The Impact of Chronic Hemolysis on Placental Angiogenesis in a Mouse Model of Sickle Cell Disease

Sickle cell disease (SCD) comprises a group of heritable autosomal recessive mutations in the β-globin gene of hemoglobin. At low oxygen levels, sickle hemoglobin polymerizes, causing red blood cells to sickle and undergo hemolysis. The complex pathophysiology of SCD is characterized by anemia, vaso-occlusive episodes, chronic inflammation and endothelial dysfunction. In women with SCD, pregnancy increases the risk of deep vein thrombosis, pre-eclampsia, pre-term birth, miscarriage, and maternal mortality. Risks to the fetus include intrauterine growth restriction and perinatal mortality. Treatment options are limited to prophylactic blood transfusion and aspirin, yet these lack evidence of improved outcomes.

The placenta is vital for fetal development and maternal-fetal nutrient exchange. Placentas from women with SCD have abnormal morphology and vascularization. Angiogenesis is critical in early placenta development. Pregnant women with SCD have an angiogenic imbalance in circulating levels of vascular endothelial growth factor (VEGF), placental growth factor (PlGF) and their receptor VEGFR1. It is unknown if SCD pregnancy complications result from this imbalance. This study aimed to investigate the role of angiogenic factors in SCD pregnancy and placental health in the Townes mouse model of SCD.

Townes sickle and control female mice were mated with control males, and placentas and blood were collected on gestation day 13.5. Sickle dams had fewer pups (2 ± 1 vs 6 ± 1, p<0.05) and higher fetal resorption (59 ± 12% vs 2 ± 2%, p<0.05) compared to control dams. Histopathology showed sickled RBCs, occlusions, and large areas of fibrin deposition in the maternal decidua and placental labyrinth of sickle dams. Sickle dams showed a trend towards reduced plasma PlGF (48.4 ± 7.5 vs 89 ± 26.9, ns) and higher soluble VEGFR1 levels (1807 ± 1088 vs 1239 ± 271) compared to controls. The sVEGFR1:PlGF ratio was significantly elevated in sickle mice (109.9 ± 49.8 vs 23.19 ±9.36, p<0.05) reflecting an anti-angiogenic imbalance.

We hypothesized that hemolysis-dependent heme release affected placental development. To test this, HTR-8/SVneo human trophoblast cells were treated with hemin (0, 2.5, 5, 10 and 20 µM) for 24 hours. Compared to untreated cells, 20 µM hemin significantly reduced PlGF levels in supernatant by 48 ± 9% (p<0.001), and total protein levels of PlGF in cell lysates by 90% (p<0.05). Interestingly, 20 µM heme modestly, but not significantly, increased total VEGFA and the anti-angiogenic splice variant VEGFA165b in the supernatant, yet VEGFR1 levels were not affected. The reduced PlGF and elevated VEGFA165b suggest that heme causes an imbalance in angiogenic factors produced by trophoblasts.

Data from in vivo and in vitro experiments suggest that chronic hemolysis causes angiogenic imbalance in pregnant sickle dams. Placental angiogenesis, orchestrated by PlGF and VEGFA binding to VEGFR1, is crucial for neo-vascularization via trophoblast invasion of the spiral arteries. Hemin treatment increased VEG165b and decreased PlGF in cultured trophoblasts, indicating that hemolysis may disrupt this balance. PlGF and VEGF work synergistically to form the vascular network in the villous tree, and our study suggests that heme might disrupt this process. Additionally, fibrin deposition, hemorrhage, and occlusion in the placentas of sickle dams indicate vascular damage and reduced perfusion at the maternal-fetal interface. Although hemin did not affect VEGFR1 levels in HTR8/SVneo trophoblast cells, its effect on VEGFR1 expression in microvascular endothelial cells remains to be tested. Future studies will further investigate the role of hemolysis and disrupted angiogenesis in this model to improve outcomes for women with SCD.

No relevant conflicts of interest to declare.