**2. The endothelium and the endoplasmic reticulum homeostasis**

According to the most accredited theory that indicates inflammation as the first pathogenic mechanism of atherosclerosis, the endothelium is really the crucial target of circulating mol‐ ecules or cells and constitutes the main entrance for LDL during the initial step of asympto‐ matic artery wall changes that end into plaques or atheromata and their dramatic clinical evolution [20-23].

Recent studies have outlined that in atherosusceptible sites in the artery tree, endothelial cells acquire a proinflammatory phenotype which is permissive in the plaque development by expressing pro-inflammatory sensors such as Toll-like receptors (TLRs), that in turn at‐ tract leukocytes adhesion in the intima layer. Mainly TLR 2 and TLR4 are active in mouse in the progression of atherosclerosis and their signals stimulate a downstream adaptor mole‐ cule, called Toll/IL-1 receptor domain-related adaptor protein that induces interferon or TRIF. Indeed also in human vascular tree, by immunostaining and mRNA survey TLR2 and TLR4 have been well characterized in selected sites, including the aorta, subclavia, carotid, mesenteric, iliac and temporal arteries [24].

Nevertheless an important concept to remind here is that the relationship between the vas‐ cular endothelium and the blood is not only "passive" in receiving inflammatory or meta‐ bolic stimuli, but instead "active", with pleiotropic activities like the secretion of regulatory factors for cholesterol and lipid homeostasis, platelets recruitment, and the adaptation to lo‐ cal changes of blood flow and pressure [25,26].

Moreover the artery wall, in particular in healthy resistance arteries, is not a static but a dy‐ namic and plastic structure, able to remodel its diameter and structure, adapting to rapid changes in the systemic pressure [27].

Indeed also artery geometry directly influences the athero-susceptibility and the distribution of mechanical forces associated to blood flux, that impair the endothelium [25,28,29].

In particular during unstable hemodynamic flux and changes in blood direction, mainly in arterial branches and bifurcations, it is particularly evident the heterogeneity of endothelial phenotypes that change their common flat shape and assume a polygonal morphology to‐ gether with a different turnover. These events are linked to the susceptibility of a specific vessel to develop atherosclerosis and to the onset of valve calcification in the heart [30-32].

tension both in animal models and in clinical patients [11,12]. Remarkably this event is early detectable in the endothelial cells [13], sometimes concurrent with other well-known athero‐

Nevertheless considering the focal distribution of plaques and their cumulative progression during the whole lifespan [14], it is mandatory to consider the role of ER stress signaling in the circulatory bed, in order to maintain the proper ER function, so preventing or reducing the progression into irreversible cardiovascular dysfunctions, such as atherosclerosis, hyper‐

We firmly believe that focusing integrated basic and applied research on ER stress in the ar‐ tery tree and in the heart might open new avenues in the treatment and management of in‐

According to the most accredited theory that indicates inflammation as the first pathogenic mechanism of atherosclerosis, the endothelium is really the crucial target of circulating mol‐ ecules or cells and constitutes the main entrance for LDL during the initial step of asympto‐ matic artery wall changes that end into plaques or atheromata and their dramatic clinical

Recent studies have outlined that in atherosusceptible sites in the artery tree, endothelial cells acquire a proinflammatory phenotype which is permissive in the plaque development by expressing pro-inflammatory sensors such as Toll-like receptors (TLRs), that in turn at‐ tract leukocytes adhesion in the intima layer. Mainly TLR 2 and TLR4 are active in mouse in the progression of atherosclerosis and their signals stimulate a downstream adaptor mole‐ cule, called Toll/IL-1 receptor domain-related adaptor protein that induces interferon or TRIF. Indeed also in human vascular tree, by immunostaining and mRNA survey TLR2 and TLR4 have been well characterized in selected sites, including the aorta, subclavia, carotid,

Nevertheless an important concept to remind here is that the relationship between the vas‐ cular endothelium and the blood is not only "passive" in receiving inflammatory or meta‐ bolic stimuli, but instead "active", with pleiotropic activities like the secretion of regulatory factors for cholesterol and lipid homeostasis, platelets recruitment, and the adaptation to lo‐

Moreover the artery wall, in particular in healthy resistance arteries, is not a static but a dy‐ namic and plastic structure, able to remodel its diameter and structure, adapting to rapid

Indeed also artery geometry directly influences the athero-susceptibility and the distribution

In particular during unstable hemodynamic flux and changes in blood direction, mainly in arterial branches and bifurcations, it is particularly evident the heterogeneity of endothelial

of mechanical forces associated to blood flux, that impair the endothelium [25,28,29].

genic processes, like inflammation, oxidative damage and endothelial cell death.

**2. The endothelium and the endoplasmic reticulum homeostasis**

tension and ischemic heart disease [15-17].

evolution [20-23].

28 Current Trends in Atherogenesis

validating cardiovascular complications [18,19].

mesenteric, iliac and temporal arteries [24].

cal changes of blood flow and pressure [25,26].

changes in the systemic pressure [27].

So endothelial dysfunctions may have serious consequences and a direct impact on the en‐ dothelial cells' role and activities, mainly on the resistance to dangerous stimuli that pro‐ mote the onset of pro-atherogenic vascular damage recently reviewed by [33].

Indeed they involve different structural and functional aspects of the endothelium, that is classified as a monocellular squamous type of epithelium [34], lining human vascular and lymphatic tree, poorly detectable by traditional light microscopy but well characterized by electron microscopy and related techniques [35-37].

Nevertheless the real consideration of the endothelium by physicians has begun about 50 years ago, but only in the last decade, it has obtained more importance in the cardiovascular community, with the rediscovery of Weibel-Palade bodies and caveole signals, the role of transcytosis mechanism, and the active participation into vascular permeability [38-40].

Among most critical structural changes linked to endothelial dysfunctions, there are the reduc‐ tion of glycocalix, which is the external component necessary to react against toxic apoB LDL, and the over-development of fundamental organelles like Golgi complex and the ER [41,42].

Remarkably the ER signaling in the vascular wall is the main topic of this chapter, because much more attention must be given to ER homeostasis in atheroprone sites in the artery tree, resulting from a chronic adaptive reaction to flow disturbance, concurrent with oxidative damage and inflammation [43,44].

Abnormal ER activity has been recently reported in coronary arteries during altered he‐ modynamic changes, diagnosed by genetic techniques as an abnormal transcription of se‐ lected genes; while, in contrast, the transcriptional activity is lacking in more resistant arterial beds [45,46].

Remarkably it must be pointed out that, in mammalian epithelial cells, the ER is commonly depicted by ultrastructural analysis as a perinuclear network of tubules and membranes, and by tomography as a dynamic assembly of tridimensional stacks associated to mitochon‐ dria [47-49].

Moreover it is well-known that the ER has different specialization and structure, called rough or smooth, if associated or not to ribosomes in the same cell, but in specialized cardiac and smooth muscle cells in the vascular wall it is called the sarcoplasmic reticulum [50,51].

Anyway, this dynamic organelle represents the elective site where nascent polypeptide chains are gradually converted in a stable tertiary structure, that is associated to a specific protein [52].

Among the main ER functions have been comprised the folding of neo-synthesized secreto‐ ry and trans-membrane proteins, the regulation of calcium balance and the synthesis of lip‐ ids, like steroids and cholesterol [53].

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

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

ther deposition of dysfunctional proteins called ERAD [11].

and by lectin chaperones calnexin, calreticulin and calmegin [70,71].

to focal adhesion in the endothelial cells upon long-term shear stress [76].

eventually death [64].

the start of the UPR cascade.

[66,67].

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

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

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

31

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‐

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

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

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

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

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

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

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

**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. Adapted by [63].
