биохимия атеросклероза

TLR4

CD36

PTK

Proteasomal degradation

IL-1Rα

IL-1β

IL-6

TNF-α/β

IFN-γ/β

FIGURE 5.1. Hypothetical model of CD36–TLR4 interactions and NF-κB activation. NF-κB is an evolutionarily conserved transcription factor required for many aspects of the immune and inflammatory response. In atherosclerosis, NF-κB coordinates the expression of a wide array of inflammatory genes (reviewed in [100]) and is thus central to the pathology of this disease. The activity of NF-κB is tightly regulated by I-κB inhibitory proteins, which bind to NF-κB, sequestering it in the cytoplasm. When the NF-κB pathway is activated, I-κBs are phosphorylated by kinases (IKKs) leading to the dissociation of the NF-κB:I-κB complex and NF-κB translocation to the nucleus. In the nucleus, NF-κB binds to the promoter of target genes and stimulates gene expression. An important regulator of the NF-κB pathway is the toll-like receptor (TLR) family of pattern recognition receptors. TLRs are crucial to the innate host response to infectious agents such as bacteria due to their ability to bind endotoxin/LPS. The growing body of data linking such pathogens with atherosclerosis has cast a new light on the role of TLRs in lesion development and progression. In particular, the TLR4 pathway has garnered much attention (reviewed in [101]). Ligand engagement of TLR4 triggers the recruitment of intracellular adapter proteins such as MyD88, which results in the activation of numerous kinases, phosphorylation of I-κB and thus elevated NF-κB activity. We hypothesize that CD36–TLR4 aggregation results in the recruitment of CD36associated protein tyrosine kinases (PTKs), which further enhance IKK and NF-κB activity. Potential sites of PTK activity are indicated by dashed arrows as are some of the genes which may be affected by CD36 expression levels [45].

and c-Src, has also been suggested [48]. Together, the data strongly argue for a functional effect of CD36 on NF-κB activation, perhaps via intracellular kinases associated with the C-terminal cytoplasmic tail of CD36.

Physical interactions between CD36 and a major regulatory protein in the NF-κB pathway have also been detected. As noted in Fig. 5.1, the toll-like receptor (TLR)-4 is an important mediator of NF-κB and has been well characterized as a major receptor for LPS, a glycolipid found on the outer membrane of gram-negative bacteria [49–51]. Similar to other immunoreceptors, the first step in TLR4 signaling is ligand-dependent receptor aggregation, which initiates signaling cascades and thus leads to the appropriate inflammatory response. CD36 appears to participate in this receptor aggregation. In human monocytes, stimulation with LPS induced the aggregation of TLR4 and CD36 with CD14 [52] (another cell surface LPS receptor which facilitates interactions between LPS and TLR4). Receptor aggregation/colocalization between CD36 and TLR4 was also detected in retinal pigment epithelial cells [53, 54], the cells that lie between the retina and the choroids (the vascular bed which oxygenates the retina), and are adjacent to the retinal photoreceptors. These examples of CD36–TLR4 interactions provide one possible mechanism whereby CD36 may regulate the NF-κB pathway.

The data above can be summarized as follows: (i) CD36 physically interacts with Src-related PTKs, (ii) CD36 expression modulates the magnitude of oxLDL-mediated NF-κB activation, and (iii) ligand engagement of TLR4 (which mediates NF-κB activation by LPS) induces aggregation with CD36. In addition, we have observed that CD36−/− mice display a muted inflammatory response following LPS injection (V.A. Drover and N.A. Abumrad, unpublished results). Thus, we propose a model (Fig. 5.1) whereby CD36 and its associated PTKs aggregate with TLR4 upon ligand binding. The PTKs may modulate TLR4 signaling to NF-κB via phosphorylation of downstream effector molecules or even the regulatory kinases directly as has been demonstrated previously [55, 56]. The report that CD36 is a signaling bridge for microbial diacylglyceride-stimulated TLR2 [57] supports this model. Further studies in lesions and macrophages from WT and CD36−/− mice are required to test the validity of this model and the potential role of CD36 in mediating atherosclerosis and inflammation.

Scavenger Receptor BI

Like CD36, SR-BI is a class B receptor and a type III transmembrane protein. SR-BI appears to have a hairpin topology, is heavily glycosylated, and binds numerous ligands (reviewed in [58]). SR-BI has a broad expression pattern including intestine, macrophages, endothelial cells, and SMCs but is most highly expressed in the liver and steroidogenic tissues where it plays a major role in cholesterol catabolism and hormone synthesis, respectively.

SR-BI is best known as the most physiologically relevant receptor for highdensity lipoprotein (HDL) particles [59]. SR-BI-mediated cholesterol uptake

80 Daisy Sahoo and Victor A. Drover

from HDL occurs by a process called selective uptake; the cholesteryl ester in the core of the HDL particle is selectively extracted and delivered to the cell for subsequent hydrolysis and/or storage. Mice lacking SR-BI have elevated plasma cholesterol, increased HDL particle size, and reduced cholesterol disposal in the liver [60–62]. In vitro, SR-BI also facilitates cholesterol efflux to lipid acceptors such as small unilamellar vesicles and small HDL particles [17, 63]. Together, the biochemical data would predict that SR-BI plays an atheroprotective role by promoting cholesterol efflux into the plasma as HDL and then facilitating hepatic HDL cholesterol disposal. Indeed, numerous studies in atherogenic strains of mice show that SR-BI deficiency promotes atherosclerosis [64–66] while hepatic overexpression is protective [67] (reviewed in [68]; see also Chapter 4).

While structure–function relationships in SR-BI have been extensively characterized, the role of SR-BI in cell signaling and inflammation remains poorly described. However, a few reports use various techniques to directly implicate SR-BI in cell signaling and inflammatory pathways, which may be relevant to atherosclerosis. For instance, Vishnyakova et al. [69] demonstrated that SR-BI is also a receptor for LPS in murine RAW and HeLa cells. Like SR-A, SR-BI may be involved in the clearance of LPS and attenuating the inflammatory response, thus protecting against atherosclerosis.

Serum amyloid A (SAA), a major acute-phase reactant typically transported on HDL, appears to be a ligand for SR-BI. SAA engagement of SRBI in transfected HeLa cells results in enhanced secretion of IL-8 [70], a proinflammatory and atherogenic chemokine (reviewed in [71]). This SR- BI-dependent, SAA-induced increase in IL-8 secretion requires the activation of ERK1/2 and p38 MAPK [70] and suggests a role for SR-BI in these signaling pathways. The observation that ERK1/2 activation by HDL is reduced by SR-BI neutralizing antibodies [72] further support this notion. One scenario whereby this pathway may be physiologically relevant is the ability of dietary cholesterol to raise serum SAA levels [73], which in turn can increase inflammation/IL-8 secretion and thus contribute to lesion formation. However, it is difficult to reconcile the concept that SR-BI mediates an inflammatory role in SAA/MAPK signaling with the observations from genetically modified mice illustrating an atheroprotective role for SR-BI in vivo (discussed earlier). One possibility is that SR-BI is atherogenic in the early stages of lesion development where inflammation may play a more prominent role in disease pathology. This is in agreement with a recent bone marrow transplantation study showing that SR-BI increased lesion development in LDL-R-deficient mice fed a Western diet for only 4 weeks [74].

The best evidence for SR-BI-dependent cell signaling in atherogenesis comes from the nitric oxide (NO) field. NO is critical for arterial tonicity and is an important mediator of atherosclerosis since chronic inhibition of NO production enhances lesion development in hypercholesterolemic rabbits [75]. NO is produced by endothelial NO synthase (eNOS) and several groups have reported that HDL stimulates eNOS activity in both epithelial and CHO cells

in an SR-BI-dependent fashion [76, 77]. As a physiologic measure of NO production, Yuhanna et al. [77] measured arterial relaxation in aortic rings ex vivo and found that HDL-mediated relaxation was highly attenuated in aortic rings from SR-BI−/− mice. Although the mechanism of action is unclear, intracellular ceramide levels covary with eNOS activation [76], suggesting that SR-BI may be mediating eNOS activity by regulating levels of this sphingolipid.

Cross talk between SR-BI-mediated eNOS activation and apoptotic cell death adds another layer of complexity to the role of SR-BI in cellular signaling mechanisms. Li et al. [78] recently reported that in the absence of ligand, SR-BI expression activates caspase-8-mediated apoptosis in CHO cells, mouse embryonic fibroblasts, and endothelial cells. Cotransfection with eNOS or incubation with HDL reversed this SR-BI-dependent cell death. The authors suggest that in healthy cells, HDL-SR-BI ligation inhibits inappropriate apoptosis via NO production. Cells in stressful, inflammatory environments have reduced eNOS activity and apoptosis occurs in an attempt to reduce further inflammation and damage to neighboring cells. Elevated mRNA levels of inflammatory markers in the artery in SR-BI−/− mice [79] are consistent with uncontrolled inflammation and support the authors’ hypothesis.

The Lectin-Like oxLDL Receptor-1 (LOX-1)

Also known as OLR1, this SR displays a type II membrane topology and is expressed on endothelial cells, macrophages, SMCs, and platelets. In addition to mLDL, bacteria, and apoptotic cells, LOX-1 can also bind advanced glycation end products and fibronectin, thus suggesting a role in cell adhesion (discussed later). Ligand engagement to LOX-1 appears to be linked to a host of intracellular signaling processes (discussed later) that may affect atherosclerotic progression but prospective studies in genetically modified mice have not been reported. However, the observations that (i) humans and rabbits exhibit LOX-1 accumulation in atherosclerotic lesions [17, 80] and (ii) LOX- 1 mutations in humans are associated with increased cardiovascular disease [55] suggest that LOX-1 is an important mediator of atherosclerosis. As mice lacking LOX-1 are not currently available, most of the definitive studies on the role of LOX-1 in inflammation/signaling utilize a mixed approach of heterologous gene expression, antisense knockdown technology, and specific α- LOX-1 antibodies which block oxLDL binding to the receptor.

One mechanism whereby LOX-1 may mediate atherosclerotic lesion development is through its ability to stimulate leukocyte adhesion following exposure to inflammatory stimuli. In human coronary artery endothelial cells (HCAEC), oxLDL-induced LOX-1 expression caused elevated monocyte chemoattractant protein-1 expression and monocyte adhesion. These effects were attenuated by transfection of the cells with antisense LOX-1, which abrogated LOX-1 expression [81]. Similarly, CHO-K1 cells transfected with a plasmid encoding LOX-1 exhibited increased leukocyte and THP-1 cell adhesion which could be blocked by oxLDL or an α-LOX-1 antibody [82]. In

82 Daisy Sahoo and Victor A. Drover

rats, leukocytes accumulated at sites of inflammation/damage in a LOX-1- dependent fashion with concomitant increases in adhesion molecule expression [83–85]. Although the mechanism(s) responsible for these LOX-1-mediated effects are not known, both MAPK (p38 and p42/p44) and PKC-β pathways appear to be involved in modulating changes in the expression of adhesion molecules [81, 84, 86].

The ability of oxLDL to stimulate other signaling pathways may also be involved in the activation of endothelial cells. Most notable is the ability of oxLDL to elevate levels of reactive oxygen species (ROS). Cominacini et al. [87] documented an oxLDL and LOX-1-dependent increase in intracellular ROS in bovine arterial endothelial cells (BAEC). Levels of NF-κB in the nucleus were also elevated by oxLDL exposure as determined by a qualitative mobility shift assay. Although a causal relationship between ROS production and nuclear NF-κB translocation was not shown in this study, the concept that ROS are upstream signals for NF-κB activation is well documented in the literature [88–92]. The additional observation that ROS levels following oxLDL stimulation rise well before NF-κB activation is detected [87] forms a strong circumstantial case for a LOX-1/ROS-mediated link between oxLDL and the NF-κB pathway in endothelial cells and other cell types such as articular chondrocytes [93].

The LOX-1-dependent rise of ROS in oxLDL-treated BAECs also results in a concomitant reduction of intracellular NO [94]. The authors speculate that oxLDL ligation of LOX-1 in atherosclerotic lesions may explain the impaired endothelium-dependent vasodilation commonly observed in this type of vascular disease. Since ROS are thought to increase oxLDL production in vivo, we see the potential for a vicious loop of LDL oxidation, ROS formation, NO inactivation, and NF-κB-dependent inflammation—all dependent on endothelial LOX-1. Perhaps not surprisingly, the situation may be further exacerbated by the feed-forward upregulation of LOX-1 expression by oxLDL [95, 96].

In addition to ROS signaling, LOX-1 appears to play some role in a myriad of signaling pathways that may be relevant to atherosclerosis. In addition to the role of p38 MAPK in adhesion molecule expression (discussed earlier), oxLDL induces apoptosis in neonatal rat cardiac myocytes in a LOX-1- and p38 MAPK-dependent fashion [97]. In cultured rat chondrocytes and HCAECs, oxLDL induces cell death via PI3 kinase/Akt [98] and NF-κB pathways [99], respectively; in each instance, cell death could be blocked by α-LOX-1 antibodies or abrogating LOX-1 expression. Given the complexity of the signaling pathways stimulated by oxLDL via LOX-1, significant cross talk appears likely.

Since LOX-1 is thought to be a major SR on endothelial cells, much of the work described herein has used primary or cultured endothelial cells. However, it is important to note that many of the pathways implicated in LOX-1 signaling are present in macrophages. LOX-1 is expressed on macrophages and oxLDL ligation may affect inflammation/apoptosis in this cell type in addition to endothelial cell activation. It is also likely that some of these signaling pathways terminate at the genes encoding adhesion molecules and other cell typespecific genes in the inflammatory/atherosclerotic expression profile (Fig. 5.2).

LOX-1

Nitric oxide

LOX-1, TNF-α, CD40/CD40L (inflammation)

MCP-1, ICAM, VCAM (adhesion)

MMP-1, MMP-3, MMP-9 (plaque rupture)

FIGURE 5.2. Intracellular signaling pathways following LOX-1 engagement by oxLDL in endothelial cells. LOX-1 is a type II integral membrane protein and contains a C-type lectin-like domain (CLD). Oxidized LDL engagement of LOX-1 causes the increased expression of numerous genes whose protein products are involved in inflammation, monocyte adhesion, and plaque rupture. These changes in gene expression occur as a result of the activation of numerous intracellular signaling pathways including mitogen-activated protein kinase (MAPK), protein kinase C (PKC), and PI3 kinase (PI3-K), as well as through the production of reactive oxygen species (ROS). It is likely that many of these signaling pathways act directly or indirectly via the NF-κB pathway. In addition, increased ROS production inactivates intracellular nitric oxide (NO), which may result in impaired vasodilation commonly observed in patients with atherosclerosis.

A major hurdle in the elucidation of these pathways is the lack of mice with a disrupted LOX-1 gene. Tissue/cell type-specific gene ablation may be necessary to disentangle the plethora of intracellular signals initiated by LOX-1 ligand engagement.

84 Daisy Sahoo and Victor A. Drover

Concluding Remarks

As the field of SR biology expands, our understanding of the functions of these proteins moves beyond the clearance and utilization of a particular ligand. As should now be evident, SRs are as promiscuous in their use of intracellular signaling pathways as they are in their cell surface ligands. Our understanding of these pathways and their role in the pathology of atherosclerosis is still a developing area of research, especially as many SRs have only recently been classified in this protein family. In addition, determining the net effect of engaging multiple SRs with a common ligand such as mLDL/oxLDL will be a challenging task. This will be further complicated by the fact that multiple SRs can affect the same signaling pathways. For instance, LOX-1 shares a functional link to NF-κB and NO with CD36 and SR-BI, respectively. Similarly, CD36 and SR-A both interact with Src-family PTKs.

While examining the effects of multilocus gene disruption may provide an overall measure of the relative contribution of the SRs to atherogenesis, determining the relevant mechanisms will require careful experimental design and attention to the effective cell type. In addition to the relative distribution of the SRs in critical cell types such as endothelial cells and monocytes/macrophages, the ability of a ligand or nutritional regimen to regulate SR levels and concomitant signaling pathways must be considered. The continued use of genetically modified mice and tissue-specific gene ablation are thus likely to play important roles in answering the remaining questions in this field.

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