ultrastructural studies on macromolecular permeabil ity in...

ATHEROSCLEROSIS

Atherosclerosis I I8 ( 1995) 89- 104

Ultrastructural studies on macromolecular permeabil relation to endothelial cell turnover

Yuh-Lien CherFb, Kung-Ming Jan”,“, Huai-San Linb, Shu Chien*’ “Institute of’ Biomedical Sciences, Academiu Sinica, Taipei, T&WI 1,529, ROC

ity in

L,C,d

“Institute of Anatomy, College of Medicine. Nationul Taiwan University, Taipei, Taiwan, ROC ‘Department of’ Physiology and Cellular Biophysics, College of Physicians and Surgeons, Columhiu Uniuersity New York,

NY 10032, USA “Department of Bioengineering und Institute fbr Biomedical Engineering. Unicersity of Cal@rniu, San Diego, La Jolla,

CA 92093, USA

Received 18 July 1994; revision received 6 March 1995; accepted 18 April 1995

Abstract

Our previous light microscopic studies demonstrated the correlation of focal arterial uptake of macromolecules with the mitosis or death of endothelial cells (ECs). To investigate horseradish peroxidase (HRP) permeability associated with the clefts surrounding these ECs at the ultrastructural level, experiments were performed on rat thoracic aortae by using transmission electron microscopy. In en face preparations of aortic specimens, light microscopy was used first to detect mitotic ECs by hematoxylin staining prior to electron microscopy. Dying (or dead) ECs containing cytoplasmic immunoglobulin G (IgG) were identified by an indirect immunogold technique. HRP was found to permeate from the vessel lumen through the widened junctions around the mitotic and dying cells, as well as some non-widened junctions and the plasma membrane of the dying cells. The transiently open junctions during cell turnover lead to an increased transendothelial permeability to macromolecules. In addition to its enhanced passage through the leaky junctions around EC turnover and through the damaged membrane of dying cells, HRP can also traverse many normal intercellular clefts into the subendothelial space of the aorta. These observations show that normal intercellular junctions can provide a significant pathway for the transport of macromolecules with the size of HRP, and that HRP transport is enhanced in transiently open junctions surrounding ECs undergoing turnover. The widened junctions around the mitotic and dying cells provide the pathway for macromolecules larger than HRP, e.g., the low density lipoproteins (LDLs).

Keywords: Cell death; Cell mitosis; Endothelium; Horseradish peroxidase (HRP); Permeability; Ultrastructure

*Corresponding author. Tel.: 619 534 5195; Fax: 619 534 5722.

0021-9150/95/$09.50 0 1995 Elsevier Science Ireland Ltd. All rights reserved SSDlOO21-9150(95)05596-O

90 Y.-L. Chen et al. / Atherosclerosis I I8 (199.5) 89- 104

1. Introduction

The vascular endothelium provides a dynamic interface between the blood plasma and the in- terstitial fluid. It has been suggested that the permeability of the arterial endothelium to macromolecules such as lipoproteins is an important factor in the initiation and progression of atherosclerosis [ l-31. Electron microscopic studies [4-l l] with the use of various types of tracers suggest that there are two modes of transport by which macromolecules can traverse the arterial endothelium to enter the subendothelium, i.e., the transcellular pathway via the endothelial plasmalemmal vesicles and the intercellular pathway between adjacent endothelial cells (ECs). The roles of the plasmalemmal vesicles and the interendothelial junctions as the transport pathways for macromolecules have recently been critically examined by functional and ultrastructural studies [ 12- 161.

Light microscopic studies on the endothelial lining of the aorta and large arteries have shown that both the permeability to plasma proteins and the rate of cell turnover are non-homogeneous in the arterial tree, with focal areas of increased permeability [ 17-201 and clusters of cell replication [21]. Gerrity et al. [22] presented data to indicate that these two phenomena are spatially associated with each other and suggested that EC dysfunction and injury at the site of cell loss may be responsible for the elevated permeability. Based on theoretical modeling and experimental findings, Weinbaum et al. [23] proposed a hypothesis that the regional variations in the frequency of transiently open junctions during cell turnover resulted in the local differences in macromolecular permeability. Light microscopic investigations [24-281 have provided experimental support for this hypothesis. Although the frequencies of endothelial mitosis (0.01%) and death (0.1%) were very low, nearly all of the mitotic ECs and dying ECs were associated with light microscopically demonstrable leakage of macromolecular tracers such as horseradish peroxidase (HRP), Evans-Blue-labeled albumin, and Lu- cifer-Yellow-labeled low density lipoprotein (LDL). It was noted, however, that some 80% of total HRP leakages were not associated with mitotic or dead ECs. Electron microscopic studies

have been conducted to investigate the permeability of large arteries, but there have been no previous ultrastructural investigations on the regional variations in aortic permeability in association with cell mitosis or cell death, except for our report of one leaky junction of EC in mitosis [29]. Such ultrastructural studies are critical to the interpreta- tion of the light microscopic studies on macromolecular passage in terms of particular sites of transit across the arterial endothelium in normal and pathological states.

The present study was performed to examine, at the ultrastructural level, the route of passage of the 5 nm HRP particles across the aortic endothelium into the underlying intima, with particular attention focused on its correlation with cell turnover.

2. Materials and methods

2.1. Experimental protocol for studies on mitotic cells

Eighteen adult male Sprague-Dawley rats weighing 250-350 g were used to study the ultrastructural relationships between HRP leakage and EC mitosis. The experiments were performed under pentobarbital anesthesia (30 mg/kg body weight intraperitoneally). The right carotid artery was cannulated with a 20-gauge needle catheter, using PE-90 polyethylene tubing. The right femoral artery and the left femoral vein were cannulated with 23-gauge needle catheters, using PE-50 polyethylene tubings. The needle catheter in the femoral vein was used for the injection of macromolecular tracer, and the needle catheters in the femoral artery and the femoral vein both served as egress routes during perfusion. HRP (Type II, Sigma Chemical Co., St. Louis, Mis- souri) at a dose of 10 mg/lOO g body weight (dissolved in 0.5 ml saline) was injected into the left femoral vein. After a predetermined circulation time (1, 3, 5 min in different experiments) following the HRP injection, an overdose of pentobarbital was given following the intravenous injection of 0.2 ml of heparin (5000 U.S.P units/ ml; China Chemical and Pharmaceutical, Taipei, Taiwan, R.O.C.) to prevent intravascular coagula- tion. The carotid artery catheter was connected to

Y.-L. Ctten et al. ,’ Atherosclerosis 118 (1995) 89- 104 91

a pressure reservoir, and perfusion was begun immediately with a heparinized phosphate- buffered saline solution (PBS) at a pressure of 110 mmHg until clear fluid emerged from the egress sites (approximately 20 s). The perfusate was then switched to the fixative (1% freshly prepared paraformaldehyde and 1.25% purified glutaraldehyde in 0.1 M sodium cacodylate buffer at pH 7.4), which was perfused at the same pressure and a flow of approximately 15 ml/min for 10 min for preliminary perfusion fixation. The aorta was then excised between the aortic root and the di- aphragm. The thoracic aorta was immersed in a fixative containing 2% paraformaldehyde and 2.5% glutaraldehyde in the same buffer for 2-3 h at room temperature. The cane-shaped thoracic aorta was longitudinally cut open, pinned onto a dental wax plate with the endothelial cells facing up, and incubated for 1 h at room temperature in a medium containing 15 mg of 3,3-diaminoben- zidine tetrahydrochloride (DAB), 10 ml Tris-HCl buffer (0.05 M, pH 7.6) and 0.01% of freshly prepared hydrogen peroxide prior to 0~0, fixation. After DAB reaction and hematoxylin staining, the specimen was subjected to embedding and sectioning for electron microscopy.

2.2. Electron microscopy The fixed specimens were washed in several

changes of 0.1 M sodium cacodylate buffer (pH 7.4) and subjected to post-fixation for 60 min in 1% 0~0, buffered with 0.1 M cacodylate at pH 7.4. The post-fixed specimens were block-stained for 2 h at 4°C with 2% uranyl acetate [30] in 0.05 M sodium hydrogen maleate-NaOH buffer (pH 6.0) and washed in maleate buffer (pH 5.2). All tissues were then dehydrated in a graded series of ethanol and embedded in Epon 8 12. Silver-to-grey sections were cut with a diamond knife on a Reichert ultramicrotome, and lightly stained with lead citrate for examination in the electron microscope (Jeol TEM 1200-EX). The intensity of HRP reaction products in the subendothelial space of the mitotic cell and its neighboring normal cells was quantified by using a digital image analyzer system (IRIS 3120, Silicon Graphics, Monhtain View, CA, and IP-512, Imaging Technology Inc., Woburn, MA).

2.3. IdentiJication of mitotic cells in thin-section electron microscopy

The ultrastructure of the endothelium and its junctions was observed on random sections. The following procedure was used to identify areas containing mitotic cells in order to examine the morphology of the junctions around these cells for comparison with normal ECs. Because the frequency of cell mitosis was very low, aortic segments containing mitotic cells and/or HRP- stained brown spots (brown areas) were first identified on hematoxylin-stained thick sections, and a specimen with a dimension of l-2 mm was then carefully excised for the cutting of thin sections, instead of random sectioning for EM observation. Consecutive thin sections ( - 70 nm) were cut from the trimmed specimen in each block. One thin section from every 100 was transferred to a fomvar grid, and ten grids obtained from 1000 thin sections were stained and examined under the electron microscope. This procedure was continued until the mitotic cell was obtained. After the finding of a mitotic cell a different block was examined by using the same method. Using this approach, brown areas (HRP leakage) with mitotic cells, brown areas without mitotic cells, and white areas (no HRP leakage) were excised, stained with lead citrate, and examined in the electron microscope.

2.4. Experimental protocol for studies on dying cells

Six adult male Sprague-Dawley rats weighing 250-350 g were used to study the dying or dead ECs identified by the indirect immunoglobulin G (IgG) immunocytochemical technique [3 l] after intravenous injection of HRP as described above. Following the clearing of the aorta by perfusion with heparinized PBS, 1% freshly prepared paraformaldehyde and 0.5% purified glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) were perfused through the carotid artery catheter at a pressure of 110 mmHg. The thoracic aorta was dissected out and immersed in the same fixative for 1 h. After its tunica adventitia had been peeled off, the aorta was dissected into several segments, pinned onto a dental wax board, and then incubated with the goat anti-rat IgG (Sigma Chemi-

92 Y.-L. Chen et al. i Atherosclerosis I18 (1995) 89-104

Fig. 1. Transmission electron micrographs of a region of the thoracic aorta 3 min following the intravenous injection of HRP. (A) HRP reaction products are present in the widened junction (arrow) around the mitotic EC (E) in the anaphase, in the subendothehal space, and deep into the first layer of smooth muscle cells (SM). (B) A higher magnification of the widened leaky junction in Fig. 1A (arrow). A and B, Bar, 1 pm. IEL internal elastic lamina; L, lumen.

cal, St Louis, MO) at an optimal dilution of 1: 1000 in 0.1 M PBS containing 1% ovalbumin (Sigma). After 90 min of incubation at room temperature, the specimen was repeatedly rinsed

in PBS for 30 min and then incubated for 90 min with the 5 nm or 10 nm gold-conjugated rabbit anti-goat IgG as a secondary antibody at an optimal dilution of 1: 1000 in PBS containing 1%

Fig. 2. Transmission electron micrographs showing a different section of the same mitotic cell as in Fig. 1. (A) An intercellular junction around the mitotic cell (E) shows unusual widening and is filled with the electron-dense reaction products. (B) A higher magnification of the widened leaky junction in Fig. 2A (arrow). The minimum width is about 220 nm. A and B. Bar, I lrm. IEL. internal elastic lamina; L, lumen; SM, smooth muscle cell.

ovalbumin. After the second incubation, the aortic pieces were rinsed three times (IO min each) with PBS. Further, the tissue blocks were incubated with DAB solution as described above. The regions of the aorta with dead ECs could not be identified under light microscope. According to our previous study [24]. the brown spots (HRP leakages) with IgG-containing cells, usually near

an intercostal orifice, were identified on thick sections and carefully excised, postfixed, dehydrated in a graded series of ethanol, and embedded in Epon 812. Control experiments were performed by incubation in PBS containing 1% ovalbumin without goat anti-rat IgG for 90 min and treatment with gold-conjugated rabbit anti- goat IgG.

94 Y.-L. Chen et al. / Atherosclerosis I 18 (1995) 89- 104

Fig. 3. Transmission electron micrographs showing a region of the thoracic aorta 5 min following the injection of HRP. (A) There is HRP staining in the intercellular junction of a mitotic cell (E) in the metaphase, in the subendothelial space, and in the first smooth muscle layer (SM). (B) A higher magnificaiion of the widened leaky junction in Fig. 3A. (C) An even higher magnification of the leaky junction on the left side of the endothelial cell in Fig. 3B (arrow). Fig. 3A-C, Bar, 0.67 pm. IEL, internal elastic lamina; L, lumen.

3. Results

3.1. Studies of HRP permeability in relation to cell mitosis

Transmission electron micrographs in Figs. 1 and 2 show two different thin sections of the same dividing EC following 3 min of circulation time of HRP. The nuclear pattern of this cell indicates that it was undergoing mitosis. The junction to the right of this cell shows abnormal widening and is filled with the electron-dense HRP reaction product. HRP permeated through the junction to the subendothelial space and then penetrated through the fenestra of the discontinuous internal elastic lamina into the first smooth muscle layer of the tunica media. Figs. 1B and 2B are enlarge- ments of the leaky junction in Fig. 1A and Fig. 2A, respectively. The smallest width of the leaky junction was about 220 nm (Fig. 2B). Fig. 3

shows the transmission electron micrograph of a mitotic cell in the metaphase following 5 min of circulation time of HRP. HRP was again found in the junction around the mitotic cell and the whole subendothelial space, and then penetrated into the first smooth muscle layer. Fig. 4A shows the transmission electron micrograph montage of a dividing EC. Densitometry by image analysis gen- erated the profile of the relative density of HRP reaction product in the subendothelial space (Fig. 4B). The HRP density underlying the mitotic cell was higher than that of neighboring cells, and it decreased progressively away from the mitotic cell in both directions.

In the present study, six mitotic cells from different rats were studied; the minimum and the maximum widths of the junctions around these mitotic cells are shown in Table 1. The narrowest width of the leaky junction was 80 nm, which was

Y.-L. Chen et al. i Atherosderosis II8 (1995) 89- 104 95

Fig. 3 (continued).

more than three times wider than the LDL molecular dimension.

Our previous studies [24, 251 showed that the average diameter of the HRP spots increased as the HRP circulation was prolonged by light microscopy. There was also a considerable degree of heterogeneity for the spot size with increasing HRP circulation time. After an HRP circulation time as short as 1 min, small brown spots and diffuse HRP staining regions co-existed in the same sample of an en face observation. At 1 min circulation time after HRP injection, the average diameter of HRP brown spots is approximately 150 pm, with an area of 2000 + 1800 pm2 (mean + S.D.) [25]. As time increased, the spot size increased rapidly, and finally the spots fused to-

gether, forming large diffuse HRP staining of the aorta at about 5- 10 min after its intravenous injection [24,25]. In the brown spots and areas, HRP was found to cross most of the normal, unwidened intercellular junctions, permeated the internal elastic lamina, and appeared in the first smooth muscle layer at 1 and 5 min circulation after HRP administration in the present electron microscopy study (Fig. 5). Some vesicles on the luminal and abluminal surfaces, as well as along the junction, were labeled with HRP (Fig. 5B). In contrast to such finding in the brown area, HRP was not found to penetrate through the normal endothelium in the white area, leaving the abluminal intercellular cleft and subendothelial space free of HRP (Fig. 6).

96 Y.-L. Chen et al. / Atherosclerosis 118 (1995) 89-104

0 15 30 45 60 75 90 105 120135150 The locot~on along the subendothellol space

B urn

Fig. 4. (A) Montage of four transmission electron micrographs made from overlapping prints. The EC in the center is in mitosis at the anaphase. (B) The intensity profile of HRP reaction product in the subendothelial space determined by using the image analyzer. The ordinate depicts the intensity of HRP reaction product and the abscissa indicates the location in the subendothelial space. A, Bar, IO pm. L, lumen.

3.2. Studies of HRP permeability in relation to cell death

in the thoracic aorta specimens of rats which had received HRP injection prior to processing for gold-conjugated anti-IgG reaction, a few sites of HRP leakages were found to contain IgG-posi- tive cells (Figs. 7 and 8). The cytoplasmic matrix of some of the dying cells was found to contain not only the IgG but also the HRP reaction product, giving the appearance of dark cells. The intercellular junction of the dying cell in Fig. 7 shows unusual widening and is filled with the HRP reaction product. The junctions around the dying cell in Fig. 8 do not show widening; one junction of this cell is permeable to HRP and another one is not permeable to HRP. In the present study, four dying or dead cells from different rats were studied, the minimum and the maximum widths of junctions around these cells are shown in Table 2. Although there was a considerable degree of heterogeneity for the widths of these junctions, the narrowest width of the junction was 30 nm or wider in three of these cells.

In the brown areas without cell death, HRP could also be found in intercellular junctions and the underlying subendothelial space of normal cells. In the white area, HRP was prevented by the tight junctions from entering the subendothelial space of the endothelium (Fig. 9). Gold-conjugated IgG was found occasionally at the luminal aspect of the endothelium, but not in the cytoplasmic matrix. Two aortic pieces from each rat were used as controls by omitting the goat anti-rat IgG in the first incubation; no gold particles were observed in these specimens.

4. Discussion

The results presented herein provide direct evidence in support of the ‘leaky junction-cell turnover’ hypothesis [23] that macromolecular leakage occurs through open junctions in specific time windows of the cell cycle. Although mitotic ECs were infrequent in occurrence (O.Ol%), the great majority of these ECs (over 90Y0) were associated with local HRP leakage in our previous study [24]. In the present study, we demonstrated

Y.-L. Chen rr 01. : Aiherosderosis I18 (199.5) 89- 104 97

Table I The minimum and maximum widths of the leaky junctions around different mitotic ECs

Mitotic cell Minimum width Maximum width no. Mm) (nm)

1 140 290 2 440 1330 3 100 1100 4 100 900 5 80 150 6 220 1000

the presence of leaky junctions around mitotic cells as evidenced by the widening of the junction and the filling of HRP at the ultrastructural level. The subendothelial space at the exits of such junctions and under the mitotic cell was heavily labeled by HRP. Densitometric analysis indicated

that the further the subendothelial space was away from the abluminal exit of the cleft, the lesser was the density of HRP. This provides evidence for the lateral spread of HRP from its focal leakage to the subendothelial space, as predicted by theoretical modeling [32]. Huttner et al. [33] :jhowed that a major feature of the regenerating endothelium of rat thoracic aortae is the continuous morphological remodeling of stress fibers and the condensation of subplas- malemmal microfilaments at cell junctions. A critical period in the morphological remodeling is in the M phase, when the chromosomes are segre- gated and the cell shape undergoes its most dra- matic changes. The cell would have open or poorly organized junctions, whose junctional proteins either are disrupted or have not yet fully formed.

Due to the rare occurrence and the difficulty of obtaining cells with turnover, we only obtained six mitotic cells and measured the width of the leaky junctions around these cells. The width of the leaky junction around these six mitotic cells varied from a minimum of 80 nm to a maximum of 1330 nm (Table 1). The minimum width of 80 nm was considerably wider than that needed for the transendothelial passage of LDL (23 nm) and larger lipoprotein molecules. Unlike the present

tracer HRP, LDL and larger macromolecules can- not pass through the normal junction clefts, and such widened junctions would provide the pri- mary transport pathways for their entry to the subendothelial space resulting in their accumulation in the intima and media.

Earlier studies with the use of the electron- dense tracer technique suggested the presence of at least two ultrastructural bases for the transendothelial transport of macromolecules: plasmalemmal vesicles and intercellular clefts [4- 111. Although plasmalemmal vesicles are the most abundant organelles in the EC and they have long been hypothesized to provide a shuttling mechanism for transendothelial transport of macromolecules, this has been questioned in recent studies [12- 16,341. Serial sectioning has shown that seemingly free vesicles are in reality parts of surface membrane invaginations in capillary endothelium [13] and in aortic endothelium [16]. In the present study luminal, abluminal, and appar- ently free vesicles were labeled with HRP in the brown area. In the white area, however, only the luminal vesicles were labeled. Our data suggest that the difference of HRP permeability between brown and white areas is related to their difference in junctional permeability, but the data do not allow a critical assessment of the role of vesicular transport of HRP.

It has been demonstrated qualitatively and quantitatively that the aortic endothelial intercellular clefts could be one of the determinants of permeability to macromolecules [22]. The length and complexity of interendothelial junctions are quite diverse [9,16,35]. Junctional complexes associated with the endothelial contacts were classified into three types: tight junctions, gap junctions, and junctionless intercellular contacts. The func- tion of gap junctions is probably related to bio- chemical communications between neighboring arterial ECs, and these structures probably do not play a critical role in macromolecular transport across the endothelial layer [36]. The complexity and distribution of the slits in the tight junctions and junctionless intercellular contacts may deter- mine the permeability characteristics of normal aortic interendothelial clefts [5,35]. By freeze fracture, Okuda and Yamamoto [S] demonstrated

98 Y.-L. Chen et al. / Atherosclerosis I I8 (1995) 89- 104

Fig. 5. Transmission electron micrographs of rat thoracic aortae I min (A) and 5 min (B) after HRP administration. HRP reaction products are present in the entire junction (arrow) between two normal ECs (E) and in the subendothelial space. The morphology of the nuclei of the ECs indicates that they are not in mitosis, and the junction is not widened. Labelled vesicles are visible on both the luminal and abluminal surfaces and along the junction. A and B, Bar, 0.5 pm. IEL, internal elastic lamina; L, lumen; SM, smooth muscle cell.

that the tight junctions of the thoracic aortic endothelium are heterogeneous; these junctions consist of one to two junctional strands in some areas of the cleaved planes and show discontinu- ities in some places. Such intercellular clefts may facilitate the permeation of HRP into the subendothelial space. Our light microscopic studies showed that approximately 80% of the total HRP leaky sites were associated with cells not morpho- logically identified as being in the M phase or in the dying stage [24]. The present electron microscopic study showed that HRP molecules can pass through the normal intercellular clefts in the brown areas, but not those in the white area. This can be correlated with the finding that the intercellular clefts of the brown areas (which consist of discontinuous junctional strands, end-to-end contacts, and simple overlap) were less complex in structure than those in the white areas.

Injured and/or dead ECs have been identified by their inability to exclude dyes such as trypan blue, Evans blue and nigrosin [17]. It is generally agreed that aortic endothelial permeability to macromolecules is not homogeneous, and that spontaneously occurring areas of enhanced permeability can be demarcated in vivo by their uptake of the protein-bound Evans blue dye. By using supravital staining to study endothelial integrity and viability in rabbit and rat aortae, Bjiikerud and Bondjaers [17] showed that the blue areas of Evans blue uptake were associated with injured and/or dead ECs, while nonstained areas were covered with normal ECs. In HHutchen preparations of the aortic endothelium with silver nitrate impregnation, Gerrity et al. [22] demonstrated that EC injury or death occurs with a significantly greater frequency in the blue than the white areas. A number of functional and struc-

Fig. 6. Transmission electron micrographs of the thoracic aorta 3 min after HRP injection. (A) HRP reaction products are present in the luminal side of the endothehal cleft, but not in the abluminal side beyond the tight junction (arrow). The subendothelium contains no reaction product. (B) A higher magnification of the tight junction in Fig. 6A (arrow). (C) A different section of the same tight junction as in Figs. 6A and B. HRP reaction product is again present only in the luminal side of the cleft. A, Bar, 0.2 pm; B and C, Bar, I pm. E endothehal cell; IEL, internal elastic lamina; L, lumen; SM smooth muscle cell

tural features, including enhanced EC turnover rate, lipid accumulation, and altered en face endothelial morphology and intimal ultrastructure, have been shown in these focal areas of enhanced macromolecular leakage, which occurred prefer- entially in the branching points of the aorta. The existence of these dying or dead ECs in discrete regions of the normal aorta may have pathophys- iological significance in the focal nature of atherogenesis.

With a double staining technique using fluores- cent antibodies and a dye exclusion test, Hansson et al. [37] found that injured ECs allowed the entry of IgG, while uninjured ECs did not. The accumulation of IgG in the cytoplasmic matrix of injured or dead ECs can be identified by antibodies labeled with an electron-dense material, such as peroxidase, for electron microscopy [38]. This relationship between IgG uptake and cell death has been verified by a highly significant correla-

100 Y.-L. Chen et al. /Atherosclerosis I18 (1995) 89-104

Fig. 7. A transmission electron micrograph showing a region of the thoracic aorta incubated with gold anti-IgG antibody after 3 min circulation of HRP injection. Gold particles are present in the cytoplasm of the EC (E). HRP reaction product is present in the cytoplasm of the EC and its underlying subendothelial space. An intercellular junction (arrow) shows unusual widening and is filled with the HRP reaction product. Bar, I pm. IEL, internal elastic lamina; L, lumen.

tion between IgG accumulation and intracellular calcium deposit which is a characteristic phe- nomenon in cell death [39,40]. Therefore, immunocytochemical detection of IgG in en face preparations has been regarded as a reliable and quantitative method for detecting cell death in the aortic endothelium. In our previous study, indi-

rect immunofluorescence techniques have been used to detect IgG and hence EC death in en face preparations of rat thoracic aorta at the light microscopic level [24]. Although dying ECs were infrequent in occurrence (O.l%), the great majority of these cells (over 900/,) were associated with focal HRP uptakes. In the present study we some- times found the junctions around the dying cells did not show widening. The permeable junction may result from changes of junctional complex during an early phase of cell death. The junctional complexes around the dying EC have to be disrupted before the detachment of dead ECs, and new junctions have to be formed between the surrounding viable ECs. Therefore, it is conceiv- able that during the processes of cell death and replacements, junctions surrounding the dying or dead ECs would be transiently leaky and provide the pathway for macromolecules to enter the intima.

In the thoracic aorta, the majority of dying cells are found to be located near the orifices of intercostal arteries, which are the sites prone to the development of atherosclerosis. This suggests that EC death is mediated directly or indirectly by

focal disturbances in the patterns of aortic blood flow. It has been shown that this regional varia- tion in macromolecular permeability is partly determined by hemodynamic factors, e.g. disturbed flow pattern at these areas of the vessel wall [41-431. It seems reasonable to postulate that the local pattern of disturbed flow causes a regional enhanced rate of EC turnover, and that the resulting increased frequencies of cell mitosis and death in turn lead to the focal nature of lipid accumulation in the arterial tree.

It has been reported that HRP has a pharmaco- logic activity due to the release of vasoactive amines in different species including the rat, thus producing endothelial leakage in venular endothelium [44,45]. In pulmonary capillaries, the fine structural alteration of cell junctions observed following HRP injection may also depend on the concentration used [46]. While a high dose of HRP may produce subtle modification of cell junctions, intravenous injection of the optimal dose of HRP did not change the level of the arterial blood pressure or vascular permeability [6]. The dose of HRP used in the present study on arterial endothelium did not attain the high level needed to exert pharmacological actions. Further- more, studies on arterial endothelium indicate that there is no difference in the ultrastructural features of arterial endothelial junctions between rats with and without prior intravenous injection of HRP [16]. We found widened junctions around the EC undergoing turnover also in rats without

Y.-L. Chen et al. 1 Atherosclerosis 118 (1995) 89-104 101

Fig. 8. Transmission electron micrographs showing a region of the thoracic aorta incubated with gold anti-IgG antibody after 3 min circulation of HRP injection. (A) HRP reaction products are present in the cytoplasm of a dying EC (E) and its underlying subendothelial space. (B) A higher magnification of the left junction of the dying cell in Fig. 8A (arrowhead). HRP reaction product is present in the junction. Gold particles are also present in the cytoplasm of the dying EC. (C) A higher magnification of the right junction of the dying cell in Fig. 8A (arrow). HRP reaction product is not present in the junction. Fig. 8A to C, Bar, I pm. IEL, internal elastic lamina; L lumen; SM, smooth muscle cell.

receiving HRP injection, similar to those reported in this study on rats receiving HRP injection. All these indicate that our studies were not artifacts resulting from a pharmacological action of the HRP injected.

In summary, our present results show that the transendothelial pathways of HRP include most intercellular junctions of normal endothelial cells, the transiently open junctions around mitotic cells and dying cells, and the cytoplasm of dying cells.

102 Y.-L. Chen et al. / Atherosclerosis 118 (1995) 89-104

Fig. 8 (continued).

Table 2 The minimum and maximum widths of the junctions around different dying or dead ECs

Dying or dead cell Minimum width Maximum width no. (nm) (nm)

1 520 1000 2 30 250 3 15 30 4 30 100

Fig. 9. A transmission electron micrograph of a white area of the thoracic aorta incubated with gold anti-IgG antibody after I min circulation of HRP injection. HRP reaction products and gold particles are present on the luminal surface of the plasmalemmal membrane. HRP is excluded by the tight junction (arrow) from entering into the subendothelial space. Bar, I pm. E, endothelial cell; IEL, internal elastic lamina: L lumen.

The major pathway of HRP transport is through normal intercellular junctions, which can be con- sidered as a medium-sized pore system that allows the entry of macromolecules up to the size of small proteins of 5-6 nm. The leaky junctions around mitotic and dying ECs, however, are probably the predominant pathway for large macromolecules such as LDL.

Acknowledgments

The authors wish to thank Professor Wen-Pin Chen and Dr. Mary M.L. Lee for their helpful discussions. This work was supported by research grants NSC 76-0412-BOOl-09 and NSC 80-0412- BOlO-43 from the National Science Council, R.O.C., and HL-19454 from the National Heart, Lung, and Blood Institute, U.S.A.

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[4] Florey L, Sheppard BL. The permeability of arterial endothelium to horseradish peroxidase. Proc R Sot Lond Biol 1970; 174435.

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