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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:534-542.)
© 1995 American Heart Association, Inc.

Articles

Selective Retention of VLDL, IDL, and LDL in the Arterial Intima of Genetically Hyperlipidemic Rabbits In Vivo

Molecular Size as a Determinant of Fractional Loss From the Intima–Inner Media

Børge G. Nordestgaard; Richard Wootton; Barry Lewis

From the Department of Chemical Pathology and Metabolic Disorders, St Thomas's Hospital, London, UK (B.G.N.); the Department of Clinical Biochemistry, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark (B.G.N.); the University of London, London, UK (B.L.); and the Department of Medical Physics, Hammersmith Hospital, London, UK (R.W.).

	Abstract

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Abstract To explore possible mechanisms whereby the triglyceride-rich lipoproteins IDL and VLDL may promote atherosclerosis, fractional loss of these lipoproteins from the intima–inner media was measured in vivo in genetically hyperlipidemic rabbits of the St Thomas's Hospital strain and compared with the fractional loss of LDL, HDL, and albumin. These rabbits exhibit elevated plasma levels of VLDL, IDL, and LDL. In each rabbit, two aliquots of the same macromolecule, one iodinated with ¹²⁵I and the other with ¹³¹I, respectively, were injected intravenously on average 24 and 3 hours, respectively, before removal of the aortic intima–inner media. The fractional loss from the intima–inner media of newly entered macromolecules was then calculated. The average fractional losses for VLDL, IDL, LDL, HDL, and albumin in lesioned aortic arches were 0.1%/h (n=4), -0.2%/h (n=3), 1.8%/h (n=4), 11.4%/h (n=3), and 26.3%/h (n=1), respectively; in nonlesioned aortic arches fractional losses for IDL, LDL, HDL, and albumin were 1.7%/h (n=1), 0.6%/h (n=2), 14.6%/h (n=3), and 25.9%/h (n=3). In both lesioned and nonlesioned aortic arches, the logarithms of these fractional loss values were inversely and linearly dependent on the diameter of the macromolecules (R²=.57, P=.001 and R²=.84, P<.001), as determined from electron photomicrographs of negatively stained lipoproteins. These results suggest that after uptake into the arterial intima, VLDL and IDL as well as LDL are selectively retained in comparison with HDL and albumin.

Key Words: arterial wall • familial combined hyperlipidemia • IDL • remnant lipoproteins • triglyceride-rich lipoproteins

	Introduction

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Hypercholesterolemia is an important causal risk factor for atherosclerosis.^{1 2} Cholesterol in plasma is distributed between four lipoprotein fractions: HDL, LDL, IDL, and VLDL, in order of increasing particle diameter. In the postprandial state, chylomicrons and chylomicron remnants may also be present in plasma. LDL is recognized as the major atherogenic lipoprotein; atherogenesis most likely involves increased accumulation of LDL in the intima of arteries, followed by oxidation of the lipoproteins, uptake of LDL by macrophages, and finally, development of fatty streak lesions.³ There is evidence that IDL, too, may cause atherosclerosis^{4 5 6 7 8} ; the status of VLDL, however, is less clear, and evidence for the atherogenicity of these triglyceride-rich lipoprotein fractions is lacking.

For lipoproteins to cause atherosclerosis, they must enter and possibly accumulate in the intima of arteries. This accumulation of lipoproteins in the intima is dependent on the balance between the rates at which they enter and leave the arterial wall and the rate at which they are degraded within it. Studies in humans, rabbits, and pigs suggest that influx of lipoproteins into the intima increases directly with increasing lipoprotein concentration in plasma^{9 10} and decreases inversely with increasing lipoprotein diameter.^{10 11 12 13 14} Very large lipoproteins, with diameters >75 nm, seem to be excluded from the intima.¹⁵ Very little is known about the loss of lipoproteins from the intima and the determinants of this process.

In the present study, which used genetically hyperlipidemic rabbits of the St Thomas's Hospital strain, the in vivo fractional losses of VLDL, IDL, LDL, HDL, and albumin from the intima–inner media were compared. When fed ordinary rabbit chow, this rabbit strain exhibits elevated plasma levels of VLDL, IDL, and LDL¹⁶ and develops arterial lesions that resemble human atherosclerosis.¹⁷

	Methods

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Animals
In this study, we used 24 rabbits, 13 females and 11 males, weighing 3.2 to 5.6 kg and aged 10 to 29 months, which were descendants of the founder male of the St Thomas's Hospital rabbit strain.¹⁶ Arterial influx and the fractional loss of VLDL, IDL, LDL, HDL, and albumin were measured in groups of 4, 4, 6, 6, and 4 rabbits, respectively. All rabbits were fed SG 1 pellets (Grain Harvesters Ltd) ad libitum. Plasma levels of cholesterol and triglyceride in the 24 rabbits ranged from 1.2 to 17.8 mmol/L and from 0.4 to 3.1 mmol/L, respectively. To test the assumption that the arterial wall behaves as a single, well-mixed compartment in nonlesioned aortas, 5 white rabbits of the Danish Country strain (Statens Serum Institute) were maintained on an individualized cholesterol feeding schedule to clamp for 2 weeks the plasma cholesterol level at 8.0±0.3 mmol/L. Therefore, these rabbits developed plasma cholesterol levels similar to those in rabbits of the St Thomas Hospital strain, but all five rabbits in the former group had aortas without lesions. To test the same assumption in lesioned aortas, data from 4 of the 6 St Thomas's Hospital strain rabbits used in LDL experiments were examined. Experimental protocols were in accordance with the Danish and UK Home Office guidelines for experiments with animals.

Labeling
VLDL, IDL, and LDL were prepared from freshly drawn nonfasted rabbit blood containing Na₂EDTA, chloramphenicol, gentamicin sulfate, -amino-n-caproic acid, benzamidine, and aprotinin (all from Sigma Chemical Co) as described.¹⁰ HDL (density range, 1.063 to 1.21 g/mL) was isolated and washed in a similar manner.

One of two separate aliquots of the same lipoprotein was iodinated¹⁰ with ¹²⁵I and the other with ¹³¹I according to the modification by Bilheimer et al¹⁸ of McFarlane's iodine monochloride method.¹⁹ To minimize possible self-irradiation damage²⁰ mainly from ¹³¹I, 100 to 200 mg rabbit albumin (Sigma) was added to the labeled lipoproteins. For albumin experiments, one of two samples of 5 mg crystallized and lyophilized 99.6% pure rabbit albumin (Sigma) was dissolved in saline and then iodinated with ¹²⁵I and the other with ¹³¹I as described¹⁰ but without addition of the glycine pH 10 buffer. The iodination efficiencies for ¹²⁵I and ¹³¹I were 38±5% (n=18) and 37±5% (n=18).

To reduce the amount of exchangeable iodine label, ie, label not attached to apo B in labeled VLDL or IDL, each of these lipoprotein fractions was incubated with a rabbit plasma fraction containing unlabeled HDL and plasma lipid transfer protein (d>1.063 g/mL) for 48 hours at 37°C. The lipoprotein fractions were subsequently reisolated by ultracentrifugation at their upper density limits. The 48-hour incubation increased the percentage of label attached to apo B, ie, isopropanol-precipitable counts, from 36±10% to 62±8% (n=4) for labeled VLDL and from 76±2% to 91±2% (n=5) for labeled IDL. This incubation at 37°C for 48 hours has been validated previously.¹⁰

Protocol
Because fasting plasma concentrations of IDL and VLDL may not represent the average lipoprotein levels to which the rabbit arterial wall is exposed, experiments were performed on nonfasted animals that were fed ordinary chow ad libitum. Of the 20 rabbits of the St Thomas Hospital strain that were used for the experiments with lipoproteins, 11 were injected with labeled autologous lipoproteins; the remaining 9 were injected with labeled lipoproteins similar to their own; all labeled lipoproteins were from rabbits of the St Thomas Hospital strain.

At 24.1±1.2 hours (n=24) before the animals were killed, ¹³¹I-labeled VLDL, IDL, LDL, HDL, or albumin (3.2±0.3 mL; 34±8 µCi) was injected intravenously, and 3.2±0.1 hours before the animals were killed, an identical sample iodinated with ¹²⁵I (2.7±0.3 mL; 253±49 µCi) was injected or vice versa. Distribution volumes of these injected doses were 28.2±1.5 mL/kg and 28.1±1.5 mL/kg for ¹³¹I- and ¹²⁵I-labeled macromolecules, respectively. The distribution volume was calculated as injected dose (counts per minute [cpm]) divided by the radioactivity concentration in plasma (cpm per milliliter) at the time of injection (obtained by extrapolation) and divided by the weight of the rabbit (in kilograms).

Nonfasting blood samples were drawn at timed intervals after injection of the two doses; the rabbits were killed by intravenous injection of pentobarbitone sodium (50 to 100 mg/kg; May & Baker Ltd); the intima–inner media of the aortic arch, the thoracic aorta, and the abdominal aorta were isolated¹⁰ ; and the extent of lesion was evaluated visually.¹⁰ The area of the arch, thoracic aorta, and abdominal aorta was 6.2±0.2, 7.6±0.2, and 7.6±0.2 cm², respectively (n=24).

Lipoprotein fractions were isolated from plasma samples by sequential ultracentrifugation.¹⁰ Enzymatic methods were used to measure cholesterol (CHOD-PAP, Boehringer Mannheim) and triglyceride (GPO-PAP, Wako Chemicals) in plasma and lipoprotein fractions.

Analyses
Two different approaches with identical tissue pretreatments were used: isopropanol precipitation and trichloroacetic acid (TCA) precipitation. In experiments with VLDL and IDL and in two of the LDL experiments, isopropanol precipitation²¹ of apo B in minced arterial tissue, in aliquots of the doses with added cold plasma, and in aliquots of lipoprotein fractions from plasma samples was performed exactly as described previously.¹⁰ The radioactivity in the intima–inner media included that in the minced arterial tissue. Therefore, the radioactivity in other apolipoproteins and lipids and the radioactivity due to free iodine were excluded; only the radioactivity in apo B was used in the calculations.

In the remaining experiments (with albumin, HDL, and some LDL experiments), calculations were based on the total radioactivity in minced arterial tissue, in aliquots of the doses, and in plasma aliquots after TCA precipitation; the radioactivity in the intima–inner media included that in the minced arterial tissue. Therefore, radioactivity due to free iodine was excluded but radioactivity in lipids was included. Although this could have been a potential technical problem in the HDL experiments in which 8% of the radioactivity was in lipids, it did not appear to be so, because as much as 97% of the plasma radioactivity in these experiments remained in the HDL fraction throughout the 24-hour experiment (Table 1). Minced arterial intima–inner medias, aliquots of the doses (with added cold plasma), and aliquots of plasma samples were precipitated at 4°C with 10% TCA (final concentration). After mixing, centrifugation, and removal of the supernatant, the precipitates were washed with 10% TCA.

View this table: [in this window] [in a new window]   Table 1. Distribution of Radiolabel Among Plasma Lipoprotein and Protein Fractions

Calculations
In the present study it was necessary to assume that lipoproteins iodinated with ¹²⁵I or ¹³¹I are transferred to the arterial intima–inner media at the same rate; this has previously been validated.^{10 13} Under experimental conditions very similar to those in this study, the contribution of adhering plasma to the radioactivity in the arterial intima–inner media was estimated by using iodinated LDL, IDL, and VLDL and was found to be 13.7±3.6 (n=6), 7.6±2.5, and 8.8±2.0 nL/cm² for the aortic arch, thoracic aorta, and abdominal aorta, respectively.¹⁰ In the present study, intimal clearance, influx, and fractional loss in all rabbits were calculated on the basis of uncorrected radioactivity values for the intima–inner media as well as on radioactivity values corrected for contamination, using the values cited above. After correction for plasma contamination for the short-term exposure, the average radioactivity in the arch, thoracic aortic, and abdominal aortic intima–inner media of the 4, 4, 6, 6, and 4 rabbits used for VLDL, IDL, LDL, HDL, and albumin experiments, respectively, was 93±1% (n=11), 87±3% (n=12), 83±2% (n=18), 85±2% (n=18), and 91±2% (n=12) of the uncorrected values. For the long-term exposure, the corresponding values were 99±1% (n=11), 97±1% (n=12), 98±0.4% (n=18), 95±1% (n=18), and 94±1% (n=12). Although contamination-corrected values are reported in this article, our conclusions are similar whether we used uncorrected or plasma contamination–corrected values.

Calculations were based on either the total plasma and intima–inner media apo B radioactivity (after isopropanol precipitation) or the total plasma and intima–inner media TCA-precipitable radioactivity (see "Analyses"). A typical data set is illustrated in Fig 1. The mathematical method and its assumptions have been described previously in detail.^{22 23} Briefly, an equation with two unknowns, k_i, the fractional uptake of plasma lipoproteins into the intima–inner media (in cm^-2 · h^-1), and k_e, the fractional loss of newly entered lipoproteins from the same intima–inner media sample (in h^-1), was derived from the following equation:

(1)

where q_a (in cpm · cm^-2) is the content of labeled lipoproteins in the excised arterial intima–inner media; q_p (in cpm), the content of labeled lipoproteins in the plasma volume; t (in hours), the time after injection of labeled lipoproteins; and T, a "dummy" variable of integration. Because this equation is valid for both short- (3.2±0.1 hours) and long- (24.1±1.2 hours) term exposures to ¹³¹I- and ¹²⁵I-labeled macromolecules, respectively, two equations were derived and solved for each arterial tissue as described.^{22 23} The feasibility of the method was tested previously by computer simulation, and it was found that measurement uncertainties on the order of those likely to be seen in practice (ie, a coefficient of variation [CV] of 1.5% for the plasma data and a CV of 5% for the arterial data) produced uncertainties with a CV of 7% in the calculated intimal clearance and 16% in the calculated fractional loss.²²

View larger version (29K): [in this window] [in a new window]   Figure 1. Line plots of typical raw data for calculation of fractional uptake of plasma LDL into the intima–inner media and fractional loss of newly entered LDL from the same arterial specimen. 125I-LDL and 131I-LDL were injected intravenously into the rabbit 3 and 22 hours, respectively, before removal of arterial tissue.

Equation 1 assumes that the intima–inner media behaves as a single, well-mixed compartment for newly entered macromolecules. To test this assumption, 5 very similar rabbits, all with plasma cholesterol levels close to 8 mmol/L (similar to cholesterol levels in rabbits of the St Thomas's Hospital strain²⁴ ) and with nonlesioned aortas, were used in the following experiment. ¹²⁵I- and ¹³¹I-labeled, pooled, autologous LDLs were injected intravenously into 1 rabbit 3 hours and 1 day, into 2 rabbits 3 hours and 2 days, and into 2 rabbits 3 hours and 3 days, respectively, before removal of the aorta. The average fractional losses for the aortic arch and for the thoracic and abdominal aortas combined were, in 1 rabbit, 6.3%/h for 3 hours versus 1 day, in 2 rabbits, 4.3%/h and 4.4%/h for 3 hours versus 2 days, and in 2 rabbits, 5.7%/h and 5.4%/h for 3 hours versus 3 days. Thus, using Equation 1 to calculate fractional loss in nonlesioned aortas does not appear to depend on whether the earliest injection is made 1, 2, or 3 days before the tissue is harvested. Furthermore, in rabbits of the St Thomas Hospital strain with lesioned aortas that were used in LDL experiments, ¹²⁵I- and ¹³¹I-labeled LDLs were injected intravenously into 1 rabbit 2 hours and 2 days and into 3 rabbits 3 hours and 1 day, respectively, before removal of the aorta. The average fractional losses for the aortic arch, thoracic aorta, and abdominal aorta were in 1 rabbit 5.5%/h for 2 hours versus 2 days and in 3 rabbits 7.1%/h, 3.9%/h, and 4.5%/h for 3 hours versus 1 day. Thus, even for lesioned aortas, using Equation 1 to calculate fractional loss also does not appear to depend on whether the earliest injection is made 1 or 2 days before tissue harvest. Therefore, for these relatively long exposures for both lesioned and nonlesioned aortas, these validation experiments fail to exclude the "single, well-mixed compartment" assumption for the arterial intima–inner media. Finally, Ghosh and coworkers (Ghosh et al²⁵ and Brasch et al²⁶ ) have provided evidence that the single, well-mixed compartment assumption is appropriate in lesioned and nonlesioned monkey aortas for iodinated LDL and in nonlesioned rabbit aortas for iodinated albumin.

Intimal clearance of lipoproteins (nL · cm^-2 · h^-1) was calculated as the product of fractional uptake (k_i in cm^-2 · h^-1) and plasma volume (nL). Plasma volume in each rabbit was taken as the average volume of distribution of ¹²⁵I- and ¹³¹I-labeled macromolecules. The influx of lipoprotein cholesterol was the intimal clearance multiplied by the plasma concentration of lipoprotein cholesterol (mmol/L). This calculation assumes that the intimal clearance value measured for the lipoprotein protein is also valid for the cholesterol moiety of the lipoprotein.

Intimal clearance was also calculated by the "sink method," using the values for short-term exposure; the amount of radioactivity in the intima–inner media (cpm/cm²) was divided by the time-averaged radioactivity in plasma (cpm/nL) and by the length of the exposure time (in hours). The sink method assumes that efflux or degradation of labeled macromolecules in the intima–inner media is negligible compared with the amount of labeled macromolecules entering the intima–inner media during the short-term exposure.

Fractional loss was also calculated as "crude fractional loss." Intimal clearances (ICs) were calculated by the sink method, using the values for both short-term (IC_3h) and long-term (IC_24h) exposures; then crude fractional loss (%/h) was calculated as:

(2)

where t₂₄ (in hours) is the period of long-term exposure. Because some loss of macromolecules from the intima–inner media may have already occurred after 3 hours, crude fractional loss is the minimum estimate of "true" fractional loss calculated in Equation 1.

For all animals, fractional loss was also calculated by the method of Schwenke and Zilversmit,²⁷ a similar but slightly different mathematical approach, in which plasma radioactivity curves are fitted to double-exponential functions. In contrast, for calculations with Equation 1, cubic spline functions were used to fit plasma radioactivity curves.^{22 23} Linear regression analyses of fractional losses, calculated from identical data but with the two different mathematical approaches,^{22 27} gave an R² of .96 (n=24, P<.001), .87 (n=23, P<.001), and .96 (n=24, P<.001) for the aortic arch, thoracic aorta, and abdominal aorta, respectively.

In VLDL and IDL experiments, some of the radioactivity in plasma was in the IDL and LDL fractions, respectively (Table 1), and accordingly, the combined movement of VLDL and IDL and their product lipoproteins was studied when total plasma apo B radioactivity was used in the calculations. Therefore, additional calculations were performed as follows. Using values for "lumped" IDL intimal clearance in individual rabbits injected with labeled IDL (Table 2), values of IDL and LDL intimal clearance were estimated on the basis that IDL intimal clearance was 71% of LDL intimal clearance¹⁰ and arterial radioactivity after IDL injection was corrected on the basis of estimated values for IDL and LDL influx and fractional loss of LDL determinations in other animals (Table 2). Similar calculations were done for arterial radioactivity after injection of VLDL, with corrections for the contributions of influx and loss of IDL (radioactivity in LDL was only 1% of total plasma radioactivity in these experiments and therefore, did not require correction). Finally, on the basis of these corrected arterial apo B radioactivities of only IDL or VLDL origin and on time-averaged apo B radioactivity in plasma IDL and VLDL, respectively, intimal clearance and fractional loss were calculated.²⁷

View this table: [in this window] [in a new window]   Table 2. Intimal Clearance and Influx of Plasma Macromolecules

Electron Microscopy of Negatively Stained Lipoproteins
Average diameters of HDL, LDL, IDL, and VLDL from rabbits of the St Thomas Hospital strain were determined from electron photomicrographs of negatively stained lipoproteins,²⁸ as described previously.¹⁰

Statistics
Values are presented as mean±SE. Linear regression analysis was performed with the MINITAB program.²⁹

	Results

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Labeled Lipoproteins and Albumin in Plasma
In the VLDL, IDL, LDL, HDL, and albumin experiments, an average of 96%, 98%, 98%, 98%, and 98%, respectively, of the labeled material in plasma was precipitable with TCA. Of this TCA-precipitable material, on average 72%, 95%, and 95% were in apo B (precipitable with isopropanol) in the VLDL, IDL, and LDL experiments, and 20%, 4%, 3%, 8%, and 0% were lipid soluble (extractable with chloroform/methanol) in the VLDL, IDL, LDL, HDL, and albumin experiments, respectively. As much as 97% to 98% of the labeled material in the LDL, HDL, and albumin experiments remained in the LDL, HDL, and plasma protein (d>1.21 g/mL) fractions, respectively, throughout the 24-hour exposure period (Table 1). However, in the VLDL and IDL experiments, after 24-hour exposure 11% and 30% of labeled apo B were found in the IDL and LDL fractions, respectively.

Intimal Clearance and Influx
In a previous study that used the sink assumption to calculate intimal clearance and influx,¹⁰ arterial influx of LDL, IDL, and VLDL appeared to be linearly dependent on the respective plasma lipoprotein concentration and on the extent of intimal lesions, independent of the other factor, ie, extent of intimal lesions and lipoprotein concentration, respectively. Lipoprotein influx also depended inversely on lipoprotein size. Therefore, Table 2 shows intimal clearance and influx values for plasma lipoproteins and albumin for animals with and without lesioned aortas; lesion extent as well as lipoprotein cholesterol concentration in plasma is also shown. In the VLDL, IDL, and LDL experiments, intimal clearances calculated with Equation 1 were very similar to those calculated with the sink assumption after a 3.2-hour exposure. However, in experiments with labeled HDL and albumin, the intimal clearance calculated with the sink assumption underestimated true intimal clearance calculated with Equation 1 by an average of 20% and 30%, respectively.

Fractional Loss
Mean fractional losses of newly entered VLDL, IDL, LDL, HDL, and albumin were 0.1%/h, -0.2%/h, 1.8%/h, 11.4%/h, and 26.3%/h, respectively, in lesioned aortic arches (Table 3). In nonlesioned aortic arches the values for IDL, LDL, HDL, and albumin were 1.7%/h, 0.6%/h, 14.6%/h, and 25.9%/h. In Table 3 some individual values of fractional loss are negative, which probably represents detection error. Because single fractional loss values for a given macromolecule for lesioned aortas completely overlapped those for nonlesioned aortas, there is no evidence to suggest that lesion extent affects fractional loss. Plasma cholesterol and triglyceride concentrations are also shown in Table 3.

View this table: [in this window] [in a new window]   Table 3. Intimal Fractional Loss of Newly Entered Macromolecules and Plasma Lipid Levels

As assessed from photomicrographs of negatively stained plasma lipoproteins from rabbits of the St Thomas's Hospital strain (Fig 2), the mean diameter of VLDL, IDL, LDL, and HDL was 46.1, 35.1, 27.2, and 11.4 nm, respectively. In 1 rabbit with plasma triglyceride and cholesterol values of 6.5 and 15.1 mmol/L, the diameters of VLDL, IDL, and LDL were 46.8±1.2, 34.7±0.3, and 27.4±0.3 nm, respectively. In another rabbit with plasma triglyceride and cholesterol values of 3.0 and 23.1 mmol/L, the diameters of VLDL, IDL, and LDL were 45.4±1.4, 35.5±0.6, and 27.0±0.3 nm, and in 3 rabbits with plasma triglyceride and cholesterol values of 0.7 and 2.9, 1.3 and 1.5, and 0.5 and 5.7 mmol/L, the diameters of HDL were 11.1±0.3, 11.4±0.3, and 11.7±0.4 nm, respectively (n=100 for all measurements). The average diameter of albumin is 7 nm.³⁰

View larger version (130K): [in this window] [in a new window]   Figure 2. Transmission electron photomicrographs of negatively stained plasma lipoproteins from rabbits of the St Thomas's Hospital strain. Average diameters of 100 randomly selected lipoproteins in each lipoprotein class were determined.

Fig 3 shows an inverse linear association between the diameter of VLDL, IDL, LDL, HDL, and albumin and the logarithm of the fractional loss from the intima–inner media of these same macromolecules for nonlesioned and lesioned aortas at all three sites. When both fractional loss and macromolecule diameter were plotted on linear scales or when both parameters were plotted on logarithmic scales, all six associations were also significantly inversely related.

View larger version (28K): [in this window] [in a new window]   Figure 3. Line plots of macromolecule diameter versus the logarithm of fractional loss from the intima–inner media of these same macromolecules. Because some fractional losses were negative, 20 was added to all fractional loss values before logarithmic transformation. Lipoprotein diameters were determined from electron photomicrographs of negatively stained lipoproteins. Fractional loss was calculated by the method of Wootton et al.22

For comparison with fractional loss values calculated with Equation 1, a much simpler calculation of crude fractional loss was performed with Equation 2 (Table 3). Like true fractional loss values, these crude fractional losses show an inverse relation between fractional loss from the intima–inner media and particle size.

Because some apo B radioactivity in plasma in the VLDL and IDL experiments was in IDL and LDL, respectively, fractional losses of VLDL and IDL, calculated on the basis of total plasma apo B radioactivity, could differ from true fractional losses of these triglyceride-rich lipoproteins. Therefore, arterial radioactivity values in VLDL and IDL experiments were corrected for possible contributions from plasma IDL and LDL, respectively (see "Methods"). Subsequently, VLDL and IDL fractional losses were calculated on the basis of VLDL and IDL apo B radioactivity in plasma and arterial tissue. The logarithm of these fractional loss values was still found to be inversely linearly related to macromolecule diameter (Fig 4).

View larger version (29K): [in this window] [in a new window]   Figure 4. Line plots of macromolecule diameter versus the logarithm of corrected fractional losses from the intima–inner media of these same macromolecules; arterial radioactivity in VLDL and IDL experiments was subtracted the radioactivity in IDL and LDL before calculation of fractional loss. Fractional loss was calculated by the method of Schwenke and Zilversmit.27

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The aim of the present study was to identify possible mechanisms whereby triglyceride-rich VLDL and IDL may promote atherosclerosis. Genetically hyperlipidemic rabbits of the St Thomas's Hospital strain are well suited for this purpose, because their VLDL, IDL, and LDL concentrations vary widely, and any or all may be elevated in plasma.²⁴ The defect in these rabbits appears to be overproduction of apo B–containing lipoproteins,¹⁶ as it is in humans with familial combined hyperlipidemia. When fed ordinary rabbit chow, these rabbits develop arterial lesions that resemble human atherosclerosis.¹⁷

It has previously been demonstrated that in the St Thomas Hospital rabbit strain, intimal clearance of VLDL and IDL is smaller than that of LDL.¹⁰ This accords with other data that have demonstrated an inverse relation between macromolecule size and intimal clearance of lipoproteins,^{11 12 13 14} but it seems to contrast with the present finding of higher intimal clearance for VLDL than IDL or LDL in lesioned aortas. However, this apparent discrepancy most likely reflects a larger lesion extent in aortas in the VLDL than the IDL or LDL experiments in the present study. The previous study demonstrated a positive linear relation between intimal clearance of VLDL, IDL, and LDL and lesion extent.¹⁰

True intimal clearance (and thus, influx) of VLDL, IDL, and LDL as calculated with Equation 1 was very similar to that calculated using the sink assumption after an average 3-hour exposure. This suggests that the sink assumption is appropriate for such short exposure periods when transfer of iodinated apo B–containing lipoproteins into lesioned or nonlesioned rabbit aorta is under study. This interpretation is in accordance with previous validation of the sink assumption for transfer of iodinated LDL into lesioned rabbit aortas.^{10 15} However, in our recent human study²³ with iodinated lipoproteins, the sink assumption calculation was found to underestimate the true intimal clearance of LDL and Sf 12 to 60 lipoproteins (IDL and smaller VLDL) by 19% to 27%. In that study, the sink assumption calculation was based on a somewhat longer (3- to 6-hour) exposure, but more important, calculation of influx and fractional loss was based only on the radioactivity in lipoproteins isolated from the arterial intima and not on radioactivity in the whole arterial intima as in the present study. Thus, lipoproteins that had entered the intima and had been attached to arterial wall components would not have contributed to the intimal clearance value but would have increased the fractional loss value. That the sink assumption for HDL and albumin after a 3-hour exposure underestimates their true intimal clearance by an average of 20% and 30%, respectively, points to a major loss of these macromolecules from the intima–inner media during these 3 hours.

The fractional loss documented in the present study is a combination of (1) efflux of macromolecules from the intima–inner media into the aortic lumen or outer media and (2) degradation of lipoproteins by the cells in the intima–inner media. In our previous study in humans,²³ fractional loss was also due to lipoproteins that were irreversibly attached to arterial wall components, most likely glycosaminoglycans. This may explain the larger mean fractional loss for LDL and Sf 12 to 60 lipoproteins of 12%/h and 18%/h, respectively, compared with 2%/h to 3%/h and 1%/h for LDL and IDL in the present study. Species difference, however, is another possibility. Ghosh et al²⁵ found an 8%/h fractional loss for LDL in lesioned monkey aortas. That the fractional loss of large VLDL particles is minimal and not significantly different from zero in the present study is in accordance with the findings by Schwenke and Zilversmit, who concluded that the fractional loss of labeled cholesterol ester in noninjured aortas from cholesterol-fed rabbits was not statistically significantly different from zero at 3.5%/h³¹ or even not detectable.^{27 32} In their studies in cholesterol-fed rabbits, most of the plasma cholesterol ester was in the large ß-VLDL particles. The present estimate of a large fractional loss of 25%/h for albumin from the intima–inner media agrees with previous findings that the fractional loss for albumin from arterial tissue is large, ie, 20%/h²⁶ and 45%/h.³¹

A novel observation in the present study is the linear inverse relation between macromolecule diameter and the fractional loss of newly entered macromolecules from the intima–inner media. Even when arterial radioactivity in the VLDL and IDL experiments was corrected for influx of IDL and LDL, respectively, a similar linear inverse relation was still demonstrated. This means that VLDL and IDL as well as LDL, relative to HDL and albumin, become "trapped" in the arterial intima–inner media.

Interestingly, the extent of aortic lesion did not appear to affect the fractional loss of macromolecules from the intima–inner media, in accordance with previous results in monkeys.²⁵ However, a recent study in pigeons found a smaller fractional loss of LDL from lesioned compared with nonlesioned arteries.³³

It may be interesting to calculate the relative intima–inner media content of VLDL, IDL, and LDL by using the measured respective mean fractional loss values of 0.4%/h, 1.3%/h, and 3.3%/h for lesioned aortas and assuming similar plasma concentrations for each lipoprotein fraction (eg, 3 mmol/L) and the same lesion size. Previously measured values for the relative intimal clearance of IDL and VLDL (ie, 71% and 85% of that of LDL¹⁰ ) could then be used. Under the steady-state assumption that influx of lipoproteins equals loss from the intima–inner media, the intima–inner media content of lipoprotein cholesterol (nmol/cm²) can be calculated as lipoprotein cholesterol influx (nmol · cm^-2 · h^-1) divided by fractional loss (h^-1); influx is intimal clearance (nL · cm^-2 · h^-1) multiplied by plasma concentration (mmol/L). The intima–inner media content of VLDL and IDL would then be 7.0-fold and 1.8-fold that of LDL. If the measured mean values for VLDL, IDL, and LDL of 2.3, 2.0, and 4.1 mmol/L in rabbits of the St Thomas's Hospital strain²⁴ were used in the same calculation, the intima–inner media content of VLDL and IDL cholesterol would be 3.9-fold and 0.9-fold that of LDL. That VLDL- and IDL-sized particles accumulate in the arterial intima is in accordance with previous reports of human arterial samples.^{23 34 35 36}

What is the mechanism behind the possible atherogenicity of IDL and VLDL? First, it is worth noting that only small VLDL particles may be involved, because large VLDLs with diameters >75 nm are excluded from the intima.¹⁵ IDL and small VLDL particles in the intima could be taken up directly by macrophages to produce foam cells, as has been suggested from cell culture studies.^{37 38} Alternatively, these triglyceride-rich, intimal lipoproteins could interact with lipoprotein lipase synthesized by macrophages, foam cells, and some smooth muscle cells³⁹ to produce additional lipolytic surface remnants; such remnants are cytotoxic to macrophages in vitro.⁴⁰

What is the relative atherogenicity of LDL, IDL, and VLDL? The present results suggest that when IDL and small VLDL are present in plasma, as they are in humans with familial combined hyperlipidemia,⁴¹ type III hyperlipidemia,⁴² chronic renal failure,^{43 44} and non–insulin-dependent diabetes mellitus⁴⁵ or in rabbits of the St Thomas Hospital strain,^{10 24} these triglyceride-rich lipoproteins are at least as atherogenic as LDL. This notion is supported by our previous study with rabbits of the St Thomas Hospital strain, which demonstrated that the cholesterol in IDL and the IDL plus small VLDL fraction (Sf 12 to 60 lipoproteins) was a better predictor of the extent of atherosclerosis than was LDL cholesterol.²⁴ Similarly, in humans without major genetic forms of hyperlipidemia, IDL and small VLDL have been shown to be independent predictors of the presence, severity, or progression of atherosclerosis^{5 6 7 8} ; in two of these studies, levels or changes in IDL were stronger predictors than those in LDL.^{7 8} Data from the CLAS (Cholesterol Lowering Atherosclerosis Study) also point to an important role for triglyceride-rich lipoproteins in the progression and regression of human atherosclerosis.⁴⁶ Finally, the often-demonstrated univariate and multivariate association between plasma triglycerides and coronary heart disease (CHD)⁴⁷ may reflect the effect of elevated plasma levels of IDL and small VLDL particles in the promotion of atherosclerosis and thereby of CHD. In the Helsinki Heart Study, plasma triglycerides had prognostic value, both for assessing CHD risk and in predicting the effect of gemfibrozil treatment.⁴⁸

In conclusion, the data presented in this article suggest a simple mechanism whereby smaller, triglyceride-rich lipoproteins may promote atherogenesis: after uptake of small VLDL and IDL into the arterial intima, these lipoproteins, relative to HDL and albumin, become trapped in the intima. Whether small VLDL and IDL are trapped to a greater extent than is LDL cannot be concluded from the present data, but the inverse relation between macromolecule diameter and fractional loss from the intima suggests that this is a possibility.

	Acknowledgments

 
These studies were supported by the Danish Heart Foundation and the Danish Medical Research Council. We thank Peter Lumb, Hanne Damm, and Kurt S. Jensen for skillful technical assistance; Ian Chrystie for performing the electron microscopy; Birthe Brüel and Erna Quist for typing the manuscript; and Jørgen Hilden and Lars Bo Nielsen for in-depth advice.

	Footnotes

 
Reprint requests to Dr Børge G. Nordestgaard, Department of Clinical Chemistry, Herlev University Hospital, DK-2730 Herlev, Denmark.

Received August 9, 1994; accepted February 7, 1995.

	References

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