Limits...
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.


Biophysical properties of HUVEC cell membrane lipids. Analysis of TMA-DPH (A) amplitude-weighted (τav)- and intensity-weighted 〈τ〉 mean fluorescence lifetime, (B) steady-state fluorescence anisotropy (〈r〉), (C) rotational correlation time (ϕ) and (D) infinite anisotropy () from HUVEC cultured in gelatin or laminin showed that the ECMs had no effect on water penetration and induced no changes on the global order and dynamics at the membrane surface. Analysis of t-PnA (E) long fluorescence lifetime component (τ3), amplitude (τav)- and intensity 〈τ〉-weighted mean fluorescence lifetime, (F) steady-state fluorescence anisotropy (〈r〉), (G) rotational correlation time (ϕ) and (H) infinite anisotropy () from HUVEC cultured in gelatin or laminin showed that the ECMs had no effect on water penetration but induced subtle changes in the packing and dynamics of some lipids in ordered membrane microdomains (values are the mean and SEM; N=3–5; two-tailed Student's t test, *p<0.02, ***p<0.00002).
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4588420&req=5

f0010: Biophysical properties of HUVEC cell membrane lipids. Analysis of TMA-DPH (A) amplitude-weighted (τav)- and intensity-weighted 〈τ〉 mean fluorescence lifetime, (B) steady-state fluorescence anisotropy (〈r〉), (C) rotational correlation time (ϕ) and (D) infinite anisotropy () from HUVEC cultured in gelatin or laminin showed that the ECMs had no effect on water penetration and induced no changes on the global order and dynamics at the membrane surface. Analysis of t-PnA (E) long fluorescence lifetime component (τ3), amplitude (τav)- and intensity 〈τ〉-weighted mean fluorescence lifetime, (F) steady-state fluorescence anisotropy (〈r〉), (G) rotational correlation time (ϕ) and (H) infinite anisotropy () from HUVEC cultured in gelatin or laminin showed that the ECMs had no effect on water penetration but induced subtle changes in the packing and dynamics of some lipids in ordered membrane microdomains (values are the mean and SEM; N=3–5; two-tailed Student's t test, *p<0.02, ***p<0.00002).

Mentions: The fluorescence lifetime of DPH family probes, such as TMA-DPH, is very sensitive to membrane perturbations that increase membrane hydration, which causes a strong reduction of the probe fluorescence lifetime and quantum yield [20]. Since alterations in lipid organization and composition of cellular membranes reflect H2O2 permeability [21], we measured fluorescence lifetime values of TMA-DPH of HUVEC cultured in gelatin or laminin. Our results showed a remarkable similarity between all of the fluorescence lifetime values obtained for TMA-DPH, either amplitude-weighted (τav) or intensity-weighted (〈τ〉) (2.96 ns τav and 4.79 ns 〈τ〉 for gelatin; 3.01 ns τav and 4.77 ns 〈τ〉 for laminin), a strong indication that there was no difference in membrane water penetration (Fig. 2A). The steady-state fluorescence anisotropy 〈r〉 is the most common parameter used to assess membrane fluidity. The values of the steady-state anisotropy for TMA-DPH (0.215 for gelatin and 0.216 for laminin) indicated a fluid lipid bilayer and that there were no significant differences on the global fluidity of the membrane surface induced by the ECM proteins (Fig. 2B). The fluorescence anisotropy decay allows to decompose 〈r〉 into several contributions, with a direct molecular meaning and nanosecond time-resolution. The rotational correlation time (ϕ) and infinite anisotropy values () contain information on the variety, speed and hindering of rotational modes of the probe wobbling. Both of these parameters were also similar for cells cultured in the two ECMs analyzed (4.61 ns ϕ and 0.170 for gelatin; 4.22 ns ϕ and 0.169 for laminin) (Fig. 2C, D). Altogether, the results obtained with the TMA-DPH probe suggest that there were no differences in membrane water permeability between cells cultured in gelatin and laminin. Therefore, the observed differences in hydrogen peroxide consumption were probably not caused by differences in the rate of passive permeation.


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)

Biophysical properties of HUVEC cell membrane lipids. Analysis of TMA-DPH (A) amplitude-weighted (τav)- and intensity-weighted 〈τ〉 mean fluorescence lifetime, (B) steady-state fluorescence anisotropy (〈r〉), (C) rotational correlation time (ϕ) and (D) infinite anisotropy () from HUVEC cultured in gelatin or laminin showed that the ECMs had no effect on water penetration and induced no changes on the global order and dynamics at the membrane surface. Analysis of t-PnA (E) long fluorescence lifetime component (τ3), amplitude (τav)- and intensity 〈τ〉-weighted mean fluorescence lifetime, (F) steady-state fluorescence anisotropy (〈r〉), (G) rotational correlation time (ϕ) and (H) infinite anisotropy () from HUVEC cultured in gelatin or laminin showed that the ECMs had no effect on water penetration but induced subtle changes in the packing and dynamics of some lipids in ordered membrane microdomains (values are the mean and SEM; N=3–5; two-tailed Student's t test, *p<0.02, ***p<0.00002).
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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

f0010: Biophysical properties of HUVEC cell membrane lipids. Analysis of TMA-DPH (A) amplitude-weighted (τav)- and intensity-weighted 〈τ〉 mean fluorescence lifetime, (B) steady-state fluorescence anisotropy (〈r〉), (C) rotational correlation time (ϕ) and (D) infinite anisotropy () from HUVEC cultured in gelatin or laminin showed that the ECMs had no effect on water penetration and induced no changes on the global order and dynamics at the membrane surface. Analysis of t-PnA (E) long fluorescence lifetime component (τ3), amplitude (τav)- and intensity 〈τ〉-weighted mean fluorescence lifetime, (F) steady-state fluorescence anisotropy (〈r〉), (G) rotational correlation time (ϕ) and (H) infinite anisotropy () from HUVEC cultured in gelatin or laminin showed that the ECMs had no effect on water penetration but induced subtle changes in the packing and dynamics of some lipids in ordered membrane microdomains (values are the mean and SEM; N=3–5; two-tailed Student's t test, *p<0.02, ***p<0.00002).
Mentions: The fluorescence lifetime of DPH family probes, such as TMA-DPH, is very sensitive to membrane perturbations that increase membrane hydration, which causes a strong reduction of the probe fluorescence lifetime and quantum yield [20]. Since alterations in lipid organization and composition of cellular membranes reflect H2O2 permeability [21], we measured fluorescence lifetime values of TMA-DPH of HUVEC cultured in gelatin or laminin. Our results showed a remarkable similarity between all of the fluorescence lifetime values obtained for TMA-DPH, either amplitude-weighted (τav) or intensity-weighted (〈τ〉) (2.96 ns τav and 4.79 ns 〈τ〉 for gelatin; 3.01 ns τav and 4.77 ns 〈τ〉 for laminin), a strong indication that there was no difference in membrane water penetration (Fig. 2A). The steady-state fluorescence anisotropy 〈r〉 is the most common parameter used to assess membrane fluidity. The values of the steady-state anisotropy for TMA-DPH (0.215 for gelatin and 0.216 for laminin) indicated a fluid lipid bilayer and that there were no significant differences on the global fluidity of the membrane surface induced by the ECM proteins (Fig. 2B). The fluorescence anisotropy decay allows to decompose 〈r〉 into several contributions, with a direct molecular meaning and nanosecond time-resolution. The rotational correlation time (ϕ) and infinite anisotropy values () contain information on the variety, speed and hindering of rotational modes of the probe wobbling. Both of these parameters were also similar for cells cultured in the two ECMs analyzed (4.61 ns ϕ and 0.170 for gelatin; 4.22 ns ϕ and 0.169 for laminin) (Fig. 2C, D). Altogether, the results obtained with the TMA-DPH probe suggest that there were no differences in membrane water permeability between cells cultured in gelatin and laminin. Therefore, the observed differences in hydrogen peroxide consumption were probably not caused by differences in the rate of passive permeation.

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.