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Liver Biology and Pathobiology
Age-related changes in the hepatic sinusoidal endothelium impede lipoprotein transfer in the rat†
Article first published online: 29 NOV 2005
Copyright © 2005 American Association for the Study of Liver Diseases
Volume 42, Issue 6, pages 1349–1354, December 2005
How to Cite
Hilmer, S. N., Cogger, V. C., Fraser, R., McLean, A. J., Sullivan, D. and Le Couteur, D. G. (2005), Age-related changes in the hepatic sinusoidal endothelium impede lipoprotein transfer in the rat. Hepatology, 42: 1349–1354. doi: 10.1002/hep.20937
Potential conflict of interest: Nothing to report.
- Issue published online: 29 NOV 2005
- Article first published online: 29 NOV 2005
- Manuscript Accepted: 2 SEP 2005
- Manuscript Received: 21 OCT 2004
- National Health and Medical Research Council
- Department of Veterans Affairs
- Ageing and Alzheimers Research Foundation
- Australian Association of Gerontology RM Gibson grant
- RACP McCaughey Fellowship
The mechanisms for the association of old age with post-prandial hyperlipidemia and atherosclerosis are not well understood. Post-prandial hyperlipidemia has emerged as a significant risk for atherosclerosis. The liver is the central organ for lipoprotein metabolism. The initial step in the hepatic uptake of post-prandial lipoproteins is their transfer from the hepatic sinusoidal capillary lumen across the hepatic sinusoidal endothelium into the space of Disse. Here, they access hepatocytes for receptor-mediated uptake. We proposed that fenestrations (pores) within the hepatic sinusoidal endothelium filter lipoproteins on the basis of size. Recently we discovered age-related changes in the sinusoidal endothelium (pseudocapillarization), including reduction in the porosity of the endothelium. Using the impulse response technique in perfused rat livers, we found that aging is associated with impaired hepatic transendothelial transfer of chylomicrons with diameters smaller than those of fenestrations. In conclusion, age-related pseudocapillarization of the hepatic sinusoidal endothelium provides a novel mechanism for the association of old age with impaired hepatic lipoprotein metabolism and with atherosclerosis. (HEPATOLOGY 2005.)
The liver is the major organ for the metabolism of lipoproteins1 and is critical for the uptake and clearance of chylomicron remnants.2, 3 It has been well established that chylomicron remnants are highly atherogenic4, 5 and therefore, any impairment in the clearance of chylomicron remnants will have a major impact on the development of atherosclerosis.
The incidence and prevalence of atherosclerosis increases dramatically with old age and its clinical manifestations are present in the majority of older people.6 As the population ages, coronary syndromes are occurring primarily in older people, rather than in middle-aged patients with standard vascular risk factors such as smoking, fasting hyperlipidemia and hypertension.7 The prevalence of such risk factors does increase in old age; however, their overall impact on susceptibility to cardiovascular disease decreases as old age itself becomes the predominant influence.8 The pathogenesis of the association between aging and vascular disease remains unclear and is “a conundrum of significant proportions to clinicians and basic scientists alike.”9
The clearance of chylomicron remnants is significantly impaired in older people10, 11 and in people aged 65 years and older remnant-like lipoprotein cholesterol is strongly associated with the development of coronary artery disease.12 We hypothesized that age related-changes in the liver sinusoid impair the hepatic uptake of chylomicron remnants, providing a mechanism for the association of old age with post-prandial hyperlipidemia and atherosclerosis.13
The initial step in the hepatic disposition of postprandial lipoproteins is their transfer from the sinusoidal lumen, across the sinusoidal endothelium, into the extravascular space of Disse. Here, remnants contact hepatocytes and undergo receptor-mediated uptake. We coined the term “liver sieve” to describe the fenestrated hepatic sinusoidal endothelium because the structural features of the fenestrations indicate that they act as a filter between the sinusoidal blood and the extracellular space of Disse for plasma and larger substrates.14 Fenestrations are membrane-bound cytoplasmic pores, approximately 100-150 nm in diameter, that occupy approximately 5% of the endothelial surface.15 We postulated that the liver sieve filters macromolecules, including lipoproteins, according to size. The fenestrations are thought to be too small to allow passage of chylomicrons (diameter 100-1000 nm) but permit smaller remnants (diameter 30-80 nm) to enter the space of Disse.13 There is accumulating evidence that the liver traps lipoproteins according to size. Radiolabeled lipoproteins with diameters less than 100 nm are taken up by the intact liver to a greater extent than those with diameters greater than 100 nm.16 The differential trapping is abolished when fenestrations in the sinusoidal endothelium are disrupted by elevated portal perfusion pressure.17
We reported that old age is associated with marked ultrastructural changes in the sinusoidal endothelium, fenestrations and space of Disse in all species studied: rats,18 humans,19 the nonhuman primate Papio hamadryas20 and mice.21 We termed these changes pseudocapillarization because the aging sinusoidal endothelium becomes more like capillaries seen in other nonfenestrated vascular beds.18 Electron microscopy reveals a 40%-80% increase in endothelial thickness and a 60%-80% reduction in porosity and fenestrations. Basal lamina deposition was observed in 25%-40% of old livers. Although morphology determined by light microscopy was unremarkable, there were age-related increases in expression of von Willebrand factor in all species, and collagen and laminin in humans and rats.18–20
We hypothesized that these endothelial changes contribute to age-related impairment of hepatic lipoprotein metabolism because they would be expected to impair the transfer of lipoproteins across the sinusoidal endothelium.13 The effect would only be expected for small chylomicrons and their remnants because only lipoproteins <100 nm in diameter are small enough to cross the normal fenestrations. This study assessed the impact of age-related pseudocapillarization of the sinusoidal endothelium on the hepatic disposition of small chylomicrons with diameters less than those of fenestrations.
Materials and Methods
Young mature adult (aged 4-6 months, weight 300-400 g) and old adult (aged 24-26 months, weight 360-440 g) Fisher 344 male rats were obtained from the National Institute of Aging (Bethesda, MD). The animals were allowed free access to water and commercial rat pellets. The study was approved by the Central Sydney Area Health Service Animal Welfare Committee. We chose Fisher F344 rats because liver weight does not change substantially in old age, which enables comparison of liver perfusion conditions.
Preparation of Radiolabeled Chylomicrons.
Rats were fasted overnight and gavaged with 3 mL skim milk and 50 μL3H-oleic acid. One hour after gavage, rats were anesthetized with pentobarbitone sodium (60 mg/kg, intraperitoneally), and chyle was collected from the thoracic duct via a midline laparotomy incision, as previously described.16 The diameter of the chylomicrons was examined using transmission electron microscopy (Philips CM120, Philips Electronics, Eindhoven, the Netherlands). Diameters were calculated using ScanPro image analysis software (Jandel Scientific, San Rafael, CA).
Impulse Response Experiments
Liver perfusions and impulse response experiments were performed as previously described.22 The perfusate was Krebs-Henseleit bicarbonate buffer (10 mmol/L glucose, pH 7.4, saturated with 95% O2/5% CO2, 2% bovine serum albumin, 37°C). The perfusate flow rate was maintained at approximately 1 mL/min/g of liver using a cartridge pump (Masterflex L/S, model 794-32; Cole-Palmer, Extech Equipment, Boronia, Australia) in a nonrecirculating system. Viability was confirmed by macroscopic appearance, oxygen consumption, portal venous pressure, light microscopy and electron microscopy.
The injectate, made up to total 100 μL volume with Krebs-Henseleit bicarbonate buffer, contained3H-oleic–labeled small chylomicrons (15 μL) and14C-sucrose (1 μL). Immediately after administration of each injectate as a bolus into the portal vein catheter, 30 outflow samples were collected using a Universal Fraction Collector (Extech Equipment, Boronia, Australia) at 2-second intervals. Outflow samples were analyzed for14C- and3H-specific activity (Wallac 1410 Liquid Scintillation Counter, Pharmacia, Sydney, Australia).
Dose-normalized outflow time-activity curves were constructed so that outflow concentrations were expressed as the fraction of the dose per mL of outflow.23 The mean transit time was estimated from the ratio of the area under the first moment of the curve and area under the curve normalized for dose (AUC).24 Mean transit time was corrected for the catheter and nonexchanging vessel transit time (t0), estimated from the time of first appearance of radioactivity above background levels. Hepatic extraction (E) of the indicators and their recovery (R) from the liver were determined from AUC by the
If there is no hepatic extraction of an indicator (i.e., its AUC is not statistically different from the AUC of the nonextracted substrate, sucrose), then the volume of distribution (V) can be determined from the product of the mean transit time and the flow rate (Q).
The ratio of the volume of distribution of small chylomicrons to that of sucrose was determined for each liver. Sucrose is a nonmetabolized tracer that travels freely throughout the vascular and extracellular spaces of the liver but does not enter the hepatocytes. If this ratio of hepatic volumes of distribution is unity, the marker enters the entire extracellular space. If this ratio is less than unity, the marker travels through a smaller space; when the ratio is less than approximately 0.9, the marker is confined to the vascular space.25
After completion of the impulse response experiments, liver specimens were fixed for light microscopy in 4% buffered paraformaldehyde and for electron microscopy with 2% glutaraldehyde/3% paraformaldehyde in 0.1 mol/L sodium cacodylate buffer (0.1 mol/L sucrose, 2 mmol/L CaCl2). Light microscopy was used to detect underlying disease and animals found to have any pathology were excluded from further analysis. Immunohistochemistry was used to detect expression of von Willebrand factor in young and aged rats, as previously described.18 Randomly selected specimens were prepared for and examined with transmission electron microscopy using a Philips CM12 or CM120 microscope, and under scanning electron microscopy using a Philips XL30 microscope.
The results are expressed as mean ± standard deviation. Comparison of the impulse response data for young and old rats was performed using a two-tailed Student t test. Comparison of the microscopy data for young and old animals was performed using the z test.
The livers from the old rats had the same morphological changes of pseudocapillarization that we have previously described.18–21 We noted increased perisinusoidal expression of von Willebrand factor in the livers of old rats. All young rat livers displayed minimal staining intensity while all old rat livers showed intense staining in the peri-sinusoidal regions (P < .001; Fig. 1A -B). Transmission and scanning electron microscopy demonstrated thickening of the sinusoidal endothelium with increased collagen deposition and basement membrane formation (P < .001; Fig. 1C-D) and reduced numbers of fenestrations (Fig. 1E-F) in old rat livers.
Electron microscopic examination showed that the small chylomicrons used in the impulse response experiments had an average diameter of 56 ± 6 nm.
The outflow curves from experiments in livers from young and old rats are shown in Fig. 2. In the young liver, the outflow curve for the small chylomicrons is superimposed upon that of the extracellular marker, sucrose. This is consistent with the small chylomicrons having a similar volume of distribution to that of sucrose. In the old liver, the outflow curve for the small chylomicrons precedes that of sucrose. This suggests that the small chylomicrons have a smaller volume of distribution than sucrose.
The results of the individual experiments are summarized in Table 1. In the livers of young rats, the ratio of the volume of distribution of small chylomicrons to that of sucrose was 1.02 ± 0.14 (n = 12) and the recovery ratio to sucrose was 1.05 ± 0.08. However, in livers of old rats the ratio of the volumes of distribution was significantly reduced compared with that of the young livers (0.92 ± 0.07, n = 11, P = .02), with no age-related change in recovery (0.99 ± 0.13). This indicates that in old livers, the access of small chylomicrons into the space of Disse is impeded. The volume of distribution of sucrose in the young rats was 0.12 ± 0.06 mL/g and in the old rats was 0.17 ± 0.08 mL/g (P = .06).
|Experiment||Liver Weight (g)||Flow rate (mL/s/g)||Recovery*||Volume of Distribution†|
|P (young vs. old)||ns||ns||ns||.02|
In this study we used small chylomicrons rather than chylomicron remnants in order to study the effect of lipoprotein diameter on hepatic disposition without the confounding effects of receptor-binding that would take place using chylomicron remnants. We also deliberately chose to use a relatively slow flow rate to maximize the difference between the sinusoidal lumen volume and the extracellular volume. At high flow rates, these volumes become similar as the endothelium as forced against the hepatocyte membranes. However at low flow rates, de-recruitment and hypoxia may occur. Hypoxia is most likely to be associated with increased fenestration secondary to the formation of large gaps.26 This would be expected to increase the transfer of particles across the endothelium thus any possible age-related hypoxia of endothelial cells would have the opposite effects to those we observed in the older livers.
The livers from old rats had developed the morphological features of pseudocapillarization, which we have described previously in rats.18 These livers demonstrated increased expression of von Willebrand factor in the sinusoidal endothelium, which is associated with endothelial thickening, defenestration, increased collagen deposition and basement membrane formation. In the past we and others have provided evidence suggesting that some lipoproteins traverse the liver endothelium via the fenestrations14 and accordingly, this ultrafiltration process was termed the liver sieve. More recently we have postulated that defenestration of the liver sinusoidal endothelial cell, in both cirrhosis27 and old age,13 might impede the transfer of lipoproteins from the blood to the hepatocytes. Through application of the principles of ultrafiltration, this should theoretically effect mainly lipoproteins with diameters in the range of 30-60 nm.21
Our study supports this hypothesis. Impulse response experiments performed with small chylomicrons and the extracellular marker, sucrose, showed that in young rat livers, small chylomicrons distributed into the entire extracellular space. However, in livers from old rats the small chylomicrons were limited to a significantly smaller space than sucrose was, which is consistent with distribution to the vascular space but reduced access to the space of Disse. Although the reduction in the volume of distribution of the small chylomicrons in old livers was numerically small (i.e., a reduction of approximately 10% compared with that found in the young livers), the implications for lipoprotein metabolism are very significant. The data suggest that some lipoproteins are excluded from the extracellular space in old age and remain confined to the vascular compartment. Such lipoproteins will not have access to hepatocellular receptors and will not be hepatically metabolized. In addition, it is conceivable that the transport in the opposite direction of some lipoproteins manufactured by hepatocytes will be impaired. Notably, the sucrose volume as a fraction of liver weight was not reduced in old age—indeed, it was slightly increased. We have observed a tendency toward an age-related increase in the sucrose volume previously,28 indicating that the pseudocapillarized aging endothelium does not impede the distribution of sucrose into the extracellular space.
These findings in livers of aged rats with pseudocapillarization of the sinusoidal endothelium have some parallels to observations made using impulse response methodology in the isolated perfused livers of rats with cirrhosis, which have capillarized sinusoidal endothelium.29 The impulse response technique has been used in the livers of rats with cirrhosis using albumin (diameter 3-7 nm) as a sieved extracellular marker. It was found that the calculated extravascular space available to albumin was a measure of sinusoidal capillarization and was the main determinant of hepatic function in cirrhosis.30 In cirrhosis, the capillarization of the sinusoidal endothelium also impedes the transendothelial transfer of drugs bound to albumin and other plasma proteins, which normally cross the fenestrations to enter the space of Disse. This has been demonstrated using the impulse response technique with indocyanine green,31 lignocaine,32 and propranolol.33 Reduced transfer of lipoproteins across the capillarized sinusoidal endothelium in cirrhosis has also been reported. The dimethylnitrosamine-fed rat, which is a model of defenestration and cirrhosis, was injected with radiolabeled chylomicrons and had reduced hepatic uptake of label compared with untreated controls.34 We proposed that the hyperlipoproteinemia observed in alcoholic patients is the result of defenestration. When alcoholic patients abstained from alcohol, the fenestrated hepatic sinusoidal endothelium was restored and the hyperlipoproteinemia resolved.35
In conclusion, in old age the endothelium becomes defenestrated and thickened, associated with some fibrosis and basal lamina deposition. In this study, we have shown that these changes impair the transfer of small chylomicrons from sinusoidal blood into the extracellular space of Disse for subsequent hepatic uptake and metabolism. Thus, pseudocapillarization of the hepatic sinusoidal endothelium provides a mechanism for postprandial hyperlipidemia in old age. Age-related change in the liver sinusoidal endothelial cell might prove to be a therapeutic target for prevention of postprandial hyperlipidemia and vascular disease in older subjects.
We thank the electron microscopy unit at Concord RG Hospital for technical assistance.
- 27Defenestration of hepatic sinusoids as a cause of hyperlipoproteinaemia in alcoholics. Lancet 1988; ii: 1225–1227., , , , , .
- 35Relationship between endothelial structure and capillarisation in fatty liver. In: KnookDL, WisseE, eds. Cells of the Hepatic Sinusoids. Vol. 4. Leiden: Kupffer Cell Foundation, 1993: 191–192., , , , , , et al.