**2. Multiple sclerosis**

MS is a demyelinating inflammatory and neurodegenerative disease of the CNS with heterogeneous pathology (see below) and clinical outcomes. More than 80% of MS patients present initially with acute attacks (relapses) of neurological dysfunction (follow by variable degree of recovery and periods of "remission"), characterizing the relapsing-remitting phenotype (RR-MS). Most of these patients develop a disabling progressive course independently of eventual relapses (secondary progressive MS). A small percentage of patients (10-15%) presents initially with a progressive disease course (primary progressive MS). Patients with clinical isolated syndrome (CIS) have an isolated episode suggestive of MS. The investigation of CIS patients is of special theoretical and practical interest because of their increased risk to develop the disease.

A possible involvement of plasma lipoprotein in MS pathogenesis was suggested in 1953 by the work of Swank [4]. This author presented evidence for a favorable disease course in patients taking a diet poor in animal fat [5]. Sinclair, in 1956, called attention for the importance of a deficiency in polyunsaturated fatty acids and remarked similar epidemiological aspects of MS and cerebrovascular disease [6]. A landmark work, providing a clear potential involvement of plasma lipoproteins in disease was published by Shore *et al*, in 1987 [7]. These authors studied the animal model of MS, experimental autoimmune encephalomyelitis (EAE) and concluded that "*major changes in apoE-containing lipoproteins are undoubtedly significant in the altered immune function in EAE*". Supporting this prediction, it was observed higher plasma apoE concentrations in MS patients during relapses in comparison to remission states and lower levels in patients under remission in comparison to normal controls [8-10]. Studying EAE induction in apoE-deficient female mice, Karussis in 2003, found that apoE deficiency might be connected with a defective neuronal repair mechanism and enhanced immune reactivity and worse course of the disease [11]. These results could indicate that plasma apoE may have an immunosuppressive role in MS [9]. In agreement with this concept, our group observed that lower levels of plasma apoE might promote immune reactivity in these patients [12].

In their work, Shore *et al* observed higher concentrations of total LDL and HDL cholesterol after onset of clinical symptoms. Giubilei *et al*, in 2002, studied plasma lipoproteins and magnetic resonance imaging (MRI) in patients with a first clinical episode suggestive of MS (CIS), supporting the findings in EAE [13]. These authors observed high total and HDL-cholesterol in these patients and a significant correlation between disease activity (as assessed by MRI) and both total and LDL-cholesterol levels. Jamroz-Wisniewska *et al* found high total cholesterol levels in patients (RR and progressive forms) and also higher LDL-cholesterol in RR patients in remission and in progressive forms than in healthy subjects [14]. Serum paraoxonase 1 (a HDL associated enzyme) activity in relapses was significantly lower in RR patients in comparison to other MS groups. An epidemiological survey based on almost 9000 patients with MS found that the presence of hypercholesterolemia, among other vascular co-morbidities, increased the risk of a more rapid disabling progression of the disease [15]. Recently, Weinstock-Guttman *et al* studied the serum lipid profiles in association with clinical disability and MRI measures in 492 MS patients [16]. They found that worsening disability was associated with higher total and LDL cholesterol, and triglycerides. Higher HDL levels were associated with lower probability for the presence of acute inflammatory lesions (assessed by MRI). Other authors have found higher HDL-cholesterol (and total blood homocysteine) levels in MS patients during a phase of clinical inactivity in comparison to normal controls [17].

564 Lipoproteins – Role in Health and Diseases

pathological states.

**2. Multiple sclerosis** 

of their increased risk to develop the disease.

promote immune reactivity in these patients [12].

In this chapter we do not pretend to make an exhaustive review on the vast literature related to this theme. Rather, we intend to incorporate some relevant studies in a comprehensive framework addressed to open new avenues of research. With this purpose, we will mainly focus on two frequent and disabling conditions, multiple sclerosis (MS) and Alzheimer disease (AD), and discuss the involvement of plasma lipoproteins in brain inflammatory and neurodegenerative mechanisms. With this approach we expect that useful insights may emerge regarding the contribution of plasma lipoproteins in CNS physiology and

MS is a demyelinating inflammatory and neurodegenerative disease of the CNS with heterogeneous pathology (see below) and clinical outcomes. More than 80% of MS patients present initially with acute attacks (relapses) of neurological dysfunction (follow by variable degree of recovery and periods of "remission"), characterizing the relapsing-remitting phenotype (RR-MS). Most of these patients develop a disabling progressive course independently of eventual relapses (secondary progressive MS). A small percentage of patients (10-15%) presents initially with a progressive disease course (primary progressive MS). Patients with clinical isolated syndrome (CIS) have an isolated episode suggestive of MS. The investigation of CIS patients is of special theoretical and practical interest because

A possible involvement of plasma lipoprotein in MS pathogenesis was suggested in 1953 by the work of Swank [4]. This author presented evidence for a favorable disease course in patients taking a diet poor in animal fat [5]. Sinclair, in 1956, called attention for the importance of a deficiency in polyunsaturated fatty acids and remarked similar epidemiological aspects of MS and cerebrovascular disease [6]. A landmark work, providing a clear potential involvement of plasma lipoproteins in disease was published by Shore *et al*, in 1987 [7]. These authors studied the animal model of MS, experimental autoimmune encephalomyelitis (EAE) and concluded that "*major changes in apoE-containing lipoproteins are undoubtedly significant in the altered immune function in EAE*". Supporting this prediction, it was observed higher plasma apoE concentrations in MS patients during relapses in comparison to remission states and lower levels in patients under remission in comparison to normal controls [8-10]. Studying EAE induction in apoE-deficient female mice, Karussis in 2003, found that apoE deficiency might be connected with a defective neuronal repair mechanism and enhanced immune reactivity and worse course of the disease [11]. These results could indicate that plasma apoE may have an immunosuppressive role in MS [9]. In agreement with this concept, our group observed that lower levels of plasma apoE might

In their work, Shore *et al* observed higher concentrations of total LDL and HDL cholesterol after onset of clinical symptoms. Giubilei *et al*, in 2002, studied plasma lipoproteins and magnetic resonance imaging (MRI) in patients with a first clinical episode suggestive of MS (CIS), supporting the findings in EAE [13]. These authors The possible influence of apoE allele polymorphism in MS susceptibility and disease severity has been addressed in many studies. Overall, literature does not suggest a role of apoE alleles as risk factor of developing MS [18-19]. An association of apoE polymorphism with disease severity in MS patients has been more controversial. Using MRI methodology, some studies have shown an association between the apoE4 isoform and more severe brain tissue destruction in these patients [20-22]. However, an influence of this isoform on the clinical course of the disease is not established and the interaction with potential confounders should be considered. For example, it was suggested that an influence of apoE polymorphism on the clinical course, and even the risk of MS, could particularly exist in women [23]. Our group has provided evidence for an influence of cigarette smoking in apoE4-carriers, in modulating the clinical severity of RR-MS patients [24]. Some studies have suggested an association of apoE4 allele and apoA1 promoter polymorphism with cognitive impairment in these patients, which may occur very early in the clinical course of the disease [25-26].

As mentioned above, MS is a heterogeneous clinical entity. RR-MS has a higher prevalence in women (which is increasing) and the course of the disease is in general more disabling in men. It is not unreasonable to hypothesize that gender-related and other genetic influences could implicate different impacts of lipoprotein metabolism in MS. Few studies have analyzed the influence of MS therapies in plasma lipoproteins of these patients. However, these studies could provide useful insights on the pathogenic role of this metabolism. Our group first suggested that interferon beta therapy changes this metabolism in RR-MS patients. In particular, we found that at 12 month of therapy, lower apoA1 and higher apoE levels were associated with the presence of relapses and/or progression of the disease [27]. Others authors have found that MS therapy is associated with a decrease of plasma total cholesterol [28-29]. Overall, the reviewed data strongly support a role of plasma lipoproteins metabolism in the pathophysiology of the disease, as discussed below.

#### **2.1. Pathophysiological mechanisms**

A major link between plasma lipoproteins and MS concerns the immune system. It is well known that immune reactivity interacts with adaptive alterations of lipoprotein metabolism [30-31]. Recent reports have showed that distinct metabolic programs are essential for survival and functional specialization of different lymphocyte cell populations. For example, lipid oxidation is essential for Treg generation while Th1 differentiation and cytokine production by differentiated Th1, Th2 and Th17 cells are suppressed by lipids and require glucose metabolism [32]. Although the immunopathogenesis of MS lesions (demyelinating plaques) is heterogeneous and may differ in different patients, an imbalance favoring a Th1 effector cell activation is generally accepted [33]. Therefore, it would not be unexpected if an abnormal lipid modulation of immune functions could contribute for MS pathogenesis. However, a primary role of lymphocytes (T cells and B cells) in mediating CNS injury in this disease (at least in all patients) is controversial [32]. Myeloid cells play a pivotal role in the regulation of infiltrating lymphocyte cell activities and are involved in myelin breakdown and axonal injury [33-34]. Macrophages of M1 phenotype are characterized by high production of pro-inflammatory mediators and are crucial in Th1 cell response, while M2 phenotypes are associated with tissue remodeling/repair and expression of antiinflammatory molecules [35]. In MS lesions, myelin phagocytosis by myeloid cells induces a foamy appearance. Foamy macrophages are originated from resident myeloid cells (microglia) and infiltrating monocytes and are suggested to be of M2-type macrophages and to contribute to the resolution of brain inflammation [36-37].

Macrophage polarization is modulated by different factors. For example, the M2 antinflammatory phenotype is induced by HDLs and apoE [35, 38], and fatty acid and phospholipid synthesis is essential for phagocytic differentiation of human monocytes [39]. ApoE is one ligand for the LDL-receptor-related-protein-1 (LRP1). Quite interesting, LRP1 mediates the downregulation of microglial inflammatory activity by apoE [40] and is essential for phagocytosis of degraded myelin in mice with EAE [41]. Moreover, LRP1 is also expressed in neurons and astrocytes and regulates BBB permeability [42]. This scenario is consistent with a reduction of inflammatory infiltrates and clinical disability by apoEderived peptides in EAE [43] and immunosuppressive and neuroprotective effects of plasma apoE in EAE [11] and MS patients [8-10, 12].

Among the transcriptional factors regulating macrophage polarization, peroxisome proliferator-activated receptor (PPAR) γ is known to promote M2 macrophages [35]. This is of potential interest in the context of preliminary evidence implicating PPARs in MS pathogenesis and as therapeutic targets for the disease [44-45].

In brain, apoE is associated with HDL-like particles, also containing the second major apolipoprotein, apoA-I. These apolipoproteins are primarily located on separated lipoproteins particles [1]. Although apoE in the brain is predominantly synthetized by glial cells, plasma HDL/apoA-I may cross the BBB and influence its levels in the brain [2]. HDL effects include an inhibition of cytocine-induced expression of adhesion molecules in endothelial cells, which could further depress brain parenchyma immune reactivity [46]. As mentioned, higher levels of plasma HDL were found in CIS and RR-MS and were associated with a lower probability in development of acute inflammatory lesions in these patients [13, 16-17]. Recently, preliminary evidence from our group suggests that higher plasma HDL levels are associated with an increased intrathecal IgG synthesis in these patients [47]. Because low plasma HDL-cholesterol is associated with a predominance of proinflammatory phenotype of monocyte-derived macrophage [48], these findings suggest an immunosuppressive role of HDL in the development of MS lesions. This interpretation is further supported by the beneficial therapeutical effects of fingolimod in the disease [49].

566 Lipoproteins – Role in Health and Diseases

**2.1. Pathophysiological mechanisms** 

to contribute to the resolution of brain inflammation [36-37].

pathogenesis and as therapeutic targets for the disease [44-45].

apoE in EAE [11] and MS patients [8-10, 12].

A major link between plasma lipoproteins and MS concerns the immune system. It is well known that immune reactivity interacts with adaptive alterations of lipoprotein metabolism [30-31]. Recent reports have showed that distinct metabolic programs are essential for survival and functional specialization of different lymphocyte cell populations. For example, lipid oxidation is essential for Treg generation while Th1 differentiation and cytokine production by differentiated Th1, Th2 and Th17 cells are suppressed by lipids and require glucose metabolism [32]. Although the immunopathogenesis of MS lesions (demyelinating plaques) is heterogeneous and may differ in different patients, an imbalance favoring a Th1 effector cell activation is generally accepted [33]. Therefore, it would not be unexpected if an abnormal lipid modulation of immune functions could contribute for MS pathogenesis. However, a primary role of lymphocytes (T cells and B cells) in mediating CNS injury in this disease (at least in all patients) is controversial [32]. Myeloid cells play a pivotal role in the regulation of infiltrating lymphocyte cell activities and are involved in myelin breakdown and axonal injury [33-34]. Macrophages of M1 phenotype are characterized by high production of pro-inflammatory mediators and are crucial in Th1 cell response, while M2 phenotypes are associated with tissue remodeling/repair and expression of antiinflammatory molecules [35]. In MS lesions, myelin phagocytosis by myeloid cells induces a foamy appearance. Foamy macrophages are originated from resident myeloid cells (microglia) and infiltrating monocytes and are suggested to be of M2-type macrophages and

Macrophage polarization is modulated by different factors. For example, the M2 antinflammatory phenotype is induced by HDLs and apoE [35, 38], and fatty acid and phospholipid synthesis is essential for phagocytic differentiation of human monocytes [39]. ApoE is one ligand for the LDL-receptor-related-protein-1 (LRP1). Quite interesting, LRP1 mediates the downregulation of microglial inflammatory activity by apoE [40] and is essential for phagocytosis of degraded myelin in mice with EAE [41]. Moreover, LRP1 is also expressed in neurons and astrocytes and regulates BBB permeability [42]. This scenario is consistent with a reduction of inflammatory infiltrates and clinical disability by apoEderived peptides in EAE [43] and immunosuppressive and neuroprotective effects of plasma

Among the transcriptional factors regulating macrophage polarization, peroxisome proliferator-activated receptor (PPAR) γ is known to promote M2 macrophages [35]. This is of potential interest in the context of preliminary evidence implicating PPARs in MS

In brain, apoE is associated with HDL-like particles, also containing the second major apolipoprotein, apoA-I. These apolipoproteins are primarily located on separated lipoproteins particles [1]. Although apoE in the brain is predominantly synthetized by glial cells, plasma HDL/apoA-I may cross the BBB and influence its levels in the brain [2]. HDL effects include an inhibition of cytocine-induced expression of adhesion molecules in endothelial cells, which could further depress brain parenchyma immune reactivity [46]. As Fingolimod (FTY720) is a structural analog of sphingosine, which down modulates sphingosine 1-phosphate (S1P) receptors. S1P is a major component of HDL, including in the CNS and induces an anti-inflammatory phenotype in macrophages. SP1 receptors are widespread in CNS cells and a defect of sphingolipid and phospholipid metabolism is observed early in normal appearing white and grey matter in MS patients. Moreover, S1P is reduced in affected white matter and is increased in CSF of these patients [49]. Importantly, FTY720 treatment has been shown to have neuroprotective effects independent of immunomodulatory mechanisms [50]. These data suggest a protective role of endogenous HDL components not only in the genesis of acute inflammatory lesions but also in the neurodegenerative process of MS.

An involvement of oxidative stress in MS, including of lipid peroxidation has recently received much support [51]. Newcombe *et al*, in 1994, demonstrated for the first time the presence of oxidized LDL (ox-LDL) and their peroxidative end-products in early and actively demyelinating plaques in post-mortem MS brain [36]. They suggested that plasma LDL enters (through a damaged BBB) the parenchyma and is oxidatively modified in the lesions. More recent data supports an important involvement of oxidative damage including oxidized phospholipids in myelin and axon injury in MS [52]. Several studies have also demonstrated that measures of oxidative stress and lipid peroxidation are consistently increased in the blood of these patients [51]. Our group reported increased levels of serum oxLDL in RR-MS patients in remission in comparison to normal controls and higher levels during relapses [53]. These findings are consistent with a contribution of plasma ox-LDL in promotion BBB permeability and acute inflammatory CNS lesions in the disease. However, increased plasma lipid peroxidation or oxidative stress is probably not associated with disability progression in these patients [54]. The pathophysiology of acute lesions (MS plaques) and disability progression are indeed thought to be mediated by different mechanisms. In fact, it was suggested that low oxygen radical formation in peripheral leukocytes may be associated with a increased severity of the disease [55]. These findings indicate that the role of oxidative stress in MS is complex. An oral formulation of dimethylfumarate (BG-12) activates the Nrf2 antioxidant pathway and was recently observed to be of clinical benefit in RR-MS patients, possibly in disease progression also [56]. These recent promising results should stimulate future research to clarify the involvement of lipid peroxidation in the disease. It should be noted that this involvement further supports a role of plasma HDL in disease pathogenesis, as discussed above. Plasma HDL-associated α-tocopherol is transcytosed across the BBB and may have antioxidant as well as anti-inflammatory effects [3].

Ludewig and Laman (2004) remarked the similarities that may exist between the atherosclerotic plaque development and MS lesions and suggested: "*Systematic comparison of these two diseases involving foam cells in chronic lesions may prove fruitful*" [57]. As we have reviewed, recent research clearly supports this prediction. Moreover, patients with MS have several vascular abnormalities and a higher risk for ischemic stroke [58]. In 2003, our group first reported a pilot trial suggesting a benefit of statin monotherapy in the pathogenesis process (assessed by MRI) and clinical activity of RR-MS patients [59]. These beneficial effects were confirmed by Vollmer *et al* trial in 2004 [60] and in a long-term follow-up of our patients [61]. Very recently, beneficial effects of statin monotherapy were reported in patients with a first clinical episode (CIS) suggestive of MS [62-63]. A synthesis of some shared pathophysiological factors involved in MS and atherosclerosis is presented on Table 1. As we will discuss below, the presence of similar mechanisms involving plasma lipoprotein metabolism in the pathogenesis of atherosclerosis/ischemic and demyelinating lesions may be extensive to other chronic inflammatory and neurodegenerative pathologies.



**Table 1.** Some pathogenic similarities between multiple sclerosis and atherosclerosis.
