**5. Scavenger function of LSEC in clearance of oxidized lipoproteins**

Another unique feature of LSECs is extraordinary endocytic activity. LSECs are rich in coated pits and vesicles and other organelles associated with endocytosis. Although LSECs constitute only 2.8 % of the total liver volume, they contain about 15% of the total lysosomal volume and about 45% of the pinocytic vesicle volume of the liver (Blouin et

extracellular space whereas in old livers, the lipoproteins were confined to the vascular space. These results strongly suggest that age-related pseudocapillarization impairs the hepatic disposition of lipoproteins and thus plays a role in age-related dyslipidaemia. Matrix heparan sulfate proteoglycans bind and sequester lipoprotein remnants (Williams, 2008). In old age, formation of basal lamina beneath LSECs leads to a change in the proportions of extracellular matrix components and may result in impaired passage of lipoproteins across the space of Disse. However, the importance of this pathway in lipoprotein turnover has not been studied sufficiently, especially in connection to aging. A reduction in caloric intake by about 40% increases maximum life expectancy and is associated with a delay in the onset of most age-associated disorders and pathology (Everitt et al., 2005). It has also been demonstrated that caloric restriction delays the onset of pseudocapillarization in rats. In the old caloric restricted rats, endothelial thickness was significantly less and fenestrae porosity was significantly greater than in the old ad libitum fed rats. Moreover, caloric restriction prevented the age-related increase in perisinusoidal collagen IV staining (Jamieson et al., 2007). The finding that caloric restriction influences pseudocapillarization suggests that the latter is secondary to the aging process and thus potentially reversible. As a consequence, modulation of LSEC fenestrations might be a therapeutic target for the treatment of age-related dyslipidemia and prevention of vascular disease. On the other hand, early onset of pseudocapillarization and dyslipidemia occur in a transgenic mouse model of Werner

Another hallmark of old age - the reduction in liver size as a fraction of body weight - is usually in the order of 25-35% (Le Couteur and McLean, 1998) and is associated with a decrease in the number of hepatocytes. In addition, several studies have shown that the total hepatic blood flow is reduced by about 30-50% (Le Couteur and McLean, 1998). Liver perfusion, which is the blood flow per mass of liver, is also reduced in old age but to a lesser extent than total blood flow. Mechanisms for these changes remain unclear; however, a recent study using high resolution *in vivo* microscopy has shown how pseudocapillarization might contribute to these phenomena. There was a 14% reduction in the numbers of perfused sinusoids with old age and a 35% reduction in sinusoidal blood flow (Ito et al., 2007). Narrower sinusoids with thickened LSECs and swollen stellate cells with abundant lipid droplets were also observed. It was concluded that these changes caused age-related reduction in hepatic perfusion and hepatic blood flow by blocking the sinusoids (Ito et al., 2007). The clearance of highly extracted substrates from the circulation is dependent on blood flow, therefore the age-related reduction in hepatic blood flow has a dramatic effect on the liver's overall function (Le Couteur and McLean, 1998), including the clearance of

It is reasonable to conclude that pseudocapillarization, in combination with a reduction in

Another unique feature of LSECs is extraordinary endocytic activity. LSECs are rich in coated pits and vesicles and other organelles associated with endocytosis. Although LSECs constitute only 2.8 % of the total liver volume, they contain about 15% of the total lysosomal volume and about 45% of the pinocytic vesicle volume of the liver (Blouin et

hepatic blood flow, are two major factors contributing to age-related dyslipidemia.

**5. Scavenger function of LSEC in clearance of oxidized lipoproteins**

syndrome, a rare premature aging syndrome in humans.

lipoproteins.

al., 1977). Moreover, specific activities of several lysosomal enzymes are higher in LSECs than in other liver cells (Knook and Sleyster, 1980). LSECs express a set of high-affinity endocytic receptors for soluble macromolecular waste products, generated during normal tissue turnover, blood clotting, inflammatory processes and pathological conditions (McCourt et al., 1999; Skogh et al., 1985; Smedsrod, 2004; Smedsrod et al., 1994; Smedsrod et al., 2006; Smedsrod et al., 1997; Smedsrod et al., 1990). Connective tissue macromolecules including hyaluronan, chondroitin sulphate, collagen α-chain, Procollagen Propeptides (PICP, PINP and PIIINP), products released during cell death such as lysosomal enzymes and metabolic byproducts including oxidized low density lipoproteins (oxLDLs), advanced glycation end products, and immune complexes and microbial CpG motifs are exclusively cleared from the blood circulation by mannose receptor-mediated or scavenger receptor-mediated endocytosis in LSECs (Elvevold et al., 2008; Malovic et al., 2007; Martin-Armas et al., 2006; Skogh et al., 1985; Smedsrod, 2004; Smedsrod et al., 1997; Smedsrod et al., 1990).

LSECs express several different scavenger receptors including scavenger receptors –A, scavenger receptors-B, and scavenger receptors-H (Hughes et al., 1995; Malerod et al., 2002). However, stabilin-1 and stabilin-2 have been recognised as the main scavenger receptors on LSECs (Hansen et al., 2005; Hansen et al., 2002; McCourt et al., 1999; Politz et al., 2002; Zhou et al., 2000). Following receptor mediated endocytosis in LSECs most of the ligands are rapidly degraded intra-lysosomally. Thus, LSECs represent a major site of scavenging and degradation of harmful waste macromolecules from the circulation and have therefore been termed 'scavenger endothelial cells' (Seternes et al., 2002).

LSEC endocytosis of oxLDL may also be implicated in the development of atherosclerosis. Atherosclerosis begins as a progressive, chronic inflammatory condition characterized by thickening of the arterial intima through proliferation of intimal smooth muscle cells, which has been shown to be precipitated by cholesterol-rich LDL and triglycerides derived from chylomicron remnants (Fischer-Dzoga et al., 1976). This may then advance to a complex plaque, which can ultimately lead to serious cardiovascular complications, such as myocardial infarction and stroke from occluded arteries. The oxidative modification of LDL has been suggested to play an important role in the development of these events (Steinberg, 1997, 2009). LDL can undergo *in vivo* oxidation in the arterial walls (Yla-Herttuala et al., 1989) and in plasma (Avogaro et al., 1988; Holvoet et al., 1998b). The process starts within the LDL particle with oxidation of polyunsaturated fatty acids which generates a great number of various intermediate and end-products. Formation of free and organic radicals launches a chain reaction that causes fragmentation of both lipid and protein constituents of LDL. Formation of reactive aldehydes, such as malondialdehyde, 4-hydroxynonenal and glyoxal results in chemical modification of side chain amino groups of the lysine residues of apoB-100, which in turn leads to an increased net negative surface charge of the molecule (Baynes and Thorpe, 1999; Fu et al., 1996; Jialal and Devaraj, 1996; Oorni et al., 2000; Witztum and Steinberg, 1991; Young and McEneny, 2001). Therefore, the oxidative modification of LDL involves changes in both the protein and the lipid components of the LDL-particle. This in turn induces changes in surface charge and conformation, which renders LDL a ligand for scavenger receptors, and reduces or abolishes its affinity to the LDL receptor (Berliner and Heinecke, 1996; Li et al., 2011).

Liver Sinusoidal Endothelial Cells and Regulation of Blood Lipoproteins 271

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26.

In arterial intima, oxLDLs are taken up by macrophages via scavenger receptors. This induces foam cell formation and subsequent atheroma development (Henriksen et al., 1981, 1983; Steinbrecher et al., 1984). Oxldls are commonly present in atherosclerotic lesions of experimental animals and humans (Palinski et al., 1989; Yla-Herttuala et al., 1989). OxLDL has also been identified in plasma of healthy individuals (Avogaro et al., 1988; Ehara et al., 2001; Itabe and Takano, 2000). In patients with cardiovascular disease, plasma levels of oxLDL have been reported to be approximately fourfold higher than in healthy subjects (Ehara et al., 2001; Holvoet et al., 1998b). In addition to cardiovascular disease, increased levels of oxLDL are associated with ageing (Brinkley et al., 2009) and certain age-related pathologies, such as Alzheimer's disease (Kankaanpaa et al., 2009), glomerulosclerosis (Lee, 1999), and diabetes mellitus (Lopes-Virella et al., 1999).

Therefore, timely clearance and maintenance of low circulatory levels of oxLDLs appear to be important for the prevention of atherosclerosis (Holvoet et al., 1998b; Itabe, 2003). Previously, it has been shown that intravenously injected radiolabeled oxLDLs are rapidly removed from blood by uptake in Kupffer cells and LSECs (Ling et al., 1997; Van Berkel et al., 1991). However, a recent study demonstrated that Kupffer cells are only active in uptake of heavily oxidized LDL (Li et al., 2011). which is mainly present in atherosclerotic plaques (Yla-Herttuala et al., 1989) or formed as an artifact during *in vitro* oxLDL preparation. At the same time, LSECs hold an exclusive role in the uptake of mildly oxidized LDL from the circulation (Li et al., 2011). Mildly oxidized LDL is the major form of oxLDL found in the blood (Chang et al., 1997; Holvoet et al., 1998a; Holvoet et al., 1998b), and has proatherogenic properties (Berliner et al., 1990; Watson et al., 1997; Witztum and Steinberg, 1991). Both stabilin-1 and stabilin-2 are involved in the endocytic uptake of oxLDL by LSECs. Stabilin-1, however, appears to be more important for the uptake of mildly oxidized LDL, which represents physiological blood-borne oxLDL, while stabilin-2 is important for uptake when there is greater LDL modification (Li et al., 2011).

The morphological changes in the LSEC in old age might also affect its role in endocytosis. Recently, *in vivo* microscopy was used to examine the real time uptake of scavenger receptor ligands by LSECs (Ito et al., 2007). Endocytosis was clearly diminished in old mice, particularly in the pericentral zone which may indicate hypoxic liver damage. The effect of old age on clearance of oxLDL by LSEC has not been examined yet. However, involvement of stabilin 1 and 2, the two major LSEC scavenger receptors, in the process of oxLDL uptake (Li et al., 2011) makes it likely that oxLDL clearance would be diminished in old age. This change would increase the level of oxLDL in the circulation, thereby promoting its extrahepatic concentration and increasing the risk of the development of atherosclerosis.
