ P < .05 vs the ratio of Ac p53 K381 and total p53 protein levels in the concurrent control group; & P < .05 vs vWF protein level in the concurrent control group. (B) Magnification of SEM of HSECs in the CTR group and the H 2 O 2 (10 μM) group on Day 1 and Day 2, revealing the fenestrae structures (Scale bar: 2 μm). The black triangles indicated fenestrae in HSECs. The total fenestral diameter was quantified in the graph, right. * P < .05 vs the CTR group on Day 1; # P < .05 vs the CTR group on Day 2. (C) The immunocytochemical co-localization of NOX2 (green) with F-actin (red) in primary HSECs on Day 2 visualized by confocal microscopy (Scale bar: 5 μm). Nuclear was showed by DAPI (blue). (D) The SAβ-Gal activity in primary HSECs on Day 1 and Day 2 was observed by SAβ-Gal staining (Scale bar: 25 μm). The black triangles indicated the SAβ-Gal-positive cells. The SAβ-Gal-positive cells were quantified in the graph, right. * P < .05 vs the CTR group on Day 1; # P < .05 vs the CTR group on Day 2. (E) Representative immunoblots of progerin, Lamin A/C and Lamin B1 of HSECs in 12 hours, 24 hours and 48 hours. The relative protein expression was quantified in the graph, right. * P < .05 vs progerin protein level in the concurrent control group; # P < .05 vs Lamin A/C protein level in the concurrent control group; $ P < .05 vs Lamin B1 protein level in the concurrent control group. (F) The immunocytochemical co-localization of Ac p53 K381 (green) with progerin (red) and F-actin (purple) of primary HSECs on Day 2 visualized by confocal microscopy (Scale bar: 5 μm). Nuclear was showed by DAPI (blue) F I G U R E 5 Inhibiting NOX2-dependent oxidative stress reduces progerin-associated premature senescence to maintain fenestrae in HSECs. Freshly primary HSECs, isolated from normal rats and cultured in vitro, were transfected with NOX2 siRNA or nontarget siRNA (called NC), and then administered with H 2 O 2 (10 μM) for two days. (A) Real-time PCR analysis of NOX2 mRNA level in HSECs on Day 2.

| Co-immunoprecipitation (Co-IP)
Primary HSECs were transfected with SIRT1 adenovirus vectors and were subsequently stimulated with H 2 O 2 for two days. IP and immunoblotting (IB) were performed as previously described. 5 The antibodies for IP included anti-progerin and non-specific IgG; the antibodies for IB included anti-progerin, anti-Ac p53 K381 and anti-p53.

| Western blotting
Primary HSECs were isolated from normal rats and were treated with various stimulators, or were isolated from the model rats. HSECs

| Statistical analysis
The data were reported as the mean ± standard deviation (SD) and were analysed by SPSS17.0 software. In the statistical analysis of two groups, a two-tailed Student's t test was utilized, whereas, in the statistical analysis of more than two groups, one-way ANOVA was performed. P < .05 was considered significant.

| Premature senescence is induced by oxidative damage, with the decrease of SIRT1 during defenestration in HSECs of CCl 4 -induced liver fibrogenesis
In our present study, fenestrae in hepatic sinusoidal endothelium disappeared entirely on the 6th day ( Figure 1A), along with the high expression of α-SMA and vWF in CCl 4 -induced rat models on the 28th day (Appendix Figure S1A-C, E). Meanwhile, the serum ALT and AST levels increased, along with the augment of NOX2 protein expression in primary HSECs which were isolated from CCl 4 -induced rat models (Appendix Figure S1D,E). These data indicated that CCl 4 induced defenestration and capillarization in HSECs via oxidative damage.
Interestingly, the senescence-associated β-galactosidase (SAβ-Gal)positive cells increased in primary HSECs; moreover, the Western blotting and immunofluorescence showed that a time-dependent elevation of the progerin protein expression in HSECs of CCl 4 -induced rat models ( Figure 1B-D). The data implied that progerin might be closely associated to premature senescence in CCl 4 -induced defenestrated HSECs. However, the SIRT1 expression was down-regulated, with the enhancement of the protein levels of Ac p53 K381 and total p53 in primary HSECs of CCl 4 -induced rat models ( Figure 1C); the immunofluorescence and the immunohistochemical staining showed less expression of SIRT1 but much expression of Ac p53 K381 in vWF-positive hepatic sinusoidal endothelium ( Figure 1E,F). Hence, these results confirmed that in the process of CCl 4 -induced defenestration in HSECs and liver fibrosis, oxidative damage triggered progerin-associated premature senescence, with the decrease of SIRT1-mediated deacetylation.

| Overexpression of SIRT1 inhibits progerinassociated premature senescence to alleviate CCl 4induced HSECs defenestration and liver fibrogenesis
To evaluate the role of SIRT1-mediated deacetylation in premature senescence and HSECs defenestration in vivo, the SIRT1 adenovi- Taken together, these results demonstrated that activating SIRT1-mediated deacetylation relieved progerin-associated premature senescence and maintained cytoskeleton to attenuate CCl 4induced defenestration in hepatic sinusoidal endothelium and liver fibrogenesis.

| Progerin-associated premature senescence emerges in the process of defenestration in HSECs in vitro
In vitro, the fenestrae in freshly primary HSECs, which were isolated from normal rats and were cultured without growth factors for 5 days (Figure 3A), shrank rapidly from the 1st day till the 3rd day and disappeared completely on the 5th day ( Figure 3B). Interestingly, the SAβ-Gal-positive cells increased gradually with time ( Figure 3C), along with the elevated protein levels of vWF, progerin and Lamin A/C. In contrast, Lamin B1 protein expression was down-regulated ( Figure 3D). These results indicated that the defenestration in HSECs was probably related to progerin-associated premature senescence.

| Oxidative stress aggravates progerinassociated premature senescence to facilitate defenestration in HSECs via acetylation of p53
Freshly primary HSECs, isolated from normal rats, were stimulated with  Figure 4B); meanwhile, the flow cytometry and immunocytochemistry showed that CD31, which labelled continuous HSECs, was highly expressed in H 2 O 2 -treated primary HSECs (Appendix Figure S3B,C), with the augment of vWF protein level ( Figure 4A). Furthermore, the immunofluorescence showed that compared with the control group, the co-localization of NOX2 with F-actin was highly expressed, accompany with the accumulation of F-actin in the nuclear envelope of H 2 O 2treated HSECs on the 2nd day ( Figure 4C). These data suggested that H 2 O 2 -induced oxidative damage might trigger the activation of acetylation of p53 and F-actin remodelling to accelerate defenestration and capillarization in HSECs via NOX2.
Besides, the SAβ-Gal staining, and the protein levels of progerin, Lamin A/C and Lamin B1, showed that H 2 O 2 -induced oxidative stress promoted progerin-associated premature senescence in HSECs, with the decrease of Lamin B1 expression ( Figure 4D,E). Compared to the control group, more progerin and Ac p53 K381 also were co-localized with F-actin in the nuclear envelope of H 2 O 2 -treated HSECs on the 2nd day ( Figure 4F). Hence, these results indicated that H 2 O 2 -induced oxidative stress triggered progerin-associated premature senescence through acetylation of p53 at lysine 381, and subsequently contributed to F-actin remodelling to aggravate defenestration in HSECs.
In addition, primary HSECs were transfected with p53 siRNA, progerin siRNA or nontarget siRNA (called NC), and then administered with H 2 O 2 (10 μM) for 2 days. We found that H 2 O 2 -induced strengthened activity of SAβ-Gal was significantly reduced by silencing p53 with p53 siRNA (Appendix Figure S4A,B), suggested p53-mediated premature senescence in H 2 O 2 -treated HSECs. As expected, the data of SEM showed that silencing progerin with progerin siRNA attenuated H 2 O 2 -induced HSECs defenestration on the 2nd day (Appendix Figure S4C and D), implied that inhibiting progerin-associated premature senescence could maintain HSECs fenestrae.
In consequence, H 2 O 2 -induced oxidative stress induced acetylation of p53 and progerin-associated premature senescence, and then brought about abnormal cytoskeleton remodelling to aggravate defenestration in HSECs.

| Inhibiting NOX2-dependent oxidative stress reduces progerin-associated premature senescence to maintain fenestrae in HSECs
To further delineate the molecular mechanism of oxidative stressinduced premature senescence and defenestration in HSECs, primary HSECs, isolated from normal rats and cultured in vitro, were transfected with NOX2 siRNA or nontarget siRNA (called NC), and F I G U R E 6 Activating SIRT1 with resveratrol reduces NOX2-dependent oxidative stress and relieves progerin-associated premature senescence. Freshly primary HSECs, isolated from normal rats and cultured in vitro, were treated with H 2 O 2 (10 μM) and were simultaneously administered with resveratrol (a specific chemical activator of SIRT1, 1 μM) for two days. on the contrary, knockdown of NOX2 down-regulated the protein levels of progerin and Lamin A/C, but up-regulated the Lamin B1 level ( Figure 5D and E), implied that inhibiting NOX2-dependent oxidative damage attenuated progerin-associated premature senescence. Additionally, the immunofluorescence and the SEM showed that accumulation of F-actin in the nuclear envelope of H 2 O 2 -treated HSECs and its defenestration, were triggered by oxidative stress, which were rescued by knockdown of NOX2 ( Figure 5F and G). In short, inhibition of NOX2-dependent oxidative stress alleviates progerin-associated premature senescence to maintain fenestrae in HSECs.

| SIRT1-mediated deacetylation relieves NOX2dependent oxidative stress to maintain fenestrae in HSECs via attenuating progerin-associated premature senescence
Firstly, to reveal the role of SIRT1 in oxidative stress-triggered premature senescence in HSECs, primary HSECs, isolated from normal rats, were treated with H 2 O 2 (10 μM) and were simultaneously administered with resveratrol (a specific chemical activator of SIRT1, 1 μM), or selisistat (a potent chemical inhibitor of SIRT1, 1 μM) for 2 days. The activation of SIRT1 was inhibited by H 2 O 2 , but was recovered by resveratrol ( Figure 6A). Compared with the control group, the protein expression of vWF and NOX2, the H 2 O 2 content, and mito-ROS increased in the H 2 O 2 group; in contrast, these effects were inhibited by resveratrol ( Figure 6A-C). Moreover, the activity of SAβ-Gal, the protein levels of Ac p53 K381, total p53, progerin, Lamin A/C were enhanced by H 2 O 2 , along with the decrease of Lamin B1; on the contrary, the effects were relieved by resveratrol ( Figure 6D,E). The data indicated that activating SIRT1 with resveratrol could attenuate NOX2-dependent oxidative stress and progerin-associated premature senescence.
Besides, H 2 O 2 down-regulated the SIRT1 protein expression and enhanced the vWF protein level, and this effect was aggravated by selisistat, which implied that blocking the activity of SIRT1 might directly promote defenestration and capillarization in HSECs.
However, selisistat did not alter the NOX2 protein expression, suggested that selisistat did not influence NOX2-dependent oxidative stress (Appendix Figure S5A). As expected, the activity of SAβ-Gal, the protein levels of Ac p53 K381, total p53, progerin, Lamin A/C, and Lamin B1 showed that inhibiting SIRT1 with selisistat could directly mediate acetylation of p53 to exacerbate progerin-associated premature senescence (Appendix Figure S5B,C).  Figure 8E). Therefore, these results confirmed that SIRT1-mediated deacetylation of p53 relieved NOX2-dependent oxidative stress and inhibited progerin-associated premature senescence to maintain fenestrae in HSECs.

| D ISCUSS I ON
Our present study discovers that NOX2-dependent oxidative stress triggers progerin-associated premature senescence via acetylation of p53, and subsequently aggravates cytoskeleton remodelling to promote defenestration in HSECs; both activating SIRT1 with resveratrol and overexpressing SIRT1 with adenovirus vector strongly activates deacetylation of p53, and then relieves progerin-related premature senescence to maintain fenestrae in HSECs and reverse liver fibrogenesis.
There is mounting evidence that premature senescence, induced by harmful stimuli, is a permanently deteriorated process about cell cycle arrest in advance, which involves in the dysfunction of endothelial cells and its related diseases as diverse as diabetes, lipodystrophy, and atherosclerosis, to name a few. 15,16 In our previous findings, premature senescence emerges in liver tissue due to acute liver injury and liver fibrogenesis. 17 In addition, in older animal models and premature ageing-related disease cases, abnormal differentiation and dysfunction in HSECs are attributed to premature senescence. 2,3 In this study, we similarly found that premature senescence in HSECs occurred in the process of fenestrae disappearance in vitro; and oxidative stress enhanced premature senescence to aggravate defenestration in H 2 O 2 -treated HSECs and in HSECs of CCl 4 -induced fibrogenesis. Nevertheless, the underlying mechanism about how oxidative stress-induced premature senescence triggers HSECs defenestration is still elusive.
Lamins and its associated protein, especially Lamin A and Lamin B1, forms an interface with the nuclear membrane and nuclear pore complexes, 18 while alteration of lamins involves in cellular premature senescence and age-associated diseases. Interestingly, some literature regarding the influence of lamins on premature senescence and liver diseases are controversial. For instance, the disruption or mutation of lamins led to hepatocytes' abnormal differentiation and premature ageing to induce steatohepatitis. 7 Progerin, a mutant of Lamin A, is deemed a moderator for premature ageing and dysfunction in endothelial cells. 19 The depletion of Lamin B contributes to chronic inflammation. 20 Our intriguing discoveries revealed that pre- and NOX4 are expressed in HSECs. 22 During chronic liver diseases, NOX1, NOX2 and NOX4 are highly expressed in HSECs. 23 During liver fibrogenesis, the redox signalling of different NOX isoforms and its effects on phenotypes and function of HSECs are unclear.
Recent reports indicated that the augment of NOX1 in HSECs reduced the bioavailability of NO to induce cellular inflammation and liver injury. 24 We previously found that activation of NOX4 in HSECs of either CCl 4 -or BDL-induced liver fibrosis rat models generated abundant ROS to promote HSECs defenestration and liver fibrosis. 5,6 NOX2 activation and its derived ROS are implicated in oxidative damage of vascular function in ageing-related diseases 25 ; activating NOX2 accelerates liver fibrosis during ageing. 26 It is reported that NOX2 contributed to cytoskeleton rearrangement and capillarization of HSECs and its loss of scavenging function. 27,28 Little is known about the exact mechanism of NOX2 in HSECs defenestration and its premature senescence. Our findings showed that H 2 O 2 elevated the NOX2 protein level and mitochondrial dysfunction to trigger progerin-associated premature senescence, leading to defenestration in HSECs; silencing NOX2 could rescue mitochondrial function and premature senescence, and subsequently maintain HSECs fenestrae. These data demonstrated that premature senescence and defenestration in HSECs, which were induced by oxidative damage, attributed to NOX2 activation.
Besides, emerging reports show that lamins play a critical role in nucleoskeleton and cytoskeleton; whereas, accumulation of progerin contributes to perturbations in actin organization, 19 implying that progerin-associated premature senescence modulates cytoskeleton to affect cell phenotype. Some novel studies report that lamins-related ageing is regulated by transcription factors (such as p53, NF-κB, etc). 29,30 In our present study, more co-localization of NOX2 and F-actin displayed in the nuclear envelope of H 2 O 2 -treated HSECs, whose fenestrae disappeared; on the contrary, knockdown of NOX2, which reduced oxidative stress-induced premature senescence, inhibited F-actin remodelling to rescue defenestration in HSECs. In brief, NOX2-dependent oxidative stress triggered progerin-associated premature senescence to result in defenestration in HSECs via F-actin remodelling. SIRT1, a class Ⅲ histone deacetylase, serves as an important modulator of metabolism, cellular survival and lifespan. 10 Some recent evidence reports that the activation of SIRT1 confers protective effects on hepatic senescence and HSCs activation to ameliorate liver fibrosis. 11,12 The previous investigations reveal that SIRT1 protects vascular endothelial cells against age-related endothelial dysfunction, whereas little research explores the role of SIRT1 in the morphology and function of HSECs. In our present study, oxidative damage activated acetylation of p53 in H 2 O 2treated HSECs; in parallel, the Ac p53 K381 protein expression highly expressed in nuclei and co-localized with more progerin and F-actin in the nuclear envelope of H 2 O 2 -treated HSECs. However, activating SIRT1 with resveratrol or overexpression of SIRT1 with adenovirus vector inhibited NOX2-dependent oxidative stress and relieved premature senescence via deacetylation of p53; whereas inhibiting SIRT1 aggravated progerin-associated premature senescence. Furthermore, overexpression of SIRT1 inhibited abnormal accumulation of progerin and F-actin remodelling, to attenuate H 2 O 2 -treated and CCl 4 -induced HSECs defenestration and liver fibrogenesis. It follows that activation of SIRT1 effectively protects against defenestration in HSECs through inhibiting progerinassociated premature senescence.
Although this study provides new insights into the mechanisms of premature senescence in HSECs defenestration, there are still some limitations to our present study. The mechanism about the reduction of Lamin B1 in HSECs defenestration needs to lucubrate.

ACK N OWLED G EM ENTS
The authors are much grateful to Mr Litao Qin, Dr Shasha Bian and Dr Yonghui Dong for essential helps in this study. This study is spon-

CO N FLI C T O F I NTE R E S T S
The authors declare that they have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data sets generated during and/or analysed during the current study are available from the corresponding author upon request.