Luteal ANGPT-TIE system during selected stages of pregnancy, and normal and antigestagen-induced luteolysis in the dog.

Abstract: Rapid establishment of a vascular network is essential for normal functionality of the corpus luteum (CL). The early luteal phase is associated with increased expression of the VEGF system in canine CL. Acting in synchrony with angiopoietins (ANGPTs), VEGF system plays major roles in stabilization of blood vessels. However, the expression of the ANGPT system has not yet been investigated in the dog. Therefore, here, we investigated the luteal expression of ANGPT1, -2, and of their receptors TIE1 and -2, in pregnant dogs at selected time points during pregnancy and at normal and antigestagen-induced luteolysis. Additionally, luteal cells from early CL were incubated with PGE2 and its effects on the ANGPT system were assessed. Whereas the luteal ANGPT1 was stable until mid-gestation, TIE1 was elevated post-implantation, their expression decreased toward prepartum luteolysis. The ANGPT2- and TIE2-mRNA did not vary during pregnancy. The ANGPT2/ANGPT1 ratio was elevated during prepartum luteolysis. PGE2 increased ANGPT2, but suppressed ANGPT1 levels. None of the ANGPT-system members was affected by antigestagen treatment in mid-pregnancy. Localization of ANGPT1 was predominantly found in the tunica intima and media of vessels and ANGPT2 stained strongly in luteal cells. Both ANGPTs were localized in macrophages. TIE1 stained in the vascular tunica media, in luteal cells and macrophages, whereas TIE2 was colocalized with ANGPT1 in vascular components. In conclusion, high expression of ANGPT1 during the increased presence of VEGFA in early canine CL implies its contribution to vascular network development. The upregulation of the ANGPT2/ANGPT1 ratio during prepartum luteolysis indicates involvement of the ANGPT system in PGF2α-mediated vascular destabilization.

Expression and localization of angiopoietin family in corpus luteum during different stages of oestrous cycle and modulatory role of angiopoietins on steroidogenesis, angiogenesis and survivability of cultured buffalo luteal cells.

The objective of this study was to document the expression and localization of angiopoietin (ANGPT) family members comprising of angiopoietin (ANGPT1 and ANGPT2), and their receptors (Tie1 and Tie2) in buffalo corpus luteum (CL) obtained from different stages of the oestrous cycle, and the modulatory role of ANGPT1 and ANGPT2 alone or in combinations on progesterone (P4 ) secretion and mRNA expression of phosphotidylinositide-3kinase-protein kinase B (PI3K-AKT), phosphoinositide-dependent kinase (PDK), protein kinase B (AKT), Bcl2 associated death promoter (BAD), caspase 3 and von willebrand factor (vWF) in luteal cells obtained from midluteal phase (MLP) of oestrous cycle in buffalo. Real-time RT-PCR (qPCR), Western blot and immunohistochemistry were applied to investigate mRNA expression, protein expression and localization of examined factors whereas, the P4 secretion was assessed by RIA. The mRNA and protein expression of ANGPT1 and Tie2 was maximum (p < .05) in mid luteal phase (MLP) of oestrous cycle. The ANGPT2 mRNA and protein expression was maximum (p < .05) in early luteal phase, decreased in MLP and again increased in late luteal phase of oestrous cycle. ANGPT family members were localized in luteal cells and endothelial cells with a stage specific immunoreactivity. P4 secretion was highest (p < .05) with 100 ng/ml at 72 hr when luteal cells were treated with either protein alone. The mRNA expression of PDK, AKT and vWF was highest (p < .05) and BAD along with caspase 3 were lowest (p < .05) at 100 ng/ml at 72 hr of incubation period, when cultured luteal cells were treated with either protein alone or in combination. To conclude, our study explores the steroidogenic potential of angiopoietins to promote P4 secretion, luteal cell survival and angiogenesis through an autocrine and paracrine actions in buffalo CL.

Tie-mediated signal from apoptotic cells protects stem cells in Drosophila melanogaster.

Many types of normal and cancer stem cells are resistant to killing by genotoxins, but the mechanism for this resistance is poorly understood. Here we show that adult stem cells in Drosophila melanogaster germline and midgut are resistant to ionizing radiation (IR) or chemically induced apoptosis and dissect the mechanism for this protection. We find that upon IR the receptor tyrosine kinase Tie/Tie-2 is activated, leading to the upregulation of microRNA bantam that represses FOXO-mediated transcription of pro-apoptotic Smac/DIABLO orthologue, Hid in germline stem cells. Knockdown of the IR-induced putative Tie ligand, Pvf1, a functional homologue of human Angiopoietin, in differentiating daughter cells renders germline stem cells sensitive to IR, suggesting that the dying daughters send a survival signal to protect their stem cells for future repopulation of the tissue. If conserved in cancer stem cells, this mechanism may provide therapeutic options for the eradication of cancer.

Elk3 deficiency causes transient impairment in post-natal retinal vascular development and formation of tortuous arteries in adult murine retinae.

Serum Response Factor (SRF) fulfills essential roles in post-natal retinal angiogenesis and adult neovascularization. These functions have been attributed to the recruitment by SRF of the cofactors Myocardin-Related Transcription Factors MRTF-A and -B, but not the Ternary Complex Factors (TCFs) Elk1 and Elk4. The role of the third TCF, Elk3, remained unknown. We generated a new Elk3 knockout mouse line and showed that Elk3 had specific, non-redundant functions in the retinal vasculature. In Elk3(-/-) mice, post-natal retinal angiogenesis was transiently delayed until P8, after which it proceeded normally. Interestingly, tortuous arteries developed in Elk3(-/-) mice from the age of four weeks, and persisted into late adulthood. Tortuous vessels have been observed in human pathologies, e.g. in ROP and FEVR. These human disorders were linked to altered activities of vascular endothelial growth factor (VEGF) in the affected eyes. However, in Elk3(-/-) mice, we did not observe any changes in VEGF or several other potential confounding factors, including mural cell coverage and blood pressure. Instead, concurrent with the post-natal transient delay of radial outgrowth and the formation of adult tortuous arteries, Elk3-dependent effects on the expression of Angiopoietin/Tie-signalling components were observed. Moreover, in vitro microvessel sprouting and microtube formation from P10 and adult aortic ring explants were reduced. Collectively, these results indicate that Elk3 has distinct roles in maintaining retinal artery integrity. The Elk3 knockout mouse is presented as a new animal model to study retinal artery tortuousity in mice and human patients.

Granulocyte/macrophage colony-stimulating factor influences angiogenesis by regulating the coordinated expression of VEGF and the Ang/Tie system.

Granulocyte/macrophage colony-stimulating factor (GM-CSF) can accelerate wound healing by promoting angiogenesis. The biological effects of GM-CSF in angiogenesis and the corresponding underlying molecular mechanisms, including in the early stages of primitive endothelial tubule formation and the later stages of new vessel maturation, have only been partially clarified. This study aimed to investigate the effects of GM-CSF on angiogenesis and its regulatory mechanisms. Employing a self-controlled model (Sprague-Dawley rats with deep partial-thickness burn wounds), we determined that GM-CSF can increase VEGF expression and decrease the expression ratio of Ang-1/Ang-2 and the phosphorylation of Tie-2 in the early stages of the wound healing process, which promotes the degradation of the basement membrane and the proliferation of endothelial cells. At later stages of wound healing, GM-CSF can increase the expression ratio of Ang-1/Ang-2 and the phosphorylation of Tie-2 and maintain a high VEGF expression level. Consequently, pericyte coverages were higher, and the basement membrane became more integrated in new blood vessels, which enhanced the barrier function of blood vessels. In summary, we report here that increased angiogenesis is associated with GM-CSF treatment, and we indicate that VEGF and the Ang/Tie system may act as angiogenic mediators of the healing effect of GM-CSF on burn wounds.

Regulation of endometrial vascular remodelling: role of the vascular endothelial growth factor family and the angiopoietin-TIE signalling system.

Angiogenesis, lymphangiogenesis and vascular maturation occur on a regular, physiological basis in human endometrium. These processes form part of a continuum of vascular remodelling involving numerous regulatory factors. Key factors include vascular endothelial growth factor (VEGF)A, VEGFC and VEGFD, and their associated receptors VEGFR1, VEGFR2 and VEGFR3. A second group of vascular regulatory proteins belongs to the angiopoietin (ANG)-TIE system. Although members of the VEGF family and the ANG-TIE system are represented in the endometrium, our understanding of how these different molecules interact to regulate remodelling of the blood and lymphatic vasculature present in the endometrium is still limited. A review of the current information is provided.

Expression of angiopoietin (ANPT)-1, ANPT-2 and their receptors in dominant follicles during periovulatory period in GnRH-treated cow.

Ovarian follicular vasculature is involved in follicular development and ovulation. Angiopoietin (ANPT)-Tie system is important for vascularization of the tissue surrounding the developing follicles and corpus luteum (CL). To determine how the expression of ANPT-1, ANPT-2 and their receptors in the follicles would be associated with the ovulatory process, the present study was conducted to examine mRNA expressions of ANPT-1, ANPT-2 and their receptors during the periovulatory phase in gonadotrophin-releasing hormone (GnRH)-treated cows. The ovaries were collected by transvaginal ovariectomy (n = 5, cows/group) and the follicles (n = 5, one follicle/cow) were classified into following groups: before GnRH administration [before luteinizing hormone (LH) surge]; 3-5 h after GnRH (during LH surge); 10 h after GnRH; 20 h after GnRH; 25 h after GnRH (peri-ovulation); and early CL (days 2-3). The mRNA expression was analysed by quantitative real-time PCR (rotor-gene 3000). Angiopoietin-1 expression rapidly decreased at 3-5 h and kept low level at 10 h after GnRH treatment compared with that before GnRH, and returned to the level before LH surge in the follicles >20 h after GnRH treatment. The levels of ANPT-2 mRNA decreased at 10 and 25 h after treatment compared with other periods. The ratio of ANPT-2/ANPT-1 (an index for destabilization of blood vessels) increased in the follicles at 3-5 h after GnRH treatment only. Both of Tie-1 and Tie-2 receptor expressions decreased in the follicles at 25 h after GnRH treatment. The results of the present study indicated that mRNA expressions of ANPT-1, ANPT-2 and their receptors changed in the bovine follicles during periovulatory period. These results suggest that angiopoietin-Tie system is associated with the initiation of vasculature of follicle that grows towards ovulation.

Molecular profile and proliferative responses of rat lymphatic endothelial cells in culture.

BACKGROUND: Lymphangiogenesis plays an important role in metastasis of many solid tumors. To study lymphangiogenesis under controlled conditions, an in vitro model is needed. The goal of this work was to establish such an in vitro model by determining a molecular profile of rat mesenteric lymphatic endothelial cells (RMLEC) and characterizing their proliferative responses to angiogenic and lymphangiogenic factors, such as vascular endothelial growth factor A and C (VEGF-A and VEGF-C). METHODS AND RESULTS: RMLEC strongly expressed most lymphatic-specific markers, including Prox-1, LYVE-1, and VEGFR-3. Proliferation of RMLEC was serum and heparin dependent. In the presence of low (2%) serum concentration, exogenously added VEGF-A and VEGFC stimulated RMLEC in a linear and dose-dependent manner. This effect was abrogated by anti-VEGF-A and VEGF-C antibodies, as well as by soluble Tie-2 and Flt-4 fusion proteins. Abrogation was reversed by VEGF-A, suggesting that this factor as an important regulator of lymphangiogenesis. CONCLUSIONS: Cultured RMLEC preserved a molecular profile consistent with the phenotype of lymphatic endothelium in vivo and respond to either VEGF-A or VEGF-C factors. VEGFA was able to rescue RMLEC proliferation inhibited by a neutralizing VEGF-C antibody or soluble Tie-2 fusion protein. These results support the existence of cross-talk among angiogenic and lymphangiogenic factors. This work established experimental conditions that allow in vitro modeling of lymphatic endothelial responses to lymphangiogenic regulators. Preliminary results using this model suggest that VEGF-A, VEGF-C, and angiopoietins work in concert to promote lymphangiogenesis in vivo.

Expression of angiogenic factors during distraction osteogenesis.

Distraction osteogenesis is a unique and effective way to treat limb length inequality resulting from congenital and posttraumatic skeletal defects. However, despite its widespread clinical use, the cellular and molecular mechanisms by which this surgical treatment promotes new bone formation are not well understood. Previous studies in distraction osteogenesis have noted increased blood flow and vessel formation within the zone of distraction. These observations suggest that distraction osteogenesis may be driven in part by an angiogenic process. Using immunohistological analysis, the expression of two different angiogenic factors (VEGF and bFGF) was shown to localize at the leading edge of the distraction gap, where nascent osteogenesis was occurring. These cells were spatially adjacent to new vessels that were identified by staining for factor VIII. Microarray analysis detected maximal mRNA expression for a wide variety of angiogenic factors including angiopoietin 1 and 2, both Tie receptors, VEGF-A and -D, VEGFR2, and neuropilin 1. Expression of these factors was found to be maximal during the phase of active distraction. Expression of mRNA for extracellular matrix proteins and BMPs was also maximal during this period. A comparison between the patterns of gene expression in fracture healing and distraction osteogenesis revealed similarities; however, the expression of a number of genes showed selective expression in these two types of bone healing. These data suggest that bone formation during distraction osteogenesis is accompanied by the robust induction of factors associated with angiogenesis and support further investigations to elucidate the mechanisms by which angiogenic events promote bone repair and regeneration.

Involvement of angiopoietin-tie system in bovine follicular development and atresia: messenger RNA expression in theca interna and effect on steroid secretion.

Angiogenesis is involved in the local mechanisms that regulate follicular development and ovulation. Recently, the angiopoietin (ANPT)-Tie system has been shown to be required to regulate angiogenesis and blood vessel regression. Expression of the ANPT-Tie system in the cyclic ovary suggests that the relative changes in the expression of ANPT-1 and ANPT-2 influence the stability of ovarian blood vessels. In this study, we investigated 1) the mRNA expression for ANPT-1, ANPT-2, and endothelial cell-specific receptors Tie1 and Tie2 in the theca interna (TI) of the bovine developing, mature, and atretic follicles by using a semiquantitative reverse transcription polymerase chain reaction assay and 2) the effect of ANPT on the secretion of steroid hormones from bovine preovulatory follicles in vitro using a microdialysis system (MDS) implanted in the thecal layer. Bovine follicles were classified as developing, mature, and atretic according to size, follicular fluid content of estradiol (E2) and progesterone (P4), and characteristics of granulosa cells (GCs). Both ANPT and Tie mRNA were expressed in the TI, whereas GCs expressed ANPT mRNA only. The expression of ANPT-2 mRNA was decreased in the mature follicles. This decrease resulted in a decrease in the ANPT-2:ANPT-1 ratio (an index of instability of blood vessels), indicating that the blood vessels became more stable or mature. The early atretic follicles showed a higher ANPT-2:ANPT-1 ratio and higher Tie2 mRNA expression than did other follicles at healthy or later atretic stages. This finding may imply that blood vessels become unstable at the initial stage of follicular atresia. In both mid and late atretic follicles, Tie2 mRNA expression dramatically decreased, indicating a disruption of the ANPT-Tie system. In the MDS experiment, an infusion of ANPT-1 or ANPT-2 increased P4 release, whereas both ANPTs inhibited the release of androstenedione. ANPT-1 also increased E2 release. These results showed that the mRNA expression for ANPT-1, ANPT-2, Tie1, and Tie2 changes during follicular development, maturation, and atresia in bovine follicles and that ANPTs affect steroidogenesis in the preovulatory follicle. The results suggest that the ANPT-Tie system is involved the structural (angiogenesis) and secretory changes that occur during follicular development and atresia.