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Discovery of antiangiogenic factors in the pathogenesis of preeclampsia

      To the Editors:
      Click Supplemental Materials under article title in Contents at ajog.org
      We report here the historic transcriptomics data that led to the discovery and characterization of antiangiogenic factors in the pathogenesis of preeclampsia. In 2001 and 2002, to identify novel secreted factors playing a pathologic role in preeclampsia, we performed gene expression profiling of placental tissue from 19 women with preeclampsia and 15 normotensive pregnant women using Affymetrix U95A microarray chips (Affymetrix, Santa Clara, California). Among the preeclamptic women, 89% were nulliparous compared with 19% among the normotensive women. The data were analyzed using Bayesian Analysis of Differential Gene Expression (BADGE method, version 1.0), a computer program implementing a Bayesian approach to identify differentially expressed genes across experimental conditions.
      • Ramoni M.F.
      • Sebastiani P.
      • Kohane I.S.
      Cluster analysis of gene expression dynamics.
      We identified a gene set of 127 genes; 65 of them were up-regulated and 62 were down-regulated, at a false positive rate of 1% (Supplemental Table). The Figure depicts the heatmap of the up- and down-regulated genes, many of which code for proteins regulating angiogenesis and metabolism. Among the most up-regulated transcripts were the endogenous inhibitor of vascular endothelial growth factor (VEGF) signaling, referred to as fms-like tyrosine kinase 1 (FLT1 or VEGFR1) and its soluble isoforms (sFLT1 or sVEGFR1). We characterized the sFLT1 pathway and noted that in animals, sFLT1 was sufficient to induce hypertension and proteinuria, and phenocopied several features of preeclampsia.
      • Maynard S.E.
      • Min J.Y.
      • Merchan J.
      • et al.
      Excess placental soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia.
      Taken together with the findings that anti-VEGF drugs were inducing preeclampsia-like phenotypes in cancer patients,
      • Yang J.C.
      • Haworth L.
      • Sherry R.M.
      • et al.
      A randomized trial of bevacizumab, an anti-vascular endothelial growth factor antibody, for metastatic renal cancer.
      we hypothesized that excess sFLT1 was responsible for the maternal syndrome of preeclampsia.
      • Maynard S.E.
      • Min J.Y.
      • Merchan J.
      • et al.
      Excess placental soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia.
      ,
      • Maynard S.E.
      • Venkatesha S.
      • Thadhani R.
      • Karumanchi S.A.
      Soluble Fms-like tyrosine kinase 1 and endothelial dysfunction in the pathogenesis of preeclampsia.
      Soluble endoglin, a second antiangiogenic protein in this list, was also further characterized by our group to play a synergistic role in the pathogenesis of preeclampsia.
      • Venkatesha S.
      • Toporsian M.
      • Lam C.
      • et al.
      Soluble endoglin contributes to the pathogenesis of preeclampsia.
      Nearly 20 years after the initial transcriptomic studies, the characterization of these antiangiogenic protein pathways have led to an improved understanding of the pathophysiology of preeclampsia, new biomarkers for early detection of the disease, and new therapeutic targets for this condition.
      • Rana S.
      • Burke S.D.
      • Karumanchi S.A.
      Imbalances in circulating angiogenic factors in the pathophysiology of preeclampsia and related disorders.
      Further characterization of other gene products described in the Supplemental Table may lead to a better understanding of the processes leading to preeclampsia.
      Figure thumbnail gr1
      FigureHeatmap of up-regulated and down-regulated genes in preeclampsia
      Colormap of the predictive gene set for placental mRNA expression in normal vs preeclampsia patients. The rows represent the predictive genes for preeclampsia, whereas the columns represent the expression levels for a given patient relative to the average gene expression. Significantly up-regulated genes include those coding for the fms-like tyrosine kinase 1 family of mRNAs and follistatin-related protein, whereas the significantly down-regulated genes include those coding for cytochrome P450 (CYP11B1). Genes with no names are left blank. However, their gene accession IDs are included in the .
      Karumanchi. Discovery of antiangiogenic factors in the pathogenesis of preeclampsia. Am J Obstet Gynecol 2022.

      Acknowledgments

      We thank Dr Marco Ramoni, PhD, (deceased) and Dr Jose Aleman, MD, for help with BADGE analysis.

      Appendix

      Supplemental TableDifferential expression of genes by Bayesian analysis (upregulated in preeclampsia in yellow and downregulated in preeclampsia in blue)
      Affy ProbeProbabilityFoldGene SymbolGene Name
      33900_at0.999923.849FSTL3follistatin-like 3 (secreted glycoprotein)
      990_at0.999903.233FLT1fms-related tyrosine kinase 1 (vascular endothelial growth factor/vascular permeability factor receptor)
      991_g_at0.999892.727FLT1fms-related tyrosine kinase 1 (vascular endothelial growth factor/vascular permeability factor receptor)
      1601_s_at0.999863.254IGFBP5insulin-like growth factor binding protein 5
      36317_at0.999823.767CORO2Acoronin, actin binding protein, 2A
      1389_at0.999822.299MMEmembrane metallo-endopeptidase (neutral endopeptidase, enkephalinase, CALLA, CD10)
      501_g_at0.999802.293CYP2J2cytochrome P450, family 2, subfamily J, polypeptide 2
      37657_at0.999793.089PALMparalemmin
      HUMGAPDH0.999783.647GAPDglyceraldehyde-3-phosphate dehydrogenase
      159_at0.999693.343VEGFCvascular endothelial growth factor C
      31754_at0.999663.737ABCA12ATP-binding cassette, sub-family A (ABC1), member 12
      1149_at0.999603.241D16154Transcription Factor Eb
      1545_g_at0.999592.692FLT1fms-related tyrosine kinase 1 (vascular endothelial growth factor/vascular permeability factor receptor)
      FLT1, sFLT1 and Endoglin genes.
      34129_at0.999532.211STXBP5Lsyntaxin binding protein 5-like
      1103_at0.999523.141ANGangiogenin, ribonuclease, RNase A family, 5
      255_s_at0.999502.761INHAinhibin, alpha
      1650_g_at0.999482.745SMOXspermine oxidase
      1964_g_at0.999462.331FLT1Soluble fms-related tyrosine kinase 1 (vascular endothelial growth factor/vascular permeability factor receptor)
      FLT1, sFLT1 and Endoglin genes.
      32298_at0.999402.894ADAM2a disintegrin and metalloproteinase domain 2 (fertilin beta)
      33995_at0.999395.997GUCA2Aguanylate cyclase activator 2A (guanylin)
      32892_at0.999372.014RPS6KA2ribosomal protein S6 kinase, 90kDa, polypeptide 2
      41577_at0.999102.361PPP1R16Bprotein phosphatase 1, regulatory (inhibitor) subunit 16B
      40790_at0.999032.169BHLHB2basic helix-loop-helix domain containing, class B, 2
      41024_f_at0.998912.617GYPEglycophorin E
      36426_g_at0.998791.981NEBLnebulette
      34800_at0.998682.943LRIG1leucine-rich repeats and immunoglobulin-like domains 1
      36979_at0.998682.389SLC2A3solute carrier family 2 (facilitated glucose transporter), member 3
      31382_f_at0.998512.065UGT2B28UDP glycosyltransferase 2 family, polypeptide B28
      40357_at0.998313.380INHBAinhibin, beta A (activin A, activin AB alpha polypeptide)
      1963_at0.998222.714FLT1Soluble fms-related tyrosine kinase 1 (vascular endothelial growth factor/vascular permeability factor receptor)
      FLT1, sFLT1 and Endoglin genes.
      35865_at0.998152.632NR5A2nuclear receptor subfamily 5, group A, member 2
      39051_at0.998141.805NNATneuronatin
      33642_s_at0.998073.236SLC6A8solute carrier family 6 (neurotransmitter transporter, creatine), member 8
      33182_at0.998042.698NTRK2neurotrophic tyrosine kinase, receptor, type 2
      33639_g_at0.998021.694ERBB3v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 (avian)
      34483_at0.997932.234SIGLEC6sialic acid binding Ig-like lectin 6
      1511_at0.997931.771SHC3src homology 2 domain containing transforming protein C3
      38280_s_at0.997873.286NTRK2neurotrophic tyrosine kinase, receptor, type 2
      41420_at0.997852.479IGFBP5insulin-like growth factor binding protein 5
      34088_at0.997832.009NXPH4neurexophilin 4
      36284_at0.997812.978LY6Dlymphocyte antigen 6 complex, locus D
      33825_at0.997772.575SERPINA3serine (or cysteine) proteinase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 3
      36533_at0.997422.354PTGISprostaglandin I2 (prostacyclin) synthase
      37813_at0.997352.073DDX51DEAD (Asp-Glu-Ala-Asp) box polypeptide 51
      39202_at0.997311.667TRAF3IP1TNF receptor-associated factor 3 interacting protein 1
      368_at0.997211.904TPBGtrophoblast glycoprotein
      500_at0.997161.751CYP2J2cytochrome P450, family 2, subfamily J, polypeptide 2
      38078_at0.996991.774FLNBfilamin B, beta (actin binding protein 278)
      41608_at0.996932.906CRHBPcorticotropin releasing hormone binding protein
      1734_at0.996562.200TGFB3Human transforming growth factor beta-3 gene
      1945_at0.996441.747CCNB1cyclin B1
      31990_at0.996361.496KIF17kinesin family member 17
      36933_at0.996182.050NDRG1N-myc downstream regulated gene 1
      32562_at0.996101.941ENGendoglin (Osler-Rendu-Weber syndrome 1)
      FLT1, sFLT1 and Endoglin genes.
      32565_at0.996062.098SMARCD3SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily d, member 3
      1369_s_at0.996013.111IL8interleukin 8
      1678_g_at0.995892.334IGFBP5insulin-like growth factor binding protein 5
      37887_at0.995721.887CHEK2CHK2 checkpoint homolog (S. pombe)
      40690_at0.995681.913CKS2CDC28 protein kinase regulatory subunit 2
      40926_at0.995592.068SLC6A8solute carrier family 6 (neurotransmitter transporter, creatine), member 8
      34898_at0.995582.179AREGamphiregulin (schwannoma-derived growth factor)
      33748_at0.995462.523HA-1minor histocompatibility antigen HA-1
      35940_at0.995362.086POU4F1POU domain, class 4, transcription factor 1
      32632_g_at0.995262.108GBAPglucosidase, beta; acid, pseudogene
      33792_at0.995182.318PSCAprostate stem cell antigen
      38566_at0.004950.730COL10A1collagen, type X, alpha 1(Schmid metaphyseal chondrodysplasia)
      31740_s_at0.004880.637PAX4paired box gene 4
      33359_at0.004850.547LPHN3latrophilin 3
      38519_at0.004760.483NR1H4nuclear receptor subfamily 1, group H, member 4
      33046_f_at0.004730.492EMX1empty spiracles homolog 1 (Drosophila)
      39108_at0.004720.616LSSlanosterol synthase (2,3-oxidosqualene-lanosterol cyclase)
      33693_at0.004510.499DSG3desmoglein 3 (pemphigus vulgaris antigen)
      834_at0.004360.615ZNFN1A1zinc finger protein, subfamily 1A, 1 (Ikaros)
      34575_f_at0.004160.480MAGEA5melanoma antigen, family A, 5
      33379_at0.004070.432SSX2IPsynovial sarcoma, X breakpoint 2 interacting protein
      31599_f_at0.003900.420MAGEA3melanoma antigen, family A, 3
      32935_at0.003890.512WDR21WD repeat domain 21
      33072_at0.003610.809HCRTR2hypocretin (orexin) receptor 2
      36777_at0.003570.525KLRK1killer cell lectin-like receptor subfamily K, member 1
      36269_at0.003560.538ADAMTS3a disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif, 3
      38095_i_at0.003510.596HLA-DPB1major histocompatibility complex, class II, DP beta 1
      36272_r_at0.003190.335PMP2peripheral myelin protein 2
      494_at0.003070.610IL13interleukin 13
      34698_at0.003000.522GNAO1guanine nucleotide binding protein (G protein), alpha activating activity polypeptide O
      39646_at0.002910.414CACNB2calcium channel, voltage-dependent, beta 2 subunit
      36049_at0.002780.497W27899CDNA clone IMAGE:4940887, partial cds
      37039_at0.002770.602HLA-DRAmajor histocompatibility complex, class II, DR alpha
      37588_s_at0.002620.621MAPK8IP2mitogen-activated protein kinase 8 interacting protein 2
      33846_at0.002600.522AA620377Cluster Incl. AA620377:ae57a07.s1 Homo sapiens cDNA, 3 end /clone=IMAGE-950964
      36416_g_at0.002590.512CASKcalcium/calmodulin-dependent serine protein kinase (MAGUK family)
      1298_at0.002560.447X86816Human estrogen receptor mRNA, alternatively spliced transcript H, partial cds.
      40646_at0.002350.562CX3CR1chemokine (C-X3-C motif) receptor 1
      37108_at0.002290.529X72755MRNA; cDNA DKFZp779B1535 (from clone DKFZp779B1535)
      32997_at0.002280.363GAGEB1G antigen, family B, 1 (prostate associated)
      35028_at0.002270.438GABRB1gamma-aminobutyric acid (GABA) A receptor, beta 1
      40679_at0.002130.458SLC6A12solute carrier family 6 (neurotransmitter transporter, betaine/GABA), member 12
      39498_at0.002130.497AA044810Cluster Incl. X86400:H.sapiens mRNA for gamma subunit of sodium potassium ATPase
      38833_at0.001990.670HLA-DPA1major histocompatibility complex, class II, DP alpha 1
      35031_r_at0.001830.281KIAA0316KIAA0316 gene product
      36911_at0.001800.433TYRP1tyrosinase-related protein 1
      31494_at0.001750.434L12691Cluster Incl. D25272:Homo sapiens mRNA, clone-RES4-16
      37782_at0.001700.654SSTsomatostatin
      36767_at0.001640.302CYP1A1cytochrome P450, family 1, subfamily A, polypeptide 1
      35539_at0.001590.386IMPG1interphotoreceptor matrix proteoglycan 1
      38330_at0.001590.371FRDAFriedreich ataxia
      35061_at0.001520.272CXCL11chemokine (C-X-C motif) ligand 11
      34002_at0.001390.627HSD3B2hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 2
      32017_at0.001390.531PARD6Bpar-6 partitioning defective 6 homolog beta (C. elegans)
      31398_at0.001320.440ABCB11ATP-binding cassette, sub-family B (MDR/TAP), member 11
      32451_at0.001310.556MS4A3membrane-spanning 4-domains, subfamily A, member 3 (hematopoietic cell-specific)
      34045_at0.001310.503LOC196993hypothetical protein LOC196993
      36428_at0.001300.569VMD2vitelliform macular dystrophy (Best disease, bestrophin)
      AFFX-DapX-3_a0.001220.469M33197L38424 B subtilis dapB, jojF, jojG genes corresponding to nucleotides 1358-3197 of L38424
      31324_at0.001160.484AF016492U82303:Homo sapiens unknown protein mRNA
      32474_at0.001110.644PAX7paired box gene 7
      37219_at0.000980.395CXCL9chemokine (C-X-C motif) ligand 9
      31506_s_at0.000970.288DEFA1defensin, alpha 1, myeloid-related sequence
      378_s_at0.000750.529GMLGPI anchored molecule like protein
      41820_s_at0.000730.570CDC2L5cell division cycle 2-like 5 (cholinesterase-related cell division controller)
      31310_at0.000610.523GLRA1glycine receptor, alpha 1 (startle disease/hyperekplexia, stiff man syndrome)
      39502_at0.000460.553DPYSL4dihydropyrimidinase-like 4
      35024_at0.000310.272OPRK1opioid receptor, kappa 1
      36220_at0.000300.346DDAH1dimethylarginine dimethylaminohydrolase 1
      204_at0.000220.601HOXA4homeo box A4
      750_at0.000210.389TRHRthyrotropin-releasing hormone receptor
      33478_at0.000090.296TNP2transition protein 2 (during histone to protamine replacement)
      1412_g_at0.000080.560CYP11B1cytochrome P450, family 11, subfamily B, polypeptide 1
      Genes selected with a 1.0% false positive error rate for a total of 127 genes, 65 of these are upregulated. Genes with no names are labeled with Genbank accession numbers.
      Karumanchi. Discovery of antiangiogenic factors in the pathogenesis of preeclampsia. Am J Obstet Gynecol 2022.
      a FLT1, sFLT1 and Endoglin genes.

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