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The new anti-actin agent dihydrohalichondramide reveals fenestrae-forming centers in hepatic endothelial cells.

Braet F, Spector I, Shochet N, Crews P, Higa T, Menu E, de Zanger R, Wisse E - BMC Cell Biol. (2002)

Bottom Line: In this study, we investigated the effects of two new actin-binding agents on fenestrae dynamics.Dihydrohalichondramide induces fenestrae-forming centers, whereas halichondramide only revealed fenestrae-forming centers without attached rows of fenestrae with increasing diameter.Comparable experiments on umbilical vein endothelial cells and bone marrow sinusoidal endothelial cells revealed cell contraction without the appearance of fenestrae or fenestrae-forming centers. (I) A comparison of all anti-actin agents tested so far, revealed that the only activity that misakinolide and dihydrohalichondramide have in common is their barbed end capping activity; (II) this activity seems to slow down the process of fenestrae formation to such extent that it becomes possible to resolve fenestrae-forming centers; (III) fenestrae formation resulting from microfilament disruption is probably unique to LSECs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory for Cell Biology and Histology, Free University of Brussels (VUB), Laarbeeklaan 103, 1090 Brussels-Jette, Belgium. filipbra@cyto.vub.ac.be

ABSTRACT

Background: Liver sinusoidal endothelial cells (LSECs) react to different anti-actin agents by increasing their number of fenestrae. A new structure related to fenestrae formation could be observed when LSECs were treated with misakinolide. In this study, we investigated the effects of two new actin-binding agents on fenestrae dynamics. High-resolution microscopy, including immunocytochemistry and a combination of fluorescence- and scanning electron microscopy was applied.

Results: Halichondramide and dihydrohalichondramide disrupt microfilaments within 10 minutes and double the number of fenestrae in 30 minutes. Dihydrohalichondramide induces fenestrae-forming centers, whereas halichondramide only revealed fenestrae-forming centers without attached rows of fenestrae with increasing diameter. Correlative microscopy showed the absence of actin filaments (F-actin) in sieve plates and fenestrae-forming centers. Comparable experiments on umbilical vein endothelial cells and bone marrow sinusoidal endothelial cells revealed cell contraction without the appearance of fenestrae or fenestrae-forming centers.

Conclusion: (I) A comparison of all anti-actin agents tested so far, revealed that the only activity that misakinolide and dihydrohalichondramide have in common is their barbed end capping activity; (II) this activity seems to slow down the process of fenestrae formation to such extent that it becomes possible to resolve fenestrae-forming centers; (III) fenestrae formation resulting from microfilament disruption is probably unique to LSECs.

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Effect of HALI and di-h-HALI on the number of fenestrae per micrometer squared in time. From this graph, we can conclude that both agents increase the number of fenestrae, although at a different rate and different maximum. Data are means plus or minus S.E.M. of triplicate determinations. Note the significant differences between control LSECs (0 minutes) and treated LSECs, as indicated by asterisks (p < 0.01) or by triangles (p < 0.05) (Mann-Whitney U test, two-sided). No significant difference in the number of fenestrae was observed at the 0.05 confidence level between 60 and 120 minutes of treatment with HALI or di-h-HALI.
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Figure 3: Effect of HALI and di-h-HALI on the number of fenestrae per micrometer squared in time. From this graph, we can conclude that both agents increase the number of fenestrae, although at a different rate and different maximum. Data are means plus or minus S.E.M. of triplicate determinations. Note the significant differences between control LSECs (0 minutes) and treated LSECs, as indicated by asterisks (p < 0.01) or by triangles (p < 0.05) (Mann-Whitney U test, two-sided). No significant difference in the number of fenestrae was observed at the 0.05 confidence level between 60 and 120 minutes of treatment with HALI or di-h-HALI.

Mentions: Computer-assisted analysis of endothelial fenestration, using digitized SEM images, showed that fenestrae occur at a frequency of 3.0 ± 0.2 per micrometer squared in control LSEC. By comparing the effects of both microfilament-disrupting agents (at 100 nM) as a function of time, it becomes clear that HALI increases the number of fenestrae faster than di-h-HALI (Fig. 3). A significant difference in the number of fenestrae between control and HALI-treated LSECs could be discerned as early as 10 minutes after HALI application. In the case of di-h-HALI, a significant difference in the number of fenestrae was observed 20 minutes later than HALI (Fig. 3), corresponding with the observation of FFCs with connected fenestrae rows (Fig. 2B). After 120 minutes in the presence of HALI or di-h-HALI, the number of fenestrae per micrometer squared increased to 7.7 ± 0.5, and 6.0 ± 0.2 respectively. Lowering the concentrations of HALI or di-h-HALI to 25 or 50 nM gave insignificant changes in the number of fenestrae (data not shown).


The new anti-actin agent dihydrohalichondramide reveals fenestrae-forming centers in hepatic endothelial cells.

Braet F, Spector I, Shochet N, Crews P, Higa T, Menu E, de Zanger R, Wisse E - BMC Cell Biol. (2002)

Effect of HALI and di-h-HALI on the number of fenestrae per micrometer squared in time. From this graph, we can conclude that both agents increase the number of fenestrae, although at a different rate and different maximum. Data are means plus or minus S.E.M. of triplicate determinations. Note the significant differences between control LSECs (0 minutes) and treated LSECs, as indicated by asterisks (p < 0.01) or by triangles (p < 0.05) (Mann-Whitney U test, two-sided). No significant difference in the number of fenestrae was observed at the 0.05 confidence level between 60 and 120 minutes of treatment with HALI or di-h-HALI.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC101387&req=5

Figure 3: Effect of HALI and di-h-HALI on the number of fenestrae per micrometer squared in time. From this graph, we can conclude that both agents increase the number of fenestrae, although at a different rate and different maximum. Data are means plus or minus S.E.M. of triplicate determinations. Note the significant differences between control LSECs (0 minutes) and treated LSECs, as indicated by asterisks (p < 0.01) or by triangles (p < 0.05) (Mann-Whitney U test, two-sided). No significant difference in the number of fenestrae was observed at the 0.05 confidence level between 60 and 120 minutes of treatment with HALI or di-h-HALI.
Mentions: Computer-assisted analysis of endothelial fenestration, using digitized SEM images, showed that fenestrae occur at a frequency of 3.0 ± 0.2 per micrometer squared in control LSEC. By comparing the effects of both microfilament-disrupting agents (at 100 nM) as a function of time, it becomes clear that HALI increases the number of fenestrae faster than di-h-HALI (Fig. 3). A significant difference in the number of fenestrae between control and HALI-treated LSECs could be discerned as early as 10 minutes after HALI application. In the case of di-h-HALI, a significant difference in the number of fenestrae was observed 20 minutes later than HALI (Fig. 3), corresponding with the observation of FFCs with connected fenestrae rows (Fig. 2B). After 120 minutes in the presence of HALI or di-h-HALI, the number of fenestrae per micrometer squared increased to 7.7 ± 0.5, and 6.0 ± 0.2 respectively. Lowering the concentrations of HALI or di-h-HALI to 25 or 50 nM gave insignificant changes in the number of fenestrae (data not shown).

Bottom Line: In this study, we investigated the effects of two new actin-binding agents on fenestrae dynamics.Dihydrohalichondramide induces fenestrae-forming centers, whereas halichondramide only revealed fenestrae-forming centers without attached rows of fenestrae with increasing diameter.Comparable experiments on umbilical vein endothelial cells and bone marrow sinusoidal endothelial cells revealed cell contraction without the appearance of fenestrae or fenestrae-forming centers. (I) A comparison of all anti-actin agents tested so far, revealed that the only activity that misakinolide and dihydrohalichondramide have in common is their barbed end capping activity; (II) this activity seems to slow down the process of fenestrae formation to such extent that it becomes possible to resolve fenestrae-forming centers; (III) fenestrae formation resulting from microfilament disruption is probably unique to LSECs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory for Cell Biology and Histology, Free University of Brussels (VUB), Laarbeeklaan 103, 1090 Brussels-Jette, Belgium. filipbra@cyto.vub.ac.be

ABSTRACT

Background: Liver sinusoidal endothelial cells (LSECs) react to different anti-actin agents by increasing their number of fenestrae. A new structure related to fenestrae formation could be observed when LSECs were treated with misakinolide. In this study, we investigated the effects of two new actin-binding agents on fenestrae dynamics. High-resolution microscopy, including immunocytochemistry and a combination of fluorescence- and scanning electron microscopy was applied.

Results: Halichondramide and dihydrohalichondramide disrupt microfilaments within 10 minutes and double the number of fenestrae in 30 minutes. Dihydrohalichondramide induces fenestrae-forming centers, whereas halichondramide only revealed fenestrae-forming centers without attached rows of fenestrae with increasing diameter. Correlative microscopy showed the absence of actin filaments (F-actin) in sieve plates and fenestrae-forming centers. Comparable experiments on umbilical vein endothelial cells and bone marrow sinusoidal endothelial cells revealed cell contraction without the appearance of fenestrae or fenestrae-forming centers.

Conclusion: (I) A comparison of all anti-actin agents tested so far, revealed that the only activity that misakinolide and dihydrohalichondramide have in common is their barbed end capping activity; (II) this activity seems to slow down the process of fenestrae formation to such extent that it becomes possible to resolve fenestrae-forming centers; (III) fenestrae formation resulting from microfilament disruption is probably unique to LSECs.

Show MeSH
Related in: MedlinePlus