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Evaluation of canine 2D cell cultures as models of myxomatous mitral valve degeneration
The utility of cells cultured from the mitral valve as models of myxomatous diseases needs to be properly validated. In this study valve interstitial cells (VICs) and valve endothelial cells (VECs) were cultured from normal and diseased canine mitral valves in 2% (v/v) or 10% FBS media, in the prese...
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| Published in: | PloS one 2019-08, Vol.14 (8), p.e0221126-e0221126 |
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| creator | Tan, Karen Markby, Greg Muirhead, Rhona Blake, Rachel Bergeron, Lisa Fici, Greg Summers, Kim Macrae, Vicky Corcoran, Brendan |
| description | The utility of cells cultured from the mitral valve as models of myxomatous diseases needs to be properly validated. In this study valve interstitial cells (VICs) and valve endothelial cells (VECs) were cultured from normal and diseased canine mitral valves in 2% (v/v) or 10% FBS media, in the presence of TGFβ1, 2 and 3, the TGFβ RI kinase inhibitor SB431542 and TGFβ neutralising antibodies, 5HT and the 5HT2RB antagonist LY272015. Cultures were examined by morphology, transcriptomic profiling, protein expression of the cell specific markers αSMA and SM22α (VICs), and CD31 (VECs), deposition of proteoglycans (PG), the PG versican, and the TGFβs themselves. VECs derived from normal valves were CD31+/αSMA-, but those from diseased valves were αSMA+, indicating endothelial-to-mesenchymal (EndoMT) transition had occurred. The TGFβs induced EndoMT in normal VECs, and this was abolished by SB431542, with significant changes in αSMA, CD31 and HAS2 expression (P |
| doi_str_mv | 10.1371/journal.pone.0221126 |
| format | article |
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In this study valve interstitial cells (VICs) and valve endothelial cells (VECs) were cultured from normal and diseased canine mitral valves in 2% (v/v) or 10% FBS media, in the presence of TGFβ1, 2 and 3, the TGFβ RI kinase inhibitor SB431542 and TGFβ neutralising antibodies, 5HT and the 5HT2RB antagonist LY272015. Cultures were examined by morphology, transcriptomic profiling, protein expression of the cell specific markers αSMA and SM22α (VICs), and CD31 (VECs), deposition of proteoglycans (PG), the PG versican, and the TGFβs themselves. VECs derived from normal valves were CD31+/αSMA-, but those from diseased valves were αSMA+, indicating endothelial-to-mesenchymal (EndoMT) transition had occurred. The TGFβs induced EndoMT in normal VECs, and this was abolished by SB431542, with significant changes in αSMA, CD31 and HAS2 expression (P<0.05). Normal VICs cultured in 10% FBS media were αSMA+ (activated myofibroblast (disease) phenotype), but were αSMA- when grown in 2% FBS. VICs from diseased dogs were αSMA+ in 2% FBS (retention of the activated myofibroblast disease phenotype), with significantly increased TGFβ1 expression (P<0.05) compared to normal cells. Treatment of normal and diseased VICs with the TGFβs significantly increased expression of αSMA, SM22α, versican, the TGFβs themselves, and deposition of PGs (P<0.05), with TGFβ1 being the most potent activator. These effects were either abolished or markedly reduced by SB431542 and a pan-TGFβ neutralizing antibody (P<0.05). SB431542 also markedly reduced αSMA expression in VICs from diseased valves, but 5HT and LY272015 had no effect on VIC phenotype. Transcriptomic profiling identified clear differences in gene expression for the different conditions and treatments that partially matched that seen in native diseased valve tissue, including changes in expression of ACTA2 (αSMA), 5HTR2B, TAGLN (SM22α) and MYH10 (SMemb), gene ontology terms and canonical signalling pathways. Normal and diseased VICs and normal VECs from canine mitral valves can be successfully grown in culture with retention of phenotype, which can be manipulated using TGFβ1 and the TGFβ RI kinase inhibitor SB431542. This optimized cell system can now be used to model MMVD to elucidate disease mechanisms and identify key regulators of disease progression.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0221126</identifier><identifier>PMID: 31415646</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Animals ; Antibodies ; Antigens, Differentiation - metabolism ; Biology and Life Sciences ; Catheters ; Cell adhesion & migration ; Cell culture ; Cell Culture Techniques ; Criminal investigation ; Cytology ; Degeneration ; Deposition ; Development and progression ; Dog diseases ; Dog Diseases - metabolism ; Dog Diseases - pathology ; Dogs ; Endothelial cells ; Endothelial Cells - metabolism ; Endothelial Cells - pathology ; Endothelium ; Enzyme inhibitors ; Extracellular matrix ; Gene expression ; Genes ; Genotype & phenotype ; Growth factors ; Heart ; Heart valve diseases ; Heart valves ; Inhibitors ; Interstitial cells ; Kinases ; Medical treatment ; Medicine and Health Sciences ; Mesenchyme ; Mitral valve ; Mitral Valve - metabolism ; Mitral Valve - pathology ; Mitral Valve Prolapse - metabolism ; Mitral Valve Prolapse - pathology ; Models, Cardiovascular ; Morphology ; Myofibroblasts - metabolism ; Myofibroblasts - pathology ; Pathogenesis ; Phenotypes ; Proteoglycans ; Regulators ; Research and Analysis Methods ; Retention ; Signal transduction ; Stem cells ; Studies ; Transforming growth factor-b1 ; Transforming growth factors ; Two dimensional models ; Versican ; Veterinary research</subject><ispartof>PloS one, 2019-08, Vol.14 (8), p.e0221126-e0221126</ispartof><rights>COPYRIGHT 2019 Public Library of Science</rights><rights>2019 Tan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2019 Tan et al 2019 Tan et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c692t-c0ed8662141b788155629edd0086dc314acf58ffae8a3ed2369014f443130e433</citedby><cites>FETCH-LOGICAL-c692t-c0ed8662141b788155629edd0086dc314acf58ffae8a3ed2369014f443130e433</cites><orcidid>0000-0001-7453-9808</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.proquest.com/docview/2273744312/fulltextPDF?pq-origsite=primo$$EPDF$$P50$$Gproquest$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.proquest.com/docview/2273744312?pq-origsite=primo$$EHTML$$P50$$Gproquest$$Hfree_for_read</linktohtml><link.rule.ids>230,314,727,780,784,885,25753,27924,27925,37012,37013,44590,53791,53793,75126</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/31415646$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Lionetti, Vincenzo</contributor><creatorcontrib>Tan, Karen</creatorcontrib><creatorcontrib>Markby, Greg</creatorcontrib><creatorcontrib>Muirhead, Rhona</creatorcontrib><creatorcontrib>Blake, Rachel</creatorcontrib><creatorcontrib>Bergeron, Lisa</creatorcontrib><creatorcontrib>Fici, Greg</creatorcontrib><creatorcontrib>Summers, Kim</creatorcontrib><creatorcontrib>Macrae, Vicky</creatorcontrib><creatorcontrib>Corcoran, Brendan</creatorcontrib><title>Evaluation of canine 2D cell cultures as models of myxomatous mitral valve degeneration</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>The utility of cells cultured from the mitral valve as models of myxomatous diseases needs to be properly validated. In this study valve interstitial cells (VICs) and valve endothelial cells (VECs) were cultured from normal and diseased canine mitral valves in 2% (v/v) or 10% FBS media, in the presence of TGFβ1, 2 and 3, the TGFβ RI kinase inhibitor SB431542 and TGFβ neutralising antibodies, 5HT and the 5HT2RB antagonist LY272015. Cultures were examined by morphology, transcriptomic profiling, protein expression of the cell specific markers αSMA and SM22α (VICs), and CD31 (VECs), deposition of proteoglycans (PG), the PG versican, and the TGFβs themselves. VECs derived from normal valves were CD31+/αSMA-, but those from diseased valves were αSMA+, indicating endothelial-to-mesenchymal (EndoMT) transition had occurred. The TGFβs induced EndoMT in normal VECs, and this was abolished by SB431542, with significant changes in αSMA, CD31 and HAS2 expression (P<0.05). Normal VICs cultured in 10% FBS media were αSMA+ (activated myofibroblast (disease) phenotype), but were αSMA- when grown in 2% FBS. VICs from diseased dogs were αSMA+ in 2% FBS (retention of the activated myofibroblast disease phenotype), with significantly increased TGFβ1 expression (P<0.05) compared to normal cells. Treatment of normal and diseased VICs with the TGFβs significantly increased expression of αSMA, SM22α, versican, the TGFβs themselves, and deposition of PGs (P<0.05), with TGFβ1 being the most potent activator. These effects were either abolished or markedly reduced by SB431542 and a pan-TGFβ neutralizing antibody (P<0.05). SB431542 also markedly reduced αSMA expression in VICs from diseased valves, but 5HT and LY272015 had no effect on VIC phenotype. Transcriptomic profiling identified clear differences in gene expression for the different conditions and treatments that partially matched that seen in native diseased valve tissue, including changes in expression of ACTA2 (αSMA), 5HTR2B, TAGLN (SM22α) and MYH10 (SMemb), gene ontology terms and canonical signalling pathways. Normal and diseased VICs and normal VECs from canine mitral valves can be successfully grown in culture with retention of phenotype, which can be manipulated using TGFβ1 and the TGFβ RI kinase inhibitor SB431542. This optimized cell system can now be used to model MMVD to elucidate disease mechanisms and identify key regulators of disease progression.</description><subject>Animals</subject><subject>Antibodies</subject><subject>Antigens, Differentiation - metabolism</subject><subject>Biology and Life Sciences</subject><subject>Catheters</subject><subject>Cell adhesion & migration</subject><subject>Cell culture</subject><subject>Cell Culture Techniques</subject><subject>Criminal investigation</subject><subject>Cytology</subject><subject>Degeneration</subject><subject>Deposition</subject><subject>Development and progression</subject><subject>Dog diseases</subject><subject>Dog Diseases - metabolism</subject><subject>Dog Diseases - pathology</subject><subject>Dogs</subject><subject>Endothelial cells</subject><subject>Endothelial Cells - metabolism</subject><subject>Endothelial Cells - pathology</subject><subject>Endothelium</subject><subject>Enzyme inhibitors</subject><subject>Extracellular matrix</subject><subject>Gene expression</subject><subject>Genes</subject><subject>Genotype & phenotype</subject><subject>Growth factors</subject><subject>Heart</subject><subject>Heart valve diseases</subject><subject>Heart valves</subject><subject>Inhibitors</subject><subject>Interstitial cells</subject><subject>Kinases</subject><subject>Medical treatment</subject><subject>Medicine and Health Sciences</subject><subject>Mesenchyme</subject><subject>Mitral valve</subject><subject>Mitral Valve - metabolism</subject><subject>Mitral Valve - pathology</subject><subject>Mitral Valve Prolapse - metabolism</subject><subject>Mitral Valve Prolapse - pathology</subject><subject>Models, Cardiovascular</subject><subject>Morphology</subject><subject>Myofibroblasts - metabolism</subject><subject>Myofibroblasts - pathology</subject><subject>Pathogenesis</subject><subject>Phenotypes</subject><subject>Proteoglycans</subject><subject>Regulators</subject><subject>Research and Analysis Methods</subject><subject>Retention</subject><subject>Signal transduction</subject><subject>Stem cells</subject><subject>Studies</subject><subject>Transforming growth factor-b1</subject><subject>Transforming growth factors</subject><subject>Two dimensional models</subject><subject>Versican</subject><subject>Veterinary research</subject><issn>1932-6203</issn><issn>1932-6203</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2019</creationdate><recordtype>article</recordtype><sourceid>PIMPY</sourceid><sourceid>DOA</sourceid><recordid>eNqNkl1r2zAUhs3YWLtu_2BshsHoLpLpw5Llm0Hpui1QKOzzUijyUaIgW5lkh_bfV27cEo9eDF_YHD3n1Tmv3yx7jdEc0xJ_3Pg-tMrNt76FOSIEY8KfZMe4omTGCaJPD76PshcxbhBiVHD-PDuiuMCMF_w4-3OxU65XnfVt7k2uVWtbyMnnXINzue5d1weIuYp542twcYCam2vfqM73qWi7oFyeNHaQ17CCFsKd2MvsmVEuwqvxfZL9-nLx8_zb7PLq6-L87HKmeUW6mUZQp5FImmdZCoEZ46SCukZI8FqnMZU2TBijQCgKNaG8QrgwRUExRVBQepK93etunY9y9CRKQkpaDhRJxGJP1F5t5DbYRoUb6ZWVdwUfVlKFzmoHkoBAQmPBDBEFILYErDRCuuCUYyOqpPVpvK1fNlBraIf1J6LTk9au5crvJOcVw7hMAqejQPB_e4idbGwcrFYtJD-HuRkpOKcooe_-QR_fbqRWKi1gW-PTvXoQlWes4oywkotEzR-h0lNDY3UKkLGpPmn4MGlITAfX3Ur1McrFj-__z179nrLvD9g1KNeto3f9EJk4BYs9qIOPMYB5MBkjOeT_3g055F-O-U9tbw5_0EPTfeDpLUtg_rA</recordid><startdate>20190815</startdate><enddate>20190815</enddate><creator>Tan, Karen</creator><creator>Markby, Greg</creator><creator>Muirhead, Rhona</creator><creator>Blake, Rachel</creator><creator>Bergeron, Lisa</creator><creator>Fici, Greg</creator><creator>Summers, Kim</creator><creator>Macrae, Vicky</creator><creator>Corcoran, Brendan</creator><general>Public Library of Science</general><general>Public Library of Science (PLoS)</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>IOV</scope><scope>ISR</scope><scope>3V.</scope><scope>7QG</scope><scope>7QL</scope><scope>7QO</scope><scope>7RV</scope><scope>7SN</scope><scope>7SS</scope><scope>7T5</scope><scope>7TG</scope><scope>7TM</scope><scope>7U9</scope><scope>7X2</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8AO</scope><scope>8C1</scope><scope>8FD</scope><scope>8FE</scope><scope>8FG</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABJCF</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>ARAPS</scope><scope>ATCPS</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BGLVJ</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>D1I</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>KB.</scope><scope>KB0</scope><scope>KL.</scope><scope>L6V</scope><scope>LK8</scope><scope>M0K</scope><scope>M0S</scope><scope>M1P</scope><scope>M7N</scope><scope>M7P</scope><scope>M7S</scope><scope>NAPCQ</scope><scope>P5Z</scope><scope>P62</scope><scope>P64</scope><scope>PATMY</scope><scope>PDBOC</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PTHSS</scope><scope>PYCSY</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope><scope>DOA</scope><orcidid>https://orcid.org/0000-0001-7453-9808</orcidid></search><sort><creationdate>20190815</creationdate><title>Evaluation of canine 2D cell cultures as models of myxomatous mitral valve degeneration</title><author>Tan, Karen ; Markby, Greg ; Muirhead, Rhona ; Blake, Rachel ; Bergeron, Lisa ; Fici, Greg ; Summers, Kim ; Macrae, Vicky ; Corcoran, Brendan</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c692t-c0ed8662141b788155629edd0086dc314acf58ffae8a3ed2369014f443130e433</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2019</creationdate><topic>Animals</topic><topic>Antibodies</topic><topic>Antigens, Differentiation - 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Academic</collection><collection>PubMed Central (Full Participant titles)</collection><collection>DOAJ Directory of Open Access Journals</collection><jtitle>PloS one</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Tan, Karen</au><au>Markby, Greg</au><au>Muirhead, Rhona</au><au>Blake, Rachel</au><au>Bergeron, Lisa</au><au>Fici, Greg</au><au>Summers, Kim</au><au>Macrae, Vicky</au><au>Corcoran, Brendan</au><au>Lionetti, Vincenzo</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Evaluation of canine 2D cell cultures as models of myxomatous mitral valve degeneration</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2019-08-15</date><risdate>2019</risdate><volume>14</volume><issue>8</issue><spage>e0221126</spage><epage>e0221126</epage><pages>e0221126-e0221126</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>The utility of cells cultured from the mitral valve as models of myxomatous diseases needs to be properly validated. In this study valve interstitial cells (VICs) and valve endothelial cells (VECs) were cultured from normal and diseased canine mitral valves in 2% (v/v) or 10% FBS media, in the presence of TGFβ1, 2 and 3, the TGFβ RI kinase inhibitor SB431542 and TGFβ neutralising antibodies, 5HT and the 5HT2RB antagonist LY272015. Cultures were examined by morphology, transcriptomic profiling, protein expression of the cell specific markers αSMA and SM22α (VICs), and CD31 (VECs), deposition of proteoglycans (PG), the PG versican, and the TGFβs themselves. VECs derived from normal valves were CD31+/αSMA-, but those from diseased valves were αSMA+, indicating endothelial-to-mesenchymal (EndoMT) transition had occurred. The TGFβs induced EndoMT in normal VECs, and this was abolished by SB431542, with significant changes in αSMA, CD31 and HAS2 expression (P<0.05). Normal VICs cultured in 10% FBS media were αSMA+ (activated myofibroblast (disease) phenotype), but were αSMA- when grown in 2% FBS. VICs from diseased dogs were αSMA+ in 2% FBS (retention of the activated myofibroblast disease phenotype), with significantly increased TGFβ1 expression (P<0.05) compared to normal cells. Treatment of normal and diseased VICs with the TGFβs significantly increased expression of αSMA, SM22α, versican, the TGFβs themselves, and deposition of PGs (P<0.05), with TGFβ1 being the most potent activator. These effects were either abolished or markedly reduced by SB431542 and a pan-TGFβ neutralizing antibody (P<0.05). SB431542 also markedly reduced αSMA expression in VICs from diseased valves, but 5HT and LY272015 had no effect on VIC phenotype. Transcriptomic profiling identified clear differences in gene expression for the different conditions and treatments that partially matched that seen in native diseased valve tissue, including changes in expression of ACTA2 (αSMA), 5HTR2B, TAGLN (SM22α) and MYH10 (SMemb), gene ontology terms and canonical signalling pathways. Normal and diseased VICs and normal VECs from canine mitral valves can be successfully grown in culture with retention of phenotype, which can be manipulated using TGFβ1 and the TGFβ RI kinase inhibitor SB431542. This optimized cell system can now be used to model MMVD to elucidate disease mechanisms and identify key regulators of disease progression.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>31415646</pmid><doi>10.1371/journal.pone.0221126</doi><tpages>e0221126</tpages><orcidid>https://orcid.org/0000-0001-7453-9808</orcidid><oa>free_for_read</oa></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 1932-6203 |
| ispartof | PloS one, 2019-08, Vol.14 (8), p.e0221126-e0221126 |
| issn | 1932-6203 1932-6203 |
| language | eng |
| recordid | cdi_plos_journals_2273744312 |
| source | NCBI_PubMed Central（免费）; Publicly Available Content Database |
| subjects | Animals Antibodies Antigens, Differentiation - metabolism Biology and Life Sciences Catheters Cell adhesion & migration Cell culture Cell Culture Techniques Criminal investigation Cytology Degeneration Deposition Development and progression Dog diseases Dog Diseases - metabolism Dog Diseases - pathology Dogs Endothelial cells Endothelial Cells - metabolism Endothelial Cells - pathology Endothelium Enzyme inhibitors Extracellular matrix Gene expression Genes Genotype & phenotype Growth factors Heart Heart valve diseases Heart valves Inhibitors Interstitial cells Kinases Medical treatment Medicine and Health Sciences Mesenchyme Mitral valve Mitral Valve - metabolism Mitral Valve - pathology Mitral Valve Prolapse - metabolism Mitral Valve Prolapse - pathology Models, Cardiovascular Morphology Myofibroblasts - metabolism Myofibroblasts - pathology Pathogenesis Phenotypes Proteoglycans Regulators Research and Analysis Methods Retention Signal transduction Stem cells Studies Transforming growth factor-b1 Transforming growth factors Two dimensional models Versican Veterinary research |
| title | Evaluation of canine 2D cell cultures as models of myxomatous mitral valve degeneration |
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