Gestational Loss and Growth Restriction by Angiogenic Defects in Placental Growth Factor Transgenic Mice Min-cheol Kang, Seo Jin Park, Hei Jung Kim, Jinhee Lee, Dong Hoon Yu, Ki Beom Bae, Young Rae Ji, Si Jun Park, Jain Jeong, Woo Young Jang, Jung-Hak Kim, Myung-Sook Choi, Dong-Seok Lee, Hyun-Shik Lee, Sanggyu Lee, Sung Hyun Kim, Myoung Ok Kim, Gyeongsin Park, Yeon Sik Choo, Je-Yoel Cho,* ZaeYoung Ryoo* Objective—Angiogenesis is an important biological process during development, reproduction, and in immune responses. Placental growth factor (PlGF) is a member of vascular endothelial growth factor that is critical for angiogenesis and vasculogenesis. We generated transgenic mice overexpressing PlGF in specifically T cells using the human CD2-promoter to investigate the effects of PlGF overexpression. Approach and Results—Transgenic mice were difficult to obtain owing to high lethality; for this reason, we investigated why gestational loss occurred in these transgenic mice. Here, we report that placenta detachment and inhibition of angiogenesis occurred in PlGF transgenic mice during the gestational period. Moreover, even when transgenic mice were born, their growth was restricted. Conclusions—Conclusively, PlGF overexpression prevents angiogenesis by inhibiting Braf, extracellular signal–regulated kinase activation, and downregulation of HIF-1α in the mouse placenta. Furthermore, it affected regulatory T cells, which are important for maintenance of pregnancy. (Arterioscler Thromb Vasc Biol. 2014;34:2276-2282.) Key Words: BRAF kinases
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extracellular signal-regulated kinases ■ regulatory T-cells
A
ngiogenesis, the process by which new blood vessels form from pre-existing vessels, differs from vasculogenesis in that mesoderm precursor cells differentiate into new endothelial cells in angiogenesis. Vasculogenesis is the process by which the first vessels form during embryonic development.1,2 Placental growth factor (PlGF), a subfamily of the vascular endothelial growth factor (VEGF), is a key molecule in angiogenesis and vasculogenesis, especially during embryogenesis. VEGF binds to VEGF receptor-1, a key receptor involved in vessel formation during embryogenesis. VEGF-B and PlGF can also bind VEGF receptor-1.3–5 PlGF has been found in the placenta,6,7 where it was shown to control trophoblast growth and differentiation and to exhibit high expression in placenta from women with fetal growth restriction.8 Many tissues and organs, including the villous trophoblast and heart, express PlGF, and endothelial cells express PlGF during vascular development.9–13 Moreover, the placental trophoblast is the main source of PlGF during pregnancy, and its expression is significantly upregulated at an early gestational age after implantation.9 PlGF knockout mice do not have defects in vascular development
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miscarriage
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placenta growth factor
and show normal embryogenesis. However, reduced collateral vascular growth has been observed in PlGF knockout mice under pathological conditions such as inflammation or ischemia.14 Presently, the function of PlGF and its related signaling mechanisms remain poorly understood. PlGF is related to survival and the angiogenesis pathway through phosphatidylinositol 3-kinase, downstream of VEGF receptor-1.15 PlGF also significantly upregulates extracellular signal–regulated kinase (ERK)-1 and ERK-2 activity in endothelial cells. Moreover, PlGF activates ERK1/2 kinases, which are associated with cellular survival and proliferation. ERK activation in the placenta is essential to vascular development. Mice in which the gene encoding this pathway member has been deleted often show embryonic death because of abnormal development of the placenta.16 Accordingly, it is possible that abnormal expression of PlGF during pregnancy influences trophoblast function as much as vascularity in the placental bed.17 The purpose of this study was to determine the biological function of PlGF in placental development from fetus under increased PlGF expression in T cell.
Received on: January 22, 2014; final version accepted on: August 8, 2014. From the School of Life Sciences and Biotechnology (M.K., S.J.P., H.J.K., J.L., D.H.Y., K.B.B., Y.R.J., S.J.P., J.J., W.Y.J., J.-H.K., D.-S.L., H.-S.L., S.L., S.H.K., M.O.K., Z.Y.R.), Department of Food Science and Nutrition (M.S.C.), and School of Biology (Y.S.C.), Kyungpook National University, Daegu, Korea; Department of Pathology, Seoul St. Mary’s Hospital, The Catholic University of Korea, Seoul, Korea (G.P.); and Department of Biochemistry, College of Veterinary Medicine, Seoul National University, Seoul, Korea (J.-Y.C.). *These authors contributed equally to this article. The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi: 10.1161/ATVBAHA.114.303693/-/DC1. Correspondence to Zae Young Ryoo, PhD, Kyungpook National University, 1370 Sankyuk-dong, Buk-ku, Daegu, 702-701, Korea. E-mail jaewoong64@ hanmail.net © 2014 American Heart Association, Inc. Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org
DOI: 10.1161/ATVBAHA.114.303693
Downloaded from http://atvb.ahajournals.org/ 2276 at CONS CALIFORNIA DIG LIB on April 1, 2015
Kang et al A Critical Role of PlGF in the Development 2277
Nonstandard Abbreviations and Acronyms eNOS ERK PlGF VEGF WT
endothelial nitric oxide synthase extracellular signal–regulated kinase placenta growth factor vascular endothelial growth factor wild type
Materials and Methods Materials and Methods are available in the online-only Supplement.
Results Generation of a Transgenic Mouse With T-Cell–Specific Overexpression of the PlGF PlGF is a subfamily of VEGF, which is the gene responsible for angiogenesis. Many studies have suggested that angiogenesis is closely associated with T cells18; therefore, we generated transgenic mice that overexpress PlGF in T cells using the human CD-2 promoter (Figure 1A). To evaluate the transgenic founder, we performed polymerase chain reaction reactions with genomic DNA (Figure 1B). We examine the number of copies by Fluorescence in situ hybridization analysis. The result shows that all 3 lines have 5 to 8 copies. And all 3 lines are shown similar phenotype of placenta and embryo development (data not shown). Moreover, we isolated mRNA from the spleen of 8-week-old littermates to determine whether PlGF mRNA is overexpressed in transgenic mice. All transgenic mice showed appropriate insertion into genomic DNA,
and PlGF mRNAs were overexpressed in transgenic mice (≥3-fold), whereas PlGF knockout mice did not exhibit PlGF mRNA expression (Figure 1C). In addition, when splenocytes were cultured with anti-CD3/CD28 antibody, secretion of PlGF was increased significantly compared with wild-type (WT) mice, whereas the serum PlGF level was unchanged (Figure 1D).
Defects of Litter Size and Weight Gain in PlGF Overexpressing Mice When hetero transgenic males were crossed with WT females, the females occasionally gave birth to dead fetuses on postnatal day 1. Genomic DNA analysis of the dead fetuses revealed that they were transgenic mice (Figure 2A). Furthermore, analysis of the litter size and birth rate revealed that littermates of PlGF transgenic mice were smaller than WT mice and control transgenic mice that expressed another gene19 by the human CD-2 promoter (Figure 2B and 2C). Interestingly, the birth rate of transgenic mice did not follow Mendelian inheritance. We suggest the possibility that the human CD-2 promoter generates nonaffected in transgenic mice. Because sizes of littermates were smaller compared with those of the WT, we measured the body weight of littermates every 2 weeks to examine whether PlGF can also affect growth. The body weights of PlGF transgenic mice were considerably lower than those of WT mice, the differences increased gradually (Figure 2D). Taken together, these findings indicate that overexpression of PlGF may cause death during gestation and that growth is restricted even after birth.
Figure 1. Targeted overexpression of placental growth factor (PlGF) in the T cells of transgenic mice. A, Transgenic construct for T-cell–specific expression of the PlGF gene. The primers used for tail biopsy polymerase chain reaction (PCR) are indicated by arrows. B, Identification of transgenic mice harboring the recombinant human CD-2 (hCD2)-PlGF gene. Three mice were determined to be positive by PCR and used as founders. C, We confirmed mRNA expression of PlGF in the spleen of 8-week-old mice. Real-time PCR of indicated genes from the spleens of wild-type (WT), knockout (KO), and transgenic (Tg) mice with T-cell activation via CD3/CD28 ex vivo. Splenocytes from WT, KO, and Tg mice were cultured for 12 hours in the presence of anti-CD3/CD28, after which the cultured cells were analyzed for mRNA level by real-time PCR. D, Splenocytes from WT, KO, and Tg mice were cultured for 48 hours in the presence of anti-CD3/CD28, after which the supernatant was analyzed for secreted PlGF using an ELISA kit. Serum PlGF concentrations in WT and Tg mice. Sera were obtained from 8-week-old mice. ***P
■
extracellular signal-regulated kinases ■ regulatory T-cells
A
ngiogenesis, the process by which new blood vessels form from pre-existing vessels, differs from vasculogenesis in that mesoderm precursor cells differentiate into new endothelial cells in angiogenesis. Vasculogenesis is the process by which the first vessels form during embryonic development.1,2 Placental growth factor (PlGF), a subfamily of the vascular endothelial growth factor (VEGF), is a key molecule in angiogenesis and vasculogenesis, especially during embryogenesis. VEGF binds to VEGF receptor-1, a key receptor involved in vessel formation during embryogenesis. VEGF-B and PlGF can also bind VEGF receptor-1.3–5 PlGF has been found in the placenta,6,7 where it was shown to control trophoblast growth and differentiation and to exhibit high expression in placenta from women with fetal growth restriction.8 Many tissues and organs, including the villous trophoblast and heart, express PlGF, and endothelial cells express PlGF during vascular development.9–13 Moreover, the placental trophoblast is the main source of PlGF during pregnancy, and its expression is significantly upregulated at an early gestational age after implantation.9 PlGF knockout mice do not have defects in vascular development
■
miscarriage
■
placenta growth factor
and show normal embryogenesis. However, reduced collateral vascular growth has been observed in PlGF knockout mice under pathological conditions such as inflammation or ischemia.14 Presently, the function of PlGF and its related signaling mechanisms remain poorly understood. PlGF is related to survival and the angiogenesis pathway through phosphatidylinositol 3-kinase, downstream of VEGF receptor-1.15 PlGF also significantly upregulates extracellular signal–regulated kinase (ERK)-1 and ERK-2 activity in endothelial cells. Moreover, PlGF activates ERK1/2 kinases, which are associated with cellular survival and proliferation. ERK activation in the placenta is essential to vascular development. Mice in which the gene encoding this pathway member has been deleted often show embryonic death because of abnormal development of the placenta.16 Accordingly, it is possible that abnormal expression of PlGF during pregnancy influences trophoblast function as much as vascularity in the placental bed.17 The purpose of this study was to determine the biological function of PlGF in placental development from fetus under increased PlGF expression in T cell.
Received on: January 22, 2014; final version accepted on: August 8, 2014. From the School of Life Sciences and Biotechnology (M.K., S.J.P., H.J.K., J.L., D.H.Y., K.B.B., Y.R.J., S.J.P., J.J., W.Y.J., J.-H.K., D.-S.L., H.-S.L., S.L., S.H.K., M.O.K., Z.Y.R.), Department of Food Science and Nutrition (M.S.C.), and School of Biology (Y.S.C.), Kyungpook National University, Daegu, Korea; Department of Pathology, Seoul St. Mary’s Hospital, The Catholic University of Korea, Seoul, Korea (G.P.); and Department of Biochemistry, College of Veterinary Medicine, Seoul National University, Seoul, Korea (J.-Y.C.). *These authors contributed equally to this article. The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi: 10.1161/ATVBAHA.114.303693/-/DC1. Correspondence to Zae Young Ryoo, PhD, Kyungpook National University, 1370 Sankyuk-dong, Buk-ku, Daegu, 702-701, Korea. E-mail jaewoong64@ hanmail.net © 2014 American Heart Association, Inc. Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org
DOI: 10.1161/ATVBAHA.114.303693
Downloaded from http://atvb.ahajournals.org/ 2276 at CONS CALIFORNIA DIG LIB on April 1, 2015
Kang et al A Critical Role of PlGF in the Development 2277
Nonstandard Abbreviations and Acronyms eNOS ERK PlGF VEGF WT
endothelial nitric oxide synthase extracellular signal–regulated kinase placenta growth factor vascular endothelial growth factor wild type
Materials and Methods Materials and Methods are available in the online-only Supplement.
Results Generation of a Transgenic Mouse With T-Cell–Specific Overexpression of the PlGF PlGF is a subfamily of VEGF, which is the gene responsible for angiogenesis. Many studies have suggested that angiogenesis is closely associated with T cells18; therefore, we generated transgenic mice that overexpress PlGF in T cells using the human CD-2 promoter (Figure 1A). To evaluate the transgenic founder, we performed polymerase chain reaction reactions with genomic DNA (Figure 1B). We examine the number of copies by Fluorescence in situ hybridization analysis. The result shows that all 3 lines have 5 to 8 copies. And all 3 lines are shown similar phenotype of placenta and embryo development (data not shown). Moreover, we isolated mRNA from the spleen of 8-week-old littermates to determine whether PlGF mRNA is overexpressed in transgenic mice. All transgenic mice showed appropriate insertion into genomic DNA,
and PlGF mRNAs were overexpressed in transgenic mice (≥3-fold), whereas PlGF knockout mice did not exhibit PlGF mRNA expression (Figure 1C). In addition, when splenocytes were cultured with anti-CD3/CD28 antibody, secretion of PlGF was increased significantly compared with wild-type (WT) mice, whereas the serum PlGF level was unchanged (Figure 1D).
Defects of Litter Size and Weight Gain in PlGF Overexpressing Mice When hetero transgenic males were crossed with WT females, the females occasionally gave birth to dead fetuses on postnatal day 1. Genomic DNA analysis of the dead fetuses revealed that they were transgenic mice (Figure 2A). Furthermore, analysis of the litter size and birth rate revealed that littermates of PlGF transgenic mice were smaller than WT mice and control transgenic mice that expressed another gene19 by the human CD-2 promoter (Figure 2B and 2C). Interestingly, the birth rate of transgenic mice did not follow Mendelian inheritance. We suggest the possibility that the human CD-2 promoter generates nonaffected in transgenic mice. Because sizes of littermates were smaller compared with those of the WT, we measured the body weight of littermates every 2 weeks to examine whether PlGF can also affect growth. The body weights of PlGF transgenic mice were considerably lower than those of WT mice, the differences increased gradually (Figure 2D). Taken together, these findings indicate that overexpression of PlGF may cause death during gestation and that growth is restricted even after birth.
Figure 1. Targeted overexpression of placental growth factor (PlGF) in the T cells of transgenic mice. A, Transgenic construct for T-cell–specific expression of the PlGF gene. The primers used for tail biopsy polymerase chain reaction (PCR) are indicated by arrows. B, Identification of transgenic mice harboring the recombinant human CD-2 (hCD2)-PlGF gene. Three mice were determined to be positive by PCR and used as founders. C, We confirmed mRNA expression of PlGF in the spleen of 8-week-old mice. Real-time PCR of indicated genes from the spleens of wild-type (WT), knockout (KO), and transgenic (Tg) mice with T-cell activation via CD3/CD28 ex vivo. Splenocytes from WT, KO, and Tg mice were cultured for 12 hours in the presence of anti-CD3/CD28, after which the cultured cells were analyzed for mRNA level by real-time PCR. D, Splenocytes from WT, KO, and Tg mice were cultured for 48 hours in the presence of anti-CD3/CD28, after which the supernatant was analyzed for secreted PlGF using an ELISA kit. Serum PlGF concentrations in WT and Tg mice. Sera were obtained from 8-week-old mice. ***P
