2.1 ER stress and the unfolded protein response (UPR)

In eukaryotic cells, the ER is the largest membrane-bound organelle and performs pivotal cellular functions such as membrane biogenesis, protein synthesis and modification, lipid synthesis and trafficking, and calcium storage and secretion.^{13, 14} The ER is essential for proteostasis, as more than one-third of cellular proteins are synthesized and folded in the ER. Under a wide range of pathophysiological conditions, the accumulation of unfolded or misfolded proteins overwhelms the protein folding capability of the ER, leading to ER stress and the activation of an adaptive response called the UPR¹⁵ to preserve proteostasis, which includes protein overaccumulation, improved protein folding, and the degradation of misfolded proteins.¹¹ Three main signaling branches mediate the UPR, known as IRE1 (α and β), activating transcription factor 6α (ATF6α) and ATF6β, and protein kinase RNA (PKR)-like ER kinase (PERK). Under severe or consistent ER stress, homeostasis is disrupted in the ER, and overactivation of the UPR results in the activation of cell death programs.^{13, 16} Work over the last 3 decades has pinpointed the UPR as a driving force for the progression of a variety of diseases, including cancer,^17-19 central nervous system diseases,^{20, 21} metabolic diseases,^22-24 and CVD.¹¹

2.2 The structure of IRE1α

IRE1 is the most ancient branch of the UPR-related signaling pathway and is evolutionarily conserved from yeast to humans.²⁵ IRE1 has two isoforms: IRE1α (encoded by the gene ER to nucleus signaling 1 (Ern1)) and IRE1β (encoded by the gene Ern2). IRE1α is abundant and prevalent in almost all tissues, while IRE1β is expressed only in airway mucous cells and intestinal epithelial cells.²⁶ IRE1 consists of two domains: the N-terminal, ER luminal domain, and the C-terminal cytoplasmic region, serine/threonine kinase and endoribonuclease (RNase) domains (Figure 1).²⁷ The LD is used to sense unfolded or misfolded proteins and monitor ER homeostasis, and the C-terminal domain triggers the UPR.²⁷ The unfolded protein can directly bind to the LD, which triggers IRE1 oligomerization and allows trans-autophosphorylation of the kinase domain28. The only known substrate of the kinase is IRE1 itself, and the RNase of IRE1 is regulated by the intrinsic kinase module of IRE1.²⁸

2.3 The activation of IRE1α

ER plays a critical role in protein and lipid synthesis, and regulate lipid and glucose metabolism.²⁹ As a branch of UPR, IRE1α has been reported to be activated by several types of signals, including nutrients, metabolisms, and hormones.¹³ Glucose and lipid have been considered as stressors for activating IRE1α-signaling pathway. It has been well-documented that high glucose stimulated the hyperactivation and phosphorylation of IRE1α in pancreatic β cells,^{30, 31} and the prolonged activation of IRE1α contributes to β -cell apoptosis in type 1 diabetes.³² Moreover, high glucose activated IRE1α-signaling pathway in retinal muller cells and renal tubular epithelial cells, which further results in diabetic retinopathy and diabetic nephropathy, respectively.^{33, 34} In turn, the highly phosphorylated IRE1α in the liver drives hyperglycemia under metabolic ER stress.³⁵ Lipid also stimulates the activation of IRE1α. IRE1α could be activated by lipid independently of its lumenal unfolded protein stress-sensing domain.³⁶ Under metabolic ER stress conditions, activation of IRE1α underlies the dysregulation of thermogenic fat in obese mice.³⁷ Besides, IRE1α has been reported to be activated in both human and rodent obese adipose tissue.^{38, 39} In addition, high fat diet induced the activation of IRE1α in macrophages, hepatocytes, and ECs.^40-43

Numerous studies have shown that IRE1α could be activated by metabolic hormones. Insulin is an anabolic hormone, which is a participant in protein synthesis and lipogenesis, and results in increased load of protein folding in the ER.¹³ The phosphorylation of IRE1α was observed in mice with hyperinsulinemia and insulin resistance induced by high fat diet.⁴⁴ Targeting IRE1α improves insulin resistance and glucose intolerance in obese mice.³⁹ Besides, mice with β cell-specific deletion of IRE1α exhibited a diabetic phenotype, with less insulin secretion and elevated blood glucose level after feeding.⁴⁵ In addition, IRE1α is metabolically activated by glucagon in the liver, and glucagon or epinephrine could stimulate protein kinase A, which further phosphorylated IRE1α at Ser⁷²⁴.³⁵

2.4 The signaling pathways of IRE1α

Under resting conditions, IRE1α binds to Bip/GRP78 in the ER.²⁴ Upon stress, IRE1α disintegrates with Bip/GRP78 and is activated via autophosphorylation and dimerization/oligomerization, leading to RNase activation .¹³ In mammalian cells, the active RNase cleaves 26 nucleotides from the unspliced form of X-box binding protein-1 (XBP1u) mRNA and produces the spliced form of XBP1 (XBP1s) mRNA.⁴⁶ XBP1u contains a nuclear C-terminus called hydrophobic region 2 and a degradation domain, while the C-terminus of XBP1s has a transcriptional activation domain.⁴⁶ This difference makes XBP1s a crucial transcription factor, which could translocate into the nucleus and initiate transcriptional processes, thus participating in ER biogenesis, protein folding, and ER-associated degradation.²⁴ After being translocated to the nucleus, XBP1s binds to ER stress-responsive element I and promotes the transcription of mesencephalic astrocyte-derived neurotrophic factor (MANF).⁴⁷ Inactivation of MANF contributes to the upregulation of a pro-apoptotic component of the UPR, C/EBP-homologous protein (CHOP).⁴⁸ In addition, IRE1α cleaves a subset of ER-associated mRNAs, resulting in their degradation through an activity named regulated IRE1-dependent decay (RIDD) (Figure 2).

IRE1α is associated with well-known risk factors for vascular diseases, such as inflammation, autophagy, and apoptosis. IRE1α has been shown to trigger inflammation in vascular diseases. The dimerization of IRE1α stimulates its protein kinase domain, and induces the binding of tumor necrosis factor receptor associated factor 2 (TRAF2) to the IRE1α complex.⁴⁹ This thereby directly activates the apoptosis-signaling kinase 1 (ASK1) and IκB kinase (IKK) pathways, and subsequently activates the c-Jun NH2-terminal kinase (JNK) and nuclear factor-kappa B (NF-κB) pathways.^49-51 IKK is a crucial kinase that responds to inflammatory stimuli and can increase the activity of XBP1s.⁵² IRE1α can promote inflammatory cytokine generation through the NF-κB pathway.⁵³ IRE1α/NF-κB can activate the JNK-signaling pathway, and therefore induce the transcription of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-8.⁵⁴ IRE1α also regulates the expression of proinflammatory cytokine genes through the activation of XBP1s and glycogen synthase kinase (GSK)-3β.⁵⁵

IRE1α can promote both apoptosis and autophagy, which are essential processes that mediate vascular diseases.⁵⁶ IRE1α has been reported to be the main switch between these autophagy and apoptosis.⁵⁷ Some researchers believe that the real cell autophagic-signaling pathway induced by ER-stress involves IRE1α, not PERK or ATF6, as IRE1α is essential for the autophagosome formation and LC3-II conversion.^57-59 As we have mentioned above, upon ER-stress, IRE1α binds to TRAF2 and ASK1, and the formation of the IRE1α-TRAF2-ASK1 complex activates the JNK pathway, and mediates ER stress-induced cell autophagy.^{58, 60} Active JNK mediates Bcl-2 phosphorylation and disassociates the Beclin-1/Bcl-2 complex, thus releasing free Beclin-1.⁶¹ The IRE1α-JNK pathway also mediates autophagic cell death through the CHOP-signaling pathway.^{62, 63} The IRE1α/XBP1s pathway also regulates autophagy through Beclin-1.^{64, 65} Chromatin immunoprecipitation assays revealed that XBP1s can directly bind to the promoter of Beclin-1 at the region from nt −537 to −755.⁶⁴ XBP1s also triggers autophagy via the Forkhead Box O1 (FoxO1) pathway, while XBP1u serves as a negative regulator because it recruits FoxO1 to the 20S proteasome and promotes FoxO1 degradation.^{66, 67}

In the early stage of ER-stress, the phosphorylation of Bcl-2 mediated by JNK triggers autophagy, while sustained JNK activation induces apoptosis.⁶⁸ The IRE1α-TRAF2-JNK axis enhances pro-apoptotic Bax and Bak activity and represses anti-apoptotic Bcl-2 activity.^69-72 In addition, the TRAF2 not only facilitates the activation of caspase-3 and promotes apoptosis through the JNK pathway^{73, 74} but also directly activates caspase-12, which is specifically localized at the ER and can activate cleaved-caspase-3.^{57, 73} IRE1α can induce apoptosis through CHOP, and CHOP downregulates Bcl-2 and Mcl-1, and releases Bax to form pores in the mitochondrial outer membrane, which subsequently activates caspase3 and induces apoptosis.^{54, 73} In addition, IRE1α mediates ER stress-induced apoptosis through the RIDD pathway.⁷⁵

3.1 Atherosclerosis

Atherosclerosis is the most common vascular disease worldwide.^76-78 Chronic inflammation, imbalanced lipid metabolism, and an aberrant immune system response contribute to atherosclerosis through acting on vascular cells.^{79, 80} The ECs, macrophages, monocytes, and foam cells are involved in atherosclerotic lesion initiation.⁸¹ The risk factors for atherosclerosis, such as hyperhomocysteinaemia, oxidative stress, reactive nitrogen species, and free cholesterol accumulation in macrophages, contribute to IRE1α activation, which in turn promotes atherosclerosis.¹¹

Phosphorylation of IRE1α is observed in human atherosclerotic lesions but is not detected in normal arteries.⁸² Moreover, the expression of p-IRE1α was greater in the cores of advanced carotid atherosclerotic lesions than in those of stable aortic plaques, suggesting a key role for IRE1α in atherogenesis.⁸² This notion is also supported by the results from rodent studies. The phosphorylation of IRE1α is induced in the aortic intima and atherosclerotic lesions of ApoE^−/− mice, and is significantly greater in ApoE^−/− mice subjected to 5/6 nephrectomy than in controls.^{71, 83, 84} Furthermore, XBP1s, the downstream of IRE1α, was abundantly detected at branch points and in atherosclerotic lesions of ApoE^−/− mice and spontaneously hypertensive rats (SHRs) fed a cholesterol-enriched diet.^{65, 85, 86}

The initiation of atherosclerosis begins with the EC dysfunction; ECs facilitate the transport of low-density lipoprotein (LDL) into the subendothelial space, and the accumulation of LDL provokes an immune response.⁸⁷ In the atherosclerotic model of swine, the expression of both p-IRE1α and XBP1 was upregulated in the endothelium from the athero-susceptible arterial region compared with the athero-protected region.⁸⁸ In addition, overexpression of XBP1s induces atherosclerotic lesions in a thoracic aorta isograft model.⁸⁵ Numerous studies have reported that the activation of IRE1α in human umbilical vein ECs (HUVECs) and human aortic ECs (HAECs) is associated with risk factors for atherosclerosis, including LDL, homocysteine, and high glucose levels (Figure 3).^89-92 In addition, exposure to arsenite, an inducer of atherosclerosis, can activate IRE1α/XBP1s to promote hypoxia inducible factor 1α (HIF1α) accumulation.⁹³ The XBP1s/HIF1α complex further regulates the transcription of angiotensinogen, angiotensin-converting enzyme, and angiotensin II type 1 receptor, which play a role in endothelial dysfunction and inflammatory reactions.⁹³ Inhibition of the IRE1α/XBP1s/HIF1α pathway attenuated arsenite-induced oxidative stress and the proinflammatory response.⁹³

EC apoptosis plays a pivotal role in the initiation and progression of atherosclerosis. IRE1α participates in the apoptosis of ECs, which promotes atherosclerotic plaque formation and destabilization.⁹⁴ The IRE1α-JNK pathway plays a role in the apoptotic effect in oxidized LDL (oxLDL)-treated ECs, and inhibiting the IRE1α/JNK/caspase-3 pathway could protect HUVECs against apoptosis.^{74, 82} Sustained activation of XBP1s results in HUVEC apoptosis, endothelial denudation, and lesion progression through downregulation of the VE-cadherin and activation of multiple caspases.⁸⁵ The IRE1α-XBP1s pathway also regulates the autophagy of ECs in atherosclerosis and induces an autophagic response and death in ECs through the transcriptional activation of Beclin-1.⁶⁴ IRE1 also triggers inflammatory pathways. The IRE1α/NF-κB pathway results in lipopolysaccharide-induced HUVEC injury through the promotion of inflammatory cytokine production.⁵³ In addition, XBP1 is an essential mediator of inflammatory factors in ECs, including IL-6, IL-8, monocyte chemoattractant protein (MCP)-1, and Chemokine (CXC) motif ligand 3 (CXCL3).⁹⁵

Atherosclerosis tends to occur in arteries with bifurcations, which exhibit turbulent blood flow, where shear stress is low.^{81, 96} Low shear stress regulates inflammation in HAECs through the activation of XBP1, which together with interferon regulatory factor 1, upregulates the expression of the vascular cell adhesion molecule-1.⁹⁷ Disturbed flow leads to the disturbances in CLOCK expression and the activation of the IRE1α-XBP1 pathway, which results in endothelial-to-mesenchymal transition and inflammation, and ultimately vulnerable plaque progression.⁹⁸ In contrast, steady laminar flow can downregulate XBP1s through the phosphoinositide 3-kinase (PI3K)/Akt pathway.⁹⁹

The macrophages in advanced atherosclerotic lesions contribute to lesion necrosis, plaque rupture, and acute atherothrombotic vascular occlusion.⁷¹ Transcriptome analysis revealed that IRE1α regulates the expression of proatherogenic genes in primary mouse bone marrow-derived macrophages (BMDMs).¹⁰⁰ In the peritoneal macrophages of ApoE^−/− mice with hyperlipidemia, the phosphorylation of IRE1α is observed.¹⁰¹ In vitro, cholesterol can induce the expression of both p-IRE1α and XBP1 in macrophages.^{71, 72, 102} Stimulation of macrophages with angiotensin II also induces the phosphorylation of IRE1α in a dose- and time-dependent manner.⁸³ In addition, XBP1 splicing is enhanced in oxLDL-induced human leukemic monocytic THP-1 cells and the mouse macrophage-like cell line J774A1^103-105 (Figure 4).

The apoptosis of macrophages is one of the main consequences of prolonged IRE1α activation. Activated IRE1α in macrophages affects the TRAF2-ASK1-JNK pathway, and the subsequent apoptosis.^{71, 72} In addition, the activation of the IRE1α-XBP1-JNK axis leads to the apoptosis of foam cells.¹⁰⁶ As for XBP1s, Tian et al. revealed that transient overexpression of XBP1s in macrophages induces autophagy through the upregulation of Beclin-1, and enhances cell proliferation, while sustained overexpression of XBP1s induces apoptosis, thus decreasing cell proliferation.⁶⁵ In addition to apoptosis, many other pathways are involved in IRE1α-mediated atherosclerosis. Recently, Yildirim reported that IRE1 kinase activation induces fragile X mental retardation protein (FMRP) phosphorylation, and suppresses the macrophage cholesterol efflux and efferocytosis.¹⁰¹ IRE1α also plays a role in macrophage-derived foam cell formation by taking up more oxLDL, which is partly mediated by CD36.¹⁰⁷ In addition, IRE1α regulates the IL-1β secretion in lipid-stimulated mouse BMDMs and human peripheral blood monocytes.¹⁰⁰ In addition, XBP1s promotes macrophage-mediated inflammation through TNF-α and IL-8 production, and foam cell accumulation.¹⁰⁸

In addition to its role in ECs and macrophages, the IRE1α-JNK pathway is also involved in oxLDL-induced apoptosis in VSMCs.¹⁰⁹ In addition, XBP1 drives the plasma cell maturation and protective antibody responses.¹¹⁰ B-cell-specific XBP1 deficiency increases the apoptotic cell accumulation and antibody deposition in atherosclerotic plaques in Ldlr^−/− mice and increases the necrotic core area.¹¹⁰

3.2 Systemic hypertension and pulmonary arterial hypertension (PAH)

Currently, the role of IRE1α in hypertension remains largely unknown. In animal models, the protein levels of IRE1α, p-IRE1α, and XBP1 are elevated in SHR arteries.^111-113 The oxidation of IRE1α is partly regulated by nicotinamide adenine dinucleotide phosphate oxidase (Nox)-4, and may serve as a counterregulatory mechanism against oxidative stress in hypertension.¹¹¹ Moreover, the administration of an IRE1α inhibitor reduces the proliferation of VSMCs in SHRs.¹¹¹

PAH is a life-threatening disease characterized by increased pulmonary vascular resistance and vascular remodeling.¹¹⁴ In a rat model of PAH, the protein expression of IRE1α, p-IRE1α, and XBP1s was significantly greater in the lung tissues of rats with PAH than in those without PAH,^115-119 and knocking down XBP1s ameliorated PAH in rats.^{115, 116} Vascular remodeling is largely due to the proliferation and resistance of pulmonary artery smooth muscle cells (PASMCs) to apoptosis.¹¹⁶ The upregulation of p-IRE1α and XBP1s is also observed in PASMCs in PAH.¹²⁰ Recently, Jiang et al. reported that XBP1s promotes the proliferation, migration, and apoptotic resistance of PASMCs through the p-JNK/mitogen-activated protein kinase (MAPK) pathway.¹¹⁵ The overexpression of XBP1s also increases the expression of cell cycle-related proteins.¹²⁰

3.3 Aortic aneurysm and aortic dissection

Aneurysm is a life-threatening vascular disease that is characterized by a permanent aortic dilatation.¹²¹ A study analyzing the expression of ER stress markers in abdominal aorta samples from abdominal aortic aneurysm (AAA) patients (n = 96) and healthy controls (n = 17) revealed that IRE1α/XBP1s levels are significantly greater in patients with AAA than in healthy controls, and the differences remained statistically significant after adjusting for age, sex, smoking status, hypertension status, and diabetes status.¹²² Moreover, strong immunostaining for XBP1 is observed mainly in VSMCs in the medial layer and, to a lesser extent, in lymphocytes in the inflammatory infiltration area of the aneurysmatic wall.¹²² In animal models, the expression of XBP1s is increased in AAAs in angiotensin II-treated ApoE^−/− mice.¹²³ Interestingly, Zhao et al. reported that XBP1u, not XBP1s, is repressed in the early stage of aneurysm formation in angiotensin II-infused ApoE^−/− mice, which is correlated with VSMC dedifferentiation.¹²¹ XBP1u maintains VSMC homeostasis through FoxO4 interactions.¹²¹ In addition, XBP1u deficiency facilitates VSMC switching to a proinflammatory and proteolytic phenotype, and stimulates both thoracic and abdominal aortic aneurysm formation in mice.¹²¹ Recently, Chen et al. analyzed the functional enrichment of differentially expressed genes on the basis of Gene Expression Omnibus microarray datasets, and the results revealed that the expression of the IRE1α pathway is significantly upregulated in intracranial aneurysms and that the IRE1α pathway is highly related to FKBP14, Bax, and SEC61B expression.¹²⁴

Aortic dissection is a rare but catastrophic disease with an extremely high mortality rate.¹²⁵ The protein expression of p-IRE1α and XBP1s is significantly greater in aortic specimens from patients with aortic dissection than in those from patients without aortic dissection.^{125, 126} Moreover, the phosphorylation of IRE1α is also increased in animal models of aortic dissection and angiotensin II-treated rat aortic SMCs.¹²⁶ Angiotensin II can promote the nuclear translocation of XBP1s, further resulting in the gene transcription of CHOP, cleaved caspase 3, Bax, and Bcl-2.¹²⁵ In addition, Shi et al. revealed that the IRE1-XBP1-CHOP axis accelerates the apoptosis of VSMCs in the aortic media and promotes aortic dissection.¹²⁶

3.4 IRE1α is involved in the pathological process related to vascular diseases

Neointimal hyperplasia is a universal response to vascular injury and is the leading cause of vascular restenosis.¹²⁷ The known risk factors for neointimal hyperplasia include vascular injury, systemic inflammation, diabetes, and turbulent flow.¹²⁸ p-IRE1α and XBP1s are increased in rat carotid artery balloon-injured, mouse wire-injured, and disturbed flow-induced neointimal hyperplasia models.^129-131 In addition, the IRE1α/XBP1s axis is involved in increased endothelial-mesenchymal transition and subsequent carotid artery stenosis.⁹⁸ The proliferation and migration of VSMCs play essential roles in the progression of neointimal hyperplasia. In vitro, both p-IRE1α and XBP1s are upregulated in the platelet-derived growth factor (PDGF)-BB stimulated human and rodent VSMCs.^129-131 Inhibiting the IRE1α pathway reduces the hypertrophy and proliferation of VSMCs, which is partly mediated via inhibition of the NF-κB pathway.¹³² XBP1s increases the proliferation of VSMCs through the PI3K/Akt pathway and enhances the proliferation of VSMCs by suppressing calponin h1 and modulating the PDGF/transforming growth factor (TGF)-β pathway.¹³¹ Moreover, Angbohang et al. reported that XBP1s in VSMCs upregulates type IV collagen alpha 1/2 (COL4A1/2) transcription and induces soluble COL4A1 secretion, further recruiting stem cell antigen 1-positive-vascular progenitor cells (VPCs) and leading to neointimal formation.¹³³

Vascular calcification is considered as a pathological process associated with CVD, including atherosclerosis, hypertension, and coronary artery disease.¹³⁴ It has been well-documented that VSMCs play indispensable roles in the progression of vascular calcification.³ The phosphorylation of IRE1α and increased expression of XBP1s are observed in calcified VSMCs.^{46, 135} The expression of p-IRE1α and XBP1 is increased in bone morphogenetic protein-2 (BMP2)-induced human coronary artery smooth muscle cells (HCSMCs).¹³⁶ XBP1s is also upregulated in stearate-stimulated VSMC osteoblastic differentiation and mineralization.¹³⁷ XBP1s not only directly binds to the promoter of Runx2 in HCSMCs,¹³⁶ but also promotes the activation of β-catenin/T-cell factor, the key regulator of vascular calcification, and facilitates Runx2 and Msx2 transcription.⁴⁶ Overexpression of XBP1s accelerates high phosphate-induced VSMC calcification, and inhibition of XBP1s through IRE1α siRNA alleviates VSMC calcification²⁹. In addition, OxLDL promotes osteoblastic differentiation through the IRE1α/XBP1s/Runx2 pathway and activates inflammation through the IRE1α/JNK and NF-κB pathways.¹³⁸ The IRE1α/JNK axis is also activated in parathyroid hormone-induced HASMCs.¹³⁹ Recently, Yang et al. reported that XBP1u protects against calcification via an ER stress-independent signaling pathway.⁴⁶ The XBP1u level is lower in the radial arteries of chronic kidney disease patients with calcification than in those without calcification.⁴⁶ Smooth muscle-specific knockout of XBP1u aggravated 5/6 nephrectomy and adenine diet-induced vascular calcification.⁴⁶ Interactome analysis revealed that XBP1u binds directly to β-catenin, promotes its ubiquitin-proteasomal degradation, and inhibits Runx2 and Msx2 transcription.⁴⁶

3.5 IRE1α is involved in vascular diseases mediated by common risk factors

Age is considered as an important risk factor for vascular diseases. Bioinformatic analysis of transcriptome datasets revealed that the expression of XBP1 is upregulated in aged ApoE^−/− mice.¹⁴⁰ The phosphorylation of IRE1α is also observed in the aortas of aged mice and aged ApoE^−/− mice.^{140, 141} In addition, the protein levels of p-IRE1α and XBP1s are increased in aged HUVECs.^{141, 142} Compared with that in young fibroblasts, the IRE1α/XBP1s axis is also activated in aged fibroblasts.¹⁴³ In aging cells, activated Nox4 is involved in the dissociation of heat shock protein 90 from IRE1α.¹⁴² Additionally, IRE1α-mediated RIDD facilitates premature senescence through the degradation of Id1 mRNA.¹⁴⁴

Smoking is a leading cause of CVD. Smoking increases the risk of mostly CVD subtypes, and the risk increases with smoking intensity.¹⁴⁵ Cigarette smoke extracts significantly activate the IRE1α/XBP1s axis.^{146, 147} The phosphorylation of IRE1α is also induced in nicotine-induced human coronary artery ECs.¹⁴⁸

Previous studies have demonstrated that increased levels of circulating advanced glycation end products (AGEs) promote vascular diseases.^149-151 IRE1α/JNK have been reported to participate in AGE-induced cell apoptosis.^{152, 153} AGEs induce the phosphorylation of IRE1α and the JNK-signaling pathway in a time- and dose-dependent manner in HUVECs.¹⁵⁴ AGEs also directly induce XBP1 expression in HAECs and play a role in apoptosis, and further lead to endothelial dysfunction.¹⁴⁹ IRE1α knockdown inhibits AGE-induced NF-κB p65 nuclear translocation and the inflammatory response.^{154, 155} In addition, IRE1α knockdown suppresses Bax upregulation, Bcl-2 downregulation, and caspase-3 activation, thereby ameliorating apoptosis.¹⁵³