Mechanisms of Vascular Dysfunction

### **Chapter 1**

## Endothelial Dysfunction and Disruption in Pulmonary Hypertension

*Rajamma Mathew*

### **Abstract**

A number of systemic diseases lead to pulmonary hypertension (PH), a serious disorder with a high morbidity and mortality rate. Irrespective of the underlying disease, endothelial dysfunction or disruption plays a key role in the initiation and progression of PH. Endothelial dysfunction and disruption result in impaired vascular relaxation response, activation of proliferative pathways leading to medial hypertrophy and PH. Endothelial cells (EC) play a crucial role in regulating vascular tone and maintaining homeostasis. Caveolin-1, a 21-22 kD membrane protein, interacts with a number of transducing factors and maintains them in a negative conformation. Disruption of EC results in endothelial caveolin-1 loss and reciprocal activation of proliferative pathways leading to PH, and the accompanying loss of PECAM1 and vascular endothelial cadherin results in barrier dysfunction. These changes lead to the irreversibility of PH. Hypoxia-induced PH is not accompanied by endothelial disruption or caveolin-1 loss but is associated with caveolin-1 dysfunction and the activation of proliferative pathways. Removal of hypoxic exposure results in the reversal of the disease. Thus, EC integrity is an important factor that determines irreversibility vs. reversibility of PH. This chapter will discuss normal EC function and the differences encountered in PH following EC disruption and EC dysfunction.

**Keywords:** caveolin-1, endothelial cells, membrane integrity, smooth muscle cells, pulmonary hypertension

### **1. Pulmonary hypertension**

A number of systemic diseases such as cardiopulmonary, infectious, inflammatory and autoimmune diseases, hematological disorders, drug toxicity and several genetic mutations lead to pulmonary hypertension (PH), a devastating disease with a high morbidity and mortality rate. Based on clinical diagnosis, PH has been classified into five major groups that were updated in 2013 [1]. Group 1, labeled as pulmonary arterial hypertension (PAH), includes idiopathic and heritable PAH, PAH associated with congenital heart defect (CHD), connective tissue diseases, portal hypertension, HIV, schistosomiasis and drug-/toxin-induced PAH. In addition, mutation of several genes such as *BMPRII* (bone morphogenetic protein receptor type 2)*, CAV1* (caveolin-1), *ENG* (endoglin)*, SMAD9* (SMAD family member 9), *ACVRL1* (activin A receptor like type 1) and *KCNK3* (potassium two

pore domain channel subfamily K member 3) are among the well-documented causes of PAH. Pulmonary veno-occlusive disease (PVOD)/pulmonary capillary hemangioma and persistent pulmonary hypertension of the newborn (PPHN) are included in Group 1 as subcategories 1′ and 1″, respectively. Recently, mutation of *EIF2AK4* (eukaryotic translation initiation factor 2a kinase 4) has been shown to be associated with PVOD and capillary hemangioma [2]. Included in Group 2 are PH associated with congenital and acquired left heart diseases; Group 3 comprises PH due to lung diseases and/or hypoxia. Group 4 includes chronic thromboembolic pulmonary hypertension (CTEPH). PH associated with hematological disorders, myeloproliferative diseases, splenectomy and a number of miscellaneous systemic and metabolic disorders is included in Group 5. Up until recently, the diagnosis of PAH was considered when the mean pulmonary artery pressure (PAP) of ≥25 mmHg, pulmonary capillary wedge pressure of ≤ 15 mmHg, and a pulmonary vascular resistance (PVR) of > 3 Wood units were observed at rest. During the 6th World Symposium on PH, the mean PAP of threshold was lowered to >20 mmHg and PVR was maintained as >3 Wood units [3]. These changes are based on the evaluation of 47 studies from 13 countries, which showed that independent of age the normal mean PAP rarely exceeded 20 mmHg [4]. The worldwide prevalence of PH is estimated to be 1%, increasing to 10% in patients older than 65 years of age. Globally, left heart diseases (Gr 2) and lung diseases (Gr 3) are considered the most common causes of PH. About 80% of patients are from developing countries; the common causes of PH in these patients are CHD, rheumatic heart disease and infection such as HIV and schistosomiasis. These patients tend to be younger than 65 years [5]. With modern therapy, the survival in patients with idiopathic and heritable PAH and PAH associated with anorexigen drugs has improved to 92%, 75% and 66% at 1, 3 and 5 years, respectively [6]. However, the underlying vascular changes remain progressive [7]. There is still a significant delay between the onset of symptoms and the final diagnosis. A recent retrospective study revealed a delay of 3.9 years between the onset of symptoms and the diagnosis of idiopathic PAH [8]. Thus, by the time the diagnosis is made, patients often have significant pulmonary vascular disease, which is a serious challenge to therapy. Interestingly, in an animal model of PH, significant disruption of endothelial cells and the activation of pro-proliferative pathways have been shown to occur before the onset of PH, indicating that the vascular pathology is already present by the time the symptoms appear [9].

The major causes of PH in children are CHD, PPHN, PH associated with disruption of normal pulmonary vascular and alveolar development in preterm infants, and congenital defects associated with hypoplasia of the lungs [10, 11]. A significant number of pediatric patients (>80%) have transient PH. These include resolution of PPHN and the majority of CHD cases that become free of PAH after the surgical correction of the defect [12]. However, preterm birth itself has an increased risk of developing PH even after adjusting for known factors such as heart and lung diseases, congenital diaphragmatic hernia and chromosomal abnormalities [13]. Furthermore, poor outcome has been reported in children with *BMPRII* mutation associated with idiopathic or heritable PAH [14]. Mutation of *TBX4* (T-box transcription factor 4 gene) is associated with skeletal, cardiac and neurologic defects. It also leads to a form of developmental lung disease that has been shown to be associated with severe PH during infancy and childhood [15]. It is worth noting that infants over the age of 2 years who had CHD exhibited increased pulmonary artery pressure and PVR even after surgical correction of the defect [16]. Pulmonary vascular lesions found in PAH associated with CHD are reported to be similar to what is found in idiopathic PAH [17]. However, the plexiform lesions in idiopathic PAH have monoclonal cell population, whereas Eisenmenger disease (PAH associated

### *Endothelial Dysfunction and Disruption in Pulmonary Hypertension DOI: http://dx.doi.org/10.5772/intechopen.92177*

with CHD) displays polyclonal cells [18], indicating a distinct difference between two forms of PAH.

Endothelial cells (EC) play a key role in maintaining vascular homeostasis in response to various stimuli and regulate vascular tone, permeability, coagulation, inflammation through mediators such as nitric oxide (NO), endothelium-derived hyperpolarization factor (EDHF), endothelin-1 (ET1), cell adhesion molecules, cytokines and chemokines. Regardless of the underlying disease, endothelial dysfunction/ disruption plays a key role in the pathogenesis of PH. The genetic and environmental factors act as an initial trigger leading to endothelial cell injury and impaired regeneration resulting in vascular remodeling and loss of small pulmonary arteries [19]. Endothelial dysfunction, impaired vascular dilatation, alterations in the expression of NO, ET1 and serotonin, increased expression of inflammatory cytokines and chemokines, loss of endothelial caveolin-1 and disordered proteolysis of extracellular matrix contribute to the pathogenesis of PAH [20, 21]. Increased expression of chemokines such as CX(3)C (fractalkine) and RANTES (CCL5) has been reported in PAH; importantly, both these chemokines are produced in EC [22, 23]. In sugen + hypoxia model (mice), the deletion of CCL5 resulted in reduction in PH via caveolin-1–dependent amplification of BMPR2 signaling. It stabilized surface caveolin-1, restored BMPR2 signaling and enhanced BMPR2 and caveolin-1 interaction [24]. This observation further supports the role for inflammation in PH. In addition, perivascular infiltration with inflammatory cells (T and B cells) is present in plexiform lesions [25, 26]. Increased expression of interleukin-1 (IL-1) and IL-6 occurs in human PAH and monocrotaline (MCT)-induced PH, and inhibition of IL-6 expression and bioactivity as a preventive measure results in the abrogation of MCT-induced PH [27, 28].

In addition to the imbalance of vasoactive mediators and vascular remodeling, abnormality in ion channels (Ca2+, K+ ) and growth factors such as VEGF, EGF, TGF beta, MMPs, BMPR2 and Notch1 has been implicated in pathophysiology of PAH, leading to vasoconstriction, abnormal remodeling and plexiform lesions [29]. Proliferative EC reveals increased expression of angiogenesis and survival-related molecules such as VEGF, VEGFR2, Hif-1 α, and 1β and reduced expression of p27/ kip1. Signal transducer and activator of transcription (STAT3) is essential for cell proliferation and survival, and antiapoptotic function [30]. In the MCT model of PH, the loss of endothelial caveolin-1 was shown to be associated with reciprocal activation of STAT3 (PY-STAT3) and increased proliferating cell nuclear antigen (PCNA) [31]. Furthermore, EC in plexiform and concentric lesions exhibits increased expression of PY-STAT3 [32]. Importantly, the inhibition of STAT3 prevents neointima formation by inhibiting cell proliferation and promoting the apoptosis of neointimal SMC [33]. *BMPRII* mutations linked to PAH are associated with the activation of STAT3. Furthermore, BMPR2 deficiency promotes inflammatory response resulting in increased IL-6 levels and PY-STAT3 activation [34]. BMPR2, a cell surface receptor, is essential for differentiation and proliferation of EC and SMC. Without altering the *BMPRII* mRNA levels, miR-17/92 modulates BMPR2 protein levels. Importantly, IL-6 regulates the expression of miR-17/92 in human pulmonary arterial EC via STAT3 signaling. Persistent activation of STAT3 results in the upregulation of miR-20, which leads to the reduction in the expression of BMPR2 protein [35]. BMPR2 expression is decreased also in patients with heritable and idiopathic PAH, without associated mutation [36]. Importantly, levels of SMAD-specific E3 ubiquitin protein ligase 1 (Smurf1), a key negative regulator of BMPPR2, has been shown to be increased in hypoxia and MCT models of PH in rats [37]. Increased Smurf1 immunoreactivity has also been reported in EC and SMC in the explanted lungs from patients with PAH. Furthermore, Smurf1 deletion protects mice from sugen + hypoxia-induced PH [38]. Interestingly, elafin reverses obliterative changes in pulmonary arteries via elastase inhibition

and caveolin-1–dependent amplification of BMPR2. In addition, elafin promotes angiogenesis via increasing interaction of BMPR2 and caveolin-1 via mediating stabilization of endothelial surface caveolin-1 [39].

Recent studies have shown the involvement of Notch1 signaling in PAH. Increased expression of Notch1 has been reported in the lungs of patients with IPAH and in rats with sugen + hypoxia-induced PH. Notch1 positively regulates EC proliferation by downregulating p21 and negatively regulating apoptosis via Bcl2 and survivin. *In-vitro* studies with human pulmonary arterial EC revealed increased expression of Notch1 during hypoxia exposure, and Notch1 downregulation decreased cell proliferation [40]. Furthermore, Notch1 under hypoxia contributes to increased proliferation, migration and survival in cancer cells [41]. Notch1 is essential for VEGF-induced proliferation, migration and survival of EC [42]. Thus, Notch1 plays a significant role in the pathogenesis of PH. However, Notch1 also plays a key role in vascular morphogenesis, EC quiescence, junction stability and vascular homeostasis. Reduction in Notch1 activity destabilizes cellular junction and triggers EC proliferation and results in the loss of arterial identity and incorporation of these cells into veins. Notch1 is sensitive to shear stress and it requires VEGFA and VEGFR2 for growth [43, 44]. Interestingly, Notch-mediated inhibition of proliferation requires phosphatase-tensin homolog (PTEN), a dual lipid/protein phosphatase. PTEN localization is cell cycle dependent, negatively regulates cell cycle progression and has a restrictive role on angiogenesis [45]. Recent studies have shown significant loss of PTEN concomitant with caveolin-1 dysfunction in hypoxia-induced PH [46]. Fibroblasts from idiopathic pulmonary fibrosis lungs exhibit low membrane PTEN associated with low membrane caveolin-1 levels, and overexpression of caveolin-1 restores membrane PTEN levels. PTEN contains a caveolin-1–binding motif and, in part, colocalizes in caveolae [47]. Thus, caveolin-1 expression determines the membrane PTEN levels through its binding sequence. Furthermore, PTEN has also been shown to negatively regulate STAT3 and its activation, and importantly, membrane localization of PTEN is considered responsible for the inactivation of STAT3 [48].

### **2. Endothelial cell function**

EC forms a monolayer in contact with blood flow and mechanical forces and underlying SMC. It is a non-thrombogenic and a selective barrier to circulating macromolecules. Juxtaposition of EC and SMC facilitates cross talk, and EC maintains SMC in quiescent state. Myoendothelial gap junction plays an important role in Ca2+ exchange between EC and SMC. EC is crucial for delivery of O2 and nutrients to underlying organs. EC maintains a balance between vasodilatation and vasoconstriction, apoptosis and cell proliferation, participate in immune and metabolic function, and maintain anticoagulant state [21, 49]. In addition, EC converts mechanical information into biological responses through mechanotransduction processes. EC adapts to mechanical inputs while maintaining crucial vascular barrier function. Failure of EC to adapt to changes has effects on vascular permeability, an important cause of vascular diseases [50].

### **2.1 Caveolae, caveolin-1 and cavin-1**

Caveolae, a subset of specialized lipid rafts (50–100 nm), first described in 1950s by Palade [51] and Yamada [52], is found on plasma membranes of a variety of cell types including EC, SMC, fibroblasts and adipocytes. Caveolae are non-clathrin– coated plasma membrane vesicles (50–100 nm) enriched in glycosphingolipids,

### *Endothelial Dysfunction and Disruption in Pulmonary Hypertension DOI: http://dx.doi.org/10.5772/intechopen.92177*

cholesterol, sphingomyelin and lipid-anchored membrane proteins. They form an important signaling platform that compartmentalizes and integrates a number of signaling molecules and allows cross talk between different signaling pathways, and mediates and integrates signaling events at the cell surface. EC contains 5000–10,000 caveolae per cell [53]. In addition, caveolae act as plasma membrane sensors and respond to stress. Caveolae flatten in response to membrane stretch. The flattening is a protective mechanism; it buffers the membrane and prevents its rupture [54, 55]. Caveolin-1 is a major protein (21–22 kDa) constituent of caveolae that maintains the shape of caveolae; EC has the highest levels of caveolin-1 [56]. Caveolin-1 is involved in multiple cellular processes such as molecular transport, cell proliferation, adhesion, migration and signal transduction. Caveolin-1 has an integral role in endocytosis. However, overexpression of caveolin-1 inhibits endocytosis [57, 58]. Caveolin-1 is synthesized in endoplasmic reticulum and then transported to Golgi complex. During its biosynthesis, it is associated with lipid rafts and become detergent resistant. From a structural standpoint, caveolin-1 contains a hairpin loop structure and three palmitoylation sites and a scaffolding domain that facilitates interaction with the plasma membrane [59, 60]. Caveolin-1 functions through protein-protein interaction and regulates and stabilizes several proteins including Src family of kinases, G proteins (α-subunits), G protein-coupled receptors, H-Ras, PKC, endothelial NO synthase (eNOS), integrins, and growth factor receptors such as VEGFR2, EGFR and PDGFR in an inhibitory conformation. Importantly, a 20-amino acid membrane proximal region of the cytosolic amino-terminal domain, termed caveolin-scaffolding domain (residue 82–101), is sufficient to mediate these interactions [61, 62]. Caveolin-1 also functions as a suppressor of cytokine signaling (SOCS), the family of proteins that are upregulated by cytokines and that in turn inhibit cytokine signaling via modulating JAK-STAT pathway [63]. Caveolin-2 is present associated with caveolin-1 in all cell types. It requires caveolin-1 for its transport from Golgi body to the plasma membrane. Caveolin-2 is not necessary for caveolae formation or caveolar localization of caveolin-1, but the coexpression results in a more efficient formation of caveolae [64]. In the absence of caveolin-1, caveolin-2 is degraded, and the decreased expression of caveolin-2 promotes increased cell proliferation [65, 66]. Furthermore, caveolin-2 knockout mice display increased proliferation of endothelial cells, hyper-cellular lung parenchyma and cell cycle progression [67].

In addition to caveolin-1, caveolae require polymerase 1 and transcript release factor (PTRF) also known as cavin-1. It is an essential component of caveolae; it regulates membrane curvature by stabilizing caveolin-1 in caveolae. The loss of cavin-1 results in the loss of caveolae and the release of caveolin-1 into the plasma membrane. Importantly, caveolin-1 is required for cavin-1 recruitment to plasma membrane [68, 69]. Loss of caveolin-1 is accompanied by a marked loss of caveolin-2 and partial reduction in cavin-1 expression in the lungs. The re-expression of caveolin-1 rescues both caveolin-2 and cavin-1 [70]. In a carotid artery-injury model, the local loss of cavin-1 is reported to promote neointima formation. Furthermore, in cultured vascular SMC, the overexpression of cavin-1 suppresses SMC proliferation and migration, whereas its inhibition promotes cell proliferation and migration [71]. Cavin-1 knockout mice display lung pathological changes such as remodeled pulmonary vessels, PH and right ventricular hypertrophy. In addition, these mice have altered metabolic phenotype with insulin resistance [72, 73].

Recent studies have shown other accessory proteins required in caveolae biogenesis. The accessory protein pacsin2 also known as syndapin2 contains F-BAR domain associated with generation and maintenance of caveolae. It partially colocalizes with caveolin-1 at plasma membrane level. Loss of pacsin2 function results in the loss of caveolae and accumulation of caveolin-1 within the plasma membrane. Interestingly, overexpression of F-BAR domain can cause loss of caveolae. Another

protein EH 15 homology domain 2 (EHD2) is present in caveolae, and it binds to pacsin2 that partially colocalizes with caveolin-1. It is a dynamin-related ATPase that confines caveolae to cell surface. Furthermore, regulation of EHD2 oligomerization in a membrane-bound state is crucial in order to restrict caveolar dynamics in cells [74, 75]. Importantly, caveolar coat controls a large number of signaling circuits; a defect in any of these pathways can lead to several systemic diseases such as vascular dysfunction, cardiomyopathy, cancer, muscular dystrophy and lipodystrophy [76].

The role of caveolin-1 is well established in the pathogenesis of PH. Caveolin-1 knockout mice are viable but have dysregulated NO synthesis, impaired NO and Ca2+ signaling, cell proliferation, increased vascular permeability accompanied by cardiomyopathy and PH. Reconstituting endothelial caveolin-1 has been shown to recover dysregulated NO synthesis, cardiomyopathy and PH [77, 78]. In addition, caveolin-1 knockout mice exhibit low-grade systemic pro-inflammatory status and moderately increased IL-6 and TNFα levels [79]. EC-specific *CAV1* knockout mice and LPS-treated wild-type mice exhibit reduced BMPR2 expression and eNOS uncoupling, accompanied by increased TGF-β–promoted TGFβRI-dependent SMAD-2/3 phosphorylation. In addition, human lung sections from patients with ARDS reveal reduced endothelial caveolin-1 expression, increased TGF-β levels and severe pulmonary vascular remodeling. These results further support the view that the loss of endothelial caveolin-1 promotes pulmonary vascular remodeling in inflamed lungs via oxidative stress-mediated reduction in BMPR2 expression [80]. Furthermore, endothelial dysfunction during inflammation leads to endotheliummesenchymal transition (End MT). These cells lose endothelial characteristics and acquire mesenchymal phenotypes and express mesenchymal specific markers such as smooth muscle α-actin, fibroblast-specific protein 1 and Notch1 [81]*.* In addition, caveolin-1 is a determinant of oxidative stress and is a regulator of metabolic switch and autophagy [82].

### **2.2 Vascular relaxation**

NO, EDHF and prostacyclin (PGI2) induce endothelium-dependent vascular relaxation. NO is produced by eNOS via its action on L-arginine and oxygen. NO activates guanylate cyclase, which catalyzes the conversion of guanosine triphosphate to cyclic guanylate monophosphate. eNOS expressed in endothelial cells and cardiac myocytes is targeted to caveolae. It directly binds to caveolin-1 scaffolding domain and is held in an inhibitory state. This interaction prevents eNOS activation leading to inappropriate NO production under basal conditions. The eNOS/ caveolin-1 regulatory cycle is a reversible protein-protein interaction controlled by Ca2+/calmodulin and by enzyme palmitoylation. Increase in intracellular Ca2+ with calmodulin disrupts the caveolin-1/eNOS complex resulting in eNOS activation and NO production leading to vascular relaxation. Calmodulin is a direct allosteric competitor promoting the caveolin-1 and eNOS dissociation. Heat shock protein (HSP) 90 binds to eNOS in Ca2+/calmodulin-dependent manner and it reduces the inhibitory effects of caveolin scaffolding domain on eNOS, thus promoting eNOS activation [83–85]. Furthermore, increase in vascular flow and pressure rapidly activates caveolar eNOS with its dissociation from caveolin-1 and association with calmodulin [86]. Thus, caveolin-1 and eNOS have a dynamic relationship. Importantly, caveolin-1 contained within non-caveolar lipid rafts fails to exert its inhibitory effect on eNOS [87]. The loss of endothelial caveolin-1 leads to eNOS uncoupling, oxidative stress and endothelial injury [88]. Interestingly, under conditions of stress, caveolin-1 increases eNOS trafficking in plasma membrane and primes eNOS for flow-mediated activation. Caveolin-1 plays a positive role in shear-induced

### *Endothelial Dysfunction and Disruption in Pulmonary Hypertension DOI: http://dx.doi.org/10.5772/intechopen.92177*

eNOS activation by targeting eNOS to plasma membrane. Importantly, the coupling of flow stimulus to activate eNOS is lost in the absence of caveolin-1 and caveolae. Thus, caveolin-1 exerts dual role of post-translational regulation of eNOS activity [89]. In addition, caveolin-1 plays a critical role in VEGFR2 stimulation and downstream mediators of angiogenesis, but higher levels of caveolin-1 repress this function [90]. Interestingly, EC migration, tube formation and angiogenesis are impaired both in caveolin-1 and eNOS knockout mice but are fully restored by double knockout [91].

The transient receptor potential (TRP) channels are the link between caveolae and EDHF. TRP channels facilitating the capacitive Ca2+ entry directly interact with caveolin-1 in EC. Ca2+-activated K+ channels play a key role in endothelium-dependent hyperpolarization and vascular tone regulation. Absence of caveolin-1 impairs Ca2+ homeostasis in EC and decreases the activity of TPRV4 cation channels that participate in NO and EDHF activation. Caveolin-1 is required for EDHF-related relaxation, modulating TRPV4 and connexins. Caveolin-1 knockout arteries exhibit fewer gap junctions and altered myoendothelial communication. Furthermore, caveolin-1 deficiency is associated with impaired EDHF-mediated vascular relaxation in response to shear stress and acetylcholine [92–94]. Colocalization of PGI2 synthase and caveolin-1 regulates angiogenesis [95]. Thus, caveolin-1 interacts with relaxing factors to maintain homeostasis.

### **2.3 Barrier function**

Endothelial barrier controls the passage of fluids, nutrients and cells through vascular wall. Glycocalyx coats the luminal surface of EC and forms an important barrier. It modulates permeability, prevents leukocyte and platelet adhesion to EC, serves as an anti-inflammatory, anti-adhesive and anti-coagulant barrier, and allows selective permeability. In addition, it mediates mechanotransduction of shear stress. Under normal conditions, the apoptosis rate in EC is very low, but the activated EC exhibits a reduction in the EC surface layer, the glycocalyx, and an increased rate of apoptosis [96, 97]. Disturbed flow has been shown to inhibit glycocalyx expression as well as to reduce caveolin-1 expression in systemic arterial EC [98]. Inflammatory mediators lead to the disruption of glycocalyx resulting in the weakening of vascular protection. Integrity of vascular glycocalyx is inversely related to the degree of inflammation. Inflammatory mediators lead to the loss of glycocalyx resulting in the weakening of vascular protection. Furthermore, destruction of glycocalyx has been reported in the MCT model of PH [99].

Ca2+-dependent vascular endothelial cadherin (VE-Cad) and its associated catenins control cell-cell adhesion and paracellular barrier function and are important for the tight junction complexes. VE-Cad is tissue specific for EC and is expressed at the intercellular clefts and mediates cell-cell adhesion, maintains barrier function, and contributes to the inhibition of cell growth. Association of caveolin-1 and VE-Cad catenin complex is essential for barrier function [100–102]. Depletion of caveolin-1 reduces VE-Cad levels and facilitates endothelial cell permeability [103]. Furthermore, VE-Cad interacts with various growth receptors, regulates endothelial proliferative signaling and mediates contact inhibition of cell growth [104, 105]. In adult EC, VE-Cad and VEGFR2 are physically linked. This maintains VEGFR2 stable and prevents its endocytosis. VEGF-induced permeability is facilitated by decoupling of VE-Cad and VEGFR2 [106]. Loss of VE-Cad and PECAM1 has been shown to occur in the MCT-induced PH [31, 107]. PECAM-1 supports EC integrity and maintains barrier function [108]. Importantly, BMPR2 also plays a role in maintaining vascular integrity by dampening inflammatory signals in pulmonary vasculature [109].

### **3. Endothelial disruption/dysfunction**

Vascular injury from different conditions such as inflammation, hypoxia, increased flow and pressure, and shear stress leads to endothelial dysfunction. Injury can lead to disruption of EC and endothelial caveolin-1 loss or endothelial dysfunction without EC disruption. Both lead to the activation of proliferative pathways, vascular remodeling and PH [96, 110]. Recent studies have shown the loss of myocyte enhancer factor 2 (MEF2) in dysfunctional EC from PAH patients. MEF2 regulates a number of transcription factors involved in pulmonary vascular homeostasis [111]. Furthermore, these dysfunctional ECs exhibit increased production of leptin, and SMCs overexpress leptin receptor contributing to SMC proliferation [112]. In addition, pulmonary arterial ECs from PAH patients have been shown to produce increased FGF2 leading to increased proliferation and survival response by constitutive activation of ERK1/2 and decreased apoptosis associated with the activation of Bcl2 and Bcl-xL. It is thought that FGF2 in PAH may contribute to abnormal EC phenotype [113]. Furthermore, there is evidence that pro-inflammatory cytokine macrophage migration inhibitory factor and its receptor CD74 are markedly increased in idiopathic PAH, which may contribute to pro-inflammatory phenotype of EC [114].

### **3.1 EC disruption and pulmonary hypertension**

Endothelial disruption accompanied by the loss of endothelial caveolin-1 has been reported in several forms of experimental models of PH such as MCT, myocardial infarction and sugen + hypoxia [31, 115, 116]. In the MCT model, progressive loss of endothelial caveolin-1 and reciprocal activation of proliferative and anti-apoptotic pathways such as PY-STAT3 and Bcl-xL occur before the onset of PH. Loss of other membrane proteins such as PECAM-1, Tie2 and soluble guanylate cyclase (α and β) occurs in tandem with caveolin-1 loss indicating extensive disruption of endothelial cell membrane. At 2 weeks, a further loss of endothelial caveolin-1 is accompanied by the loss of cytosolic proteins such as HSP90 and IκB-α and increased pulmonary artery pressure. However, at this stage, eNOS expression is relatively well preserved. In the presence of significant loss of endothelial caveolin-1 and HSP90, eNOS gets uncoupled resulting in an increased production of reactive oxygen species (ROS). By 3 and 4 weeks, there is a significant reduction in eNOS levels, leading to normalization of ROS levels [9, 31, 117]. At 4 weeks post-MCT, extensive endothelial caveolin-1 loss is accompanied by the loss of von Willebrand factor (vWF) in 29% of the arteries; and 23% of arteries exhibit enhanced expression of caveolin-1 in SMC. Enhanced expression of caveolin-1 in SMC occurs only in the arteries with extensive endothelial caveolin-1 and vWF loss. At this stage, the expression of total caveolin-1 in the lungs remains low. In addition, there is a progressive increase in MMP2 expression and activation [117]. The rescue of endothelial caveolin-1 as a preventive measure abrogates MCT-induced PH, but once the PH is established, the treatment does not alter the progression of the disease [118–120]. Exposing MCT-treated rats to hypoxia accelerates the disease process, and by 4 weeks, extensive endothelial disruption and endothelial caveolin-1 loss are accompanied by enhanced expression of caveolin-1 in SMC in 61% of the arteries, near normalization of lung caveolin-1 expression, and neointima formation. Importantly, neointimal cells exhibit low to no caveolin-1 expression [121, 122]. Extensive loss of endothelial caveolin-1, enhanced expression of caveolin-1 in SMC and neointima formation are also observed in idiopathic and hereditary PAH, PAH associated with CHD and drug toxicity [122–125]. In *in-vitro* studies, pulmonary arterial SMCs from idiopathic PAH exhibit increased caveolin-1 expression accompanied by increased capacitive Ca2+ entry and DNA synthesis, which could be abrogated by silencing caveolin-1 [125]. Loss of EC exposes SMC to direct pressure and shear stress,

### *Endothelial Dysfunction and Disruption in Pulmonary Hypertension DOI: http://dx.doi.org/10.5772/intechopen.92177*

which is likely to result in flattening of caveolae leading to displacement of caveolin-1 to non-caveolar site on the plasma membrane. Recently, it has been shown that in the MCT + hypoxia model, at 4 weeks, the extensive loss of endothelial caveolin-1 as well as VE-Cad loss and enhanced expression of caveolin-1 in SMC are accompanied by cavin-1 loss, tyrosine phosphorylation of caveolin-1 and neointima formation. Loss of VE-Cad is indicative of loss of EC attachment to the junction [126]. Interestingly, p-caveolin-1 in cancer has been shown to make cells pro-migratory [127, 128]. As PH progresses, SMC phenotype changes from contractile to synthetic, facilitating cell migration, and neointima formation resulting in arterial occlusion. Neointima formation leads to the irreversibility of the disease [129]. In addition, increasing pulmonary blood flow either by pneumonectomy or by a shunt procedure (left subclavian and pulmonary artery) in rats treated with MCT leads to the development of neointimal lesions. Pneumonectomy or shunt alone does not lead to neointima formation [130, 131]. Furthermore, in children with significant left to right cardiac shunt, reversal of pulmonary vascular changes were seen after they underwent pulmonary artery banding to restrict the pulmonary flow. Medial hypertrophy and early intimal changes seem reversible, but not during the later stages [132, 133]. These studies demonstrate that EC injury and disruption associated with increased flow or pressure play an important role in determining the pattern of pulmonary vascular remodeling.

Apoptosis of EC in PAH is followed by proliferation of antiapoptotic EC. This concept has been confirmed in *in-vitro* studies. Sugen (VEGFR antagonist) causes initial apoptosis, and the surviving cells become hyperproliferative [134]. Importantly, increased levels of circulating EC (CEC) have been reported in PAH, and 50% of these cells expressed CD36, a marker of microvascular origin, and 25% exhibited E selectin, a marker of EC activation [135]. In children with CHD and PAH, the increased levels of CEC are reported to be associated with worse prognosis. Pulmonary ECs exhibited high expression of antiapoptotic protein Bcl-2 in cases of irreversible PAH but not in cases of reversible PAH, or in controls. Interestingly, intimal proliferation was observed only in irreversible PAH cases, but not in the reversible PAH [136, 137]*.* In addition, increased vWF levels in patients with PAH were reported to be associated with worse survival [138]. Interestingly, increased CEC levels were observed in PAH, but not in CTEPH [139]. These studies strongly support the view that the disruption and loss of EC are associated with severe PAH and poor prognosis.

Endothelial mesenchymal transition (EndMT) is a process by which ECs exhibit phenotype alteration. These cells lose endothelial characteristics and acquire the properties of myofibroblasts or mesenchymal cells. They exhibit loss of PECAM-1 and VE-Cad, in addition to caveolin-1, and express smooth muscle α-actin, fibroblast-specific protein 1 and Notch1. PECAM-1 and VE-Cad support EC integrity and junctional stability and maintain barrier function. Thus, their loss leads to the loss of barrier function and junction stability. These transformed ECs also acquire pro-inflammatory phenotype and are primed for proliferation, migration and tissue generation [81, 140]. Neointimal cells exhibit low levels of caveolin-1, but normal eNOS expression in the experimental model of PH and also in human PAH [96, 122], and sustained NO production has been shown to degrade caveolin-1 [141]. Importantly, caveolin-1 deficiency has been shown to induce spontaneous EndMT in pulmonary EC [142]. EndMT plays an important role in vascular remodeling, and it is also linked to the loss of BMPR2 in PH [143–145]. Furthermore, TGFβ1 plays a significant role in EndMT [146]. In addition, endothelial caveolin-1 depletion leads to eNOS uncoupling and oxidative stress that switches from BMPR2 signaling to TGFβR1 and thus may promote EndMT [80]. Plexogenic lesions contain increased VEGFA and VEGFR expression indicating misguided angiogenesis involving cells of EC origin. EC dysfunction in PAH model is shown to act through DNA methylation, histone protein modification and non-coding RNA [19]. Thus,

the initial apoptosis followed by the proliferation of dysfunctional and antiapoptotic EC leads to deregulation of a number of pathways resulting in neointima and plexiform lesion formation and irreversible PAH.

### **3.2 EC dysfunction without EC disruption and pulmonary hypertension**

Exposure to acute hypoxia results in pulmonary arterial contraction and elevated pulmonary artery pressure, while sustained hypoxia leads to pulmonary vascular remodeling [147]. Hypoxia impairs endothelium-dependent relaxation response [148, 149]. In the MCT model of PH, the progressive loss of endothelial caveolin-1 is accompanied by a significant reduction in the expression of HSP90 (2 weeks post-MCT) and eNOS (3 weeks post-MCT) [9]. However, hypoxia does not alter the protein expression of caveolin-1, eNOS or HSP90 in the lungs. During hypoxia, caveolin-1 and eNOS have been shown to form a tight complex *in vivo* and *in vitro*, resulting in their dysfunction [110, 150]. Normally, in response to Ca2+ agonists, eNOS dissociates from caveolin-1 and binds to HSP90. Ca2+ activated calmodulin further aids in recruitment of HSP90, thus facilitating the release of eNOS from caveolin-1 [151]. However, hypoxia disrupts eNOS/HSP90 binding [152]. Furthermore, normally functioning caveolin-1 is required for the plasma membrane localization of TRPC4 and endothelial Ca2+ entry [153], and introduction of caveolin-1 scaffolding domain restores Ca2+ entry during chronic hypoxia [154]. Thus, the hypoxia-induced caveolin-1 and eNOS complex formation may in part be responsible for the deregulation of Ca2+ entry and disruption of HSP90/ eNOS binding leading to impaired vascular relaxation. Statins have been shown to disrupt hypoxia-induced abnormal coupling of eNOS and caveolin-1, thus restoring eNOS function and attenuating hypoxia-induced PH [155]. Recent studies of hypoxia-induced PH in rats and cows showed no disruption of EC or any alterations in the expression of caveolin-1, VE-Cad or vWF [46, 126]. Not surprisingly, there was no enhanced expression of caveolin-1 in SMC as seen in the MCT model. However, there was evidence of caveolin-1 dysfunction, such as the activation of proliferative pathways such as PY-STAT3, β-catenin and pERK1/2, and a loss of PTEN. PTEN contains a Cav-1–binding motif and, in part, colocalizes in caveolae. Caveolin-1 determines the membrane PTEN levels through its binding sequence. The loss of PTEN during hypoxia further confirms caveolin-1 dysfunction [46].

People living at high altitude develop PH and right ventricular hypertrophy as an adaptive mechanism. Upon return to sea level, PH reverts to normal slowly [156]. These observations suggest that the absence of physical disruption of EC observed in the hypoxia model may be the reason why hypoxia-induced PH is reversible. Although hypoxia plays a role, inflammation and endothelial dysfunction are important factors that determine the development of PH in chronic obstructive pulmonary disease (COPD). The outflow obstruction in COPD results from inflammatory processes affecting airways, lung parenchyma and pulmonary vasculature. PH in COPD can develop independently of underlying parenchymal destruction and loss of lung vessels [157, 158]. Endothelial dysfunction has been observed in mild cases of COPD, and the loss of endothelium-dependent relaxation in the pulmonary vasculature correlates with the severity of the disease [159]. Importantly, the loss of endothelial caveolin-1 accompanied by enhanced expression of caveolin-1 in SMC is reported in COPD associated with PH. COPD without PH had preserved endothelial caveolin-1 [160]. In addition, severe pulmonary arterial lesions such as plexiform and angiomatoid lesions have been documented in explanted lungs after transplantation in COPD associated with severe PH. These lesions were similar to what are seen in IPAH [161]. In infants with respiratory distress syndrome, despite significantly elevated pulmonary artery pressure and significant medial thickening, pulmonary arteries

*Endothelial Dysfunction and Disruption in Pulmonary Hypertension DOI: http://dx.doi.org/10.5772/intechopen.92177*

exhibit well-preserved endothelial caveolin-1, without any evidence of EC disruption or enhanced expression of Cav-1 in SMC. In contrast, loss of endothelial Cav-1 and disruption/loss of EC coupled with enhanced expression of Cav-1 in SMC were observed in infants with bronchopulmonary dysplasia and associated inflammation [123]. These results indicate that irrespective of the underlying disease, EC disruption leads to the loss of endothelial caveolin-1 and subsequent enhanced expression of Cav-1 in SMC, followed by neointima formation and irreversible PH. Thus, the EC disruption puts the patients at a higher risk of developing irreversible PH.

### **4. Conclusions**

Under normal conditions, ECs play a key role in maintaining SMCs in quiescent state and vascular homeostasis. Caveolin-1, a major protein constituent of caveolae on the cell membrane, regulates multiple cellular processes including inflammation, molecular transport, cell proliferation, adhesion, migration and signal transduction. Caveolin-1 interacts with protein molecules that are in or are recruited to caveolae and maintains them in inhibitory confirmation. Endothelial caveolin-1 loss and caveolin-1 dysfunction lead to PH.

### **4.1 EC disruption and caveolin-1 loss**

Injury such as inflammation, increased pulmonary blood flow associated with increased pressure, drugs and toxins can cause endothelial disruption, which is usually progressive. Endothelial disruption leads to the progressive loss of endothelial membrane proteins including caveolin-1, PECAM-1 and VE-Cad. These alterations lead to deregulation of multiple pathways. As depicted in **Figure 1**, (a) the loss of caveolin-1 is accompanied by reciprocal activation of proliferative and antiapoptotic pathways leading to SMC hypertrophy and proliferation. (b) Further loss of EC exposes SMCs to direct pressure resulting in enhanced expression of caveolin-1 in SMCs. Tyrosine phosphorylated caveolin-1 could alter the phenotype and facilitate cell migration leading to neointima formation. (c) Loss of PECAM-1 and VE-Cad results in the loss of barrier function and junction stability. These alterations lead to EndMT. These cells lose endothelial properties and acquire pro-inflammatory phenotype and are primed for proliferation, migration and tissue generation and participate in neointima formation, thus leading to irreversible PH.

### **Figure 1.**

*This figure depicts the pathway leading from endothelial cell disruption to irreversible PH. Cav-1 = caveolin-1, EC = endothelial cells, EndMT = endothelial mesenchymal transformation, PECAM-1 = platelet endothelial cell adhesion molecule 1, pCav-1 = tyrosine phosphorylated caveolin-1, PH = pulmonary hypertension, SMC = smooth muscle cells.*

### **4.2 EC and caveolin-1 dysfunction**

Hypoxia exposure to EC leads to a tight complex formation between caveolin-1 and eNOS, resulting in the dysfunction of both factors (**Figure 2**). Importantly, there is no EC disruption or the loss of caveolin-1 or any other membrane proteins. Since there is no loss of EC, medial layer is not exposed to shear stress and pressure. Not surprisingly, there is no enhanced expression of caveolin-1 in SMCs. However, caveolin-1 and eNOS dysfunction lead to SMC proliferation, medial hypertrophy and loss of endothelial-dependent vascular relaxation. Removal of hypoxia results in the disruption of caveolin-1/eNOS tight complex leading to reversal of PH. Slowly, the pulmonary artery pressure and medial hypertrophy return to normal as seen in experimental animals and in people returning to sea level from high altitude. Hypoxia-induced PH is reversible. However, associated inflammation/shear stress in hypoxia-induced PH, resulting in EC disruption, would lead to irreversible PH.

In conclusion, EC integrity and caveolin-1 function are important factors that determine reversible vs. irreversible PH.

### **Figure 2.**

*This figure shows the effect of hypoxia on EC leading to eNOS/caveolin-1 complex formation, endothelial dysfunction and subsequent medial hypertrophy and PH. EC = endothelial cells, Cav-1 = caveolin-1, eNOS = endothelial nitric oxide synthase, SMC = smooth muscle cells, PH = pulmonary hypertension.*

### **Acknowledgements**

This work is in part supported by Cardiovascular Medical Research and Education Fund.

*Endothelial Dysfunction and Disruption in Pulmonary Hypertension DOI: http://dx.doi.org/10.5772/intechopen.92177*

### **Author details**

Rajamma Mathew Department of Pediatrics, Section of Pediatric Cardiology, New York Medical College, Valhalla, NY, USA

\*Address all correspondence to: rajamma\_mathew@NYMC.edu

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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### **Chapter 2**

## Pathogenesis of Abdominal Aortic Aneurysm

*Michael Patel, Daniel Braga, Brad Money, Andres Pirela, Adam Zybulewski, Brandon Olivieri and Robert Beasley*

### **Abstract**

Abdominal aortic aneurysms (AAAs) are encountered by many healthcare providers such as interventional radiologists, vascular surgeons, cardiologists, and general practitioners. Much effort has been placed in the screening, diagnosis, and treatment of AAA with somewhat little understanding of its pathophysiology. AAA is a complex disease typically segmented into a process of proteolysis, inflammation, and vascular smooth muscle cell (VSMC) apoptosis with oxidative stress balancing its components. AAA and other aortic syndromes such as aortic dissection share this same process. On the other hand, AAA formation and aortic pathology may be acquired through infection like in mycotic aneurysm or may be genetic in origin such as seen with Ehlers-Danlos and Marfan syndromes.

**Keywords:** abdominal, aortic, aneurysm, dissection, mycotic, atherosclerosis, proteolysis, inflammation, oxidative stress, VSMC apoptosis, Marfan, Ehlers-Danlos, endovascular, vascular

### **1. Introduction and background of AAA**

Abdominal aortic aneurysm (AAA) is a complex disease comprised of multifactorial molecular processes that carry a host of players yet to be solidified in literature. Although options continue to expand in the treatment of AAA, understanding the pathophysiology is pivotal for the development of screening tests and pharmacological treatment modalities.

In this chapter, we will go beyond the clinical context of AAA and discuss the various pathologic pathways that lead to its creation. Some of these pathways overlap with other aortic pathologies such as aortic dissection as well as mycotic aneurysm. Lastly, we will discuss common genetic disorders that are predisposed to aortic aneurysm and aortic dissection.

### **2. Abdominal aortic aneurysm**

### **2.1 Normal anatomy and histology**

The aorta is the main artery of the body that carries oxygenated blood from the heart to the remaining major arteries of the body. It may be segmented into the thoracic aorta and abdominal aorta based on its location to the diaphragmatic hiatus.

There are three sheaths that make up the aortic wall: tunica intima, tunica media, and tunica adventitia. The intimal layer is thin and mainly composed of endothelial cells, while the tunica media is the largest component of the aortic wall and consist of elastic fibers, smooth muscle cells, and collagenous tissue. Connective tissue makes up the most outer layer called the tunica adventitia and contains small blood vessels known as the vasa vasorum, which supply the cells of the arterial wall.

An aneurysm is defined as the localized dilatation of a vessel exceeding 1.5 times the normal diameter of the vessel, which is defined as greater than 3 cm in the abdominal aorta. As the abdominal aorta dilates, it becomes prone to rupture or tearing within the layers of its wall, otherwise known as aortic dissection (AD). In AAA and AD, patients may present with low blood pressure and a tearing sensation in the chest or back. When blood rushes into the medial layer forming a new, "false" lumen, further expansion can compress the "true" lumen causing downstream ischemia.

### **2.2 Role of aortic atherosclerosis**

Atherosclerosis is often present in the setting of aortic pathology and although no causal pathway has been established, understanding the bridge between atherosclerosis and the inflammatory response in AAA remains essential. Early atherogenesis begins with subendothelial retention of circulating lipoproteins on proteoglycans within the extracellular matrix of the arterial wall. Aggregation and oxidation of retained lipoproteins further leads to a maladaptive immune response with circulating monocytes entering the subendothelium, differentiating into macrophages, ingesting the modified lipoproteins, and transforming into the classic "foam cell." The release of cytokines in this process, such as tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), leads to immune cell infiltration and inflammation [1, 2].

### **2.3 Pathogenesis of abdominal aortic aneurysm**

Much of our understanding of AAA until this point arises from histopathological studies dating back to 1972 when the "Inflammatory" variant of AAA was described [3]. Since then, the most prevalent features of studied AAA segments have demonstrated elastin degeneration, immune cell infiltration, and apoptosis of vascular smooth muscle cells (VSMCs). Although the complete pathogenesis is unknown, information from animal models, histopathological studies and genomewide association studies (GWAS) may be used to partition this intricate process into proteolysis, inflammation, and vascular smooth muscle cell (VSMC) apoptosis. Oxidative stress appears to be a major player as well and balances different facets of AAA development and growth.

### *2.3.1 Proteolysis*

During aneurysmal growth, significant proteolytic degradation of elastin, collagen, laminin, fibronectin, and many other extracellular matrix (ECM) proteins occurs in the arterial wall. Upregulation of proteolytic enzymes in the aortic wall is stimulated by the presence of oxidized LDL and cytokines such as TNF-α, IL-1, and IL-3 [4]. The most famous set of proteolytic enzymes are known as the matrix metalloproteinases (MMPs), which have been implicated in cancer, wound healing, and many other processes. During normal homeostasis, regulation of MMPs in the aortic wall is carried out by tissue inhibitors of MMPs (TIMPs), but a higher MMP/ TIMP ratio is typically observed in aneurysms [5, 6].

### *Pathogenesis of Abdominal Aortic Aneurysm DOI: http://dx.doi.org/10.5772/intechopen.91670*

Of the multiple MMPs, MMP-9 and MMP-2 are considered crucial participants in AAA and aortic dissection (AD) development, both significantly upregulated in AAA segments with MMP-9 expression correlating with aneurysm diameter. MMP-9 is derived from macrophages and neutrophils and MMP-2 is produced by smooth muscle cells and fibroblasts; together, they both balance ECM remodeling, inflammation, and VSMC apoptosis through various signaling pathways such as the MAPK (mitogen-activated protein kinase)/ERK pathway [7]. TGF-beta signaling pathways help balance MMP-2 and MMP-9 in addition to creating protection against AAA formation by increasing type I and III collagen production and upregulating protease inhibitors [8].

### *2.3.2 Inflammation*

Innate and adaptive arms of the immune system are involved in the development and growth of AAA. Neutrophil infiltration and release of elastase induces early degradation of the ECM in the aortic wall. Elastin breakdown products trigger either pro-inflammatory or anti-inflammatory macrophages in the adventitial layer of the aortic wall. T-cell derived interferon gamma and B cells are also involved in AAA formation. B cells provide a source of immunoglobulins, complement pathway, and cytokines, which add to the complexity of AAA formation [9–11].

### *2.3.3 VSMC apoptosis*

Necrosis and apoptosis have traditionally been deemed different mechanisms [12]. Apoptosis is considered an organized and instructed route of cell death while necrosis is regarded as an unorganized disruption of a cell with an additional immune response. With this in mind, VSMC apoptosis occurring in the tunica media of the aortic wall in AAA formation is not a recent discovery, although the exact phenomenon is not known.

In addition to a review on cell death nomenclature, Wang et al. describe receptor-interacting protein kinase 3 (RIP3) on VSMCs as a key player in a structured form of necrosis, also known as necroptosis [12]. It is believed that RIP3 mediating inflammatory cytokine production by smooth muscle cells is mediated in the aortic wall through TNF-α signaling pathways. Additionally, protein kinase C-delta (PKC) is upregulated in aneurysmal tissues which lack VSMCs and murine models have demonstrated decreased RIP3 levels in aortic tissue that lacks PKC, further validating the role of PKC in AAA formation [13, 14].

### **2.4 Oxidative stress**

### *2.4.1 Defining oxidative stress*

Oxidative stress is defined as cellular injury induced by reactive oxygen species (ROS) and reactive nitrogen species (RNS), taken as a combined ROS/RNS system [15]. Pathologic oxidative stress to the vasculature occurs via a multi-faceted, highly complex mechanism that is thought to occur in part by the ROS/RNS system. The ROS/RNS system is defined as a group of molecules consisting of free radicals or molecules which predispose to free radical formation. A free radical is any species that contains one or more unpaired electrons that is capable of existing independently, which makes them highly unstable and will react readily with lipids, cellular proteins, and nucleic acids [15, 16].

### *2.4.2 Normal pathophysiology and regulation*

The production of ROS/RNS is highly regulated and occurs naturally to some degree in all normal cells due to the incomplete reduction of molecular oxygen to water during cellular respiration and within phagosomes of phagocytic cells (chiefly neutrophils and macrophages) [15]. ROS/RNS are typically short-lived, owing to their instability and prompt removal/inactivation by endogenous cellular antioxidants and antioxidant enzymes such as catalase, glutathione peroxidase, and superoxide dismutase. Dysregulation occurs when ROS/RNS production exceeds clearance.

### *2.4.3 The various ROS/RNS entities and their chemical reactions*

The principal ROS/RNS involved in cellular injury include superoxide, hydrogen peroxide, hydroxyl radicals, and peroxynitrite. These entities mediate vascular damage either directly or indirectly by conversion to more reactive substances [15, 16].


### *2.4.4 Reactive nitrogen species*

In addition to ROS, reactive nitrogen species (RNS) also play a role in the pathophysiology of vascular dysfunction. Nitric oxide (NO) is produced via an l-arginine precursor by nitric oxide synthases with multiple cofactors. Encoded isozymes of mammalian NOS include endothelial, neuronal, and inducible subtypes (eNOS, nNOS, and iNOS, respectively). Endothelial homeostasis rests firmly upon tight regulation of endogenous NO production, but pathologic uncoupling of NOS isozymes or IFN-γ mediated NO production by macrophages can lead to excess NO and/or ROS. Peroxynitrite (ONOO<sup>−</sup>), a powerful non-radical nitrosative stressor, is formed by the reaction of O2 •<sup>−</sup> and NO and serves as the basis for other RNS derivatives such as nitrogen dioxide (NO2) [16, 17].

Peroxynitrite is a highly reactive proatherogenic mediator and readily reacts with protein side chains and carbon dioxide to cause cellular injury [16]. A notable example is 3-nitrotyrosine, formed by the reaction of peroxynitrite with tyrosine, which has been suggested as a local marker of oxidative stress in immunostained samples of aneurysmal aortas [19]. Additionally, Kotlarczyk et al. revealed a pathway consisting of oxidative and hemodynamic stress on the aortic wall leading to increased superoxide production and NO bioavailability. This linkage corresponded to an increased rate of asymmetric thoracic aorta dilatation in patients with bicuspid aortopathy versus their tricuspid counterparts [20].

### *2.4.5 Role of oxidative stress in aortic pathology*

The role of oxidative stress in the pathogenesis of aortic pathologies such as aortic aneurysm involves pathologic vascular remodeling along with dysfunctional balancing of connective tissue breakdown and synthesis by VSMCs [19–21]. This is thought to occur due to several mechanisms, including ROS/RNS induced VSMC apoptosis and enhanced matrix metalloproteinase (MMP) activity, which leads to progressive weakening of the aortic wall, dilatation, and eventual aneurysm formation via the breakdown of collagen, elastin, and laminin. Oxidative stress is a major modulator of MMP formation and can disrupt the corresponding balance of TIMPs that are otherwise crucial to the structural integrity of the extracellular matrix of the arterial wall [21, 22].

Additionally, ROS can disrupt VSMC proliferation via a mechanism linked to the relative local redox microenvironment concentrations of hydrogen peroxide and lipid hydroperoxides [16, 18, 23]. Hydrogen peroxide is involved in various pathways and serves as a mediator of vascular inflammation, upregulating various chemotactic and adhesion molecules such as ICAM-1, IL-8, and P-selectin which facilitate leukocyte migration into the aortic wall. In a study of patients undergoing elective infrarenal AAA repair, tissue samples of aneurysmal aortic segments demonstrated superoxide levels 2.5 times that of adjacent non-aneurysmal aortic segments, as well as increased expression and activity of NADPH oxidase [18]. In addition, changes in normal local blood flow hemodynamics in aneurysmal aortas may also induce ROS production and contribute to aortic remodeling and dissection [20, 24, 25].

Xanthine oxidoreductase (XOR) is a famous complex molybdoflavin protein known to healthcare providers as a catalyzer in the terminal steps of purine degradation, and when therapeutically inhibited, a target for treatment of hyperuricemia and gout. Although XOR may be involved in the pro-inflammatory state associated with crystal formation in gout, it may have an antioxidant role when under the optimal conditions, which necessitates further studies [26].

### **2.5 Aortic dissection**

Similar to AAA, the physiology of aortic dissection (AD) entails a complex multifactorial process consisting of proteolysis, inflammation, and VSMC apoptosis. The differentiating factor is that hemodynamic stress results in intimal tearing of the aortic wall allowing blood to rush into the medial layer. This process creates a "true" and "false" lumen that may propagate in either direction to occlude the true lumen and/or cause a variety of issues resulting in significant morbidity and mortality.

### **2.6 Mycotic aneurysm**

A mycotic aortic aneurysm is characterized by a local, irreversible dilatation of the aorta which is secondary to a direct bacterial or fungal inoculation of the vessel wall. The term mycotic aneurysm is actually a misnomer, as these "infective" aneurysms are most commonly bacterial in nature and fungal to a lesser extent. Staphylococcus and Salmonella species are the two most commonly cultured organisms in mycotic aneurysms, however, improved bacteriologic techniques have led to the detection of anaerobic bacteria (mostly Bacteroides, and Clostridium spp.). Mycotic aneurysms are rare as they only represent 1–2.6% of all aortic aneurysms [27, 28].

The formation of mycotic aneurysms is initiated by a microbial induced proinflammatory cascade of cytokines, such as TNF-α, IL-1, IL-6, invading the aortic vessel wall [28]. The recruitment of inflammatory cells within the vessel causes functional changes to VSMCs and endothelial cells with subsequent loss of integrity in the tunica media. This intense cytokine cascade causes mycotic aneurysms to progress more rapidly and aggressively than inflammatory aneurysms and thus have a higher mortality rate when compared [29].

Mycotic aneurysms most commonly affect diseased aortic endothelium in the setting of bacteremia and may present as nonspecific back pain or abdominal pain depending on the location of the lesion. Patients will typically be febrile, indicating a systemic infection, and lab values will show signs of leukocytosis and elevated ESR. Importantly, Gram negative organisms tend to cause a more virulent arterial infection than Gram positive bacteria, which makes the resultant aneurysm even more prone to rupture and further increases the risk of mortality [30–32].

Treatment of mycotic aneurysm focuses on empiric antibiotic therapy while waiting for blood culture susceptibility panel with individualized duration, surgical excision with wide debridement of infected tissues, and revascularization as needed.

### **2.7 Screening and diagnosis of AAA**

The primary role of AAA screening and surveillance is mortality reduction, primarily through one-time and/or periodic non-invasive imaging. The initial workup of any aortic pathology begins with a focused history and physical examination. Classically, AAA may present with a pulsatile epigastric abdominal mass, but many patients are asymptomatic and lack this finding. However, the physical exam may assist in identifying more distal aneurysmal disease, particularly those occurring in the femoropopliteal distribution, which may be predictive of coexisting AAA. The physical examination is also crucial to determine a patient's baseline status in terms of perioperative risk with regards to a future surgical or endovascular intervention [33]. A number of serum biomarkers and genetic factors are known to be associated with AAA, and despite being an exciting area of developing research, the prognostic and diagnostic value of these factors has not yet been validated clinically, and therefore do not yet play a significant role in the diagnosis and management of AAA [33].

*Pathogenesis of Abdominal Aortic Aneurysm DOI: http://dx.doi.org/10.5772/intechopen.91670*

### *2.7.1 Screening*

Ultrasound (US) and computed tomography (CT) angiography are the two primary imaging modalities used for AAA and are both highly accurate and reproducible. Transabdominal US is relatively inexpensive and can be performed in minutes without the use of ionizing radiation or iodinated contrast media. US carries a sensitivity and specificity approaching 100% in asymptomatic patients with AAA, making it the modality of choice for AAA screening and surveillance. Both the Society for Vascular Surgery (SVS) and the US Preventive Services Task Force (USPSTF) recommend a one-time screening ultrasound for AAA in men or women 65–75 years of age with a history of tobacco use [33, 34]. Various additional recommendations exist for that of first-degree relatives of those presenting with AAA, follow-up US examinations based on initial aortic diameter at initial screening, and screening in non-smokers or females, but these recommendations are supported by lower-level data or are of unclear benefit. Obesity, overlying bowel gas, and user dependence are recognized limitations ultrasound evaluation for AAA, and US may underestimate AAA size by 2 mm [33, 34]. However, limitations are invariably offset by the aforementioned benefits, and US can be useful evaluating other causes of abdominal pain, particularly in the emergent setting, resulting in a reduction in time to diagnosis and treatment. When AAA repair is indicated in an otherwise stable patient, CT offers more precise pre-operative planning via multiplanar orthogonal measurements.

### *2.7.2 Surveillance*

Aside from baseline screening, AAA surveillance also plays a significant role in mortality reduction, by monitoring changes in AAA size over time and subsequent timely identification of patients whose risk of rupture begins to approach or outweigh the risks of intervention. Despite multiple large-scale clinical research trials comparing AAA size versus risk of rupture, vascular and radiology literature has yet to produce a single unifying surveillance parameter, but several evidence-based criteria allow patients to be safely observed over time despite a relatively small background risk of rupture. The SVS recently provided updated guidelines for the surveillance of patients with AAA, including recommended surveillance imaging at 3-year intervals for patients with AAA between 3.0 and 3.9 cm in diameter, 12-month intervals for 4.0–4.9 cm, and 6-month intervals for 5.0 and 5.4 cm [33]. The American College of Radiology (ACR) appropriateness criteria designates duplex ultrasound of the aorta/abdomen, CTA of the abdomen and pelvis with intravenous contrast, or MRA of the abdomen and pelvis with intravenous contrast as "usually appropriate" (a rating of 7, 8, or 9) for surveillance of asymptomatic AAA without previous repair [35].

The remaining aortic pathologies, including that of aortic dissection, present with a wide range of clinical, laboratory, and imaging findings, and therefore are similarly evaluated with CT or CT angiography for prompt diagnosis.

### **2.8 Treatment of AAA**

### *2.8.1 Medical therapy*

Hypertensive disease is the main major risk factor for aortic thoracic disease with genetic predisposition as second major risk factor [36, 37]. Although, this notion is based on studies mostly including Marfan disease patients [38]. For patients with asymptomatic AA, anti-hypertensive therapy with beta blockers is recommended for blood pressure control with the goal being to limit aortic wall expansion. Angiotensin converting enzyme inhibitors or angiotensin receptor blockers (ARBs) are preferred as well due to their role as modifiers of inflammatory mediators and by decreasing vascular smooth muscle apoptosis [38, 39].

Beta blockers have been the traditional treatment for thoracic aortic disease. It was originally demonstrated more than 70 years ago when turkeys eating sweet pea seed, *Lathyrus odoratus*, which contains the lysis oxidase inhibitor, B-aminopropionitrile, die of acute aortic dissections. The beta blocker propranolol was found to decrease deaths from dissection in B-aminopropionitrile fed turkeys [40–43]. Beta-blockers such as propranolol benefit the aortic wall through negative inotropic and chronotropic effects. Through these effects, the elastic fibers of the wall are protected from further damage and is further supported by reduction in left ventricular pressure of the heart and heart rate [44]. However, the benefit of beta-adrenergic blockade is better established in aortic dissection than in AAA because beta blockers provide theoretical benefit on blood pressure and left ventricular pressure reduction [45].

Angiotensin converting enzyme inhibition may have a beneficial role by modifying inflammatory mediators and decreasing vascular smooth muscle apoptosis. Angiotensin receptor blockers such as Losartan have been shown to prevent expansion of aneurysms by downregulation of transforming growth factor B [38, 46, 47]. Angiotensin II type 1 receptor blockade within the reninangiotensin-aldosterone system causes a decrease in TGF-B signaling further reducing levels of intracellular mediators within the TGF-B signaling cascade, such as phosphorylated SMAD [37, 48]. By this mechanism, there is a reduced proliferation of vascular smooth-muscle cells, fibrosis, and expression of matrix metalloproteinases [49]. Overactivation of the angiotensin II type 2 receptor pathway by ARBs causes antiproliferative and anti-inflammatory effects that are beneficial in aortic wall homeostasis [50]. In contrast, ACE inhibitors limit the production of angiotensin II, producing a negative effect on Angiotensin II type 1 and type 2 receptor pathways which do not influence alternative mechanisms [46].

Medical treatment with statins (3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors) power many of the inflammatory pathways of the formation of aortic aneurysms. Statins provide a protective effect by inhibition of matrix metalloproteinases (MMPs) and plasminogen activator due to the aforementioned proteolytic enzymes involved in the pathophysiology of aortic aneurysm formation [39, 51, 52].

### *2.8.2 Surgical and endovascular treatment*

Historically, abdominal aortic aneurysm was treated with open surgical repair once aneurysmal growth reached a certain size or rate. Recent advancements have allowed endovascular repair to be used as the primary modality of repair with open surgical repair reserved for emergency/unconventional situations.

### **3. Genetic etiologies of aortic aneurysms and dissections**

Genetics play an important role in the pathogenesis of various diseases. While multifactorial processes have been described in the development of AAA and AD, there are two Mendelian disorders which may lead to AAA or AD, Marfan syndrome and Ehlers-Danlos syndrome (EDS). Both have been connected to the development of AAs and ADs lacking classic risk factors such as smoking, hypertension, and old age. By definition, both of these syndromes result from mutations in a single gene

and inheritance pattern. Overall, both Marfan syndrome and EDS share deleterious effects on connective tissues in the body which consequently have major ramifications on the integrity of major blood vessels.

### **3.1 Ehlers-Danlos syndrome**

Ehlers-Danlos syndromes (EDS) are a rare group of inherited disorders of collagen which ultimately impair the integrity of the extracellular matrix of supporting structures such as connective tissues. Clinically, people with EDS usually feature remarkable hyperelastic skin, hypermobile joints, and often a bleeding diathesis. At least six clinical and genetic variants of EDS have been established and they all share a generalized defect in collagen, including abnormalities in its structure, synthesis, secretion and degradation.

In this discussion, the vascular subtype of EDS, previously referred to as type IV EDS, is the focus of this section. The vascular subtype of EDS (vEDS) is manifested from a key mutation affecting the COL3A1 gene, which subsequently causes deficient synthesis of type III procollagen. The diagnosis of vEDS is made from major and minor clinical criteria and can be confirmed by abnormalities in procollagen production as seen in protein gel electrophoresis and molecular genetic testing.

### *3.1.1 Epidemiology of EDS*

The incidence of vEDS is roughly 1:100,000 with a total of 1500 affected individuals in the United States having been identified on the basis of biochemical and genetic testing and analysis of family pedigrees [53].

### *3.1.2 Molecular genetics of EDS*

The COL3A1 gene is found on chromosome locus 2q32.2 and encodes for type III pro-collagen. The COL3A1 is estimated to be over 44 kb in size [54]. The vEDS subtype is inherited in an autosomal dominant pattern.

### *3.1.3 Pathogenesis of EDS*

Type III collagen is extremely prevalent in skin, vessel walls and reticular fibers of most tissues such as the lungs, liver, and spleen. Mutations in the COL3A1 gene responsible for vEDS can take various forms. These include point mutations, deletions, insertions, splicing mutations, and missense mutations. The most common genetic mutation associated with vEDS is a missense mutation of a crucial glycine residue in the triple helical domain of the alpha-1 (III)-chains of type III procollagen. The mutation almost always occurs in a particular region of the protein that is used to bind to other collagen proteins. Three collagen proteins always bind together into a trimer, which is required for collagen functionality; when not bound in a trimer, collagen is useless, as it cannot provide functional or structural support [55].

The most common missense mutation recognized in the literature is a substitution of glycine to glutamic acid or lysine (Glu>Lys), both leading to the production of a defective polypeptide and disrupted (Gly-X-Y)n collagen motif [56]. This leads to the development of severely malformed collagen fibrils and reticulin fibers in the extracellular matrix of dermal and arterial tissues.

Type III procollagen is a major structural protein in hollow organs and vessel walls. An altered structure of the protein makes it dysfunctional in large elastic arteries such as the aorta causing them to be more prone to rupture or dissection. The mechanisms by which mutant type III collagen molecules create vascular

fragility are not well understood in humans, though clinically vEDS is characterized by weakness of tissues rich in type III collagen, such as blood vessels, thus predisposing them to aneurysm and dissection [57].

### **3.2 Marfan syndrome**

Marfan syndrome is caused by an inherited mutation of the FBN1 gene coding for the extracellular glycoprotein Fibrillin-1. The mutation in FBN1 initiates instability of connective tissue extracellular matrix, manifesting broadly as changes to the skeleton, eyes, and cardiovascular system. There have been more than 1800 distinct causative mutations in the FBN1 gene which complicates the diagnosis by DNA sequencing alone. As a result, the diagnosis of Marfan syndrome is mainly based on clinical findings. Classically you will see ectopia lentis, tall stature with coinciding arachnodactyly, and hyperlaxity of joints.

### *3.2.1 Epidemiology of Marfan syndrome*

The prevalence of Marfan syndrome is estimated to be 1 in 5000. According to National Human Genome Research Institute, roughly 75% of cases are familial and the remaining 25% of cases are a result of a new (de novo) mutation in the FBN1 gene. In a 2015 study involving 412 people confirmed as having Marfan syndrome, the median age at diagnosis is found to be 19.0 years [58].

### *3.2.2 Molecular genetic of Marfan syndrome*

Fibrillin-1 is encoded for by the FBN1 gene (chromosome locus 15q21) which is estimated to be 235 kb in size [59]. Marfan syndrome is inherited in an autosomal dominant pattern.

### *3.2.3 Pathogenesis of Marfan syndrome*

Fibrillin-1 is secreted by fibroblasts, is modified post-translationally by glycosylation, and is the major component of microfibrils found in the extracellular matrix of connective tissue. Microfibrils are widely distributed in the body, more specifically they are abundant in the aorta, ligaments, and the ciliary zonules that support the ocular lens. This distribution of microfibrils gives rise to the unique clinical presentation classically known as Marfanoid habitus.

More recently, microfibril-associated glycoprotein 4 (MFAP4) has been linked to the pathogenesis of Marfan syndrome. Yin et al. using a glycoproteomic analysis of aortic extracellular matrix in Marfan patients, found an increased and more diverse N-glycosylation of MFAP4 in patients with Marfan syndrome compared with control patients. Most importantly in our discussion of AA and AD, this increased N-glycosylation was particularly in the aneurysmal stages [60, 61].

The defective Fibrillin-1 protein and subsequent faulty microfibrils are fundamental in the progression to an aortic aneurysm or an aortic dissection seen in Marfan syndrome. Not only do microfibrils provide structural integrity of specific organ systems, but they also provide a scaffold for elastogenesis in elastic tissues, most notably in elastic arteries such as the aorta [62]. In a way, malfunctioning FBN1 gene inserts malware into microfibrils, thus dismantling the scaffold needed for elastogenesis. This defective elasticity in the tunica media of elastic arteries such as the aorta weakens the vessel wall predisposing to early aneurysm. Weakening of the media also predisposes to any intimal tear, which may initiate an intramural hematoma that cleaves the layers of the media to produce aortic dissection.

### *Pathogenesis of Abdominal Aortic Aneurysm DOI: http://dx.doi.org/10.5772/intechopen.91670*

Interestingly, the loss of microfibrils also gives rise to abnormal and excessive activation of transforming growth factor-B (TGF-B). Normally sequestered by wellfunctioning microfibrils, excessive TGF-B signaling has deleterious effects on both vascular smooth muscle development and the overall integrity of the extracellular matrix at a cellular level. Excessive TGF-B signaling in the adventitia of large elastic arteries causes increased deposition of weak fibrotic tissue leading to aneurysm development [58].

### **4. Conclusion**

The pathogenesis of AAA formation is complex and multidimensional. The traditional atherosclerotic or inflammatory variant of AAA may be segmented into a process of proteolysis, inflammation, and VSMC apoptosis. Oxidative stress acts as a fulcrum throughout this process which is also involved in other acquired aortic pathologies such as mycotic aneurysm and aortic dissection. Classically, aortic pathology is affiliated with connective tissue disorders like seen in Marfan and Ehlers-Danlos syndromes. With further studies and eventual development, the understanding of AAA formation as well as other aortic pathologies will lead to additional treatment tools for vascular specialists and other healthcare providers alike.

### **Author details**

Michael Patel\*, Daniel Braga, Brad Money, Andres Pirela, Adam Zybulewski, Brandon Olivieri and Robert Beasley Mount Sinai Medical Center, Nova Southeastern University College of Osteopathic Medicine, Miami, FL, USA

\*Address all correspondence to: michaelpatel813@gmail.com

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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### **Chapter 3**

## The Importance of Autophagy and Proteostasis in Metabolic Cardiomyopathy

*María Cristina Islas-Carbajal, Ana Rosa Rincón-Sánchez, Cesar Arturo Nava-Valdivia and Claudia Lisette Charles-Niño*

### **Abstract**

Metabolic cardiomyopathy and other heart disorders are associated with proteostasis derailment and subsequent autophagy. Proteostasis is a process of protein homeostasis, and autophagy is a mechanism of self-degradation for surviving cells facing stressful conditions. Metabolic challenges have been linked to excess reactive oxygen species. Cardiomyocyte proteotoxicity, an important underlying pathologic mechanism in cardiac disease, is characterized by chronic accumulation of misfolded or unfolded proteins that can lead to proteotoxic formation or aggregation of soluble peptides. Autophagic processes are mediated by the ubiquitinproteasome and autophagy-lysosome systems, fundamental for cardiac adaptation to physiological and pathological stress. Cellular proteostasis alterations in cardiomyopathy are represented by myocardial remodeling and interstitial fibrosis with reduced diastolic function and arrhythmias. Autophagy regulation may be a potential therapeutic strategy for metabolic cardiomyopathy necessary for the treatment of fibrosis and cardiac tissue remodeling alterations. Furthermore, autophagy has been shown to be active in the perimeter of cardiovascular fibrotic tissue as mechanism of fibrosis recovery and scarring secondary to cell apoptosis. In the present work, we review the current knowledge on the role of autophagy and proteostasis in the pathogenesis of heart failure to resolve the ever-expanding epidemic of metabolic cardiomyopathy and heart failure associated with substantial morbidity and mortality.

**Keywords:** autophagy, proteostasis, cardiac hypertrophy, metabolic cardiomyopathy, myocardial interstitial fibrosis, heart failure

### **1. Introduction**

Metabolic cardiomyopathies can be caused by disturbances in metabolism and may develop in the context of a broad spectrum of pathological conditions. These disorders include a number of inherited metabolic diseases in early childhood affecting the heart and other organs. Cardiomyopathies are associated with systemic metabolic diseases acquired during adulthood, such as metabolic syndrome, dyslipidemia, obesity, hypertension, diabetes mellitus and cardiomyopathy by alcoholism [1], which are also considered important causes of cardiovascular diseases. Furthermore, abnormal mitochondrial function related to mitochondrial ATP-producing capacity and high cardiac energy demand is linked to several of these cardiovascular diseases. The heart is a high-energy-demanding organ and mitochondria are important organelles that provide its source of cellular energy by oxidative phosphorylation; however, enzyme deficiency related to mitochondrial beta-oxidation leads to cardiac disorders. Another key point is that autophagic activity has been found to decrease with age resulting in intracellular protein aggregate accumulation, unfolded protein response activation and subsequent cardiomyocyte apoptosis, likely contributing to the accumulation of damaged macromolecules and organelles during aging. Equally important, several forms of heart failure are progressive disorders associated with substantial morbidity and mortality, and of these, cardiovascular pathologies are the leading cause of death in the elderly. Autophagy, a lysosome-mediated degradation pathway, plays a critical role in proteostasis by removing potentially toxic cytosolic protein aggregates and damaged organelles within cells [2]. Cardiomyocyte proteostasis is the gradual derailment of cellular protein homeostasis important to protein quality control [3]. The dysfunction in proteostasis leading to the accumulation of protein aggregates is the hallmark of cardiovascular disease and many chronic and age-related diseases [4].

The metabolic syndrome has become one of the most important topics in recent decades because of the marked increase in cardiovascular risk associated with the clustering of risk factors [5]. Obesity is a major independent risk factor for cardiovascular disease, including cardiac hypertrophy and heart failure. Leptin, an adipocyte-derived hormone, acts through its receptors (LepRs) on hypothalamic neurons that regulate body weight and energy homeostasis. LepRs are also expressed on cardiovascular cells, and leptin has also been shown to promote cardiomyocyte hypertrophy, endothelial proliferation, migration and angiogenesis, and fibrosis.

The effects of the mechanistic target of rapamycin (mTOR) are mediated through its activity as a central inhibitor of autophagy, a highly conserved cell survival mechanism. Cardiac hypertrophy is associated with increased energy demands, and cellular stressors like ischemia or nutrient deprivation, which result in the rapid regulation of myocardial autophagy. In this context, endothelial cells are particularly sensitive to metabolic stress, and defective or maladaptive endothelial autophagy may contribute to the rarefaction of the cardiac microvasculature during hypertrophy, a critical event in the transition toward heart failure [6]. In the present work, we review the current understanding of the role of autophagy and proteostasis in the pathogenesis of heart disease, considering the essential involvement of both degradation processes to find a novel therapeutic target to resolve the ever-expanding epidemic of metabolic cardiomyopathy and heart failure associated with significant morbidity and mortality.

### **2. Proteostasis**

A complex proteostasis network functions to ensure the maintenance of proteostasis, consisting of molecular chaperones and proteolytic machineries and their regulators in healthy cells. Each type of these molecules with a precise amino acid sequence has important physical properties to determine specific protein structure and a three-dimensional conformation to proteins, which is important in order to regulate cellular performance and balance. Protein structures are made by *The Importance of Autophagy and Proteostasis in Metabolic Cardiomyopathy DOI: http://dx.doi.org/10.5772/intechopen.92727*

the formation of peptide bonds that build the polypeptide long chains of alphaamino acids, a common property of all proteins. Disturbed proteostasis in postmitotic cell types, such as cardiomyocytes and neurons, produces an accumulation of misfolded and aggregated proteins resulting in disease. These factors coordinate protein synthesis with polypeptide folding for the conservation of protein conformation and protein degradation. In particular, maintaining proteome balance is a challenging task against external and endogenous stresses that accumulate during chronic cardiovascular disease and aging, which lead to the decline of the proteostasis network capacity and proteome integrity [4].

### **2.1 Balance and integrity of the proteome**

The protein flux of the cell must remain in balance to ensure proper cell and tissue function. The protein homeostasis, also known as proteostasis, leads to the accumulation of protein aggregates and it is the cause of several diseases. In view of this, protein aggregation is a common characteristic of many chronic diseases. Proteome balance is a task in defiance of external and endogenous stresses that accumulate in a lifetime, such as chronic cardiovascular diseases and aging. Moreover, regulated proteolysis mediated by proteases of damaged proteins is fundamental for protein quality control of eukaryotic cells that require the ubiquitinproteasome system (UPS). The UPS activity can be executed by ubiquitin-protein ligases or chaperones and the first crucial step is recognition of a specific degradation signal (degron). Degrons are portions of a protein that when exposed create a signal that is recognized by target proteins to the UPS pathway [7].

From this perspective, after several steps of substrate polyubiquitylation followed by substrate unfolding and degradation, proteins with specific degrons are recognized by the proteasome and targeted for degradation.

The cellular proteome is exceedingly complex and large-scale proteomic studies have identified thousands of modification sites (common modifications include phosphorylation, ubiquitylation, methylation and acetylation) in roughly 50% of proteins in humans, the combinatorial nature of which is mostly unknown [8]. Individual proteins often exist in several modified forms and they also engage in numerous dynamically regulated protein complexes during their life cycle. It is estimated that about 100,000 distinct protein isoforms can be generated through alternative splicing from all the pool of protein-coding genes. Nonetheless, the mechanisms that underlie the dynamics, interactions, stoichiometry and turnover of most individual protein species are poorly understood at the global level [8].

### **2.2 Proteostasis and its network**

In the cell, the proteome is a wide surveillance and regulatory network of the biogenesis process and protein degradation, which intervenes when these processes develop in a suboptimal way [8]. Proteome imbalance often results in complex and chronic diseases; therefore, it is a continuous process in order to meet the dynamic of proteomic needs of the cell [8]. In healthy cells, a complex proteostasis network (PN), comprised of molecular chaperones and proteolytic machineries and their regulators, operates to ensure the maintenance of proteostasis. These factors coordinate protein synthesis with polypeptide folding, the conservation of protein conformation and protein degradation [4]. The PN is performed by mechanisms controlling protein biosynthesis, cotranslational folding process, trafficking, neofunctionalization and degradation of proteins in vivo, among others, to maintain proteome balance and conform to the PN [9].

The proteome must have the ability to generate adequate synthesis, folding and protein expression and at the same time to detect abnormalities during this process by identifying the characteristics that force protective degradation when a component lacks quality. Human cells have more than 10<sup>3</sup> proteins per cell, and 5% of these are involved only in protein synthesis and turnover, and 60–80% of the etiologies of some diseases are associated with misfolding proteins. Therefore, it is clear that the constantly dynamic and complex eukaryote proteome requires a tightly regulated process [10].

The description of cellular proteomes requires an understanding not only of how proteins and their multimeric assemblies are built and their mechanisms established but also of the rules that determine how proteins are selected for degradation when they are unable to assemble properly with components of cognate networks. The network is constantly regulating the proteome, but it responds to conditions of proteotoxic stress by addressing the triage decision of fold, hold or degrade [11]. Consequently, the PN is constantly regulating the proteome and influences several cellular functions by affecting their physiology and readapting through transcriptional and translational changes within the biology of the cell [10, 11].

Numerous biological pathways affecting protein synthesis, folding, misfolding, trafficking, disaggregation and degradation may adapt the PN by using proteostasis regulators that can partially correct protein impairment, resulting in human diseases by cell stress and aging. The main PN components include several modules like protein synthesis machinery and the major mammalian protein degradation: UPS that is central to the unfolding protein response (UPR), which is activated when unfolded or misassembled proteins accumulate in the endoplasmic reticulum (ER), and the armada of intra- and extracellular chaperones including proteases, which detoxify cells from nonrepairable proteins [10, 11].

The structure of a determined protein is crucial for its function; hence, molecular chaperones are important components of the PN. Chaperones and other proteins like oxidoreductases and glycosylating enzymes bind nascent proteins and assist in proper folding into the correct structure and cellular location throughout their life cycle [12].

Diverse agents modify the structure of proteins like aging, oxidative, and thermal stress or misfolding-prone mutations. In this context, misfolded, damaged, unnecessary or aggregated proteins should be degraded, or their interactions could cause cell instability. There are two major intracellular proteolysis pathways: the autophagy-lysosomal pathway and UPS [13]. The difference between these two processes is the nature of the targeted protein degradation: in the case of autophagy, it mainly acts in the cytoplasm, and for UPS, considered the main route of protein degradation in mammalian cells, it acts on both cytoplasm and nucleus [14].

A deficient PN allows the disruption of cellular membranes by damaged proteins or toxic aggregates, which interfere with cell function, and as a result, many metabolic, oncological, cardiovascular and neurodegenerative disorders could appear in the individual [15].

The UPS is a complex machine formed by numerous subunits that degrade ubiquitin-attached proteins. This proteolysis pathway is critical for the quality control of proteins by eliminating damaged proteins and also maintaining the concentration of many regulatory proteins of apoptosis, inflammation, signal transduction and cell cycle [12]. The other proteolysis pathway, autophagy that is in charge of degraded proteins, is not detected by the UPS, and it has an important role in the immune response and starvation stage [16]. Autophagy eliminates several dysfunctional cell components or catabolizes them when the cell is under starvation and stress to maintain optimal levels of energy and nutrients [12]. ROS, DNA damage or starvation activates this autoproteolysis pathway engulfing organelles in

### *The Importance of Autophagy and Proteostasis in Metabolic Cardiomyopathy DOI: http://dx.doi.org/10.5772/intechopen.92727*

the autophagosome that are later fused with lysosomes, and by doing so, the amino acids and fatty acids produced by the catabolism of the organelles are recycled in the cytoplasm. However, three ways of delivering target proteins to the lysosome have been identified, and based on this, autophagy is classified into three distinct types: microautophagy, chaperone-mediated autophagy (CMA) and macroautophagy [16].

In microautophagy, the cellular contents are invaginated directly by the lysosome. The major cytosolic chaperone systems are HSP70 and HSP90, which are connected to the UPS pathway. The proteasome complex contains the proteolytic active sites in the core particle (20S) and the regulatory activity of the holo-complex in the regulatory particle (19S). The UPS pathway only recognizes polyubiquitination proteins, a process that requires three enzymes: E1 ubiquitin activator, E2 conjugase and E3 ligase, which act sequentially. The polyubiquitylated proteins are recognized by the core particle for their degradation by the regulatory particle (19S) [17]. Meanwhile, CMA uses the molecular chaperone, known as heat shock cognate 71 kDa protein (Hsc70), for recognition of the KFERQ sequence motif in cytosolic proteins that must be degraded, and drives them to the lysosome membrane [18]. The transmembrane receptor or docking protein is a lysosomal-associated membrane protein-2A (LAMP-2A) that transports the unfolded cytosolic proteins into the lysosome [18].

Macroautophagy involves the formation of the autophagosomes, defined as special structures that invaginate cellular contents or target proteins and then transport them to the lysosome. Besides eliminating pathogens, autophagy is also required for antigen presentation by the major histocompatibility complex (MHC) class II. The major autophagy pathway used by cells is the MHC class II [16].

### **2.3 Cardiomyopathy, cellular proteostasis alterations and myocardial remodeling with interstitial fibrosis**

Diabetic cardiomyopathy (DC) is a specific heart muscle disease that increases the risk of heart failure and mortality in diabetic patients independent of vascular pathology. Basal level autophagy plays a housekeeping role to maintain cellular homeostasis. However, autophagy mechanisms are impaired in diabetic hearts. In this sense, diminished autophagy limits cardiac injury in type 1 diabetes and inhibited autophagy contributes to cardiac injury in type 2 diabetes. In this context, protein homeostasis is a necessity for the correct function of the cell, in other words, an interaction between protein synthesis, transport, post-translational modification and degradation [17]. However, an accumulation of defective proteins results in proteotoxicity or disturbed proteostasis. Progression of cardiovascular diseases due to proteostasis alterations has been related with interstitial fibrosis and altered myocardial remodeling. Recent evidence indicates that the progression of ventricular dysfunction may be associated with changes in the process of autophagy and impaired proteostasis.

Autophagy in the mitochondria is a necessary process for maintaining a healthy mitochondrial network, also known as mitophagy. Under pathological conditions, mitochondrial dysfunction and enhanced ROS generation associated to cardiac hypertrophy and impaired left ventricular function with increased aggregation of abnormal proteins and enlarged or collapsed mitochondria can be found, such as structural and functional remodeling with changes in composition of the extracellular matrix, which are characterized by fibrotic tissue, impaired vascular and coronary microvascular function or effects on subcellular cardiomyocyte composition (**Figure 1**). Thus, mitophagy has been shown to be essential for myocardial protection [19]. In addition, calorie restriction is sufficient to accelerate cardiac

### **Figure 1.**

*Factors involved in autophagy and proteostasis in metabolic cardiomyopathy. Many proteins participate in the activation and formation of the autophagosome. TGF-β1 induces the activation of signaling pathways such as Smad, which in turn activates the formation of fibrogenic proteins such as type 1 collagen and fibronectin, and these induce hypertrophy and cardiac fibrosis generating cardiac damage and activating autophagy in cardiomyocytes. Modified of Kobayashi et al. [19].*

autophagic flux and reduce mitochondrial oxidative damage in the heart, results that suggest the important role of autophagy for maintaining optimal mitochondrial structure and function [20].

Proteostasis and autophagy are related to various heart diseases; however, both mechanisms can be beneficial or harmful depending on age and pathology. From this standpoint, heart diseases linked to autophagy due to degradation of contractile heart proteins are associated with cardiac aging, inherited cardiomyopathy, diabetic cardiomyopathy (DC), atherosclerosis, heart failure (HF) and atrial fibrillation (AF) [20]. The quality of cardiomyocytes depends on the efficient elimination of damaged proteins by autophagy. The mechanism performed by chaperone proteins, particularly heat shock proteins (HSP70/HSP40/HSP110) and chaperonins like the T-complex protein 1 ring complex (TRiC), takes place to a greater extent in the heart in response to oxidative stress [21]. HSPs are found in specific protein regions to prevent aggregation; these HSPs regulate oxidative stress (OS) and metabolism and maintain proper cell proliferation. The imbalance in the degradation of damaged intracellular proteins induces aging of the heart muscle fibers as a result of OS, the deterioration of the Ca+2 transits and the excessive generation of ROS. This process affects remodeling, favoring hypertrophy and cardiac fibrosis [22].

In cardiomyopathies, the accumulation of incorrectly folded proteins or acquired dysfunction of protein quality control has been implicated in impaired proteostasis. The cellular function in the myocardium follows the regulation of proteostasis and autophagy in order to control the quality of new synthesized proteins and removal of unfolded/misfolded proteins. When UPS targets are too large to be degraded by the proteasome, the autophagy system must control degradation through the selection between UPS and autophagy. Among autophagy regulators, the endosomal sorting complex required for transport protein complexes (ESCRT) affects the lysosome-autophagosome fusion. Part of ESCRT is the charged multivesicular body protein 2B (CHMP2B), which is required for autophagy. The work of Zaglia et al., in 2014, identified a novel link between UPS and autophagy and showed that the muscle-specific ubiquitin ligase atrogin-1 controls turnover of the ESCRT-III family protein CHMP2B, which controls the autophagy signaling pathways [23].

Transforming growth factor β1 (TGF-β1) is an important regulator of fibrogenesis. Its expression is regulated by biochemical stimuli, as a humoral response to infections, glucose and pH [24]. Binding of TGF-β1 to specific cellular

### *The Importance of Autophagy and Proteostasis in Metabolic Cardiomyopathy DOI: http://dx.doi.org/10.5772/intechopen.92727*

receptors, such as TGF-β type II and RII, activates phosphorylation for intracellular signaling pathways, such as Smad2 and Smad3 [25], which induce the expression of fibrogenic proteins like type I collagen and fibronectin [26]. These pathways trigger an inappropriate deposition of collagen in cardiac fibers, causing impaired heart function [27]. However, in other cell lineages, TGF-β1 is also capable of inducing autophagy, so the regulatory mechanisms between the two events are unknown [26].

Many target molecules are involved in fibrosis including the multiprotein complex formed by phosphoinositide 3-kinase class III (PI3K) dependent on Beclin 1, which regulates vesicular autophagy by activation of signaling pathways, such as Akt, and, in turn, increases the expression of TGF-β for the development of fibrosis [28]. HSP25 and alpha B-crystallin are expressed to a lesser extent in the heart; however, they fulfill the function of chaperone proteins that favor stability between actin and desmin, thus avoiding cardiotoxicity [29]. PINK1 (PTEN-induced putative kinase 1) is another protein involved in autophagy. It is located in the outer membrane of defective mitochondria, and it favors autophagy through the recruitment of the Parkin protein to depolarized mitochondria of cardiomyocytes [30].

These mechanisms can induce deregulation of autophagy by apoptosis in type II cells in cardiac tissue, which leads to the development of myocardial infarction (MI) [31]. Moreover, autophagy has been shown to be active in the perimeter of cardiovascular fibrotic tissue as a mechanism for fibrosis recovery and scarring secondary to cell apoptosis [32]. Many molecules protect against type 1 diabetes–induced cardiac dysfunction by activating autophagy. Lastly, the inhibition of autophagy has a beneficial effect on type 2 diabetes–induced cardiomyopathy [33].

### **2.4 Mitophagy and redox dysregulation**

Cardiomyopathies as a result of excessive ROS production and protein modifications in the mitochondria involve abnormal mitochondrial function resulting in cardiac disorders due to the high energy demand of the heart through this organelle, considered as a source of cellular energy production and mitochondrial ATP production achieved by oxidative phosphorylation and beta-oxidation. As a result of mitochondrial damage, the process of autophagy, known as mitophagy, is essential for myocardial function and protection [19].

The physiological performance of endothelial nitric oxide synthase enzyme (eNOS) is important for NO production, which is dependent on L-arginine through its reaction with O2 and the constitutive eNOS dependent on Ca2+/calmodulin, as well as the cofactors (6R-)5,6,7,8-tetrahydrobiopterin (BH4), nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN). Nitric oxide (NO) produced by the endothelium from eNOS, which is oxidized to L-citrulline and NO, works through the transference of electrons from NADPH via FAD and FMN. Both eNOS constitutive activation events are dependent, and in caveolae, they are Ca2+/calmodulin concentration dependent [34].

Under pathological situations and in the presence of uncoupled eNOS, increased OS is produced, instead of producing NO after eNOS activation due to the reaction with reduced BH4 levels and upregulated NADPH. As a result of these cardiovascular (CV) risk factors, NO is not produced, but there is ROS production. These abnormal reactions due to CV risk factors reduce bioactive NO [34].

The biological abnormalities produced by excessive ROS production such as superoxide anion (dO2), hydrogen peroxide (H2O2) and hydroxyl radical (dOH) species [35], including the rapid interaction of O2 with NO, result in the loss of NO bioavailability and increased production of peroxynitrite (ONOO) [34].

The harmful overproduction of these ROS and protein mitochondrial modifications as a result of impaired redox and pathological signaling in the CV system mediate regulation of the most important ion channels, transporters and kinases related to heart diseases.

These mechanisms lead to selective cardiac dysfunction and decreased energy production due to reductions in mitochondrial respiration, increased OS and defective contractile Ca2+ regulatory proteins. These types of changes and alterations in mitochondrial biogenesis, content and function related to an heterogeneous group of cardiovascular disease risk factors like metabolic syndrome, have been documented. Damaged mitochondria are degraded through mitophagy, the main protective function of autophagy that is for myocardial protection and the target of successful drug development emerging in the cardiovascular space. These strategies may be applied upon several redox targets, such as the membrane caveolae region where key cardiovascular redox proteins, such as eNOS, calmodulin and NADPH oxidase, among other important cardiovascular-related receptors, are located. Thus, calorie restriction is sufficient to accelerate cardiac autophagic flux to help improve mitochondrial oxidative damage and to maintain a healthy mitochondrial network [11, 18, 19].

### **3. Autophagy**

Dr. Christian de Duve was the first to use the term "autophagy," meaning "selfeating" in Greek, at the Ciba Foundation Symposium on Lysosomes, which took place in London on February 12–14, 1963 [36]. When there is a functional decline in the cardiovascular system and aging, cardiomyocytes need a cellular control mechanism to minimize damage and prevent cardiac malfunction. In this context, autophagy may degrade and recycle long-lived proteins, cytoplasmic components and organelles [37]. The notion of autophagy as cell death is a phenomenon that has been controversial and remains mechanistically undefined. It should be noted that when autophagy promotes cell death, there is an association of autophagy with the different cell death pathways [38].

The biogenesis of an autophagosome is orchestrated by the so-called autophagyrelated (ATG) proteins, which act in a hierarchical order to first generate the phagophore and then expand it into an autophagosome. The mammalian homologs of ATG1 are the uncoordinated-51-like kinases 1 and 2 (ULKA1 and ULK2) ULK complex, the ATG9A cycling system and the autophagy-specific class III phosphatidylinositol 3-kinase (PtdIns3K) complex, which are key in generating the phagophore upon induction of autophagy [39]. Besides, knockdown of EP300 and the inhibition of histone acetylases potentially induce autophagy indicating that protein deacetylation may play a role in the autophagic cascade. EP300 acetylates several autophagy-related proteins, including autophagy-related 5 (ATG5), ATG7, ATG12 and microtubule-associated protein 1 light chain 3 β (LC3). Lastly, protein deacetylation influenced by several proteins controls autophagy at diverse levels from the modification of autophagy core proteins to transcriptional factors controlling autophagic genes [39].

### **3.1 Autophagy basics**

Autophagy is also considered an evolutionarily conserved process critical for cellular homeostasis [3]. Implication of either the pathogenesis or the response to a wide variety of diseases by autophagy has been related to the pathogenesis of various disease states and to the basic molecular pathways that regulate autophagy [40].

### *The Importance of Autophagy and Proteostasis in Metabolic Cardiomyopathy DOI: http://dx.doi.org/10.5772/intechopen.92727*

Basal levels of autophagy maintain cellular homeostasis, and under stress conditions, high levels of autophagy are induced. However, the pro-death role of autophagy is complicated due to the extensive cross-talk between different signaling pathways [38]. Autophagy is a process by which cytoplasmic components are sequestered in double membrane vesicles and degraded upon fusion with lysosomal compartments. Depending on the stimulus, autophagy can degrade cytoplasmic contents nonspecifically or it can target the degradation of specific cellular components. Higher eukaryotes have adopted both of these mechanisms and account for the expanding role of autophagy in various cellular processes, as well as they contribute to the variation in cellular outcomes after induction of autophagy. As the basic molecular pathways that regulate autophagy are elucidated, the relationship of autophagy to the pathogenesis of various disease states becomes apparent [40].

Autophagy is a highly conserved eukaryotic pathway responsible for the lysosomal degradation (and subsequent recycling) that is rapidly growing and elucidating an intriguing mechanistic complexity as well as a tremendous range of cargo substrates. Imbalances in proteostasis are connected to aging and multiple (ageassociated) disorders [41]. Several pathologies including cardiovascular disease and stress-related disorders are associated with autophagy dysregulation. Moreover, excessive or insufficient levels of autophagic flux have been characterized in cardiomyocytes, cardiac fibroblasts, endothelial cells and vascular smooth muscle cells within the cardiovascular system [42]. Damaged and potentially cytotoxic mitochondria elicit an autophagic response termed mitophagy. Depending on the initiating stimulus, the substrate selection could differ. Thus, mitophagy takes part in physiological processes like the removal of paternal mitochondria during egg fertilization, and it is also a key process for the removal of damaged mitochondria in toxic conditions [43]. Furthermore, autophagy stimulation may result in reduced accumulation of misfolded and aggregated proteins; however, the overactivation of autophagy can trigger autophagy-mediated apoptosis.

### **3.2 Autophagy and heart diseases**

The cardiovascular system has the ability to adapt to a wide range of environmental stresses. The myocardium itself manifests robust plasticity for both physiological and pathological stimuli. From this perspective, autophagy is an intracellular process required to maintain cardiovascular homeostasis, and it is also an evolutionarily ancient process of intracellular catabolism in response to a wide variety of stresses. In the case of postmitotic cells, where cell replacement is not an option, finely tuned quality control of cytoplasmic constituents and organelles is especially critical [41]. Mitochondrial DNA has an important role at inducing and maintaining inflammation in the heart that escapes from autophagy. These autophagic mechanisms degrade damaged mitochondria through fusion of autophagosomes and lysosomes. Lastly, the impairment of mitochondrial cristae affecting cardiac morphology and function is induced by pressure overload [44].

### **3.3 Regulation of autophagy in the heart**

Excessive caloric intake results in obesity, a major independent risk factor for cardiovascular disease, including cardiac hypertrophy and heart failure. From this standpoint, cardiac remodeling is modulated by overnutrition or starvation. The adipokine leptin mediates energy balance between adipose tissue and the brain. Leptin and its receptors (LepRs) are expressed in the heart. LepRs belong to the class I cytokine receptor family signaling via JAK (Janus kinase)-2 and signal transducer and activator of transcription (STAT)-3. In addition, nutrient signaling

mediators, such as mTOR (mammalian target of rapamycin), induce LepRmediated activation of Akt. Cellular hypertrophy, proliferation and survival play an important role in cardiovascular function and pathology mediated by the Akt/ mTOR pathway [45]. To examine the importance of endothelial leptin signaling in cardiac hypertrophy, transverse aortic constriction was used in mice with inducible endothelium-specific deletion of leptin receptors (End.LepR-KO) or littermate controls (End.LepR-WT). Histology and quantitative polymerase chain reaction analysis confirmed reduced cardiomyocyte hypertrophy. STAT3 activation was reduced, and Akt (protein kinase B) and mTOR phosphorylation after transverse aortic constriction were blunted in End.LepR-KO mice hearts [46].

For normal cardiac physiology in response to pressure overload (PO), mTORC2 is also required to ensure cardiomyocyte survival. It has been observed that dysregulation of autophagy in cardiomyocytes is implicated in various heart disease conditions. In these cases, vigorous protein quality control (PQC) systems are essential for maintaining the long-term well-being of nonproliferating mammalian cells, such as neurons and cardiomyocytes (CMs) [47]. Similarly, PO activates autophagy in at least an acute phase and the suppression of PO-induced autophagy that alleviates pathological cardiac remodeling. Recent investigations revealed that enhancing autophagy ameliorates desmin-related cardiomyopathies, which are inherited cardiomyopathies that result in severe heart failure due to protein aggregation and myofibrillar disarray in CMs [47].

### **3.4 New therapeutic objective of metabolic cardiomyopathy in autophagy**

Perturbations in autophagy are involved in virtually all stages of cardiovascular disease. Research in the last decade has revealed that autophagy in cardiomyocytes plays a protective role, but not only during hemodynamic stress, but also in homeostasis during aging, resulting in mitochondrial damage. These damaged mitochondria are degraded through mitophagy and this process could be the main protective function of autophagy in the heart. From this standpoint, the mTORC1 complex regulates numerous biological processes, including proliferation, protein synthesis and autophagy inhibition. In addition, the mTORC1 pathway inhibits phosphorylation of the ULK1 protein (Ser 757) [48] considered an important element of autophagy activation.

The effects of mTOR are mediated through its activity as a central inhibitor of autophagy, a highly conserved cellular survival mechanism by which nutrientdeprived cells refresh the bioavailability of metabolic precursors [6]. In the cardiovascular system, the mTOR pathway regulates the physiological and pathological processes in the heart. In this regard, mTORC2 is necessary to maintain normal cardiac physiology and it ensures the survival of cardiomyocytes that have been subjected to PO. However, partial genetic or pharmacological inhibition of mTORC1 has been shown to reduce cardiac remodeling and heart failure in response to PO and chronic myocardial infarction. Therefore, mTOR may be a therapeutic strategy to confer cardioprotection [45].

Nonetheless, depending on the context, autophagic flux may be biased up or down. A large body of preclinical evidence suggests that autophagy is a doubleedged sword in cardiovascular disease, acting in either beneficial or maladaptive ways, depending on the context. Modulation of Beclin 1 significantly influences both autophagy and apoptosis, thereby deeply affecting the survival and death of cardiomyocytes in the heart. This is the reason why it is important to discuss the signaling mechanism of autophagy modulation through Beclin 1, including the therapeutic potential of Beclin 1 in heart diseases [49]. In light of this, the autophagic machinery in cardiomyocytes and other cardiovascular cell types has

been proposed as potential therapeutic targets. Autophagy mediators hold promise as targets for cardiovascular disease therapy; however, recent evidence suggesting that titration of autophagic flux holds potential as a new therapeutic goal for cardiovascular diseases, and heart failure, needs to be analyzed further [40].

### **3.5 Treatment for autophagy**

The use of pharmacological modulators can be beneficial for the treatment and prevention of autophagy. It is known that many agents or procedures induce or reduce autophagy activity; among these are spermidine, carvedilol, trehalose, resveratrol, metformin, caloric restriction, exercise training, intermittent fasting and ischemia/reperfusion.

Fasting and calorie restriction are the most potent nongenetic autophagy stimulators related to autophagy promotion. Regarding the upregulation of autophagy, the evidence overwhelmingly suggests that autophagy has to be induced in a wide variety of tissues and organs in response to food deprivation. From a mechanistic point of view, age-related vascular remodeling is driven by a greater accumulation of ROS. Thus, the induction of autophagy per se is sufficient to extend the shelf life in various species ranging from yeast to mammals [50]. Therefore, in addition to preserving the homeostasis of organisms in baseline physiological conditions, autophagy also contributes to metabolic fitness and the adaptation to stressful conditions, such as nutrient deprivation, hypoxia, OS or physical exercise.

Autophagy is a critical process for cell homeostasis and survival, and it is also implicated in the reduction of OS and inflammation. Furthermore, autophagic processes have been associated with a greater expression of eNOS and bioavailability of the protein. Long-lived, damaged, dysfunctional and potentially harmful cellular components break down for detoxification, energy production and cell renewal, providing building components and stimulating anabolic processes for effective cell recycling.

Vascular induction of NO production as a response to shear stress during exercise with augmented blood flow and increased flux sanguin over endothelial cells (EC) result in eNOS activation and NO production. Autophagic process has been related to greater expression of eNOS and bioavailability of the protein. ATG3 is an important autophagy pathway mediator; in contrast with a reduction of 85% by knockdown of ATG3 protein expression using control siRNA upon exposure to shear stress showed impairment of eNOS activation and as a result were incapable of produce NO as a response to shear stress. Autophagy is a critical process for cell homeostasis and survival is also implicated. Long-lived, damaged, dysfunctional and potentially harmful cellular components break down for detoxification, energy production and cell renewal, providing building components and stimulating anabolic processes for effective cell recycling as a result of autophagy [51].

### *3.5.1 Exercise-mediated regulation of autophagy*

Substantial evidence indicates that exercise training plays a beneficial role in the prevention and treatment of CV diseases. The regulation of autophagy during exercise is a bidirectional process. Autophagy is a physiologic process that is a defense mechanism for cells in adverse environments and it is also involved in several pathological processes [52]. Autophagy normal levels confer cell protection versus environmental stimuli to balance and protect organisms [53]. In this context, various diseases are the response to excessive or insufficient autophagy. Exercise training, referring generally to the cardiac adaptation to exercise, which has to be in an appropriate intensity as a chronic stimulation process, can reduce the risk of CV

diseases and improve the prognosis of patients after CV events. This type of training can also reduce the production of ROS, reduce the inflammatory response, regulate collagen metabolism, moderate the imbalance of extracellular matrix synthesis and degradation, and alleviate cardiac fibrosis [54].

### *3.5.2 Intermittent fasting*

Calorie restriction and stimulation of autophagy have healthy effects on the lifespan and cardioprotection in humans. Intermittent fasting induces adverse ventricular remodeling and cardiomyocyte death in null mice with LAMP2 (lysosomeassociated membrane protein 2) associated with an impaired autophagic flow. The study of Godar et al. [54] highlights that intermittent fasting conferred cardioprotection in wild-type female mice, with an 50% reduction in infarct size compared to controls matched without fasting, and this cardioprotection was lost in heterozygous null mice for LAMP2. One of the characteristics of these heterozygous null mice is the accumulation of damaged mitochondria with a deteriorated basal autophagic flux even on a fed day fed after 6 weeks, which probably results in the loss of cardioprotection observed with this regimen in wild-type mice. Intermittent fasting modulates OS from the myocardium through the effects on the mitochondria, where it is lost in the context of LAMP2 ablation due to the deterioration of mitochondrial autophagy [55]. Recent studies have discovered a potential mechanism for transcriptional replacement of autophagy-lysosome machinery with starvation. In addition, a central role was attributed to dephosphorylation and the cytoplasm induced by rapid hunger to nuclear translocation of TFEB (EB transcription factor) [55]. The endogenous TFEB-mediated stimulation of the autophagic flow is essential for the cytoprotective effects of repetitive hunger in hypoxiareoxygenation injury. The research group suggests the hypothesis that the transcriptional replenishment of the autophagy-lysosome machinery by fasting (and hunger as described earlier) may be a critical determinant of beneficial autophagy, which allows living organisms to survive in what has probably been one of the first evolutionary stresses that accompanied the origin of life [56].

Therefore, starvation (total caloric restriction) is a potent stimulus for the induction of myocardial macroautophagy (called "autophagy") [57–59]. It is already known that autophagy is essential for cardiac homeostasis in the period of perinatal hunger at birth; this effect is observed before the establishment of breast milk supply [60]. In experiments using mice with genetic ablation of autophagy proteins ATG5 and ATG7, autophagosomes could not be formed and fatal myocardial ischemia developed [60, 61]. In this respect, autophagy is also essential for the maintenance of cardiac structure and function during prolonged starvation in mice, since the concomitant deterioration of autophagy with FOXo1 genetic ablation, Becn1 haplo-insufficiency [57] or pharmacological inhibition with bafilomycin A1 [62], an inhibitor of acidification and lysosome function, produces a rapid development of cardiomyopathy with starvation.

### *3.5.3 Ischemia/reperfusion*

The different roles of autophagy in cardiomyocytes exposed to varying degrees of ischemia/reperfusion injury (I/R) or severe anoxia (S/A) were explored, and it was observed that the autophagic activity of cardiomyocytes increased with an increment in ischemia that was dependent on the duration of anoxia, undergoing ischemia, or severe ischemia [63].

During the process of cardiac ischemia, the restriction in the blood supply and the reduction of ATP leads to an imbalance in the amount of blood and energy,

### *The Importance of Autophagy and Proteostasis in Metabolic Cardiomyopathy DOI: http://dx.doi.org/10.5772/intechopen.92727*

causing cell heart dysfunction and myocardial damage, inflammation and excess of ROS production leading to cardiomyocyte death. It should be noted that ATP levels can be monitored by adenosine monophosphate-activated protein kinase (AMPK), which functions as a nutrient deprivation sensor in response to a decreased ATP level during cardiac ischemia [64].

In the initial phase of ischemia, a low level of ATP activates AMPK in cardiomyocytes. Once activated, AMPK directly phosphorylates and activates ULK1 resulting in the induction of autophagy by modifying ULK1 directly or indirectly [48]. The pathway by which AMPK activates autophagy is through AMPK/ mTORC1 signaling. AMPK inhibits mTORC1 through phosphorylation of TSC2 and the raptor site, followed by indirect activation of ULK1 [48]. Recent studies revealed new pathways through which AMPK activated autophagy. Also, AMPK directly phosphorylates and activates activated ULK1, allowing the onset of autophagy [65–67]. Also, in the early I/R process, ROS modify the function of Ca2+ channels and exchangers, which triggers a decrease in available ATP, and thus, directly affect the autophagy process [68].

Beclin 1 is an important autophagic protein that has been shown to regulate both the formation and processing of autophagosomes, especially in the reperfusion phase. An in vitro study revealed that autophagic response to nutrient deprivation mediated by Beclin 1 is modulated by the Bcl-2 protein in cardiac cells [69]. Moreover, it has been observed that ROS can also be strong inducers of Beclin 1 in mediating autophagy during the reperfusion phase [70]. In addition to regulating Beclin 1 expression, ROS could also oxidize and decrease ATG4 activity, contributing to LC3 lipidation at the start of autophagy [71].

Cellular stress by ischemia, hypoxia, depletion of intracellular Ca2+ stores, induced OS and ROS, and the accumulation of unfolded/misfolded proteins induce ER dysfunction known as ER stress, and then the unfolded protein response (UPR) is generated to deal and play a critical role in cell death after myocardial I/R injury. Several transcription factors are induced by ER stress and the UPR whose branch includes ATF6, inositol-requiring enzyme 1 (IRE1) and PKR-like ER kinase (PERK) activated by I/R injury, which is the mediated signal pathway of UPR. The activating transcription factor 6 alpha (ATF6) is an ER transmembrane protein and most ATF6-induced proteins localize to the ER [72].

Catalase is an enzyme that has been shown to decrease damaging ROS in the heart. ATF6 induces catalase known to decrease ROS and reduce I/R damage in the heart. Catalase is a component of peroxisomes that has also been found in the cytosol and cardiac mitochondria, and it neutralizes H2O2 and also serves to oxidize ONOOd, NO and organic peroxides; however, it has not be found in the ER. In the study by Jin et al. [72], they examined the effects of blocking ATF6-induced proteins in the ER stress response on I/R injury in cardiac myocytes and mouse hearts. The role of ATF6 as a link between ER stress and OS and its effect on I/R myocardial injury show an important function for ATF6, which binds to specific elements in the regulatory elements of the catalase gene inducing its transcription [72].

Myocardial I/R injury negatively regulates protein synthesis, leading to the activation of signaling pathways from the ER to the cytosol and nucleus, representing UPR and ER-associated protein degradation (ERAD). Most of I/R damage is caused by ROS generated outside the ER. The study by Zhang et al. revealed that all the three branches of UPR pathway are involved. Moreover, they demonstrated reduced myocardium damage in I/R surgery, while the activation of UPR had opposite effects. The results of this study were shown after the inhibition using a standardized animal model with Sprague-Dawley rats that were pretreated with UPR stimulator dithiothreitol (DTT) and UPR inhibitor 4-phenylbutyrate (4PBA) and then subjected to myocardial I/R surgery [73].

Under the cardiac I/R condition, increased autophagic activity compensated for impaired UPS function, thereby maintaining proteolysis at an appropriate level. However, cooperation between UPS (short-lived proteins) and autophagy (longlived proteins) is considered a housekeeping mechanism for protein quality control in I/R injury. Thus, this increased autophagic response helps to maintain an adequate proteolysis level and proteostasis in order to compensate impaired UPS function under cardiac I/R condition, which ultimately results in degradation by the proteasome as well as autophagy.

### *3.5.4 Spermidine*

Spermidine (SPD) is a type of polyamine that has been shown to enhance heart function to delay cellular and organismal aging and provide cardiovascular protection in humans. Initially, the cardioprotective effects of SPD were explored in rodent models of physiological cardiac aging (mice) and congestive heart failure induced by high salt concentration (rats) [74]. SPD in the diet of mice delays cardiac aging by improving diastolic function.

Furthermore, the evidence demonstrated that a high intake of SPD in the diet was correlated with a reduction in the incidence of cardiovascular diseases. In humans, high levels of SPD (natural polyamine) in the diet, as assessed by food questionnaires, correlated with reduced blood pressure and a lower incidence of cardiovascular disease. Subsequently, SPD was identified as a potent inducer of autophagy [74]. SPD by increasing autophagic and mitophageal activity improves mitochondrial respiratory function. SPD also inhibited kidney damage and fibrosis. It is suggested that the effect of SPD on the improvement of cardiac function is mediated by the promotion of autophagy and mitophagy in the heart and by the reduction of the systemic chronic inflammatory response. This natural polyamine is importantly involved in maintaining cellular homeostasis, and it affects several processes including cell growth, proliferation and tissue regeneration; it also stimulates the antineoplastic immune response and anti-aging properties, including transcriptional and transductional modulation through several enzymes and nucleic acid enzymes. Moreover, SPD promotes chaperone activity and ensures proteostasis through anti-inflammatory and antioxidant properties, and it also enhances mitochondrial function and cellular respiration [75].

Therefore, the effect of exogenous SDP administration was examined in aged rat hearts [76]. SPD was shown to improve mitochondrial biogenesis by increasing nuclear expression of PGC-1α (peroxisome proliferator-activated receptor gamma coactivator alpha), which is mediated by enhanced NAD<sup>+</sup> -dependent deacetylase activity of SIRT1 (sirtuin-1). These results suggest that SIRT1 is an essential intermediary in the mechanism by which SPD stimulates mitochondrial biogenesis and function in cardiac cells. In addition, findings showed that the administration of SPD in vivo increased the activity of the antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT), and improved mitochondrial respiratory activity in the myocardium [76]. To date, there are not enough clinical trials to evaluate the effects of SPD in reducing cardiovascular diseases. These findings could guide new therapeutic strategies to counteract cardiac aging and prevent agerelated cardiovascular disease and, as a result, lay the foundation for better heart disease treatments related to mitochondrial dysfunction [76].

### *3.5.5 Carvedilol*

Carvedilol (CVL) belongs to the so-called α, β blockers, used to treat high blood pressure and congestive heart failure, which are generally used for the treatment of

### *The Importance of Autophagy and Proteostasis in Metabolic Cardiomyopathy DOI: http://dx.doi.org/10.5772/intechopen.92727*

cardiovascular disorders. CVL blocks sympathetic neural activation through antagonism of the β1, β2 and α1 adrenoceptors and it has demonstrated greater cardiovascular benefits than traditional β blockers in both humans and animals. However, some benefits beyond decreased blood pressure were observed clinically, suggesting the potential anti-inflammatory activity of CVL [77]. In addition, CVL is a known membrane "fluidizer" that alters membrane structure and protein-lipid interactions [78]. The most widely characterized inflammasome sensor in the heart is activated in response to noninfectious stimuli, such as cell debris during acute myocardial infarction. The NOD-like receptor (NLR) family, pyrin domain–containing protein 3 (NLRP3) inflammasome is a component of the inflammatory process. Activation of the NLRP3 inflammasome triggers further myocardial damage indirectly through the release of IL-1β and directly through the promotion of inflammatory cell death via pyroptosis [79]. Pyroptosis is a type of caspase-1–dependent cell death, which is often associated with inflammasome activation and IL-1β production characterized by a loss of cell membrane integrity that leads to fluid influx and cell swelling [77]. Experimental studies have shown that strategies inhibiting the activation of the NLRP3 inflammasome in the early reperfusion period after acute myocardial infarction reduce the overall size of the infarct and preserve normal cardiac function [79]. There is also evidence supporting the therapeutic value of NLRP3 inflammasometargeted strategies in experimental models and data supporting the role of the NLRP3 inflammasome in AMI and its consequences on adverse cardiac remodeling, cytokine-mediated systolic dysfunction and heart failure [79]. Mechanistic analysis revealed that CVL prevented lysosomal and mitochondrial damage and reduced apoptosis-associated speck-like protein containing a CARD (ASC) oligomerization. Additionally, CVL caused autophagic induction through a SIRT1-dependent pathway, which inhibited the NLRP3 inflammasome [77].

CVL activates survival signaling of p-AKT and pluripotential markers in cardiomyocytes (CM) after I/R. Cardioprotective actions of CVL are associated with higher levels of the miR-199a-3p and miR-214 cardioprotective miRNAs [79]. CVL stimulates the processing of microRNA (MIR)-199a-3p and miR-214 in the heart through β-arrestin-1–biased β-1 adrenergic receptor (β1 AR) for cardioprotective signaling. Studies show that using cultured cardiomyocyte and primary cardiomyocyte cell lines, carvedilol is regulated by an increase in miR-199a-3p and miR-214 in ventricular and atrial cardiomyocytes undergoing reperfusion ischemia (I/R) injury.

### *3.5.6 Trehalose*

It is known that trehalose, a natural disaccharide, protects cells against various stresses. Trehalose is a natural disaccharide formed from two glucose molecules with an α-type glycosidic junction. It is widely distributed in nonmammalian species, such as fungi, yeasts, bacteria, invertebrates, insects and plants. Trehalose acts to provide energy sources and protect the integrity of cells exposed to various environmental stresses. Furthermore, it has also been shown that trehalose protects against apoptosis in an autophagy-dependent manner. This natural disaccharide improves cardiac remodeling, fibrosis and apoptosis after myocardial infarction and attenuated heart dysfunction [80]. The cardioprotective effect of trehalose was not observed in the heterozygous elimination of Beclin 1 in mice, indicating that these protective effects are mediated by autophagy [81]. In this connection, trehalose induces autophagy by facilitating the recruitment of LC3B to the autophagosomal membranes in an mTOR-independent manner. The basal level of autophagy plays a unique housekeeping role in the regulation of cardiac geometry and impaired autophagy function and may contribute to various end-organ complications in

insulin resistance and diabetes, including cardiomyopathy and nephropathy [82]. Autophagy is usually regulated by both mTOR-dependent and -independent mechanisms. The mTOR pathway is considered the classic autophagy regulation route, which negatively regulates autophagy involving two functional complexes: mTORC1 and mTORC2, with a much more predominant role for mTORC1. Research findings suggest that trehalose may rescue the contractile myocardial defect induced by insulin resistance and apoptosis, through autophagy associated with the dephosphorylation of p38 MAPK and FOXo1 without affecting the phosphorylation of Akt [82]. Moreover, it was observed that trehalose not only activated autophagy but also increased the expression of p62. In addition, the expression of antioxidant genes regulated by trehalose through enhanced nuclear translocation of Nrf2 in a p62-dependent manner leads to the suppression of OS. Therefore, a new antioxidant action target for trehalose was proposed [83].

### *3.5.7 Lysosomal inhibitors blocking autophagy*

Several lysosomal inhibitors such as bafilomycin A1 (BafA1), protease inhibitors and chloroquine (CQ) have been used interchangeably to block autophagy in vitro for lysosomal degradation. Only CQ and its derivate hydroxychloroquine (HCQ) are FDA-approved drugs currently considered the principal compounds used in clinical trials aimed for treating tumors through autophagy inhibition by impairing autophagosome fusion [84]. They focus on how CQ inhibits autophagy and directly compare its effects to those of BafA1. CQ mainly inhibits autophagy by impairing autophagosome fusion with lysosomes rather than by affecting the acidity and/or degradative activity of this organelle. Furthermore, CQ induces an autophagyindependent severe disorganization of the Golgi and endolysosomal systems, which impair autophagosome fusion. These results of Mauthe et al. suggest not using these compounds (CQ and HCQ) for in vivo experiments because of multiple cellular alterations caused by these drugs [84].

### *3.5.8 Resveratrol*

Human clinical studies differ markedly in terms of the administered doses of resveratrol, as well as in the duration of treatment. Overall, the most pronounced effects of resveratrol include reduced body weight in obese patients and a partial decrease in systolic blood pressure, as well as fasting blood glucose levels and HbA1c in patients with diabetes mellitus in some clinical trials. Studies show that resveratrol attenuates high glucose-induced cardiomyocyte apoptosis through AMPK, a serine/ threonine kinase that detects the state of cellular energy and regulates energy homeostasis [85]. Activation of AMPK is involved in the determination of multiple cellular processes including cell growth, apoptosis [86] and autophagy [87]. It is known that AMPK activation could inhibit mTOR, the best characterized protein kinase that negatively regulates autophagy [88]. Diabetic cardiomyopathy has shown inhibition of autophagy and increased apoptosis in cardiac cells. The study of Xu et al. demonstrated that using resveratrol in H9c2 cardiac myoblast cells exposed to high glucose combined with palmitate suppressed autophagic activity and increased apoptotic cell death. The H9c2 cells showed restored autophagy and attenuated apoptosis in cells with diabetic stimuli when treated with resveratrol [89, 90].

### *3.5.9 Metformin*

Metformin is a first-line antidiabetic drug that also activates autophagy and it has cardiovascular protective effects [91], although a recent study reported

### *The Importance of Autophagy and Proteostasis in Metabolic Cardiomyopathy DOI: http://dx.doi.org/10.5772/intechopen.92727*

otherwise, since metformin did not achieve the cardioprotective effect in an I/R model in nonaged pigs [92]. This was proven because the protective effect of metformin was abolished by treatment with chloroquine. This treatment inhibits the fusion of lysosomes with autophagosomes and a high lysosomal pH, avoids the final digestion stage and inhibits lysosomal activity [93].

However, a recent study by Chen Li et al. showed protection with metformin on both cellular and animal models of aging and I/R injury. During aging, failure of organelles results in the accumulation of macromolecules and impaired proteostasis that result in the death of cardiac tissue. Necroptosis is a programmed cell death involving receptor-interacting protein kinases 1 and 3 (RIP1, RIP3) that form the necrosome and mixed lineage kinase domain-like protein (MLKL), which are subsequently phosphorylated [94]. Besides, metformin treatment was able to restore autophagy and reduce the accumulation of p62 in the aged myocardium, as well as decrease the cardiac junction of p62-RIP1-RIP3 complexes and the RIP3 and MLKLinduced phosphorylation. Therefore, metformin can break the unfavorable chain mechanism of aging-related autophagy decrease that induces necroptosis [94].

### **3.6 Development of new autophagy modulators**

Diabetes is a metabolic disorder that contributes to the development of cardiac fibrosis and cardiomyopathy. Aminoguanidine (AG) inhibits advanced glycation end products (AGEs) and advanced oxidation protein products (AOPP) accumulated as a result of excessive oxidative stress in diabetes. In a recent work, we investigated whether AG supplementation mitigates oxidative-associated cardiac fibrosis in rats with type 2 diabetes mellitus (T2DM). In vivo experiments were performed in a model of T2DM, and in vitro we used primary rat myofibroblasts to confirm the antioxidant and antifibrotic effects of AG to determine if blocking the receptor for AGEs (RAGE) prevents the fibrogenic response in myofibroblasts. Diabetic rats exhibited an increase in cardiac fibrosis resulting from a high-fat, high-carbohydrate diet (HFCD) and streptozotocin (STZ) injections. In contrast, AG treatment significantly reduced cardiac fibrosis, alfa-smooth muscle actin (αSMA) and oxidative-associated NOX4 and NOS2 mRNA expression [95]. In vitro challenge of myofibroblasts with AG under T2DM conditions reduced intra- and extracellular collagen type I expression and platelet-derived growth factor (PDGF), transforming growth factor beta (TGFβ1) and collagen type 1 a 1 (COL1A1) mRNAs, albeit with a similar expression of tumor necrosis factor alpha (TNFα) and interleukin-6 (IL-6) mRNAs. This was accompanied by reduced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and SMAD2/3 but not of AKT1/2/ 3 and signal transducer and activator of transcription (STAT) pathways. RAGE blockade further attenuated collagen type I expression in AG-treated myofibroblasts. Thus, AG reduces oxidative stress-associated cardiac fibrosis by reducing pERK1/2, pSMAD2/3 and collagen type I expression via AGE/RAGE signaling in T2DM [95]. However, clinical studies need to be performed in order to evaluate if AG treatment is useful and well-tolerated in human cardiac disease and leads to a significant reduction in cardiac fibrosis as well as it modulates the expression of oxidative and fibrogenic response in myofibroblasts like in this disease model.

Although the autophagy modulators described above have great potential, there are currently no interventions aimed at modulating autophagy for human use. Despite this, there are already licensed medicines for use in humans, which activate or inhibit autophagy, such as rapamycin, chloroquine and HCQ, among others, that were not developed for this purpose [96]. The main clinical obstacle is that they have low pharmacological specificity for their objective, which is the autophagic


### **Table 1.**

*Autophageal processes susceptible to therapeutic modulation.*

*Beclin 1; H2S, hydrogen sulfide; mTORC1, target of rapamycin complex 1.*

process [84]. However, they have allowed us to know the main pathways by which the autophagy process is activated or inactivated. Several pharmacological and nutritional interventions are available to inhibit autophagy in the initiation, nucleation, elongation, fusion or degradation phase [97]. In addition, several agents modulate autophagy through multiple molecular mechanisms that are not yet characterized (**Table 1**).

### **4. Conclusions**

Alteration of proteostasis in heart tissue leads to diabetic cardiomyopathy characterized by myocardial remodeling and interstitial fibrosis. Cardiomyocyte proteotoxicity frequently faces the chronic accumulation of misfolded or unfolded proteins that can lead to proteotoxic formation or aggregation of soluble peptides with reduced cardiac function and arrhythmias. However, under pathological conditions, autophagic flux may be an important strategy to prevent the progression of various cardiovascular diseases due to risk of dysfunctional endothelial cells. Autophagy is insufficient in endothelial cells isolated from individuals with diabetes *The Importance of Autophagy and Proteostasis in Metabolic Cardiomyopathy DOI: http://dx.doi.org/10.5772/intechopen.92727*

mellitus. Moreover, it has been demonstrated that intact autophagy is essential for eNOS signaling in endothelial cells. Nitric oxide-mediated vasodilation was promoted by the induction of autophagy.

Autophagy has been shown to be a mechanism of fibrosis recovery and scarring secondary to cell apoptosis and active in the perimeter of cardiovascular fibrotic tissue. Autophagy inhibition has a beneficial effect on type 2 diabetes–induced cardiomyopathy. These findings suggest that autophagy is diversely altered in different types of diabetes-induced cardiac pathologies. Therefore, targeting autophagy regulation may be a potential therapeutic strategy for diabetic cardiomyopathy.

Moreover, the animal model of T2DM induced by STZ plus HFCD whose diabetic response evokes pro-oxidative and profibrotic attenuated reactions in the presence of AG suggests that this molecule may be part of autophagy therapy for diabetic cardiomyopathy. Thus, AG reduces oxidative stress-associated cardiac fibrosis by decreasing pERK1/2, pSMAD2/3 and collagen type I expression via AGE/RAGE signaling in T2DM.

The knowledge of the molecules involved in mechanisms of proteostasis and autophagy in cardiac cells and the role they play in various signaling pathways will serve as an opportunity for the future design of therapeutic targets for the treatment of fibrosis, alterations of cardiac tissue remodeling and cardiomyopathy.

### **Acknowledgements**

This work was partially supported by grant from the academic group, SEP/PRODEP and CONACYT/PRO-SNI.

### **Conflict of interest**

The authors declare no conflict of interest.

*Cardiovascular Risk Factors in Pathology*

### **Author details**

María Cristina Islas-Carbajal<sup>1</sup> \*, Ana Rosa Rincón-Sánchez<sup>2</sup> , Cesar Arturo Nava-Valdivia<sup>3</sup> and Claudia Lisette Charles-Niño<sup>3</sup>

1 Department of Physiology, Institute of Experimental and Clinical Therapeutics, University Center of Health Sciences, Guadalajara University, Guadalajara, Jalisco, Mexico

2 Department of Molecular Biology and Genomics, Institute of Molecular Biology and Gene Therapy, University Center of Health Sciences, Guadalajara University, Guadalajara, Jalisco, Mexico

3 Department of Microbiology and Pathology, University Center of Health Sciences, Guadalajara University, Guadalajara, Jalisco, Mexico

\*Address all correspondence to: islascarbajal@yahoo.com; cristina.islas@academicos.udg.mx

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*The Importance of Autophagy and Proteostasis in Metabolic Cardiomyopathy DOI: http://dx.doi.org/10.5772/intechopen.92727*

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Section 2
