Exosomal lncRNA-H19 promotes osteogenesis and angiogenesis through mediating Angpt1/Tie2-NO signaling in CBS-heterozygous mice.

Rationale: Emerging evidence indicates that the growth of blood vessels and osteogenesis is tightly coordinated during bone development. However, the molecular regulators of intercellular communication in the bone microenvironment are not well studied. Therefore, we aim to investigate whether BMMSC-Exo promotes osteogenesis and angiogenesis via transporting lnc-H19 in the CBS- heterozygous mouse model. Methods: Using RT2 lncRNA PCR array screening, we identify a bone-specific, long noncoding RNA-H19 (lncRNA-H19/lnc-H19) in exosomes derived from bone marrow mesenchymal stem cells (BMMSC-Exo) during osteogenesis. Using bioinformatics analysis, we further discovered the seed sequence of miR-106a that could bind to lnc-H19. A luciferase reporter assay was performed to demonstrate the direct binding of miR-106a to the target gene angiopoietin 1 (Angpt1). We employed an immunocompromised Nude mouse model, to evaluate the effects of BMMSC-Exo on angiogenesis in vivo. Using a micro-CT scan, we monitored microstructural changes of bone in the experimental mice. Results: BMMSC-Exo possessed exosomal characteristics including exosome size, and typical markers including CD63, CD9, and TSD101. In vitro, BMMSC-Exo significantly promoted endothelial angiogenesis and osteogenesis. Mechanistic studies have shown that exosomal lnc-H19 acts as "sponges" to absorb miR-106 and regulate the expression of angiogenic factor, Angpt1 that activates lnc-H19/Tie2-NO signaling in mesenchymal and endothelial cells. Both of these effects on osteogenesis and angiogenesis are inhibited by antagonizing Tie2 signaling. Treatment of BMMSC-Exo also restored the bone formation and mechanical quality in vivo. Conclusion: These findings provide a novel insight into how the extracellular role of exosomal lnc-H19 affects osteogenesis and angiogenesis through competing endogenous RNA networks.

Angiopoietin-1 accelerates Alzheimer's disease via FOXA2/PEN2/APP pathway in APP/PS1 mice.

Angiopoietin-1 (Ang-1), a regulatory angiogenesis protein and it has been found to be involved in the occurrence and progression of Alzheimer's disease. However, it was still to be addressed the distinctly role and the molecular mechanisms of Ang-1 affects Alzheimer's disease. Our data suggest that Ang-1 aggravated the accumulation of Aß42 and cognitive decline in APP/PS1 mice. The upregulation of APPß is essential for Aß42 production in N2a cells overexpressing the mutational human APP gene (N2a/APP695 cells), while downregulation of PEN2 could reduce APP expression. Silencing of FOXA2 lead to inhibition of APP expression, as well as decrease of Aß42 contents. In conclusion, Ang-1 has an accelerative effect on Alzheimer's disease by increasing the secretion of Aß42 via FOXA2/PEN2/APP pathway.

Metformin coordinates osteoblast/osteoclast differentiation associated with ischemic osteonecrosis.

In this study, we aimed to identify a candidate drug that can activate endogenous Angiopoietin 1 (Ang1) expression via drug repositioning as a pharmacological treatment for avascular osteonecrosis. After incubation with 821 drugs from the Food and Drug Administration (FDA)-approved drug library, Ang1 expression in U2OS cell culture media was examined by ELISA. Metformin, the first-line medication for treatment of type 2 diabetes, was selected as a candidate for in vitro and in vivo experimental evaluation. Ang1 was induced, and alkaline phosphatase activity was increased by metformin treatment in U2OS and MG63 cells. Wound healing and migration assay showed increased osteoblastic cell mobility by metformin treatment in U2OS and MG63 cells. Metformin upregulated expression of protein markers for osteoblastic differentiation in U2OS and MG63 cells but inhibited osteoclastic differentiation in Raw264.7 cells. Metformin (25 mg/kg) protected against ischemic necrosis in the epiphysis of the rat femoral head by maintaining osteoblast/osteocyte function and vascular density but inhibiting osteoclast activity in the necrotic femoral head. These findings provide novel insight into the specific biomarkers that are targeted and regulated by metformin in osteoblast differentiation and contribute to understanding the effects of these FDA-approved small-molecule drugs as novel therapeutics for ischemic osteonecrosis.

Rapid Development of Glaucoma Via ITV Nonselective ANGPT 1/2 Antibody: A Potential Role for ANGPT/TIE2 Signaling in Primate Aqueous Humor Outflow.

Purpose: Investigate a significant, dose-related increase in IOP, leading to glaucomatous damage to the neuroretina and optic nerve following intravitreal (ITV) administration of a bispecific F(ab')2 [anti-VEGF/Angiopoietins [ANGPT]F(ab')2] molecule in adult monkeys. Methods: ITV ocular tolerability and investigation of anti-VEGF/ANGPT F(ab')2 (blocking both ANGPT1 and ANGPT2) was done in monkeys; mechanistic studies were done in neonatal mice. Results: Following the second ITV dose of anti-VEGF/ANGPT F(ab')2, all 1.5- and 4-mg/eye treated monkeys developed elevated IOP, which eventually was associated with optic disc cupping and thinning of the neuroretinal rim. Histopathologic examination showed nonreversible axonal degeneration in the optic nerves of animals administered 1.5 mg/eye and higher that was considered secondary to high IOP. Anti-ANGPT Fab also caused elevated IOP in monkeys, but anti-VEGF Fab did not contribute to the IOP increase. In addition, an anti-ANGPT2-selective antibody did not change IOP. In mice simultaneous blockade of ANGPT1 and ANGPT2 impaired the expansion and formation of Schlemm's canal (SC) vessels, similar to genetic ablation of Angpt1/Angpt2 and their receptor TIE2. As previously reported, blocking ANGPT2 alone did not affect SC formation in mice. Conclusions: Dual inhibition of ANGPT1/ANGPT2, but not ANGPT2 alone, leads to increased IOP and glaucomatous damage in monkeys. This confirms a role for TIE2/ANGPT signaling in the control of IOP in adults, a finding initially identified in transgenic mice. Dual pharmacologic inhibition of ANGPT1/ANGPT2 may affect aqueous drainage and homeostasis in adult monkeys and may be useful in developing novel models of glaucoma.

Angiopoietin 1 influences ischemic reperfusion renal injury via modulating endothelium survival and regeneration.

BACKGROUND: Damage to the endothelium due to ischemia reperfusion injury (IRI) leads to a disruption of the microvasculature, which could be influenced by angiopoietin 1 via its effects on endothelium. We investigated the physiological and therapeutic roles of angiopoietin 1 in renal IRI using angiopoietin 1 knockout and over-expression mice. METHODS: Renal IRI was induced by clamping the right renal artery seven days after left uninephrectomy for 25 min followed by reperfusion. A whole body angiopoietin 1 knockout was achieved by induction with tamoxifen. The renal tubule over-expression of angiopoietin 1 was induced by doxycycline. RESULTS: In the normal mice, the renal expression of angiopoietin 1 increased 7 days to 14 days after IRI. The angiopoietin 1 knockout caused a delay in the recovery of renal function, less tubular regeneration and more residual tubular necrosis. The endothelial density was lower and the VE-cadherin protein loss was greater in the knockout mice. The over-expression of angiopoietin 1 attenuated the tubular necrosis and renal function impairment 1 and 3 days after IRI. The loss of the endothelium was ameliorated in the over-expression mice. This protective effect was associated with the up-regulation of the gene expression of epidermal growth factor, hepatocyte growth factor, and insulin like growth factor-1 and less tubular apoptosis. The over-expression of angiopoietin 1 stimulated tumor necrosis factor-α, C-C chemokine receptor type 2 and CX3C chemokine receptor 1 inflammatory gene expression, but did not influence macrophage infiltration. CONCLUSIONS: Altogether, the augmentation and downregulation of angiopoietin 1 attenuated renal damage and impaired renal recovery, respectively, by influencing the survival/regeneration of the endothelium. The manipulation of angiopoietin 1 represents a novel therapeutic approach for the treatment of ischemic kidney injury.

Angiopoietin-1 deficiency increases renal capillary rarefaction and tubulointerstitial fibrosis in mice.

Presence of tubulointerstitial fibrosis is predictive of progressive decline in kidney function, independent of its underlying cause. Injury to the renal microvasculature is a major factor in the progression of fibrosis and identification of factors that regulate endothelium in fibrosis is desirable as they might be candidate targets for treatment of kidney diseases. The current study investigates how loss of Angipoietin-1 (Angpt1), a ligand for endothelial tyrosine-kinase receptor Tek (also called Tie2), affects tubulointerstitial fibrosis and renal microvasculature. Inducible Angpt1 knockout mice were subjected to unilateral ureteral obstruction (UUO) to induce fibrosis, and kidneys were collected at different time points up to 10 days after obstruction. Staining for aSMA showed that Angpt1 deficient kidneys had significantly more fibrosis compared to wildtype mice 3, 6, and 10 days after UUO. Further investigation 3 days after UUO showed a significant increase of Col1a1 and vimentin in Angpt1 deficient mice, as well as increased gene expression of Tgfb1, Col1a1, Fn1, and CD44. Kidney injury molecule 1 (Kim1/Havcr1) was significantly more increased in Angpt1 deficient mice 1 and 3 days after UUO, suggesting a more severe injury early in the fibrotic process in Angpt1 deficient mice. Staining for endomucin showed that capillary rarefaction was evident 3 days after UUO and Angpt1 deficient mice had significantly less capillaries 6 and 10 days after UUO compared to UUO kidneys in wildtype mice. RNA sequencing revealed downregulation of several markers for endothelial cells 3 days after UUO, and that Angpt1 deficient mice had a further downregulation of Emcn, Plvap, Pecam1, Erg, and Tek. Our results suggest that loss of Angpt1 is central in capillary rarefaction and fibrogenesis and propose that manipulations to maintain Angpt1 levels may slow down fibrosis progression.

Ascending Vasa Recta Are Angiopoietin/Tie2-Dependent Lymphatic-Like Vessels.

Urinary concentrating ability is central to mammalian water balance and depends on a medullary osmotic gradient generated by a countercurrent multiplication mechanism. Medullary hyperosmolarity is protected from washout by countercurrent exchange and efficient removal of interstitial fluid resorbed from the loop of Henle and collecting ducts. In most tissues, lymphatic vessels drain excess interstitial fluid back to the venous circulation. However, the renal medulla is devoid of classic lymphatics. Studies have suggested that the fenestrated ascending vasa recta (AVRs) drain the interstitial fluid in this location, but this function has not been conclusively shown. We report that late gestational deletion of the angiopoietin receptor endothelial tyrosine kinase 2 (Tie2) or both angiopoietin-1 and angiopoietin-2 prevents AVR formation in mice. The absence of AVR associated with rapid accumulation of fluid and cysts in the medullary interstitium, loss of medullary vascular bundles, and decreased urine concentrating ability. In transgenic reporter mice with normal angiopoietin-Tie2 signaling, medullary AVR exhibited an unusual hybrid endothelial phenotype, expressing lymphatic markers (prospero homeobox protein 1 and vascular endothelial growth factor receptor 3) as well as blood endothelial markers (CD34, endomucin, platelet endothelial cell adhesion molecule 1, and plasmalemmal vesicle-associated protein). Taken together, our data redefine the AVRs as Tie2 signaling-dependent specialized hybrid vessels and provide genetic evidence of the critical role of AVR in the countercurrent exchange mechanism and the structural integrity of the renal medulla.

Differential regulation of blood flow-induced neovascularization and mural cell recruitment by vascular endothelial growth factor and angiopoietin signalling.

KEY POINTS: Combining nitric oxide (NO)-mediated increased blood flow with angiopoietin-1-Tie2 receptor signalling induces arteriolargenesis - the formation of arterioles from capillaries - in a model of physiological angiogenesis. This NO-Tie-mediated arteriolargenesis requires endogenous vascular endothelial growth factor (VEGF) signalling. Inhibition of VEGF signalling increases pericyte coverage in microvessels. Together these findings indicate that generation of functional neovasculature requires close titration of NO-Tie2 signalling and localized VEGF induction, suggesting that the use of exogenous VEGF expression as a therapeutic for neovascularization may not be successful. ABSTRACT: Signalling through vascular endothelial growth factor (VEGF) receptors and the tyrosine kinase with IgG and EGF domains-2 (Tie2) receptor by angiopoietins is required in combination with blood flow for the formation of a functional vascular network. We tested the hypothesis that VEGF and angiopoietin-1 (Ang1) contribute differentially to neovascularization induced by nitric oxide (NO)-mediated vasodilatation, by comparing the phenotype of new microvessels in the mesentery during induction of vascular remodelling by over-expression of endothelial nitric oxide synthase in the fat pad of the adult rat mesentery during inhibition of angiopoietin signalling with soluble Tie2 (sTie2) and VEGF signalling with soluble Fms-like tyrosine kinase receptor-1 (sFlt1). We found that NO-mediated angiogenesis was blocked by inhibition of VEGF with sFlt1 (from 881 ± 98% increase in functional vessel area to 279 ± 72%) and by inhibition of angiopoietin with sTie2 (to 337 ± 67%). Exogenous angiopoietin-1 was required to induce arteriolargenesis (8.6 ± 1.3% of vessels with recruitment of vascular smooth muscle cells; VSMCs) in the presence of enhanced flow. sTie2 and sFlt1 both inhibited VSMC recruitment (both 0%), and VEGF inhibition increased pericyte recruitment to newly formed vessels (from 27 ± 2 to 54 ± 3% pericyte ensheathment). We demonstrate that a fine balance of VEGF and angiopoietin signalling is required for the formation of a functional vascular network. Endogenous VEGF signalling prevents excess neovessel pericyte coverage, and is required for VSMC recruitment during increased nitric oxide-mediated vasodilatation and angiopoietin signalling (NO-Tie-mediated arteriogenesis). Therapeutic vascular remodelling paradigms may therefore require treatments that modulate blood flow to utilize endogenous VEGF, in combination with exogenous Ang1, for effective neovascularization.

Targeting Tie2 for Treatment of Diabetic Retinopathy and Diabetic Macular Edema.

Tie2 is a tyrosine kinase receptor located predominantly on vascular endothelial cells that plays a central role in vascular stability. Angiopoietin-1 (Angpt1), produced by perivascular cells, binds, clusters, and activates Tie2, leading to Tie2 autophosphorylation and downstream signaling. Activated Tie2 increases endothelial cell survival, adhesion, and cell junction integrity, thereby stabilizing the vasculature. Angiopoietin-2 (Angpt2) and vascular endothelial-protein tyrosine phosphatase (VE-PTP) are negative regulators increased by hypoxia; they inactivate Tie2, destabilizing the vasculature and increasing responsiveness to vascular endothelial growth factor (VEGF) and other inflammatory cytokines that stimulate vascular leakage and neovascularization. AKB-9778 is a small-molecule antagonist of VE-PTP which increases phosphorylation of Tie2 even in the presence of high Angpt2 levels. In preclinical studies, AKB-9778 reduced VEGF-induced leakage and ocular neovascularization (NV) and showed additive benefit when combined with VEGF suppression. In two clinical trials in diabetic macular edema (DME) patients, subcutaneous injections of AKB-9778 were safe and provided added benefit to VEGF suppression. Preliminary data suggest that AKB-9778 monotherapy improves diabetic retinopathy. These data suggest that Tie2 activation may be a valuable strategy to treat or prevent diabetic retinopathy.

New Insights into the Pro-Inflammatory Activities of Ang1 on Neutrophils: Induction of MIP-1ß Synthesis and Release.

We reported the expression of angiopoietin Tie2 receptor on human neutrophils and the capacity of angiopoietins (Ang1 and Ang2) to induce pro-inflammatory activities, such as platelet-activating factor synthesis, ß2-integrin activation and neutrophil migration. Recently, we observed differential effects between both angiopoietins, namely, the capacity of Ang1, but not Ang2, to promote rapid interleukin-8 synthesis and release, as well as neutrophil viability. Herein, we addressed whether Ang1 and/or Ang2 could modulate the synthesis and release of macrophage inflammatory protein-1ß (MIP-1ß) by neutrophils. Neutrophils were isolated from blood of healthy volunteers; intracellular and extracellular MIP-1ß protein concentrations were assessed by ELISA. After 24 hours, the basal intracellular and extracellular MIP-1ß protein concentrations were ≈500 and 100 pg/106 neutrophils, respectively. Treatment with Ang1 (10 nM) increased neutrophil intracellular and extracellular MIP-1ß concentrations by 310 and 388% respectively. Pretreatment with PI3K (LY294002), p38 MAPK (SB203580) and MEK (U0126) inhibitors completely inhibited Ang1-mediated increase of MIP-1ß intracellular and extracellular protein levels. Pretreatment with NF-κB complex inhibitors, namely Bay11-7085 and IKK inhibitor VII or with a transcription inhibitor (actinomycin D) and protein synthesis inhibitor (cycloheximide), did also abrogate Ang1-mediated increase of MIP-1ß intracellular and extracellular protein levels. We validated by RT-qPCR analyses the effect of Ang1 on the induction of MIP-1ß mRNA levels. Our study is the first one to report Ang1 capacity to induce MIP-1ß gene expression, protein synthesis and release from neutrophils, and that these effects are mediated by PI3K, p38 MAPK and MEK activation and downstream NF-κB activation.

Disruption of vascular endothelial homeostasis in systemic juvenile idiopathic arthritis-associated macrophage activation syndrome: The dynamic roles of angiopoietin-1 and -2.

To assess the role of angiopoietin (Ang)-1 and Ang-2 and to investigate the clinical significance of serum levels of them in systemic juvenile idiopathic arthritis (s-JIA)-associated macrophage activation syndrome (MAS), we determined these levels in 51 patients with s-JIA, 11 patients with polyarticular JIA (poly-JIA), 12 patients with virus associated hemophagocytic syndrome (VAHS), 12 patients with Kawasaki disease (KD), and 15 age-matched healthy controls (HC). The results were compared with clinical features of MAS. During the MAS phase, serum Ang-1 levels were significantly decreased compared with those during the active and inactive phases. Serum Ang-2/1 ratio were significantly elevated during the MAS phase, compared with those during the active and inactive phases. There was a rapid increase in the Ang-2/1 ratio at the onset of MAS. Serum Ang-1 and the Ang-2/1 ratio significantly correlated with measures of disease activity, including AST and LDH. Ang-2/1 dysregulation was also observed in patients with VAHS, whereas not observed in most cases of KD. The homeostasis of vascular endothelial function by Ang-1 and Ang-2 is disrupted in MAS. Serum Ang-1 levels and the Ang-2/1 ratio might represent promising indicators of disease activity for MAS.

Phosphorylation of Threonine 794 on Tie1 by Rac1/PAK1 Reveals a Novel Angiogenesis Regulatory Pathway.

The endothelial receptor tyrosine kinase (RTK) Tie1 was discovered over 20 years ago, yet its precise function and mode of action remain enigmatic. To shed light on Tie1's role in endothelial cell biology, we investigated a potential threonine phosphorylation site within the juxtamembrane domain of Tie1. Expression of a non-phosphorylatable mutant of this site (T794A) in zebrafish (Danio rerio) significantly disrupted vascular development, resulting in fish with stunted and poorly branched intersomitic vessels. Similarly, T794A-expressing human umbilical vein endothelial cells formed significantly shorter tubes with fewer branches in three-dimensional Matrigel cultures. However, mutation of T794 did not alter Tie1 or Tie2 tyrosine phosphorylation or downstream signaling in any detectable way, suggesting that T794 phosphorylation may regulate a Tie1 function independent of its RTK properties. Although T794 is within a consensus Akt phosphorylation site, we were unable to identify a physiological activator of Akt that could induce T794 phosphorylation, suggesting that Akt is not the physiological Tie1-T794 kinase. However, the small GTPase Ras-related C3 botulinum toxin substrate 1 (Rac1), which is required for angiogenesis and capillary morphogenesis, was found to associate with phospho-T794 but not the non-phosphorylatable T794A mutant. Pharmacological activation of Rac1 induced downstream activation of p21-activated kinase (PAK1) and T794 phosphorylation in vitro, and inhibition of PAK1 abrogated T794 phosphorylation. Our results provide the first demonstration of a signaling pathway mediated by Tie1 in endothelial cells, and they suggest that a novel feedback loop involving Rac1/PAK1 mediated phosphorylation of Tie1 on T794 is required for proper angiogenesis.

Topical Application of Insulin Accelerates Vessel Maturation of Wounds by Regulating Angiopoietin-1 in Diabetic Mice.

Reestablishment of the structural and functional microvasculature would be beneficial to promote healing of diabetic wounds. We explored the role of insulin application on microvascular maturation of diabetic wounds to determine whether it is associated with insulin-induced wound healing. We adopted the multiple injections of streptozotocin (STZ) to establish a diabetic animal model. The effect of insulin on microvessel formation, especially the effect of insulin on microvascular maturation was observed by transmission electron microscopy and laser scanning confocal microscopy. The pivotal protein regulated by insulin during healing processes was explored by tropical application neutralizing antibodies to these proteins; the specific protein was further confirmed using immunoblotting. On days 7 and 11, the blood vessel in insulin-treated wounds was surrounded by more α-smooth muscle actin (α-SMA) expressing cells. The blockage of angiopoietin-1 (Ang-1), but not angiopoietin-2 (Ang-2) or platelet-derived growth factor-B (PDGF-B), resulted in reduced maturation of newly formed blood vessels despite the presence of insulin in vivo. Further analysis showed that insulin induced an increased expression of Ang-1. The blood vessels in insulin-treated wounds showing advanced coverage of pericytes and reconstruction of new vascular basement membrane suggest that insulin is a potent accelerator of microvascular maturation, which may be involved in the mechanisms of insulin-induced wound healing.

[Effects of adenovirus-delivered angiopoietin-1 siRNA on expression of matrix metalloproteinases in rats with acute lung injury induced by phosgene].

OBJECTIVE: To investigate the effects of adenovirus-delivered angiopoietin-1 siRNA (Ad. Ang-1siRNA) on the expression of matrix metalloproteinase-2, 9 (MMP-2, 9) and tissue inhibitor of metallopro-teinase-1 (TIMP-1) in rats with acute lung injury (ALI) induced by phosgene (Psg). METHODS: We first established a rat model of Psg-induced acute lung injury (ALI). The rats were randomly divided into 6 groups: air control group with exposure to air, air+adenovirus (air+Ad) group with caudal vein injection of 1×10(8) pfu/ml adenovirus 1 h after air exposure, air+Ad/Ang1 group with caudal vein injection of 1×10(8) pfu/ml Ad.Ang-1siRNA 1 h after air exposure, Psg group with exposure to 8.33 mg/L Psg (purity 100%, of the same volume as the inhaled air in the air control group) for 5 min, Psg+Ad group with caudal vein injection of 1×10(8) pfu/ml adenovirus 1 h after exposure to the same dose of Psg, and Psg+Ad/Ang1 group with caudal vein injection of 1×10(8) pfu/ml Ad.Ang-1siRNA 1 h after exposure to the same dose of Psg. Serum, bronchoalveolar lavage fluid (BALF), and lung tissue were collected 36 h after exposure. The protein expression of Ang-1, MMP-2, 9, and TIMP-1 in serum and BALF was determined by double-antibody sandwich ELISA. RT-PCR was used to determine the mRNA levels of Ang-1, MMP-2, 9, and TIMP-1 in lung tissue. The protein expression of MMP-2, 9 and TIMP-1 in lung tissue was determined by Western blot. RESULTS: A rat model of Psg-induced ALI was successfully established. The levels of MMP-2, 9 in serum, BALF, and lung tissue were significantly increased in the Psg group and Psg+Ad/Ang1 group as compared with the control group (P<0.01); no significant change was observed in serum TIMP-1 protein expression (P>0.05); interestingly, TIMP-1 protein expression in BALF and lung tissue was significantly increased (P<0.01). Compared with the Psg group, the Psg+Ad/Ang1 group showed a significant decrease in MMP-2, 9 expression in BALF, serum, and lung tissue (P<0.05), but no significant change in protein expression of TIMP-1 was discovered (P>0.05). CONCLUSION: Ad.Ang-1siRNA has a potential beneficial effect in rats with Psg-induced ALI through inhibition of MMP-2, 9 expression, but has no significant effect on the expression of TIMP-1.

Regulation of decidualization and angiogenesis in the human endometrium: mini review.

AIM: The human endometrium is a dynamic tissue that undergoes regular cycles of menstruation, menstrual repair, proliferation and secretory differentiation in response to hypoxia and the female sex hormones. METHODS: We identified new target genes that are regulated by progesterone during the decidualization of human endometrial stromal cells (ESC), including interleukin-15 (IL-15), fibulin-1 (FBLN-1), and heart and neural crest derivatives expressed transcript 2 (HAND2). RESULTS: IL-15 is deeply involved in the hormonal control of the human endometrium by progesterone and may be important in embryo implantation. FBLN-1 has been shown to be an important extracellular matrix that mediates progesterone action in human ESC differentiation toward implantation. Moreover, progestin-induced HAND2 is a transcription factor that contributes to the increased levels of FBLN-1 in human ESC. Several mediators, including vascular endothelial growth factor (VEGF), angiopoietin (ANGPT) and stromal cell-derived factor 1 (SDF-1), regulate human endometrial angiogenesis. Hypoxia increased the expression of VEGF and decreased the expression of SDF-1 in ESCs. Furthermore, hypoxia reduced ANGPT1 levels in ESC; however, ANGPT2 levels were unaffected. Estradiol simultaneously induced the expressions of VEGF and SDF-1, suppressing ANGPT1 production. Therefore, hypoxia and estradiol caused an increase in the ANGPT2/ANGPT1 ratio. CONCLUSION: Hypoxia and female sex hormones are involved in the regulation of angiogenic factors in an independent manner in human ESC. Analysis of the process of decidualization and angiogenesis in the human endometrium would provide useful information for the fields of reproductive biology, regenerative medicine and tissue engineering.

Angiopoietin1 inhibits mast cell activation and protects against anaphylaxis.

Since morbidity and mortality rates of anaphylaxis diseases have been increasing year by year, how to prevent and manage these diseases effectively has become an important issue. Mast cells play a central regulatory role in allergic diseases. Angiopoietin1 (Ang-1) exhibits anti-inflammatory properties by inhibiting vascular permeability, leukocyte migration and cytokine production. However, Ang-1's function in mast cell activation and anaphylaxis diseases is unknown. The results of our study suggest that Ang-1 decreased lipopolysaccharide (LPS)-induced pro-inflammatory cytokines production of mast cells by suppressing IκB phosphorylation and NF-κB nuclear translocation. Ang-1 also strongly inhibited compound 48/80 induced and FcεRI-mediated mast cells degranulation by decreasing intracellular calcium levels in vitro. In vivo lentivirus-mediated delivery of Ang-1 in mice exhibited alleviated leakage in IgE-dependent passive cutaneous anaphylaxis (PCA). Furthermore, exogenous Ang-1 intervention treatment prevented mice from compound 48/80-induced mesentery mast cell degranulation, attenuated increases in pro-inflammatory cytokines, relieved lung injury, and improved survival in anaphylaxis shock. The results of our study reveal, for the first time, the important role of Ang-1 in the activation of mast cells, and identify a therapeutic effect of Ang-1 on anaphylaxis diseases.

Angiopoietin-1 regulates microvascular reactivity and protects the microcirculation during acute endothelial dysfunction: role of eNOS and VE-cadherin.

The growth factor angiopoietin-1 (Ang-1) plays an essential role in angiogenesis and vascular homeostasis. Nevertheless, the role of Ang-1 in regulating vascular tone and blood flow is largely unexplored. Endothelial nitric oxide synthase (eNOS) and the junctional protein VE-cadherin are part of the complex signalling cascade initiated by Ang-1 in endothelial cells. In this study, we aimed to investigate the mechanisms underlying acute effects of Ang-1 on microvascular reactivity, permeability and blood flow, and hypothesise that eNOS and VE-cadherin underpin Ang-1 mediated vascular effects that are independent of angiogenesis and proliferation. Myography of isolated microarterioles from male C3H/HeN mice (7-10 weeks) was employed to measure vascular reactivity in vitro. Microcirculatory function in vivo was evaluated by intravital microscopy and Doppler fluximetry in dorsal window chambers. Ang-1 and its stable variant MAT.Ang-1 induced a concentration-dependent vasodilation of arterioles in vitro, which was blocked with nitric oxide (NO) synthesis inhibitor l-NAME. In vivo, MAT.Ang-1 restored to control levels l-NAME induced peripheral vasoconstriction, decreased blood flow and microvascular hyperpermeability. Tissue protein expression of VE-cadherin was reduced by NOS inhibition and restored to control levels by MAT.Ang-1, whilst VE-cadherin phosphorylation was increased by l-NAME and subsequently reduced by MAT.Ang-1 administration. Moreover, MAT.Ang-1 alone did not modulate systemic levels of angiogenetic factors. Our novel findings report that Ang-1 induces arteriolar vasodilation via release of NO, suggesting that Ang-1 is an important regulator of microvascular tone. As MAT.Ang-1 ameliorates detrimental effects on the microcirculation induced by inhibition of NO synthesis and stabilizes the endothelial barrier function through VE-cadherin, we propose that this Ang-1 variant may serve as a novel therapeutic agent to protect the microcirculation against endothelial dysfunction.

Angiopoietin 1 and angiopoietin 2 are associated with medial thickening of hepatic arterial branches in biliary atresia.

BACKGROUND: Biliary atresia (BA) is an infantile disorder characterized by progressive sclerosing cholangiopathy leading to biliary obstruction. First-line treatment of BA is hepatoportoenterostomy, the prognosis of which is related to age at surgery and to histological variables such as extent of fibrosis and ductular reaction. Hepatic arterial medial thickening (MT) suggests an arteriopathy in BA pathogenesis. We evaluated the expression of angiopoietin (ANGPT)/tyrosine kinase with immunoglobulin-like and epidermal growth factor-like domains 2 (TIE2) system in liver samples obtained from patients with BA, correlating it with MT, variables associated with disease severity, and postoperative prognosis. METHODS: ANGPT1, ANGPT2, and TIE2 expression levels were assessed by quantitative PCR in liver samples obtained from BA patients (n = 23) at portoenterostomy and age-matched infants with intrahepatic cholestasis (IHC; n = 7). Histological variables were morphometrically assessed. RESULTS: ANGPT1 and ANGPT2 were overexpressed in BA in comparison with IHC (P = 0.024 and P = 0.029, respectively). In BA, ANGPTs expression was positively correlated with MT (ANGPT1: rs = 0.59, P = 0.013; ANGPT2: rs = 0.52, P = 0.032), not with the variables associated with disease severity. TIE2 and ANGPTs expression levels were negatively correlated (ANGPT1: rs = -0.73, P < 0.001; ANGPT2: rs = -0.54, P = 0.007). CONCLUSION: In BA, there is overexpression of both ANGPT1 and ANGPT2, which is correlated with MT but not with age at portoenterostomy or with the histological variables associated with disease severity at the time of procedure.

Expression and function of Angiopoietins and their tie receptors in human basophils and mast cells.

The Angiopoietin/Tie system is a key regulator of vascular remodeling, maturation, angiogenesis and lymphangiogenesis. In humans there are three angiopoietins: Angiopoietin-1 (Ang1), Angiopoietin-2 (Ang2), and Angiopoietin-4 (Ang4). Ang1 and Ang2 are the best characterized angiopoietins. The angiopoietin receptor system consists of two type I tyrosine kinase receptors (Tie1 and Tie2). Tie2 binds all known angiopoietins. We sought to characterize Ang1, Ang2, Tie1 and Tie2 expression and functions in human basophils and mast cells. Basophils, LAD-2 cells and Human Lung Mast Cells (HLMCs) constitutively express Ang1 and Ang2 mRNA. Intracellular staining for Ang1 and Ang2 was stronger in basophils than in mast cells. Immunoelectron microscopy demonstrated Ang1 in cytoplasmic vesicles of basophils. The protein kinase C activators phorbol diester (PMA) and bryostatin 1 (Bryo1) stimulated basophils to rapidly release a large amount of Ang1. PMA-induced Ang1 release was inhibited by brefeldin A. Tie1 and Tie2 mRNAs were expressed in basophils, LAD-2 and HLMCs. Basophils, LAD-2 and HLMCs expressed Tie1 on the cell surface. HLMCs and LAD-2 expressed Tie2 on the cell surface, whereas basophils did not. Ang1, but not Ang2, induced migration of mast cells through the engagement of Tie2. Neither Ang1 nor Ang2 induced basophil chemotaxis. We have identified a novel mechanism of cross-talk between human basophils and mast cells mediated by the Ang1/Tie2 system that might be relevant in the orchestration of inflammatory and neoplastic angiogenesis.