Tie2 Phosphorylation Research Articles

Angiopoietin/Tie2 Signaling Transforms Capillaries into Venules Primed for Leukocyte Trafficking in Airway Inflammation

Vascular endothelial growth factor (VEGF) is a key angiogenic factor in tumors, but less is known about what drives vascular remodeling in inflammation, where plasma leakage and leukocyte influx are prominent features. In chronic airway inflammation in mice infected by the bacterium Mycoplasma pulmonis (M. pulmonis), the segment of the microvasculature that supports leukocyte adhesion and migration expands through remodeling of capillaries into vessels with features of venules. Here, we report that the angiopoietin/Tie2 pathway is an essential driving force for capillary remodeling into venules in M. pulmonis-infected mouse airways. Similar to M. pulmonis infection, systemic overexpression of angiopoietin-1 (Ang1) resulted in remodeling of airway capillaries into venular-like vessels that expressed venous markers like P-selectin, ICAM-1, and EphB4 and were sites of leukocyte adhesion during lipopolysaccharide-induced acute inflammation. Ang1 and Ang2 protein increased in M. pulmonis-infected mouse airways but came from different cellular sources: Ang1 was expressed in infiltrating neutrophils and Ang2 in endothelial cells. Indeed, systemic administration of soluble Tie2 inhibited capillary remodeling, induction of venous markers, and leukocyte influx in M. pulmonis-infected mouse airways. Together, these findings suggest that blockade of the Ang/Tie2 pathway may represent a therapeutic approach in airway inflammation.

The role of the Angiopoietins in vascular morphogenesis

The Angiopoietin/Tie system acts as a vascular specific ligand/receptor system to control endothelial cell survival and vascular maturation. The Angiopoietin family includes four ligands (Angiopoietin-1, Angiopoietin-2 and Angiopoietin-3/4) and two corresponding tyrosine kinase receptors (Tie1 and Tie2). Ang-1 and Ang-2 are specific ligands of Tie2 binding the receptor with similar affinity. Tie2 activation promotes vessel assembly and maturation by mediating survival signals for endothelial cells and regulating the recruitment of mural cells. Ang-1 acts in a paracrine agonistic manner inducing Tie2 phosphorylation and subsequent vessel stabilization. In contrast, Ang-2 is produced by endothelial cells and acts as an autocrine antagonist of Ang-1-mediated Tie2 activation. Ang-2 thereby primes the vascular endothelium to exogenous cytokines and induces vascular destabilization at higher concentrations. Ang-2 is strongly expressed in the vasculature of many tumors and it has been suggested that Ang-2 may act synergistically with other cytokines such as vascular endothelial growth factor to promote tumor-associated angiogenesis and tumor progression. The better mechanistic understanding of the Ang/Tie system is gradually paving the way toward the rationale exploitation of this vascular signaling system as a therapeutic target for neoplastic and non-neoplastic diseases.

Vascular endothelial growth factor activates the Tie family of receptor tyrosine kinases

The ability of cells to respond appropriately to changes in their environment requires integration and cross-talk between relevant signalling pathways. The vascular endothelial growth factor (VEGF) and angiopoietin families of ligands are key regulators of blood vessel formation. VEGF binds to receptor tyrosine kinases of the VEGF-receptor family to activate signalling pathways leading to endothelial migration, proliferation and survival whereas the angiopoietins interact with the Tie receptor tyrosine kinases to control vessel stability, survival and maturation. Here we show that VEGF can also activate the angiopoietin receptor Tie2. Activation of human endothelial cells with VEGF caused a four-fold stimulation of tyrosine phosphorylation of Tie2. This stimulation was not due to VEGF-induction of Tie2 ligands as soluble ligand binding domain of Tie2 failed to inhibit VEGF activation of the receptor. Immunoprecipitation analysis demonstrated no physical interaction between VEGF receptors and Tie2. However Tie2 does interact with the related receptor tyrosine kinase Tie1 and this receptor was found to be essential for VEGF activation of Tie2. VEGF stimulated proteolytic cleavage of Tie1 generating a truncated Tie1 intracellular domain. Similarly, phorbol ester also both stimulated Tie1 truncation and activated Tie2 phosphorylation. Inhibition of Tie1 cleavage with the metalloprotease inhibitor TAPI-2 suppressed VEGF- and phorbol ester-induced phosphorylation of Tie2. Truncated Tie1 formed in response to VEGF was also found to be tyrosine phosphorylated and this was independent of Tie2, though Tie2 could enhance Tie1 intracellular domain phosphorylation. Together these data demonstrate that VEGF activates Tie2 via a mechanism involving proteolytic cleavage of the associated tyrosine kinase Tie1 leading to trans-phosphorylation of Tie2. This novel mechanism of receptor tyrosine kinase activation is likely to be important in integrating signalling between two of the key receptor groups regulating angiogenesis.

Angiopoietin 2 Is a Partial Agonist/Antagonist of Tie2 Signaling in the Endothelium

Angiopoietin 2 (Ang2) was originally shown to be a competitive antagonist for Ang1 of the receptor tyrosine kinase Tie2 in endothelial cells (ECs). Since then, reports have conflicted on whether Ang2 is an agonist or antagonist of Tie2. Here we show that Ang2 functions as an agonist when Ang1 is absent but as a dose-dependent antagonist when Ang1 is present. Exogenous Ang2 activates Tie2 and the promigratory, prosurvival PI3K/Akt pathway in ECs but with less potency and lower affinity than exogenous Ang1. ECs produce Ang2 but not Ang1. This endogenous Ang2 maintains Tie2, phosphatidylinositol 3-kinase, and Akt activities, and it promotes EC survival, migration, and tube formation. However, when ECs are stimulated with Ang1 and Ang2, Ang2 dose-dependently inhibits Ang1-induced Tie2 phosphorylation, Akt activation, and EC survival. We conclude that Ang2 is both an agonist and an antagonist of Tie2. Although Ang2 is a weaker agonist than Ang1, endogenous Ang2 maintains a level of Tie2 activation that is critical to a spectrum of EC functions. These findings may reconcile disparate reports of Ang2's effect on Tie2, impact our understanding of endogenous receptor tyrosine kinase signal transduction mechanisms, and affect how Ang2 and Tie2 are targeted under conditions such as sepsis and cancer.

Mechanism of inhibition of tumor angiogenesis by β‐hydroxyisovalerylshikonin

Shikonin and beta-hydroxyisovalerylshikonin (beta-HIVS) from Lithospermum erythrorhizon inhibit angiogenesis via inhibition of vascular endothelial growth factor receptors (VEGFR) in an adenosine triphosphate-non-competitive manner, although the underlying molecular mechanism has not been fully understood. In the present study, we found that beta-HIVS inhibited angiogenesis within chicken chorioallantoic membrane approximately threefold more efficiently than shikonin. beta-HIVS also significantly inhibited angiogenesis in two other assays, induced either by Lewis lung carcinoma cells implanted in mouse dorsal skin or by VEGF in s.c. implanted Matrigel plugs and metastasis of Lewis lung carcinoma cells to lung. Therefore, using beta-HIVS as a bioprobe, we investigated the molecular mechanism of shikonin's anti-angiogenic actions. beta-HIVS inhibited the phosphorylation and expression of VEGFR2 and Tie2 without affecting VEGFR1 and fibroblast growth factor receptor 1 levels. beta-HIVS suppressed the phosphorylation but not the expression of extracellular signal-regulated kinase, and an Sp1-dependent transactivation of the VEGFR2 and Tie2 promoters, thereby suppressing the proliferation of vascular endothelial and progenitor cells. This was mimicked by an Sp1 inhibitor mithramycin A and partially rescued by Sp1 overexpression. These results implicate potential use of shikonin and beta-HIVS as leading compounds for clinical application in the future by virtue of their unique properties including: (i) inhibition of VEGFR2 and Tie2 phosphorylation in an adenosine triphosphate-non-competitive manner; (ii) simultaneous inhibition of the phosphorylation and expression of VEGFR2 and Tie2; and (iii) bifunctional inhibition of the growth in endothelial cells and vascular remodeling.

Tyrosine phosphatase beta regulates angiopoietin-Tie2 signaling in human endothelial cells

The endothelial cell (EC)-selective receptor tyrosine kinase, Tie2, and its ligands angiopoietin Ang-1 and Ang-2, are essential for blood vessel maintenance and repair. Ang-1 is an agonist of Tie2 receptor activation, whereas Ang-2 is a context-dependent antagonist/agonist. Therefore, we investigated the role of the EC-selective phosphatase, human protein tyrosine phosphatase beta (HPTPbeta), in regulating Tie2 activity. siRNA silencing of HPTPbeta enhanced Ang-1 and Ang-2-induced Tie2 phosphorylation at 10 min (2.5-fold, P < 0.001; and 1.8-fold, P < 0.05, respectively). The cell survival response to Ang-1, but not Ang-2, was enhanced by HPTPbeta silencing as measured by flow cytometry (0.85-fold to 0.66-fold, P < 0.05) and ELISA (0.88-fold to 0.53-fold, P < 0.01). Hypoxia, which upregulated HPTPbeta expression in endothelial cells, impaired Ang-1-induced Tie2 phosphorylation. These results reveal a novel role for HPTPbeta in modulating Ang-1-Tie2 signaling and endothelial cell survival.

A nuclease-resistant RNA aptamer specifically inhibits angiopoietin-1-mediated Tie2 activation and function

Tie2 is a receptor tyrosine kinase that is expressed predominantly in the endothelium and plays key roles in both physiological and pathological angiogenesis. The ligands for Tie2, the angiopoietins (Ang), perform opposing functions in vascular maintenance and angiogenesis; Ang1 regulates vascular quiescence, while Ang2 is thought to promote vascular destabilization and facilitate angiogenesis. However, the mechanisms responsible for these differences are not understood. To begin to elucidate the molecular differences between the angiopoietins, we previously developed a specific RNA aptamer inhibitor of Ang2. Here, we used the same iterative in vitro selection process, termed SELEX (Systematic Evolution of Ligands by EXponential enrichment), to screen a library of 2'-fluoro-modified ribonucleotides for Ang1-binding aptamers. After nine rounds of selection, we identified a single clone, ANG9-4, that bound with high affinity to human Ang1 (K ( d ) 2.8 nM) but not Ang2 (K ( d ) > 1 microM), demonstrating specificity for Ang1. ANG9-4 blocked Ang1-mediated Tie2 phosphorylation and downstream Akt activation. Moreover, ANG9-4 inhibited Ang1-induced endothelial cell survival. Together, these findings demonstrate the feasibility of developing an Ang1-inhibitory aptamer. ANG9-4 and its derivatives may provide useful tools for elucidating the biology of Ang1 and for treating certain angiogenic diseases.

Gene expression profiling of breast cancer

Whole-body radiation therapy can cause severe injury to the hematopoietic system, and therefore it is necessary to identify a novel strategy for overcoming this injury.Mice were irradiated with 4.5 Gy after heat shock protein 25 (HSP25) gene transfer using an adenoviral vector. Then, peripheral blood cell counts, histopathological analysis, and Western blotting on bone marrow (BM) cells were performed. The interaction of HSP25 with Tie2 was investigated with mouse OP9 and human BM-derived mesenchymal stem cells to determine the mechanism of HSP25 in the hematopoietic system.HSP25 transfer increased BM regeneration and reduced apoptosis following whole-body exposure to ionizing radiation (IR). The decrease in Tie2 protein expression that followed irradiation of the BM was blocked by HSP25 transfer, and Tie2-positive cells were more abundant among the BM cells of HSP25-transferred mice, even after IR exposure. Following systemic RNA interference of Tie2 before IR, HSP25-mediated radioprotective effects were partially blocked in both mice and cell line systems. Stability of Tie2 was increased by HSP25, a response mediated by the interaction of HSP25 with Tie2. IR-induced tyrosine phosphorylation of Tie2 was augmented by HSP25 overexpression; downstream events in the Tie2 signaling pathway, including phosphorylation of AKT and EKR1/2, were also activated.HSP25 protects against radiation-induced BM damage by interacting with and stabilizing Tie2. This may be a novel strategy for HSP25-mediated radioprotection in BM.

Angiopoietin-1 Protects against Airway Inflammation and Hyperreactivity in Asthma

The angiopoietins (Ang) comprise a family of growth factors mainly known for their role in blood vessel formation and remodeling. The best-studied member, Ang-1, exhibits antiapoptotic and antiinflammatory effects. Although the involvement of Ang-1 in angiogenesis is well recognized, little information exists about its role in respiratory physiology and disease. On the basis of its ability to inhibit vascular permeability, adhesion molecule expression, and cytokine production, we hypothesized that Ang-1 administration might exert a protective role in asthma. To determine changes in the expression of Ang and to assess the ability of Ang-1 to prevent the histologic, biochemical, and functional changes observed in an animal model of asthma. To test our hypothesis, a model of allergic airway disease that develops after ovalbumin (OVA) sensitization and challenge was used. Ang-1 expression was reduced at the mRNA and protein levels in lung tissue of mice sensitized and challenged with OVA, leading to reduced Tie2 phosphorylation. Intranasal Ang-1 treatment prevented the OVA-induced eosinophilic lung infiltration, attenuated the increase in IL-5 and IL-13, and reduced eotaxin and vascular cell adhesion molecule 1 expression. These antiinflammatory actions of Ang-1 coincided with higher levels of IkappaB and decreased nuclear factor-kappaB binding activity. More importantly, Ang-1 reversed the OVA-induced increase in tissue resistance and elastance, improving lung function. We conclude that Ang-1 levels are decreased in asthma and that administration of Ang-1 might be of therapeutic value because it prevents the increased responsiveness of the airways to constrictors and ameliorates inflammation.

Angiopoietin‐1 modifies multiple microvessel permeability coefficients in non‐inflamed vessels

Angiopoietin‐1 (Ang1) is widely expressed by pericytes and induces constitutive phosphorylation of the endothelial cell receptor Tie2. Ang1 reduces neutrophil adhesion and solute flux following inflammatory stimulation. We examined the hitherto untested hypothesis that Ang1 modifies permeability coefficients in non‐inflamed microvessels. Continuous microvessel hydraulic conductivity (LP: x107 cm.s−1.cmH2O−1) and reflection coefficient (σ) were measured in frog mesenteric microvessels with the Landis‐Michel assay. Ang1 (200 ng.ml−1) caused a significant rise in σ (vehicle: 0.78±0.03; Ang1: 0.91±0.03; p&lt;0.05, paired t‐test (n=10)) and a significant reduction in LP (vehicle: 2.0±0.9; Ang1: 1.2±0.9; p&lt;0.05, Wilcoxon (n=11)). Fenestrated microvessel LP‐area product (LPA: nl.min−1.mmHg−1), measured in isolated rat glomeruli with an oncometric assay, also reduced significantly in response to Ang1 (vehicle: 1.0±0.1 (n=31); Ang1: 0.8±0.1(n=30); p&lt;0.05, unpaired t‐test). Ang1 may induce a plethora of changes in different microvessels that alter resistance to water and albumin movement, or may modify a determinant of microvascular permeability coefficients that is common to both continuous and fenestrated microvessels.

Anti‐angiogenic properties of a sulindac analogue

Angiopoietins (Ang) are crucial for new blood vessel formation and exert their effects by acting on the Tie2 receptor. We have recently described a sulindac analogue 2-((1E,Z)-1-benzylidene-5-bromo-2-methyl-1H-inden-3-yl)acetic acid; termed C-18 from now onwards) that inhibits Tie2 receptor activity in kinase assays in vitro. Here, we have assessed the ability of C-18 to inhibit angiogenesis-related properties of endothelial cells and tested its selectivity for the Tie2 receptor. For in vitro experiments human umbilical vein endothelial cells (HUVEC) were used. Proliferation was measured using the MTT assay; migration assays were performed in a modified Boyden chamber and tube-like structure formation was determined on matrigel. The effects of C-18 in vivo were evaluated in the chicken chorioallantoic membrane (CAM). Pre-treatment of HUVEC with C-18 blocked Ang-1-stimulated migration, but also abolished vascular endothelial cell growth factor (VEGF)- and fibroblast growth factor 2-induced responses. Incubation with C-18 inhibited serum-induced proliferation in a concentration-dependent manner; C-18 was, however, without effect on Ang-1-induced survival. In addition, we observed that C-18 did not inhibit ligand-induced receptor phosphorylation of Tie2 or VEGFR2. On the other hand, C-18 blocked activation of members of the mitogen-activated protein kinase family and of the Ser/Thr kinase Akt induced by both VEGF and Ang-1. Furthermore, incubation of CAMs with C-18 led to a dose-dependent inhibition of vascular length. C-18 did not act as a Tie2 inhibitor, as originally thought, but rather inhibited growth factor-stimulated signalling pathways that regulate endothelial cell migration and potently reduces neovascularization in vivo.

BMPs Promote Proliferation and Migration of Endothelial Cells via Stimulation of VEGF-A/VEGFR2 and Angiopoietin-1/Tie2 Signalling

The differentiation, growth, and survival of endothelial cells (ECs) are regulated by multiple signalling pathways, such as vascular endothelial growth factors (VEGFs) and angiopoietins through their receptor tyrosine kinases, VEGF receptor (VEGFR) 2 and Tie2, respectively. Bone morphogenetic proteins (BMPs), members of the transforming growth factor (TGF)-beta family, have been implicated in the development and maintenance of vascular systems. However, their effects on EC proliferation remain to be elucidated. In the present study, we show that BMPs induce the proliferation and migration of mouse embryonic stem cell (ESC)-derived endothelial cells (MESECs) and human microvascular endothelial cells (HMECs). Addition of BMP-4 to culture induced significant proliferation and migration of both types of ECs. BMP-4 also increased the expression and phosphorylation of VEGFR2 and Tie2. These findings suggest that BMP signalling activates endothelium via activation of VEGF/VEGFR2 and Angiopoietin/Tie2 signalling.

Angiopoietin-mediated endothelial P-selectin translocation: cell signaling mechanisms

Recently identified, angiopoietin-1 (Ang1) and -2 (Ang2) bind to the tyrosine kinase receptor Tie2 and contribute to orchestrate blood vessel formation during angiogenesis. Ang1 mediates vessel maturation and integrity by favoring the recruitment of pericytes and smooth muscle cells. Ang2, initially identified as a Tie2 antagonist, may under certain circumstances, induce Tie2 phosphorylation and biological activities. As inflammation exists in a mutually dependent association with angiogenesis, we sought to determine if Ang1 and/or Ang2 could modulate proinflammatory activities, namely P-selectin translocation, in bovine aortic endothelial cells (EC) and dissect the mechanisms implicated. P-selectin, an adhesion molecule found in the Weibel-Palade bodies of EC, is translocated rapidly to the cell surface upon EC activation during inflammatory processes. Herein, we report that Ang1 and Ang2 (1 nM) are capable of mediating a rapid Tie2 phosphorylation as well as a rapid and transient endothelial P-selectin translocation maximal within 7.5 min (125% and 100% increase, respectively, over control values). In addition, we demonstrate for the first time that angiopoietin-mediated endothelial P-selectin translocation is calcium-dependent and regulated through phospholipase C-gamma activation.

Inhibition of In Vivo Tumor Angiogenesis and Growth Via Systemic Delivery of an Angiopoietin 2-Specific RNA Aptamer

Cellular events mediated by the Tie2 receptor are important to tumor neovascularization. Despite the complex interplay of the best-characterized Tie2 ligands, angiopoietins 1 and 2, Ang2 is purportedly "proangiogenic" in the presence of vascular endothelial growth factor. We examined whether in vivo administration of an RNA aptamer that specifically blocks Ang 2 would inhibit tumor angiogenesis and growth. Ang2-mediated Tie2 receptor phosphorylation was assessed in vitro in the absence and presence of aptamer coupled to polyethylene glycol. IN VIVO ANGIOGENESIS ASSAY: CT26 murine colon carcinoma cells expressing green fluorescent protein were delivered into mouse dorsal skinfold window chambers. Animals received daily intraperitoneal injections of phosphate-buffered saline, low-dose (Ang2 aptamer-LD; 1 mg/kg/d), or high-dose aptamer (Ang2 aptamer-HD; 10 mg/kg/d). Vascular length density was measured under fluorescence microscopy. PRIMARY TUMOR GROWTH: CT26 cells expressing luciferase were injected into flanks of BALB/c mice to allow tumor growth monitoring by bioluminescence imaging. Animals received continuous phosphate-buffered saline or aptamer (1 mg/kg/d) via ALZET pumps. Tumors were assessed for CD31/PECAM-1 immunostaining and Hoechst dye uptake. Pegylated aptamer inhibited Tie2 phosphorylation. Systemic aptamer administration reduced vascular length density (P < or = 0.03) and decreased bioluminescence emission (P < 0.04), corresponding to 50% decrease in tumor volume (P = 0.04). Control tumors displayed abundant vascular marker staining, in contrast to tumors from aptamer-treated animals. in vivo administration of a clinically relevant, pegylated RNA aptamer specifically designed against Ang2 inhibited tumor angiogenesis and growth. These findings support targeted Ang2 inhibition as a relevant anti-angiogenic, anti-neoplastic strategy.

Abstract 442: Flow-Dependent Regulation of Angiopoietin-2

Background: Endothelial cells are constantly exposed to high or low shear stress in arteries and veins by the flowing blood. Angiopoietin-2 (Ang-2) is acting as a critical regulator of vessel maturation and endothelial cell quiescence. In this study, we determined the effect of low and high laminar shear stress on the expression and release of angiopoietin (Ang-)2 in human endothelial cells, studied the role of nitric oxide and protein kinases in this context, determined the impact of VEGF on Ang-2, studied the expression, activity and translocation of the forkhead box O transcription factor FOXO1 by low and high shear stress, and analyzed the vessel- and flow-dependent expression of Ang-2 in vivo . Methods and Results: Primary cultures of human umbilical vein endothelial cells (HUVEC) were subjected to laminar shear stress at different physiological levels of laminar shear stress of 1, 5, 10, 15, and 30 dyne/cm 2 in a cone-and-plate viscometer. Ang-2 mRNA, protein expression and release was upregulated by 24 h of low (1 dyne/cm 2 ), but downregulated by high flow (30 dyne/cm 2 ) in human endothelial cells. Increased endothelial NO synthase expression and NO formation was not affecting regulation of Ang-2 by low or high flow. Tie2 protein expression, but not Tie2 phosphorylation was induced by high flow. Furthermore, low and high flow increased VEGF-A expression. Inhibition of VEGFR-2 prevented upregulation of Ang-2 by low flow, but not downregulation of Ang-2 by high flow. Upregulation of Ang-2 by VEGF was reduced by application of high flow. Forkhead box O (FOXO) transcription factor FOXO1 has been shown to regulate Ang-2 expression in endothelial cells. FOXO1 binding activity was reduced by high flow. Nuclear localization of transcription factor FOXO1 was not changed by low flow, but reduced by high flow. In vivo , Ang-2 was higher expressed in veins compared to arteries. Arterial ligation augmented Ang-2 expression in distal arterial low flow areas. Conclusion: Our results support a VEGF-dependent induction of Ang-2 in low flow areas, and FOXO1-dependent downregulation of Ang-2 in high flow areas. These data suggest a new mechanism of flow-dependent regulation of vessel stability and differentiation.

VEGF Induces Tie2 Shedding via a Phosphoinositide 3-Kinase/Akt–Dependent Pathway to Modulate Tie2 Signaling

Tie2 and its ligands, the angiopoietins (Ang), are required for embryonic and postnatal angiogenesis. Previous studies have demonstrated that Tie2 is proteolytically cleaved, resulting in the production of a 75-kDa soluble receptor fragment (sTie2). We investigated mechanisms responsible for Tie2 shedding and its effects on Tie2 signaling and endothelial cellular responses. sTie2 bound both Ang1 and Ang2 and inhibited angiopoietin-mediated Tie2 phosphorylation and antiapoptosis. In human umbilical vein endothelial cells, Tie2 shedding was both constitutive and induced by treatment with PMA or vascular endothelial growth factor (VEGF). Constitutive and VEGF-inducible Tie2 shedding were mediated by PI3K/Akt and p38 MAPK. Tie2 shedding was blocked by pharmacological inhibitors of either PI3K or Akt as well as by overexpression of the lipid phosphatase PTEN. In contrast, sTie2 shedding was enhanced by overexpression of either dominant negative PTEN, which increased Akt phosphorylation, or constitutively active, myristoylated Akt. These findings demonstrate that VEGF regulates angiopoietin-Tie2 signaling by inducing proteolytic cleavage and shedding of Tie2 via a novel PI3K/Akt-dependent pathway. These results suggest a previously unrecognized mechanism by which VEGF may inhibit vascular stabilization to promote angiogenesis and vascular remodeling.

P021: Expressions of Angiopoietin 1 and 2 in Recurrent Oral Cancer

This study aims to evaluate the clinical significance of Ang 1 and 2 in recurrent but operable squamous cell carcinoma of oral cavity (OCSCC). The formalin-fixed, paraffin-block tissues from 40 patients who underwent surgical intervention for local and localregional recurrent OCSCC were retrieved for IHC study. The results showed that the actuarial 3-year disease-free survival rate was 43.1% and high expression of Ang 1 was correlated significantly with positive nodal stage (p =0.041). Patients with higher recurrent tumor stage (p =0.0036) or higher expression of Ang 2 (p =0.045) had a poorer actuarial 3-year disease-free survival, respectively. However, Cox’s regression analysis showed that only the recurrent tumor stage (rT) was the independent prognostic factor for survival. There were 1 woman and 39 men. The mean age was 51.4 years (range, 31 73 years). The mean duration from the initial surgery to the detected recurrence was 11.3 months (3 58 months). Among these patients, their recurrent tumor (rT) stages were 5 patients (12.5 %) in rT1, 5 (12.5%) in rT2, 3 in rT3 (7.5%), and 27 (67.5%) in rT4. Twenty-seven patients (67.5%) were classified as N0, 5 (12.5%) as rN1, 7 (17.5%) as rN2, and 1 (2.5%) as rN3. The recurrent TNM stage revealed that 5 patients were classified as stage I, 4 as stage II, 2 as stage III, and 29 as stage IV. The actuarial 3-year disease-free survival rate was 43.1% with mean follow-up of 18.4 (ranged 1-70) months. We found that high expression of Angiopoietin 1 was correlated significantly with positive nodal stage (p =0.041). Those patients with higher recurrent tumor stage (p =0.0036) or higher expression of Angiopoietin 2 (p =0.045) had a poorer actuarial 3-year disease-free survival, respectively. However, Cox’s regression analysis revealed that only the recurrent tumor stage (rT) was the independent prognostic factor for survival (p=0.019, 95% CI: 1.473-82.017, Relative Risk: 10.992). Results 1. Schwartz GJ, Mehta RH, Wenig BL, et al.: Salvage treatment for recurrent squamous cell carcinoma of the oral cavity. Head Neck 2000;22:34-41. 2. Yuen AP, Wei WI, Wong SH, Ng RW.: Local recurrence of carcinoma of the tongue after glossectomy: patient prognosis. Ear Nose Throat J 1998;77:181-4. 3. Nathanson A, Agren K, Biorklund A, et al.: Evaluation of some prognostic factors in small squamous cell carcinoma of the mobile tongue: a multicenter study in Sweden. Head Neck 1989;11:387-92. 4. Pearlman NW.: Treatment outcome in recurrent head and neck cancer. Arch Surg 1979;114:39-42. 5. Jones KR, Lodge-Rigal RD, Reddick RL, et al.: Prognostic factors in the recurrence of stage I and II squamous cell cancer of the oral cavity. Arch Otolaryngol Head Neck Surg 1992;118:483-5. 6. Meyza JW, Towpik E.: Surgical and cryosurgical salvage of oral and oropharyngeal cancer recurring after radical radiotherapy. Eur J Surg Oncol 1991;17:567-70. Bibliography Between 1995 and 2005, the formalin-fixed, paraffin-embedded tissues from forty patients who underwent surgical intervention for local and local-regional recurrent OCSCC were retrieved for immunohistochemistry study. Fisher’s exact test was used to evaluate the correlation between the clinicopathological variables and the expressions of Ang1 and 2. Scaling for the intensity of immunostaining with Ang-1 and Ang-2 A four-tier scaling system was used to grade the intensity of immunoreactivity for Ang-1 and Ang-2. The sections were examined under low-power-field (100x) with microscopy to evaluate the percentage of tumor cells positive for Ang-1 or Ang-2. The tumor with less than 1% positive tumor cells was graded as score 0, 1 - 25% as 1+, 26 50% as 2+, and over 50% as 3+. Both 0 and 1+ were defined as low expression and both 2+ and 3+ as high expression for Ang-1 or Ang-2 immunostaining. Survival analysis was based on Kaplan-Meier method. To determine the effect of distinct prognosis factors on survival, a multivariate analysis was performed according to the Cox’s regression model. Methods and Materials The recurrent OCSCC with a high expression of Ang-1 or Ang-2 in the tumor bed have a more aggressive tumor behavior. Furthermore, the expression of Angiopoietin-1 correlates with the nodal status in recurrent but operable OCSCC. However, the salvage outcome depends on the recurrent tumor stage. Conclusions Introduction The recurrence of squamous cell carcinoma of oral cavity (OCSCC) following surgical treatment is usually encountered with a frequency of 19-48%. Salvage treatment for recurrent OCSCC is a challenge with an overall salvage cure rate of 9% to 67%[1-5]. The success of salvage for a recurrent disease depends on the extent of the tumor and the health status of the patient at the time of recurrence. Tumors develop angiogenesis by secreting growth factors, such as platelet-derived growth factor, vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1), to stimulate endothelial migration and proliferation . Ang-2 is the antagonist to Ang-1 and inhibits Ang-1-mediated Tie2 phosphorylation. Significant Ang-1 and Ang-2 expressions in the tumor tissue may contribute to the progression of malignancy.

Angiopoietin-1 Requires p190 RhoGAP to Protect against Vascular Leakage in Vivo

Angiopoietin-1 (Ang-1), a ligand of the endothelium-specific receptor Tie-2, inhibits permeability in the mature vasculature, but the mechanism remains unknown. Here we show that Ang-1 signals Rho family GTPases to organize the cytoskeleton into a junction-fortifying arrangement that enhances the permeability barrier function of the endothelium. Ang-1 phosphorylates Tie-2 and its downstream effector phosphatidylinositol 3-kinase. This induces activation of one endogenous GTPase, Rac1, and inhibition of another, RhoA. Loss of either part of this dual effect abrogates the cytoskeletal and anti-permeability actions of Ang-1, suggesting that coordinated GTPase regulation is necessary for the vessel-sealing effects of Ang-1. p190 RhoGAP, a GTPase regulatory protein, provides this coordinating function as it is phosphorylated by Ang-1 treatment, requires Rac1 activation, and is necessary for RhoA inhibition. Ang-1 prevents the cytoskeletal and pro-permeability effects of endotoxin but requires p190 RhoGAP to do so. Treatment with p190 RhoGAP small interfering RNA completely abolishes the ability of Ang-1 to rescue endotoxemia-induced pulmonary vascular leak and inflammation in mice. We conclude that Ang-1 prevents vascular permeability by regulating the endothelial cytoskeleton through coordinated and opposite effects on the Rho GTPases Rac1 and RhoA. By linking Rac1 activation and RhoA inhibition, p190 RhoGAP is critical to the protective effects of Ang-1 against endotoxin. These results provide mechanistic evidence that targeting the endothelium through Tie-2 may offer specific therapeutic strategies in life-threatening endotoxemic conditions such as sepsis and acute respiratory distress syndrome.

Evolution of a Highly Selective and Potent 2-(Pyridin-2-yl)-1,3,5-triazine Tie-2 Kinase Inhibitor

Inhibition of angiogenesis is a promising and clinically validated approach for limiting tumor growth and survival. The receptor tyrosine kinase Tie-2 is expressed almost exclusively in the vascular endothelium and is required for developmental angiogenesis and vessel maturation. However, the significance of Tie-2 signaling in tumor angiogenesis is not well understood. In order to evaluate the therapeutic utility of inhibiting Tie-2 signaling, we developed a series of potent and orally bioavailable small molecule Tie-2 kinase inhibitors with selectivity over other kinases, especially those that are believed to be important for tumor angiogenesis. Our earlier work provided pyridinyl pyrimidine 6 as a potent, nonselective Tie-2 inhibitor that was designed on the basis of X-ray cocrystal structures of KDR inhibitors 34 (triazine) and 35 (nicotinamide). Lead optimization resulted in pyridinyl triazine 63, which exhibited >30-fold selectivity over a panel of kinases, good oral exposure, and in vivo inhibition of Tie-2 phosphorylation.

Lymphangiogenesis following obstruction of large postnodal lymphatics in sheep

We examined the impact of lymph flow obstruction in large post-nodal lymphatic vessels in sheep. A silk ligature was placed 2 cm downstream from the prescapular or popliteal lymph node and tightened to interrupt flow. At 6, 12 and 16 weeks after lymph flow blockage, a network of small interconnecting lymphatics (∼ 10–40 μm in diameter) could be observed in the vicinity of the ligature. These were identified using antibodies to the lymphatic endothelial markers LYVE-1 or VEGFR-3 or unequivocally, with the upstream intraluminal injection of the non-specific cell dye CFDA-SE. The observed lymphangiogenesis coincided with increased levels of Prox1, Tie2 (Y992) phosphorylation, MAPK activation, and decreased Akt activition. In the popliteal preparations, saline was infused into the prenodal ducts upstream of the regeneration site. The slopes of the inflow pressure versus flow relationships were 17.3 ± 3.6, immediately after vessel obstruction, 36.2 ± 9.6 at 6 weeks and 15.0 ± 5.3 at 12–16 weeks. For comparison, the average slope in a completely intact popliteal system was 3.1 ± 0.3 (from a previous publication). The resistance to flow remained high up to 12–16 weeks after flow obstruction suggesting that normal flow parameters had not been achieved over this time. The lymph node appeared to have some role in limiting the impact of post-nodal lymph obstruction, a function that appeared to be compromised by lymph stasis.