Growth Of Human Placenta Research Articles

A PlGF-1 Derived Peptide Inhibits Angiogenesis via HIF-1β/VEGF Pathway

Many diseases are associated with angiogenesis. Therefore, inhibition of angiogenesis is deemed as a treatment for those diseases. To date, many angiogenesis inhibitors are from large plasma proteins. In this study we identified a 21-aa peptide (named peptide ZY1) from human placenta growth factor-1 and it may serve as a potent angiogenesis inhibitor. Our study demonstrated ZY1 inhibited angiogenesis in vitro by suppressing proliferation, migration and tube formation of HUVECs. The in vivo inhibition activity of ZY1 was observed in chicken chorioallantoic membrane assays and tumor-bearing mouse models. Moreover, we found ZY1 inhibited angiogenesis by decreasing the expression of HIF-1β and subsequently reducing its downstream molecule VEGF. In conclusion, peptide ZY1 can inhibit angiogenesis and may serve as an anti-angiogenesis drug candidate.

Inhibition of ocular neovascularization by a novel peptide derived from human placenta growth factor‐1

To evaluate the effect of ZY1, a novel 21-amino acid peptide from human placenta growth factor-1 (PlGF-1), against ocular neovascularization, and to study its possible toxicity to the retina and the underlying mechanism of antiangiogenic effect. MTS assays, a modified Boyden chamber and Matrigel(™) were used to evaluate the effect of ZY1 on the proliferation, migration and tube formation of RF/6A rhesus macaque choroid-retina endothelial cells induced by vascular endothelial growth factor (VEGF) in vitro. The antiangiogenic effect of ZY1 was also studied with corneal micropocket angiogenesis assays and oxygen-induced retinopathy (OIR) assays in mice. Electrophysiological tests and histological examinations were used to study the possible toxicity of ZY1 against mouse neuroretina. Competitive ELISA and Western blotting were performed to elucidate the underlying mechanism of ZY1. ZY1 inhibited VEGF-induced RF/6A proliferation, migration and tube formation. It also inhibited ocular neovascularization when applied to the corneal micropocket angiogenesis assays and OIR assays in mice. Electrophysiological tests and histological examinations revealed no evident functional or morphologic abnormalities in mouse neuroretina after ZY1 injection. ZY1 competed for binding to VEGFR-1 against PlGF and VEGF and inhibited VEGFR-1/ERK/AKT activation. It is concluded that the novel peptide ZY1 is an effective inhibitor of ocular pathologic angiogenesis and may provide a promising alternative for ocular antiangiogenic therapy.

1141584386 Cell type specific regulation of human placenta growth factor

Background: Maternal serum levels of PlGF are significantly lower in preeclamptic patients than in normal pregnant women. PlGF mRNA expression is cell type specific with high levels in trophoblast but comparatively low levels in most nontrophoblast. Despite two consensus hypoxia response elements (HREs), PlGF expression is down regulated by hypoxia in trophoblast, but increased in other cell types. These observations suggest unique regulatory mechanisms control PlGF expression. We sought to characterize PlGF promoter regions which function to regulate transcription rates in different cell types and under hypoxia.Methods: Regions of human PlGF gene (−1521/+34), (−828/+34), (−698/+34) were cloned into a reporter vector and (−844/−677) was cloned into a minimal TK promoter containing expression vectors. Trophoblast (JEG3 and JAR), and nontrophoblast (HeLa and hEK‐293) were transiently transfected to determine functional responsiveness of the regions. A PlGF gene region containing two HREs was cloned upstream of a TK containing vector to determine hypoxia (1% O2) responsiveness.Results: PlGF (−1521/+34), (−828/+34) regions showed high promoter activity in trophoblast cells, but little activity in nontrophoblast cells. PlGF (−844/−677) cloned downstream of the TK reporter, augmented promoter activity in trophoblast but not nontrophoblast cells. When cloned upstream to the TK promoter, this region significantly decreased activity in nontrophoblast cells. Surprisingly, the PlGF region containing HREs produced little increase in minimal promoter activity during hypoxia in any of the cells.Conclusions: PlGF transcription is cell type specific and a region of PlGF (−828/−698) may act as an enhancer in trophoblast yet mediates repressor activity in nontrophoblast. The putative HREs in PlGF 5’UTR do not seem to increase PlGF transcription under hypoxia suggesting that they are not essential for governing PlGF expression. Further understanding of transcriptional regulation of PlGF in trophoblast may facilitate approaches to increase expression during preeclampsia.

Possible Involvement of Placental Peptidases that Degrade Gonadotropin-releasing Hormone (GnRH) in the Dynamic Pattern of Placental hCG Secretion via GnRH Degradation

The presence of an extrahypothalamic gonadotropin releasing hormone (GnRH) in human placenta is well known and this decapeptide is presumed to play an important role in the regulation of the function and growth of human placenta. Immunohistochemistry showed that neutral endopeptidase 24.11 (NEP), a candidate of the responsible enzyme of GnRH degradation, is highly expressed on the cell surface of trophoblasts. Hydrolysis of GnRH by human villi was studied by measuring liberated amino acids using high performance liquid chromatography. The GnRH degrading activity was 1.53 times higher after incubation with the membrane fraction of first trimester villi than that after incubation with the membrane fraction of term villi. Phosphoramidon, a potent inhibitor of NEP, reduced the liberated amino acids to about a half, suggesting that NEP is a responsible enzyme for GnRH degradation. Ubenimex, which can inhibit several aminopeptidases, also reduced the liberated amino acids to about 50 per cent. O-phenanthroline, EDTA, and thiorphan could inhibit GnRH degradation but inhibitors of post proline endopeptidase could not. Furthermore, GnRH degrading activity of the membrane fraction was reduced remarkably after the membrane fraction was immunotitrated by anti NEP and anti placental leucine aminopeptidase (P-LAP) IgG. In conclusion, NEP and P-LAP are responsible enzymes for GnRH degradation in human villi.

The Crystal Structure of Human Placenta Growth Factor-1 (PlGF-1), an Angiogenic Protein, at 2.0 Å Resolution

The angiogenic molecule placenta growth factor (PlGF) is a member of the cysteine-knot family of growth factors. In this study, a mature isoform of the human PlGF protein, PlGF-1, was crystallized as a homodimer in the crystallographic asymmetric unit, and its crystal structure was elucidated at 2.0 A resolution. The overall structure of PlGF-1 is similar to that of vascular endothelial growth factor (VEGF) with which it shares 42% amino acid sequence identity. Based on structural and biochemical data, we have mapped several important residues on the PlGF-1 molecule that are involved in recognition of the fms-like tyrosine kinase receptor (Flt-1, also known as VEGFR-1). We propose a model for the association of PlGF-1 and Flt-1 domain 2 with precise shape complementarity, consider the relevance of this assembly for PlGF-1 signal transduction, and provide a structural basis for altered specificity of this molecule.

Endogenous regulation of the GnRH receptor by GnRH in the human placenta.

Gonadotrophin releasing hormone (GnRH) plays an important regulatory role in the function and growth of human placenta, but its placental expression sites and co-localization with GnRH receptor (GnRH-R) are not well known. GnRH and GnRH-R expression has been found in both placenta and cultured trophoblasts; however, cultured trophoblastic cells very quickly lose GnRH-R message and subsequently receptor protein. Speculating that endogenously released GnRH induced this down-regulation, we determined GnRH-R in cultures of trophoblastic cells in the presence of a neutralizing anti-GnRH antibody. Cells incubated with this antibody showed a strong signal for the GnRH-R, while those cultured without did not (as analysed by immunofluorescence, reverse transcription-polymerase chain reaction, protein 'dot blot' and Western blotting). Furthermore, addition of the GnRH agonist buserelin led to a reduction of the receptor protein. We have therefore shown that GnRH released from trophoblastic cells down-regulates GnRH-R in these trophoblastic cells. Having previously shown that trophoblast layers were synchronously positive for GnRH and GnRH-R, these new findings support the hypothesis of an ultrashort feedback regulation of trophoblasts by GnRH involving autocrine regulation of GnRH-R.

A Mutant Form of Vascular Endothelial Growth Factor (VEGF) That Lacks VEGF Receptor-2 Activation Retains the Ability to Induce Vascular Permeability

Vascular endothelial growth factor (VEGF) is a major mediator of vasculogenesis and angiogenesis both during development and in pathological conditions. VEGF has a variety of effects on vascular endothelium, including the ability to stimulate endothelial cell mitogenesis, and the potent induction of vascular permeability. These activities are at least in part mediated by binding to two high affinity receptors, VEGFR-1 and VEGFR-2. In this study we have made mutations of mouse VEGF in order to define the regions that are required for VEGFR-2-mediated functions. Development of a bioassay, which responds only to signals generated by cross-linking of VEGFR-2, has allowed evaluation of these mutants for their ability to activate VEGFR-2. One mutant (VEGF0), which had amino acids 83-89 of VEGF substituted with the analogous region of the related placenta growth factor, demonstrated significantly reduced VEGFR-2 binding compared with wild type VEGF, indicating that this region was required for VEGF-VEGFR-2 interaction. Intriguingly, when this mutant was evaluated in a Miles assay for its ability to induce vascular permeability, no difference was found when compared with wild type VEGF. In addition we have shown that the VEGF homology domain of the structurally related growth factor VEGF-D is capable of binding to and activating VEGFR-2 but has no vascular permeability activity, indicating that VEGFR-2 binding does not correlate with permeability activity for all VEGF family members. These data suggest different mechanisms for VEGF-mediated mitogenesis and vascular permeability and raise the possibility of an alternative receptor mediating vascular permeability.

Vascular endothelial growth factor can substitute for macrophage colony-stimulating factor in the support of osteoclastic bone resorption.

We demonstrated previously that a single injection of recombinant human macrophage colony-stimulating factor (rhM-CSF) is sufficient for osteoclast recruitment and survival in osteopetrotic (op/op) mice with a deficiency in osteoclasts resulting from a mutation in M-CSF gene. In this study, we show that a single injection of recombinant human vascular endothelial growth factor (rhVEGF) can similarly induce osteoclast recruitment in op/op mice. Osteoclasts predominantly expressed VEGF receptor 1 (VEGFR-1), and activity of recombinant human placenta growth factor 1 on osteoclast recruitment was comparable to that of rhVEGF, showing that the VEGF signal is mediated through VEGFR-1. The rhM-CSF-induced osteoclasts died after injections of VEGFR-1/Fc chimeric protein, and its effect was abrogated by concomitant injections of rhM-CSF. Osteoclasts supported by rhM-CSF or endogenous VEGF showed no significant difference in the bone-resorbing activity. op/op mice undergo an age-related resolution of osteopetrosis accompanied by an increase in osteoclast number. Most of the osteoclasts disappeared after injections of anti-VEGF antibody, demonstrating that endogenously produced VEGF is responsible for the appearance of osteoclasts in the mutant mice. In addition, rhVEGF replaced rhM-CSF in the support of in vitro osteoclast differentiation. These results demonstrate that M-CSF and VEGF have overlapping functions in the support of osteoclastic bone resorption.

Detection of gonadotrophin releasing hormone and its receptor mRNA in human placental trophoblasts using in-situ reverse transcription-polymerase chain reaction.

Neuropeptides such as gonadotrophin releasing hormone (GnRH) are presumed to play an important role in the regulation of the function and growth of human placenta. Knowledge about the placental site of GnRH expression and the eventual co-localization of its peptide with the GnRH receptor (GnRH-R) is crucial for a better understanding of possible autocrine/paracrine mechanisms. We therefore investigated these questions by use of in-situ reverse transcription-polymerase chain reaction (RT-PCR) alone or in combination with immunocytochemistry in human first and third trimester placentae. Paraffin-embedded placental sections (7 microm thick), or single trophoblasts in monolayer cultures for up to 3 days, were treated with proteinase K. Following RT with GnRH or GnRH-R specific oligoprimers, PCR was performed employing primers with exon-exon overlaps to exclude non-specific DNA amplification. Detection of the amplicons was accomplished by nested PCR which was performed with digoxigenin-labelled dUTP and nitroblue tetrazolium/5-bromo-4-chloro-3-indoyl-phosphate (NBT/BCIP) for substrate visualization. The GnRH peptide was detected using a sandwich-antibody assay. GnRH and GnRH-R gene expression was found in all first and third trimester placentae, with abundant signals for the GnRH and GnRH-R message both in the cyto- and syncytiotrophoblasts. Single trophoblasts of different gestational ages in culture also displayed GnRH expression in individual cytotrophoblasts and in syncytiotrophoblast-like fusionates. Additional immunostaining revealed GnRH peptide to be co-localized with GnRH-R message in trophoblast layers. Since messages for GnRH and GnRH-R were found in virtually all trophoblasts, we infer that GnRH and GnRH-R are co-expressed in identical cells. These data strongly suggest that the trophoblasts are the source of GnRH, and that there is autocrine/ paracrine regulation by GnRH in human placenta.

Induction by cortisol of aminopeptidases production from the human placenta in tissue culture.

Human placental aminopeptidase M and A and post-proline endopeptidase are known to act as degrading enzymes of bioactive peptides such as angiotensin II, oxytocin and endogenous opioids. We tested the effects of cortisol on the activities of human placental aminopeptidase A and M and post-proline endopeptidase using short-cultured placental tissues. From 34.5 nM to 3.45 microM of cortisol significantly increased the activities of 3 enzymes. Our present data suggest a possible important role of cortisol in the growth of human placenta via induction of placental aminopeptidases.

Hormone secretion by human placenta grown in the eyes of rabbits

Successful growth of human placenta in tissue culture in a previous study showed that the Langhans cells secreted gonadotropin.1 The amount of gonadotropin in the tissue culture fluid was directly proportional to the number of the Langhans cells present. Syncytial cells grew sparingly and degenerated early. The study was inconclusive as to the production of estrogens and progesterone due to the failure of syncytial cells to grow.Since estrone, progesterone, and their metabolic derivatives attain the highest concentrations in the blood and urine during the last month of pregnancy, it would be desirable to grow the full-term placenta. In all attempts, tissue cultures of the term placenta were unsuccessful, and resulted only in an over-growth of fibrous elements. Syncytial cells, as well as Langhans cells, failed to grow.Intraocular transplants in monkeys, guinea pigs, hamsters, and rabbits have been successful in the study of normal tissues of the male and female genital systems, as well as embryonic organ implants and benign and malignant tumors. Implantation of the mature human placental tissue into the eyes of rabbits offers a method of study where tissue cultures in vitro have been inconclusive. If Langhans cells are present in the term placenta, will they grow in intraocular explants with evidence of gonadotropin secretion? Will syncytial cells grow with evidence of estrogen and progestin secretion? The present study developed with these questions in mind.