Critical Review Endoplasmic Reticulum Stress and Endothelial Dysfunction

Stefania Lenna Rong Han Maria Trojanowska*

Arthritis Center, Boston University School of Medicine, Boston, MA, USA

Abstract Prolonged perturbation of the endoplasmic reticulum (ER) leads to ER stress and unfolded protein response (UPR) and contributes to the pathogenesis of various chronic disorders. This review focuses on the role of ER stress and UPR in endothelial cells and the relevance of these processes to vascular diseases. Chronic activation of ER stress and UPR pathways in endothelial cells leads to increased oxidative stress and inflammation and often results in cell death. Because endothelial cells play a pivotal role in maintaining vascular homeostasis, various pathological conditions interfering with this

homeostasis including homocysteinemia, hyperlipidemia, high glucose, insulin resistance, disturbed blood flow, and oxidative stress can lead to endothelial dysfunction in part through the activation of ER stress. We discuss recently discovered aspects of the role of ER stress/UPR in those pathological conditions. We also summarize recent findings implicating ER stress and UPR in systemic hypertension as well as pulmonary arterial hypertension. Finally, this review will highlight a novel role of C 2014 IUBMB UPR mediators in the process of angiogenesis. V Life, 00(00):000–000, 2014

Keywords: cell death; complex diseases; JNK; reactive oxygen species; NF-kB/AP-1; oxidative stress

Introduction The endoplasmic reticulum (ER) plays essential roles in physiologic regulation of many cellular processes. Accumulating evidence indicates that perturbations of the normal functions of the ER trigger a signaling network that coordinates adaptive and/or apoptotic responses. Pathologic conditions that interfere with ER homeostasis can lead to chronic activation of the unfolded protein response (UPR) pathways, which contribute to the pathogenesis of many diseases. It has been established that prolonged ER stress is involved in the development and progression of a number of diseases including neurodegeneration, atherosclerosis, type 2 diabetes, liver disease, and cancer (1,2). Recent work has highlighted the significance of ER homeostasis in endothelial cell (EC) function.

C 2014 International Union of Biochemistry and Molecular Biology V

Volume 00, Number 00, Month 2014, Pages 00–00 Address correspondence to: Maria Trojanowska, Arthritis Center, Boston University, 72 East Concord St, Boston, Massachusetts 02118, USA. E-mail: [email protected] Received 11 June 2014; Accepted 8 July 2014 DOI 10.1002/iub.1292 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com)

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Despite the essential beneficial functions of the UPR during transient ER stress, pathologically chronic ER stress can lead to EC injury and subsequently to apoptosis and inflammation, resulting in endothelial impairment and disease (3). This review will focus on the mechanisms involving ER stress and the UPR in endothelial cells and the relevance of these processes to vascular diseases.

Endothelial Cell Dysfunction It is generally accepted that endothelial dysfunction plays an early pathogenic role in several diseases such as hypertension, coronary artery disease, chronic heart failure, peripheral artery disease, systemic sclerosis (scleroderma), diabetes, and chronic renal failure (4–6). Vascular endothelial cells not only provide a physical barrier between the vessel wall and lumen but also maintain vascular homeostasis and facilitate the passage of substances such as nutrients and leukocytes across the vessel wall. The endothelium secretes numerous mediators necessary for normal vascular functioning including those that regulate vascular tone and coagulation, modulate immune responses, and control vascular cell growth. Under physiological conditions, the endothelium maintains a fine balance between vasodilation and vasoconstrictions. The term endothelial dysfunction is most often used to denote impairment of

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endothelium-dependent vasodilatation in favor of vasoconstriction, including proinflammatory and prothrombotic effects. Nitric oxide (NO), prostacyclin, and thrombomodulin, which dominate under basal conditions, inhibit platelet aggregation, smooth muscle cell proliferation, and expression of leukocyteadhesion molecules. Part of the dysfunction is related to decreased endothelial NO synthase (eNOS) activity, reduced anticoagulant properties, increased adhesion molecule expression, chemokine and cytokine release, as well as reactive oxygen species (ROS) production by the endothelium. Various stimuli such as high glucose, insulin resistance, disturbed blood flow, and oxidative stress can lead to endothelial dysfunction in part through the activation of ER stress.

ER Stress and the Unfolded Protein Response ER has crucial roles in cell homeostasis and survival, including protein folding, lipid biosynthesis, and calcium and redox homeostasis. Under physiologic conditions, there is an equilibrium between the ER’s protein load and its folding capacity. Alterations in ER homeostasis due to increased protein synthesis, accumulation of misfolded proteins, or alterations in the calcium or redox balance of the ER lead to a condition called ER stress. Under such circumstances the cell responds by activating the UPR, which is aimed at restoring ER homeostasis as well as other cellular functions. The cell fate depends on the successful resolution of the ER stress, which, if left unresolved, leads to cell death (7,8). There are three UPR branches activated through the transmembrane sensor proteins: PERK (protein kinase RNA-like ER kinase), IRE1 (inositol requiring protein-1), and ATF6 (activating transcription factor-6). These three ER-stress sensors are usually maintained in their inactive forms through an interaction with the immunoglobulin heavy chain-binding protein (BiP/ GRP78). The accumulation of unfolded proteins in the ER leads to the release of BiP and activation of the UPR. PERK is activated through transautophosphorylation and oligomerization and in turn phosphorylates the eukaryotic translation initiation factor 2 alpha (eIF2a), resulting in global translational attenuation with the exception of a small number of proteins, including ATF4 (activating transcription factor-4). ATF4 regulates genes involved in restoring ER homeostasis; however, during prolonged ER stress the PERK-ATF4 pathway induces a proapoptotic mediator CHOP (C/EBPa-homologous protein, also known as GADD153) and its downstream target gene, DNA damageinducible protein-34 (GADD34) (9). IRE1a, the ubiquitously expressed isoform of IRE1, is a bifunctional kinase/ribonuclease activated by oligomerization and transautophosphorylation, which leads to splicing of the mRNA encoding X-box binding protein-1 (XBP1). The spliced isoform of XBP1 (XBP1s) functions as a transcription factor and inducer of many essential UPR genes leading to increased ER folding capacity and expansion of the ER membrane surface area. During prolonged

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stress, XBP1 cooperates with the ATF4-CHOP pathway in the induction of apoptosis (7). During ER stress, ATF6 translocates to the Golgi apparatus where it is cleaved by S1P and S2P proteases followed by translocation to the nucleus and activation of the ER stress response genes. ATF6 together with XBP1 is also involved in activation of the ER associated protein degradation (ERAD) system, whose role is to eliminate misfolded proteins through the ubiquitin-proteasome pathway (10). Recent evidence indicates that autophagy, which is primarily activated by the IRE1-mediated c-Jun N-terminal kinases (JNK) pathway in cooperation with PERK-ATF4, serves as another cytoprotective mechanism during severe ER stress (10). The intent of the UPR is to restore protein homeostasis. This adaptive mechanism involves suppression of protein translation, induction of ER-related molecular chaperones to promote refolding of unfolded proteins, elimination of unfolded proteins by activating the ERAD and autophagy mechanisms, and promoting cell survival. Excessive and prolonged ER stress is often associated with increased inflammation and ultimately triggers the switch from a prosurvival to a proapoptotic mode. The UPR can activate distinct ER stress-specific proapoptotic signaling cascades, which are integrated with the cellular apoptotic machinery. Under stress conditions, CHOP/GADD153 downregulates expression of the antiapoptotic factor BCL-2, while upregulating some proapoptotic members of the BCL-2 family, leading to mitochondrial membrane depolarization, cytochrome c release, and activation of caspases (9). Increasing evidence links the ER stress/UPR pathways with other major cellular networks, including inflammatory and stress signaling networks. For example, during ER stress, IRE1a and PERK-ATF4 trigger activation of JNK/AP1, a key inflammatory signaling pathway, which regulates many inflammatory genes. The UPR signaling pathways have also been shown to activate another crucial inflammatory mediator, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), through several independent mechanisms resulting in elevated levels of proinflammatory cytokines such as interleukin (IL) 8, IL-6, monocyte chemotactic protein 1 (MCP1), and tumor necrosis factor (TNF)-a (11). Furthermore, during ER stress the ER generates increased amounts of ROS coupled with decreased production of antioxidants due to the inhibition of protein translation by the PERK-ATF4 pathway. This is partially mitigated by ATF4 induction of the antioxidant pathways including glutathione as well as activation of the NFE2-related factor 2-mediated antioxidant pathway (10). The ER is also closely associated with mitochondria forming the ER-mitochondria unit via specialized protein complexes termed ER-associated mitochondria membranes (MAMs) (12). Dysfunctional mitochondria represent another significant source of ROS. MAMs are also the sites of formation and regulation of inflammasomes, which can be induced by ROS. Inflammasomes promote the maturation of IL-1b and IL-18, thus further contributing to the inflammatory response (13). Another important source of ROS during ER stress are NADPH oxidases, primarily Nox2 and Nox4 (14) (Fig. 1).

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FIG 1

ER stress triggers a signaling network that coordinates adaptive and/or apoptotic responses. In response to ER stress, BiP dissociates from the ER stress sensors, including PERK, IRE1, and ATF6, resulting in their activation. The intent of the UPR is to restore protein homeostasis. This adaptive mechanism involves suppression of protein translation, elimination of unfolded proteins through the ERAD and autophagy mechanisms, and returning to normal ER function. Excessive and prolonged ER stress is associated with increased inflammation and apoptosis contributing to the development and progression of diseases. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

ER Stress-Mediated Endothelial Dysfunction A number of studies in cultured endothelial cells and animal models provided insights into the molecular mechanisms linking induction of ER stress and the UPR to EC dysfunction. In this section, we will highlight some of the recent studies with relevance to vascular diseases that involve ER stress related alterations in EC function.

Homocysteinemia One of the well-studied ER stress inducers is homocysteine (HHc) (15). Elevation of plasma levels of HHc (hyperhomocysteinemia, HHcy) is considered an independent risk factor for cardiovascular diseases (16). Although the mechanisms of HHc-induced EC dysfunction are not fully understood, studies have shown that activation of JNK and ATF3 through the IRE1/TRAF2 pathway and subsequent upregulation of CHOP contribute to cell death of vascular ECs (17). Another proapoptotic mechanism associated with HHcy involves impairment of cell adhesion, which is mediated by eIF2a induction of the Tcell associated gene 51 (TDAG51) (18). Importantly, TDGA51 is elevated in endothelial cells and macrophages in vivo during

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the process of atherogenesis, and TDGA51 deficiency reduces the growth of atherosclerotic lesions in ApoE2/2 mice (19).

Hemodynamic Shear Stress The relationship between the disturbed blood flow and activation of the ER stress/UPR pathways has recently been reviewed by Davies et al. (20). The sites of disturbed blood flow are characterized by chronic partial activation of the ER stress and UPR pathways as evidenced by extensive in vivo gene expression studies. These sites are also prone to low-grade chronic inflammation. Corresponding protein analyses indicated that although the IRE1a and ATF6 pathways were increased, the PERK-ATF4 branch of the UPR was not activated. Thus, these UPR-related changes may indicate activation of protective mechanisms to mitigate the effects of disturbed flow. However, in the presence of additional stimuli, including hypertension, hypercholesterolemia, or diabetes, these sites are at risk to develop atherosclerotic lesions.

Hyperlipidemia Modified lipoproteins, in particular oxidized low density lipoproteins (OxLDL) are the key early contributors to the atherogenic process in part through the induction of endothelial cell apoptosis (21). Recent studies indicate that the ER stress/UPR pathways may contribute to this process through activation of

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the IRE1a-JNK axis (22). The involvement of this pathway was confirmed by the demonstration of phospho-IRE1a in lipid-rich areas of advanced atherosclerotic lesions (22). In a related study, Gora et al. observed activation of all three branches of the UPR, as well as IL-6 and IL-8, in human umbilical vein endothelial cells (HUVECs) treated with phospholipolyzed LDL (LDL-X). The proinflammatory LDL-X is a product of LDL hydrolysis by the human group X phospholipase A2, which is elevated in human atherosclerotic lesions (23). High lipid content in triglyceride-rich lipoproteins (TGRL) also correlates with increased risk for cardiovascular diseases. Interestingly, TGRL particles produced by hypertriglyceridemic subjects were shown to induce expression of vascular cell adhesion 1 molecule (VCAM-1) in human aortic endothelial cells, while TGRL particles from healthy subjects lowered VCAM-1 expression (24). Likewise, VCAM-1 upregulation by TNFa was further enhanced by proatherogenic TGRL but attenuated by antiatherogenic TGRL. Additional experiments demonstrated that proatherogenic TGRL activated the IRE1a-XBP1 and PERKeIF2a-CHOP pathways, as well as a transcriptional regulator of VCAM-1, interferon regulatory factor 1. Furthermore, small interfering RNA (siRNA)-mediated blockade of CHOP, but not IRE1a, ameliorated induction of VCAM-1 by TNFa (24). In contrast to LDLs, high-density lipoproteins (HDLs) have anti-inflammatory and antioxidant functions and can counterbalance the proinflammatory effects of oxLDLs. These beneficial effects of HDL include suppression of oxLDL-induced ER stress as well as autophagy and prevention of cell death induced by these pathways (25). Although the precise mechanism of the protective effect of HDL is not fully elucidated, the authors of the latter study suggest that HDL might prevent a rise of cytosolic Ca21 levels (25). ER stress accompanied by increased cytosolic Ca21 and ROS leading to impairment of sarcomplasmic/endoplasmic reticulum Ca21 ATPase function was also observed in response to oxidized and glycated LDL (26). Importantly, activation of AMP-activated protein kinase (AMPK) reversed these effects (26). A causal link between exacerbated ER stress and AMPK dysfunction was also confirmed in vivo using AMPKa2 knockout mice, suggesting that AMPK may play an important role in vivo by maintaining ER and Ca21 homeostasis (27). Metformin, an activator of AMPK, currently used to treat type 2 diabetes, was shown to inhibit ER stress and restore endothelial cell function in high fat dietinduced obese mice (28).

Hyperglycemia High glucose-induced ER stress has been closely linked to various aspects of endothelial cell dysfunction in patients with diabetes. Diabetes is associated with microvascular complications, including retinopathy and nephropathy as well as macrovascular manifestations such as ischemia and peripheral vascular disease (29). Notably, hyperglycemia also induces oxidative stress in parallel with ER stress, and both pathways are likely to contribute to excessive ROS production and downstream pathological effects (29,30). A number of studies implicated ER

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stress in endothelial cell inflammatory response and apoptosis in diabetic retinopathy (31). Recent work by Chen et al. (32) demonstrated that activation of STAT3, which is mediated by ATF4, is a key pathway responsible for inflammation and vascular leakage in the streptozotocin (STZ) induced model of type I diabetes. Conversely, Atf4 deficiency ameliorated these effects. Interestingly, complementary cell culture studies also showed that inhibition of STAT3 attenuated ER stress activation in response to high glucose, suggesting a reciprocal regulation of the ER stress and inflammatory pathways. Specific mechanisms involved in ATF4 activation of STAT3 as well as STAT3 contribution to the activation of ER stress pathways are not yet known. The mechanism of cardiac complications in the STZ model of diabetes was addressed in a recent study (33). The authors demonstrated that activation of the epidermal growth factor receptor tyrosine kinase (EGFR) contributed to ER stress induction and microvascular dysfunction, as well as cardiac fibrosis, in this model. Specifically, elevated EGFR phosphorylation was responsible for activation of the PERKeIF2a-ATF4 branch but had no effect on activation of ATF6. Blockade of EGFR’s kinase activity with a pharmacological inhibitor improved endothelium-dependent relaxation and decreased Nox2 and Nox4 in mesenteric resistance arteries. This interesting study implicates EGFR as a novel upstream regulator of ER stress in endothelial cells. ER stress has also been shown to contribute to type 2 diabetes. A recent study has investigated the role of ER stress in ischemia-induced neovascularization in type 2 diabetic db/db mice (34). The authors demonstrated that the ER stress inhibitor tauroursodeoxycholic acid (TUDCA), as well as the IL-1 inhibitor, Anakinra, had similar beneficial effects on hind-limb ischemia after femoral artery ligation. Both inhibitors reduced ATF4 and CHOP levels, improved blood flow recovery, and upregulated angiogenic factors. This study further underscores a close relationship between the inflammatory and the ER stress pathways in endothelial cell function. Interestingly, however, only TUDCA decreased body weight, blood glucose and insulin levels, suggesting that IL-1 is not involved in regulating obesity and insulin resistance.

The Role of ER Stress in Hypertension Hypertension Hypertension is a complex multifactorial disorder, characterized by chronic elevation of arterial blood pressure; however, despite significant research efforts, the pathogenesis of hypertension remains poorly understood. Recent studies suggest that ER stress and the UPR may play an important role in the development of this chronic disorder. Young et al. (35) were the first to implicate ER stress in the Ang-II induced hypertension model. Their study that focused on brain ER stress demonstrated that Ang-II upregulated the typical ER stress markers, including BiP/GRP78, PERK, and CHOP. Importantly, treatment with the chemical chaperone TUDCA or the local

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FIG 2

SSc-PAH lungs show upregulation of BiP and CHOP levels. Human lung specimens from healthy controls and SSc-PAH were analyzed by immunostaining to determine protein levels of BiP and CHOP. BiP and CHOP antibodies were obtained from Abcam (Cambridge, MA) and used at 1:100 and 1:150 dilutions, respectively. Representative staining is shown. Red arrows indicate endothelial cells and green arrows indicate macrophages. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

supplementation of the ER chaperone, BiP/GRP78, ameliorated ER stress and prevented Ang-II induced hypertension (35). Similar results were obtained in a study that investigated the effects of Ang-II on cardiac damage and vascular endothelial dysfunction (36). In addition, the investigators showed that inhibition of ER stress by TUDCA or 4-phenylbutyric acid (4PBA) reduced cardiac fibrosis and improved macrovascular endothelial function by reducing TGFa activity and by increasing eNOS phosphorylation (36). Similar beneficial effects of reducing ER stress were observed in hypertensive rats treated with TUDCA or 4-PBA (37). Mechanistically, it was shown that ER stress contributes to elevated COX-1 expression and prostanoid production via the enhanced phosphorylation of ERK1/2 and phospholipase A2. Suppression of ER stress improved endothelium-dependent contractile responses in part by inhibiting the production of vasoactive prostanoids (37).

Pulmonary Arterial Hypertension Pulmonary arterial hypertension (PAH) is a life-threatening condition characterized by a progressive increase in pulmonary vascular resistance that can eventually lead to right ventricular failure and death. The pathological process underpinning vascular remodeling in PAH is complex and involves interactions between different cell types within the vascular wall with further contribution from the immune and circulating progenitor cells. Accumulating evidence links dysregulated ROS and NO signaling to the pathogenesis of PAH. Consistent with a central role for oxidative stress in PAH, therapies targeting deficiencies in either NO (phosphodiesterase type 5

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inhibitors), prostaglandin I2 (prostacyclin analogs), or excessive endothelin-1 (ET-1) (ET receptor blocker) are routinely used for treatment of PAH (38). However, in contrast to other vascular diseases the role of the ER stress and UPR pathways in PAH has only recently been appreciated. Using two established models of PAH, hypoxia induced PAH in mice and monocrotaline induced PAH in rats, Dromparis et al. (39) found that 4-PBA prevented and also reversed pulmonary hypertension as measured by decreased pulmonary artery pressure and vascular remodeling, as well as reduced right ventricular (RV) hypertrophy. The authors observed activation of the ATF6 pathway in the vasculature in the hypoxia model in vivo and in cultured vascular smooth muscle cells, but, surprisingly, they did not observe activation of the CHOP pathway in their studies (39). However, in a subsequent report Koyama et al. (40) demonstrated activation of all three UPR branches in hypoxia-induced PAH in mice. Consistent with the previous study, administration of 4-PBA significantly attenuated right ventricular systolic pressure and prevented RV remodeling, which correlated with suppressed expression of the UPR markers, including ATF6, BiP, sXBP1, PDI, ATF4, and CHOP. Furthermore, 4-PBA treatment resulted in a reduced expression of proinflammatory markers such as MCP1, IL-6, and TNFa (40). Additional experiments using cultured pulmonary smooth muscle cells showed activation of UPR pathways by platelet-derived growth factor (PDGF)-BB treatment, suggesting that PDGF-BB may contribute to the activation of UPR pathways in PAH. While both studies explored the role of ER stress in pulmonary smooth muscle cells, the potential role of this pathway in activation of endothelial cells in PAH has not yet been addressed. To determine whether UPR pathways are activated in endothelial cells, we examined expression of the UPR markers in lung vasculature of patients with systemic sclerosis-associated PAH (SSc-PAH). We observed significantly elevated levels of ER stress markers, BiP and CHOP, in the vasculature and in macrophages of SSc-PAH lungs compared to healthy subjects, where only a few positively stained cells were observed in control lungs. Interestingly, elevated CHOP levels were mainly present in endothelial cells in SSc-PAH lungs (unpublished data) (Fig. 2). Similarly, elevated levels of ER stress markers were observed in a mouse model of SSc-PAH, Gata6-KO mice (41). Mice with conditional Gata6 deletion in endothelial cells (Gata6-KO) spontaneously develop moderately elevated pulmonary arterial pressure. After exposure to hypoxia, Gata6-KO mice display marked worsening in pulmonary pressure that is paralleled by extensive vascular remodeling and right ventricular hypertrophy. Analyses of lung tissues from control and Gata6-KO mice showed upregulation of BiP and CHOP in normoxic Gata6-KO mice, when compared to normoxic WT mice, whereas WT mice showed increased staining for BiP and CHOP after 1 month of hypoxia challenge (Fig. 3) (unpublished observations). Elevated levels of BiP and CHOP were also observed in sorted endothelial cells and macrophages obtained from hypoxic lungs (data not shown). Our observations are

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FIG 3

Upregulation of BiP and CHOP in Gata6-KO mice. Lung samples from Gata6-KO mice and control mice (WT) exposed to either normoxic or hypoxic conditions were analyzed by immunostaining to determine protein levels of BiP and CHOP. Representative staining is shown. Red arrows indicate endothelial cells and green arrows indicate macrophages. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

consistent with the results reported by Koyama et al. (40) and suggest that macrophages and endothelial cells may be primarily responsible for the elevation of ER stress/UPR in hypoxic mice. The relevance of ER stress to the pathogenesis of PAH is further supported by the findings of elevated expression levels of BiP and DNAJB1 as well as other ER stress/UPR markers in PBMCs isolated from patients with SSc-PAH. Furthermore, ER stress markers positively correlated with inflammation (IL-6) and severity of PAH (pulmonary arterial pressure) in those patients (42).

ER Stress and Angiogenesis Uncontrolled angiogenesis contributes to the pathogenesis of a number of diseases, including ischemia, cancer, PAH, and various inflammatory and fibrotic disorders. There is growing evidence that the ER stress and UPR pathways play an important role not only in regulating expression of proangiogenic factors, but also as novel mediators of the angiogenic process.

UPR Mediators Regulate Expression of Proangiogenic Factors Increasing evidence implicates the UPR pathways in upregulation of angiogenic factors such as vascular endothelial growth factor (VEGF)-A, FGF, IL-8, and ET-1 [reviewed in (43)]. Notably, it was shown that ER stress-mediated upregulation of VEGF-A was dependent on the IRE1a/XBP1, PERK/ATF4, as well as ATF6 pathways, but it was independent of a key inducer of VEGF-A, hypoxia-inducible factor (HIF)-1a (44). The regulation of VEGF-A expression by the UPR mediators was observed during normal development of labyrinthine trophoblast cells in the placenta, but also in cancer cells, suggesting the involvement of the UPR pathways beyond the conditions of ER stress (44). ET-1, a potent vasoconstrictor and a mitogen for endothelial cells, was also shown to be regulated by the ER

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stress pathways. Lenna et al. (45) reported that thapsigargin upregulated ET-1 expression in endothelial cells through increased formation of ATF4/c-Jun transcriptional complexes. Similarly, upregulation of ET-1 by a Toll-like receptor 3 ligand, poly(I:C), was mediated by the ATF4/c-Jun complexes (45). Endothelial cell-specific ER stress-dependent upregulation of ET-1 was also found in rat aortic rings; however, the ER stress pathway responsible for this effect was not investigated (46).

ER Stress/UPR Mediators as Novel Components of the Angiogenic Machinery Recent studies have uncovered an unexpected role of the UPRrelated molecules in the process of angiogenesis not related to the ER stress response. Dong et al. (47) investigated the contribution of BiP/GRP78 to tumor-induced angiogenesis. They found that heterozygous knockout of GRP78 in host endothelial cells resulted in severe reduction of tumor angiogenesis. Additional experiments in cultured endothelial cells demonstrated that knockdown of GRP78 resulted in reduced cell proliferation and migration (47). In line with this result, BiP/GRP78 overexpression was reported in the synovium of rheumatoid arthritis (RA) patients, where active angiogenesis occurs (48). Furthermore, BiP/GRP78 activation induced angiogenesis in an animal model of RA (48). GRP78 was linked to VEGF-induced angiogenesis in in vitro studies and Grp78 haploinsufficiency reduced the extent of angiogenesis in mice with antibodyinduced arthritis (48). A recent in-depth study on the role of the UPR mediators in angiogenesis revealed a critical role for ATF6 and PERK in VEGF-mediated signaling (49). In an elegant series of experiments, Karali and colleagues showed activation of all three UPR sensors, including IRE1a, ATF6, and PERK via the phospholipase C (PLC)c and mammalian target of rapamycin complex 1 (mTORC1) by a mechanism independent of dissociation of BiP. The activation of PERK and ATF6, but not IRE1a, was

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important for the VEGF-mediated antiapoptotic pathway that also involved mTORC2-dependent Ser473 phosphorylation of AKT. This physiological activation of UPR by VEGF did not lead to induction of proapototic CHOP, which is usually associated with prolonged ER stress, but instead ensured a prosurvival advantage by maintaining a high level of AKT phosphorylation (49).

Concluding Remarks Very active research during the past decade led to a better understanding of the role of ER stress and UPR in the pathogenesis of vascular diseases. While much still remains to be learned about cellular interactions of each of the UPR components and the specific pathways that are activated by the ER stress and UPR in different vascular beds and in association with different pathogenic triggers, there is a growing consensus that attenuating excessive ER stress may prove beneficial for the treatment of vascular disorders. FDA-approved chemical chaperones, 4-PBA and TUDCA, were shown to be effective in alleviating ER stress and mitigating disease symptoms in a number of animal models and also in small clinical trials (50). However, for the effective treatment of diverse vascular disorders more specific inhibitors will be needed. A better understanding of the components of the ER stress related pathways in different pathogenic condition would be critical to developing therapies for diverse vascular disorders.

Acknowledgements The authors thank Paul Haines for the critical proofreading of the manuscript. The work in the author’s laboratory is supported by the National Institutes of Health Grants P50 AR060780 and RO1 AR04334 (M.T.) and T32 AR007598 (R.H.).

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ER Stress and Endothelial Dysfunction

Endoplasmic reticulum stress and endothelial dysfunction.

Prolonged perturbation of the endoplasmic reticulum (ER) leads to ER stress and unfolded protein response (UPR) and contributes to the pathogenesis of...
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