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• W2053976090 abstract "Human placental development combines elements of tumorigenesis and vasculogenesis. The organ's specialized epithelial cells, termed cytotrophoblasts, invade the uterus where they reside in the interstitial compartment. They also line uterine arteries and veins. During invasion, ectodermally derived cytotrophoblasts undergo pseudovasculogenesis, switching their adhesion molecule repertoire to mimic that of vascular cells. Failures in this transformation accompany the pregnancy complication preeclampsia. Here, we used a combination of in situ and in vitro analyses to characterize the cell's expression of vascular endothelial growth factor (VEGF) family ligands and receptors, key regulators of conventional vasculogenesis and angiogenesis. Cytotrophoblast differentiation and invasion during the first and second trimesters of pregnancy were associated with down-regulation of VEGF receptor (VEGFR)-2. Invasive cytotrophoblasts in early gestation expressed VEGF-A, VEGF-C, placental growth factor (PlGF), VEGFR-1, and VEGFR-3 and, at term, VEGF-A, PlGF, and VEGFR-1. In vitro the cells incorporated VEGF-A into the surrounding extracellular matrix; PlGF was secreted. We also found that cytotrophoblasts responded to the VEGF ligands they produced. Blocking ligand binding significantly decreased their expression of integrin α1, an adhesion molecule highly expressed by endovascular cytotrophoblasts, and increased apoptosis. In severe preeclampsia and hemolysis, elevated liver enzymes, and low platelets syndrome, immunolocalization on tissue sections showed that cytotrophoblast VEGF-A and VEGFR-1 staining decreased; staining for PlGF was unaffected. Cytotrophoblast secretion of the soluble form of VEGFR-1 in vitro also increased. Together, the results of this study showed that VEGF family members regulate cytotrophoblast survival and that expression of a subset of family members is dysregulated in severe forms of preeclampsia. Human placental development combines elements of tumorigenesis and vasculogenesis. The organ's specialized epithelial cells, termed cytotrophoblasts, invade the uterus where they reside in the interstitial compartment. They also line uterine arteries and veins. During invasion, ectodermally derived cytotrophoblasts undergo pseudovasculogenesis, switching their adhesion molecule repertoire to mimic that of vascular cells. Failures in this transformation accompany the pregnancy complication preeclampsia. Here, we used a combination of in situ and in vitro analyses to characterize the cell's expression of vascular endothelial growth factor (VEGF) family ligands and receptors, key regulators of conventional vasculogenesis and angiogenesis. Cytotrophoblast differentiation and invasion during the first and second trimesters of pregnancy were associated with down-regulation of VEGF receptor (VEGFR)-2. Invasive cytotrophoblasts in early gestation expressed VEGF-A, VEGF-C, placental growth factor (PlGF), VEGFR-1, and VEGFR-3 and, at term, VEGF-A, PlGF, and VEGFR-1. In vitro the cells incorporated VEGF-A into the surrounding extracellular matrix; PlGF was secreted. We also found that cytotrophoblasts responded to the VEGF ligands they produced. Blocking ligand binding significantly decreased their expression of integrin α1, an adhesion molecule highly expressed by endovascular cytotrophoblasts, and increased apoptosis. In severe preeclampsia and hemolysis, elevated liver enzymes, and low platelets syndrome, immunolocalization on tissue sections showed that cytotrophoblast VEGF-A and VEGFR-1 staining decreased; staining for PlGF was unaffected. Cytotrophoblast secretion of the soluble form of VEGFR-1 in vitro also increased. Together, the results of this study showed that VEGF family members regulate cytotrophoblast survival and that expression of a subset of family members is dysregulated in severe forms of preeclampsia. The embryo's acquisition of a supply of maternal blood is a critical hurdle in pregnancy maintenance. In humans, the mechanics of this process (diagrammed in Figure 1) are accomplished by the placenta's specialized epithelial cells, termed “cytotrophoblasts.” In a subset of chorionic villi, these cells form aggregates that attach the conceptus to the uterine wall. Cytotrophoblasts that emanate from these sites invade the interstitium of the decidua, the inner third of the myometrium, and uterine blood vessels. Although there are many circumstances in which normal and abnormal cells carry out interstitial invasion, several aspects of cytotrophoblast vascular invasion are unique. During this process, placental cells of fetal origin intravasate the superficial portions of uterine vessels that lie near the implantation site. Initially these vessels are plugged by cytotrophoblast aggregates. As invasion proceeds, the placental cells form heterotypic interactions with, then replace, the endothelium. They also intercalate within the muscular tunica, a process that substantially increases the vessel diameter while decreasing its resistance. In this manner uterine arterioles, and to a lesser extent veins, are remodeled into unique hybrid vessels composed of fetal and maternal cells. Once the lumina reform, uterine blood flow is diverted to the intervillous space where nutrient, gas, and waste exchange occur. Although the histological features of this process have been studied for decades, the molecular aspects, which are likely to be complex, have only recently been addressed. Our previous work suggests that invading cytotrophoblasts execute a novel epithelial-to-endothelial phenotypic transformation we termed “pseudovasculogenesis.”1Damsky CH Fisher SJ Trophoblast pseudo-vasculogenesis: faking it with endothelial adhesion receptors.Curr Opin Cell Biol. 1998; 10: 660-666Crossref PubMed Scopus (221) Google Scholar, 2Zhou Y Fisher SJ Janatpour M Genbacev O Dejana E Wheelock M Damsky CH Human cytotrophoblasts adopt a vascular phenotype as they differentiate. A strategy for successful endovascular invasion?.J Clin Invest. 1997; 99: 2139-2151Crossref PubMed Scopus (835) Google Scholar This switch is exemplified by changes in the cell's adhesion molecule expression that take place during uterine invasion. For example, cytotrophoblast stem cells, which express adhesion molecules typical of many epithelial cells, eg, E-cadherin and α6β4 integrin, dramatically alter this repertoire as they invade the uterus. In essence, they replace their epithelial-like receptors with adhesion molecules typical of endothelial cells, eg, vascular endothelial (VE)-cadherin, vascular cell adhesion molecule-1, platelet-endothelial cell adhesion molecule-1, and αVβ3 integrin. Other aspects of the cell surfaces of invasive cytotrophoblasts also resemble vascular cells. For example, they express urokinase plasminogen activator3Queenan Jr, JT Kao LC Arboleda CE Ulloa-Aguirre A Golos TG Cines DB Strauss JFD Regulation of urokinase-type plasminogen activator production by cultured human cytotrophoblasts.J Biol Chem. 1987; 262: 10903-10906Abstract Full Text PDF PubMed Google Scholar and the thrombin receptor.4Even-Ram S Uziely B Cohen P Grisaru-Granovsky S Maoz M Ginzburg Y Reich R Vlodavsky I Bar-Shavit R Thrombin receptor overexpression in malignant and physiological invasion processes.Nat Med. 1998; 4: 909-914Crossref PubMed Scopus (409) Google Scholar The functional importance of this transformation program is highlighted by the fact that failures in executing portions of it are associated with a subset of pregnancy complications—for example, preeclampsia, the leading cause of maternal mortality in the Western world, which also increases perinatal mortality fivefold.5Walker JJ Pre-eclampsia.Lancet. 2000; 356: 1260-1265Abstract Full Text Full Text PDF PubMed Scopus (548) Google Scholar The clinical diagnostic criteria of this syndrome include the new onset of hypertension and the appearance of proteinuria and edema during pregnancy, all of which could be explained by functional alterations in the maternal vascular endothelium.6Roberts JM Taylor RN Musci TJ Rodgers GM Hubel CA McLaughlin MK Preeclampsia: an endothelial cell disorder.Am J Obstet Gynecol. 1989; 161: 1200-1204Abstract Full Text PDF PubMed Scopus (1680) Google Scholar Preeclampsia and approximately half the cases of intrauterine growth restriction are associated with particular placental pathologies. The extent of interstitial invasion by cytotrophoblasts is variable, but frequently shallow, and endovascular invasion is consistently rudimentary, making it extremely difficult to find any maternal vessels that contain cytotrophoblasts.7Brosens IA Robertson WB Dixon HG The role of the spiral arteries in the pathogenesis of preeclampsia.Obstet Gynecol Annu. 1972; 1: 177-191PubMed Google Scholar, 8Zhou Y Damsky CH Fisher SJ Preeclampsia is associated with failure of human cytotrophoblasts to mimic a vascular adhesion phenotype. One cause of defective endovascular invasion in this syndrome?.J Clin Invest. 1997; 99: 2152-2164Crossref PubMed Scopus (810) Google Scholar These anatomical defects suggested to us that in preeclampsia, cytotrophoblast differentiation along the invasive pathway is abnormal. Biopsies of the uterine wall of women with this syndrome showed that invasive cytotrophoblasts retain expression of adhesion receptors characteristic of stem cells and fail to turn on receptors that promote invasion and/or assumption of an endothelial phenotype.8Zhou Y Damsky CH Fisher SJ Preeclampsia is associated with failure of human cytotrophoblasts to mimic a vascular adhesion phenotype. One cause of defective endovascular invasion in this syndrome?.J Clin Invest. 1997; 99: 2152-2164Crossref PubMed Scopus (810) Google Scholar The concept that failed cytotrophoblast invasion and pseudovasculogenesis are linked to the maternal vascular pathology seems plausible, but has yet to be proved. Thus, the chain of events that links a defect in placentation to the maternal systemic disorder is under intense investigation. We are very interested in understanding the molecular pathways that regulate cytotrophoblast pseudovasculogenesis, as well as the possible defects that occur in pregnancy complications such as preeclampsia. A vast array of physiological factors (eg, hypoxia, shear stress) and effector pathways (eg, transcription factors, growth factors, chemokines, cytokines, and protein fragments) governs blood vessel development and growth, either directly or indirectly.9Bussolino F Mantovani A Persico G Molecular mechanisms of blood vessel formation.Trends Biochem Sci. 1997; 22: 251-256Abstract Full Text PDF PubMed Scopus (419) Google Scholar To focus the proposed investigation, we have been considering cytotrophoblast pseudovasculogenesis, which involves cells derived from the extraembryonic lineages, in the context of master regulatory pathways that govern de novo differentiation and assembly of blood vessels within the embryo. Gene deletion studies in mice have pointed to the particular importance of three families of ligands and their tyrosine kinase receptors—vascular endothelial growth factors and their receptors (VEGF/VEGFRs), angiopoietins and their Tie receptors (Ang/Tie), and ephrins and their Eph receptors (ephrin/Ephs)—in vasculogenesis and angiogenesis.10Hanahan D Signaling vascular morphogenesis and maintenance.Science. 1997; 277: 48-50Crossref PubMed Scopus (1049) Google Scholar, 11Neufeld G Cohen T Gengrinovitch S Poltorak Z Vascular endothelial growth factor VEGF and its receptors.FASEB J. 1999; 13: 9-22Crossref PubMed Scopus (3171) Google Scholar, 12Risau W Mechanisms of angiogenesis.Nature. 1997; 386: 671-674Crossref PubMed Scopus (4891) Google Scholar, 13Yancopoulos GD Klagsbrun M Folkman J Vasculogenesis, angiogenesis, and growth factors: ephrins enter the fray at the border.Cell. 1998; 93: 661-664Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar The discrete phenotypes of the null animals suggest distinct roles for individual families, with VEGF family members and their receptors having important actions during the initial stages of vasculogenesis and angiogenesis, both during development and as a result of pathological processes.14Veikkola T Karkkainen M Claesson-Welsh L Alitalo K Regulation of angiogenesis via vascular endothelial growth factor receptors.Cancer Res. 2000; 60: 203-212PubMed Google Scholar, 15Carmeliet P Jain RK Angiogenesis in cancer and other diseases.Nature. 2000; 407: 249-257Crossref PubMed Scopus (7607) Google Scholar In accord with our hypothesis that cytotrophoblast pseudovasculogenesis co-opts a subset of the ligand-receptor interactions that govern conventional vasculogenesis, we studied the expression of VEGF family members and their receptors during this process in vivo. Immunolocalization experiments performed on tissue sections of the maternal-fetal interface showed that the expression of many of these ligands and receptors is modulated as cytotrophoblasts invade the uterus and its blood vessels in normal pregnancy. We also used an in vitro model of this process to understand the effects of interfering with interactions between VEGF and its receptors on cytotrophoblast differentiation/invasion and apoptosis. The results of these experiments, together with data showing that defects in VEGF and VEGFR expression exist in severe forms of preeclampsia, suggest that VEGF-VEGFR interactions play an important role in differentiation and survival of the unique cytotrophoblast subpopulation that invades the uterine wall, occupies the maternal vessels, and channels maternal blood to the placenta during pregnancy. A mouse anti-VEGF-A monoclonal antibody (mAb) (M293; R & D Systems, Minneapolis, MN) and a goat anti-VEGF-C polyclonal antibody (pAb) (R&D Systems) were used at a dilution of 1:50 for immunolocalization analyses of paraffin-embedded tissues. A goat anti-PlGF-1, 2 pAb (R&D Systems) was used at a dilution of 1:25 for immunolocalization on tissues that were embedded in optimal cutting temperature (OCT) medium (Miles Scientific, Naperville, IL). A goat anti-VEGF-B pAb and a mouse anti-VEGF-D mAb, both from R&D Systems, were also used for immunolocalization analyses of OCT-embedded tissues at dilutions of 1:10 and 1:50, respectively. VEGF-A and PlGF levels in conditioned medium were measured separately using ELISA kits (R&D Systems). A mouse mAb (A4.6.1) that recognizes all isoforms of human VEGF-A (gift of Dr. N. Ferrara, Genentech, South San Francisco, CA) was used in immunoblotting experiments. The 190.11 anti-VEGFR-1 mouse mAb (diluted 1:400)16Simon M Rockl W Hornig C Grone EF Theis H Weich HA Fuchs E Yayon A Grone HJ Receptors of vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) in fetal and adult human kidney: localization and [125I] VEGF binding sites.J Am Soc Nephrol. 1998; 9: 1032-1044PubMed Google Scholar and the rabbit N-931 anti-VEGFR-2 antibody (diluted 1:200; Santa Cruz Biotechnology Inc., Santa Cruz, CA) were used for immunostaining. The 9D9 anti-VEGFR-3 mouse mAb (Molecular/Cancer Biology Laboratory, University of Helsinki, Finland) was used for immunostaining and immunoblotting (diluted 1:500). The C-17 rabbit anti-human VEGFR-1 antibody (diluted 1:1000, Santa Cruz Biotechnology Inc.) and the 2-10−1 anti-VEGFR-2 mAb (diluted 1:200; gift of Dr. K. Chwalisz, Scherring AG, Berlin, Germany) were used for immunoblotting. Soluble VEGFR-1 (sVEGFR-1) in conditioned medium was quantified with an ELISA kit that was developed using the 190.11 mAb.17Hornig C Behn T Bartsch W Yayon A Weich HA Detection and quantification of complexed and free soluble human vascular endothelial growth factor receptor-1 (sVEGFR-1) by ELISA.J Immunol Methods. 1999; 226: 169-177Crossref PubMed Scopus (48) Google Scholar The TS2/7 mAb that specifically recognizes integrin α1/β1 (Endogen, Woburn, MA) was used for immunostaining and immunoprecipitation at a dilution of 1:50. mAbs BV6, BV9, and TEA against VE-cadherin (gifts of Dr. Elisabetta Dejana, Mario Negri Institute, Milan, Italy) were all used at a dilution of 1:10 for immunostaining and immunoblotting. The OV-TL 12/30 mAb against cytokeratin 7 (diluted 1:25; DAKO, Carpinteria, CA) was used for immunolocalization analyses of paraffin-embedded tissues. Rat anti-human cytokeratin mAb 7D3, produced in the Fisher laboratory, was used at a dilution of 1:50 for immunolocalization analyses of OCT-embedded tissues.18Damsky CH Fitzgerald ML Fisher SJ Distribution patterns of extracellular matrix components and adhesion receptors are intricately modulated during first trimester cytotrophoblast differentiation along the invasive pathway, in vivo.J Clin Invest. 1992; 89: 210-222Crossref PubMed Scopus (621) Google Scholar Placentas were obtained from normal pregnant women and patients with preeclampsia. We analyzed 19 control samples from patients with no evidence of preeclampsia, gestational hypertension, or a medical history that suggested an increased risk of developing preeclampsia. Where applicable, the labor status is indicated with L denoting women who experienced labor and NL denoting women who did not. The gestational ages at which these samples were collected were as follows: 6 weeks (n = 1), 10 weeks (n = 1), 12 weeks (n = 1), 14 weeks (n = 1), 16 weeks (n = 1), 17 weeks (n = 1), 19 weeks (n = 1), 20 weeks (n = 1), 22 weeks (n = 1), 24 weeks (n = 1; NL), 26 weeks (n = 2; NL), 32 weeks (n= 1; NL), 36 weeks (n = 2; 1 L and 1 NL), 38 weeks (n = 3; L), and 40 weeks (n = 1; NL). We analyzed 13 samples from patients with preeclampsia diagnosed according to the classic criteria originally recommended by Dr. Leon Chesley and modified by the National Institutes of Health:19Gifford Jr RW, August PA, Cunningham G, Green LA, Lindheimer MD, McNellis D, Roberts JM, Roccella EJ, Sibai BM, Taler SJ:, National Institutes of Health, publication no: 00-3029, 2000Google Scholar no history of hypertension before pregnancy; increase in diastolic pressure of 15 mm Hg or systolic pressure of 30 mm Hg compared with blood pressure obtained before 20 weeks of gestation; proteinuria ≥0.5 g/24 hours or ≥30 mg/dl (or 1+ on urine dipstick) in a catheterized specimen; hyperuricemia >5.5 mg/dl (or 1 SD greater than the normal mean value before term); return to normal blood pressure and resolution of proteinuria by 12 weeks postpartum. Severe preeclampsia (SPE) was diagnosed according to the following criteria:20Sibai BM Treatment of hypertension in pregnancy women.N Engl J Med. 1996; 335: 257-265Crossref PubMed Scopus (323) Google Scholar systolic blood pressure ≥160 mm Hg and/or diastolic pressure ≥110 mm Hg; proteinuria of ≥5 g in a 24-hour period or 3+ on urine dipstick; presence of cerebral or visual disturbances. The criteria used to diagnose the syndrome of hemolysis, elevated liver enzymes, and low platelets (HELLP) have been published.21Sibai BM The HELLP syndrome (hemolysis, elevated liver enzymes, and low platelets): much ado about nothing?.Am J Obstet Gynecol. 1990; 162: 311-316Abstract Full Text PDF PubMed Scopus (545) Google Scholar Samples were collected for embedding in OCT from women with the diagnoses indicated at the following weeks of gestation: 26 weeks (n = 3; 1 SPE, 2 HELLP; NL), 28 weeks (n = 1 SPE; NL), 29 weeks (n = 1 SPE; NL), 30 weeks (n = 1 HELLP; L), 31 weeks (n = 1 SPE; L), 32 weeks (n = 1 SPE; L), 33 weeks (n = 1 SPE; L), 35 weeks (n = 1 SPE; L), 36 weeks (n = 1 SPE; L), 38 weeks (n = 2; 1 PE, 1 HELLP; L). Samples were collected for embedding in paraffin from women with the diagnoses indicated at the following weeks of gestation: 25 weeks (n = 1 HELLP; NL), 29 weeks (n = 1 HELLP; NL), 29 weeks (n = 1 SPE; L), 30 weeks (n = 1 HELLP; L), 31 weeks (n = 1 SPE; L), 32 weeks (n = 1 SPE; L), 33 weeks (n = 1 SPE; L), 35 weeks (n = 1 SPE; L), 36 weeks (n = 1 SPE; L), 38 weeks (n = 2; 1 PE, 1 HELLP; L). Placental tissues were processed for double indirect immunolocalization as previously described.22Damsky CH Librach C Lim KH Fitzgerald ML McMaster MT Janatpour M Zhou Y Logan SK Fisher SJ Integrin switching regulates normal trophoblast invasion.Development. 1994; 120: 3657-3666PubMed Google Scholar, 23Zhou Y Damsky CH Chiu K Roberts JM Fisher SJ Preeclampsia is associated with abnormal expression of adhesion molecules by invasive cytotrophoblasts.J Clin Invest. 1993; 91: 950-960Crossref PubMed Scopus (520) Google Scholar For detection of VEGF-B and VEGF-D and the VEGFRs, tissues were fixed in 3% paraformaldehyde for 30 minutes, washed three times in phosphate-buffered saline (PBS), infiltrated with 5 to 15% sucrose followed by OCT medium, and frozen in liquid nitrogen. Sections (5 μm) were prepared using a cryostat (Slee International, Tiverton, RI) and collected on charged and precleaned microscope slides (Fisher Scientific, Pittsburgh, PA). For detection of PlGF, tissues were directly embedded in OCT and frozen without previous fixation. Sections, prepared as described above, were fixed in cold acetone for 5 minutes before staining as follows. The sections were incubated in a mixture of anti-cytokeratin (to localize trophoblasts) and another primary antibody for 1 hour to overnight. Then the sections were rinsed, incubated with the appropriate species-specific secondary antibodies conjugated to rhodamine or fluorescein, washed three times in PBS for 10 minutes, and mounted with Vectashield medium (Vector, South San Francisco, CA). Samples were examined with a Zeiss Axiophot Epifluorescence microscope (Thornwood, NY) equipped with filters to selectively view the rhodamine and fluorescein fluorescence. For detection of VEGF-A, tissues were fixed in 10% neutral buffered formalin for 24 hours and embedded in paraffin. Sections (5 μm) were cut on a Leica microtome and stained as described by Zhang and colleagues.24Zhang L Scott PA Turley H Leek R Lewis CE Gatter KC Harris AL Mackenzie IZ Rees MC Bicknell R Validation of anti-vascular endothelial growth factor (anti-VEGF) antibodies for immunohistochemical localization of VEGF in tissue sections: expression of VEGF in the human endometrium.J Pathol. 1998; 185: 402-408Crossref PubMed Scopus (74) Google Scholar For detection of VEGF-C, sections cut from paraffin blocks were incubated with the primary antibody for 3 hours and washed in PBS (three times for 10 minutes). Bound antibody was detected by incubation with diaminobenzoate (Vector). The sections were examined using a Zeiss microscope. As a control for each experiment, the staining patterns of the primary and secondary antibodies alone were assessed. For immunolocalization of antigens expressed by cultured cytotrophoblasts, isolated cells were plated on coverslips coated with Matrigel (Collaborative Biomedical Products, Bedford, MA) for various periods of time, then fixed in 3% paraformaldehyde for 5 minutes, and permeabilized with cold acetone or methanol for another 5 minutes. Samples were stained and analyzed as described above. Cytotrophoblasts were isolated from chorionic villi of 7- to 24-week human placentas by routine procedures established in our laboratory.25Librach CL Werb Z Fitzgerald ML Chiu K Corwin NM Esteves RA Grobelny D Galardy R Damsky CH Fisher SJ 92-kD type IV collagenase mediates invasion of human cytotrophoblasts.J Cell Biol. 1991; 113: 437-449Crossref PubMed Scopus (647) Google Scholar Briefly, the placentas were obtained immediately after elective pregnancy terminations. After a series of collagenase and trypsin digestions, cytotrophoblasts were separated from contaminating cell types on Percoll gradients. Purified cells were used immediately or cultured in serum-free Dulbecco's modified Eagle's medium-high glucose, with 2% Nutridoma (Boehringer Mannheim Biochemicals, Indianapolis, IN), on Matrigel-coated or human placental laminin-coated (Life Technologies, Inc., Rockville, MD) substrates for the times indicated. Total RNA was extracted from purified first and second trimester cytotrophoblasts either immediately on isolation or after 12 hours in culture according to published methods.26Chomczynski P Sacchi N Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63290) Google Scholar Blots were prepared as previously described.27McMaster MT Librach CL Zhou Y Lim KH Janatpour MJ DeMars R Kovats S Damsky C Fisher SJ Human placental HLA-G expression is restricted to differentiated cytotrophoblasts.J Immunol. 1995; 154: 3771-3778PubMed Google Scholar Before transfer, gels were stained with acridine orange to ensure integrity of the RNA samples and confirm equal loading. The probes were generated by random priming with [32P]CTP and the Klenow fragment of DNA polymerase I28Tabor S Sruhl K Scharf SJ Gelfand DH Enzymatic manipulation of DNA and RNA.in: Ausubel FM Current Protocols in Molecular Biology. John Wiley & Sons, New York1993: 3.0.1-3.19.18Google Scholar of the following fragments: bp 57 to 638 of the VEGF165 cDNA (GenBank accession number M32977), bp 1 to 362 of the VEGF-B167 cDNA (GenBank accession number U48801), bp 494 to 1661 of the VEGF-C cDNA (GenBank accession number X94216), the entire coding regions of the VEGF-D (1064 bp) gene (GenBank accession number AJ000185), bp 304 to 944 of the PlGF cDNA (GenBank accession number X54936), bp 706 to 2310 of the Flt1 (VEGFR-1) cDNA (GenBank accession number X51602), bp 6 to 715 of the KDR (VEGFR-2) cDNA (GenBank accession number L04947), and bp 1 to 595 of the Flt4 (VEGFR-3) cDNA (GenBank accession number X68203). Probes had a specific activity of 2 × 109 dpm/pg. The final posthybridization washes were conducted in 0.3× standard saline citrate (150 mmol/L NaCl, 15 mmol/L sodium citrate, pH 7.4) and 0.1% sodium dodecyl sulfate (SDS) at 65°C. Blots were stripped in 0.1× standard saline citrate with 0.5% SDS at 95°C. Levels of VEGF-A, PlGF, and sVEGFR-1 in first (n = 5), second (n = 5), and third trimester (n = 5) cytotrophoblast-conditioned medium were assessed by ELISA. Patients with preeclampsia whose placentas were used as sources of cells had the diagnoses indicated at the following weeks of gestation: 26 weeks (n = 2; 1 SPE, 1 HELLP), 31 weeks (n = 1 SPE), 32 weeks (n= 1 SPE), and 36 weeks (n = 1 SPE). Equal numbers of cytotrophoblasts (1 × 106/ml) were cultured for 48 hours on Matrigel substrates as described above, and then medium was collected and centrifuged at 12,000 ×g for 5 minutes. The ELISAs were performed according to the manufacturer's instructions (VEGF-A and PlGF) or published methods (sVEGFR-117Hornig C Behn T Bartsch W Yayon A Weich HA Detection and quantification of complexed and free soluble human vascular endothelial growth factor receptor-1 (sVEGFR-1) by ELISA.J Immunol Methods. 1999; 226: 169-177Crossref PubMed Scopus (48) Google Scholar). The results were analyzed on a Vmax kinetic microplate reader (Molecular Devices Corporation, Sunnyvale, CA). The statistical significance of the data was analyzed by using an analysis of variance test. Freshly isolated cytotrophoblasts or cytotrophoblasts cultured on either Matrigel-coated or human placental laminin-coated wells were washed twice with PBS and extracted with 200 μl of lysis buffer (50 mmol/L Tris buffer, pH 7.6, containing 1% Nonidet P-40, 0.1% SDS, 120 mmol/L NaCl, 100 μmol/L phenylmethylsulfonyl fluoride, 20 μg/ml aprotinin, 10 μg/ml leupeptin, 10 μg Na2VCO3). Cell extracts were centrifuged at 12,000 × g for 5 minutes to remove insoluble materials. Samples containing equal amounts of protein were mixed with SDS sample buffer and separated by SDS-polyacrylamide gel electrophoresis under nonreducing conditions (VEGF-A and VEGFR-2) or reducing conditions (VEGFR-1 and VEGFR-3 and VE-cadherin). After the proteins were transferred to nitrocellulose, the membranes were incubated first with primary antibody, then with peroxidase-conjugated secondary antibodies (Jackson Immuno Research Labs, Inc., West Grove, PA), by using procedures standard in our laboratory.27McMaster MT Librach CL Zhou Y Lim KH Janatpour MJ DeMars R Kovats S Damsky C Fisher SJ Human placental HLA-G expression is restricted to differentiated cytotrophoblasts.J Immunol. 1995; 154: 3771-3778PubMed Google Scholar Immune complexes were visualized using enhanced chemiluminescence and Hyperfilm (Amersham Life Sciences-USB, Arlington Heights, IL). The entire experiment was done three times using different batches of cytotrophoblasts. Fusion proteins consisting of the first three repeats of VEGFR-1/Flt1 or VEGFR-3/Flt-4 fused to the Fc portion of IgG [VEGFR-1(1-3)-Fc and VEGFR-3(1-3)-Fc] were produced in the Alitalo laboratory.29Pajusola K Aprelikova O Armstrong E Morris S Alitalo K Two human FLT4 receptor tyrosine kinase isoforms with distinct carboxy terminal tails are produced by alternative processing of primary transcripts.Oncogene. 1993; 8: 2931-2937PubMed Google Scholar, 30Hirashima M Kataoka H Nishikawa S Matsuyoshi N Maturation of embryonic stem cells into endothelial cells in an in vitro model of vasculogenesis.Blood. 1999; 93: 1253-1263Crossref PubMed Google Scholar As a control, nonimmune IgG was added to some of the wells. Initially, the activity of the fusion and control proteins was tested at concentrations ranging from 500 ng/ml to 50 μg/ml. Thereafter, 15 μg/ml, the lowest protein concentration that had maximum effects on cell morphology and integrin α1 expression, was used routinely. The entire experiment was done three times using different batches of cytotrophoblasts. Cytotrophoblasts cultured on Matrigel-coated wells were washed twice with PBS and then incubated for 90 minutes in a 0.1% solution of freshly prepared biotin (Pierce, Rockford, IL) in ice-cold PBS. The cells were washed three times, also in ice-cold PBS, then extracted with 200 μl of the lysis buffer described above. Cell lysates were centrifuged at 12,000 × g for 5 minutes. Samples of e" @default.
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• W2053976090 date "2002-04-01" @default.
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• W2053976090 title "Vascular Endothelial Growth Factor Ligands and Receptors That Regulate Human Cytotrophoblast Survival Are Dysregulated in Severe Preeclampsia and Hemolysis, Elevated Liver Enzymes, and Low Platelets Syndrome" @default.
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