Advanced Glycation End Product Induced Endothelial Dysfunction through ER Stress: Unravelling the Role of Paraoxonase 2

Abstract

Hyperglycemia accelerates the formation of advanced glycation end products (AGEs). AGEs are a heterogeneous group of compounds generated by non-enzymatic glycation of proteins or lipids with glucose through Amadori rearrangement and its accumulation increases with aging in diabetes. AGEs augments ROS generation, diminishes the antioxidant defense of the cells, decreases mitochondrial membrane potential, ATP production, and elevates the levels of mitochondrial fission protein (Drp1) and mitophagic proteins (Parkin and PTEN) leading to dysfunction of mitochondria. In this chapter, we have discussed how AGEs trigger the endoplasmic reticulum stress and inflammation and mediate endothelial dysfunction in diabetes and also have discussed the role played by endogenous Paraoxonase 2 (PON2) in mitigating endothelial dysfunction by inhibiting the adverse effects of AGE.

1. Introduction

Diabetes mellitus (DM) is a growing metabolic health problem, and around 346 million people are currently affected by diabetes worldwide, and it is anticipated to double by 2030 [http://www.who.int/mediacentre/factsheets/fs312/en/index.html]. Macro and microvascular complications are the major cause of morbidity and mortality in patients with type 2 diabetes [1]. The important pathology associated with vascular diseases is the formation and accumulation of atherosclerotic plaques leading to the process of atherosclerosis, which ultimately results in the narrowing of the blood vessels [2].

Vascular endothelial cells (ECs) are monolayers of cells that line the inner surface of the luminal vessel wall. It acts as a physical barrier between the bloodstream and the luminal wall [3, 4]. The term endothelial cell dysfunction (ECD) is referred to as loss/dysregulation of EC cells function: such as impairment of the barrier function, reduction of the anti-coagulants, disturbance in the balance between the vasodilation and vasoconstrictions, increased pro-inflammatory response, attachment, and adhesion of leukocytes to the endothelial cell surface, increase the production of pro-oxidant molecules [5, 6]. These points highlight the importance of ECs in maintaining normal vascular homeostasis.

During endothelial injury, oxidized lipids and protein adducts (advanced glycation end products) accumulate in the arterial walls. Then the circulating monocytes adhere to the endothelial cells that express adhesion molecules, such as vascular adhesion molecule-1 (VCAM-1) and selectins, and then migrate into the sub-endothelial space. These monocytes that infiltrate the arterial wall get differentiated into macrophages, and this macrophage also accumulates oxidized lipids to form foam cells. Then the foam cells attract the T-lymphocytes, which in turn induce the proliferation of smooth muscle cells in arterial walls. The entire process leads to the formation of a lipid-rich atherosclerotic lesion and rupture of this lesion leads to vascular infarction. Additionally, increased platelet aggregation and coagulation are also observed due to impaired nitric oxide generation, free-radical generation from the platelet, and elevated levels of plasminogen activator inhibitor. All these factors contribute to the vascular wall occlusion and further increase the risk of cardiovascular events as shown in Figure 1 [2, 7].

Figure 1.

Mechanism of AGEs-induced oxidative stress in the formation of atherosclerotic plaque.

Numerous studies have demonstrated the existence of an association between elevated AGE levels and cardiovascular disease in DM patients. In line with this, clinical studies reported that CML and pentosidine levels are increased in the progression of the disease and act as a predictor of cardiovascular events [8, 9, 10, 11, 12, 13, 14]. A fourfold increase in the incidence of coronary artery disease, a 10-fold increase in peripheral vascular disease and a 3–4-fold higher mortality rate with as much as 75% of diabetics ultimately dying from vascular disease have been reported [15].

Vascular complications are classified into two types, namely macrovascular and microvascular. Wherein the large vessels such as arteries and veins get affected in macrovascular disease and microvascular involves small vessels such as capillaries. Chronic hyperglycemia initiates the production of AGE and turns on diabetic vascular complications through elevated production of reactive oxygen species, which instigates various signaling cascades such as Receptor for advanced glycation end product receptor (RAGE) activation, protein kinase C (PKC) and Mitogen activated protein kinases (MAPK) pathways. Macrovascular complications associated with diabetes are arteriosclerotic cardiovascular diseases (ASCVDs) such as coronary heart disease (CHD), peripheral artery disease (PAD), and stroke [2]. Microvascular complications associated with diabetes are diabetic retinopathy (DR), neuropathy, and nephropathy [16, 17] as shown in Figure 2.

Figure 2.

AGEs-induced macrovascular and microvascular complications.

The pathogenic mechanisms underlying diabetic nephropathy involve the generation of reactive oxygen species (ROS), accumulation of AGEs, and activation of intracellular signaling molecules such as PKC [18, 19]. Studies suggest that increased formation of AGEs in the vitreous may be involved in the development of diabetic retinopathy by inducing the production of interleukins −6 (IL-6) from retinal Müller cells [20]. AGEs interaction RAGE plays an important role in the pathogenesis of DR [21]. In DR, AGEs (both early and late Amadori products) have been localized to vascular cells (endothelial and pericytes), neurons, glia, and also in the vitreous [22, 23]. Pentosidine and Nε-(carboxymethyl)lysine (CML) levels are increased in aqueous, vitreous, and serum of DR patients when compared with non-diabetic controls especially increased in proliferative DR (PDR) patients than non-proliferative DR (NPDR) and referred to as biomarker for microvascular complications [24, 25] with the progression of DR with decreased visual acuity, emphasizing that AGEs are novel biomarkers/risk markers for type 2 DR [26, 27]. In addition to the above AGEs, methylglyoxal derivative hydroimidazolone was also increased in DR patients [28, 29].

AGEs are a heterogeneous group of compounds generated by non-enzymatic glycation of proteins or lipids with glucose through Amadori rearrangement and its accumulation increases with aging and in diabetes [30]. The term AGEs has been applied to a broad range of advanced glycation end products such as CML, N^ε-(carboxymethyl)hydroxylysine, pyrraline, and pentosidine [31]. Among these, CML is the predominant epitope in the AGE adducts detected in tissue proteins of diabetic patients [32, 33, 34]. Its level is found to be increased in serum and aqueous humor of type 2 diabetic patients with retinopathy (DR) [2]. It is also used as a biomarker to predict the progression of different stages of DR. AGEs-stimulated cell response is initiated by its engagement with the receptors present on the cell surface [35]. The most studied AGE receptor is RAGE. Several other AGE receptors identified so far consist of AGE-receptor complex (AGER1/OST-48, AGER2/80 K-H, AGER3/Gal-3), some members of the Toll-like Receptor (TLRs) family (TLR4, TLR2), and scavenging receptors (SRs) family (SR-AI, SR-AII, CD36, LOX-1, FEEL-1, and FEEL-2). The expression of these AGE-activated receptors depends on the cell/tissue type [36].

RAGE and TLRs are well-known Pattern Recognition Receptors (PRRs), which recognize molecules found in pathogens (Pathogen-Associated Molecular Patterns–PAMPs, ex-LPS) or molecules released from damaged or stressed cells such as high mobility group B (HMGB1) and serum amyloid proteins referred as Damage-Associated Molecular patterns (DAMPs) [37]. It is expressed on the surface of various cells, including endothelial, epithelial, and fibroblast mediates multiple signaling pathways such as MAPK kinase and Nuclear factor kappa B (NFĸB), which activate a pro-inflammatory response [38, 39]. Among various TLRs, TLR-2 and 4 are reported to be increased in monocytes of type 1 and type 2 diabetic patients [40, 41] associated with microvascular complications such as retinopathy, nephropathy, and neuropathy [42].

Although various pathways are involved in the pathogenesis of endothelial dysfunction such as activation of the polyol pathway, auto-oxidation of glucose, PKC pathway activation, and formation of AGEs, all these pathways may intersect at several points to increase the complexity of the disease [43]. Among these, increased formations of AGEs (advanced glycation end product) is one of the causes of ECs dysfunction, which has been implicated in the pathogenesis of diabetes-induced vascular complications, as evident by their in vivo accumulation as reported in numerous diseases [44]. In this chapter, we would focus on how AGEs induce an inflammatory response, mitochondrial dysfunction, and endothelial dysfunction through ER stress.

2. AGE-induced effect in endothelial cells

The high glucose-induced “oxidative stress” and “endoplasmic reticulum (ER) stress” of the endothelium may play major roles in the initiation and progression of cardiovascular clinical manifestations in diabetes [45]. While diabetes management has largely focused on the control of hyperglycemia, the rising burden of this disease is mainly correlated to its vascular complications [46]. This is reflected by a type II diabetes differs principally from type I diabetes in that it is accompanied by a period of hyperinsulinemia and is characterized by late as opposed to early onset of hyperglycemia. In type I DM, vascular involvement (through endothelial dysfunction) occurs as a result of metabolic insult/hyperglycemia, while in type II DM, endothelial dysfunction plays a more direct role and is aggravated by, rather than caused by, hyperglycemia [6].

2.1 AGE and oxidative stress

One of the best-characterized actions of AGEs on ECs is the induction of ROS [47, 48]. There is considerable evidence to show that AGE induces ROS generation and diminishes the antioxidant defense of the cells [49, 50, 51].

Oxidative stress is induced either by the abundant production of ROS or the failure of the antioxidative machinery mechanism. The main source of ROS is the electrons present in the mitochondrial respiratory chain, which results in the formation of superoxide anion (O2-), hydroxyl radical (OH.) hydrogen peroxide (H₂O₂). AGEs induce mitochondrial dysfunction through the generation of ROS production.

Mitochondria are complex organelle that undergoes complete fusion and division under physiological or pathological conditions. Mitochondrial fission is defined as the division of a mitochondrion from two or more separate mitochondrial compartments, and this process is essential in the distribution of mtDNA during cell division and helps in the removal of damaged mitochondria through mitophagy. Mitochondrial fusion is merging two or more mitochondria. It is regulated by at least three proteins: optic atrophy 1 (OPA1), mitofusin 1 (MFN1), and mitofusin 2 (MFN2), and mitochondrial fission are controlled by dynamin-related protein 1 (DRP1/DLP1/DNM1), fission 1 (FIS1), and mitochondrial fission factor (MFF) [52, 53]. Studies show that in diabetes, mitochondrial fission is increased [54, 55, 56]. Diabetic animal model (diabetic-STZ model) study demonstrated that HG alters the mitochondrial respiration and alters glomerular bioenergetics, whereas podocytes isolated from those mice showed fragmented mitochondria [57]. Another study in Sprague-Dawley rats (STZ induced) reported an increase in neuronal pyknosis with increased DRP1 expression in neurons when compared with Normal control (NC) group associating that High glucose (HG) aggravated ischemic brain damage and alteration in mitochondrial dynamics [58]. Very importantly, AGEs such as AGE-BSA promoted mitochondrial fission, loss of membrane potential, and apoptosis through the RAGE pathway, whereas blocking the RAGE signaling reduces the events of mitochondrial abnormalities in endothelial and osteoblastic cells [59, 60]. In a rodent model, AGE-BSA disturbs the mitochondrial respiratory chain and induces mitochondrial pore formation with an increase in RAGE expression suggesting the fact that AGE-induced mitochondrial dysfunction plays a major role in diabetic neuropathy [61]. Proteomics study by tandem mass spectrometry (nanoLC-ESI-ETD MS/MS) revealed that renal tubular cells exposed to HG increased phosphorylation and oxidation of mitochondrial proteins when compared with the NG [62]. CML-BSA, another AGE, produces detrimental effects in diabetic db/db mice inducing mitophagy. In this study, CML-BSA treatment in pancreatic β-cells increased RAGE and ROS with a decrease in membrane potential and ATP production with elevated levels of mitochondrial fission (Drp1) and mitophagic proteins (Parkin and PTEN) supporting the factor that increases the concentration of AGEs damage the β-cells and reduces the insulin cells function by making vulnerable to AGEs-induced damages [63].

Under physiological conditions, low concentrations of ROS are needed to maintain cellular proliferation, migration, and survival [64]. However, during the pathological condition, overproduction of ROS induces deleterious effects on the cells and tissues, and to overcome this, the cellular environment has an antioxidative defense system that is capable of scavenging the ROS. The antioxidant defense system includes low-molecular ROS scavengers, antioxidative enzymes, and degrading or repairing proteins [50]. The low-molecular ROS scavengers include glutathione (GSH), vitamin C, and D. Secondly, the antioxidative enzymes superoxide dismutases (SOD) and catalase, which can convert ROS into less reactive ions. The last part of the defense system includes the proteasome and protease system, which will degrade the damaged proteins. The impairments in the antioxidant defense system lead to oxidative stress, which eventually activates various cellular signaling pathways.

2.2 Pro-inflammatory response by AGE

Further, AGEs also increase pro-inflammatory response, by augmenting endoplasmic reticulum (ER) stress and amplifying the angiogenic potential of ECs, which has been observed in a myriad of human diseases such as atherosclerosis, acute/chronic inflammatory diseases, vascular complication, and aging [65, 66, 67, 68].

Numerous AGEs-induced vascular diseases display elevated levels of pro-inflammatory cytokine either by directly activating ROS or through engaging with RAGE receptor. AGE-RAGE is a prominent axis that facilitates the activation of NFĸB and MAPK signaling pathways, which subsequently induces the expression of cytokines, chemokines, and adhesion molecules [38, 69]. Elevated levels of AGEs enhanced the release of pro-inflammatory cytokines such as interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor (TNF-α), and interferon-beta (IL-1β). RAGE also binds HMGB1, commonly referred to as damage-associated molecules (DAMPs), and acts as an inflammatory mediator and is released into circulation when there is an injury [70]. Recognition of HMGB1 by RAGE induces the NFĸB activation and its downstream cytokines IL-6, IL-8, and TNF-α. [70, 71] Accumulating evidence shows that DAMPs are reported to mediate the pathogenesis of atherosclerosis and diabetic vascular complications.

Another important feature of inflammatory response is the recruitment and attachment of circulating leukocytes to the endothelium, which is the initial stage in the development of atherosclerosis. Increased adhesion of leukocytes to ECs is facilitated by enhanced expression of chemokines such as monocyte chemoattractant protein (MCP-1) and adhesion molecules such as intercellular adhesion molecules (ICAM), vascular cell adhesion molecules (VCAM), and E-selectins. Studies also demonstrate that direct interaction exists between the ECs adhesion molecules and lymphocyte-function-associated antigen 1 (LFA-1), macrophage-1 antigen (Mac-1), and very late antigen-1 (VLA-1), which are the major counter receptors present on the surface of the leukocytes [39, 72]. Abundant studies show that AGEs elevate the expression of adhesion molecules on the surface of the endothelial cells [73, 74].

2.3 AGE increases ER stress

Numerous studies provided molecular insights, highlighting the functional link existing between endothelial dysfunction and endoplasmic reticulum (ER) stress [75]. ER is the vital organelle that plays important role in protein folding, lipid biosynthesis, and calcium (Ca²⁺) regulation. When the ER homeostasis is disturbed either due to increased synthesis of protein, accumulation of misfolded proteins, increased oxidative stress, or alteration in calcium load leads to a condition termed as ER stress. It is closely monitored by the evolutionarily conserved quality control system called unfolded protein response (UPR) [75, 76].

The UPR system is activated by three transmembrane proteins: 1) RNA-dependent protein kinase-like ER eukaryotic initiation factor-2α kinase (PERK), 2) inositol-requiring ER-to-nucleus signaling protein 1 (IRE1), and 3) activating transcription factor 6 (ATF6). These three sensors are inactive form when they are bound with glucose-regulated protein kinase-78 (GRP78) and released upon induction of ER stress. These sensors resolve the ER stress by 1) activating the induction of UPR genes, which enhances the protein folding; 2) attenuation of protein translation, therefore, it reduces the workload of ER; and 3) activating ER-associated degradation (ERAD) pathway activation, which eliminates the unfolded protein through proteasome degradation pathway [75, 77].

During ER stress, activation of the PERK pathway leads to phosphorylation of the eukaryotic translation initiation factor 2 alpha (eIF2α), resulting in the attenuation of protein translation and activating the ATF4 (activating transcription factor-4). ATF4 subsequently induces the activation of pro-apoptotic mediator CHOP (C/EBPα-homologous protein, also known as GADD153) and its downstream target gene, DNA-damage-inducible protein-34 (GADD34). Activation of IRE1 promotes splicing of the endoribonuclease unconventional splicing of an mRNA encoding the transcription factor X-box-binding protein 1 (XBP1s). XBP1s activation further induces the activation of UPR genes, which either increases the ER folding capacity or intersects with AFT4-CHOP to activate the apoptosis. Another UPR pathway is the activation of AFT6, which translocates to the Golgi apparatus. It alleviates misfolded proteins through activation of the ERAD pathway mediated by ATF6-XBP1as shown in Figure 3 [75, 76].

Figure 3.

AGEs-induced ER stress in endothelial cells.

Studies reported that under stress conditions, ER show an unusual morphological pattern called ER whorls, which can be used as a biomarker for ER stress [78]. Further compared with the normal cells, DTT treatment (inducer of ER stress) activated the formation of ER whorls, which are seen beneath the plasma membrane, which is in turn imported into the vacuole and proceeds for autophagy [79]. Earlier a study has shown the presence of whorl-like sER in the Leydig cells of STZ-induced mouse model indicating that diabetes can induce morphological changes in ER. Till now there are no studies that have described the effect of AGE on ER morphology. Whereas there are studies that have highlighted the distorted mitochondrial morphology (mitochondrial fission) seen during mitochondrial fission in diabetic animal models [80, 81].

ER and mitochondria are connected through mitochondrial- associated ER membranes (MAMs), which help in the transfer of Ca²⁺, ATP, and metabolites [82, 83]. As ER stress magnitudes, it increases the releases of Ca²⁺ from ER to mitochondria, which leads to the opening of mitochondrial membrane pore and releases of cytochrome C, which further intensifies the ROS production. Taken together, it creates a vicious cycle of ER stress and mitochondrial dysfunction, which progresses toward apoptotic signaling [84].

A vast number of studies link ER stress with inflammatory and oxidative signaling pathways, which play a putative role in the development and progression of endothelial dysfunction. Several lines of evidence support the fact that ER stress acts as a potent inflammatory activator because each of the UPR arms activates NFĸB pathway, which in turn releases an array of inflammatory cytokines IL-6, IL-8, and TNF-α [85, 86]. A lot of in vivo and in vitro studies also support the notion that AGEs-induced ER stress promotes the activation of ROS, autophagy, pro-inflammatory cytokines response, and apoptosis in most of diabetic vascular complications [85, 87, 88, 89, 90, 91].

Although several studies have identified an array of molecular entities and pathways that activate endothelial dysfunction, the cellular processes underlying endothelial dysfunction are majorly oxidative stress and inflammation. Increasing the intake of fruits and vegetables rich in polyphenols and flavonoids (anti-inflammatory and antioxidant properties) protects the endothelium and reduces the risk of cardiovascular complications [92, 93]. Increasing the endogenous antioxidant may pay way to develop a more effective and safer option. One such endogenous molecule with antioxidative, anti-apoptotic, and anti-inflammatory properties is paraoxonase [94]. Decreased serum paraoxonase (PON) activity is seen in both diabetes, and its complications have been reported, which is attributed to its glycation [95, 96]. The human PON enzyme consists of three family members, paraoxonase (PON1), paraoxonase 2 (PON2), and paraoxonase 3 (PON3). These genes are located on the long arm of chromosome no 7 (7q21-22) with nine exons. Based on the structural homology and from the evolutionary point of view, PON2 is reported to be the oldest member of the family followed by PON3 and PON1 [97]. PON1 is HDL associated and secreted in serum, whereas PON2 and PON3 are located intracellularly mainly in the endoplasmic reticulum, mitochondria, and the nuclear membrane and are ubiquitously expressed in most of the tissues including the liver, kidney, intestine, placenta, etc. [98]. Its role has been explored in many cells including epithelial, endothelial, macrophages, and smooth muscle cells.

PON2 is a ubiquitously expressed antioxidant and anti-inflammatory protein, where its expression and regulation during diabetes- induced complication have not been studied so far. In our recent study, we have established in HUVECs that AGE treatment decreases mRNA, protein, and activity of PON2, whereas overexpression of PON2 alleviates the GA and CML-induced oxidative stress, ER stress, and inflammation through NFκB and ERK1/2 phosphorylation and thereby mitigates pro-inflammatory response [99]. Further silencing of PON2 aggravates GA and CML-induced oxidative stress, ER stress, and pro-inflammatory cytokines expression in HUVEC cells. We found that in diabetic retina PON2 expression was significantly downregulated and HRECs treatment with CML increased mitochondrial fission and aggravates the mitochondrial-dependent apoptosis [100]. Conversely, overexpression of PON2 inhibits the JNK1/2-mediated signaling pathway and rescues the cells from mitochondrial fission and apoptosis as shown in Figure 4 [100].

Figure 4.

Possible mechanistic role of PON2 in mitigating AGEs-induced ER stress, pro-inflammation, and mitochondrial dysfunction in endothelial cells.

3. Conclusion

There are a large number of studies that have established the deleterious effects of AGE on various cellular models. In this chapter, we have discussed in detail how AGEs induce mitochondrial dysfunction, inflammation and endothelial dysfunction through augmenting ER stress. We have also highlighted the association between mitochondrial stress and ER stress. The role of antioxidant PON in inhibiting these deleterious effects has also been discussed.

Acknowledgments

We thank the Council of Scientific and Industrial Research (CSIR-27 (0310)/14-EMR-11) and Indian Council of Medical Research (ICMR-ID no: 2017-0976/CMB-BMS) for providing the funding and the fellowship.

Conflict of interest

The authors declare no conflict of interest.