Abstract 004: Pro-angiogenic Effects Of The Thrombospondin-1 Inhibitor Lskl In Preeclampsia Models

Abstract

Introduction: Preeclampsia (PE) is characterized by high blood pressure, proteinuria and/or multi-organ damage, reaching 10% of all pregnancies globally. To date, no medication can treat PE. We recently identified an upregulation of the antiangiogenic protein Thrombospondin-1 (THBS1) in human and experimental PE. THBS1 is a main activator of latent TGF-β. We postulate that THBS1-mediated TGF-β activation can contribute to impair placental angiogenesis during PE. In this study, we aim to test the effects of LSKL, an inhibitor of TGF-β activation by THBS1, on angiogenesis in placentas of a mouse model of PE and in human placental endothelial cells exposed to PE-like conditions. Methods: Heterozygous female mice overexpressing human angiotensinogen and renin (PE) or C57BL/6 (control) were treated with LSKL or control peptide SLLK (280 μM, sc) at gestational day (GD) 14.5 (n=3 pregnant mouse/group). Placentas (n=4-9/pregnancy) were collected at GD 18.5. Placental expressions of TGF-β1, β2, total/phospho SMAD2 were assessed by western blot and angiogenesis by immunohistochemistry. Endothelial cells were extracted by magnetic sorting from human placentas (n=4) of healthy pregnancies. Cells were treated with LSKL or SLLK (30 μM) under hypoxia (0.5% vs. 8% O 2 ) or thrombin (10 u/mL). THBS1 expression in cells was assessed by immunofluorescence. Cell angiogenesis was quantified by the number of closed tubes formed on matrigel (per 15,000 cells, 4h). Results: LSKL treatment reduced the expression of TGF-β2 (P<0.01) and phospho-SMAD2 (P<0.05) and significantly improved angiogenesis (P<0.05) in placentas of PE mouse versus control. In cells, both thrombin (P<0.05) and hypoxia (P<0.05) significantly increased THBS1 expression. Thrombin (P<0.01) and hypoxia (P<0.05) significantly impaired cell angiogenesis on matrigel (P<0.01), which was prevented by LSKL treatment (P<0.01). LSKL similarly reduced TGB-β2 (p<0.05) and phospho-SMAD2 (P<0.05) expressions in endothelial cells under thrombin and hypoxia cell exposure. Conclusion: Our results describe a significant pro-angiogenic effect of LSKL treatment by regulating THBS1 and TGF-β2 mechanisms in an experimental model of PE and in human placental endothelial cells exposed to PE-like conditions.