Nitric oxide inhibition increases matrix metalloproteinase–9 expression by rat aortic smooth muscle cells in vitro
Objective: The hypothesis to be tested was that diminished bioavailable nitric oxide (NO) affects matrix metalloproteinase (MMP) expression and activation in vascular smooth muscle cells (SMCs). Methods: Cultivated rat aortic SMCs (RA-SMCs) were exposed to increasing concentrations of L-N-monomethyl...
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| Veröffentlicht in: | Journal of vascular surgery 2001-07, Vol.34 (1), p.76-83 |
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| creator | Upchurch, Gilbert R. Ford, John W. Weiss, Steven J. Knipp, Brian S. Peterson, David A. Thompson, Robert W. Eagleton, Matthew J. Broady, Autumn J. Proctor, Mary C. Stanley, James C. |
| description | Objective: The hypothesis to be tested was that diminished bioavailable nitric oxide (NO) affects matrix metalloproteinase (MMP) expression and activation in vascular smooth muscle cells (SMCs). Methods: Cultivated rat aortic SMCs (RA-SMCs) were exposed to increasing concentrations of L-N-monomethyl arginine (L-NMMA), a nonselective inhibitor of NO synthase, in the presence of proinflammatory cytokines (50 ng/mL interleukin [IL]–1β, 50 ng/mL interferon-γ, and 30 μg/mL lipopolysaccharide). Nitrite and nitrate, two of the final end products of NO metabolism, were measured in media collected at 48 hours with the use of the Saville assay (n = 4). MMP activity was measured with 1% gelatin zymography (n = 4). In separate experiments in which 2 ng/mL of IL-1β and L-NMMA was used, MMP protein and messenger RNA (mRNA) levels were determined with Western blot analysis (n = 3) and semiquantitative reverse transcriptase-polymerase chain reaction (n = 3), respectively. Data were analyzed with nonparametric analysis of variance. Results: Increasing concentrations of the NO synthase inhibitor L-NMMA caused a dose-dependent decrease (P |
| doi_str_mv | 10.1067/mva.2001.115598 |
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Methods: Cultivated rat aortic SMCs (RA-SMCs) were exposed to increasing concentrations of L-N-monomethyl arginine (L-NMMA), a nonselective inhibitor of NO synthase, in the presence of proinflammatory cytokines (50 ng/mL interleukin [IL]–1β, 50 ng/mL interferon-γ, and 30 μg/mL lipopolysaccharide). Nitrite and nitrate, two of the final end products of NO metabolism, were measured in media collected at 48 hours with the use of the Saville assay (n = 4). MMP activity was measured with 1% gelatin zymography (n = 4). In separate experiments in which 2 ng/mL of IL-1β and L-NMMA was used, MMP protein and messenger RNA (mRNA) levels were determined with Western blot analysis (n = 3) and semiquantitative reverse transcriptase-polymerase chain reaction (n = 3), respectively. Data were analyzed with nonparametric analysis of variance. Results: Increasing concentrations of the NO synthase inhibitor L-NMMA caused a dose-dependent decrease (P <.05) in nitrite and nitrate production by RA-SMCs after cytokine exposure. Zymography documented an early dosedependent increase (P <.05 compared with cytokines alone) in 92-kd MMP activity, with no significant changes in 72-kd MMP activity after treatment with L-NMMA (P >.05 compared with cytokines alone). Reverse transcriptase-polymerase chain reaction and Western blot analysis revealed that the addition of L-NMMA to IL-1β–stimulated RA-SMCs led to significant increases in MMP-9 mRNA (n = 3, P <.01 for 1.0 mmol/L L-NMMA) and MMP-9 protein levels (n = 3, P <.05), respectively. No differences in MMP-2 mRNA or protein levels were demonstrated. Conclusions: Inhibition of cytokine-induced NO expression in RA-SMCs is associated with a selective, dose-dependent increase in MMP-9 expression and synthesis. These findings suggest that alterations in local NO synthesis may influence MMP-9–dependent vessel wall damage. (J Vasc Surg 2001;34:76-83.)</description><identifier>ISSN: 0741-5214</identifier><identifier>EISSN: 1097-6809</identifier><identifier>DOI: 10.1067/mva.2001.115598</identifier><identifier>PMID: 11436078</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>Animals ; Aorta - cytology ; Blotting, Western ; Cells, Cultured ; Electrophoresis, Polyacrylamide Gel ; Enzyme Inhibitors - pharmacology ; Male ; Matrix Metalloproteinase 9 - metabolism ; Muscle, Smooth, Vascular - cytology ; Nitric Oxide - physiology ; omega-N-Methylarginine - pharmacology ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; RNA, Messenger - metabolism</subject><ispartof>Journal of vascular surgery, 2001-07, Vol.34 (1), p.76-83</ispartof><rights>2001 Society for Vascular Surgery and The American Association for Vascular Surgery</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c384t-37e3076ae4442b6fb6646a58dd66a41c6f43fc5bce188da5306997b6af077efd3</citedby><cites>FETCH-LOGICAL-c384t-37e3076ae4442b6fb6646a58dd66a41c6f43fc5bce188da5306997b6af077efd3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1067/mva.2001.115598$$EHTML$$P50$$Gelsevier$$Hfree_for_read</linktohtml><link.rule.ids>315,781,785,3551,27929,27930,46000</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/11436078$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Upchurch, Gilbert R.</creatorcontrib><creatorcontrib>Ford, John W.</creatorcontrib><creatorcontrib>Weiss, Steven J.</creatorcontrib><creatorcontrib>Knipp, Brian S.</creatorcontrib><creatorcontrib>Peterson, David A.</creatorcontrib><creatorcontrib>Thompson, Robert W.</creatorcontrib><creatorcontrib>Eagleton, Matthew J.</creatorcontrib><creatorcontrib>Broady, Autumn J.</creatorcontrib><creatorcontrib>Proctor, Mary C.</creatorcontrib><creatorcontrib>Stanley, James C.</creatorcontrib><title>Nitric oxide inhibition increases matrix metalloproteinase–9 expression by rat aortic smooth muscle cells in vitro</title><title>Journal of vascular surgery</title><addtitle>J Vasc Surg</addtitle><description>Objective: The hypothesis to be tested was that diminished bioavailable nitric oxide (NO) affects matrix metalloproteinase (MMP) expression and activation in vascular smooth muscle cells (SMCs). Methods: Cultivated rat aortic SMCs (RA-SMCs) were exposed to increasing concentrations of L-N-monomethyl arginine (L-NMMA), a nonselective inhibitor of NO synthase, in the presence of proinflammatory cytokines (50 ng/mL interleukin [IL]–1β, 50 ng/mL interferon-γ, and 30 μg/mL lipopolysaccharide). Nitrite and nitrate, two of the final end products of NO metabolism, were measured in media collected at 48 hours with the use of the Saville assay (n = 4). MMP activity was measured with 1% gelatin zymography (n = 4). In separate experiments in which 2 ng/mL of IL-1β and L-NMMA was used, MMP protein and messenger RNA (mRNA) levels were determined with Western blot analysis (n = 3) and semiquantitative reverse transcriptase-polymerase chain reaction (n = 3), respectively. Data were analyzed with nonparametric analysis of variance. Results: Increasing concentrations of the NO synthase inhibitor L-NMMA caused a dose-dependent decrease (P <.05) in nitrite and nitrate production by RA-SMCs after cytokine exposure. Zymography documented an early dosedependent increase (P <.05 compared with cytokines alone) in 92-kd MMP activity, with no significant changes in 72-kd MMP activity after treatment with L-NMMA (P >.05 compared with cytokines alone). Reverse transcriptase-polymerase chain reaction and Western blot analysis revealed that the addition of L-NMMA to IL-1β–stimulated RA-SMCs led to significant increases in MMP-9 mRNA (n = 3, P <.01 for 1.0 mmol/L L-NMMA) and MMP-9 protein levels (n = 3, P <.05), respectively. No differences in MMP-2 mRNA or protein levels were demonstrated. Conclusions: Inhibition of cytokine-induced NO expression in RA-SMCs is associated with a selective, dose-dependent increase in MMP-9 expression and synthesis. These findings suggest that alterations in local NO synthesis may influence MMP-9–dependent vessel wall damage. (J Vasc Surg 2001;34:76-83.)</description><subject>Animals</subject><subject>Aorta - cytology</subject><subject>Blotting, Western</subject><subject>Cells, Cultured</subject><subject>Electrophoresis, Polyacrylamide Gel</subject><subject>Enzyme Inhibitors - pharmacology</subject><subject>Male</subject><subject>Matrix Metalloproteinase 9 - metabolism</subject><subject>Muscle, Smooth, Vascular - cytology</subject><subject>Nitric Oxide - physiology</subject><subject>omega-N-Methylarginine - pharmacology</subject><subject>Rats</subject><subject>Rats, Sprague-Dawley</subject><subject>Reverse Transcriptase Polymerase Chain Reaction</subject><subject>RNA, Messenger - metabolism</subject><issn>0741-5214</issn><issn>1097-6809</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2001</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp1kL9OwzAQhy0EoqUwsyFPbGntxrGTEVX8kypYYLYc56IaJXGxnardeAfekCfBUSoxMfkkf_e7uw-ha0rmlHCxaHdqviSEzinNsiI_QVNKCpHwnBSnaEoEo0m2pGyCLrz_iBzNcnGOJpSylBORT1F4McEZje3eVIBNtzGlCcZ2sdQOlAePWxWJPW4hqKaxW2cDmC7-_Hx9Fxj2WwfeDx3lATsVsLIuxEDfWhs2uO29bgBraBofM_EujrOX6KxWjYer4ztD7w_3b6unZP36-Ly6Wyc6zVlIUgEpEVwBY2xZ8rrknHGV5VXFuWJU85qltc5KDTTPK5WlhBeFKLmqiRBQV-kM3Y65cenPHnyQrfHDKqoD23spoivGmYjgYgS1s947qOXWmVa5g6REDqJlFC0H0XIUHTtujtF92UL1xx_NRqAYAYgH7gw46bWBTkNlHOggK2v-Df8FAjeQfA</recordid><startdate>20010701</startdate><enddate>20010701</enddate><creator>Upchurch, Gilbert R.</creator><creator>Ford, John W.</creator><creator>Weiss, Steven J.</creator><creator>Knipp, Brian S.</creator><creator>Peterson, David A.</creator><creator>Thompson, Robert W.</creator><creator>Eagleton, Matthew J.</creator><creator>Broady, Autumn J.</creator><creator>Proctor, Mary C.</creator><creator>Stanley, James C.</creator><general>Elsevier Inc</general><scope>6I.</scope><scope>AAFTH</scope><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>7X8</scope></search><sort><creationdate>20010701</creationdate><title>Nitric oxide inhibition increases matrix metalloproteinase–9 expression by rat aortic smooth muscle cells in vitro</title><author>Upchurch, Gilbert R. ; Ford, John W. ; Weiss, Steven J. ; Knipp, Brian S. ; Peterson, David A. ; Thompson, Robert W. ; Eagleton, Matthew J. ; Broady, Autumn J. ; Proctor, Mary C. ; Stanley, James C.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c384t-37e3076ae4442b6fb6646a58dd66a41c6f43fc5bce188da5306997b6af077efd3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2001</creationdate><topic>Animals</topic><topic>Aorta - cytology</topic><topic>Blotting, Western</topic><topic>Cells, Cultured</topic><topic>Electrophoresis, Polyacrylamide Gel</topic><topic>Enzyme Inhibitors - pharmacology</topic><topic>Male</topic><topic>Matrix Metalloproteinase 9 - metabolism</topic><topic>Muscle, Smooth, Vascular - cytology</topic><topic>Nitric Oxide - physiology</topic><topic>omega-N-Methylarginine - pharmacology</topic><topic>Rats</topic><topic>Rats, Sprague-Dawley</topic><topic>Reverse Transcriptase Polymerase Chain Reaction</topic><topic>RNA, Messenger - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Upchurch, Gilbert R.</creatorcontrib><creatorcontrib>Ford, John W.</creatorcontrib><creatorcontrib>Weiss, Steven J.</creatorcontrib><creatorcontrib>Knipp, Brian S.</creatorcontrib><creatorcontrib>Peterson, David A.</creatorcontrib><creatorcontrib>Thompson, Robert W.</creatorcontrib><creatorcontrib>Eagleton, Matthew J.</creatorcontrib><creatorcontrib>Broady, Autumn J.</creatorcontrib><creatorcontrib>Proctor, Mary C.</creatorcontrib><creatorcontrib>Stanley, James C.</creatorcontrib><collection>ScienceDirect Open Access Titles</collection><collection>Elsevier:ScienceDirect:Open Access</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Journal of vascular surgery</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Upchurch, Gilbert R.</au><au>Ford, John W.</au><au>Weiss, Steven J.</au><au>Knipp, Brian S.</au><au>Peterson, David A.</au><au>Thompson, Robert W.</au><au>Eagleton, Matthew J.</au><au>Broady, Autumn J.</au><au>Proctor, Mary C.</au><au>Stanley, James C.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Nitric oxide inhibition increases matrix metalloproteinase–9 expression by rat aortic smooth muscle cells in vitro</atitle><jtitle>Journal of vascular surgery</jtitle><addtitle>J Vasc Surg</addtitle><date>2001-07-01</date><risdate>2001</risdate><volume>34</volume><issue>1</issue><spage>76</spage><epage>83</epage><pages>76-83</pages><issn>0741-5214</issn><eissn>1097-6809</eissn><abstract>Objective: The hypothesis to be tested was that diminished bioavailable nitric oxide (NO) affects matrix metalloproteinase (MMP) expression and activation in vascular smooth muscle cells (SMCs). Methods: Cultivated rat aortic SMCs (RA-SMCs) were exposed to increasing concentrations of L-N-monomethyl arginine (L-NMMA), a nonselective inhibitor of NO synthase, in the presence of proinflammatory cytokines (50 ng/mL interleukin [IL]–1β, 50 ng/mL interferon-γ, and 30 μg/mL lipopolysaccharide). Nitrite and nitrate, two of the final end products of NO metabolism, were measured in media collected at 48 hours with the use of the Saville assay (n = 4). MMP activity was measured with 1% gelatin zymography (n = 4). In separate experiments in which 2 ng/mL of IL-1β and L-NMMA was used, MMP protein and messenger RNA (mRNA) levels were determined with Western blot analysis (n = 3) and semiquantitative reverse transcriptase-polymerase chain reaction (n = 3), respectively. Data were analyzed with nonparametric analysis of variance. Results: Increasing concentrations of the NO synthase inhibitor L-NMMA caused a dose-dependent decrease (P <.05) in nitrite and nitrate production by RA-SMCs after cytokine exposure. Zymography documented an early dosedependent increase (P <.05 compared with cytokines alone) in 92-kd MMP activity, with no significant changes in 72-kd MMP activity after treatment with L-NMMA (P >.05 compared with cytokines alone). Reverse transcriptase-polymerase chain reaction and Western blot analysis revealed that the addition of L-NMMA to IL-1β–stimulated RA-SMCs led to significant increases in MMP-9 mRNA (n = 3, P <.01 for 1.0 mmol/L L-NMMA) and MMP-9 protein levels (n = 3, P <.05), respectively. No differences in MMP-2 mRNA or protein levels were demonstrated. Conclusions: Inhibition of cytokine-induced NO expression in RA-SMCs is associated with a selective, dose-dependent increase in MMP-9 expression and synthesis. These findings suggest that alterations in local NO synthesis may influence MMP-9–dependent vessel wall damage. (J Vasc Surg 2001;34:76-83.)</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>11436078</pmid><doi>10.1067/mva.2001.115598</doi><tpages>8</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Animals Aorta - cytology Blotting, Western Cells, Cultured Electrophoresis, Polyacrylamide Gel Enzyme Inhibitors - pharmacology Male Matrix Metalloproteinase 9 - metabolism Muscle, Smooth, Vascular - cytology Nitric Oxide - physiology omega-N-Methylarginine - pharmacology Rats Rats, Sprague-Dawley Reverse Transcriptase Polymerase Chain Reaction RNA, Messenger - metabolism |
| title | Nitric oxide inhibition increases matrix metalloproteinase–9 expression by rat aortic smooth muscle cells in vitro |
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