Activation of the orphan endothelial receptor Tie1 modifies Tie2-mediated intracellular signaling and cell survival

A critical role for Tie1, an orphan endothelial receptor, in blood vessel morphogenesis has emerged from mutant mouse studies. Moreover, it was recently demonstrated that certain angiopoietin (Ang) family members can activate Tie1. We report here that Ang1 induces Tie1 phosphorylation in endothelial cells. Tie1 phosphorylation was, however, Tie2 dependent because 1) Ang1 failed to induce Tie1 phosphorylation when Tie2 was down-regulated in endothelial cells; 2) Tie1 phosphorylation was induced in the absence of Ang1 by either a constitutively active form of Tie2 or a Tie2 agonistic antibody; 3) in HEK 293 cells Ang1 phosphorylated a form of Tie1 without kinase activity when coexpressed with Tie2, and Ang1 failed to phosphorylate Tie1 when coexpressed with kinase-defective Tie2. Ang1-mediated AKT and 42/44MAPK phosphorylation is predominantly Tie2 mediated, and Tie1 down-regulates this pathway. Finally, based on a battery of in vitro and in vivo data, we show that a main role for Tie1 is to modulate blood vessel morphogenesis by virtue of its ability to down-regulate Tie2-driven signaling and endothelial survival. Our new observations help to explain why Tie1 null embryos have increased capillary densities in several organ systems. The experiments also constitute a paradigm for how endothelial integrity is fine-tuned by the interplay between closely related receptors by a single growth factor.

Endothelial survival factors and spatial completion, but not pericyte coverage of retinal capillaries determine vessel plasticity

Pericyte loss and capillary regression are characteristic for incipient diabetic retinopathy. Pericyte recruitment is involved in vessel maturation, and ligand-receptor systems contributing to pericyte recruitment are survival factors for endothelial cells in pericyte-free in vitro systems. We studied pericyte recruitment in relation to the susceptibility toward hyperoxia-induced vascular remodeling using the pericyte reporter X-LacZ mouse and the mouse model of retinopathy of prematurity (ROP). Pericytes were found in close proximity to vessels, both during formation of the superficial and the deep capillary layers. When exposure of mice to the ROP was delayed by 24 h, i.e., after the deep retinal layer had formed [at postnatal (p) day 8], preretinal neovascularizations were substantially diminished at p18. Mice with a delayed ROP exposure had 50% reduced avascular zones. Formation of the deep capillary layers at p8 was associated with a combined up-regulation of angiopoietin-1 and PDGF-B, while VEGF was almost unchanged during the transition from a susceptible to a resistant capillary network. Inhibition of Tie-2 function either by soluble Tie-2 or by a sulindac analog, an inhibitor of Tie-2 phosphorylation, resensitized retinal vessels to neovascularizations due to a reduction of the deep capillary network. Inhibition of Tie-2 function had no effect on pericyte recruitment. Our data indicate that the final maturation of the retinal vasculature and its resistance to regressive signals such as hyperoxia depend on the completion of the multilayer structure, in particular the deep capillary layers, and are independent of the coverage by pericytes.

Roles of ephrinB ligands and EphB receptors in cardiovascular development: demarcation of arterial/venous domains, vascular morphogenesis, and sprouting angiogenesis

Eph receptor tyrosine kinases and their cell-surface-bound ligands, the ephrins, regulate axon guidance and bundling in the developing brain, control cell migration and adhesion, and help patterning the embryo. Here we report that two ephrinB ligands and three EphB receptors are expressed in and regulate the formation of the vascular network. Mice lacking ephrinB2 and a proportion of double mutants deficient in EphB2 and EphB3 receptor signaling die in utero before embryonic day 11.5 (E11.5) because of defects in the remodeling of the embryonic vascular system. Our phenotypic analysis suggests complex interactions and multiple functions of Eph receptors and ephrins in the embryonic vasculature. Interaction between ephrinB2 on arteries and its EphB receptors on veins suggests a role in defining boundaries between arterial and venous domains. Expression of ephrinB1 by arterial and venous endothelial cells and EphB3 by veins and some arteries indicates that endothelial cell-to-cell interactions between ephrins and Eph receptors are not restricted to the border between arteries and veins. Furthermore, expression of ephrinB2 and EphB2 in mesenchyme adjacent to vessels and vascular defects in ephB2/ephB3 double mutants indicate a requirement for ephrin-Eph signaling between endothelial cells and surrounding mesenchymal cells. Finally, ephrinB ligands induce capillary sprouting in vitro with a similar efficiency as angiopoietin-1 (Ang1) and vascular endothelial growth factor (VEGF), demonstrating a stimulatory role of ephrins in the remodeling of the developing vascular system.

Pericyte migration: a novel mechanism of pericyte loss in experimental diabetic retinopathy

OBJECTIVE: The mechanism underlying pericyte loss during incipient diabetic retinopathy remains controversial. Hyperglycemia induces angiopoietin-2 (Ang-2) transcription, which modulates capillary pericyte coverage. In this study, we assessed loss of pericyte subgroups and the contribution of Ang-2 to pericyte migration. RESEARCH DESIGN AND METHODS: Numbers of total pericytes and their subgroups were quantified in retinal digest preparations of spontaneous diabetic XLacZ mice. Pericytes were divided into subgroups according to their localization, their position relative to adjacent endothelial cells, and the expression of LacZ. The contribution of Ang-2 to pericyte migration was assessed in Ang-2 overexpressing (mOpsinhAng2) and deficient (Ang2LacZ) mice. RESULTS: Pericyte numbers were reduced by 16% (P < 0.01) in XLacZ mice after 6 months of diabetes. Reduction of pericytes was restricted to pericytes on straight capillaries (relative reduction 27%, P < 0.05) and was predominantly observed in LacZ-positive pericytes (-20%, P < 0.01). Hyperglycemia increased the numbers of migrating pericytes (69%; P < 0.05), of which the relative increase due to diabetes was exclusively in LacZ-negative pericytes, indicating reduced adherence to the capillaries (176%; P < 0.01). Overexpression of Ang-2 in nondiabetic retinas mimicked diabetic pericyte migration of wild-type animals (78%; P < 0.01). Ang-2 deficient mice completely lacked hyperglycemia-induced increase in pericyte migration compared with wild-type littermates. CONCLUSIONS: Diabetic pericyte loss is the result of pericyte migration, and this process is modulated by the Ang-Tie system.

Angiopoietins assemble distinct Tie2 signalling complexes in endothelial cell-cell and cell-matrix contacts

The receptor tyrosine kinase Tie2, and its activating ligand Angiopoietin-1 (Ang1), are required for vascular remodelling and vessel integrity, whereas Ang2 may counteract these functions. However, it is not known how Tie2 transduces these different signals. Here, we show that Ang1 induces unique Tie2 complexes in mobile and confluent endothelial cells. Matrix-bound Ang1 induced cell adhesion, motility and Tie2 activation in cell-matrix contacts that became translocated to the trailing edge in migrating endothelial cells. In contrast, in contacting cells Ang1 induced Tie2 translocation to cell-cell contacts and the formation of homotypic Tie2-Tie2 trans-associated complexes that included the vascular endothelial phosphotyrosine phosphatase, leading to inhibition of paracellular permeability. Distinct signalling proteins were preferentially activated by Tie2 in the cell-matrix and cell-cell contacts, where Ang2 inhibited Ang1-induced Tie2 activation. This novel type of cellular microenvironment-dependent receptor tyrosine kinase activation may explain some of the effects of angiopoietins in angiogenesis and vessel stabilization.

The systemic angiogenic response during bone healing

INTRODUCTION: Angiogenesis is known to be a critical and closely regulated step during bone formation and fracture healing driven by a complex interaction of various cytokines. Delays in bone healing or even nonunion might therefore be associated with altered concentrations of specific angiogenic factors. These alterations might in turn be reflected by changes in serum concentrations. METHOD: To determine physiological time courses of angiogenic cytokines during fracture healing as well as possible changes associated with failed consolidation, we prospectively collected serum samples from patients who had sustained surgical treatment for a long bone fracture. Fifteen patients without fracture healing 4 months after surgery (nonunion group) were matched to a collective of 15 patients with successful healing (union group). Serum concentrations of angiogenin (ANG), angiopoietin 2 (Ang-2), basic fibroblast growth factor (bFGF), platelet derived growth factor AB (PDGF-AB), pleiotrophin (PTN) and vascular endothelial growth factor (VEGF) were measured using enzyme linked immunosorbent assays over a period of 24 weeks. RESULTS: Compared to reference values of healthy uninjured controls serum concentrations of VEGF, bFGF and PDGF were increased in both groups. Peak concentrations of these cytokines were reached during early fracture healing. Serum concentrations of bFGF and PDGF-AB were significantly higher in the union group at 2 and 4 weeks after the injury when compared to the nonunion group. Serum concentrations of ANG and Ang-2 declined steadily from the first measurement in normal healing fractures, while no significant changes over time could be detected for serum concentrations of these factures in nonunion patients. PTN serum levels increased asymptotically over the entire investigation in timely fracture healing while no such increase could be detected during delayed healing. CONCLUSION: We conclude that fracture healing in human subjects is accompanied by distinct changes in systemic levels of specific angiogenic factors. Significant alterations of these physiologic changes in patients developing a fracture nonunion over time could be detected as early as 2 (bFGF) and 4 weeks (PDGF-AB) after initial trauma surgery.

Apelin signaling modulates splanchnic angiogenesis and portosystemic collateral vessel formation in rats with portal hypertension

BACKGROUND/AIMS: Angiogenesis is a pathological hallmark of portal hypertension. Although VEGF is considered to be the most important proangiogenic factor in neoangiogenesis, this process requires the coordinated action of a variety of factors. Identification of novel molecules involved in angiogenesis is highly relevant, since they may represent potential new targets to suppress pathological neovascularization in angiogenesis-related diseases like portal hypertension. The apelin/APJ signaling pathway plays a crucial role in angiogenesis. Therefore, we determined whether the apelin system modulates angiogenesis-driven processes in portal hypertension. METHODS: Partial portal vein-ligated rats were treated with the APJ antagonist F13A for seven days. Splanchnic neovascularization and expression of angiogenesis mediators (Western blotting) was determined. Portosystemic collateral formation (microspheres), and hemodynamic parameters (flowmetry) were also assessed. RESULTS: Apelin and its receptor APJ were overexpressed in the splanchnic vasculature of portal hypertensive rats. F13A effectively decreased, by 52%, splanchnic neovascularization and expression of proangiogenic factors VEGF, PDGF and angiopoietin-2 in portal hypertensive rats. F13A also reduced, by 35%, the formation of portosystemic collateral vessels. CONCLUSIONS: This study provides the first experimental evidence showing that the apelin/APJ system contributes to portosystemic collateralization and splanchnic neovascularization in portal hypertensive rats, presenting a potential novel therapeutic target for portal hypertension.

VE-PTP controls blood vessel development by balancing Tie-2 activity

Vascular endothelial protein tyrosine phosphatase (VE-PTP) is an endothelial-specific receptor-type tyrosine phosphatase that associates with Tie-2 and VE-cadherin. VE-PTP gene disruption leads to embryonic lethality, vascular remodeling defects, and enlargement of vascular structures in extraembryonic tissues. We show here that antibodies against the extracellular part of VE-PTP mimic the effects of VE-PTP gene disruption exemplified by vessel enlargement in allantois explants. These effects require the presence of the angiopoietin receptor Tie-2. Analyzing the mechanism we found that anti-VE-PTP antibodies trigger endocytosis and selectively affect Tie-2-associated, but not VE-cadherin-associated VE-PTP. Dissociation of VE-PTP triggers the activation of Tie-2, leading to enhanced endothelial cell proliferation and enlargement of vascular structures through activation of Erk1/2. Importantly, the antibody effect on vessel enlargement is also observed in newborn mice. We conclude that VE-PTP is required to balance Tie-2 activity and endothelial cell proliferation, thereby controlling blood vessel development and vessel size.

Myc regulates embryonic vascular permeability and remodeling

Previous work has shown that c-Myc is required for adequate vasculogenesis and angiogenesis. To further investigate the contribution of Myc to these processes, we conditionally expressed c-Myc in embryonic endothelial cells using a tetracycline-regulated system. Endothelial Myc overexpression resulted in severe defects in the embryonic vascular system. Myc-expressing embryos undergo widespread edema formation and multiple hemorrhagic lesions. They die between embryonic days 14.5 and 17.5. The changes in vascular permeability are not caused by deficiencies in vascular basement membrane composition or pericyte coverage. However, the overall turnover of endothelial cells is elevated as is revealed by increased levels of both proliferation and apoptosis. Whole-mount immunohistochemical analysis revealed alterations in the architecture of capillary networks. The dermal vasculature of Myc-expressing embryos is characterized by a reduction in vessel branching, which occurs despite upregulation of the proangiogenic factors vascular endothelial growth factor-A and angiopoietin-2. Thus, the net outcome of an excess of vascular endothelial growth factor-A and angiopoietin-2 in the face of an elevated cellular turnover appears to be a defect in vascular integrity.

Bone marrow-derived mesenchymal stromal cells improve vascular regeneration and reduce leukocyte-endothelium activation in critical ischemic murine skin in a dose-dependent manner

BACKGROUND AIMS Stem cells participate in vascular regeneration following critical ischemia. However, their angiogenic and remodeling properties, as well as their role in ischemia-related endothelial leukocyte activation, need to be further elucidated. Herein, we investigated the effect of bone marrow-derived mesenchymal stromal cells (BM-MSCs) in a critically ischemic murine skin flap model. METHODS Groups received either 1 × 10(5), 5 × 10(5), or 1 × 10(6) BM-MSCs or cell-free conditioned medium (CM). Controls received sodium chloride. Intravital fluorescence microscopy was performed for morphological and quantitative assessment of micro-hemodynamic parameters over 12 days. RESULTS Tortuosity and diameter of conduit-arterioles were pronounced in the MSC groups (P < 0.01), whereas vasodilation was shifted to the end arteriolar level in the CM group (P < 0.01). These effects were accompanied by angiopoietin-2 expression. Functional capillary density and red blood cell velocity were enhanced in all treatment groups (P < 0.01). Although a significant reduction of rolling and sticking leukocytes was observed in the MSC groups with a reduction of diameter in postcapillary venules (P < 0.01), animals receiving CM exhibited a leukocyte-endothelium interaction similar to controls. This correlated with leukocyte common antigen expression in tissue sections (P < 0.01) and p38 mitogen-activated protein kinase expression from tissue samples. Cytokine analysis from BM-MSC culture medium revealed a 50% reduction of pro-inflammatory cytokines (interleukin [IL]-1β, IL-6, IL-12, tumor necrosis factor-α, interferon-γ) and chemokines (keratinocyte chemoattractant, granulocyte colony-stimulating factor) under hypoxic conditions. DISCUSSION We demonstrated positive effects of BM-MSCs on vascular regeneration and modulation of endothelial leukocyte adhesion in critical ischemic skin. The improvements after MSC application were dose-dependent and superior to the use of CM alone.

Bovine coccygeal intervertebral discs contain multipotent Tie2+ cells which can differentiate into osteogenic and adipogenic lineages

Question: The intervertebral disc (IVD) has a limited regenerative potential and low back pain represents a leading cause of disability [1]. IVD repair strategies require an appropriate cell source that is able to regenerate the damaged tissue such as progenitor stem cells. Recently, progenitor cells that are positive for the angiopoietin re- ceptor (Tie2) in the nucleus pulposus were identified [2]. Here we isolated primary cells from bovine IVD and sorted bovine nucleus pulposus progenitor cells (NPPC) for the marker Tie2. Furthermorewe tested whether Tie2 expressing cells can differentiate into os- teogenic and adipogenic lineages in vitro. Methods: NP cells were obtained from 1 year old bovine tails by sequential digestion with pronase for 1 h and collagenase over- night. Sorted Tie2- and Tie2+ cells were cultured in osteogenic and adipogenic medium for 3 weeks. The formed cell layers from both subpopulations were stained for calcium deposition and fat droplets. Colony forming units were prepared for both cell sus- pensions in methylcellulose-based medium and formed colonies ([10 cells) were analyzed macroscopically after 8 days. Results: After 3 weeks of culture, sorted Tie2+ cells were able to differentiate into osteocytes and adipocytes as characterized by cal- cium deposition and fat droplet formation. By contrast, Tie2- cells generated a weak staining for calcium and no fat droplets were ob- tained (Fig. 1). Sorted Tie2- and Tie2+ subpopulations of cells both formed colonies, however with different morphologies. The colonies formed from Tie2+ cells were spheroid in shape whereas those from Tie2- cells were spread and fibroblastic. Conclusion: Our data showed that Tie2+ cells of the nucleus pul- posus cells are progenitor-like cells that are able to differentiate into osteogenic and adipogenic lineages. Sorting of NPPC for Tie2 may represent a promising strategy with the potential to be used in the clinics for treatment of intervertebral disc damage. References 1. Freemont AJ (2009) The cellular pathobiology of the degenerate intervertebral disc and discogenic back pain. Rheumatology (Oxford) 48:5–10 2. Sakai D, Nakamura Y, Nakai T et al (2012) Exhaustion of nucleus pulposus progenitor cells with ageing and degeneration of the intervertebral disc. Nat Commun 3:1264 Acknowledgments: This project was funded by two projects of the Swiss National Science Foundation grant number #IZK0Z3_154384 and #310030_153411.

Biochemical and genetic analysis of two protein kinases JNKK2 and MEKK3

Mitogen-activated protein kinase (MAPK) cascades are conserved eukaryotic signaling modules consisting of a MAPK, a MAPKK and a MAP3K. MAPK cascades are involved in many cellular responses including proliferation, differentiation, apoptosis, stress and immune responses. ^ The first part of this thesis describes the cloning and biochemical analysis of JNKK2, a member of MAPKK gene family. Our results demonstrate that JNKK2 is a specific JNK activator and activates the JNK-dependent signal transduction pathway in vivo by inducing c-Jun and ATF2-mediated gene expression. We also found that JNKK2 is specifically activated by a MAP3K MEKK2 through formation of MEKK2-JNKK2-JNK1 triple complex module. JNKK2 is likely to mediate specific upstream signals to activate JNK cascade. ^ The second part of this thesis describes biochemical and gene disruption analysis of MEKK3, a member of MAP3K gene family. We showed that overexpression of MEKK3 strongly activates both JNK and p38 MAPKs but only weakly activates ERK. MEKK−/− embryos die at about embryonic day (E) 11. MEKK3−/− embryos displayed defects in blood vessel development in the yolk sacs, and in the myocardium and endocardium development at E9.5. The angiogenesis in the head, intersomitic region and placenta was also abnormal. These results demonstrate that MEKK3, a member of MAP3K MEKK/STE11 subgene family, is essential for early embryonic cardiovascular development. Furthermore, it was found that disruption of MEKK3 did not alter the expression of vascular endothelial growth factor-1 (VEGF-1), angiopoietin-1, -2 and their respective receptors Flt-1, Flk-1, Tie-1, Tie-2. Finally, MEKK3 was shown to activate myocyte-specific enhancer factor 2C (MEF2C), a crucial transcription factor for early embryonic cardiovascular development through the p38 MAPK cascade, suggesting that MEF2C is one of the key targets of the MEEKK3 signaling pathway during early embryonic cardiovascular development. ^

Angiopoietins 3 and 4: Diverging gene counterparts in mice and humans

The angiopoietins have recently joined the members of the vascular endothelial growth factor family as the only known growth factors largely specific for vascular endothelium. The angiopoietins include a naturally occurring agonist, angiopoietin-1, as well as a naturally occurring antagonist, angiopoietin-2, both of which act by means of the Tie2 receptor. We now report our attempts to use homology-based cloning approaches to identify new members of the angiopoietin family. These efforts have led to the identification of two new angiopoietins, angiopoietin-3 in mouse and angiopoietin-4 in human; we have also identified several more distantly related sequences that do not seem to be true angiopoietins, in that they do not bind to the Tie receptors. Although angiopoietin-3 and angiopoietin-4 are strikingly more structurally diverged from each other than are the mouse and human versions of angiopoietin-1 and angiopoietin-2, they appear to represent the mouse and human counterparts of the same gene locus, as revealed in our chromosomal localization studies of all of the angiopoietins in mouse and human. The structural divergence of angiopoietin-3 and angiopoietin-4 appears to underlie diverging functions of these counterparts. Angiopoietin-3 and angiopoietin-4 have very different distributions in their respective species, and angiopoietin-3 appears to act as an antagonist, whereas angiopoietin-4 appears to function as an agonist.

Redundant roles of Sox17 and Sox18 in postnatal angiogenesis in mice

Sox7, Sox17 and Sox18 constitute group F of the Sox family of HMG box transcription factor genes. Dominant-negative mutations in Sox18 underlie the cardiovascular defects observed in ragged mutant mice. By contrast, Sox18(-/-) mice are viable and fertile, and display no appreciable anomaly in their vasculature, suggesting functional compensation by the two other SoxF genes. Here, we provide direct evidence for redundant function of Sox17 and Sox18 in postnatal neovascularization by generating Sox17(+/-)-Sox18(-/-) double mutant mice. Whereas Sox18(-/-) and Sox17(+/-)-Sox18(+/)-mice showed no vascular defects, approximately half of the Sox17(+/-)-Sox18(-/-) pups died before postnatal day 21 (P21). They showed reduced neovascularization in the liver sinusoids and kidney outer medulla vasa recta at P7, which most likely caused the ischemic necrosis observed by P14 in hepatocytes and renal tubular epithelia. Those that survived to adulthood showed similar, but milder, vascular anomalies in both liver and kidney, and females were infertile with varying degrees of vascular abnormalities in the reproductive organs. These anomalies corresponded with sites of expression of Sox7 and Sox17 in the developing postnatal vasculature. In vitro angiogenesis assays, using primary endothelial cells isolated from the P7 livers, showed that the Sox17(+/-)-Sox18(-/-)endothelial cells were defective in endothelial sprouting and remodeling of the vasculature in a phenotype-dependent manner. Therefore, our findings indicate that Sox17 and Sox18, and possibly all three SoxF genes, are cooperatively involved in mammalian vascular development.

Loss of Akt activity increases circulating soluble endoglin release in preeclampsia:identification of inter-dependency between Akt-1 and heme oxygenase-1

Aims - Endothelial dysfunction is a hallmark of preeclampsia. Desensitization of the phosphoinositide 3-kinase (PI3K)/Akt pathway underlies endothelial dysfunction and haeme oxygenase-1 (HO-1) is decreased in preeclampsia. To identify therapeutic targets, we sought to assess whether these two regulators act to suppress soluble endoglin (sEng), an antagonist of transforming growth factor-ß (TGF-ß) signalling, which is known to be elevated in preeclampsia. Methods and results - Vascular endothelial growth factor-A (VEGF-A), fibroblast growth factor (FGF-2), angiopoietin-1 (Ang-1), and insulin, which all activate the PI3K/Akt pathway, inhibited the release of sEng from endothelial cells. Inhibition of the PI3K/Akt pathway, by overexpression of phosphatase and tensin homolog (PTEN) or a dominant-negative isoform of Akt (Aktdn) induced sEng release from endothelial cells and prevented the inhibitory effect of VEGF-A. Conversely, overexpression of a constitutively active Akt (Aktmyr) inhibited PTEN and cytokine-induced sEng release. Systemic delivery of Aktmyr to mice significantly reduced circulating sEng, whereas Aktdn promoted sEng release. Phosphorylation of Akt was reduced in preeclamptic placenta and this correlated with the elevated level of circulating sEng. Knock-down of Akt using siRNA prevented HO-1-mediated inhibition of sEng release and reduced HO-1 expression. Furthermore, HO-1 null mice have reduced phosphorylated Akt in their organs and overexpression of Aktmyr failed to suppress the elevated levels of sEng detected in HO-1 null mice, indicating that HO-1 is required for the Akt-mediated inhibition of sEng. Conclusion - The loss of PI3K/Akt and/or HO-1 activity promotes sEng release and positive manipulation of these pathways offers a strategy to circumvent endothelial dysfunction.