American Journal of Pathology, Vol. 139, No. 6, December 1991 Copyright © American Association of Pathologists
Heterogeneous Distribution of Weibel-Palade Bodies and von Willebrand Factor Along the Porcine Vascular Tree Jeannine Gebrane-Younes,* Ludovic Drouet,t Jacques P. Caen,t and Louis Orcel* From the Faculte de Medecine Saint-Antoine,* Universite Pierre et Marie Curie; and INSERM U150, I.VS.,t Hopital Laziboisiere, Paris, France
Vessels obtainedfrom different levels ofpig vascular tree were examined by transmission electron microscope, with the aim of determining whether or not their endothelial cells contain Weibel-Palade bodies (WPB). As these organelles are knoum to store the von Willebrand factor (vWF), a two-step immunogold labeling of this protein also was performed Our results showed for the first time a heterogeneous distribution of WPB along the vascular tree of the normal pig: These structures were absent from the thoracic aortg rare in the abdominal aorta, present in myocardial capillaries, and numerous in the inferior vena cava and pulmonary artery. Atypical WPB devoid of tubules were seen in all endothelial cells. The ultrastructural labeling of vWF demonstrated its presence only in the WPB, being absent in the subendothelium, and showed the same variation in its distribution along the vascular tree as for its storage organelle. Pigs homozygous for the von Willebrand disease werefound to have only the atypical WPB, and do not express the vWF. (Am JPathol
for the WPB as storage organelles for the von Willebrand factor (vWF). von Willebrand factor is a large glycoprotein that mediates the attachment of platelets to the subendotheHum.6 It is synthesized by the endothelial cell,7 which produces a series of disulfide-bounded multimers with a size ranging from 0.5 to 20 million daltons.8 A majority of the smallest multimers are secreted constitutively, without stimulation,9 whereas the largest multimers are predominantly stored in WPB,10 and released after physiologic stimuli such as thrombin11 and fibrin,12 or after treatment with secretagogues such as phorbol myristate acetate, 13 and the calcium ionophore A 23187.10,14 As endothelial cells synthesize vWF, this factor was used to establish the endothelial origin of cells in histopathologic diagnosis.15'16 In 1983, Giddings et al17 were unable to visualize vWF in porcine aortic endothelium, in contrast to venous endothelium. Using the immunofluorescence technique, Wu et al18 showed that the distribution of vWF in the porcine vessels varies according to the origin of cultured endothelial cells. Comparable results were obtained by Rand et al,19 who worked on porcine fresh endothelia. The differential expression of vWF in porcine vessels was correlated to vWF mRNA levels.2O As vWF is stored in WPB, the distribution of WPB and immunoreactive vWF along the vascular tree was investigated by electron microscope. This characterization was performed in the pig for three reasons:
1991, 139:1471-1484)
Described in 1964,1 Weibel-Palade bodies (WPB) were considered as a key marker for the identification of endothelial cells. Quantitative studies on the distribution of these bodies in various parts of the vascular system in the rat2 and frog3 show that these are ubiquitous bodies found in vessels of all calibers as well as in the endocardium. The function of these granules was difficult to define for a long time, but Weibel already suggested in 19644 that they could be involved in blood coagulation because of their resemblance to ot-granules of thrombocytes. It was not until 1982 that Wagner et a15 established a role
1) Weibel-Palade bodies were observed in human endothelial cells, as in many other animals species, but they have never been described in pig. 2) We had the opportunity to use pigs homozygous for the von Willebrand disease (vWD), in comparison with normal pigs. 3) Vascular tissues from various levels of the vascular tree are easily available. Support was provided by School of Medicine, Saint-Antoine and INSERM U150. Accepted for publication August 2, 1991. Address reprnt requests to Dr. Jeannine Gebrane-Younbs, Service Central d'Anatomie et Cytologie Pathologiques, Facult6 de M6decine Saint-Antoine, 27, Rue Chaligny, F-75571, Paris Cedex-12, France.
1471
1472
Gebrane-Younes et al
AJP December 1991, Vol. 139, No. 6
We studied five different locations of blood vessels: thoracic aorta, abdominal aorta, inferior vena cava, pulmonary artery, and myocardial capillaries. Our results showed, for the first time, a heterogeneous distribution of WPB along the vascular tree of the normal pigs. This distribution coincides with a heterogeneous immunoultrastructural labeling of vWF in endothelial cells, the latter correlating with the previous immunofluorescence data. 18,19 The vWD pigs showed an absence of WPB in all the endothelial cells examined, concomitant with a negative immunolabeling for the vWF. A brief report of this work was recently published.21
pigs within 15 to 20 minutes after death, and those from piglets were resected under general anesthesia (intramuscular injection of nesdonal 5 ml from a 5% solution). The following samples were treated either for standard electron microscopy or for the immunoultrastructural labeling of vWF:
1) Distal thoracic aorta, above the celiac trunk origin 2) Distal abdominal aorta, before iliac bifurcation 3) Pulmonary artery 4) Inferior vena cava, at the level of coming out of the extrahepatic vein 5) Myocardium (for the study of myocardial capillaries) at the level of the upper third of interventricular coronary artery
Material and Methods
Normal and von Willebrand Pig Colonies
Standard Electron Microscopy
Normal 'large white' pigs were maintained at the experimental farm of the Institut National de la Recherche Agronomique (INRA, Jouy en-Josas, France). Von Willebrand diseased pigs were obtained by artificial insemination of 'large white' with semen from homozygous vWD pigs (crossbred of Yorkshire-Hampshire and PolandChina, provided by E. J. W. Bowie from Mayo-Clinic, Rochester, MN). Animals used in this experiment were taken from the third to the fifth generation of crossbreeding. Normal and vWD pigs were fed the same diet, with regular veterinarian follow-up. All animal manipulation, animal facilities, and investigators involved with animals comply with national regulation and institutional pol-
Samples of 1 mm3 of ten normal, nine vWD adult pigs, and three normal piglets were immersed in the fixative solution (2% wt/vol paraformaldehyde [PF] + 2.5% vol/ vol glutaraldehyde [GA] in 0.1 mol/l [molar] sodium cacodylate buffer pH 7.2) at + 40C. After 1 hour 30 minutes, the fragments were washed in 0.1 mol/l cacodylate buffer, postfixed in 1% wt/vol osmium tetroxide in the same buffer, block-stained in 1% wt/vol aqueous uranyl acetate, dehydrated, and embedded in Epon.
Immuno-ultrastructural Labeling of vWF
icy.
Diagnosis of von Willebrand Pigs Diagnosis of vWD in the porcine colony was assessed by the 'ratio of bleeding time,'22 as well as levels of vWF antigen (vWF Ag),23 using a specific enzyme-linked inmunosorbent assay (ELISA) technique,18 and vWF activity in the absence of ristocetin,24 by comparison with a reference pool of plasma from 15 normal large white pigs.
Collection of Vessel Segments Normal and vWD pigs that were killed were 6 to 12 months old (100 to 180 kg body weight). Three normal piglets aged 2 to 4 days (1.2 to 2 kg) were also used for this study. Fragments of blood vessels were removed from adult
To increase antibody penetration and the specificity of the immunocytochemical labeling, an hydrophilic embedding medium: lowicryl K4M was used.25 Four normal and two vWD adult pigs were treated as follows after killing: the resected samples were minced into 0.5 mm3 and immersed in one of the two fixatives (a mixture of 2% wt/vol PF and 0.05% vol/vol GA in 0.1 moVI sodium phosphate buffer pH 7.4; or 3% wt/vol PF and 0.25% vol/vol GA in the same buffer). After 3 hours' fixation at + 40C, the tissues were rinsed in three baths of 0.1 mol/l phosphate buffer pH 7.4, with an admixture of 10% sucrose, during 3 hours; they then were incubated in 0.05 mol/l ammonium chloride in phosphate-buffered saline (PBS) at + 40C throughout the night, to quench nonreactive aldehyde groups. Fragments were again washed in PBS. For low-temperature embedding, a lowicryl K4M (Balzers Union) protocol was employed.26'27 Briefly, after dehydration in a graded series of ethanol: 30% (at 00C), 50% (at -200C), 70%, 95%, and 100% (at -350C), the
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AJP December 1991, Vol. 139, No. 6
tissues were infiltrated with increasing concentrations of lowicryl at -35°C. Polymerization was started with ultraviolet light at - 35°C during one whole night, and continued for 2 to 3 days at room temperature. Thin sections (60 to 90 nm) were picked up on nickel grids for immunocytochemical reactions. a-Antibodies Rabbit polyclonal antiserum and the IgG fraction to porcine vWF were prepared in collaboration with D. Meyer, INSERM (U.143) and Diagnostica Stago, Franconville, France. These polyclonal antibodies were rendered monospecific by adsorption on a solid phase as described for human vWF.28 Briefly, the fractions obtained from the porcine extract of factor Vil devoided of vWF were rendered insoluble on agarose gel. After removing the unwanted antibodies, the specificity was verified by a twodimensional gel electrophoresis in which increasing quantities of antiserum were incorporated in the gel of the second migration (with a concentration reaching 20%, so that to eliminate a possible contaminant). The tested material was either normal porcine plasma or plasma of pigs with vWD. In any case, just one arc should be obtained using normal plasma, whereas no arc should be seen with the plasma of vWF pigs. Goat anti-rabbit IgG coupled to 5 or 10 nm colloidal gold (GAR G5 or GAR Glo) was from Janssen Pharmaceutical (Belgium).
b-Procedure of On-grid Immunomarking With both the antiserum and the IgG fraction, a simple two-step reaction was employed. The sections were first incubated in TRIS-HCI buffer (pH 8.2) admixed with 1% gelatin for 40 minutes. The grids then were transferred to a drop of the first antibody (in concentrations of 1/50 for the antiserum, and 1/100 for the IgG fraction) and left for 2 hours at room temperature. After washing in TRIS buffer, the sections were exposed to a drop of anti-rabbit IgG linked to colloidal gold (GAR G5 or G10) for 1 hour at room temperature. The grids were again washed with TRIS buffer and treated with 1% GA in PBS for 5 minutes to fix the antibodies to the tissue sites. The fixative was washed out with three changes of distilled water and the grids contrasted with uranyl acetate and lead citrate before being examined on a transmission electron microscope Zeiss EM10 (Oberkochen, Federal Republic of
Germany). To demonstrate the specificity of the staining, control sections were incubated with nonimmune rabbit serum or IgG. They were otherwise incubated in an identical manner.
Results Standard Electron Microscopy Vessels from Normal Adult Pigs Whatever the level of sampling aorta (thoracic or abdominal), the endothelium showed structures having the size and the shape of WPB. These structures appeared rounded, elongated, or oval, with a granular matrix varying from moderately to highly electron dense. They failed, however, to display the tubules originally described by Weibel and Palade (Figure 1a). In the pulmonary artery (Figure 2a), the inferior vena cava (Figure 3a), and the myocardial capillaries, two types of granules were observed in the endothelial cells, the typical WPB with tubules, and a second type resembling the organelle present in the aortic endothelium and devoided of tubules. Although these granules were present throughout the cytoplasm, more were located at the periphery of the endothelial cells, sometimes very close to the plasma membrane (Figure 3c). Weibel-Palade bodies were more numerous in the pulmonary artery and the inferior vena cava, than in the myocardial capillaries. The diameter of these bodies ranges from 0.1 to 0.2 ,u, and that of tubules from 15 to 18 nm.
Vessels from von Willebrand Adult Pigs The endothelium of vWD pigs did not show WPB in any of the investigated localizations: thoracic and abdominal aorta, pulmonary artery, inferior vena cava, and myocardial capillaries. These structures were vainly searched for in the cytoplasm, especially in the Golgi zone, from which it has been demonstrated that they developed' (Figure 2b). Occasionally we observed in the endothelial cells of the aorta (Figure 1 b), the pulmonary artery and the inferior vena cava (Figure 3b), the same structures devoided of tubules described in the aortic endothelium of the normal pig. Vessels from Normal Piglets Weibel-Palade bodies were absent from the endothelial cells of pulmonary artery, and more numerous in the inferior vena cava (Figure 3c) and the myocardial capillaries (Figure 4), as compared with the adult pigs.
Immuno-ultrastructural Localization of vWF Vessels from Normal Adult Pigs Thoracic Aorta When the sections were incubated with the first antibody anti-vWF followed by immunogold, there was no
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AJP December 1991, Vol. 139, No. 6
Figure 1. a: Aortic endothelium ofa normalpig showing a structure having the size and the shape of WP.B., but devoided of tubules (arrow). Cytoplasmic microtubules 25 nm in diameter (arrowheads), uranyl acetate and lead citrate (Ur-Pb), x45,000. b: Aortic endothelium of a vWD pig. As in the normal pig aorta, the endothelium contains granules with a dense matrix devoided of tubules (arrows). Ur-Pb, x 45,000. Figure 2. a: Pulmonary artety of a normalpig. In this endothelium we can observe two types of organelles: the typical WP.B. with internal tubules of 18 nm (arrows) and granules without tubules (curved arrow); cytoplasmic microtubules (arrowheads) and vessel lumen a); Ur-Pb, X55,400. b: Pulmonaiy artery of a vWD pig. Even in the area where the Golgi apparatus (G) is well developed, we did not observe WP.B. lumen (L); Ur-Pb, X35,400.
Weibel-Palade Bodies and vWF
1475
AJP December 1991, Vol. 139, No. 6
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6% il -.~ Figure 3. a: Inferior vena cava of a normalpig. WP.B. with tubules longitudinallv cut (vertical arrows) or tranversally cut (horizontal arrow); granules uithout tubules (double arrows). Elastic lamellae (EL) in the subendothelium. LTr-Pb, x 45,000. b: Inferior vena cava of a vWD pig. The endothelium shows three granules u'ith a dense matrix dev,oided of tubules (arrows). Subendothelium (SE). Ur-Pb, x55,400. c: Inferior vena cava of a normal piglet. In this endothelial cell, all the WP.B. seen (arrows) contain tubules u'hich sometimes shou' a spiral configuration (curved arrow). There is apparentfusion of a WP.B. uith the basalplasma membrane (double arrow). Vessel lumen (L). Ur-Pb, x 70,400. .
1476
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AJP December 1991, Vol. 139, No. 6
Figure 4. Myocardial capillary of a normal piglet. Endothelial cell uith four WP.B. (arrows). The horizontal arrou' indicates a WP.B. merging uith a vesicle and containing tubules not clearly defined ur-Ph, x 55,400.
labeling of the endothelial cells and the subendothelium (Figure 5)
Abdominal Aorta In the distal portion of this vessel, rare granules contained the vWF, whereas the subendothelium was showed negative (Figure 6).
Pulmonary Artery All endothelial cells examined showed an intense labeling of the WPB. The distribution pattern of gold particles delineated the rounded or elongated shapes of these organelles, which sometimes appeared to fuse with the plasma membrane (Figure 7). Von Willebrand factor was absent from any other cytoplasmic organelle as well as from the subendothelial space.
Inferior Vena Cava Endothelial cells were strongly positive for the vWF, which was only located in WPB. The occasional gold particles observed in the extracellular matrix were indistinguishable from the background staining (Figures 8a, b).
These organelles were showed consistently negative in sections incubated with control IgG (Figure 9).
Myocardial Capillaries The gold particles concentrated in WPB (Figure 10) help to locate these granules, which are few in the endothelium of adult pigs' myocardial capillaries. A comparison of the immunolabeling results obtained with two types of fixatives (PF 2% + GA 0.05% and PF 3% + GA 0.25%) showed a good preservation of antigenicity in both cases (See photo's legends). Although the IgG fraction showed a lower background staining than the antiserum, this latter gave also a specific localization of vWF in the WPB (Figure 1 1). Vessels from von Willebrand Adult Pigs The endothelial cells obtained from the five localizations (thoracic aorta, abdominal aorta, pulmonary artery, inferior vena cava, myocardial capillaries) did not show any concentration of gold particles (Figures 12, 13). This absence of labeling for vWF coincides with the absence of true WPB in the vWD pigs. A summary of the results is shown in Table 1.
.:f"it1;,V*F. :\,tA'. .':
~
Weibel-Palade Bodies and vWF 1477 AJP December 1991, Vol. 139, No. 6
~~~~~~~~~~~M
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Heterogeneous Distribution of Weibel-Palade Bodies and von Willebrand Factor Along the Porcine Vascular Tree Jeannine Gebrane-Younes,* Ludovic Drouet,t Jacques P. Caen,t and Louis Orcel* From the Faculte de Medecine Saint-Antoine,* Universite Pierre et Marie Curie; and INSERM U150, I.VS.,t Hopital Laziboisiere, Paris, France
Vessels obtainedfrom different levels ofpig vascular tree were examined by transmission electron microscope, with the aim of determining whether or not their endothelial cells contain Weibel-Palade bodies (WPB). As these organelles are knoum to store the von Willebrand factor (vWF), a two-step immunogold labeling of this protein also was performed Our results showed for the first time a heterogeneous distribution of WPB along the vascular tree of the normal pig: These structures were absent from the thoracic aortg rare in the abdominal aorta, present in myocardial capillaries, and numerous in the inferior vena cava and pulmonary artery. Atypical WPB devoid of tubules were seen in all endothelial cells. The ultrastructural labeling of vWF demonstrated its presence only in the WPB, being absent in the subendothelium, and showed the same variation in its distribution along the vascular tree as for its storage organelle. Pigs homozygous for the von Willebrand disease werefound to have only the atypical WPB, and do not express the vWF. (Am JPathol
for the WPB as storage organelles for the von Willebrand factor (vWF). von Willebrand factor is a large glycoprotein that mediates the attachment of platelets to the subendotheHum.6 It is synthesized by the endothelial cell,7 which produces a series of disulfide-bounded multimers with a size ranging from 0.5 to 20 million daltons.8 A majority of the smallest multimers are secreted constitutively, without stimulation,9 whereas the largest multimers are predominantly stored in WPB,10 and released after physiologic stimuli such as thrombin11 and fibrin,12 or after treatment with secretagogues such as phorbol myristate acetate, 13 and the calcium ionophore A 23187.10,14 As endothelial cells synthesize vWF, this factor was used to establish the endothelial origin of cells in histopathologic diagnosis.15'16 In 1983, Giddings et al17 were unable to visualize vWF in porcine aortic endothelium, in contrast to venous endothelium. Using the immunofluorescence technique, Wu et al18 showed that the distribution of vWF in the porcine vessels varies according to the origin of cultured endothelial cells. Comparable results were obtained by Rand et al,19 who worked on porcine fresh endothelia. The differential expression of vWF in porcine vessels was correlated to vWF mRNA levels.2O As vWF is stored in WPB, the distribution of WPB and immunoreactive vWF along the vascular tree was investigated by electron microscope. This characterization was performed in the pig for three reasons:
1991, 139:1471-1484)
Described in 1964,1 Weibel-Palade bodies (WPB) were considered as a key marker for the identification of endothelial cells. Quantitative studies on the distribution of these bodies in various parts of the vascular system in the rat2 and frog3 show that these are ubiquitous bodies found in vessels of all calibers as well as in the endocardium. The function of these granules was difficult to define for a long time, but Weibel already suggested in 19644 that they could be involved in blood coagulation because of their resemblance to ot-granules of thrombocytes. It was not until 1982 that Wagner et a15 established a role
1) Weibel-Palade bodies were observed in human endothelial cells, as in many other animals species, but they have never been described in pig. 2) We had the opportunity to use pigs homozygous for the von Willebrand disease (vWD), in comparison with normal pigs. 3) Vascular tissues from various levels of the vascular tree are easily available. Support was provided by School of Medicine, Saint-Antoine and INSERM U150. Accepted for publication August 2, 1991. Address reprnt requests to Dr. Jeannine Gebrane-Younbs, Service Central d'Anatomie et Cytologie Pathologiques, Facult6 de M6decine Saint-Antoine, 27, Rue Chaligny, F-75571, Paris Cedex-12, France.
1471
1472
Gebrane-Younes et al
AJP December 1991, Vol. 139, No. 6
We studied five different locations of blood vessels: thoracic aorta, abdominal aorta, inferior vena cava, pulmonary artery, and myocardial capillaries. Our results showed, for the first time, a heterogeneous distribution of WPB along the vascular tree of the normal pigs. This distribution coincides with a heterogeneous immunoultrastructural labeling of vWF in endothelial cells, the latter correlating with the previous immunofluorescence data. 18,19 The vWD pigs showed an absence of WPB in all the endothelial cells examined, concomitant with a negative immunolabeling for the vWF. A brief report of this work was recently published.21
pigs within 15 to 20 minutes after death, and those from piglets were resected under general anesthesia (intramuscular injection of nesdonal 5 ml from a 5% solution). The following samples were treated either for standard electron microscopy or for the immunoultrastructural labeling of vWF:
1) Distal thoracic aorta, above the celiac trunk origin 2) Distal abdominal aorta, before iliac bifurcation 3) Pulmonary artery 4) Inferior vena cava, at the level of coming out of the extrahepatic vein 5) Myocardium (for the study of myocardial capillaries) at the level of the upper third of interventricular coronary artery
Material and Methods
Normal and von Willebrand Pig Colonies
Standard Electron Microscopy
Normal 'large white' pigs were maintained at the experimental farm of the Institut National de la Recherche Agronomique (INRA, Jouy en-Josas, France). Von Willebrand diseased pigs were obtained by artificial insemination of 'large white' with semen from homozygous vWD pigs (crossbred of Yorkshire-Hampshire and PolandChina, provided by E. J. W. Bowie from Mayo-Clinic, Rochester, MN). Animals used in this experiment were taken from the third to the fifth generation of crossbreeding. Normal and vWD pigs were fed the same diet, with regular veterinarian follow-up. All animal manipulation, animal facilities, and investigators involved with animals comply with national regulation and institutional pol-
Samples of 1 mm3 of ten normal, nine vWD adult pigs, and three normal piglets were immersed in the fixative solution (2% wt/vol paraformaldehyde [PF] + 2.5% vol/ vol glutaraldehyde [GA] in 0.1 mol/l [molar] sodium cacodylate buffer pH 7.2) at + 40C. After 1 hour 30 minutes, the fragments were washed in 0.1 mol/l cacodylate buffer, postfixed in 1% wt/vol osmium tetroxide in the same buffer, block-stained in 1% wt/vol aqueous uranyl acetate, dehydrated, and embedded in Epon.
Immuno-ultrastructural Labeling of vWF
icy.
Diagnosis of von Willebrand Pigs Diagnosis of vWD in the porcine colony was assessed by the 'ratio of bleeding time,'22 as well as levels of vWF antigen (vWF Ag),23 using a specific enzyme-linked inmunosorbent assay (ELISA) technique,18 and vWF activity in the absence of ristocetin,24 by comparison with a reference pool of plasma from 15 normal large white pigs.
Collection of Vessel Segments Normal and vWD pigs that were killed were 6 to 12 months old (100 to 180 kg body weight). Three normal piglets aged 2 to 4 days (1.2 to 2 kg) were also used for this study. Fragments of blood vessels were removed from adult
To increase antibody penetration and the specificity of the immunocytochemical labeling, an hydrophilic embedding medium: lowicryl K4M was used.25 Four normal and two vWD adult pigs were treated as follows after killing: the resected samples were minced into 0.5 mm3 and immersed in one of the two fixatives (a mixture of 2% wt/vol PF and 0.05% vol/vol GA in 0.1 moVI sodium phosphate buffer pH 7.4; or 3% wt/vol PF and 0.25% vol/vol GA in the same buffer). After 3 hours' fixation at + 40C, the tissues were rinsed in three baths of 0.1 mol/l phosphate buffer pH 7.4, with an admixture of 10% sucrose, during 3 hours; they then were incubated in 0.05 mol/l ammonium chloride in phosphate-buffered saline (PBS) at + 40C throughout the night, to quench nonreactive aldehyde groups. Fragments were again washed in PBS. For low-temperature embedding, a lowicryl K4M (Balzers Union) protocol was employed.26'27 Briefly, after dehydration in a graded series of ethanol: 30% (at 00C), 50% (at -200C), 70%, 95%, and 100% (at -350C), the
Weibel-Palade Bodies and vWF
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AJP December 1991, Vol. 139, No. 6
tissues were infiltrated with increasing concentrations of lowicryl at -35°C. Polymerization was started with ultraviolet light at - 35°C during one whole night, and continued for 2 to 3 days at room temperature. Thin sections (60 to 90 nm) were picked up on nickel grids for immunocytochemical reactions. a-Antibodies Rabbit polyclonal antiserum and the IgG fraction to porcine vWF were prepared in collaboration with D. Meyer, INSERM (U.143) and Diagnostica Stago, Franconville, France. These polyclonal antibodies were rendered monospecific by adsorption on a solid phase as described for human vWF.28 Briefly, the fractions obtained from the porcine extract of factor Vil devoided of vWF were rendered insoluble on agarose gel. After removing the unwanted antibodies, the specificity was verified by a twodimensional gel electrophoresis in which increasing quantities of antiserum were incorporated in the gel of the second migration (with a concentration reaching 20%, so that to eliminate a possible contaminant). The tested material was either normal porcine plasma or plasma of pigs with vWD. In any case, just one arc should be obtained using normal plasma, whereas no arc should be seen with the plasma of vWF pigs. Goat anti-rabbit IgG coupled to 5 or 10 nm colloidal gold (GAR G5 or GAR Glo) was from Janssen Pharmaceutical (Belgium).
b-Procedure of On-grid Immunomarking With both the antiserum and the IgG fraction, a simple two-step reaction was employed. The sections were first incubated in TRIS-HCI buffer (pH 8.2) admixed with 1% gelatin for 40 minutes. The grids then were transferred to a drop of the first antibody (in concentrations of 1/50 for the antiserum, and 1/100 for the IgG fraction) and left for 2 hours at room temperature. After washing in TRIS buffer, the sections were exposed to a drop of anti-rabbit IgG linked to colloidal gold (GAR G5 or G10) for 1 hour at room temperature. The grids were again washed with TRIS buffer and treated with 1% GA in PBS for 5 minutes to fix the antibodies to the tissue sites. The fixative was washed out with three changes of distilled water and the grids contrasted with uranyl acetate and lead citrate before being examined on a transmission electron microscope Zeiss EM10 (Oberkochen, Federal Republic of
Germany). To demonstrate the specificity of the staining, control sections were incubated with nonimmune rabbit serum or IgG. They were otherwise incubated in an identical manner.
Results Standard Electron Microscopy Vessels from Normal Adult Pigs Whatever the level of sampling aorta (thoracic or abdominal), the endothelium showed structures having the size and the shape of WPB. These structures appeared rounded, elongated, or oval, with a granular matrix varying from moderately to highly electron dense. They failed, however, to display the tubules originally described by Weibel and Palade (Figure 1a). In the pulmonary artery (Figure 2a), the inferior vena cava (Figure 3a), and the myocardial capillaries, two types of granules were observed in the endothelial cells, the typical WPB with tubules, and a second type resembling the organelle present in the aortic endothelium and devoided of tubules. Although these granules were present throughout the cytoplasm, more were located at the periphery of the endothelial cells, sometimes very close to the plasma membrane (Figure 3c). Weibel-Palade bodies were more numerous in the pulmonary artery and the inferior vena cava, than in the myocardial capillaries. The diameter of these bodies ranges from 0.1 to 0.2 ,u, and that of tubules from 15 to 18 nm.
Vessels from von Willebrand Adult Pigs The endothelium of vWD pigs did not show WPB in any of the investigated localizations: thoracic and abdominal aorta, pulmonary artery, inferior vena cava, and myocardial capillaries. These structures were vainly searched for in the cytoplasm, especially in the Golgi zone, from which it has been demonstrated that they developed' (Figure 2b). Occasionally we observed in the endothelial cells of the aorta (Figure 1 b), the pulmonary artery and the inferior vena cava (Figure 3b), the same structures devoided of tubules described in the aortic endothelium of the normal pig. Vessels from Normal Piglets Weibel-Palade bodies were absent from the endothelial cells of pulmonary artery, and more numerous in the inferior vena cava (Figure 3c) and the myocardial capillaries (Figure 4), as compared with the adult pigs.
Immuno-ultrastructural Localization of vWF Vessels from Normal Adult Pigs Thoracic Aorta When the sections were incubated with the first antibody anti-vWF followed by immunogold, there was no
1474
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AJP December 1991, Vol. 139, No. 6
Figure 1. a: Aortic endothelium ofa normalpig showing a structure having the size and the shape of WP.B., but devoided of tubules (arrow). Cytoplasmic microtubules 25 nm in diameter (arrowheads), uranyl acetate and lead citrate (Ur-Pb), x45,000. b: Aortic endothelium of a vWD pig. As in the normal pig aorta, the endothelium contains granules with a dense matrix devoided of tubules (arrows). Ur-Pb, x 45,000. Figure 2. a: Pulmonary artety of a normalpig. In this endothelium we can observe two types of organelles: the typical WP.B. with internal tubules of 18 nm (arrows) and granules without tubules (curved arrow); cytoplasmic microtubules (arrowheads) and vessel lumen a); Ur-Pb, X55,400. b: Pulmonaiy artery of a vWD pig. Even in the area where the Golgi apparatus (G) is well developed, we did not observe WP.B. lumen (L); Ur-Pb, X35,400.
Weibel-Palade Bodies and vWF
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AJP December 1991, Vol. 139, No. 6
A
*;
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It
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;461.
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6% il -.~ Figure 3. a: Inferior vena cava of a normalpig. WP.B. with tubules longitudinallv cut (vertical arrows) or tranversally cut (horizontal arrow); granules uithout tubules (double arrows). Elastic lamellae (EL) in the subendothelium. LTr-Pb, x 45,000. b: Inferior vena cava of a vWD pig. The endothelium shows three granules u'ith a dense matrix dev,oided of tubules (arrows). Subendothelium (SE). Ur-Pb, x55,400. c: Inferior vena cava of a normal piglet. In this endothelial cell, all the WP.B. seen (arrows) contain tubules u'hich sometimes shou' a spiral configuration (curved arrow). There is apparentfusion of a WP.B. uith the basalplasma membrane (double arrow). Vessel lumen (L). Ur-Pb, x 70,400. .
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Figure 4. Myocardial capillary of a normal piglet. Endothelial cell uith four WP.B. (arrows). The horizontal arrou' indicates a WP.B. merging uith a vesicle and containing tubules not clearly defined ur-Ph, x 55,400.
labeling of the endothelial cells and the subendothelium (Figure 5)
Abdominal Aorta In the distal portion of this vessel, rare granules contained the vWF, whereas the subendothelium was showed negative (Figure 6).
Pulmonary Artery All endothelial cells examined showed an intense labeling of the WPB. The distribution pattern of gold particles delineated the rounded or elongated shapes of these organelles, which sometimes appeared to fuse with the plasma membrane (Figure 7). Von Willebrand factor was absent from any other cytoplasmic organelle as well as from the subendothelial space.
Inferior Vena Cava Endothelial cells were strongly positive for the vWF, which was only located in WPB. The occasional gold particles observed in the extracellular matrix were indistinguishable from the background staining (Figures 8a, b).
These organelles were showed consistently negative in sections incubated with control IgG (Figure 9).
Myocardial Capillaries The gold particles concentrated in WPB (Figure 10) help to locate these granules, which are few in the endothelium of adult pigs' myocardial capillaries. A comparison of the immunolabeling results obtained with two types of fixatives (PF 2% + GA 0.05% and PF 3% + GA 0.25%) showed a good preservation of antigenicity in both cases (See photo's legends). Although the IgG fraction showed a lower background staining than the antiserum, this latter gave also a specific localization of vWF in the WPB (Figure 1 1). Vessels from von Willebrand Adult Pigs The endothelial cells obtained from the five localizations (thoracic aorta, abdominal aorta, pulmonary artery, inferior vena cava, myocardial capillaries) did not show any concentration of gold particles (Figures 12, 13). This absence of labeling for vWF coincides with the absence of true WPB in the vWD pigs. A summary of the results is shown in Table 1.
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Weibel-Palade Bodies and vWF 1477 AJP December 1991, Vol. 139, No. 6
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