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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Integrin-α9 is required for fibronectin matrix assembly
during lymphatic valve morphogenesis</title><author><name sortKey="Bazigou, Eleni" sort="Bazigou, Eleni" uniqKey="Bazigou E" first="Eleni" last="Bazigou">Eleni Bazigou</name><affiliation><nlm:aff id="A1"> Lymphatic Development Laboratory, Cancer Research UK London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom</nlm:aff></affiliation></author><author><name sortKey="Xie, Sherry" sort="Xie, Sherry" uniqKey="Xie S" first="Sherry" last="Xie">Sherry Xie</name><affiliation><nlm:aff id="A1"> Lymphatic Development Laboratory, Cancer Research UK London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom</nlm:aff></affiliation></author><author><name sortKey="Chen, Chun" sort="Chen, Chun" uniqKey="Chen C" first="Chun" last="Chen">Chun Chen</name><affiliation><nlm:aff id="A2"> Lung Biology Center, UCSF, San Francisco CA 94143-2922, USA</nlm:aff></affiliation></author><author><name sortKey="Weston, Anne" sort="Weston, Anne" uniqKey="Weston A" first="Anne" last="Weston">Anne Weston</name><affiliation><nlm:aff id="A3"> Electron Microscopy Unit, Cancer Research UK London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom</nlm:aff></affiliation></author><author><name sortKey="Miura, Naoyuki" sort="Miura, Naoyuki" uniqKey="Miura N" first="Naoyuki" last="Miura">Naoyuki Miura</name><affiliation><nlm:aff id="A4"> Department of Biochemistry, Hamamatsu University School of Medicine, Hamamatsu 431-3192, Japan</nlm:aff></affiliation></author><author><name sortKey="Sorokin, Lydia" sort="Sorokin, Lydia" uniqKey="Sorokin L" first="Lydia" last="Sorokin">Lydia Sorokin</name><affiliation><nlm:aff id="A5"> Institute for Physiological Chemistry and Pathobiochemistry, Münster University, 48149 Münster, Germany</nlm:aff></affiliation></author><author><name sortKey="Adams, Ralf" sort="Adams, Ralf" uniqKey="Adams R" first="Ralf" last="Adams">Ralf Adams</name><affiliation><nlm:aff id="A6"> Vascular Development Laboratory, Cancer Research UK London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom</nlm:aff></affiliation><affiliation><nlm:aff id="A7"> Max–Planck–Institute for Molecular Biomedicine, Department of Tissue Morphogenesis, and University of Münster, Faculty of Medicine, 48149 Münster, Germany</nlm:aff></affiliation></author><author><name sortKey="Muro, Andres F" sort="Muro, Andres F" uniqKey="Muro A" first="Andrés F." last="Muro">Andrés F. Muro</name><affiliation><nlm:aff id="A8"> International Centre for Genetic Engineering and Biotechnology, 34012 Trieste, Italy</nlm:aff></affiliation></author><author><name sortKey="Sheppard, Dean" sort="Sheppard, Dean" uniqKey="Sheppard D" first="Dean" last="Sheppard">Dean Sheppard</name><affiliation><nlm:aff id="A2"> Lung Biology Center, UCSF, San Francisco CA 94143-2922, USA</nlm:aff></affiliation></author><author><name sortKey="Makinen, Taija" sort="Makinen, Taija" uniqKey="Makinen T" first="Taija" last="Makinen">Taija Makinen</name><affiliation><nlm:aff id="A1"> Lymphatic Development Laboratory, Cancer Research UK London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">19686679</idno><idno type="pmc">2747264</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2747264</idno><idno type="RBID">PMC:2747264</idno><idno type="doi">10.1016/j.devcel.2009.06.017</idno><date when="2009">2009</date><idno type="wicri:Area/Pmc/Corpus">004688</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">004688</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Integrin-α9 is required for fibronectin matrix assembly
during lymphatic valve morphogenesis</title><author><name sortKey="Bazigou, Eleni" sort="Bazigou, Eleni" uniqKey="Bazigou E" first="Eleni" last="Bazigou">Eleni Bazigou</name><affiliation><nlm:aff id="A1"> Lymphatic Development Laboratory, Cancer Research UK London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom</nlm:aff></affiliation></author><author><name sortKey="Xie, Sherry" sort="Xie, Sherry" uniqKey="Xie S" first="Sherry" last="Xie">Sherry Xie</name><affiliation><nlm:aff id="A1"> Lymphatic Development Laboratory, Cancer Research UK London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom</nlm:aff></affiliation></author><author><name sortKey="Chen, Chun" sort="Chen, Chun" uniqKey="Chen C" first="Chun" last="Chen">Chun Chen</name><affiliation><nlm:aff id="A2"> Lung Biology Center, UCSF, San Francisco CA 94143-2922, USA</nlm:aff></affiliation></author><author><name sortKey="Weston, Anne" sort="Weston, Anne" uniqKey="Weston A" first="Anne" last="Weston">Anne Weston</name><affiliation><nlm:aff id="A3"> Electron Microscopy Unit, Cancer Research UK London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom</nlm:aff></affiliation></author><author><name sortKey="Miura, Naoyuki" sort="Miura, Naoyuki" uniqKey="Miura N" first="Naoyuki" last="Miura">Naoyuki Miura</name><affiliation><nlm:aff id="A4"> Department of Biochemistry, Hamamatsu University School of Medicine, Hamamatsu 431-3192, Japan</nlm:aff></affiliation></author><author><name sortKey="Sorokin, Lydia" sort="Sorokin, Lydia" uniqKey="Sorokin L" first="Lydia" last="Sorokin">Lydia Sorokin</name><affiliation><nlm:aff id="A5"> Institute for Physiological Chemistry and Pathobiochemistry, Münster University, 48149 Münster, Germany</nlm:aff></affiliation></author><author><name sortKey="Adams, Ralf" sort="Adams, Ralf" uniqKey="Adams R" first="Ralf" last="Adams">Ralf Adams</name><affiliation><nlm:aff id="A6"> Vascular Development Laboratory, Cancer Research UK London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom</nlm:aff></affiliation><affiliation><nlm:aff id="A7"> Max–Planck–Institute for Molecular Biomedicine, Department of Tissue Morphogenesis, and University of Münster, Faculty of Medicine, 48149 Münster, Germany</nlm:aff></affiliation></author><author><name sortKey="Muro, Andres F" sort="Muro, Andres F" uniqKey="Muro A" first="Andrés F." last="Muro">Andrés F. Muro</name><affiliation><nlm:aff id="A8"> International Centre for Genetic Engineering and Biotechnology, 34012 Trieste, Italy</nlm:aff></affiliation></author><author><name sortKey="Sheppard, Dean" sort="Sheppard, Dean" uniqKey="Sheppard D" first="Dean" last="Sheppard">Dean Sheppard</name><affiliation><nlm:aff id="A2"> Lung Biology Center, UCSF, San Francisco CA 94143-2922, USA</nlm:aff></affiliation></author><author><name sortKey="Makinen, Taija" sort="Makinen, Taija" uniqKey="Makinen T" first="Taija" last="Makinen">Taija Makinen</name><affiliation><nlm:aff id="A1"> Lymphatic Development Laboratory, Cancer Research UK London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom</nlm:aff></affiliation></author></analytic><series><title level="j">Developmental cell</title><idno type="ISSN">1534-5807</idno><idno type="eISSN">1878-1551</idno><imprint><date when="2009">2009</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><title>Summary</title><p id="P1">Dysfunction of lymphatic valves underlies human lymphedema, yet the
process of valve morphogenesis is poorly understood. Here, we show that during
embryogenesis lymphatic valve leaflet formation is initiated by upregulation of
integrin-α9 expression and deposition of its ligand, fibronectin-EIIIA
(FN-EIIIA), in the extracellular matrix. Endothelial cell specific deletion of
<italic>Itga9</italic> (encoding integrin-α9) in mouse embryos
results in the development of rudimentary valve leaflets, characterized by
disorganized FN matrix, short cusps and retrograde lymphatic flow. Similar
morphological and functional defects are observed in mice lacking the EIIIA
domain of FN. Mechanistically, we demonstrate that in primary human lymphatic
endothelial cells the integrin-α9-EIIIA interaction directly regulates
FN fibril assembly, which is essential for the formation of the extracellular
matrix core of valve leaflets. Our findings reveal an important role for
integrin-α9 signaling during lymphatic valve morphogenesis and implicate
it as a candidate gene for primary lymphedema caused by valve defects.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Alitalo, K" uniqKey="Alitalo K">K Alitalo</name></author><author><name sortKey="Tammela, T" uniqKey="Tammela T">T Tammela</name></author><author><name sortKey="Petrova, Tv" uniqKey="Petrova T">TV Petrova</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Armstrong, Ej" uniqKey="Armstrong E">EJ Armstrong</name></author><author><name sortKey="Bischoff, J" uniqKey="Bischoff J">J Bischoff</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Astrof, S" uniqKey="Astrof S">S Astrof</name></author><author><name sortKey="Crowley, D" uniqKey="Crowley D">D Crowley</name></author><author><name sortKey="George, El" uniqKey="George E">EL George</name></author><author><name sortKey="Fukuda, T" uniqKey="Fukuda T">T Fukuda</name></author><author><name sortKey="Sekiguchi, K" uniqKey="Sekiguchi K">K Sekiguchi</name></author><author><name sortKey="Hanahan, D" uniqKey="Hanahan D">D Hanahan</name></author><author><name sortKey="Hynes, Ro" uniqKey="Hynes R">RO Hynes</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Avraamides, Cj" uniqKey="Avraamides C">CJ Avraamides</name></author><author><name sortKey="Garmy Susini, B" uniqKey="Garmy Susini B">B Garmy-Susini</name></author><author><name sortKey="Varner, Ja" uniqKey="Varner J">JA Varner</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Dumont, Dj" uniqKey="Dumont D">DJ Dumont</name></author><author><name sortKey="Jussila, L" uniqKey="Jussila L">L Jussila</name></author><author><name sortKey="Taipale, J" uniqKey="Taipale J">J Taipale</name></author><author><name sortKey="Lymboussaki, A" uniqKey="Lymboussaki A">A Lymboussaki</name></author><author><name sortKey="Mustonen, T" uniqKey="Mustonen T">T Mustonen</name></author><author><name sortKey="Pajusola, K" uniqKey="Pajusola K">K Pajusola</name></author><author><name sortKey="Breitman, M" uniqKey="Breitman M">M Breitman</name></author><author><name sortKey="Alitalo, K" uniqKey="Alitalo K">K Alitalo</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Dzamba, Bj" uniqKey="Dzamba B">BJ Dzamba</name></author><author><name sortKey="Jakab, Kr" uniqKey="Jakab K">KR Jakab</name></author><author><name sortKey="Marsden, M" uniqKey="Marsden M">M Marsden</name></author><author><name sortKey="Schwartz, Ma" uniqKey="Schwartz M">MA Schwartz</name></author><author><name sortKey="Desimone, Dw" uniqKey="Desimone D">DW DeSimone</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ferrell, Re" uniqKey="Ferrell R">RE Ferrell</name></author><author><name sortKey="Levinson, Kl" uniqKey="Levinson K">KL Levinson</name></author><author><name sortKey="Esman, Jh" uniqKey="Esman J">JH Esman</name></author><author><name sortKey="Kimak, Ma" uniqKey="Kimak M">MA Kimak</name></author><author><name sortKey="Lawrence, Ec" uniqKey="Lawrence E">EC Lawrence</name></author><author><name sortKey="Barmada, Mm" uniqKey="Barmada M">MM Barmada</name></author><author><name sortKey="Finegold, Dn" uniqKey="Finegold D">DN Finegold</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Finegold, Dn" uniqKey="Finegold D">DN Finegold</name></author><author><name sortKey="Kimak, Ma" uniqKey="Kimak M">MA Kimak</name></author><author><name sortKey="Lawrence, Ec" uniqKey="Lawrence E">EC Lawrence</name></author><author><name sortKey="Levinson, Kl" uniqKey="Levinson K">KL Levinson</name></author><author><name sortKey="Cherniske, Em" uniqKey="Cherniske E">EM Cherniske</name></author><author><name sortKey="Pober, Br" uniqKey="Pober B">BR Pober</name></author><author><name sortKey="Dunlap, Jw" uniqKey="Dunlap J">JW Dunlap</name></author><author><name sortKey="Ferrell, Re" uniqKey="Ferrell R">RE Ferrell</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Fogerty, Fj" uniqKey="Fogerty F">FJ Fogerty</name></author><author><name sortKey="Akiyama, Sk" uniqKey="Akiyama S">SK Akiyama</name></author><author><name sortKey="Yamada, Km" uniqKey="Yamada K">KM Yamada</name></author><author><name sortKey="Mosher, Df" uniqKey="Mosher D">DF Mosher</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Forsberg, E" uniqKey="Forsberg E">E Forsberg</name></author><author><name sortKey="Hirsch, E" uniqKey="Hirsch E">E Hirsch</name></author><author><name sortKey="Frohlich, L" uniqKey="Frohlich L">L Frohlich</name></author><author><name sortKey="Meyer, M" uniqKey="Meyer M">M Meyer</name></author><author><name sortKey="Ekblom, P" uniqKey="Ekblom P">P Ekblom</name></author><author><name sortKey="Aszodi, A" uniqKey="Aszodi A">A Aszodi</name></author><author><name sortKey="Werner, S" uniqKey="Werner S">S 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uniqKey="Yutzey K">KE Yutzey</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ma, Gc" uniqKey="Ma G">GC Ma</name></author><author><name sortKey="Liu, Cs" uniqKey="Liu C">CS Liu</name></author><author><name sortKey="Chang, Sp" uniqKey="Chang S">SP Chang</name></author><author><name sortKey="Yeh, Kt" uniqKey="Yeh K">KT Yeh</name></author><author><name sortKey="Ke, Yy" uniqKey="Ke Y">YY Ke</name></author><author><name sortKey="Chen, Th" uniqKey="Chen T">TH Chen</name></author><author><name sortKey="Wang, Bb" uniqKey="Wang B">BB Wang</name></author><author><name sortKey="Kuo, Sj" uniqKey="Kuo S">SJ Kuo</name></author><author><name sortKey="Shih, Jc" uniqKey="Shih J">JC Shih</name></author><author><name sortKey="Chen, M" uniqKey="Chen M">M Chen</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Makinen, T" uniqKey="Makinen T">T Makinen</name></author><author><name sortKey="Adams, Rh" uniqKey="Adams R">RH 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<pmc-dir>properties manuscript</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-journal-id">101120028</journal-id><journal-id journal-id-type="pubmed-jr-id">22411</journal-id><journal-id journal-id-type="nlm-ta">Dev Cell</journal-id><journal-id journal-id-type="iso-abbrev">Dev. Cell</journal-id><journal-title-group><journal-title>Developmental cell</journal-title></journal-title-group><issn pub-type="ppub">1534-5807</issn><issn pub-type="epub">1878-1551</issn></journal-meta><article-meta><article-id pub-id-type="pmid">19686679</article-id><article-id pub-id-type="pmc">2747264</article-id><article-id pub-id-type="doi">10.1016/j.devcel.2009.06.017</article-id><article-id pub-id-type="manuscript">NIHMS141065</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Integrin-α9 is required for fibronectin matrix assembly
during lymphatic valve morphogenesis</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Bazigou</surname><given-names>Eleni</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Xie</surname><given-names>Sherry</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Chen</surname><given-names>Chun</given-names></name><xref ref-type="aff" rid="A2">2</xref></contrib><contrib contrib-type="author"><name><surname>Weston</surname><given-names>Anne</given-names></name><xref ref-type="aff" rid="A3">3</xref></contrib><contrib contrib-type="author"><name><surname>Miura</surname><given-names>Naoyuki</given-names></name><xref ref-type="aff" rid="A4">4</xref></contrib><contrib contrib-type="author"><name><surname>Sorokin</surname><given-names>Lydia</given-names></name><xref ref-type="aff" rid="A5">5</xref></contrib><contrib contrib-type="author"><name><surname>Adams</surname><given-names>Ralf</given-names></name><xref ref-type="aff" rid="A6">6</xref><xref ref-type="aff" rid="A7">7</xref></contrib><contrib contrib-type="author"><name><surname>Muro</surname><given-names>Andrés F.</given-names></name><xref ref-type="aff" rid="A8">8</xref></contrib><contrib contrib-type="author"><name><surname>Sheppard</surname><given-names>Dean</given-names></name><xref ref-type="aff" rid="A2">2</xref></contrib><contrib contrib-type="author"><name><surname>Makinen</surname><given-names>Taija</given-names></name><xref ref-type="aff" rid="A1">1</xref><xref rid="FN1" ref-type="author-notes">*</xref></contrib></contrib-group><aff id="A1"><label>1</label> Lymphatic Development Laboratory, Cancer Research UK London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom</aff><aff id="A2"><label>2</label> Lung Biology Center, UCSF, San Francisco CA 94143-2922, USA</aff><aff id="A3"><label>3</label> Electron Microscopy Unit, Cancer Research UK London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom</aff><aff id="A4"><label>4</label> Department of Biochemistry, Hamamatsu University School of Medicine, Hamamatsu 431-3192, Japan</aff><aff id="A5"><label>5</label> Institute for Physiological Chemistry and Pathobiochemistry, Münster University, 48149 Münster, Germany</aff><aff id="A6"><label>6</label> Vascular Development Laboratory, Cancer Research UK London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom</aff><aff id="A7"><label>7</label> Max–Planck–Institute for Molecular Biomedicine, Department of Tissue Morphogenesis, and University of Münster, Faculty of Medicine, 48149 Münster, Germany</aff><aff id="A8"><label>8</label> International Centre for Genetic Engineering and Biotechnology, 34012 Trieste, Italy</aff><author-notes><corresp id="FN1"><label>*</label> Corresponding author:
<email>taija.makinen@cancer.org.uk</email>, Phone: +44 207 269 3459,
Fax: +44 207 269 3417</corresp></author-notes><pub-date pub-type="nihms-submitted"><day>10</day><month>9</month><year>2009</year></pub-date><pub-date pub-type="ppub"><month>8</month><year>2009</year></pub-date><pub-date pub-type="pmc-release"><day>01</day><month>8</month><year>2010</year></pub-date><volume>17</volume><issue>2</issue><fpage>175</fpage><lpage>186</lpage><pmc-comment>elocation-id from pubmed: 10.1016/j.devcel.2009.06.017</pmc-comment>
<permissions><license xlink:href="https://creativecommons.org/licenses/by-nc-nd/3.0/"><license-p>Open Access under <ext-link ext-link-type="uri" xlink:href="https://creativecommons.org/licenses/by-nc-nd/3.0/">CC BY-NC-ND 3.0</ext-link> license.</license-p></license></permissions><abstract><title>Summary</title><p id="P1">Dysfunction of lymphatic valves underlies human lymphedema, yet the
process of valve morphogenesis is poorly understood. Here, we show that during
embryogenesis lymphatic valve leaflet formation is initiated by upregulation of
integrin-α9 expression and deposition of its ligand, fibronectin-EIIIA
(FN-EIIIA), in the extracellular matrix. Endothelial cell specific deletion of
<italic>Itga9</italic> (encoding integrin-α9) in mouse embryos
results in the development of rudimentary valve leaflets, characterized by
disorganized FN matrix, short cusps and retrograde lymphatic flow. Similar
morphological and functional defects are observed in mice lacking the EIIIA
domain of FN. Mechanistically, we demonstrate that in primary human lymphatic
endothelial cells the integrin-α9-EIIIA interaction directly regulates
FN fibril assembly, which is essential for the formation of the extracellular
matrix core of valve leaflets. Our findings reveal an important role for
integrin-α9 signaling during lymphatic valve morphogenesis and implicate
it as a candidate gene for primary lymphedema caused by valve defects.</p></abstract></article-meta></front><body><sec sec-type="intro" id="S1"><title>Introduction</title><p id="P2">The lymphatic vasculature forms a network of blind-ended capillaries that
collect protein rich fluid from the interstitial space and drain it via collecting
vessels first into lymph nodes and then to larger lymphatic ducts, which connect to
the venous system (<xref rid="R1" ref-type="bibr">Alitalo et al., 2005</xref>; <xref rid="R17" ref-type="bibr">Jurisic and Detmar, 2009</xref>). The major functions
of the lymphatic vasculature are to maintain tissue fluid balance, provide immune
surveillance through transport of leukocytes and antigen-presenting dendritic cells
and participate in fat absorption (<xref rid="R1" ref-type="bibr">Alitalo et al.,
2005</xref>; <xref rid="R17" ref-type="bibr">Jurisic and Detmar,
2009</xref>).</p><p id="P3">Congenital malformation of the lymphatic system such as vessel hypoplasia and
valve defects cause primary lymphedema, which is usually a progressive and lifelong
condition, characterized by gross swelling of the affected limb accompanied by
fibrosis and susceptibility to infections (<xref rid="R1" ref-type="bibr">Alitalo et
al., 2005</xref>; <xref rid="R17" ref-type="bibr">Jurisic and Detmar,
2009</xref>). The management of symptoms is based on physiotherapy and
compression garments as at present no effective treatment for lymphedema exists.
Several genes, including <italic>Vegfr3</italic>, <italic>Foxc2 and Sox18</italic>,
which were shown to regulate developmental lymphangiogenesis in mice (<xref rid="R11" ref-type="bibr">Francois et al., 2008</xref>; <xref rid="R19" ref-type="bibr">Karkkainen et al., 2001</xref>; <xref rid="R33" ref-type="bibr">Petrova et al., 2004</xref>), have been implicated in human lymphedema. Kinase
inactivating mutations in the <italic>VEGFR-3</italic> gene lead to Milroy's
disease (<xref rid="R7" ref-type="bibr">Ferrell et al., 1998</xref>), while
mutations in the transcription factors <italic>FOXC2</italic> and
<italic>SOX18</italic> are the underlying genetic causes of
lymphedema-distichiasis and hypotrichosis-lymphedema-telangiectasia, respectively
(<xref rid="R8" ref-type="bibr">Finegold et al., 2001</xref>; <xref rid="R16" ref-type="bibr">Irrthum et al., 2003</xref>). Interestingly, defective luminal
valves observed as a consequence of loss of <italic>FOXC2</italic> function (<xref rid="R33" ref-type="bibr">Petrova et al., 2004</xref>) highlights the critical
role of valves in maintaining unidirectional lymphatic flow. While the mechanisms of
lymphatic valve morphogenesis remain poorly characterized, it has been well
established that the interactions between the blood endothelial cells and their
surrounding extracellular matrix (ECM), organized to facilitate valve function, are
of key importance in regulating heart valve development and cardiac function, and
consequently, heart valve disease (<xref rid="R2" ref-type="bibr">Armstrong and
Bischoff, 2004</xref>; <xref rid="R25" ref-type="bibr">Lincoln et al.,
2006</xref>). Similarly, ultrastructural analyses of lymphatic valves have
demonstrated a close association between ECM and lymphatic endothelial cells in the
valve leaflets (<xref rid="R21" ref-type="bibr">Lauweryns and Boussauw, 1973</xref>;
<xref rid="R30" ref-type="bibr">Navas et al., 1991</xref>). These findings
suggest that the ECM has important functions in controlling endothelial cell
signalling and could also provide structural integrity during lymphatic valve
morphogenesis.</p><p id="P4">Cell-matrix adhesion receptors, such as integrins, play essential roles in
developmental processes that involve close interactions between the cells and their
surrounding ECM. Integrins are heterodimeric transmembrane receptors composed of
α and β subunits. Their extracellular domains bind to the ECM
molecules while the cytoplasmic domains associate with the actin cytoskeleton and
affiliated proteins, thereby providing a link between the external and internal
environment of the cell (<xref rid="R12" ref-type="bibr">Geiger et al.,
2001</xref>). In addition to mediating attachment to their respective ECM ligand(s),
integrins have specialized signaling functions and they can regulate gene expression
as well as cell shape, migration, proliferation and survival. Furthermore, integrin
binding to ECM is not only required for transducing signals from the matrix to cells
but this interaction also initiates responses that allow the cells to organize and
remodel the matrix (<xref rid="R22" ref-type="bibr">Leiss et al., 2008</xref>).
Despite an apparent redundancy in their ligand-binding specificities, with several
ECM molecules being ligands for more than one integrin, genetic studies have
demonstrated distinct functions for individual integrins. Fibronectin (FN) receptors
integrin-α5β1 and -α4β1, as well as components of
the ECM, such as FN, play critical roles in the development of the blood vasculature
(<xref rid="R15" ref-type="bibr">Hynes, 2007</xref>). However, knowledge of the
expression and function of integrins and the ECM in the lymphatic vasculature is
limited (<xref rid="R4" ref-type="bibr">Avraamides et al., 2008</xref>).</p><p id="P5">Here we show that a member of the integrin-family, integrin-α9
(encoded by <italic>Itga9</italic>), was predominantly expressed in the endothelial
cells of the lymphatic valve. Interestingly, <italic>Itga9</italic> deficiency in
mice led to specific defects in the formation of luminal valves, which resulted in
retrograde lymphatic flow and impaired fluid transport. We provide <italic>in
vivo</italic> and <italic>in vitro</italic> evidence for the requirement of
integrin-α9 interaction with its specific ligand, fibronectin-EIIIA
(FN-EIIIA, also called EDA), in regulating FN matrix assembly and thereby identify
an unexpected <italic>in vivo</italic> function for an integrin-EIIIA interaction.
Collectively, our findings demonstrate an important role for integrin signaling in
lymphatic valve development and provide novel insight into the previously
undescribed morphogenetic process of lymphatic valve morphogenesis. As lymphatic
valve defects manifested in adults are likely to have origins in valve development,
integrin-α9 is a candidate gene for primary human lymphedema caused by
lymphatic valve defects.</p></sec><sec sec-type="results" id="S2"><title>Results</title><sec id="S3"><title>Integrin-α9 is expressed in mature and developing lymphatic
valves</title><p id="P6">We surveyed expression of several integrins by immunofluorescence to
determine which of these molecules might function during lymphatic valve
formation. Whole-mount staining of adult skin using antibodies against specific
α-subunits revealed low levels of integrin-α5 and -α6
expression in lymphatic endothelia and in the valve, while no apparent staining
was detected using antibodies against integrin-α1, -α2 or
-α4 (data not shown). In contrast, staining with integrin-α9
antibodies strongly highlighted the cells constituting the luminal valve (<xref rid="F1" ref-type="fig">Figures 1A and 1B</xref>). In agreement with
previous studies (<xref rid="R14" ref-type="bibr">Huang et al., 2000</xref>), no
integrin-α9 expression was detected in the blood vessel endothelia (data
not shown).</p><p id="P7">Since integrin-α9 appeared to be the predominant
α-subunit expressed in lymphatic endothelia and valves, we further
characterized its expression pattern in developing vessels and aimed our study
at determining its function in valve morphogenesis. During mouse development,
the formation of valved collecting vessels occurs in late embryonic and early
postnatal life through remodelling of a primitive lymphatic capillary network
(<xref rid="F1" ref-type="fig">Figures 1C-1E</xref>; (<xref rid="R27" ref-type="bibr">Makinen et al., 2005</xref>; <xref rid="R31" ref-type="bibr">Norrmen et al., 2009</xref>)). At embryonic day (E)16, mesenteric lymphatic
vessels form a vascular plexus that is LYVE-1 positive and does not contain
valves (<xref rid="F1" ref-type="fig">Figures 1C and 1F</xref>). However,
defined clusters of cells expressing high levels of Prox1 (<xref rid="F1" ref-type="fig">Figure 1F</xref>) and FoxC2 (data not shown) transcription
factors are indicative of initiation of valve formation (<xref rid="R31" ref-type="bibr">Norrmen et al., 2009</xref>). At this stage
integrin-α9 expression was low in lymphatic endothelia (<xref rid="F1" ref-type="fig">Figures 1F and 1G</xref>), while higher expression was
detected in scattered cells in the surrounding tissue (data not shown) and in
vascular SMCs around the blood vessels (<xref rid="F1" ref-type="fig">Figures 1F
and 1G</xref>, arrowheads). At E17, the lymphatic vessels formed
constrictions that showed elevated <italic>Vegfr3</italic>-lacZ reporter
activity (<xref rid="F1" ref-type="fig">Figure 1D</xref>) and contained
endothelial cells expressing high levels of FoxC2 (<xref rid="F1" ref-type="fig">Figure 1H</xref>), Prox1 (data not shown) and integrin-α9 (<xref rid="F1" ref-type="fig">Figures 1H and 1I</xref>). At E18,
integrin-α9 was present in the endothelial cells of developed valve
leaflets (<xref rid="F1" ref-type="fig">Figures 1J and 1K</xref>). Taken
together, these expression data suggest that integrin-α9 upregulation in
lymphatic endothelia correlated with the initiation of valve leaflet
formation.</p></sec><sec id="S4"><title><italic>Itga9</italic> deficient mice display abnormal lymphatic
valves</title><p id="P8">To investigate the physiological function of integrin-α9 in
lymphatic valves we analysed <italic>Itga9</italic> deficient mice. These mice
were reported to die perinatally of respiratory failure, caused by the presence
of bilateral chylothorax (<xref rid="R14" ref-type="bibr">Huang et al.,
2000</xref>), however the potential lymphatic vascular defects remained
unknown. Analysis of chyle-filled mesenteric collecting vessels revealed
characteristic V-shaped valves in wild-type neonates (<xref rid="F2" ref-type="fig">Figures 2A and 2A</xref>′). In contrast,
<italic>Itga9</italic> mutant mice displayed fewer and morphologically
abnormal valves (<xref rid="F2" ref-type="fig">Figures 2B and
2B</xref>′). Staining with an antibody against PECAM-1, which is
strongly expressed in endothelial cells forming the valve leaflets (<xref rid="F2" ref-type="fig">Figure 2C</xref>), demonstrated that most of the
mutant valves appeared as horizontal constrictions rather than V-shaped
structures (<xref rid="F2" ref-type="fig">Figures 2D, 2E and 2F</xref>). Leakage
of chylous fluid from the mesenteric vessels further indicated that they were
dysfunctional (arrowhead in <xref rid="F2" ref-type="fig">Figure 2B</xref>). The
defects in <italic>Itga9</italic> deficient mice were specific to lymphatic
valves since we observed apparently normal development and gross morphology of
the lymphatic vasculature in all tissues that were examined (<xref rid="SD1" ref-type="supplementary-material">Figures S1A-S2H</xref>).</p><p id="P9">Examination of longitudinal transmission electron microscopy sections of
the valves of mesenteric lymphatic vessels revealed leaflets consisting of two
endothelial layers with a central connective tissue core (<xref rid="F2" ref-type="fig">Figures 2G, 2I, 2K</xref>; (<xref rid="R21" ref-type="bibr">Lauweryns and Boussauw, 1973</xref>; <xref rid="R30" ref-type="bibr">Navas
et al., 1991</xref>)). The endothelial cells of the wild-type valve leaflets
formed long, overlapping cell-cell junctions, and they were tightly attached to
the matrix core (<xref rid="F2" ref-type="fig">Figures 2G and 2I</xref>). In the
<italic>Itga9<sup>-/-</sup></italic> vessels the cusps of the valves
were shorter with fewer endothelial cells (<xref rid="F2" ref-type="fig">Figures
2H and 2K</xref>). The disorganized or absent matrix core in between the
basal sides of the endothelial cells was associated with the separation of the
opposite endothelial surfaces (<xref rid="F2" ref-type="fig">Figure 2J</xref>).
These results suggest that integrin-α9 function is essential for the
formation of the defined ECM core of a valve leaflet.</p></sec><sec id="S5"><title>Retrograde lymph flow and impaired fluid transport in
<italic>Itga9<sup>-/-</sup></italic> mice</title><p id="P10">Luminal valves have an important role in establishing unidirectional
lymphatic flow. To evaluate if lymphatic function is compromised in the
<italic>Itga9<sup>-/-</sup></italic> mice, we investigated uptake and
transport of subcutaneously injected large-molecular-weight fluorescent dextran.
In wild-type mice the FITC-dextran injected into the forelimb footpad was
rapidly drained into the valved dermal collecting lymphatic vessels, which were
visualized proximal to the injection site (<xref rid="F3" ref-type="fig">Figure
3A</xref>). In contrast, in the <italic>Itga9</italic> mutant skin the dye
labeled a vessel network (<xref rid="F3" ref-type="fig">Figure 3B</xref>),
suggesting retrograde lymphatic flow from the collecting vessels to the
pre-collector vessel branches (<xref rid="R27" ref-type="bibr">Makinen et al.,
2005</xref>; <xref rid="R33" ref-type="bibr">Petrova et al., 2004</xref>).
In addition, the dye showed leakage into the surrounding tissue and the
transport from the injection site to the lymph nodes and the thoracic duct was
impaired (<xref rid="SD1" ref-type="supplementary-material">Figures
S2A-S2D</xref>). We also analyzed the lymphatic drainage after subcutaneous
injection of FITC-conjugated <italic>Lycopersicum Esculentum</italic> lectin
(LEL), which binds to the surface of lymphatic endothelial cells, in particular
in the valves (<xref rid="R42" ref-type="bibr">Tammela et al., 2007</xref>)
(<xref rid="F3" ref-type="fig">Figure 3C</xref>). In the
<italic>Itga9</italic> mutants valve regions were identified by stronger
lectin staining, however, the valves were abnormal and often lacked obvious
leaflets (<xref rid="F3" ref-type="fig">Figures 3D and 3E</xref>). Together, the
above data show that <italic>Itga9</italic> deficient mice display specific
defects in valve morphogenesis, which result in retrograde lymphatic flow and
impaired fluid transport.</p></sec><sec id="S6"><title>Integrin-α9 is required tissue-autonomously in endothelia for
lymphatic valve development</title><p id="P11">To examine if integrin-α9 has a tissue-autonomous function in
lymphatic endothelia during valve development we deleted its expression
specifically in endothelial cells by crossing the floxed
<italic>Itga9<sup>lx</sup></italic> mice (<xref rid="R37" ref-type="bibr">Singh et al., 2008</xref>) with <italic>Tie2-Cre</italic>animals (<xref rid="R20" ref-type="bibr">Koni et al., 2001</xref>). The
homozygous <italic>Tie2-Cre;Itga9<sup>lx/lx</sup></italic> mice displayed
chylothorax (data not shown) and reduced number of lymphatic valves (<xref rid="SD1" ref-type="supplementary-material">Figures S3A and S3B</xref>). The
few valves that were detected displayed abnormal leaflets, as visualized by
staining for an ECM molecule Laminin-α5 (<xref rid="F4" ref-type="fig">Figures 4A-4D</xref>).</p><p id="P12">We next asked whether integrin-α9 is also essential for the
maintenance of the valves. We used
<italic>VE-cadherin-CreER<sup>T2</sup></italic> mice, which allow
tamoxifen-regulated activation of Cre in both blood and lymphatic endothelia (R.
Adams, unpublished; <xref rid="SD1" ref-type="supplementary-material">Figures
S3C and S3D</xref>). To induce <italic>Itga9</italic> gene deletion in
mature valves, 4-hydroxytamoxifen (4-OHT) was injected into control
<italic>Itga9<sup>lx/</sup></italic><sup>+</sup> (<xref rid="F4" ref-type="fig">Figures 4E-4H</xref>) and
<italic>VE-cadherin-CreER<sup>T2</sup></italic>;<italic>Itga9<sup>lx/lx</sup></italic>(<xref rid="F4" ref-type="fig">Figures 4I-4L</xref>) mice at P1, when most
of the valves are fully developed, and the vessels were analysed at P7. In 4-OHT
treated
<italic>VE-cadherin-CreER<sup>T2</sup></italic>;<italic>Itga9<sup>lx/lx</sup></italic>vessels integrin-α9 expression was lost from lymphatic endothelia (<xref rid="F4" ref-type="fig">Figure 4K</xref>). However, the valve leaflets
appeared normal (<xref rid="F4" ref-type="fig">Figures 4I and 4J</xref>), and
integrin-α9 negative endothelial cells remained attached to the matrix
core (<xref rid="F4" ref-type="fig">Figures 4I, 4K and 4L</xref>), suggesting
that integrin-α9 is dispensable for stable adhesion of endothelial cells
in fully developed valves. Together, these results suggest that
integrin-α9 is required tissue-autonomously in endothelia for the
development of lymphatic valve leaflets, while it is not essential for
maintaining valve structure and endothelial cell adhesion in mature valves.</p></sec><sec id="S7"><title>Integrin-α9 ligand, fibronectin-EIIIA, is expressed in the developing
lymphatic valves</title><p id="P13">To examine the function of integrin-α9 in the formation of valve
leaflets we investigated the expression and localization of its ligands,
including Tenascin-C (TNC), Osteopontin (OPN) and fibronectin containing the
EIIIA domain (FN-EIIIA) (<xref rid="R23" ref-type="bibr">Liao et al.,
2002</xref>; <xref rid="R47" ref-type="bibr">Yokosaki et al., 1998</xref>;
<xref rid="R48" ref-type="bibr">Yokosaki et al., 1999</xref>) in the
developing lymphatic vessels. Confocal longitudinal cross section of a valve
from E18.5 mesenteric vessels revealed expression of integrin-α9 and its
ligand TNC on both luminal and abluminal sides of the valve endothelial cells
(<xref rid="SD1" ref-type="supplementary-material">Figures S4A-S4C</xref>),
while no staining was seen for OPN (data not shown). Interestingly, unlike FN,
which was detected in all lymphatic vessel basement membrane (<xref rid="SD1" ref-type="supplementary-material">Figures S4D-S4F</xref>), FN-EIIIA showed
localization restricted to the valve matrix core (<xref rid="SD1" ref-type="supplementary-material">Figures S4G-S4I</xref>).</p><p id="P14">To gain further insight into the role of FN-EIIIA we analysed its
expression during different stages of lymphatic valve development so as to
determine the relationship between integrin-α9 expression and ECM
deposition. At E16, prior to upregulation of integrin-α9 expression
(<xref rid="F5" ref-type="fig">Figure 5A</xref>), Laminin-α5 was
found deposited at specific sites along the vessel (<xref rid="F5" ref-type="fig">Figures 5A</xref>), whereas no fibrous staining for FN-EIIIA
was detected (data not shown). Induction of integrin-α9 expression
correlated with appearance of continuous FN-EIIIA fibers at sites of developing
valves (<xref rid="F5" ref-type="fig">Figures 5B</xref>), which were identified
by Laminin-α5 positive matrix (<xref rid="F5" ref-type="fig">Figure
5C</xref>) and the presence of cells expressing high levels of Prox1 (<xref rid="F5" ref-type="fig">Figure 5D</xref>). During initial steps of valve
development FN-EIIIA and Laminin-α5 showed a similar localization (<xref rid="F5" ref-type="fig">Figure 5C</xref>). However, during leaflet
elongation Laminin-α5 was found in the entire valve matrix core while
FN-EIIIA fibers were concentrated on the free edges of the leaflets (<xref rid="F5" ref-type="fig">Figures 5E and 5E</xref>′). Similar to what
has been reported for other tissues (<xref rid="R32" ref-type="bibr">Pagani et
al., 1991</xref>), FN-EIIIA expression was progressively down-regulated in
postnatal lymphatic vasculature. While prominent FN-EIIIA fibers were detected
in mesenteric lymphatic valves at P3 (data not shown), only weak staining was
observed at P9 (<xref rid="SD1" ref-type="supplementary-material">Figures
S4J-S4L</xref>) and no fibers were seen in adult valves (see <xref rid="F6" ref-type="fig">Figure 6D</xref>). These expression data suggest that
FN-EIIIA is primarily involved during embryogenesis and that it has a role in
the formation and extension of the valve leaflets.</p></sec><sec id="S8"><title>Defective FN-EIIIA matrix assembly and valve leaflet formation in
<italic>Itga9<sup>-/-</sup></italic> mice</title><p id="P15">We next examined valve morphogenesis in
<italic>Itga9<sup>-/-</sup></italic> embryos. X-Gal staining of mesenteric
lymphatic vessels in E16
<italic>Itga9<sup>-/-</sup>;Vegfr3<sup>lz/</sup></italic><sup>+</sup>embryos revealed normal vascular networks (<xref rid="SD1" ref-type="supplementary-material">Figure S5A</xref>). Consistent with the
reduced number of valves observed in postnatal
<italic>Itga9<sup>-/-</sup></italic> vessels, at E17 the number of
<italic>Vegfr3</italic>-lacZ positive constrictions (<xref rid="SD1" ref-type="supplementary-material">Figures S5B-S5E</xref>), that contained
clusters of cells expressing high levels of Prox1 and FoxC2 was reduced when
compared to the wild-type (<xref rid="SD1" ref-type="supplementary-material">Figures S5F and S5G</xref> and data not shown). In addition, staining of
mutant valves for FN-EIIIA revealed only few short fibers (<xref rid="F5" ref-type="fig">Figures 5F-5G</xref>′). Cross section view through
the valve showed a fibrous matrix in wild-type vessels (<xref rid="F5" ref-type="fig">Figure 5H</xref>). In contrast, the underdeveloped leaflets
in <italic>Itga9</italic> deficient vessels displayed predominantly a disrupted
punctuate and discontinuous pattern (<xref rid="F5" ref-type="fig">Figures
5I</xref>), suggesting that FN-EIIIA failed to assemble into continuous
fibers. Taken together, the disorganized FN-EIIIA matrix in
<italic>Itga9</italic> deficient vessels and the subsequent arrest in valve
development suggest that the defect in valve formation in
<italic>Itga9<sup>-/-</sup></italic> mice is due to interaction between
integrin-α9 and its specific ligand (<xref rid="F5" ref-type="fig">Figure 5J</xref>).</p></sec><sec id="S9"><title>Fibronectin-EIIIA is required for normal development of lymphatic valve
leaflets</title><p id="P16">To test directly if FN-EIIIA, or the other integrin-α9 binding
ECM molecules, has a function in valve formation, we analyzed the lymphatic
vessels in <italic>Tnc, Opn</italic> and <italic>Fn-EIIIA</italic> deficient
mice (<xref rid="R10" ref-type="bibr">Forsberg et al., 1996</xref>; <xref rid="R24" ref-type="bibr">Liaw et al., 1998</xref>; <xref rid="R29" ref-type="bibr">Muro et al., 2003</xref>). Both <italic>Tnc</italic> and
<italic>Opn</italic> deficient mice displayed apparently normal lymphatic
vasculature and lymphatic valves (<xref rid="SD1" ref-type="supplementary-material">Figures S6A-S6F</xref>), however, mice
deficient for <italic>Fn-EIIIA</italic> partially recapitulated the
integrin-α9 mutant phenotype. Quantification of Laminin-α5
positive valve structures in mesenteric lymphatic vessels of newborn mice showed
a 1.5-fold reduction in the number of valves in
<italic>Fn-EIIIA<sup>-/-</sup></italic> mice when compared to the wild-type
(p = 0.0026 (Mann-Whitney test), <xref rid="F6" ref-type="fig">Figure
6A</xref>; <xref rid="SD1" ref-type="supplementary-material">Table
S1</xref>). Notably, 76% of the valves in
<italic>Fn-EIIIA<sup>-/-</sup></italic> mice displayed abnormal matrix ring
appearance or underdeveloped leaflets and disorganized Laminin-α5 matrix
(<xref rid="F6" ref-type="fig">Figures 6B and 6C</xref>; <xref rid="SD1" ref-type="supplementary-material">Table S1</xref>), similar to the valves
observed in <italic>Itga9</italic> mutants. In wild-type controls at this
developmental stage only a minor proportion of immature valves with rudimentary
leaflets were observed, while in <italic>Itga9<sup>-/-</sup></italic> mice
approximately 90% of the valves were abnormal (<xref rid="F6" ref-type="fig">Figure 6A</xref>; <xref rid="SD1" ref-type="supplementary-material">Table S1</xref>). While lymphatic valve
defects were pronounced in the <italic>Fn-EIIIA<sup>-/-</sup></italic> mice
during early postnatal life, they diminished during progression into adulthood,
coinciding with when the expression of FN-EIIIA is down-regulated. In the ear
skin of three weeks old wild-type animal, punctuate staining, but no FN-EIIIA
fibers, were detected in the lymphatic vessels and the valves (<xref rid="F6" ref-type="fig">Figure 6D</xref>). At the same age the majority of the valves
in <italic>Fn-EIIIA</italic> mutant animals had apparently normal leaflets when
compared to the control (<xref rid="F6" ref-type="fig">Figures 6E and
6F</xref>). The mice also exhibited otherwise normal lymphatic vasculature,
including vessel diameter and smooth muscle cell coverage (<xref rid="F6" ref-type="fig">Figures 6E and 6F</xref>). However, subcutaneous injection of
FITC-dextran revealed reflux of dye from the collecting vessels, suggesting mild
valve defects, and some morphologically abnormal valves in
<italic>Fn-EIIIA</italic> deficient mice (<xref rid="F6" ref-type="fig">Figures 6G-6I</xref>, 4/4 mice analyzed). These results suggest that
<italic>Fn-EIIIA</italic> deficiency leads to lymphatic valve defects during
embryonic and early postnatal life.</p></sec><sec id="S10"><title>Integrin-α9-EIIIA interaction regulates FN assembly in lymphatic
endothelial cells</title><p id="P17">The defective FN matrix observed in the
<italic>Itga9<sup>-/-</sup></italic> valves prompted us to examine the
possibility that integrin-α9-EIIIA interaction contributes to FN
assembly in lymphatic endothelial cells (LECs), despite the previous findings
suggesting that the EIIIA domain is dispensable for matrix assembly in
fibroblasts (<xref rid="R43" ref-type="bibr">Tan et al., 2004</xref>). We
therefore tested whether primary human LECs are able to assemble their
endogenously produced FN, which contains the EIIIA domain (<xref rid="F7" ref-type="fig">Figure 7A</xref>). To avoid exogenous plasma FN lacking the
EIIIA domain, the cells were grown in the presence of FN-depleted serum and
fibril assembly was assessed by immunofluorescence for the EIIIA. After 24h in
culture, a fibrillar FN-EIIIA network was detected on the surface of the LECs
(<xref rid="F7" ref-type="fig">Figure 7A</xref>). Inhibition of
integrin-α9 function, or integrin-α9-EIIIA interaction, using
blocking antibodies against integrin-α9 (Y9A2) or EIIIA (IST-9) (<xref rid="F7" ref-type="fig">Figures 7A and 7B</xref>), or a blocking EIIIA
peptide EDGIHEL (<xref rid="R23" ref-type="bibr">Liao et al., 2002</xref>)(data
not shown), led to a significant decrease in FN-EIIIA fibril formation.
<italic>FN-EIIIA</italic> levels were not reduced (<xref rid="F7" ref-type="fig">Figures 7C</xref>), indicating that the defect was not due to
impaired mRNA synthesis. A similar effect was seen when integrin-α9
expression was silenced using siRNA oligos (<xref rid="F7" ref-type="fig">Figures 7A-7D</xref>). In contrast, inhibition of RGD-dependent integrin
interactions, which have been considered essential for FN assembly via
α5β1 and αv integrins, had no effect (<xref rid="F7" ref-type="fig">Figure 7B</xref>), while siRNA-mediated knock-down of
integrin-α5 (<xref rid="F7" ref-type="fig">Figure 7D</xref>) partially
blocked LEC mediated FN-EIIIA assembly (<xref rid="F7" ref-type="fig">Figure 7A
and 7B</xref>). Deoxycholate (DOC) differential solubilization assay and
Western blot analysis were further used to analyse the conversion of DOC-soluble
fibrils into insoluble stable matrix containing high molecular mass FN
multimers. No DOC-insoluble material was associated with the LECs in which
integrin-α9 expression or the integrin-α9-EIIIA interaction was
blocked (<xref rid="F7" ref-type="fig">Figure 7E</xref>, upper panel). Instead,
similar quantities of DOC-soluble FN matrices were found in all cells (<xref rid="F7" ref-type="fig">Figure 7E</xref>, lower panel). Consistent with the
immunofluorescence data, silencing of integrin-α5 expression partially
blocked the formation of DOC-insoluble FN-EIIIA matrix (<xref rid="F7" ref-type="fig">Figure 7E</xref>). These results suggest that although
integrin-α5 cannot functionally compensate for the loss of
integrin-α9, it contributes to FN fibrillogenesis in the LECs. However,
the low levels of integrin-α5 detected in the developing lymphatic
vessels (<xref rid="F7" ref-type="fig">Figure 7F</xref>) suggest that it does
not play a major role during valve morphogenesis. The above results demonstrate
that integrin-α9-EIIIA interaction can directly regulate FN matrix
assembly, suggesting a functionally indispensable, integrin-specific mechanism
for FN fibrillogenesis in the LECs.</p></sec></sec><sec sec-type="discussion" id="S11"><title>Discussion</title><p id="P18">The present study establishes integrin-α9 as an essential regulator
of the morphogenetic process controlling the formation of lymphatic valve leaflets,
which allow opening and closing of the valve in response to pressure changes. This
action of the valve is critical for the maintenance of unidirectional lymphatic flow
and the functionality of the entire lymphatic vascular system, highlighted by the
lack or insufficient function of lymphatic valves as an underlying cause of human
lymphedema (<xref rid="R1" ref-type="bibr">Alitalo et al., 2005</xref>).</p><p id="P19">The leaflets of a mature valve consist of a well-defined matrix in between
two sheets of lymphatic endothelial cells, which forms a strong but elastic
connective tissue core (<xref rid="F1" ref-type="fig">Figure 1</xref>). During
embryogenesis, the formation of the lymphatic valve is initiated by upregulation of
FoxC2 and Prox1 transcription factors in distinct clusters of endothelial cells,
which define the positions of future valves ((<xref rid="R31" ref-type="bibr">Norrmen et al., 2009</xref>), see <xref rid="F5" ref-type="fig">Figure
5J</xref>). We found that the development of the valve leaflet is subsequently
initiated by upregulation of integrin-α9 expression and deposition of ECM
containing its ligand, the EIIIA splice isoform of FN. The localization of FN-EIIIA
at the distal tip of the developing valve suggested its function in regulating valve
leaflet elongation. However, from previous studies the precise function of FN-EIIIA
has remained elusive. Despite strong expression in the angiogenic blood vessels, no
vascular phenotypes were reported in <italic>Fn-EIIIA</italic> deficient mice (<xref rid="R3" ref-type="bibr">Astrof et al., 2004</xref>). <italic>In vitro</italic>studies demonstrated that inclusion of the EIIIA segment enhanced the ability of FN
to incorporate into existing matrix (<xref rid="R13" ref-type="bibr">Guan et al.,
1990</xref>). This suggests that FN-EIIIA may participate in FN fibrillogenesis,
which is a highly regulated, multistep process initiated by binding of integrins to
specific sites in the FN molecule (<xref rid="R22" ref-type="bibr">Leiss et al.,
2008</xref>; <xref rid="R46" ref-type="bibr">Wierzbicka-Patynowski and
Schwarzbauer, 2003</xref>). Studies in cultured cells have revealed that
generation of cytoskeletal tension through integrin-mediated cell attachment and
anchoring via focal adhesions causes a change in integrin-bound FN conformation that
exposes cryptic self-association sites to allow FN fiber assembly (<xref rid="R22" ref-type="bibr">Leiss et al., 2008</xref>; <xref rid="R46" ref-type="bibr">Wierzbicka-Patynowski and Schwarzbauer, 2003</xref>). The small GTPases,
including Rho and Rac, play critical roles in cytoskeletal tension generation via
regulation of actin polymerization and myosin phosphorylation (<xref rid="R6" ref-type="bibr">Dzamba et al., 2009</xref>; <xref rid="R49" ref-type="bibr">Zhong et al., 1998</xref>). <italic>In vivo</italic>, tissue tension and FN
fibrillogenesis can also be generated by cell-cell adhesion and cohesivity (<xref rid="R6" ref-type="bibr">Dzamba et al., 2009</xref>). The observation that FN
assembly proceeded normally in <italic>Fn-EIIIA</italic> deficient fibroblasts
(<xref rid="R43" ref-type="bibr">Tan et al., 2004</xref>) led to the conclusion
that EIIIA domain is not required for matrix assembly (<xref rid="R22" ref-type="bibr">Leiss et al., 2008</xref>). Instead, fibril formation in these
cells was most likely mediated through binding of the major FN receptor,
integrin-α5β1, to the Arg-Gly-Asp (RGD) motif located in the type
III-10 module of FN (<xref rid="R9" ref-type="bibr">Fogerty et al., 1990</xref>),
which has been considered the most important mechanism of FN assembly (<xref rid="R22" ref-type="bibr">Leiss et al., 2008</xref>).</p><p id="P20">Surprisingly, we found that in lymphatic endothelial cells the
integrin-α9-EIIIA interaction directly regulated FN fibril assembly, while
RGD-dependent interactions appeared dispensable for matrix formation. Although the
EIIIA domain has not been previously implicated in FN fibrillogenesis, other
RGD-independent mechanisms for FN assembly have been observed. In fact, the
requirement of the RGD motif for the formation of a functional fibrillar FN network
<italic>in vivo</italic> was recently challenged as it was found that mice
expressing FN with a non-functional RGD motif assembled an apparently normal FN
matrix (<xref rid="R41" ref-type="bibr">Takahashi et al., 2007</xref>). The assembly
of the RGD-deficient FN was mediated via integrin-αvβ3 binding to
NGR sequence in the fifth N-terminal type I module of FN, which is converted to a
high affinity binding site through deamidation (<xref rid="R41" ref-type="bibr">Takahashi et al., 2007</xref>). In addition, binding of Mn<sup>2+</sup>activated integrin-α4β1 to the CS-1 site of the alternatively
spliced V region was shown to promote FN assembly <italic>in vitro</italic> (<xref rid="R36" ref-type="bibr">Sechler et al., 2000</xref>).</p><p id="P21">Our findings suggest that the defective FN matrix organization in
<italic>Itga9</italic> mutant valves is a direct consequence of the lack of a
specific integrin-matrix interaction and contributes to the observed defects in
valve leaflet formation. In agreement, we found that a large proportion of lymphatic
valves in neonatal <italic>Fn-EIIIA</italic> deficient mice displayed defective
leaflets similar to those observed in the <italic>Itga9</italic> mutants. However,
the absence of chylothorax and only partial recapitulation of the
<italic>Itga9<sup>-/-</sup></italic> phenotype suggest that the defect in
lymphatic development in mice deficient of <italic>Itga9</italic> cannot be solely
explained by a defect in EIIIA-containing FN matrix assembly. In particular, the
observation that the valve defect in <italic>Fn-EIIIA<sup>-/-</sup></italic> mice
diminished in adulthood suggests that the animals can eventually overcome the
requirement of FN-EIIIA, coinciding with when its expression is down-regulated. This
may imply that during postnatal development other integrin-α9 ligand(s)
become involved, or that FN fibrillogenesis can be mediated via alternative
integrin-FN interactions. In agreement, we found that acute deletion of
integrin-α9 postnatally in mature valves did not lead to degeneration of the
leaflets within the one-week period investigated, suggesting that once the stable
matrix fibrils are assembled, integrin-α9-EIIIA interaction is not required
to maintain ECM organization and valve structure. However, our results do not
exclude that integrin-α9-EIIIA is required for long-term maintenance of
valves. Notably, although not normally present in adult tissues, FN-EIIIA is
upregulated in various pathological situations, such as cancer, atherosclerosis and
thrombosis (<xref rid="R29" ref-type="bibr">Muro et al., 2003</xref>; <xref rid="R43" ref-type="bibr">Tan et al., 2004</xref>; <xref rid="R44" ref-type="bibr">Villa et al., 2008</xref>). It is therefore likely that it is
reactivated and plays a role also in adult lymphatic vessels under specific
circumstances, for example during repair processes or when the vasculature is
challenged by inflammation.</p><p id="P22">Why is integrin-α9 important specifically in valves? Unlike other
parts of the lymphatic vasculature, which have thin or absent basement membranes,
the discrete architecture of the connective tissue of a valve leaflet suggests
specific requirement for highly regulated mechanism of matrix organization. Indeed,
the development of a related structure, the heart valve, is known to rely on the
formation of a highly organized ECM, and consequently, dysregulation of ECM, often
caused by genetic defects in matrix protein structure or expression, is linked to
heart valve disease (<xref rid="R2" ref-type="bibr">Armstrong and Bischoff,
2004</xref>; <xref rid="R25" ref-type="bibr">Lincoln et al., 2006</xref>). FN
assembly is likely to play a key role in coordinating the formation of complex
matrices as it has been shown to initiate the organization of other ECM proteins,
such as collagens, and control the stability of matrix fibers (<xref rid="R18" ref-type="bibr">Kadler et al., 2008</xref>; <xref rid="R39" ref-type="bibr">Sottile and Hocking, 2002</xref>). Our observations demonstrate that
integrin-α9 is the predominant alpha subunit in lymphatic endothelia.
Although the major FN receptor, integrin-α5, appeared to contribute to FN
fibrillogenesis in the LECs <italic>in vitro</italic>, it was not able to
functionally compensate for the loss of integrin-α9 either in cultured cells
or <italic>in vivo</italic>. Low expression levels of integrin-α5 in the
developing valves may provide one explanation for the dependency of these cells on
alternative FN assembly pathways.</p><p id="P23">FN matrix assembly, as well as integrin-α9 signaling, promote
adhesion-dependent cell growth and migration (<xref rid="R22" ref-type="bibr">Leiss
et al., 2008</xref>; <xref rid="R23" ref-type="bibr">Liao et al., 2002</xref>;
<xref rid="R37" ref-type="bibr">Singh et al., 2008</xref>; <xref rid="R40" ref-type="bibr">Sottile et al., 1998</xref>) and defects in these processes may
therefore underlie the disrupted valve leaflet formation in
<italic>Itga9<sup>-/-</sup></italic> and
<italic>Fn-EIIIA<sup>-/-</sup></italic> mice. Although we cannot exclude a
contribution of integrin-α9-mediated adhesion during the early events of
valve formation, the observation that acute deletion of integrin-α9 in
postnatal valves did not lead to cell detachment and degeneration of the valves
argue against a role of integrin-α9 in mediating stable adhesion of valve
endothelial cells <italic>in vivo</italic>. Furthermore, an <italic>in vivo</italic>BrdU incorporation assay showed that the valve endothelial cells expressing high
levels of FoxC2, Prox1 and integrin-α9 do not proliferate during leaflet
formation (E.B. and T.M., unpublished). These results suggest that endothelial cell
adhesion and proliferation are unlikely to be controlled by integrin-α9 or
involved in valve leaflet formation, respectively. Finally, the observed defect in
valve leaflet elongation may be caused by failure in morphogenetic cell movements,
which were shown to rely on proper organization of FN matrices in other
developmental processes (<xref rid="R6" ref-type="bibr">Dzamba et al., 2009</xref>;
<xref rid="R35" ref-type="bibr">Rozario et al., 2009</xref>). Therefore, we
propose that the key function of the integrin-α9-EIIIA interaction is to
provide an integrin-specific mechanism of FN matrix assembly and thereby coordinate
ECM organization to allow formation of a leaflet of necessary length and strength,
capable of supporting valve function.</p><p id="P24">In summary, we demonstrate that integrin-α9 and its ligand,
FN-EIIIA, play specific roles in lymphatic valve development and remodeling into
functional leaflets. To our knowledge, the only other mouse mutants identified
to-date, which show a failure of valve formation, show additional lymphatic defects;
mice lacking the C-terminal PDZ binding domain of ephrinB2 display vessel
hyperplasia (<xref rid="R27" ref-type="bibr">Makinen et al., 2005</xref>) while
<italic>FoxC2</italic> deficient mice have patterning defects (<xref rid="R31" ref-type="bibr">Norrmen et al., 2009</xref>; <xref rid="R33" ref-type="bibr">Petrova et al., 2004</xref>). The specific valve defects observed in the
<italic>Itga9</italic> mutant mice therefore reveal a previously undescribed
morphogenetic process and provide potential insights into the development of valves
in other parts of the vascular system, such as in the veins and the heart, which are
likely to be regulated by similar molecular mechanisms. Lack of integrin-α9
signaling in mice results ultimately in chylothorax and postnatal death (<xref rid="R14" ref-type="bibr">Huang et al., 2000</xref>). Recent identification of
mutations in the <italic>ITGA9</italic> gene in fetuses with congenital chylothorax
(<xref rid="R26" ref-type="bibr">Ma et al., 2008</xref>) suggests conservation
of the signaling pathway from mice to human, making integrin-α9 a candidate
gene for primary lymphedema caused by valve defects.</p></sec><sec sec-type="methods" id="S12"><title>Experimental Procedures</title><sec id="S13"><title>Antibodies</title><p id="P25">The antibodies were rat antibodies to mouse PECAM-1 (MEC3.1, BD
Biosciences), Tenascin-C (MTn-12, Abcam) and FoxC2 (<xref rid="R33" ref-type="bibr">Petrova et al., 2004</xref>); rabbit antibodies to mouse
Fibronectin (Millipore), Laminin-α5 (<xref rid="R34" ref-type="bibr">Ringelmann et al., 1999</xref>) and LYVE-1 (Reliatch); rabbit antibodies to
human Prox1 (Reliatech) and Fibronectin (Abcam); mouse antibodies to human
cellular Fibronectin recognizing the EIIIA domain ((<xref rid="R23" ref-type="bibr">Liao et al., 2002</xref>), FN-3E2, Sigma and IST-9, Abcam),
hamster antibody to mouse PECAM-1, goat antibodies to mouse integrin-α9
and Osteopontin (R&D Systems) and Cy3-conjugated mouse antibody against
α-smooth muscle actin (Sigma). The monoclonal hamster antibody to mouse
Podoplanin (8.1.1) developed by Andrew Farr was obtained from the Developmental
Studies Hybridoma Bank. Secondary antibodies conjugated to Cy2, Cy3 or Cy5 were
obtained from Jackson ImmunoResearch.</p></sec><sec id="S14"><title>Mouse lines</title><p id="P26"><italic>Itga9<sup>-/-</sup></italic> (<xref rid="R14" ref-type="bibr">Huang et al., 2000</xref>), <italic>Itga9<sup>lx</sup></italic> (<xref rid="R37" ref-type="bibr">Singh et al., 2008</xref>),
<italic>Vegfr3<sup>lz/</sup></italic><sup>+</sup> (<xref rid="R5" ref-type="bibr">Dumont et al., 1998</xref>),
<italic>Fn-EIIIA<sup>-/-</sup></italic> (<xref rid="R29" ref-type="bibr">Muro et al., 2003</xref>), <italic>Tnc<sup>-/-</sup></italic> (<xref rid="R10" ref-type="bibr">Forsberg et al., 1996</xref>),
<italic>Opn<sup>-/-</sup></italic> (<xref rid="R24" ref-type="bibr">Liaw
et al., 1998</xref>), <italic>Tie2-Cre</italic> (<xref rid="R20" ref-type="bibr">Koni et al., 2001</xref>) and <italic>Rosa26R</italic>(<xref rid="R38" ref-type="bibr">Soriano, 1999</xref>) mice have been
described previously. <italic>VE-cadherin-CreER<sup>T2</sup></italic> mice will
be described elsewhere (R. Adams, unpublished). For the induction of Cre
mediated recombination in newborn
<italic>VE-cadherin-CreER<sup>T2</sup></italic> mice, 4-hydroxytamoxifen
(4-OHT; 2 μl of 10 mg/ml dissolved in ethanol) was injected i.p. at P1
and P2 and the vessels were analyzed at P7-8. All animal experiments were
performed in accordance with UK Home Office and institutional guidelines.</p></sec><sec id="S15"><title>Immunostaining and X-Gal staining</title><p id="P27">For whole-mount staining, the tissue was fixed in 4%
paraformaldehyde (PFA), permeabilized in 0.3% Triton-X100 in PBS (PBSTx)
and blocked in 5% milk or serum. Primary antibodies were added to the
blocking buffer and incubated with the tissue overnight at 4°C. After
washes in PBSTx, the tissue was incubated with fluorochrome-conjugated secondary
antibodies in the blocking buffer for 2h at RT, followed by washing in PBSTx and
mounting in Mowiol. The samples were analyzed using Zeiss LSM 510 laser scanning
confocal microscope. All confocal images, except <xref rid="F5" ref-type="fig">Figures 5H, 5I</xref> and <xref rid="SD1" ref-type="supplementary-material">S4A-S4I</xref>, represent 2D projections of Z-stacks. Alternatively,
biotin-conjugated secondary antibodies and ABC staining kit (Vector
Laboratories) were used and the bound antibodies were visualized using
diaminobenzidine (DAB) as a substrate. For histological analyses, skin biopsies
were fixed in 4% PFA overnight, dehydrated and embedded in paraffin.
Sections (5μm) were stained after heat-induced epitope retrieval using
Tyramide Signal Amplification kit (TSA, NEN Life Sciences) and
3-Amino-9-Ethylcarbazole (AEC) as a substrate. For the visualization of
lymphatic vessels in <italic>Vegfr3<sup>lz/</sup></italic><sup>+</sup>reporter mice, the tissues were fixed with 0.2% glutaraldehyde and
stained by the β-galactosidase substrate X-gal (Promega).</p></sec><sec id="S16"><title>Visualization of lymphatic vessel function</title><p id="P28">FITC-dextran (Sigma, 8 mg/mL in PBS) or FITC-conjugated
<italic>Lycopersicum Esculentum</italic> lectin (Vector Laboratories, 1
mg/ml in PBS) was injected subcutaneously into an anesthetized mouse and the
lymphatic vessels were analyzed by fluorescence microscopy.</p></sec><sec id="S17"><title>Electron microscopy</title><p id="P29">The intestines were dissected from P5 mice and immediately fixed in
4% PFA/2.5% glutaraldehyde in 0.1M phosphate buffer pH 7.4. The
samples were post fixed in reduced osmium tetroxide for 1 hour followed by
1% tannic acid in 0.05M sodium cacodylate for 45 minutes. Samples were
then dehydrated through a graded series of ethanol and embedded in Araldite
(Agar Scientific). Semi-thin sections were cut on a UCT ultramicrotome (Leica
Microsystems UK), stained with 1% Toluidine Blue in 1% Borax and
viewed under a III RS light microscope (Carl Zeiss UK) to locate the area of
interest. Ultra-thin sections were cut and stained with lead citrate before
being examined in a JEOL 1010 microscope and imaged with a Bioscan CCD (Gatan
UK).</p></sec><sec id="S18"><title>Cell culture</title><p id="P30">Human lymphatic endothelial cells (LEC) were isolated from primary
dermal microvascular endothelial cell cultures (PromoCell) using rat antibody to
human Podoplanin (NZ-1, Angiobio) and Mini/MidiMACS magnetic separation system
(Miltenyi Biotech), as previously described (<xref rid="R28" ref-type="bibr">Makinen et al., 2001</xref>). The cells were cultured on fibronectin
(Sigma) coated plates in the presence of 10 ng/ml VEGF-C (R&D Systems)
and used at passages 4-6. For siRNA mediated knock-down, LECs were transfected
twice during 48h using calcium phosphate (Dharmacon) in DMEM, supplemented with
20% FBS, followed by recovery in Endothelial Cell Medium (PromoCell) for
24h before the cells were used for experiments. ON-TARGETplus siRNAs were
obtained from Dharmacon. The following targeting sequences were used:
<italic>ITGA9</italic>_1: GAAGAAAGUCGUACUAUAG, <italic>ITGA9</italic>_2
GUGCAGAGAUGUUUCAUGU and <italic>ITGA5</italic>: UCACAUCGCUCUCAACUUC. Control
transfections were carried out using ON-TARGETplus siControl Non-targeting siRNA
from Dharmacon.</p></sec><sec id="S19"><title>Analysis of FN fibril assembly</title><p id="P31">LECs in 5% FN-depleted serum were plated on glass coverslips.
Similar results were obtained when the coverslips were coated with 0.2%
gelatin (no exogenous FN) or with extracellular matrices extracted from
<italic>FN-EIIIA</italic><sup>+</sup><italic><sup>/</sup></italic><sup>+</sup>(<xref rid="R29" ref-type="bibr">Muro et al., 2003</xref>) embryonic
fibroblasts (containing small quantities of exogenous FN, all of which contains
the EIIIA domain). The function-blocking antibodies (mouse anti-human
integrin-α9 (Y9A2; Millipore) and EIIIA (IST-9; Abcam)) and the blocking
peptides were used at a concentration of 10 μg/ml. 24 hours after
plating the cells were fixed in 4% PFA and FN fibril assembly was
examined using immunofluorescence. Images from five randomly chosen view fields
from two independent experiments were acquired. Image processing and analysis
was performed using MetaMorph Imaging software (Molecular Devices). Scaled
images were thresholded and filtered so that only FN-EIIIA-specific fibers
greater than 4 pixels (1.8 μm) were recorded. The total fiber length per
cell was calculated by dividing the total value recorded with the number of
cells present in each image. Isolation of DOC-soluble and – insoluble
matrix was done as previously described (<xref rid="R45" ref-type="bibr">Wierzbicka-Patynowski et al., 2004</xref>). Equal amounts of total protein
were separated by non-reducing 5% SDS-PAGE, transferred to
nitrocellulose and probed with anti-EIIIA antibody (FN-3E2).</p></sec><sec id="S20"><title>Western blot analysis</title><p id="P32">The cells were lysed in Triton-X100 lysis buffer (50 mM Tris pH 7.5, 120
mM NaCl, 10% glycerol, 1% TritonX-100) supplemented with
Complete Protease Inhibitors (Roche). Lysates were clarified by centrifugation
and subjected to immunoprecipitation and/or Western blot analysis using standard
protocols. The antibodies were mouse antibody to human integrin-α5
(GBS5; Millipore) and human integrin-α9 (Y9A2; Millipore, for IP),
chicken antibody to human integrin-α9 (GenWay, for WB) and mouse
antibody to human α-tubulin (TAT-1). The bound antibodies were detected
using HRP-conjugated secondary antibodies (Jackson ImmunoResearch) and ECL
chemiluminescence.</p></sec><sec id="S21"><title>Relative quantitative PCR</title><p id="P33">RNA from LECs was reverse transcribed using random hexamers and an avian
myeloblastosis virus reverse transcriptase (Promega). cDNA was amplified by
quantitative real-time PCR (ABI 7900HT) using SYBR Green PCR master mix reagent
(Qiagen). Each primer was used at a concentration of 0.5 μM. Cycling
conditions were as follows: step 1, 15 min at 95°C; step 2, 20 s at
94°C; step 3, 20 s at 60°C; step 4, 20 s at 72°C, with
repeat from step 2 to step 4 35 times. Data from the reaction were collected and
analyzed by the complementary computer software. Relative quantitations of gene
expression were normalized to the endogenous control (<italic>GAPDH</italic>).
The primers were <italic>ITGA9</italic>:
5′-CGGAATCATGTCTCCAACCT-3′ and
5′-TCTCTGCACCACCAGATGAG-3′, <italic>EIIIA</italic>:
5′-TTGATCGCCCTAAAGGACTG-3′ and
5′-ACCATCAGGTGCAGGGAATA-3′ and <italic>GAPDH</italic>:
5′-GAAGATGGTGATGGGATTTC-3′ and
5′-GAAGGTGAAGGTCGGAGT-3′.</p></sec><sec id="S22"><title>Statistical analysis</title><p id="P34">P values were calculated using the non-parametric Mann-Whitney test,
unpaired two-tailed Student T-test or χ<sup>2</sup> test as
indicated.</p></sec></sec><sec sec-type="supplementary-material" id="S23"><title>Supplementary Material</title><supplementary-material content-type="local-data" id="SD1"><label>01</label><media xlink:href="NIHMS141065-supplement-01.doc" mimetype="application" mime-subtype="msword" xlink:type="simple" id="d37e1268" position="anchor"></media></supplementary-material></sec></body><back><ack id="S25"><p>We are grateful to K. Alitalo for the
<italic>Vegfr3<sup>lz/</sup></italic><sup>+</sup> mice and R.
Fässler, A. Faissner and T. Czopka for the <italic>Tnc</italic> mice and
tissues. We thank I. Rosewell for help with the establishment of mouse colonies; H.
Chapman, A. Horwood, E. Murray, S. Lighterness and C. Watkins for animal husbandry;
and E. Nye for assistance with the histological analyses. We would like to thank M.
Way, N. Hogg, M. Graupera and I. Ferby for helpful discussions and critical comments
on the manuscript. This work has been supported by Cancer Research UK (E.B., S.X.,
A.W., R.A., T.M.) and by grant HL64353 from the National Heart Lung and Blood
Institute (USA) (D.S.). The authors declare no financial conflict of interest.</p></ack><fn-group><fn id="FN2" fn-type="con"><p>Author contributions: E.B. and T.M. designed research; E.B., S.X., C.C. and A.W.
performed research; N.M., L.S., R.A., A.M. and D.S. contributed reagents; E.B.,
S.X., C.C., A.W. and T.M. analysed data; and E.B. and T.M. wrote the paper.</p></fn><fn id="FN3"><p content-type="publisher-disclaimer">This is a PDF file of an unedited manuscript
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disclaimers that apply to the journal pertain.</p></fn></fn-group><ref-list><ref id="R1"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Alitalo</surname><given-names>K</given-names></name><name><surname>Tammela</surname><given-names>T</given-names></name><name><surname>Petrova</surname><given-names>TV</given-names></name></person-group><year>2005</year><article-title>Lymphangiogenesis in development
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Biol</source><volume>141</volume><fpage>539</fpage><lpage>551</lpage><pub-id pub-id-type="pmid">9548730</pub-id></element-citation></ref></ref-list></back><floats-group><fig id="F1" position="float"><label>Figure 1</label><caption><title>Expression of integrin-α9 in mature and developing lymphatic
valves</title><p>(A, B) Immunofluorescence staining of adult ear skin with antibodies against
integrin-α9 (green), FoxC2 (red) and α-smooth muscle actin
(α-SMA, blue). Arrow in (B) points to a luminal valve. (C-E)
Development of mesenteric lymphatic vessels. Whole-mount X-Gal staining of
mesenteric lymphatic vessels from
<italic>Vegfr3<sup>lz/</sup></italic><sup>+</sup> embryos. The
tissues were taken from embryos at the indicated ages (E16.5-E18.5).</p><p>(F-K) Immunofluorescence staining of developing mesenteric lymphatic vessels
of E16.5 (F, G), E17.5 (H, I) and E18.5 (J, K) with antibodies against
integrin-α9 (green), Prox1 (F, red) or FoxC2 (H, J, red) and LYVE-1
(F, H, J, blue). Arrowhead in (F, G) points to a blood vessel, the smooth
muscle coverage of which is positive for integrin-α9 staining.
Arrows point to clusters of cells expressing high levels of Prox1 and
FoxC2.</p><p>Scale bars; A, B, H-K: 50 μm, C-G: 1 mm.</p></caption><graphic xlink:href="nihms141065f1"></graphic></fig><fig id="F2" position="float"><label>Figure 2</label><caption><title>Abnormal valves in <italic>Itga9</italic> deficient mice</title><p>(A-B′) Luminal valves in chyle-filled mesenteric lymphatic vessels of
wild-type (A, A′) and <italic>Itga9</italic> mutant mice (B,
B′). Note the difference in the shape of a wild-type in comparison
to a mutant valve (A′, B′, arrows in A, B) and leakage of
chyle from the mutant vessels (arrowhead in B). BF = bright
field.</p><p>(C, D) PECAM-1 immunohistochemistry of P5 mesenteric vessels and luminal
valves (arrow) in wild-type (C) and <italic>Itga9<sup>-/-</sup></italic> (D)
mice.</p><p>(E) Quantification of the number of luminal valves in P5 wild-type and
<italic>Itga9<sup>-/-</sup></italic> mesenteric lymphatic vessels
(mean ± s.d., n = 4 animals per genotype, 3 vessels each).
Black bar = normal V-shaped valves; white bar = abnormal
valves with ring appearance. *** p < 0.0001
(Mann-Whitney test).</p><p>(F) Schematic representation of luminal valves (arrows) in the collecting
lymphatic vessels of
<italic>Itga9</italic><sup>+</sup><italic><sup>/</sup></italic><sup>+</sup>and <italic>Itga9<sup>-/-</sup></italic> mice.</p><p>(G-J) Transmission electron micrographs of wild-type (G, I) and
<italic>Itga9<sup>-/-</sup></italic> (H, J) valves in mesenteric
lymphatic vessels of P6 mice. Arrows in (G, H) point to the matrix core
(red) anchored into the vessels wall, arrowheads mark the free edges of the
valve leaflets. (I) shows the valve leaflet with a connective tissue core
(red). Note the rudimentary (arrows in H) or absent (J) matrix core in the
mutant valves and the gaps in between the two endothelial sheets (red
asterisks in J).</p><p>(K) Schematic representation of luminal valves in the
<italic>Itga9</italic><sup>+</sup><italic><sup>/</sup></italic><sup>+</sup>and <italic>Itga9<sup>-/-</sup></italic> mice. Matrix core is indicated in
red.</p><p>Scale bars; A, B: 100 μm, C, D: 50 μm, G, H: 10 μm,
I, J: 1 μm.</p></caption><graphic xlink:href="nihms141065f2"></graphic></fig><fig id="F3" position="float"><label>Figure 3</label><caption><title>Defective lymphatic drainage in <italic>Itga9</italic> deficient
mice</title><p>(A, B) Visualisation of dermal collecting vessels following injection of
FITC-dextran into the footpads of P6 wild-type (A) and
<italic>Itga9<sup>-/-</sup></italic> mice (B). Note the presence of
an abnormal vessel network (arrowheads in B) and a valve in a vessel branch
point in the <italic>Itga9</italic> mutants (arrow in B).</p><p>(C, D) FITC-lectin (LEL) staining of the valves in dermal lymphatic vessels
following footpad injection. No valve leaflets are seen in the
<italic>Itga9</italic> mutant (D). Arrows in (C) point to the two valve
leaflets seen from the 90° angle when compared to <xref rid="F2" ref-type="fig">Figure 2A, D</xref>.</p><p>(E) Schematic representation of a side view of luminal valves as visualized
by FITC-LEL staining in the collecting lymphatic vessels of
<italic>Itga9</italic><sup>+</sup><italic><sup>/</sup></italic><sup>+</sup>(left) and <italic>Itga9<sup>-/-</sup></italic> (right) mice. Arrows
indicate the direction of the flow.</p><p>Scale bars; A, B: 100 μm, C, D: 50 μm.</p></caption><graphic xlink:href="nihms141065f3"></graphic></fig><fig id="F4" position="float"><label>Figure 4</label><caption><title>Endothelial cell specific deletion of <italic>Itga9</italic> during
development and in mature valves</title><p>Lymphatic vessels of <italic>Tie2-Cre;Itga9<sup>lx/lx</sup></italic> mouse
(A-D), and of 4-OHT treated
<italic>Itga9<sup>lx/</sup></italic><sup>+</sup> (E-H) and
<italic>VEcad-CreER<sup>T2</sup>; Itga9<sup>lx/lx</sup></italic>(I-L) mice stained with antibodies against Laminin-α5 (red),
integrin-α9 (green) and PECAM-1 (blue). Expression of
integrin-α9 is detected in the vascular SMC (arrowhead in A, C, E,
G, I, K) but is lost from the endothelial cells in
<italic>Tie2-Cre;Itga9<sup>lx/lx</sup></italic> (C) and from most
endothelial cells of the valves of <italic>VEcad-CreER<sup>T2</sup>;
Itga9<sup>lx/lx</sup></italic> mutant animals (I, K, open arrowhead
points to a single integrin-α9 expressing cell). Note the abnormal
valve in <italic>Tie2-Cre;Itga9<sup>lx/lx</sup></italic> mouse (arrow in B),
which has undergone embryonic deletion of <italic>Itga9</italic> allele, but
intact valve leaflets (arrow in J) and the attachment of LECs on the
leaflets in the <italic>VEcad-CreER<sup>T2</sup>;
Itga9<sup>lx/lx</sup></italic> mutant (arrow in L), which has undergone
postnatal deletion of <italic>Itga9</italic> allele, as compared to a
control (arrows in F, G).</p><p>Scale bars; A-L: 20 μm.</p></caption><graphic xlink:href="nihms141065f4"></graphic></fig><fig id="F5" position="float"><label>Figure 5</label><caption><title>Development of lymphatic valve leaflets in wild-type and
<italic>Itga9<sup>-/-</sup></italic> mice</title><p>(A-C) Immunofluorescence staining of developing mesenteric lymphatic vessels
of E16 (A) and E17 (B, C) wild-type embryo using antibodies against
Laminin-α5, integrin-α9 and FN-EIIIA (colors as indicated).
The dotted lines outline the vessels.</p><p>(D-G) Immunolabeling of lymphatic valves in wild-type (D-E′) and
<italic>Itga9<sup>-/-</sup></italic> (F-G′) mesenteric
vessels for Prox1 (green) and FN-EIIIA (red; E, F; at E17) or for
Laminin-α5 (red), FN-EIIIA (green) and the endothelial marker
PECAM-1 (blue; E, G; at P2). The arrows in (D, E′, F, G′)
point to FN-EIIIA fibers.</p><p>(H, I) View through the opening of the valve in P0 wild-type (H) and
<italic>Itga9<sup>-/-</sup></italic> (I) vessels, labelled for FN
(red) and FN-EIIIA (green). Note the punctuate localization of FN-EIIIA in
the <italic>Itga9<sup>-/-</sup></italic> valve (arrow in I) compared to the
fibrous staining in the wild-type (arrow in H).</p><p>(J) Schematic model of lymphatic valve formation. Upregulation of Prox1 and
FoxC2 transcription factors (blue nuclei) in lymphatic vessels define the
positions of future valves. Deposition of extracellular matrix (red)
containing Laminin-α5 and FN-EIIIA and re-orientation of cells
expressing high levels of Prox1 and FoxC2 perpendicular to the vessel wall
is followed by upregulation of integrin-α9 (green) on the outflow
side of the future valve. <italic>Itga9<sup>-/-</sup></italic> mice (below)
display defective organization of the extracellular matrix and failure of
leaflet formation.</p><p>Scale bars; A-G: 50 μm, H, I: 10 μm.</p></caption><graphic xlink:href="nihms141065f5"></graphic></fig><fig id="F6" position="float"><label>Figure 6</label><caption><title>Abnormal lymphatic valves in mice lacking the integrin-α9 ligand,
FN-EIIIA</title><p>(A) Luminal valve numbers in newborn wild-type,
<italic>Fn-EIIIA<sup>-/-</sup></italic> and
<italic>Itga9<sup>-/-</sup></italic> mesenteric lymphatic vessels
(mean ± s.d., n ≥ 4 animals per genotype, ≥ 2
vessels each, see <xref rid="SD1" ref-type="supplementary-material">Suppl.
Table 1</xref>). The percentage of abnormal valves is indicated:
*** p < 0.0001 (χ<sup>2</sup>test).</p><p>(B, C) Visualization of lymphatic valves in P1 wild-type (B) and
<italic>Fn-EIIIA<sup>-/-</sup></italic> (C) mesenteric vessels using
antibodies against Laminin-α5. Note the incomplete development of
the valve as evident by lack of leaflets (arrow in C) in the
<italic>Fn-EIIIA<sup>-/-</sup></italic> vessels.</p><p>(D) Immunofluorescence staining of three weeks old ear skin for
integrin-α9 (green) and EIIIA (red).</p><p>(E, F) Dermal lymphatic vessels in the ears of three weeks old wild-type (E)
and <italic>Fn-EIIIA<sup>-/-</sup></italic> (F) mice labeled for
Laminin-α5 (green), podoplanin (blue) and α-SMA (red).</p><p>(G-I) FITC-dextran assay in three weeks old wild-type (G) and
<italic>Fn-EIIIA<sup>-/-</sup></italic> mice (H, I). Note the reflux
of dye (arrows in H) and an abnormal valve (arrow in I) in the mutant
skin.</p><p>Scale bars; B, C: 20 μm, D:50μm, E, F, I: 100 μm, G,
H: 400 μm.</p></caption><graphic xlink:href="nihms141065f6"></graphic></fig><fig id="F7" position="float"><label>Figure 7</label><caption><title>Integrin-α9-EIIIA interaction regulates FN fibril assembly in
primary human lymphatic endothelial cells</title><p>(A) FN fibrils in primary human lymphatic endothelial cells (LECs).
Integrin-α9-EIIIA interaction was blocked using antibodies against
EIIIA (IST-9) or integrin-α9β1 (Y9A2), or siRNA against
integrin-α9 or -α5, and stained with EIIIA antibodies.</p><p>(B) Quantification of FN fibrillogenesis in the LECs, in which
integrin-α9-EIIIA interaction (IST-9, Y9A2, α9 siRNA) or
integrin-α5/RGD-dependent integrin interactions (RGDSP peptide,
α5 siRNA) were inhibited, in comparison to the control cells
(untreated, ctrl siRNA or RGESP peptide). Data represent mean FN-EIIIA fiber
length per cell (± s.d) from five randomly chosen view fields in two
independent experiments. *** p< 0.003,
n.s.= non-significant, p = 0.881 (Student T-test).</p><p>(C) qPCR of <italic>ITGA9</italic> and <italic>FN-EIIIA</italic> in human
LECs. Data represent mean ± s.d. of triplicates.</p><p>(D) siRNA mediated knock-down of integrin expression in primary human LECs.
Western blot analysis of immunoprecipitated (IP) cell lysates using
integrin-α9 or -α5 antibodies (upper panels). For the
loading control, the total cell lysates (TCL) were blotted against
α-tubulin and EIIIA.</p><p>(E) Conversion of DOC-soluble FN fibrils into insoluble stable matrix.
DOC-insoluble (upper panel) and -soluble matrix (lower panel) isolated from
the LECs were separated in non-reducing SDS-PAGE and probed for EIIIA.</p><p>(F) Immunofluorescent staining of wild type E18 mesenteric vessels using
antibodies against integrin-α9 (left panel) and integrin-α5
(right panel). Note low levels of integrin-α5 expression in the
valve (arrows) in comparison to strong staining in the blood vessel
endothelia (arrowhead).</p><p>Scale bar = 20 μm.</p></caption><graphic xlink:href="nihms141065f7"></graphic></fig></floats-group></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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