Introduction: Preeclampsia (PE) is a life-threatening hypertensive disorder of pregnancy. Soluble fms-like tyrosine kinase 1 (sFlt-1) is elevated and known to contribute to PE. However, molecular basis underlying its up-regulation in PE remains unclear.
Hypothesis: We hypothesized that HIF-1α, an important transcriptional factor, and U2AF65, a key factor involved in alternative splicing, may work collaboratively underlying elevation of sFlt-1 in the placentas of PE.
Methods: To test these possibilities, immunohistochemistry and Western blot were used to determine the protein level of HIF-1α and U2AF65 in the placental tissue from PE patients. Two animal models of PE including angiotensin II type 1 receptor agonistic autoantibody (AT1-AA) and LIGHT (a TNF-α superfamily member)-induced PE mouse model coupled with in vivo nanoliposome-delivery system to specifically knockdown HIF-1α and U2AF65 were used to determine the specific biological function of HIF-1α and U2AF65 in PE. Finally, splice-specific PCR assay were used to determinewhether HIF-1α and U2AF65 underlyingsFlt-1 production at both transcriptional and post- transcriptional level in PE.
Results: We found that HIF-1α and U2AF65 were significantly increased in the placental tissue from PE patients. Then, we demonstrated knockdown of HIF-1α or U2AF65 by siRNA significantly attenuated AT1-AA-induced hypertension, proteinuria and circulating sFlt-1 level. Similarly, we found that HIF-1α and U2AF65 knockdown significantly reduced sFlt-1 levels, hypertension and proteinuria in LIGHT-infused pregnant mice. Finally, we found that knockdown HIF-1α significantly reduced total Flt-1 mRNA level in the placentas of both AT1-AA and LIGHT-infused pregnant mice but no effect on the ratio of sFlt-1/ Flt-1. In contrast, U2AF65 knock down significantly reduced the ratio of sFlt-1/ Flt-1 without an effect on total Flt-1 mRNA level in these two PE mouse models.
Conclusions: Overall, we have revealed two molecular bases underlying sFlt-1 inductions in PE: 1) elevated HIF-1α directly increases sFlt-1 levels under transcriptional levels; 2) elevated U2AF65 enhances alternative splicing to increase sFlt-1 levels. These studies immediately suggest novel therapeutics for PE.