**4. ER stress induced-cell death in the vascular wall**

substrates for producing energy and thiol repairing, so the regulation of the autophagic machi‐ nery may offer promising therapeutic opportunities to treat ischemia/reperfusion damage and heart hypertrophy [88,89]. Recently this interesting eventuality has been also demonstrated in experimental studies in genetic murine models, notably beclin 1(+/-) and Atg5 deficient mice,

In this scenario it is intriguing the proposed role of macrophages, able to remove apoptotic cell debris in the advanced atherosclerotic plaque by a mechanism called efferocytosis. It is well-known the active role of these cells in the inflammatory cascade inside the vascular wall, where they enter as adherent monocytes then become macrophages and foam cells, ac‐ cording to the progression of atherosclerosis [91]. The efferocytosis process seems necessary to limit atherosclerosis, because only a selective fully-operative efferocytosis retards the pro‐

The apolipoprotein E (ApoE) family comprises crucial lipoproteins present in the blood to transport the cholesterol and also to modulate several metabolic diseases like atherosclerosis and Alzheimer's [95]. The human ApoE gene is composed by different isoforms with different metabolic properties and the most studied are apoE3 that is protective and apoE4 that, in con‐ trast, accelerates atherosclerosis and coronary damage. A recent study demonstrated that peri‐ toneal macrophages isolated from ApoE4 mice were defective in the efferocytosis mechanism and if stimulated by inflammatory molecules, such as oxidized lipoproteins (ox-LDLs), were sensitive to apoptosis throughout the abnormal intensification of ER stress pathway [96]. How‐ ever the above condition was greatly ameliorated by chemical stimulation of ER signaling, that

Anyway if the UPR involvement in pathological complications has been largely outlined, it is important to remind that this signaling is commonly evoked during the heart morphogen‐

Really the strict association between the UPR signaling and pathology has been reported since about 20 years ago, in different pioneering papers [98,99] that discussed the relation‐ ship between dysfunctional ER and proteotoxicity, and its direct role in neurodegenerative conditions characterized by abnormal protein deposition, like Parkinson's and Alzheimer's

Seminal studies have then elucidated the crucial role of the disruption of the regular ER ac‐ tivity in several metabolic disorders like obesity, diabetes insipidus up to neurodegenerative diseases like Creuzfeld-Jacob, Hungtington's, Parkinson's [9,100]. Moreover this mechanism has been actually involved in the pathogenesis of chronic disorders, including cancer, liver

Interestingly ER sensing may contribute to atherogenic damage by four ways: 1) by connect‐ ing lipid metabolism and UPR; 2) by promoting abnormal glucose metabolism and insulin activation that serve as a bridge-mechanism between metabolic dysfunctions and atheroscle‐ rosis; 3) by driving macrophages cell death after cholesterol loading; 4) by controlling auto‐

immunity based on the processing and presentation of MHC-1 associated peptides.

even if its application in clinical trials is still an hypothesis [90].

reduced inflammation linked to apoE4, and balanced ER stress response.

diseases, heart failure and in particular atherosclerosis [101,102].

gression of this inflammatory disease [92-94].

34 Current Trends in Atherogenesis

esis and in healthy physiological conditions [97].

diseases.

A growing body of evidence indicates that prolonged ER stress, due to the persistent accu‐ mulation in the ER of misfolded proteins beyond the ability of transient UPR, causes cell death in the vascular wall and may contribute to the pathogenesis of atherosclerosis and other cardiovascular disorders, such as cardiac hypertrophy, and acute coronary syndrome [78,105-107].

Intriguingly, the three arms of UPR act together to resolve the prolonged ER stress but if they fail to reduce the amount of unfolded or misfolded proteins, an ER-driven pro-apoptot‐ ic signal starts.

The most common apoptosis-triggering molecule associated to UPR signaling is called C/EBP homologous protein, or CHOP, known also as GADD153 [108].

In the endothelial cells, different atherosclerotic-relevant UPR inducers have been identified in many *in vitro* and *in vivo* studies. In particular, the strict association between ER stress marker GRP78 and CHOP has been reported in patients and in coronary artery samples, mainly in thin-wall or ruptured plaques associated with unstable angina respect to stable plaques. Evident localization of two ER-stress signals was further correlated to mRNA ex‐ pression by in situ hybridization in thin-walled plaques and results indicated a positive rela‐ tionship between these markers and plaque vulnerability in human coronary arteries [109].

Among murine models, many studies have been performed in apolipoprotein E deficient mice (ApoE-/-), fed a standard chow diet that developed atherosclerosis during the life-span up to ne‐ crotic plaques [110]. In this murine model, ER stress markers such as GRP78 and CHOP are upregulated in macrophages at all stages of lesion development in the aortic root [111]. However it is important to remark that in the aorta of ApoE-/- mice at 9 weeks of age, corresponding to early atherosclerotic phase, no apoptosis was detected, but this event occurred in macrophages and foam cells in advanced lesions at 23 weeks of age. Remarkably strong GRP78-immunos‐ taining was also localized in the fibrous cap surface in hyperhomocysteinemic ApoE-/- [112]. Furthermore in transgenic CHOP-deficient mice less macrophages have been found in ad‐ vanced atherogenic lesions, such as instable plaques, respect to wild-type mice.

Intriguingly in double knockout mice (CHOP and ApoE-deficient) the rupture of athero‐ sclerotic plaques was significantly reduced despite their high-cholesterol diet [113]. Indeed also in primary cultured macrophages free cholesterol accumulated in the ER and stimulat‐ ed apoptosis in a CHOP-dependent pathway, so CHOP probably contributed *in vivo* and *in vitro* to instability of plaques due to macrophage cell death.

It is emerging that, in crucial artery wall sites, the IRE1 branch of the canonical UPR mecha‐ nism and its downstream CHOP signaling are activated also by various factors like distur‐ bed blood flow and hypertension [19,114,115] and modified LDL.

In particular during this advanced step, it has been reported that increased apoptotic endo‐ thelial cells may act as a pro-coagulant and favor the increase of platelet adhesion during the

Endoplasmic Reticulum Stress in the Endothelium: A Contribution to Athero-Susceptibility

http://dx.doi.org/10.5772/53024

37

In human coronary arteries plaque vulnerability is associated to the expression of ER stress

Unlike clinical patients, murine animal model of atherosclerosis are unsuited for studying plaque disruption or acute thrombosis, so they are currently studied to characterize early

Recently in CHOP-deficient mice mated with ApoE-/- atherosclerotic mice, it has been dem‐ onstrated a direct causal link between reduced CHOP-induced apoptosis and plaque ne‐ crosis [122]. In this double transgenic model, ER stress has different impacts on the vascular damage, according to the lesion stage of the artery. Indeed it is possible that in an early athe‐ rogenic phase, the UPR mechanism may be protective in macrophages and smooth muscle cells, but after persistent damage, the UPR-induced apoptosis is associated to plaque vulner‐

Besides the endothelium, also smooth muscle cells in artery wall can be susceptible to ER stress-UPR and compromise plaque integrity by reducing the protective fibrous cap in ad‐

Moreover atherogenic stressors like cholesterol and homocysteine are able to up-regulate CHOP and apoptosis in smooth muscle cells *in vitro* [124]. Indeed in human aortic cells the delivery of 7-ketocholesterol, an oxysterol linked in patients to high cardiovascular risk and

However unfortunately clear molecular evidences of pathways linking UPR to smooth mus‐ cle cells in atherosclerosis are still lacking. This is not true for macrophages, and the role of

Remarkably in atherosclerosis dual impact of macrophages resistance to apoptosis has been related to different stages of the disease: it may be beneficial in early lesions, where they hin‐ der inflammation, but is detrimental in advanced phases, where they contribute to a signifi‐ cant increase in the lesion size associated to elevated chemokines expression and monocytes

Furthermore it is important to point out that if inflammatory foam cells in the sub-endotheli‐ um space are cleared by active macrophages to prevent further secondary necrosis, in paral‐ lel many inflammatory pathways are activate to potentiate atherogenic damage, including nuclear factor k-B (NFkB) and mitogen-activated protein kinase (MAPK), in particular p38-

As commonly accepted, the chronic activation of the three canonical UPR pathways in the ER, triggers different pro-apoptotic mechanisms in the vascular wall, that may be mitochon‐

dria-dependent or independent, but largely complementary and integrated [128].

proteins like CHOP and macrophage apoptosis [119,120].

atherosclerosis, activated UPR pathway up to apoptosis [125].

UPR in macrophages apoptosis is an emerging field of investigation [91].

atherosclerotic phases up to necrotic plaque [121].

plaque erosion [118].

ability and rupture.

recruitment [126].

MAPK cascade [127].

vanced atherosclerosis [123].

High level of XBP1 splicing was detected in atherosclerosis prone areas, and in a mouse iso‐ graft model mimicking XBP1 overexpression, peculiar signs of atherogenic damage have been detected, like neointima formation and monocytes infiltration [116].

In particular in the endothelium multiple UPR pathways are activated by phospholipolized LDL that stimulate ER stress associated to cytoskeleton stress fibers formation, inflammation and dysregulation of calcium homeostasis, even if strictly related to the intensity and dura‐ tion of the lipidic stress [117] (Figure 3)

**Figure 3.** Multiple signaling triggered by lipoproteins in endothelial cells. ATF- activating transcription factors 2, 3, 4, 6; LDL- low density lipoproteins; CHOP- C/EBP homologous protein; XBP1- x-box binding protein 1; eIF2alpha- initia‐ tion factor 2 alpha; MAPK- mitogen activated protein kinase. Adapted by [117].

However not only in the initial pro-atherogenic phase but also in advanced phase, associat‐ ed to the plaque rupture, it is crucial that the endothelium maintains its integrity, so ham‐ pering the diffusion in the blood of the circulating plaque.

In particular during this advanced step, it has been reported that increased apoptotic endo‐ thelial cells may act as a pro-coagulant and favor the increase of platelet adhesion during the plaque erosion [118].

It is emerging that, in crucial artery wall sites, the IRE1 branch of the canonical UPR mecha‐ nism and its downstream CHOP signaling are activated also by various factors like distur‐

High level of XBP1 splicing was detected in atherosclerosis prone areas, and in a mouse iso‐ graft model mimicking XBP1 overexpression, peculiar signs of atherogenic damage have

In particular in the endothelium multiple UPR pathways are activated by phospholipolized LDL that stimulate ER stress associated to cytoskeleton stress fibers formation, inflammation and dysregulation of calcium homeostasis, even if strictly related to the intensity and dura‐

**Figure 3.** Multiple signaling triggered by lipoproteins in endothelial cells. ATF- activating transcription factors 2, 3, 4, 6; LDL- low density lipoproteins; CHOP- C/EBP homologous protein; XBP1- x-box binding protein 1; eIF2alpha- initia‐

However not only in the initial pro-atherogenic phase but also in advanced phase, associat‐ ed to the plaque rupture, it is crucial that the endothelium maintains its integrity, so ham‐

tion factor 2 alpha; MAPK- mitogen activated protein kinase. Adapted by [117].

pering the diffusion in the blood of the circulating plaque.

bed blood flow and hypertension [19,114,115] and modified LDL.

tion of the lipidic stress [117] (Figure 3)

36 Current Trends in Atherogenesis

been detected, like neointima formation and monocytes infiltration [116].

In human coronary arteries plaque vulnerability is associated to the expression of ER stress proteins like CHOP and macrophage apoptosis [119,120].

Unlike clinical patients, murine animal model of atherosclerosis are unsuited for studying plaque disruption or acute thrombosis, so they are currently studied to characterize early atherosclerotic phases up to necrotic plaque [121].

Recently in CHOP-deficient mice mated with ApoE-/- atherosclerotic mice, it has been dem‐ onstrated a direct causal link between reduced CHOP-induced apoptosis and plaque ne‐ crosis [122]. In this double transgenic model, ER stress has different impacts on the vascular damage, according to the lesion stage of the artery. Indeed it is possible that in an early athe‐ rogenic phase, the UPR mechanism may be protective in macrophages and smooth muscle cells, but after persistent damage, the UPR-induced apoptosis is associated to plaque vulner‐ ability and rupture.

Besides the endothelium, also smooth muscle cells in artery wall can be susceptible to ER stress-UPR and compromise plaque integrity by reducing the protective fibrous cap in ad‐ vanced atherosclerosis [123].

Moreover atherogenic stressors like cholesterol and homocysteine are able to up-regulate CHOP and apoptosis in smooth muscle cells *in vitro* [124]. Indeed in human aortic cells the delivery of 7-ketocholesterol, an oxysterol linked in patients to high cardiovascular risk and atherosclerosis, activated UPR pathway up to apoptosis [125].

However unfortunately clear molecular evidences of pathways linking UPR to smooth mus‐ cle cells in atherosclerosis are still lacking. This is not true for macrophages, and the role of UPR in macrophages apoptosis is an emerging field of investigation [91].

Remarkably in atherosclerosis dual impact of macrophages resistance to apoptosis has been related to different stages of the disease: it may be beneficial in early lesions, where they hin‐ der inflammation, but is detrimental in advanced phases, where they contribute to a signifi‐ cant increase in the lesion size associated to elevated chemokines expression and monocytes recruitment [126].

Furthermore it is important to point out that if inflammatory foam cells in the sub-endotheli‐ um space are cleared by active macrophages to prevent further secondary necrosis, in paral‐ lel many inflammatory pathways are activate to potentiate atherogenic damage, including nuclear factor k-B (NFkB) and mitogen-activated protein kinase (MAPK), in particular p38- MAPK cascade [127].

As commonly accepted, the chronic activation of the three canonical UPR pathways in the ER, triggers different pro-apoptotic mechanisms in the vascular wall, that may be mitochon‐ dria-dependent or independent, but largely complementary and integrated [128].

The most common death-sensors activated in the mitochondria are: 1) the stimulation of ino‐ sitol requiring protein-1 (IRE1) that can further regulate B-cell lymphoma-2 (BCL-2) family of proteins and 2) PERK and ATF6 signals that directly induce CHOP/GADD153 protein. Remarkably, CHOP is also involved in the activation of a mitochondria-independent mecha‐ nism of apoptosis that relies on inositol-1,4,5-triphosphate receptor (IP3R), able to trigger ab‐ normal calcium (Ca2+) flux from the ER and the death receptor Fas [129].

bosis, it may be due to an inability to resist to PERK and eIF2alpha signaling and to reduce

Endoplasmic Reticulum Stress in the Endothelium: A Contribution to Athero-Susceptibility

http://dx.doi.org/10.5772/53024

39

In Figure 4 we resumed complex relationships between ER signaling and apoptosis in athe‐

**Figure 4.** Different ER signals leading to successful apoptosis. IP3R- inositol -1,4, 5,-triphosphate receptor; PERK- pro‐ tein kinase-like ER kinase; ATF6- activating transcription factor 6; IRE1- inositol requiring protein1; BCL2- B cell lympho‐ ma/leukemia 2; Bak- Bcl2-homologous antagonist; Bax- Bcl2-associated x protein; RIDD- regulated IRE1-dependent decay; CaMK II- calcium/calmodulin-dependent protein kinase; FAS- tumor necrosis factor receptor superfamily mem‐

In metabolic diseases such as atherosclerosis, hypertension, diabetes and related cardiovas‐ cular complications, improved understanding of ER stress pathways and their relationship with inflammation and apoptosis represents the basis on which to try novel drugs, to test

**5. ER stress as a therapeutic target in atherosclerosis and metabolic**

therapeutic interventions and to identify targets for different therapeutic options.

ber 6; CHOP- C/EBP homologous protein; BH3- homology domain. Adapted by [107].

downstream ATF4-CHOP associated apoptosis *in vivo* as recently hypothesized [136].

rogenesis.

**diseases**

Although three branches may be activated by any prolonged stressful event, the timing of each pathway can differ and persistent ER stress leads to sequential progression of IRE1, then ATF6, finally PERK respectively. Moreover it is important to outline that each proapoptotic mechanism is strictly cell-type and stimulus-specific.

IRE 1 isoforms are activated by auto-phosphorylation and trigger the splicing and transla‐ tion of mRNA transcript for a specific transcription factor, called XBP1s, that induces chap‐ erones and other molecules able to limit ER stress.

However in mammalian cells, IRE1 stimulates also another mechanism known as regulated IRE1 dependent decay (RIDD) [130], that may directly lead to apoptosis even if this branch is still controversial in cardiovascular diseases.

Nevertheless the major downstream effector of IRE1 signaling is the BCL-2 family of pro‐ teins, that includes both anti-apoptotic and pro-apoptotic members able to regulate the ac‐ tivity of ER and mitochondria [131].

In human and mice anti-apoptotic domains are called Bcl-2 and Bcl-XL, while the most well characterized pro-apoptotic are Bcl2-associated x protein (BAX) and Bcl2-homologous an‐ tagonist (BAK) proteins. When these last two members become activated in the mitochon‐ dria, release cytochrome c and other death factors that may amplify the caspases cascade up to overt cell death. Despite *in vitro* observations on IRE1 signaling, actually there is not yet *in vivo* evidence for apoptosis along this pathway [132].

Remarkably CHOP signaling is common also to PERK and ATF6 pathways in the ER stress response, where it may act like in the IRE1, even if there is the possibility to by-pass the mi‐ tochondria and to stimulate calcium flux, working on the ER calcium channel called inosi‐ tol-1, 4, 5-triphosphate or IP3R [133].

Many recent studies point to the apoptotic mechanism driven by calcium release from the ER lumen, able to stimulate the calcium-sensing enzyme called calcium/calmodulin-depend‐ ent protein kinase, CaMK II, which in turn regulates other apoptotic pathways, like FAS ac‐ tivation but also caspase 12 [134,135].

In advanced atherosclerosis, the level of ER stress-CHOP expression in macrophages is very high despite the presence of TRLs ligands and the activation of TRIF-signaling. A crucial concept in the regulation of macrophage apoptosis in atherosclerosis is called "the two-hit concept", that consists in the eventuality of a milder ER stress *in vivo* respect to in *vitro*. So different cumulative sub-apoptotic stimuli may lead to a synergic more effective response in the artery vessel, and in particular because generally TLRs act as a second pro-apoptotic stimuli. If this eventuality is lost as evident in advanced ruptured plaque and related throm‐ bosis, it may be due to an inability to resist to PERK and eIF2alpha signaling and to reduce downstream ATF4-CHOP associated apoptosis *in vivo* as recently hypothesized [136].

The most common death-sensors activated in the mitochondria are: 1) the stimulation of ino‐ sitol requiring protein-1 (IRE1) that can further regulate B-cell lymphoma-2 (BCL-2) family of proteins and 2) PERK and ATF6 signals that directly induce CHOP/GADD153 protein. Remarkably, CHOP is also involved in the activation of a mitochondria-independent mecha‐ nism of apoptosis that relies on inositol-1,4,5-triphosphate receptor (IP3R), able to trigger ab‐

Although three branches may be activated by any prolonged stressful event, the timing of each pathway can differ and persistent ER stress leads to sequential progression of IRE1, then ATF6, finally PERK respectively. Moreover it is important to outline that each pro-

IRE 1 isoforms are activated by auto-phosphorylation and trigger the splicing and transla‐ tion of mRNA transcript for a specific transcription factor, called XBP1s, that induces chap‐

However in mammalian cells, IRE1 stimulates also another mechanism known as regulated IRE1 dependent decay (RIDD) [130], that may directly lead to apoptosis even if this branch

Nevertheless the major downstream effector of IRE1 signaling is the BCL-2 family of pro‐ teins, that includes both anti-apoptotic and pro-apoptotic members able to regulate the ac‐

In human and mice anti-apoptotic domains are called Bcl-2 and Bcl-XL, while the most well characterized pro-apoptotic are Bcl2-associated x protein (BAX) and Bcl2-homologous an‐ tagonist (BAK) proteins. When these last two members become activated in the mitochon‐ dria, release cytochrome c and other death factors that may amplify the caspases cascade up to overt cell death. Despite *in vitro* observations on IRE1 signaling, actually there is not yet *in*

Remarkably CHOP signaling is common also to PERK and ATF6 pathways in the ER stress response, where it may act like in the IRE1, even if there is the possibility to by-pass the mi‐ tochondria and to stimulate calcium flux, working on the ER calcium channel called inosi‐

Many recent studies point to the apoptotic mechanism driven by calcium release from the ER lumen, able to stimulate the calcium-sensing enzyme called calcium/calmodulin-depend‐ ent protein kinase, CaMK II, which in turn regulates other apoptotic pathways, like FAS ac‐

In advanced atherosclerosis, the level of ER stress-CHOP expression in macrophages is very high despite the presence of TRLs ligands and the activation of TRIF-signaling. A crucial concept in the regulation of macrophage apoptosis in atherosclerosis is called "the two-hit concept", that consists in the eventuality of a milder ER stress *in vivo* respect to in *vitro*. So different cumulative sub-apoptotic stimuli may lead to a synergic more effective response in the artery vessel, and in particular because generally TLRs act as a second pro-apoptotic stimuli. If this eventuality is lost as evident in advanced ruptured plaque and related throm‐

normal calcium (Ca2+) flux from the ER and the death receptor Fas [129].

apoptotic mechanism is strictly cell-type and stimulus-specific.

erones and other molecules able to limit ER stress.

is still controversial in cardiovascular diseases.

*vivo* evidence for apoptosis along this pathway [132].

tivity of ER and mitochondria [131].

38 Current Trends in Atherogenesis

tol-1, 4, 5-triphosphate or IP3R [133].

tivation but also caspase 12 [134,135].

In Figure 4 we resumed complex relationships between ER signaling and apoptosis in athe‐ rogenesis.

**Figure 4.** Different ER signals leading to successful apoptosis. IP3R- inositol -1,4, 5,-triphosphate receptor; PERK- pro‐ tein kinase-like ER kinase; ATF6- activating transcription factor 6; IRE1- inositol requiring protein1; BCL2- B cell lympho‐ ma/leukemia 2; Bak- Bcl2-homologous antagonist; Bax- Bcl2-associated x protein; RIDD- regulated IRE1-dependent decay; CaMK II- calcium/calmodulin-dependent protein kinase; FAS- tumor necrosis factor receptor superfamily mem‐ ber 6; CHOP- C/EBP homologous protein; BH3- homology domain. Adapted by [107].
