Full-length human placental sFlt-1-e15a isoform induces distinct maternal phenotypes of preeclampsia in mice

<div><p>Objective</p><p>Most anti-angiogenic preeclampsia models in rodents utilized the overexpression of a truncated soluble fms-like tyrosine kinase-1 (sFlt-1) not expressed in any species. Other limitations of mouse preeclampsia models included stressful blood pressure measurements and the lack of postpartum monitoring. We aimed to 1) develop a mouse model of preeclampsia by administering the most abundant human placental sFlt-1 isoform (hsFlt-1-e15a) in preeclampsia; 2) determine blood pressures in non-stressed conditions; and 3) develop a survival surgery that enables the collection of fetuses and placentas and postpartum (PP) monitoring.</p><p>Methods</p><p>Pregnancy status of CD-1 mice was evaluated with high-frequency ultrasound on gestational days (GD) 6 and 7. Telemetry catheters were implanted in the carotid artery on GD7, and their positions were verified by ultrasound on GD13. Mice were injected through tail-vein with adenoviruses expressing hsFlt-1-e15a (n = 11) or green fluorescent protein (GFP; n = 9) on GD8/GD11. Placentas and pups were delivered by cesarean section on GD18 allowing PP monitoring. Urine samples were collected with cystocentesis on GD6/GD7, GD13, GD18, and PPD8, and albumin/creatinine ratios were determined. GFP and hsFlt-1-e15a expression profiles were determined by qRT-PCR. Aortic ring assays were performed to assess the effect of hsFlt-1-e15a on endothelia.</p><p>Results</p><p>Ultrasound predicted pregnancy on GD7 in 97% of cases. Cesarean section survival rate was 100%. Mean arterial blood pressure was higher in hsFlt-1-e15a-treated than in GFP-treated mice (∆MAP = 13.2 mmHg, p = 0.00107; GD18). Focal glomerular changes were found in hsFlt-1-e15a -treated mice, which had higher urine albumin/creatinine ratios than controls (109.3±51.7μg/mg vs. 19.3±5.6μg/mg, p = 4.4x10^-2; GD18). Aortic ring assays showed a 46% lesser microvessel outgrowth in hsFlt-1-e15a-treated than in GFP-treated mice (p = 1.2x10^-2). Placental and fetal weights did not differ between the groups. One mouse with liver disease developed early-onset preeclampsia-like symptoms with intrauterine growth restriction (IUGR).</p><p>Conclusions</p><p>A mouse model of late-onset preeclampsia was developed with the overexpression of hsFlt-1-e15a, verifying the <i>in vivo</i> pathologic effects of this primate-specific, predominant placental sFlt-1 isoform. HsFlt-1-e15a induced early-onset preeclampsia-like symptoms associated with IUGR in a mouse with a liver disease. Our findings support that hsFlt-1-e15a is central to the terminal pathway of preeclampsia, and it can induce the full spectrum of symptoms in this obstetrical syndrome.</p></div

Tyska immigranter i New Knoxville, OH

America is a nation of immigrants and all immigrants brought culture with them. There is not one American culture. Many Ohioans are descended from German ancestors, their German heritage is still present in the cultural and social landscapes. German immigrants came to America in search of farmland and independence.In the period between 1830 and 1890 approximately 1,300 people left Ladbergen, a village of 2,700, with hopes of a better life in America. The main reason for emigration from Germany was changes in the political landscape that led to financial hardship for those who did not own farmland. During the 1800’s German immigrants settled in Ohio, Pennsylvania and along the northern East coast. Choice of location was influenced by proximity to other German immigrants and the possibility for successful agriculture. All immigrants in New Knoxville came from the same village, Ladbergen, which provides a unique example of “chain immigration”.The village was platted by an Irishman, James Lyle, in 1836 and the German immigrants purchased the platted lots. A copy of the map has been preserved.Close to 80% of immigrants had been tenant farmers in Ladbergen. The life of farmers and tenant farmers in Ladbergen has been documented by local historians as is life among early immigrants in New Knoxville. Immigrant families were farmers in Ladbergen and became farmers in New Knoxville. Soil in New Knoxville was fertile and gave good yields of corn,beans, wheat, rye and buckwheat. I visited the area at different seasons and walked in the farmland with the assistance of a local guide. In the US this requires permits from the landowner.This is a flat agricultural area with large open corn and soybean fields with patches of old pineand deciduous forest penetrated by narrow dirt roads.In New Knoxville immigrant families were free to design their farms and living space according to their own preference. Immigrants built and settled in cabins while barns were built to house farm animals. Larger homes inspired by British/American architecture were soon erected and a few of these buildings are on display at the German Heritage Center in New Knoxville. Barns around New Knoxville are usually large gambrel roof barns for storage.People in New Knoxville are proud of their German roots and have an extensive cultural exchange with Ladbergen. German language, for example, was used in newspapers and church services in New Knoxville and in other areas with many German immigrants until the 1950's,some early immigrants never learnt to speak English. An important observation was that the immigrants adjusted very quickly to an American way of life when they depended on it for better housing and food supply but were not eager to mingle with Americans but kept a closed knit community.Amerika är en nation av invandrare och alla invandrare tog med sig kultur. Det finns inte en amerikansk kultur. Många Ohioans härstammar från tyska förfäder, deras tyska arv är fortfarande närvarande i de kulturella och sociala landskapen. Tyska immigranter kom till Amerika på jakt efter jordbruksmark och självständighet.Under perioden mellan 1830 och 1890 lämnade cirka 1300 människor Ladbergen, en by med 2700 invånare, med hopp om ett bättre liv i Amerika. Den främsta orsaken till emigrationen från Tyskland var förändringar i det politiska landskapet som ledde till ekonomiska svårigheter för dem som inte ägde jordbruksmark. Under 1800-talet bosatte sig tyska immigranter i Ohio,Pennsylvania och längs den norra östkusten. Immigranterna föredrog att bosätta sig i närheten av andra tyska invandrare och i områden med möjligheter till framgångsrikt jordbruk. Alla invandrare i New Knoxville kom från samma by, Ladbergen, vilket är ett unikt exempel på "kedje invandring".Byn mutades in av en irländare, James Lyle, 1836 och de tyska immigranterna köpte tomter. En kopia av den första kartan av New Knoxville finns bevarad.Närmare 80% av invandrarna hade varit arrendatorer i Ladbergen. Bönders och arrendatorers liv i Ladbergen har dokumenterats av lokala historiker, livet bland tidiga invandrare i New Knoxville har också studerats eftersom det är ett exempel på kedje invandring.Invandrarfamiljer var bönder i Ladbergen och blev bönder i New Knoxville. Det krävdes hjälp av en lokal guide för att utforska området utanför byn, det krävs tillstånd av landägaren att uppehålla sig på all typ av privatägd mark i USA. Gårdarna ligger i ett platt jordbruks område med stora öppna majs- och soja-fält som korsas av smala grus vägar. Det finns skogspartier med barr och lövskog på alla gårdar, och immigranterna sparade skog för virke. I New Knoxville var invandrarfamiljer fria att designa sina gårdar efter sina egna önskemål. Invandrare byggde och bosatte sig i stugor medan lador byggdes för att hysa husdjur.Större hem inspirerade av brittisk/amerikansk arkitektur uppfördes snart och några av dessa byggnader visas på German Heritage Centre i New Knoxville. Ladugårdar runt New Knoxville är vanligtvis stora byggnader med brutet tak för maximalt förvarings utrymme.Människor i New Knoxville är stolta över sina tyska rötter och har ett omfattande kulturellt utbyte med Ladbergen. Tyska språket, till exempel, användes i tidningar och gudstjänster i New Knoxville och i andra områden med många tyska invandrare fram till 1950-talet, några av de tidiga invandrarna lärde sig aldrig att tala engelska. En viktig observation var att invandrarna mycket snabbt anpassade sig till ett amerikanskt sätt att leva när de var beroende av det för bättre bostäder och matförsörjning men inte var ivriga att mingla med amerikaner utan levde i ett slutet samhälle.

Tyska immigranter i New Knoxville, OH

America is a nation of immigrants and all immigrants brought culture with them. There is not one American culture. Many Ohioans are descended from German ancestors, their German heritage is still present in the cultural and social landscapes. German immigrants came to America in search of farmland and independence.In the period between 1830 and 1890 approximately 1,300 people left Ladbergen, a village of 2,700, with hopes of a better life in America. The main reason for emigration from Germany was changes in the political landscape that led to financial hardship for those who did not own farmland. During the 1800’s German immigrants settled in Ohio, Pennsylvania and along the northern East coast. Choice of location was influenced by proximity to other German immigrants and the possibility for successful agriculture. All immigrants in New Knoxville came from the same village, Ladbergen, which provides a unique example of “chain immigration”.The village was platted by an Irishman, James Lyle, in 1836 and the German immigrants purchased the platted lots. A copy of the map has been preserved.Close to 80% of immigrants had been tenant farmers in Ladbergen. The life of farmers and tenant farmers in Ladbergen has been documented by local historians as is life among early immigrants in New Knoxville. Immigrant families were farmers in Ladbergen and became farmers in New Knoxville. Soil in New Knoxville was fertile and gave good yields of corn,beans, wheat, rye and buckwheat. I visited the area at different seasons and walked in the farmland with the assistance of a local guide. In the US this requires permits from the landowner.This is a flat agricultural area with large open corn and soybean fields with patches of old pineand deciduous forest penetrated by narrow dirt roads.In New Knoxville immigrant families were free to design their farms and living space according to their own preference. Immigrants built and settled in cabins while barns were built to house farm animals. Larger homes inspired by British/American architecture were soon erected and a few of these buildings are on display at the German Heritage Center in New Knoxville. Barns around New Knoxville are usually large gambrel roof barns for storage.People in New Knoxville are proud of their German roots and have an extensive cultural exchange with Ladbergen. German language, for example, was used in newspapers and church services in New Knoxville and in other areas with many German immigrants until the 1950's,some early immigrants never learnt to speak English. An important observation was that the immigrants adjusted very quickly to an American way of life when they depended on it for better housing and food supply but were not eager to mingle with Americans but kept a closed knit community.Amerika är en nation av invandrare och alla invandrare tog med sig kultur. Det finns inte en amerikansk kultur. Många Ohioans härstammar från tyska förfäder, deras tyska arv är fortfarande närvarande i de kulturella och sociala landskapen. Tyska immigranter kom till Amerika på jakt efter jordbruksmark och självständighet.Under perioden mellan 1830 och 1890 lämnade cirka 1300 människor Ladbergen, en by med 2700 invånare, med hopp om ett bättre liv i Amerika. Den främsta orsaken till emigrationen från Tyskland var förändringar i det politiska landskapet som ledde till ekonomiska svårigheter för dem som inte ägde jordbruksmark. Under 1800-talet bosatte sig tyska immigranter i Ohio,Pennsylvania och längs den norra östkusten. Immigranterna föredrog att bosätta sig i närheten av andra tyska invandrare och i områden med möjligheter till framgångsrikt jordbruk. Alla invandrare i New Knoxville kom från samma by, Ladbergen, vilket är ett unikt exempel på "kedje invandring".Byn mutades in av en irländare, James Lyle, 1836 och de tyska immigranterna köpte tomter. En kopia av den första kartan av New Knoxville finns bevarad.Närmare 80% av invandrarna hade varit arrendatorer i Ladbergen. Bönders och arrendatorers liv i Ladbergen har dokumenterats av lokala historiker, livet bland tidiga invandrare i New Knoxville har också studerats eftersom det är ett exempel på kedje invandring.Invandrarfamiljer var bönder i Ladbergen och blev bönder i New Knoxville. Det krävdes hjälp av en lokal guide för att utforska området utanför byn, det krävs tillstånd av landägaren att uppehålla sig på all typ av privatägd mark i USA. Gårdarna ligger i ett platt jordbruks område med stora öppna majs- och soja-fält som korsas av smala grus vägar. Det finns skogspartier med barr och lövskog på alla gårdar, och immigranterna sparade skog för virke. I New Knoxville var invandrarfamiljer fria att designa sina gårdar efter sina egna önskemål. Invandrare byggde och bosatte sig i stugor medan lador byggdes för att hysa husdjur.Större hem inspirerade av brittisk/amerikansk arkitektur uppfördes snart och några av dessa byggnader visas på German Heritage Centre i New Knoxville. Ladugårdar runt New Knoxville är vanligtvis stora byggnader med brutet tak för maximalt förvarings utrymme.Människor i New Knoxville är stolta över sina tyska rötter och har ett omfattande kulturellt utbyte med Ladbergen. Tyska språket, till exempel, användes i tidningar och gudstjänster i New Knoxville och i andra områden med många tyska invandrare fram till 1950-talet, några av de tidiga invandrarna lärde sig aldrig att tala engelska. En viktig observation var att invandrarna mycket snabbt anpassade sig till ett amerikanskt sätt att leva när de var beroende av det för bättre bostäder och matförsörjning men inte var ivriga att mingla med amerikaner utan levde i ett slutet samhälle.

Survival cesarean section.

<p>(A) After a 1cm–1.5cm midline abdominal incision, a short segment of one uterine horn was exteriorized, and a 3mm–5mm longitudinal hysterectomy was performed on the opposite side of the mesometrial arterial arcade. (B-C) As the uterine wall could be easily dilated, this minimal incision enabled the delivery of two to three fetuses and their placentas using gentle fingertip pressure. An arrowhead depicts a placenta, stars depict umbilical cords, and arrows point to the fetuses. (D) Hysterectomies were closed with a single 4/0 absorbable multifilament suture. (E) After abdominal lavage with 0.9% sterile saline, the abdominal wall was closed with an absorbable multifilament continuous suture, and the skin was closed with 7mm-wide staples. The image shows a surgical field before euthanization on PPD8. (F) One uterus harvested after euthanization on PPD8. Arrowheads show hysterectomy sutures. (G) H&E staining of a uterine cross-section of a control mouse euthanized on PPD8 shows granulation tissue with enlarged vessels, foam cells, myofibroblasts, macrophages, and hemosiderin deposition. The image inside the black box is magnified in Subfigure H. (I) SMA immunostaining of the same uterus. The arrow depicts the cesarean section incision site with the disruption of the two-layered myometrium. (J) H&E staining of a uterine cross-section of a control mouse euthanized on PPD77 shows granulation tissue and the complete healing of the two-layered myometrium. The image inside the black box is magnified in Subfigure K. (L) SMA immunostaining of the same uterus. The arrow depicts a suture site. (G, I, J and L: 40x magnifications, H and K: 100x magnifications).</p

The possible mechanisms of distinct preeclampsia phenotypes induced by hsFlt-1-e15a.

<p>Blood pressure peaks (blue arrows) occurred after a ten-day-period following the first and second Ad-CMV-hsFlt-1-e15a injections in all but one mice that developed late-onset or postpartum preeclampsia without growth restriction. In these cases the growth potential of the fetuses was not compromised because they have already reached the top of their growth curve when hsFlt-1-e15a effect reached its plateau (blue stick crossing the curve). In case of “EM35” mouse, blood pressure elevations already peaked at six days after the first Ad-CMV-hsFlt-1-e15a injection (red arrow). In this case the fetal growth potential was severely compromised because the hsFlt-1-e15a effect was the highest when fetuses had reached only about one third or fourth of their growth curve (red stick crossing the curve). Preeclampsia phenotypes are described below the growth curve of CD-1 mouse embryos, which was adapted from a figure in a previous publication of Mu et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119547#pone.0119547.ref240" target="_blank">240</a>]. Permission for reuse of this original figure was obtained from BioMed Central.</p

<i>FLT1</i> protein isoforms and mRNA transcript variants.

<p>(A) Flt-1 contains seven extracellular Ig-like domains and an intracellular tyrosine kinase. The first three extracellular Ig-like domains are essential for ligand-binding, while the 4–7th extracellular Ig-like domains for receptor dimerization. The truncated mouse sFlt-1 mutant [msFlt-1(1–3)] contains only 1–329 amino acids of Flt-1, corresponding to the first three Ig-like domains. Mouse and human sFlt-1-i13 contains the first six Ig-like domains corresponding to 1–657 amino acids of Flt-1, as well as a unique 31-amino-acid tail. This unique C-terminus is evolutionarily highly conserved among mammals; the mouse and human amino acid sequences of this tail are only different in two positions (shown with blue letters). Among the human placental expressed sFlt-1 isoforms, hsFlt-1-i14, hsFlt-1-e15a and hsFlt-1-e15b diverge from Flt-1 after amino acid 706, and contain a 31-, 28- and 13-amino-acid unique tails, respectively. (B) Among the placental expressed <i>FLT1</i> transcripts, the abundance of the mRNA encoding for the transmembrane receptor is about 2.5% in preeclampsia. <i>FLT1</i> transcript expression data was retrieved from Jebbink et al. and is shown as transcript level divided by total <i>FLT1</i> transcript level [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119547#pone.0119547.ref154" target="_blank">154</a>]. HsFlt-1-i13, the second most abundant placental <i>FLT1</i> transcript in preeclampsia, is generated by skipped splicing and extension of exon 13. Similarly, hsFlt-1-i14 is generated by skipped splicing and extension of exon 14. HsFlt-1-e15a and hsFlt-1-e15b contain alternatively spliced exons derived from intronic sequences (exon 15a and exon 15b, respectively). The most abundant placental transcript, hsFlt-1-e15a contains exon 15a, which is located within a primate-specific AluSeq retrotransposon. The graph was adapted with permission from figures in publications of Heydarian et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119547#pone.0119547.ref153" target="_blank">153</a>] and Shibuya M. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119547#pone.0119547.ref243" target="_blank">243</a>]. Permissions for reuse of these original figures were obtained from Elsevier Ltd. and from the Proceedings of the Japan Academy, Series B, respectively.</p

Implantation with a telemetric blood pressure monitoring system.

<p>(A) On GD8, after isolation and ligation of the left common carotid artery at the level of bifurcation, a small arteriotomy was prepared with a 25G tip needle, (B) and the blood pressure monitoring catheter was positioned into the aortic arch with the assistance of the vessel cannulation forceps. (C) The transmitter was placed into a subcutaneous pocket in the left flank and preformed with blind dissection. (D) On GD13, the position of the telemetry catheter was determined with a 55MHz ultrasound probe. The catheter tip is situated in the aortic arch, and the intra-aortic part of the catheter reaches the optimal 2mm length. (E) On GD18, a pregnant mouse is shown before a cesarean section. The incision line of the telemetry surgery healed completely. The projected graph illustrates the position of the intra-aortic catheter and the subcutaneous telemetric blood pressure transmitter. (F) On PPD8, the catheter position, aortic arch, and main arterial branches were visualized after autopsy in the mouse mediastinum and chest. The dotted lines show the heart and main arteries of the mediastinum. (A-F) Head orientations are shown with asterisks.</p

Litter sizes, maternal, placental and fetal weights, and placental/fetal weight ratios.

<p>Controls (n = 17) and msFlt-1(1-3)-treated mice (n = 6) had a litter size consistent with the strain average published by the vendor (n = 11.5). The number of pups (13.8±0.4, p = 0.046) and living pups (13.6±0.45, p = 0.05) were higher in hsFlt-1-e15a-treated mice (n = 18) than in controls. The total weight of living pups (14.2±0.56 g, p = 0.04) and maternal weights (56.3±1.1 g, p = 0.04) were also higher in hsFlt-1-e15a-treated mice than in controls. Truncated msFlt-1(1-3)-treated mice did not differ in any parameters from the controls. Among hsFlt-1-e15a-treated mice, the total weights of living pups was higher in the Ad-RGD-CYP-hsFlt-1-e15a treated mice than in controls (15±0.48 g, p = 0.043). The number of pups (14.3±0.42, p = 0.047) and the number of living pups (14.1±0.46, p = 0.039) was higher in the Ad-RGD-CMV-hsFlt-1-e15a treated mice than in controls. Stars denote statistical significance.</p