Background: Pregnant women with Type 1 diabetes mellitus develop vascular lesions similar to atheroma in the placental bed. These lesions are characterised by an accumulation of activated macrophages around spiral arteries, where they can stimulate a vigorous inflammatory reaction and impair vascular integrity and threaten blood flow. Diabetic pregnancies carry a high risk of development and rapid progression of preeclampsia and decreased placental blood flow causing fetal growth restriction.
In the development of the vascular lesions, peripheral blood monocytes are recruited, adhere to endothelium and migrate to subendothelial space where they initiate an inflammatory response. These events are mediated by surface adhesion molecules on both monocytes and endothelial cells. Earlier work from our research group had shown increased in vitro adherence of monocytes from diabetic pregnancy to decidual endothelial cells from normal pregnancies,
Experiments described in this thesis explore further the mechanisms subserving this abnormal monocyte adherence to decidual endothelium in diabetic pregnancy. After initial studies comparing adhesion molecule expression by monocytes from normal and diabetic pregnancies, the role of monocyte-derived Mac-1 in monocyte-decidual endothelial cell adherence was further investigated by the use of a blocking antibody.
Methods: Mixed peripheral blood mononuclear cells from three groups of pregnant 3rd trimester subjects, (i) normal, (ii) gestational diabetes, and (iii) type I diabetes, were isolated by density gradient centrifugation through Ficoll-Paque. Monocytes were identified (CD 14+) and their adhesion molecule expression (LFA-1, Mac-1, VLA-4) determined by double staining flow cytometry.
DECs were isolated from decidual biopsies collected from normally pregnant women at elective caesarean section. For subsequent flow cytometry, DECs were identified CD45-, and their ICAM-1 expression was measured by double staining flow cytometry. A highly enriched population of monocytes was then isolated (by density gradient centrifugation through iodixanol) from the blood of normally and type I diabetic pregnant women. Monocytes underwent incubation under basal and stimulated conditions followed by a further incubation with or without a blocking antibody to Mac-1. Monocytes were then co-cultured with DECs, and their adhesion and expression of Mac-1 were assessed.
Results: Monocytes from type I diabetic pregnancies displayed increased expression of Mac-1 and LFA-1 but not VLA-4. Adhesion molecule expression in gestational diabetes was between normal and type 1 diabetes. There was no significant direct correlation with adequacy of diabetic control as assessed by concomitantly measured blood glucose or HbA1c levels across the three groups in this study.
LPS pre-treatment of monocytes from normal pregnancy caused an increase in Mac-1 expression and was associated with an increase in their adherence to normal DECs.
ICAM-1 expression by normal DECs was stimulated by the presence of adherent monocytes, but no further increase in this ICAM-1 expression was seen when the monocytes had been pre-stimulated by LPS.
Exposure of monocytes from normal pregnancy to high levels of glucose also caused an increase in Mac-1 expression, associated with an increase in their adherence to normal DECs. The cause-and-effect nature of this Mac-1 expression was shown by monocyte pre-treatment with a Mac-1 monoclonal antibody, which prevented their adhesion to DECs in both basal and stimulated conditions, both in normal and in type I diabetic pregnancy.
Conclusion: The increased adhesion molecule expression of monocytes from type I diabetic pregnancy is paralleled by their increased adhesion to decidual endothelium, and may be causally involved in production of placental bed vascular disease in diabetic pregnancy .Pre-treatment of monocytes with stimuli that increased Mac-1 expression resulted in an increase in their adhesion to decidual endothelium, an effect prevented by specific Mac-1 antibody blockade. The experiments conducted in this study examine only a part of the adhesion interaction of monocytes and decidual endothelial cells. While monocyte Mac-1 expression is clearly of central importance, other factors are involved, some resulting from the initiation of monocyte adherence, such as the observed DEC-derived ICAM-1 which may amplify the adhesion. Other adhesion molecules could be involved, as could be the release of chemo-attractant peptides.
These results add significantly to our earlier report of increased adhesion of monocytes from pregnant woman with type I diabetes to normal decidual endothelial cells, and provide evidence related to the mechanisms subserving this increased adhesion.