In Vivo Transfer of Lipoprotein(a) Into Human Atherosclerotic Carotid Arterial Intima
Abstract The aim of this study was to compare the atherogenic potential of lipoprotein(a) [Lp(a)] and LDL by measuring the intimal clearance of these two plasma lipoproteins in the atherosclerotic intima of the human carotid artery in vivo. Autologous 131I-Lp(a) and 125I-LDL were mixed and reinjected intravenously 3 hours before elective surgical removal of the arterial intima in four patients. The intimal clearance of Lp(a) and LDL was 229±48 and 405±127 nL/cm2 per hour, respectively (paired t test; P=.12). The mass accumulation of Lp(a) (114±32 ng/cm2 per hour) was on average one 15th that of LDL (paired t test; P=.06), mainly reflecting a low plasma concentration of Lp(a) compared with LDL in the human subjects studied. In accordance with our previous observation in rabbits, there was a positive association between the intimal clearance of LDL and that of Lp(a) (r=.97, P=.03). Accordingly, high plasma levels of Lp(a) may share with LDL the potential for causing lipid accumulation in the arterial intima in humans.
Reprint requests to Dr Børge G. Nordestgaard, Department of Clinical Biochemistry, DK-2730 Herlev, Denmark, or Dr Lars B. Nielsen, Gladstone Institute of Cardiovascular Disease, PO Box 419100, San Francisco, CA 94141-9100.
- Received October 27, 1995.
- Accepted July 24, 1996.
High plasma levels of LDL are causally related to the development of atherosclerosis.1 The importance of another plasma lipoprotein fraction, the lipoprotein(a) [Lp(a)] particle, is less well understood.1 2 Lp(a) can be taken up by macrophages in vivo3 and in vitro4 and may thus contribute to foam cell formation in atherosclerotic plaque. Additionally, Lp(a) may stimulate growth of smooth muscle cells5 and inhibit fibrinolysis,6 both of which could be important in atherosclerosis. In most case-control studies, high plasma levels of Lp(a) are associated with an increased risk of atherosclerosis-related diseases, and in five of seven prospective studies, plasma Lp(a) levels were higher in atherosclerosis-affected individuals than in control subjects (for review see Reference 22 ). Transgenic mice expressing human apolipoprotein (apo) (a) also develop more atherosclerosis than control mice when fed an atherogenic diet.7
LDL- and Lp(a)-like particles are detected predominantly in the vessel wall at sites of atherosclerosis.8 9 The concentration of the Lp(a) apolipoprotein, apo(a), in vein grafts was 240% that of apoB compared with the concentrations of the two apolipoproteins in plasma.10 More Lp(a) than LDL was extractable after plasmin digestion of human arterial intima,11 and autoradiographic studies in mice suggest that Lp(a) accumulates preferentially to LDL in the arterial intima.12 Whether these observations reflect increased rates of transfer into or decreased rates of transfer out of the vessel wall remains to be determined.
In a previous study we compared the transfer into rabbit aortic intima of simultaneously injected human Lp(a) and LDL.3 The results of that study suggest that Lp(a) and LDL enter the arterial wall by a similar mechanism in rabbits. The extrapolation of these findings to humans, however, may be flawed by the absence of endogenous Lp(a) in the rabbit. To gain insight into the mechanism by which Lp(a) enters human arterial tissue under in vivo conditions, the present study compared the transfer of Lp(a) and LDL into carotid arterial intima in human subjects undergoing elective carotid endarterectomy for atherosclerotic stenosis.
Seventeen human subjects who suffered from transient ischemic attacks, minor stroke, or amaurosis fugax and were scheduled for elective carotid endarterectomy for atherosclerotic stenosis had plasma Lp(a) concentrations determined. Of these individuals, only four, who had a plasma Lp(a) concentration >15 mg/mL, ie, a sufficiently high level for the isolation of Lp(a) for iodination, participated. Each participant gave informed consent; the study protocol was approved by the Ethical Committee for Copenhagen and Frederiksberg (file No. KF 02-235/94).
Radioiodination of Lp(a) and LDL
Autologous 131I-Lp(a) and 125I-LDL for reinjection were prepared under sterile and nonpyogenic conditions. The procedures and facilities for the production of labeled lipoproteins were inspected and approved by the Isotope Pharmacy, the National Board of Health, Denmark.
Blood (≈200 mL) was drawn into tubes containing Na2EDTA (final plasma concentration 1.2 mg/mL), benzamidine (10 μg/mL), and aprotinin (10 kallikrein units per milliliter) (all from Sigma). Lp(a) was isolated essentially as described previously.3 13 Briefly, the d<1.12-g/mL plasma fraction was equilibrated with PBS (pH 7.4) containing 0.1 mg/mL Na2EDTA (PBS-EDTA) and loaded onto a lysine 4B Sepharose column (Pharmacia). After a wash with PBS-EDTA, purified Lp(a) was eluted with 10 mmol/L ε-amino-n-caproic acid and 0.5 mol/L NaCl in the same PBS-EDTA buffer. In purified LDL3 13 (d=1.019 to 1.050 g/mL), only 0.8% to 1.6% of the total lipoprotein mass was in Lp(a).
Purified Lp(a) [2 mL, 0.6 to 2.9 mg total Lp(a) mass] was labeled with 185 MBq 131I and purified LDL (2 mL, 5 mg LDL protein) with 185 MBq 125I by the use of the iodine monochloride method,14 15 as described previously.3 Preparations of labeled lipoproteins were immediately added to 3 mL of a human albumin solution (200 g/L; Statens Seruminstitut). The specific activities were 4.5±2.0×108 cpm 131I per milligram total mass of Lp(a) and 6.9±2.6×107 cpm 125I per milligram LDL protein. Aliquots of 131I-Lp(a) and 125I-LDL were mixed and kept at 4°C for a maximum of 14 hours before injection. Labeled lipoprotein preparations were passed through a 0.22-μm filter (Millipore SA) immediately before injection.
A mixture of autologous 131I-Lp(a) and 125I-LDL (37 MBq to 55 MBq of each isotope) was injected intravenously 3.1±0.2 hours before the carotid endarterectomy specimen was removed during surgery. Blood samples were taken at regular intervals from the time of injection until removal of the carotid endarterectomy specimen. Each participant was given 200 mg potassium iodide per day (Kaliumjodid, Nycomed DAK) from 1 day before until 6 days after the injection of labeled lipoproteins.
The carotid endarterectomy specimen was immediately placed on ice and thoroughly rinsed with 1 L of cold saline. Only arterial tissue with moderate atherosclerosis was studied: a part of the common carotid arterial intima (1.2 to 3.5 cm2 surface area) 1 to 2 cm caudal to the external/internal carotid artery bifurcation was separated from the cranial part of the endarterectomy specimen and used for radioactivity determination. Another segment of the endarterectomy specimen immediately cranial to the specimen used for radioactivity determination was fixed in formalin, paraffin embedded, sectioned, and stained with Verhoeff's elastic stain. The remaining tissue including the stenotic area with severe atherosclerosis was used for histological evaluation as part of a plaque morphology study (results to be published elsewhere).
Tissue and Plasma Radioactivity
The part of the endarterectomy specimen taken for radioactivity analysis was washed with an additional 1 L of saline to remove further contaminating plasma. The rinsed tissue was then counted for 5 to 10 minutes to determine the total amount of radioactivity in the tissue before 2 mL of cold PBS-EDTA was added and the tissue thoroughly minced with a pair of scissors. The tube containing minced tissue and PBS-EDTA was gently turned for 2 minutes and centrifuged at low speed for 10 minutes at 4°C before 1.8 mL of the supernatant (the first wash) was transferred to another tube. Another 1.8 mL of cold PBS-EDTA was then added to the minced tissue. This washing procedure was repeated to obtain four successive PBS-EDTA washes of 1.8 mL each. Finally, 1.8 mL of cold PBS-EDTA containing 10 mmol/L ε-amino-n-caproic acid and 0.5 mol/L NaCl was added to the minced tissue as a final wash.
Proteins in the four PBS-EDTA washes, the ε-amino-n-caproic acid wash, and the washed arterial tissue were precipitated with trichloroacetic acid (TCA) (final concentration 15% wt/vol) after the addition of human albumin (final concentration 20 mg/mL). Plasma samples (100 μL) and preparations used for injections (100 μL diluted 1:50) were likewise precipitated with TCA.
Radioactivity in an aliquot of the supernatant and in the precipitate was assayed twice in a double-channel gamma counter (LKB Compugamma 1282, Wallac); tissues and tissue washes were counted twice for a period of 42 minutes, and plasma samples and diluted doses were counted twice for a period of 10 minutes. 131I and 125I were determined after corrections for background, spillover, and decay of 131I during counting. Presented values are the mean of the two radioactivity assays. The average deviation between the duplicate values of total and tightly bound influx or intimal clearance generally was <10%; however, the duplicate determinations of tightly bound intimal clearance of Lp(a) in subject 2 deviated by 25%.
Fixed-density ultracentrifugation of plasma samples at 1.019 and 1.063 g/mL was performed as previously described.3 Cholesterol concentrations in plasma and lipoprotein fractions were determined using an enzymatic method (CHOD-PAP, Boehringer Mannheim). The protein concentration in isolated LDL was estimated from the absorbance at 220 nm, using serum albumin as a standard16 ; we found that this method for LDL protein gives results that are similar to those obtained with the method of Lowry et al17 (data not shown). Lp(a) concentrations in plasma and purified Lp(a) were determined using a turbidimetric assay (DAKO A/S), which measures the total Lp(a) mass. Apo(a) isoforms were determined by Dr M. Jauhiainen, National Public Health Institute, Helsinki, Finland, essentially as described by Utermann et al.18
Two-tier rocket immunoelectrophoresis of plasma containing 131I-Lp(a) and 125I-LDL was performed as previously described3 ; polyclonal rabbit antibodies to Lp(a) and apoB were from DAKO A/S. Anti-Lp(a) did not cross-react with apoB or plasminogen. Briefly, labeled lipoproteins migrated through anti-Lp(a)–containing agarose gel, where Lp(a) will precipitate, and then into anti-apoB–containing agarose gel, where LDL and Lp(a)− [ie, Lp(a) that has lost apo(a)] will precipitate. Rockets were cut out and counted, and the relative amounts of 131I and 125I in Lp(a) and Lp(a)−/LDL were determined. The recovery of the radioactivity applied was on average 97%.
Agarose gel electrophoresis (0.5% agarose) was conducted in Tris/barbital buffer (73 mmol/L Tris, 24 mmol/L barbital, pH 8.6) at 90 volts for 60 minutes in a Wide Mini Sub Cell (Bio Rad). Lipoproteins were fixed in methanol/H2O, 1:1 (vol/vol) and visualized by staining with 0.2 g Sudan black (Merck) and 2 g zinc acetate dissolved in 100 mL ethanol/H2O, 6:4 (vol/vol). Cronex 4 x-ray film (DuPont) was used for autoradiography.
Gradient density ultracentrifugation, gel filtration chromatography, and nondenaturing polyacrylamide gel electrophoresis were performed as described previously.3
TCA precipitable radioactivity in tissue and plasma was used in the calculations. The accumulation of labeled Lp(a) and LDL in the arterial intima is expressed as intimal clearance, ie, the plasma volume cleared of these lipoproteins per unit intimal surface area and per unit of time. The assumptions for this expression have been extensively addressed previously.19 20 21 The intimal clearance of labeled LDL, ILDL, in nanoliters per square centimeter intimal surface per hour, was calculated by solving the equation: (Equation 1) in which A125I is the 125I radioactivity in the arterial intima in counts per minute per square centimeter, t is the duration of the experimental period in hours, and Cavg,125I-LDL is the time-averaged concentration of 125I-LDL in plasma in counts per minute per nanoliter (calculated by numeric integration of the plasma radioactivity-versus-time curve).
On average, 7% to 19% of 131I in plasma was in apoB-containing lipoproteins not containing apo(a), presumably Lp(a)− or contaminating LDL. Lp(a)− is similar to LDL22 and may thus interact with the arterial wall like LDL. The contribution of 131I in Lp(a)−/LDL to the total amount of 131I in the arterial intima was taken into account in the calculations of intimal clearance of Lp(a), ILp(a), by solving the equation: (Equation 2) in which A131I is the 131I radioactivity in the arterial intima in counts per minute per square centimeter, t is the duration of the experimental period in hours, Cavg,131I-Lp(a) and Cavg,131I-Lp(a)−/LDLare mean plasma radioactivity concentrations of 131I-Lp(a) and 131I-Lp(a)−/LDL (in counts per minute per nanoliter), respectively, and ILDL is the intimal clearance of LDL determined for 125I-LDL using Equation 1. If intimal clearance of Lp(a) was calculated from total amounts of TCA-precipitable 131I in plasma and arterial tissue, the estimated intimal clearance of total lipoproteins and tightly bound lipoproteins were respectively on average 109% and 101% of that calculated using Equation 2; ie, corrected for 131I-Lp(a)−/LDL. Therefore, whether the Lp(a) intimal clearance was calculated with or without corrections for radioactivity in Lp(a)−/LDL, the results and conclusions of the present study remain the same.
The mass accumulation of Lp(a) and LDL, M, in nanograms total lipoprotein mass per square centimeter per hour was calculated as: (Equation 3) where I is the intimal clearance (in nanoliters per square centimeter per hour) and Cavg is the mean plasma lipoprotein concentration in plasma (in nanograms lipoprotein per nanoliter). Total LDL mass in nanograms was calculated by assuming that cholesterol contributed 47% of the total lipoprotein mass (see Reference 2323 , Table V).
Intimal clearance and mass accumulation of Lp(a) and LDL were both calculated on the basis of total amounts of TCA-precipitable radioactivity in the arterial intima and the amounts of tightly bound lipoproteins, ie, labeled lipoproteins remaining in the arterial wall after four preceding washes with PBS-EDTA and a final wash including ε-amino-n-caproic acid.
The volume of distribution (milliliters per kilogram) for labeled Lp(a) and LDL was calculated as the amount of radioactivity injected intravenously (counts per minute) divided by the concentration of radioactivity in plasma after 5 to 10 minutes and by the body weight (in kilograms).
Values are mean±SE. Differences between Lp(a) and LDL were analyzed using paired Student's t tests.
Labeled Lp(a) and Labeled LDL Used for Injection
In accordance with our previous studies,3 13 131I-Lp(a) and 125I-LDL were well separated on gel filtration chromatography and gradient density ultracentrifugation. On agarose gel electrophoresis, 131I-Lp(a) was more electronegative than 125I-LDL, and 131I-Lp(a) and 125I-LDL were also well separated on nondenaturing polyacrylamide gel electrophoresis (data not shown). In neither of the validation experiments just mentioned was there any evidence for the presence of significant amounts of labeled free apo(a) or any other disintegration products of 131I-Lp(a) and 125I-LDL.
Labeled Lp(a) and Labeled LDL in Plasma
After intravenous injection, the volume of distribution of 131I-Lp(a) was 43.3±2.9 mL/kg and that of 125I-LDL 42.9±1.6 mL/kg. Using two-tier rocket immunoelectrophoresis, 7% to 19% of 131I in plasma was in LDL and/or Lp(a)− and 100% of 125I was in LDL; plasma radioactivity and specific activity of 131I-Lp(a) and intimal clearance of Lp(a) were corrected for the contribution from 131I in Lp(a)−/LDL (see calculations). Fig 1⇓ shows plasma decay of 131I-Lp(a) [corrected for 131I in Lp(a)−/LDL] and 125I-LDL. The time-averaged specific activity in plasma was on average 92% of the initial value, and there was no statistically significant difference between mean values of 131I-Lp(a) and 125I-LDL. During the experimental period of 3.1±0.2 hours, an average of 94±3% 131I and 98±1% 125I in plasma was associated with lipoproteins, as determined by TCA precipitation.
Transfer of Lp(a) and LDL Into the Arterial Intima
Fig 2⇓ shows histological sections of the carotid endarterectomy specimens immediately adjacent to the segment used for intimal clearance measurement; the endarterectomy specimens primarily consisted of atherosclerotic intima and only minimal tissue from the media.
The Table⇓ shows intimal clearance and mass accumulation of Lp(a) and LDL in the arterial intima; basic characteristics of the four patients studied are also shown in this table. The intimal clearance (in nanoliters per square centimeter per hour) was on average 35% lower for Lp(a) than for LDL (paired t test; P=.12). On linear regression analysis of the intimal clearance of LDL as a predictor of the intimal clearance of Lp(a), there was a positive association between the two variables (Fig 3⇓); the slope of the regression line was significantly lower than 1 (95% confidence interval: 0.10 to 0.63). Total mass accumulation of Lp(a) (in nanograms lipoprotein per square centimeter per hour) was on average one 15th that of LDL (paired t test; P=.06), mainly reflecting the lower plasma concentration of Lp(a) compared with LDL in the human subjects studied.
To estimate the amount of tightly bound lipoproteins in the intima, arterial tissues from three human subjects were successively washed four times with PBS-EDTA and finally once with PBS-EDTA and added ε-amino-n-caproic acid (see “Methods”). The intimal clearance of tightly bound lipoproteins, ie, labeled lipoproteins in tissue after the five washes, was similar for Lp(a) and LDL (Table⇑). A similar fraction of the total amount of 131I-Lp(a) and 125I-LDL in the tissue was extractable with ε-amino-n-caproic acid, ie, on average 3% to 4% for both lipoproteins.
Different lines of evidence have established that the mass flux of LDL from plasma into the arterial wall (in milligrams LDL per square centimeter intimal surface area per hour) is determined by the plasma concentration of LDL (in milligrams LDL per nanoliter) multiplied by the permeability at the plasma–arterial wall interface (in nanoliters per square centimeter intimal surface per hour).24 25 26 A series of studies in rabbits and humans furthermore suggests an inverse relationship between lipoprotein size and arterial wall lipoprotein permeability in normal as well as atherosclerotic arteries.25 27 28 29 These observations support the idea of molecular sieving as the main transport mechanism for the transfer of plasma lipoproteins such as VLDL, IDL, LDL, and HDL from plasma into the arterial wall. In contrast, only little is known about the transfer of Lp(a) into the arterial wall. A recent study in rabbits suggested that human Lp(a) and LDL enter the aortic wall by a similar mechanism.3 This result, however, was possibly affected by the absence of endogenous Lp(a) in the rabbit, and we have therefore now studied the transfer of Lp(a) and LDL into human carotid arterial intima. The present findings are in accordance with our previous results in rabbits: after 3 hours' exposure to simultaneously injected iodinated Lp(a) and LDL, there was a close association between the simultaneously measured intimal clearances of Lp(a) and LDL in human atherosclerotic carotid arteries.
Histological examination of arterial specimens adjacent to arterial tissue used for intimal clearance measurements in the present study showed a different lesion morphology in the four human subjects studied. Atherosclerosis severity is an important determinant of arterial influx of plasma lipoproteins.25 26 27 29 30 31 32 Accordingly, the intimal clearance of LDL and Lp(a) was highest in subjects 2 and 4, who had the most severe atherosclerotic lesions. It was not possible, however, to obtain serial sections for a more thorough morphological examination of lesion severity because the major part of the arterial tissues were processed for radioactivity determinations, and any extrapolations from lesion morphology shown in Fig 2⇑ and relative ratio of intimal clearance of lipoproteins in the human subjects studied should be done with utmost caution. Importantly, however, the relative rates by which plasma macromolecules such as VLDL, IDL, LDL, HDL, and albumin are transferred into the arterial wall appear to be independent of lesion severity.25 27 28 This observation justifies the comparison between the relative intimal clearances of Lp(a) and LDL in human subjects in the present study.
Intimal clearance of labeled Lp(a) and LDL was determined by dividing arterial wall radioactivity by the mean plasma radioactivity concentration and by the 3-hour duration of the experimental period; intimal clearance reflects the combined contributions of influx of labeled lipoproteins and loss of label from the arterial wall, due either to efflux of intact lipoproteins or to degradation of the labeled apolipoprotein moiety followed by subsequent diffusion of labeled tyrosine from the arterial intima. A previous study of human carotid arterial intima suggests that the intimal clearance of iodinated LDL after 3 hours' exposure underestimates the LDL permeability by only 10%.33 Hence, the intimal clearance of labeled LDL after 3 hours' exposure presumably represents a reasonable estimate of the arterial wall permeability to LDL in the present study.
The loss of labeled Lp(a) from human carotid intima during the 3-hour exposure period due to efflux or degradation is not known. However, in recent studies from our laboratory, we have observed a 73% lower fractional loss of newly entered Lp(a) compared with newly entered LDL in balloon-injured rabbit aorta13 and a 27% (P=.07) lower fractional loss of Lp(a) compared with LDL in normal and atherosclerotic rabbit aortic intima (Nielsen et al, J Clin Invest. 1996;98:563-567). These data provide in vivo support to the hypothesis that Lp(a) under some circumstances may bind more strongly than LDL to components of the arterial intima.34 35 Accordingly, the loss of newly entered Lp(a) from human carotid arterial intima, like that of LDL, is most likely also small during a 3-hour exposure period. Therefore, the close association of the intimal clearance of Lp(a) and LDL conceivably reflects a close association of permeabilities of the arterial wall to Lp(a) and LDL. If true, this implies a similar mechanism for the transport of Lp(a) and LDL from plasma into arterial wall.
When intimal clearances of Lp(a) and LDL were determined simultaneously, the intimal clearance of Lp(a) was 57% that of LDL. Although this difference in intimal clearance between Lp(a) and LDL did not reach statistical significance, linear regression analysis of the intimal clearance of LDL as a predictor of the intimal clearance of Lp(a) had a slope that was significantly lower than one, and the ratio of arterial wall mass accumulation of Lp(a) and LDL was significantly lower than the ratio of plasma concentration of Lp(a) and LDL. These observations suggest that the intimal clearance of Lp(a) may in fact be lower than that of LDL after 3 hours' exposure. This is similar to our previous observation in rabbits after 3 hours' exposure to labeled Lp(a) and LDL.3 In rabbits, however, the intima clearance of Lp(a) was higher than that of LDL after 1 hour's exposure. This puzzling opposite finding on the relative intimal clearances of labeled Lp(a) and LDL after 1 and 3 hours in rabbits has been discussed extensively in our previous study3 and most likely relates to variation between batches of labeled Lp(a) in our initial studies.
As discussed above, the intimal clearance of LDL and Lp(a) during 3 hours presumably reflects the arterial wall permeability to these two lipoprotein species. Therefore, the larger intimal clearance of labeled LDL than of labeled Lp(a) in the present study, if real, most likely relates to a larger arterial wall permeability to LDL than to Lp(a) rather than to a larger loss of newly entered labeled Lp(a) than LDL from the arterial intima.
The presently measured mass accumulation of LDL in carotid arterial intima was on average 1.4 μg lipoprotein per square centimeter per hour (≈1.7 nmol LDL cholesterol per square centimeter per hour), which is in accordance with previously reported values of LDL flux into human aortic intima27 and human carotid arterial intima.33 This finding supports the validity of the present data and suggests further that the extensive washing of the arterial surface before radioactivity determination did not remove a large amount of labeled lipoproteins from the intima.
Despite the fact that the present studies were performed in human subjects with relatively high plasma concentrations of Lp(a), the mass accumulation of Lp(a) in the arterial intima was on average only one 15th that of LDL. This observation suggests that the contribution of Lp(a) cholesteryl ester to the total flux of cholesteryl ester into the intima is relatively small compared with the contribution from LDL, even in individuals with a relatively high plasma Lp(a) concentration. Comparisons between cholesteryl ester influx and cholesteryl ester content in human aortic intima27 suggest, however, that most of the cholesteryl ester that enters the arterial intima is removed and only a small fraction is deposited in the intima. Therefore, if Lp(a) is retained preferentially to LDL in the intima after entry, it could still contribute significantly to deposition of cholesterol during the development of atherosclerotic lesions. Lp(a) binds with a larger affinity than LDL to fibrin,34 and a larger affinity of Lp(a) than LDL to glycosaminoglycans in the arterial intima may also facilitate specific accumulation of Lp(a) in human arterial intima.35
This study was supported by the Danish Hospital Foundation for Medical Research, Region of Copenhagen, The Faroe Islands and Greenland; the Danish Heart Foundation; the Danish Medical Research Council; and the Danish foundations Novo's Fond Komite and Overlæge Johan Boserup og Lise Boserups Legat. We are thankful to Dr Matti Jauhiainen, National Public Health Institute, Helsinki, Finland, who measured apo(a) isoforms. Dr Henning Laursen, Laboratory of Neuropathology, Rigshospitalet, Copenhagen, Denmark, kindly prepared the histological sections. Karen Rasmussen provided excellent technical assistance.
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