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Class II phosphoinositide 3-kinases contribute to endothelial cells morphogenesis.

Tibolla G, Piñeiro R, Chiozzotto D, Mavrommati I, Wheeler AP, Norata GD, Catapano AL, Maffucci T, Falasca M - PLoS ONE (2013)

Bottom Line: In this study we investigated the relative contribution of distinct PI3K isoforms to endothelial cells (EC) functions specifically regulated by the sphingolipid sphingosine-1-phosphate (S1P) and by high density lipoproteins (HDL), the major carrier of S1P in human plasma.Here we show that a co-ordinated action of different PI3Ks is required to tightly regulate remodelling of EC on Matrigel, a process dependent on cell proliferation, apoptosis and migration.The contribution of each isoform to this process appears to be distinct, with the class II enzyme PI3K-C2β and the class IB isoform p110γ mainly regulating the S1P- and HDL-dependent EC migration and PI3K-C2α primarily controlling EC survival.

View Article: PubMed Central - PubMed

Affiliation: Queen Mary University of London, Barts and The London School of Medicine and Dentistry, Blizard Institute, Centre for Diabetes, Inositide Signalling Group, London, United Kingdom.

ABSTRACT
The question of whether the distinct isoforms of the family of enzymes phosphoinositide 3-kinases (PI3Ks) play redundant roles within a cell or whether they control distinct cellular processes or distinct steps within the same cellular process has gained considerable importance in the recent years due to the development of inhibitors able to selectively target individual isoforms. It is important to understand whether inhibition of one PI3K can result in compensatory effect from other isoform(s) and therefore whether strategies aimed at simultaneously blocking more than one PI3K may be needed. In this study we investigated the relative contribution of distinct PI3K isoforms to endothelial cells (EC) functions specifically regulated by the sphingolipid sphingosine-1-phosphate (S1P) and by high density lipoproteins (HDL), the major carrier of S1P in human plasma. Here we show that a co-ordinated action of different PI3Ks is required to tightly regulate remodelling of EC on Matrigel, a process dependent on cell proliferation, apoptosis and migration. The contribution of each isoform to this process appears to be distinct, with the class II enzyme PI3K-C2β and the class IB isoform p110γ mainly regulating the S1P- and HDL-dependent EC migration and PI3K-C2α primarily controlling EC survival. Data further indicate that PI3K-C2β and p110γ control distinct steps involved in cell migration supporting the hypothesis that different PI3Ks regulate distinct cellular processes.

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Class II and class IB PI3Ks regulate remodelling of HUVEC.HUVEC transfected with the indicated siRNAs were serum starved in M199 containing 0.5% FBS overnight before being detached and plated on growth factor reduced Matrigel in the presence of 1 µM S1P or 200 µg/ml HDL3. EC rearrangement was visualised after 4 to 6 h using an Axiovert200 microscope. (A) Representative images of branching points formation in HUVEC transfected with the indicated siRNAs (Sequences 1) in the absence or presence of S1P or HDL3. (B,C) Results from quantitative analysis in HUVEC transfected with the indicated siRNAs (Sequences 2) in the absence or presence of S1P or HDL3. Data indicate the total number of branching points and are means ± SEM from 4 (B) and 3 (C) independent experiments. *p<0.01; **p<0.001.
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pone-0053808-g004: Class II and class IB PI3Ks regulate remodelling of HUVEC.HUVEC transfected with the indicated siRNAs were serum starved in M199 containing 0.5% FBS overnight before being detached and plated on growth factor reduced Matrigel in the presence of 1 µM S1P or 200 µg/ml HDL3. EC rearrangement was visualised after 4 to 6 h using an Axiovert200 microscope. (A) Representative images of branching points formation in HUVEC transfected with the indicated siRNAs (Sequences 1) in the absence or presence of S1P or HDL3. (B,C) Results from quantitative analysis in HUVEC transfected with the indicated siRNAs (Sequences 2) in the absence or presence of S1P or HDL3. Data indicate the total number of branching points and are means ± SEM from 4 (B) and 3 (C) independent experiments. *p<0.01; **p<0.001.

Mentions: Since migration is a crucial process in EC shape change and morphogenic rearrangement on Matrigel we then analysed the effect of downregulation of the PI3K isoforms on this process. Images of unstimulated, S1P- and HDL3-stimulated HUVEC from one representative experiment are shown in Figure 4A. Downregulation of either PI3K-C2α or PI3K-C2β using two distinct siRNAs reduced HUVEC morphogenesis upon stimulation with S1P and HDL3 (Figure 4B, Figure S4A). Inhibition on the basal remodelling of the cells (induced by interaction with Matrigel and likely by factors released by the cells) was also consistently detected in cells upon downregulation of PI3K-C2α (Figure 4B, Figure S4A). Parallel experiments also indicated a reduction in the number of branching points in cells upon downregulation of p110γ using two distinct siRNAs following stimulation with either S1P or HDL3 but not under basal conditions (Figure 4C, Figure S4B,C). In addition, consistent with data obtained in migration experiments, treatment of HUVEC with 1 µM A66 significantly inhibited both the HDL3- and S1P-induced remodelling on Matrigel (Figure S4D,E).


Class II phosphoinositide 3-kinases contribute to endothelial cells morphogenesis.

Tibolla G, Piñeiro R, Chiozzotto D, Mavrommati I, Wheeler AP, Norata GD, Catapano AL, Maffucci T, Falasca M - PLoS ONE (2013)

Class II and class IB PI3Ks regulate remodelling of HUVEC.HUVEC transfected with the indicated siRNAs were serum starved in M199 containing 0.5% FBS overnight before being detached and plated on growth factor reduced Matrigel in the presence of 1 µM S1P or 200 µg/ml HDL3. EC rearrangement was visualised after 4 to 6 h using an Axiovert200 microscope. (A) Representative images of branching points formation in HUVEC transfected with the indicated siRNAs (Sequences 1) in the absence or presence of S1P or HDL3. (B,C) Results from quantitative analysis in HUVEC transfected with the indicated siRNAs (Sequences 2) in the absence or presence of S1P or HDL3. Data indicate the total number of branching points and are means ± SEM from 4 (B) and 3 (C) independent experiments. *p<0.01; **p<0.001.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3539993&req=5

pone-0053808-g004: Class II and class IB PI3Ks regulate remodelling of HUVEC.HUVEC transfected with the indicated siRNAs were serum starved in M199 containing 0.5% FBS overnight before being detached and plated on growth factor reduced Matrigel in the presence of 1 µM S1P or 200 µg/ml HDL3. EC rearrangement was visualised after 4 to 6 h using an Axiovert200 microscope. (A) Representative images of branching points formation in HUVEC transfected with the indicated siRNAs (Sequences 1) in the absence or presence of S1P or HDL3. (B,C) Results from quantitative analysis in HUVEC transfected with the indicated siRNAs (Sequences 2) in the absence or presence of S1P or HDL3. Data indicate the total number of branching points and are means ± SEM from 4 (B) and 3 (C) independent experiments. *p<0.01; **p<0.001.
Mentions: Since migration is a crucial process in EC shape change and morphogenic rearrangement on Matrigel we then analysed the effect of downregulation of the PI3K isoforms on this process. Images of unstimulated, S1P- and HDL3-stimulated HUVEC from one representative experiment are shown in Figure 4A. Downregulation of either PI3K-C2α or PI3K-C2β using two distinct siRNAs reduced HUVEC morphogenesis upon stimulation with S1P and HDL3 (Figure 4B, Figure S4A). Inhibition on the basal remodelling of the cells (induced by interaction with Matrigel and likely by factors released by the cells) was also consistently detected in cells upon downregulation of PI3K-C2α (Figure 4B, Figure S4A). Parallel experiments also indicated a reduction in the number of branching points in cells upon downregulation of p110γ using two distinct siRNAs following stimulation with either S1P or HDL3 but not under basal conditions (Figure 4C, Figure S4B,C). In addition, consistent with data obtained in migration experiments, treatment of HUVEC with 1 µM A66 significantly inhibited both the HDL3- and S1P-induced remodelling on Matrigel (Figure S4D,E).

Bottom Line: In this study we investigated the relative contribution of distinct PI3K isoforms to endothelial cells (EC) functions specifically regulated by the sphingolipid sphingosine-1-phosphate (S1P) and by high density lipoproteins (HDL), the major carrier of S1P in human plasma.Here we show that a co-ordinated action of different PI3Ks is required to tightly regulate remodelling of EC on Matrigel, a process dependent on cell proliferation, apoptosis and migration.The contribution of each isoform to this process appears to be distinct, with the class II enzyme PI3K-C2β and the class IB isoform p110γ mainly regulating the S1P- and HDL-dependent EC migration and PI3K-C2α primarily controlling EC survival.

View Article: PubMed Central - PubMed

Affiliation: Queen Mary University of London, Barts and The London School of Medicine and Dentistry, Blizard Institute, Centre for Diabetes, Inositide Signalling Group, London, United Kingdom.

ABSTRACT
The question of whether the distinct isoforms of the family of enzymes phosphoinositide 3-kinases (PI3Ks) play redundant roles within a cell or whether they control distinct cellular processes or distinct steps within the same cellular process has gained considerable importance in the recent years due to the development of inhibitors able to selectively target individual isoforms. It is important to understand whether inhibition of one PI3K can result in compensatory effect from other isoform(s) and therefore whether strategies aimed at simultaneously blocking more than one PI3K may be needed. In this study we investigated the relative contribution of distinct PI3K isoforms to endothelial cells (EC) functions specifically regulated by the sphingolipid sphingosine-1-phosphate (S1P) and by high density lipoproteins (HDL), the major carrier of S1P in human plasma. Here we show that a co-ordinated action of different PI3Ks is required to tightly regulate remodelling of EC on Matrigel, a process dependent on cell proliferation, apoptosis and migration. The contribution of each isoform to this process appears to be distinct, with the class II enzyme PI3K-C2β and the class IB isoform p110γ mainly regulating the S1P- and HDL-dependent EC migration and PI3K-C2α primarily controlling EC survival. Data further indicate that PI3K-C2β and p110γ control distinct steps involved in cell migration supporting the hypothesis that different PI3Ks regulate distinct cellular processes.

Show MeSH
Related in: MedlinePlus