Endometrial VEGF induces placental sFLT1 and leads to pregnancy complications
Xiujun Fan, Anshita Rai, Neeraja Kambham, Joyce F. Sung, Nirbhai Singh, Matthew Petitt, Sabita Dhal, Rani Agrawal, Richard E. Sutton, Maurice L. Druzin, Sanjiv S. Gambhir, Balamurali K. Ambati, James C. Cross, Nihar R. Nayak
Xiujun Fan, Anshita Rai, Neeraja Kambham, Joyce F. Sung, Nirbhai Singh, Matthew Petitt, Sabita Dhal, Rani Agrawal, Richard E. Sutton, Maurice L. Druzin, Sanjiv S. Gambhir, Balamurali K. Ambati, James C. Cross, Nihar R. Nayak
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Research Article

Endometrial VEGF induces placental sFLT1 and leads to pregnancy complications

Abstract

There is strong evidence that overproduction of soluble fms-like tyrosine kinase-1 (sFLT1) in the placenta is a major cause of vascular dysfunction in preeclampsia through sFLT1-dependent antagonism of VEGF. However, the cause of placental sFLT1 upregulation is not known. Here we demonstrated that in women with preeclampsia, sFLT1 is upregulated in placental trophoblasts, while VEGF is upregulated in adjacent maternal decidual cells. In response to VEGF, expression of sFlt1 mRNA, but not full-length Flt1 mRNA, increased in cultured murine trophoblast stem cells. We developed a method for transgene expression specifically in mouse endometrium and found that endometrial-specific VEGF overexpression induced placental sFLT1 production and elevated sFLT1 levels in maternal serum. This led to pregnancy losses, placental vascular defects, and preeclampsia-like symptoms, including hypertension, proteinuria, and glomerular endotheliosis in the mother. Knockdown of placental sFlt1 with a trophoblast-specific transgene caused placental vascular changes that were consistent with excess VEGF activity. Moreover, sFlt1 knockdown in VEGF-overexpressing animals enhanced symptoms produced by VEGF overexpression alone. These findings indicate that sFLT1 plays an essential role in maintaining vascular integrity in the placenta by sequestering excess maternal VEGF and suggest that a local increase in VEGF can trigger placental overexpression of sFLT1, potentially contributing to the development of preeclampsia and other pregnancy complications.

Authors

Xiujun Fan, Anshita Rai, Neeraja Kambham, Joyce F. Sung, Nirbhai Singh, Matthew Petitt, Sabita Dhal, Rani Agrawal, Richard E. Sutton, Maurice L. Druzin, Sanjiv S. Gambhir, Balamurali K. Ambati, James C. Cross, Nihar R. Nayak

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Figure 7

Effective sFlt1 knockdown in placentas by trophoblast-specific sFLT1 shRNA expression.

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Effective sFlt1 knockdown in placentas by trophoblast-specific sFLT1 shR...
(A and B) Blastocysts transduced with lentiviruses expressing GFP control (A) or both sFLT1 shRNA and GFP (B) expressed GFP (denoted by #) in the trophectoderm, but not the inner cell mass. (C and D) Full-thickness placenta on GD18 showing GFP expression in trophoblast lineages from blastocysts transduced with control (C) and sFLT1 shRNA (D) viruses. (E and F) Magnified images from C and D showing GFP expression in the labyrinth layer. (G and H) ISH showing dramatic reduction of sFlt1 signal in sFLT1 shRNA–expressing placentas (H) relative to controls (G); signals were found primarily in GlyTCs and spongiotrophoblasts in the JZ (arrows) and the invading GlyTCs below the giant cell layer (arrowheads). Ch, chorionic plate; La, labyrinth layer. (I) Representative Western blots showing a substantial sFLT1 shRNA–mediated decrease in placental sFLT1 levels on GD18 without changes in VEGF levels. (J–M) qPCR of placental sFlt1 mRNA (J), quantification of placental sFLT1 protein by Western blot (K), and ELISA of maternal serum sFLT1 protein (M) revealed significant reductions at GD18 in placental sFlt1 knockdown animals, whereas (L) placental VEGF/sFLT1 protein ratio was significantly increased by sFlt1 knockdown. Results are mean ± SD. *P < 0.05 (n = 5). Scale bars: 20 μm (A and B); 500 μm (C, D, G, and H); 100 μm (E and F).