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Breakdown of the blood-brain barrier is a feature of acute and chronic neurodegenerative changes, yet the relationship between astrocytes and the mature barrier remains unclear. We studied this role of astrocytes in vivo using a gliotoxin and evaluated changes in three vascular tight junction markers. Male Fisher F344 rats given systemic 3-chloropropanediol showed astrocytic loss in the inferior colliculus from 12-24 h until the lesion was repopulated 8-28 days later. Within 6 h of astrocyte loss, microvessels in this area began to demonstrate a loss of the normal paracellular localization of the transmembrane proteins occludin and claudin-5 and cytoplasmic zonula occludens-1, which correlated with focal vascular leak of dextran (10 kDa) and fibrinogen. Platelet endothelial adhesion molecule-1 staining revealed that there was no loss of the endothelial lining. Between 4-8 days, severe downregulation of tight junction protein expression was observed, which subsequently returned over the same time period as astrocytes repopulated the lesion. Unexpectedly, dextran and fibrinogen leak from vessels had ceased at 6 days, well before the return of occludin and claudin-5 to appropriate paracellular domains. Control nonvulnerable cortical tissue showed no change in astrocyte morphology and tight junction expression over the same time course. Our data supports a primary role for astrocytic contact in the expression of occludin, claudin-5, and zonula occludens-1 in the mature brain vasculature in vivo. However, barrier integrity to dextran (10 kDa) and fibrinogen can be restored in the absence of astrocytes and tight junction proteins (occludin, claudin-5, and zonula occludens-1).

In the human placenta, the angioarchitecture of fetal vessels lying in maternal blood is useful for nutrient uptake, but it makes the synthesis, maturation and functioning of placental vessels vulnerable to any alterations in the fetal and maternal environment. This review discusses how the maternal diabetic milieu, and the resultant fetal hyperglycemia and hyperinsulinemia, may act together to produce an altered placental vascular phenotype, which includes increased angiogenesis, altered junctional maturity, increased vascular endothelial-like growth factor (VEGF), altered VEGF and insulin receptor profiles, and upregulation of genes involved in signal transduction, transcription and mitosis in placental endothelial cells. The placental vascular dysfunction does extend to other fetal vascular beds including endothelial cells from umbilical vessels, where there are reports of elevated basal iNOS activity and altered sensitivity to insulin. There is emerging evidence of epigenetic modulation of fetal endothelial genes in diabetes and long-term vascular consequences of this. Thus, placental vascular dysfunction in diabetes may be contributing to and describing disturbances in the fetal vasculature, which may produce an overt pathological response in later life if challenged with additional cardiovascular stresses.

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The development and functioning of the human fetoplacental vascular system are vulnerable to the maternal diabetic milieu. These vessels are in direct continuum with the fetal vascular system and are therefore also vulnerable to fetal endocrine derangements. Increased angiogenesis, altered junctional maturity and molecular occupancy, together with increased leakiness, constitute a well-described phenotype of vessels in the Type 1 diabetic human placenta and can be related to increased levels of placental vascular endothelial growth factor. The causes of these observed changes, whether maternal hyperglycaemia or fetal hyperinsulinaemia, still remain to be shown in the human placenta. Mechanistic studies using different vascular systems have shown high glucose and insulin to have profound vascular effects, with elevations in vascular endothelial growth factor, nitric oxide and protein kinase C being behind alterations in junctional adhesion molecules such as occludin and vascular endothelial-cadherin and vascular leakage of albumin. The role of advanced glycation products and oxidative stress in this vascular pathology is also discussed. The altered molecular mechanisms underlying the vascular changes in the diabetic human placenta may reflect similar consequences of high glucose and hyperinsulinaemia.

The outer blood-retinal barrier is composed of a monolayer of retinal pigment epithelium, Bruch's membrane and the choriocapillaris which is fenestrated. Endothelial proliferation and breaching of Bruch's membrane leads to the neovascular form of age-related macula degeneration (ARMD). The aim of this study was to generate an in vitro model that mimics more faithfully the phenotype of the choriocapillaris and the trilayer architecture in vitro. A trilayer culture model was generated with retinal pigment epithelium (ARPE-19) cell cultures on the epithelial surface of amniotic membrane and with human umbilical vein-derived endothelial cells on the other surface. A control model for the effect of retinal pigment epithelium on endothelial changes was generated with corneal epithelial cells replacing the ARPE-19. Both human umbilical vein-derived endothelial and ARPE-19 cells formed confluent monolayers on respective surfaces of the amnion. The human umbilical vein-derived endothelial cells in the trilayer became fenestrated when co-cultured with the ARPE-19 cells, but not with corneal epithelial cells, or when grown as monolayers on the amnion, showing a loss of fidelity of origin in the presence of ARPE-19 cells. These cells also revealed VE-cadherin and ZO-1 at cell-cell contacts from 24 h in the trilayer. The tight junctional molecules, occludin and ZO-1, were localized to cell-cell contact regions in the retinal pigment epithelium, both in the monolayer and in the trilayer system. Permeability of the trilayer was tested by using fluorescein and fluorescein-conjugated tracers under flow. At 72 h the trilayer severely restricted transfer of sodium fluorescein (NaF) (ten-fold reduction) whilst transfer of a 4 kDa FITC-conjugated dextran was virtually occluded, confirming a restrictive barrier. Ultrastructural studies showed the retinal pigment epithelium monolayer was polarized with microvilli present on the apical surface. Paracellular clefts showed numerous tight junctional-like appositions, similar to that seen on amnion alone. This study demonstrates that ARPE-19 and human umbilical vein-derived endothelial cells can be co-cultured on the amniotic membrane and that the resultant cross-talk leads to formation of a fenestrated endothelium, whilst maintaining a polarized restrictive epithelial layer. The fenestrated endothelial phenotype achieved in this human in vitro trilayer model is a first and offers an outer-retinal barrier which approaches the in vivo state and has potential for studies into induced junctional disruption, endothelial proliferation and migration: features of ARMD.

Aims/hypothesis. Increased angiogenesis of fetoplacental vessels is a feature of pregnancies complicated by Type 1 diabetes mellitus, but the underlying molecular mechanisms are unknown. This investigation tests whether the diabetic maternal environment alters the phenotypic expression of placental vascular endothelial cadherin and β-catenin, which have been implicated as key molecules in barrier formation and angiogenesis in the endothelium. Methods. Term placental microvessels from normal pregnancies (n=8) and from those complicated by Type 1 diabetes (n=8) were perfused with 76-M r dextran tracers (1 mg/ml) and subjected to immunocytochemistry, immunoblotting and microscopy. Junctional integrity, localisation and phosphorylation were investigated along with total protein levels of vascular endothelial cadherin, β-catenin and vascular endothelial growth factor. Stereological sampling and estimation tools were used to quantify aspects of angiogenesis and endothelial proliferation. Results. In the Type 1 diabetic placentae, junctional localisations of vascular endothelial cadherin and β-catenin altered significantly, with more than 50% of microvessels showing complete loss of immunoreactivity and with no overall loss of total protein. Tracer leakage was associated with these vessels. There was a two-to three-fold increase in vessels showing junctional phospho-tyrosine immunoreactivity and hyperphosphorylated β-catenin. Vascular endothelial growth factor levels were higher in these placentae. A fourfold increase in endothelial proliferation was observed, along with an increase in total length of capillaries without any change in luminal diameter. Conclusions/interpretation. Molecular perturbations of vascular endothelial cadherin and β-catenin occur in fetoplacental vessels of pregnancies complicated by Type 1 diabetes. Phosphorylation and loss of these molecules from the adherens junctional domains may be influenced in part by the elevated levels of vascular endothelial growth factor in the placenta. Perturbations of the junctional proteins may explain the observed breach in barrier integrity and may contribute to the mechanisms that drive proliferation and increases in capillary length.