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.

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.

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.

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.

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.

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.

[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.

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.

COMP-Ang1 inhibits apoptosis as well as improves the attenuated osteogenic differentiation of mesenchymal stem cells induced by advanced glycation end products.

BACKGROUND: In the present study, we have investigated the possibility that cartilage oligomeric matrix protein angiopoietin1 (COMP-Ang1), important factor in angiogenesis, osteogenesis and the survival of mesenchymal stem cells (MSCs) through the Ang1/Tie2 pathway has beneficial effects on osteogenic differentiated cells (ODCs) from MSCs treated by advanced glycation end products (AGE), which are pathological factors of diabetes. METHODS: Primary culture of MSCs was used. For comparison analysis of AGE and COMP-Ang1 effects, we performed cell viability assay with each treated variety concentration for 24h. Apoptosis rate and Caspase-3 activity were measured by each ELISA assay. To make sure with Ang1/Tie2 pathway, we performed small interfering RNA transfected to MSCs. Real-time RT-PCR was performed to identify ODCs marker genes. Immunoblotting was used to evaluate the expression of Tie2, AKT, p38 and ERK. RESULTS: Our results clearly demonstrate that COMP-Ang1 upregulates the phosphorylation of AKT and p38 by activating the Ang1/Tie2 signaling pathway, indicating that COMP-Ang1 affects both AGE-induced apoptosis and the attenuated osteogenic differentiation of MSCs through the p38/MAPK and PI3K/AKT pathways. CONCLUSIONS: COMP-Ang1 improves cell viability and differentiation function of ODCs against AGE via Ang/Tie2 signaling pathway. GENERAL SIGNIFICANCE: Our results suggest the potential importance of COMP-Ang1 as a new therapy for impaired bone formation that is associated with diabetes and advanced age.

COMP-Ang1 promotes chondrogenic and osteogenic differentiation of multipotent mesenchymal stem cells through the Ang1/Tie2 signaling pathway.

Mesenchymal stem cells (MSCs) are pleiotrophic cells that differentiate to chondrocytes, osteoblasts, or adipocytes, as a result of crosstalk by specific signaling pathways including MAPK pathway. Recently cartilage oligomeric matrix protein angiopoietin1 (COMP-Ang1), an Ang1 variant which is more potent than native Ang1 in phosphorylating Tie2 receptor was developed. The Ang1/Tie2 signaling system not only plays a pivotal role in vessel growth, remodeling, and maturation, but also protective and recruit effect on MSCs. Thus, the aim of the present study was to investigate the differentiate effect of Ang1/Tie2 signaling on MSCs in the presence of chondrogenic, osteogenic and adipogenic induction medium, and to determine the possible mechanisms. Our results clearly demonstrated that MSCs cultured in each induction medium with COMP-Ang1 revealed strongly chondrogenic and osteogenic morphological change (3.5- and 2-fold, respectively) as well as up-regulate each gene, except for adipogenic differentiation. Accordingly, we found that phosphorylation of Tie2 expression lead to phosphorylation of p38 and AKT and then accelerating each differentiation of MSCs to chondrocytes and osteoblasts. Therefore, our findings suggest that COMP-Ang1 present a portal to promote MSCs differentiation to chondrocytes and osteoblasts through Ang1/Tie2 signaling pathway and provide insights into novel therapies for bone diseases.

Potential protective effect of angiopoietin-1 on the leakage of rat choroidal neovascularization.

OBJECTIVES: To observe the potential protective effect of angiopoietin-1 (Ang-1) on rat choroidal neovascularization (CNV) leakage. METHODS: The study was conducted at the Eye Institute of Shandong University of Traditional Chinese Medicine, Jinan, China from June 2012 to June 2013. Thirty CNV model rats were induced by laser. In vivo, fluorescein fundus angiography and pathological techniques were applied to detect the effect of vascular endothelial growth factor (VEGF) and Ang-1 intravitreous injection. In vitro, 3-(4, 5-dimethylthiazole-2-yl)-2, 5-biphenyl tetrazolium bromide (MTT) assay was applied to detect the proliferation of cultured bovine retinal endothelial cells (BRECs) after treatment with VEGF and Ang-1. Transmission electron microscopy (TEM) was used to detect the morphological changes under VEGF and Ang-1. RESULTS: In the CNV rat model, less late leakage was found in the Ang-1 group than the vehicle control or the VEGF group. The MTT assay showed Ang-1 administration inhibited the proliferation of BRECs. The VEGF promoted proliferation at low concentrations and inhibited the proliferation when its concentration reached 50 ng/ml. The administration of VEGF+Ang-1 rescued the inhibition effect of Ang-1 alone. The TEM results showed that there were less intercellular junctions in the VEGF group compared with the vehicle control. In the VEGF + Ang-1 group, the intercellular junctions were nearly normal. CONCLUSION: The Ang-1 can induce intercellular junction formation and decrease the CNV leakage.

VEGF and Angiopoietin-1 exert opposing effects on cell junctions by regulating the Rho GEF Syx.

Vascular endothelial growth factor (VEGF) and Ang1 (Angiopoietin-1) have opposing effects on vascular permeability, but the molecular basis of these effects is not fully known. We report in this paper that VEGF and Ang1 regulate endothelial cell (EC) junctions by determining the localization of the RhoA-specific guanine nucleotide exchange factor Syx. Syx was recruited to junctions by members of the Crumbs polarity complex and promoted junction integrity by activating Diaphanous. VEGF caused translocation of Syx from cell junctions, promoting junction disassembly, whereas Ang1 maintained Syx at the junctions, inducing junction stabilization. The VEGF-induced translocation of Syx from EC junctions was caused by PKD1 (protein kinase D1)-mediated phosphorylation of Syx at Ser(806), which reduced Syx association to its junctional anchors. In support of the pivotal role of Syx in regulating EC junctions, syx(-/-) mice had defective junctions, resulting in vascular leakiness, edema, and impaired heart function.

Exhaustion of nucleus pulposus progenitor cells with ageing and degeneration of the intervertebral disc.

Despite the high prevalence of intervertebral disc disease, little is known about changes in intervertebral disc cells and their regenerative potential with ageing and intervertebral disc degeneration. Here we identify populations of progenitor cells that are Tie2 positive (Tie2+) and disialoganglioside 2 positive (GD2+), in the nucleus pulposus from mice and humans. These cells form spheroid colonies that express type II collagen and aggrecan. They are clonally multipotent and differentiated into mesenchymal lineages and induced reorganization of nucleus pulposus tissue when transplanted into non-obese diabetic/severe combined immunodeficient mice. The frequency of Tie2+ cells in tissues from patients decreases markedly with age and degeneration of the intervertebral disc, suggesting exhaustion of their capacity for regeneration. However, progenitor cells (Tie2+GD2+) can be induced from their precursor cells (Tie2+GD2-) under simple culture conditions. Moreover, angiopoietin-1, a ligand of Tie2, is crucial for the survival of nucleus pulposus cells. Our results offer insights for regenerative therapy and a new diagnostic standard.

Unique angiogenic and vasculogenic properties of renal cell carcinoma in a xenograft model of bone metastasis are associated with high levels of vegf-a and decreased ang-1 expression.

Management of various tumor metastases to bone has dramatically improved, but this is not so for renal cell carcinoma (RCC), which is a difficult surgical problem due to its great vascularity. Furthermore, the unique mechanisms that mediate RCC vasculogenesis in bone remain unknown. To understand this process we developed a xenograft model that recapitulates highly vascular RCC versus less vascular tumors that metastasize to bone. Human tumor cell lines of RCC (786-O), prostate cancer (PC3), lung cancer (A549), breast cancer (MDA-MB231), and melanoma (A375) were transduced with firefly luciferase (Luc), injected into the tibiae of nude mice, and differences in growth, osteolysis, and vascularity were assessed by longitudinal bioluminescent imaging, micro-CT for measurement of calcified tissues and vascularity and histology. The results showed that while RCC-Luc has reduced growth and osteolytic potential versus the other tumor lines, it displayed a significant increase in vascular volume (p < 0.05). This expansion was due to 3- and 5-fold increases in small and large vessel numbers respectively. In vitro gene expression profiling revealed that RCC-Luc expresses significantly (p < 0.05) more vegf-a (10-fold) and 20- to 30-fold less ang-1 versus the other lines. These data demonstrate the utility of this model to study the unique vasculogenic properties of RCC bone metastases.