Am J Reprod Immunol. 2018;e12989. wileyonlinelibrary.com/journal/aji | 1 of 10 https://doi.org/10.1111/aji.12989 © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 1 | INTRODUCTION During placental development, macrophages are present in both the maternal (decidual macrophages) and fetal (Hofbauer cells) sides of the placenta. Decidual macrophages are recruited primarily from peripheral monocytes that are derived from bone marrow hemato- poietic stem cells. 1,2 In early pregnancy during trophoblast invasion, these macrophages are located near the spiral arteries and play a role in spiral artery remodeling. 3 They secrete cytokines, growth factors, angiogenic factors, and hormones, and may thus coordinate immune responses and spiral artery remodeling, which are import- ant for a successful term pregnancy. 4 Hofbauer cells are placental tissue macrophages and located primarily in the chorionic villous mesenchyme. 5 They are found in close contact with endothelial pro- genitor cells and primitive vessels, suggesting their critical role in the development of the placental villous tree. 6 Abnormal numbers or Received: 21 February 2018 | Accepted: 6 May 2018 DOI: 10.1111/aji.12989 ORIGINAL ARTICLE Hypoxia regulates placental angiogenesis via alternatively activated macrophages Hui Zhao | Flora S. Kalish | Ronald J. Wong | David K. Stevenson Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, USA Correspondence Hui Zhao, Department of Pediatrics, Division of Neonatology & Developmental Medicine, Stanford University School of Medicine, Stanford, CA, USA. Email: huizhao2@stanford.edu Funding information The Prematurity Research Fund; the March of Dimes Prematurity Research Center at Stanford; the Mary L. Johnson Research Fund; the Christopher Hess Research Fund; Stanford Child Health Research Institute Problem: Uterine and placental macrophages play critical roles in maintaining a nor- mal pregnancy. The majority of these macrophages are believed to be alternatively activated macrophages (M2). Method of Study: Mouse bone marrow cells were differentiated into macrophages and polarized to M2 in vitro by treatment with IL-4 [M2a] or IL-10 [M2c] and M1 with LPS/IFN-γ as controls. Macrophage subtypes were confirmed by surface markers and characterized by gene expression profiles. Results: Compared to M1, M2 showed strong pro-angiogenic activity by expressing higher mRNA for angiogenic-associated factors (Cxcl12, Vegfa, PlGF, Mmp2). M2c produced the highest levels of PlGF, Mmp2, and Cxcr4. To mimic the normal placental microenvironment, M2 were exposed to hypoxia and sex hormones (progesterone, estrogen). In both M2c and M2a, severe hypoxic (1%-3% O 2 ) exposure significantly suppressed PlGF, Cxcl12, and Mmp2 mRNA, but not Vegfa, compared to normoxia (21% O 2 ) or physiological hypoxia (5% O 2 ). mRNA expression returned to normal when hypoxic cells were returned to normoxia. Hypoxia (1%) reduced antioxidant levels in M2 and re-exposure to normoxia significantly increased superoxide dis- mutase (Sod1, Sod2) and heme oxygenase-1 (HO-1) levels in M2a, and only glu- tathione peroxidase (Gpx1, Gpx3, Gpx4) in M2c. However, progesterone and estrogen treatment had minimal effects on angiogenic factor expression in M2. Conclusion: M2, particularly M2c, displayed strong pro-angiogenic potential, which decreased under severe hypoxia such as in early pregnancy. Thus, conditions that alter the placental oxygen microenvironment or macrophage response in early preg- nancy might cause aberrant angiogenesis and vascular remodeling, and lead to ab- normal placental vascular development. KEYWORDS angiogenesis, cytokines, oxygen, placenta, polarization