Pravastatin for early-onset pre-eclampsia: a randomised, blinded, placebo-controlled trial.

OBJECTIVE: Women with pre-eclampsia have elevated circulating levels of soluble fms-like tyrosine kinase-1 (sFlt-1). Statins can reduce sFlt-1 from cultured cells and improve pregnancy outcome in animals with a pre-eclampsia-like syndrome. We investigated the effect of pravastatin on plasma sFlt-1 levels during pre-eclampsia. DESIGN: Blinded (clinician and participant), proof of principle, placebo-controlled trial. SETTING: Fifteen UK maternity units. POPULATION: We used a minimisation algorithm to assign 62 women with early-onset pre-eclampsia (24+0 -31+6  weeks of gestation) to receive pravastatin 40 mg daily (n = 30) or matched placebo (n = 32), from randomisation to childbirth. PRIMARY OUTCOME: Difference in mean plasma sFlt-1 levels over the first 3 days following randomisation. RESULTS: The difference in the mean maternal plasma sFlt-1 levels over the first 3 days after randomisation between the pravastatin (n = 27) and placebo (n = 29) groups was 292 pg/ml (95% CI -1175 to 592; P = 0.5), and over days 1-14 was 48 pg/ml (95% CI -1009 to 913; P = 0.9). Women who received pravastatin had a similar length of pregnancy following randomisation compared with those who received placebo (hazard ratio 0.84; 95% CI 0.50-1.40; P = 0.6). The median time from randomisation to childbirth was 9 days (interquartile range [IQR] 5-14 days) for the pravastatin group and 7 days (IQR 4-11 days) for the placebo group. There were three perinatal deaths in the placebo-treated group and no deaths or serious adverse events attributable to pravastatin. CONCLUSIONS: We found no evidence that pravastatin lowered maternal plasma sFlt-1 levels once early-onset pre-eclampsia had developed. Pravastatin appears to have no adverse perinatal effects. TWEETABLE ABSTRACT: Pravastatin does not improve maternal plasma sFlt-1 or placental growth factor levels following a diagnosis of early preterm pre-eclampsia #clinicaltrial finds.

Effects of glycol-split low molecular weight heparin on placental, endothelial, and anti-inflammatory pathways relevant to preeclampsia.

Low molecular weight heparin (LMWH) is being investigated as a potential preventative therapy against preeclampsia. There is evidence suggesting that LMWH may prevent preeclampsia through anticoagulation-independent mechanisms. In this study, we compared the in vitro placental, endothelial, and anti-inflammatory effects of an LMWH (dalteparin) with a nonanticoagulant, glycol-split heparin derivative (gsHep). In contrast with dalteparin, gsHep did not interact with antithrombin III, possess significant anti-Factor Xa activity, or significantly prolong in vitro plasma clotting time. However, dalteparin and gsHep were otherwise mechanistically similar, both interacting with soluble fms-like tyrosine kinase-1 (sFlt1) and promoting release of the pro-angiogenic protein placental growth factor, but not the antiangiogenic sFlt1, from healthy placental villous explants. Placental explant media pretreated with dalteparin or gsHep significantly stimulated endothelial cell tube formation compared to untreated explants. Lastly, dalteparin and gsHep both significantly suppressed inflammation by inhibiting complement activation and leukocyte adhesion to endothelial cells that were activated using serum from preeclamptic women. Our data suggest that nonanticoagulant heparin derivatives may be utilized as a tool to distinguish the anticoagulation-independent mechanisms of LMWH, and provide insight into the role of anticoagulation in the prevention of preeclampsia.

Effects of simvastatin, rosuvastatin and pravastatin on soluble fms-like tyrosine kinase 1 (sFlt-1) and soluble endoglin (sENG) secretion from human umbilical vein endothelial cells, primary trophoblast cells and placenta.

BACKGROUND: Preeclampsia is associated with the placental release of soluble fms-like tyrosine kinase 1 (sFlt-1) and soluble endoglin (sENG). These anti-angiogenic factors cause hypertension and multi-organ injury. Pravastatin decreases placental secretion of sFlt-1 in vitro and is currently being examined in clinical trials as a potential treatment for preeclampsia. However, it is possible that different classes of statins may be more potent at decreasing sFlt-1 secretion. We compared the relative potency of three different generations of statins on sFlt-1 and sENG secretion from human endothelial cells, trophoblast cells, and placenta explants. METHODS: We performed functional experiments using primary human umbilical vein endothelial cells, trophoblast cells and preterm preeclamptic placental explants to assess the affect of simvastatin, rosuvastatin and pravastatin on sFlt-1 and sENG secretion and compared the relative potency of each statin at reducing these factors (Inhibitory Concentration 50). Furthermore we assessed the effect of each statin on the antioxidant and cytoprotective enzyme, heme-oxygenase 1. RESULTS: All statins reduced sFlt-1 secretion from endothelial cells, trophoblasts and preterm preeclamptic placental explants. Simvastatin was the most potent inhibitor of sFlt-1 secretion from endothelial cells (IC 50 3.2 µM), trophoblast cells (IC 50 61.4 µM) and placental explants. Simvastatin was 28 times and 3 times more potent at reducing sFlt-1 secretion from endothelial cells and 85 times and 33 times more potent at reducing sFlt-1 secretion from trophoblast cells than pravastatin or rosuvastatin respectively. All statins increased sENG secretion from endothelial cells however did not change secretion from placental explants. While all statins up-regulated heme-oxygenase 1 in endothelial cells, only simvastatin up-regulated its expression in placenta from patients with preterm preeclampsia. CONCLUSION: Simvastatin may be a more potent inhibitor of sFlt-1 secretion from endothelial cells, trophoblast cells and placenta from women with preterm preeclampsia than either pravastatin or rosuvastatin.

Inhibition of lectin-like oxidized low-density lipoprotein receptor 1 protects against plasma/hypoxia-mediated trophoblast dysfunction associated with preeclampsia.

BACKGROUND: Preeclampsia (PE) is associated with oxidative stress in the maternal circulation and placenta. This study aimed to determine if inhibition of lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1) gives protection against oxidative stress-mediated trophoblast dysfunction. METHODS: Plasma and placenta samples were obtained from 106 women with PE and 106 women with normal pregnancy (NP). Oxidized low-density lipoprotein (oxLDL) and soluble LOX-1 levels were determined by enzyme-linked immunoassay. Placental LOX-1 expression was determined by western blotting. Trophoblasts were subjected to hypoxia and treated with pooled plasma from patients with PE. Expression levels of placenta growth factor (PIGF) and the soluble form of the PIGF receptor (sFlt-1) in trophoblasts were determined. RESULTS: Plasma concentrations of oxLDL and sLOX-1 were significantly over-expressed and LOX-1 protein expression in the placenta was significantly increased in PE patients compared with matched NP controls (both p < 0.05). Exposure of trophoblasts to hypoxia and pooled PE plasma induced overexpression of sFlt-1 and downregulation of PIGF. These effects were inhibited by the LOX-1 inhibitor TS20. CONCLUSION: LOX-1 accumulation may contribute to the pathogenesis of PE by promoting sFlt-1 production in trophoblasts, suggesting that oxidative stress may be an important mediator regulating angiogenic pathways in trophoblasts.

VEGF receptor-1 involvement in pericyte loss induced by Escherichia coli in an in vitro model of blood brain barrier.

The key aspect of neonatal meningitis is related to the ability of pathogens to invade the blood-brain barrier (BBB) and to penetrate the central nervous system. In the present study we show that, in an in vitro model of BBB, on the basis of co-culturing primary bovine brain endothelial cells (BBEC) and primary bovine retinal pericytes (BRPC), Escherichia coli infection determines changes of transendothelial electrical resistance (TEER) and permeability (Pe) to sodium fluorescein. In the co-culture model, within BBEC, bacteria are able to stimulate cytosolic and Ca(2+)-independent phospholipase A2 (cPLA2 and iPLA2 ) enzyme activities. In supernatants of E. coli-stimulated co-cultures, an increase in prostaglandins (PGE2) and VEGF production in comparison with untreated co-cultures were found. Incubation with E. coli in presence of AACOCF3 or BEL caused a decrease of PGE2 and VEGF release. SEM and TEM images of BBEC and BRPC showed E. coli adhesion to BBEC and BRPC but only in BBEC the invasion occurs. VEGFR-1 but not VEGFR-2 blockade by the specific antibody reduced E. coli invasion in BBEC. In our model of BBB infection, a significant loss of BRPC was observed. Following VEGFR-1, but not VEGFR-2 blockade, or in presence of AACOCF3 or BEL, elevated TEER values, reduced permeability and BRPC loss were found. These data suggest that VEGFR-1 negatively regulates BRPC survival and its blockade protects the barrier integrity. PGs and VEGF could exert a biological effect on BBB, probably by BRPC coverage ablation, thus increasing BBB permeability. Our results show the role played by the BBEC as well as BRPC during a bacterial attack on BBB. A better understanding of the mechanisms by which E. coli enter the nervous system and how bacteria alter the communication between endothelial cells and pericytes may provide exciting new insight for clinical intervention.

Hyperglycemia impairs cytotrophoblast function via stress signaling.

OBJECTIVE: Diabetes mellitus is a risk factor for preeclampsia. Cytotrophoblast (CTB) invasion is facilitated from the conversion of plasminogen to plasmin by urokinase plasminogen activator (uPA), regulated by plasminogen activator inhibitor 1 (PAI-1), and may be inhibited in preeclampsia. This study assessed signaling mechanisms of hyperglycemia-induced CTB dysfunction. STUDY DESIGN: Human CTBs were treated with 45, 135, 225, 495, or 945 mg/dL glucose for 48 hours. Some cells were pretreated with a p38 inhibitor (SB203580) or a peroxisome proliferator-activated receptor-gamma (PPAR-γ) ligand (rosiglitazone). Expression of uPA, PAI-1, and PPAR-γ levels and p38 mitogen-activated protein kinase phosphorylation were measured by Western blot in cell lysates. Messenger ribonucleic acid of uPA and PAI-1 was measured by quantitative polymerase chain reaction. Levels of interleukin-6, angiogenic (vascular endothelial growth factor [VEGF], placenta growth factor [PlGF]) and antiangiogenic factors (soluble fms-like tyrosine kinase-1 [sFlt-1], soluble endoglin [sEng]) were measured in the media by enzyme-linked immunosorbent assay kits. Statistical comparisons were performed using analysis of variance with a Duncan's post-hoc test. RESULTS: Both uPA and PAI-1 protein and messenger ribonucleic acid were down-regulated (P < .05) in CTBs treated with 135 mg/dL glucose or greater compared with basal (45 mg/dL). The sEng, sFlt-1, and interleukin-6 were up-regulated, whereas the VEGF and PlGF were down-regulated by 135 mg/dL glucose or greater. p38 phosphorylation and PPAR-γ were up-regulated (P < .05) in hyperglycemia-treated CTBs. The SB203580 or rosiglitazone pretreatment showed an attenuation of glucose-induced down-regulation of uPA and PAI-1. CONCLUSION: Hyperglycemia disrupts the invasive profile of CTB by decreasing uPA and PAI-1 expression; down-regulating VEGF and PlGF; and up-regulating sEng, sFlt-1, and interleukin-6. Attenuation of CTB dysfunction by SB203580 or rosiglitazone pretreatment suggests the involvement of stress signaling.

Heparin use during dialysis sessions induces an increase in the antiangiogenic factor soluble Flt1.

BACKGROUND: Soluble Flt1 (sFlt1) is a potent inhibitor of vascular endothelial growth factor, secreted mainly by the placenta, endothelial cells and monocytes. Increased sFlt1 serum levels correlate with endothelial dysfunction and cardiovascular complications in dialysis patients. However, the impact of dialysis by itself on sFlt1 serum levels remains unknown. METHODS: We assessed sFlt1 kinetics during dialysis and the impact of different dialysis techniques [high-flux haemodialysis (HD), haemodiafiltration (HDF)] and heparinization procedures on sFlt1 serum levels in 48 patients on regular dialysis. RESULTS: sFlt1 serum levels increased as early as 1 min after the start of dialysis and peaked at 15 min before returning to baseline at 4 h [mean peak level 2551 pg/mL, versus 102 before dialysis (P < 0.0001)]. sFlt1 kinetics were similar with two different dialysis membranes. In contrast, when unfractionated heparin (UH) and low-molecular-weight heparin (LMWH) were omitted during dialysis (HD or pre-dilution HDF), no significant increase in sFlt1 levels occurred. Conversely, delayed administration of LMWH (after 30 min of a heparin-free HD) induced a sharp increase in sFlt1. Similarly, when UH and LMWH were omitted and citrate-based dialysate or a heparin-coated membrane was used, sFlt1 levels remained unchanged. When heparinization procedures were the same, no difference in sFlt1 levels was noted between HD and HDF. In vitro, UH and LMWH failed to induce sFlt1 release by monocytes from controls or HD patients. These findings suggest that priming of monocytes on the extracorporeal circuit is required for heparin-induced sFlt1 release or that endothelial cells contribute to this increase. CONCLUSIONS: Our results indicate that heparin-based HD induces a major sFlt1 release, which may exacerbate the anti-angiogenic state and thus endothelial dysfunction, commonly found in dialysis patients.

Angiogenic growth factors augment K-Cl cotransporter expression in erythroid cells via hypoxia-inducible factor-1α.

The potassium chloride cotransporters (KCC) family of proteins are widely expressed and are involved in the transepithelial movement of potassium and chloride ions and the regulation of cell volume. KCC activity is high in reticulocytes, and contributes to the dehydration of sickle red blood cells. Because plasma levels of both vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) are elevated in sickle cell individuals, and VEGF has been shown to increase KCC expression in other cells, we hypothesized that VEGF and PlGF influence KCC expression in erythroid cells. Both VEGF and PlGF treatment of human erythroid K562 cells increased both mRNA and protein levels of KCC1, KCC3b, and KCC4. VEGF- and PlGF-mediated cellular signaling involved VEGF-R1 and downstream effectors, specifically, PI-3 kinase, p38 MAP kinase, mTOR, NADPH-oxidase, JNK kinase, and HIF-1α. VEGF and PlGF-mediated transcription of KCC3b and KCC4 involved hypoxia response element (HRE) motifs in their promoters, as demonstrated by promoter analysis, EMSA and ChiP. These results were corroborated in vivo by adenoviral-mediated overexpression of PlGF in normal mice, which led to increased expression of mKCC3 and mKCC4 in erythroid precursors. Our studies show that VEGF and PlGF regulate transcription of KCC3b and KCC4 in erythroid cells via activation of HIF-1α, independent of hypoxia. These studies provide novel therapeutic targets for regulation of cell volume in RBC precursors, and thus, amelioration of dehydration in RBCs in sickle cell disease.

An Examination of the Effect of Aspirin and Salicylic Acid on Soluble Fms-like Tyrosine Kinase-1 Release from Human Placental Trophoblasts.

Low-dose aspirin (LDA) is efficacious in preventing preeclampsia, but its mechanism of action is unclear. Conflicting evidence suggests that it may inhibit placental trophoblast release of soluble fms-like tyrosine kinase-1 (sFlt1), a key mediator of preeclampsia. We examined whether, and at what concentrations, aspirin and its principal metabolite, salicylic acid, modulate sFlt1 release and/or expression in trophoblasts. Human trophoblast lines BeWo and HTR-8/SVneo were cultured; BeWo cells were also treated with 1% oxygen vs. normoxia to mimic hypoxia in preeclamptic placentas. Cells were treated with aspirin or salicylic acid vs. vehicle for 24 h at concentrations relevant to LDA and at higher concentrations. Protein concentrations (ELISA) and mRNA expression (RT-PCR) of sFlt1 were determined. Under normoxia, LDA-relevant concentrations of aspirin (10-50 µmol/L) or salicylic acid (20-100 µmol/L) had no significant effect on sFlt1 protein release or mRNA expression in BeWo cells. However, inhibition was observed at higher concentrations (1 mmol/L for aspirin and ≥200 µmol/L for salicylic acid). Hypoxia enhanced sFlt1 protein release and mRNA expression in BeWo cells, but these responses were not significantly affected by either aspirin or salicylic acid at LDA concentrations. Similarly, neither drug altered sFlt1 protein secretion or mRNA expression in normoxic HTR-8/SVneo cells at LDA concentrations. We suggest that direct modulation of trophoblast release or expression of sFlt1 is unlikely to be a mechanism underlying the clinical efficacy of LDA in preeclampsia.

AICAR administration ameliorates hypertension and angiogenic imbalance in a model of preeclampsia in the rat.

Previous studies suggest restoration of angiogenic balance can lower blood pressure and improve vascular endothelium function in models of preeclampsia. Our laboratory has recently reported exercise training mitigates hypertension in an animal model of preeclampsia, but the mechanisms are unknown. AMP-activated protein kinase (AMPK) is stimulated during exercise and has been shown to increase expression of VEGF. Therefore, the purpose of this study was to determine whether AICAR (5-aminoimidazole-4-carboxamide-3-ribonucleoside), a potent AMPK stimulator, would increase circulating VEGF, improve angiogenic potential, decrease oxidative stress, and abrogate placental ischemia-induced hypertension. In rats, reduced uteroplacental perfusion pressure (RUPP) was induced on day 14 of gestation by introducing silver clips on the inferior abdominal aorta and ovarian arteries. AICAR was administered intraperitoneally (50 mg/kg b.i.d.) days 14-18, and blood pressure and tissues were collected on day 19. RUPP-induced hypertension was ameliorated (P < 0.05) with AICAR versus RUPP. AICAR increased (P < 0.05) plasma VEGF and decreased (P < 0.05) plasma soluble VEGF receptor-1 in the RUPP + AICAR versus RUPP. Antioxidant capacity was restored (P < 0.05) by AICAR in RUPP placenta. Renal and placental catalase activity was decreased (P < 0.05) in RUPP + AICAR versus RUPP. Angiogenic potential was increased (P < 0.05) in RUPP + AICAR versus RUPP. Fetal and placental weights were unaffected by AICAR. Placental AMPK phosphorylation was increased (P < 0.05) in RUPP + AICAR versus normal pregnant and RUPP. These findings suggest AICAR may be useful to mitigate angiogenic imbalance, renal, and placental oxidative stress and increase in blood pressure associated with RUPP hypertension. Furthermore, placental AMPK phosphorylation was observed only in the setting of ischemia.

KH902 suppresses high glucose-induced migration and sprouting of human retinal endothelial cells by blocking VEGF and PIGF.

AIMS: Vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) are upregulated in many ocular neovascular diseases such as diabetic retinopathy (DR). KH902 is a recombinant fusion protein with its binding ligand taken from the domains of VEGF receptor-1 (VEGFR-1) and VEGF receptor-2 (VEGFR-2) and can bind all VEGF-A isoforms and PlGF. The aim of this study was to investigate the underlying mechanisms of anti-angiogenic effects of KH902. METHODS: The toxic effect of KH902 on cultured human retinal endothelial cells (HRECs) was measured by Annexin V/PI staining and MTT assay. The concentrations of secreted VEGF and PlGF were measured by ELISA. The migration of HRECs was assessed by scratch wound and transwell assay. The sprouting of HRECs was determined by tube formation assay. The protein levels of Src, p-Src, PI3K, Akt1, p-Akt1, Erk1/2 and p-Erk1/2 were measured by Western blot. RESULTS: KH902 at the concentrations from 100 ng/ml to 100 µg/ml had no cytotoxicity to cultured HRECs. KH902 bound not only VEGF165, but also PlGF that were secreted by HRECs under high glucose condition. A 500 ng/ml of KH902 significantly suppressed high glucose-induced migration and sprouting of HRECs through downregulating the expression of PI3K and inhibiting the activation of Src, Akt1 and Erk1/2. CONCLUSION: Our study indicates that KH902 suppresses high glucose-induced migration and sprouting of HRECs through not only binding VEGF, but also PlGF to inhibit the activation of Src-Akt1-Erk1/2 pathway. KH902 is a drug that potentially inhibits angiogenic pathways involving in DR or other ocular neovascular diseases.

[Investigation of the venodilatory effect of vascular endothelial growth factor (VEGF) in rat gingiva]. / A vaszkularis endoteliális növekedési faktor (VEGF) venulákra kifejtett hatásának vizsgálata patkány fogínyben.

VEGF induces proliferation of endothelial cells, stimulates angiogenesis, and increases vascular permeability in many organs. Nevertheless, we have only limited information about its role on gingival hemodynamics, especially in venules. Therefor the aim of this study was to assess the acute circulatory effects of VEGF on rat gingival venules by means of the following protocol. Wister rats (n=63) were devided into five study groups after anesthesia; each animal received 10 microl of experimental solution dripped onto the lower interincisal gingiva. The groups included: 1) saline control (after the experiment, gingiva was excised for VEGF receptor 2 [VEGFR2] immunohistochemistry); 2) VEGF (0.1, 1, 10, or 50 microg/ml); 3) VEGF2 receptor antagonist 5-((7-benzyloxyquinazolin-4-yl)amino)-4-fluoro-2-methyl-phenol-hydrochloride (ZM323881; 20 microg/ml); 4) ZM323881 (20 microg/ml) followed by VEGF application (50 microg/ml after 15 minutes); and 5) VEGF (10 microg/ml), these rats were premedicated with nitric oxide (NO) synthase blocker (NG-nitro-L-arginine-methyl-ester [L-NAME]; 1 mg/ml in drinking water) for 1 week before the experiment. Changes in gingival superficial venule diameter were measured by vital microscopy prior to and 1, 5, 15, 30, and 60 minutes after the administration of the experimental solutions. According to our findings, VEGF dose-dependently increased the venular diameter compared to saline. ZM323881 alone did not cause any alteration. Premedication with ZM323881 or L-NAME decreased the dilatory effects of VEGF. Occassionally moderate VEGFR2 immunohistochemical labeling was observed in the wall components of the venules. Concluding our results we can say, that there is no remarkable VEGF production under physiologic circumstances in rat gingiva, but VEGF is able to increase gingival blood flow through the activation of VEGF2 receptors and consequent NO release.

Hemodynamic, vascular, and reproductive impact of FMS-like tyrosine kinase 1 (FLT1) blockade on the uteroplacental circulation during normal mouse pregnancy.

To investigate the role of FMS-like tyrosine kinase 1 (FLT1, also known as VEGFR1) signaling during pregnancy, mice were injected with anti-FLT1 neutralizing antibody (Ab) beginning on Gestational Day 8 or 12 and every other day thereafter until Day 18; vehicle-only injected mice served as controls. Uterine artery blood flow was measured with ultrasound on Days 13 and 18, and morphometric measurements of the uterine arcade were carried out on Day 19 to provide a measure of gestational vascular remodeling; reproductive performance was evaluated by determining litter size, resorption rates, and pup and placental weights. Ab injections beginning on Day 8 or Day 12 resulted in significant reductions of uterine artery peak systolic and diastolic flows at Days 13 and 18. In addition, normal reproductive function was compromised, as evidenced by a significant reduction in average number of viable pups along with enhanced resorption rates. Reproductive performance was also significantly compromised in this group, although less severely. There was no evidence of a reduction in main uterine artery diameters, though arterial distensibility was reduced, and the diameter of the main uterine vein was significantly smaller in the Ab-injected mice. Significant reductions in main uterine artery and segmental artery length were also noted. Placental and pup weights were similar in all the groups. FLT1 inhibition during murine pregnancy impaired blood flow to the fetal-placental unit, compromised several indices of vascular remodeling, reduced fecundity, and increased fetal reabsorptions. The effects of FLT1 inhibition are most pronounced when targeted during early pregnancy.

Effects of intravitreal injection of KH902, a vascular endothelial growth factor receptor decoy, on the retinas of streptozotocin-induced diabetic rats.

AIMS: KH902 is a fusion protein that can bind vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) through its binding ligand taken from the domains of VEGF receptor 1 and VEGF receptor 2 (VEGFR2). This study was to investigate the effects of intravitreal injection of KH902 on the retinas of streptozotocin-induced diabetic rats. METHODS: Two weeks after induction of diabetes, the left eyes of diabetic rats in each group received an intravitreal injection of phosphate-buffered saline (PBS), Avastin or KH902 solution, respectively. Four weeks after intravitreal injection, retinal electrophysiological function and the integrity of inner blood retinal barrier (iBRB) were measured by electroretinogram and Evans blue perfusion. The protein levels of VEGF signal pathway were assayed by western blot. The expression and distribution of claudin-5 and occludin were analysed by double immunofluorescent staining under confocal microscope. The expression of VEGFR2 and PlGF was measured by immunohistochemistry. RESULTS: Four weeks after intravitreal injection, KH902-treated rats had better retinal electrophysiological function, less retinal vessel leakage and lower levels of VEGFR2, PI3K, AKT, p-AKT, p-ERK and p-SRC than PBS or Avastin-treated rats. The distribution of claudin-5 and occludin in the retinal vessels of diabetic rats treated by KH902 was smoother and more uniform than those of diabetic rats treated by PBS or Avastin. The expression of PlGF and VEGFR2 in KH902-treated rats was decreased compared with those in PBS or Avastin-treated rats. CONCLUSIONS: KH902 could improve retinal electrophysiological function and inhibit the breakdown of iBRB by inhibiting the expression of VEGFR2, PlGF and PI3K, and the activation of SRC, AKT and ERK.

Uncaria tomentosa extract (AC-11) improves pregnancy hypertension together with suppression of sFlt-1 and sEng.

Disruption of well-controlled reproductive functions leads to pregnancy complications such as hypertensive disorders of pregnancy (HDP). Uncaria tomentosa (Wild), known as cat's claw, is widely used for the treatment of a various types of health problems; AC-11 (AC-11®, hot-water extract of U. tomentosa) is unique phytochemical compound and has potential roles as anti-inflammatory or anti-oxidant processes. We investigated whether AC-11 has a protective effect on pathogenesis of HDP in vivo and production of anti-angiogenic factors (sFlt-1 and sEng, major factors for the onset of HDP) in in vitro. Non-pregnant or pregnant mice were administered AC-11 (4 mg/mL), then, angiotensin II (Ang II) was subcutaneously infused to increase blood pressure. Human placental tissues or human umbilical vein endothelial cells (HUVECs) were incubated with or without AC-11. Treatment with AC-11 significantly reduced blood pressure induced by Ang II infusion. The population of CD8+T cells, the ratio of CD8/CD4, and plasma interleukin-6 levels were increased by Ang II infusion, and were decreased by AC-11 both in pregnant and non-pregnant mice. In pregnant mice, plasma levels of sFlt-1 and sEng were decreased by AC-11. In in vitro cell culture of HUVECs or placental tissue culture, treatment with AC-11 significantly inhibited secretion of sFlt-1 and sEng. We suggest a novel role of AC-11 in regulating blood pressure by controlling the balance of T cell population and inflammatory cytokine production both in non-pregnant and pregnant conditions. In addition, AC-11 inhibits HDP-related factors, including sFlt-1 and sEng, suggesting that AC-11 may useful for relieving HDP.

Esomeprazole and sulfasalazine in combination additively reduce sFlt-1 secretion and diminish endothelial dysfunction: potential for a combination treatment for preeclampsia.

Development and repurposing of therapies that show promise in the prevention or treatment of preeclampsia would be a major advance for the obstetrics field. We recently identified esomeprazole and sulfasalazine as potential candidates for the treatment of preeclampsia. Both reduce placental and endothelial secretion of sFlt-1 and sENG and mitigate endothelial dysfunction in vitro. Here we assessed whether esomeprazole and sulfasalazine in combination would additively attenuate the elevated release of anti-angiogenic factors and markers of endothelial dysfunction, key characteristics of preeclampsia. Primary placental tissue and cells, and primary endothelial cells were treated with esomeprazole and sulfasalazine alone and in combination. We assessed secretion of sFlt-1 and sENG and performed in vitro assays of endothelial dysfunction. Combining esomeprazole and sulfasalazine in lower concentrations caused an additive reduction in sFlt-1 secretion in primary cytotrophoblasts, placental explants and endothelial cells. No additive reduction was observed in sENG secretion when esomeprazole and sulfasalazine were combined. Together, esomeprazole and sulfasalazine additively reduced TNF-α-induced VCAM and ET-1 mRNA expression, and monocyte adhesion to endothelial cells. In conclusion, combining esomeprazole and sulfasalazine additively reduced secretion of sFlt-1 and markers of endothelial dysfunction. Combined administration of esomeprazole and sulfasalazine may provide a more effective treatment or prevention for preeclampsia compared to either as single agents.

Vandetanib (ZD6474), a dual inhibitor of vascular endothelial growth factor receptor (VEGFR) and epidermal growth factor receptor (EGFR) tyrosine kinases: current status and future directions.

Vandetanib is a novel, orally available inhibitor of different intracellular signaling pathways involved in tumor growth, progression, and angiogenesis: vascular endothelial growth factor receptor-2, epidermal growth factor receptor, and REarranged during Transfection tyrosine kinase activity. Phase I clinical trials have shown that vandetanib is well tolerated as a single agent at daily doses < or =300 mg. In the phase II setting, negative results were observed with vandetanib in small cell lung cancer, metastatic breast cancer, and multiple myeloma. In contrast, three randomized phase II studies showed that vandetanib prolonged the progression-free survival (PFS) time of patients with non-small cell lung cancer (NSCLC) as a single agent when compared with gefitinib or when added to chemotherapy. Rash, diarrhea, hypertension, fatigue, and asymptomatic QTc prolongation were the most common adverse events. Antitumor activity was also observed in medullary thyroid cancer. Four randomized phase III clinical trials in NSCLC are exploring the efficacy of vandetanib in combination with docetaxel, the Zactima in cOmbination with Docetaxel In non-small cell lung Cancer (ZODIAC) trial, or with pemetrexed, the Zactima Efficacy with Alimta in Lung cancer (ZEAL) trial, or as a single agent, the Zactima Efficacy when Studied versus Tarceva (ZEST) and the Zactima Efficacy trial for NSCLC Patients with History of EGFR-TKI chemo-Resistance (ZEPHYR) trials. Based on a press release by the sponsor of these trials, the PFS time was longer with vandetanib in the ZODIAC and ZEAL trials; the ZEST trial was negative for its primary superiority analysis, but was successful according to a preplanned noninferiority analysis of PFS. Ongoing phase II and III clinical trials will better define the appropriate schedule, the optimal setting of evaluation, and the safety of long-term use of vandetanib.

VEGF is a chemoattractant for FGF-2-stimulated neural progenitors.

Migration of undifferentiated neural progenitors is critical for the development and repair of the nervous system. However, the mechanisms and factors that regulate migration are not well understood. Here, we show that vascular endothelial growth factor (VEGF)-A, a major angiogenic factor, guides the directed migration of neural progenitors that do not display antigenic markers for neuron- or glia-restricted precursor cells. We demonstrate that progenitor cells express both VEGF receptor (VEGFR) 1 and VEGFR2, but signaling through VEGFR2 specifically mediates the chemotactic effect of VEGF. The expression of VEGFRs and the chemotaxis of progenitors in response to VEGF require the presence of fibroblast growth factor 2. These results demonstrate that VEGF is an attractive guidance cue for the migration of undifferentiated neural progenitors and offer a mechanistic link between neurogenesis and angiogenesis in the nervous system.

Vascular endothelial growth factor receptor-1 contributes to resistance to anti-epidermal growth factor receptor drugs in human cancer cells.

PURPOSE: The resistance to selective EGFR inhibitors involves the activation of alternative signaling pathways, and Akt activation and VEGF induction have been described in EGFR inhibitor-resistant tumors. Combined inhibition of EGFR and other signaling proteins has become a successful therapeutic approach, stimulating the search for further determinants of resistance as basis for novel therapeutic strategies. EXPERIMENTAL DESIGN: We established human cancer cell lines with various degrees of EGFR expression and sensitivity to EGFR inhibitors and analyzed signal transducers under the control of EGFR-dependent and EGFR-independent pathways. RESULTS: Multitargeted inhibitor vandetanib (ZD6474) inhibited the growth and the phosphorylation of Akt and its effector p70S6 kinase in both wild-type and EGFR inhibitor-resistant human colon, prostate, and breast cancer cells. We found that the resistant cell lines exhibit, as common feature, VEGFR-1/Flt-1 overexpression, increased secretion of VEGF and placental growth factor, and augmented migration capabilities and that vandetanib is able to antagonize them. Accordingly, a new kinase assay revealed that in addition to VEGF receptor (VEGFR)-2, RET, and EGFR, vandetanib efficiently inhibits also VEGFR-1. The contribution of VEGFR-1 to the resistant phenotype was further supported by the demonstration that VEGFR-1 silencing in resistant cells restored sensitivity to anti-EGFR drugs and impaired migration capabilities, whereas exogenous VEGFR-1 overexpression in wild-type cells conferred resistance to these agents. CONCLUSIONS: This study shows that VEGFR-1 contributes to anti-EGFR drug resistance in different human cancer cells. Moreover, vandetanib inhibits VEGFR-1 activation, cell proliferation, and migration, suggesting its potential utility in patients resistant to EGFR inhibitors.