- Email: [email protected]
Necklace-like detachment of endothelial cell layer from arterial wall under low-calcium condition
Exp Toxic Pathol 1994; 46: 307-313 Gustav Fischer Verlag Jena
Department of Pathology, School of Medicine, Keio University, Japan Tokyo Electric Power HospitaP)
Necklace-like detachment of endothelial cell layer from arterial wall under low-calcium condition HISAO YAMAGUCHI, MASARU MORISADA'), HIROKUNI KAKU, TAKA-AKI ONODERA and Ryu KUROKAWA With 5 figures Received: September 28, 1993; Revised: January 4, 1994; Accepted: February 1, 1994 Address for correspondence: Prof Dr. H. YAMAGUCHI, 35 Shinano-machi, Shinjuku-ku, Tokyo 160, Japan Key words: Endothelial cell; Arterial wall, endothelium; Calcium, low condition; Detachment, arterial endothelium; Anchoring villi
Summary
Introduction
The diversified morphological manifestations in various tissues and organs obtained by administration of differing amounts of calcium chelating agents were reported in previous papers (YAMAGUCHI et al. 1981 a & b; 1982; 1990; 1993). In our recent research described here, administration of a moderate dose of Na2EDTA over the short term demonstrated necklace-like detachment from the arterial wall without disruption of the endothelial cell chain. Intercellular spaces in the media just beneath the detached endothelial cell layer was stained strongly with colloidal iron staining. Electron microscopic observation revealed that the detached endothelial cells showed a lot of elongated anchoring villi from the basal surface, usually seen at the luminal surface, adhered to the degenerative and thin flattened internal elastic lamellae. The alteration of the colloidal iron staining of the vascular wall under the low-calcium condition is suggested to be induced by loosening of the molecular structure of glycosaminoglycans (GAGs) as well as glycoproteins (GPs), comprising the important component ofthe intercellular matrix and elastic lamellae, which would induce a change in their pasty or viscous character. This would be an accelerative factor for detachment of endothelial cells. Moreover, the lack of the waving of the internal elastic lamellae, trapping of endothelial cytoplasmic processes among them, would play the decisive role in the total detachment of the endothelial cell layer. On the other hand, the low-calcium condition did not adversely influence the joining of endothelial cells. The pathognomatic mechanism will be discussed, with a comparison made to the angiolytic changes provoked by a large amount of Na2EDTA.
Desquamation or detachment of individual endothelial cells is usually seen as a physiologic turnover. In inflammation or under some conditions, such phenomena as subendothelial edema, vacuolarization of the individual endothelial cells, pseudopodia formation, the lift up phenomenon followed by desquamation (Y AMAGUCHI et al. 1972 and 1973) were increased in frequency. However, these kinds of incidences were demonstrated on an individual cell basis. Also noted in previous studies in which various amounts and types of calcium chelating agents were administered were the diversified morphological manifestations such as angiolytic changes, aneurysmal formation and severe constriction of the arteries resulting in pulmonary hypertension (YAMAGUCHI al. 1981 a & b; 1993 a & b). In our recent research presented herein, necklace-like detachment of the endothelial cell layer in mesenterial arteries was demonstrated with repeated administration of a moderate dose of Na2EDT A over the short term. In this paper, the pathognomatic mechanism of this kind of total detachment of the endothelial cell layer without disruption and the underlying relation between detachment of the endothelial cell layer and the morpho-functional attitude of vascular media will be discussed.
Material and methods Ten guinea pigs of both sexes weighing 300 - 350 gm were used in this experiment. Two ml of 6 % Na2EDT A were administered intraperitoneally for 5 days once a day repeatedly, with the animals being sacrificed at day 6. Mesenteria were removed after sacrifice. Light microscopic specimens of mesenterium were prepared following routine techniques and were stained with H&E, colloidal iron and elastica Van-Gieson staining. Exp Toxic Pathol46 (1994) 4-5
307
, r
,"
-'"
4
~
'.
.,
or-
~
"
I'
1a'" ,
.~:;-~,
.
.1, I~
.' '".
',:
-
•
~ ~,. ~ '
.,
..
.'
,.
.,""''' - ,
. I
" I
.. .. " ~
\.
, I
·1
.~
~
'. ~
,I , ,
. I
I
.
"
,
.
,
'
/
.
;
. ("
;
.
- ~
To identify the mesenterial vasculatures for electron microscopic observation, the chopped mesenterial tissues were prepared following a routine procedure, with the cross-sectioning of resin embedded specimens across the mesenterial vasculatures being subsequently fixed to the resin-made holder by Alon Alpha. This kind of resin-embedded specimen was treated following routine procedure. Tannic acid treatment was employed to facilitate examination of the internal elastic lamellae. 308
Exp Toxic Pathol46 (1994) 4-5
,. ~
.
Fig. la. Mesenteric artery of a control guinea pig. X1S0, H&E Fig. lb. Colloidal iron staining of the above case. No staining in the control artery. X1S0, colloidal iron staining.
Results Administration of different amounts and types of Na2EDTA dearly demonstrated the diversified morphological manifestations of the vasculature in a range of tissues and organs such as angiolytic changes, aneurysmal variations and severe constriction resulting in pulmonary hypertension (YAMAGUCHI et al. 1981 a & b; 1983 a & b).
..
I
.
,-
,.
--
~
.
J
I
..
-'" " , - - -.
2·a
•
• , I
.~ I
"
Fig. 2a. Almost all of the endothelial cells are detached from the vascular wall. The smooth muscle cells change their shape into a round or oval configuration. X150, H&E.
, II
"
. ,
.
,
,, '
Fig. 2b. Colloidal iron stained strongly in this stage. In fact, however, colloidal iron is not stained on the site where the endothelial cells are partially adhered to the wall. X150, colloidal iron staining.
In our recent research described here, 2 ml of 6 % Na2EDTA was injected intraperitoneally once a day for 5 days. No tetanic shock leading to the death of the animals was observed with this administration. In the controls, however, no detachment of endothelial cells (fig. la) and no stainability by colloidal iron dye in vacular media was noted (fig. Ib). Light microscopic studies of the mesenterial arteries of the experimental group (fig. 2a) revealed that with slight
"
" ,
\1
,...
., .
.<
) 2b ~
, i
f
I
I
•
arterial constriction, which is expected to occur under low-calcium condition, the smooth muscle cells in the arterial media changed their shape into a round or oval configuration and the smooth muscle cell layers became slightly disarrayed. The intercellular tissues surrounding them became expansive and distinct. These morphological manifestations are less than that of angiolytic cases induced by administration of a large amount of Na2EDTA. The total detachment of the endothelial cell Exp Toxic Pathol46 (1994) 4-5
309
'. •
...
.
•
. _"
..
' ( ~, '
0:
layer from the arterial wall was noted which began to be partially lifted up and dispersed to complete the total desquamation of the endothelial cell layer. Furthermore, no disruption of the endothelial cell chain resembling a necklace was noted. The detached endothelial cell chains were seen to be floating in the vascular lumina (fig. 3). No degenerative changes in these endothelial cells were noted. Thus the low-calcium condition only slightly influences the endothelial junction. The vascular media beneath the detached endothelial cell was stained strongly positive by colloidal iron dye (fig. 2b). The intensity of stainability became stronger as time passed. Electron microscopic observation revealed that the detached endothelial cells showed no degenerative changes (figs. 4 & 5), but featured elongated anchoring villi from the cell bottom into (fig. 4) or sometimes away from the anchoring site (fig. 5) where the internal elastic lamellae showed wide and frequent disruption with slight degenerative changes. The internal elastic lamellae were slightly slender and flattened, covered with flocculated materials around them through which anchoring villi attached to the elastic lamellae. The smooth muscle cells in the vascular media changed their shape into the round or oval configuration and sometimes arranged themselves perpendicularly (fig. 5). Notably, they exhibited some degenerative changes such as vacuolar formation and concentrated cytoplasm. Intercellular spaces became slightly expanded. The flocculated materials around the elastic lamellae and intercellular matrix were assumed to be the similar components such as GAGs and GPs or a mixture of them. 310
Exp Toxic Pathol46 (1994) 4-5
Fig. 3. The detached endothelial cell chain without disruption is floating in the vascular lumen which takes on a necklace-like appearance. X150, H&E.
The detachment of endothelial cells from the vascular wall began to be noticeable as the subendothelial edema with elongated anchoring processes (figs. 4 & 5) proceeding up to the total detachment (fig 5). Subsequently, the detached lumen became a balloon-like expansion, and the anchoring villi were elongated (fig. 5). No platelet accumulation was demonstrated. Through gaps between disrupted internal elastic lamellae, the medial smooth muscles elongated their cytoplasmic processes up to the subendothelial spaces (fig. 4).
Discussion Necklace-like detachment of the endothelial cell layer without disruption was demonstrated with repeated administration of 2 ml of 6 % Na2EDTA intraperitoneally for 5 days. With administration of this dose of Na2EDTA, the animals showed unrest without tetanic death. As is already known, the endothelial cells are desquamated individually from the vascular wall, a phenomenon referred to as physiologic turnover. In fact, however, the necklace-like detachment of the endothelial cells seen in our experimental result has never been previously reported. Even though total detachment of endothelial cells from the vascular wall occurred, no disruption of the endothelial chain was seen. This means that the induced low-calcium condition does not adversely affect the joining of endothelial cells but that it does very severely influence the adherence of endothelial cells to the vascular wall.
Fig. 4. Extended and frequent gap fonnation of internal elastic lamellae (iel) and slight expansion of intercellular spaces (*) in the vascular media are demonstrated. The anchoring villi of endothelial cells are elongated (black arrows). Anchoring viIi are usually attached to the flocculated materials around the internal elastic lamellae. Concerning this adhesion, a crucial role is played by type IV collagen, fibronectin and laminin (COUCHMAN et al. 1983; GRINNEL et al. 1977 & 1979; HOOK et al. 1977; ROUOSHLATI et al. 1979; VLODASKY et al. 1980) whose important component is glycoprotein (GPs) as well as glycosaminoglycans (GAGs). However, the detailed mechanism underlying this adhesion remains to be clarified. In this report, constriction of the vascular walls should inevitably have occurred, of course, with the administration of Na2EDTA. Accompanying this, the medial smooth muscle cells assumed a round or oval shape and a perpendicular arrangement. Expansion of the intercellular spaces was slight and the internal elastic lamellae were fragmented and were flattened by elongation, covered with surrounding flocculated materials. Anchoring villi fonned with elongated endothelial cytoplasmic processes were attached to the flocculated materials and they would be extremely extended and then elongated follo-
wing detachment of the endothelial cell layer from the vascular wall. However, as has already been mentioned in the previous papers (YAMAGUCHI et al. 1981 a & b), with the administration of a large amount of N a2EDT A, angiolytic changes in the arterial wall were induced by severe expansion of the intercellular spaces as well as the severe waving of the internal elastic lamellae pulling the endothelial cytoplasmic processes deeper into the vascular wall where these waved elastic lamellae and enveloped elongated endothelial cytoplasmic processes were packed tightly from the outside by constricted smooth muscle cells. In these cases, total detachment was not demonstrated even though the severe expansion of the intercellular spaces was provoked. As mentioned previously, since the common theoretical mechanism underlying the colloidal iron staining (PEARSE 1985) involves GPs as well as GAGs having a considerable abundance of anionic charges in their moleExp Toxic Pathol46 (1994) 4--5
311
Fig. S. The smooth muscle cells (sm) are arranged perpendicularly. Total detachment of the endothelial cells (end) with elongated anchoring villi (black arrows) is observed. Internal elastic lamellae (iel) are somewhat degenerative in appearance.
cules, staining results with a fixing cationic dye being responsive to these anionic charges. The intensity of staining is dependent not only on the amount of these substances but also on how many anionic charges would be unsaturated by positively charged materials. Therefore, the staining intensity is sometimes independent of the quantitative existence of these substances but is related to the number of open anionic charges. When these substances were stained strongly, their molecular structure became loosened and elongated. As far as the colloid chemical property is concerned, they change their character 312
Exp Toxic Patho146 (1994) 4-5
into sol indicating that their viscosity would be decreased and their sticky character reduced. The vascular media of these cases showed intensively positive with colloid iron staining. As far as the staining attitude of colloidal iron dye, the positivity, based on the sol-diversion of GAGs and GPs which is induced by the existence of a lot of negative charges in their molecules, is commonly observed under the low-calcium condition. This kind of changes in the matrix would have occurred not only in the intercellular matrix but also in the GAGs and GPs composed elements throughout the body.
In cases of angiolysis, provoked by the administration of a large dose of Na2EDT A, in spite of the less sticky character of these materials to be expected, total detachment of the endothelial cell layer was not demonstrated. On the contrary, with administration of repeated but moderate doses of Na2EDTA, necklace-like detachment of the endothelial cell layer was observed. Considering the phenomenon from the morphological differences between the above two kinds of low-calcium conditions, all of the phenomena are basically the same but the severity of several phenomena induce the differences. Most of them constitute the severity of the waving of the internal elastic lamellae by constriction which would induce the trapping of the endothelial cell layer to the vascular wall. The alteration of colloid chemical properties in the matrical materials as well as the degenerative changes in the internal elastic lamellae are suggested to be more or less the same in the above two cases, considering the staining attitude of colloid iron dye and also electron microscopic observations. Trapping of endothelial cytoplasm with severely waved internal elastic lamellae on the one hand and attachment to them via the anchoring villi on the other hand would result in either attachment or detachment of the endothelial cell layer. Considering the necklace-like detachment of the endothelial cell layer, the alteration of colloid chemical properties of the matrix and also the degenerative changes occurring in the internal elastic lamellae would be inevitable. In addition to these causes, however, the lack of waving of the internal elastic lamellae induced by the severe constriction which provoked the trapping of endothelial cytoplasmic processes should play the important role.
References 1. COUCHMAN JR, HOOK M, REES DA, et al.: Adhesion,
growth and matrix production by fibroblasts on laminin substrates. J Cell Bioi 1983; 96: 177-183. 2. GRINNELL F, HAYS DG, MINTER D: Cell adhesion and spreading factor - partial purification and properties. Exp Cell Res 1977; 110: 175-190.
3. GRINNELL F, FELD MK: Intial adhesion of human fibroblasts in serum free medium; possible role of secreted fibronectin. Cell 1979; 17: 117-129. 4. HOOK M, RUBIN K, OLDBERG A, et al.: Cold insoluble globulin mediates the adhesion of rat liver cells to plastic petri dishes. Biochem Biophys Res commun 1977, 79: 726-733. 5. PEARSE AGE: Histopathology, Theoretical and Applied. Churchill Livingstone, 1985. 6. RUOSLAHTI E and HAYMAN EG: Two active site with different characteristics in fibronectin FEBS Letters. 1979;97:221-224. 7. VLODAVSKY I and GOSPONDAROWICZ D: Morphological appearance, growth behavior and migratory activity of human tumor cells maintained in extracellular matrix versus plastic. Cell 1980; 19: 607-616. 8. YAMAGUCHI H, NAKAJIMA S, TORIKATA C, et al.: Studies on the morphological changes of artery caused by exudation - Initial changes of arteritis. Acta Path Jap 1972;22:441-455. 9. YAMAGUCHI H, TORIKATA C, TAKEUCHI H, et al.: Studies on the morphological changes of the endothelium in lung with administration of soluble immune complexes. Acta Path Jap 1973; 23: 51-58. 10. YAMAGUCHI H, USUI Hand TAJIMA T: Multiple angitis of mesenterial arteries induced by intraperitoneal administration of vast amount of Na2EDTA - light microscopic study. Exp Patholl98la, 20: 26-30. 11. YAMAGUCHI H, USUI H, TAJIMA T, et al.: Multiple angitis of mesenterial arteries induced by intraperitoneal administration of vast amount of Na2EDT A - electron microscopic study. Exp Pathol198lb; 20: 31-40. 12. YAMAGUCHI H, SAKAGUCHI Hand MORISADA M: Renal lesions in hypocalcemic condition - phenotypic manifestation of genetical background and its renal expresion. Exp Patho11990; 41: 10-20. l3. YAMAGUCHI H, KAKU Hand MORIS ADA M: Experimental pulmonary hypertension induced by constriction of pulmonary arteries, resulted in rupture or extraordinary dilatation of right ventricle with administration of a little amount of Na2EDTA. Exp Toxic Pathol 1993a; 45: 21-27. 14. YAMAGUCHI H, KAKU Hand MORIS ADA M: Study of morphological manifestations of pulmonary arteries in experimental pulmonary hypertension provoked by administration of long-term, low-dose Na2EDTA - especially on aneurysmal changes and plexiform-like lesions. Exp Toxic Pathol1993b; 45: 329-335.
Exp Toxic Pathol46 (1994) 4--5
313
Department of Pathology, School of Medicine, Keio University, Japan Tokyo Electric Power HospitaP)
Necklace-like detachment of endothelial cell layer from arterial wall under low-calcium condition HISAO YAMAGUCHI, MASARU MORISADA'), HIROKUNI KAKU, TAKA-AKI ONODERA and Ryu KUROKAWA With 5 figures Received: September 28, 1993; Revised: January 4, 1994; Accepted: February 1, 1994 Address for correspondence: Prof Dr. H. YAMAGUCHI, 35 Shinano-machi, Shinjuku-ku, Tokyo 160, Japan Key words: Endothelial cell; Arterial wall, endothelium; Calcium, low condition; Detachment, arterial endothelium; Anchoring villi
Summary
Introduction
The diversified morphological manifestations in various tissues and organs obtained by administration of differing amounts of calcium chelating agents were reported in previous papers (YAMAGUCHI et al. 1981 a & b; 1982; 1990; 1993). In our recent research described here, administration of a moderate dose of Na2EDTA over the short term demonstrated necklace-like detachment from the arterial wall without disruption of the endothelial cell chain. Intercellular spaces in the media just beneath the detached endothelial cell layer was stained strongly with colloidal iron staining. Electron microscopic observation revealed that the detached endothelial cells showed a lot of elongated anchoring villi from the basal surface, usually seen at the luminal surface, adhered to the degenerative and thin flattened internal elastic lamellae. The alteration of the colloidal iron staining of the vascular wall under the low-calcium condition is suggested to be induced by loosening of the molecular structure of glycosaminoglycans (GAGs) as well as glycoproteins (GPs), comprising the important component ofthe intercellular matrix and elastic lamellae, which would induce a change in their pasty or viscous character. This would be an accelerative factor for detachment of endothelial cells. Moreover, the lack of the waving of the internal elastic lamellae, trapping of endothelial cytoplasmic processes among them, would play the decisive role in the total detachment of the endothelial cell layer. On the other hand, the low-calcium condition did not adversely influence the joining of endothelial cells. The pathognomatic mechanism will be discussed, with a comparison made to the angiolytic changes provoked by a large amount of Na2EDTA.
Desquamation or detachment of individual endothelial cells is usually seen as a physiologic turnover. In inflammation or under some conditions, such phenomena as subendothelial edema, vacuolarization of the individual endothelial cells, pseudopodia formation, the lift up phenomenon followed by desquamation (Y AMAGUCHI et al. 1972 and 1973) were increased in frequency. However, these kinds of incidences were demonstrated on an individual cell basis. Also noted in previous studies in which various amounts and types of calcium chelating agents were administered were the diversified morphological manifestations such as angiolytic changes, aneurysmal formation and severe constriction of the arteries resulting in pulmonary hypertension (YAMAGUCHI al. 1981 a & b; 1993 a & b). In our recent research presented herein, necklace-like detachment of the endothelial cell layer in mesenterial arteries was demonstrated with repeated administration of a moderate dose of Na2EDT A over the short term. In this paper, the pathognomatic mechanism of this kind of total detachment of the endothelial cell layer without disruption and the underlying relation between detachment of the endothelial cell layer and the morpho-functional attitude of vascular media will be discussed.
Material and methods Ten guinea pigs of both sexes weighing 300 - 350 gm were used in this experiment. Two ml of 6 % Na2EDT A were administered intraperitoneally for 5 days once a day repeatedly, with the animals being sacrificed at day 6. Mesenteria were removed after sacrifice. Light microscopic specimens of mesenterium were prepared following routine techniques and were stained with H&E, colloidal iron and elastica Van-Gieson staining. Exp Toxic Pathol46 (1994) 4-5
307
, r
,"
-'"
4
~
'.
.,
or-
~
"
I'
1a'" ,
.~:;-~,
.
.1, I~
.' '".
',:
-
•
~ ~,. ~ '
.,
..
.'
,.
.,""''' - ,
. I
" I
.. .. " ~
\.
, I
·1
.~
~
'. ~
,I , ,
. I
I
.
"
,
.
,
'
/
.
;
. ("
;
.
- ~
To identify the mesenterial vasculatures for electron microscopic observation, the chopped mesenterial tissues were prepared following a routine procedure, with the cross-sectioning of resin embedded specimens across the mesenterial vasculatures being subsequently fixed to the resin-made holder by Alon Alpha. This kind of resin-embedded specimen was treated following routine procedure. Tannic acid treatment was employed to facilitate examination of the internal elastic lamellae. 308
Exp Toxic Pathol46 (1994) 4-5
,. ~
.
Fig. la. Mesenteric artery of a control guinea pig. X1S0, H&E Fig. lb. Colloidal iron staining of the above case. No staining in the control artery. X1S0, colloidal iron staining.
Results Administration of different amounts and types of Na2EDTA dearly demonstrated the diversified morphological manifestations of the vasculature in a range of tissues and organs such as angiolytic changes, aneurysmal variations and severe constriction resulting in pulmonary hypertension (YAMAGUCHI et al. 1981 a & b; 1983 a & b).
..
I
.
,-
,.
--
~
.
J
I
..
-'" " , - - -.
2·a
•
• , I
.~ I
"
Fig. 2a. Almost all of the endothelial cells are detached from the vascular wall. The smooth muscle cells change their shape into a round or oval configuration. X150, H&E.
, II
"
. ,
.
,
,, '
Fig. 2b. Colloidal iron stained strongly in this stage. In fact, however, colloidal iron is not stained on the site where the endothelial cells are partially adhered to the wall. X150, colloidal iron staining.
In our recent research described here, 2 ml of 6 % Na2EDTA was injected intraperitoneally once a day for 5 days. No tetanic shock leading to the death of the animals was observed with this administration. In the controls, however, no detachment of endothelial cells (fig. la) and no stainability by colloidal iron dye in vacular media was noted (fig. Ib). Light microscopic studies of the mesenterial arteries of the experimental group (fig. 2a) revealed that with slight
"
" ,
\1
,...
., .
.<
) 2b ~
, i
f
I
I
•
arterial constriction, which is expected to occur under low-calcium condition, the smooth muscle cells in the arterial media changed their shape into a round or oval configuration and the smooth muscle cell layers became slightly disarrayed. The intercellular tissues surrounding them became expansive and distinct. These morphological manifestations are less than that of angiolytic cases induced by administration of a large amount of Na2EDTA. The total detachment of the endothelial cell Exp Toxic Pathol46 (1994) 4-5
309
'. •
...
.
•
. _"
..
' ( ~, '
0:
layer from the arterial wall was noted which began to be partially lifted up and dispersed to complete the total desquamation of the endothelial cell layer. Furthermore, no disruption of the endothelial cell chain resembling a necklace was noted. The detached endothelial cell chains were seen to be floating in the vascular lumina (fig. 3). No degenerative changes in these endothelial cells were noted. Thus the low-calcium condition only slightly influences the endothelial junction. The vascular media beneath the detached endothelial cell was stained strongly positive by colloidal iron dye (fig. 2b). The intensity of stainability became stronger as time passed. Electron microscopic observation revealed that the detached endothelial cells showed no degenerative changes (figs. 4 & 5), but featured elongated anchoring villi from the cell bottom into (fig. 4) or sometimes away from the anchoring site (fig. 5) where the internal elastic lamellae showed wide and frequent disruption with slight degenerative changes. The internal elastic lamellae were slightly slender and flattened, covered with flocculated materials around them through which anchoring villi attached to the elastic lamellae. The smooth muscle cells in the vascular media changed their shape into the round or oval configuration and sometimes arranged themselves perpendicularly (fig. 5). Notably, they exhibited some degenerative changes such as vacuolar formation and concentrated cytoplasm. Intercellular spaces became slightly expanded. The flocculated materials around the elastic lamellae and intercellular matrix were assumed to be the similar components such as GAGs and GPs or a mixture of them. 310
Exp Toxic Pathol46 (1994) 4-5
Fig. 3. The detached endothelial cell chain without disruption is floating in the vascular lumen which takes on a necklace-like appearance. X150, H&E.
The detachment of endothelial cells from the vascular wall began to be noticeable as the subendothelial edema with elongated anchoring processes (figs. 4 & 5) proceeding up to the total detachment (fig 5). Subsequently, the detached lumen became a balloon-like expansion, and the anchoring villi were elongated (fig. 5). No platelet accumulation was demonstrated. Through gaps between disrupted internal elastic lamellae, the medial smooth muscles elongated their cytoplasmic processes up to the subendothelial spaces (fig. 4).
Discussion Necklace-like detachment of the endothelial cell layer without disruption was demonstrated with repeated administration of 2 ml of 6 % Na2EDTA intraperitoneally for 5 days. With administration of this dose of Na2EDTA, the animals showed unrest without tetanic death. As is already known, the endothelial cells are desquamated individually from the vascular wall, a phenomenon referred to as physiologic turnover. In fact, however, the necklace-like detachment of the endothelial cells seen in our experimental result has never been previously reported. Even though total detachment of endothelial cells from the vascular wall occurred, no disruption of the endothelial chain was seen. This means that the induced low-calcium condition does not adversely affect the joining of endothelial cells but that it does very severely influence the adherence of endothelial cells to the vascular wall.
Fig. 4. Extended and frequent gap fonnation of internal elastic lamellae (iel) and slight expansion of intercellular spaces (*) in the vascular media are demonstrated. The anchoring villi of endothelial cells are elongated (black arrows). Anchoring viIi are usually attached to the flocculated materials around the internal elastic lamellae. Concerning this adhesion, a crucial role is played by type IV collagen, fibronectin and laminin (COUCHMAN et al. 1983; GRINNEL et al. 1977 & 1979; HOOK et al. 1977; ROUOSHLATI et al. 1979; VLODASKY et al. 1980) whose important component is glycoprotein (GPs) as well as glycosaminoglycans (GAGs). However, the detailed mechanism underlying this adhesion remains to be clarified. In this report, constriction of the vascular walls should inevitably have occurred, of course, with the administration of Na2EDTA. Accompanying this, the medial smooth muscle cells assumed a round or oval shape and a perpendicular arrangement. Expansion of the intercellular spaces was slight and the internal elastic lamellae were fragmented and were flattened by elongation, covered with surrounding flocculated materials. Anchoring villi fonned with elongated endothelial cytoplasmic processes were attached to the flocculated materials and they would be extremely extended and then elongated follo-
wing detachment of the endothelial cell layer from the vascular wall. However, as has already been mentioned in the previous papers (YAMAGUCHI et al. 1981 a & b), with the administration of a large amount of N a2EDT A, angiolytic changes in the arterial wall were induced by severe expansion of the intercellular spaces as well as the severe waving of the internal elastic lamellae pulling the endothelial cytoplasmic processes deeper into the vascular wall where these waved elastic lamellae and enveloped elongated endothelial cytoplasmic processes were packed tightly from the outside by constricted smooth muscle cells. In these cases, total detachment was not demonstrated even though the severe expansion of the intercellular spaces was provoked. As mentioned previously, since the common theoretical mechanism underlying the colloidal iron staining (PEARSE 1985) involves GPs as well as GAGs having a considerable abundance of anionic charges in their moleExp Toxic Pathol46 (1994) 4--5
311
Fig. S. The smooth muscle cells (sm) are arranged perpendicularly. Total detachment of the endothelial cells (end) with elongated anchoring villi (black arrows) is observed. Internal elastic lamellae (iel) are somewhat degenerative in appearance.
cules, staining results with a fixing cationic dye being responsive to these anionic charges. The intensity of staining is dependent not only on the amount of these substances but also on how many anionic charges would be unsaturated by positively charged materials. Therefore, the staining intensity is sometimes independent of the quantitative existence of these substances but is related to the number of open anionic charges. When these substances were stained strongly, their molecular structure became loosened and elongated. As far as the colloid chemical property is concerned, they change their character 312
Exp Toxic Patho146 (1994) 4-5
into sol indicating that their viscosity would be decreased and their sticky character reduced. The vascular media of these cases showed intensively positive with colloid iron staining. As far as the staining attitude of colloidal iron dye, the positivity, based on the sol-diversion of GAGs and GPs which is induced by the existence of a lot of negative charges in their molecules, is commonly observed under the low-calcium condition. This kind of changes in the matrix would have occurred not only in the intercellular matrix but also in the GAGs and GPs composed elements throughout the body.
In cases of angiolysis, provoked by the administration of a large dose of Na2EDT A, in spite of the less sticky character of these materials to be expected, total detachment of the endothelial cell layer was not demonstrated. On the contrary, with administration of repeated but moderate doses of Na2EDTA, necklace-like detachment of the endothelial cell layer was observed. Considering the phenomenon from the morphological differences between the above two kinds of low-calcium conditions, all of the phenomena are basically the same but the severity of several phenomena induce the differences. Most of them constitute the severity of the waving of the internal elastic lamellae by constriction which would induce the trapping of the endothelial cell layer to the vascular wall. The alteration of colloid chemical properties in the matrical materials as well as the degenerative changes in the internal elastic lamellae are suggested to be more or less the same in the above two cases, considering the staining attitude of colloid iron dye and also electron microscopic observations. Trapping of endothelial cytoplasm with severely waved internal elastic lamellae on the one hand and attachment to them via the anchoring villi on the other hand would result in either attachment or detachment of the endothelial cell layer. Considering the necklace-like detachment of the endothelial cell layer, the alteration of colloid chemical properties of the matrix and also the degenerative changes occurring in the internal elastic lamellae would be inevitable. In addition to these causes, however, the lack of waving of the internal elastic lamellae induced by the severe constriction which provoked the trapping of endothelial cytoplasmic processes should play the important role.
References 1. COUCHMAN JR, HOOK M, REES DA, et al.: Adhesion,
growth and matrix production by fibroblasts on laminin substrates. J Cell Bioi 1983; 96: 177-183. 2. GRINNELL F, HAYS DG, MINTER D: Cell adhesion and spreading factor - partial purification and properties. Exp Cell Res 1977; 110: 175-190.
3. GRINNELL F, FELD MK: Intial adhesion of human fibroblasts in serum free medium; possible role of secreted fibronectin. Cell 1979; 17: 117-129. 4. HOOK M, RUBIN K, OLDBERG A, et al.: Cold insoluble globulin mediates the adhesion of rat liver cells to plastic petri dishes. Biochem Biophys Res commun 1977, 79: 726-733. 5. PEARSE AGE: Histopathology, Theoretical and Applied. Churchill Livingstone, 1985. 6. RUOSLAHTI E and HAYMAN EG: Two active site with different characteristics in fibronectin FEBS Letters. 1979;97:221-224. 7. VLODAVSKY I and GOSPONDAROWICZ D: Morphological appearance, growth behavior and migratory activity of human tumor cells maintained in extracellular matrix versus plastic. Cell 1980; 19: 607-616. 8. YAMAGUCHI H, NAKAJIMA S, TORIKATA C, et al.: Studies on the morphological changes of artery caused by exudation - Initial changes of arteritis. Acta Path Jap 1972;22:441-455. 9. YAMAGUCHI H, TORIKATA C, TAKEUCHI H, et al.: Studies on the morphological changes of the endothelium in lung with administration of soluble immune complexes. Acta Path Jap 1973; 23: 51-58. 10. YAMAGUCHI H, USUI Hand TAJIMA T: Multiple angitis of mesenterial arteries induced by intraperitoneal administration of vast amount of Na2EDTA - light microscopic study. Exp Patholl98la, 20: 26-30. 11. YAMAGUCHI H, USUI H, TAJIMA T, et al.: Multiple angitis of mesenterial arteries induced by intraperitoneal administration of vast amount of Na2EDT A - electron microscopic study. Exp Pathol198lb; 20: 31-40. 12. YAMAGUCHI H, SAKAGUCHI Hand MORISADA M: Renal lesions in hypocalcemic condition - phenotypic manifestation of genetical background and its renal expresion. Exp Patho11990; 41: 10-20. l3. YAMAGUCHI H, KAKU Hand MORIS ADA M: Experimental pulmonary hypertension induced by constriction of pulmonary arteries, resulted in rupture or extraordinary dilatation of right ventricle with administration of a little amount of Na2EDTA. Exp Toxic Pathol 1993a; 45: 21-27. 14. YAMAGUCHI H, KAKU Hand MORIS ADA M: Study of morphological manifestations of pulmonary arteries in experimental pulmonary hypertension provoked by administration of long-term, low-dose Na2EDTA - especially on aneurysmal changes and plexiform-like lesions. Exp Toxic Pathol1993b; 45: 329-335.
Exp Toxic Pathol46 (1994) 4--5
313