Upregulation of urotensin II receptor in preeclampsia causes in vitro placental release of soluble vascular endothelial growth factor receptor 1 in hypoxia

Preeclampsia is a hypertensive disorder of pregnancy caused by abnormal placental function, partly because of chronic hypoxia at the utero-placental junction. The increase in levels of soluble vascular endothelial growth factor receptor 1, an antiangiogenic agent known to inhibit placental vascularization, is an important cellular factor implicated in the onset of preeclampsia. We investigated the ligand urotensin II (U-II), a potent endogenous vasoconstrictor and proangiogenic agent, for which levels have been reported to increase in patients with preeclampsia. We hypothesized that an increased sensitivity to U-II in preeclampsia might be achieved by upregulation of placental U-II receptors. We further investigated the role of U-II receptor stimulation on soluble vascular endothelial growth factor receptor 1 release in placental explants from diseased and normal patients. Immunohistochemistry, real-time PCR, and Western blotting analysis revealed that U-II receptor expression was significantly upregulated in preeclampsia placentas compared with controls (P<0.01). Cellular models of syncytiotrophoblast and vascular endothelial cells subjected to hypoxic conditions revealed an increase in U-II receptor levels in the syncytiotrophoblast model. This induction is regulated by the transcriptional activator hypoxia-inducible factor 1a. U-II treatment is associated with increased secretion of soluble vascular endothelial growth factor receptor 1 only in preeclamptic placental explants under hypoxia but not in control conditions. Interestingly, normal placental explants did not respond to U-II stimulation.

Hydrogen sulphide rescues preeclampsia-like phenotype aggravated by high sFlt-1 in Placenta Growth Factor (PlGF) deficiency

INTRODUCTION: Low circulating levels of placenta growth factor (PlGF) is strongly associated with the onset of preeclampsia, a maternal hypertensive disorder characterized by high blood pressure and proteinuria after 20 weeks of gestation. Although, PlGF-deficient mice are born healthy and fertile at a Mendelian ratio, the physiological importance of PlGF in the pathogenesis of preeclampsia is unclear. We hypothesised that decreased levels of PlGF in pregnancy exacerbates the fetal growth restriction associated with preeclampsia in the presence of high sFlt-1. METHODS: Pregnant PlGF-/- mice were injected with adenovirus encoding sFlt-1 (Ad-sFlt-1) at high (i) 1.5x109 pfu/ml and low (ii) 0.5x109 pfu/ml doses. Mean arterial blood pressure (MBP), biochemical and histological assessments of maternal kidney, placenta and embryos were performed. RESULTS: Ad-sFlt-1 significantly increased MBP and induced severe glomerular endotheliosis in PlGF-/- mice at E10.5 gestation compared to wild-type animals. High sFlt-1 also significantly elevated albumincreatinine ratio and increased levels of urinary kidney injury molecule-1, a marker for proximal tubule injury.At a high dose of sFlt-1, there was complete fetal resorption in the pregnant PlGF-/- mice, and even the lower dose of sFlt-1 induced severe fetal resorption and abnormal placental vascularization. Hydrogen sulphide-releasing agent, GYY4137, significantly reduced resorption, hypertension and proteinuria in Ad-sFlt-1 treated pregnant PlGF-/- mice. To determine if placental PlGF is critical for preventing fetal growth restriction associated with preeclampsia, we generated haploinsufficient PlGF+/- placentas and embryos were generated in wild-time dams and exposed to high sFlt-1 environment. This resulted in reduced fetal resorption, gestational hypertension and proteinuria when compared to pregnant PlGF-/- mice. CONCLUSIONS: Placental PlGF is a critical protective factor against the damaging effects of high sFlt-1 in preeclampsia and the hydrogen sulphide pathway may rescue preeclampsia phenotypes.

Dysregulation of hydrogen sulfide producing enzyme cystathionine γ-lyase contributes to maternal hypertension and placental abnormalities in preeclampsia

Background—The exact etiology of preeclampsia is unknown, but there is growing evidence of an imbalance in angiogenic growth factors and abnormal placentation. Hydrogen sulfide (H2S), a gaseous messenger produced mainly by cystathionine ?-lyase (CSE), is a proangiogenic vasodilator. We hypothesized that a reduction in CSE activity may alter the angiogenic balance in pregnancy and induce abnormal placentation and maternal hypertension. Methods and Results—Plasma levels of H2S were significantly decreased in women with preeclampsia (P<0.01), which was associated with reduced placental CSE expression as determined by real-time polymerase chain reaction and immunohistochemistry. Inhibition of CSE activity by DL-propargylglycine reduced placental growth factorproduction from first-trimester (8–12 weeks gestation) human placental explants and inhibited trophoblast invasion in vitro. Knockdown of CSE in human umbilical vein endothelial cells by small-interfering RNA increased the release of soluble fms-like tyrosine kinase-1 and soluble endoglin, as assessed by enzyme-linked immunosorbent assay, whereas adenoviral-mediated CSE overexpression in human umbilical vein endothelial cells inhibited their release. Administration of DL-propargylglycine to pregnant mice induced hypertension and liver damage, promoted abnormal labyrinth vascularization in the placenta, and decreased fetal growth. Finally, a slow-releasing H2S-generating compound, GYY4137, inhibited circulating soluble fms-like tyrosine kinase-1 and soluble endoglin levels and restored fetal growth in mice that was compromised by DL-propargylglycine treatment, demonstrating that the effect of CSE inhibitor was attributable to inhibition of H2S production. Conclusions—These results imply that endogenous H2S is required for healthy placental vasculature and that a decrease in CSE/H2S activity may contribute to the pathogenesis of preeclampsia. (Circulation. 2013;127:2514-2522.)

Dysregulation of hydrogen sulfide producing enzyme cystathionine γ-lyase contributes to maternal hypertension and placental abnormalities in preeclampsia

Background-The exact etiology of preeclampsia is unknown, but there is growing evidence of an imbalance in angiogenic growth factors and abnormal placentation. Hydrogen sulfide (H2S), a gaseous messenger produced mainly by cystathionine γ-lyase (CSE), is a proangiogenic vasodilator. We hypothesized that a reduction in CSE activity may alter the angiogenic balance in pregnancy and induce abnormal placentation and maternal hypertension. Methods and Results-Plasma levels of H2S were significantly decreased in women with preeclampsia (P<0.01), which was associated with reduced placental CSE expression as determined by real-time polymerase chain reaction and immunohistochemistry. Inhibition of CSE activity by DL-propargylglycine reduced placental growth factorproduction from first-trimester (8-12 weeks gestation) human placental explants and inhibited trophoblast invasion in vitro. Knockdown of CSE in human umbilical vein endothelial cells by small-interfering RNA increased the release of soluble fms-like tyrosine kinase-1 and soluble endoglin, as assessed by enzyme-linked immunosorbent assay, whereas adenoviral-mediated CSE overexpression in human umbilical vein endothelial cells inhibited their release. Administration of DL-propargylglycine to pregnant mice induced hypertension and liver damage, promoted abnormal labyrinth vascularization in the placenta, and decreased fetal growth. Finally, a slow-releasing H2S-generating compound, GYY4137, inhibited circulating soluble fms-like tyrosine kinase-1 and soluble endoglin levels and restored fetal growth in mice that was compromised by DL-propargylglycine treatment, demonstrating that the effect of CSE inhibitor was attributable to inhibition of H2S production. Conclusions-These results imply that endogenous H2S is required for healthy placental vasculature and that a decrease in CSE/H2S activity may contribute to the pathogenesis of preeclampsia. © 2013 American Heart Association, Inc.

Induction of placental heme oxygenase-1 is protective against TNFα-induced cytotoxicity and promotes vessel relaxation

Background: Pregnancy is characterized by an inflammatory-like process and this may be exacerbated in preeclampsia. The heme oxygenase (HO) enzymes generate carbon monoxide (CO) that induces blood vessel relaxation and biliverdin that acts as an endogenous antioxidant. Materials and Methods: We examined the expression and localization of HO-1 and HO-2 in normal and preeclamptic placenta using reverse transcription polymerase chain reaction (RT-PCR), RNase protection assay, immunoblotting and immunohistochemistry. In addition, the effect of HO activation on tumor necrosis factor-alpha (TNF) induced placental damage and on feto-placental circulation was studied. Results: We provide the first evidence for the role of HO as an endogenous placental factor involved with cytoprotection and placental blood vessel relaxation. HO-1 was significantly higher at term, compared with first trimester placentae indicating its role in placental vascular development and regulation. HO-1 predominantly localized in the extravascular connective tissue that forms the perivascular contractile sheath around the developing blood vessels. HO-2 was localized in the capillaries, as well as the villous stroma, with weak staining of trophoblast. Induction of HO-1 caused a significant attenuation of TNF-mediated cellular damage in placental villous explants, as assessed by lactate dehydrogenase leakage (p 0.01). HO-1 protein was significantly reduced in placentae from pregnancies complicated with preeclampsia, compared with gestationally matched normal pregnancies. This suggests that the impairment of HO-1 activation may compromise the compensatory mechanism and predispose the placenta to cellular injury and subsequent maternal endothelial cell activation. Isometric contractility studies showed that hemin reduced vascular tension by 61% in U46619-preconstricted placental arteries. Hemininduced vessel relaxation and CO production was inhibited by HO inhibitor, tin protoporphyrin IX. Conclusions: Our findings establish HO-1 as an endogenous system that offers protection against cytotoxic damage in the placenta, identifies the HO-CO pathway to regulate feto-placental circulation and provides a new approach to study the disease of preeclampsia.

The presence of pleiotrophin in the human intervertebral disc is associated with increased vascularization:an immunohistologic study

Study Design. An immunohistological study of surgical specimens of human intervertebral disc.Objective.To examine the presence of pleiotrophin in diseased or damaged intervertebral disc tissue and the association between its presence and the extent of tissue vascularization and innervation.Summary of Background Data. Increased levels of pleiotrophin, a growth and differentiation factor that is active in various pathophysiologic processes, including angiogenesis, has been associated with osteoarthritic changes of human articular cartilage. The association between pleiotrophin expression and pathologic conditions of the human intervertebral disc is unknown.Methods. Specimens of human lumbar intervertebral discs, obtained following surgical discectomy, were divided into 3 groups: nondegenerated discs (n = 7), degenerated discs (n = 6), and prolapsed discs (n = 11). Serial tissue sections of each specimen were immunostained to determine the presence of pleiotrophin, blood vessels (CD34-positive endothelial cells), and nerves (neurofilament 200 kDa [NF200]-positive nerve fibers).Results. Pleiotrophin immunoreactivity was seen in disc cells, endothelial cells, and in the extracellular matrix in most specimens of intervertebral disc but was most prevalent in vascularized tissue in prolapsed discs. There was a significant correlation between the presence of pleiotrophin-positive disc cells and that of CD34-positive blood vessels. NF200-positive nerves were seen in vascularized areas of more degenerated discs, but nerves did not appear to codistribute with blood vessels or pleiotrophin positivity in prolapsed discs.Conclusions. Pleiotrophin is present in pathologic human intervertebral discs, and its prevalence and distribution suggest that it may play a role in neovascularization of diseased or damaged disc tissue.

Elevated placental soluble vascular endothelial growth factor receptor-1 inhibits angiogenesis in preeclampsia

Preeclampsia is an inflammatory disorder in which serum levels of vascular endothelial growth factor (VEGF) and its soluble receptor-1 (sVEGFR-1, also known as sFlt-1) are elevated. We hypothesize that VEGF and placenta growth factor (PlGF) are dysregulated in preeclampsia due to high levels of sVEGFR-1, which leads to impaired placental angiogenesis. Analysis of supernatants taken from preeclamptic placental villous explants showed a four-fold increase in sVEGFR-1 than normal pregnancies, suggesting that villous explants in vitro retain a hypoxia memory reflecting long-term fetal programming. The relative ratios of VEGF to sVEGFR-1and PlGF to sVEGFR-1 released from explants decreased by 53% and 70%, respectively, in preeclampsia compared with normal pregnancies. Exposure of normal villous explants to hypoxia increased sVEGFR-1 release compared with tissue normoxia (P<0.001), as did stimulation with tumor necrosis factor-α (P<0.01). Conditioned medium (CM) from normal villous explants induced endothelial cell migration and in vitro tube formation, which were both attenuated by pre-incubation with exogenous sVEGFR-1 (P<0.001). In contrast, endothelial cells treated with preeclamptic CM showed substantially reduced angiogenesis compared withnormal CM (P<0.001), which was not further decreased by the addition of exogenous sVEGFR-1, indicating a saturation of the soluble receptor.Removal of sVEGFR-1 by immunoprecipitation from preeclamptic CM significantly restored migration (P<0.001) and tube formation (P<0.001) to levels comparable to that induced by normal CM, demonstrating that elevated levels of sVEGFR-1 in preeclampsia are responsible for inhibiting angiogenesis. Our finding demonstrates the dysregulation of the VEGF/PlGF axis in preeclampsiaand offers an entirely new therapeutic approach to its treatment.

Antiangiogenic effect of soluble vascular endothelial growth factor receptor-1 in placental angiogenesis

Differential splicing of the flt-1 mRNA generates soluble variant of vascular endothelial growth factor (VEGF) receptor-1 (sVEGFR-1, also known as sFlt-1). The action of VEGF is antagonized by sVEGFR-1. Soluble VEGFR-1 binds to VEGF with a high affinity and therefore works to modulate VEGF and VEGF signaling pathway. In this study, the authors tested the hypothesis that VEGF-mediated endothelial cell angiogenesis is tightly modulated by the release of sVEGFR-1 and placental expression of sVEGFR-1 is upregulated by hypoxia. Immunolocalization studies showed progressively intense staining for sVEGFR-1 and VEGF in the trophoblast of placental villous explants throughout gestation. Endothelial cell migration studies using a modified Boyden's chamber showed a significant increase in cell migration in response to VEGF that was significantly attenuated in the presence of exogenous sVEGFR-1. Furthermore, stimulation of endothelial cells with VEGF led to a dose-dependent increase in the release of sVEGFR-1 as determined by enzyme-linked immunosorbent assay (ELISA). Exposure of normal placental villous explants to hypoxia (1% pO2) increased trophoblast expression of sVEGFR-1 when compared with tissue normoxia (5% pO2). In addition, conditioned media from hypoxia treated placental villous explants induced a significant increase in endothelial cell migration that was significantly reduced in presence of sVEGFR-1. Our study demonstrates that hypoxia positively regulates sVEGFR-1 protein expression in ex vivo trophoblasts, which control VEGF-driven angiogenesis.

Dysregulation of placental cystathionine-β-synthase promotes fetal growth restriction

Fetal growth restriction (FGR) is characterized by the birth weight and body mass below the tenth percentile for gestational age. FGR is a major cause of perinatal morbidity and mortality and babies born with FGR are prone to develop cardiovascular diseases later in life. The underlying pathology of FGR is inadequate placental transfer of nutrients from mother to fetus, which can be caused by placental insufficiency. Hydrogen sulfide (H2S), a gaseous messenger is produced endogenously by cystathionine-lyase (Cth), cystathionine-β-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (3-MST), which are present in human placenta. Recently, we demonstrated that the dysregulation of H2S/Cth pathway is associated with preeclampsia and blockade of CSE activity induces preeclampsia-like condition in pregnant mice. We hypothesized that defect in H2S pathways promote FGR and H2S donor restores fetal growth in mice where CBS or CSE activity has been compromised. Western blotting and qPCR revealed that placental CBS expressions were significantly reduced in women with FGR. ELISA analysis showed reduced placental growth factor production (PlGF) from first trimester (8–12 weeks gestation) human placental explants following inhibition of CBS activity by aminooxyacetic acid (AOA). Administration of AOA to pregnant mice had no effects on blood pressure, but caused fetal growth restriction. This was associated with reduced PlGF production. Histological analysis revealed a reduction in the placental junction zone, within which trophoblast giant cells and glycogen cells were less prominent in CBS inhibitor treated mice. These results imply that placental CBS is required for placental development and that dysregulation of CBS activity may contribute to the pathogenesis of FGR but not preeclampsia.

Dysregulation of placental cystathionine-β-synthase impact on fetal growth restriction

INTRODUCTION: Fetal growth restriction (FGR), which causes perinatal morbidity and mortality, is characterized by birth weight and body mass being below 10th percentile for gestational age. FGR babies are prone to develop cardiovascular diseases later in life. Inadequate placental transfer of nutrients from mother to fetus due to placental insufficiency is considered the underlying cause of FGR. Recently, we demonstrated that blockade of cystathionine-γ-lyase (CSE) activity induces preeclampsia-like condition in pregnant mice. We hypothesized that defect in cystathionine-β-synthase (CBS) / H2S pathway may promote FGR. METHODS: Placental CBS expressions were determined in women with FGR (n=9) and normal controls (n=14) by Western blotting and real-time qPCR. ELISA was used to determine angiogenic factors levels in plasma and first-trimester (8–12 weeks gestation) human placental explants. Time pregnant mice were treated with CBS inhibitor, aminooxyacetic acid (AOA). Mean arterial blood pressure (MBP), histological assessments of placenta and embryos were performed. RESULTS: Placental CBS expressions were significantly reduced in women with FGR. Inhibition of CBS activity by AOA reduced PlGF production from first-trimester human placental explants, Administration of AOA to pregnant mice had no effects on blood pressure, but caused fetal growth restriction, which was associated with reduced placental PlGF production. Histological analysis revealed a reduction in the placental junction zone, within which trophoblast giant cells and glycogen cells were less prominent in CBS inhibitor-treated animals. Furthermore, H2S donor GYY4137 treatment restored fetal growth in pregnant mice exposed to high level of sFlt-1. CONCLUSIONS: These results imply that placental CBS is required for placental development and that dysregulation of CBS activity may contribute to the pathogenesis of FGR but not preeclampsia opening up the therapeutic potentials of H2S therapy in this condition.

Articular cartilage glycosaminoglycans inhibit the adhesion of endothelial cells

Articular cartilage undergoes severe loss of proteoglycan and its constituent glycosaminoglycans (GAGs) in osteoarthritis. We hypothesize that the low GAG content of osteoarthritic cartilage renders the tissue susceptible to pathological vascularization. This was investigated using an in vitro angiogenesis model assessing endothelial cell adhesion to GAG-depleted cartilage explants. Bovine cartilage explants were treated with hyaluronidase to deplete GAG content and then seeded with fluorescently tagged human endothelial cells (HMEC-1). HMEC-1 adherence was assessed after 4 hr and 7 days. The effect of hyaluronidase treatment on GAG content, chondrocyte viability, and biochemical composition of the extracellular matrix was also determined. Hyaluronidase treatment reduced the GAG content of cartilage explants by 78 ± 3% compared with that of controls (p <0.0001). GAG depletion was associated with significantly more HMEC-1 adherence on both the surface (superficial zone) and the underside (deep zone) of the explants (both p <0.0001). The latter provided a more favorable environment for extended culture of HMEC-1 compared with the articulating surface. Hyaluronidase treatment altered the immunostaining for chondroitin sulfate epitopes, but not for lubricin. Our results support the hypothesis that articular cartilage GAGs are antiadhesive to endothelial cells and suggest that chondroitin sulfate and/or hyaluronan are responsible. The loss of these GAGs in osteoarthritis may allow osteochondral angiogenesis resulting in disease progression.

Mechanical stimulation alters pleiotrophin and aggrecan expression by human intervertebral disc cells and influences their capacity to stimulate endothelial migration

Study Design. The influence of mechanical load on pleiotrophin (PTM) and aggrecan expression by intervertebral disc (IVD) cells, and the effects of disc cell conditioned medium on endothelial cell migration was investigated. Objective. To examine possible interactions of mechanical loads and known pro- and antiangiogenic factors, which may regulate disc angiogenesis during degeneration. Summary of Background Data. Pleiotrophin expression can be influenced by mechanical stimulation and has been associated with disc vascularization. Disc aggrecan inhibits endothelial cell migration, suggesting an antiangiogenic role. A possible interplay between these factors is unknown. Methods. The influence of the respective predominant load (cyclic strain for anulus fibrosus and hydrostatic pressure for nucleus pulposus cells) on PTN and aggrecan expression by IVD cells was determined by real-time RT-PCR and Western blotting (PTN only). The effects of IVD cell conditioned medium on endothelial cell migration were analyzed in a bioassay using human microvascular endothelial (HMEC-1) cells. Results. Application of both mechanical loads resulted in significant alterations of gene expression of PTN (+67%, P = 0.004 in anulus cells; +29%, P = 0.03 in nucleus cells) and aggrecan (+42%, P = 0.03 in anulus cells, -25%, P = 0.03 in nucleus cells). These effects depended on the cell type, the applied load, and timescale. Conditioned media of nucleus pulposus cells enhanced HMEC-1 migration, but this effect was diminished after 2.5 MPa hydrostatic pressure, when aggrecan expression was diminished, but not 0.25 MPa, when expression levels were unchanged. Conclusion. Mechanical loading influences PTN expression by human IVD cells. Conditioned media from nucleus pulposus cell cultures stimulated HMEC-1 endothelial cell migration. This study demonstrates that the influence of mechanical loads on vascularization of the human IVD is likely to be complex and does not correlate simply with altered expression of known pro- and antiangiogenic factors.

Characterizing the hierarchical structures of bioactive sol-gel silicate glass and hybrid scaffolds for bone regeneration

Bone is the second most widely transplanted tissue after blood. Synthetic alternatives are needed that can reduce the need for transplants and regenerate bone by acting as active temporary templates for bone growth. Bioactive glasses are one of the most promising bone replacement/regeneration materials because they bond to existing bone, are degradable and stimulate new bone growth by the action of their dissolution products on cells. Sol-gel-derived bioactive glasses can be foamed to produce interconnected macropores suitable for tissue ingrowth, particularly cell migration and vascularization and cell penetration. The scaffolds fulfil many of the criteria of an ideal synthetic bone graft, but are not suitable for all bone defect sites because they are brittle. One strategy for improving toughness of the scaffolds without losing their other beneficial properties is to synthesize inorganic/organic hybrids. These hybrids have polymers introduced into the sol-gel process so that the organic and inorganic components interact at the molecular level, providing control over mechanical properties and degradation rates. However, a full understanding of how each feature or property of the glass and hybrid scaffolds affects cellular response is needed to optimize the materials and ensure long-term success and clinical products. This review focuses on the techniques that have been developed for characterizing the hierarchical structures of sol-gel glasses and hybrids, from atomicscale amorphous networks, through the covalent bonding between components in hybrids and nanoporosity, to quantifying open macroporous networks of the scaffolds. Methods for non-destructive in situ monitoring of degradation and bioactivity mechanisms of the materials are also included. © 2012 The Royal Society.

Placenta growth factor-1 exerts time-dependent stabilization of adherens junctions following VEGF-induced vascular permeability

Increased vascular permeability is an early event characteristic of tissue ischemia and angiogenesis. Although VEGF family members are potent promoters of endothelial permeability the role of placental growth factor (PlGF) is hotly debated. Here we investigated PlGF isoforms 1 and 2 and present in vitro and in vivo evidence that PlGF-1, but not PlGF-2, can inhibit VEGF-induced permeability but only during a critical window post-VEGF exposure. PlGF-1 promotes VE-cadherin expression via the trans-activating Sp1 and Sp3 interaction with the VE-cadherin promoter and subsequently stabilizes transendothelial junctions, but only after activation of endothelial cells by VEGF. PlGF-1 regulates vascular permeability associated with the rapid localization of VE-cadherin to the plasma membrane and dephosphorylation of tyrosine residues that precedes changes observed in claudin 5 tyrosine phosphorylation and membrane localization. The critical window during which PlGF-1 exerts its effect on VEGF-induced permeability highlights the importance of the translational significance of this work in that PLGF-1 likely serves as an endogenous anti-permeability factor whose effectiveness is limited to a precise time point following vascular injury. Clinical approaches that would pattern nature's approach would thus limit treatments to precise intervals following injury and bring attention to use of agents only during therapeutic windows.