SUMMARY Sonic hedgehog (Shh), a soluble ligand overexpres sed by neoplastic cells in pancreatic ductal adenocarcinoma (PDAC), drives formation of a fibroblast-rich desmoplastic stroma. To better understand its role in malignant progression, we deleted Shh in a well-defined mouse model of PDAC. As predicted, Shh-deficient tumors had reduced stromal content. Surprisingly, such tumors were more aggressive and exhibited undifferentiated histology, increased vascularity, and heightened proliferation – features that were fully recapitulated in control mice treated with a Smoothened inhibitor. Furthermore, administration of VEGFR blocking antibody selectively improved survival of Shh-deficient tumors, indicating that Hedgehog-driven stroma suppresses tumor growth in part by restraining tumor angiogenesis. Together, these data demonstrate that some components of the tumor stroma can act to restrain tumor growth.

A pro-angiogenic role for Jagged-dependent activation of Notch signaling in the endothelium has yet to be described. Using proteins that encoded different NOTCH1 EGF-like repeats, we identified unique regions of DLL-class and JAG-class ligand/receptor interactions, and developed Notch decoys that function as ligand-specific Notch inhibitors. N110-24 decoy blocked JAG1/JAG2-mediated NOTCH1 signaling, angiogenic sprouting in vitro and retinal angiogenesis, demonstrating JAG-dependent Notch signal activation promotes angiogenesis. In tumors, N110-24 decoy reduced angiogenic sprouting, vessel perfusion, pericyte coverage, and tumor growth. JAG/NOTCH signaling uniquely inhibited expression of anti-angiogenic sVEFGFR-1/sFlt-1. N11-13 decoy interfered with DLL1/DLL4-mediated NOTCH1 signaling and caused endothelial hypersprouting in vitro, in retinal angiogenesis and in tumors. Thus, blockade of JAG- or DLL-mediated Notch signaling inhibits angiogenesis by distinct mechanisms. JAG/Notch signaling positively regulates angiogenesis by suppressing sVEGFR-1/sFlt-1 and promoting mural/endothelial cell interactions. Blockade of JAG-class ligands represents a novel, viable therapeutic approach to block tumor angiogenesis and growth.

Notch is a critical regulator of angiogenesis, vascular differentiation, and vascular integrity. We investigated whether Notch signaling affects macrophage function during retinal angiogenesis in mice. Retinal macrophage recruitment and localization in mice with myeloid-specific loss of Notch1 was altered, as these macrophages failed to localize at the leading edge of the vascular plexus and at vascular branchpoints. Furthermore, these retinas were characterized by elongated endothelial cell sprouts that failed to anastomose with neighboring sprouts. The role of Notch signaling in macrophages in the developing retina has not previously been assessed.In this study, we show that Notch signaling is important for macrophage recruitment and localization in the developing retina. We also found an increased frequency of elongated sprouts that did not anastomose with neighboring sprouts in retinas in mice with myeloid-specific loss of Notch1. Furthermore, we show Notch signal activation in macrophages that interact with Dll4-positive tip cells, and that macrophages with Notch signaling are found predominately at the vascular front and in association with vascular branchpoints. These data suggest a novel way that Notch signaling regulates retinal angiogenesis. MethodsNotch1 mutant mice have been described. 5 Mice with a conditional allele of Notch1 (Notch1 flox ) 6 and the myeloid-specific Cre recombinase driver line (LysMCre) 7 were obtained from The Jackson Laboratory. Transgenic Notch reporter mice (TNR), harboring an enhanced GFP sequence under the control of 4 tandem copies of the CBF1 binding site consensus sequence 8 were also obtained from The Jackson Laboratory. All procedures were carried out according to approved protocols and guidelines established by the Columbia University Institutional Animal Care and Use Committee. Eyes from postnatal day 5 mice were fixed for 2 hours in 4% paraformaldehyde. Retinas were permeabilized in 1% BSA and 0.5% Triton X-100 overnight at 4°C and washed in PBLEC buffer (1% Triton X-100, 0.1mM MgCl 2 , 0.1mM CaCl 2 , 0.1mM MnCl 2 in PBS pH 6.8), then incubated overnight in PBLEC plus isolectin-B4 (Sigma-Aldrich), anti-F4/80 (Abcam), anti-GFP (Invitrogen), or anti-Dll4 (R&D Systems). After washing, retinas were incubated with Alexa Fluor-conjugated secondary antibodies (Invitrogen), washed, and mounted on slides with Vectashield (Dako) mounting medium for visualization using a LSM Meta 510 or Nikon A1R MP Multiphoton confocal microscope. Results/discussionPrevious studies have shown that loss of Dll4 in mice leads to excessive sprouting during retinal angiogenesis. 3,4 We first investigated whether decreased expression of Notch1 would lead to a similar defect. We found increased vascular density in retinas from Notch1 ϩ/Ϫ mice compared with control littermates ( Figure 1A,C and data not shown), supporting the model where Notch1 and Dll4 in endothelial cells regulate sprouting. Because macrophages are present in the retina and are known to express Notch1, 9,10 we assessed macrophage r...

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Objective Plasma high-density-lipoproteins (HDL) have several putative anti-atherogenic effects, including preservation of endothelial functions. This is thought to be mediated, in part, by the ability of HDL to promote cholesterol efflux from endothelial cells (ECs). The ATP binding cassette transporters A1 and G1 (ABCA1 and ABCG1) interact with HDL to promote cholesterol efflux from ECs. To determine the impact of endothelial cholesterol efflux pathways on atherogenesis, we prepared mice with endothelial-specific knockout of Abca1 and/or Abcg1. Approach and Results Generation of mice with EC-ABCA1 and ABCG1 deficiency required crossbreeding Abca1fl/flAbcg1fl/flLdlr−/− mice with the Tie2Cre strain, followed by irradiation and transplantation of Abca1fl/flAbcg1fl/fl bone marrow to abrogate the effects of macrophage ABCA1 and ABCG1 deficiency induced by Tie2Cre. After 20–22 weeks of Western type diet (WTD), both single EC-Abca1 and Abcg1 deficiency increased atherosclerosis in the aortic root and whole aorta. Combined EC-Abca1/g1 deficiency caused a significant further increase in lesion area at both sites. EC-Abca1/g1 deficiency dramatically enhanced macrophage lipid accumulation in the branches of the aorta that are exposed to disturbed blood flow, decreased aortic eNOS activity, and increased monocyte infiltration into the atherosclerotic plaque. Abca1/g1 deficiency enhanced LPS-induced inflammatory gene expression in mouse aortic ECs, which was recapitulated by ABCG1 deficiency in human aortic ECs. Conclusions These studies provide direct evidence that endothelial cholesterol efflux pathways mediated by ABCA1 and ABCG1 are non-redundant and athero-protective, reflecting preservation of eNOS activity and suppression of endothelial inflammation, especially in regions of disturbed arterial blood flow.