Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Instruction for Examination in Biotechnology January 2011 Attached is the text of a draft article, which the inventors wish to submit shortly for publication. You are instructed to draft a priority application to be filed in Israel- and drafted according to Israeli patent practice (note that Figure 3 and Fig 4(c) were deleted by me from the text in order to avoid confusion) . The draft application you are preparing should contain two types of comments: “Comments to researchers” requesting them to supply information or verify statement. If you need the researchers to supply additional experimental data during the priority year- please draft a short letter explaining what additional data is needed. “Comments to examiner” (to me) explaining your drafting strategy, why you are choosing to protect (or not protect) certain aspects in the way appearing in your examination. Understanding the logic behind your choices may affect your final grade. In addition the comments to me should mention sections of the Israeli Law, Registrar’s circulars, or decisions which are relevant to the manner you drafted the claims (both as regards subject matter and phrasing) The “claims” will account for 85% of your grade and the following will be checked : understanding what is patentable in view of the prior art, drafting the broadest patentable claims, protecting all aspects of the invention (leave aside at this stage the issue of unity), using correct claim language and claim format for a patent application in Israel. The “description” will account for 15% of your grade and the following will be checked:having all the correct specification parts, and having good definitions for terms used in the claims. GOOD LUCK. A HUMAN-SPECIFIC SOLUBLE VEGF-RECEPTOR 1: ABSTRACT A human-specific splicing variant of VEGF receptor 1 (Flt1) was discovered, producing a soluble receptor (designated” sFlt1-14”) that is qualitatively different from the previously described soluble receptor (sFlt1), and functioning as a potent VEGF inhibitor. sFlt1-14 is generated in a cell type-specific fashion, primarily in non-endothelial cells.. Increased production of soluble VEGF receptors during pregnancy is entirely due to induced expression of placental sFlt1-14 starting by the end of the first trimester. Expression is dramatically elevated in the placenta of women with preeclampsia (PE), specifically induced in abnormal clusters of degenerative syncytiotrophoblasts known as syncytial knots where it may undergo further mRNA editing. sFlt1-14 is the predominant VEGF-inhibiting protein accumulating in the circulation of preeclampsic women and hence capable of neutralizing VEGF in distant organs affected in PE. Together, these findings revealed a new natural VEGF inhibitor that has evolved in humans, possibly to protect non-endothelial cells from adverse VEGF signaling. Further, the study uncovered the exact identity of the VEGF-blocking protein implicated in PE. 2 INTRODUCTION VEGF is the key factor promoting vasculogenesis and angiogenesis in the embryo and the factor orchestrating most, if not all processes of adult neovascularization. In addition, VEGF performs non-vascular developmental functions and plays a number of homeostatic roles in the adult. The later includes maintenance of endothelial fenestrations, a role in vasodilatation, neurogenic and neurotrophic activities, and others 1,2. Not surprisingly, therefore, VEGF is under a tight spatial and temporal regulation and even a moderate deviation from its exquisite dosage control or perturbation of its precise morphogenetic gradients are detrimental for proper development and organ homeostasis3-6. Among the multiple layers of VEGF control, natural VEGF inhibitors, primarily soluble VEGF receptors, are likely to be important. Indeed, VEGF receptor-1 (Flt1) mRNA may undergo alternative splicing in a way that the encoded protein retains the ligand-binding domain but is devoid of the membrane spanning- and intracellular kinase domains, hence functioning as a VEGF-trapping soluble receptor. This secreted protein (designated sFlt1) is a potent inhibitor of both VEGF-A, VEGF-B and PLGF 2 . It is widely used as a research tool for VEGF inhibition and more recently also harnessed in clinical trials of VEGF blockade. Surprisingly, however, little is known concerning natural sFlt1 regulation. Likewise, the different areas where it may act in physiologic titration of VEGF or in pathologic neutralization of VEGF activity remain to be uncovered. A remarkable recent example for a physiologic role of sFlt1 is the identification of sFlt1 as the factor responsible for preventing vascular invasion onto the cornea 7. The best documented case of a pathological role is the involvement of sFlt1 in preeclampsia 8,9. 3 Preeclampsia (PE), affecting 5% of pregnant women is characterized by severe maternal hypertension, proteinuria, glomerular endotheliosis and neurological symptoms. While the underlying pathogenetic mechanism of PE is largely unknown, sFlt1 was implicated in PE causation because of its abnormally high levels in the circulation of preeclempsic women 9. Further, a seminal study by Maynard et. al. has shown that systemic sFlt1 causes a PE-like syndrome in rats, presumably through negating constitutive VEGF function in the kidney 8. Initially considered as an endothelial cell-specific receptor, Flt1 was subsequently detected also in a number of non-endothelial cells, thus extending the spectrum of cells directly responsive to VEGF. We reason that the splicing ratio of Flt1/sFlt1 should determine the extent to which a given cell will transmit or, conversely, resist VEGF signaling. We propose that dynamic changes in the splicing ratio may serve a regulatory function, in general, and that predominance of the soluble receptor might protect non-endothelial cells from adverse effects of VEGF, in particular. Experiments reported here are compatible with the proposition of a role for sFlt1 in the EC/VSMC inter-phase of the mature vessel wall. We describe a novel splice variant of Flt1 functioning as a potent VEGF-blocking soluble receptor. This natural VEGF inhibitor designated sFlt1-14, evolved during primate evolution and is the predominant VEGF inhibitor produced by human non-endothelial cells. Importantly, we show that sFlt1-14 is also the VEGF-neutralizing protein produced by the placenta of women with preeclampsia. 4 MATERIALS & METHODS cDNA cloning. Placental RNA was prepared and converted to cDNA using standard procedures. Rapid Amplification of cDNA Ends (RACE) was performed using a BD Smart RACE cDNA Amplification Kit (Cat. no. 634914). For cloning/sequencing of sFlt1-14 3’-UTRs, RNAs of PE placentas were reverse-transcribed using a primer complementary to the beginning of exon 14 of Flt1. Recombinant sFlt1 and sflt1-14 proteins. cDNAs encompassing the entire coding region of both soluble receptor isoforms were sub-cloned into Bluescript expression vectors and transfected onto T7 polymerase-expressing Hela cells. 20-24 hours later, media were collected and the cells were harvested. Secreted and cell associated proteins were immuno precipitated with the FLT11 (sigma) antibody and analyzed by immuno blotting with the ab9540 antibody (abcam), both antibodies directed against the extra cellular domain of Flt1. VEGF inhibition assay- Porcine Aortic Endothelial (PAE) cells engineered to express high levels of human VEGF-R2 were exposed to VEGF pre-incubated (or not) with sFlt1 or sFlt1-14 proteins. A reduction in VEGF-R1 phosphorylation was determined using antibodies detecting phospho-VEGF-R2 (Cell-signaling, Cat. #2478) and standardized to total VEGF-R2 protein visualized by immunoblotting with anti-VEGF-R2 antibody (Santa Cruz Cat. SC-504). RNA and protein analyses. For Northern-blot analysis, total RNA (5−20 μg) was resolved by formaldehyde−agarose (1%) denaturing gels, blotted onto positively charged nylon membrane by capillary elution and hybridized with 32 P-labeled specific cDNA probes prepared with the aid of a Rediprime kit (Amersham). Probes derived from different regions of FLT1 transcripts were used, as specified in the text and figure legends. For in situ detection of sFlt1-14 RNA, paraffin- 5 embedded sections of normal and PE placenta were hybridized with a 35S-labeled riboprobe composed of intron 14 sequences as previously described (Motro et al., PNAS, 1990, 87(8),3092-6). For IP/IB analysis, placentas were homogenized and immuno-precipitated with one of three antibodies: CHFK produced in our laboratory , CESS produced in our laboratory and FLT11 (Sigma Cat. #V4262) and incubated overnight with Protein A-coated beads (P3391, Sigma). Precipitates were than dissolved, proteins separated on 6% acrylamide gel, electrophoreticaly transferred to a membrane, and detected with the CESS antibody. For analysis of serum proteins, 20 ml serum from PE patients was first concentrated through capture on FLT11-coated beads and affinity-purified proteins analysed by Western-blotting as described above. Placental samples were taken with IRB approval. Preeclampsia samples met ACOG criteria for severe preeclampsia. Mass spectrometry – Protein homogenate of a PE placenta was incubated for 3 hours with a rabbit pre-immune serum (20 l), incubated overnight with Protein A beads and precipitated by through 3 hrs incubation with 15l of the CESS antibody. Preciptated proteins were subsequently separated on a 6% acrylamide gel and visualized by staining with h coomassie blue. A 115 Kd band was cut-out, digested with trypsin and was subjected to mass spectrometry analysis by LC-MS/MS on DECA/LCQ. Peptides were identified and analyzed by Pep-Miner and Sequest software against nr database of Human, mouse, rat bovine and Rabbit. ELISA- A commercial ELISA kit for the detection of all soluble VEGF receptors isoforms was used (R&D systems, DVR 100B). Cells and culture- Human primary endothelial and vascular smooth muscle cells were a gift from Dr. ,,,, 6 RESULTS Identification of a novel splice variant of a soluble VEGF receptor-1 (sFlt1-14). The majority of mRNA molecules transcribed from VEGF-R1 DNA encodes a transmembrane signaling receptor but a small fraction of primary transcripts is usually alternatively spliced generating a truncated mRNA devoid of the transmembrane- and intracellular-encoding domains (Fig.1) and hence capable of sequestering VEGF and block signaling.. 3’-RACE methodology was thus used for cloning truncated Flt1 mRNAs present in human placental RNA. These studies culminated in the isolation and characterization of a novel human-specific splice variant of VEGF-R1, designated therein as sFlt1-14 (Fig.1A).. sFLT1-14 sequence new data have been submitted to the GenBank databases under accession No. EU368830. We have found a new soluble sFlt1(which we called sFlt14)variant different in sequence from the sFlt-1of the prior art (Maynard et al) and produced by the Flt gene by a previously undescribed mode of alternative splicing . This variant excludes intron 13 (present in the prior art sFlt-1 but not in the full membranal receptor); includes exon 14 (present in the full membranal Flt1 protein but not in the prior art soluble sFlt-1(-marked in the figure below as UNDERLINED) and in addition includes sequences derived from intron 14 which are not present either in the prior art sFlt -1 nor in the full membranal receptor(sequences from expressed intron 14 are marked in the sequence below as BOLD ) Met V S Y W D T G V L L C A L L S C L L L T G S S S G S K L K D P E L S L K G T Q H I MQAGQTLHLQCRGEAAHKWSLPEMVSKESERLSITKSACGRN GKQFCSTLTLNTAQANHTGFYSCKYLAVPTSKKKETESAIYIFI SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFP 7 LDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYK TNYLTHRQTNTIIDVQISTPRPVKLLRGHTLVLNCTATTPLNTR VQMTWSYPDEKNKRASVRRRIDQSNSHANIFYSVLTIDKMQN KDKGLYTCRVRSGPSFKSVNTSVHIYDKAFITVKHRKQQVLET VAGKRSYRLSMKVKAFPSPEVVWLKDGLPATEKSARYLTRGY SLIIKDVTEEDAGNYTILLSIKQSNVFKNLTATLIVNVKPQIYEK AVSSFPDPALYPLGSRQILTCTAYGIPQPTIKWFWHPCNHNHSE ARCDFCSNNEESFILDADSNMGNRIESITQRMAIIEGKNKMAST LVVADSRISGIYICIASNKVGTVGRNISFYITDVPNGFHVNLEK MPTEGEDLKLSCTVNKFLYRDVTWILLRTVNNRTMHYSISKQK MAITKEHSITLNLTIMNVSLQDSGTYACRARNVYTGEEILQKK EITIRDQEAPYLLRNLSDHTVAISSSTTLDCHANGVPEPQITWF K N N H K I Q Q E P E L Y T S T S P S S S S S S P L S S S S S S S S S S S S Stop The following is the cDNA sequence coding for the full sFlt14 is: (Bold and underlinedsequence coding for the expressed intron 14 ) ATGGTCAGCTACTGGGACACCGGGGTCCTGCTGTGCGCGCTGCTCAGCTGTC TGCTTCTCACAGGATCTAGTTCAGGTTCAAAATTAAAAGATCCTGAACTGAGTTT AAAAGGCACCCAGCACATCATGCAAGCAGGCCAGACACTGCATCTCCAATGCA 8 GGGGGGAAGCAGCCCATAAATGGTCTTTGCCTGAAATGGTGAGTAAGGAAAGC GAAAGGCTGAGCATAACTAAATCTGCCTGTGGAAGAAATGGCAAACAATTCTGC AGTACTTTAACCTTGAACACAGCTCAAGCAAACCACACTGGCTTCTACAGCTGC AAATATCTAGCTGTACCTACTTCAAAGAAGAAGGAAACAGAATCTGCAATCTATA TATTTATTAGTGATACAGGTAGACCTTTCGTAGAGATGTACAGTGAAATCCCCG AAATTATACACATGACTGAAGGAAGGGAGCTCGTCATTCCCTGCCGGGTTACG TCACCTAACATCACTGTTACTTTAAAAAAGTTTCCACTTGACACTTTGATCCCTG ATGGAAAACGCATAATCTGGGACAGTAGAAAGGGCTTCATCATATCAAATGCA ACGTACAAAGAAATAGGGCTTCTGACCTGTGAAGCAACAGTCAATGGGCATTTG TATAAGACAAACTATCTCACACATCGACAAACCAATACAATCATAGATGTCCAAA TAAGCACACCACGCCCAGTCAAATTACTTAGAGGCCATACTCTTGTCCTCAATT GTACTGCTACCACTCCCTTGAACACGAGAGTTCAAATGACCTGGAGTTACCCTG ATGAAAAAAATAAGAGAGCTTCCGTAAGGCGACGAATTGACCAAAGCAATTCCC ATGCCAACATATTCTACAGTGTTCTTACTATTGACAAAATGCAGAACAAAGACAA AGGACTTTATACTTGTCGTGTAAGGAGTGGACCATCATTCAAATCTGTTAACAC CTCAGTGCATATATATGATAAAGCATTCATCACTGTGAAACATCGAAAACAGCA GGTGCTTGAAACCGTAGCTGGCAAGCGGTCTTACCGGCTCTCTATGAAAGTGA AGGCATTTCCCTCGCCGGAAGTTGTATGGTTAAAAGATGGGTTACCTGCGACT GAGAAATCTGCTCGCTATTTGACTCGTGGCTACTCGTTAATTATCAAGGACGTA ACTGAAGAGGATGCAGGGAATTATACAATCTTGCTGAGCATAAAACAGTCAAAT GTGTTTAAAAACCTCACTGCCACTCTAATTGTCAATGTGAAACCCCAGATTT ACGAAAAGGCCGTGTCATCGTTTCCAGACCCGGCTCTCTACCCACTGGGCAGC AGACAAATCCTGACTTGTACCGCATATGGTATCCCTCAACCTACAATCAAGTGG TTCTGGCACCCCTGTAACCATAATCATTCCGAAGCAAGGTGTGACTTTTGTTCC AATAATGAAGAGTCCTTTATCCTGGATGCTGACAGCAACATGGGAAACAGAATT GAGAGCATCACTCAGCGCATGGCAATAATAGAAGGAAAGAATAAGATGGCTAG CACCTTGGTTGTGGCTGACTCTAGAATTTCTGGAATCTACATTTGCATAGCTTCC AATAAAGTTGGGACTGTGGGAAGAAACATAAGCTTTTATATCACAGATGTGCCA AATGGGTTTCATGTTAACTTGGAAAAAATGCCGACGGAAGGAGAGGACCTGAA ACTGTCTTGCACAGTTAACAAGTTCTTATACAGAGACGTTACTTGGATTTTACTG CGGACAGTTAATAACAGAACAATGCACTACAGTATTAGCAAGCAAAAAATGGCC 9 ATCACTAAGGAGCACTCCATCACTCTTAATCTTACCATCATGAATGTTTCCCTGC AAGATTCAGGCACCTATGCCTGCAGAGCCAGGAATGTATACACAGGGGAAGAA ATCCTCCAGAAGAAAGAAATTACAATCAGAGATCAGGAAGCACCATACCT CCTGCGAAACCTCAGTGATCACACAGTGGCCATCAGCAGTTCCA CCACTTTAGACTGTCATGCTAATGGTGTCCCCGAGCCTCAGATC ACTTGGTTTAAAAACAACCACAAAATACAACAAGAGCCTGAACT GTATACATCAACGTCACCATCGTCATCGTCATCATCACCATTGTC ATCATCATCATCATCGTCATCATCATCATCATCATAG .Notably, sFlt1-14 contains unique coding, as well as non-coding sequences shared neither by the full-length receptor nor by the known soluble receptor, sFlt1. This was utilized for preparing sFlt1-14-specific RNA probes that could be used for in situ hybridization having the following sequence: AACTGTATACATCAACGTCACCATCGTCATCGTCATCATCACCATTGTCATCATCATC ATCATCGTCATCATCATCATCATCATAGCTATCATCATTATCATCATCATCATCATCA TCATCATAGCTACCATTTATTGAAAACTATTATGTGTCAACTTCAAAGAACTTATCCT TTAGTTGGAGAGCCAAGACAATCATAACAATAACAAA and for preparation of sFlt1-14-specific antibodies directed against predicted carboxy-terminal unique epitopes One antibody was directed against the following sequence : QEAPYL L R N L S D H T V A I S S S T T L D C H A N G V P E P Q I T W F K N N H K IQQEP which is the sequence of exon 14 (present in the full membranal Flt1 protein but not in the prior art soluble sFlt-1 A second antibody was prepared against sequence 10 ELYTSTSPSSSSSSPL Which is part of the sequence expressed intron 14 which are not present either in the prior art sFlt -1 nor in the full membranal receptor These antibodies were subsequently used to demonstrate that sFlt1-14 mRNA is naturally translated into a 732 amino acid-long soluble receptor protein (see Fig.6 below). sFlt1-14 is a natural VEGF inhibitor. Considering that sFlt1 and sFlt1-14 are qualitatively different proteins (with sFlt1-14 containing 75 amino acids not present in sFlt1 and with sFlt1 containing 31 highly-conserved amino acids not present in sFlt1-14), it was essential to demonstrate that sFlt1-14 functions as a VEGF inhibitor. To this end, we first expressed a recombinant sFlt1-14 protein in human Hela cells (and for comparison, also produced sFlt1-expressing Hela cells). Amounts of the respective protein released to growth medium were then monitored by ELISA directed against a shared extra-cellular epitope. For both proteins, concentrations of 100-200ng/ml were detected in the medium. Media were further analyzed by immuno-precipitation and western blotting with two different shared (extra-cellular) antibodies, confirming the mutually exclusive production of either sFlt1-14 or sFlt1. Two sFlt1-14 proteins were detected - a cell-associated 115Kd isoform and a secreted 130Kd isoform, likely representing additional post-translational modifications. As expected from the size of the respective coding region, sFlt1-14 proteins were larger than the corresponding secreted- and cellular sFlt1 isoforms (Fig.2A). To determine whether sFlt1-14 inhibits VEGF signaling, increasing amounts were pre-incubated with a constant amount of VEGF (20ng/ml) prior to adding to the growth medium of Porcine Aortic Endothelial (PAE) cells engineered to express high levels of human VEGF-R2. Levels of VEGF-R2 11 phosphorylation were than measured as a function of the sFlt1-14/VEGF ratio. As shown in Fig.2B, sFlt1-14 inhibited VEGF-R2 phosphorylation almost completely already at a 1:1 ratio. For comparison, at this ratio, sFlt1 did not significantly inhibit VEGF-R2 phosphorylation, which was only evident at higher sFlt1/VEGF ratios. We conclude that sFlt1-14 is a potent inhibitor of VEGF signaling, at least as good, if not better than sFlt1. sFlt1-14 is upregulatd in syncytial knots of the preclampsic placenta Previous studies highlighted the role of sFlt1 in preeclampsia (PE) causation, based on findings that sFlt1 accumulates the circulation of women with PE to substantially higher levels than in normal pregnancies and on findings that when expressed in rats, a chimeric protein containing ligand-binding domains of sFlt1 produces the PE-like symptoms of hypertension and proteinuria 8 . Yet, the placental cellular sources of circulating VEGF receptors and factors responsible for its deregulated expression in the human preeclampsia are not known. Furthermore, with the discovery of sFlt1-14, the question arises which soluble receptor is associated with PE. To address these questions, Placental RNAs from different stages of normal pregnancies and from term placentas of both normal and PE pregnancies were first examined by northern blot analysis with shared- and specific riboprobes. While expression of total soluble VEGF-R1 increases progressively, particularly towards the third trimester of pregnancy, the relative contribution of the two sFlt1 isoforms changed dramatically, with sFlt1-14 becoming the dominant isoform over time. Thus, while the ‘classical’ sFlt1 accounted for approximately half of placenta-produced soluble receptors during the first trimester, further increase at later times was entirely due to upregulated expression of sFlt1-14 (Fig.4A). Transition to almost exclusive production of sFlt1- 12 14 took place by the beginning of the second trimester and was clearly evident at term (Fig.4A). sFlt1-14 mRNA was also the dominant, if not the sole transcript encoding soluble Flt1 in the PE placenta, reaching steady state levels exceedingly higher than in the term normal placenta (Fig.4B). Identity of the respective mRNAs was secured through re-probing with a sFlt1-14specific riboprobe (data not shown) and was further confirmed by direct sequencing of cDNA clones prepared from placenta RNA of 7 different PE patients. To identify which cells in the PE placenta produce sFlt1-14, we carried-out in situ hybridization analysis using a sFlt1-14-specific riboprobe AACTGTATACATCAACGTCACCATCGTCATCGTCATCATCACCATTGTCATCATCATC ATCATCGTCATCATCATCATCATCATAGCTATCATCATTATCATCATCATCATCATCA TCATCATAGCTACCATTTATTGAAAACTATTATGTGTCAACTTCAAAGAACTTATCCT TTAGTTGGAGAGCCAAGACAATCATAACAATAACAAA (Fig.5). The major sites of placental sFlt1-14 expression were found to be syncytial knots, i.e., clusters of degenerative syncytiotropoblasts that are a histological hallmark of the PE placenta. Noteworthy, sFlt1-14 is also produced by seemingly healthy syncytiotrophoblasts but at significantly lower levels and, importantly, not at all in vascular endothelial cells (Fig.5 Right and at a higher magnification in the image on the right), thus identifying syncytial knots as the predominant site of sFlt1-14 production. It should be pointed-out that syncytial knots are often found in aged normal placenta and we could indeed detect sFlt1-14 expression in syncytial knots of normal placenta (data not shown). We conclude that the much increased abundance of syncytial knot in PE accounts for the greatly elevated levels of sFlt1-14 produced in the PE placenta. 13 sFlt1-14 is the predominant circulating VEGF-inhibiting protein in PE. Findings that the PE placenta expresses exclusively sFlt1-14 argue that this is the major soluble receptor isoform capable of accumulating in the serum of PE patients. To show that sFlt1-14 protein is produced in the PE placenta and subsequently released into the circulation of PE patients, the following experiments were performed. First, term placentas from normal and PE pregnancies were examined for the relative abundance of soluble receptors, using immunoprecipitations (IP) and immuno-blotting (IB) with isoform-specific antibodies. As expected from the mRNA expression data, sFlt1-14 protein was abundantly produced in term placenta (Fig.6A). Second, proteins from a preeclampsia placenta were immuno-precipitated with the sFLT1-14 specific antibody directed against the expressed intron 14 (E L Y T S T S P S S S S S S P L) and the eluted 115 Kd band was unequivocally identified as sFlt1-14 using mass-spectrometry. Particularly informative in this regard was a peptide spanning the exon13-exon 14 junction which is not present in sFlt1 (Fig.6B). Third, sera of PE women were analyzed for soluble Flt1 receptors. ELISA with antibody directed against a shared extra-cellular domain confirmed earlier report of significantly elevated levels of circulating soluble receptor in PE (800-900 pg/ml vs. 8000-10000 pg/ml in control and PE third trimester sera, respectively). To identify the molecular identity of circulating soluble receptors (whether it is the Sftl1 or the new Sflt14), PE serum specimens were passed through FLT1 antibody-coated columns and bound proteins were subsequently analyzed by western blotting (Fig.6C). Again, sFlt1-14 protein was readily detected in the PE serum, visualized as two bands identical in size to those produced by the cells transfected with sFlt1-14 expression plasmid and detected with the sFlt1-14-specific antibody . 14 Notably, we failed to detect appreciable levels of sFlt1 in these sera in the limited set of patients analyzed, indicating that sflt1-14 is the major VEGF –inhibiting protein in the PE circulation. 15 DISCUSSION The study uncovered a new player in VEGF regulation namely, a novel variant of soluble VEGF receptor-1 (designated sFlt1-14) functioning as a potent VEGF inhibitor. Evolutionarily, sFlt1-14 represents a relatively recent addition to the multiple layers of VEGF control, as the unique splicing pattern generating it, utilizing a previously unrecognized intron 14 splice acceptor (SA) site, only takes place in humans and possibly also in non-human primate harboring Alu insertion within the Flt1 locus. .. The newly-discovered sflt1-14 is qualitatively different from sFlt1 by both missing 31 amino acids, and by containing 28 unique amino acids. Although the 31 amino acids missing in sFlt114 are evolutionary highly conserved, arguing for their functional importance, inhibition of VEGF signaling was not compromised. Conversely, it is possible that addition of extra amino acids (e.g. a polyserine stretch) confer unique biological properties to sFlt1-14. . The most significant pathology in which a soluble VEGF-R1 has been causally implicated is preecempsia. This was based on detecting abnormally high levels of soluble receptor in circulation of preecelmpsic women and on demonstrating that its systemic administration in rats produces a PE-like syndrome 8,9. Here we provide evidence that the soluble receptor produced by the placenta, accumulating in the circulation of PE women, and hence capable of affecting distant organs is predominantly, if not exclusively, sFlt1-14 and not the previously recognized sFlt1. The discrepancy with published work is explained by the fact that, unlike in our study, previous studies have only used probes and antibodies directed against the extra-cellular domain of Flt1, i.e. against a sequence or an epitope shared by all mRNA and proteins encoded by Flt1 gene. The facts that PE is a human-specific disease and that sFlt1-14 is a human-specific protein raise the 16 speculation that a unique aspect in sflt1-14 regulation or a unique, yet unidentified biological property of this protein are responsible for disease. Yet, the reason why rodents do not develop PE could also be attributed to inherent differences in placentation in rodents and primates. A sFlt1-14-expressing mouse (currently under construction) should aid addressing this question. Here we identified syncytial knots as the major source of local and circulating sFlt1-14. The fact that these structures increase in number in the normal aging placenta but are much more abundant in the degenerating PE placenta, a phenomenon known as the "Tenney-Parker change"18, may explain findings that serum levels of soluble receptors (now identified as mostly sFlt1-14) increase in the second and third trimesters of normal pregnancies but to dramatically higher levels in PE pregnancies. These findings place syncytial knotting, a phenomenon common to several placental pathologies, as central to PE pathogenesis. We note in this regard that hypoxia, often considered to be an ethiological cause of PE, is known to induce syncytial knotting 19 and that syncytial knotting is also increased in placental malaria 20 known to be associated with increasing levels of circulating VEGF-R1 receptors 21. Finally, we note that our finding provide another example for a required caution in extrapolating from mouse systems to human disease, as the later might also involve humanspecific mRNAs and proteins. 17 REFERENCES .1 Cao L, Jiao X, Zuzga DS, Liu Y, Fong DM, Young D, During MJ. VEGF links hippocampal activity with neurogenesis, learning and memory. Nat Genet. 2004;36:82735. .2 Olsson AK, Dimberg A, Kreuger J, Claesson-Welsh L. VEGF receptor signalling - in control of vascular function. Nat Rev Mol Cell Biol. 2006;7:359-71. .3 Carmeliet P, Ferreira V, Breier G, Pollefeyt S, Kieckens L, Gertsenstein M, Fahrig M, Vandenhoeck A, Harpal K, Eberhardt C, Declercq C, Pawling J, Moons L, Collen D, Risau W, Nagy A. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature. 1996;380:435-9. .4 Ferrara N, Carver-Moore K, Chen H, Dowd M, Lu L, O'Shea KS, Powell-Braxton L, Hillan KJ, Moore MW. Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature. 1996;380:439-42. .5 Miquerol L, Langille BL, Nagy A. Embryonic development is disrupted by modest increases in vascular endothelial growth factor gene expression. Development. 2000;127:3941-6. .6 Carmeliet P, Ng YS, Nuyens D, Theilmeier G, Brusselmans K, Cornelissen I, Ehler E, Kakkar VV, Stalmans I, Mattot V, Perriard JC, Dewerchin M, Flameng W, Nagy A, Lupu F, Moons L, Collen D, D'Amore PA, Shima DT. Impaired myocardial angiogenesis and ischemic cardiomyopathy in mice lacking the vascular endothelial growth factor isoforms VEGF164 and VEGF188. Nat Med. 1999;5:495-502. 18 .7 Ambati BK, Nozaki M, Singh N, Takeda A, Jani PD, Suthar T, Albuquerque RJ, Richter E, Sakurai E, Newcomb MT, Kleinman ME, Caldwell RB, Lin Q ,Ogura Y, Orecchia A, Samuelson DA, Agnew DW, St Leger J, Green WR, Mahasreshti PJ, Curiel DT, Kwan D, Marsh H, Ikeda S, Leiper LJ, Collinson JM, Bogdanovich S, Khurana TS, Shibuya M, Baldwin ME, Ferrara N, Gerber HP, De Falco S, Witta J, Baffi JZ, Raisler BJ, Ambati J. Corneal avascularity is due to soluble VEGF receptor-1. Nature. 2006;443:993-7. .8 Maynard SE, Min JY, Merchan J, Lim KH, Li J, Mondal S, Libermann TA, Morgan JP, Sellke FW, Stillman IE, Epstein FH, Sukhatme VP, Karumanchi SA. Excess placental soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia. J Clin Invest. 2003;111:649-58. .9 Levine RJ, Maynard SE, Qian C, Lim KH, England LJ, Yu KF, Schisterman EF, Thadhani R, Sachs BP, Epstein FH, Sibai BM, Sukhatme VP, Karumanchi SA. Circulating angiogenic factors and the risk of preeclampsia. N Engl J Med. 2004;350:672-83. .10 Sorek R, Lev-Maor G, Reznik M, Dagan T, Belinky F, Graur D, Ast G. Minimal conditions for exonization of intronic sequences: 5' splice site formation in alu exons. Mol Cell. 2004;14:221-31. .11 Chandra A, Angle N. Vascular endothelial growth factor stimulates a novel calciumsignaling pathway in vascular smooth muscle cells. Surgery. 2005;138:780-7. .12 Wang Z, Castresana MR, Newman WH. Reactive oxygen and NF-kappaB in VEGFinduced migration of human vascular smooth muscle cells. Biochem Biophys Res Commun. 2001;285:669-74. 19 .13 Lee S, Chen TT, Barber CL, Jordan MC, Murdock J, Desai S, Ferrara N, Nagy A, Roos KP, Iruela-Arispe ML. Autocrine VEGF signaling is required for vascular homeostasis. Cell. 2007;130:691-703. .14 Iacono M, Mignone F, Pesole G. uAUG and uORFs in human and rodent 5'untranslated mRNAs. Gene. 2005;349:97-105. .15 Hughes TA. Regulation of gene expression by alternative untranslated regions. Trends Genet. 2006;22:119-22. .16 Levanon K, Eisenberg E, Rechavi G, Levanon EY. Letter from the editor: Adenosine-toinosine RNA editing in Alu repeats in the human genome. EMBO Rep. 2005;6:8315.- .17 Yang JH, Luo X, Nie Y, Su Y, Zhao Q, Kabir K, Zhang D, Rabinovici R. Widespread inosine-containing mRNA in lymphocytes regulated by ADAR1 in response to inflammation. Immunology. 2003;109:15-23. .18 Tenney B, Parker F. The Placenta in Toxemia of Pregnancy. Am J Obstet Gynecol. 1940;39:1000-1005. .19 Heazell AE, Moll SJ, Jones CJ, Baker PN, Crocker IP. Formation of syncytial knots is increased by hyperoxia, hypoxia and reactive oxygen species. Placenta. 2007;28 Suppl A:S33-40. .20 Crocker IP, Tanner OM, Myers JE, Bulmer JN, Walraven G, Baker PN. Syncytiotrophoblast degradation and the pathophysiology of the malaria-infected placenta. Placenta. 2004;25:273-82. .21 Muehlenbachs A, Mutabingwa TK, Edmonds S, Fried M, Duffy PE. Hypertension and maternal-fetal conflict during placental malaria. PLoS Med. 2006;3:e446. 20 FIGURE LEGENDS Figure 1. Structure of the novel VEGF-R1 soluble receptor (sFlt1-14). A. A schematic representation of mRNAs encoding the full-size trans-membrane receptor (Flt1), the known soluble receptor (sFlt1) and the new alternatively-spiced soluble receptor (sFlt1-14). Translation start and stop codons are indicated. Boxes mark the three different RNA probes used in this work : A probe encompassing exons 1-5 detecting all three receptor isoforms, a probe derived from exon 13 specific for sFlt1 and an intron 14-based probe specific for sFlt1-14. B. The predicted amino acid sequence of sFlt1-14 protein, starting at exon 13 (aa 623 in NP_002010). First raw depicts exon 13 amino acids (shared by all three receptors), second raw show exon 14 amino acids (shared by flt1 and sFlt1-14 but absent in sFlt1), third raw show exonized amino acids derived from intron 14 (unique to sFlt1-14). Note the unusual polyserine stretch at the carboxy-terminus. The underlined amino acids served for developing exons 14 and exon 14a specific antibodies, designated CHFK and CESS, respectively. Figure 2. sFlt1-14 is a potent inhibitor of VEGF signaling. A. Recombinant sFlt1 and sFlt1-14 proteins were produced in HeLa cells transfected with the respective expression vector. Soluble flt1 receptors found in the cell (c) or released to the medium (m) were detected by Western blotting using antibodies directed against shared epitopes. Note the presence of a 130Kd secreted sFlt1-14 protein and a 115Kd sflt1-14 protein in the cellular fraction. 21 B. Porcine aortic Endotelial cells (PAE) stably expressing Flk1 were exposed to 20ng/ml VEGF and Flk1 phosphorylation was determined as readout for the extent of VEGF signaling. Inhibitory effect of pre-incubation of VEGF with the indicated amounts of recombinant sFlt1 or sFlt1-14 was measured, presented as the pFlk1/Flk1 ratio relative to the non-inhibited ratio. Figure 3. Entire figure and related text in article were taken out Figure 4. Prefferential expression of sFlt1-14 in the maturing normal and PE placenta. A. (Left) Northern blot analysis of placental RNAs from the indicated weeks of gestation with a probe detecting shared sequences of sFlt1-14 and sFlt1. (Right) The sFlt1-14/sFlt1 expression ratio calculated on the basis of hybridization intensities. Note dominance of sFlt1-14 from the second trimester and onwards. B. Northern blot analysis with the shared extra-cellular part of Flt1 identifies sflt1-14 as the predominant receptor isoform in the PE placenta Figure 4 (c )was taken out of the text Figure 5. Identification of sFlt1-14-producing cell in the placenta. In situ hybridization with a sFlt1-14-specific riboprobe. Note that syncytial knots (SK) are the major source of placental sFlt1-14, with a lower expression by apparently healthy syncytiotrophoblasts (STB) and no sflt114 expression by placental endothelial cells (EC). 22 Figure 6. sFlt1-14 protein produced by the placenta accumulates in the circulation of preecempsia patients. A. Protein extracts from term human placenta were immuno-precipitated separately with the three indicated antibodies detecting each a different epitope of sFlt1-14 and immuno-blotted with the antibody unique to sFlt1-14. This procedure identified the 130Kd and 115Kd placental proteins as sFlt1-14. B. The 115 Kd protein band immuno-precipitated from a preeclamptic placental biopsy with the CESS antibody and visualized by coomassie blue staining, eluted and analyzed by mass spectrometry. Highlighted in red are three identified peptides that map either to the first 13 exons or to the exon13/exon14 junction (Junction indicated by the green vertical line). The latter ruleout that the protein is sFlt1, while the CESS immuno-precipitation and size considerations ruleout that it is Flt1, thus identifying the 115kd protein unequivocally as sFlt1-14. C. 20ml serum of a PE patient were passed through a column of beads coated with anti-Flt1 antibodies (against a shared extra-cellular epitope), eluted at a low pH and immuno-blotted with the sFlt1-14-specific CESS antibody. For comparison, a CESS immuno-precipitate of the placenta was run in parallel and immunoblotted . Note that the same two protein bands detected in the placenta (and identified above as sFlt1-14 proteins) are also found in the PE serum. 23