Maternal anti-platelet β3 integrins impair angiogenesis and cause intracranial hemorrhage

Abstract

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.

Figures

Figure 7. Anti-β3 antibody decreased AKT phosphorylation and increased apoptosis in HUVECs.

(A) Human anti–HPA-1a IgG and murine anti-β3 sera significantly reduced AKT phosphorylation in HUVECs. (B) Human anti–HPA-1a IgG significantly increased caspase-3 activation. Results are pooled from 4 independent experiments. Statistical analysis was performed using an unpaired, 2-tailed Student’s t test. Mean ± SEM. n = 3 wells per group. ***P < 0.001. Scale bar: 200 μm. Original magnification, ×10.

Figure 6. Mouse anti-β3 sera and human anti–HPA-1a IgG inhibit EC proliferation and network formation on Matrigel.

(A) Anti–HPA-1a IgG binding to HUVECs was tested with MAIPA assay. (B and C) Anti-β3 sera significantly inhibited HUVEC proliferation compared with naive control sera. HUVECs seeded onto Matrigel-coated wells were incubated with EGM-2 media containing murine sera (D) or containing human IgG (E), as indicated. HUVECs treated with naive sera or anti-GPIbα sera and control IgG formed capillary-like networks, while anti-β3 sera and anti–HPA-1a inhibited this process in a dose-dependent manner. Results are pooled from 4 independent experiments. Statistical analysis was performed using unpaired, 2-tailed Student’s t test. Mean ± SEM; n = 3 wells per group. *P < 0.05; **P < 0.01; ***P < 0.001. Scale bars: 200 μm (D); 500 μm (E). Original magnification, ×10 (D); ×4(E).

Figure 5. IVIG reverses the impairment in retinal and brain vascular development in anti- β3–mediated FNAIT pups.

(A) Representative images and quantification of brain sections stained by peroxidase immunohistochemistry for vWF. (B) Representative fluorescent images and quantification of isolectin (IB4 conjugated to Alexa Fluor 594) immunostaining of retinas from P2 pups. (C) Brain and (D) retina vessel length were quantified. Blood vessel development in the retina and brain of pups from immunized β3–/– mice treated with albumin was significantly impaired compared with that in pups from naive β3–/– mice. IVIG significantly reversed the inhibition of vessel growth. Statistical analysis was performed using 1-way ANOVA followed by Bonferroni’s post-hoc test. Mean ± SEM; n = 4–6 mice per group. **P < 0.01; ***P < 0.001. Scale bars: 200 μm (A) 500 μm (B). Original magnification, ×10 (A); ×4 (B).

Figure 4. Anti-β3 antibody decreased proangiogenic and AKT signaling in brain.

(A) Brain homogenates from heterozygote pups with FNAIT were analyzed with the mouse angiogenesis antibody cytokine array kit (R&D Systems). Anti-β3–mediated FNAIT significantly downregulated angiogenic growth factors, but upregulated angiogenic inhibitor TSP-1. Representative images and densitometry quantification of Western blotting of P2 brain lysates from FNAIT pups (B) and sera-injected pups (C) are shown. Phosphorylated AKT was normalized to total AKT and expressed as a percentage of the naive control group. Statistical analysis was performed using an unpaired, 2-tailed Student’s t test (A and C) and 1-way ANOVA followed by Bonferroni’s post-hoc test (B). Results are representative of at least 3 independent experiments of Western blots. Mean ± SEM. n = 3 mice per group. **P < 0.01; ***P < 0.001.

Figure 3. Increased apoptosis in the brain vessels of anti-β3–mediated FNAIT pups.

(A) Representative images of TUNEL (green) and vWF (red) costaining are presented. (B) Area of colocalized fluorescence (yellow) was quantified using ImageJ. Increased apoptosis was detected in the brain blood vessels of anti-β3–mediated FNAIT pups compared with naive controls and anti-GPIbα–mediated FNAIT pups. Statistical analysis was performed using 1-way ANOVA followed by Bonferroni’s post-hoc test. Mean ± SEM. n = 3–6 mice per group. ***P < 0.001. Scale bar: 50 μm. Original magnification, ×40.

Figure 2. Severely impaired vascularization observed in brain and retina of anti-β3–mediated FNAIT pups.

(A) Representative images and (B) quantification of IB4 staining of brain blood vessels are presented. Anti-β3–mediated FNAIT pups had reduced blood vessel density compared with anti-GPIbα–mediated FNAIT pups or naive controls. Representative images and quantification of FNAIT (C and D) or sera-injected (E and F) pup retinas immunostained by anti-collagen IV antibody and revealed with anti-rabbit conjugated to Alexa Fluor 488. Vessel development in the retina from anti-β3–mediated FNAIT and anti-β3 sera–injected pups was significantly impaired compared with that of naive controls and anti-GPIbα–mediated FNAIT and anti-GPIbα sera–injected pups. Results are representative of at least 4 independent experiments. All of the statistical comparisons were performed using 1-way ANOVA followed by Bonferroni’s post-hoc test. Mean ± SEM. n = 4–6 mice per group. **P < 0.01. Scale bars: 200 μm (A); 500 μm (C, E). Original magnification, ×10 (A); ×4 (C, E).

Figure 1. ICH in anti-β3–mediated FNAIT neonates and pups.

(A) Anti-platelet antibodies were detected in both immunized β3–/– and GPIbα–/– mice. (B) Platelet counts were lower in neonates delivered from immunized β3–/– and GPIbα–/– mice compared with those delivered from naive mice. (C, D, and F) ICH was observed in anti-β3–mediated FNAIT pups (black arrows), but not in naive controls or anti-GPIbα–mediated FNAIT pups. Frequency of ICH increased with the number of maternal immunizations in anti-β3–mediated FNAIT fetuses. 2× and 3× indicates the number of immunizations. (E) Representative images of high-frequency ultrasound of ICH (red arrow) in utero at E15.5. (G and H) P2 pups were injected with naive sera and anti-sera. ICH was only observed in β3 heterozygote and αIIb pups after anti-β3 sera injection. Statistical analysis was performed using unpaired, 2-tailed Student’s t test (A and B) and 1-way ANOVA followed by Bonferroni’s post-hoc test (C and D). Data were collected from more than 5 pregnancies per group. Mean ± SEM. **P < 0.01; ***P < 0.001. Original magnification, ×1 (H&E staining of brain sections ). Scale bar: 100 μm.