**4. Ageing of the LSEC and regulation of blood lipids**

Many important diseases, particularly cardiovascular diseases, that result in disability and death, occur late in life, indicating that aging itself is a key risk factor. Old age is associated with significant changes in the cells of the hepatic sinusoid. Previously, it has been considered that the liver does not undergo significant aging changes because of its large functional reserve, regenerative capacity and dual blood supply (Popper, 1986). Only a few descriptions of the aging liver have been generally established, such as an increase in the number of polyploid and binucleate hepatocytes (Schmucker, 1998) and "brown atrophy", which is a reduction in liver mass accompanied by the deposition of the aging pigment, lipofuscin (Popper, 1986). Today it has become clear that age-related changes in hepatic structure and function are significant and influence systemic exposure to xenobiotics, endogenous substances associated with disease and medications (McLean and Le Couteur, 2004). Thus, such changes in the liver have implications for many diseases of aging and the aging process itself.

It has now been reported that old age is associated with substantial ultrastructural changes in the LSECs and space of Disse in intact livers of the rat (Jamieson et al., 2007; Le Couteur et

Multiple microvilli from the the sinusoidal surface of the hepatocytes protrude into the space of Disse and increase the available surface area for the recognition, transport and diffusion of substrates to and from the liver (Cogger and Le Couteur, 2009; Fraser et al.,

There are three other cell types residing in the liver sinusoids apart from the LSECs: Kupffer cells (resident liver macrophages), stellate cells and pit cells. Kupffer cells represent only 20% of all the population of liver sinusoidal cells but 80-90% of all tissue macrophages in the body (Knook and Sleyster, 1976). They generally reside within the lumen of the liver sinusoids and take up bacteria and other large particles, such as cell debris, from the circulation by phagocytosis. In response to bacterial infection, Kupffer cells produce cytokines and a number of soluble pro-inflammatory factors that promote influx and activation of neutrophils (Smedsrod et al., 1994; Smedsrod et al., 2009) and may alter the porosity of the sinusoids to promote cirrhosis (Dobbs et al., 1994). Together, LSECs (pinocytosis) and Kupffer cells (phagocytosis) constitute the hepatic reticuloendothelial system (RES), the most powerful scavenger system of mammals and other terrestrial

Because of the LSECs fenestrae, all lipoproteins except large chylomicrons have unimpeded access to the hepatocytes. After delivering triglycerides to peripheral tissues, chylomicrons become processed into so-called "chylomicron remnants" that carry significant amounts of cholesterol and are highly pro-atherogenic (Fujioka and Ishikawa, 2009; Karpe et al., 1994). At the same time they become small enough to pass through LSECs fenestrae (Fujioka and Ishikawa, 2009) and can be taken up by hepatocytes, which allows liver parenchyma to be the major site for removal of pro-atherogenic chylomicron remnants from the blood (Cooper, 1997; Dietschy et al., 1993) under normal circumstances. However, fast and efficient blood clearance of highly atherogenic chylomicron-remnants by hepatocytes

Many important diseases, particularly cardiovascular diseases, that result in disability and death, occur late in life, indicating that aging itself is a key risk factor. Old age is associated with significant changes in the cells of the hepatic sinusoid. Previously, it has been considered that the liver does not undergo significant aging changes because of its large functional reserve, regenerative capacity and dual blood supply (Popper, 1986). Only a few descriptions of the aging liver have been generally established, such as an increase in the number of polyploid and binucleate hepatocytes (Schmucker, 1998) and "brown atrophy", which is a reduction in liver mass accompanied by the deposition of the aging pigment, lipofuscin (Popper, 1986). Today it has become clear that age-related changes in hepatic structure and function are significant and influence systemic exposure to xenobiotics, endogenous substances associated with disease and medications (McLean and Le Couteur, 2004). Thus, such changes in the liver have implications for many diseases of aging and the

It has now been reported that old age is associated with substantial ultrastructural changes in the LSECs and space of Disse in intact livers of the rat (Jamieson et al., 2007; Le Couteur et

1995; Wisse et al., 1996).

vertebrates (Aschoff, 1924; Kawai et al., 1998).

requires well fenestrated LSECs.

aging process itself.

**3. The normal function of the LSEC in regulation of blood lipids** 

**4. Ageing of the LSEC and regulation of blood lipids** 

al., 2001), human (McLean et al., 2003), the mouse (Ito et al., 2007; Warren et al., 2005) and the non-human primate, *Papio hamadryas* (Cogger et al., 2003). The findings have been replicated in at least three separate centres around the world (Furrer et al.; Ito et al., 2007; Le Couteur et al., 2001; Stacchiotti et al., 2008). These changes have been termed 'pseudocapillarization' because the aging sinusoids become similar to capillaries seen in other non-fenestrated vascular beds (Le Couteur et al., 2001). Unlike 'capillarization' seen in the hepatic sinusoid in cirrhosis of the liver, aging is not associated with any of the typical changes apparent on light microscopy, such as bridging fibrosis and nodular regeneration (Le Couteur et al., 2001; Le Couteur et al., 2008). In old age, LSEC thickness is increased by approximately 50% and there is a similar reduction of about 50% in the porosity and number of fenestrae (Figure 2). These changes are associated with perisinusoidal basal lamina deposition in many old livers and some scattered collagen in the space of Disse. The effect of aging on the diameter of fenestrae has been inconsistent between species, however there is a trend towards a reduction in diameter of around 5-10% (Le Couteur et al., 2008). Isolated LSECs typically retain some of these ultrastructural changes. Fenestrae diameter was reduced in old age from 194±1 nm to 185±1 nm in isolated rat LSECs (O'Reilly et al., 2009).

Fig. 2. Scanning electron micrographs of the liver sinusoid of a young (A) and old (B) rat. The loss of fenestrae perforating the endothelial cell surface in the old liver is apparent. (Preparations performed by A Warren).

Fenestrations have a role in the transfer of lipoproteins from blood to the hepatocyte, therefore it is likely that pseudocapillarization of sinusoids will impair lipoprotein clearance by the liver and contribute to dyslipidaemia in older people (Le Couteur et al., 2002). Atherosclerosis increases dramatically with old age and its complications affect most older people (Lakatta and Levy, 2003). The clearance of chylomicron remnants is significantly impaired in older people (Borel et al., 1998; Krasinski et al., 1990) and in those aged 65 years and older, remnant-like lipoprotein cholesterol is associated with the development of coronary artery disease (Simons et al., 2001). To determine whether age-related defenestration impairs the transfer of lipoproteins across the LSECs, the multiple indicator dilution method was used to study lipoprotein disposition in perfused rat livers (Hilmer et al., 2005b). In young livers, lipoproteins (approximately 50 nm diameter) entered the entire

Liver Sinusoidal Endothelial Cells and Regulation of Blood Lipoproteins 269

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;

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

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

Smedsrod et al., 1997; Smedsrod et al., 1990).

et al., 2011).

termed 'scavenger endothelial cells' (Seternes et al., 2002).

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 syndrome, a rare premature aging syndrome in humans.

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 lipoproteins.

It is reasonable to conclude that pseudocapillarization, in combination with a reduction in hepatic blood flow, are two major factors contributing to age-related dyslipidemia.
