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In vivo experiments reveal the good, the bad and the ugly faces of sFlt-1 in pregnancy.

Szalai G, Xu Y, Romero R, Chaiworapongsa T, Xu Z, Chiang PJ, Ahn H, Sundell B, Plazyo O, Jiang Y, Olive M, Wang B, Jacques SM, Qureshi F, Tarca AL, Erez O, Dong Z, Papp Z, Hassan SS, Hernandez-Andrade E, Than NG - PLoS ONE (2014)

Bottom Line: Truncated msFlt-1(1-3) simulated the preeclampsia-promoting effects of full-length hsFlt-1.MsFlt-1(1-3) had strong effect on maternal endothelium but not on placentas and embryos.In accord with the predominant placental expression of hsFlt-1-e15a and msFlt-1-i13, full-length sFlt-1 may have a role in the regulation of embryonic development.

View Article: PubMed Central - PubMed

Affiliation: Perinatology Research Branch, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Department of Health and Human Services, Bethesda, MD, and Detroit, MI, United States of America.

ABSTRACT

Objective: Soluble fms-like tyrosine kinase (sFlt)-1-e15a, a primate-specific sFlt-1-isoform most abundant in the human placenta in preeclampsia, can induce preeclampsia in mice. This study compared the effects of full-length human (h)sFlt-1-e15a with those of truncated mouse (m)sFlt-1(1-3) used in previous preeclampsia studies on pregnancy outcome and clinical symptoms in preeclampsia.

Methods: Mice were injected with adenoviruses or fiber-mutant adenoviruses overexpressing hsFlt-1-e15a, msFlt-1(1-3) or control GFP under the CMV or CYP19A1 promoters on gestational day 8 (GD8) and GD11. Placentas and pups were delivered by cesarean section, and dams were monitored postpartum. Blood pressure was telemetrically recorded. Urine samples were collected with cystocentesis and examined for albumin/creatinine ratios. Tissue specimens were evaluated for transgene as well as endogenous mFlt-1 and msFlt-1-i13 expression. H&E-, Jones- and PAS-stained kidney sections were histopathologically examined. Placental GFP expression and aortic ring assays were investigated with confocal microscopy.

Results: Mean arterial blood pressure (MAP) was elevated before delivery in hsFlt-1-e15a-treated mice compared to controls (GD18: ΔMAP = 7.8 mmHg, p = 0.009), while ΔMAP was 12.8 mmHg (GD18, p = 0.005) in msFlt-1(1-3)-treated mice. Urine albumin/creatinine ratio was higher in hsFlt-1-e15a-treated mice than in controls (GD18, p = 0.04; PPD8, p = 0.03), and msFlt-1(1-3)-treated mice had marked proteinuria postpartum (PPD8, p = 4 × 10(-5)). Focal glomerular changes were detected in hsFlt-1-e15a and msFlt-1(1-3)-treated mice. Aortic ring microvessel outgrowth was decreased in hsFlt-1-e15a (p = 0.007) and msFlt-1(1-3)-treated (p = 0.02) mice. Full-length msFlt-1-i13 expression was unique for the placenta. In hsFlt-1-e15a-treated mice, the number of pups (p = 0.046), total weight of living pups (p = 0.04) and maternal weights (p = 0.04) were higher than in controls. These differences were not observed in truncated msFlt-1(1-3)-treated mice.

Conclusions: Truncated msFlt-1(1-3) simulated the preeclampsia-promoting effects of full-length hsFlt-1. MsFlt-1(1-3) had strong effect on maternal endothelium but not on placentas and embryos. In contrast, hsFlt-1-e15a induced preeclampsia-like symptoms; however, it also increased litter size. In accord with the predominant placental expression of hsFlt-1-e15a and msFlt-1-i13, full-length sFlt-1 may have a role in the regulation of embryonic development. These observations point to the difference in the biological effects of full-length and truncated sFlt-1 and the changes in the effect of full-length sFlt-1 during pregnancy, and may have important implications in the management of preeclampsia.

No MeSH data available.


Related in: MedlinePlus

The development of viral constructs for the various treatment groups.(A) Human and mouse Flt-1 contains seven extracellular Ig-like domains and an intracellular tyrosine kinase, from which the first three Ig-like domains are important in ligand-binding, while the 4–7th Ig-like domains in receptor dimerization. The most abundant placental sFlt-1 variant in humans, sFlt-1-e15a, contains six Ig-like domains and a unique C-terminus encoded by exon 15a, which is located within a primate-specific AluSeq retrotransposon. The truncated mouse sFlt-1 mutant [msFlt-1(1-3)] contains only the first three Ig-like domains of Flt-1. (B) Besides the replication deficient human adenovirus Type5 (dE1/E3), “RGD fiber-mutant” adenoviruses were also used. (C) Besides the CMV promoter that has a strong promoter activity, the human CYP19A1 promoter was also used. CYP19A1 is strongly and predominantly expressed in the placenta among 40 human tissues (left), and its expression increases during trophoblast differentiation (right). Relative gene expressions are shown on the Y-axes. (D–E) Various combinations of viruses, promoters and transgenes used in this study.
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pone-0110867-g002: The development of viral constructs for the various treatment groups.(A) Human and mouse Flt-1 contains seven extracellular Ig-like domains and an intracellular tyrosine kinase, from which the first three Ig-like domains are important in ligand-binding, while the 4–7th Ig-like domains in receptor dimerization. The most abundant placental sFlt-1 variant in humans, sFlt-1-e15a, contains six Ig-like domains and a unique C-terminus encoded by exon 15a, which is located within a primate-specific AluSeq retrotransposon. The truncated mouse sFlt-1 mutant [msFlt-1(1-3)] contains only the first three Ig-like domains of Flt-1. (B) Besides the replication deficient human adenovirus Type5 (dE1/E3), “RGD fiber-mutant” adenoviruses were also used. (C) Besides the CMV promoter that has a strong promoter activity, the human CYP19A1 promoter was also used. CYP19A1 is strongly and predominantly expressed in the placenta among 40 human tissues (left), and its expression increases during trophoblast differentiation (right). Relative gene expressions are shown on the Y-axes. (D–E) Various combinations of viruses, promoters and transgenes used in this study.

Mentions: In order to test the biological effects of differences in hsFlt-1-e15a expression patterns, we used three different viral vectors constructed from replication deficient adenovirus (Type5, dE1/E3) and an “RGD fiber-mutant” adenovirus with distinct tissue-tropism as well as two different gene promoters that differ in terms of tissue-specific promoter activity (Figure 2). The “RGD fiber-mutant” adenovirus that contains an RGD (Arg-Gly-Asp) motif on the fiber knob was developed in conjunction with Vector BioLabs (Philadelphia, PA, USA) according to that described by Mizuguchi et al., 2001 [133]. Adenoviruses and fiber-mutant adenoviruses expressing the full-length hsFlt-1-e15a, the truncated msFlt-1(1-3&^rpar; or green fluorescent protein (GFP) were constructed and titered by Vector BioLabs. HsFlt-1-e15a was overexpressed by 1) a wild-type adenovirus under the control of the cytomegalovirus promoter (Ad-CMV-hsFlt-1-e15a; n = 6), 2) an RGD fiber-mutant adenovirus under the control of the cytomegalovirus promoter (Ad-RGD-CMV-hsFlt-1-e15a; n = 6), or 3) an RGD fiber-mutant adenovirus under the control of the human CYP19A1 promoter (Ad-RGD-CYP-hsFlt-1-e15a; n = 5). Truncated msFlt-1(1-3) was overexpressed by the RGD fiber-mutant adenovirus under the control of the cytomegalovirus promoter [Ad-RGD-CMV-msFlt-1(1-3); n = 6]. GFP was overexpressed by 1) the RGD fiber-mutant adenovirus under the control of the cytomegalovirus promoter (Ad-RGD-CMV-GFP; n = 12), or 2) by the RGD fiber-mutant adenovirus under the control of the human CYP19A1 promoter (Ad-RGD-CYP-GFP; n = 4). According to the technique described by our parallel study [120], mice in these treatment and control groups were injected via the tail vein with 2.5×109 plaque-forming units (PFU) of adenovirus constructs (in 100 µl saline) on GD8 and then repeatedly with 2.5×109 PFU adenoviral constructs or saline on GD11 (Figure 1). A group of mice (n = 9) that was used only for the expression profiling of endogenous mouse transmembrane Flt-1 and sFlt-1-i13 received only 100 µl saline injection on GD8 and GD11 via the tail vein.


In vivo experiments reveal the good, the bad and the ugly faces of sFlt-1 in pregnancy.

Szalai G, Xu Y, Romero R, Chaiworapongsa T, Xu Z, Chiang PJ, Ahn H, Sundell B, Plazyo O, Jiang Y, Olive M, Wang B, Jacques SM, Qureshi F, Tarca AL, Erez O, Dong Z, Papp Z, Hassan SS, Hernandez-Andrade E, Than NG - PLoS ONE (2014)

The development of viral constructs for the various treatment groups.(A) Human and mouse Flt-1 contains seven extracellular Ig-like domains and an intracellular tyrosine kinase, from which the first three Ig-like domains are important in ligand-binding, while the 4–7th Ig-like domains in receptor dimerization. The most abundant placental sFlt-1 variant in humans, sFlt-1-e15a, contains six Ig-like domains and a unique C-terminus encoded by exon 15a, which is located within a primate-specific AluSeq retrotransposon. The truncated mouse sFlt-1 mutant [msFlt-1(1-3)] contains only the first three Ig-like domains of Flt-1. (B) Besides the replication deficient human adenovirus Type5 (dE1/E3), “RGD fiber-mutant” adenoviruses were also used. (C) Besides the CMV promoter that has a strong promoter activity, the human CYP19A1 promoter was also used. CYP19A1 is strongly and predominantly expressed in the placenta among 40 human tissues (left), and its expression increases during trophoblast differentiation (right). Relative gene expressions are shown on the Y-axes. (D–E) Various combinations of viruses, promoters and transgenes used in this study.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4230935&req=5

pone-0110867-g002: The development of viral constructs for the various treatment groups.(A) Human and mouse Flt-1 contains seven extracellular Ig-like domains and an intracellular tyrosine kinase, from which the first three Ig-like domains are important in ligand-binding, while the 4–7th Ig-like domains in receptor dimerization. The most abundant placental sFlt-1 variant in humans, sFlt-1-e15a, contains six Ig-like domains and a unique C-terminus encoded by exon 15a, which is located within a primate-specific AluSeq retrotransposon. The truncated mouse sFlt-1 mutant [msFlt-1(1-3)] contains only the first three Ig-like domains of Flt-1. (B) Besides the replication deficient human adenovirus Type5 (dE1/E3), “RGD fiber-mutant” adenoviruses were also used. (C) Besides the CMV promoter that has a strong promoter activity, the human CYP19A1 promoter was also used. CYP19A1 is strongly and predominantly expressed in the placenta among 40 human tissues (left), and its expression increases during trophoblast differentiation (right). Relative gene expressions are shown on the Y-axes. (D–E) Various combinations of viruses, promoters and transgenes used in this study.
Mentions: In order to test the biological effects of differences in hsFlt-1-e15a expression patterns, we used three different viral vectors constructed from replication deficient adenovirus (Type5, dE1/E3) and an “RGD fiber-mutant” adenovirus with distinct tissue-tropism as well as two different gene promoters that differ in terms of tissue-specific promoter activity (Figure 2). The “RGD fiber-mutant” adenovirus that contains an RGD (Arg-Gly-Asp) motif on the fiber knob was developed in conjunction with Vector BioLabs (Philadelphia, PA, USA) according to that described by Mizuguchi et al., 2001 [133]. Adenoviruses and fiber-mutant adenoviruses expressing the full-length hsFlt-1-e15a, the truncated msFlt-1(1-3&^rpar; or green fluorescent protein (GFP) were constructed and titered by Vector BioLabs. HsFlt-1-e15a was overexpressed by 1) a wild-type adenovirus under the control of the cytomegalovirus promoter (Ad-CMV-hsFlt-1-e15a; n = 6), 2) an RGD fiber-mutant adenovirus under the control of the cytomegalovirus promoter (Ad-RGD-CMV-hsFlt-1-e15a; n = 6), or 3) an RGD fiber-mutant adenovirus under the control of the human CYP19A1 promoter (Ad-RGD-CYP-hsFlt-1-e15a; n = 5). Truncated msFlt-1(1-3) was overexpressed by the RGD fiber-mutant adenovirus under the control of the cytomegalovirus promoter [Ad-RGD-CMV-msFlt-1(1-3); n = 6]. GFP was overexpressed by 1) the RGD fiber-mutant adenovirus under the control of the cytomegalovirus promoter (Ad-RGD-CMV-GFP; n = 12), or 2) by the RGD fiber-mutant adenovirus under the control of the human CYP19A1 promoter (Ad-RGD-CYP-GFP; n = 4). According to the technique described by our parallel study [120], mice in these treatment and control groups were injected via the tail vein with 2.5×109 plaque-forming units (PFU) of adenovirus constructs (in 100 µl saline) on GD8 and then repeatedly with 2.5×109 PFU adenoviral constructs or saline on GD11 (Figure 1). A group of mice (n = 9) that was used only for the expression profiling of endogenous mouse transmembrane Flt-1 and sFlt-1-i13 received only 100 µl saline injection on GD8 and GD11 via the tail vein.

Bottom Line: Truncated msFlt-1(1-3) simulated the preeclampsia-promoting effects of full-length hsFlt-1.MsFlt-1(1-3) had strong effect on maternal endothelium but not on placentas and embryos.In accord with the predominant placental expression of hsFlt-1-e15a and msFlt-1-i13, full-length sFlt-1 may have a role in the regulation of embryonic development.

View Article: PubMed Central - PubMed

Affiliation: Perinatology Research Branch, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Department of Health and Human Services, Bethesda, MD, and Detroit, MI, United States of America.

ABSTRACT

Objective: Soluble fms-like tyrosine kinase (sFlt)-1-e15a, a primate-specific sFlt-1-isoform most abundant in the human placenta in preeclampsia, can induce preeclampsia in mice. This study compared the effects of full-length human (h)sFlt-1-e15a with those of truncated mouse (m)sFlt-1(1-3) used in previous preeclampsia studies on pregnancy outcome and clinical symptoms in preeclampsia.

Methods: Mice were injected with adenoviruses or fiber-mutant adenoviruses overexpressing hsFlt-1-e15a, msFlt-1(1-3) or control GFP under the CMV or CYP19A1 promoters on gestational day 8 (GD8) and GD11. Placentas and pups were delivered by cesarean section, and dams were monitored postpartum. Blood pressure was telemetrically recorded. Urine samples were collected with cystocentesis and examined for albumin/creatinine ratios. Tissue specimens were evaluated for transgene as well as endogenous mFlt-1 and msFlt-1-i13 expression. H&E-, Jones- and PAS-stained kidney sections were histopathologically examined. Placental GFP expression and aortic ring assays were investigated with confocal microscopy.

Results: Mean arterial blood pressure (MAP) was elevated before delivery in hsFlt-1-e15a-treated mice compared to controls (GD18: ΔMAP = 7.8 mmHg, p = 0.009), while ΔMAP was 12.8 mmHg (GD18, p = 0.005) in msFlt-1(1-3)-treated mice. Urine albumin/creatinine ratio was higher in hsFlt-1-e15a-treated mice than in controls (GD18, p = 0.04; PPD8, p = 0.03), and msFlt-1(1-3)-treated mice had marked proteinuria postpartum (PPD8, p = 4 × 10(-5)). Focal glomerular changes were detected in hsFlt-1-e15a and msFlt-1(1-3)-treated mice. Aortic ring microvessel outgrowth was decreased in hsFlt-1-e15a (p = 0.007) and msFlt-1(1-3)-treated (p = 0.02) mice. Full-length msFlt-1-i13 expression was unique for the placenta. In hsFlt-1-e15a-treated mice, the number of pups (p = 0.046), total weight of living pups (p = 0.04) and maternal weights (p = 0.04) were higher than in controls. These differences were not observed in truncated msFlt-1(1-3)-treated mice.

Conclusions: Truncated msFlt-1(1-3) simulated the preeclampsia-promoting effects of full-length hsFlt-1. MsFlt-1(1-3) had strong effect on maternal endothelium but not on placentas and embryos. In contrast, hsFlt-1-e15a induced preeclampsia-like symptoms; however, it also increased litter size. In accord with the predominant placental expression of hsFlt-1-e15a and msFlt-1-i13, full-length sFlt-1 may have a role in the regulation of embryonic development. These observations point to the difference in the biological effects of full-length and truncated sFlt-1 and the changes in the effect of full-length sFlt-1 during pregnancy, and may have important implications in the management of preeclampsia.

No MeSH data available.


Related in: MedlinePlus