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The extracellular matrix modulates H2O2 degradation and redox signaling in endothelial cells.

Bagulho A, Vilas-Boas F, Pena A, Peneda C, Santos FC, Jerónimo A, de Almeida RF, Real C - Redox Biol (2015)

Bottom Line: Instead, we found that the ECM regulated GPx activity, a known H2O2 scavenger.Thus, our results unraveled a new mechanism by which the ECM regulates endothelial cell function by altering redox balance.These results pinpoint the ECM as an important component of redox-signaling.

View Article: PubMed Central - PubMed

Affiliation: Centro de Química e Bioquímica, Faculdade de Ciências, Universidade de Lisboa, 1749-016 Lisboa, Portugal. Electronic address: anabagulho@gmail.com.

No MeSH data available.


PTEN oxidation levels in HUVEC on different ECMs. Western blots for PTEN after non-reducing SDS-PAGE and subsequent quantification of PTEN oxidation levels in HUVEC on different ECMs showed that in the presence of H2O2, PTEN was oxidized in an ECM-dependent manner, since endothelial cells cultured in laminin showed increased PTEN oxidation when compared with cells cultured in gelatin. Percentage of oxidized/reduced PTEN obtained for each ECM in each experiment was normalized by the percentage obtained for gelatin (values show the mean and SEM; N=7; Mann–Whitney U test, *p<0.03).
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f0020: PTEN oxidation levels in HUVEC on different ECMs. Western blots for PTEN after non-reducing SDS-PAGE and subsequent quantification of PTEN oxidation levels in HUVEC on different ECMs showed that in the presence of H2O2, PTEN was oxidized in an ECM-dependent manner, since endothelial cells cultured in laminin showed increased PTEN oxidation when compared with cells cultured in gelatin. Percentage of oxidized/reduced PTEN obtained for each ECM in each experiment was normalized by the percentage obtained for gelatin (values show the mean and SEM; N=7; Mann–Whitney U test, *p<0.03).

Mentions: H2O2, either from extracellular or intracellular sources, is able to alter the function of several proteins regulating cell signaling pathways in a process called redox signaling [22]. Phosphatase and tensin homolog (PTEN), a phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase (EC 3.1.3.67), is a redox-regulated protein that is reversibly inactivated by H2O2 through the formation of a disulfide between Cys71 and Cys124 [23]. In order to test whether the extracellular matrix is able to modulate redox-signaling, we quantified the oxidation level of PTEN in endothelial cells cultured in gelatin or laminin in the presence or absence of H2O2. For this, we took advantage of the difference in the electrophoretic mobility of the reduced vs. the oxidized form of PTEN in non-reducing conditions (Fig. 4). Our results showed that, while in the absence of H2O2, PTEN was mainly in the reduced form, in the presence of 50 µM of H2O2, PTEN oxidation increased. Importantly, this oxidation occurred in an ECM-dependent manner, since endothelial cells cultured in laminin showed nearly 2.5 times increase in PTEN oxidation levels when compared with cells cultured in gelatin. These results show that the ECM is able to modulate H2O2-dependent protein oxidation, which might be highly relevant for the regulation of redox-signaling.


The extracellular matrix modulates H2O2 degradation and redox signaling in endothelial cells.

Bagulho A, Vilas-Boas F, Pena A, Peneda C, Santos FC, Jerónimo A, de Almeida RF, Real C - Redox Biol (2015)

PTEN oxidation levels in HUVEC on different ECMs. Western blots for PTEN after non-reducing SDS-PAGE and subsequent quantification of PTEN oxidation levels in HUVEC on different ECMs showed that in the presence of H2O2, PTEN was oxidized in an ECM-dependent manner, since endothelial cells cultured in laminin showed increased PTEN oxidation when compared with cells cultured in gelatin. Percentage of oxidized/reduced PTEN obtained for each ECM in each experiment was normalized by the percentage obtained for gelatin (values show the mean and SEM; N=7; Mann–Whitney U test, *p<0.03).
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4588420&req=5

f0020: PTEN oxidation levels in HUVEC on different ECMs. Western blots for PTEN after non-reducing SDS-PAGE and subsequent quantification of PTEN oxidation levels in HUVEC on different ECMs showed that in the presence of H2O2, PTEN was oxidized in an ECM-dependent manner, since endothelial cells cultured in laminin showed increased PTEN oxidation when compared with cells cultured in gelatin. Percentage of oxidized/reduced PTEN obtained for each ECM in each experiment was normalized by the percentage obtained for gelatin (values show the mean and SEM; N=7; Mann–Whitney U test, *p<0.03).
Mentions: H2O2, either from extracellular or intracellular sources, is able to alter the function of several proteins regulating cell signaling pathways in a process called redox signaling [22]. Phosphatase and tensin homolog (PTEN), a phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase (EC 3.1.3.67), is a redox-regulated protein that is reversibly inactivated by H2O2 through the formation of a disulfide between Cys71 and Cys124 [23]. In order to test whether the extracellular matrix is able to modulate redox-signaling, we quantified the oxidation level of PTEN in endothelial cells cultured in gelatin or laminin in the presence or absence of H2O2. For this, we took advantage of the difference in the electrophoretic mobility of the reduced vs. the oxidized form of PTEN in non-reducing conditions (Fig. 4). Our results showed that, while in the absence of H2O2, PTEN was mainly in the reduced form, in the presence of 50 µM of H2O2, PTEN oxidation increased. Importantly, this oxidation occurred in an ECM-dependent manner, since endothelial cells cultured in laminin showed nearly 2.5 times increase in PTEN oxidation levels when compared with cells cultured in gelatin. These results show that the ECM is able to modulate H2O2-dependent protein oxidation, which might be highly relevant for the regulation of redox-signaling.

Bottom Line: Instead, we found that the ECM regulated GPx activity, a known H2O2 scavenger.Thus, our results unraveled a new mechanism by which the ECM regulates endothelial cell function by altering redox balance.These results pinpoint the ECM as an important component of redox-signaling.

View Article: PubMed Central - PubMed

Affiliation: Centro de Química e Bioquímica, Faculdade de Ciências, Universidade de Lisboa, 1749-016 Lisboa, Portugal. Electronic address: anabagulho@gmail.com.

No MeSH data available.