Tie2 Signaling Research Articles

Hyperoxia-induced S1P1 signaling reduced angiogenesis by suppression of TIE-2 leading to experimental bronchopulmonary dysplasia.

We have earlier shown that hyperoxia (HO)-induced sphingosine kinase 1 (SPHK1)/sphingosine-1-phosphate (S1P) signaling contribute to bronchopulmonary dysplasia (BPD). S1P acts through G protein-coupled receptors, S1P1 through S1P5. Further, we noted that heterozygous deletion of S1pr1 ameliorated the HO-induced BPD in the murine model. The mechanism by which S1P1 signaling contributes to HO-induced BPD was explored. S1pr1+/+ and S1pr1+/- mice pups were exposed to either room air (RA) or HO (75% oxygen) for 7 days from PN 1-7. Lung injury and alveolar simplification was evaluated. Lung protein expression was determined by Western blotting and immunohistochemistry (IHC). In vitro experiments were performed using human lung microvascular endothelial cells (HLMVECs) with S1P1 inhibitor, NIBR0213 to interrogate the S1P1 signaling pathway. HO increased the expression of S1pr1 gene as well as S1P1 protein in both neonatal lungs and HLMVECs. The S1pr1+/- neonatal mice showed significant protection against HO-induced BPD which was accompanied by reduced inflammation markers in the bronchoalveolar lavage fluid. HO-induced reduction in ANG-1, TIE-2, and VEGF was rescued in S1pr1+/- mouse, accompanied by an improvement in the number of arterioles in the lung. HLMVECs exposed to HO increased the expression of KLF-2 accompanied by reduced expression of TIE-2, which was reversed with S1P1 inhibition. HO induces S1P1 followed by reduced expression of angiogenic factors. Reduction of S1P1 signaling restores ANG-1/ TIE-2 signaling leading to improved angiogenesis and alveolarization thus protecting against HO-induced neonatal lung injury.

Compensatory Vascular Remodeling in Stage 5 Chronic Kidney Disease in Response to Chronic Inflammation: An Interplay between Platelets and Lymphocytes

INTRODUCTION: Widespread chronic inflammation in patients with stage 5 chronic kidney disease on hemodialysis (CKD5-HD) has been demonstrated by elevated levels of circulating inflammatory biomarkers, including CD40 ligand (CD40L), angiopoietin-1 (Ang-1), and angiopoietin-2 (Ang-2). As CD40L and Ang-2 are indicators of damage due to inflammation, Ang-1 demonstrates protective vascular effects, such as inflammation reduction and vascular remodeling. In CKD5-HD patients, renal death increases levels of circulating inflammatory mediators, which may be damaging to endothelial cells throughout the vascular system. Despite this chronic inflammation, complex activation of the immune system may be involved in upregulated Ang-1-mediated compensatory mechanisms to maintain vascular integrity and counter the Ang-2 response.METHODS: Plasma levels of CD40L, Ang-2, Ang-1, and PAI-1 in CKD5-HD patients (n=97) and commercially acquired controls (n=17-50) were measured using sandwich ELISA technique. Complete blood counts (CBC) and electronic medical records (EMR) were reviewed for blood values. WBC count between 4-11 K/UL and platelet count between 150-450 K/UL were considered within normal range. GraphPad Prism software was used for Mann-Whitney t-tests, Kruskal-Wallis nonparametric ANOVAs and Spearman r statistical analyses.RESULTS: CD40L (0.32 ± 0.04 ng/mL), Ang-2 (10.46 ± 0.67 ng/mL), and Ang-1 (0.62 ± 0.08 ng/mL) levels were significantly increased in CKD5-HD patients compared to controls (p<0.0001), although there was no significant difference between PAI-1 levels (p=0.9764). WBC count (6.798 ± 0.244 K/UL) was normal in 87.63% of CKD5-HD patients, and platelet count (199.1 ± 7.51 K/UL) was normal in 73.20% of patients. Correlation between platelet and WBC counts revealed strong statistical significance (r=0.5331; p<0.0001). Correlation between CD40L and Ang-1 was also significant (r=0.209; p=0.040).SUMMARY AND CONCLUSION: Elevated levels of CD40L and Ang-2 are suggestive of chronic inflammation related to CKD5-HD. As inflammatory mediators, such as these, are continually elevated in this condition, Ang-1 may be upregulated as a compensatory mechanism to dampen the inflammatory response and promote vascular remodeling of damaged vessels. Strong correlation between CD40L and Ang-1 may be indirectly related to platelet activation. Besides activating lymphocytes, CD40L activates platelets, which, in turn, release Ang-1. This may be another tactic to limit inflammation. Within the endothelium, Ang-1 reduces recruitment of lymphocytes to damaged tissue by downregulating adhesion molecules and reducing vascular leak. Additionally, Ang-1 has been demonstrated to enhance HSC quiescence in bone marrow niches. These combined effects of Ang-1 activation of the Tie2 signaling pathway may counteract chronic inflammation-mediated stress signals to help maintain homeostatic WBC and platelet counts seen in most of these patients. The overall increase in CD40L, Ang-1, and Ang-2 in CKD5-HD suggests the homeostatic role of Ang-1 in maintaining the vasculature damaged by chronic inflammation, participation in maintaining normal levels of WBC and platelets, and demonstrates its potential to be utilized for therapeutic management of CKD5-HD patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Mucopolysaccharide polysulfate promotes microvascular stabilization and barrier integrity of dermal microvascular endothelial cells via activation of the angiopoietin-1/Tie2 pathway

BackgroundMucopolysaccharide polysulfate (MPS) is a heparinoid and MPS-containing formulations are widely used as moisturizers for dry skin and to treat peripheral vascular insufficiency. Although MPS has therapeutic effects in skin diseases with microvascular abnormalities, the effects of MPS on microvascular function remain incompletely understood. ObjectiveThe aim of this study was to evaluate the functional activities of MPS on human pericytes (HPC) and human dermal microvascular endothelial cells (HDMEC) in vitro, and on microvascular permeability of the skin. MethodsThe protein expression of angiopoietin (Ang)-1 in HPC, and platelet-derived growth factor-BB (PDGF-BB) and phosphorylated tyrosine-protein kinase receptor 2 (Tie2) in HDMEC were measured in the presence or absence of MPS. The vascular barrier was evaluated by the expressions of claudin-5 and vascular endothelial (VE)-cadherin, and transendothelial electrical resistance (TEER). ResultsIn HPC, MPS dose-dependently enhanced Ang-1 secretion, which activated Tie2 in HDMEC. In HDMEC, MPS significantly increased the production of PDGF-BB, which is important for the recruitment of HPC to the vascular endothelium, and significantly increased the phosphorylation of Tie2, which results in the activation of the Ang-1/Tie2 signaling . MPS significantly increased the expression of tight junction protein claudin-5 and TEER in the HDMEC. Moreover, the intradermal injection of MPS prevented vascular endothelial growth factor-induced increase in vascular permeability in mouse skin. ConclusionWe found that MPS promoted microvascular stabilization and barrier integrity in HDMEC via Ang-1/Tie2 activation. These results suggest that MPS might improve microvascular abnormalities in various diseases accompanied by disturbances in Ang-1/Tie2 signaling.

Vascular Endothelial Growth Factor Antagonists: Promising Players in the Treatment of Neovascular Age-Related Macular Degeneration.

Neovascular age-related macular degeneration (nAMD) treatment has been revolutionized by the introduction of vascular endothelial growth factor antagonists (anti-VEGF), but the need for frequent intravitreal injections poses a heavy burden to patients and physicians. Evolving anti-VEGF therapies include longer duration agents, approaches that target multiple pathways, topical anti-VEGF agents, sustained-release, and genetic therapies. Abicipar pegol, a designed ankyrin repeat protein (DARPin), demonstrated the ability to maintain stable visual acuity with 12-week dosing, but was not approved by the FDA due to higher than usual rates of intraocular inflammation. Conbercept, a recombinant anti-VEGF fusion protein, has been approved in China, and is in Phase 3 trials globally. KSI-301 is an anti-VEGF antibody biopolymer conjugate that allowed 66% of nAMD patients to maintain at least a 6-month treatment-free interval in Phase 1b studies. OPT-302, an inhibitor of VEGF-C/D, will be tested in phase 3 studies that compare anti-VEGF-A monotherapy against combination therapy with OPT-302. Faricimab is a bispecific anti-VEGF/Ang-2 antibody that upregulates the Tie-2 signaling pathway and promotes vascular stability; it is undergoing phase 3 trials with potential for 12- or 16-week dosing. PAN-90806 is a topical anti-VEGF agent that showed the ability to reduce injection frequency by 79% compared to ranibizumab monotherapy in a phase 1/2a trial. Sustained-release anti-VEGF therapies include the ranibizumab Port Delivery System (in phase 3 studies), GB-102 (Phase 2b), OTX-TKI (phase 1), and Durasert (preclinical). Suprachoroidal delivery of the tyrosine kinase inhibitor, axitinib, is in preclinical studies. Genetic therapies in phase 1 studies include RGX-314 and ADVM-022, which introduce a viral vector that modifies the retina’s cellular apparatus to create an anti-VEGF biofactory, potentially serving as a one-time treatment. Further investigation is warranted for drugs and delivery systems that hope to advance visual outcomes and reduce treatment burden of nAMD.

DPSCs treated by TGF-\u03b21 regulate angiogenic sprouting of three-dimensionally co-cultured HUVECs and DPSCs through VEGF-Ang-Tie2 signaling

BackgroundMaintaining the stability and maturation of blood vessels is of paramount importance for the vessels to carry out their physiological function. Smooth muscle cells (SMCs), pericytes, and mesenchymal stem cells (MSCs) are involved in the maturation process of the newly formed vessels. The aim of this study was to investigate whether transforming growth factor beta 1 (TGF-β1) treatment could enhance pericyte-like properties of dental pulp stem cells (DPSCs) and how TGF-β1-treated DPSCs for 7 days (T-DPSCs) stabilize the newly formed blood vessels.MethodsWe utilized TGF-β1 to treat DPSCs for 1, 3, 5, and 7 days. Western blotting and immunofluorescence were used to analyze the expression of SMC markers. Functional contraction assay was conducted to assess the contractility of T-DPSCs. The effects of T-DPSC-conditioned media (T-DPSC-CM) on human umbilical vein endothelial cell (HUVEC) proliferation and migration were examined by MTT, wound healing, and trans-well migration assay. Most importantly, in vitro 3D co-culture spheroidal sprouting assay was used to investigate the regulating role of vascular endothelial growth factor (VEGF)-angiopoietin (Ang)-Tie2 signaling on angiogenic sprouting in 3D co-cultured spheroids of HUVECs and T-DPSCs. Angiopoietin 2 (Ang2) and VEGF were used to treat the co-cultured spheroids to explore their roles in angiogenic sprouting. Inhibitors for Tie2 and VEGFR2 were used to block Ang1/Tie2 and VFGF/VEGFR2 signaling.ResultsWestern blotting and immunofluorescence showed that the expression of SMC-specific markers (α-SMA and SM22α) were significantly increased after treatment with TGF-β1. Contractility of T-DPSCs was greater compared with that of DPSCs. T-DPSC-CM inhibited HUVEC migration. In vitro sprouting assay demonstrated that T-DPSCs enclosed HUVECs, resembling pericyte-like cells. Compared to co-culture with DPSCs, a smaller number of HUVEC sprouting was observed when co-cultured with T-DPSCs. VEGF and Ang2 co-stimulation significantly enhanced sprouting in HUVEC and T-DPSC co-culture spheroids, whereas VEGF or Ang2 alone exerted insignificant effects on HUVEC sprouting. Blocking Tie2 signaling reversed the sprouting inhibition by T-DPSCs, while blocking VEGF receptor (VEGFR) signaling boosted the sprouting inhibition by T-DPSCs.ConclusionsThis study revealed that TGF-β1 can induce DPSC differentiation into functional pericyte-like cells. T-DPSCs maintain vessel stability through Ang1/Tie2 and VEGF/VEGFR2 signaling.

Synaptotagmin-Like Protein 2a Regulates Angiogenic Lumen Formation via Weibel-Palade Body Apical Secretion of Angiopoietin-2.

[Figure: see text].

Angiopoietin 2 stimulates trophoblast invasion via a mechanism associated with JNK signaling.

Extravillous trophoblast cell (EVT) invasion is tightly controlled, and its dysregulation can lead to altered spiral artery remodeling and contribute to a number of different pregnancy complications. Angiopoietin-2 (Ang-2) is expressed by trophoblast cells and various cells in the decidua, and trophoblast cells express its receptor, Tie2. Ang-2 has been shown to play roles in tumor progression and metastasis but it is not known if it also regulates EVT invasion. Here, we show that both the HTR-8/SVneo cell line and primary isolates of human EVT expressed various integrins and the Tie2 receptor, and Ang-2 stimulated their migration and/or invasion. Ang-2 increased expression of matrix metalloproteinase (MMP)2 and MMP9, altered the cytoskeleton of HTR-8/SVneo cells and also induced phosphorylation of Tie2, JNK and c-Jun. Inhibition of p-JNK (using SP600125) blocked the Ang-2 induced invasion of HTR-8/SVneo cells. In addition, inhibition of Tie2 (pexmetinib) and integrin signaling (RGDS and ATN-161) also blocked Ang-2-induced invasion. In conclusion, we demonstrate that Ang-2 can stimulate EVT invasion via a mechanism associated with activation of both the Tie2 receptor and integrins, which appear to work through different pathways; Tie2 through the JNK/c-JUN pathway and integrins through an as yet unidentified pathway(s). We therefore propose that any alterations in Ang-2 expression in the decidua would lead to an imbalance in pro- and anti-invasive factors, disrupting regulation of EVT invasion and spiral artery remodeling and thereby contribute to the etiology of several complications of pregnancy.

Caveolae-mediated Tie2 signaling contributes to CCM pathogenesis in a brain endothelial cell-specific Pdcd10-deficient mouse model

Cerebral cavernous malformations (CCMs) are vascular abnormalities that primarily occur in adulthood and cause cerebral hemorrhage, stroke, and seizures. CCMs are thought to be initiated by endothelial cell (EC) loss of any one of the three Ccm genes: CCM1 (KRIT1), CCM2 (OSM), or CCM3 (PDCD10). Here we report that mice with a brain EC-specific deletion of Pdcd10 (Pdcd10BECKO) survive up to 6-12 months and develop bona fide CCM lesions in all regions of brain, allowing us to visualize the vascular dynamics of CCM lesions using transcranial two-photon microscopy. This approach reveals that CCMs initiate from protrusion at the level of capillary and post-capillary venules with gradual dissociation of pericytes. Microvascular beds in lesions are hyper-permeable, and these disorganized structures present endomucin-positive ECs and α-smooth muscle actin-positive pericytes. Caveolae in the endothelium of Pdcd10BECKO lesions are drastically increased, enhancing Tie2 signaling in Ccm3-deficient ECs. Moreover, genetic deletion of caveolin-1 or pharmacological blockade of Tie2 signaling effectively normalizes microvascular structure and barrier function with attenuated EC-pericyte disassociation and CCM lesion formation in Pdcd10BECKO mice. Our study establishes a chronic CCM model and uncovers a mechanism by which CCM3 mutation-induced caveolae-Tie2 signaling contributes to CCM pathogenesis.

Adaptor Protein ShcD/SHC4 Interacts with Tie2 Receptor to Synergistically Promote Glioma Cell Invasion.

Gliomas are characterized by diffuse infiltration of tumor cells into surrounding brain tissue, and this highly invasive nature contributes to disease recurrence and poor patient outcomes. The molecular mechanisms underlying glioma cell invasion remain incompletely understood, limiting development of new targeted therapies. Here, we have identified phosphotyrosine adaptor protein ShcD as upregulated in malignant glioma and shown that it associates with receptor tyrosine kinase Tie2 to facilitate invasion. In human glioma cells, we find that expression of ShcD and Tie2 increases invasion, and this significant synergistic effect is disrupted with a ShcD mutant that cannot bind Tie2 or hyperphosphorylate the receptor. Expression of ShcD and/or Tie2 further increases invadopodia formation and matrix degradation in U87 glioma cells. In a coculture model, we show that U87-derived tumor spheroids expressing both ShcD and Tie2 display enhanced infiltration into cerebral organoids. Mechanistically, we identify changes in focal adhesion kinase phosphorylation in the presence of ShcD and/or Tie2 in U87 cells upon Tie2 activation. Finally, we identify a strong correlation between transcript levels of ShcD and Tie2 signaling components as well as N-cadherin in advanced gliomas and those with classical or mesenchymal subtypes, and we show that elevated expression of ShcD correlates with a significant reduction in patient survival in higher grade gliomas with mesenchymal signature. Altogether, our data highlight a novel Tie2-ShcD signaling axis in glioma cell invasion, which may be of clinical significance. IMPLICATIONS: ShcD cooperates with Tie2 to promote glioma cell invasion and its elevated expression correlates with poor patient outcome in advanced gliomas.

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.

Decreased lymphatic HIF-2α accentuates lymphatic remodeling in lymphedema.

Pathologic lymphatic remodeling in lymphedema evolves during periods of tissue inflammation and hypoxia through poorly defined processes. In human and mouse lymphedema, there is a significant increase of hypoxia inducible factor 1 α (HIF-1α), but a reduction of HIF-2α protein expression in lymphatic endothelial cells (LECs). We questioned whether dysregulated expression of these transcription factors contributes to disease pathogenesis and found that LEC-specific deletion of Hif2α exacerbated lymphedema pathology. Even without lymphatic vascular injury, the loss of LEC-specific Hif2α caused anatomic pathology and a functional decline in fetal and adult mice. These findings suggest that HIF-2α is an important mediator of lymphatic health. HIF-2α promoted protective phosphorylated TIE2 (p-TIE2) signaling in LECs, a process also replicated by upregulating TIE2 signaling through adenovirus-mediated angiopoietin-1 (Angpt1) gene therapy. Our study suggests that HIF-2α normally promotes healthy lymphatic homeostasis and raises the exciting possibility that restoring HIF-2α pathways in lymphedema could mitigate long-term pathology and disability.

Identification of specific Tie2 cleavage sites and therapeutic modulation in experimental sepsis.

Endothelial Tie2 signaling plays a pivotal role in vascular barrier maintenance at baseline and after injury. We previously demonstrated that a sharp drop in Tie2 expression observed across various murine models of critical illnesses is associated with increased vascular permeability and mortality. Matrix metalloprotease (MMP)-14-mediated Tie2 ectodomain shedding has recently been recognized as a possible mechanism for Tie2 downregulation in sepsis. Here, we identified the exact MMP14-mediated Tie2 ectodomain cleavage sites and could show that pharmacological MMP14 blockade in experimental murine sepsis exerts barrier protective and anti-inflammatory effects predominantly through the attenuation of Tie2 cleavage to improve survival both in a pre-treatment and rescue approach. Overall, we show that protecting Tie2 shedding might offer a new therapeutic opportunity for the treatment of septic vascular leakage.

Suppression of Ocular Vascular Inflammation through Peptide-Mediated Activation of Angiopoietin-Tie2 Signaling.

Persistent inflammation is a complication associated with many ocular diseases. Changes in ocular vessels can amplify disease responses and contribute to vision loss by influencing the delivery of leukocytes to the eye, vascular leakage, and perfusion. Here, we report the anti-inflammatory activity for AXT107, a non-RGD, 20-mer αvβ3 and α5β1 integrin-binding peptide that blocks vascular endothelial growth factor (VEGF)-signaling and activates tyrosine kinase with immunoglobulin and EGF-like domains 2 (Tie2) using the normally inhibitory ligand angiopoietin 2 (Ang2). Tumor necrosis factor α (TNFα), a central inflammation mediator, induces Ang2 release from endothelial cells to enhance its stimulation of inflammation and vascular leakage. AXT107 resolves TNFα-induced vascular inflammation in endothelial cells by converting the endogenously released Ang2 into an agonist of Tie2 signaling, thereby disrupting both the synergism between TNFα and Ang2 while also preventing inhibitor of nuclear factor-κB α (IκBα) degradation directly through Tie2 signaling. This recovery of IκBα prevents nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) nuclear localization, thereby blocking NF-κB-induced inflammatory responses, including the production of VCAM-1 and ICAM-1, leukostasis, and vascular leakage in cell and mouse models. AXT107 also decreased the levels of pro-inflammatory TNF receptor 1 (TNFR1) without affecting levels of the more protective TNFR2. These data suggest that AXT107 may provide multiple benefits in the treatment of retinal/choroidal and other vascular diseases by suppressing inflammation and promoting vascular stabilization.

Tumor Cell-Derived Angiopoietin-2 Promotes Metastasis in Melanoma.

The angiopoietin (Angpt)-TIE signaling pathway controls vascular maturation and maintains the quiescent phenotype of resting vasculature. The contextual agonistic and antagonistic Tie2 ligand ANGPT2 is believed to be exclusively produced by endothelial cells, disrupting constitutive ANGPT1-TIE2 signaling to destabilize the microvasculature during pathologic disorders like inflammation and cancer. However, scattered reports have also portrayed tumor cells as a source of ANGPT2. Employing ISH-based detection of ANGPT2, we found strong tumor cell expression of ANGPT2 in a subset of patients with melanoma. Comparative analysis of biopsies revealed a higher fraction of ANGPT2-expressing tumor cells in metastatic versus primary sites. Tumor cell-expressed Angpt2 was dispensable for primary tumor growth, yet in-depth analysis of primary tumors revealed enhanced intratumoral necrosis upon silencing of tumor cell Angpt2 expression in the absence of significant immune and vascular alterations. Global transcriptional profiling of Angpt2-deficient tumor cells identified perturbations in redox homeostasis and an increased response to cellular oxidative stress. Ultrastructural analyses illustrated a significant increase of dysfunctional mitochondria in Angpt2-silenced tumor cells, thereby resulting in enhanced reactive oxygen species (ROS) production and downstream MAPK stress signaling. Functionally, enhanced ROS in Angpt2-silenced tumor cells reduced colonization potential in vitro and in vivo. Taken together, these findings uncover the hitherto unappreciated role of tumor cell-expressed ANGPT2 as an autocrine-positive regulator of metastatic colonization and validate ANGPT2 as a therapeutic target for a well-defined subset of patients with melanoma. SIGNIFICANCE: This study reveals that tumor cells can be a source of ANGPT2 in the tumor microenvironment and that tumor cell-derived ANGPT2 augments metastatic colonization by protecting tumor cells from oxidative stress.

Essential Contribution of Macrophage Tie2 Signalling in a Murine Model of Laser-Induced Choroidal Neovascularization

Wet age-related macular degeneration (AMD), which can cause progressive blindness, is characterised by choroid neovascularization (CNV) in the macular area. Although close attention has been paid to AMD, and anti-vascular endothelial growth factor (VEGF) drugs are available, its complex pathogenesis is still elusive. Tie2-expressing macrophages (TEMs) have been found to promote angiogenesis in remodel tissues and tumours. This study aimed to elucidate the role of macrophage Tie2 signalling in laser-induced CNV (LCNV). We observed that TEMs were responsible for the severity of CNV. Mechanistically, TEM deletion resulted in impaired LCNV due to the suppression of inflammatory angiogenesis and the promotion of apoptosis. We also observed that TEMs prevented apoptosis of b.End3 cells, but promoted their migration, proliferation and tube formation via VEGF, extracellular signal-regulated kinase (ERK) and v-akt murine thymoma viral oncogene (AKT)-dependent signalling pathways. The flow cytometry results comparing dry AMD patients and healthy controls with wet AMD patients showed that the percentage of Tie2+CD14+ cells was higher in the wet AMD patients’ peripheral blood. This study demonstrates that Tie2 expression by macrophages intensifies CNV in LCNV murine models, thereby proposing an additional intervention option to inhibit CNV.

Aliskiren Improved the Endothelial Repair Capacity of Endothelial Progenitor Cells from Patients with Hypertension via the Tie2/PI3k/Akt/eNOS Signalling Pathway.

Background Studies show that aliskiren exerts favourable effects not only on endothelial progenitor cells (EPCs) but also on endothelial function. However, the mechanism of the favourable effect of aliskiren on EPCs from patients with hypertension is unclear and remains to be further studied. Methods The object of this study was to investigate and assess the in vitro function of EPCs pretreated with aliskiren. After treated with aliskiren, the human EPCs were transplanted into a nude mouse model of carotid artery injury, and the in vivo reendothelialization of injured artery was estimated by staining denuded areas with Evans blue dye via tail vein injection. Results We found that aliskiren increased the in vitro migration, proliferation, and adhesion of EPCs from patients with hypertension in a dose-dependent manner and improved the reendothelialization capability of these EPCs. Furthermore, aliskiren increased the phosphorylation of Tie2, Akt, and eNOS. After the blockade of the Tie2 signalling pathway, the favourable effects of aliskiren on the in vitro function and in vivo reendothelialization capability of EPCs were suppressed. Conclusions This study demonstrates that aliskiren can improve the in vitro function and in vivo reendothelialization capability of EPCs from patients with hypertension via the activation of the Tie2/PI3k/Akt/eNOS signalling pathway. These findings further indicate that aliskiren is an effective pharmacological treatment for cell-based repair in hypertension-related vascular injury.

Targeting the ER\u03b2/Angiopoietin-2/Tie-2 signaling-mediated angiogenesis with the FDA-approved anti-estrogen Faslodex to increase the Sunitinib sensitivity in RCC

Sunitinib has been used as the main therapy to treat the metastatic clear cell renal cell carcinoma (ccRCC) as it could function via suppressing the tumor growth and angiogenesis. Yet most ccRCC tumors may still regrow due to the development of sunitinib-resistance, and detailed mechanisms remain to be further investigated. The angiopoietin family includes angiopoietin-1 and angiopoietin-2 (ANGPT-1 and -2). It was reported that estradiol regulates expression of ANGPT-1, but not ANGPT-2, through estrogen receptor α (ERα) in an experimental stroke model. To date, there is no finding to link the E2/ER signal on regulating ANGPT-2. Our study is the first to explore (i) how estrogen receptor β (ERβ) can up-regulate ANGPT-2 in RCC cells, and (ii) how ERβ-increased ANGPT-2 can promote the HUVEC tube formation and reduce sunitinib sensitivity. Mechanistic studies revealed that ERβ could function via transcriptional regulation of the cytokine ANGPT-2 in the ccRCC cells. We found the up-regulated ANGPT-2 of RCC cells could then increase the Tie-2 phosphorylation to promote the angiogenesis and increase sunitinib treatment resistance of endothelial cells. In addition to the endothelial cell tube formation and aortic ring assay, preclinical studies with a mouse RCC model also confirmed the finding. Targeting this newly identified ERβ/ANGPT-2/Tie-2 signaling pathway with the FDA-approved anti-estrogen, Faslodex, may help in the development of a novel combined therapy with sunitinib to better suppress the ccRCC progression.

Role of Angiopoietins in Development of Cancer and Neoplasia Associated with Viral Infection.

Angiopoietin/tyrosine protein kinase receptor Tie-2 signaling in endothelial cells plays an essential role in angiogenesis and wound healing. Angiopoietin-1 (Ang-1) is crucial for blood vessel maturation while angiopoietin-2 (Ang-2), in collaboration with vascular endothelial growth factor (VEGF), initiates angiogenesis by destabilizing existing blood vessels. In healthy people, the Ang-1 level is sustained while Ang-2 expression is restricted. In cancer patients, Ang-2 level is elevated, which correlates with poor prognosis. Ang-2 not only drives tumor angiogenesis but also attracts infiltration of myeloid cells. The latter rapidly differentiate into tumor stromal cells that foster tumor angiogenesis and progression, and weaken the host’s anti-tumor immunity. Moreover, through integrin signaling, Ang-2 induces expression of matrix metallopeptidases (MMPs) to promote tumor cell invasion and metastasis. Many oncogenic viruses induce expression of Ang-2 to promote development of neoplasia associated with viral infection. Multiple Ang-2 inhibitors exhibit remarkable anti-tumor activities, further highlighting the importance of Ang-2 in cancer development.

Essential contribution of macrophage Tie2 signal mediated autophagy in laser-induced choroidal neovascularization

Autophagy plays critical roles in various ocular diseases, including age-related macular degeneration (AMD). Tie2-expressing macrophages (TEMs) play crucial roles in angiogenesis. To investigate the role of TEMs and autophagy in the development of AMD, we employed macrophage-specific Tie2 knockout mice and used a laser-induced choroidal neovascularization (CNV). The results showed that TEMs can promote CNV formation by up-regulating the level of autophagy. These results were further verified by in vitro cell experiments that peritoneal macrophages from Tie2 knockout mice can inhibit the expression of autophagy-related factors and inhibit the expression of angiogenic factor of VEGF by activating AMPK signaling pathway. Our results suggest that TEMs and macrophage Tie2 signal mediated-autophagy play critical role in experimental CNV, and they may be novel preventive targets for AMD treatment.

Angiopoietin-1/Tie-2 signal after focal traumatic brain injury is potentiated by BQ788, an ETB receptor antagonist, in the mouse cerebrum: Involvement in recovery of blood-brain barrier function.

Angiopoietin-1, an angiogenic factor, stabilizes brain microvessels through Tie-2 receptor tyrosine kinase. In traumatic brain injury, blood-brain barrier (BBB) disruption is an aggravating factor that induces brain edema and neuroinflammation. We previously showed that BQ788, an endothelin ETB receptor antagonist, promoted recovery of BBB function after lateral fluid percussion injury (FPI) in mice. To clarify the mechanisms underlying BBB recovery mediated by BQ788, we examined the involvements of the angiopoietin-1/Tie-2 signal. When angiopoietin-1 production and Tie-2 phosphorylation were assayed by quantitative reverse transcription polymerase chain reaction and western blotting, increased angiopoietin-1 production and Tie-2 phosphorylation were observed in 7-10days after FPI in the mouse cerebrum, whereas no significant effects were obtained at 5days. When BQ788 (15nmol/day, i.c.v.) were administered in 2-5days after FPI, increased angiopoietin-1 production and Tie-2 phosphorylation were observed. Immunohistochemical observations showed that brain microvessels and astrocytes contained angiopoietin-1 after FPI, and brain microvessels also contained phosphorylated Tie-2. Treatment with endothelin-1 (100nM) decreased angiopoietin-1 production in cultured astrocytes and the effect was inhibited by BQ788 (1μM). Five days after FPI, increased extravasation of Evans blue dye accompanied by reduction in claudin-5, occludin, and zonula occludens-1 proteins were observed in mouse cerebrum while these effects of FPI were reduced by BQ788 and exogenous angiopoietin-1 (1μg/day, i.c.v.). The effects of BQ788 were inhibited by co-administration of a Tie-2 kinase inhibitor (40nmol/day, i.c.v.). These results suggest that BQ788 administration after traumatic brain injury promotes recovery of BBB function through activation of the angiopoietin-1/Tie-2 signal.