**5. Lipoprotein oxidation and disease states**

#### *5.1. Atherosclerosis*

Atherosclerosis is one of the leading causes of death worldwide, and is commonly associated with coronary and cerebrovascular diseases (Rocha & Libby, 2009; Moore & Tabas, 2011). Originally considered purely a lipid-storage disease, it is now recognized that atherosclerosis is intrinsically linked to inflammation, particularly with respect to involvement of innate and adaptive immunity.

398 Lipoproteins – Role in Health and Diseases

is also associated with the loss in ability of HDL to activate LCAT (McCall et al., 1995). Acrolein modifies apoAI specifically at Lys226 in helix 10 converting it to Nε-(3 methylpyridinium)lysine (MP-Lys). A corresponding decrease in ABCA1-mediated cholesterol efflux was also noted. In addition, immunohistochemical analysis demonstrated co-localization of acrolein-adducts and apoAI in human atherosclerotic lesions, **Figure 8**, confirming previous studies (Uchida et al., 1998b). Further chlorination of Tyr192 and oxidation of specific Met residues in apoAI via the MPO pathway impairs its ability to promote cholesterol efflux (Shao et al., 2010). Taken together, it appears that oxidative

modification of HDL apoAI may be one of the contributory factors to atherogenesis.

**Figure 9.** Distribution cholesterol mediated by apoB-100, apoE and apoAI. The green block arrow shows the general direction of the RCT process. The red stars draw attention to processes that are

Atherosclerosis is one of the leading causes of death worldwide, and is commonly associated with coronary and cerebrovascular diseases (Rocha & Libby, 2009; Moore & Tabas, 2011). Originally considered purely a lipid-storage disease, it is now recognized that

affected by oxidative modification of protein and lipid components of lipoproteins.

**5. Lipoprotein oxidation and disease states** 

*5.1. Atherosclerosis* 

are likely to be affected because of oxidative modification of lipoproteins.

**Figure 9** provides a simplified overview of the roles of apoB-100, apoAI and apoE in lipid distribution between liver and peripheral tissues. It also shows potential functional sites that Atherosclerosis is a progressive disorder marked by several stages with varying extent of lesions marking each stage. It is initiated by accumulation of lipoproteins in the subendothelium, typically at arterial branch points, which tend to have impaired laminar flow. The physiological response to this retention includes: (i) chemical modification (due to the production of ROS and other oxidative factors from intimal macrophages) and aggregation of the lipoproteins, (ii) early inflammatory response such as T cell recruitment, (iii) cytokine secretion, and, (iv) endothelial alterations. In response to the chemokines, the circulating monocytes enter the arterial wall and eventually differentiate to macrophages under the influence of macrophage colony stimulating factors.

The macrophages internalize oxidized and modified lipoproteins via several types of scavenger receptors, including class A and B scavenger receptors (e.g. CD36) and lectin-like oxidized LDLr-1 (LOX-1). Mice lacking CD36, platelet-activating-factor receptor, and tolllike receptors 2 and 4 show decreased atherosclerosis. The internalized cholesterol is stored in the cytoplasm as cholesterylester lipid droplets, surrounded by a monolayer of phospholipids. When viewed by electron microscopy, these cells have a foamy appearance, and are therefore called foam cells. Foam cell formation marks the earliest pathological lesion in atherosclerosis called 'fatty streaks'.

As the fatty streaks progress, they induce migration of smooth muscle cells from media to the intima of the arterial wall, which is accompanied by secretion of collagen and matrix proteins and macrophage proliferation. As lipid accumulation continues to occur in macrophages, smooth muscle cells also take up lipids. Collectively, these processes give rise to fibrous lesions. As the lesion progresses, foam cells die and release lipids, which aggregate with lipoproteins trapped in the matrix, eventually leading to advanced lesions with calcification and hemorrhage.

Ox-LDL also plays a role in modulating smooth muscle function, by increasing their adhesion to macrophages and foam cells in the plaques. At low concentrations, Ox-LDL stimulates proliferation of smooth muscle cells, whereas at higher concentrations, they cause smooth muscle apoptosis by up-regulating levels of the pro-apoptotic lipid ceramide. During this process, a necrotic core is formed in the vessel lumen. The necrotic plaque is unstable and subject to disintegration or rupture, leading to formation of a thrombus. Ox-LDL are key stimulators of the coagulation pathway, and promote the secretion of tissue factor from endothelial cells, which is required to form a clot. At various stages of the lesions unstable angina, heart attack or stroke may occur.

In summary, our understanding of the atherogenesis process has progressed rapidly since the deleterious nature of Ox-LDL was first pointed out about 3 decades back. We now recognize it as a multifactorial disease with inflammation and oxidative stress playing key roles. The last decade has seen the additional role of HDL in mitigating the severity of atherosclerosis; in this context, the role of HDL (not HDL levels *per se*, but the robustness of HDL function) is currently under intense scrutiny.

#### **5.2. Alzheimer's disease**

Alzheimer's disease is a neurodegenerative disorder that is clinically characterized by progressive cognitive decline and dementia. The major neuropathological hallmarks of this age-related disease are the extra cellular accumulation of amyloid beta peptide (Aβ), the intracellular presence of neurofibrillary tangles composed of hyperphosphorylated tau and the loss of cholinergic neurons in the brain.

Several hypotheses have been put forward to explain the pathogenesis of Alzheimer's disease, of which the oxidative stress hypothesis is of relevance here. In general, the brain of Alzheimer's disease subjects is believed to be in a heightened state of oxidative stress and is characterized by higher levels of lipid peroxidation products (acrolein, 4-HNE and MDA) (Arlt et al., 2002; Butterfield et al., 2010). The brain is particularly susceptible to oxidative stress due to the high oxygen and metal content, lipid levels, and paucity of antioxidants (such as vitamins C and E) compared to normal tissues (Schippling et al., 2000). The Alzheimer's disease brain is also rich in isoprostanes (a stable marker of peroxidation of arachidonic acid) and neuroprostanes (Lovell et al., 2001).

Accumulation of soluble and insoluble assemblies of Aβ (a peptide composed of 39-43 residues) in the brain parenchyma and in the cerebral vasculature is considered the primary event in Alzheimer's disease pathogenesis by the amyloid hypothesis. Aβ is derived from the ubiquitously expressed transmembrane protein amyloid precursor protein by regulated intramembranous proteolysis. The oligomeric form of Aβ is considered as the most toxic species; one of the reasons for its toxicity is its ability to act as an oxidative stress agent in a lipid environment. The oxidative nature of Aβ has been attributed to the presence of Met35, which is capable of undergoing one-electron oxidation to form a sulfuranyl radical cation (S+). This in turn is able to abstract a H-atom from a fatty acyl chain as shown in **Figure 7**, and initiate a series of free radical-mediated events in a lipid milieu such as a membrane bilayer or a lipoprotein particle (Butterfield et al., 2005).

In the CNS, apoE is localized on HDL-sized spherical and discoidal particles. Lipoproteins isolated from CSF of Alzheimer's disease patients were shown to have a higher sensitivity to *in vitro* oxidation compared to those from normal subjects. It has also been shown that CSF lipoproteins can be oxidized through transition metal ions such as Cu2+ or Fe3+. These metal ions are present in CSF as complexes with metal-binding proteins like ceruloplasmin or transferrin (Schippling et al., 2000); under pathological conditions, they are released in a catalytically active form. Furthermore, Aβ itself can induce oxidation through interactions with metal ions. Aβ contains three histidines and one tyrosine, all of which can chelate transition metal ions. This promotes Aβ aggregation and a pro-oxidative state of Aβ through reduction of the transition metal (Artl, et al., 2002). The peptide can then produce ROS or induce lipid peroxidation, both of which strongly affect lipoprotein stability.

Conversion of cholesterol to 24S-hydroxycholesterol is believed to be a mechanism by which the brain maintains cholesterol homeostasis. Plasma concentrations of 24S-

hydroxycholesterol are utilized as a biomarker and a diagnostic tool for neurological disorders. Neuronal damage is accompanied by destruction of neuronal membranes, which causes more cholesterol to be converted into 24S-hydroxycholesterol. In agreement significantly higher peripheral concentrations of 24S-hydroxycholesterol were found in Alzheimer's disease and vascular demented patients (Lutjohann at al., 2000). Importantly, the latter study showed that the apoE genotype does not contribute significantly to the elevated plasma levels of 24S-hydroxycholesterol in Alzheimer's disease patients (Bretillon, 2000).

The CSF levels of 24S-hydroxycholesterol appears to be sensitive to changes in the brain (possibly because they are not affected by hepatic clearance rates of this oxysterol) and may represent better markers both for neurodegenerative diseases and for disturbances in the blood brain barrier (Leoni & Caccia, 2011). In early stages of Alzheimer's disease, there are significantly higher CSF concentrations of 24S-hydroxycholesterol suggesting increased cholesterol turnover in the CNS during degeneration (Leoni & Caccia, 2011). These levels decrease as the disease advances, possibly reflecting the loss of cells expressing cholesterol 24S-hydroxylase, the enzyme responsible for the conversion of brain cholesterol into 24Shydroxycholesterol. In Alzheimer's disease and mild cognitive impairment, but not in normal individuals, the levels of 24S-hydroxycholesterol significantly correlate with CSF levels of apoE (Shafaati et al., 2007).

The elevation of 24S-hydroxycholesterol in CSF is consistent with a significant role for this oxysterol as a signaling molecule during neuronal degeneration. It has been shown that 24Shydroxycholesterol is able to induce expression of apoE and ABC transporters in astrocytes through activation of LXR, and to stimulate cellular cholesterol efflux (Baldan et al., 2009).

#### **5.3. Diabetes**

400 Lipoproteins – Role in Health and Diseases

the loss of cholinergic neurons in the brain.

arachidonic acid) and neuroprostanes (Lovell et al., 2001).

bilayer or a lipoprotein particle (Butterfield et al., 2005).

Alzheimer's disease is a neurodegenerative disorder that is clinically characterized by progressive cognitive decline and dementia. The major neuropathological hallmarks of this age-related disease are the extra cellular accumulation of amyloid beta peptide (Aβ), the intracellular presence of neurofibrillary tangles composed of hyperphosphorylated tau and

Several hypotheses have been put forward to explain the pathogenesis of Alzheimer's disease, of which the oxidative stress hypothesis is of relevance here. In general, the brain of Alzheimer's disease subjects is believed to be in a heightened state of oxidative stress and is characterized by higher levels of lipid peroxidation products (acrolein, 4-HNE and MDA) (Arlt et al., 2002; Butterfield et al., 2010). The brain is particularly susceptible to oxidative stress due to the high oxygen and metal content, lipid levels, and paucity of antioxidants (such as vitamins C and E) compared to normal tissues (Schippling et al., 2000). The Alzheimer's disease brain is also rich in isoprostanes (a stable marker of peroxidation of

Accumulation of soluble and insoluble assemblies of Aβ (a peptide composed of 39-43 residues) in the brain parenchyma and in the cerebral vasculature is considered the primary event in Alzheimer's disease pathogenesis by the amyloid hypothesis. Aβ is derived from the ubiquitously expressed transmembrane protein amyloid precursor protein by regulated intramembranous proteolysis. The oligomeric form of Aβ is considered as the most toxic species; one of the reasons for its toxicity is its ability to act as an oxidative stress agent in a lipid environment. The oxidative nature of Aβ has been attributed to the presence of Met35, which is capable of undergoing one-electron oxidation to form a sulfuranyl radical cation (S+). This in turn is able to abstract a H-atom from a fatty acyl chain as shown in **Figure 7**, and initiate a series of free radical-mediated events in a lipid milieu such as a membrane

In the CNS, apoE is localized on HDL-sized spherical and discoidal particles. Lipoproteins isolated from CSF of Alzheimer's disease patients were shown to have a higher sensitivity to *in vitro* oxidation compared to those from normal subjects. It has also been shown that CSF lipoproteins can be oxidized through transition metal ions such as Cu2+ or Fe3+. These metal ions are present in CSF as complexes with metal-binding proteins like ceruloplasmin or transferrin (Schippling et al., 2000); under pathological conditions, they are released in a catalytically active form. Furthermore, Aβ itself can induce oxidation through interactions with metal ions. Aβ contains three histidines and one tyrosine, all of which can chelate transition metal ions. This promotes Aβ aggregation and a pro-oxidative state of Aβ through reduction of the transition metal (Artl, et al., 2002). The peptide can then produce ROS or

Conversion of cholesterol to 24S-hydroxycholesterol is believed to be a mechanism by which the brain maintains cholesterol homeostasis. Plasma concentrations of 24S-

induce lipid peroxidation, both of which strongly affect lipoprotein stability.

**5.2. Alzheimer's disease** 

Diabetes mellitus is a disease characterized by hyperglycemia and insufficiency or resistance to insulin. In general, diabetes is associated with increased generation of free radicals, heightened state of oxidative stress, and attenuated antioxidant response. Studies have revealed greater levels of TBARS in the plasma of diabetic patients compared to controls (Kawamura et al., 1994). Plasma lipoproteins isolated from diabetic rats have been shown to be cytotoxic *in vitro* in cultured cells, suggesting they may have been oxidatively modified *in vivo* (Morel & Chisolm, 1989)*.* Other studies have shown that glucose autoxidation and protein glycation can result in the formation of radicals like superoxide anions that promote lipoprotein oxidation.

Hyperglycemia promotes glycation of the protein and lipid components of lipoproteins leading to generation of advanced glycation end-products (AGE) (Sun et al., 2009). Glycation is the process whereby glucose attaches to the ε-amino group of lysines or the αamino group of an N-terminal amino acid in a non-enzymatic manner (Maritim et al., 2003). Biochemically, glucose attachment to the protein results in the formation of an unstable Schiff base, which then rearranges to an Amadori product. These Amadori products then

undergo dehydration reactions and rearrange themselves to finally form AGE, which are responsible for many of the irreversible pathological effects seen in diabetes. The receptors for AGE (RAGE) are abundantly expressed on vascular endothelial cells, smooth muscle cells and macrophages, which are enhanced in atherosclerotic lesions in diabetes. Since Ox-LDL has AGE epitopes, it binds RAGE on macrophages and enhances macrophage proliferation and oxidative stress.

Like oxidation, glycation of LDL prevents LDLr-mediated cellular uptake of lipoproteins and promotes scavenger receptor-mediated uptake. *In vivo*, small, dense, which is more atherogenic than large buoyant LDL, appears to be preferentially glycated; also, *in vitro* studies suggest that it is more susceptible to glycation (Soran & Durrington, 2011). Diabetic individuals display higher plasma concentrations of glycated LDL than non-diabetic individuals.

Type 1 diabetes subjects have lipid disorders (diabetic dyslipidemia), with a pro-atherogenic lipid profile: increased concentration of TG and LDL cholesterol, low HDL levels (Verges, 2009). In addition, they display increased cholesterol-triglyceride ratio within their VLDL, increased triglyceride in their LDL and HDL, glycation of apolipoproteins, increased oxidation of LDL and an increase in small dense LDL (relatively more atherogenic). HDL from Type 1 diabetes subjects is less effective in promoting cholesterol efflux and has reduced antioxidant properties.

Subjects with Type 2 diabetes also have a proatherogenic lipid profile with quantitative and qualitative differences in their lipoproteins (Verges, 2005). Typically, they have increased triglyceride levels, increased VLDL production and decreased VLDL catabolism, and decreased HDL cholesterol levels. They have large VLDL particles that are richer in triglyceride, small dense LDL particles, increase in triglyceride content of LDL and HDL, Ox- and glycated-LDL, glycation of apolipoproteins and increased susceptibility of LDL to oxidation.
