Control of soluble fms-like tyrosine-1 (sFlt-1) production response to placental ischemia/hypoxia: role of tumor necrosis factor-α

Abstract

Although abnormal soluble fms-like tyrosine kinase-1 (sFlt-1) production is thought to be an important factor in the pathogenesis of preeclampsia (PE), the mechanisms that regulate the production of sFlt-1 during PE are unclear. While our laboratory has shown tumor necrosis factor-α (TNF-α) and sFlt-1 to be elevated in pregnant rats in response to placental ischemia, the importance of TNF-α in the regulation of sFlt-1 production is unknown. Therefore, the purpose of this study was to determine the role of TNF-α in mediating the increase in sFlt-1 in response to placental ischemia or hypoxia. Reductions in uterine perfusion pressure in pregnant rats significantly increased plasma levels of sFlt-1 and tended to increase TNF-α, an effect markedly attenuated by pretreatment with a TNF-α inhibitor etanercept (0.4 mg/kg). To further assess chronic interactions between TNF-α and sFlt-1, we examined a chronic effect of TNF-α infusion (50 ng/day) into normal pregnant rats to increase plasma sFlt-1 levels, as well as the effects of acute hypoxia on placental sFlt-1 production in the absence and presence of TNF-α blockade. Placental explants exposed to hypoxic conditions had enhanced TNF-α levels versus normoxic conditions, as well as increased sFlt-1 production. Pretreatment of placental explants with etanercept (15 μM) significantly reduced TNF-α levels in response to hypoxia but did not attenuate sFlt-1 production. These data suggest that while TNF-α may not play an important role in stimulating sFlt-1 production in response to acute hypoxia, a more chronic hypoxia, or placental ischemia may be an important stimulus for enhanced sFlt-l production.

A Maternally Sequestered, Biopolymer‐Stabilized Vascular Endothelial Growth Factor (VEGF) Chimera for Treatment of Preeclampsia

“…We found that spiking the ELISA standard curves with ELP‐VEGF reduced the signal, suggesting that ELP‐VEGF might bind the Flt‐1 in the assay and prevent binding of 1 or both antibodies, thus confirming this as an assay for free sFlt‐1. Again, RUPP surgery resulted in a significant increase in free plasma sFlt‐1 (Figure 4C), consistent with previous reports 32, 36, 53, 56, 58, 63, 68. In contrast to the Western blot data, the ELISA data showed that ELP‐VEGF‐treated rats (both sham and RUPP surgical groups) had markedly reduced free sFlt‐1 levels (Figure 4C).…”

“…These bands correspond to the report of Rajakumar,67 who describes sFlt‐1 bands of 145, 100, and 60 kDa in plasma from preeclamptic patients, though considerable species and glycosylation variation could exist. The RUPP surgery leads to a significant increase in plasma sFlt‐1, as has previously been reported 32, 36, 53, 56, 58, 68. This was confirmed in a separate cohort of rats, treated entirely independently with the sham or RUPP surgeries, and analyzed in a separate Western blot (Figure S1).…”

“…Studies using viral overexpression33, 57, 59, 60, 61 or direct infusion of sFlt‐132, 55, 62, 63 have demonstrated a critical role for this anti‐angiogenic factor for induction of hypertension and renal damage during pregnancy. Previous studies using exogenous VEGF infusion have reported reduced circulating sFlt‐1 levels 36, 64.…”

“…In addition to Western blotting for total sFlt‐1 levels, we also assessed plasma sFlt‐1 by ELISA. The ELISA used has been previously reported to be specific for free, non‐VEGF‐bound, sFlt‐1 32, 53, 55, 56. We found that spiking the ELISA standard curves with ELP‐VEGF reduced the signal, suggesting that ELP‐VEGF might bind the Flt‐1 in the assay and prevent binding of 1 or both antibodies, thus confirming this as an assay for free sFlt‐1.…”

“…Unable to meet the metabolic demands of the developing fetus because of this compromised arterial blood supply, the placenta enters a chronically hypoxic/ischemic state,27 which activates pathways that increase the production of inflammatory cytokines,20 reactive oxygen species,28 and angiogenic imbalance factors including the soluble form of the vascular endothelial growth factor (VEGF) receptor Flt‐1 (sFlt‐1),29 all of which lead to systemic endothelial dysfunction that is manifested as hypertension 30. Among these, sFlt‐1 has been implicated as a major driver of the preeclampsia syndrome because of its high levels in the plasma of preeclamptic mothers31 and the fact that exogenously administered sFlt‐1 induces a preeclampsia‐like syndrome in pregnant rodents 32, 33, 34, 35…”