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Atherosclerosis 189 (2006) 273–281

Hyperlipidemia and surfactants: The liver sieve is a link

Victoria C. Cogger a,∗, Sarah N. Hilmer a, David Sullivan b, Michael Muller a,Robin Fraser a,c, David G. Le Couteur a

a Centre for Education and Research on Ageing and ANZAC Research Institute, University of Sydney and Concord RG Hospital,Concord, NSW 2139, Australia

b Department of Clinical Biochemistry, Royal Prince Alfred Hospital, Sydney, Australiac Department of Pathology, Christchurch School of Medicine and Health Sciences, University of Otago, Christchurch, New Zealand

Received 11 July 2005; received in revised form 22 November 2005; accepted 15 December 2005Available online 2 February 2006

bstract

Poloxamer 407 is a ubiquitous synthetic surfactant that causes massive hyperlipidemia and atherosclerosis in the rodent. The initial step inepatic metabolism of lipoproteins is their transfer through 100–200 nm pores (fenestrations) in the liver sinusoidal endothelial cell, prior toeceptor-mediated uptake. The ‘liver sieve hypothesis’ emphasizes the role of these fenestrations in the regulation of lipoprotein disposition.ere we show that P407 causes dramatic defenestration of the liver sinusoidal endothelium in vivo. By 24 h after intraperitoneal administration

n mice, fenestrations were reduced by approximately 80% coincident with a 10-fold increase in plasma lipids. Moreover impulse–responsexperiments in the perfused rat liver showed that P407 prevented the passage of small chylomicrons across the liver sinusoidal endothelium.efenestration was also induced acutely with P407 in isolated liver sinusoidal endothelial cells, indicating this is a direct effect of P407 on

enestrations. The results establish the role of the porosity of the liver sinusoidal endothelial cell as a pivotal yet relatively unrecognisedechanism for hyperlipidemia. Furthermore, the results establish an intriguing mechanism for surfactant-induced hyperlipidemia. Thus the

iver sieve is a new and untapped target for the treatment and prevention of hyperlipidemia.2006 Elsevier Ireland Ltd. All rights reserved.

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eywords: Hyperlipidemia; Liver sieve; Liver sinusoidal endothelial cells;

. Introduction

The sinusoidal endothelium has a unique structure. It isxtremely thin and perforated with pores 100–200 nm iniameter called fenestrations [1–4]. We were pioneers in theemonstration that these fenestrations act as a sieve allowinghe passage of lipoproteins with diameters less than the fen-strations, such as chylomicron remnants, into the extravas-

ular space of Disse for subsequent receptor-mediated uptakey hepatocytes [5]. Larger substrates such as chylomicronsnd liposomal drug formulations are excluded from enter-ng the extracellular space of Disse on the basis of their

Abbreviation: P407, Poloxamer 407∗ Corresponding author. Tel.: +61 2 9767 6929; fax: +61 2 9767 5419.

E-mail address: victoric@physiol.usyd.edu.au (V.C. Cogger).

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021-9150/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.atherosclerosis.2005.12.025

stration

ize [5–8]. Consequently, we attributed the impaired clear-nce of lipoproteins and resultant hyperlipidemia observedn ageing and cirrhosis to changes in the liver sinusoidalndothelium, specifically loss of fenestrations (‘defenestra-ion’) [3,6]. More recently it has been shown in a neonatalEGF knockdown model, which causes abnormal develop-ent of hepatic vasculature and the absence of endothelial

enestrations, that lipoprotein transfer across the endotheliums impaired [9].

Poloxamers are a family of synthetic block co-polymersonsisting of alternating units of hydrophobic propylenexide and hydrophilic ethylene oxide [10]. Developed in the950s, there are a number of polymers in this class, in partic-

lar poloxamer 407 (P407) and poloxamer 188 (P188) haveained widespread use as components of many pharmaceuti-al and cosmetic preparations. In addition these polymersre being adapted for more specialised applications such

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s sealing permeabilized cell membranes [11,12], vascularcclusion procedures [13] and drug delivery systems [14,15].t has recently been shown that P407 generates massive hyper-ipidemia after acute administration [16] and atherosclerosisfter chronic administration [17,18] in rodents. The possi-ility that P407 maybe a novel environmental risk factor foryperlipidemia and atherosclerosis in humans has not beennvestigated, perhaps because reports to date have investi-ated the actions of P407 following parenteral rather thanral administration. The mechanisms for the hyperlipidemicffects of P407 remain unclear.

To determine whether hyperlipidemia associated with407 is mediated by changes to the liver sinusoidal endothe-

ial cell, we have examined the effects of P407 on these cellsn in vitro and in vivo models.

. Experimental procedures

.1. Animals

This study was completed using both mice and rats. Dueo ethical constraints, the P407 time course experiments wereompleted using 10-week-old male BL57/6 mice, which werebtained from the in-house animal-breeding program at theNZAC Research Institute (Sydney, Australia). For technical

easons male Sprague-Dawley rats (aged 8–12 weeks) weredded to the protocol for the impulse–response experimentsnd the liver sinusoidal endothelial cell (LSEC) isolationxperiments. These animals were obtained from the Animalesearch Centre (Perth, Australia). All animals were allowed

ree access to water and commercial rat pellets. The study waspproved by the Central Sydney Area Health Service Animalelfare Committee.

.2. Materials

Oleic acid 9,10-3H (5 mCi/ml), sucrose UL-14C0.1 mCi/ml) and bovine serum albumin were obtained fromigma Chemical Company (Sydney, Australia). 95% O2/5%O2 gas was obtained from BOC Gases (North Ryde,ustralia). Poloxamer 407 was kindly donated by BASFustralia Ltd (Sydney, Australia).

.3. Induction of hyperlipidemia

Mice and rats were injected with either 1 g/kg of P407 tonduce hyperlipidemia or volume-matched saline. Mice andats were maintained under standard conditions until sam-ling. We chose the dose of 1 g/kg by intraperitoneal injec-ion because this dose has been shown to generate massiveyperlipidemia. As observed by other researchers (personal

orrespondence, Dr. Neal Davies), some rodents are non-esponders, presumably related to technical factors such asailure to inject the substance into the peritoneal cavity orelated to the unusual gel properties of P407 which might

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sis 189 (2006) 273–281

nfluence the injection or its uptake into the peritoneal cavityfter injection.

.4. Time course experiments/liver sampling

At 12, 24, 36, 48, 72 and 96 h after administration of407 or saline, mice were anaesthetised with Ketamine andylazine (50 and 5 mg/kg, Troy Laboratories, Smithfield,ustralia) and a midline laparotomy was performed. Theortal vein was cannulated with a 22G cannula and the liveras immediately perfused with Krebs–Henseleit bicarbon-

te buffer at 10 cm H2O, with the abdominal and thoracicena cava severed to prevent high-pressure artefact. Once theiver was cleared of blood the perfusate was replaced withxative for electron microscopy, containing 2% glutaralde-yde/3% paraformaldehyde in 0.1 M Na-cacodylate buffer0.1 M sucrose, 2 mM CaCl2, pH 7.4). Following perfusionxation the liver was excised and randomly selected spec-

mens were post-fixed in fresh fixative for 2 h followed byashing in 0.1 M Na-cacodylate buffer (pH 7.4).

.5. Lipoprotein analysis

Prior to portal cannulation 1 ml of blood was removedrom the inferior vena cava for determining lipoprotein pro-le, cholesterol and triglycerides. Plasma triglyceride andholesterol levels were determined enzymatically on a 917utoanalyzer (Roche Pty Ltd, Basel, Switzerland) by theentral Sydney Area Health Service Biochemistry NATAccredited laboratories for all samples. For lipoprotein anal-sis 0.8 �l of plasma taken from animals 24 h after P407reatment was loaded onto a 6% agarose gel and electrophore-is (Corning, Palo Alto, CA) performed at 90 V for 45 min.taining was performed with fat red 7B.

.6. Preparation of radiolabelled chylomicrons

Small radiolabelled chylomicrons were collected fromhoracic ducts of fasted rats. For collection of small chylomi-rons, rats were fasted overnight and gavaged with 3 ml skimilk and 50 �l 3H-oleic acid. One hour after gavage rats were

naesthetized with pentobarbitone sodium (60 mg/kg, i.p.,hone Merieux, Pinkenba, Australia), and chyle collected

rom the thoracic duct via a midline laparotomy incision, ase have described previously [5].

.7. Impulse–response experiments

Liver perfusions and impulse–response experiments wereerformed as described previously [19]. The perfusate wasrebs–Henseleit bicarbonate buffer (10 mM glucose, pH 7.4,

aturated with 95% O2/5% CO2, 2% bovine serum albumin,

7 ◦C). The perfusate flow rate was maintained at approxi-ately 1 ml/min/g of liver using a cartridge pump (Masterflex/S, model 794-32, Cole-Palmer, Extech Equipment, Boro-ia, Australia) in a non-recirculating system. Viability was

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onfirmed by macroscopic appearance, oxygen consumptionnd portal venous pressure.

The injectate used for the impulse–response experimentsas 3H-oleic small chylomicrons (15 �l) and 14C-sucrose

1 �l), made up to total 100 �l. After administration of thenjectate, 30 outflow samples were collected using a Uni-ersal Fraction Collector (Extech Equipment, Boronia, Aus-ralia) at 2-s intervals. Outflow samples were analysed for 14Cnd 3H specific activity (Packard 1600TR liquid scintillationounter, Sydney, Australia).

The mean transit time was estimated from the ratio ofhe area under the first moment of the curve to the areander the curve. The mean transit time was corrected forhe catheter and non-exchanging vessel transit time (t0), esti-

ated from the time of first appearance of radioactivity aboveackground levels. The volume of distribution was deter-ined from the product of the mean transit time and the flow

ate.

.8. Isolation of liver sinusoidal endothelial cells

Our method for the isolation of LSECs has been describedreviously [2,20]. The livers of male Sprague-Dawley ratsere perfused with collagenase A (Sigma, Sydney, Australia)

nd the cell suspension centrifuged at 100 g for 5 min. Theupernatant, containing a mixture of sinusoidal liver cells,as layered on a two-step Percoll gradient (25–50%) and

entrifuged for 20 min at 900 g. The intermediate zone wasnriched in sinusoidal endothelial cells. Purity was enhancedy selective adherence of Kupffer cells. Endothelial cellsere cultivated in 24-multiwell plates on collagen-coatedhermanox cover slips for scanning electron microscopy.ndothelial cells were cultured for 16 h (37 ◦C/5% CO2) inerum-free culture medium consisting of RPMI-1640 withmmol/l l-glutamine, 100 U/ml penicillin, and 100 g/ml

treptomycin.Coverslips with liver sinusoidal endothelial cells were

llocated to one of five treatment groups: normal saline;.05 mg/ml P407; 0.005 mg/ml P407; and 0.001 mg/ml P407,.0005 mg/ml P407 and 0.0001 mg/ml P407. Cells werereated for 1 h, rinsed twice with PBS and fixed with 2% glu-araldehyde in 0.1 mol/l Na-cacodylate buffer (with 0.1 mol/lucrose) at pH 7.4 for 12 h. They were subsequently treatedith 1% tannic acid in 0.15 mol/l Na-cacodylate at pH 7.4

or 1 h and post-fixed with 1% osmium tetroxide in 0.1 mol/la-cacodylate at pH 7.4 for 1 h. Samples were dehydrated ingraded ethanol series, dried with hexamethyldisilazane, andputter coated with gold. The samples were examined with ahilips XL-30. Experiments were performed in triplicate asescribed [20].

.9. ATP assay and mitochondrial function

ATP was determined using a CellTiter-Glo luminescentell viability assay kit (Promega) and a Flurostar Optima plateeader (BMG Labtech, Australia). Mitochondrial function

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sis 189 (2006) 273–281 275

as determined by incubating the cells with the mitochon-rial specific fluorescent dye rhodamine 123 [21].

.10. Electron microscopy

Liver tissue samples from all experimental time pointsere prepared for scanning electron microscopy, while only

iver tissue samples from 24 h post P407 injection were pre-ared for transmission electron microscopy. All samples wererepared according to methods we have described previ-usly [1,4,22]. For transmission electron microscopy twoechnically eligible blocks per liver were studied. Electron

icroscopy was also performed to determine the diameterf the lipoproteins in the blood of the P407-treated micend to determine the diameter of the chylomicrons used inmpulse–response experiments.

Intact liver tissue and isolated endothelial cells were exam-ned using a Philips XL30 scanning microscope. From eachiver perfusion five blocks of technically eligible tissue and alloverslips from the isolated endothelial cell experiments werexamined. Following visual assessment at 5000× magnifica-ion, 10 random images were taken at 20,000× magnification.nalysis of fenestration diameter and endothelial porosityas performed using the Zeiss KS Image Analysis program.

.11. Analysis

Results are presented as mean ± standard error of theean. ANOVA with post-hoc Bonferroni corrections of t-

ests (SigmaStat version 2.03, SPSS Inc.) were used to com-are the control and treatment group data from the lipideasurements, impulse–response experiments and scanning

lectron microscopy. The significance level was P < 0.05.

. Results

.1. Induction of hyperlipidemia by P407

Intraperitoneal injection of P407 led to dramatic hyper-ipidemia in BL57/6 mice. Hypertriglyceridemia andypercholesterolemia were first observed at 12 h, andontinued until 36 h. The hypertriglyceridemia and hyper-holesterolemia was completely resolved by 48 h (Fig. 1).s observed by others (personal correspondence, Dr. Nealavies), a few animals (12%) did not respond to the P407 and

hese were excluded from further analysis. These animalsid not develop hyperlipidemia, and interestingly, thereere no changes in the sinusoidal endothelium (data not

hown).

.2. Intraperitoneal injection of P407 is associated with

efenestration of the liver sieve

No differences between the livers from the P407 treatedr sham injected mice could be seen at the macroscopic or

276 V.C. Cogger et al. / Atherosclero

Fig. 1. (A) Plasma triglyceride and cholesterol levels following intraperi-toneal P407 injection in mice. (B) Porosity of liver sinusoidal endothelialcp

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ight microscopic level, in particular, there was no evidencef steatosis (data not shown).

However scanning electron microscopy of the liver sinu-oidal endothelium at each time point (four control and six407-treated animals) revealed a number of dramatic changesFig. 2). The endothelium had reduced numbers of fenestra-ions 12, 24 and 36 h after P407 treatment, followed by refen-stration, at 48, 72 and 96 h. These changes were quantifiednd were highly statistically significant (P < 0.001, Fig. 1).ransmission electron microscopy of the endothelium, under-

ying space of Disse and hepatocytes at 24 h (four control andix P407-treated animals) confirmed defenestration and alsoevealed deposits, presumably of P407 in the space of DisseFig. 2).

.3. Treatment of isolated liver sinusoidal endothelialells with P407 causes defenestration

There was a concentration-dependent relationshipetween fenestration number and acute P407 exposure

n the isolated liver sinusoidal endothelial cells (Fig. 3).ncreasing concentration of P407 led to a corresponding lossf fenestrations, with complete defenestration of the LSECseen at the highest concentration of P407 (0.05 g/ml). Loss

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sis 189 (2006) 273–281

f fenestrations was independent of changes in ATP levelsFig. 3) and mitochondrial function (data not shown).

.4. Small chylomicrons are unable to traverse thendothelium after P407 administration

Impulse–response experiments were performed in the per-used livers of rats to determine the disposition of smallipoproteins. The marker of extracellular space was sucrose.he ratio of the volume of distribution of small chylomicrons

45 ± 4 nm) to sucrose was reduced from 1.03 ± 0.02 (n = 5)n the untreated controls to 0.83 ± 0.08 (n = 5, P < 0.05)n the perfused livers from rats that had received an i.p.njection of P407 24 h previously (Fig. 3). This indicateshat small chylomicrons are prevented from entering thentire extracellular space after P407. There was no differ-nce between the recovery of small chylomicrons (92 ± 28%)nd sucrose (ratio 1 ± 0.1) or between the small chylomi-rons from the P407 treated rats (97 ± 13%) and control rats90 ± 27%). This indicates that the liver does not take upppreciable quantities of small chylomicrons during a singleass.

.5. P407 is associated with elevated blood levels ofhylomicron remnants

Electrophoretograms of the plasma lipid profiles fromamples taken 24 h after P407 injection in mice (n = 6)howed high levels of chylomicrons and chylomicron rem-ants with no �, � or pre-� particles. Electrophoretogramsrom control mice (n = 3) indicated a predominance of � par-icles together with pre-� and � particles and low levels ofhylomicrons and chylomicron remnants (Fig. 4).

. Discussion

The recent observation that P407 induces massive hyper-ipidemia has led to investigations into possible mecha-isms, focussing primarily on enzymes involved in lipopro-ein metabolism [23–25]. In particular, it has been reportedhat P407 directly inhibits the heparin-releasable fraction ofipoprotein lipase and hepatic lipase, and after long term treat-

ent, P407 inhibited cholesterol 7� hydroxylase (C7�H).here is no effect of P407 on HMG CoA reductase, and the

ncrease in the activity of cholesterol-ester-transfer-proteinCETP) and lecithin cholesterol acyltransferase (LCAT) ishought to be indirect. Although it has been concluded fromhese observations that P407 might act through inhibitionf LPL and C7�H, the mechanism remains controversial23]. Interestingly, it has been established that P407 is local-zed to the liver without hepatic lipid accumulation [26]

et in most other conditions associated with hyperlipidemiahe liver is steatotic. Defenestration of the liver sinusoidalndothelium provides a plausible mechanism for the absencef such steatosis in P407-induced hyperlipidemia, because

V.C. Cogger et al. / Atherosclerosis 189 (2006) 273–281 277

Fig. 2. Electron micrographs of the hepatic sinusoidal endothelium in mice. Scanning electron micrographs in control mice (A) and 12 h (B), 24 h (C), 36 h( smissioa transm( ling the

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D), 48 h (E) and 96 h (F) after intraperitoneal injection of P407. (G) Tranfter passage through a fenestration [F]. (H) 24 h after P407 administration,indicated by → in high magnification inlay) lining the endothelium and fil

ipoproteins are impeded in their transfer from blood intoepatocytes.

Coincident with the increase in plasma lipids, we notedramatic ultrastructural changes in the liver sinusoidalndothelial cell. On scanning electron microscopy, webserved that following P407 injection, the number of fen-

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n electron microscopy shows the endothelium [E] with a lipoprotein [L]ission electron microscopy reveals electron dense material, probably P407,space of Disse (original magnification 20,000×, scale bar = 1 �m).

strations was significantly reduced in all treatment groupsnd this was most dramatic at 24 h (Fig. 1). There was par-

ial re-fenestration by 96 h. The time course for defenestrationnd subsequent re-fenestration corresponded with the plasmaevels of triglycerides and cholesterol (Fig. 1). The resultsstablish the intimate relationship between hyperlipidemia

278 V.C. Cogger et al. / Atherosclerosis 189 (2006) 273–281

Fig. 3. Scanning electron microscopy of hepatic sinusoidal endothelial cells isolated from rat liver. (A) In control endothelial cells there are numerousf thelial c( epatic sP l endoth

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enestrations grouped into sieve plates. (B) Isolated hepatic sinusoidal endoC) The relationship between the dose of P407 and the porosity of isolated h< 0.001, one-way ANOVA). (D) ATP content of isolated hepatic sinusoida

nd liver sinusoidal endothelial fenestrations. It is of notehat defenestration of the liver sinusoidal endothelial celllso occurs in ageing [1,3,4], alcoholic cirrhosis [27,28] andiabetes mellitus [29]. These conditions are associated withmpaired lipoprotein metabolism [27,30–32] and increasedusceptibility to atherosclerosis [3].

On transmission electron microscopy, sinusoidal endothe-ial thickening, defenestration and deposition of electronense material in the space of Disse were observed 24 h afterhe administration of P407 (Fig. 2). While the identity of this

eposited material is unproven, it probably represents P407hat has extruded through the fenestrations into the spacef Disse. This is consistent with a report that most P407 isocalised to the liver within 24 h [26].

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ig. 4. (A) The volume of distribution of small chylomicrons in the perfused livhe volume of distribution was determined using the impulse–response techniquehylomicrons in the livers of rats treated with P407. (B) Electrophoretograms of bl

ells following incubation with 0.05 g/ml of P407 for 1 h (scale bar = 1 �m).inusoidal endothelial cells (all groups are significantly different to Control,elial cells following P407 administration. There was no significant change.

To further examine how P407 induces defenestration of theiver sinusoidal endothelial cells we exposed liver sinusoidalndothelial cells isolated from rat livers to P407 in vitro.gain we noted marked defenestration (Fig. 3). There was a

trong dose-dependent relationship between P407 exposurend defenestration of the isolated liver sinusoidal endothelialells, and complete defenestration was seen at the highestose (P < 0.001, Fig. 3). The results indicate that P407 hasdirect effect on endothelial cells that is not mediated by

ther hepatic cells (Kupffer cells, stellate cells, hepatocytes),

ipoproteins, blood constituents or metabolites. Because weave previously shown a relationship between endothelialioenergetics and defenestration [33], we measured ATP lev-ls in sinusoidal endothelial cells isolated from the rat liver.

ers of rats, presented as a fraction of the extracellular (sucrose) volume.. There was a significant reduction in the volume of distribution of small

ood from P407 treated and control mice.

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e found that P407 did not influence ATP or mitochondrialunction. Thus it is most likely that P407 has a direct physi-al effect on the fenestrations and cell membranes of the liverndothelial cells.

To determine which class of lipoproteins was increasedollowing P407 exposure, we performed lipoprotein elec-rophoretograms on the blood of mice treated with P407.here was neither � nor � electrophoretic mobility in thelasma lipids of P407 treated animals (Fig. 4) indicating thebsence of HDLs and LDLs and consistent with other stud-es of P407 [34]. Furthermore, lipoproteins from the P407reated animals remained at the origin, or migrated in a neg-tive direction, which is characteristic of chylomicrons andhylomicron remnants. Analysis of transmission electronicrographs of blood showed that the average size of

hese lipoproteins was 40 ± 2 nm, with only 20% between0 and 200 nm. It has been found using Western blottinghat the concentration of apolipoprotein-E, relative topolipoprotein-A–I was increased after administration of407, which is also consistent with impaired metabolism ofhylomicrons and chylomicron remnants [34]. Although thelectrophoretogram and electron microscopy are consistentith an increase in circulating chylomicron remnants other

xplanations are possible. For example, the particles couldepresent small chylomicrons generated through P407-nhibition of lipoprotein lipase, circulating P407 micelles, oripoproteins altered by their interaction with P407. Together,hese findings suggest, but do not confirm that there iseduced metabolism of chylomicron remnants following407 administration. Given that chylomicron remnantsass through fenestrations [6], this result is mechanisticallyonsistent with defenestration.

To determine whether P407 restricts the trans-endothelialransfer of small lipoproteins in the liver, we used impulse–esponse methodologies to study the disposition of small chy-omicrons in the perfused livers of male Sprague-Dawley rats.H-small chylomicrons (mean diameter 45 ± 4 nm, similar tohe usual diameter of chylomicron remnants) were generatedn vivo by administration of 3H-oleic acid to starved ratsnd collection of chyle as described previously [5]. Thesexperiments showed that the ratio of the volume of distribu-ion of small chylomicrons to that of sucrose (a marker ofhe extracellular space) was reduced from 1.03 ± 0.02 in theontrols (n = 5) to 0.83 ± 0.08 in the P407 treated animalsn = 5, P < 0.001, Fig. 4). The results show that in normalivers, small chylomicrons travel through the entire extracel-ular (sucrose) space while in the livers of rats treated with407, small chylomicrons were restricted to 83% of the extra-ellular space. Given that in the liver, the vascular space ispproximately 80–90% the size of the entire extracellularpace [35], the results are consistent with the conclusion thathe small chylomicrons are confined to the vascular com-

artment after P407 administration. The recovery of smallhylomicrons was 92 ± 28%, which was not significantly dif-erent from the non-metabolised substrate sucrose nor washere any difference between the controls and the treatment

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sis 189 (2006) 273–281 279

roups. This shows that, as expected, there is no extractionf the small chylomicrons by the liver because they lackpoE, which is required for binding to the LDL receptorn the hepatocellular membrane. Thus P407 prevents therans-endothelial transfer of lipoproteins from the sinusoidallood into the extracellular space of Disse for subsequenteceptor-mediated uptake by hepatocytes. This provides airect mechanism for the massive hyperlipidemia inducedy P407 and confirms the concept that porosity of the liverinusoidal endothelial cell influences systemic lipoproteinevels.

We therefore suggest that P407 induced hyperlidemia aseen in rats [16] and mice [17], and its amelioration bytatins [36,37] is related to changes induced not only tohe systemic endothelium [25] but also that of the hepaticinusoids. P407 induces profound and acute defenestrationf the hepatic sinusoidal endothelium, leading to impairedrans-endothelial transfer of lipoproteins from the sinusoidallood into the extracellular space of Disse. By analogy, renalunction is impaired following P407 secondary to loss oflomerular fenestrations [26,38]. Although the mechanismor the morphological changes induced by P407 has not beenetermined, we noted electron dense material coating thendothelium and plugging the fenestrations, in the absence ofny ATP changes. This suggests that P407 directly coats thendothelial cell membrane and is consistent with in vitro stud-es in cancer cells showing direct adsorption of poloxamernto cell membranes with subsequent changes in structurend fluidity [10]. In addition, poloxamers form micelles withiameters of 20–80 nm [39], which could cause plugging ofenestral lumens analogous to the process of ‘fouling’ seenn synthetic ultrafiltration systems.

Importantly, our studies establish the role of the fen-strated liver sinusoidal endothelial cell in the regulationf hepatic disposition of lipoproteins. This function wasrst proposed by Fraser et al. [5] and Wisse et al. [40] andas greatest significance for those conditions associatedith defenestration of the endothelium such as old age [3]

nd liver disease [27]. In these conditions, modulation ofndothelial fenestrations may provide a novel pharmacolog-cal target for the management of hyperlipidemia [22,41].wo very recent publications lend further support to rela-

ionship between fenestrations and lipoproteins. We recentlyeported that the transfer of lipoproteins across the hepaticinusoidal endothelium is impeded in old age, presumptivelys a result of defenestration [42]. In addition, it has beenound that defenestration associated with a transgenic mousexpressing a hepatocyte-specific, tetracycline-regulatableEGF receptor that can sequester VEGF but cannot

elay its signal, is associated with marked hyperlipidemia9].

In conclusion, P407 caused marked defenestration of liver

inusoidal endothelial cells both in vivo and in isolatedells. This was associated with hyperlipidemia and impairedransfer of small chylomicrons across the hepatic sinusoidalndothelium. Such results suggest that the hyperlipidemic

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ffects of P407 are mediated by a direct effect on fenestra-ions in the liver sinusoidal endothelial cell.

cknowledgements

The authors would like to thank Drs. Laurent Rivorynd Neal Davies for their assistance with the poloxamer07 model. This study was supported by the Ageing andlzheimer’s Research Foundation and grants from theational Health and Medical Research Council and Aus-

ralian Association of Gerontology.

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[4] McLean AJ, Cogger VC, Chong GC, et al. Age-related pseudocapillar-ization of the human liver. J Pathol 2003;200:112–7.

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[6] Fraser R, Dobbs BR, Rogers GWT. Lipoproteins and the liversieve: the role of the fenestrated sinusoidal endothelium in lipopro-tein metabolism, atherosclerosis, and cirrhosis. Hepatology 1995;21:863–74.

[7] Fraser R, Bowler LM, Day WA, et al. High perfusion pressure damagesthe sieving ability of sinusoidal endothelium in rat livers. Br J ExpPathol 1980;61:222–8.

[8] Hilmer SN, Cogger VC, Muller M, Le Couteur DG. The hepatic phar-macokinetics of doxorubicin and liposomal doxorubicin. Drug MetabDispos 2004;32:794–9.

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