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

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 therapeutic interventions and to identify targets for different therapeutic options.

In cardiovascular diseases but also in the endothelial cells and cardiomyocytes *in vitro*, the regulation of the UPR arms can lead to an adaptation phase and survival or to a detrimental phase that ends into cell death.

thionine-L-lyase in the cardiovascular system, as an effective anti-atherosclerotic compound. Indeed it reduces oxidative damage in the aorta but also potentiates the adaptive beneficial role of ER signaling by increasing GRP78 expression in the intima layer. This effect might be related to the reduction of plasma level of LDL and lipids deposition in the aorta [148].

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

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41

Furthermore up-regulation of T-cadherin has emerged as an effective tool that limits the progression of atherosclerotic lesions in endothelial cells *in vitro*. This molecule is a glycosyl‐ phosphatidylinositol-anchored element belonging to the cadherin family, that colocalizes with GRP78 on the plasma membrane [149]. Its over-expression or silencing by genetic ma‐ nipulations selectively attenuates or amplifies the PERK branch of the UPR cascade obtained by ER stressors like homocysteine, thapsigargin and brefeldin A, so influencing apoptosis [150]. Indeed T-cadherin up-regulation is able to directly limit the phosphorylation of the eukaryotic translation initiation factor 2 alpha (phospho-eIF2alpha) and CHOP-driven cell death, even if how it communicates with ER-stress machinery *in vitro* is not yet known.

Salubrinal is another chemical chaperone that modulates the dephosphorylation of eIF2alpha, so reducing abnormal protein load on the ER and prolonged UPR, and it has been demonstrat‐ ed to limit ischemia-reperfusion damage in the mice brain [151]. Despite some promising re‐ ports, it is important to consider that there are different commercial preparations of the drug

Finally among ER-resident chemical chaperones oxygen-regulated protein150 (ORP150), a 150 kDa oxygen-regulated protein, has been implicated not only in reducing apoptosis dur‐ ing oxidative damage but also in preventing ox-LDLs induced ER stress in transfected vas‐ cular endothelial cells. In particular, by immune-precipitation assay it has been demonstrated that ORP150 is bound to three ER stress sensors IRE1alpha, PERK and ATF6 so maintaining them in an inactive status and contributing to delay UPR activation. Further‐ more ORP150 and IRE1alpha were also linked in situ in atherosclerotic lesions from human carotid plaque, but no ORP150-IRE1 alpha association was detected in normal human mam‐

A growing body of evidence indicates that LDLs, modified by oxidation, enzymatic attack, glycation and aggregation in ox-LDL, trigger local vascular inflammation and toxic events implicated in atherosclerosis, but in contrast high density lipoproteins (HDLs) have antiatherogenic properties that have been linked to reduced ER stress and autophagy [153].

In endothelial cells *in vitro* HDLs pretreatment was able to prevent detrimental UPR path‐ ways inhibiting IRE1 alpha activation and phosphorylation in the PERK arm and the nucle‐ ar translocation of ATF6 that triggered the pro-apoptotic CHOP signaling. All these mechanisms were stimulated by prolonged ER stress induced by ox-LDLs that in parallel ac‐ tivated also autophagy then overwhelmed by apoptosis if the vascular stress lasted too much. However, calcium deregulation was a common upstream signal for two parallel path‐ ways in this *in vitro* model, where ER stress-UPR but also autophagy are involved. Indeed HDLs were able to prevent the increase in autophagic markers like LC3-II and beclin-1 in the endothelial cells that, silenced for beclin 1 and then stimulated by toxic ox-LDLs, dis‐

played less ability to be recognized by macrophages.

providing different level of protection, so the real efficacy is currently debated.

mary artery [152].

These opposite effects, considered as "the double-edged sword", are an important issue for vascular biology, even if the molecular mechanisms that differentially regulate survival or cell death are yet to be clarified [137,138].

Anyway, it is possible to resume ER-regulatory interventions into two types: 1) one directly targeting ER stress-UPR by interfering with UPR branches with the use of chemical chaper‐ ones or inhibitors; 2) others indirectly targeting ER stress-UPR by regulation of related apoptosis, autophagy, oxidative or inflammatory signaling.

It is established that diabetic retinopathy is a major complication of diabetes, associated to inflammation and leukocyte adhesion in the endothelium of retinal vasculature, that impairs the inner blood-retinal barrier necessary to normal visual activity [139].

Recently ER stress has been involved in the pathogenesis of this invalidating disease [140]. However when used as a preconditioning tool, it may provide therapeutic benefits.

In particular the activation of XBP1s in endothelial cells, negatively regulates IRE1-alpha phosphorylation and suppresses inflammation. So, improving this branch of ER stress path‐ way may be useful to prevent or limit retinopathy in diabetes [141].

Furthermore emerging data on angiotensin II-induced cardiac hypertrophy in mice, have demonstrated a direct involvement of ER stress and related markers, GRP78 and CHOP, in cardiac remodeling and fibrosis [18].

ER chaperones represent a group of low-molecular compounds able to increase ER folding capacity and alleviate the accumulation of dysfunctional proteins, so maintaining ER ho‐ meostasis [142]. Different chaperones like 4-phenyl butyrate (PBA) and taurine-conjugated deoxycholic acid (TUDCA) have been successfully tested *in vivo* in different murine models of atherosclerosis, diabetes and leptin resistance where ER stress was attenuated [143].

Moreover PBA and TUDCA, have been successfully tested against endothelium-dependent relaxation and oxidative damage in the aorta and mesenteric artery in hypertensive mice [19]. Indeed ER signaling might represent a potential target to reverse hypertension-induced vascular and cardiac dysfunctions.

In particular ER stress was linked also to oxidative damage, due to abnormal calcium flux from the ER driven by protein misfolding and its uptake into the mitochondria where calci‐ um disrupted the electron transport chain [144]. Nevertheless further studies are required to elucidate how these two mechanisms can activate each other [145].

In a mouse model of type 2 diabetes chemical chaperones increased insulin sensitivity acting by antioxidant properties, this finding is particularly interesting because ER stress may also induce insulin-resistance [146,147].

A recent study performed in transgenic ApoE-/- mice, fed a Western diet, has supported the protective role of hydrogen sulfide, a product generated from L-cysteine catalyzed by cysta‐ thionine-L-lyase in the cardiovascular system, as an effective anti-atherosclerotic compound. Indeed it reduces oxidative damage in the aorta but also potentiates the adaptive beneficial role of ER signaling by increasing GRP78 expression in the intima layer. This effect might be related to the reduction of plasma level of LDL and lipids deposition in the aorta [148].

In cardiovascular diseases but also in the endothelial cells and cardiomyocytes *in vitro*, the regulation of the UPR arms can lead to an adaptation phase and survival or to a detrimental

These opposite effects, considered as "the double-edged sword", are an important issue for vascular biology, even if the molecular mechanisms that differentially regulate survival or

Anyway, it is possible to resume ER-regulatory interventions into two types: 1) one directly targeting ER stress-UPR by interfering with UPR branches with the use of chemical chaper‐ ones or inhibitors; 2) others indirectly targeting ER stress-UPR by regulation of related

It is established that diabetic retinopathy is a major complication of diabetes, associated to inflammation and leukocyte adhesion in the endothelium of retinal vasculature, that impairs

Recently ER stress has been involved in the pathogenesis of this invalidating disease [140].

In particular the activation of XBP1s in endothelial cells, negatively regulates IRE1-alpha phosphorylation and suppresses inflammation. So, improving this branch of ER stress path‐

Furthermore emerging data on angiotensin II-induced cardiac hypertrophy in mice, have demonstrated a direct involvement of ER stress and related markers, GRP78 and CHOP, in

ER chaperones represent a group of low-molecular compounds able to increase ER folding capacity and alleviate the accumulation of dysfunctional proteins, so maintaining ER ho‐ meostasis [142]. Different chaperones like 4-phenyl butyrate (PBA) and taurine-conjugated deoxycholic acid (TUDCA) have been successfully tested *in vivo* in different murine models of atherosclerosis, diabetes and leptin resistance where ER stress was attenuated [143].

Moreover PBA and TUDCA, have been successfully tested against endothelium-dependent relaxation and oxidative damage in the aorta and mesenteric artery in hypertensive mice [19]. Indeed ER signaling might represent a potential target to reverse hypertension-induced

In particular ER stress was linked also to oxidative damage, due to abnormal calcium flux from the ER driven by protein misfolding and its uptake into the mitochondria where calci‐ um disrupted the electron transport chain [144]. Nevertheless further studies are required to

In a mouse model of type 2 diabetes chemical chaperones increased insulin sensitivity acting by antioxidant properties, this finding is particularly interesting because ER stress may also

A recent study performed in transgenic ApoE-/- mice, fed a Western diet, has supported the protective role of hydrogen sulfide, a product generated from L-cysteine catalyzed by cysta‐

However when used as a preconditioning tool, it may provide therapeutic benefits.

phase that ends into cell death.

40 Current Trends in Atherogenesis

cell death are yet to be clarified [137,138].

cardiac remodeling and fibrosis [18].

vascular and cardiac dysfunctions.

induce insulin-resistance [146,147].

apoptosis, autophagy, oxidative or inflammatory signaling.

the inner blood-retinal barrier necessary to normal visual activity [139].

way may be useful to prevent or limit retinopathy in diabetes [141].

elucidate how these two mechanisms can activate each other [145].

Furthermore up-regulation of T-cadherin has emerged as an effective tool that limits the progression of atherosclerotic lesions in endothelial cells *in vitro*. This molecule is a glycosyl‐ phosphatidylinositol-anchored element belonging to the cadherin family, that colocalizes with GRP78 on the plasma membrane [149]. Its over-expression or silencing by genetic ma‐ nipulations selectively attenuates or amplifies the PERK branch of the UPR cascade obtained by ER stressors like homocysteine, thapsigargin and brefeldin A, so influencing apoptosis [150]. Indeed T-cadherin up-regulation is able to directly limit the phosphorylation of the eukaryotic translation initiation factor 2 alpha (phospho-eIF2alpha) and CHOP-driven cell death, even if how it communicates with ER-stress machinery *in vitro* is not yet known.

Salubrinal is another chemical chaperone that modulates the dephosphorylation of eIF2alpha, so reducing abnormal protein load on the ER and prolonged UPR, and it has been demonstrat‐ ed to limit ischemia-reperfusion damage in the mice brain [151]. Despite some promising re‐ ports, it is important to consider that there are different commercial preparations of the drug providing different level of protection, so the real efficacy is currently debated.

Finally among ER-resident chemical chaperones oxygen-regulated protein150 (ORP150), a 150 kDa oxygen-regulated protein, has been implicated not only in reducing apoptosis dur‐ ing oxidative damage but also in preventing ox-LDLs induced ER stress in transfected vas‐ cular endothelial cells. In particular, by immune-precipitation assay it has been demonstrated that ORP150 is bound to three ER stress sensors IRE1alpha, PERK and ATF6 so maintaining them in an inactive status and contributing to delay UPR activation. Further‐ more ORP150 and IRE1alpha were also linked in situ in atherosclerotic lesions from human carotid plaque, but no ORP150-IRE1 alpha association was detected in normal human mam‐ mary artery [152].

A growing body of evidence indicates that LDLs, modified by oxidation, enzymatic attack, glycation and aggregation in ox-LDL, trigger local vascular inflammation and toxic events implicated in atherosclerosis, but in contrast high density lipoproteins (HDLs) have antiatherogenic properties that have been linked to reduced ER stress and autophagy [153].

In endothelial cells *in vitro* HDLs pretreatment was able to prevent detrimental UPR path‐ ways inhibiting IRE1 alpha activation and phosphorylation in the PERK arm and the nucle‐ ar translocation of ATF6 that triggered the pro-apoptotic CHOP signaling. All these mechanisms were stimulated by prolonged ER stress induced by ox-LDLs that in parallel ac‐ tivated also autophagy then overwhelmed by apoptosis if the vascular stress lasted too much. However, calcium deregulation was a common upstream signal for two parallel path‐ ways in this *in vitro* model, where ER stress-UPR but also autophagy are involved. Indeed HDLs were able to prevent the increase in autophagic markers like LC3-II and beclin-1 in the endothelial cells that, silenced for beclin 1 and then stimulated by toxic ox-LDLs, dis‐ played less ability to be recognized by macrophages.

Remarkably, even if autophagy is not involved in apoptosis, probably contributes like a ben‐ eficial "eat-me" signal on the cell surface by exposing phosphatydylserine, necessary to the clearance by efferocytosis of apoptotic cells [154] (Figure 5). All these important findings suggest a potential efficacy for HDLs-based therapeutic opportunities in atherosclerosis.

tosis, even if the detailed mechanism by which it regulates GRP78/IRE1 activation is still un‐ known. Anyway even if more studies on transgenic Gipie-deficient animals will improve the understanding of the proper function in circulatory system, Gipie may be considered a

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

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

43

Actually the pivotal role of ER stress response in atherosclerosis and cardiovascular diseases is widely accepted. Nevertheless it remains much work to do in particular to discover the multiple relationship between different integrated pathways associated to ER signaling and to maintain the best ER stress modulation in the endothelium and vascular wall. Indeed it is important to point out that in biology the UPR is considered a surviving mechanism, so its complete deregulation may not be useful but dangerous. However additional experimental studies are required to help identify novel therapies to restore proper ER homeostasis but in particular, those to stabilize the minority of dangerous plaques associated with acute cardio‐

Human Anatomy Division, Department of Biomedical Sciences and Biotechnology, Univer‐

[1] Hansson G, Robertson A, Soderberg-Naucler C. Inflammation and atherosclerosis.

[2] Packard R, Libby P. Inflammation in atherosclerosis: from vascular biology to bio‐

[3] Mulligan-Kehoe M. The vasa vasorum in diseased and non diseased arteries. Am J

marker discovery and risk prediction. Clin Chem 2008;54(1) 24-38.

Physiol Heart Circ Physiol 2010;298 H-295-H305

This chapter is supported by academic grants (ex MIUR 60% 2011-2012).

Alessandra Stacchiotti, Gaia Favero and Rita Rezzani

\*Address all correspondence to: stacchio@med.unibs.it

Ann Rev Pathol 2006;1 297-329.

reliable therapeutic target in atherosclerosis yet.

**6. Conclusions**

vascular damage.

**Author details**

**References**

**Acknowledgements**

sity of Brescia, Brescia, Italy

**Figure 5.** Beneficial role of high density lipoproteins upstream ER stress and autophagy in endothelial cells. HDL- high density lipoprotein; LDL: low density lipoprotein; ox-LDL- oxidized lipoproteins; LC3-II- microtubule-associated pro‐ tein1 light chain 3; CHOP- C/EBP homologous protein; JNK- c-Jun N-terminal kinase. Adapted from [154].

Recently an interesting study reported the peculiar expression on endothelial cells and macro‐ phages of a novel GRP78-interacting protein induced by ER stress, called Gipie [155]. Gipie be‐ longs to the Girdin family protein and is localized in the ER and Golgi apparatus in the endothelial cell lines (human umbilical vein endothelial cell-HUVEC and human coronary ar‐ tery endothelial cells-HCAEC), but not in epithelial or mesenchymal cells *in vitro*. The transfec‐ tion of Gipie into HUVEC cells exposed to ER inducer thapsigargin, a specific blocker of ER calcium ATP-ase pumps, was able to decrease CHOP expression and apoptosis.

Moreover the same protection was demonstrated by Gipie's over-expression in rat carotid artery endothelial cells after baloon injury, a well-known *in vivo* model of endothelial dam‐ age and restenosis. Finally also in adult P65 mice aorta, Gipie was superimposed with GRP78 in atheroprone sites like the inner curvature of the aortic arch, but not in the outer curvature or in the ascending aorta, less sensitive to hemodynamic stress. By interaction with GRP78, Gipie modulates IRE1/JNK signaling and CHOP expression, so reducing apop‐ tosis, even if the detailed mechanism by which it regulates GRP78/IRE1 activation is still un‐ known. Anyway even if more studies on transgenic Gipie-deficient animals will improve the understanding of the proper function in circulatory system, Gipie may be considered a reliable therapeutic target in atherosclerosis yet.
