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Circulation Research. 1997;80:37-44

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(Circulation Research. 1997;80:37-44.)
© 1997 American Heart Association, Inc.

Articles

Exogenous Oxidized Low-Density Lipoprotein Injures and Alters the Barrier Function of Endothelium in Rats In Vivo

Shanthini Rangaswamy, Marc S. Penn, Gerald M. Saidel, Guy M. Chisolm

the Department of Cell Biology, Cleveland Clinic Foundation, and the Department of Biomedical Engineering, Case Western Reserve University, Cleveland, Ohio.


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Oxidation converts low-density lipoprotein (LDL) into a cytotoxin in vitro. Oxidized LDL exists in vivo in atherosclerotic lesions and possibly in plasma. Many cell functions are altered in vitro by oxidized LDL, but few have been examined in vivo. To test whether oxidized LDL could injure endothelial cells and alter endothelial permeability to macromolecules in vivo, we infused oxidized LDL, native LDL, or their solvent intravenously into rats. Subsequently, endothelial cell injury and proliferation were measured, and the transport into the aorta wall of the macromolecule horseradish peroxidase (HRP) was quantified. Transport data were analyzed using mathematical models of macromolecular transport; parameters were estimated by optimally fitting model-predicted HRP concentrations to experimental data. Compared with native LDL or solvent control infusion, oxidized LDL infusion increased (1) the number of injured aortic endothelial cells fivefold to sixfold at 36 hours, (2) proliferation of endothelial cells at 48 hours, (3) intimal and medial accumulations of HRP twofold to threefold at 48 hours, and (4) the permeability coefficient of the endothelium to HRP fourfold to fivefold at 48 hours. Hence, oxidized LDL administered in vivo can injure the endothelium, despite the presence of endogenous antioxidants, compromising the function of the endothelium as a permeability barrier.


Key Words: atherosclerosis • lipoprotein, low-density, modified • cytotoxicity • biological transport • vascular permeability


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Elevated plasma LDL is known to be a risk factor for atherosclerosis. However, the cellular events believed to be associated with atherogenesis, such as injury to endothelial cells, adhesion of monocytes to endothelial cells, and unregulated uptake of lipoproteins by macrophages, cannot be easily mimicked by exposing vascular cells in culture to high concentrations of native LDL. These events, however, can readily be observed in vitro after the oxidative modification of LDL.1 2 3 Furthermore, in vitro studies also show that the various cell types associated with early lesions in the arterial wall, smooth muscle cells, monocytes, macrophages, and endothelial cells can mediate the oxidation of LDL.4 5 6 7 Recent studies have offered evidence of oxidized LDL in vivo, both in plasma8 and in arterial lesions.9 10 11 Data showing the protective effect of various antioxidants against the progression of atherosclerosis or risk of coronary disease12 13 14 15 16 17 18 indirectly suggest that lipoprotein oxidation may be linked to the pathogenesis of the disease.

The capacity of oxidized LDL to injure cultured cells does not appear dependent on the protocol used to oxidize the lipoprotein in vitro.5 6 19 20 21 22 Even the putatively oxidized lipoprotein forms obtained from in vivo sources, including diabetic rat plasma23 and monkey plasma,8 have been shown to injure cells in culture. Antioxidants can inhibit cell injury by oxidized LDL in vitro.22 24 25 Yet, it is unknown whether oxidized LDL can in fact injure endothelium in vivo. In addition, it is unknown what form such an injury might take in vivo. The widely accepted response-to-injury hypothesis of atherosclerosis, in its recent iterations, proposes oxidized LDL as possibly responsible for the endothelial injury, activation, or dysfunction associated with lesion initiation.26 In functional terms, the "injury" could be sublethal; it could be an activation of endothelium, as some have proposed, increasing atherogenic properties such as monocyte adhesion.27 Furthermore, it could lead to more general dysfunction,26 and it could shorten the average life span of the cell population, leading to the increased permeability to macromolecules that has been associated with cell injury or increased turnover.28 29 30

Hence, an important next step toward examining a role for oxidized LDL–induced injury in the development of vascular lesions is to determine whether oxidized LDL can, in fact, injure endothelial cells and alter their function in vivo, in the presence of protective antioxidant systems present in plasma and tissue. In the present study, exogenous oxidized LDL was introduced into the blood of rats, and endothelial injury and changes in endothelial function were quantified. Endothelial injury was defined in its extreme manifestation, ie, injury leading to a shortened cell life span and increased cell turnover.


*    Materials and Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Lipoprotein Preparation and Modification
LDL (solvent density, 1.019 to 1.063 g/mL) was prepared by sequential ultracentrifugation of citrated human plasma as modified19 from Hatch and Lees.31 EDTA (final concentration, 1 mmol/L) was present throughout the procedure. Protein,32 total cholesterol (using cholesterol esterase/cholesterol oxidase, Sigma catalog No. 352-100; with standards, Sigma catalog No. C0534), relative electrophoretic mobility (Ciba-Corning catalog No. E4635-11A), thiobarbituric acid reactivity,19 and endotoxin (limulus assay-QCL1000, Whittaker catalog No. 50-647 U) were determined in all LDL preparations. The average cholesterol-to-protein ratio of the LDL preparations used in the present study was 1.63±0.21 (n=38). LDL (5 to 8 mg LDL cholesterol/mL) was oxidized by dialysis against 0.9% NaCl, pH 7 to 8, with FeSO4 (5 µmol/L) at room temperature.19 The preparation underwent a characteristic color change from golden, to yellow, to translucent.33 Oxidation was stopped, and bound metal ion was removed by extensive dialysis against 0.9% NaCl with 0.5 mmol/L EDTA at pH 8 to 9 at 4°C, which retards oxidation.34 Cholesterol, total LPO,35 relative electrophoretic mobility, thiobarbituric acid reactivity, and, selectively, endotoxin were determined in lipoprotein preparations after oxidation. LDL oxidation increased thiobarbituric acid reactivity from <0.068 to 4 to 8 nmol malondialdehyde/mg LDL cholesterol, total LPO from <10 to 550 nmol/mg LDL cholesterol, and relative electrophoretic mobility by twofold to fourfold. We found only trace amounts of endotoxin in our lipoprotein preparations (<0.01 endotoxin units/mg LDL cholesterol). Our lipoproteins were prepared and oxidized by procedures designed to minimize endotoxin contamination36 ; endotoxin levels did not increase during the manipulations associated with oxidation, as assessed in selective preparations.

Toxicity to Cells In Vitro
Twenty-four–well tissue culture plates were coated with fibronectin (10 g/m2) and left undisturbed at room temperature for 1 hour. RAECs, isolated similarly to the method previously described for human aortic endothelial cells,37 were plated with MCDB107 media (MCDB105 [Sigma] with 15 mg/L KCl and 15 mg/L glycine, pH 7.4), containing crude endothelial cell growth substance (150 mg/mL), heparin (90 mg/mL), and 15% FBS. When the cells were confluent, they were washed twice with media and made quiescent by incubation for 48 hours in media with 1% FBS. [14C]Adenine (0.2 µCi/mL) was added overnight. The cells were washed twice with media and oxidized, or native LDL at various concentrations was added in media with 1% FBS. After 24 hours, aliquots of the media were counted in a liquid scintillation counter (TR1900, Packard) to determine 14C-labeled products released as a measure of cell injury.38 The toxicity data were expressed as percent specific release of 14C, which was determined using wells treated with detergent (Triton X-100, 0.5%) to obtain maximum (100%) release and wells treated with native LDL (1.5 mg cholesterol/mL; cholesterol-to-protein ratio, 1.57) to obtain basal (0%) release.

Lipoprotein Infusion
Oxidized LDL and native LDL preparations were concentrated twofold to threefold using a 30K Centriprep (Amicon). Male Sprague-Dawley rats (334±60 g) were anesthetized with ether. Infusions were via the tail vein (0.1 mL/minute, for 30 to 40 minutes) using a butterfly infusion set. To determine disappearance of native or oxidized LDL from plasma (four animals, Fig 2Down), doses of 40 and 43 mg cholesterol per animal for single injections (2 mL via the tail vein) and 81 and 85 mg cholesterol per animal for infusions were given. For experiments to obtain endothelial cell proliferation measurements, a dose response was evaluated using oxidized or native lipoprotein doses ranging from 0 to 90 mg LDL cholesterol/300 g body wt. Expressed as LPO, these doses ranged from 0 to 50 µmol/300 g body wt. For doses of <22 mg oxidized or native LDL cholesterol, single injections of 1.5 mL via the tail vein were used. The dose of lipoprotein infused in cell injury and HRP transport experiments ranged from 75 to 89 mg oxidized or native LDL cholesterol per animal. Two solvents were used with lipoprotein preparations: PBS and isotonic saline with 0.5 mmol/L EDTA. Thus, in each experiment, solvent controls were matched as indicated with the solvent of the lipoproteins.



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  		Figure 2. Disappearance from plasma of oxidized (n=2, filled symbols) or native (n=2, open symbols) human LDL in representative animals after intravenous injection (diamonds, n=2) of 40 mg oxidized LDL and 43 mg native LDL cholesterol per animal or infusion (circles, n=2) of 81 mg oxidized LDL and 85 mg native LDL cholesterol per animal. Plasma disappearance was inferred by measuring plasma total cholesterol as a function of time after infusion. The preinjection cholesterol levels, expressed as a function of the initial postinjection or postinfusion levels, were 0.10 ({bullet}), 0.12 ({circ}), 0.23 ({diamondsuit}), and 0.23 ({diamond}).

Endothelial Cell Injury In Vivo
The assessment of cell injury in vivo was performed using a technique based on the observation that significant amounts of HRP accumulate in the cytoplasm of injured cells within 1 minute after intravenous HRP injection. Injured cells are readily identified and quantified after treatment of cells with HRP substrate.39 40 Endothelial cell injury was assessed 36 hours after infusion of lipoproteins, a period found previously to be optimal for observing endothelial cell injury by other intravenously administered injurious agents.41 Animals were anesthetized with sodium pentobarbital (50 mg/kg body wt IP) 36 hours after lipoprotein or PBS (Dulbecco's, Bio Whittaker catalog No. 17-512F) was infused and catheters were inserted in the left femoral vein and right carotid artery. Heparin (600 U/kg body wt) in 0.5 mL PBS was injected via the femoral vein, followed 1 minute later by HRP (80 mg/kg body wt, type II, Sigma) in PBS (0.5 mL/350 g body wt). After a 1-minute circulation of HRP, an intravenous bolus of sodium pentobarbital (50 mg/kg body wt) was given via the carotid artery. PBS at 37°C was perfused via the carotid artery to wash out blood. This was followed by 10% buffered formalin (15 mL, 37°C). The inferior vena cava was cut below the level of the kidney for efflux. Fixation of the aorta was continued by formalin perfusion at 100 mm Hg for 15 minutes. The thoracic aorta, distal to the first pair of intercostals, was excised from the animal, further fixed in formalin for 15 minutes, and stored in PBS. A proximal sample (15 mm in length) was cut longitudinally between the intercostal ostea, rinsed, and reacted for 30 minutes with HRP substrate, 1 mg/mL 3,3'-diaminobenzidine, and 0.015% H2O2 in imidazole buffer (0.1N, pH 7.6) to yield a brown HRP reaction product. The tissue was washed and stored in formalin for further processing to obtain endothelial monolayers.

Endothelial monolayers were transferred to glass microscope slides as previously described42 and modified.41 Injured endothelial cells were identified by their brown cytoplasmic staining and quantified by cell enumeration using an Olympus microscope with a x20 objective and x10 eyepiece. Positive endothelial cells were counted in microscope fields lying along the longitudinal axis of the anterior thoracic aortas, equidistant from the pairs of intercostal ostea. Occasional cells adhering to monolayers, which were morphologically distinct from endothelial cells, were excluded from quantification. Fourteen animals were studied for cell injury. Typically, positive cells in 19 to 20 fields per aorta, of 648±85 (n=8 fields from representative animals of all groups) endothelial cells per field, were counted by each of three trained observers who were unaware of the experimental groups. From one aortic sample from each animal, anatomically matched to the other animals, each observer independently chose the 19 to 20 nonoverlapping fields to be counted.

Endothelial Cell Proliferation In Vivo
Since endothelial cell injury in vivo by other damaging agents, such as endotoxin, is followed by a compensatory proliferation,41 43 44 we quantified endothelial cell proliferation 48 hours after lipoprotein infusion. Rats were given lipoproteins or their respective solvent controls (saline with 0.5 mmol/L EDTA for lipoproteins of <22 mg LDL cholesterol, PBS for others) as detailed above. Forty-eight hours later, animals were anesthetized as described above, and endothelial cell proliferation in the proximal section of the descending thoracic aorta was quantified over the final 24-hour period by injecting [3H]thymidine (500 µCi/kg body wt, ICN) intraperitoneally at 17, 9, and 1 hour before termination.44 After autoradiography, nuclei labeled with silver grains in endothelial monolayers42 were quantified in seven or eight fields per aorta, as detailed above by three trained observers. Six separate experiments were performed, each with multiple animals receiving no injection or one of the following: native LDL, oxidized LDL, or solvent (either PBS or saline with EDTA). Proliferation of endothelial cells in the thoracic aorta of each animal was normalized by the average value determined for the solvent-injected and uninjected control animals for a given experiment (since the results were the same for solvent and no injection). Labeled cells in the control (solvent-injected or uninjected) animals averaged 8.3±2.7 (±SEM) per field among the six experiments. The SD values of the labeled cell counts from each of the six control groups averaged 45% of their means, likely reflecting in part the heterogeneity of endothelial cell turnover in the thoracic aorta.45 46 The average number of cells per field was 648; thus, the endothelial cell turnover in control aortas was determined to be 1.3% per day.

HRP Transport In Vivo
Changes in transport from plasma into the aortic wall of the macromolecular marker HRP (44 kD) were used as an indicator of altered function of endothelium after injury. The following technique has been reported in more detail previously.47 Briefly, 48 hours after the lipoprotein or solvent infusions (PBS or saline with EDTA), animals were anesthetized as described above. HRP (50 mg/kg body wt, type II, Sigma) was dissolved in PBS (0.5 mL/350 g body wt), and an aliquot of 125I-labeled HRP (labeling grade, ICN) that was labeled using Iodo-beads (Pierce) as described previously47 was added as tracer, such that the final injection volume consisted of 0.1% by weight 125I-HRP. The HRP was injected as a bolus via an exposed femoral vein and allowed to circulate for 15 minutes. Blood samples were taken at various times after the HRP injection via a catheter in the contralateral femoral artery catheter to determine the disappearance of 125I-HRP. This was used to calculate Cpo. The thoracic cavity was then cut open, and 3 mL of ice-cold PBS was injected into the left ventricle to clear blood. This was followed immediately by 10 mL of ice-cold 2.5% glutaraldehyde in PBS. Vascular drainage was via the femoral artery catheter. Fixation in situ continued for 5 to 10 minutes, after which the descending thoracic aorta distal to the first pair of intercostals was removed and cut into three sections. The middle section was processed for HRP accumulation by reacting with substrate, 3,3'-diaminobenzidine, and H2O2 and by quantifying HRP accumulation in 4-µm-thick sections using an image-processing system. From aortic cross sections, HRP accumulation was quantified by comparing the gray-scale values with those of standards prepared by equilibrating for 36 to 48 hours fresh aortic tissue with solutions of known HRP concentration47 and then processing analogously. Using this technique, we obtained HRP concentration profiles in the arterial wall as a function of radial distance from the endothelium. Medial accumulation of HRP was measured as a gray-scale image of the HRP reaction product at 20 discrete sites, equally spaced along a radial line from the IEL to the external elastic lamina. An average medial accumulation of HRP for each animal was obtained by averaging 16 profiles (four profiles from each of four tissue cross sections). HRP accumulation in the neighboring intimal region was measured by computer-assisted planimetry at the intimal site in line with each radial (medial) profile. The intimal planimetry scrupulously avoided endothelial cells and the IEL. Each of these was a single value, because HRP accumulation was found previously to be uniform across the intima; ie, it exhibited a flat transintimal HRP concentration profile.47 This single value was thus obtained for each aorta by averaging 16 data values, where an intimal data value was obtained from each of the four profiles from each of the four tissue sections. Fifteen animals were studied for HRP transport. Statistical analyses were performed using Student's t test. In limited ancillary experiments, we determined that oxidized LDL infusions did not alter the blood pressure compared with the control condition during the HRP infusion period 48 hours later (data not shown). Blood pressure was measured via a transducer (Micron) and recorder (Gould) connected to a femoral artery cannula. The procedures followed for animal experiments were in accordance with the Cleveland Clinic Foundation guidelines for the care and use of animals in research.

Mathematical Determination of Transport Parameters
Mathematical models of macromolecular transport in the arterial wall developed and described in detail previously48 were used in the present study to determine parameters of interest, including PE and PIEL. To evaluate these permeability coefficients, the fractional distribution volumes of the tissue for HRP had to be defined for intima and media. We have previously measured these in rat aorta, defined to exclude the tissue of the elastic layers as required for the present model.48 They are .155 and .375 for intima and media, respectively. We have assumed in the permeability calculations that these values are unchanged by the transiently increased lipoprotein levels; this assumption is consistent with the assumptions we made after transiently exposing arteries in vivo to endotoxin.41 49 The model follows classic concepts of Fick's law of diffusion treating anatomic layers of the artery wall as sequential porous layers. Parameters were estimated by a least-squares best fit of the model output to experimental data of intimal and medial HRP concentration values as previously described.48


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
Two considerations influenced our choice of a dose range for oxidized LDL: (1) the concentration of oxidized LDL required to injure confluent RAEC in culture and (2) the rate of disappearance of oxidized LDL from the plasma after intravenous administration in rats. Confluent RAECs grown in culture, preloaded with [14C]adenine, released 14C in response to oxidized LDL. As shown in Fig 1Down, cytotoxicity, expressed as the specific 14C released during 24 hours of oxidized LDL exposure, increased significantly between 300 and 600 µg LDL cholesterol/mL in media containing 1% serum. These data suggested the minimum target concentration range that we wished to achieve in plasma in vivo.



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  		Figure 1. Toxicity to quiescent confluent RAECs in culture 24 hours after the addition of oxidized LDL was measured as specific 14C release after loading cells with [14C]adenine. Native LDL at 1.5 mg cholesterol/mL added to replicate dishes of cells served as control (0% release). Data are mean±SD of three wells.

From previous studies of others, we knew the disappearance of LDL from plasma to be accelerated after its oxidation,50 51 depending on the degree of oxidation.51 From these studies and the data in Fig 1Up, we estimated an effective dose range for oxidized LDL. The disappearance rate of intravenously administered exogenous LDL from plasma is shown in Fig 2Up for four representative animals, as inferred from the decline in total plasma cholesterol. As expected, the decline was significantly more rapid for oxidized LDL than native LDL. As examples, in two representative animals, the estimated time-averaged plasma concentrations of exogenous LDL over 24 hours for an 81 mg dose of oxidized LDL and an 85 mg dose of native LDL were {approx}2.1 and 4.1 mg/mL, respectively. The former concentration is comfortably above the concentration range of oxidized LDL that caused injury to confluent cultured RAECs in 1% serum over a 24-hour period.

We quantified endothelial cell injury in vivo 36 hours after infusion of native LDL, oxidized LDL, or PBS. Injury was measured by counting the number of aortic endothelial cells that accumulated HRP in their cytoplasm 1 minute after an intravenous bolus of HRP.39 40 Table 1Down shows that aortic endothelial cell injury was significantly elevated (P<.01) in animals treated with oxidized LDL compared with control animals infused with native LDL or PBS.


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  		Table 1. Aortic Endothelial Cell Injury 36 Hours After Lipoprotein or PBS Infusion

We quantified endothelial cell proliferation in response to oxidized LDL or control solutions 48 hours after infusion. The number of endothelial cells traversing the DNA synthesis phase of the cell cycle between 24 and 48 hours after infusion correlated with the dose of total lipoprotein administered for oxidized LDL (r=.52, P<.039) but not for native LDL (r=.17, P<.69). Native LDL did not significantly increase proliferation above solvent or no-injection control values. As displayed in Fig 3Down, proliferation in all animals showed a significant correlation with the dose when it was expressed as the total lipid peroxide administered, with a correlation coefficient of .69 (P<.0001). Although these correlation coefficients are based on an assumption of linearity, the data are insufficient to confirm that the relationship is in fact linear rather than another functional form.



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  		Figure 3. Aortic endothelial cell proliferation as a function of the dose of lipoprotein-borne lipid peroxides, measured 48 hours after infusion of oxidized LDL ({bullet}, n=16), native LDL ({circ}, n=8), and solvent (PBS or saline with EDTA) or no infusion (x, n=19). Dose of lipoprotein infused was varied from 0 to 90 mg LDL cholesterol per animal. Doses of oxidized LDL and native LDL were matched on the basis of LDL cholesterol. [3H]Thymidine was given intraperitoneally at 17, 9, and 1 hour before termination, and its incorporation into DNA of proliferating cells was quantified by autoradiography. Each data point represents one animal. Data are from six separate experiments and are expressed relative to the average labeled cells per field in the control (solvent-injected and uninjected) animals of each respective experiment. The control values averaged 8.3±2.7 (mean±SEM) labeled cells per field. Three individuals counted labeled nuclei from seven or eight fields in endothelial monolayers of a proximal section of descending thoracic aorta of each animal (by least-squares regression, r=.69, P<.0001).

To determine the influence of oxidized LDL on endothelial function, the transport past the endothelium of a macromolecule, HRP, was determined. Concentration profiles of HRP were obtained as a function of radial distance through the media of the aortic wall 15 minutes after the injection of HRP and 48 hours after the infusion of oxidized LDL or control solutions. The averages of these for all animals are shown in Fig 4Down. The accumulation of HRP in the arterial media was expressed relative to Cpo to allow data from multiple animals to be averaged.52 Transmural concentration profiles in the media were plotted versus the normalized distance from the endothelium (0.0) to the adventitia (1.0). The data in Fig 4Down show an increased HRP accumulation in the media, particularly in the luminal half, when compared with the control animals infused with native LDL or solvent (PBS or saline with EDTA).



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  		Figure 4. Averages of transmural medial profiles of HRP accumulation 48 hours after infusions of oxidized LDL ({bullet}, n=6 animals), native LDL ({circ}, n=3), solvent (x, PBS or saline with EDTA, n=5), or no infusion (+, n=1). Dose of lipoprotein infused ranged from 75 to 89 mg LDL cholesterol per animal. Data for each animal were obtained by averaging 16 profiles (four profiles from each of four tissue cross sections). Data are mean±SEM.

When the medial accumulation of HRP was integrated from the IEL to the external elastic lamina by the trapezoidal rule, we found that the accumulation of HRP after oxidized LDL treatment was significantly elevated (P<.05) compared with that after native LDL or solvent infusion, as shown in Table 2Down. The intimal accumulation of HRP was determined separately from the media. It was also significantly elevated (P<.01) in aortas of oxidized LDL–treated animals compared with those exposed to native LDL or control infusions (Table 2Down).


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  		Table 2. Accumulation of HRP and HRP Permeability Coefficients 48 Hours After Lipoprotein or Solvent Infusion (PBS or Saline/EDTA)

As shown in Fig 5Down, for a representative aorta, the output of the mathematical model was successfully fit to the experimental data to determine PE and PIEL as previously described.48 Table 2Up shows that PE was significantly elevated (P<.05) after oxidized LDL treatment compared with PE after native LDL or control infusion; however, PIEL was unaltered by oxidized LDL treatment.



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  		Figure 5. Experimental HRP accumulation data ({bullet}) with least-squares mathematical model fit (solid line) 48 hours after oxidized LDL infusion in a representative aorta. Experimental data were obtained by averaging four profiles from each of four tissue sections. The best fit of the mathematical model output to experimental data provided estimates for the parameters PE and PIEL. IEL and EEL denote the internal and external elastic lamina, respectively.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
Our results show oxidized LDL to be capable of causing endothelial injury in vivo (Table 1Up) despite the antioxidants known to be present in plasma and aorta. A consequence of the injury is increased endothelial cell proliferation (Fig 3Up), likely to be a compensatory response to the injury, and enhanced transport of plasma-borne macromolecules into the intima and inner media (Table 2Up).

We chose to study the effects of oxidized LDL in animals using LDL oxidized before injection in the presence of metal ion. The extent to which LDL oxidized in vitro resembles oxidized LDL found in vivo is unknown. This form of oxidized LDL has been well characterized in its interactions with cells, and perhaps more important, the principal cytotoxic component of metal ion oxidized LDL, 7ß-hydroperoxycholesterol, has also been shown to be a component of human atherosclerotic lesions.53 Hence, this in vitro form of oxidized LDL carries at least one lesion constituent capable of causing cell injury. It is also relevant in this context to recognize that oxidized LDL injures cultured cells whether the LDL is oxidized in vitro by a variety of oxidation initiators or by in vivo sources.5 6 8 19 20 21 22 23

The pathophysiological relevance of the injurious effects of oxidized LDL on arterial endothelium likely relates to its focal accumulation in the intima during lesion formation. In the present experiments, oxidized LDL was removed from plasma more rapidly than native LDL (Fig 2Up); thus, we presume that the endothelium was exposed to oxidized LDL initially from plasma and secondarily from the intima, since it has been shown to accumulate in the intima-media rapidly after intravenous injection.54 Others have used oxidized LDL injections or infusions to study its influence in vivo, in particular, on the induction of cytokines55 and the promotion of leukocyte adhesion to endothelium.56 In these studies and ours, an inherent limitation is that the interactions between endothelium and oxidized LDL that may occur in disease are being simulated by oxidized LDL carried in plasma or acutely accumulating in the intima. In disease states, it is more likely that the abluminal face of the endothelium is exposed over a protracted period of time to oxidized LDL. It is unknown whether the cellular responses evoked by exposure of oxidized LDL to the luminal and abluminal faces of the cells are the same.

The intimal oxidized lipoprotein concentrations that pertain in atherogenesis are unknown, although attempts have been made to speculate on these.57 Whether the oxidized LDL injury to endothelium in our experiments is relevant to other species or to actual pathophysiological situations can currently be approached only hypothetically. Although in the present studies we examined these effects in rats, endothelial cells from human and other species have also been shown to be susceptible to oxidized LDL injury in vitro.58 59 The lipoprotein infusions we used acutely and transiently increased the total plasma cholesterol by up to 7 mg/mL from a basal level of {approx}0.8 to 1.0 mg/mL. Over the subsequent 24-hour period, the time-averaged plasma concentration of oxidized LDL exposed to endothelium was {approx}2 mg/mL and was associated with marked injury to the endothelium. Interestingly, the same or even higher acutely elevated levels of native LDL did not cause injury. Early studies of LDL cytotoxicity to endothelium in vitro58 60 were later attributed to LDL oxidation.34 59 61 Early studies in vivo also suggested that hypercholesterolemia is injurious to endothelium,62 63 although it is not known if the injury depends on prolonged exposure or oxidation. Drawing on other studies, one can compare the levels of oxidized LDL to which we exposed the endothelium with the intimal concentrations of oxidized LDL that could theoretically pertain in vivo to early lesion sites. In normal animals, elevated plasma LDL proportionally increases interstitial LDL.64 Estimates of the concentration of LDL in the intima of human arterial lesion sites have suggested levels at least as high as those in plasma.65 66 Thus, if LDL could reach this level in a lesion-prone site, an LDL concentration of 1 to 2 mg cholesterol/mL could persist on the abluminal side of the endothelium. Our results suggest that if this LDL was to be oxidized and if the endothelial cells were as susceptible on their abluminal aspects as their luminal aspects, a condition capable of injuring endothelium could result and perhaps be made worse by prolonged exposure. These effects may be more pronounced at sites of arterial branching, which have been shown to accumulate LDL more readily67 68 and which are known to be predilection sites for lesion development.

We have performed these in vivo experiments on the basis of our in vitro findings of cell injury by oxidized LDL, and it is tempting to interpret that the cell injury we quantified in vivo occurred by a direct mechanism analogous to what we have proposed in vitro.24 25 From the present experiments, however, we cannot be certain that the endothelial effects of oxidized LDL are in fact analogous. Other indirect mechanisms could be involved in vivo. For example, endothelial injury could take place secondary to cytokine actions evoked by the influence of oxidized LDL on other cells, including macrophages.55 69 70

To determine the functional consequences of oxidized LDL–induced injury in vivo, we evaluated macromolecular transport across the endothelium and IEL using a high-resolution technique for assessing the transport and accumulation of the 44-kD macromolecular marker HRP.47 Our results (Fig 4Up and Table 2Up) correlate oxidized LDL exposure to endothelium with an increase in endothelial passage of HRP from plasma to tissue. It is interesting to speculate whether the increased transport of this macromolecule from plasma to intima could have implications relevant to that of the larger LDL. Stemerman et al71 have shown that labeled LDL concentration was 47 times higher in regions of rabbit aorta that showed increased permeability to HRP than in areas that did not. Both HRP and LDL are likely transported across normal endothelium from plasma to intima by bulk phase vesicular processes. Increases in HRP passage across endothelium after oxidized LDL injury may be related to enhanced vesicular transport such as that shown to occur in proliferating cells,28 increased movement through enlarged junctions of injured cells,29 or increased transport allowed as junctions reform at sites of proliferating cells.30 If the HRP transport increases that we observed were via one of these pathways, one could speculate that any of these would be likely to increase LDL transport analogously, since the altered transport rates for either molecule would be proportional to the product of its concentration difference between plasma and intima and the altered permeability coefficient for the respective molecule crossing the endothelial cell monolayer. Whether this analogy actually pertains awaits further experimentation using a high-resolution quantifiable LDL marker. It is possible that the oxidized LDL injury enhances a mode of HRP transport unavailable to LDL.

We used a mathematical model to quantify processes that contribute to the increase in HRP accumulation in the intima and media under the influence of oxidized LDL. This allowed us to consider the implications of the non–steady state intimal accumulation we observed by determining the changes in well-defined physiological parameters. Parameter values obtained to characterize these processes were those that provided the best fit of the model-predicted concentrations to the experimental data (Fig 5Up). By resolving separate estimates for the permeability coefficients of the endothelium and IEL, we were able to show a diminished macromolecular barrier function of the endothelium and the absence of a change in the permeability of the IEL by oxidized LDL (Table 2Up). We have recently demonstrated that in the normal rat aorta, the IEL constitutes a sizable resistance to the transport of even a macromolecule as small as HRP from the plasma to the arterial media (25% of the total resistance).48 A marked increase in endothelial permeability to macromolecules, such as is brought about for HRP by oxidized LDL, coupled with no change in the IEL permeability puts the arterial intima in a situation particularly prone to increased accumulation of larger macromolecules, including (potentially) LDL, a scenario predicted mathematically by Fry72 in an earlier theoretical study.

In summary, our results demonstrate that if oxidized LDL, which is known to accumulate in lesion sites, is present in high enough concentrations, it can injure endothelium, increase cell turnover, and interfere with normal function of this important cell layer. The compromised barrier function of the endothelium could lead to an increase in the entry and accumulation of LDL and exacerbate lesion development. Since the permeability of the IEL was not altered, the focal accumulation of macromolecules could increase further in the intima. If this occurred in the case of LDL, this could accelerate lipoprotein accumulation, oxidation, and lesion formation. Whether endogenously generated oxidized LDL can accumulate to an extent that it could mimic this injury is still undetermined, but the finding that such injury is possible, even in the potentially antioxidant-rich environment of plasma and intima, suggests that the concept warrants further consideration.


*    Selected Abbreviations and Acronyms
 
Cpo = initial concentration of HRP in plasma
HRP = horseradish peroxidase
IEL = internal elastic lamina
LDL = low-density lipoprotein
LPO = lipid peroxide
PE, PIEL = permeability coefficients of endothelium and IEL
RAEC = rat aortic endothelial cell


*    Acknowledgments
 
This study was funded by a grant from the National Institutes of Health (HL-29582).


*    Footnotes
 
Reprint requests to Guy M. Chisolm, PhD, Department of Cell Biology, Cleveland Clinic Foundation, NC-10, 9500 Euclid Ave, Cleveland, OH 44195. E-mail chisolg@cesmtp.ccf.org

Received April 24, 1996; accepted October 15, 1996.


*    References
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 *References
 

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