**2. The structure of the hepatic sinusoid**

The hepatic sinusoids are small blood vessels, comparable to capillaries in size, that perfuse the hepatocytes. However, unlike the capillaries in other tissues, sinusoids are formed by a discontinuous endothelium that lacks any significant underlying basement membrane. Walls of sinusoids are formed by the liver sinusoidal endothelial cells (LSECs). LSECs are separated from liver parenchyma by the perisinusoidal extravascular space, known as the space of Disse (Figure 1A). LSECs contribute only 15-20% of all liver cells but comprise 70% of the population of sinusoidal cells in the liver (Arias, 1990; Arii and Imamura, 2000; Blouin et al., 1977; Knook and Sleyster, 1976).

The LSECs are perforated by trans-cytoplasmic pores called fenestrae, which do not have any intervening diaphragmatic membrane, and thus are fully patent holes through the cell (Figure 1A-B). This specialized lace-like morphology of the LSECs minimizes any barrier to

Liver Sinusoidal Endothelial Cells and Regulation of Blood Lipoproteins 265

Fig. 1. A: Transmission electron micrograph showing liver sinusoidal endothelial cell (LSEC) perforated by fenestrae (Fen). (Hep) – hepatocyte, (SoD) - space of Disse, (SC) - stellate cells with lipid droplet in space of Disse. B: Scanning electron micrograph of an isolated liver sinusoidal endothelial cell showing fenestrae clustered into sieve plates (SP). C: Scanning electron micrograph of a vascular cast showing branches of the portal vein (TPV) with surrounding sinusoidal network (Sin). (Preparations performed by A Warren).

the bi-directional transfer of solutes and particulate substrates between the sinusoidal blood and hepatocytes, whilst retaining the capacity and substantial surface area to undergo interactions with circulating blood cells including immune cells (Cogger and Le Couteur, 2009; Fraser et al., 1995; Wisse et al., 1996). Fenestrae are not uniformly distributed over the LSEC surface but are aggregated into groups of tens to hundreds in gossamer thin areas of cytoplasm and towards the periphery of the cell. These areas of fenestral aggregations are termed sieve plates (Figure 1B). Between 60-75% of fenestrae are found within sieve plates in rats (Vidal-Vanaclocha and Barbera-Guillem, 1985) but isolated fenestrae are also frequently observed on the LSEC surface.

The LSEC becomes fenestrated at an early gestational stage (Enzan et al., 1997; Martinez-Hernandez and Amenta, 1993; Nonaka et al., 2007; Smedsrod et al., 2009). The porosity of the sinusoids depends on the number and especially the size or diameter of the fenestrae. The diameter of fenestrae has a normal distribution curve of about 50-200nm (Cogger and Le Couteur, 2009). Gaps larger than about 200nm are regarded as artifacts formed during specimen preparation for electron microscopy (Akinc et al., 2009; Fraser et al., 1978; Fraser et al., 1980; Hilmer et al., 2005a).

Fenestrae have been found in many species including such diverse species as man, rat, mouse, guinea pig, sheep, goat, rabbit, fowl, monkey, baboon, bat, kitten, dog, turtle and fish (Cogger and Le Couteur, 2009).

The fenestrated endothelium was first suggested as a filter of chylomicrons by Wisse in 1970 (Wisse, 1970) from their reported diameters (Fraser et al., 1968) and termed the "liver sieve" once sieving was confirmed (Fraser et al., 1978; Naito and Wisse, 1978). Fenestrae allow the transfer of a wide range of substrates including plasma and plasma molecules, such as plasma proteins, some lipoproteins and colloidal particles (Le Couteur et al., 2005). The latter also include artificial chylomicron-like nanospheres such as Intralipid, but also small viruses leading to hepatitis, viral vectors for DNA manipulation of hepatocytes.

The fenestrated LSEC can be defined as an ultrafiltration system because it is a low pressure system with pores approximately 100nm in diameter. Specifically, the liver sieve can be described as a Loeb–Sourirajan ultrafiltration system, with the LSECs providing the thin porous layer (Baker, 2004). The transfer of fluid across an ultrafiltration system can be calculated using the Hagen Poiseuille equation for ultrafiltration where the flux of fluid is proportional to the number of pores and the radius of the pores to the power of four (Baker, 2004; Le Couteur et al., 2006; Warren et al., 2005). Therefore small changes in the size of fenestrae has profound effects on the size and number of substrates and macromolecules that can gain passage into the space of Disse. Indeed, manipulation of fenestrae diameter might have a role in regulating the transfer of substrates in response to physiological changes, such as feeding and fasting (O'Reilly et al., 2010).

The space of Disse is the extravascular space lying between the hepatocytes and LSECs. It contains some components of extracellular matrix and most components of blood plasma filtered through LSEC's sieve plates. Extracellular matrix in the space of Disse contains fibronectin and collagen type I ,III, V, and VI. Collagen type IV is also present but unlike its sheet-like polymeric presentation in typical basement membranes, here it appears in the form of discontinuous aggregates (Martinez-Hernandez and Amenta, 1993). Of note, basement membrane has not been identified in liver sinusoids in any non-pathological state or developmental stage until old age (Enzan et al., 1997; Martinez-Hernandez and Amenta, 1993; Nonaka et al., 2007; Smedsrod et al., 2009), where its appearance is believed to be a sign of age-related degeneration and – possibly – a cause of pathology.

the bi-directional transfer of solutes and particulate substrates between the sinusoidal blood and hepatocytes, whilst retaining the capacity and substantial surface area to undergo interactions with circulating blood cells including immune cells (Cogger and Le Couteur, 2009; Fraser et al., 1995; Wisse et al., 1996). Fenestrae are not uniformly distributed over the LSEC surface but are aggregated into groups of tens to hundreds in gossamer thin areas of cytoplasm and towards the periphery of the cell. These areas of fenestral aggregations are termed sieve plates (Figure 1B). Between 60-75% of fenestrae are found within sieve plates in rats (Vidal-Vanaclocha and Barbera-Guillem, 1985) but isolated fenestrae are also

The LSEC becomes fenestrated at an early gestational stage (Enzan et al., 1997; Martinez-Hernandez and Amenta, 1993; Nonaka et al., 2007; Smedsrod et al., 2009). The porosity of the sinusoids depends on the number and especially the size or diameter of the fenestrae. The diameter of fenestrae has a normal distribution curve of about 50-200nm (Cogger and Le Couteur, 2009). Gaps larger than about 200nm are regarded as artifacts formed during specimen preparation for electron microscopy (Akinc et al., 2009; Fraser et al., 1978; Fraser et

Fenestrae have been found in many species including such diverse species as man, rat, mouse, guinea pig, sheep, goat, rabbit, fowl, monkey, baboon, bat, kitten, dog, turtle and

The fenestrated endothelium was first suggested as a filter of chylomicrons by Wisse in 1970 (Wisse, 1970) from their reported diameters (Fraser et al., 1968) and termed the "liver sieve" once sieving was confirmed (Fraser et al., 1978; Naito and Wisse, 1978). Fenestrae allow the transfer of a wide range of substrates including plasma and plasma molecules, such as plasma proteins, some lipoproteins and colloidal particles (Le Couteur et al., 2005). The latter also include artificial chylomicron-like nanospheres such as Intralipid, but also small

The fenestrated LSEC can be defined as an ultrafiltration system because it is a low pressure system with pores approximately 100nm in diameter. Specifically, the liver sieve can be described as a Loeb–Sourirajan ultrafiltration system, with the LSECs providing the thin porous layer (Baker, 2004). The transfer of fluid across an ultrafiltration system can be calculated using the Hagen Poiseuille equation for ultrafiltration where the flux of fluid is proportional to the number of pores and the radius of the pores to the power of four (Baker, 2004; Le Couteur et al., 2006; Warren et al., 2005). Therefore small changes in the size of fenestrae has profound effects on the size and number of substrates and macromolecules that can gain passage into the space of Disse. Indeed, manipulation of fenestrae diameter might have a role in regulating the transfer of substrates in response to physiological

The space of Disse is the extravascular space lying between the hepatocytes and LSECs. It contains some components of extracellular matrix and most components of blood plasma filtered through LSEC's sieve plates. Extracellular matrix in the space of Disse contains fibronectin and collagen type I ,III, V, and VI. Collagen type IV is also present but unlike its sheet-like polymeric presentation in typical basement membranes, here it appears in the form of discontinuous aggregates (Martinez-Hernandez and Amenta, 1993). Of note, basement membrane has not been identified in liver sinusoids in any non-pathological state or developmental stage until old age (Enzan et al., 1997; Martinez-Hernandez and Amenta, 1993; Nonaka et al., 2007; Smedsrod et al., 2009), where its appearance is believed to be a

viruses leading to hepatitis, viral vectors for DNA manipulation of hepatocytes.

changes, such as feeding and fasting (O'Reilly et al., 2010).

sign of age-related degeneration and – possibly – a cause of pathology.

frequently observed on the LSEC surface.

al., 1980; Hilmer et al., 2005a).

fish (Cogger and Le Couteur, 2009).

Fig. 1. A: Transmission electron micrograph showing liver sinusoidal endothelial cell (LSEC) perforated by fenestrae (Fen). (Hep) – hepatocyte, (SoD) - space of Disse, (SC) - stellate cells with lipid droplet in space of Disse. B: Scanning electron micrograph of an isolated liver sinusoidal endothelial cell showing fenestrae clustered into sieve plates (SP). C: Scanning electron micrograph of a vascular cast showing branches of the portal vein (TPV) with surrounding sinusoidal network (Sin). (Preparations performed by A Warren).

Liver Sinusoidal Endothelial Cells and Regulation of Blood Lipoproteins 267

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.,

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.

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

(Preparations performed by A Warren).

2009).

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., 1995; Wisse et al., 1996).

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