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Endotoxin-induced vascular endothelial injury and repair
EXPERIMENTAL
AND
MOLECULAR
Endotoxin-Induced
PATHOLOGY
Vascular
59-49 ( 1976)
24,
Endothelia~ Injury and Repair
II. Focal Injury, en Face Morphology, PHlThymidine Uptake and Circulating Endothelial Cells in the Dog 1
R. G, GERRITY,~ M. RICHARDSON,~ B. A. CAPLAN, J. HIRSH, AND C. J. SCHWARTZ Depaltmerats
J. F. CADE,
of Pathology and Medicine, Faculty of Health Sciences, McMaster and Chedoke ~o~~i~a~, P.O. Box 590, ~a.m~Zton, Ontario, Canada
University
Received June 2, 1975; and in revised form August 18, 1975 In this study we have examined the iniluence of intravenous Esc~erichia coli endotoxin on the induction of endothelial injury and repair in the aorta and pulmonary artery of the dog, with special reference to areas of Evans blue dye uptake. The sequential changes were monitored by quantitation of the numbers of circulating endothelial cells in three vascular beds, by incorporation of [3H] thymidine by endotheliai cell nuclei, and by light microscopy of silver ~~at~stained H&utchen preparations. Circulating endothelial cells were observed as early as 5 min after endotoxin injection, being most numerous in the pulmonary circulation. By 60 min, most circulating cefls had disappeared. In the aorta, uptake of ~Hl~ymidine by endothelial cell nuclei was maximal in the arch and, in particular, in those areas showing Evans blue dye accumulation. Light microscopy of silver-stained HPutchen preparations from blue areas of the aorta and pulmonary artery revealed foci of intense silver staining, which have been interpreted as indicating damage or denuded endothelium. Blue areas also exhibited darkly staining globular structures of variable size and shape apparently overlying the endothelial surface. Neither the foci of intense silver staining nor the globular structures were observed in specimens from adjoining areas of no dye uptake (white areas), which were essentially similar in appearance to control samples, except that in white areas from endotoxin-treated animals, endothelial cells were more granular, and silver-stained cell boundaries showed less separation. The results confirm the findings of an earlier study in which endotoxin-induced endothelial injury in the rabbit aorta was found to be most severe in the arch. The en face morphologic features of endotoxin-induced endothdial injury are shown to be largely confined to areas of enhanced endothelial permeability to protein as demarcated by in uivo Evans blue dye uptake.
INTRODUCTION In the pig and the dog, intravenous injection of the protein-binding azo dye, Evans blue, results in the focal uptake of dye by specific areas in the aortic arch and pulmonary artery. Such areas of dye uptake (blue areas> in the pig aortic arch have been we11 documented and are associated with enhanced uptake of 1 Supported by Medical Research Council of Canada grants, No. MA and MT 3067, and the Ontario Heart Foundation. 2 Ontario Heart Foundation Research Fellow. 59 Copyright All rights
1976 by Academic Press, Inc. oP reproduction in any form reserved.
60
GERRITY
ET AL.
radioiodinated albumin (Packham et al., 1967; Bell et al., 1974a), fibrinogen ( Bell et al., 1974b) and unesterified [“HIcholesterol (Somer and Schwartz, 1971). These areas also exhibit an increased rate of endothelial cell turnover (Caplan and Schwartz, 1973) and altered endothelial cell morphology as seen both in Haiutchen preparations (Caplan et al., 1974) and in the electron microscope (Gerrity et al., 1975a). Additionally, Gerrity et al. ( 197513) have demonstrated an increased endothelial cell turnover in the aortic arch of rabbits after intravenous endotoxin injection. Endotoxin has been shown to elicit multiple and diverse pathophysiological responses, and the subject has been exhaustively reviewed (Bennett and Cluff, 1957, Martin et al., 1965, McCabe, 1974). Although endotoxemia induces profound effects on the cardiovascular system (Gilbert, 1967) and blood elements (Stetson, 1961), relatively little attention has been given to the influence of endotoxin on the vascular endothelium. McKay et aZ. (1966, 1967) examined the ultrastructure of the microvascular endothelium in rats and concluded that endothelial injury was secondary to the ischemia produced by occlusive fibrin thrombi. They did not examine large arteries. Gaynor et al. (1968) and Gaynor (1971, 1972) demonstrated that endotoxemia in rabbits produced an increase in I13H]thymidine uptake by endothelium in both large and small vessels and also an increase in the numbers of circulating endothelial cells in aortic blood, changes that were not prevented by pretreatment with heparin. Endotoxin-induced endothelial damage in the mesenteric artery of rabbits has been demonstrated by McGrath and Stewart (1969) and Stewart and Anderson (1971) using both light and electron microscopy. In view of these findings, the current studies were undertaken to determine whether areas of enhanced permeability in the aortic arch and pulmonary artery, as demarcated by Evans blue uptake, are more susceptible to endotoxin-induced injury and to define the nature of endotoxin-induced endothelial injury in the large arteries as a baseline for further studies into the mechanisms involved. MATERIALS
AND METHODS
Six mature mongrel dogs, weighing 11-22 kgm, were anesthetized with sodium pentabarbital (Diabutal, Diamond Laboratories, Des Moines, Iowa), and indwelling cannulae were placed in the right atrium, aortic arch, and pulmonary artery. Three hours before killing, 50 ml of Evans blue, prepared as described previously (Caplan et al., 1974), were injected into the right atrium. One hour before death, Escherichia coli endotoxin (Difco), 10 mgm/ml, was injected into four dogs at a dosage of 2 mgm/kgm in pyrogen-free isotonic saline. Two control dogs were injected with an equivalent volume of saline. Blood samples for quantitation of the numbers of circulating endothelial cells were obtained through each of the indwelling cannulae (aortic arch, right atrium, and pulmonary artery) 10 and 20 min before the injection of endotoxin or saline, and subsequently at intervals of 5, 15, and 60 min after injection. Endothelial cells were concentrated according to the method of Herbeuval and Fourot (1964), and the results expressed as the number of circulating endothelial cells/1000 leucocytes. One hour after injection of endotoxin, the anesthetized animals were killed by exsanguination, and samples of pulmonary artery and aortic arch were excised
ENDOTOXIN-INDUCED
ENDOTHELIAL
INJURY
61
from areas with (blue areas) and without (white areas) Evans blue accumulation. Similar samples were excised for white areas in the thoracic and abdominal aorta. For light microscopy, samples having an approximate surface area of 1 cm2 were removed and used to make silver-stained HPutchen preparations, employing the method of Pugatch and Saunders ( 1968), as modified by Caplan and Schwartz ( 1973). Additional samples of aortic arch and thoracic and abdominal aorta were also incubated in [3H]thymidine, and autoradiographs were prepared from these by the technique described previously by Caplan and Schwartz ( 1973). A minimum of 1000 nuclei were counted from each sampling site in each of the six animals. For each sampling site, the numbers of labeled nuclei were expressed as a percentage of the total number of nuclei counted. Student’s t test was used to determine the statistical significance of differences between blue and white areas in both treated and control animals. RESULTS
Circulating
Endotiielial
Cells
Figure 1 depicts the numbers of circulating endotheIia1 cells observed in blood samples from the pu~ona~ artery (PA), right atrium (RA ), and aorta ( AO) of four dogs 10 min before the administration of endotoxin and at intervals of Circulating endothelial cells were not observed 5, 15, and 60 min after injection. prior to endotoxin injection. A maximum increase in the number of circulating endothelial cells was observed in each site at 5 min, the increase being greatest in the pulmonary artery blood and least in the aortic blood. Endothelial ceils were still present in the circulation at 15 min after injection, and by 60 min their numbers had declined further, with none detected in the aortic samples.
[ 3H] Thymidine
Uptake
The influence of endotoxin on incorporation of [311]thymidine by endothelial nuclei of the aortic arch and thoracic and abdominal aorta is shown in Table I.
.lO
;
10
20
30
40
50
60
ENDOTOXIN TIME IMINt
FIG.
1. The numbers of circuIating endothelial (PA), right atrium (RA), and aorta (AO) injection of endotoxin.
celis in blood sampIes at various times before
from and
puImonary artery after in~aveno~
CERRI’IY
G2
ET
TABLE
AL. I
PFt] Tl~~i~~i(li~~~ Ill~~)~j~o~~t,i(~n into Actrtk: ~~l~(~(~t,h~!li~~(Xl in t*he I jog aft.er
~ildotoxin
N~~doi
AdmiIlist,ratioI~
-Site of sample
Labelled
nuclei
Endotoxin-treated
(S)O cor1tro1
----
Aortic arch (blue) Aortic arch (white) Thoracic aorta (white) Abdominal aorta (white) a Values are t,he means 6 Significantly different animaLs (P < 0.05).
with from
0.29 0.03 0.09 0.02
(0.16)6 (0.03) (0.02) (0.04)
SD in parentheses; n = four animals. control blue and from white areas derived
0.03 0.00 0.03 0.01
(0.04) (0.00) (0.02)
from
treated
(0.01)
and control
In the aortic arch, areas of Evans bIue dye accumulation (blue) are compared with adjacent areas showing no dye accumulation (white) in treated and control animals. The effect of endotoxin was most prominent in the aortic arch, and in particular, in areas showing Evans blue accumulation, where increased endothelial cell nuclear labeling was significantly greater than in control blue areas and in white areas from both treated and control animals. There was no significant difference in the uptake of [ sH]thymidine into the endothelial nuclei of white areas from aortic arch and thoracic and abdominal aorta. Moreover, uptake in
FIG 2. Hautchen preparation of endothelium Endothelial cell boundaries (B) are stained, are elongate and fairly regular in outline, with x450.
from the thoracic aorta of a control dog. resulting in pavement-like appearance. Cells a faint granularity to the cytoplasm (arrows).
ENDOTOXIN-INDUCED
these segments did not differ significantly trol animals ( TabIe I).
ENDOTHELIAL
INJURY
from corresponding
63 segments
in con-
Light microscopic ex~ination of silver ni~ate-stail~ed Hautchen preparations from control and endotox~-treated animals was undertaken by using endothelium from blue and white areas in both the pulmonary artery and the aortic arch and from white areas in the thoracic and abdominal aorta. In no experiment did endotoxin visibly modify the pattern or intensity of Evans blue uptake. Endothelium from control animals (Fig. 2) showed the morphology typical of Htiutchen preparations, with silver-stained cell boundaries and a faint granularity to the cytoplasm. Endothelium from white areas in endotoxin-treated animals was essentially similar regardless of the sampling site but did exhibit certain features common to all sites that were not observed in control animals. In particular, the endothelial cytoplasm of all white areas in endotoxin-treated dogs exhibited a much greater degree of granularity than was observed in control animals (Fig. 3), and in certain areas (Fig. 4) many larger, more prominent silver granules were visible along the cell borders. Additionally, the cell boundaries in endotoxin-treated animals were often extremely irregular, and frequently cell borders appeared compressed, such that they ran parallel to each other (Fig, 5). Although these features were common to all treated animals, the thoracic aorta of one dog was particularly affected and, additionally, revealed large globular elements overlying the endothelium (Fig. 6).
FIG
treated
3. H&&hen preparation of endothdium dog. Cytoplasm of endothdial ce1l.s shows
from the abdominal aorta a high degree of granularity
of an endotoxin(arrows). XlooO.
64
GERRITY
ET AL.
FIG. 4. Hautchen preparation of endothelium from a white area in the aortic arch of an endotoxin-treated dog. Large, prominent silver granules (arrows) are visible, usually associated with cell boundaries (B ). X1000.
FIG. 5. Hkiutchen preparation of endothelium from a white area in the pulmonary artery of an endotoxin-treated dog. Note irregularity of cellular outlines and compression (arrows) of cell borders (B ). Compare with Fig. 2. X700.
ENDOTOXIN-INDUCED
ENDOTHELIAL
INJURY
65
FIG. 6. HLutchen preparation of endothelium from a white area in the thoracic aorta of an endotoxin-treated dog. Severe distortion of cell boundaries (B) is evident, and large, prominent silver granules (G) can be seen. x450.
FIG. 7. Hautchen preparation of endothelium from a blue area in the pulmonary artery of an endotoxin-treated dog. Foci (F) of silver deposition follow outlines of cells but appear to involve several cells within one focus. Cell boundaries (B) are irregular in outline and compressed (arrows) as compared to control specimens (Fig. 2). x450.
66
GERRITY
ET
AL.
Hlutchcn preparations of cndothelium from blue areas of pulmonary artery and aortic arch of endotoxin-treated dogs showed the same features as those described for white areas. In addition, however, foci of intense silver-staining were observed within the larger confines of areas of Evans Blue accumulation. While these were most numerous in blue areas from the pulmonary artery (Fig. 7), they were also frequent in the aortic arch (Fig. 8) and less so in white areas of the thoracic aorta (Fig. 6). These foci of intense silver deposition were usually sharply delineated by cell boundaries (Fig. 8) and frequently involved many cells (Fig. 7). Dense globular structures of variable size and shape (Figs. 8 and 9) were usually associated with foci of intense silver deposition, and appeared to overlie the endothelium, but were not confined within the outlines of individual endothelial cells. DISCUSSION Our results demonstrate that endothelium from areas of enhanced permeability to protein, as demarcated by the uptake of Evans blue, is more susceptible to endotoxin-induced injury than endothelium from continguous areas of lesser permeability showing no dye uptake. Endothelial Hautchen preparations of blue areas from both the aorta and pulmonary artery revealed focal regions of intense silver staining that were frequently covered by globular elements of variable size and shape. The areas of intense silver staining, because of their sharp delineation by silver-stained cell boundaries, are considered to be regions
FIG. 8. Hlutchen preparation of endothelium from a blue area in the aortic arch of an endotoxin-treated dog. Foci (F) of silver deposition are confined within cellular boundaries ( B ) . Cytoplasm is granular ( arrows), and dense globules (G) of variable size and shape overlie the surface. X1000.
ENDOTOXIN-INDUGED
ENDOTHELIAL
INJURY
67
FIG. 9. Butchen preparation of endothelium from a blue area in the aortic arch of an endotoxin-treated dog. Densely staining globules (G) of variable size and shape overlie endothelium. Silver-stained cell boundaries (B) in this area are irregular and compressed. x 1000.
of endothehal damage and/or denudation involving severa cells in any given focus. The dense gIobular elements may represent platelets and Ieucocytes adherent to these foci of endotheIia1 injury. The above features were not observed in white areas from treated dogs, al~ough, in one dog, a region in the upper thoracic aorta did contain globular elements and appeared to be more severely damaged than other white areas. Blue areas in the aortic arch also showed significantly enhanced endothefial nuclear [3H]thymidine uptake relative to white areas from different sites in the aorta in treated animals and to blue and white areas from control animals. These results are consistent with the findings of Gerrity et al. (1975), which demonstrated a greater nuclear [“H] thymidine uptake in the aortic arch than in other aortic sites of the rabbit after endotoxin injection, and indicate that the enhanced cell turnover in the arch is most likely due to the greater susceptibility of blue areas to endotoxin-induced injury. In this respect, blue areas in the normal pig aorta (Caplan and Schwartz, 1973) and in subscorbutic guinea pigs both before and after cholesterol feeding and stress (Payling-Wright et al., 1975) have been shown to exhibit an increased [ SH]thymidine uptake compared with adjacent white areas. These findings, together with the present results, indicate that blue areas not only exhibit greater endothelial cell turnover under normal conditions but also are more susceptible to experimentally induced injury. The significantly enhanced nuclear [3H]thymidine uptake in bIue areas, which aIso exhibit foci of endothelial cellular denudation, suggests that circulating en-
68
GEHRITY
ET AL.
dothelial cells isolated from aortic blood samples are largely derived from blue areas. A similar conclusion may be drawn with respect to endothelial cells in the pulmonary circulation, although one cannot exclude the possibility that some of these may be derived from the endothelium of the microvasculature (Gaynor, 1971). The presence of circulating endothelial cells in all three sampling sites as early as 5 min after injection does indicate, however, that endotoxin induces an almost immediate denudation of endothelium. The greater numbers of circulating cells recovered from the pulmonary atery and right atrium could possibly reflect a greater susceptibility of endothelium in these areas to endotoxin, but, as the endotoxin was injected into the right atrium, the findings may alternatively be due to a greater concentration of endotoxin in the pulmonary circulation. The relatively rapid clearance of these cells from the circulation may reflect rapid lysis of denuded cells and a progressive sequestration in the reticuloendothelial system. On the basis of the above findings, it is interesting to speculate on the possibility that the presence of circulating endothelial cells may have some clinical relevance in the diagnosis and management of patients with endotoxemia. REFERENCES BELL, F. P., ADAMSON, I. L., and SCHWARTZ, C. J. (1974a). Aortic endothelial permeability to albumin: Focal and regional patterns of uptake and transmural distribution of ?-albumin in the young pig. Exp. Mol. Pathol. 20, 57-68. BELL, F. P., CALLUS, A., and SCHWARTZ, C. J. (197413). Aortic endothelial permeability to fibrinogen: Focal and regional patterns of uptake and transmural distribution of l”I-fibrinogen in the young pig. Exp. Mol. Pathol. 20, 281-292. BENNETT, I. L., and CLUFF, L. E. (1957). Bacterial pyrogens. Pharmacol. Reu. 9, 427-475. CAPLAN, B. A., and SCHWARTZ, C. J. (1973). Increased endothelial cell turnover in areas of in uioo Evans blue uptake in the pig aorta. Atherosclerosis 17, 401417. CAPLAN, B. A., GERRITY, R. G., and SCHWARTZ, C. J. (1974). Endothelial cell morphology in focal areas of in uiuo Evans blue uptake in the young pig aorta. Exp. Mol. PathoE. 21, 102-117. GAYNOR, E. (1971). Increased mitotic activity in rabbit endothelium after endotoxin. An autoradiographic study. Lab. Invest. 24, 318-320. GAYNOR, E. (1972). Vascular Iesions in endotoxemia. In “Advances in Experimental Medicine and Biology,” Vol. 23, “The Fundamental Mechanisms of Shock” (L. B. Hinshaw and B. G. Cox, eds.), pp. 337-345. Plenum, New York. GAYNOR, E., BOWER, C. A., and SPAET, T. H. (1968). C irculating endothelial cells in endotoxin-treated rabbits. Clin. Res. 16, 535 (abstract). GERRITY, R. G., RICHARDSON, M. A., and SCHWARTZ, C. J. (1975a). Endothelial cell morphology in focal areas of in vivo Evans blue uptake in the young pig aorta. III. Electron microscope observations. In preparation. GERRITY, R. G., CAPLAN, B. A., RICHARDSON, M., CADE, J. F., HIRSH, J., and SCHWARTZ, C. J. (197533). Endotoxin-induced endothelial injury and repair. 1. Endothelial cell turnover in the aorta of the rabbit. Exp. Mol. Pathol. 23, 379-385. GILBERT, R. P. (1960). Mechanisms of the hemodynamic effects of endotoxin. Physiol. Rev. 40, 245-279. HERBEIJVAL, H., and FOUROT, M. ( 1964). Etude comparative de l’endothelium vasculaire et de sa basale sur coupe et en Ieucoconcentration. Sot. Biol. Nancy 158, 137-141. MARTIN, D. S., CASSISI, N. J., and PICKENS, J. L. (1965). Endotoxin shock: A collective review. Reu. SUTg. 22, 311-319. M&ABE, W. R. ( 1974). Gram-negative bacteremia. In “Advances in Internal Medicine” (G. H. Stollerman, ea.), Vol. 19, pp. 135-158. Year Book Medical, Chicago.
ENDOTO~IN-INDUCED
ENDOT~ELIAL
INJURY
69
MCGRATH, J. M., and STEWART, G. J. (1969). The effects of endotoxin on vascular endothelium. 1. Exp. Med. 129, 833439. MCKAY, D. G., MARGARETTEN, W., and CSAVOSSY, I. (1966). An electron microscope study of the effects of bacterial endotoxin on the blood-vascular system. Lab. Invest. 15, 18151829. MCKAY, D. G., MARGARETTEN, W., and CSAVOSSY, I. ( 1967). The role of the leukocyte in the generalized Shwartzman reaction. cab:lever. 16, 511-515. PACKHAM, M. A., ROWSELX., H, C., J@GENSEN, L., and MUSTARD, J. F. (1967). Localized protein accumulation in the wall of the aorta. Exp. MOE. Pathol. 7, 214-232. PUGATCH, E. M. J., and SAUNDERS, A. M. (1968). A new technique for making Hgutchen preparations of unfixed aortic endothelium. J. Atheroscler. Res. 8, 735-738. SOMER, J. B., and Scnwan, C. J. ( 1971). Focal ‘H-cholesterol uptake in the pig aorta. Atherosclerosis 13, 293-304. STETSON, C. A. (1961). Vascular effects of endotoxins. Bull. N.Y. Acud. Med. 37, 48&492. STEWART, G. J,, and ANDERSON, M. J. (1971). An ultrastructural study of endotoxin-induced damage in rabbit mesenteric arteries. B&t. J. Exp. Pathos. 52, 75-80.
AND
MOLECULAR
Endotoxin-Induced
PATHOLOGY
Vascular
59-49 ( 1976)
24,
Endothelia~ Injury and Repair
II. Focal Injury, en Face Morphology, PHlThymidine Uptake and Circulating Endothelial Cells in the Dog 1
R. G, GERRITY,~ M. RICHARDSON,~ B. A. CAPLAN, J. HIRSH, AND C. J. SCHWARTZ Depaltmerats
J. F. CADE,
of Pathology and Medicine, Faculty of Health Sciences, McMaster and Chedoke ~o~~i~a~, P.O. Box 590, ~a.m~Zton, Ontario, Canada
University
Received June 2, 1975; and in revised form August 18, 1975 In this study we have examined the iniluence of intravenous Esc~erichia coli endotoxin on the induction of endothelial injury and repair in the aorta and pulmonary artery of the dog, with special reference to areas of Evans blue dye uptake. The sequential changes were monitored by quantitation of the numbers of circulating endothelial cells in three vascular beds, by incorporation of [3H] thymidine by endotheliai cell nuclei, and by light microscopy of silver ~~at~stained H&utchen preparations. Circulating endothelial cells were observed as early as 5 min after endotoxin injection, being most numerous in the pulmonary circulation. By 60 min, most circulating cefls had disappeared. In the aorta, uptake of ~Hl~ymidine by endothelial cell nuclei was maximal in the arch and, in particular, in those areas showing Evans blue dye accumulation. Light microscopy of silver-stained HPutchen preparations from blue areas of the aorta and pulmonary artery revealed foci of intense silver staining, which have been interpreted as indicating damage or denuded endothelium. Blue areas also exhibited darkly staining globular structures of variable size and shape apparently overlying the endothelial surface. Neither the foci of intense silver staining nor the globular structures were observed in specimens from adjoining areas of no dye uptake (white areas), which were essentially similar in appearance to control samples, except that in white areas from endotoxin-treated animals, endothelial cells were more granular, and silver-stained cell boundaries showed less separation. The results confirm the findings of an earlier study in which endotoxin-induced endothelial injury in the rabbit aorta was found to be most severe in the arch. The en face morphologic features of endotoxin-induced endothdial injury are shown to be largely confined to areas of enhanced endothelial permeability to protein as demarcated by in uivo Evans blue dye uptake.
INTRODUCTION In the pig and the dog, intravenous injection of the protein-binding azo dye, Evans blue, results in the focal uptake of dye by specific areas in the aortic arch and pulmonary artery. Such areas of dye uptake (blue areas> in the pig aortic arch have been we11 documented and are associated with enhanced uptake of 1 Supported by Medical Research Council of Canada grants, No. MA and MT 3067, and the Ontario Heart Foundation. 2 Ontario Heart Foundation Research Fellow. 59 Copyright All rights
1976 by Academic Press, Inc. oP reproduction in any form reserved.
60
GERRITY
ET AL.
radioiodinated albumin (Packham et al., 1967; Bell et al., 1974a), fibrinogen ( Bell et al., 1974b) and unesterified [“HIcholesterol (Somer and Schwartz, 1971). These areas also exhibit an increased rate of endothelial cell turnover (Caplan and Schwartz, 1973) and altered endothelial cell morphology as seen both in Haiutchen preparations (Caplan et al., 1974) and in the electron microscope (Gerrity et al., 1975a). Additionally, Gerrity et al. ( 197513) have demonstrated an increased endothelial cell turnover in the aortic arch of rabbits after intravenous endotoxin injection. Endotoxin has been shown to elicit multiple and diverse pathophysiological responses, and the subject has been exhaustively reviewed (Bennett and Cluff, 1957, Martin et al., 1965, McCabe, 1974). Although endotoxemia induces profound effects on the cardiovascular system (Gilbert, 1967) and blood elements (Stetson, 1961), relatively little attention has been given to the influence of endotoxin on the vascular endothelium. McKay et aZ. (1966, 1967) examined the ultrastructure of the microvascular endothelium in rats and concluded that endothelial injury was secondary to the ischemia produced by occlusive fibrin thrombi. They did not examine large arteries. Gaynor et al. (1968) and Gaynor (1971, 1972) demonstrated that endotoxemia in rabbits produced an increase in I13H]thymidine uptake by endothelium in both large and small vessels and also an increase in the numbers of circulating endothelial cells in aortic blood, changes that were not prevented by pretreatment with heparin. Endotoxin-induced endothelial damage in the mesenteric artery of rabbits has been demonstrated by McGrath and Stewart (1969) and Stewart and Anderson (1971) using both light and electron microscopy. In view of these findings, the current studies were undertaken to determine whether areas of enhanced permeability in the aortic arch and pulmonary artery, as demarcated by Evans blue uptake, are more susceptible to endotoxin-induced injury and to define the nature of endotoxin-induced endothelial injury in the large arteries as a baseline for further studies into the mechanisms involved. MATERIALS
AND METHODS
Six mature mongrel dogs, weighing 11-22 kgm, were anesthetized with sodium pentabarbital (Diabutal, Diamond Laboratories, Des Moines, Iowa), and indwelling cannulae were placed in the right atrium, aortic arch, and pulmonary artery. Three hours before killing, 50 ml of Evans blue, prepared as described previously (Caplan et al., 1974), were injected into the right atrium. One hour before death, Escherichia coli endotoxin (Difco), 10 mgm/ml, was injected into four dogs at a dosage of 2 mgm/kgm in pyrogen-free isotonic saline. Two control dogs were injected with an equivalent volume of saline. Blood samples for quantitation of the numbers of circulating endothelial cells were obtained through each of the indwelling cannulae (aortic arch, right atrium, and pulmonary artery) 10 and 20 min before the injection of endotoxin or saline, and subsequently at intervals of 5, 15, and 60 min after injection. Endothelial cells were concentrated according to the method of Herbeuval and Fourot (1964), and the results expressed as the number of circulating endothelial cells/1000 leucocytes. One hour after injection of endotoxin, the anesthetized animals were killed by exsanguination, and samples of pulmonary artery and aortic arch were excised
ENDOTOXIN-INDUCED
ENDOTHELIAL
INJURY
61
from areas with (blue areas) and without (white areas) Evans blue accumulation. Similar samples were excised for white areas in the thoracic and abdominal aorta. For light microscopy, samples having an approximate surface area of 1 cm2 were removed and used to make silver-stained HPutchen preparations, employing the method of Pugatch and Saunders ( 1968), as modified by Caplan and Schwartz ( 1973). Additional samples of aortic arch and thoracic and abdominal aorta were also incubated in [3H]thymidine, and autoradiographs were prepared from these by the technique described previously by Caplan and Schwartz ( 1973). A minimum of 1000 nuclei were counted from each sampling site in each of the six animals. For each sampling site, the numbers of labeled nuclei were expressed as a percentage of the total number of nuclei counted. Student’s t test was used to determine the statistical significance of differences between blue and white areas in both treated and control animals. RESULTS
Circulating
Endotiielial
Cells
Figure 1 depicts the numbers of circulating endotheIia1 cells observed in blood samples from the pu~ona~ artery (PA), right atrium (RA ), and aorta ( AO) of four dogs 10 min before the administration of endotoxin and at intervals of Circulating endothelial cells were not observed 5, 15, and 60 min after injection. prior to endotoxin injection. A maximum increase in the number of circulating endothelial cells was observed in each site at 5 min, the increase being greatest in the pulmonary artery blood and least in the aortic blood. Endothelial ceils were still present in the circulation at 15 min after injection, and by 60 min their numbers had declined further, with none detected in the aortic samples.
[ 3H] Thymidine
Uptake
The influence of endotoxin on incorporation of [311]thymidine by endothelial nuclei of the aortic arch and thoracic and abdominal aorta is shown in Table I.
.lO
;
10
20
30
40
50
60
ENDOTOXIN TIME IMINt
FIG.
1. The numbers of circuIating endothelial (PA), right atrium (RA), and aorta (AO) injection of endotoxin.
celis in blood sampIes at various times before
from and
puImonary artery after in~aveno~
CERRI’IY
G2
ET
TABLE
AL. I
PFt] Tl~~i~~i(li~~~ Ill~~)~j~o~~t,i(~n into Actrtk: ~~l~(~(~t,h~!li~~(Xl in t*he I jog aft.er
~ildotoxin
N~~doi
AdmiIlist,ratioI~
-Site of sample
Labelled
nuclei
Endotoxin-treated
(S)O cor1tro1
----
Aortic arch (blue) Aortic arch (white) Thoracic aorta (white) Abdominal aorta (white) a Values are t,he means 6 Significantly different animaLs (P < 0.05).
with from
0.29 0.03 0.09 0.02
(0.16)6 (0.03) (0.02) (0.04)
SD in parentheses; n = four animals. control blue and from white areas derived
0.03 0.00 0.03 0.01
(0.04) (0.00) (0.02)
from
treated
(0.01)
and control
In the aortic arch, areas of Evans bIue dye accumulation (blue) are compared with adjacent areas showing no dye accumulation (white) in treated and control animals. The effect of endotoxin was most prominent in the aortic arch, and in particular, in areas showing Evans blue accumulation, where increased endothelial cell nuclear labeling was significantly greater than in control blue areas and in white areas from both treated and control animals. There was no significant difference in the uptake of [ sH]thymidine into the endothelial nuclei of white areas from aortic arch and thoracic and abdominal aorta. Moreover, uptake in
FIG 2. Hautchen preparation of endothelium Endothelial cell boundaries (B) are stained, are elongate and fairly regular in outline, with x450.
from the thoracic aorta of a control dog. resulting in pavement-like appearance. Cells a faint granularity to the cytoplasm (arrows).
ENDOTOXIN-INDUCED
these segments did not differ significantly trol animals ( TabIe I).
ENDOTHELIAL
INJURY
from corresponding
63 segments
in con-
Light microscopic ex~ination of silver ni~ate-stail~ed Hautchen preparations from control and endotox~-treated animals was undertaken by using endothelium from blue and white areas in both the pulmonary artery and the aortic arch and from white areas in the thoracic and abdominal aorta. In no experiment did endotoxin visibly modify the pattern or intensity of Evans blue uptake. Endothelium from control animals (Fig. 2) showed the morphology typical of Htiutchen preparations, with silver-stained cell boundaries and a faint granularity to the cytoplasm. Endothelium from white areas in endotoxin-treated animals was essentially similar regardless of the sampling site but did exhibit certain features common to all sites that were not observed in control animals. In particular, the endothelial cytoplasm of all white areas in endotoxin-treated dogs exhibited a much greater degree of granularity than was observed in control animals (Fig. 3), and in certain areas (Fig. 4) many larger, more prominent silver granules were visible along the cell borders. Additionally, the cell boundaries in endotoxin-treated animals were often extremely irregular, and frequently cell borders appeared compressed, such that they ran parallel to each other (Fig, 5). Although these features were common to all treated animals, the thoracic aorta of one dog was particularly affected and, additionally, revealed large globular elements overlying the endothelium (Fig. 6).
FIG
treated
3. H&&hen preparation of endothdium dog. Cytoplasm of endothdial ce1l.s shows
from the abdominal aorta a high degree of granularity
of an endotoxin(arrows). XlooO.
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FIG. 4. Hautchen preparation of endothelium from a white area in the aortic arch of an endotoxin-treated dog. Large, prominent silver granules (arrows) are visible, usually associated with cell boundaries (B ). X1000.
FIG. 5. Hkiutchen preparation of endothelium from a white area in the pulmonary artery of an endotoxin-treated dog. Note irregularity of cellular outlines and compression (arrows) of cell borders (B ). Compare with Fig. 2. X700.
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FIG. 6. HLutchen preparation of endothelium from a white area in the thoracic aorta of an endotoxin-treated dog. Severe distortion of cell boundaries (B) is evident, and large, prominent silver granules (G) can be seen. x450.
FIG. 7. Hautchen preparation of endothelium from a blue area in the pulmonary artery of an endotoxin-treated dog. Foci (F) of silver deposition follow outlines of cells but appear to involve several cells within one focus. Cell boundaries (B) are irregular in outline and compressed (arrows) as compared to control specimens (Fig. 2). x450.
66
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AL.
Hlutchcn preparations of cndothelium from blue areas of pulmonary artery and aortic arch of endotoxin-treated dogs showed the same features as those described for white areas. In addition, however, foci of intense silver-staining were observed within the larger confines of areas of Evans Blue accumulation. While these were most numerous in blue areas from the pulmonary artery (Fig. 7), they were also frequent in the aortic arch (Fig. 8) and less so in white areas of the thoracic aorta (Fig. 6). These foci of intense silver deposition were usually sharply delineated by cell boundaries (Fig. 8) and frequently involved many cells (Fig. 7). Dense globular structures of variable size and shape (Figs. 8 and 9) were usually associated with foci of intense silver deposition, and appeared to overlie the endothelium, but were not confined within the outlines of individual endothelial cells. DISCUSSION Our results demonstrate that endothelium from areas of enhanced permeability to protein, as demarcated by the uptake of Evans blue, is more susceptible to endotoxin-induced injury than endothelium from continguous areas of lesser permeability showing no dye uptake. Endothelial Hautchen preparations of blue areas from both the aorta and pulmonary artery revealed focal regions of intense silver staining that were frequently covered by globular elements of variable size and shape. The areas of intense silver staining, because of their sharp delineation by silver-stained cell boundaries, are considered to be regions
FIG. 8. Hlutchen preparation of endothelium from a blue area in the aortic arch of an endotoxin-treated dog. Foci (F) of silver deposition are confined within cellular boundaries ( B ) . Cytoplasm is granular ( arrows), and dense globules (G) of variable size and shape overlie the surface. X1000.
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FIG. 9. Butchen preparation of endothelium from a blue area in the aortic arch of an endotoxin-treated dog. Densely staining globules (G) of variable size and shape overlie endothelium. Silver-stained cell boundaries (B) in this area are irregular and compressed. x 1000.
of endothehal damage and/or denudation involving severa cells in any given focus. The dense gIobular elements may represent platelets and Ieucocytes adherent to these foci of endotheIia1 injury. The above features were not observed in white areas from treated dogs, al~ough, in one dog, a region in the upper thoracic aorta did contain globular elements and appeared to be more severely damaged than other white areas. Blue areas in the aortic arch also showed significantly enhanced endothefial nuclear [3H]thymidine uptake relative to white areas from different sites in the aorta in treated animals and to blue and white areas from control animals. These results are consistent with the findings of Gerrity et al. (1975), which demonstrated a greater nuclear [“H] thymidine uptake in the aortic arch than in other aortic sites of the rabbit after endotoxin injection, and indicate that the enhanced cell turnover in the arch is most likely due to the greater susceptibility of blue areas to endotoxin-induced injury. In this respect, blue areas in the normal pig aorta (Caplan and Schwartz, 1973) and in subscorbutic guinea pigs both before and after cholesterol feeding and stress (Payling-Wright et al., 1975) have been shown to exhibit an increased [ SH]thymidine uptake compared with adjacent white areas. These findings, together with the present results, indicate that blue areas not only exhibit greater endothelial cell turnover under normal conditions but also are more susceptible to experimentally induced injury. The significantly enhanced nuclear [3H]thymidine uptake in bIue areas, which aIso exhibit foci of endothelial cellular denudation, suggests that circulating en-
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dothelial cells isolated from aortic blood samples are largely derived from blue areas. A similar conclusion may be drawn with respect to endothelial cells in the pulmonary circulation, although one cannot exclude the possibility that some of these may be derived from the endothelium of the microvasculature (Gaynor, 1971). The presence of circulating endothelial cells in all three sampling sites as early as 5 min after injection does indicate, however, that endotoxin induces an almost immediate denudation of endothelium. The greater numbers of circulating cells recovered from the pulmonary atery and right atrium could possibly reflect a greater susceptibility of endothelium in these areas to endotoxin, but, as the endotoxin was injected into the right atrium, the findings may alternatively be due to a greater concentration of endotoxin in the pulmonary circulation. The relatively rapid clearance of these cells from the circulation may reflect rapid lysis of denuded cells and a progressive sequestration in the reticuloendothelial system. On the basis of the above findings, it is interesting to speculate on the possibility that the presence of circulating endothelial cells may have some clinical relevance in the diagnosis and management of patients with endotoxemia. REFERENCES BELL, F. P., ADAMSON, I. L., and SCHWARTZ, C. J. (1974a). Aortic endothelial permeability to albumin: Focal and regional patterns of uptake and transmural distribution of ?-albumin in the young pig. Exp. Mol. Pathol. 20, 57-68. BELL, F. P., CALLUS, A., and SCHWARTZ, C. J. (197413). Aortic endothelial permeability to fibrinogen: Focal and regional patterns of uptake and transmural distribution of l”I-fibrinogen in the young pig. Exp. Mol. Pathol. 20, 281-292. BENNETT, I. L., and CLUFF, L. E. (1957). Bacterial pyrogens. Pharmacol. Reu. 9, 427-475. CAPLAN, B. A., and SCHWARTZ, C. J. (1973). Increased endothelial cell turnover in areas of in uioo Evans blue uptake in the pig aorta. Atherosclerosis 17, 401417. CAPLAN, B. A., GERRITY, R. G., and SCHWARTZ, C. J. (1974). Endothelial cell morphology in focal areas of in uiuo Evans blue uptake in the young pig aorta. Exp. Mol. PathoE. 21, 102-117. GAYNOR, E. (1971). Increased mitotic activity in rabbit endothelium after endotoxin. An autoradiographic study. Lab. Invest. 24, 318-320. GAYNOR, E. (1972). Vascular Iesions in endotoxemia. In “Advances in Experimental Medicine and Biology,” Vol. 23, “The Fundamental Mechanisms of Shock” (L. B. Hinshaw and B. G. Cox, eds.), pp. 337-345. Plenum, New York. GAYNOR, E., BOWER, C. A., and SPAET, T. H. (1968). C irculating endothelial cells in endotoxin-treated rabbits. Clin. Res. 16, 535 (abstract). GERRITY, R. G., RICHARDSON, M. A., and SCHWARTZ, C. J. (1975a). Endothelial cell morphology in focal areas of in vivo Evans blue uptake in the young pig aorta. III. Electron microscope observations. In preparation. GERRITY, R. G., CAPLAN, B. A., RICHARDSON, M., CADE, J. F., HIRSH, J., and SCHWARTZ, C. J. (197533). Endotoxin-induced endothelial injury and repair. 1. Endothelial cell turnover in the aorta of the rabbit. Exp. Mol. Pathol. 23, 379-385. GILBERT, R. P. (1960). Mechanisms of the hemodynamic effects of endotoxin. Physiol. Rev. 40, 245-279. HERBEIJVAL, H., and FOUROT, M. ( 1964). Etude comparative de l’endothelium vasculaire et de sa basale sur coupe et en Ieucoconcentration. Sot. Biol. Nancy 158, 137-141. MARTIN, D. S., CASSISI, N. J., and PICKENS, J. L. (1965). Endotoxin shock: A collective review. Reu. SUTg. 22, 311-319. M&ABE, W. R. ( 1974). Gram-negative bacteremia. In “Advances in Internal Medicine” (G. H. Stollerman, ea.), Vol. 19, pp. 135-158. Year Book Medical, Chicago.
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MCGRATH, J. M., and STEWART, G. J. (1969). The effects of endotoxin on vascular endothelium. 1. Exp. Med. 129, 833439. MCKAY, D. G., MARGARETTEN, W., and CSAVOSSY, I. (1966). An electron microscope study of the effects of bacterial endotoxin on the blood-vascular system. Lab. Invest. 15, 18151829. MCKAY, D. G., MARGARETTEN, W., and CSAVOSSY, I. ( 1967). The role of the leukocyte in the generalized Shwartzman reaction. cab:lever. 16, 511-515. PACKHAM, M. A., ROWSELX., H, C., J@GENSEN, L., and MUSTARD, J. F. (1967). Localized protein accumulation in the wall of the aorta. Exp. MOE. Pathol. 7, 214-232. PUGATCH, E. M. J., and SAUNDERS, A. M. (1968). A new technique for making Hgutchen preparations of unfixed aortic endothelium. J. Atheroscler. Res. 8, 735-738. SOMER, J. B., and Scnwan, C. J. ( 1971). Focal ‘H-cholesterol uptake in the pig aorta. Atherosclerosis 13, 293-304. STETSON, C. A. (1961). Vascular effects of endotoxins. Bull. N.Y. Acud. Med. 37, 48&492. STEWART, G. J,, and ANDERSON, M. J. (1971). An ultrastructural study of endotoxin-induced damage in rabbit mesenteric arteries. B&t. J. Exp. Pathos. 52, 75-80.