The human placenta nourishes and protects the developing foetus whilst influencing maternal physiology for fetal advantage. It expresses several members of the vascular endothelial growth factor (VEGF) family including the pro-angiogenic/pro-permeability VEGF-A 165 a isoform, the anti-angiogenic VEGF-A 165 b, placental growth factor (PIGF) and their receptors, VEGFR1 and VEGFR2. Alterations in the ratio of these factors during gestation and in complicated pregnancies have been reported; however, the impact of this on feto-placental endothelial barrier integrity is unknown. The present study investigated the interplay of these factors on junctional occupancy of VE-cadherin and macromolecular leakage in human endothelial monolayers and the perfused placental microvascular bed. Whilst VEGF-A 165 a (50 ng/ml) increased endothelial monolayer albumin permeability ( P <0.0001), equimolar concentrations of VEGF-A 165 b ( P >0.05) or PlGF ( P >0.05) did not. Moreover, VEGF-A 165 b (100 ng/ml; P <0.001) but not PlGF (100 ng/ml; P >0.05) inhibited VEGF-A 165 a-induced permeability when added singly. PlGF abolished the VEGF-A 165 b-induced reduction in VEGF-A 165 a-mediated permeability ( P >0.05); PlGF was found to compete with VEGF-A 165 b for binding to Flt-1 at equimolar affinity. Junctional occupancy of VE-cadherin matched alterations in permeability. In the perfused microvascular bed, VEGF-A 165 b did not induce microvascular leakage but inhibited and reversed VEGF-A 165 a-induced loss of junctional VE-cadherin and tracer leakage. These results indicate that the anti-angiogenic VEGF-A 165 b isoform does not increase permeability in human placental microvessels or HUVEC primary cells and can interrupt VEGF-A 165 a-induced permeability. Moreover, the interplay of these isoforms with PIGF (and s-flt1) suggests that the ratio of these three factors may be important in determining the placental and endothelial barrier in normal and complicated pregnancies.

Diabetic retinopathy (DR) is one of the leading causes of blindness in the developed world. Characteristic features of DR are retinal neurodegeneration, pathological angiogenesis and breakdown of both the inner and outer retinal barriers of the retinal vasculature and retinal pigmented epithelial (RPE)–choroid respectively. Vascular endothelial growth factor (VEGF-A), a key regulator of angiogenesis and permeability, is the target of most pharmacological interventions of DR. VEGF-A can be alternatively spliced at exon 8 to form two families of isoforms, pro- and anti-angiogenic. VEGF-A 165 a is the most abundant pro-angiogenic isoform, is pro-inflammatory and a potent inducer of permeability. VEGF-A 165 b is anti-angiogenic, anti-inflammatory, cytoprotective and neuroprotective. In the diabetic eye, pro-angiogenic VEGF-A isoforms are up-regulated such that they overpower VEGF-A 165 b. We hypothesized that this imbalance may contribute to increased breakdown of the retinal barriers and by redressing this imbalance, the pathological angiogenesis, fluid extravasation and retinal neurodegeneration could be ameliorated. VEGF-A 165 b prevented VEGF-A 165 a and hyperglycaemia-induced tight junction (TJ) breakdown and subsequent increase in solute flux in RPE cells. In streptozotocin (STZ)-induced diabetes, there was an increase in Evans Blue extravasation after both 1 and 8 weeks of diabetes, which was reduced upon intravitreal and systemic delivery of recombinant human (rh)VEGF-A 165 b. Eight-week diabetic rats also showed an increase in retinal vessel density, which was prevented by VEGF-A 165 b. These results show rhVEGF-A 165 b reduces DR-associated blood–retina barrier (BRB) dysfunction, angiogenesis and neurodegeneration and may be a suitable therapeutic in treating DR.

Diabetic peripheral neuropathy affects up to half of diabetic patients. This neuronal damage leads to sensory disturbances, including allodynia and hyperalgesia. Many growth factors have been suggested as useful treatments for prevention of neurodegeneration, including the vascular endothelial growth factor (VEGF) family. VEGF-A is generated as two alternative splice variant families. The most widely studied isoform, VEGF-A 165 a is both pro-angiogenic and neuroprotective, but pro-nociceptive and increases vascular permeability in animal models. Streptozotocin (STZ)-induced diabetic rats develop both hyperglycaemia and many of the resulting diabetic complications seen in patients, including peripheral neuropathy. In the present study, we show that the anti-angiogenic VEGF-A splice variant, VEGF-A 165 b, is also a potential therapeutic for diabetic neuropathy. Seven weeks of VEGF-A 165 b treatment in diabetic rats reversed enhanced pain behaviour in multiple behavioural paradigms and was neuroprotective, reducing hyperglycaemia-induced activated caspase 3 (AC3) levels in sensory neuronal subsets, epidermal sensory nerve fibre loss and aberrant sciatic nerve morphology. Furthermore, VEGF-A 165 b inhibited a STZ-induced increase in Evans Blue extravasation in dorsal root ganglia (DRG), saphenous nerve and plantar skin of the hind paw. Increased transient receptor potential ankyrin 1 (TRPA1) channel activity is associated with the onset of diabetic neuropathy. VEGF-A 165 b also prevented hyperglycaemia-enhanced TRPA1 activity in an in vitro sensory neuronal cell line indicating a novel direct neuronal mechanism that could underlie the anti-nociceptive effect observed in vivo . These results demonstrate that in a model of Type I diabetes VEGF-A 165 b attenuates altered pain behaviour and prevents neuronal stress, possibly through an effect on TRPA1 activity.

Pre-eclampsia is a pregnancy-related condition characterized by hypertension, proteinuria and endothelial dysfunction. VEGF 165 b, formed by alternative splicing of VEGF (vascular endothelial growth factor) pre-mRNA, inhibits VEGF 165 -mediated vasodilation and angiogenesis, but has not been quantified in pregnancy. ELISAs were used to measure means±S.E.M. plasma VEGF 165 b, sEng (soluble endoglin) and sFlt-1 (soluble fms-like tyrosine kinase-1). At 12 weeks of gestation, the plasma VEGF 165 b concentration was significantly up-regulated in plasma from women who maintained normal blood pressure throughout their pregnancy (normotensive group, 4.90±1.6 ng/ml; P <0.01, as determined using a Mann-Whitney U test) compared with non-pregnant women (0.40±0.22 ng/ml). In contrast, in patients who later developed pre-eclampsia, VEGF 165 b levels were lower than in the normotensive group (0.467±0.209 ng/ml), but were no greater than non-pregnant women. At term, plasma VEGF 165 b concentrations were greater than normal in both pre-eclamptic (3.75±2.24 ng/ml) and normotensive (10.58 ng/ml±3.74 ng/ml; P >0.1 compared with pre-eclampsia) pregnancies. Patients with a lower than median plasma VEGF 165 b at 12 weeks had elevated sFlt-1 and sEng pre-delivery. Concentrations of sFlt-1 (1.20±0.07 and 1.27±0.18 ng/ml) and sEng (4.4±0.18 and 4.1±0.5 ng/ml) were similar at 12 weeks of gestation in the normotensive and pre-eclamptic groups respectively. Plasma VEGF 165 b levels were elevated in pregnancy, but this increase is delayed in women that subsequently develop pre-eclampsia. In conclusion, low VEGF 165 b may therefore be a clinically useful first trimester plasma marker for increased risk of pre-eclampsia.

PET (pre-eclamptic toxaemia) has recently been linked with alterations in production of a VEGFR1 [VEGF (vascular endothelial growth factor) receptor 1] splice variant that acts as a circulating inhibitor. We have recently described a family of naturally occurring splice variants of VEGF, termed VEGF xxx b, that also appear to act as inhibitors of conventional VEGF xxx -mediated angiogenesis. To determine whether alteration in splicing of VEGF–VEGFR family members extended beyond VEGFR1, we investigated the effect of pre-eclampsia on placental VEGF xxx b mRNA and protein expression. VEGF xxx and VEGF xxx b mRNA and protein were both found in normal human term placentae. VEGF xxx protein formed the majority of the total VEGF protein (980±195 pg/mg), whereas VEGF xxx b (11.5 pg/mg) was found to form a small part of the total VEGF protein expression (1.5±0.24%). Evidence for VEGF 165 b, VEGF 121 b and VEGF 145 b expression was found. In pre-eclamptic placentae, there was a significant down-regulation of VEGF xxx b isoforms, but a small up-regulation of VEGF xxx isoforms. In normal placenta VEGF xxx b and VEGF xxx concentrations were positively correlated ( r =0.69, P <0.02), whereas in pre-eclamptic placentae, there was a significant negative correlation between VEGF xxx b and VEGF xxx protein expression ( r =−0.8, P <0.02), indicating that there was a significant uncoupling of the splicing regulation of the VEGF isoforms. Combined with previous studies showing increased soluble VEGFR1 isoforms in human pre-eclampsia, these data suggest that there may be a common mechanism in pre-eclampsia that involves dysregulation of mRNA splicing of members of the VEGF–VEGFR axis.

Pre-eclampsia results in oedema, hypertension and proteinuria, and is associated with increased vascular permeability. A number of studies have pointed to the existence of a circulating macromolecule that induces this endothelial dysfunction. To test whether this circulating factor could increase vascular permeability, we have measured the effect of dialysed human plasma from pregnant women with mild or severe pre-eclampsia (pre-eclamptic toxaemia). Plasma was collected from patients with mild or severe pre-eclampsia and from normotensive women. Plasma was dialysed against frog Ringer's solution using a 12–14 kDa molecular-mass cut-off dialysis tubing. π c (colloid osmotic pressure) was measured with a modified Hansen oncometer. L p (hydraulic conductivity) and σ (oncotic reflection coefficient) were measured in individually perfused frog mesenteric microvessels using the Landis–Michel technique during perfusion with dialysed plasma. Perfusion of vessels with normal plasma or plasma from patients with mild pre-eclampsia did not alter either L p or σ. However, plasma from patients with severe pre-eclampsia resulted in a 3.8±0.3-fold increase in L p and a reduction in σ from 0.96±0.03 to 0.80±0.11. There was a significant correlation between the change in σ and the change in L p , suggesting that the increase in permeability was due to an increase in pore size in these vessels. A circulating macromolecule in human plasma in severe pre-eclampsia is therefore able to increase vascular permeability in an animal model. The nature of the circulating macromolecule is not known, except that it is, or is bound to, a molecule greater than 12 kDa.