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Proc Natl Acad Sci U S A. May 19, 2009; 106(20): 8326–8331.
Published online May 1, 2009. doi:  10.1073/pnas.0903197106
PMCID: PMC2676169
Medical Sciences

Sustained activation of XBP1 splicing leads to endothelial apoptosis and atherosclerosis development in response to disturbed flow

Abstract

X-box binding protein 1 (XBP1) is a key signal transducer in endoplasmic reticulum stress response, and its potential role in the atherosclerosis development is unknown. This study aims to explore the impact of XBP1 on maintaining endothelial integrity related to atherosclerosis and to delineate the underlying mechanism. We found that XBP1 was highly expressed at branch points and areas 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 endothelial cells (HUVECs) indicated that disturbed flow increased the activation of XBP1 expression and splicing. Overexpression of spliced XBP1 induced apoptosis of HUVECs and endothelial loss from blood vessels during ex vivo cultures because of caspase activation and down-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. Importantly, 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 important role in maintaining endothelial integrity and atherosclerosis development, which provides a potential therapeutic target to intervene in atherosclerosis.

Keywords: caspase, endothelial integrity, Ve-cadherin, vessel graft, mouse 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 (36). An important 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 important role in the development of atherosclerotic lesions (7, 8).

Endothelial 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 endothelial monolayer, which causes lipid accumulation, monocyte adhesion, and inflammatory reactions that initiate atherosclerotic lesion (5, 912). 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 regions 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 endoplasmic 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 (1822), 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-old wild-type (ApoE+/+, C57BL/6J) and ApoE−/−/Tie2-LacZ (C57BL/6J) mice by en face preparation. X-gal staining showed different morphology of endothelial cells in the linear (Fig. 1A) and branching (Fig. 1B) regions. Immunostaining indicates that very little XBP1 protein was detected in normal aorta (data not shown) and the linear regions of 18-month-old ApoE−/− mice (Fig. 1C), but abundant amount of XBP1 was detected in the branch curve and lesion areas (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 tell 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 shown in Fig. 1F, the 56KDa spliced isoform was detected at a small amount in wild-type mice (18 months old), but at high levels in older ApoE−/− mice (18 or 24 months old). The 29KDa unspliced isoform was only detected in old 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 areas as shown in Fig. 1 D and E with the spliced isoform as the main one. The lack of significant differences 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.
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 showing the different morphology ...

XBP1 Splicing Is Related to EC Proliferation.

As elevated XBP1 proteins were only detected in the branch curve and lesion areas of aortas in ApoE−/− mice, it seemed that the expression of XBP1 responded 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 contrast, disturbed flow caused an increase in both isoforms of XBP1 protein (Fig. 2B).

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 ...

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 responding pattern of XBP1 expression and splicing suggests that XBP1 may be involved in EC proliferation. Indeed, Western blot analysis showed 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, knockdown 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 showed decreased spliced and unspliced XBP1 proteins by one of the XBP1 shRNA lentiviruses 72 h after infection; the upper panel showed the average of the relative 5-Bromo-2′-deoxy-Uridine (Br-dU) incorporation by 3 different XBP1 shRNA lentiviruses. Further experiments showed that knockdown 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 shown). These results suggest that transient activation of XBP1 splicing may increase EC proliferation.

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

To explore the effect of high level of XBP1 on EC, we overexpressed XBP1s in HUVECs by adenoviral gene transfer to mimic the endogenous high levels of XBP1. Morphology observation revealed that overexpression of unspliced XBP1 (Ad-XBP1u) exerted no significant effect 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 showed a significant effect on HUVEC, further experiments were mainly focused on this isoform.

We then studied the effect of overexpression of XBP1 on EC survival in intact vessel walls. Arterial vessels were isolated from Tie2-LacZ transgenic mice and cut 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 shown in Fig. 3A, Ad-XBP1s induced EC loss from the vessel wall in a dose-dependent manner compared to the same titer of empty virus.

Fig. 3.
Overexpression of spliced XBP1 induced EC apoptosis through down-regulation of VE-cadherin (A) Overexpression of spliced XBP1 induced EC loss from the vessel wall in a dose dependent manner. Artery segments from Tie2-LacZ/ApoE−/− mice ...

VE-cadherin is one of the most important molecules in the maintenance of endothelium integrity via its role in adherens junctions. Western blot analysis showed that overexpression of spliced XBP1 by Ad-XBP1s gene transfer decreased VE-cadherin protein levels in a dose-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 showed 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 Down-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 (2427). To determine which pathway might be involved, the effect 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.
Ad-XBP1s down-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) ...

RT-PCR analysis indicates that overexpression of spliced XBP1 decreased VE-cadherin mRNA level in a dose- and time-dependent manner (data not shown). Luciferase activity assay with VE-cadherin gene promoter (pGL3-VEcad-Luc reporter) showed that overexpression of XBP1s significantly decreased the reporter gene expression (Fig. 4C). Unspliced XBP1 (XBP1u) exerted a slightly inhibitory effect, while mature ATF6 (ATF6N), another ER stress transducer (28), had no effect 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 down endogenous XBP1, we infected HUVECs with Ad-XBP1 and used antiflag antibody to pull down exogenous XBP1 and its associated DNA fragments instead. Six primer sets covering the +121−2027nt promoter region (Table S1) were used to amplify the pull-down 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 shown). These results suggest XBP1 binds to the −374~−672nt region in VE-cadherin promoter. Considering histone acetylation and methylation played a switch role in controlling chromatin structure and gene transcription (2932), we performed ChIP assay with anti-acH3 (lysine 9 acetylated) and H3K4DM (lysine 4 double methylated) antibodies. As shown in Fig. 4E, the acetylation and methylation of histone H3 in VE-cadherin gene promoter region 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 shown 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 cleaved bands were detected. Although caspase-8 and -12 inhibitors could not block Ad-XBP1s' effect, the activation of both caspases was also identified (data not shown). Fig. 5C showed 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 endothelial cell apoptosis.

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 ...

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 areas. Artery isograft is an appropriate model to study EC function in atherosclerosis, as the isograft itself does not induce lesion development (33). In this model, a monolayer of endothelial cells was found in grafted vessels 4 weeks after grafting (Fig. S4). The thoracic aortas were harvested from donor 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 little (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 shows 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 area (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.
Overexpression of spliced XBP1 induced atherosclerosis development. Thoracic aortas were isolated from donor 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 × ...

Discussion

Atherosclerosis is a multistep process involving multiple genes and signal pathways. The initiation of the pathology is the perturbation of the endothelium triggered by multiple risk factors. In this study, we have found that XBP1 expression and splicing was highly increased in the atherosclerosis prone area in vessel walls and activated by disturbed flow in endothelial 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 effect and may serve as a potential therapeutic target 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 novel 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 areas in older ApoE−/− mice but not in linear regions of the vessel wall, and that both isoforms were up-regulated by disturbed flow but decreased by laminar flow. ECs in atherosclerotic lesion prone areas or under disturbed flow are believed to be undergoing proliferation and apoptosis, while cells in the linear regions 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 shown). 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 important role in the regulation of cell apoptosis in response to different stimuli. Several signal pathways have already been established. All these pathways activate the effector 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, 4145). In this study, we identify another candidate target 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 effect. Both isoforms of XBP1 have common N-terminal and internal DNA binding domain but differ in the C-terminals; the spliced isoform has a much longer C-terminal domain. Analyzing the DNA sequence of the promoter region (−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 domain of spliced XBP1 may recruit deacetylases and demethylases, as the acetylation and methylation status of histone H3 in the VE-cadherin gene promoter area is significantly decreased by overexpression of spliced XBP1. Therefore, XBP1 inhibits VE-cadherin gene transcription. The transcriptional inhibitory effect may be specific to XBP1, and not relating to the secondary effect of the ER stress response, as active ATF6 (ATF6N), another ER stress transducer (34), has no effect on VE-cadherin gene expression. Although unspliced XBP1 could also bind to VE-cadherin gene promoter and luciferase reporter analysis also showed slightly inhibitory effect, 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 novel insights into the VE-cadherin gene transcriptional regulation and offers additional evidence for its role in the maintenance of endothelial integrity.

Endothelial cell dysfunction is the initial step of atherosclerosis development. In this process, XBP1 splicing may play a very important role in endothelial cell dysfunction. High level of spliced XBP1 was detected in atherosclerosis prone areas, and overexpression of spliced XBP1 could induce EC apoptosis in vitro and EC loss from vessel wall ex vivo. Importantly, 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 areas, 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, endothelium denudation, and atherosclerotic lesion development via multiple caspases activation and down-regulation of VE-cadherin at gene transcriptional level and MMP-mediated degradation. This study provides novel insights into understanding how the atherosclerosis process is initiated, and targeting 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 flasks in M199 medium supplemented with 1 ng/ml β-endothelial 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 maintained 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 Adobe 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 described 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-target shRNA lentivirus were purchased from Sigma. Ad-tTA virus is commercially available. The viral vector construction and transduction of viruses are described 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 described in detail in SI Text.

Supplementary Material

Supporting Information:

Acknowledgments.

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

Footnotes

The authors declare no conflict of interest.

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

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