HLF/HIF-2alpha is a key factor in retinopathy of prematurity in association with erythropoietin

An HLF (HIF-1alpha-like factor)/HIF-2alpha-knockout mouse is embryonic lethal, preventing investigation of HLF function in adult mice. To investigate the role of HLF in adult pathological angiogenesis, we generated HLF-knockdown (HLF(kd/kd)) mice by inserting a neomycin gene sandwiched between two loxP sequences into exon 1 of the HLF gene. HLF(kd/kd) mice expressing 80-20% reduction, depending on the tissue, in wild-type HLF mRNA were fertile and apparently normal. Hyperoxia-normoxia treatment, used as a murine model of retinopathy of prematurity (ROP), induced neovascularization in wild-type mice, but not in HLF(kd/kd) mice, whereas prolonged normoxia following hyperoxic treatment caused degeneration of retinal neural layers in HLF(kd/kd) mice due to poor vascularization. Cre-mediated removal of the inserted gene recovered normal HLF expression and retinal neovascularization in HLF(kd/kd) mice. Expression levels of various angiogenic factors revealed that only erythropoietin (Epo) gene expression was significantly affected, in parallel with HLF expression. Together with the results from intraperitoneal injection of Epo into HLF(kd/kd) mouse, this suggests that Epo is one of the target genes of HLF responsible for experimental ROP.     

Critical Role of Endothelial Hydrogen Peroxide in Post-Ischemic Neovascularization

Background

Reactive oxygen species (ROS) play an important role in angiogenesis in endothelial cells (ECs) in vitro and neovascularization in vivo. However, little is known about the role of endogenous vascular hydrogen peroxide (H₂O₂) in postnatal neovascularization.

Methodology/Principal Findings

We used Tie2-driven endothelial specific catalase transgenic mice (Cat-Tg mice) and hindlimb ischemia model to address the role of endogenous H₂O₂ in ECs in post-ischemic neovascularization in vivo. Here we show that Cat-Tg mice exhibit significant reduction in intracellular H₂O₂ in ECs, blood flow recovery, capillary formation, collateral remodeling with larger extent of tissue damage after hindlimb ischemia, as compared to wild-type (WT) littermates. In the early stage of ischemia-induced angiogenesis, Cat-Tg mice show a morphologically disorganized microvasculature. Vascular sprouting and tube elongation are significantly impaired in isolated aorta from Cat-Tg mice. Furthermore, Cat-Tg mice show a decrease in myeloid cell recruitment after hindlimb ischemia. Mechanistically, Cat-Tg mice show significant decrease in eNOS phosphorylation at Ser1177 as well as expression of redox-sensitive vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemotactic protein-1 (MCP-1) in ischemic muscles, which is required for inflammatory cell recruitment to the ischemic tissues. We also observed impaired endothelium-dependent relaxation in resistant vessels from Cat-Tg mice.

Conclusions/Significance

Endogenous ECs-derived H₂O₂ plays a critical role in reparative neovascularization in response to ischemia by upregulating adhesion molecules and activating eNOS in ECs. Redox-regulation in ECs is a potential therapeutic strategy for angiogenesis-dependent cardiovascular diseases.     

Mammalian tumor suppressor Int6 specifically targets hypoxia inducible factor 2 alpha for degradation by hypoxia- and pVHL-independent regulation

The hypoxia-inducible factors HIF-1 alpha and HIF-2 alpha are structurally similar as regards their DNA-binding and dimerization domains, but differ in their transactivation domains and, as is shown by experiments using hif-1 alpha(-/-) and hif-2 alpha(-/-) mice, in their functions. This implies that HIF-1 alpha and HIF-2 alpha may have unique target genes. To address this discrepancy and identify HIF-2 alpha-specific target genes, we performed yeast two-hybrid analysis and identified the tumor suppressor Int6/eIF3e/p48 as a novel target gene product involved in HIF-2 alpha regulation. The int6 gene was first identified from a screen in which the mouse mammary tumor virus was employed as an insertional mutagen to identify genes whose functions are critical for breast tumor formation. Here, by using two-hybrid analysis, immunoprecipitation in mammalian cells, and HRE-reporter assays, we report the specific interaction of HIF-2 alpha (but not HIF-1 alpha or HIF-3 alpha) with Int6. The results indicate that the direct interaction of Int6 induces proteasome inhibitor-sensitive HIF-2 alpha degradation. This degradation was clearly observed in renal cell carcinoma 786-O cells, and was found to be both hypoxia- and pVHL-independent. Furthermore, Int6 protein knockdown by int6-siRNA vectors or the dominant-negative mutant Int6-Delta C increased endogenous HIF-2 alpha expression, even under normoxia, and induced sets of critical angiogenic factors comprising vascular endoplasmic growth factor, angiopoietin, and basic fibroblast growth factor mRNA. These results indicate that Int6 is a novel and critical determinant of HIF-2 alpha-dependent angiogenesis as well as cancer formation, and that int6-siRNA transfer may be an effective therapeutic strategy in pathological conditions such as heart and brain ischemia, hepatic cirrhosis, and obstructive vessel diseases.     

Ischemia-induced angiogenesis is impaired in aminopeptidase A deficient mice via down-regulation of HIF-1α

Aminopeptidase A (APA; EC 3.4.11.7) is a transmembrane metalloprotease with several functions in tumor angiogenesis. To investigate the role of APA in the process of ischemia-induced angiogenesis, we evaluated the cellular angiogenic responses under hypoxic conditions and the process of perfusion recovery in the hindlimb ischemia model of APA-deficient (APA-KO; C57Bl6/J strain) mice.Western blotting of endothelial cells (ECs) isolated from the aorta of APA-KO mice revealed that the accumulation of hypoxia-inducible factor-1α (HIF-1α) protein in response to hypoxic challenge was blunted. Regarding the proteasomal ubiquitination, a proteasome inhibitor MG-132 restored the reduced accumulation of HIF-1α in ECs from APA-KO mice similar to control mice under hypoxic conditions. These were associated with decreased growth factor secretion and capillary formation in APA-KO mice. In the hindlimb ischemia model, perfusion recovery in APA-KO mice was decreased in accordance with a significantly lower capillary density at 2 weeks. Regarding vasculogenesis, no differences were observed in cell populations and distribution patterns between wild type and APA-KO mice in relation to endothelial progenitor cells.Our results suggested that Ischemia-induced angiogenesis is impaired in APA-KO mice partly through decreased HIF-1α stability by proteasomal degradation and subsequent suppression of HIF-1α-driven target protein expression such as growth factors. APA is a functional target for ischemia-induced angiogenesis.     

In vivo enhancement of angiogenesis by adenoviral transfer of HIF-1alpha-modified endothelial progenitor cells (Ad-HIF-1alpha-modified EPC for angiogenesis)

Hypoxia inducible factor (HIF)-1alpha over-expression may have beneficial effects in cell therapy of hypoxia-induced pathophysiological processes, such as ischemic disease. Our previous study showed the feasibility of ex vivo modification of endothelial progenitor cells (EPCs) by HIF-1alpha transfection. In this study, we sought to determine if such ex vivo modified EPCs facilitated functional therapeutic neovascularization. Ad-HIF-1alpha was transduced in human EPC in vitro. HIF-1alpha-transduced EPCs were administered to nude mice with hindlimb ischemia. BrdU-labeling of these EPCs showed that they enhanced neovascularization in vivo. Limb and toe necrosis was significantly reduced in HIF-1alpha-EPC group compared to GFP-EPC group and medium control group at 14 days after transplantation (both P<0.05). A statistically significant difference was still observed in the HIF-1alpha group until 1 and 2 months of follow-up. Neovascularization was improved by both histological and physiological assessments. Exogenous EPC homing was observed. HIF-1alpha over-expression enhanced its mRNA and protein expression in the ischemia zone. The expression of genes downstream of HIF-1alpha was examined to explore the possible mechanism of EPC homing. In conclusion, HIF-1alpha-EPC gene transfer augments impaired neovascularization in experimentally induced mouse hindlimb ischemia in vivo.     

LKB1 in endothelial cells is required for angiogenesis and TGFbeta-mediated vascular smooth muscle cell recruitment

Inactivation of the tumor suppressor kinase Lkb1 in mice leads to vascular defects and midgestational lethality at embryonic day 9-11 (E9-E11). Here, we have used conditional targeting to investigate the defects underlying the Lkb1(-/-) phenotype. Endothelium-restricted deletion of Lkb1 led to embryonic death at E12.5 with a loss of vascular smooth muscle cells (vSMCs) and vascular disruption. Transforming growth factor beta (TGFbeta) pathway activity was reduced in Lkb1-deficient endothelial cells (ECs), and TGFbeta signaling from Lkb1(-/-) ECs to adjacent mesenchyme was defective, noted as reduced SMAD2 phosphorylation. The addition of TGFbeta to mutant yolk sac explants rescued the loss of vSMCs, as evidenced by smooth muscle alpha actin (SMA) expression. These results reveal an essential function for endothelial Lkb1 in TGFbeta-mediated vSMC recruitment during angiogenesis.     

The ephrins and Eph receptors in angiogenesis.

Eph receptors are a unique family of receptor tyrosine kinases that play critical roles in embryonic patterning, neuronal targeting, vascular development and adult neovascularization. Engagement of Eph receptors by ephrin ligands mediates critical steps of angiogenesis, including juxtacrine cell-cell contacts, cell adhesion to extracellular matrix, and cell migration. Recent evidence from in vitro angiogenesis assays and analysis of mice deficient for one or more members of the Eph family establishes the role of Eph signaling in sprouting angiogenesis and blood vessel remodeling during vascular development. Furthermore, elevated expression of Eph receptors and ephrin ligands is associated with tumors and associated tumor vasculature, suggesting that Eph receptors and their ephrin ligands also play critical roles in tumor angiogenesis and tumor growth. This review will focus on the relevance of Eph receptor signaling in embryonic and adult neovascularization, and possible contributions to tumor growth and metastasis.     

Eph receptor and ephrin ligand-mediated interactions during angiogenesis and tumor progression

Eph receptors comprise the largest family of receptor tyrosine kinases. They are classified into an A family and a B family on the basis of the characteristic properties of the corresponding ephrin ligands which are either GPI-anchored peripheral membrane molecules (A class ephrins) or transmembrane molecules (B class ephrins). Eph receptors and ephrin ligands were originally identified as neuronal pathfinding molecules. Yet, gene targeting experiments in mice have identified the EphB/ephrinB system as critical and rate-limiting determinant of arterio-venous differentiation during embryonic vascular development. Identification of vascular EphB/ephrinB functions has in the last few years stimulated two emerging fields of vascular biology research, namely (1) the molecular analysis of the structural and functional mechanisms of arterio-venous differentiation, and (2) the molecular study of the commonalities between vascular and neuronal guidance and patterning mechanisms. This review summarizes the current understanding of vascular Eph receptor and ephrin ligand functions and provides an overview of emerging roles of the Eph/ephrin system in controlling tumor and vascular functions during tumorigenesis and tumor progression.     

Ciliary Neurotrophic Factor Protects Mice Against Streptozotocin-induced Type 1 Diabetes through SOCS3: THE ROLE OF STAT1/STAT3 RATIO IN β-CELL DEATH*

Type 1 diabetes is characterized by a loss of islet β-cells. Ciliary neurotrophic factor (CNTF) protects pancreatic islets against cytokine-induced apoptosis. For this reason, we assessed whether CNTF protects mice against streptozotocin-induced diabetes (a model of type 1 diabetes) and the mechanism for this protection. WT and SOCS3 knockdown C57BL6 mice were treated for 5 days with citrate buffer or 0.1 mg/kg CNTF before receiving 80 mg/kg streptozotocin. Glycemia in non-fasted mice was measured weekly from days 0–28 after streptozotocin administration. Diabetes was defined as a blood glucose > 11.2 mmol/liter. Wild-type (WT) and SOCS3 knockdown MIN6 cells were cultured with CNTF, IL1β, or both. CNTF reduced diabetes incidence and islet apoptosis in WT but not in SOCS3kd mice. Likewise, CNTF inhibited apoptosis in WT but not in SOCS3kd MIN6 cells. CNTF increased STAT3 phosphorylation in WT and SOCS3kd mice and MIN6 cells but reduced STAT1 phosphorylation only in WT mice, in contrast to streptozotocin and IL1β. Moreover, CNTF reduced NFκB activation and required down-regulation of inducible NO synthase expression to exert its protective effects. In conclusion, CNTF protects mice against streptozotocin-induced diabetes by increasing pancreatic islet survival, and this protection depends on SOCS3. In addition, SOCS3 expression and β-cell fate are dependent on STAT1/STAT3 ratio.     

Mouse models for preeclampsia: disruption of redox-regulated signaling

The concept that oxidative stress contributes to the development of human preeclampsia has never been tested in genetically-defined animal models. Homozygous deletion of catechol-O-methyl transferase (Comt-/-) in pregnant mice leads to human preeclampsia-like symptoms (high blood pressure, albuminurea and preterm birth) resulting from extensive vasculo-endothelial pathology, primarily at the utero-fetal interface where maternal cardiac output is dramatically increased during pregnancy. Comt converts estradiol to 2-methoxyestradiol 2 (2ME2) which counters angiogenesis by depleting hypoxia inducible factor-1 alpha (HIF-1 alpha) at late pregnancy. We propose that in wild type (Comt++) pregnant mice, 2ME2 destabilizes HIF-1 alpha by inhibiting mitochondrial superoxide dismutase (MnSOD). Thus, 2ME2 acts as a pro-oxidant, disrupting redox-regulated signaling which blocks angiogenesis in wild type (WT) animals in physiological pregnancy. Further, we suggest that a lack of this inhibition under normoxic conditions in mutant animals (Comt-/-) stabilises HIF-1 alpha by inactivating prolyl hydroxlases (PHD). We predict that a lack of inhibition of MnSOD, leading to persistent accumulation of HIF-1 alpha, would trigger inflammatory infiltration and endothelial damage in mutant animals. Critical tests of this hypothesis would be to recreate preeclampsia symptoms by inducing oxidative stress in WT animals or to ameliorate by treating mutant mice with Mn-SOD-catalase mimetics or activators of PHD.     

Impaired tumor angiogenesis and VEG-Finduced pathway in endothelial CD146 knockout mice

CD146 is a newly identified endothelial biomarker that has been implicated in angiogenesis. Though in vitro angiogenic function of CD146 has been extensively reported, in vivo evidence is still lacking. To address this issue, we generated endothelial-specific CD146 knockout (CD146^EC-KO) mice using the Tg(Tek-cre) system. Surprisingly, these mice did not exhibit any apparent morphological defects in the development of normal retinal vasculature. To evaluate the role of CD146 in pathological angiogenesis, a xenograft tumor model was used. We found that both tumor volume and vascular density were significantly lower in CD146^EC-KO mice when compared to WT littermates. Additionally, the ability for sprouting, migration and tube formation in response to VEGF treatmentwas impairedinendothelial cells (ECs) of CD146^EC-KO mice. Mechanistic studies further confirmed that VEGFinduced VEGFR-2 phosphorylation and AKT/p38 MAPKs/ NF-κB activation were inhibited in these CD146-null ECs, whichmight present theunderlyingcause for theobserved inhibition of tumor angiogenesis in CD146^EC-KO mice. These results suggest thatCD146 plays a redundant role in physiological angiogenic processes, but becomes essential during pathological angiogenesis as observed in tumorigenesis.     

Targeted disruption of ephrin B1 in cells of myeloid lineage increases osteoclast differentiation and bone resorption in mice

Disruption of ephrin B1 in collagen I producing cells in mice results in severe skull defects and reduced bone formation. Because ephrin B1 is also expressed during osteoclast differentiation and because little is known on the role of ephrin B1 reverse signaling in bone resorption, we examined the bone phenotypes in ephrin B1 conditional knockout mice, and studied the function of ephrin B1 reverse signaling on osteoclast differentiation and resorptive activity. Targeted deletion of ephrin B1 gene in myeloid lineage cells resulted in reduced trabecular bone volume, trabecular number and trabecular thickness caused by increased TRAP positive osteoclasts and bone resorption. Histomorphometric analyses found bone formation parameters were not changed in ephrin B1 knockout mice. Treatment of wild-type precursors with clustered soluble EphB2-Fc inhibited RANKL induced formation of multinucleated osteoclasts, and bone resorption pits. The same treatment of ephrin B1 deficient precursors had little effect on osteoclast differentiation and pit formation. Similarly, activation of ephrin B1 reverse signaling by EphB2-Fc treatment led to inhibition of TRAP, cathepsin K and NFATc1 mRNA expression in osteoclasts derived from wild-type mice but not conditional knockout mice. Immunoprecipitation with NHERF1 antibody revealed ephrin B1 interacted with NHERF1 in differentiated osteoclasts. Treatment of osteoclasts with exogenous EphB2-Fc resulted in reduced phosphorylation of ezrin/radixin/moesin. We conclude that myeloid lineage produced ephrin B1 is a negative regulator of bone resorption in vivo, and that activation of ephrin B1 reverse signaling inhibits osteoclast differentiation in vitro in part via a mechanism that involves inhibition of NFATc1 expression and modulation of phosphorylation status of ezrin/radixin/moesin.