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He Y, Smith SK, Day KA, Clark DE, Licence DR, and Charnock-Jones DS. Alternative splicing of vascular endothelial growth factor (VEGF)-R1 (Flt-1) pre-mRNA is important for the regulation of VEGF activity. Mol Endocr 13: 537-545
... The object of the study is to develop an electrochemical biosensor system based on electrochemical impedance spectroscopy which will enable fast, sensitive, and practical determination of a potential cancer biomarker, VEGF, which is a protein produced by cells that stimulates vasculogenesis and angiogenesis In the current study, human vascular endothelial growth factor receptor 1 (VEGF-R1, Flt-1) was used as a biorecognition element for the first time. The recombinant soluble VEGF-R1 binds to VEGF with high affinity [4][5][6]. Glassy carbon electrodes were coated potentiometrically with thin gold film. Cysteamine monolayers were self-assembled on the gold films. ...
Problem
Antiangiogenic molecule soluble fms‐like tyrosine kinase receptor 1 (sFLT1) released from trophoblast cells is associated with pregnancy‐specific hypertensive disorder pre‐eclampsia. Cause of elevated sFLT1 in pre‐eclampsia patients is not well understood. Despite evidence of excess systemic and placental complement activation in pre‐eclampsia patients, its role in pathophysiology is not clear. If the complement activation plays a role in upregulation and secretion of sFLT1 is not known.
Method of study
Human trophoblast cells were isolated from term placentas and allowed to syncytialize. Complement was activated in vitro at sublethal levels on syncytiotrophoblast cells. Effect of complement activation on expression and release of sFLT1 was assessed by comparing its levels in these cells with and without complement activation.
Results
Sublethal level of complement activation on syncytialized human trophoblast cells induced upregulation of sFLT1 mRNA and protein. Complement also induced secretion of sFLT1 in a manner depending on degree of activation. Anaphylatoxins C3a induced upregulation but not the release of sFLT1. Release of terminal membrane attack complex (MAC) was associated with sFLT1 secretion.
Conclusion
Complement activation plays a major role in both the expression and secretion of sFLT1 from syncytial trophoblast cells. The terminal MAC complex is involved in its secretion. Increased levels of sFLT1 in pre‐eclampsia patients may be due to complement‐induced upregulation and secretion.
Background
Preeclampsia is a hypertensive syndrome that complicates 3% to 5% of pregnancies in the United States. Preeclampsia originates from an improperly vascularized and ischemic placenta that releases factors that drive systemic pathophysiology. One of these factors, soluble fms‐like tyrosine kinase‐1, is believed to sequester vascular endothelial growth factor (VEGF), leading to systemic endothelial dysfunction and hypertension. With the goal of targeting soluble fms‐like tyrosine kinase‐1 while simultaneously preventing fetal exposure to VEGF, we fused VEGF to elastin‐like polypeptide, a biopolymer carrier that does not cross the placental barrier (ELP‐VEGF).
Methods and Results
ELP‐VEGF restored in vitro endothelial cell tube formation in the presence of plasma from placental ischemic rats. Long‐term administered ELP‐VEGF in pregnant rats accumulated in maternal kidneys, aorta, liver, and placenta, but the protein was undetectable in the pups when administered at therapeutic doses in dams. Long‐term administration of ELP‐VEGF in a placental ischemia rat model achieved dose‐dependent attenuation of hypertension, with blood pressure equal to sham controls at a dose of 5 mg/kg per day. ELP‐VEGF infusion increased total plasma soluble fms‐like tyrosine kinase‐1 levels but dramatically reduced free plasma soluble fms‐like tyrosine kinase‐1 and induced urinary excretion of nitrate/nitrite, indicating enhanced renal nitric oxide signaling. ELP‐VEGF at up to 5 mg/kg per day had no deleterious effect on maternal or fetal body weight. However, dose‐dependent adverse events were observed, including ascites production and neovascular tissue encapsulation around the minipump.
Conclusions
ELP‐VEGF has the potential to treat the preeclampsia maternal syndrome, but careful dosing and optimization of the delivery route are necessary.
Pre-eclampsia is a common obstetric complication globally responsible for a significant burden of maternal and perinatal morbidity and mortality. Central to its pathophysiology is the anti-angiogenic protein, soluble fms-like tyrosine kinase-1 (sFLT-1). sFLT-1 is released from a range of tissues into the circulation, where it antagonizes the activity of vascular endothelial growth factor and placental growth factor leading to endothelial dysfunction. It is this widespread endothelial dysfunction that produces the clinical features of pre-eclampsia including hypertension and proteinuria. There are multiple splice variants of sFLT-1. One, known as sFLT-1 e15a, evolved quite recently and is only present in humans and higher order primates. This sFLT-1 variant is also the main sFLT-1 secreted from the placenta. Recent work has shown that sFLT-1 e15a is significantly elevated in the placenta and circulation of women with pre-eclampsia. It is also biologically active, capable of causing endothelial dysfunction and the end-organ dysfunction seen in pre-eclampsia. Indeed, the over-expression of sFLT-1 e15a in mice recapitulates the pre-eclamptic phenotype in pregnancy. Therefore, here we propose that sFLT-1 e15a may be the sFLT-1 variant primarily responsible for pre-eclampsia, a uniquely human disease. Furthermore, this placental-specific sFLT-1 variant provides promise for use as an accurate biomarker in the prediction or diagnosis of pre-eclampsia.
In the adult organism angiogenesis is generally rare and is usually confined to pathological conditions. However, one marked exception to this is the female reproductive tract. In both the ovary and the uterus, there is cyclic growth and regression of specialized tissues that necessitates the growth and regression of vascular structures. These events are coordinated by steroid hormones, but it is now increasingly recognized that local effectors mediate their actions. Thus the female reproductive tract permits the study of the cyclic growth and regression of blood vessels in a normal physiological setting. This is obviously of importance to reproductive biology, but is also relevant to the study of blood vessels generally.
Preeclampsia (PE) is a pregnancy-specific syndrome that causes substantial maternal and fetal morbidity and mortality. Increased production of antiangiogenic factors, soluble fms-like tyrosine kinase receptor-1 (sFlt-1) and soluble endoglin (sEng), as well as decreased circulating levels of free vascular endothelial growth factor (VEGF), contribute to the pathophysiology of PE. Our objective was to evaluate a novel placenta-related factor, tissue transglutaminase (tTG), in PE and to investigate the correlation among tTG and sFlt-1, sEng and VEGF levels in both normotensive pregnant patients and PE patients. A total of 205 pregnant primigravid women were recruited and divided into a normotensive group (n=100), a mild PE group (n=45) and a severe PE group (n=60). Circulating serum tTG, sFlt-1, sEng and free VEGF levels were determined using an enzyme-linked immunosorbent assay. The severe PE group showed higher levels of tTG, sFlt-1 and sEng than the mild PE and normotensive groups. Free VEGF levels were lower in the severe PE group than in the mild PE and normotensive groups. tTG correlated significantly with sFlt-1, sEng and VEGF in the PE groups, whereas this correlation was not observed in the normotensive group. The tTG, sFlt-1, sEng and VEGF levels showed a significant correlation with mean arterial pressure in the PE groups but not in the normotensive group. The tTG, sFlt-1, sEng and VEGF levels correlated with the degree of proteinuria. Our results reveal that tTG is associated with sFlt-1, sEng and VEGF in the maternal circulation of PE patients, suggesting that tTG may have a role in the pathogenesis of PE.Journal of Human Hypertension advance online publication, 12 May 2016; doi:10.1038/jhh.2016.32.
Aims
In developing blood vessel networks, the overall level of vessel branching often correlates with angiogenic sprout initiations, but in some pathological situations increased sprout initiations paradoxically lead to reduced vessel branching and impaired vascular function. We examine the hypothesis that defects in the discrete stages of angiogenesis can uniquely contribute to vessel branching outcomes.
Methods and Results
Time-lapse movies of mammalian blood vessel development were used to define and quantify the dynamics of angiogenic sprouting. We characterized the formation of new functional conduits by classifying discrete sequential stages - sprout initiation, extension, connection, and stability - that are differentially affected by manipulation of VEGF-A signaling via genetic loss of the receptor Flt-1 (VEGFR-1). In mouse embryonic stem cell-derived vessels genetically lacking flt-1, overall branching is significantly decreased while sprout initiations are significantly increased. Flt-1-mutant sprouts are less likely to retract, and they form increased numbers of connections with other vessels. However, loss of flt-1 also leads to vessel collapse, which reduces the number of new stable conduits. Computational simulations predict that loss of Flt-1 results in ectopic Flk-1 signaling in connecting sprouts post-fusion, causing protrusion of cell processes into avascular gaps and collapse of branches. Thus, defects in stabilization of new vessel connections offset increased sprout initiations and connectivity in flt-1-/- vascular networks, with an overall outcome of reduced numbers of new conduits.
Conclusions
These results show that VEGF-A signaling has stage-specific effects on vascular morphogenesis, and that understanding these effects on dynamic stages of angiogenesis, and how they integrate to expand a vessel network, may suggest new therapeutic strategies.
Preeclampsia is a major cause of maternal, fetal, and neonatal mortality worldwide. Although the etiology of preeclampsia is still unclear, recent studies suggest that its major phenotypes, high blood pressure and proteinuria, are due in part to excess circulating soluble fms-like tyrosine kinase-1 concentrations. Soluble fms-like tyrosine kinase-1 is an endogenous antiangiogenic protein that is made by the placenta and acts by neutralizing the proangiogenic proteins vascular endothelial growth factor and placental growth factor. High serum soluble fms-like tyrosine kinase-1 and low serum free placental growth factor and free vascular endothelial growth factor have been observed in preeclampsia. Abnormalities in these circulating angiogenic proteins are not only present during clinical preeclampsia but also antedate clinical symptoms by several weeks. Therefore, this raises the possibility of measuring circulating angiogenic proteins in the blood and the urine as a diagnostic and screening tool for preeclampsia. The availability of a test to predict preeclampsia would be a powerful tool in preventing preeclampsia-induced mortality, especially in developing nations, where high-risk specialists are limited. This review will summarize our current understanding of the role of circulating angiogenic proteins in the pathogenesis and clinical diagnosis/prediction of preeclampsia.
Current enthusiasm for the therapeutic application of angiogenesis to a wide range of disease processes derives in large part from studies of one of the most potent and biologically important growth factors, vascular permeability factor/vascular endothelial growth factor (VPF/VEGF). VPF/VEGF, also known as VEGF-A, is the foremost member of a large family of growth factors, which includes VEGF-B, VEGF-C, VEGF-D,VEGF-E, and placenta growth factor (PIGF). VPF/VEGF acts as a key regulator in the angiogenic process by inducing hyperpermeability, proliferation, and migration of endothelial cells. More recently, VPF/VEGF has become recognized for its important role in promoting endothelial cell survival. The biological actions of VPF/VEGF are mediated through two tyrosine kinase receptors, VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1), selectively expressed on vascular endothelium, together with a recently discovered receptor, neuropilin. Expression of VPF/VEGF and its receptors is regulated primarily by hypoxia, other cytokines, oncogenes, and tumor suppressor genes. The signaling mechanisms of endothelial cell proliferation, migration, and hyperpermeability and the role of the anti-apoptotic AKT pathway in endothelial survival are areas of active research. Angiogenesis mediated through VPF/VEGF is pivotal to the pathological entities of wound healing, ischemia, and tumor growth. Methods of detection and quantitation of VPF/VEGF in tissues and body fluids have become increasingly important as VPF/VEGF gains clinical importance in the diagnosis and treatment of disease.
In this study, we aimed to show how cadmium (Cd) affects the trophoblast proliferation and differentiation in the placenta and the apoptotic activity in different gestational days and, hence, its effects of placental development with immunohistochemical and TUNEL techniques. Experimental model of our study consisted of placental development of control and Cd groups on 15, 17, 19, and 21th days of the gestation. Female rats in Cd groups were subcutaneously administered a single dose of 0.5 mg Cd/kg/day dissolved in sodium chloride as 2 mL/kg Cd chloride until the day they sacrificed. Embryo and placenta of female rats were separately removed on 15, 17, 19, and 21th days of the gestation in which the placental development takes place and placentas were processed for microscopic examinations. In the placentas of the control group, all layers were observed to be formed on the 15th gestational day and thereafter a continuous growth was monitored. In the Cd group also all layers existed from the 15th gestational day. However, they were smaller in size than control groups. Frequency of proliferating cell nuclear antigen (PCNA)-positive cells was decreased and the number of apoptotic cells was increased in all the gestational days related to Cd. In conclusion, Cd administered during the pregnancy was observed to cause abnormal placental development by disrupting the normal structure of the placenta, inhibiting the proliferation of trophoblast and increasing the number of apoptotic trophoblast cells.
This chapter discusses placental vascular development during health and in disease with an emphasis on the role of placental antiangiogenic factors in the pathogenesis of the maternal syndrome of preeclampsia. Vascular development occurs through angiogenesis and vasculogenesis. Angiogenesis is the process of neovascularization from preexisting blood vessels, while vasculogenesis is the process of blood vessel generation from angioblast precursor cells. The human placenta undergoes extensive angiogenesis and vasculogenesis during development. Additionally, the developing placenta undergoes a process of vascular mimicry as cytotrophoblasts convert from an epithelial to an endothelial phenotype. When placental vascular development is deranged, the success of the pregnancy is jeopardized and serious complications such as preeclampsia and growth restriction can occur. During the first two trimesters the spiral arteries undergo extensive remodeling. This is believed to reduce maternal blood flow resistance and to increase uteroplacental perfusion. Abnormal spiral arterial remodeling is a key pathological feature of both preeclampsia and intrauterine growth retardation. Other pregnancy complications such as placental abruption and idiopathic intrauterine fetal death (IUFD) may also be due to defective placental vascular development and coagulation abnormalities; however, mechanisms mediating these complications are less well understood.
Mammalian implantation is a complex, but still poorly understood, process in which the developing embryo establishes contact with the endometrial surface epithelium before developing a haemochorial placenta. The growth and development of the placenta and its subsequent invasion into the decidua can be likened to the invasion of healthy tissue by malignancy. The establishment of an angiogenic phenotype is a critical event in the progression of benign disease to malignant. Little is known about the role of angiogenesis in implantation but in view of the importance of vascular development to the receptive endometrium, angiogenesis is likely to be critical for successful implantation. Here, the factors that control the development of blood vessels in the pre-implantation phase are reviewed and the complex interplay between the embryo and decidua that leads to the establishment of a healthy placenta is considered.
Placental villi can be classified into subtypes that differ principally in their caliber, stromal, and vessel architecture, position within the villous tree, and function (Fig. 7.1). Several classification systems have been used in the past, but the one most commonly employed today was introduced by Kaufmann and colleagues (1979). This recognizes the following villous types (Fig.7.2).KeywordsVascular Endothelial Growth FactorSpiral ArteryCytotrophoblast CellExtravillous TrophoblastVillous TreeThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.
Adipose stem/stromal cells (ASC) after isolation produce numerous angiogenic growth factors. This justifies their use to promote angiogenesis per transplantation. In parallel, local co-implantation of ASC with endothelial cells (EC) leading to formation of functional vessels by the donor cells suggests the existence of a mechanism responsible for fine-tuning ASC paracrine activity essential for vasculogenesis. As expected, conditioned media (CM) from ASC promoted EC survival, proliferation, migration, and vasculogenesis. In contrast, media from EC-ASC co-cultures had neutral effects upon EC responses. Media from co-cultures exhibited lower levels of VEGF, HGF, Angiopoietin-1, and SDF-1 compared to those in ASC CM. Activin A was induced in ASC in response to EC exposure and was responsible for overall anti-vasculogenic activity of EC-ASC CM. Except for VEGF, activin A diminished secretion of all tested factors by ASC. Activin A mediated induction of VEGF expression in ASC, but also upregulated expression of VEGF scavenger receptor FLT-1 in EC in EC-ASC co-cultures. Blocking the FLT-1 expression in EC led to an increase in VEGF concentration in CM. In vitro pre-exposure of ASC to low number of EC prior to subcutaneous co-implantation with EC resulted in decrease in vessel density in the implants. In vitro tests suggested that activin A was partially responsible for this diminished ASC activity. This study shows that neo-vessel formation is associated with induction of activin A expression in ASC; this factor, by affecting the bioactivity of both ASC and EC, directs the crosstalk between these complementary cell types to establish stable vessels. This article is protected by copyright. All rights reserved.
© 2015 AlphaMed Press.
Objective:
Most anti-angiogenic preeclampsia models in rodents utilized the overexpression of a truncated soluble fms-like tyrosine kinase-1 (sFlt-1) not expressed in any species. Other limitations of mouse preeclampsia models included stressful blood pressure measurements and the lack of postpartum monitoring. We aimed to 1) develop a mouse model of preeclampsia by administering the most abundant human placental sFlt-1 isoform (hsFlt-1-e15a) in preeclampsia; 2) determine blood pressures in non-stressed conditions; and 3) develop a survival surgery that enables the collection of fetuses and placentas and postpartum (PP) monitoring.
Methods:
Pregnancy status of CD-1 mice was evaluated with high-frequency ultrasound on gestational days (GD) 6 and 7. Telemetry catheters were implanted in the carotid artery on GD7, and their positions were verified by ultrasound on GD13. Mice were injected through tail-vein with adenoviruses expressing hsFlt-1-e15a (n = 11) or green fluorescent protein (GFP; n = 9) on GD8/GD11. Placentas and pups were delivered by cesarean section on GD18 allowing PP monitoring. Urine samples were collected with cystocentesis on GD6/GD7, GD13, GD18, and PPD8, and albumin/creatinine ratios were determined. GFP and hsFlt-1-e15a expression profiles were determined by qRT-PCR. Aortic ring assays were performed to assess the effect of hsFlt-1-e15a on endothelia.
Results:
Ultrasound predicted pregnancy on GD7 in 97% of cases. Cesarean section survival rate was 100%. Mean arterial blood pressure was higher in hsFlt-1-e15a-treated than in GFP-treated mice (∆MAP = 13.2 mmHg, p = 0.00107; GD18). Focal glomerular changes were found in hsFlt-1-e15a -treated mice, which had higher urine albumin/creatinine ratios than controls (109.3 ± 51.7 μg/mg vs. 19.3 ± 5.6 μg/mg, p = 4.4 x 10(-2); GD18). Aortic ring assays showed a 46% lesser microvessel outgrowth in hsFlt-1-e15a-treated than in GFP-treated mice (p = 1.2 x 10(-2)). Placental and fetal weights did not differ between the groups. One mouse with liver disease developed early-onset preeclampsia-like symptoms with intrauterine growth restriction (IUGR).
Conclusions:
A mouse model of late-onset preeclampsia was developed with the overexpression of hsFlt-1-e15a, verifying the in vivo pathologic effects of this primate-specific, predominant placental sFlt-1 isoform. HsFlt-1-e15a induced early-onset preeclampsia-like symptoms associated with IUGR in a mouse with a liver disease. Our findings support that hsFlt-1-e15a is central to the terminal pathway of preeclampsia, and it can induce the full spectrum of symptoms in this obstetrical syndrome.
The soluble fms-like tyrosine kinase 1 (sFlt-1), known to be increased in the serum of preeclamptic patients, is a relevant factor in causing maternal symptoms like hypertension and proteinuria. In this study, we aimed to reveal whether hypoxia is a cause of increased sFlt-1 levels and inflammation markers in vivo and whether these symptoms can be attenuated by interleukin 6 (IL-6) depletion. For this purpose, pregnant wild-type (wt) mice or IL-6(-/-) mice on embryonic day 16 were placed under either normoxic (20.9% oxygen) or hypoxic (6% oxygen) conditions for 6 hours. This led to a rise of sFlt-1 levels in maternal serum, independent of the IL-6 status of the dam. Increased maternal sFlt-1 serum levels were, however, not due to an increase in sFlt-1 messenger RNA levels in the placenta. Moreover, there was no increase in inflammatory markers in neither wt mice nor IL-6(-/-) mice. This suggests that hypoxia alone does not contribute to the induction of an inflammatory placenta. Also, the hypoxia-induced rise in sFlt-1 levels seems not to be mediated by IL-6 in vivo.
© The Author(s) 2014.
Objective
Soluble fms-like tyrosine kinase (sFlt)-1-e15a, a primate-specific sFlt-1-isoform most abundant in the human placenta in preeclampsia, can induce preeclampsia in mice. This study compared the effects of full-length human (h)sFlt-1-e15a with those of truncated mouse (m)sFlt-1(1-3) used in previous preeclampsia studies on pregnancy outcome and clinical symptoms in preeclampsia.
Methods
Mice were injected with adenoviruses or fiber-mutant adenoviruses overexpressing hsFlt-1-e15a, msFlt-1(1-3) or control GFP under the CMV or CYP19A1 promoters on gestational day 8 (GD8) and GD11. Placentas and pups were delivered by cesarean section, and dams were monitored postpartum. Blood pressure was telemetrically recorded. Urine samples were collected with cystocentesis and examined for albumin/creatinine ratios. Tissue specimens were evaluated for transgene as well as endogenous mFlt-1 and msFlt-1-i13 expression. H&E-, Jones- and PAS-stained kidney sections were histopathologically examined. Placental GFP expression and aortic ring assays were investigated with confocal microscopy.
Results
Mean arterial blood pressure (MAP) was elevated before delivery in hsFlt-1-e15a-treated mice compared to controls (GD18: ΔMAP = 7.8 mmHg, p = 0.009), while ΔMAP was 12.8 mmHg (GD18, p = 0.005) in msFlt-1(1-3)-treated mice. Urine albumin/creatinine ratio was higher in hsFlt-1-e15a-treated mice than in controls (GD18, p = 0.04; PPD8, p = 0.03), and msFlt-1(1-3)-treated mice had marked proteinuria postpartum (PPD8, p = 4×10−5). Focal glomerular changes were detected in hsFlt-1-e15a and msFlt-1(1-3)-treated mice. Aortic ring microvessel outgrowth was decreased in hsFlt-1-e15a (p = 0.007) and msFlt-1(1-3)-treated (p = 0.02) mice. Full-length msFlt-1-i13 expression was unique for the placenta. In hsFlt-1-e15a-treated mice, the number of pups (p = 0.046), total weight of living pups (p = 0.04) and maternal weights (p = 0.04) were higher than in controls. These differences were not observed in truncated msFlt-1(1-3)-treated mice.
Conclusions
Truncated msFlt-1(1-3) simulated the preeclampsia-promoting effects of full-length hsFlt-1. MsFlt-1(1-3) had strong effect on maternal endothelium but not on placentas and embryos. In contrast, hsFlt-1-e15a induced preeclampsia-like symptoms; however, it also increased litter size. In accord with the predominant placental expression of hsFlt-1-e15a and msFlt-1-i13, full-length sFlt-1 may have a role in the regulation of embryonic development. These observations point to the difference in the biological effects of full-length and truncated sFlt-1 and the changes in the effect of full-length sFlt-1 during pregnancy, and may have important implications in the management of preeclampsia.
There is strong evidence that overproduction of soluble fms-like tyrosine kinase-1 (sFLT1) in the placenta is a major cause of vascular dysfunction in preeclampsia through sFLT1-dependent antagonism of VEGF. However, the cause of placental sFLT1 upregulation is not known. Here we demonstrated that in women with preeclampsia, sFLT1 is upregulated in placental trophoblasts, while VEGF is upregulated in adjacent maternal decidual cells. In response to VEGF, expression of sFlt1 mRNA, but not full-length Flt1 mRNA, increased in cultured murine trophoblast stem cells. We developed a method for transgene expression specifically in mouse endometrium and found that endometrial-specific VEGF overexpression induced placental sFLT1 production and elevated sFLT1 levels in maternal serum. This led to pregnancy losses, placental vascular defects, and preeclampsia-like symptoms, including hypertension, proteinuria, and glomerular endotheliosis in the mother. Knockdown of placental sFlt1 with a trophoblast-specific transgene caused placental vascular changes that were consistent with excess VEGF activity. Moreover, sFlt1 knockdown in VEGF-overexpressing animals enhanced symptoms produced by VEGF overexpression alone. These findings indicate that sFLT1 plays an essential role in maintaining vascular integrity in the placenta by sequestering excess maternal VEGF and suggest that a local increase in VEGF can trigger placental overexpression of sFLT1, potentially contributing to the development of preeclampsia and other pregnancy complications.
The placental vasculature rapidly expands during the course of pregnancy in order to sustain the growing needs of the fetus. Angiogenesis and vascular growth are stimulated and regulated by a variety of growth factors expressed in the placenta or present in the fetal circulation. Like in tumors, hypoxia is a major regulator of angiogenesis because of its ability to stimulate expression of various proangiogenic factors. Chronic fetal hypoxia is often found in pregnancies complicated by maternal diabetes as a result of fetal hyperglycaemia and hyperinsulinemia. Both are associated with altered levels of hormones, growth factors, and proinflammatory cytokines, which may act in a proangiogenic manner and, hence, affect placental angiogenesis and vascular development. Indeed, the placenta in diabetes is characterized by hypervascularisation, demonstrating high placental plasticity in response to diabetic metabolic derangements. This review describes the major regulators of placental angiogenesis and how the diabetic environment in utero alters their expression. In the light of hypervascularized diabetic placenta, the focus was placed on proangiogenic factors.
The last several decades have seen intensive research into the molecular mechanisms underlying the symptoms of preeclampsia. While the underlying cause of preeclampsia is believed to be defective placental development and resulting placental ischemia, it is only recently that the links between the ischemic placenta and maternal symptomatic manifestation have been elucidated. Several different pathways have been implicated in the development of the disorder; most notably production of the anti-angiogenic protein sFlt-1, induction of auto-immunity and inflammation, and production of reactive oxygen species. While the molecular mechanisms are becoming clearer, translating that knowledge into effective therapeutics has proven elusive. Here we describe a number of peptide based therapies we have developed to target theses pathways, and which are currently being tested in preclinical models. These therapeutics are based on a synthetic polymeric carrier elastin-like polypeptide (ELP), which can be synthesized in various sequences and sizes to stabilize the therapeutic peptide and avoid crossing the placental interface. This prevents fetal exposure and potential developmental effects. The therapeutics designed will target known pathogenic pathways, and the ELP carrier could prove to be a versatile delivery system for administration of a variety of therapeutics during pregnancy.
Introduction:
Vascular endothelial growth factor (VEGF) is relevant for healthy pregnancy, and abnormalities in VEGF functions have been associated with hypertensive disorders of pregnancy. Our group recently demonstrated that VEGF genetic polymorphisms affect the susceptibility to preeclampsia (PE).
Objective:
Therefore, in this study our aim is to examine whether VEGF polymorphisms affect the antihypertensive responses in women with PE.
Methods:
We studied 113 white PE women who were stratified according to blood pressure levels after antihypertensive treatment (46 responsive, R group and 67 non-responsive, NR group). We then compared the frequencies of two VEGF genetic polymorphisms (C-2578A and G-634C) between R and NR groups.
Results:
We found no significant differences in genotype or allele distributions between R and NR groups (P > 0.05). In addition, no difference was observed in overall distribution of haplotypes (P > 0.05).
Conclusion:
Our data suggest that VEGF polymorphisms do not affect responsiveness to the antihypertensive therapy in PE.
Objective:
Preeclampsia (PE) affects 2-8% of pregnancies worldwide and is a significant source of maternal and neonatal morbidity and mortality. However, the mechanisms underlying PE are poorly understood and major questions regarding etiology and risk factors remain to be addressed. Our objective was to examine whether abnormal expression of the cardiovascular developmental transcription factor, Nkx2-5, was associated with early onset and severe preeclampsia (EOSPE).
Methods:
Using qPCR and immunohistochemical assay, we examined expression of Nkx2-5 and target gene expression in EOSPE and control placental tissue. We tested resulting mechanistic hypotheses in cultured cells using shRNA knockdown, qPCR, and western blot.
Results:
Nkx2-5 is highly expressed in racially disparate fashion (Caucasians > African Americans) in a subset of early EOSPE placentae. Nkx2-5 mRNA expression is highly correlated (Caucasians > African Americans) to mRNA expression of the preeclampsia marker sFlt-1, and of the Nkx2-5 target and RNA splicing factor, Sam68. Knockdown of Sam68 expression in cultured cells significantly impacts sFlt-1 mRNA isoform generation in vitro, supporting a mechanistic hypothesis that Nkx2-5 impacts EOSPE severity in a subset of patients via upregulation of Sam68 to increase sFlt-1 expression. Expression of additional Nkx2-5 targets potentially regulating metabolic stress response is also elevated in a racially disparate fashion in EOSPE.
Conclusions:
Expression of Nkx2-5 and its target genes may directly influence the genesis and racially disparate severity, and define a mechanistically distinct subclass of EOSPE.
Introduction:
Altered angiogenesis has been implicated in the pathogenesis of various pregnancy complications, particularly preeclampsia. At present, there is a lack of data on the possible role of angiogenesis and its molecular mechanism in preterm pregnancy. We have previously reported reduced placental global DNA methylation levels in preterm pregnancy. Now, we have extended the study to examine plasma levels of angiogenic factors from maternal and cord blood and correlate them with placental promoter CpG methylation and messenger RNA expression of these angiogenic genes in preterm pregnancies.
Methods:
We recruited 99 women delivering at term and 90 women delivering preterm. Plasma levels of angiogenic factors, vascular endothelial growth factor (VEGF), placental growth factor (PlGF), fms-related tyrosine kinase 1 (FLT-1), and kinase insert domain receptor (KDR) were analyzed by enzyme-linked immunosorbent assay. Expression levels and promoter CpG methylation of angiogenic genes in placentae were determined by quantitative real-time polymerase chain reaction and by the Sequenom EpiTYPER technology, respectively.
Results:
Maternal VEGF and PlGF levels (P < .01 for both) were lower but soluble FLT-1 (sFLT-1) levels and sFLT-1-PlGF ratio (P < .05 for both) were higher in the preterm group. Placental VEGF expression (P < .05) was lower, and CpG site 14 in the VEGF promoter was hypermethylated (P < .05) in the preterm group. The KDR expression (P < .05) was higher in women delivering preterm.
Conclusions:
Our study provides first evidence of differential placental CpG methylation patterns and expression of VEGF, FLT-1, and KDR genes in women delivering preterm. This may explain the possible mechanism for angiogenic imbalance in the pathophysiology of preterm pregnancy.
Pre-eclampsia, characterized by hypertension and proteinuria, affects approximately 3-5% of all pregnancies worldwide and is a major cause of maternal and fetal morbidity and mortality. Maternal endothelial dysfunction is associated with disease pathogenesis. Recently, reports have shown that elevated levels of circulating soluble fms-like tyrosine kinase 1 [sFlt1] and soluble endoglin [sEng] are associated with pre-eclampsia. Flt1 is a receptor for vascular endothelial growth factor receptor [VEGF], whereas endoglin [Eng] is an auxiliary receptor for transforming growth factor-β [TGF-β] super-family members. Both signaling pathways modulate angiogenesis and are involved in vascular homeostasis. Increased levels of sFlt1 and sEng dysregulate VEGF and TGF-β signaling respectively, resulting in endothelial dysfunction of maternal blood vessels. This review summarizes our current knowledge of Flt1 and endoglin and soluble forms in pre-eclampsia. Furthermore, it highlights the predictive and early-screening value of circulating levels of sFlt1 and sEng for the risk of developing pre-eclampsia.
Copyright © 2012 International Society for the Study of Hypertension in Pregnancy. Published by Elsevier B.V. All rights reserved.
Chymase is a chymotrypsin-like serine protease. It has been identified as a key angiotensin converting enzyme (ACE)-independent and endothelin converting enzyme (ECE)-independent converting enzyme that generates angiotensin II and endothelin-1 (ET-1). As an inflammatory protease, chymase participates in multiple inflammatory responses in the vasculature which drive cytokine production and adhesion molecule expression. Chymase is also involved in extracellular matrix remodeling in both vascular and non-vascular tissues. Consequently, chymase has been implicated in the pathogenesis of multiple cardiovascular, immune, and inflammatory diseases. Recent studies have shown that chymase expression and activity are increased in placental trophoblasts and in the maternal vascular endothelium in women diagnosed with preeclampsia, a hypertensive disorder in human pregnancy. The present review will address the potential roles of chymase-mediated placental and vascular dysfunction in preeclampsia. The effects of chymase on inflammatory responses associated with endothelial activation/dysfunction in preeclampsia are also discussed.
Fibroblast growth factors (FGF) and their tyrosine kinase receptors (FGFR) support cell proliferation, survival and migration during embryonic development, organogenesis and tissue maintenance and their deregulation is frequently observed in cancer development and progression. Consequently, increasing efforts are focusing on the development of strategies to target FGF/FGFR signaling for cancer therapy. Among the FGFRs the family member FGFR4 is least well understood and differs from FGFRs1-3 in several aspects. Importantly, FGFR4 deletion does not lead to an embryonic lethal phenotype suggesting the possibility that its inhibition in cancer therapy might not cause grave adverse effects. In addition, the FGFR4 kinase domain differs sufficiently from those of FGFRs1-3 to permit development of highly specific inhibitors. The oncogenic impact of FGFR4, however, is not undisputed, as the FGFR4-mediated hormonal effects of several FGF ligands may also constitute a tissue-protective tumor suppressor activity especially in the liver. Therefore it is the purpose of this review to summarize all relevant aspects of FGFR4 physiology and pathophysiology and discuss the options of targeting this receptor for cancer therapy.
FLT1 and its soluble form (sFLT1) arise as alternate transcripts from the same gene and sFLT1 can antagonize the effect of vascular endothelial growth factor (VEGF) on its cognate receptors. We investigated the effect of VEGF and protein kinase C (PKC) activation on sFLT1 abundance. We demonstrated that VEGF stimulates sFLT1 and FLT1 mRNA and protein levels in vascular endothelial cells via VEGFR2 and PKC. Using an FLT1 expression vector with N and C-terminal epitope tags, we show that PKC activation increases the cleavage of FLT1 into an N-terminal extracellular fragment and a C-terminal intracellular fragment with the cleavage occurring adjacent to the transmembrane domain. The trafficking and glycosylation inhibitors brefeldin, monensin and tunicamycin substantially reduced cleavage and release of the N-terminal ectodomain of FLT1 and inhibited secretion of the isoforms of sFLT1. The shed FLT1 ectodomain can bind VEGF and PlGF and inhibit VEGF-induced vascular tube formation thus confirming that it is functionally equivalent to the alternately spliced and secreted sFLT1 isoforms.
Inhibition of vascular endothelial growth factor (VEGF) has become the standard of care for patients presenting with wet age-related macular degeneration. However, monthly intravitreal injections are required for optimal efficacy. We have previously shown that electroporation enabled ciliary muscle gene transfer results in sustained protein secretion into the vitreous for up to 9 months. Here, we evaluated the long-term efficacy of ciliary muscle gene transfer of three soluble VEGF receptor-1 (sFlt-1) variants in a rat model of laser-induced choroidal neovascularization (CNV). All three sFlt-1 variants significantly diminished vascular leakage and neovascularization as measured by fluorescein angiography (FA) and flatmount choroid at 3 weeks. FA and infracyanine angiography demonstrated that inhibition of CNV was maintained for up to 6 months after gene transfer of the two shortest sFlt-1 variants. Throughout, clinical efficacy was correlated with sustained VEGF neutralization in the ocular media. Interestingly, treatment with sFlt-1 induced a 50% downregulation of VEGF messenger RNA levels in the retinal pigment epithelium and the choroid. We demonstrate for the first time that non-viral gene transfer can achieve a long-term reduction of VEGF levels and efficacy in the treatment of CNV.Gene Therapy advance online publication, 27 June 2013; doi:10.1038/gt.2013.36.
Although there have been many experimental studies demonstrating that bone marrow-derived mesenchymal stem cells (MSCs) have the potential to differentiate into mesenchymal tissues such as osteocytes, chondrocytes, and adipocytes in vivo and in vitro, little information is available regarding their potential to differentiate into lymphatic endothelial cells. Therefore, we chose to investigate differentiation of MSCs into lymphatic endothelial cells using stimulation with members of the vascular endothelial growth factor (VEGFs) family. Rat MSCs were isolated from bone marrow aspirate of Sprague-Dawley rats as previously described and characterized with flow cytometry for surface markers CD14, CD34, CD29, and CD90. Purified MSCs were plated and cultured in the presence of VEGF-A, VEGF-C, or the combination of both for 10 days. We examined the cells for Prox-1 and LYVE-1 by immunocytochemistry, RT-PCR, and Western blot analysis. Results demonstrated that compared to controls, cell differentiated with VEGF-A, VEGF-C and VEGF-A+VEGF-C expressed Prox-1 and LYVE-1. Our results indicate that MSCs induced by VEGFs are capable of differentiating into lymphatic endothelial-like cells in vitro, and this response has the potential to make them attractive candidates for the development of autologous tissue grafts for future therapy.
The sequential changes in the placenta from rats exposed to methotrexate were examined histopathologically. Methotrexate was intraperitoneally administered at 0.2mg/kg/day during gestation days (GDs) 11-12 or GDs 13-14, and the placentas were sampled on GDs 13, 15, 17 and 21. The fetal mortality rates tended to increase throughout the experimental period, and fetal weights were significantly decreased on GD13 in the GD11,12-treated group. A significant reduction in placental weights was detected on GDs 13 and 15 in the GD11,12-treated group. Histopathologically, in the GD11,12-treated group, a significant thinning of the basal zone was detected throughout the experimental period, whereas the thickness of the labyrinth zone decreased significantly during GDs 13-17. The severity of thinning of the basal zone was higher than that of the labyrinth zone. In addition, a marked decrease in glycogen cell-islands in the basal zone was detected on GD 15. A significant decrease in Phospho-Histone H3-positive cells and a significant increase in terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells were detected on GD 13 in the basal zone of the GD11,12-treated group. In the GD13,14-treated group, there were no other significant changes in placentas and fetuses throughout the experimental period other than thinning of the labyrinth zone on GD 17. These results suggest that component cells of the basal and labyrinth zones during GDs 11-12 are more highly sensitive for methotrexate than those during GDs 13-14 and methotrexate affects the component cells of basal zone more strongly than those of the labyrinth zone.
Thrombospondin-1 (TSP-1) is an endogenous inhibitor of angiogenesis whose expression suppresses tumor growth in vivo. Like many angiogenesis-related genes, TSP-1 expression is tightly controlled by various mechanisms, but there is little data regarding the contribution of post-transcriptional processing to this regulation. NOL7 is a novel tumor suppressor that induces an antiangiogenic phenotype and suppresses tumor growth, in part through upregulation of TSP-1. Here we demonstrate that NOL7 is an mRNA-binding protein that must localize to the nucleoplasm to exert its antiangiogenic and tumor suppressive effects. There, it associates with the RNA-processing machinery and specifically interacts with TSP-1 mRNA through its 3'UTR. Reintroduction of NOL7 into SiHa cells increases luciferase expression through interaction with the TSP-1 3'UTR at both the mRNA and protein levels. NOL7 also increases endogenous TSP-1 mRNA half-life. Further, NOL7 post-transcriptional stabilization is observed in a subset of angiogenesis-related mRNAs, suggesting that the stabilization of TSP-1 may be part of a larger novel mechanism. These data demonstrate that NOL7 significantly alters TSP-1 expression and may be a master regulator that coordinates the post-transcriptional expression of key signaling factors critical for the regulation of the angiogenic phenotype.Oncogene advance online publication, 22 October 2012; doi:10.1038/onc.2012.464.
Dendritic cell (DC) and natural killer (NK) cell interactions are important for the regulation of innate and adaptive immunity, but their relevance during early pregnancy remains elusive. Using two different strategies to manipulate the frequency of NK cells and DC during gestation, we investigated their relative impact on the decidualization process and on angiogenic responses that characterize murine implantation. Manipulation of the frequency of NK cells, DC or both lead to a defective decidual response characterized by decreased proliferation and differentiation of stromal cells. Whereas no detrimental effects were evident upon expansion of DC, NK cell ablation in such expanded DC mice severely compromised decidual development and led to early pregnancy loss. Pregnancy failure in these mice was associated with an unbalanced production of anti-angiogenic signals and most notably, with increased expression of genes related to inflammation and immunogenic activation of DC. Thus, NK cells appear to play an important role counteracting potential anomalies raised by DC expansion and overactivity in the decidua, becoming critical for normal pregnancy progression.
The objective of this study was to evaluate fetal weight, histomorphometric changes and proliferative activity, apoptosis and angiogenesis of the placenta in rats with hypothyroidism. Thirty-six adult female rats were divided into two groups with 18 animals each: control and hypothyroidism. Hypothyroidism was induced by daily administration of propylthiouracil (1 mg/animal). The administration began five days before becoming pregnant and the animals were sacrificed at 14 or 19 days of gestation. The control group received a placebo. The number and weight of fetuses and the rate of fetal death was determined, as well as the morphometric characteristics, the immunohistochemical expression of cell division control protein 47 (CDC)-47 and vascular endothelial growth factor (VEGF) and the number of apoptotic cells in the placental disk. The data were analysed by Mann-Whitney U test. Hypothyroidism reduced the weight of fetuses and of the uterus and placenta (P<0.05), altered the thickness of the placental labyrinth and spongiotrophoblast (P<0.05), increased the population of glycogen cells in the spongiotrophoblast (P<0.05), interfered with the vascular development of the placental labyrinth and decreased VEGF expression (P<0.05), reduced the expression of CDC-47 and cellularity and increased the apoptotic rate in the placental disk (P<0.05). We conclude that hypothyroidism affects fetal weight by altering the proliferative activity, apoptosis and vascularisation of the placenta.
Tumor progression and pregnancy share many common features, such as immune tolerance and invasion. The invasion of trophoblasts in the placenta into the uterine wall is essential for fetal development, and is thus precisely regulated. Its deregulation has been implicated in preeclampsia, a leading cause for maternal and perinatal mortality and morbidity. Pathogenesis of preeclampsia remains to be defined. Microarray-based gene profiling has been widely used for identifying genes responsible for preeclampsia. In this review, we have summarized the recent data from the microarray studies with preeclamptic placentas. Despite the complex of gene signatures, suggestive of the heterogeneity of preeclampsia, these studies identified a number of differentially expressed genes associated with preeclampsia. Interestingly, most of them have been reported to be tightly involved in tumor progression. We have discussed these interesting genes and analyzed their potential molecular functions in preeclampsia, compared with their roles in malignancy development. Further investigations are warranted to explore the involvement in molecular network of each identified gene, which may provide not only novel strategies for prevention and therapy for preeclampsia but also a better understanding of cancer cells. The trophoblastic cells, with their capacity for proliferation and differentiation, apoptosis and survival, migration, angiogenesis and immune modulation by exploiting similar molecular pathways, make them a compelling model for cancer research.
: Preeclampsia is characterized by an imbalance in angiogenic factors such as soluble fms-like tyrosine kinase-1 (sFlt-1) and placental growth factor (PlGF). We herein assessed whether these factors measured by a newly developed automated electrochemiluminescence immunoassay are associated with risk to develop preeclampsia.
: We performed a nested case-control study within a cohort of 230 women with singleton pregnancies. The study included all 37 women who eventually developed preeclampsia and 29 normotensive controls. Serum samples were collected at 4-week intervals (from weeks 20th to 36th). sFlt-1 and PlGF were measured using a commercial automated immunoassay (Elecsys).
: Women destined to develop preeclampsia had lower PlGF levels and higher sFlt-1 levels and sFlt-1/PlGF ratio than women with normal pregnancies. These changes became significant at 20 weeks in women destined to develop early preeclampsia (<34 weeks, P ≤ 0.003), and at 24-28 weeks in women who later developed preeclampsia (P ≤ 0.024). The risk for developing preeclampsia was higher among women with PlGF concentration values in the lowest quartile or with sFlt-1 levels and sFlt-1/PlGF ratio in the highest quartile of the control distribution. The odds ratios were higher and appeared earlier in women destined to develop early preeclampsia than in women who presented preeclampsia later. The sFlt-1/PlGF ratio was more tightly associated with risk of preterm or term preeclampsia than either angiogenic factor alone.
: Changes in circulating concentrations of PlGF, sFlt-1, and in the sFlt-1/PlGF ratio precede the onset of preeclampsia. The risk profile of circulating angiogenic factors for developing preeclampsia distinctly evolves depending on whether this condition is manifested at preterm or term.
While it is self-evident that the placenta is essential for the mammalian reproductive process, historically its role has
been confused. The precise architectural arrangement of the blood vessels has been a matter of dispute, and this in part can
be explained by the diversity of structures observed in common domestic, laboratory, and wild species (for a review of this
subject area, see Kaufmann and Burton, 1994). However, by the beginning of the last century it had been established that the
placenta served as an organ for fetal nutrition, respiration, and as an endocrine organ. All of the different placental types
show specialized adaptations to facilitate exchange between the fetal and maternal circulatory systems. Both the human and
murine placentae are of the hemochorial type, and although there are marked differences between them, knockout and transgenic
studies in the mouse have been extremely informative. It is a reasonable assumption that the requirements for exchange play
the most significant role in determining placental morphology, and therefore the adaptations of the fetal vasculature to facilitate
this process are fundamental. In a range of species as gestation advances and fetal demand increases, the capacity of the
placenta for exchange increases. In the human, there is a continual elaboration of the principal functional units of the placenta,
the terminal villi, and a progressive reduction in the mean thickness of the villous membrane separating the two circulations.
These changes, and the changes seen in other species, are dependent on continued angiogenesis, and therefore placental angiogenesis
is central for the success of pregnancy.
Pre-eclampsia, small-for-gestational-age infants, preterm birth and recurrent miscarriage complicate a significant number of pregnancies. The vascular endothelial growth factor (VEGF) family of angiogenic growth factors is implicated in the pathophysiology of these complications. We aimed to elucidate the role of these angiogenic factors in placentation and to evaluate the predictive value of their protein concentrations and genetic variations in pregnancy complications.
We performed a systematic search of PubMed, and retrieved original articles. The search included a combination of terms such as VEGF-A, placental growth factor (PlGF), kinase insert domain receptor, fms-like-tyrosine-kinase receptor 1, soluble fms-like-tyrosine-kinase receptor 1, pre-eclampsia, small-for-gestational-age infants, preterm birth, recurrent miscarriage, placenta, prediction and polymorphisms.
This review summarizes the current knowledge of the roles of the VEGF family in early placentation and of the abnormalities in maternal plasma and placental expression of angiogenic proteins in adverse pregnancy outcomes compared with normal pregnancy. PlGF and sFLT-1 in combination with other clinical and biochemical markers in late first or second trimester appear to predict early-onset pre-eclampsia with a high sensitivity and specificity. However, VEGF family proteins do not have sufficient power to accurately predict late-onset pre-eclampsia, small-for-gestational age pregnancies or preterm birth. Functional polymorphisms in these angiogenic genes are implicated in pregnancy complications, but their contribution appears to be minor.
Although the VEGF family has important roles in normal and complicated pregnancy, the current predictive value of the VEGF family as biomarkers appears to be limited to early-onset pre-eclampsia.
Through studies of transgenic and mutant mice, it is possible to describe molecular pathways that control the development of all major trophoblast cell subtypes and structures of the placenta. For example, the proliferation of trophoblast stem cells is dependent on FGF signalling and downstream transcription factors Cdx2, Eomes and Err2. Several bHLH transcription factors regulate the progression from trophoblast stem cells to spongiotrophoblast and to trophoblast giant cells (Id1/2, Mash2, Hand1, Stra13). Intercellular actions critical for maintaining stable precursor cell populations are dependent on the gap junction protein Cx31 and the growth factor Nodal. Differentiation towards syncytiotrophoblast as well as the initiation of chorioallantoic (villous) morphogenesis is regulated by the Gcm1 transcription factor, and subsequent labyrinth development is dependent on Wnt, HGF and FGF signalling. These insights suggest that most of the genes that evolved to regulate placental development are either identical to ones used in other organ systems (e.g., FGF and epithelial branching morphogenesis), were co-opted to take on new functions (e.g., AP-2gamma, Dlx3, Hand1), or arose via gene duplication to take on a specialized placental function (e.g., Gcm1, Mash2). Many of the human orthologues of these critical genes show restricted expression patterns that are consistent with a conserved function. Such information is aiding the comparison of the human and mouse placenta. In addition, the prospect of a conserved function clearly suggests potential mechanisms for explaining complications of human placental development.
This study aimed to investigate the effects of Kraussianone-2 (Kr2), a pyrano-isoflavone isolated from the roots of Eriosema kraussianum N. E. Br. (Fabaceae) on various fetal and physiological parameters in pregnant, L-NAME treated Sprague-Dawley rats. Twenty-four pregnant Sprague-Dawley dams were divided into three groups (n = 8), i.e. the control group (CON), the experimental control group (PRE), where the pre-eclampsia-like symptoms were induced using L-NAME, and the experimental group (EK2), where the pre-eclampsia-like symptoms were once again induced using L-NAME, however, these animals were treated with Kr2. On gestation day 20 the animals were sacrificed, at which time a laparotomy was performed and the number of live pups were counted and their corresponding birth and placental weights were recorded. Blood was also collected in heparin-coated tubes and the plasma samples were then analysed for specific variables using commercially available kits for rats. Kraussianone-2 administration decreased fetal mortality and demonstrated a trend toward increasing birth and placental weights in this model. Furthermore, Kr2 administration also reduced blood pressure amplification and decreased the plasma concentrations of two antiangiogenic factors, soluble fms-like tyrosine kinase1 (sFlt-1) and soluble endoglin (sEng). We speculate that Kr2, by improving uterine artery blood flow, results in improved fetal outcomes and decreased antiangiogenic factors in pregnant, L-NAME treated, Sprague-Dawley rats. Copyright © 2012 John Wiley & Sons, Ltd.
Our earlier studies, in preeclamptic women have shown altered levels of long chain polyunsaturated fatty acids (LCPUFA), essential constituents of the cell membrane lipids responsible for membrane stability as one of the key factors contributing to the pathophysiology of preeclampsia. We have also reported elevated levels of sFlt-1 in preeclampsia. The present study examines the levels of LCPUFA and their association with sFlt-1 levels in 69 pre-eclamptic women and 40 normotensive women. DHA and omega 3 fatty acid levels were lower (p < 0.001) while arachidonic acid and omega 6 fatty acid levels were higher (p < 0.05) in preeclamptic women as compared to normotensive women. Maternal plasma sFlt-1 levels were higher (p < 0.05) in preeclamptic women and were negatively associated with DHA (p=0.008) and omega 3 fatty acids concentrations (p=0.031). Our results suggest that altered placental LCPUFA may result in altered membrane lipid fatty acid composition leading to increased release of sFlt-1 in circulation.
During decidualization, uterine natural killer (uNK) cells are the most abundant immune cell types found in the uterus. Although it is well known that they play key roles in spiral arteriole modification and the maintenance of decidual integrity seen after mid-pregnancy, their roles in the differentiation of decidual cells and accompanying angiogenesis during the process of decidualization is less well characterized. To address this, we used whole-genome Illumina BeadChip analysis to compare the gene expression profiles in implantation segments of the uterus during decidualization on day 7.5 of pregnancy between wild-type and uNK cell-deficient (interleukin-15-knockout) mice. We found almost 300 differentially expressed genes and verified the differential expression of ~60 using quantitative RT-PCR. Notably, there was a lack of differential expression of genes involved in decidualization and angiogenesis and this was also verified by quantitative RT-PCR. Similar endothelial cell densities and proliferation indices were also found in the endometrium between the implantation site tissues of wild-type and knockout mice undergoing decidualization. Overall, the results of this study reveal that uNK cells likely do not play a major role in decidualization and accompanying angiogenesis during implantation. In addition, the study identifies a large number of genes whose expression in implantation-site uterine tissue during decidualization depends on interleukin-15 expression in mice.
Diseases complicated by abnormal growth of vessels or excessive leakage are the most prevalent cause of moderate or severe vision loss in developed countries. Recent progress unraveling the molecular pathogenesis of several of these disease processes has led to new drug therapies that have provided major benefits to patients. However, those treatments often require frequent intraocular injections, and despite monthly injections, some patients have a suboptimal response. Gene transfer of antiangiogenic proteins is an alternative approach that has the potential to provide long-term suppression of neovascularization (NV) and/or excessive vascular leakage in the eye. Studies in animal models of ocular NV have demonstrated impressive results with a number of transgenes, and a clinical trial in patients with advanced neovascular age-related macular degeneration has provided proof-of-concept. Two ongoing clinical trials, one using an adeno-associated viral (AAV) vector to express a vascular endothelial growth factor-binding protein and another using a lentiviral vector to express endostatin and angiostatin, will provide valuable information that should help to inform future trials and provide a foundation on which to build.
Specific proteolytic cleavages of the hormone prolactin (PRL) generate vasoinhibins, a family of peptides (including 16-kDa PRL) that are able to inhibit the pathologic increase in retinal vasopermeability (RVP) associated with diabetes. Here the authors tested the ability of an adenoassociated virus type 2 (AAV2) vasoinhibin vector to inhibit vascular endothelial growth factor (VEGF)- and diabetes-induced RVP.
AAV2 vectors encoding vasoinhibin, PRL, or soluble VEGF receptor 1 (soluble FMS-like tyrosine kinase-1 [sFlt-1]) were injected intravitreally into the eyes of rats. Four weeks later, either VEGF was injected intravitreally or diabetes was induced with streptozotocin. Tracer accumulation was evaluated as an index of RVP using fluorescein angiography or the Evans blue dye method. RT-PCR verified transgene expression in the retina, and the intravitreal injection of an AAV2 vector encoding green fluorescent protein revealed transduced cells in the retinal ganglion cell layer. In addition, Western blot analysis of AAV2-transduced HEK293 cells confirmed the expression and secretion of the vector-encoded proteins.
The AAV2-vasoinhibin vector prevented the increase in tracer accumulation that occurs 24 hours after the intravitreal injection of VEGF. Diabetes induced a significant increase in tracer accumulation compared with nondiabetic controls. This increase was blocked by the AAV2-vasoinhibin vector and reduced by the AAV2-sFlt-1 vector. The AAV2-PRL vector had no effect.
These results show that an AAV2-vasoinhibin vector prevents pathologic RVP and suggest it could have therapeutic value in patients with diabetic retinopathy.
Contents
The development of dominant follicles requires the parallel growth of a vascular network, regulated by VEGF and its receptors VEGFR‐1 and VEGFR‐2. Here, we demonstrate the presence of mRNA for the soluble forms of VEGFR‐1 and VEGFR‐2 by RT‐PCR and the respective proteins by Western blot, in bovine dominant follicles. The 3′ end of the mRNA coding region and the deduced C‐terminal amino acid sequence of the bovine VEGFR soluble forms were similar to those previously described in human and mice. The relative abundance of sVEGFR‐1 was higher in dominant follicles of day 4, decreasing on day 6 and further on day 9 of the cycle. In contrast, sVEGFR‐2 expression was low on day 4 follicles and increased as the cycle advanced, becoming greater on day 9. The changes of sVEGFR‐1 and sVEGFR‐2 with the age of the bovine dominant follicle indicate a physiological role in its growth and atresia.
Alternative intronic polyadenylation (IPA) can generate truncated protein isoforms with significantly altered functions. Here, we describe 31 dominant-negative, secreted variant isoforms of receptor tyrosine kinases (RTKs) that are produced by activation of intronic poly(A) sites. We show that blocking U1-snRNP can activate IPA, indicating a larger role for U1-snRNP in RNA surveillance. Moreover, we report the development of an antisense-based method to effectively and specifically activate expression of individual soluble decoy RTKs (sdRTKs) to alter signaling, with potential therapeutic implications. In particular, a quantitative switch from signal transducing full-length vascular endothelial growth factor receptor-2 (VEGFR2/KDR) to a dominant-negative sKDR results in a strong antiangiogenic effect both on directly targeted cells and on naive cells exposed to conditioned media, suggesting a role for this approach in interfering with angiogenic paracrine and autocrine loops.
Preeclampsia is a major cause of maternal and perinatal mortality and morbidity. The etiology of preeclampsia remains unclear. Recently, it was shown that misregulation of fms-like tyrosine kinase-1 (Flt-1) in the peripheral blood mononuclear cells of pregnant women results in over-expression of the soluble splice variant of Flt-1, sFlt-1, producing an additional (extra-placental) source of sFlt-1 that can contribute to the etiology of preeclampsia. The aim of this study was to investigate the relationship between preeclampsia and a dinucleotide (threonine-glycine; TG)n repeat polymorphism in the 3' non-coding region of the Flt-1 gene.
The number of the d(TG)n repeats was analyzed in 170 patients with preeclampsia and in 202 normotensive pregnancies. The region containing the dinucleotide repeat polymorphism of the Flt-1 gene was amplified by polymerase chain reaction (PCR) from the DNA samples and was analyzed by direct PCR sequencing.
We found 10 alleles of the dinucleotide repeat polymorphism and designated these as allele*12 (A1) through allele*23 (A12) according to the number of the TG repeats, from 12 to 23. The frequency of the 14-repeat allele (A3) was most abundant (63.82% in preeclampsia and 69.06% in controls), followed by the 21-repeat allele (A10; 28.53% in preeclampsia and 23.76% in controls). There was no significant difference in the allele frequency between patients with preeclampsia and normal controls. The most common genotype in preeclamptic and normotensive pregnancies was heterozygous (TG)14/(TG)21 (41.76%) and homozygous (TG)14/(TG)14 (45.05%), respectively. However, the genotype frequencies were not significantly different between preeclamptic patients and controls.
This is the first study to characterize the dinucleotide repeat polymorphism of the Flt-1 gene in patients with preeclampsia. We found no differences in the allele or genotype frequencies between patients with preeclampsia and normal pregnancies. Although limited by a relatively small sample size, our study suggests that the d(TG)n repeat polymorphism of the Flt-1 gene is not associated with the development of preeclampsia in Korean pregnant women.
In early pregnancy, the O(2) gradient between the maternal circulation and the gestational sac tissues modulates trophoblast biological functions. The aim was to evaluate if placental partial pressure of oxygen (PaO(2)) modulates in vivo synthesis of specific placental proteins inside the first trimester gestational sac. Matched samples of peripheral venous blood, blood from the placental bed (PB), coelomic fluid (CF) and placental tissue were obtained in 37 normal pregnancies at 6-12 weeks gestation. PaO(2) was measured in PB and CF using an IRMA blood gas monitor. Inhibin A, activin A, sEng, PlGF, sFlt-1 and free VEGF concentrations were measured in all samples. HSP 70 was measured in placental extracts. ANOVA showed approximately 60% increase in PB PaO(2) (P = 0.02) between after 10 weeks gestation. Unpaired Student's T-test between two groups (6-9 weeks vs 9-12 weeks) shows a significant increase in MS Activin A (P = 0.001), CF activin A (P<0.001), MS P1GF (P = 0.001), CF PlGF (P<0.001), MS sFLT-1 (P = 0.03), CF sFLT-1 (P = 0.01), HSP 70 in placental extracts (P = 0.04) and a significant decrease in PB inhibin A levels (P<0.001) and PB sFLT-1 (P = 0.02) . Multiple correlation analysis showed a significant negative correlation between PB inhibin A levels and gestation (r = -0.45, P<0.05) and PB PaO(2) (r = -0.5, P = 0.008) and also between sFLT-1 and PB PaO(2) (P = 0.03). There was a positive correlation (P<0.01) between PlGF, sEng and VEGF levels in the placental extracts. Our results indicate a direct relationship in the early intrauterine PaO(2) in vivo and inhibin A and sFLT-1 concentrations confirming our hypothesis that specific placental proteins are regulated by intrauterine O(2) tension.
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