Arterial Permeability and Efflux of Apolipoprotein B–Containing Lipoproteins Assessed by In Situ Perfusion and Three-Dimensional Quantitative Confocal Microscopy
Objectives— There is accumulating evidence that an increased risk of cardiovascular disease (CVD) is not simply caused by the degree of arterial exposure to plasma lipoproteins but, in addition, is determined by the affinity of the vasculature for different lipoprotein phenotypes. In this study we compare the delivery and efflux of 2 atherogenic lipoproteins to further understand the factors that regulate cholesterol accumulation in early atherogenesis.
Methods and Results— Lipoproteins containing apolipoprotein (apo) B100 (a low-density lipoprotein [LDL]) and apoB48 (chylomicron remnants) were isolated and differentially conjugated with fluorophores and simultaneously perfused at equivalent concentrations in situ through rabbit carotid vessels. Perfusion systems were established to quantify and differentiate between lipoprotein arterial delivery and efflux. The total average rate of delivery for LDL particles (23 nm) compared with chylomicron remnants (50 nm) was 4427 particles/min−1 per μm3 and 452 particles/min−1 per μm3, respectively. In contrast, the average rate of efflux was 3195 particles/min−1 per μm3 and 163 particles/min−1 per μm3 for LDL and chylomicron remnants, respectively.
Conclusions— Results indicate that although LDL particles have a higher rate of delivery, they efflux more readily from arterial tissue compared with the larger chylomicron remnants. Collectively, our findings highlight that lipoproteins permeate through arterial tissue differently and may be dependent on the phenotype and potential interactions with extracellular matrix components.
Cardiovascular disease is initiated by the entrapment and deposition of cholesterol-rich plasma lipoproteins within the intima of arterial vessels. However, there is now evidence to suggest that an increased risk of cardiovascular disease is not simply caused by the degree of arterial exposure to plasma lipoproteins but, in addition, is determined by the affinity of the vasculature for different lipoprotein phenotypes.1–7 The biophysical characteristics of lipoproteins such as size, density, and lipid and apoprotein (apo) composition appear to play an important role in determining interactions with the arterial subendothelium.2,5–10 There is accumulating evidence that the extracellular proteoglycan matrices have preferential affinity for apoE-containing and apoB-containing lipoproteins of both hepatic and intestinal origin.1–3,5,9
In recent years we have established an in situ perfusion system coupled with 3-dimensional confocal microscopy analysis to quantitatively study the arterial uptake and retention of lipoproteins.1,11,12 In comparative studies of isolated lipoprotein fractions, we have found that cholesterol-rich lipoproteins of hepatic (apoB100) and intestinal (apoB48) origin are differentially retained within the arterial intima.1,11–13 Although apoB100 (such as low-density lipoprotein [LDL]) and apoB48 (such as chylomicron remnants) lipoproteins both accumulate in sporadic focal areas within the intima, the retention of cholesterol derived from apoB48 chylomicron remnant lipoproteins was shown to be markedly greater compared with cholesterol derived from LDL particles.1,11,13 Further studies have demonstrated that in metabolic conditions in which atherosclerotic development is accelerated, such as hypercholesterolemia and diabetes, the differential arterial retention of apoB48 and apoB100 lipoproteins is exacerbated.14 The latter observations are consistent with the concept that pathological changes in arterial matrix composition may, in addition to lipoprotein phenotype, regulate vascular flux of lipoproteins.
Previous studies by several laboratories have concluded that the process of lipoprotein retention is complex and requires further investigation.1,6–9 The anchoring, tethering, and binding of lipoproteins with plasma membrane proteins and extracellular matrices collectively dictates lipoprotein delivery and efflux. In terms of atherogenesis, the arterial accumulation of lipoproteins (retention) is the sum of the permeability (delivery) of the vessel wall to lipoproteins, less particles that migrate (or efflux) from the tissue. Evidence from our laboratory suggests that focal aberrations in efflux appear to be primarily responsible for lipoprotein entrapment, because the majority of lipoproteins delivered to the subendothelial space are not retained and diffuse directly through the internal elastic laminae and tunica media.3,11,12
The delivery of lipoproteins to the subendothelial space occurs via transcytosis, which describes the formation of vesicles (of ≈80 nm) on the endothelial plasma membrane and migration to the basolateral surface. In intact arterial vessels, lipoprotein delivery is limited to particles that have a diameter that can be accommodated by the transcytotic vesicles.6,15,16 By extension, it is possible that arterial efflux of lipoproteins is also regulated by size, with smaller particles being cleared more readily than larger lipoproteins. It is our contention and the basis for this study that the differential rates of retention between lipoprotein phenotypes reported previously is in part a consequence of lipoprotein diameter limiting particle efflux (ie, chylomicron remnant 50 nm, relative to smaller LDL 23 nm).3,4,6,12,17
The 3-dimensional confocal microscopy techniques described in this and in previous studies enable us to discriminate between the efflux of lipoproteins relative to delivery. We have shown previously that at equivalent levels of lipoprotein exposure, the retention of cholesterol derived from chylomicron remnants within the arterial intima can differ compared with cholesterol derived from LDL particles. In this study, we compare the delivery and efflux of these 2 lipoproteins to further understand the factors that regulate cholesterol accumulation in early atherogenesis.
Isolation of Lipoproteins
LDLs were isolated from freshly prepared human plasma by sequential ultracentrifugation (1.019 g/mL<P<1.063g/mL) as previously described.18 LDL particles were free of apoE and apoB48, as determined by SDS-PAGE, and were homogenous in size (23.9±1.9) and charge (−2.0±1.3 mV). The generation of apoB48-containing chylomicron remnants has been detailed in earlier studies but is described briefly.4,11 A large bolus dose of nascent lymph chylomicrons was injected into eviscerated rabbits at a dose of triglyceride ≈20-fold greater than the endogenous plasma pool. After 3 hours, the rabbits are exsanguinated and remnant lipoproteins isolated by ultracentrifugation (1.006 g/mL at 2.256×106 g/h). The chylomicron remnant fraction isolated by this method is free of apoB100-containing lipoproteins (<1%), have a TG/cholesterol ratio <10.0, and are essentially homogenous in size (48.1±8.5 nm) and electrostatic charge (−16.1±1.9 mV).11,12,14 The biochemical properties of lipoprotein preparations are shown in Table 1.
Fluorescent Labeling of Lipoproteins
The preparation of fluorescent lipoproteins has been well-described.11,12 Briefly, lipoprotein isolates were conjugated with Cy3 (565 nm emission; Amersham #PA23000) or Cy5 (670 nm emission; Amersham #PA25000). 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/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. Fluorophores do not exchange with other plasma macromolecules, are bound to protein, and do not alter the clearance of lipoproteins from plasma (Figure I, available online at http://atvb.ahajournals.org).11,12
Rabbit carotid vessels were perfused in situ with equivalent and physiological concentrations of apoB-containing lipoproteins. Particle number was determined via laser particle counting (Brookhaven Industries Pty Ltd) and confirmed by apoB mass determination (on the basis of 1 apoB48/100 molecule per chylomicron remnant or LDL particle, respectively). ApoB was determined by density scanning of protein Coomassie staining based on purified standards as previously described.19 The concentration of lipoproteins perfused in these studies ranged between 8.8×1013−1.2×1014/mL perfusate, which was chosen to reflect the concentration of LDL found in normolipidemic individuals (7×1014/mL fasting plasma concentration) and the chylomicron remnant apoB48 concentration in the postabsorptive state (5×1013 mL plasma).11,17
Lipoprotein Particle Size
When generating chylomicron remnants in hepatectomized rabbits, the decline in plasma triglyceride concentration is monitored routinely as a measure of triglyceride-rich lipoprotein (TRL) hydrolysis. Earlier studies have shown that lipolysis is complete 2.5 hours after injection of lymph chylomicrons into eviscerated rabbits and no further decline in plasma triglyceride is observed.20 Compositional analysis of chylomicron remnants collected 3 hours after injection into hepatectomized rabbits show remnant preparations to be homogenous with particle size ranging from 45 to 55 nm in diameter. In this study, eviscerated rabbits were also bled at 60 and 90 minutes after injection of fluorescent chylomicrons to collect particles ranging in size from 45 to 55 nm, 80 to 100 nm, and 150 to 200 nm. Particle size was assessed by automated laser particle sizer (Brookhaven Industries Pty Ltd) and confirmed by electron microscopy (Figure II, available online at http://atvb.ahajournals.org). Note that labeled chylomicron remnant-labeled preparations are homogenous and do not aggregate.
In Situ Perfusion Studies
Rabbits were obtained from the University of Western Australia, Perth, Biological Animal Sciences Unit, and the institutional animal ethics committee approved all procedures. Fluorescent lipoproteins were perfused through the common carotid arteries of normolipidemic New Zealand White rabbits. Carotid artery segments (30 to 50 mm) were cannulated at the proximal and distal ends to create a closed circuit.12 Chylomicron remnants and LDL were perfused simultaneously in fully oxygenated Hemaccel. The flow rate and perfusion pressure were kept physiological (18 mL/min and at 50 mm Hg).
After perfusion 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 15 to 20 minutes.12 After the perfusion experiments, vessels were removed and fixed in 2% paraformaldehyde (Sigma Cat #P-6148) for 30 minutes. Carotid segments were frozen in liquid nitrogen (LN2) and sectioned by cryostat (≈50 to 100 μm) for analysis by digital confocal microscopy.
Lipoprotein delivery was determined by quantifying lipoprotein particles associated with different morphological regions of the arterial wall in vessels removed immediately after lipoprotein perfusion (ie, without further washout with buffer). We also determined the quantity of lipoproteins in carotid vessels after extensive additional perfusion with lipoprotein-free buffer (defined as “washout”). The washout procedure enables all nonbound particles to clear from the arterial wall under physiological perfusion conditions. Lipoprotein efflux was subsequently determined by calculating the difference between the quantity of lipoproteins delivered to each morphological region (tunica intima, tunica media, adventitia, and endothelial surface), less the quantity of lipoproteins remaining in vessels after extensive washout (ie, particles delivered − particles remaining after washout=lipoprotein efflux).
Fluorescent lipoproteins associated with arterial sections and corresponding morphological regions were visualized using confocal laser (Kr/Ar) scanning microscopy (BioRad MRC 1000) and viewed by Comos software. The intensity of fluorescence associated with arterial sections (intima, media, and adventitia) were identified and collected in 3 dimensions using a custom macro file developed for the National Institutes of Health Image Software and represented as the intensity of fluorescence per unit volume of arterial tissue. We have previously demonstrated linearity for Cy-fluorophores, which was used to calculate lipoprotein particle number.1,11,13 Fluorescent-specific activity of range 2.40×109 − 8.0×1010 intensity units/mL of labeled lipoproteins was used to determine the corresponding number of particles within each arterial section. In addition, conjugated lipoprotein preparations (for dilutions of up to 1/10 000) were used to generate algorithms describing linearity for both Cy5 and Cy3 fluorophores, with corresponding correlation coefficients (r2) of 0.980 and 0.950, respectively.
The intra-assay variability for any specific lipoprotein preparation was always <1.0%. The excitation and emission spectra of the 2 fluorophores used in this study do not overlap, nor does the autofluorescence from the elastin matrix. The double fluorescent-labeled method used in this technique does not allow us to distinguish lipoproteins associated with the extracellular milieu versus those associated with cells per se because of limitations of autofluorescence. However, competition studies have demonstrated that unlabeled lipoproteins compete effectively with fluorescent-labeled chylomicron remnants and LDL, consistent with the assumption that conjugation per se does not influence interaction with arterial tissue.12 Compromised endothelial integrity as a contributing factor of lipoprotein delivery was excluded on the basis of intact endothelium at focal sites of arterial lipoprotein retention and assessed routinely by phase contrast confocal microscopy. For each vessel perfused, data represent an average of at least 15 3-dimensional regions of tissue (≥40 μm3). Significant differences between values of arterial uptake were analyzed by paired analysis (2-tailed paired alternative t test). The coefficient of variation for fluorescent intensity determined repeatedly within a particular region was <5.0%.
The delivery of chylomicron remnant lipoproteins of different size (50 to 150 nm) to arterial tissue is shown in Figure 1. The fluorescence associated with larger particles (150 nm) was observed to accumulate on the luminal surface of arterial vessels, whereas smaller chylomicron remnants of 80 nm and 50 nm can be seen to penetrate to the endothelial monolayer. Similarly, images show that the smaller particles of 50 nm are delivered to deeper regions of the arterial wall compared with larger particles of 80 nm, despite equivalent conditions of lipoprotein particle exposure.
Images representing the delivery of lipoproteins within arterial vessels for differing particle size were digitally analyzed to quantify the corresponding number of lipoprotein particles as shown in Figure 2. The quantitative data depict a pattern of lipoprotein particle delivery, which is consistent with the representative images in Figure 1. Lipoprotein particle number has been calculated to compare the relative delivery with particle size under conditions of equivalent arterial exposure. The total number of chylomicron remnants that are 50 nm had ≈1.4-fold and 1.7-fold greater delivery when compared with particles 80 nm and 150 nm, respectively. The number of LDL particles (23 nm) delivered to the arterial tissue was found to be 10-fold higher than the number of chylomicron remnants (50 nm), which is consistent with the smaller lipoproteins having greater total arterial delivery.
The effect of arterial exposure (length of perfusion) on chylomicron remnant and LDL delivery is expressed as number of particles per unit tissue in Table 2. We have previously demonstrated that lipoprotein arterial delivery and subsequent retention occur within minutes after perfusion.1,11,12 In experiments that determined arterial delivery (ie, without washout), we observed that increasing length of arterial exposure (ie, length of perfusion) resulted in a marked increase in the delivery of particles to arterial tissue (Figure III, available online at http://atvb.ahajournals.org). The total number of chylomicron remnants and LDL particles uniformly increased ≈4–fold over the time of perfusion, consistent with a nonspecific transcytotic mode of delivery (Table 2). The total average rate of delivery for chylomicron remnants (50 nm) compared with LDL particles (23 nm) was 452 particles/min−1 and 4427 particles/min−1, respectively. Therefore, the rate of delivery to arterial tissue for smaller LDL particles was ≈9-fold greater than the larger chylomicron remnants, despite being perfused with an equal number of lipoprotein particles for an equivalent length of time. The data from these arterial exposure experiments indicate a reduced size, coupled with a longer circulating time in plasma, can significantly increase the delivery of lipoproteins to arterial tissue.
Analysis of the morphological distribution of chylomicron remnants and LDL lipoproteins delivered to the arterial wall of vessels is shown in Figure 3. The majority of LDL and chylomicron remnant particles were delivered to the tunica intima. However, the smaller LDL particles were shown to permeate more readily through the arterial tissue, as demonstrated by a greater number of LDL particles delivered to the tunica media and adventitia compared with the larger chylomicron remnants.
The efflux of lipoproteins represents the number of lipoprotein particles that migrated from each region of arterial tissue as shown in Figure 4. Qualitatively, the extent of lipoprotein efflux reflected that described for lipoprotein delivery (Figure 3), with the greatest efflux of lipoproteins occurring in regions that also showed the highest delivery. However, quantitatively, the rate of efflux differed significantly from the rate of delivery observed for each lipoprotein. The average rate of efflux was 3195 particles/min−1 and 163 particles/min−1 for LDL and chylomicron remnants, respectively. Thus, the efflux rate of LDL particles was ≈20-fold greater than the efflux of chylomicron remnants. Collectively, these results are consistent with the hypothesis that smaller particles (such as LDL) migrate out of vessels efficiently, whereas larger particles (such as chylomicron remnants) efflux at a comparatively slower rate.
In this study we have used an established physiological perfusion model to further delineate how the delivery and efflux of different plasma lipoproteins can influence residency time and retention within arterial vessels. We observed that the rate of arterial delivery for LDL particles was ≈9-fold greater than the rate of delivery for chylomicron remnants (based on increasing length of perfusion time). These observations are consistent with the transcytosis model of delivery and support the hypothesis that lipoprotein delivery is primarily dependent on size.6,16 We know that LDL particles (23 nm) are approximately half the size of chylomicron remnants (50 nm); therefore, if we assume that transcytotic vesicles are spherical and use the formula for a spherical volume, we can calculate that halving lipoprotein size would increase the delivery “capacity” (or rate) by 8-fold (ie, [4/3*∏*(2x)3] / [4/3*∏*(x)3]=8), which is consistent with the observations from this study.
There is ongoing debate as to whether lipoprotein size or lipoprotein number (circulating concentration) is of greater clinical significance in atherosclerotic development.21,22 Results from this study suggest that (within the physiological range of lipoprotein concentration) particle size appears to be a limiting factor of net lipoprotein delivery. There is significant evidence that small dense LDL particles are considered more atherogenic than larger LDL particles caused, in part, by their potential higher affinity for LDL receptor-independent pathways.23 Particle number appears to be less important (at least under the conditions of this study) on the basis that lipoprotein delivery and efflux are rate-limiting. If one considers a normolipidemic individual, then the number of LDL particles in circulation will always be in excess of the number of particles delivered to arterial tissue. Consequently, the size of circulating lipoproteins may more accurately predict the impact to lipoprotein delivery and efflux.
In terms of cholesterol delivery to the arterial wall, there is now further evidence that lipoprotein particle composition may also be a critical factor in determining the net mass of cholesterol delivered to arterial tissue.11,12,23 Despite the lower net delivery of particles shown in this study, the larger chylomicron remnant particles contain more cholesterol relative to LDL particles and deliver a greater mass of cholesterol to the arterial wall compared with LDL (under conditions of perfusion with equivalent particle number).
The assessment of lipoprotein delivery in this study showed that the relative distribution of lipoproteins throughout the vessel wall is consistent with the progressive migration of particles from tunica intima to adventitia. Typically, LDL particles were observed to have a greater distribution throughout the vessel wall compared with chylomicron remnants, primarily as a consequence of the increased number of LDL particles delivered. In contrast, we found that chylomicron remnants were mainly distributed within the intimal regions of the arterial wall and few particles were observed in the tunica media and adventitia. However, earlier studies by our laboratory have shown that with continuous perfusion (≤60 minutes) the delivery of chylomicron remnants to the medial and adventitial regions of the arterial wall occurs.12 These studies demonstrate that the preferential retention of cholesterol derived from chylomicron remnants relative to LDL in the subendothelial space is not necessarily caused by the greater delivery of chylomicron remnants. Rather, our findings suggest that the retention of lipoprotein cholesterol may be more dependent on the rate at which particles exit the vascular wall, as opposed to how readily they are delivered.
Efflux of Lipoproteins
The factors regulating the efflux of lipoproteins may also be dependent on the size of lipoprotein particles. The morphological composition of the vessel wall consists of an increase in the density of extracellular matrix medial to the subendothelial space. The presence of extracellular proteoglycan and collagen complexes within the tunica intima and media may be an important factor in limiting the migration and consequent efflux of particles, particularly larger particles from the vessel wall. The results of this study demonstrate for the first time to our knowledge that the larger chylomicron remnant particles have a slower rate of efflux, 20-fold less than the efflux of LDL particles. Evidence suggests that the different apoproteins may play a role in inhibiting efflux and anchoring of particles within the arterial wall. Different apoproteins, including apoB100, apoB48, apoE, and also apoCIII, have been shown to bind or exacerbate binding to extracellular arterial proteoglycans and their components, such as glycosaminoglycan side chains and Biglycan core proteoglycan structures.24–31 These interactions have been reviewed in detail previously by us and others.1,24,25 However, of particular relevance to this study are the recent observations that arterial biglycan proteoglycans avidly bind to apoB-containing and apoE-containing lipoproteins.26–29 Furthermore, Boren et al have recently demonstrated that truncated forms of the apoB100 molecule (such as apoB48 found only in chylomicron remnants) may expose otherwise masked regions that facilitate increased binding with arterial biglycan.2,24 Therefore, cumulative evidence suggests that differences between the apolipoprotein composition of LDL and chylomicron remnants may help to explain the contrasting data observed in this study. Chylomicron remnants contain apoB48, apoE, and some apoC, whereas LDL contains primarily apoB100, no apoE, and very little apoC.3,20 We propose that the reduced efflux of chylomicron remnants, relative to LDL observed in this study, may be further explained by the differing expression of apolipoproteins. In addition, the apolipoprotein complement found in chylomicron remnants may help to reconcile previous studies, in which preferential retention of chylomicron remnants rather than LDL was evidenced in different disease states and animal models predisposed to atherosclerosis.3,11,13,14
In summary, these studies have provided new insight into the delivery and efflux of lipoproteins by the arterial wall. It was shown that the rate at which lipoproteins are delivered to arterial tissue is limiting and regulated by particle size, consistent with transport via transcytosis. Results indicated that although LDL particles have a higher rate of delivery, these particles efflux more readily from arterial tissue compared with the larger chylomicron remnants, giving rise to the preferential entrapment of the latter. Findings from these studies highlight that lipoproteins permeate through arterial tissue differently, which appears dependent on the phenotype and potential interactions with extracellular matrix components.
The studies presented in this article have been supported in part by the National Heart Foundation of Australia. Dr. Spencer Proctor is a C. J. Martin Research Fellow (ID#229030) funded by the National Health and Medical Research Council of Australia.
S.D.P. and D.F.V. are presently at the Metabolic and Cardiovascular Diseases Laboratory, Department of Agriculture, Food, and Nutritional Sciences, University of Alberta, Edmonton, Alberta, Canada.
- Received March 16, 2004.
- Accepted July 26, 2004.
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