Vascular niche IL-6 induces alternative macrophage activation in glioblastoma through HIF-2α.

Spatiotemporal regulation of tumor immunity remains largely unexplored. Here we identify a vascular niche that controls alternative macrophage activation in glioblastoma (GBM). We show that tumor-promoting macrophages are spatially proximate to GBM-associated endothelial cells (ECs), permissive for angiocrine-induced macrophage polarization. We identify ECs as one of the major sources for interleukin-6 (IL-6) expression in GBM microenvironment. Furthermore, we reveal that colony-stimulating factor-1 and angiocrine IL-6 induce robust arginase-1 expression and macrophage alternative activation, mediated through peroxisome proliferator-activated receptor-γ-dependent transcriptional activation of hypoxia-inducible factor-2α. Finally, utilizing a genetic murine GBM model, we show that EC-specific knockout of IL-6 inhibits macrophage alternative activation and improves survival in the GBM-bearing mice. These findings illustrate a vascular niche-dependent mechanism for alternative macrophage activation and cancer progression, and suggest that targeting endothelial IL-6 may offer a selective and efficient therapeutic strategy for GBM, and possibly other solid malignant tumors

Genetic Dissection of Quantitative Trait Loci for Hemostasis and Thrombosis on Mouse Chromosomes 11 and 5 Using Congenic and Subcongenic Strains

<div><p>Susceptibility to thrombosis varies in human populations as well as many inbred mouse strains. Only a small portion of this variation has been identified, suggesting that there are unknown modifier genes. The objective of this study was to narrow the quantitative trait locus (QTL) intervals previously identified for hemostasis and thrombosis on mouse distal chromosome 11 (<i>Hmtb6</i>) and on chromosome 5 (<i>Hmtb4</i> and <i>Hmtb5</i>). In a tail bleeding/rebleeding assay, a reporter assay for hemostasis and thrombosis, subcongenic strain (6A-2) had longer clot stability time than did C57BL/6J (B6) mice but a similar time to the B6-Chr11^A/J consomic mice, confirming the <i>Hmtb</i>6 phenotype. Six congenic and subcongenic strains were constructed for chromosome 5, and the congenic strain, 2A-1, containing the shortest A/J interval (16.6 cM, 26.6 Mbp) in the <i>Hmtb4</i> region, had prolonged clot stability time compared to B6 mice. In the 3A-2 and CSS-5 mice bleeding time was shorter than for B6, mice confirming the <i>Hmtb5</i> QTL. An increase in bleeding time was identified in another congenic strain (3A-1) with A/J interval (24.8 cM, 32.9 Mbp) in the proximal region of chromosome 5, confirming a QTL for bleeding previously mapped to that region and designated as <i>Hmtb10</i>. The subcongenic strain 4A-2 with the A/J fragment in the proximal region had a long occlusion time of the carotid artery after ferric chloride injury and reduced dilation after injury to the abdominal aorta compared to B6 mice, suggesting an additional locus in the proximal region, which was designated <i>Hmtb11</i> (5 cM, 21.4 Mbp). CSS-17 mice crossed with congenic strains, 3A-1 and 3A-2, modified tail bleeding. Using congenic and subcongenic analysis, candidate genes previously identified and novel genes were identified as modifiers of hemostasis and thrombosis in each of the loci <i>Hmtb</i>6, <i>Hmtb</i>4, <i>Hmtb</i>10, and <i>Hmtb</i>11. </p> </div

Tail Bleeding/Rebleeding Assay.

<p>A. First bleeding time. B. Clot Stability-time between first and second bleeding. Values are the mean ± SEM, n=7-28. One-way ANOVA, *P < 0.05, ** P < 0.01.</p

Comparison of Consomic and Congenic crosses.

<p>A. First bleeding time. B. Clot Stability-time between first and second bleeding. Values are the mean ± SEM, n=9-24, one-way ANOVA, * P< 0.05.</p

Genotype of Chromosome 11 Congenic and Subcongenic Mice.

<p>A. Marker positions. White bars-A/J, grey-uncertain, black-B6. B. First bleeding. C. Time between first and second bleeding. Values are the mean ± SEM, n=10-28, one-way ANOVA, * P< 0.05, **P<0.01.</p

Genotype of Chromosome 5 Congenics and Subcongenic Mice.

<p>Marker positions. White bars-A/J, grey-uncertain, black-B6, hatched-heterozygous.</p

EMILIN2 Regulates Platelet Activation, Thrombus Formation, and Clot Retraction

<div><p>Thrombosis, like other cardiovascular diseases, has a strong genetic component, with largely unknown determinants. EMILIN2, Elastin Microfibril Interface Located Protein2, was identified as a candidate gene for thrombosis in mouse and human quantitative trait loci studies. EMILIN2 is expressed during cardiovascular development, on cardiac stem cells, and in heart tissue in animal models of heart disease. In humans, the EMILIN2 gene is located on the short arm of Chromosome 18, and patients with partial and complete deletion of this chromosome region have cardiac malformations. To understand the basis for the thrombotic risk associated with EMILIN2, EMILIN2 deficient mice were generated. The findings of this study indicate that EMILIN2 influences platelet aggregation induced by adenosine diphosphate, collagen, and thrombin with both EMILIN2-deficient platelets and EMILIN2-deficient plasma contributing to the impaired aggregation response. Purified EMILIN2 added to platelets accelerated platelet aggregation and reduced clotting time when added to EMILIN2-deficient mouse and human plasma. Carotid occlusion time was 2-fold longer in mice with platelet-specific EMILIN2 deficiency, but stability of the clot was reduced in mice with both global EMILIN2 deficiency and with platelet-specific EMILIN2 deficiency. <i>In vitro</i> clot retraction was markedly decreased in EMILIN2 deficient mice, indicating that platelet outside-in signaling was dependent on EMILIN2. EMILIN1 deficient mice and EMILIN2:EMILIN1 double deficient mice had suppressed platelet aggregation and delayed clot retraction similar to EMILIN2 mice, but EMILIN2 and EMILIN1 had opposing affects on clot retraction, suggesting that EMILIN1 may attenuate the effects of EMILIN2 on platelet aggregation and thrombosis. In conclusion, these studies identify multiple influences of EMILIN2 in pathophysiology and suggest that its role as a prothrombotic risk factor may arise from its effects on platelet aggregation and platelet mediated clot retraction.</p></div

Aggregation of platelets from E2^-/- and E2p^-/- mice.

<p>A,C. E2^-/- mice. B,D. E2p^-/- mice. A,B. Representative (3–5 experiments repeated 2–3 times with each platelet preparation) aggregometer tracing in PRP induced by 5μM ADP with platelets adjusted to 1.2x10⁸ per ml. C,D. Maximum amplitude from the platelet aggregometry tracings. The aggregation of PRP was induced with 1 μM or 5μM ADP added to PRP with platelets at adjusted to 0.8–1.2x10⁸ per mL pooled from 3 mice. Bars are mean±SEM, performed in triplicate. Statistical analysis, one-way ANOVA, Newman-Keuls post-test **P = 0.01, ***P = 0.001. E2^-/- (EMILIN2 deficient), E2p^-/- (EMILIN2 platelet deficient)</p

Bleeding and Thrombus Formation in E2^-/- and E2p^-/- Mice.

<p>A. D. Tail Bleeding/Rebleeding Assay. Bleeding time (1^st) is between the start of the bleeding and cessation of the bleeding. Clot stability (rebleeding) time (CS) is measured as the time between the cessation of the bleeding and the start of the second bleeding time (2^nd). Bars are mean±SEM, n = 9–11. Statistical Analysis, One-way ANOVA. B, E. Carotid Occlusion Time. Bars are mean±SEM, n = 9–10. Statistical analysis, t-test. C,F. Patency (percent mice with open carotid 4hr after treatment), WTc mice (4/10), E2^-/- mice (6/9). WTp mice (3/5), E2p^-/- (9/10).</p

Role of E2 in Clotting Time and Clot Retraction.

<p>A. Clotting time in WTc and E2^-/- mouse PPP and PRP was induced by CaCl₂. B. Clotting time of PRP upon addition of varying concentrations of recombinant EMILIN2 (rE2). rE2 protein was preincubated with PRP or PPP prior to addition of CaCl₂. C. Clot retraction. Plasma clotted with 1 U/mL thrombin and 5mM CaCl₂, maintained at 37°C, and photographed from 0–120 min after the thrombin addition.</p