Intimal Retention of Cholesterol Derived From Apolipoprotein B100– and Apolipoprotein B48–Containing Lipoproteins in Carotid Arteries of Watanabe Heritable Hyperlipidemic Rabbits
Objectives— The arterial retention of apolipoprotein (apo) B100- and apoB48-containing lipoproteins was simultaneously determined in a rabbit model of human hypercholesterolemia using 3D confocal microscopy.
Methods and Results— Lipoproteins containing apoB100 (LDL) and apoB48 (chylomicron remnants) were differentially conjugated with fluorophores and simultaneously perfused at equivalent concentrations under physiological conditions in situ through carotid vessels of Watanabe heritable hyperlipidemic rabbits and compared with controls. Retention of lipoproteins was defined as the amount remaining after an extensive washout phase. LDL and chylomicron remnants were both retained, primarily within the subendothelial space. Without a concomitant increase in exposure to lipoproteins, we found a marked increase in the retention of cholesterol within the intima of Watanabe heritable hyperlipidemic rabbits compared with controls, specifically because of increased entrapment of apoB48-containing lipoproteins.
Conclusions— Collectively, our data suggest that hypercholesterolemia induced as a consequence of LDL receptor deficiency differentially influences retention of LDL and of chylomicron remnants.
The intimal retention of cholesterol reflects an imbalance between delivery and the efflux of lipoproteins via the media and adventitia.1–3⇓⇓ Small quantities of lipoproteins penetrate arterial tissue via transcytosis.4 Vesicles of approximately 70 nm form on the lumenal surface of endothelial cells and migrate to the basolateral side, where the contents are expelled.4,5⇓ Transcytosis is unregulated, representing random invaginations of the plasma membrane, and is a nonspecific form of macromolecular delivery. Transcytosis is uniform and accounts for more than 85% of arterial lipoprotein delivery in healthy tissue.6 However, cholesterol may also be delivered to arterial vessels by other mechanisms. Compromised endothelial integrity may lead to lipoprotein transport via cellular gap junctions,7 and earlier studies raised the possibility of cholesterol transfer from plasma lipoproteins resident within the glycocalyx to the intima.8
Arterial entrapment of lipoprotein cholesterol occurs when delivery is exaggerated and exceeds efflux. Abnormal endothelial phenotypes have been reported in human atherosclerotic tissue,7 consistent with a history of compromised cellular integrity. However, there is little evidence that loss of endothelium is the initiating event of atherogenesis. Rather, a body of evidence suggests that cholesterol accumulation reflects sites of compromised efflux.9,10⇓
The triglyceride-rich lipoproteins, VLDLs, and chylomicrons, when secreted, are generally larger than the transytotic vesicles. VLDL and chylomicrons do not penetrate arterial tissue significantly and are not likely to contribute to early atherogenesis.5,11,12⇓⇓ However, after delipidation by endothelial lipases, the depleted lipoproteins attain sizes sufficiently small to permit arterial passage.1,2,5,9,13,14⇓⇓⇓⇓⇓ Less clear is what regulates retention of lipoproteins. Collectively, evidence suggests that lipoproteins of hepatic (apolipoprotein [apo] B100) and intestinal origin (apoB48) and at different stages of the metabolic cascade are retained.10,15,16⇓⇓ Both apoB100 and apoB48 have been reported in human plaque.17
Recent studies suggest that after delivery, lipoprotein size does not influence efflux, which is complete within minutes.9,10⇓ Therefore, focal accumulation of cholesterol must be a consequence of intimal sites where delivery exceeds efflux.10,18,19⇓⇓ It is perceived that a reduction in the plasma concentration of lipoproteins attenuates atherogenesis by limiting arterial exposure and correcting for aberrant rates of arterial delivery.2 However, the physiological significance of this mechanism is unsubstantiated, because the plasma concentration of lipoproteins exceeds the capacity of intimal transport via transcytosis by orders of magnitude. Consistent with this notion are subjects who develop coronary artery disease in the absence of dyslipidemia.20,21⇓
In this study we have used 3D confocal microscopy to quantitatively determine arterial retention of apoB100 and apoB48 lipoproteins. We chose to study Watanabe heritable hyperlipidemic rabbits, a strain devoid of functional LDL receptor activity, to discriminate between cellular internalisation and extracellular entrapment. In rabbits, the LDL receptor is responsible for more than 90% of high-affinity uptake for apoB48 and apoB100 lipoproteins.22,23⇓ Collectively, our results provide additional evidence that the affinity of arterial tissue for lipoproteins is likely to regulate the residency time.
Isolation of Lipoproteins
Human apoB100 lipoproteins (LDL) were isolated from plasma by ultracentrifugation (1.063 g/mL<ρ<1.019 g/mL). For generation in vivo of apoB48-containing postprandial remnants, an intravenous injection of nascent chylomicrons was given to eviscerated rabbits.13,14⇓ apoB48 remnants were isolated at ρ<1.006 g/mL (2.256×106 g/h). The remnant fractions are free of apoB100 lipoproteins (<1%), have a triglyceride to cholesterol ratio <10, and are homogenous in size and density.13,14⇓
Fluorescent Labeling of Lipoproteins
Lipoprotein isolates were conjugated with Cy3 (565-nm emission, Amersham No. PA23000) or Cy5 (670-nm emission, Amersham No. PA25000).9,10⇓ The excitation and emission wavelength of the far-red fluorophore is sufficiently different from yellow and green fluorophores to avoid signal contamination. Cy protein fluorophores were activated by dissolving with dimethylfluoride and incubated with lipoproteins (15 mg protein per 1 mg fluorophore, pH 7). Conjugation was quenched by addition of hydroxylamine at 45 minutes. Fluorescent lipoproteins were separated by passage through acrylamide desalting columns and dialysis against PBS.
Arterial vessels were perfused with equivalent concentrations of apoB100 and apoB48 lipoproteins. Lipoprotein particle number was determined via laser particle counting and confirmed by apoB mass analysis. ApoB48 and apoB100 were determined by immunodetection using enhanced chemiluminescence.24 The concentrations of apoB containing lipoproteins ranged between 8.8×1013 and 1.2×1014/mL perfusate and were chosen to reflect the concentration of LDL found in normolipidemic individuals (7×1014/mL fasting plasma concentration) and the apoB48 concentration in the postabsorptive state (≈5×1013/mL plasma).10
In Situ Perfusion Studies
Rabbits were obtained from the University of Western Australia, and the animal ethics committee approved all procedures. Carotid artery segments (30 to 50 mm) were cannulated at proximal and distal ends to create a closed circuit.9 Fluorescent lipoproteins were perfused simultaneously in oxygenated Hemacell. The flow rate and perfusion pressure were kept physiological for this species.
After perfusion for 20 minutes with lipoproteins, vessels were either removed immediately or perfused with buffer free of lipoproteins for an additional 20 minutes. We have previously shown that the efflux of nonbound arterial lipoproteins is complete within 5 to 10 minutes.9 Both carotid vessels were perfused; however, only 1 underwent the wash procedure. Vessels were fixed in 2% paraformaldehyde for 30 minutes. Carotid segments were frozen in liquid nitrogen and sectioned by cryostat (≈50 to 100 μm). Arterial lipoprotein interactions are described in this study as associated (no wash procedure) and retained (after washing).
Fluorescent lipoproteins associated with arterial sections were visualized using confocal laser (Kr/Ar) microscopy (BioRad MRC 1000, Comos software). The intensity of fluorescence associated with arterial sections was collected in 3D using a macro file developed for the NIH Image Software and represented as the intensity of fluorescence per unit volume of arterial tissue. For quantitative analysis, it was necessary to demonstrate linearity for both fluorophores, taking into consideration the optimal threshold window that the confocal microscopy software uses to collate pixel intensity. Using conjugated lipoprotein preparations with a fluorophore-specific activity of approximately 19 600 fluorescent intensity units per mg lipoprotein cholesterol, algorithms describing the linearity of Cy5 and Cy3 were generated for dilutions of up to 1/10 000, with correlation coefficients (r) of 0.980 and (0.950), respectively (data not shown). The intra-assay variability for any specific lipoprotein preparation was less than 1.0%.10 The excitation and emission spectra of the 2 fluorophores used in this study do not overlap, nor does the autofluorescence of the vessel. The fluorophores remain tightly bound with the lipoprotein and do not influence plasma kinetics or tissue uptake,10 and competition studies have demonstrated that unlabelled lipoproteins compete effectively with fluorescent-labeled chylomicron remnants and LDL. Compromised endothelial integrity was excluded on the basis of intact endothelium at focal sites of arterial lipoprotein retention. For each vessel, data represent an average of at least 15 3D regions of tissue (≥40 um3). Associated values for delivery were compared with retention values and analyzed by paired analysis (2-tailed, paired alternative t test). The coefficient of variation for fluorescent intensity was less than 5.0%.
Biophysical properties of the lipoproteins used are provided in the Table. Chylomicron remnants had a triglyceride to cholesterol ratio of less than 10 and were homogeneous in size with a mean diameter twice that of LDL (45 versus 23 nm). Chylomicron remnants were enriched in apoE compared with LDL but had similar sialic acid content. Chylomicron remnants had greater overall negative charge than LDL.
In this study, arterial lipoprotein association was determined as fluorescent intensity after perfusion with equivalent numbers of both apoB100- and apoB48-containing lipoproteins. The lipoproteins associated with arterial tissue represent lipoproteins delivered, bound, internalized, and effluxing from tissue. The retention of lipoproteins was defined as lipoproteins that remained bound to arterial tissue after washout. Figure 1 confirms that a large proportion of lipoproteins that penetrate the vessel migrate through the tissue, inclusive of the media and adventitia, and that retention occurs within the intimal regions.9
ApoB100 and apoB48 lipoproteins were perfused simultaneously in each vessel to explore the relative affinity of arterial tissue for the lipoprotein phenotypes. Figure 2 depicts association and retention of cholesterol within different regions of tissue in normal rabbits. For both lipoprotein types, the intimal region of the vessel consistently presented with greater lipoprotein cholesterol association and retention compared with media, adventitia, or the surface. However, we found the profile of lipoprotein retention was different for subsequent regions of the vessel and for each lipoprotein type. Surface retention of apoB100 lipoproteins was minimal (Figure 2A), whereas apoB48 lipoproteins were not significantly removed by washing (Figure 2B).
Several interesting observations of retention were made. First, efflux of cholesterol derived from apoB48 lipoproteins was clearly less complete than for apoB100 lipoproteins (ie, ≈15% and 40% of associated lipoproteins were removed, respectively, after washout). Moreover, the intimal retention of cholesterol derived from apoB48 lipoproteins was more than 2-fold that of apoB100 lipoproteins (Figure 2).
Retention of Lipoproteins in WHHR Rabbits
Arterial association and retention of cholesterol derived from apoB48 and apoB100 lipoproteins in WHHRs are given in Figure 3 and shown in Figure 4. The intimal association of cholesterol derived from apoB48 lipoproteins in WHHR rabbits was found to be almost 2-fold greater compared with controls (Figure 3B versus Figure 2B). In contrast, total cholesterol derived from apoB100 lipoproteins associated with intimal regions of the vessel was 4.5-fold less than that for normal rabbits (Figure 3A versus Figure 2A).
We observed that the mass of cholesterol retained within the intimal regions of tissue from WHHR rabbits differed between apoB100 and apoB48 lipoproteins. The retention of cholesterol from apoB48 lipoproteins in vessels from WHHRs was substantially greater than for controls (Figures 3B and 2B⇑), whereas the retention of cholesterol derived from apoB100 lipoproteins in vessels from WHHRs was just 10% of that retained within the intima in normal rabbits (Figures 3A and 2A⇑). In contrast, we observed that for other regions of arterial tissue (media and adventitia), the pattern of lipoprotein cholesterol association and retention was similar in both WHHRs and in control rabbits.
The arterial retention of cholesterol derived from apoB48 and apoB100 lipoproteins was investigated in a rabbit carotid perfusion model under physiological conditions. We found that the intimal retention of cholesterol derived from apoB48 lipoproteins was greater in WHHRs compared with controls despite evidence that up to 90% of apoB48 lipoprotein internalization is mediated via the LDL receptor.22–26⇓⇓⇓⇓ On this basis, the increased retention of apoB48 lipoproteins in LDL-deficient WHHRs would be consistent with extracellular entrapment.
The retention of cholesterol derived from apoB100 lipoproteins in WHHRs was less than controls. The paradoxical observations might be a consequence of the preexisting hypercholesterolemia caused by LDL receptor deficiency, with the result that arterial extracellular binding sites were already saturated. However, this is unlikely, because WHHRs also have substantially exaggerated concentrations of apoB48 lipoproteins, yet for this lipoprotein, intimal retention was not reduced. Rather, a reduction in retention of cholesterol derived from apoB100 lipoproteins may have been indicative of a decrease in LDL receptor–mediated uptake, a process that reflects cellular internalization as opposed to extracellular entrapment. Oorni et al27 suggested that lipoprotein modification might be a prerequisite for retention of LDL. They showed that interaction of LDL with sphingomyelinase and phospholipase A2 can induce lipoprotein aggregation and activate lysine residues that facilitate binding with proteoglycans. The perfusion model used in this study may not have been conducive to the formation of enzyme-catalyzed fused LDL. We found no evidence of aggregated lipoproteins in postperfusion samples, suggesting that modification of B48 lipoproteins was not necessary for arterial entrapment. Collectively, observations from this study suggest that hypercholesterolemia induced by LDL receptor deficiency differentially alters the retention of apoB100 and apoB48 lipoproteins independent of exposure.
Several studies suggest that lipid composition is critical. The apoB100 segment encompassing amino acids 3359 to 3367 mediates association between LDL and chondroitin proteoglycans.27 Surface phospholipids regulate the α-helical structure, which contributes to negative charge.27 Phospholipids may mask apoB epitopes that serve as the binding ligand to proteoglycans.28,29⇓ Loss of phospholipids may also result in greater exposure of core lipids that can react nonspecifically with cell membranes and extracellular matrices. Small dense LDL contain fewer phospholipids and unesterified cholesterol but greater triglyceride and protein. The surface unesterified cholesterol to phospholipid ratio decreases, as does the surface area covered by lipid.30 ApoB100 occupies a more extensive surface area in small dense LDL and may have a greater tendency to form nonsoluble complexes.31
Plasma lipoproteins are sialylated on their apo and glycolipid constituents. Some functions of sialic acid are associated with lipoprotein binding and tethering.32 The sialic acid content of plasma lipoproteins can vary, but subjects with low levels of lipoprotein sialic acid are at increased risk of developing coronary heart disease.32 Both isoforms of apoB are multisialylated, although apoB100 some 10-fold more so than apoB48.33 Studies in vitro suggest that the sialic acid content of LDL is inversely associated with the extent of complex formation with chondroitin sulfate. Millar et al33 showed that incubation of LDL with ganglioside resulted in a decreased interaction between LDL and proteoglycans, whereas incubation with asialoganglioside resulted in increased interactions. We report here that net sialic acid content of chylomicron remnants was similar for LDL when expressed per unit of protein; however, chylomicron remnants generally contain less protein than LDL. Hence, a relatively lesser proportion of chylomicron remnant sialic acid may explain the divergence patterns of retention. Packard and colleagues concluded that although specific sialic acid–containing components on lipoproteins can affect interaction, total sialic acid content of lipoproteins was not a major determinant of binding.35,36⇓
In WHHRs, the plasma concentration of apoB100 and apoB48 lipoproteins is elevated, suggesting that increased delivery of lipoproteins may exacerbate accumulation,22 a notion consistent with evidence that intimal lipoprotein concentration parallels plasma concentration. However, intimal concentration per se is not indicative of lipoproteins entrapped, and furthermore, in this study the concentrations of lipoproteins used in the perfusate were similar. Therefore, differences in lipoprotein concentration cannot explain the disparity observed for the retention of apoB100 and apoB48 lipoproteins. Whereas transcytosis accounts for approximately 85% of lipoprotein delivery, less is known about this process tissue that may possibly be diseased, as might be the case in WHHRs. It is unclear how increased rates of lipoprotein delivery could explain the divergent patterns of apoB48 and apoB10 lipoprotein retention, because we would have expected qualitatively similar results for both isoforms.
To our knowledge, this study is the first to simultaneously quantitate retention of 2 lipoprotein types using confocal microscopy. However, there are a significant number of other studies that have described the uptake of LDL.1–3,6,37–40⇓⇓⇓⇓⇓⇓⇓ The findings of this study are consistent with previous data indicating that LDL is found associated within the intima.13,14,40⇓⇓ The delivery of lipoproteins via transcytosis represents only a fraction of the plasma pool available, hence it could be argued that significant changes in plasma concentration of lipoproteins are of lesser significance to arterial cholesterol entrapment than other regulating factors.
The primary premise for the efficacy of lipid lowering is that a reduction in arterial exposure will result in lower rates of delivery and decreased entrapment. Based on the findings presented, we suggest that the intimal entrapment of lipoproteins is dependent on factors in addition to the level of arterial exposure.18,41,42⇓⇓
This study was supported in part by the National Heart Foundation of Australia. S.P. is a CJ Martin Research Fellow, funded by the National Health and Medical Research Council of Australia.
- Received March 7, 2003.
- Accepted May 20, 2003.
- ↵Bjornheden T, Bondjers G, Wiklund O. Direct assessment of lipoprotein outflow from in vivo-labelled arterial tissue as determined in an in vitro perfusion system. Arterioscler Thromb Vasc Biol. 1998; 18: 1927–1933.
- ↵Schwenke DC, Carew TE. Initiation of atherosclerotic lesions in cholesterol-fed rabbits, II: selective retention of LDL vs selective increases in LDL permeability in susceptible sites of arteries Arteriosclerosis. 1989; 9: 908–918.
- ↵Simionescu N, Simionescu M. Cellular interactions of lipoproteins with the vascular endothelium: endocytosis and transcytosis. Targital Diagn Ther. 1991; 5: 45–97.
- ↵Nordestgaard BG, Tybjaerg-Hansen A. IDL, VLDL and chylomicrons and atherosclerosis. Eur J Epidemiol. 1992; 8: 92–98.
- ↵Proctor SD, Vine DF, Mamo JCL. Arterial retention of apolipoproteinB48 and B100-containing lipoproteins in atherogenesis. Curr Opin Lipidol. 2002; 5: 461–470.
- ↵Nordestgaard BG, Zilversmit DB. Comparison of arterial intimal clearances of LDL form diabetic and non-diabetic cholesterol-fed rabbits: differences in intimal clearance explained by size differences. Arteriosclerosis. 1989; 9: 176–183.
- ↵Nordestgaard BG, Stender S, Kjeldsen K. Reduced atherogenesis in cholesterol-fed diabetic rabbits: giant lipoproteins do not enter the arterial wall. Arteriosclerosis. 1988; 8: 421–428.
- ↵Proctor SD, Mamo JCL. Arterial fatty lesions have increased uptake of chylomicron-remnants but not low-density lipoproteins. Coron Artery Dis. 1997; 7: 239–245.
- ↵Karpe F, Tornvall P, Olivecrona T, Steiner G, Calson LA, Hamsten A. Composition of human low-density lipoprotein: effects of postprandial triglyceride rich lipoproteins, lipoproteins lipase, hepatic lipase and cholesteryl ester transfer protein. Atherosclerosis. 1993; 98: 33–49.
- ↵Krauss RM. Heterogeneity of plasma low-density lipoproteins and atherosclerosis risk. Curr Opin Lipidol. 1994; 38: 339–349.
- ↵Pal S, Semorine K, Watts GF, Mamo JC. Identification of lipoproteins of intestinal origin in human atherosclerotic plaque. Clin Chem Lab Med. In press.
- ↵Weintraub MS, Grosskopf I, Rassin T, Miller H, Charach G, Rotmensch HH, Liron M, Rubinstein A, Iaina A. Clearance of chylomicron remnants in normolipidaemic patients with coronary artery disease: case control study over three years. Brit Med J. 1996; 312: 935–939.
- ↵Groot PH, van Stiphout WA, Krauss XH, Jansen H, van Tol A, van Ramshorst E, Chin-On S, Hofman A, Cresswell SR, Havekes L. Post-prandial lipoprotein metabolism in normolipidaemic men with and without coronary artery disease. Arterioscler Thromb. 1991; 11: 653–662.
- ↵Bowler A, Redgrave TG, Mamo JCL. Chylomicron remnant clearance in homozygote and heterozygote Watanabe heritable hyperlipidemic rabbits is defective: lack of evidence for an independent chylomicron-remnant receptor. Biochem J. 1991; 276: 381–386.
- ↵Smith D, Proctor SD, Mamo JCL. A highly sensitive assay for quantitation of apolipoprotein B48 using an antibody to human apolipoprotein B and enhanced chemo-luminescence. Ann Clin Biochem. 1997; 34: 185–189.
- ↵Choi SY, Cooper AD. A comparison of the roles of the low density lipoprotein (LDL) receptor and the LDL receptor-related protein/alpha 2-macroglobulin receptor in chylomicron-remnant removal in the mouse in-vivo. J Biol Chem. 1993; 268: 15804–15811.
- ↵Oorni K, Hakala JK, Annila A, Ala-Korpela M, Kovanen PT. Sphingomyelinase induces aggregation and fusion, but phospholipase A2 only aggregation, of low density lipoprotein (LDL) particles: two distinct mechanisms leading to increased binding strength of LDL to human aortic proteoglycans. J Biol Chem. 1998; 273: 29127–19134.
- ↵Kleinman Y, Krul ES, Burnes M, Aronson W, Pfleger B, Schonfeld G. Lipolysis of LDL with phospholipase A2 alters the expression of selected apo B100 epitopes and the interaction of LDL with cells. J Lipid Res. 1988; 29: 729–743.
- ↵McNamara JR, Small DM, Li Z, Schaefer EJ. Differences in LDL subspecies involve alterations in lipid composition and conformational changes in apolipoprotein B. J Lipid Res. 1996; 37: 1924–1935.
- ↵Sartipy P, Camejo G, Svensson L, Hurt-Camejo E. Phospholipase A2 modification of low density lipoproteins forms small high density particles with increased affinity for proteoglycans and glycosaminoglycans. J Biol Chem. 1999; 274: 25913–25920.
- Deleted in proof.
- ↵Anber V, Millar JS, McConnell M, Shepherd J, Packard CJ. Interaction of very low density, intermediate density, and low density lipoproteins with human arterial wall proteoglycans. Arterioscler Thromb Vasc Biol. 1997; 17: 2507–2514.
- ↵Tozer EC, Carew TE. Residence time of low-density lipoprotein in the normal and atherosclerotic rabbit aorta. Circ Res. 1997; 80: 208–218.
- ↵Rosenfeld ME, Carew TE, von Hodenberg E, Pittmen RC, Ross R, Steinberg D. Autoradiographical analysis of the distribution of 125I-tyramine-cellobiose-LDL in atherosclerotic lesions of the WHHL rabbit. Arterioscler Thromb. 1992; 12: 985–995.
- ↵Nievelstein PF, Fogelman Am, Mottino G, Frank JS. Lipid accumulation in rabbit aortic intima 2 hours after bolus infusion of low-density lipoprotein: a deep-etch and immunolocalization study of ultra-rapidly frozen tissue. Arterioscler Thromb. 1991; 11: 1795–1805.
- ↵Schwenke DC, St Clair RW. Influx, efflux and accumulation of LDL in normal arterial areas and atherosclerotic lesions of white Carneau pigeons with naturally occurring and cholesterol-aggravated aortic atherosclerosis. Arterioscler Thromb. 1993; 13: 1368–1381.