Mathias Wenes

Macrophage skewing by Phd2 haplodeficiency prevents ischaemia by inducing arteriogenesis

PHD2 serves as an oxygen sensor that rescues blood supply by regulating vessel formation and shape in case of oxygen shortage. However, it is unknown whether PHD2 can influence arteriogenesis. Here we studied the role of PHD2 in collateral artery growth by using hindlimb ischaemia as a model, a process that compensates for the lack of blood flow in case of major arterial occlusion. We show that Phd2 (also known as Egln1) haplodeficient (Phd2+/−) mice displayed preformed collateral arteries that preserved limb perfusion and prevented tissue necrosis in ischaemia. Improved arteriogenesis in Phd2+/− mice was due to an expansion of tissue-resident, M2-like macrophages and their increased release of arteriogenic factors, leading to enhanced smooth muscle cell (SMC) recruitment and growth. Both chronic and acute deletion of one Phd2 allele in macrophages was sufficient to skew their polarization towards a pro-arteriogenic phenotype. Mechanistically, collateral vessel preconditioning relied on the activation of canonical NF-κB pathway in Phd2+/− macrophages. These results unravel how PHD2 regulates arteriogenesis and artery homeostasis by controlling a specific differentiation state in macrophages and suggest new treatment options for ischaemic disorders.

Tumor stroma: a complexity dictated by the hypoxic tumor microenvironment

A lot of effort has been done to study how cancer cells react to low-oxygen tension, a condition known as hypoxia. Indeed, abnormal and dysfunctional blood vessels in the tumor are incapable to restore oxygenation, therefore perpetuating hypoxia, which, in turn, will fuel tumor progression, metastasis and resistance to antitumor therapies. Nevertheless, how stromal components including blood and lymphatic endothelial cells, pericytes and fibroblasts, as well as hematopoietic cells, respond to low-oxygen tension in comparison with their normoxic counterparts has been a matter of investigation in the last few years only and, to date, this field of research remains poorly understood. In general, opposing phenotypes can arise from the same stromal component when embedded in different tumor microenvironments, and, vice versa, different stromal components can have opposite reaction to the same tumor microenvironment. In this article, we will discuss the emerging link between tumor stroma and hypoxia, and how this complexity is translated at the molecular level.

Macrophage Metabolism Controls Tumor Blood Vessel Morphogenesis and Metastasis

Hypoxic tumor-associated macrophages (TAMs) acquire angiogenic and immunosuppressive properties. Yet it remains unknown if metabolic changes influence these functions. Here, we argue that hypoxic TAMs strongly upregulate the expression of REDD1, a negative regulator of mTOR. REDD1-mediated mTOR inhibition hinders glycolysis in TAMs and curtails their excessive angiogenic response, with consequent formation of abnormal blood vessels. Accordingly, REDD1 deficiency in TAMs leads to the formation of smoothly aligned, pericyte-covered, functional vessels, which prevents vessel leakiness, hypoxia, and metastases. Mechanistically, highly glycolytic REDD1-deficient TAMs outcompete endothelial cells for glucose usage that thwarts vascular hyperactivation and promotes the formation of quiescent vascular junctions. Tuning down glycolysis in REDD1 knockout TAMs re-establishes abnormal angiogenesis and metastases. On this basis, we prove that the anti-tumor effect of mTOR inhibitors is partly countered by the deleterious outcome of these drugs on TAMs. Our data provide a functional link between TAM metabolism and tumor angiogenesis.

1,25-Dihydroxyvitamin D3 curtails the inflammatory and T cell stimulatory capacity of macrophages through an IL-10-dependent mechanism.

The vitamin D receptor (VDR) is a hormone nuclear receptor regulating bone and calcium homeostasis. Studies revealing the expression of VDR on immune cells point toward a role for VDR-dependent signaling pathways in immunity. Here we verified the ability of the natural VDR ligand, 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) to interfere in inflammatory and T cell stimulatory capacity of macrophages, in particular within a chronic inflammatory disease features of experimental type 1 diabetes (T1D). We demonstrated that VDR is constitutively expressed in macrophages and both the levels of VDR and its downstream targets, are clearly induced by 1,25(OH)(2)D(3). In control mice, macrophage programming with 1,25(OH)(2)D(3) partially abrogated the activation-provoked expression of IL-12p40, TNFα and iNOS as well as the effector T cell-recruiting chemokines, CXCL9, CXCL10 and CXCL11. Targeting VDR signaling in macrophages counteracted their T-cell stimulatory ability despite essentially unaltered expression of antigen-presenting and costimulatory molecules. Furthermore, even in non-obese diabetic (NOD) mice, where macrophages/monocytes featured a heightened responsiveness toward danger signals and a superior T cell stimulatory capacity, 1,25(OH)(2)D(3) successfully curtailed these basic macrophage-mediated functions. Interestingly, the inhibitory action of the active compound was associated with an IL-10-dependent mechanism since 1,25(OH)(2)D(3)-treatment of IL-10-deficient macrophages failed to reproduce the characteristic repression on inflammatory mediators or T cell proliferation. Combined, these results highlight the possible therapeutic applicability of this natural immunomodulator, due to its ability to counteract macrophage inflammatory and T cell-activating pathways.

FXR agonist obeticholic acid reduces hepatic inflammation and fibrosis in a rat model of toxic cirrhosis

Hepatic inflammation drives hepatic stellate cells (HSC), resulting in liver fibrosis. The Farnesoid-X receptor (FXR) antagonizes inflammation through NF-κB inhibition. We investigated preventive and therapeutic effects of FXR agonist obeticholic acid (OCA) on hepatic inflammation and fibrosis in toxic cirrhotic rats. Cirrhosis was induced by thioacetamide (TAA) intoxication. OCA was given during or after intoxication with vehicle-treated rats as controls. At sacrifice, fibrosis, hemodynamic and biochemical parameters were assessed. HSC activation, cell turn-over, hepatic NF-κB activation, pro-inflammatory and pro-fibrotic cytokines were determined. The effect of OCA was further evaluated in isolated HSC, Kupffer cells, hepatocytes and liver sinusoidal endothelial cells (LSEC). OCA decreased hepatic inflammation and fibrogenesis during TAA-administration and reversed fibrosis in established cirrhosis. Portal pressure decreased through reduced intrahepatic vascular resistance. This was paralleled by decreased expression of pro-fibrotic cytokines (transforming growth-factor β, connective tissue growth factor, platelet-derived growth factor β-receptor) as well as markers of hepatic cell turn-over, by blunting effects of pro-inflammatory cytokines (e.g. monocyte chemo-attractant protein-1). In vitro, OCA inhibited both LSEC and Kupffer cell activation; while HSC remained unaffected. This related to NF-κB inhibition via up-regulated IκBα. In conclusion, OCA inhibits hepatic inflammation in toxic cirrhotic rats resulting in decreased HSC activation and fibrosis.

PHD2 regulates arteriogenic macrophages through TIE2 signalling

Occlusion of the main arterial route redirects blood flow to the collateral circulation. We previously reported that macrophages genetically modified to express low levels of prolyl hydroxylase domain protein 2 (PHD2) display an arteriogenic phenotype, which promotes the formation of collateral vessels and protects the skeletal muscle from ischaemic necrosis. However, the molecular mechanisms underlying this process are unknown. Here, we demonstrate that femoral artery occlusion induces a switch in macrophage phenotype through angiopoietin‐1 (ANG1)‐mediated Phd2 repression. ANG blockade by a soluble trap prevented the downregulation of Phd2 expression in macrophages and their phenotypic switch, thus inhibiting collateral growth. ANG1‐dependent Phd2 repression initiated a feed‐forward loop mediated by the induction of the ANG receptor TIE2 in macrophages. Gene silencing and cell depletion strategies demonstrate that TIE2 induction in macrophages is required to promote their proarteriogenic functions, enabling collateral vessel formation following arterial obstruction. These results indicate an indispensable role for TIE2 in sustaining in situ programming of macrophages to a proarteriogenic, M2‐like phenotype, suggesting possible new venues for the treatment of ischaemic disorders.

Genetic Deficiency in Plasma Protein HRG Enhances Tumor Growth and Metastasis by Exacerbating Immune Escape and Vessel Abnormalization

Histidine-rich glycoprotein (HRG) is a 75-kDa heparin-binding plasma protein implicated in the regulation of tumor growth and vascularization. In this study, we show that hrg(-/-) mice challenged with fibrosarcoma or pancreatic carcinoma grow larger tumors with increased metastatic properties. Compared with wild-type mice, fibrosarcomas in hrg(-/-) mice were more hypoxic, necrotic, and less perfused, indicating enhanced vessel abnormalization. HRG deficiency was associated with a suppressed antitumor immune response, with both increased infiltration of M2 marker-expressing macrophages and decreased infiltration of dendritic cells and cytotoxic T cells. Analysis of transcript expression in tumor-associated as well as peritoneal macrophages from hrg(-/-) mice revealed an increased expression of genes associated with a proangiogenic and immunoinhibitory phenotype. In accordance, expression arrays conducted on HRG-treated peritoneal macrophages showed induction of genes involved in extracellular matrix biology and immune responsiveness. In conclusion, our findings show that macrophages are a direct target of HRG. HRG loss influences macrophage gene regulation, leading to excessive stimulation of tumor angiogenesis, suppression of tumor immune response, and increased tumor growth and metastatic spread.

Tumour-educated circulating monocytes are powerful candidate biomarkers for diagnosis and disease follow-up of colorectal cancer

Objective Cancer immunology is a growing field of research whose aim is to develop innovative therapies and diagnostic tests. Starting from the hypothesis that immune cells promptly respond to harmful stimuli, we used peripheral blood monocytes in order to characterise a distinct gene expression profile and to evaluate its potential as a candidate diagnostic biomarker in patients with colorectal cancer (CRC), a still unmet clinical need. Design We performed a case-control study including 360 peripheral blood monocyte samples from four European oncological centres and defined a gene expression profile specific to CRC. The robustness of the genetic profile and disease specificity were assessed in an independent setting. Results This screen returned 43 putative diagnostic markers, which we refined and validated in the confirmative multicentric analysis to 23 genes with outstanding diagnostic accuracy (area under the curve (AUC)=0.99 (0.99 to 1.00), Se=100.0% (100.0% to 100.0%), Sp=92.9% (78.6% to 100.0%) in multiple-gene receiver operating characteristic analysis). The diagnostic accuracy was robustly maintained in prospectively collected independent samples (AUC=0.95 (0.85 to 1.00), Se=92.6% (81.5% to 100.0%), Sp=92.3% (76.9% to 100.0%). This monocyte signature was expressed at early disease onset, remained robust over the course of disease progression, and was specific for the monocytic fraction of mononuclear cells. The gene modulation was induced specifically by soluble factors derived from transformed colon epithelium in comparison to normal colon or other cancer histotypes. Moreover, expression changes were plastic and reversible, as they were abrogated upon withdrawal of these tumour-released factors. Consistently, the modified set of genes reverted to normal expression upon curative treatment and was specific for CRC. Conclusions Our study is the first to demonstrate monocyte plasticity in response to tumour-released soluble factors. The identified distinct signature in tumour-educated monocytes might be used as a candidate biomarker in CRC diagnosis and harbours the potential for disease follow-up and therapeutic monitoring.

Loss of Caveolin-1 in Metastasis-Associated Macrophages Drives Lung Metastatic Growth through Increased Angiogenesis

Summary Although it is well established that tumor-associated macrophages take part in each step of cancer progression, less is known about the distinct role of the so-called metastasis-associated macrophages (MAMs) at the metastatic site. Previous studies reported that Caveolin-1 (Cav1) has both tumor-promoting and tumor-suppressive functions. However, the role of Cav1 in bone-marrow-derived cells is unknown. Here, we describe Cav1 as an anti-metastatic regulator in mouse models of lung and breast cancer pulmonary metastasis. Among all the recruited inflammatory cell populations, we show that MAMs uniquely express abundant levels of Cav1. Using clodronate depletion of macrophages, we demonstrate that macrophage Cav1 signaling is critical for metastasis and not for primary tumor growth. In particular, Cav1 inhibition does not affect MAM recruitment to the metastatic site but, in turn, favors angiogenesis. We describe a mechanism by which Cav1 in MAMs specifically restrains vascular endothelial growth factor A/vascular endothelial growth factor receptor 1 (VEGF-A/VEGFR1) signaling and its downstream effectors, matrix metallopeptidase 9 (MMP9) and colony-stimulating factor 1 (CSF1).

P0445 : Obeticholic acid, an FXR agonist, reduces hepatic fibrosis in a rat model of toxic cirrhosis

use of ammonia lowering agent, OP, lowers portal pressure in BDL rats, through reduced HSC activation. Methods: In vitro: Primary humanHSC (hHSC) were cultured. Effects of NH4Cl challenge (0.1–10mM over 24–72hrs) on hHSC to proliferation (BrdU), metabolic activity (MTS assay), viability (Neutral-Red), ultrastructural changes (EM) and gene/protein expression were studied. To test recovery, ammonia treated cells were replenished with glutamine and in separate experiments, pre-treated with L-methionine-sulfoximine (MSO-GS inhibitor) to determine the importance of GS. In vivo: 28-day BDL rats were treated with saline or OP for 5 days and portal pressure measured at termination and tissues were harvested for studies. Results: In vitro: Hyperammonemia in primary hHSC induced time-dependent decreases in proliferation and metabolic activity, whilst inducing cell swelling and a myofibroblast-like phenotype even at 50–100umol/L. Ultrastructurally, ammonia-treated hHSC caused a dose-dependent ER enlargement and this was reversible by replenishing the culture with glutamine. NH3 inhibition of hHSC proliferation was dependent on GS activity as MSO with hyperammonemia induced cell detachment and prevention of recovery suggesting that glutamine is important for hHSC survival. In vivo: BDL rats, with hyperammonemia had increased hepatic expression of pro-fibrogenic hHSC-related genes (a-SMA, PDGFb-R, Myosin IIA/IIB and Coll1), low eNOS activity and DDAH-1 and high portal pressure (14.4±0.8mmHg), all of which were corrected by OP treatment (PP: 11.1±0.3mmHg, P < 0.01). Conclusions: These novel data suggest that hyperammonemia modifies hHSC’s and imparts a swollen myofibroblast phenotype, which is reversible upon ammonia reduction. In vivo ammonia lowering decreases pro-fibrogenic and activated HSC gene and protein expression and lowers portal pressure, highlighting ammonia as a target for portal hypertension therapy and the key role of HSC in this process.