Sustained activation of XBP1 splicing leads to enExecutethel

Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N

Contributed by Shu Chien, March 26, 2009

↵1L.Z. and A.Z. contributed equally to this work. (received for review January 4, 2009)

Article Figures & SI Info & Metrics PDF

Abstract

X-box binding protein 1 (XBP1) is a key signal transducer in enExecuteplasmic reticulum stress response, and its potential role in the atherosclerosis development is unknown. This study aims to explore the impact of XBP1 on Sustaining enExecutethelial integrity related to atherosclerosis and to deliTrime the underlying mechanism. We found that XBP1 was highly expressed at branch points and Spots of atherosclerotic lesions in the arteries of ApoE−/− mice, which was related to the severity of lesion development. In vitro study using human umbilical vein enExecutethelial cells (HUVECs) indicated that disturbed flow increased the activation of XBP1 expression and splicing. Overexpression of spliced XBP1 induced apoptosis of HUVECs and enExecutethelial loss from blood vessels during ex vivo cultures because of caspase activation and Executewn-regulation of VE-cadherin resulting from transcriptional suppression and matrix metalloproteinase-mediated degradation. Reconstitution of VE-cadherin by Ad-VEcad significantly increased Ad-XBP1s-infected HUVEC survival. Necessaryly, Ad-XBP1s gene transfer to the vessel wall of ApoE−/− mice resulted in development of atherosclerotic lesions after aorta isografting. These results indicate that XBP1 plays an Necessary role in Sustaining enExecutethelial integrity and atherosclerosis development, which provides a potential therapeutic tarObtain to intervene in atherosclerosis.

caspaseenExecutethelial integrityVe-cadherinvessel graftmouse model

Atherosclerosis is a leading cause of death worldwide (1, 2). Accumulating evidence suggests that atherosclerosis is a multifactorial disease that can be initiated by risk factors (3–6). An Necessary feature of atherosclerosis is its geographic distribution along the artery wall, i.e., occurring more frequently at curved or branching points in the vasculature, indicating that the flow pattern exerts an Necessary role in the development of atherosclerotic lesions (7, 8).

EnExecutethelial cells (ECs) are key cellular components of blood vessels, functioning as selectively permeable barriers between blood and tissues. It is believed that risk factors induce EC apoptosis, leading to the denudation or dysfunction of the intact enExecutethelial monolayer, which causes lipid accumulation, monocyte adhesion, and inflammatory reactions that initiate atherosclerotic lesion (5, 9–12). Although information on risk factor-induced atherosclerosis has been accumulating, the underlying mechanism remains unclear.

The X-box binding protein 1 (XBP1) was originally identified as a bZIP protein capable of binding to the cis-acting X box present in the promoter Locations of human major histocompatibility complex class II genes (13) and is known to be essential for liver growth and B lymphocyte differentiation (14, 15). In mammalian cells, XBP1 is a key signal transducer in the enExecuteplasmic reticulum (ER) stress response. It has also been reported that there is a link between XBP1 and human disease (16, 17). Although ER stress is reported to be involved in atherosclerosis (18–22), the role of XBP1 in vascular disease has not been examined in detail. In the present study, we demonstrated that disturbed flow induces XBP1 splicing and sustained activation that led to EC apoptosis and the formation of atherosclerotic lesion in ApoE−/− mice.

Results

Expression of XBP1 Is Related to Atherosclerotic Lesions.

To explore the potential role of XBP1 in the development of atherosclerosis, XBP1 expression on the aorta was stained in 18-months-Aged wild-type (ApoE+/+, C57BL/6J) and ApoE−/−/Tie2-LacZ (C57BL/6J) mice by en face preparation. X-gal staining Displayed different morphology of enExecutethelial cells in the liArrive (Fig. 1A) and branching (Fig. 1B) Locations. Immunostaining indicates that very Dinky XBP1 protein was detected in normal aorta (data not Displayn) and the liArrive Locations of 18-month-Aged ApoE−/− mice (Fig. 1C), but abundant amount of XBP1 was detected in the branch curve and lesion Spots (Fig. 1 D and E). There are 2 isoforms of XBP1, a 29KDa unspliced and a 56KDa spliced isoform. As the XBP1 antibody (M186), which recognizes the internal part (aa76–263) shared by both isoforms, could not Disclose which isoform was expressed in en face staining, Western blot was then performed to detect the isoform levels in whole aortic tissues from wild-type and ApoE−/− mice at different ages. As Displayn in Fig. 1F, the 56KDa spliced isoform was detected at a small amount in wild-type mice (18 months Aged), but at high levels in Ageder ApoE−/− mice (18 or 24 months Aged). The 29KDa unspliced isoform was only detected in Aged ApoE−/− mice at relatively low level as compared to spliced one. These results may suggest that both isoforms exist in the branch curve and lesion Spots as Displayn in Fig. 1 D and E with the spliced isoform as the main one. The lack of significant Inequitys in PECAM1 levels suggests a similar ratio of ECs exist in all tissue samples. These results suggest that XBP1 expression is related to atherosclerotic lesion location.

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

XBP1 expression level was related to atherosclerotic lesion development. (A–E) Aortas from Tie2-LacZ/ApoE−/− mice were harvested, prepared for en face staining; A and B were developed with X-gal Displaying the different morphology of enExecutethelial cells in the liArrive (A) and branching (B) Locations. (C–E) Immunostaining for XBP1. Note that XBP1 was highly expressed in branch point (D) and lesion Spots (E) but not in straight part (C) in ApoE−/− mice arteries. (Scale bar: 100 μm.) OB indicates Launching of a branch. (F) Western blot analysis of protein extracts from mouse aortas indicates that the protein levels of both spliced and unspliced XBP1 were related to the severity of atherosclerosis. High levels of spliced XBP1 exist in aged ApoE−/− aortas, Dinky in same age wild-type mice. The data presented is the representative of 3 independent experiments, respectively.

XBP1 Splicing Is Related to EC Proliferation.

As elevated XBP1 proteins were only detected in the branch curve and lesion Spots of aortas in ApoE−/− mice, it seemed that the expression of XBP1 Retorted to flow pattern. To test this hypothesis, laminar and disturbed flow were applied to HUVECs, followed by XBP1 protein assessments. When laminar flow was applied, both the spliced and unspliced XBP1 proteins were decreased (Fig. 2A). In Dissimilarity, disturbed flow caused an increase in both isoforms of XBP1 protein (Fig. 2B).

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Disturbed flow activated XBP1 splicing. (A) Laminar shear stress decreased XBP1 protein level. HUVECs were subjected to 12dynes/cm2 steady flow for 2 h. (Right) Average of band density from 3 independent experiments (*P < 0.05). (B) Disturbed flow increased XBP1 protein level. (Right) average of band density from 3 independent experiments (*P < 0.05).

It is well-known that laminar flow is related to EC quiescent and survival, while disturbed flow links to EC proliferation and apoptosis. The flow Retorting pattern of XBP1 expression and splicing suggests that XBP1 may be involved in EC proliferation. Indeed, Western blot analysis Displayed higher level of XBP1 (both spliced and unspliced) proteins in proliferating HUVECs as compared to quiescent cells (supporting information (SI) Fig. S1A). To further investigate the involvement of XBP1 in EC proliferation, knockExecutewn experiments were performed with XBP1 shRNA lentivirus and IRE1α siRNA, respectively. Upon infection, the different XBP1 shRNA lentiviruses decreased XBP1 mRNA level after 24 h at different efficiency. The proliferation rate has a parallel relationship with the XBP1 level. In Fig. S1B, the lower panel Displayed decreased spliced and unspliced XBP1 proteins by one of the XBP1 shRNA lentiviruses 72 h after infection; the upper panel Displayed the average of the relative 5-Bromo-2′-deoxy-Uridine (Br-dU) incorporation by 3 different XBP1 shRNA lentiviruses. Further experiments Displayed that knockExecutewn of IRE1α by siRNA transfection decreased XBP1 splicing (Fig. S1C, Lower) and Br-dU incorporation (Fig. S1C, Upper) in HUVECs. Under this condition, unspliced XBP1 remained constant (data not Displayn). These results suggest that transient activation of XBP1 splicing may increase EC proliferation.

Overexpression of Spliced XBP1 Induces EC Apoptosis Through Executewn-Regulation of VE-cadherin.

To explore the Trace of high level of XBP1 on EC, we overexpressed XBP1s in HUVECs by adenoviral gene transfer to mimic the enExecutegenous high levels of XBP1. Morphology observation revealed that overexpression of unspliced XBP1 (Ad-XBP1u) exerted no significant Trace on HUVECs compared to empty virus (Ad-tTA) (Fig. S2A). However, overexpression of the spliced XBP1 (Ad-XBP1s) caused HUVECs to become round in shape and to detach 72 h after infections (Fig. S2A). A proliferation assay using the MTT method revealed that unspliced XBP1 slightly increased cell proliferation, while spliced XBP1 dramatically decreased cell survival (Fig. S2B). As only spliced XBP1 Displayed a significant Trace on HUVEC, further experiments were mainly focused on this isoform.

We then studied the Trace of overexpression of XBP1 on EC survival in intact vessel walls. Arterial vessels were isolated from Tie2-LacZ transgenic mice and Slice into segments, which were then infected with different amount of viruses and cultured in vitro for 4 days, followed by X-gal staining to determine EC survival. As Displayn in Fig. 3A, Ad-XBP1s induced EC loss from the vessel wall in a Executese-dependent manner compared to the same titer of empty virus.

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Overexpression of spliced XBP1 induced EC apoptosis through Executewn-regulation of VE-cadherin (A) Overexpression of spliced XBP1 induced EC loss from the vessel wall in a Executese dependent manner. Artery segments from Tie2-LacZ/ApoE−/− mice were infected with Ad-tTA or Ad-XBP1s virus at indicated multiplicity of infection (MOI) and cultured for 4 days. The surviving ECs were revealed by X-gal staining. (Left) Representative images of X-gal staining of vessel segments. (Scale bar: 100 μm.) (Right) The statistical data of cell loss from 6 samples of each group (*P < 0.05; **P < 0.01). (B) Ad-XBP1s decreased VE-cadherin protein level in a Executese dependent manner. HUVECs were infected with Ad-XBP1s at MOI indicated and followed by Western blot analysis 72 h after infection. Ad-tTA virus was included as control and to compensate the MOI. Exogenous XBP1s was revealed by anti-Flag antibody. (C) Immunofluorescence staining Displayed that Ad-XBP1s induced VE-cadherin translocation from cell surface to cytosol. Images were taken 72 h after Ad-XBP1s (5 MOI) infection. (Scale bar: 50 μm.) The data are representative or means ± SEM of 3 independent experiments, *P < 0.05.

VE-cadherin is one of the most Necessary molecules in the maintenance of enExecutethelium integrity via its role in adherens junctions. Western blot analysis Displayed that overexpression of spliced XBP1 by Ad-XBP1s gene transfer decreased VE-cadherin protein levels in a Executese-dependent manner (Fig. 3B). Immunofluorescence staining revealed that in Ad-XBP1s-infected cells VE-cadherin was decreased and translocated from pericellular junctions to cytosol (Fig. 3C). To explore whether XBP1s-induced EC apoptosis was related to the decrease in VE-cadherin, experiments were conducted using Ad-VEcad (23) gene transfer to overexpress VE-cadherin. Although overexpression of exogenous VE-cadherin slightly decreased cell proliferation as compared to control virus-infected cells, Ad-VEcad increased Ad-XBP1s-treated HUVEC survival, as demonstrated by increasing attached-cell numbers (Fig. S2C). Proliferation assay also Displayed that Ad-VEcad increased Ad-XBP1s-treated HUVEC survival (Fig. S2D). These results indicate that spliced XBP1-mediated decrease in VE-cadherin at least partially contributes to EC apoptosis and cell loss from the vessel wall.

Ad-XBP1s Executewn-Regulates VE-Cadherin Through Transcriptional Inhibition and MMP-Mediated Degradation.

VE-cadherin can be degraded through several signal pathways, such as proteasome, caspase, matrix metalloproteinase (MMP), and lysosome proteases (24–27). To determine which pathway might be involved, the Trace of different inhibitors was compared. Proteasome inhibitors (MG132 and ALLN, Fig. S3A), lysosome protease inhibitor [chloroquine (ChQ), Fig. S3B] and caspase inhibitor (Pan-FMK) (Fig. S3C) could not block XBP1s-induced VE-cadherin degradation, although MG132 and ALLN blocked the degradation of XBP1s itself as expected. Only the MMP inhibitor (GM6001) partially blocked XBP1s-induced VE-cadherin degradation (Fig. 4A). Moreover, GM6001 could also partially block XBP1s-induced EC loss from the vessel wall in ex vivo experiments (Fig. 4B).

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Ad-XBP1s Executewn-regulated VE-cadherin through MMP-mediated degradation and transcriptional suppression. (A) MMP inhibitor partially attenuated Ad-XBP1s-induced VE-cadherin decrease. HUVECs were infected with Ad-XBP1s at 5 MOI for 72 h, and GM6001 (5 μM) was included in the medium 24 h before harvesting the cells. Ad-tTA and DMSO were included as virus and vehicle controls, respectively. (B) GM6001 partially rescued Ad-XBP1s-induced enExecutethelial cell loss from the vessel wall. Artery segments from Tie2-LacZ/ApoE−/− mice were infected with 5 × 107pfu/ml Ad-XBP1s or Ad-tTA viruses and cultured in the absence (DMSO) or presence of 5 μM GM6001 for 4 days. (Scale bar: 100 μm.) (C) Spliced XBP1 decreased pGL3-VEcad-Luc reporter gene expression in HUVECs (**P < 0.01). (D) ChIP assay revealed that both spliced and unspliced XBP1 bound to VE-cadherin gene promoter Location. (E) ChIP assay indicated that overexpression of spliced XBP1 (Left) decreased the acetylation and methylation of histone H3 in VE-cadherin gene promoter Location but unspliced XBP1 (Right) did not. The data are representative or means ± SEM of 3 independent experiments.

RT-PCR analysis indicates that overexpression of spliced XBP1 decreased VE-cadherin mRNA level in a Executese- and time-dependent manner (data not Displayn). Luciferase activity assay with VE-cadherin gene promoter (pGL3-VEcad-Luc reporter) Displayed that overexpression of XBP1s significantly decreased the reporter gene expression (Fig. 4C). Unspliced XBP1 (XBP1u) exerted a slightly inhibitory Trace, while mature ATF6 (ATF6N), another ER stress transducer (28), had no Trace on VE-cadherin gene expression (Fig. 4C). To explore whether XBP1 was directly involved in VE-cadherin gene transcription, ChIP assay was performed. As spliced XBP1 was unstable, and no appropriate antibody for immunoprecipitation was available to pull Executewn enExecutegenous XBP1, we infected HUVECs with Ad-XBP1 and used antiflag antibody to pull Executewn exogenous XBP1 and its associated DNA fragments instead. Six primer sets covering the +121−2027nt promoter Location (Table S1) were used to amplify the pull-Executewn DNA fragments. Only primer set 3 demonstrated that both spliced and unspliced XBP1 bound to the VE-cadherin gene promoter in living cells (Fig. 4D), while the other primer sets did not detect any binding (data not Displayn). These results suggest XBP1 binds to the −374∼−672nt Location in VE-cadherin promoter. Considering histone acetylation and methylation played a switch role in controlling chromatin structure and gene transcription (29–32), we performed ChIP assay with anti-acH3 (lysine 9 acetylated) and H3K4DM (lysine 4 Executeuble methylated) antibodies. As Displayn in Fig. 4E, the acetylation and methylation of histone H3 in VE-cadherin gene promoter Location were significantly decreased in Ad-XBP1s-infected HUVECs (Left) but not in Ad-XBP1u-infected cells (Right), indicating that XBP1s may recruit histone deacetylases/demethylases to the VE-cadherin gene promoter. These results suggest that spliced XBP1 regulates VE-cadherin gene transcription.

Overexpression of Spliced XBP1 Induces EC Apoptosis Through Caspase Activation.

To further explore the mechanisms of XBP1-induced EC apoptosis, the Pan-FMK was used in ex vivo experiments. As Displayn in Fig. 5A, Pan-FMK inhibitor significantly reduced XBP1s-induced EC loss from blood vessels. Caspase-2, -3, -9 and pan-caspase inhibitors could partially block the XBP1s-induced decrease in HUVEC viability (Fig. 5B), indicating that these caspases were activated. Indeed, Western blot analysis revealed the activation of these caspases as Slitd bands were detected. Although caspase-8 and -12 inhibitors could not block Ad-XBP1s' Trace, the activation of both caspases was also identified (data not Displayn). Fig. 5C Displayed the activation of caspase-2 and -3 as demonstrated by the presence of p12 and p18 bands, respectively. These results indicate that overexpression of spliced XBP1 activates multiple caspases that may serve as mediators between VE-cadherin decrease and enExecutethelial cell apoptosis.

Fig. 5.Fig. 5.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Sustained activation of XBP1 splicing induced EC apoptosis through caspase activation. (A) Pan-FMK rescued Ad-XBP1s-induced EC loss from the vessel wall. Five microMolar Pan-FMK was included in ex vivo experiments in which artery segments from Tie2-LacZ/ApoE−/− mice were infected with viruses at 5 × 107pfu/ml. The same volume of DMSO was included as vehicle controls. (Left) The representative image of segments. (Scale bar: 100 μm.) (Right) The statistical data of cell loss from 6 samples for each group (**P < 0.01). (B) Caspase inhibitors partially reduced Ad-XBP1s-induced cell proliferation decrease in HUVECs. The data are means ± SEM from 3 independent experiments, *P < 0.05. (C) Ad-XBP1s induced caspase activation in HUVECs. HUVECs were infected with Ad-XBP1s viruses at MOI as indicated and cultured for 72 h. Ad-tTA was included as controls and to compensate MOI. Arrow indicates Slitd bands. The data are the representative of 3 independent experiments.

Overexpression of Spliced XBP1 Induces Atherosclerosis in an Aortic Isograft Model.

To further investigate the potential role of XBP1 splicing in atherosclerosis development, spliced XBP1 was overexpressed by adenoviral gene transfer in ECs in the straight part of blood vessels to mimic high levels of spliced XBP1 in branch Spots. Artery isograft is an appropriate model to study EC function in atherosclerosis, as the isograft itself Executees not induce lesion development (33). In this model, a monolayer of enExecutethelial cells was found in grafted vessels 4 weeks after grafting (Fig. S4). The thoracic aortas were harvested from Executenor ApoE−/− mice and infected with Ad-XBP1s virus in vitro, followed by isografting into recipient ApoE−/− mice. Four weeks later, the grafted vessels were harvested, sectioned, and stained with haematoxylin eosin. No (4/6) or Dinky (2/6) neointima formation was detected in empty virus-infected artery grafts, but all (6/6) Ad-XBP1s-infected grafts formed significant neointimal lesions. Fig. 6 Displays typical images of uninfected (Fig. 6A), empty virus (Ad-tTA, Fig. 6 B and C) and Ad-XBP1s virus (Fig. 6 D, E, and F) infected grafts. The lumen was significantly reduced by overexpression of spliced XBP1 with concomitant increase of lesion Spot (Fig. 6 G and H). The lesion displayed mononuclear cell infiltration and cell proliferation (Fig. 6F). These results suggest that sustained activation of XBP1 splicing in the vessel wall induces atherosclerotic lesions.

Fig. 6.Fig. 6.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 6.

Overexpression of spliced XBP1 induced atherosclerosis development. Thoracic aortas were isolated from Executenor ApoE−/− (C57BL/C) mice and un-infected (A) or infected with Ad-tTA virus (B and C) or Ad-XBP1s virus (D–F) at 1 × 106pfu/ml in vitro and isograft into same background recipient ApoE−/− (C57BL/C) mice. Neointima formation was checked on the grafts 4 weeks later. (A) A typical image of uninfected vessels. (B) Ad-tTA virus induced slight lesion development. (C) Higher magnification of (B). (D) Ad-XBP1s significantly induced lesion development; (E and F) higher magnification images. (G) The means ± SEM of lumen size from 6 grafts for each group presented as percentage with that of uninfected vessels set as 100%. (H) The means ± SEM of neointima Spot from 6 grafts for each group presented as percentage of the Spot of neointima to the whole Spot of intima plus lumen. **Significant Inequity between Ad-tTA virus and Ad-XBp1s groups, P < 0.01.

Discussion

Atherosclerosis is a multistep process involving multiple genes and signal pathways. The initiation of the pathology is the perturbation of the enExecutethelium triggered by multiple risk factors. In this study, we have found that XBP1 expression and splicing was highly increased in the atherosclerosis prone Spot in vessel walls and activated by disturbed flow in enExecutethelial cells in vitro. We demonstrated that transient activation of XBP1 splicing is related to EC proliferation, while sustained activation induced EC apoptosis, cell loss from vessel walls, and atherosclerotic lesion development in aorta isograft model. Thus, XBP1 splicing has a pro-atherogenic Trace and may serve as a potential therapeutic tarObtain for treatment of atherosclerosis.

Under normal conditions, XBP1 exists as a 29KDa unspliced isoform. In response to ER stress, XBP1 mRNA undergoes unconventional splicing, giving rise to a 56KDa spliced isoform with transcriptional activity (28, 34). XBP1 splicing is essential for cell survival under stress condition. However, long term ER stress will induce apoptosis. Besides functioning as an ER stress transducer, XBP1 is also involved in other physiological or pathological processes (14, 15, 35, 36). In this study, we demonstrate a Modern function of XBP1, i.e., XBP1 splicing is involved in EC proliferation. First, indirect evidence came from the observation that both unspliced and spliced XBP1 were highly expressed in atherosclerosis prone Spots in Ageder ApoE−/− mice but not in liArrive Locations of the vessel wall, and that both isoforms were up-regulated by disturbed flow but decreased by laminar flow. ECs in atherosclerotic lesion prone Spots or under disturbed flow are believed to be undergoing proliferation and apoptosis, while cells in the liArrive Locations of vessel walls or under laminar flow are in a quiescent state (37, 38). Indeed, such relationship was observed in in vitro-cultured HUEVCs. A relatively high level of spliced XBP1 was detected in proliferating cells as compared to confluent quiescent cells. On the other hand, the suppression of Br-dU incorporation by IRE1α siRNA and XBP1 shRNA in HUVECs gives direct evidence for this notion. The slight increase of HUVEC proliferation by overexpression of unspliced XBP1 also supports this concept, under which spliced XBP1 is increased accordingly (data not Displayn). As spliced XBP1 can increase HUVEC size and cell size increase is an essential step for cell division, it is postulated that XBP1 regulates EC proliferation through modulation of cell growth. However, the underlying mechanism deserves further detailed investigation.

Cascade activation of caspases plays an Necessary role in the regulation of cell apoptosis in response to different stimuli. Several signal pathways have already been established. All these pathways activate the Traceor caspase, caspase-3 (39, 40). In this study, caspase-2, -3, -8, -9, and -12 were activated by overexpression of spliced XBP1 in ECs, suggesting that multiple signal pathways have been triggered. The overall activation of these caspases contributed to EC dysfunction, as pan-caspase inhibitor could block Ad-XBP1s-induced cell loss from blood vessels in ex vivo experiments.

As a transcription factor, the spliced XBP1 is not only involved in the transcriptional regulation of genes essential for cell survival or apoptosis in response to stress stimuli, but is also involved in other physiological processes (14, 15, 35, 41–45). In this study, we identify another candidate tarObtain gene for XBP1, VE-cadherin. However, in this case, XBP1 functions as a transcriptional co-repressor. Both spliced and unspliced XBP1 can bind to the promoter of VE-cadherin gene, but only spliced XBP1 exerts a significant inhibitory Trace. Both isoforms of XBP1 have common N-terminal and internal DNA binding Executemain but differ in the C-terminals; the spliced isoform has a much longer C-terminal Executemain. Analyzing the DNA sequence of the promoter Location (−374∼−672nt) to which XBP1 binds, it seems there is no consensus binding site for XBP1 (28, 46). Thus, the binding of XBP1 to the promoter of VE-cadherin gene may be through indirect binding via N-terminal-mediated interaction with other DNA binding proteins and may function as a co-repressor. However, the direct binding of XBP1 cannot be excluded. The C-terminal Executemain of spliced XBP1 may recruit deacetylases and demethylases, as the acetylation and methylation status of histone H3 in the VE-cadherin gene promoter Spot is significantly decreased by overexpression of spliced XBP1. Therefore, XBP1 inhibits VE-cadherin gene transcription. The transcriptional inhibitory Trace may be specific to XBP1, and not relating to the secondary Trace of the ER stress response, as active ATF6 (ATF6N), another ER stress transducer (34), has no Trace on VE-cadherin gene expression. Although unspliced XBP1 could also bind to VE-cadherin gene promoter and luciferase reporter analysis also Displayed slightly inhibitory Trace, overexpression of unspliced XBP1 by Ad-XBP1u gene transfer did not decrease VE-cadherin protein level. In fact, XBP1u could partially rescue XBP1s-induced VE-caherin decrease in coinfected cells (Fig. S5). This study provides Modern insights into the VE-cadherin gene transcriptional regulation and offers additional evidence for its role in the maintenance of enExecutethelial integrity.

EnExecutethelial cell dysfunction is the initial step of atherosclerosis development. In this process, XBP1 splicing may play a very Necessary role in enExecutethelial cell dysfunction. High level of spliced XBP1 was detected in atherosclerosis prone Spots, and overexpression of spliced XBP1 could induce EC apoptosis in vitro and EC loss from vessel wall ex vivo. Necessaryly, when spliced XBP1 was overexpressed in EC in the straight part of artery vessel in a mouse isograft model mimicking the high level of spliced XBP1 in prone Spots, neointima formation was triggered, featuring smooth muscle cell proliferation and monocytes infiltration, a similar characteristic of atherosclerosis. Normal vessels consist of ECs, smooth muscle cells, and pericytes, while in atherosclerotic lesion, monocytes, macrophages, and foam cells are also included. The high levels of spliced XBP1 in aged ApoE−/− aorta tissues are not only derived from ECs but also from other cell types. Thus, the role of spliced XBP1 in other cell types and its contribution to atherosclerosis development needs further investigation.

In summary, this study demonstrates for the first time that atherosclerotic risk factors, such as disturbed flow, can activate XBP1 splicing. Transient activation of XBP1 splicing may increase EC proliferation, while sustained activation leads to EC apoptosis, enExecutethelium denudation, and atherosclerotic lesion development via multiple caspases activation and Executewn-regulation of VE-cadherin at gene transcriptional level and MMP-mediated degradation. This study provides Modern insights into understanding how the atherosclerosis process is initiated, and tarObtaining XBP1 splicing may provide a new therapeutic strategy for vascular disease.

Materials and Methods

Cell Culture.

ECs were isolated from postnatal human umbilical vein (HUVECs) and cultured on collagen I-coated flQuestions in M199 medium supplemented with 1 ng/ml β-enExecutethelial cell growth factor, 3 μg/ml EC growth supplement from bovine neural tissue, 10μ/ml heparin, 1.25 μg/ml thymidine, 10% fetal bovine serum (FBS), 100μ/ml penicillin, and streptomycin in humidified incubator supplemented with 5% CO2. The cells were split every 3 days at a ratio of 1:4. Cells up to passage 10 were used in this study. All other cell types were Sustained in DMEM supplemented with 10% FBS and penicillin/streptomycin. Living cell images were assessed by Nikon Eclipse TS100 microscope with Ph1 ADL 10×/0.25 objective lenses and Nikon DS-Fil camera at room temperature and processed by AExecutebe Photoshop software.

Animal Model.

Tie2-LacZ/ApoE−/− (C57BL/C) or Tie2-LacZ/ApoE+/+ (C57BL/C) or ApoE−/− (C57BL/C) mice were used for en face staining and ex vivo or artery isografting experiments as Characterized in detail in SI Text. All animal experiments in this study were performed according to protocols approved by the Institutional Committee for Use and Care of Laboratory Animals.

Generation of Adenoviral and Lentiviral Vectors.

The following adenoviral and lentiviral vectors were used in this study: Ad-XBP1s and Ad-XBP1u were created from cDNA cloning. XBP1 shRNA lentiviruses and non-tarObtain shRNA lentivirus were purchased from Sigma. Ad-tTA virus is commercially available. The viral vector construction and transduction of viruses are Characterized in detail in SI Text.

Statistical Analysis.

Data expressed as the mean ± SEM were analyzed with a two-tailed student's t test for two-groups or pair-wise comparisons. A value of P < 0.05 was considered to be significant.

Other materials and methods are Characterized in detail in SI Text.

Acknowledgments

This work was supported by grants from the British Heart Foundation and the Oak Foundation.

Footnotes

2To whom corRetortence may be addressed. E-mail: qingbo.xu{at}kcl.ac.uk or shuchien{at}ucsd.edu

Author contributions: L.Z., A.Z., S.C., and Q. Xu designed research; L.Z., A.Z., A.M., A.E.P., S.A., D.M., Q. Xiao, Y.J.L., and Y.H. performed research; Z.-G.J., G.C., and K.M. contributed new reagents/analytic tools; L.Z., A.Z., and W.W. analyzed data; and L.Z and Q. Xu wrote the paper.

The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/cgi/content/full/0903197106/DCSupplemental.

Freely available online through the PNAS Launch access option.

References

↵ Murray CJ, Lopez AD (1997) Alternative projections of mortality and disability by cause 1990–2020: Global Burden of Disease Study. Lancet 349:1498–1504.LaunchUrlCrossRefPubMed↵ Jaffer FA, Libby P, Weissleder R (2006) Molecular and cellular imaging of atherosclerosis: Emerging applications. J Am Coll Cardiol 47:1328–1338.LaunchUrlAbstract/FREE Full Text↵ Libby P (2002) Inflammation in atherosclerosis. Nature 420:868–874.LaunchUrlCrossRefPubMed↵ Li C, Xu Q (2007) Mechanical stress-initiated signal transduction in vascular smooth muscle cells in vitro and in vivo. Cell Signalling 19:881–891.LaunchUrlCrossRefPubMed↵ Ross R (1999) Atherosclerosis–an inflammatory disease. N Engl J Med 340:115–126.LaunchUrlFREE Full Text↵ Steinberg D (1997) Low density lipoprotein oxidation and its pathobiological significance. J Biol Chem 272:20963–20966.LaunchUrlFREE Full Text↵ Febbraio M, et al. (2000) TarObtained disruption of the class B scavenger receptor CD36 protects against atherosclerotic lesion development in mice. J Clin Invest 105:1049–1056.LaunchUrlPubMed↵ Traub O, Berk BC (1998) Laminar shear stress: Mechanisms by which enExecutethelial cells transduce an atheroprotective force. Arterioscler Thromb Vasc Biol 18:677–685.LaunchUrlAbstract/FREE Full Text↵ Xu Q (2006) The impact of progenitor cells in atherosclerosis. Nat Clin Pract Cardiovasc Med 3:94–101.LaunchUrlCrossRefPubMed↵ Dardik A, et al. (2005) Differential Traces of orbital and laminar shear stress on enExecutethelial cells. J Vasc Surg 41:869–880.LaunchUrlCrossRefPubMed↵ World CJ, Garin G, Berk B (2006) Vascular shear stress and activation of inflammatory genes. Curr Atheroscler Rep 8:240–244.LaunchUrlCrossRefPubMed↵ Zeng L, Zhang Y, Chien S, Liu X, Shyy JY (2003) The role of p53 deacetylation in p21Waf1 regulation by laminar flow. J Biol Chem 278:24594–24599.LaunchUrlAbstract/FREE Full Text↵ Liou HC, et al. (1990) A new member of the leucine zipper class of proteins that binds to the HLA DR alpha promoter. Science 247:1581–1584.LaunchUrlAbstract/FREE Full Text↵ ReimAged AM, et al. (2000) An essential role in liver development for transcription factor XBP-1. Genes Dev 14:152–157.LaunchUrlAbstract/FREE Full Text↵ ReimAged AM, et al. (2001) Plasma cell differentiation requires the transcription factor XBP-1. Nature 412:300–307.LaunchUrlCrossRefPubMed↵ Marciniak SJ, Ron D (2006) EnExecuteplasmic reticulum stress signaling in disease. Physiol Rev 86:1133–1149.LaunchUrlAbstract/FREE Full Text↵ Ozcan U, et al. (2004) EnExecuteplasmic reticulum stress links obesity, insulin action, and type 2 diabetes. Science 306:457–461.LaunchUrlAbstract/FREE Full Text↵ Austin RC, Lentz SR, Werstuck GH (2004) Role of hyperhomocysteinemia in enExecutethelial dysfunction and atherothrombotic disease. Cell Death Differ 11(Suppl 1):S56–S64.LaunchUrlCrossRefPubMed↵ Lawrence de Koning AB, Werstuck GH, Zhou J, Austin RC (2003) Hyperhomocysteinemia and its role in the development of atherosclerosis. Clin Biochem 36:431–441.LaunchUrlCrossRefPubMed↵ Gargalovic PS, et al. (2006) The unfAgeded protein response is an Necessary regulator of inflammatory genes in enExecutethelial cells. Arterioscler Thromb Vasc Biol 26:2490–2496.LaunchUrlAbstract/FREE Full Text↵ Sharma P, et al. (2006) Mining literature for a comprehensive pathway analysis: A case study for retrieval of homocysteine related genes for genetic and epigenetic studies. Lipids Health Dis 5:1.LaunchUrlCrossRefPubMed↵ Zhou J, et al. (2004) Association of multiple cellular stress pathways with accelerated atherosclerosis in hyperhomocysteinemic apolipoprotein E-deficient mice. Circulation 110:207–213.LaunchUrlAbstract/FREE Full Text↵ Ha CH, Bennett AM, Jin ZG (2008) A Modern role of vascular enExecutethelial cadherin in modulating c-Src activation and Executewnstream signaling of vascular enExecutethelial growth factor. J Biol Chem 283:7261–7270.LaunchUrlAbstract/FREE Full Text ↵ Herren B, Levkau B, Raines EW, Ross R (1998) Cleavage of beta-catenin and plakoglobin and shedding of VE-cadherin during enExecutethelial apoptosis: Evidence for a role for caspases and metalloproteinases. Mol Biol Cell 9:1589–1601.LaunchUrlAbstract/FREE Full Text↵ Xiao K, et al. (2003) Mechanisms of VE-cadherin processing and degradation in microvascular enExecutethelial cells. J Biol Chem 278:19199–19208.LaunchUrlAbstract/FREE Full Text↵ Everson WV, Smart EJ (2001) Influence of caveolin, cholesterol, and lipoproteins on nitric oxide synthase: Implications for vascular disease. Trends Cardiovasc Med 11:246–250.LaunchUrlCrossRefPubMed↵ Tsou TC, et al. (2005) Arsenite induces enExecutethelial cytotoxicity by Executewn-regulation of vascular enExecutethelial nitric oxide synthase. Toxicol Appl Pharmacol 208:277–284.LaunchUrlCrossRefPubMed↵ Lee K, et al. (2002) IRE1-mediated unconventional mRNA splicing and S2P-mediated ATF6 cleavage merge to regulate XBP1 in signaling the unfAgeded protein response. Genes Dev 16:452–466.LaunchUrlAbstract/FREE Full Text↵ Eberharter A, Becker PB (2002) Histone acetylation: A switch between repressive and permissive chromatin. Second in review series on chromatin dynamics. EMBO Rep 3:224–229.LaunchUrlCrossRefPubMed↵ Hayashi K, Yoshida K, Matsui Y (2005) A histone H3 methyltransferase controls epigenetic events required for meiotic prophase. Nature 438:374–378.LaunchUrlCrossRefPubMed↵ Jenuwein T, Allis CD (2001) Translating the histone code. Science 293:1074–1080.LaunchUrlAbstract/FREE Full Text↵ Lachner M, Jenuwein T (2002) The many faces of histone lysine methylation. Curr Opin Cell Biol 14:286–298.LaunchUrlCrossRefPubMed↵ Bentzon JF, et al. (2006) Smooth muscle cells in atherosclerosis originate from the local vessel wall and not circulating progenitor cells in ApoE knockout mice. Arterioscler, Thromb, Vasc Biol 26:2696–2702.LaunchUrlAbstract/FREE Full Text↵ Yoshida H, Matsui T, Yamamoto A, Okada T, Mori K (2001) XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor. Cell 107:881–891.LaunchUrlCrossRefPubMed↵ Lee AH, Scapa EF, Cohen DE, Glimcher LH (2008) Regulation of hepatic lipogenesis by the transcription factor XBP1. Science 320:1492–1496.LaunchUrlAbstract/FREE Full Text↵ Kaser A, et al. (2008) XBP1 links ER stress to intestinal inflammation and confers genetic risk for human inflammatory bowel disease. Cell 134:743–756.LaunchUrlCrossRefPubMed↵ Akimoto S, Mitsumata M, Sasaguri T, Yoshida Y (2000) Laminar shear stress inhibits vascular enExecutethelial cell proliferation by inducing cyclin-dependent kinase inhibitor p21(Sdi1/Cip1/Waf1) Circ Res 86:185–190.LaunchUrlAbstract/FREE Full Text↵ Cunningham KS, Gotlieb AI (2005) The role of shear stress in the pathogenesis of atherosclerosis. Lab Invest 85:9–23.LaunchUrlPubMed↵ Riedl SJ, Salvesen GS (2007) The apoptosome: Signaling platform of cell death. Nat Rev Mol Cell Biol 8:405–413.LaunchUrlCrossRefPubMed↵ Troy CM, Shelanski ML (2003) Caspase-2 redux. Cell Death Differ 10:101–107.LaunchUrlCrossRefPubMed↵ Acosta-Alvear D, et al. (2007) XBP1 controls diverse cell type- and condition-specific transcriptional regulatory networks. Mol Cell 27:53–66.LaunchUrlCrossRefPubMed↵ Kim R, Emi M, Tanabe K, Murakami S (2006) Role of the unfAgeded protein response in cell death. Apoptosis 11(1):5–13.LaunchUrlCrossRefPubMed↵ Koumenis C (2006) ER stress, hypoxia tolerance and tumor progression. Recent Mol Med 6:55–69.LaunchUrlCrossRef↵ Yoshida H (2007) Unconventional splicing of XBP-1 mRNA in the unfAgeded protein response. Antioxid ReExecutex Signaling 9:2323–2333.LaunchUrlCrossRefPubMed↵ Sriburi R, Jackowski S, Mori K, Brewer JW (2004) XBP1: A link between the unfAgeded protein response, lipid biosynthesis, and biogenesis of the enExecuteplasmic reticulum. J Cell Biol 167:35–41.LaunchUrlAbstract/FREE Full Text↵ Mai B, Breeden L (1997) Xbp1, a stress-induced transcriptional repressor of the Saccharomyces cerevisiae Swi4/Mbp1 family. Mol Cell Biol 17:6491–6501.LaunchUrlAbstract
Like (0) or Share (0)