Telomeres and replicative cellular aging of the human placenta and chorioamniotic membranes.

Recent hypotheses propose that the human placenta and chorioamniotic membranes (CAMs) experience telomere length (TL)-mediated senescence. These hypotheses are based on mean TL (mTL) measurements, but replicative senescence is triggered by short and dysfunctional telomeres, not mTL. We measured short telomeres by a vanguard method, the Telomere shortest length assay, and telomere-dysfunction-induced DNA damage foci (TIF) in placentas and CAMs between 18-week gestation and at full-term. Both the placenta and CAMs showed a buildup of short telomeres and TIFs, but not shortening of mTL from 18-weeks to full-term. In the placenta, TIFs correlated with short telomeres but not mTL. CAMs of preterm birth pregnancies with intra-amniotic infection showed shorter mTL and increased proportions of short telomeres. We conclude that the placenta and probably the CAMs undergo TL-mediated replicative aging. Further research is warranted whether TL-mediated replicative aging plays a role in all preterm births.

Differential physiological response of slow- and fast-growing broiler lines to hypoxic conditions during chorioallantoic membrane development.

Ambient conditions during chicken embryogenesis, such as insufficient oxygen or changes in temperature, are expected to cause permanent phenotypic changes and affect their posthatch performance. Decades of genetic selection for high growth rate resulted with various physiological and morphological changes that can affect the broiler fitness under environmental stress. To evaluate the selection effect on responses to environmental challenge during embryonic development, and the long-term implications, we have used a unique genetic line, that was not selected for over 30 yr (since 1986), as control for the modern commercial genetic line. At embryonic day 5 (E5), broiler embryos from these 2 genetic lines were divided into 2 treatments: 1) control; 2) 15% O2 concentration for 12 h/day from E5 through E12 the embryonic period of chorioallantoic membrane formation. Embryos and hatched chicks were characterized for physiological and morphological parameters. Significant differences in relative embryo weight and yolk consumption were found between the 2 lines. The modern line was characterized by a higher metabolic rate and rapid growth, supported by higher hemoglobin levels and hematocrit concentrations, whereas the 1986 line had slower metabolism, lower levels of hematocrit and hemoglobin, higher oxygen volume per 1 g of embryonic tissue indicating higher oxygen availability. Both lines exhibited changes in heart rate, and blood parameters corresponding to cardiovascular system adaptation after hypoxic exposure, seemingly implemented to increase oxygen-carrying capacity to the embryo tissues. Our finding stand in agreement that the genetic selection for high growth rate that led to higher metabolism without a fit of the cardiovascular system, increased the imbalance between oxygen supply and demand.

A DHX9-lncRNA-MDM2 interaction regulates cell invasion and angiogenesis of cervical cancer.

Cervical cancer (CC) is the third most common carcinoma and the fourth leading cause of cancer-associated mortality in women. Here, we report that MDM2-DHX9 interaction mediates CC motility and angiogenesis in a long noncoding RNA-dependent fashion. A long noncoding RNA, named lnc-CCDST, is significantly downregulated in CC tissues, and binds to pro-oncogenic DHX9. DHX9 is upregulated in CC tissue, and promotes CC cell motility and angiogenesis. The lnc-CCDST and DHX9 interaction promotes DHX9 degradation through the ubiquitin proteasome pathway. Furthermore, DHX9 bound to E3 ubiquitin ligase MDM2, and this interaction is enhanced by lnc-CCDST. Thus, lnc-CCDST promotes DHX9 degradation by serving as a scaffold to facilitate the formation of MDM2 and DHX9 complexes. Moreover, HPV oncogenes E6 and E7 abolish the expression of lnc-CCDST resulting in the increase of DHX9. Our results have revealed a novel mechanism by which high-risk HPVs promote motility and angiogenesis of CC by inhibiting expression of lnc-CCDST to disrupt MDM2 and DHX9 interaction, and DHX9 degradation, and identified a potential therapeutic target for CC.

Kinetics of the chromosome 14 microRNA cluster ortholog and its potential role during placental development in the pregnant mare.

BACKGROUND: The human chromosome 14 microRNA cluster (C14MC) is a conserved microRNA (miRNA) cluster across eutherian mammals, reported to play an important role in placental development. However, the expression kinetics and function of this cluster in the mammalian placenta are poorly understood. Here, we evaluated the expression kinetics of the equine C24MC, ortholog to the human C14MC, in the chorioallantoic membrane during the course of gestation. RESULTS: We demonstrated that C24MC-associated miRNAs presented a higher expression level during early stages of pregnancy, followed by a decline later in gestation. Evaluation of one member of C24MC (miR-409-3p) by in situ hybridization demonstrated that its cellular localization predominantly involved the chorion and allantoic epithelium and vascular endothelium. Additionally, expression of predicted target transcripts for C24MC-associated miRNAs was evaluated by RNA sequencing. Expression analysis of a subset of predicted mRNA targets showed a negative correlation with C24MC-associated miRNAs expression levels during gestation, suggesting the reciprocal control of these target transcripts by this miRNA cluster. Predicted functional analysis of these target mRNAs indicated enrichment of biological pathways related to embryonic development, endothelial cell migration and angiogenesis. Expression patterns of selected target mRNAs involved in angiogenesis were confirmed by RT-qPCR. CONCLUSION: This is the first report evaluating C24MC kinetics during pregnancy. The findings presented herein suggest that the C24MC may modulate angiogenic transcriptional profiles during placental development in the horse.

Effects of low oxygen during chorioallantoic membrane development on post-hatch growing performance of broiler chickens.

The prenatal circulatory system is adaptive and capable of plasticity designed for the needs of the growing tissue. When a broiler embryo is faced with hypoxic stress, the process of angiogenesis in tissues begins. Exposure to hypoxic conditions of 17% oxygen during the chorioallantoic membrane (CAM) development (E5 to E12) affected the circulatory system and contributed to an increase in the blood oxygen carrying capacity. The present study aimed to evaluate the effects of hypoxic exposure during CAM development on post-hatch performance of broilers and to examine whether hypoxic exposure improved sustainability of birds exposed to acute heat stress.Two consecutive trials, with male broilers from each of the incubation treatments-optimal conditions and exposure to hypoxia of 15 or 17% oxygen, for 12 h/day, during CAM development-were conducted. In experiment 1, 60 male chicks from each group were raised in individual cages. In experiment 2, 160 male chicks from each group were raised in 40-chick pens until marketing. On d 35, 20 birds from each group were transferred to individual cages kept at a temperature of 23°C for 72 h, and then birds were exposed to 35°C for 5 hours. Body temperatures were measured at 0, 2, and 5 h of the heat exposure. In both experiments BW, feed intake, and FCR were recorded. At marketing, chicks were slaughtered, and relative weights of breast muscle, abdominal fat pad, heart, and liver were calculated.Hypoxia treatment resulted in a FCR advantage. Food intake was similar in all treatments, but groups exposed to hypoxia grew better than controls until the age of 35 days. Hypoxia-treated groups had higher relative breast, heart, and liver weights than controls. Body temperatures of hypoxia-treated chickens remained lower during heat stress exposure, and their mortality rate was lower as well. Intermittent exposure to moderate hypoxia during CAM development confers advantages to broilers in feed utilization efficiency and in coping with heat stress. It may be considered as a mitigating step in incubation to facilitate broilers in achieving their full growth potential.

The Human Mesenchymal Stem Cells and the Chick Embryo Chorioallantoic Membrane: The Key and the Lock in Revealing Vasculogenesis.

BACKGROUND/AIM: To analyze the interaction between the human mesenchymal stem cells (hMSC) and the chick embryo chorioallantoic membrane (CAM), in order to assess the still obscure process of vasculogenesis. MATERIALS AND METHODS: We implanted hMSC onto CAM and we analyzed the morphology and the immunohistochemical profile of CAM. RESULTS: hMSC adhered to CAM, few of them entered the chorionic epithelium and the mesoderm and developed a CD44-/Ki67- status. hMSC stimulated the CAM mesenchymal cells (cMSC) to acquire endothelial and pericyte-like features and to generate cord/capillary-like structures (CLS) in the chorionic epithelium and the mesoderm, but they also entered these structures (CD34+/SMA (smooth muscle actin)+ hMSC). Simultaneously, hMSC induced a process of sprouting angiogenesis in the mesoderm, CD105+ hMSC being identified in the proximity of the angiogenic areas. CONCLUSION: hMSC and CAM establish a genuine hotspot of vasculogenesis, which may evolve to a valuable experimental model for this research field.

Color-Doppler signals of blood flow in the corpus luteum and vascular perfusion index for ovarian and uterine arteries during expansion of the allantochorion in Bos taurus heifers.

Hemodynamics of the CL and each main uterine artery during expansion of the allantochorion from the ipsilateral side (CL side) to the contralateral side were studied in heifers (n = 8 nonbred, 9 pregnant). Progesterone concentration, vascular perfusion index for each uterine artery (by spectral ultrasonography), extent of blood flow in the CL (percentage of CL tissue with color-Doppler signals of blood flow), and intrauterine location of the expanding allantochorion (by gray-scale ultrasonic imaging) were determined daily from Days 14-60 (Day 0 = ovulation). In the pregnant group, but not in the nonbred group, the percentage of CL tissue with blood-flow signals increased (P < 0.003) from Days 16 (66.7 ± 4.2%) to 23 (79.4 ± 2.5) and then more slowly increased (P < 0.02) from Days 24 (76.7 ± 2.9%) to 50 (85.0 ± 2.0%). The volume of CL increased (P < 0.0001) progressively from Days 26 (6.1 ± 0.4 cm3) to 60 (7.3 ± 0.7 cm3). The vascular perfusion index in the ipsilateral uterine artery did not change in the nonbred group and progressively increased in the pregnant group beginning on Day 17 in approximate temporal association with the increase in luteal blood-flow signals. Functional increases in the CL during early pregnancy were attributed to the greater uterine arterial blood flow on the ipsilateral or CL side and the reported prominent anastomosis from a branch of the uterine artery to the ovarian artery that supplies the CL ovary. After an initial significant decrease in vascular perfusion index in each uterine artery in the pregnant group, the index began to increase on Day 17 in the ipsilateral artery and on Day 18 in the contralateral artery. The perfusion continued in the ipsilateral artery but discontinued in the contralateral artery on Day 21. Perfusion and diameter of the uterine artery were greater for the ipsilateral side until the vesicle entered the contralateral horn on Day 33 and increased for the contralateral horn between vesicle entry and filling of the horn on Day 44. During Days 35-60, the perfusion index (P < 0.04) and artery diameter (P < 0.001) were lower in the contralateral than in the ipsilateral artery. Results supported the hypotheses that (1) CL function increases during early pregnancy in temporal association with an increase in blood flow in the ipsilateral uterine artery and (2) blood flow in each of the ipsilateral and contralateral uterine arteries increases as the allantochorion expands in each uterine horn.

Luteolin inhibits angiogenesis by blocking Gas6/Axl signaling pathway.

Growth arrest-specific protein 6 (Gas6) induces the activation of Axl receptor tyrosine kinase (Axl), which plays an important role in angiogenic processes, including proliferation, migration, invasion, tube formation and pericyte recruitment of endothelial cells. The inhibition of Gas6/Axl pathway has been demonstrated to be an effective anti-angiogenic therapy. Luteolin, which is a natural active flavonoid, has been reported to possess anti-angiogenic effects. However, the underlying mechanism of luteolin in anti-angiogenesis is not fully understood. Herein, we report that luteolin significantly inhibited the Gas6-induced proliferation, migration, invasion and tube formation of human microvascular endothelial cells (HMEC­1s) in vitro, and suppressed the Gas6-induced recruitment of human brain vascular pericytes (HBVPs) to the endothelial tubes. Luteolin also suppressed Gas6-induced microvessel sprouting in aortic ring assay and neovascularization in chick chorioallantoic membrane assay. The anti-angiogenic effect of luteolin may be associated with the inhibition of the Gas6/Axl pathway and its downstream phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathways. Taken together, the present study provides new evidence regarding an anti-angiogenic mechanism of luteolin, and supports the notion that the dietary intake of luteolin contributes to the treatment of pathological angiogenesis.

Single stage incubators and Hypercapnia during incubation affect the vascularization of the chorioallantoic membrane in broiler embryos.

Incubation management can have direct effects on neonate health and consequently affect post-hatching development. The effects of incubation in multiple and single stage incubators with different concentrations of CO2 were evaluated in terms of the vessel density in the chorioallantoic membrane, hatching, heart morphology, and body development of the neonate up to the tenth day. A total of 2,520 fertile eggs were used and distributed in a completely randomized design with 4 levels of CO2 in 4 single-stage incubators (4,000; 6,000; 8,000; and 10,000 ppm) and a control treatment based on multiple-stage incubation, totaling 5 treatments. The levels of CO2 were used during the first 10 d of the incubation period, and after this period, all eggs were submitted to the same level of CO2 (4,000 ppm). Eggs that were incubated in multiple-stage incubators presented a lower percentage of vessels in the chorioallantoic membrane, lower yolk absorption by the embryo, wall depth of the right ventricle, and greater humidity losses in the eggs when compared to eggs in the single-stage incubators. The eggs submitted to hypercapnia, between 5,000 and 6,000 ppm of CO2, had a higher percentage of vessels in the chorioallantoic membrane; the embryos originating from these eggs had higher weight, with higher relative weight of the liver. However, the same levels reduced the yolk absorption. Single-stage incubation with moderate levels of hypercapnia is an efficient tool to be adopted by the hatcheries when attempting to improve chick quality.

Sympathetic Innervation Promotes Arterial Fate by Enhancing Endothelial ERK Activity.

RATIONALE: Arterial endothelial cells are morphologically, functionally, and molecularly distinct from those found in veins and lymphatic vessels. How arterial fate is acquired during development and maintained in adult vessels is incompletely understood. OBJECTIVE: We set out to identify factors that promote arterial endothelial cell fate in vivo. METHODS AND RESULTS: We developed a functional assay, allowing us to monitor and manipulate arterial fate in vivo, using arteries isolated from quails that are grafted into the coelom of chick embryos. Endothelial cells migrate out from the grafted artery, and their colonization of host arteries and veins is quantified. Here we show that sympathetic innervation promotes arterial endothelial cell fate in vivo. Removal of sympathetic nerves decreases arterial fate and leads to colonization of veins, whereas exposure to sympathetic nerves or norepinephrine imposes arterial fate. Mechanistically, sympathetic nerves increase endothelial ERK (extracellular signal-regulated kinase) activity via adrenergic α1 and α2 receptors. CONCLUSIONS: These findings show that sympathetic innervation promotes arterial endothelial fate and may lead to novel approaches to improve arterialization in human disease.

The chick embryo chorioallantoic membrane (CAM). A multifaceted experimental model.

During avian development the mesodermal layers of the allantois and chorion fuse to form the chorioallantoic membrane (CAM). This structure rapidly expands generating a rich vascular network that provides an interface for gas and waste exchange. The CAM allows to study tissue grafts, tumor growth and metastasis, wound healing, drugs delivery and toxicologic analysis, and angiogenic and anti-angiogenic molecules. The CAM is relatively simple, quick, and low-cost model that allows screening of a large number of pharmacological samples in a short time; does not require administrative procedures for obtaining ethics committee approval for animal experimentation. Moreover, being naturally immunodeficient, the chick embryo may receive transplantations from different tissues and species, without immune responses.

Dynamics of the Developing Chick Chorioallantoic Membrane Assessed by Stereology, Allometry, Immunohistochemistry and Molecular Analysis.

The chick chorioallantoic membrane (CAM) is a widely used model for the study of angiogenesis, tumour growth, as well as drug efficacy. In spite of this, little is known about the developmental alteration from its appearance to the time of hatching. In the current study the CAM has been studied by classical stereology and allometry. Expression levels of selected angiogenesis-related molecules were estimated by RT-PCR and cell dynamics assessed by proliferation and apoptosis assays. Absolute CAM volume increased from a low of 0.47 ± 0.11 cm3 at embryonic day 8 (E8) to a high of 2.05 ± 0.27 cm3 at E18, and then decreased to 1.6 ± 0.47 cm3 at E20. On allometric analysis, three growth phases were identifiable. Between E8-13 (phase I), the CAM grew fastest; moderately in phase II (E13-18) but was regressing in phase III (E18-20). The chorion, the mesenchyme and the allantoic layers grew fastest in phase I, but moderately in phase II. The mesenchyme grew slowly in phase III while the chorion and allantois were regressing. Chorionic cell volume increased fastest in phase I and was regressing in phase III. Chorionic capillaries grew steadily in phase I and II but regressed in phase III. Both the chorion and the allantois grew by intrinsic cell proliferation as well as recruitment of cells from the mesenchyme. Cell proliferation was prominent in the allantois and chorion early during development, declined after E17 and apoptosis started mainly in the chorion from E14. VEGFR2 expression peaked at E11 and declined steadily towards E20, VEGF peaked at E13 and E20 while HIF 1α had a peak at E11 and E20. Studies targeting CAM growth and angiogenesis need to take these growth phases into consideration.

Effect of Laryngeal Squamous Cell Carcinoma Tissue Implantation on the Chick Embryo Chorioallantoic Membrane: Morphometric Measurements and Vascularity.

BACKGROUND: The aim of this study was to develop chick embryo chorioallantoic membrane (CAM) model of laryngeal squamous cell carcinoma (LSCC) and to evaluate the morphological and morphometric characteristics and angiogenic features of it. METHODS: Fresh LSCC tissue samples obtained from 6 patients were implanted onto 15 chick embryo CAMs. Morphological, morphometric, and angiogenic changes in the CAM and chorionic epithelium were evaluated up to 4 days after the tumor implantation. Immunohistochemical analysis (34ßE12, CD31, and Ki67 staining) was performed to detect cytokeratins and tumor endothelial cells and to evaluate the proliferative capacity of the tumor before and after implantation on the CAM. RESULTS: The implanted LSCC tissue samples survived on the CAM in all the experiments and retained the essential morphologic characteristics and proliferative capacity of the original tumor. Implants induced thickening of both the CAM (103-417%, p = 0.0001) and the chorionic epithelium (70-140%, p = 0.0001) and increase in number of blood vessels (75-148%, p = 0.0001) in the CAM. CONCLUSIONS: This study clarifies that chick embryo CAM is a relevant assay for implanting LSCC tissue and provides the first morphological and morphometric characterization of the LSCC CAM model that opens new perspectives to study this disease.

The In Ovo Chick Chorioallantoic Membrane (CAM) Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma.

The chick chorioallantoic membrane (CAM) begins to develop by day 7 after fertilization and matures by day 12. The CAM is naturally immunodeficient and highly vascularized, making it an ideal system for tumor implantation. Furthermore, the CAM contains extracellular matrix proteins such as fibronectin, laminin, collagen, integrin alpha(v)beta3, and MMP-2, making it an attractive model to study tumor invasion and metastasis. Scientists have long taken advantage of the physiology of the CAM by using it as a model of angiogenesis. More recently, the CAM assay has been modified to work as an in vivo xenograft model system for various cancers that bridges the gap between basic in vitro work and more complex animal cancer models. The CAM assay allows for the study of tumor growth, anti-tumor therapies, and pro-tumor molecular pathways in a biologically relevant system that is both cost- and time-effective. Here, we describe the development of CAM xenograft model of hepatocellular carcinoma (HCC) with embryonic survival rates of up to 93% and reliable tumor take leading to growth of three-dimensional, vascularized tumors.

Reduced heart rate and cardiac output differentially affect angiogenesis, growth, and development in early chicken embryos (Gallus domesticus).

An increase in both vascular circumferential tension and shear stress in the developing vasculature of the chicken embryo has been hypothesized to stimulate angiogenesis in the developing peripheral circulation chorioallantoic membrane (CAM). To test this hypothesis, angiogenesis in the CAM, development, and growth were measured in the early chicken embryo, following acute and chronic topical application of the purely bradycardic drug ZD7288. At hour 56, ZD7288 reduced heart rate (f(H)) by ~30% but had no significant effect on stroke volume (~0.19 ± 0.2 µL), collectively resulting in a significant fall in cardiac output (CO) from ~27 ± 3 to 18 ± 2 µL min(-1). Mean f(H) at 72 h of development was similarly significantly lowered by acute ZD7288 treatment (250 µM) to 128 ± 0.3 beats min(-1), compared with 174.5 ± 0.3 and 174.7 ± 0.8 beats min(-1) in control and Pannett-Compton (P-C) saline-treated embryos, respectively. Chronic dosing with ZD7288-and the attendant decreases in f(H) and CO-did not change eye diameter or cervical flexion (key indicators of development rate) at 120 h but significantly reduced overall growth (wet and dry body mass decreased by 20%). CAM vessel density index (reflecting angiogenesis) measured 200-400 µm from the umbilical stalk was not altered, but ZD7288 reduced vessel numbers-and therefore vessel density-by 13%-16% more distally (500-600 µm from umbilical stalk) in the CAM. In the ZD7288-treated embryos, a decrease in vessel length was found within the second branch order (~300-400 µm from the umbilical stock), while a decrease in vessel diameter was found closer to the umbilical stock, beginning in the first branch order (~200-300 µm). Paradoxically, chronic application of P-C saline also reduced peripheral CAM vessel density index at 500 and 600 µm by 13% and 7%, respectively, likely from washout of local angiogenic factors. In summary, decreased f(H) with reduced CO did not slow development rate but reduced embryonic growth rate and angiogenesis in the CAM periphery. This study demonstrates for the first time that different processes in the ontogeny of the early vertebrate embryo (i.e., hypertrophic growth vs. development) have differential sensitivities to altered convective blood flow.

A positive feedback loop involving Gcm1 and Fzd5 directs chorionic branching morphogenesis in the placenta.

Chorioallantoic branching morphogenesis is a key milestone during placental development, creating the large surface area for nutrient and gas exchange, and is therefore critical for the success of term pregnancy. Several Wnt pathway molecules have been shown to regulate placental development. However, it remains largely unknown how Wnt-Frizzled (Fzd) signaling spatiotemporally interacts with other essential regulators, ensuring chorionic branching morphogenesis and angiogenesis during placental development. Employing global and trophoblast-specific Fzd5-null and Gcm1-deficient mouse models, combining trophoblast stem cell lines and tetraploid aggregation assay, we demonstrate here that an amplifying signaling loop between Gcm1 and Fzd5 is essential for normal initiation of branching in the chorionic plate. While Gcm1 upregulates Fzd5 specifically at sites where branching initiates in the basal chorion, this elevated Fzd5 expression via nuclear ß-catenin signaling in turn maintains expression of Gcm1. Moreover, we show that Fzd5-mediated signaling induces the disassociation of cell junctions for branching initiation via downregulating ZO-1, claudin 4, and claudin 7 expressions in trophoblast cells at the base of the chorion. In addition, Fzd5-mediated signaling is also important for upregulation of Vegf expression in chorion trophoblast cells. Finally, we demonstrate that Fzd5-Gcm1 signaling cascade is operative during human trophoblast differentiation. These data indicate that Gcm1 and Fzd5 function in an evolutionary conserved positive feedback loop that regulates trophoblast differentiation and sites of chorionic branching morphogenesis.

Does low gas permeability of rigid-shelled gekkotan eggs affect embryonic development?

Parchment-shelled eggs are characteristic of most squamates, including the basal clades of gekkotan lizards. The majority of gekkotan lizards, however, produce rigid-shelled eggs that are highly impermeable to gas exchange; eggs are laid in dry sites and experience a net loss of water during incubation. We tested the hypothesis that the 1,000-fold lower rate of oxygen diffusion through the shells of rigid- compared to parchment-shelled eggs imposes a physiological cost on development. To do this, we contrasted species with rigid and with parchment shells with regards to (1) rates of embryonic metabolism and (2) rates and patterns of development of the yolk sac and chorioallantois, the vascularized extra-embryonic membranes that transport oxygen to embryonic tissues. Metabolic rates of embryos from the rigid-shelled eggs of Gehyra variegata did not differ from those of the parchment-shelled eggs of Oedura lesueurii. Moreover, maximum metabolic rates of gekkotans with rigid shells did not differ from those of gekkotan or scincid lizards with parchment shells. In contrast, the yolk sac covered more of the surface area of the egg at oviposition, and the chorioallantois reached its full extent earlier for the species with rigid shelled eggs (Chondrodactylus turneri, G. variegata) than for the species with parchment-shelled eggs (Eublepharis macularius, O. lesueurii). Differences in the temporal patterns of yolk sac and chorioallantois development would thus serve to compensate for low rates of oxygen diffusion through rigid shells of gekkotans.

Snail mediates PDGF-BB-induced invasion of rat bone marrow mesenchymal stem cells in 3D collagen and chick chorioallantoic membrane.

We previously reported that membrane type-1 matrix metalloproteinase (MT1-MMP) enables mesenchymal stem cells (MSCs) to move through both three-dimensional (3D) type I collagen and basement membrane barriers; however, its upstream regulating factors were unidentified. Here, we report that PDGF-BB upregulates both mRNA and protein expression of snail in rat bone marrow MSCs (rBMMSCs). PDGF-BB enhances rBMMSC invasion in 3D collagen, which is blocked by snail specific siRNA transfection. Snail overexpression induced by plasmid transfection results in increased rBMMSC invasion in 3D collagen. Snail expression induced by PDGF-BB in MSCs is inhibited by LY294002 and PD98059, which are inhibitors of the PI3K/AKT and MAPK1/2/ERK1/2 signaling pathways, respectively. MT1-MMP expression in rBMMSCs, both as mRNA and protein, is decreased by snail siRNA transfection, but increased by snail overexpression, indicating that they are controlled by snail. Finally, snail controls MSC transmigration through chorioallantoic membrane of 11-day-old chick embryos. Taken together, these in vitro and in vivo data identify snail as a critical mediator for rBMMSC invasion induced by PDGF-BB.

Role of pesticides in the induction of tumor angiogenesis.

Due to their estrogen-mimicking ability, pesticides are considered as prime etiological suspects of increasing tumor incidence, although a direct link is still undefined. The present study aimed to identify the effect of xenoestrogens (lindane, propoxur and endosulfan) at 20 mg/l each on tumorigenesis, by evaluating endothelial cell proliferation, H(3) thymidine incorporation, wound healing, ascites formation and secretion, shell less Chorio Allantoic Membrane (CAM) formation using in vitro, as well as in vivo, models. The genotoxic effect of xenoestrogens in terms of DNA damage was also studied. The results showed that the endothelial cell proliferation, H(3) thymidine incorporation, wound healing, CAM formation were increased following xenoestrogen exposure, but the intensity of angiogenesis was dependent on the structural homology of these xenoestrogens to endogenous estrogen. Moreover, lindane was the most potent angiogenesis stimulator followed by propoxur and Endosulfan. Further studies were undertaken to examine lindane for its possible carcinogenicity. However, no effect was observed on the integrity of DNA after exposure to these xenoestrogens.

Tumor endothelial marker 8 amplifies canonical Wnt signaling in blood vessels.

Tumor Endothelial Marker 8/Anthrax Toxin Receptor 1 (TEM8/ANTXR1) expression is induced in the vascular compartment of multiple tumors and therefore, is a candidate molecule to target tumor therapies. This cell surface molecule mediates anthrax toxin internalization, however, its physiological function in blood vessels remains largely unknown. We identified the chicken chorioallantoic membrane (CAM) as a model system to study the endogenous function of TEM8 in blood vessels as we found that TEM8 expression was induced transiently between day 10 and 12 of embryonic development, when the vascular tree is undergoing final development and growth. We used the cell-binding component of anthrax toxin, Protective Antigen (PA), to engage endogenous TEM8 receptors and evaluate the effects of PA-TEM8 complexes on vascular development. PA applied at the time of highest TEM8 expression reduced vascular density and disrupted hierarchical branching as revealed by quantitative morphometric analysis of the vascular tree after 48h. PA-dependent reduced branching phenotype was partially mimicked by Wnt3a application and ameliorated by the Wnt antagonist, Dikkopf-1. These results implicate TEM8 expression in endothelial cells in regulating the canonical Wnt signaling pathway at this day of CAM development. Consistent with this model, PA increased beta catenin levels acutely in CAM blood vessels in vivo and in TEM8 transfected primary human endothelial cells in vitro. TEM8 expression in Hek293 cells, which neither express endogenous PA-binding receptors nor Wnt ligands, stabilized beta catenin levels and amplified beta catenin-dependent transcriptional activity induced by Wnt3a. This agonistic function is supported by findings in the CAM, where the increase in TEM8 expression from day 10 to day 12 and PA application correlated with Axin 2 induction, a universal reporter gene for canonical Wnt signaling. We postulate that the developmentally controlled expression of TEM8 modulates endothelial cell response to canonical Wnt signaling to regulate vessel patterning and density.