Sciota, Flavia
(2012)
Effect of hyperglycaemia on VEGF-A splice variants, junctional integrity and vascular leakage in human feto-placental vessels from normal and type 1 diabetic pregnancies.
PhD thesis, University of Nottingham.
Abstract
Hyperglycaemia is a main feature of diabetes, and this pathology is a main complication of pregnancy. Diabetic patients often require tighter glycaemic control in pregnancy, as glucose metabolism changes, and insulin dosages need to be adjusted. Throughout gestation, the placenta is therefore subjected to periods of hyperglycaemic insult. Thus, we wished to study how brief periods of hyperglycaemia affected the feto-placental vasculature, through study of important permeability molecules, pro-permeability VEGFa, anti-permeability VEGFb, and junctional stability molecule VE-cadherin. A 15mM concentration of glucose was chosen because it is a level seen postprandially in diabetic pregnancies. We explored this by explant and perfusion methods.
We tested two chorionic villous explants methodologies to validate them for 4h and 24h duration studies, and found that a free-floating methodology which sought to replicate the in utero flow was not appropriate, as this resulted in high basal levels of endothelial VEGF and low junctional VE-cadherin, a feature that we hypothesised to be a wound healing response. Therefore, we chose the stationary explants methodology, with no simulation of flow, for our subsequent experiments.
The stationary method, where chorionic villi were incubated with hyperglycaemia (15mM glucose) vs. euglycaemia (5mM glucose) for the two different durations (4h and 24h) revealed that 15mM glucose was affecting junctional stability and the pro-permeability molecule VEGF-A after a 24h hyperglycaemic insult, but that VEGF was not affected by 4h. Given that 4h is a physiologically important timepoint (representing a postprandial glucose peak seen in diabetic pregnancies), we continued with this timepoint in further experiments.
Whilst we found no effect of 15mM glucose insult after a 4h incubation on total VEGF expression, we found that the recently discovered anti-permeability VEGFb splice variant was decreased in hyperglycaemia compared to euglycaemic explants. Furthermore, the there was a significant negative correlation between total VEGF and VEGFb levels, indicating that the ratio of the two molecules was changing in diabetic explants compared to normal explants. The diabetic explants showed no further down-regulation of VEGFb on 15mM glucose insult, indicating a tolerance of the diabetic placental vessels to hyperglycaemia.
We then investigated whether total VEGF and/or VEGFb were important predictors of vascular dysfunction as measured by an increased in leakage to 76Mr dextran-TRITC tracer in a well established perfusion model. The vascular bed was perfused with 15mM glucose administered to the maternal circuit. After a 3h hyperglycaemic perfusion, we observed high total VEGF, low VEGFb, and a loss of VE-cadherin from the endothelial junctions. These changes corresponded to a mild increase in leakage to 76Mr dextran tracer measured by counting vessels showing 'hotspots' of tracer at perivascular regions (18% of vessels leakage in the hyperglycaemic perfusions vs. 10% leakage in the euglycaemic perfusions). The percentage of vessels exhibiting tracer leakage showed a significant negative correlation with VEGFb (Spearman r value -0.8857) but not total VEGF, indicating that the former may be an important predictor of vascular dysfunction.
It would be clinically important to be able to predict placental vascular dysfunction in diabetic pregnancies. Further experiments are needed to see whether the VEGFa/VEGFb ratio can predict vascular leakage under hyperglycaemia and the other main feature of diabetic pregnancies, hyperinsulinaemic insult, and whether the hyperglycaemic insult resulting in the diabetic phenotype is reversible upon euglycaemic conditions being restored. Our studies so far have shown that the diabetic phenotype can be partly replicated, in terms of vascular leakage, with a single 15mM hyperglycaemic insult. Chronic insult may well prove to result in the fully leaky vessels observed in diabetic placentae. VEGFb might be an important predictor of this leakage, and may be clinically used for assessment of risks in diabetic pregnancies.
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