Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a life-threatening disease in which intracranial hemorrhage (ICH) is the major risk. Although thrombocytopenia, which is caused by maternal antibodies against β3 integrin and occasionally by maternal antibodies against other platelet antigens, such as glycoprotein GPIbα, has long been assumed to be the cause of bleeding, the mechanism of ICH has not been adequately explored. Utilizing murine models of FNAIT and a high-frequency ultrasound imaging system, we found that ICH only occurred in fetuses and neonates with anti–β3 integrin–mediated, but not anti-GPIbα–mediated, FNAIT, despite similar thrombocytopenia in both groups. Only anti–β3 integrin–mediated FNAIT reduced brain and retina vessel density, impaired angiogenic signaling, and increased endothelial cell apoptosis, all of which were abrogated by maternal administration of intravenous immunoglobulin (IVIG). ICH and impairment of retinal angiogenesis were further reproduced in neonates by injection of anti–β3 integrin, but not anti-GPIbα antisera. Utilizing cultured human endothelial cells, we found that cell proliferation, network formation, and AKT phosphorylation were inhibited only by murine anti–β3 integrin antisera and human anti–HPA-1a IgG purified from mothers with FNAIT children. Our data suggest that fetal hemostasis is distinct and that impairment of angiogenesis rather than thrombocytopenia likely causes FNAIT-associated ICH. Additionally, our results indicate that maternal IVIG therapy can effectively prevent this devastating disorder.
Issaka Yougbaré, Sean Lang, Hong Yang, Pingguo Chen, Xu Zhao, Wei-She Tai, Darko Zdravic, Brian Vadasz, Conglei Li, Siavash Piran, Alexandra Marshall, Guangheng Zhu, Heidi Tiller, Mette Kjaer Killie, Shelley Boyd, Howard Leong-Poi, Xiao-Yan Wen, Bjorn Skogen, S. Lee Adamson, John Freedman, Heyu Ni
Submitter: Shahram Eisa-Beygi | firstname.lastname@example.org
Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
Published September 29, 2015
I believe Yougbaré et al. (1) have misrepresented the findings of a zebrafish paper by Liu et al. (2) twice in the following manner:
1- "In a zebra fish model, impaired angiogenic signaling that affects vascular stability resulted in development of severe hemorrhage in the brain without obvious bleeding in other organs during embryogenesis (23)."
2- "Our findings that impairment of angiogenesis is the mechanism, or at least the major mechanism, that causes ICH in fetuses are consistent with the earlier observations in zebra fish and rabbit models (23)..."
Please note that in Liu et al. (2), in contrast with the aforementioned assertion, angiogenesis is not shown to be the cause of the cerebral hemorrhages in zebrafish embryos. The hemorrhages are shown to arise between 36 and 52 hpf, during which there are no discernable changes in angiogenesis in the brain of these embryos (Figure 3). In fact, delayed angiogenesis is reported only after vascular rupture and hematoma expansion and the mutant embryos eventully 'catch up' with wild-type embryos (Figure 3). More specifically, Liu et al. (2) state that in zebrafish with hemorrhages there is "poor or no contact of endothelial cells with surrounding substratum", which clearly is a case for loss of vascular stability and not impaired angiogenesis as the primary cause for cerebral hemorrhages. The misrepresentation of the findings of Liu et al. (2) by Yougbaré et al. (1) can have implications for their hypothesis, as Yougbaré et al. twice make reference to the erroneous interpretation of zebrafish data in order to support their findings in a murine model.
1. Yougbaré et al. Maternal anti-platelet β3 integrins impair angiogenesis and cause intracranial hemorrhage. J Clin Invest. 2015;125(4):1545–1556.
2. Liu J, et al. A betaPix Pak2a signaling pathway regulates cerebral vascular stability in zebrafish. Proc Natl Acad Sci U S A. 2007;104(35):13990–13995.
Submitter: Heyu Ni | Nih@smh.ca
Authors: Issaka Yougbaré,S. Lee Adamson, and Heyu Ni
Department of Laboratory Medicine and Pathobiology, University of Toronto
Published September 29, 2015
The letter from Eisa-Beygi is trying to differentiate vessel instability from the process of angiogenesis. However, experts in the field do not think it is easy to distinguish these processes. In fact, many would consider vessel instability as the first step in angiogenesis and vessel stabilization as the late step that is required for the completion of angiogenesis. Therefore vessel stability is a component of angiogenesis.
Liu et al (1) found that “The window of time in which hemorrhages appear corresponds to a period of active angiogenesis, when many new vessels carry flow for the first time and when hemodynamic forces are rapidly changing (page 13991).” Further, their article states, "mutant and wild-type vessel pattern was identical through 48 hpf, and all major blood vessels were patterned normally (Fig. 3). However, from that point on, wild-type embryos continued to develop an elaborate pattern of small cranial vessels, whereas bbh mutants retained an immature pattern. Thus, at 77 hpf, the bbhm292 vessel pattern is similar to that of a 48-hpf embryo (page 13993).” In addition, Liu et al report, “the pattern of vessels does not increase in complexity as it does in wild-type embryos at 72 hpf. This immaturity of vascular development could be a secondary effect of hemorrhage or could reflect a defect in migration of endothelial cells (page 13994).” These findings are consistent with our hypothesis and data observed in the murine models of fetal and neonatal alloimmune thrombocytopenia (2).
The mechanism for vessel stability, however, remains to be understood but the interactions between endothelial cells and their environment including matrix proteins as well as other cells (e.g. smooth muscle cells, pericytes, neuroepithelial cells etc.) are clearly important (1). Integrins including β3 integrins (2), as the major responsible receptors for these interactions, certainly play roles in vessel stability. Whether the intracranial hemorrhage observed in our models are mainly due to the maternal antibodies targeting this stage of angiogenesis, as Eisa-Beygi mentioned, is an important question and warrants further investigation.
1. Liu J, et al. A betaPix Pak2a signaling pathway regulates cerebral vascular stability in zebrafish. Proc Natl Acad Sci U S A. 2007;104(35):13990–13995.
2. Yougbaré et al. Maternal anti-platelet β3 integrins impair angiogenesis and cause intracranial hemorrhage. J Clin Invest. 2015;125(4):1545–1556.