**3. ER stress and UPR pathways in cardiovascular diseases**

If one of these activities fails, the ER efficiency is lost and aberrant unfolded proteins accu‐ mulate within the ER membranes, causing the "ER stress". This condition has been defined as " any perturbation that compromises the protein folding functionality" in the organelle

So it has become clear that each perturbation in the ER balance interferes with folding process of different proteins, that are devoid of their intrinsic function, so unable to properly work in the cells and often degraded by a process called ER associated degradation (ERAD) [56-58].

In mammalian cells, disrupted ER homeostasis can be restored within short or long time ac‐

It is accepted that endothelial cells may tolerate acute stressors that last short time, such as circulatory ischemia or hypoxia, calcium and nutrient deprivation, adapting themselves to clear dysfunctional proteins. In doing this activity, they use a rapid process that involves a transient intracellular signaling from the ER to the nuclear transcription mechanism of genes, called "unfolded protein response" or UPR [59-62]. Indeed UPR is able to rectify and limit the cellular damage induced by metabolic, genetic, environmental factors, enhancing cell survival, but strictly related to the duration of the stress. On the contrary, if the stressful stimuli are severe or last for a long time, like the majority of chronic inflammatory and he‐ modynamic factors in atherogenesis, UPR is unable to resolve persistent ER stress so leading

**Figure 1.** ER stress balance – Schematic representation of ER homeostasis: on the left, adaptive responses to acute stress that lead to recovery and on the right, reactions to chronic vascular stress that lead to apoptosis. NF-kB-Nuclear Factor k-B; ERAD-ER-associated degradation; CHOP- C/EBP homologous protein; JNK- c JUN NH2-terminal kinase.

and implies an adaptive response to restore correct ER homeostasis [54,55].

30 Current Trends in Atherogenesis

cording to the type of stimulus, if acute and transient or chronic and prolonged.

to endothelial cell death, generally by apoptosis (Figure 1).

Adapted by [63].

Given the vital role of fundamental UPR to augment the protein folding in the ER and to reduce the pool of misfolded products, it is clear that this organelle represents an efficient checkpoint for quality control of secretory proteins that may migrate to other organelles and/or to the plasma membrane to be secreted. Indeed UPR works also in collaboration with the Golgi apparatus and plasma membrane, and only correctly-assembled molecules are driven to their final destination. Therefore the kinetic and the amplitude of UPR are emerg‐ ing as key events for combine a stress response in specific cell types to their final fate and eventually death [64].

In the heart, for example, the UPR pathway produces several proteins, that ameliorate the ER ability to cope with stress, by three separate mechanisms: 1) translational attenuation, that avoids further deposition of abnormal proteins in the ER; 2) transcriptional activation of genes for chaperones and related proteins [65]; 3) activation of a process to hamper the fur‐ ther deposition of dysfunctional proteins called ERAD [11].

Indeed to start the quality control work in the ER factory, it is crucial that about one-third of novel proteins are translocated there, because they acquire the specific configuration and as‐ sembling with the assistance of ER chaperones, then further change by post-translational modifications, like disulphide bonds or glycosylation performed by specialized enzymes [66,67].

Remarkably unlike the cytosol, where the abnormal accumulation of proteins is handled by different families of chaperones, belonging to heat shock protein (HSP) 20 and HSP70 fami‐ lies, called HSP25/27 and HSP70 [68,69], in the ER environment the UPR mechanism is sus‐ tained by specific resident chaperones, glucose-regulated protein (GRP) GRP78 /Bip, GRP94 and by lectin chaperones calnexin, calreticulin and calmegin [70,71].

In eukaryotic cells GRP78, a trans-membrane protein, is called "the master regulator" of ER stress response and usually works by binding to nascent polypeptides to ensure their proper secondary structure. In unstressed conditions, GRP78 is usually associated to three different UPR-sensors and renders them inactive through the direct interaction with their N-terminus [72]. In contrast, when unfolded proteins accumulate in the ER, GRP78 dissociates from three UPR-sensing elements, and allows their oligomerization and activation, so ensuring the start of the UPR cascade.

Currently, it is established that GRP78 is induced by chemical and inflammatory atherogen‐ ic factors, further associated to ER stress signaling, such as excess cholesterol, oxidized phos‐ pholipids, peroxynitrite, homocysteine [73,74]. In a recent *in vitro* model, that simulates human arterial shear stress waveforms, GRP78 was over-expressed in the endothelial cells as a compensatory effect before lesion development [75]. The mechanisms by which GRP78 increased were dependent on upstream alpha 2-beta1integrin linked to p38 activity localized to focal adhesion in the endothelial cells upon long-term shear stress [76].

Remarkably in the above study it was further demonstrated that inflammatory cytokines as‐ sociated to atheroprone environment, had no effect on GRP78 expression in the endothelial cells. So it is plausible that hemodynamic flow might be the earliest ER stressor and GRP78 inducer in an atheroprone environment.

ones and also for enzymes involved in the ER-associated protein degradation pathway (ERAD). The ERAD mechanism mediates the translocation of unfolded proteins from the ER into the cytosol where they are degraded by the ubiquitin-proteasome system and so allevi‐

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

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

33

Interestingly, a novel gene, called derlin-3, as a component of the ERAD induced by ATF6, was recently discovered in the mouse heart, and derlin-3 over-expression was able to protect

Besides ER chaperones involvement, dysfunctional proteins may be degraded directly in the ER in a chaperone-independent manner, by a specialized protease system called the ubiqui‐ tin-proteasome, that works independently or in synergy with the UPR. According to recent clinical and experimental studies, atherosclerosis may be considered also as a "protein-qual‐ ity disease" and the proteasome works at early phases of the disease, especially in both the coronary and carotid arteries, as a compensatory reaction to prevent complete protein dys‐

It is currently accepted that in mammalians the necessity to remove aberrant proteins that engulfed the ER environment is based upon three main ER activities: 1) the transient UPR associated to resident chaperones, 2) the ubiquitin-proteasome system and 3) the prolonged

In particular through this last process, properly called macro-autophagy, abnormal cyto‐ plasmic contents or organelles engulfed in autophagosomes, upon fusion with lysosomes,

Evidence is emerging that the ER provides membrane for autophagosome formation and that autophagy is crucial for ER homeostasis due to its ability to remove unwanted or dam‐ aged organelles like abnormal mitochondria by mitophagy [83]. Moreover the ER contrib‐ utes also lipids and specific proteins, such as beclin-1, to initiate autophagosome formation very close to itself. This physical proximity probably reflects a functional dependence be‐ tween ER and autophagy process, that in the endothelial cells often occurs in response to reactive oxygen species (ROS) by circuits localized to the ER surface [84]. However in divid‐ ing cells with high turnover, autophagy may not be so relevant, but in long-lived cells like smooth muscle cells and cardiomyocytes, it is critical to maintain optimal cellular function. Remarkably autophagy is a suitable mechanism to eliminate abnormal proteins and organ‐ elles, during fast and relatively mild ER stress conditions, but if the ER stress is severe, this

Anyway, many studies suggest that autophagy is activated in the heart and vascular tree as a defensive mechanism for survival during myocardial ischemia/reperfusion and in athero‐

If autophagy may be considered as a safeguard that protects vascular wall from rupture-prone lesions, autophagy up-regulation by recent pharmacologic modulators has been proven to be effective in short-term experimental studies on knockout atherosclerotic mice [87]. Moreover autophagy is directly involved in the acute setting of cardiac diseases by providing metabolic

cardiomyocytes from ischemia-induced apoptosis *in vitro* [80].

ates the ER over-crowding [58].

UPR linked to autophagy [82].

mechanism is overwhelmed.

sclerosis [85,86].

function [81].

are degraded.

Moreover the conservative pro-survival role of GRP78 is outlined also *in vivo*, considering that GRP78-deficient mice are embryonic lethal and present increased apoptosis [77].

The canonical UPR starting signals include three distinct pathways: the inositol-requiring kinase 1 (IRE1), the transcriptional factor activating transcription factor 6 (ATF6) and the protein kinase-like ER kinase (PERK) [15] (Figure 2).

**Figure 2.** Canonical UPR pathways. IRE1- inositol-requiring kinase 1; ATF6- ATF4- activating transcription factors 6 or 4; XBP1- x-box binding protein 1; eIF2alpha- initiation factor 2 alpha; ERAD- ER-associated protein degradation path‐ way; GRP78- glucose-regulated protein; PERK- protein kinase-like ER kinase; CHOP- C/EBP homologous protein. Modi‐ fied from [15,78].

This last enzyme is able to phosphorylate the translation initiation factor 2 alpha (eIF2alpha) after ER stress then reduces the further protein load on the ER by blocking mRNA transla‐ tion. In contrast, there are some mRNAs that require eIF2 alpha autophosphorylation for their translation, including the transcriptional factor ATF4, that is directly involved in the nuclear activation of UPR-related genes. Furthermore eIF2 alpha influences, by endonu‐ clease activity, the splicing of another transcriptional factor, called X-box binding protein 1 (XBP1), that regulates the transcription of UPR-related genes, although in the heart its func‐ tion is largely unknown [79].

ATF6 is another crucial transcriptional factor that, moving from the Golgi complex, becomes activated and able to interact with XBP1 target genes for the synthesis of molecular chaper‐ ones and also for enzymes involved in the ER-associated protein degradation pathway (ERAD). The ERAD mechanism mediates the translocation of unfolded proteins from the ER into the cytosol where they are degraded by the ubiquitin-proteasome system and so allevi‐ ates the ER over-crowding [58].

cells. So it is plausible that hemodynamic flow might be the earliest ER stressor and GRP78

Moreover the conservative pro-survival role of GRP78 is outlined also *in vivo*, considering

The canonical UPR starting signals include three distinct pathways: the inositol-requiring kinase 1 (IRE1), the transcriptional factor activating transcription factor 6 (ATF6) and the

**Figure 2.** Canonical UPR pathways. IRE1- inositol-requiring kinase 1; ATF6- ATF4- activating transcription factors 6 or 4; XBP1- x-box binding protein 1; eIF2alpha- initiation factor 2 alpha; ERAD- ER-associated protein degradation path‐ way; GRP78- glucose-regulated protein; PERK- protein kinase-like ER kinase; CHOP- C/EBP homologous protein. Modi‐

This last enzyme is able to phosphorylate the translation initiation factor 2 alpha (eIF2alpha) after ER stress then reduces the further protein load on the ER by blocking mRNA transla‐ tion. In contrast, there are some mRNAs that require eIF2 alpha autophosphorylation for their translation, including the transcriptional factor ATF4, that is directly involved in the nuclear activation of UPR-related genes. Furthermore eIF2 alpha influences, by endonu‐ clease activity, the splicing of another transcriptional factor, called X-box binding protein 1 (XBP1), that regulates the transcription of UPR-related genes, although in the heart its func‐

ATF6 is another crucial transcriptional factor that, moving from the Golgi complex, becomes activated and able to interact with XBP1 target genes for the synthesis of molecular chaper‐

that GRP78-deficient mice are embryonic lethal and present increased apoptosis [77].

inducer in an atheroprone environment.

32 Current Trends in Atherogenesis

fied from [15,78].

tion is largely unknown [79].

protein kinase-like ER kinase (PERK) [15] (Figure 2).

Interestingly, a novel gene, called derlin-3, as a component of the ERAD induced by ATF6, was recently discovered in the mouse heart, and derlin-3 over-expression was able to protect cardiomyocytes from ischemia-induced apoptosis *in vitro* [80].

Besides ER chaperones involvement, dysfunctional proteins may be degraded directly in the ER in a chaperone-independent manner, by a specialized protease system called the ubiqui‐ tin-proteasome, that works independently or in synergy with the UPR. According to recent clinical and experimental studies, atherosclerosis may be considered also as a "protein-qual‐ ity disease" and the proteasome works at early phases of the disease, especially in both the coronary and carotid arteries, as a compensatory reaction to prevent complete protein dys‐ function [81].

It is currently accepted that in mammalians the necessity to remove aberrant proteins that engulfed the ER environment is based upon three main ER activities: 1) the transient UPR associated to resident chaperones, 2) the ubiquitin-proteasome system and 3) the prolonged UPR linked to autophagy [82].

In particular through this last process, properly called macro-autophagy, abnormal cyto‐ plasmic contents or organelles engulfed in autophagosomes, upon fusion with lysosomes, are degraded.

Evidence is emerging that the ER provides membrane for autophagosome formation and that autophagy is crucial for ER homeostasis due to its ability to remove unwanted or dam‐ aged organelles like abnormal mitochondria by mitophagy [83]. Moreover the ER contrib‐ utes also lipids and specific proteins, such as beclin-1, to initiate autophagosome formation very close to itself. This physical proximity probably reflects a functional dependence be‐ tween ER and autophagy process, that in the endothelial cells often occurs in response to reactive oxygen species (ROS) by circuits localized to the ER surface [84]. However in divid‐ ing cells with high turnover, autophagy may not be so relevant, but in long-lived cells like smooth muscle cells and cardiomyocytes, it is critical to maintain optimal cellular function.

Remarkably autophagy is a suitable mechanism to eliminate abnormal proteins and organ‐ elles, during fast and relatively mild ER stress conditions, but if the ER stress is severe, this mechanism is overwhelmed.

Anyway, many studies suggest that autophagy is activated in the heart and vascular tree as a defensive mechanism for survival during myocardial ischemia/reperfusion and in athero‐ sclerosis [85,86].

If autophagy may be considered as a safeguard that protects vascular wall from rupture-prone lesions, autophagy up-regulation by recent pharmacologic modulators has been proven to be effective in short-term experimental studies on knockout atherosclerotic mice [87]. Moreover autophagy is directly involved in the acute setting of cardiac diseases by providing metabolic 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, even if its application in clinical trials is still an hypothesis [90].

Currently there is ample evidence of the involvement of the immune system in the patho‐ genesis of atherosclerosis and ER stress-driven autoimmunity may represent a novel contri‐

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

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35

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

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‐

The most common apoptosis-triggering molecule associated to UPR signaling is called

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‐

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*

vanced atherogenic lesions, such as instable plaques, respect to wild-type mice.

*vitro* to instability of plaques due to macrophage cell death.

buting factor in the progression of the disease [103,104].

[78,105-107].

ic signal starts.

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

C/EBP homologous protein, or CHOP, known also as GADD153 [108].

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‐ gression of this inflammatory disease [92-94].

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

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‐ esis and in healthy physiological conditions [97].

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 diseases.

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

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.

Currently there is ample evidence of the involvement of the immune system in the patho‐ genesis of atherosclerosis and ER stress-driven autoimmunity may represent a novel contri‐ buting factor in the progression of the disease [103,104].
