1 Université Joseph Fourier, Grenoble, France, 2 Universitäts-Frauenklinik Würzburg and 3 Humboldt Universität zu Berlin, Germany
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Abstract |
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Key words: angiogenesis/decidua basalis and parietalis/human pregnancy/spontaneous abortion
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Introduction |
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vWF is a glycoprotein synthesized in endothelial cells and megakaryocytes (Ruggeri, 1997). It participates in haemostasis by mediating the adhesion of platelets to exposed subendothelium and promoting the formation of a platelet plug at the site of vascular injury. vWF also plays an essential role as a carrier protein for Factor VIII (Wagner, 1990
; Ruggeri, 1997
). As the expression and storage of vWF is confined to endothelial cells and megakaryocytes, this marker is a useful target to detect blood vessels by immunohistochemistry. The second endothelial marker investigated is PECAM-1 (also referred to as endoCAM or CD31), which is a transmembrane endothelial cell adhesion molecule belonging to the immunoglobulin superfamily (DeLisser et al., 1994
). The expression of PECAM-1 is confined to the surface of circulating platelets, leukocytes, and the endothelial intercellular junction (Newman, 1997
). One of the roles of PECAM-1 could be to modulate the endothelial permeability, thereby modulating leukocyteendothelial transmigration (Muller et al., 1993
). The third marker we chose is CD34, which is a glycosylated transmembrane protein expressed on haematopoietic, as well as on a variety of endothelial cells (Fina et al., 1990
). Its function remains unclear, but it has a possible role in angiogenesis and leukocyte adhesion (Fina et al., 1990
; Schlingemann et al., 1990
).
The vascular density (VD) of a tissue can be modulated by angiogenesis, which is defined as the formation of new blood vessels from pre-existing vessels (Risau, 1997, 1998
). Angiogenesis is a complex, multifactorial process, which normally does not occur in the healthy adult body, except during regulated physiological situations such as wound healing and in the female reproductive tract (Folkman 1995
; Rees and Bicknell, 1998
). Many integrins are implicated in angiogenesis (Davis and Camarillo, 1995
; Friedlander et al., 1995
). These molecules are transmembrane heterodimeric cell surface receptors (Clark and Brugge, 1995
). The
vß3 integrin, especially, is likely to play an important role (Varner et al., 1997
). This integrin has a low expression in quiescent blood vessels, but its expression is highly increased in angiogenic vessels (Brooks et al., 1994a
,b
, 1995
). Experiments performed with anti-
vß3 antibodies showed that this integrin plays a role during neovascularization (Brooks et al., 1995
; Friedlander et al., 1995
).
The aim of the present study was to quantify the blood vessel density in human decidua from normal pregnancies and from spontaneous abortions, using a panel of antibodies directed against vWF, PECAM-1 and CD34. The expression of the integrin vß3 was also investigated as a putative marker of angiogenesis.
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Materials and methods |
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Immunostaining of cytokeratin (CK), vWF, PECAM-1 and CD34
Sections 2 µm thick were stained with a monoclonal antibody (mAb) against pancytokeratin (Clone KL1; Immunotech, Hamburg, Germany) to confirm the presence or absence of invasive trophoblast and proliferating endometrial glands. On this basis, the decidua was classified as decidua basalis (invasive trophoblast or huge proliferating glands present) or decidua parietalis (no trophoblast or proliferating glands present). Consecutive slides were stained with mAb against human vWF (Clone F8/86; Dako, Hamburg, Germany), PECAM-1 (Clone JC/70A; Dako) and CD34 (Clone QBEND10; Dianova, Hamburg, Germany). All subsequent procedures were conducted at room temperature, unless otherwise detailed. Tissue sections were dewaxed in Xylol (Merck, Darmstadt, Germany) and rehydrated through a descending ethanol series. Non-specific endogenous peroxidase activity was blocked by treatment with 3% hydrogen peroxide in methanol for 30 min. Antigen retrieval was performed by microwave boiling in citrate buffer at pH 6.0 for 15 min. Tissue sections were then washed with Tris-buffered saline (TBS; pH 7.4) for 2x5 min and exposed to a non-immune, serum-free protein block (Dako). Cytokeratin mAb was applied at a dilution of 1 in 100 for 60 min. PECAM-1 mAb was applied at a dilution of 1 in 30 for 60 min and vWF mAb at a dilution of 1 in 25 for 60 min. CD34 mAb was a ready-to-use solution and also incubated for 60 min. This was followed by biotinylated multilink anti-mouse mAb for 15 min, and an avidinbiotinperoxidase detection system (LSAB-Kit; Dako) for a further 15 min. Finally, the sections were developed with diaminobenzidine (DAB; Sigma, Munich, Germany) for 5 min to generate a brown-coloured product and lightly counterstained with haemalum (Roth, Munich, Germany) prior to mounting with Vitro-Clud (Langenbrinck, Emmendingen, Germany). As a positive control, tissue from umbilical cord and endometrium was used. Negative controls were performed by replacing the primary mAb with serum-free protein block (Dako) at the same concentration as the primary mAb.
Immunostaining of vß3
Unfortunately, the staining of vß3 integrin was not possible on our pool of paraffin sections and therefore performed on a different tissue pool of frozen sections. All subsequent procedures were done at room temperature. Frozen sections were allowed to air-dry for 1 h. Tissue sections were then washed with TBS, pH 7.4 for 5 min and exposed to a non-immune, serum-free protein block (Dako).
vß3 mAb (clone LM609; Immunotech) was applied at a dilution of 1 in 100 for 60 min. Tissue from endometrium was used as a positive control. The protocol was then continued as described for the paraffin sections.
Determination of the percentage of blood vessels stained for vß3 integrin
Normal pregnancy decidua parietalis and decidua parietalis from abortion were investigated. The percentage of blood vessels stained by vß3 mAb was calculated from the number of blood vessels stained for
vß3 and the total number of blood vessels. The total number of blood vessels was quantified from a CD34 stained serial section.
Vessel counting
The number of blood vessels/mm2 of tissue was evaluated by two independent observers using a light microscope (Leica, Wetzlar, Germany) with scaled eye pieces pre-calibrated with a slide micrometer, at a magnification of x250 (x25 objective, with a x10 ocular), without knowledge of the patient's outcome. Deciduas were frequently heterogeneous in their vessel density. In particular, we observed that the vascularization of the stroma was frequently poor. Thus, for each slide, the blood vessels were counted in the entire tissue section and not only in the areas of highest vascularization. Then, the surface of the tissue section was measured and the mean blood vessel density calculated for each slide. Any brown-staining endothelial cell or endothelial cell cluster that was clearly separate from adjacent vessels was considered as a single, countable vessel. Vessel lumens were not necessary for a structure to be defined as a vessel, and red blood cells were not used to define a vessel lumen. Branching structures were counted as a single vessel. PECAM-1 and CD34 immunostained plasma cells were eliminated from the counts. No statistically sgnificant difference between the counts made by observer I and observer II could be calculated. Therefore we considered the inter-observer reproducibility as satisfactory (Student's t-test, P < 0.05).
Statistical analysis
Mean scores and SEM were calculated for each group. We used Student's t-test to assess significant differences of the mean VD values. P < 0.05 was considered significant.
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Results |
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Discussion |
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Endothelial cells are phenotypically heterogeneous (Thorin et al., 1997). Therefore they are also heterogeneous in their immunoreactivity (Kuzu et al., 1992
), thus justifying the choice of a panel of different markers to assess the vascularization of a tissue. Our vascular counts obtained with different immunostainings clearly support this observation. CD34 has been investigated in the assessment of endometrial vascularization (Rees et al., 1993
; Rogers et al., 1993
; Song et al., 1995
; Hickey et al., 1996
), but very few studies used it as a vascular marker in first trimester human pregnancy (te Velde et al., 1997
). Our observation that, in each case, the blood vessel density is higher when detected by CD34 compared to vWF is in agreement with other authors (Song et al., 1995
). It is possible that when using antibodies against vWF or PECAM some vessels have been missed due to poor antibody or antigen availability. However, this does not explain the discrepancy in vessel density between our study and previous studies by Rogers' and Song's groups, because this discrepancy is exclusively observed in CD34 stainings. vWF is expressed by the decidual endothelium (Grimwood et al., 1995
), and it has been shown that in human endometrium vascular staining for vWF is heterogeneous, with some vessels devoid of any positive staining (Au and Rogers, 1993
). Furthermore, other studies have shown changes in the immunoreactivity of vWF during the menstrual cell cycle (Zhu and Gu, 1988
). However, the results we obtained with both vWF and CD34 staining showed a significant increase in the vascularization of decidua parietalis from abortion. The concentration of blood vessels stained for PECAM-1 was not different in normal pregnancy decidua parietalis and in decidua parietalis from abortion, suggesting a decrease of PECAM-1 expression in decidua from abortion. We observed a heterogeneous staining of PECAM-1 in decidua from abortion, with some weakly stained vessels. However, we did not observe such a weak staining in normal pregnancy decidua. This confirms the previous observations (Haynes et al., 1997
; Ruck et al., 1994
) where an intense immunostaining of PECAM-1 in endothelium of early normal pregnancy decidua was found, demonstrating the pertinence of the use of this marker to assess the blood vessel density in this tissue.
Our data suggest a causal link between the increased vascularization observed in decidua parietalis and the phenomenon of spontaneous abortion. We observed no differences between normal pregnancy decidua and decidua from abortion regarding the angiogenic activity assessed by vß3 integrin staining. This observation allows us to postulate that angiogenesis is not occurring in the decidua at the moment of abortion. The expression of
vß3 integrin in human endometrial vasculature and glands has been reported and investigated as a sign of angiogenesis (Hii and Rogers, 1998
). Our data suggest that the increased blood vessel density observed in decidua parietalis from abortion could reveal intrinsic vascular disorders, linked with the development of the decidua, and thus with causal effects. However, very little is known about the mechanisms of angiogenesis in the endometrium, and some arguments support the hypothesis that these mechanisms could differ from the classical mechanisms known as `sprouting angiogenesis' (Goodger and Rogers, 1995
). Furthermore, as we did not observe a significant increase in vascularization of decidua basalis, the hypothesis of a causal link between the increased vascularization of parietalis and abortion remains to be investigated in depth. If the increased vascularization we observed in decidua parietalis from abortion reflects an attempt of the mother to rescue the pregnancy, why this is not the case in decidua basalis? It was found that E-selectin was only detectable on blood vessels from normal pregnancy decidua basalis (Burrows et al., 1994
), and not in normal pregnancy decidua parietalis. We confirmed this observation (results not shown). These results strongly suggest that the vessels of decidua basalis are phenotypically different from the vessels of decidua parietalis. Even if the role of the E-selectin is still not well understood, observations suggest that this molecule is most likely involved in angiogenesis in vitro (Nguyen et al., 1993
) and in vivo (Koch et al., 1995
). Therefore, these data suggest structural and functional differences between the blood vessels of normal pregnancy decidua basalis and decidua parietalis. Thus, even if these differences remain to be characterized in depth, it can be assumed that the ability of endothelial cells from decidua basalis and decidua parietalis to react to a particular pathophysiological situation could be different.
Our results could also be considered as a consequence of the abortion. It was reported that TNF-, which belongs to the Th1 cytokine family, is present at the feto-maternal interface, (Lea et al., 1997
) and Th1 cytokines are associated with spontaneous abortion (Raghupathy, 1997
). It has been reported that PECAM-1 expression can be decreased by inflammatory cytokines such as TNF-
and IFN-
(Rival et al., 1996
; Stewart et al., 1996
). This decrease of PECAM-1 expression at endothelial cellcell contacts could result in a reduction of polymorphonuclear cell migration across the endothelial cell monolayer (Rival et al., 1996
). Furthermore, PECAM-1 could be involved in the regulation of capillary morphogenesis, because of its localization and expression in angiogenic blood vessels (Berger et al., 1993
). However, its precise role in angiogenesis in general and in reproduction is still unclear (Dejana and Lostaglio, 1996
; DeLisser et al., 1997
). The earliest event in activated endothelium is usually an increase in vascular permeability, to accelerate the delivery of nutrients, oxygen and perhaps leukocytes rather than by making new blood vessels (Risau, 1998
); this further supports the suggestion that the increased vascularization we observed is not a consequence of Th1 activity at the feto-maternal interface. Therefore, we can postulate that a decrease of PECAM-1 expression could rather be a consequence of abortion.
In conclusion, further investigation is needed to assess if the increased vascularization observed in decidua parietalis from abortion is directly linked with causal effects, for example problems during the development of the decidua, or if it is a consequence of tissue damage during the course of the abortion. Our data suggest vascular disorders that could be linked with both.
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Acknowledgments |
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Notes |
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References |
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Submitted on October 28, 1998; accepted on February 15, 1999.