Context- and Cell-Dependent Effects of Delta-Like 4 Targeting in the Bone Marrow Microenvironment

Leonor Remédio,Diana Martins,Francisco Caiado,Hideo Yagita,António Duarte,Ana Bastos-Carvalho,Tânia Carvalho,Sergio Dias

doi:10.1371/journal.pone.0052450

Abstract

Delta-like 4 (Dll4) is a ligand of the Notch pathway family which has been widely studied in the context of tumor angiogenesis, its blockade shown to result in non-productive angiogenesis and halted tumor growth. As Dll4 inhibitors enter the clinic, there is an emerging need to understand their side effects, namely the systemic consequences of Dll4:Notch blockade in tissues other than tumors. The present study focused on the effects of systemic anti-Dll4 targeting in the bone marrow (BM) microenvironment. Here we show that Dll4 blockade with monoclonal antibodies perturbs the BM vascular niche of sub-lethally irradiated mice, resulting in increased CD31+, VE-Cadherin+ and c-kit+ vessel density, and also increased megakaryocytes, whereas CD105+, VEGFR3+, SMA+ and lectin+ vessel density remained unaltered. We investigated also the expression of angiocrine genes upon Dll4 treatment in vivo, and demonstrate that IGFbp2, IGFbp3, Angpt2, Dll4, DHH and VEGF-A are upregulated, while FGF1 and CSF2 are reduced. In vitro treatment of endothelial cells with anti-Dll4 reduced Akt phosphorylation while maintaining similar levels of Erk 1/2 phosphorylation. Besides its effects in the BM vascular niche, anti-Dll4 treatment perturbed hematopoiesis, as evidenced by increased myeloid (CD11b+), decreased B (B220+) and T (CD3+) lymphoid BM content of treated mice, with a corresponding increase in myeloid circulating cells. Moreover, anti-Dll4 treatment also increased the number of CFU-M and -G colonies in methylcellulose assays, independently of Notch1. Finally, anti-Dll4 treatment of donor BM improved the hematopoietic recovery of lethally irradiated recipients in a transplant setting. Together, our data reveals the hematopoietic (BM) effects of systemic anti-Dll4 treatment result from qualitative vascular changes and also direct hematopoietic cell modulation, which may be favorable in a transplant setting.

Highlights

Hematopoiesis is the process by which new blood cells are generated and occurs mainly in the adult bone marrow (BM)
We used six different vascular markers to characterize the effects of anti-Delta-like 4 (Dll4) in the BM vascular niche: CD31, CD105 and VECadherin antibodies, widely used to identify BM endothelial cells [9,13]; VEGFR3 antibody, described as a specific marker of BM sinusoids [13]; SMA antibody, which labels pericytes in arteries and capillaries [22,23,24]; and Lycopersicon esculentum lectin, used as a pan-endothelial marker that stains perfused vessels [18,54]
By day 15 post-irradiation, increased number of CD31+ and VE-Cadherin+ vessels were scored in the BM of anti-Dll4 treated mice, with no significant changes in CD105+, VEGFR3+, SMA+, and lectin+ vessels (Figure 1B, C, S1A)

Summary

Introduction

Hematopoiesis is the process by which new blood cells are generated and occurs mainly in the adult bone marrow (BM). The importance of the BM microenvironment in regulating hematopoiesis has been amply demonstrated by studying the so-called ‘‘stem cell niches’’, in which the endosteal and vascular niches were shown to support hematopoietic stem cells (HSCs) selfrenewal, proliferation, and differentiation [1,2,3,4]. The BM endothelial cells were shown to express different ‘‘angiocrine genes’’, whose production is dependent on the activation of Akt or MAP kinase signaling pathways [29], and whose function is to restore hematopoiesis following insults such as irradiation. Targeting the BM vascular niche and angiocrine genes production to modulate hematopoietic recovery and function may be of clinical relevance. We found Delta-like 4 (Dll, a ligand of the Notch signaling pathway expressed by BM endothelial cells) targeting to potentially fulfill this aim

Methods

Results

Conclusion