FKBPL and SIRT-1 Are Downregulated by Diabetes in Pregnancy Impacting on Angiogenesis and Endothelial Function.

Diabetes in pregnancy is associated with adverse pregnancy outcomes including preterm birth. Although the mechanisms leading to these pregnancy complications are still poorly understood, aberrant angiogenesis and endothelial dysfunction play a key role. FKBPL and SIRT-1 are critical regulators of angiogenesis, however, their roles in pregnancies affected by diabetes have not been examined before in detail. Hence, this study aimed to investigate the role of FKBPL and SIRT-1 in pre-gestational (type 1 diabetes mellitus, T1D) and gestational diabetes mellitus (GDM). Placental protein expression of important angiogenesis proteins, FKBPL, SIRT-1, PlGF and VEGF-R1, was determined from pregnant women with GDM or T1D, and in the first trimester trophoblast cells exposed to high glucose (25 mM) and varying oxygen concentrations [21%, 6.5%, 2.5% (ACH-3Ps)]. Endothelial cell function was assessed in high glucose conditions (30 mM) and following FKBPL overexpression. Placental FKBPL protein expression was downregulated in T1D (FKBPL; p<0.05) whereas PlGF/VEGF-R1 were upregulated (p<0.05); correlations adjusted for gestational age were also significant. In the presence of GDM, only SIRT-1 was significantly downregulated (p<0.05) even when adjusted for gestational age (r=-0.92, p=0.001). Both FKBPL and SIRT-1 protein expression was reduced in ACH-3P cells in high glucose conditions associated with 6.5%/2.5% oxygen concentrations compared to experimental normoxia (21%; p<0.05). FKBPL overexpression in endothelial cells (HUVECs) exacerbated reduction in tubule formation compared to empty vector control, in high glucose conditions (junctions; p<0.01, branches; p<0.05). In conclusion, FKBPL and/or SIRT-1 downregulation in response to diabetic pregnancies may have a key role in the development of vascular dysfunction and associated complications affected by impaired placental angiogenesis.

Role of A Novel Angiogenesis FKBPL-CD44 Pathway in Preeclampsia Risk Stratification and Mesenchymal Stem Cell Treatment.

CONTEXT: Preeclampsia is a leading cardiovascular complication in pregnancy lacking effective diagnostic and treatment strategies. OBJECTIVE: To investigate the diagnostic and therapeutic target potential of the angiogenesis proteins, FK506-binding protein like (FKBPL) and CD44. DESIGN AND INTERVENTION: FKBPL and CD44 plasma concentration or placental expression were determined in women pre- or postdiagnosis of preeclampsia. Trophoblast and endothelial cell function was assessed following mesenchymal stem cell (MSC) treatment and in the context of FKBPL signaling. SETTINGS AND PARTICIPANTS: Human samples prediagnosis (15 and 20 weeks of gestation; n ≥ 57), or postdiagnosis (n = 18 for plasma; n = 4 for placenta) of preeclampsia were used to determine FKBPL and CD44 levels, compared to healthy controls. Trophoblast or endothelial cells were exposed to low/high oxygen, and treated with MSC-conditioned media (MSC-CM) or a FKBPL overexpression plasmid. MAIN OUTCOME MEASURES: Preeclampsia risk stratification and diagnostic potential of FKBPL and CD44 were investigated. MSC treatment effects and FKBPL-CD44 signaling in trophoblast and endothelial cells were assessed. RESULTS: The CD44/FKBPL ratio was reduced in placenta and plasma following clinical diagnosis of preeclampsia. At 20 weeks of gestation, a high plasma CD44/FKBPL ratio was independently associated with the 2.3-fold increased risk of preeclampsia (odds ratio = 2.3, 95% confidence interval [CI] 1.03-5.23, P = 0.04). In combination with high mean arterial blood pressure (>82.5 mmHg), the risk further increased to 3.9-fold (95% CI 1.30-11.84, P = 0.016). Both hypoxia and MSC-based therapy inhibited FKBPL-CD44 signaling, enhancing cell angiogenesis. CONCLUSIONS: The FKBPL-CD44 pathway appears to have a central role in the pathogenesis of preeclampsia, showing promising utilities for early diagnostic and therapeutic purposes.

Placental vascularization indices and prediction of pre-eclampsia in high-risk women.

OBJECTIVE: To assess ability of first and second trimester Placental Vascularization Indices (PVIs) to predict pre-eclampsia (PE) in high-risk pregnancies. METHOD: PVIs derived from 3-Dimensional power Doppler imaging were measured at 11+0-13 + 6 (n = 194) and 19+0-21 + 6 weeks (n = 195). Logistic regression (LR) models used PE as the outcome. To quantify added value of PVIs to baseline characteristics in predicting PE, integrated discrimination improvement (IDI) and net reclassification improvement (NRI) indices were calculated. RESULTS: Overall rate of PE was 12% (n = 26). Lower first trimester PVIs were seen in women with PE (mean, SD); Vascularization Index (VI,%): 10.0 (6.2) v 14.7 (7.6), P = 0.005, Flow Index (FI): 37.7 (9.1) v 42.9 (10.4), P = 0.03, Vascularization Flow Index (VFI): 3.8 (2.5) v 6.6 (4.0), P < 0.001). All first trimester PVIs predicted PE in LR models adjusted for covariates. IDI and NRI analyses confirmed added clinical utility of VI (IDI 0.05, P = 0.004; NRI 0.66, P < 0.001) and VFI (IDI 0.06, P = 0.004; NRI 0.53, P = 0.91). In the second trimester, FI was lower in women with PE (39.6 (9.1) v 44.4 (8.6), P = 0.01) and predicted PE in adjusted LR models (standardised OR 0.53, 95% CI 0.29-0.97, P = 0.04). FI discriminated between cases and non-cases of PE (IDI 0.04, P = 0.04). CONCLUSION: First trimester placental vascularization indices (VI, FI and VFI) have the potential to predict PE in high-risk pregnancies, with FI remaining predictive in the second trimester.

The endogenous anti-angiogenic family of splice variants of VEGF, VEGFxxxb, are down-regulated in pre-eclamptic placentae at term.

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 VEGFxxxb, that also appear to act as inhibitors of conventional VEGFxxx-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 VEGFxxxb mRNA and protein expression. VEGFxxx and VEGFxxxb mRNA and protein were both found in normal human term placentae. VEGFxxx protein formed the majority of the total VEGF protein (980+/-195 pg/mg), whereas VEGFxxxb (11.5 pg/mg) was found to form a small part of the total VEGF protein expression (1.5+/-0.24%). Evidence for VEGF165b, VEGF121b and VEGF145b expression was found. In pre-eclamptic placentae, there was a significant down-regulation of VEGFxxxb isoforms, but a small up-regulation of VEGFxxx isoforms. In normal placenta VEGFxxxb and VEGFxxx concentrations were positively correlated (r=0.69, P<0.02), whereas in pre-eclamptic placentae, there was a significant negative correlation between VEGFxxxb and VEGFxxx 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.

Soluble vascular endothelial growth factor receptor-1 (sFlt-1) is increased throughout gestation in patients who have preeclampsia develop.

OBJECTIVE: This study was undertaken to analyze prospectively circulating vascular endothelial growth factor (VEGF) and its soluble receptor, (s) Flt-1, throughout normotensive and preeclamptic pregnancies and to assess the importance of these proteins in the development of preeclampsia. STUDY DESIGN: In this longitudinal cohort study, serum samples were collected from recruited subjects throughout pregnancy at 12, 20, 30, and 37 weeks and in the 24 hours before and after delivery. Subjects were divided retrospectively into normotensive and preeclamptic groups. Circulating VEGF and sFlt-1 concentrations were analyzed by radioimmunoassay and enzyme-linked immunosorbent assay, respectively. RESULTS: Circulating sFlt-1 and VEGF significantly increased as gestation progressed and both were further elevated in preeclampsia compared with normotensive pregnancy. Soluble Flt-1 concentrations were elevated early in gestation and were significantly increased at 30 weeks' gestation in those who subsequently developed preeclampsia. CONCLUSION: These results indicate a definite association between elevated sFlt-1 concentrations and the onset of preeclampsia suggesting that sFlt-1 is linked with disease pathogenesis.

Plasma from women with severe pre-eclampsia increases microvascular permeability in an animal model in vivo.

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. pi c (colloid osmotic pressure) was measured with a modified Hansen oncometer. Lp (hydraulic conductivity) and sigma (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 Lp or sigma. However, plasma from patients with severe pre-eclampsia resulted in a 3.8+/-0.3-fold increase in Lp and a reduction in sigma from 0.96+/-0.03 to 0.80+/-0.11. There was a significant correlation between the change in sigma and the change in Lp, 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.