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PDF file - 108K, S3. VEGF levels are not changed in PHD4-overexpressing LM8 tumors.

Solid tumor growth is intimately associated with angiogenesis, a process that is efficiently triggered by hypoxia. Therefore, oxygen-sensitive signaling pathways are thought to play a critical role in tumor angiogenesis and progression. Here, the function of prolyl hydroxylase-4 (PHD4), a relative of the prolyl hydroxylase domain proteins 1–3 that promote the degradation of hypoxia-inducible factors (HIF), was interrogated. To test the hypothesis that PHD4 might inhibit tumor angiogenesis, it was overexpressed in osteosarcoma cells, and unexpectedly, this manipulation led to increased tumor blood vessel density. However, the newly formed blood vessels were smaller than normal and appeared to be partially nonfunctional, as indicated by poor vessel perfusion. PHD4 overexpression in tumor cells stimulated the expression of TGF-α, which was necessary and sufficient to promote angiogenic sprouting of endothelial cells. On the other hand, PHD4 overexpression reduced HIF-2α protein levels, which in turn inhibited in vivo tumor growth. Combined, elevated PHD4 levels deregulate angiogenesis via increased TGF-α expression in vitro and in vivo. These data support the hypothesis that tumor growth can be uncoupled from vessel density and that the individual PHD family members exert distinct functions in tumors.Implications: PHD4 influences tumor growth and vascularization through discrete mechanisms and molecular pathways that likely have therapeutic potential. Mol Cancer Res; 11(11); 1337–48. ©2013 AACR.

PDF file - 133K, S5. Lentivirus-mediated silencing of HIF-2alpha in LM8 control cells.

PDF file - 72K, S1. PHD4 overexpression in eEPCs reduces HIF-2alpha levels under hypoxia.

PDF file - 101K, S4. Lentivirus-mediated silencing of TGF-alpha in LM8-PHD4 cells.

PDF file - 40K, S2. Overexpression of PHD4 in LM8 osteosarcomas has no effect on the relative endothelial area.

Ischemic retinopathies (IR) are vision-threatening diseases that affect a substantial amount of people across all age groups worldwide. The current treatment options of photocoagulation and anti-VEGF therapy have side effects and are occasionally unable to prevent disease progression. It is therefore worthwhile to consider other molecular targets for the development of novel treatment strategies that could be safer and more efficient. During the manifestation of IR, the retina, normally an immune privileged tissue, encounters enhanced levels of cellular stress and inflammation that attract mononuclear phagocytes (MPs) from the blood stream and activate resident MPs (microglia). Activated MPs have a multitude of effects within the retinal tissue and have the potential to both counter and exacerbate the harmful tissue microenvironment. The present review discusses the current knowledge about the role of inflammation and activated retinal MPs in the major IRs: retinopathy of prematurity and diabetic retinopathy. We focus particularly on MPs and their secreted factors and cell–cell-based interactions between MPs and endothelial cells. We conclude that activated MPs play a major role in the manifestation and progression of IRs and could therefore become a promising new target for novel pharmacological intervention strategies in these diseases.

Supplementary Figures 1-5, Tables 1-2 from Inhibition of HIF Prolyl Hydroxylase-2 Blocks Tumor Growth in Mice through the Antiproliferative Activity of TGFβ

The mechanism underlying vasoproliferative retinopathies like retinopathy of prematurity (ROP) is hypoxia‐triggered neovascularisation. Nerve growth factor (NGF), a neurotrophin supporting survival and differentiation of neuronal cells may also regulate endothelial cell functions. Here we studied the role of NGF in pathological retinal angiogenesis in the course of the ROP mouse model. Topical application of NGF enhanced while intraocular injections of anti‐NGF neutralizing antibody reduced pathological retinal vascularization in mice subjected to the ROP model. The pro‐angiogenic effect of NGF in the retina was mediated by inhibition of retinal endothelial cell apoptosis. In vitro, NGF decreased the intrinsic (mitochondria‐dependent) apoptosis in hypoxia‐treated human retinal microvascular endothelial cells and preserved the mitochondrial membrane potential. The anti‐apoptotic effect of NGF was associated with increased BCL2 and reduced BAX, as well as with enhanced ERK and AKT phosphorylation, and was abolished by inhibition of the AKT pathway. Our findings reveal an anti‐apoptotic role of NGF in the hypoxic retinal endothelium, which is involved in promoting pathological retinal vascularization, thereby pointing to NGF as a potential target for proliferative retinopathies.

The replication stress inflicted on retinal endothelial cells (ECs) in the context of hypoxia-induced pathological neovascularization during proliferative retinopathy is linked with activation of the deoxyribonucleic acid (DNA) repair response. Here, we studied the effect of deficiency of the DNA damage response adaptor 53BP1, which is an antagonist of homologous recombination (HR), in the context of proliferative retinopathy. In the model of retinopathy of prematurity (ROP), 53BP1-deficient mice displayed increased hypoxia-driven pathological neovascularization and tuft formation, accompanied by increased EC proliferation and reduced EC apoptosis, as compared with 53BP1-sufficient mice. In contrast, physiological retina angiogenesis was not affected by 53BP1 deficiency. Knockdown of 53BP1 in ECs in vitro also resulted in enhanced proliferation and reduced apoptosis of the cells under hypoxic conditions. Additionally, upon 53BP1 knockdown, ECs displayed increased HR rate in hypoxia. Consistently, treatment with an HR inhibitor reversed the hyper-proliferative angiogenic phenotype associated with 53BP1 deficiency in ROP. Thus, by unleashing HR, 53BP1 deletion increases pathological EC proliferation and neovascularization in the context of ROP. Our data shed light to a previously unknown interaction between the DNA repair response and pathological neovascularization in the retina.

Aims In the pathogenesis of several diseases, neo-angiogenesis is increased (e.g. tumour growth). The peptide L-glutamyl-L-tryptophan (EW/IM862) has been claimed to exhibit inhibitory effects on tumour growth in vivo. However, the potential role of natural peptides with respect to anti-angiogenic properties is unsettled. The current study explores anti-angiogenic effects of the dipeptides WL, EW, IW and WE. Methods and results Using a bottom-up strategy, we first evaluated the effects of the peptides on VEGFR-2 signalling and quantified their effects in different angiogenesis assays. WL consistently had the strongest effects on phosphorylation of VEGFR-2 and downstream signalling. Therefore, this peptide was chosen in comparison with EW to further assess anti-angiogenic properties. However, sprout formation in three-dimensional (3D) fibrin gel bead assay was significantly inhibited by EW only. Furthermore, vessel sprouting in the mouse aortic ring assay was decreased by the presence of WL and EW compared to control. Results from a chorioallantoic membrane assay showed that under vascular endothelial growth factor (VEGF) stimulation WL and EW decreased the number of blood vessels versus control. These results were in line with those obtained in a matrigel plug assay. The VEGF-induced increase in the haemoglobin content was nearly abolished when treatment was combined with either WL or EW application. In the murine model of oxygen-induced retinopathy, WL exhibited a small albeit significant anti-angiogenic effect. Conclusion Comprehensive screening of WL suggests an anti-angiogenic effect, demonstrated in in vitro, ex vivo and in vivo models. Thus, WL is a dipeptide with potential anti-angiogenic effects and is worthy for further exploration.

SummaryWe have recently identified endothelial cell-secreted developmental endothelial locus-1 (Del-1) as an endogenous inhibitor of β2-integrin–dependent leukocyte infiltration. Del-1 was previously also implicated in angiogenesis. Here, we addressed the role of endogenously produced Del-1 in ischaemia-related angiogenesis. Intriguingly, Del-1–deficient mice displayed increased neovascularisation in two independent ischaemic models (retinopathy of prematurity and hind-limb ischaemia), as compared to Del-1–proficient mice. On the contrary, angiogenic sprouting in vitro or ex vivo (aortic ring assay) and physiological developmental retina angiogenesis were not affected by Del-1 deficiency. Mechanistically, the enhanced ischaemic neovascularisation in Del-1-deficiency was linked to higher infiltration of the ischaemic tissue by CD45+ haematopoietic and immune cells. Moreover, Del-1-deficiency promoted β2-integrin–dependent adhesion of haematopoietic cells to endothelial cells in vitro, and the homing of hematopoietic progenitor cells and of immune cell populations to ischaemic muscles in vivo. Consistently, the increased hind limb ischaemia-related angiogenesis in Del-1 deficiency was completely reversed in mice lacking both Del-1 and the β2-integrin LFA-1. Additionally, enhanced retinopathy-associated neovascularisation in Del-1-deficient mice was reversed by LFA-1 blockade. Our data reveal a hitherto unrecognised function of endogenous Del-1 as a local inhibitor of ischaemia-induced angiogenesis by restraining LFA-1–dependent homing of pro-angiogenic haematopoietic cells to ischaemic tissues. Our findings are relevant for the optimisation of therapeutic approaches in the context of ischaemic diseases.Supplementary Material to this article is available online at www.thrombosis-online.com.

A hallmark of proliferative retinopathies, such as retinopathy of prematurity (ROP), is a pathological neovascularization orchestrated by hypoxia and the resulting hypoxia-inducible factor (HIF)-dependent response. We studied the role of Hif2α in hematopoietic cells for pathological retina neovascularization in the murine model of ROP, the oxygen-induced retinopathy (OIR) model. Hematopoietic-specific deficiency of Hif2α ameliorated pathological neovascularization in the OIR model, which was accompanied by enhanced endothelial cell apoptosis. That latter finding was associated with up-regulation of the apoptosis-inducer FasL in Hif2α-deficient microglia. Consistently, pharmacological inhibition of the FasL reversed the reduced pathological neovascularization from hematopoietic-specific Hif2α deficiency. Our study found that the hematopoietic cell Hif2α contributes to pathological retina angiogenesis. Our findings not only provide novel insights regarding the complex interplay between immune cells and endothelial cells in hypoxia-driven retina neovascularization but also may have therapeutic implications for proliferative retinopathies.-Korovina, I., Neuwirth, A., Sprott, D., Weber, S., Sardar Pasha, S. P. B., Gercken, B., Breier, G., El-Armouche, A., Deussen, A., Karl, M. O., Wielockx, B., Chavakis, T., Klotzsche-von Ameln, A. Hematopoietic hypoxia-inducible factor 2α deficiency ameliorates pathological retinal neovascularization via modulation of endothelial cell apoptosis.

A new therapeutic target for sepsis Infections can sometimes unleash powerful immune responses that careen out of control, leading to sepsis, organ failure, and death. Although antibiotics can help to quash the infection, sepsis patients also need therapies that will rein in the immune response. Weber et al. now identify one potential target, the secreted protein interleukin-3 (IL-3) (see the Perspective by Hotchkiss). In sepsis patients, higher serum concentrations of IL-3 were correlated with higher rates of mortality. In septic mice, IL-3 caused the immune system to produce large amounts of cells called monocytes and neutrophils, which secrete highly inflammatory proteins. Blocking IL-3 protected mice from sepsis-induced death. Science , this issue p. 1260 ; see also p. 1201

SummaryIn proliferative retinopathies, like proliferative diabetic retinopathy and retinopathy of prematurity (ROP), the hypoxia response is sustained by the failure of the retina to revascularise its ischaemic areas. Non-resolving retina ischaemia/hypoxia results in upregulation of proangiogenic factors and pathologic neovascularisation with ectopic, fragile neovessels. Promoting revascularisation of the retinal avascular area could interfere with this vicious cycle and lead to vessel normalisation. Here, we examined the function of endothelial junctional adhesion molecule-C (JAM-C) in the context of ROP. Endothelial-specific JAM-C-deficient (EC-JAM-C KO) mice and littermate JAM-C-proficient (EC-JAM-C WT) mice were subjected to the ROP model. An increase in total retinal vascularisation was found at p17 owing to endothelial JAM-C deficiency, which was the result of enhanced revascularisation and vessel normalisation, thereby leading to significantly reduced avascular area in EC-JAM-C KO mice. In contrast, pathologic neovessel formation was not affected by endothelial JAM-C deficiency. Consistent with improved vessel normalisation, tip cell formation at the interface between vascular and avascular area was higher in EC-JAM-C KO mice, as compared to their littermate controls. Consistently, JAM-C inactivation in endothelial cells resulted in increased spreading on fibronectin and enhanced sprouting in vitro in a manner dependent on β1-integrin and on the activation of the small GTPase RAP1. Together, endothelial deletion of JAM-C promoted endothelial cell sprouting, and consequently vessel normalisation and revascularisation of the hypoxic retina without altering pathologic neovascularisation. Thus, targeting endothelial JAM-C may provide a novel therapeutic strategy for promoting revascularisation and vessel normalisation in the treatment of proliferative retinopathies.

The low-grade inflammatory state present in obesity contributes to obesity-related metabolic dysregulation, including nonalcoholic steatohepatitis (NASH) and insulin resistance. Intercellular interactions between immune cells or between immune cells and hepatic parenchymal cells contribute to the exacerbation of liver inflammation and steatosis in obesity. The costimulatory molecules, B7.1 and B7.2, are important regulators of cell-cell interactions in several immune processes; however, the role of B7 costimulation in obesity-related liver inflammation is unknown. Here, diet-induced obesity (DIO) studies in mice with genetic inactivation of both B7.1 and B7.2 (double knockout; DKO) revealed aggravated obesity-related metabolic dysregulation, reduced insulin signalling in the liver and adipose tissue (AT), glucose intolerance, and enhanced progression to steatohepatitis resulting from B7.1/B7.2 double deficiency. The metabolic phenotype of B7.1/B7.2 double deficiency upon DIO was accompanied by increased hepatic and AT inflammation, associated with largely reduced numbers of regulatory T cells (Tregs) in these organs. In order to assess the role of B7 costimulation in DIO in a non-Treg-lacking environment, we performed antibody (Ab)-mediated inhibition of B7 molecules in wild-type mice in DIO. Antibody-blockade of both B7.1 and B7.2 improved the metabolic phenotype of DIO mice, which was linked to amelioration of hepatic steatosis and reduced inflammation in liver and AT. Conclusion: Our study demonstrates a dual role of B7 costimulation in the course of obesity-related sequelae, particularly NASH. The genetic inactivation of B7.1/B7.2 deteriorates obesity-related liver steatosis and metabolic dysregulation, likely a result of the intrinsic absence of Tregs in these mice, rendering DKO mice a novel murine model of NASH. In contrast, inhibition of B7 costimulation under conditions where Tregs are present may provide a novel therapeutic approach for obesity-related metabolic dysregulation and, especially, NASH

The immune system plays an instrumental role in obesity and insulin resistance. Here, we unravel the role of the costimulatory molecule CD40 and its signaling intermediates, TNF receptor-associated factors (TRAFs), in diet-induced obesity (DIO). Although not exhibiting increased weight gain, male CD40(-/-) mice in DIO displayed worsened insulin resistance, compared with wild-type mice. This worsening was associated with excessive inflammation of adipose tissue (AT), characterized by increased accumulation of CD8(+) T cells and M1 macrophages, and enhanced hepatosteatosis. Mice with deficient CD40-TRAF2/3/5 signaling in MHCII+ cells exhibited a similar phenotype in DIO as CD40(-/-) mice. In contrast, mice with deficient CD40-TRAF6 signaling in MHCII+ cells displayed no insulin resistance and showed a reduction in both AT inflammation and hepatosteatosis in DIO. To prove the therapeutic potential of inhibition of CD40-TRAF6 in obesity, DIO mice were treated with a small-molecule inhibitor that we designed to specifically block CD40-TRAF6 interactions; this compound improved insulin sensitivity, reduced AT inflammation, and decreased hepatosteatosis. Our study reveals that the CD40-TRAF2/3/5 signaling pathway in MHCII+ cells protects against AT inflammation and metabolic complications associated with obesity whereas CD40-TRAF6 interactions in MHCII+ cells aggravate these complications. Inhibition of CD40-TRAF6 signaling by our compound may provide a therapeutic option in obesity-associated insulin resistance.

A right amount of oxygen and nutrients is essential for a tumor to develop. The role of oxygen dependent pathways and their regulators is therefore of utmost importance although little is known about the detailed impact they can have. Recently we have shown that inhibition of the oxygen sensor PHD2 in tumor cells blocks tumor growth due to the anti-proliferative activity of TGFβ. In this study, we refined these results by comparing different shPHD2 sequences in depth in the early phase of tumor growth. Our findings also reveal an intriguing role for MMP2 and MT1MMP in these settings, as these activated proteases display an anti-proliferative characteristic through the activation of downstream TGFβ targets. In conclusion, PHD2 inhibition is essential for the regulation of the anti-tumoral activity in mouse tumor cells and might bring some new insight in our understanding of tumor growth inhibition.

Developmental endothelial locus-1 (Del-1) is an endothelial cell-derived secreted protein circulating in blood and associated with the cell surface and the extracellular matrix. As we previously demonstrated, Del-1 restricts leukocyte recruitment by inhibiting the β2-integrin, LFA-1. Leukocytes and progenitor cells (PC) may contribute to angiogenesis. The role of endogenous Del-1 in angiogenesis is elusive. We found, that physiological angiogenesis of the developing retina was not affected in the Del-1-/- mice compared to the wildtype (WT) mice. Surprisingly, Del-1-/- mice displayed a significantly increased angiogenic response compared to WT mice after induction of hind limb ischemia (144 ± 6 % increase of capillary density) and retinal ischemia (retinopathy of prematurity model) suggesting that endogenous Del-1 is an inhibitor of ischemia-induced neovascularization. Silencing of Del-1 with siRNA did not affect the angiogenic sprouting of endothelial cell (EC) spheroids, indicating that Del-1 blocks angiogenesis in a non-endothelial cell autonomous pathway. Soluble Del-1 blocked the adhesion of inflammatory cells on EC monolayers. In line with these results, ischemic muscles and ischemic retinae from Del-1-/- mice displayed an enhanced infiltration with inflammatory cells compared to the WT mice. Since Del-1 blocks inflammatory cell homing by inhibiting the leukocytic LFA-1-integrin, we addressed the role of the Del-1/LFA-1-integrin interaction on the inhibitory function of endogenous Del-1 on angiogenesis. Indeed, Del-1/LFA-1-double deficiency reversed the pro-angiogenic phenotype of the Del-1-/- mice to the level of WT mice in the model of hind limb ischemia. Thus, the inhibitory role of Del-1 on neovascularization is mediated by the interaction of Del-1 with the LFA-1-integrin. Moreover, Del-1-deficiency led to an increased homing of intravenously injected murine fluorescence-labeled WT Lin- BM PC in ischemic muscles in comparison to WT mice after the induction of hind limb ischemia. Taken together, Del-1 acts as a negative regulator of ischemia-induced angiogenesis by interacting with the LFA-1-integrin expressed in hematopoietic cells, thereby inhibiting the homing of hematopoietic cells to ischemic tissues.

The immune system plays an instrumental role in obesity and insulin resistance. Here we unravel the role of the co-stimulatory molecule, CD40, and its signaling intermediates, TNF-Receptor-Associated-Factors (TRAFs), in diet-induced obesity (DIO). Although not exhibiting increased weight gain, male CD40-/- mice in DIO displayed worsened insulin resistance, as compared to wild type mice. This was associated with excessive inflammation of adipose tissue (AT), characterized by increased accumulation of CD8+ T cells and M1 macrophages, and enhanced hepatosteatosis. Mice with deficient CD40-TRAF2/3/5 signaling in MHCII+ cells exhibited a similar phenotype in DIO as CD40-/- mice. In contrast, mice with deficient CD40-TRAF6 signaling in MHCII+ cells displayed no insulin resistance, and showed a reduction in both AT inflammation and hepatosteatosis in DIO. To prove the therapeutic potential of inhibition of CD40-TRAF6 in obesity, DIO mice were treated with a small-molecule inhibitor that we designed to specifically block CD40-TRAF6 interactions; this improved insulin sensitivity, reduced AT inflammation and decreased hepatosteatosis. Our study reveals that the CD40-TRAF2/3/5 signaling pathway in MHCII+ cells protects against AT inflammation and metabolic complications associated with obesity, whereas CD40-TRAF6 interactions in MHCII+ cells aggravate these complications. Inhibition of CD40-TRAF6 signaling by our newly developed compound may provide a novel therapeutic option in obesity-associated insulin resistance.

Solid tumor growth is intimately associated with angiogenesis, a process that is efficiently triggered by hypoxia. Therefore, oxygen-sensitive signaling pathways are thought to play a critical role in tumor angiogenesis and progression. Here, the function of prolyl hydroxylase-4 (PHD4), a relative of the prolyl hydroxylase domain proteins 1–3 that promote the degradation of hypoxia-inducible factors (HIF), was interrogated. To test the hypothesis that PHD4 might inhibit tumor angiogenesis, it was overexpressed in osteosarcoma cells, and unexpectedly, this manipulation led to increased tumor blood vessel density. However, the newly formed blood vessels were smaller than normal and appeared to be partially nonfunctional, as indicated by poor vessel perfusion. PHD4 overexpression in tumor cells stimulated the expression of TGF-α, which was necessary and sufficient to promote angiogenic sprouting of endothelial cells. On the other hand, PHD4 overexpression reduced HIF-2α protein levels, which in turn inhibited in vivo tumor growth. Combined, elevated PHD4 levels deregulate angiogenesis via increased TGF-α expression in vitro and in vivo. These data support the hypothesis that tumor growth can be uncoupled from vessel density and that the individual PHD family members exert distinct functions in tumors. Implications: PHD4 influences tumor growth and vascularization through discrete mechanisms and molecular pathways that likely have therapeutic potential. Mol Cancer Res; 11(11); 1337–48. ©2013 AACR.

Obese adipose tissue (AT) inflammation contributes critically to development of insulin resistance. The complement anaphylatoxin C5a receptor (C5aR) has been implicated in inflammatory processes and as regulator of macrophage activation and polarization. However, the role of C5aR in obesity and AT inflammation has not been addressed. We engaged the model of diet-induced obesity and found that expression of C5aR was significantly upregulated in the obese AT, compared with lean AT. In addition, C5a was present in obese AT in the proximity of macrophage-rich crownlike structures. C5aR-sufficient and -deficient mice were fed a high-fat diet (HFD) or a normal diet (ND). C5aR deficiency was associated with increased AT weight upon ND feeding in males, but not in females, and with increased adipocyte size upon ND and HFD conditions in males. However, obese C5aR−/− mice displayed improved systemic and AT insulin sensitivity. Improved AT insulin sensitivity in C5aR−/− mice was associated with reduced accumulation of total and proinflammatory M1 macrophages in the obese AT, increased expression of IL-10, and decreased AT fibrosis. In contrast, no difference in β cell mass was observed owing to C5aR deficiency under an HFD. These results suggest that C5aR contributes to macrophage accumulation and M1 polarization in the obese AT and thereby to AT dysfunction and development of AT insulin resistance.

Virtually all solid tumors are dependent on a vascular network to provide them with the right amount of nutrients and oxygen. In that sense, low oxygen tension or hypoxia leads to an adaptive response that is transcriptionally regulated by the hypoxia-inducible factors (HIF), which are tightly controlled by the HIF prolyl hydroxylases (PHD). In this study, we show that inhibition of the oxygen sensor PHD2 in tumor cells stimulates vessel formation but paradoxically results in a profound reduction of tumor growth. This effect relies on the antiproliferative nature of the TGFβ signaling pathway, in a largely HIF-independent manner. Moreover, our findings reveal that PHD2 has an essential function in controlling the dual nature of TGFβ during tumorigenesis and may offer an alternative opportunity for anticancer therapy. Cancer Res; 71(9); 3306–16. ©2011 AACR.