**5. Biological function**

Many cellular events are initiated and modulated by biologically active oxidized phospholipids. OxPLs were initially characterized as an active principle of minimally modified LDL (MM-LDL), responsible for its ability to stimulate EC to bind the leukocytes (Watson et al 1995). MM-LDL and OxPLs has the characteristic feature of inflammatory agonist i.e., their ability to activate binding of monocytes but not neutrophils (Watson et al 1997). In contrast to lipopolysaccride (LPS), tumor necrosis factor α (TNFα), or interleukin 1 (IL-1), MM-LDL does not up-regulate the expression of ICAM-1, VCAM-1 and E-Selectin on EC (Kim et al 1994), but promotes surface deposition of CS-1-containing variant of fibronectin (CS-1 FN) serving as ligand for the α4β1 (VLA-4) integrin expressed on the surface of monocytes (Shih et al 1999). Similar to MM-LDL, OxPLs selectively stimulate adhesion of monocytes by CS-1 FN-dependent mechanism. Likewise other inflammatory agonists, OxPLs also stimulate the production of cyto- and chemokines. OxPLs are known to up-regulate expression IL-6, IL-8, MCP-1, GROα, MIP-1α, MIP-1β and CXCL3 (Subbanagounder et al 2002, Furnkranz et al 2005, Lee et al 2000, Reddy et al 2002, Kadl et al 2002, Gargalovic et al 2006, Huo et al 2001).

Expression of a number of genes related to angiogenesis, atherosclerosis, inflammation and wound healing are modulated by oxidized phospholipids in human aortic endothelial cells (Berliner and Gharavi, 2008; Gargalovic et al., 2006). Bochkov and colleagues (2002, 2007) have made known that OxPLs counteract the lipopolysaccride (LPS) pathway. Considering anti-inflammatory role of OxPLs, they reported that oxidized 1-palmitoyl-2arachidonoyl-*sn*glycero-3-phosphocholine (OxPAPC) interfered with the ability of LPS to bind to the LPSbinding protein (LBP) and to CD-14, thus suppressing LPS-induced nuclear factor-κB (NFκB)-mediated up-regulation of inflammatory genes.

Knapp and coworkers (2007) found that OxPAPC inhibits the interaction of LPS with LPSbinding protein and CD14. This also reduces phagocytotic activity of neutrophils and macrophages by a CD-14-independent mechanism. However, in these experiments, administration of OxPAPC rendered mice highly susceptible to *Escherichia coli* peritonitis, which may cause mortality during gram-negative sepsis *in vivo*. Thus the overall harmful profile of phospholipid oxidation products includes the impairment of host response to bacterial infections.

Recently, Gharavi and colleagues (2007) have reported the activation of JAK2/STAT3 pathway by phospholipids and implicated their role in atherogenesis. 1-Palmitoyl-2 epoxyisoprostane-*sn*-glycero-3-phosphocholine, an oxidation product of -1palmitoyl-2 arachidonoyl-*sn*-glycero-3-phosphocholine, induces c-Src kinase-dependent activation of JAK2 in endothelial cells and synthesis of chemotactic factors, such as interleukin (IL)-8. In turn, STAT3 activation and regulation of IL-8 transcription is dependent on JAK2 leading to the enhanced levels of STAT3 activity in inflammatory regions of human atherosclerotic lesions. Since STAT3 activation is involved in other chronic inflammatory diseases such as rheumatoid arthritis, psoriasis etc, it has been suggested that STAT3 activation by oxidized phospholipids could be an important interventional target for atherosclerosis and other diseases with inflammatory components.

#### **5.1. Regulation of vascular cell function**

OxPLs have multiple effects on endothelial cells. After 4h treatment with 50μg/ml of OxPAPC ~1000 genes are regulated amongst which ~600 are up-regulated and ~400 are down-regulated (Gargalovic et al 2006). Also, a major difference in responsiveness to specific effects of Ox-PAPC of endothelial cells from different human donors has been documented (Gargalovic et al 2006). The atherogenic pathways which were found to be upregulated include inflammation, cholesterol synthesis, coagulation and decrease in cell division. Some important effects of OxPAPC on endothelial cell function independent of gene regulation have been reported. OxPAPC has been shown to increase monocytes but not neutrophils binding by activating β-1 integrin (Berlin et al 2008, Leitinger et al 2005).

Many effects of OxPLs are mediated by its interaction with CD36. Several studies have indicated that LDL supplemented with OxPAPC or vesicles supplemented with fragmented α/β unsaturated fatty acids at the sn-2 position, such as KOdiA or HODA PC, bind to CD36 (Podrez et al 2002, Greenberg et al 2006). Another important phagocytic function of macrophages is the uptake of apoptotic cells, which are abundant in atherosclerotic plaques. OxPLs including oxidized phosphatidyl serine and phosphatidyl choline derivatives were shown to serve as ligands for macrophage uptake of apoptotic cells (Chou et al 2008, Greenberg et al 2006).

OxPLs also interact and bind with other recognition receptors in macrophages such as TLRs, CD14, LPS binding protein and C-reactive protein competing with negative ligands (Bochkov et al 2007, Bochkov et al 2002, Erridge et al 2008, Miller et al 2003). Thus, the formation of OxPLs during inflammation may represent an important feedback mechanism to limit further tissue damage. OxPLs have also been shown to activate macrophages. Currently conducted studies have revealed the role of OxPAPC in inducing lung injury and cytokine production by lung macrophages (Imai et al 2008).

The role of OxPLs in adaptive immune response can't be overlooked where they modulate the maturation process of dendritic cells (DCs). OxPLs also regulate innate immunity in human leprosy (Cruz et al 2008). In addition to the effects on DCs, OxPLs have also been shown to affect and induce T-cells (Seyerl et al 2008).

Phenotypic switching of smooth muscle cells (SMCs) involving increased proliferation; enhanced migration and down-regulation of SMC differentiation marker genes play a critical role in atherogenesis. Many studies have shown that OxPLs stimulate differentiation and cell division of SMCs (Heery et al 1995, Pidkovka et al 2007) while others have shown activation of apoptotic signaling pathways (Fruhwirth et al 2008).

### **5.2. Gene expression**

418 Lipoproteins – Role in Health and Diseases

bacterial infections.

Greenberg et al 2006).

κB)-mediated up-regulation of inflammatory genes.

diseases with inflammatory components.

**5.1. Regulation of vascular cell function** 

have made known that OxPLs counteract the lipopolysaccride (LPS) pathway. Considering anti-inflammatory role of OxPLs, they reported that oxidized 1-palmitoyl-2arachidonoyl-*sn*glycero-3-phosphocholine (OxPAPC) interfered with the ability of LPS to bind to the LPSbinding protein (LBP) and to CD-14, thus suppressing LPS-induced nuclear factor-κB (NF-

Knapp and coworkers (2007) found that OxPAPC inhibits the interaction of LPS with LPSbinding protein and CD14. This also reduces phagocytotic activity of neutrophils and macrophages by a CD-14-independent mechanism. However, in these experiments, administration of OxPAPC rendered mice highly susceptible to *Escherichia coli* peritonitis, which may cause mortality during gram-negative sepsis *in vivo*. Thus the overall harmful profile of phospholipid oxidation products includes the impairment of host response to

Recently, Gharavi and colleagues (2007) have reported the activation of JAK2/STAT3 pathway by phospholipids and implicated their role in atherogenesis. 1-Palmitoyl-2 epoxyisoprostane-*sn*-glycero-3-phosphocholine, an oxidation product of -1palmitoyl-2 arachidonoyl-*sn*-glycero-3-phosphocholine, induces c-Src kinase-dependent activation of JAK2 in endothelial cells and synthesis of chemotactic factors, such as interleukin (IL)-8. In turn, STAT3 activation and regulation of IL-8 transcription is dependent on JAK2 leading to the enhanced levels of STAT3 activity in inflammatory regions of human atherosclerotic lesions. Since STAT3 activation is involved in other chronic inflammatory diseases such as rheumatoid arthritis, psoriasis etc, it has been suggested that STAT3 activation by oxidized phospholipids could be an important interventional target for atherosclerosis and other

OxPLs have multiple effects on endothelial cells. After 4h treatment with 50μg/ml of OxPAPC ~1000 genes are regulated amongst which ~600 are up-regulated and ~400 are down-regulated (Gargalovic et al 2006). Also, a major difference in responsiveness to specific effects of Ox-PAPC of endothelial cells from different human donors has been documented (Gargalovic et al 2006). The atherogenic pathways which were found to be upregulated include inflammation, cholesterol synthesis, coagulation and decrease in cell division. Some important effects of OxPAPC on endothelial cell function independent of gene regulation have been reported. OxPAPC has been shown to increase monocytes but not neutrophils binding by activating β-1 integrin (Berlin et al 2008, Leitinger et al 2005).

Many effects of OxPLs are mediated by its interaction with CD36. Several studies have indicated that LDL supplemented with OxPAPC or vesicles supplemented with fragmented α/β unsaturated fatty acids at the sn-2 position, such as KOdiA or HODA PC, bind to CD36 (Podrez et al 2002, Greenberg et al 2006). Another important phagocytic function of macrophages is the uptake of apoptotic cells, which are abundant in atherosclerotic plaques. OxPLs including oxidized phosphatidyl serine and phosphatidyl choline derivatives were shown to serve as ligands for macrophage uptake of apoptotic cells (Chou et al 2008, OxPLs have profound effect on gene expression. OxPAPC have been shown to modulate the expression of approximately 1000 genes in human aortic ECs which include both upregulated and down-regulated mRNAs (Gargalovic et al 2006). OxPLs regulate genes related to inflammation, lipid metabolism, cellular stress, proliferation, and differentiation. These include VEGF-A and IL-8, which are induced by OxPLs independent of their transcription factors.

### **5.3. Pathophysiological functions**

Pathophysiologically OxPLs are involved in various proinflammatory and cardiovascular disorder; details are being described below (FIGURE-3).

#### **5.4. Atherosclerosis**

Quantification of OxPLs using liquid chromatography coupled with mass spectrometry has indicated that atherosclerotic vessels contain high concentrations of OxPCs. Different species of OxPCs were detected in atherosclerotic vessels including PL-hydroperoxides and hydroxides (Waddington et al 2001). In addition to elevated levels of OxPLs, atherosclerotic vessels express high amounts of proteins known to be induced by OxPLs in vitro. The latter includes MKP-1 (Reddy et al 2004), ATF3, ATF4 (Gargalovic et al 2006), SREBP-1 (Yeh et al 2004), HO-1 and IL-8 (Cheng et al 2009), MCP-1 and COX-2 (Ma et al 2008). OxPLs act on all major cell types involved in atherogenesis including monocytes, endothelial and vascular smooth muscle cells, lymphocytes, and platelets.

**Figure 3.** Oxidized phospholipids present in oxidized LDL induce various Diseases.

### **5.5. Lung injury**

The epithelial lining pulmonary surfactant is permanently exposed to high concentrations of oxygen and other oxidants present in the air. Ozone gas also plays a role in generating oxidatively truncated PLs (Uhlson et al 2002). Under normal healthy conditions surfactant is protected from oxidation by maintaining low contents of PUFAs, antioxidant action of glutathione present in the lining fluid and surfactant proteins A and D (Kuzmenko et al 2004). However, the accumulation of biologically active OxPLs products occurs in pathological states due to the oxidation of surfactant PCs, membrane lipids and apoptosis of bronchial cells. Studies conducted with animal models have shown that OxPLs protect lungs from acute lung injury. Ma et al. (2004) showed that OxPAPC inhibits elevation of TNFa in mice upon intratracheal or systemic administration of LPS or CpG DNA. Hence the available data shows that OxPLs may induce either beneficial or detrimental effects on lungs. The action of OxPLs on the lungs may depend upon their concentrations, lower levels of OxPLs protect endothelial barrier whereas high concentrations of the same OxPLs induce disruptive effects (Birukov et al 2004, DeMaio et al 2006).

#### **5.6. Ischemia**

420 Lipoproteins – Role in Health and Diseases

**5.5. Lung injury** 

**Figure 3.** Oxidized phospholipids present in oxidized LDL induce various Diseases.

The epithelial lining pulmonary surfactant is permanently exposed to high concentrations of oxygen and other oxidants present in the air. Ozone gas also plays a role in generating oxidatively truncated PLs (Uhlson et al 2002). Under normal healthy conditions surfactant is protected from oxidation by maintaining low contents of PUFAs, antioxidant action of glutathione present in the lining fluid and surfactant proteins A and D (Kuzmenko et al 2004). However, the accumulation of biologically active OxPLs products occurs in pathological states due to the oxidation of surfactant PCs, membrane lipids and apoptosis of bronchial cells. Studies conducted with animal models have shown that OxPLs protect lungs from acute lung injury. Ma et al. (2004) showed that OxPAPC inhibits elevation of TNFa in mice upon intratracheal or systemic administration of LPS or CpG DNA. Hence the available data shows that OxPLs may induce either beneficial or detrimental effects on lungs. The action of OxPLs on the lungs may depend upon their concentrations, lower levels Ischemia/reperfusion results in elevated levels of OxPLs both in tissues and systemic levels. PAF like (alkyl-acyl) OxPLs were detected within the first minutes after reperfusion of kidneys after warm ischemia (Lloberas et al 2002). Plasma concentrations of fragmented OxPCs were increased in patients during the reperfusion period after coronary surgery with cardiopulmonary bypass (Frey et al 2000). Hence available data shows that ischemia/reperfusion is a pathological state characterized by elevated local and circulating levels of OxPLs.

### **5.7. Inflammation**

Inflammation is characterized by a massive production of ROS. The elevation of circulating levels of OxLDL in response to inflammatory stimuli has already been shown. The OxPLs production in response to inflammation is induced by different cell types including leukocytes. Phorbol ester-stimulated neutrophils and monocytes incubated with PUFA-PCs produced mono- and bishydroperoxides of PC, as well as isoP–PC, thus suggesting that activated phagocytes can oxidize lipids in the surrounding medium (Jerlich et al 2003).

#### **5.8. Radiation stress**

Formation of OxPLs can be activated by visual and UV-light. OxPLs accumulating in retinas serve as ligands for CD36-dependent phagocytosis of shed photoreceptor outer segments by retinal pigment epithelium; this process is necessary for normal function of the retina (Sun et al 2006). Generation of OxPLs by light exposure has also been shown in skin cells. UVA-1 irradiated PAPC containing several OxPLs species induced expression of antioxidant and anti-inflammatory enzyme heme oxygenase-1 in dermal fibroblasts, keratinocytes, and in a three-dimensional epidermal equivalent model (Gruber et al 2007). Therefore, OxPLs are likely to play a protective role in UVA irradiated skin by inducing HO-1.

#### **5.9. Leprosy**

Oxidized PCs have been detected in lepromatous (disseminated) leprosy lesions, but not in tuberculous leprosy characterized by stronger host immune response and self-contained infection (Cruz et al 2008). Lepromatous leprosy lesions are characterized by the accumulation of OxPLs, which can counteract innate and specific immune responses, thereby promoting survival.

#### **5.10. Multiple sclerosis**

Multiple sclerosis (MS) is an autoimmune disease of the brain that causes neurodegeneration. Role of OxPLs in MS is supported by Qin et al. (2007), demonstrating the presence of OxPLs (alone and conjugated to a 15 KDa protein) in extracts of MS lesions directly by Western blot analyses using the E06 antibody. OxPLs might be promoting the inflammatory process in MS lesions.
