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EC4, a truncation of soluble N-cadherin, reduces vascular smooth muscle cell apoptosis and markers of atherosclerotic plaque instability.

Lyon CA, Johnson JL, White S, Sala-Newby GB, George SJ - Mol Ther Methods Clin Dev (2014)

Bottom Line: Interaction with FGFR in the extracellular (EC) 4 domain of SNC was essential for the antiapoptotic effect.EC4 significantly reduced smooth muscle cell, macrophage, and endothelial cell apoptosis in vitro by ~70%, similar to SNC.Elevation of plasma levels of EC4 in male apolipoprotein E-deficient mice with existing atherosclerosis significantly reduced apoptosis in brachiocephalic artery plaques by ~50%.

View Article: PubMed Central - PubMed

Affiliation: Bristol Heart Institute, Bristol Royal Infirmary , Bristol, UK.

ABSTRACT
Atherosclerotic plaque instability is precipitated by vascular smooth muscle cell apoptosis in the fibrous cap, weakening it and leading to plaque rupture. We previously showed that reducing smooth muscle cell apoptosis with soluble N-cadherin (SNC) increased features of plaque stability. We have now identified the active site of SNC and examined whether a truncated form containing this site retains the antiapoptotic effect. SNC was mutated to prevent interaction with N-cadherin or fibroblast growth factor receptor (FGFR). Interaction with FGFR in the extracellular (EC) 4 domain of SNC was essential for the antiapoptotic effect. Therefore, we made a truncated form consisting of the EC4 domain. EC4 significantly reduced smooth muscle cell, macrophage, and endothelial cell apoptosis in vitro by ~70%, similar to SNC. Elevation of plasma levels of EC4 in male apolipoprotein E-deficient mice with existing atherosclerosis significantly reduced apoptosis in brachiocephalic artery plaques by ~50%. EC4 reduced plaque size and the incidence of buried fibrous layers and the macrophage:smooth muscle cell ratio (surrogate markers of plaque instability). Interaction of EC4 with FGFR induced potent antiapoptotic signaling in vitro and in vivo. EC4 modulates atherosclerosis in mice demonstrating its therapeutic potential for retarding plaque size and instability.

No MeSH data available.


Related in: MedlinePlus

The effect of SNC-Fc mutation and truncation on vascular smooth muscle cell (VSMC) apoptosis. (a) Diagrammatic representation of the SNC-Fc molecule to show the locations of the fibroblast growth factor receptor (FGFR) and N-cadherin binding sites, the mutations to prevent binding, and truncation of SNC-Fc to EC4-Fc. (b,c) The percentage of apoptotic VSMCs (assessed by cleaved caspase-3 immunocytochemistry) 24 hours after induction of apoptosis with Fas-L and treatment with the purified proteins (mean ± SEM, n = 3, *P < 0.05 versus Fc, #P < 0.05 versus FGFR mutated, $P < 0.05 versus EC4 mutant). (d) EC4 activated Akt signaling to the same extent as SNC-Fc. Analysis of phospho-Akt immunocytochemistry following 24-hour treatment with the purified proteins (mean ± SEM, n = 4, *P < 0.05 versus Fc, FGFR mutated and EC4 mutant). EC, extracellular; SNC, soluble N-cadherin.
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fig1: The effect of SNC-Fc mutation and truncation on vascular smooth muscle cell (VSMC) apoptosis. (a) Diagrammatic representation of the SNC-Fc molecule to show the locations of the fibroblast growth factor receptor (FGFR) and N-cadherin binding sites, the mutations to prevent binding, and truncation of SNC-Fc to EC4-Fc. (b,c) The percentage of apoptotic VSMCs (assessed by cleaved caspase-3 immunocytochemistry) 24 hours after induction of apoptosis with Fas-L and treatment with the purified proteins (mean ± SEM, n = 3, *P < 0.05 versus Fc, #P < 0.05 versus FGFR mutated, $P < 0.05 versus EC4 mutant). (d) EC4 activated Akt signaling to the same extent as SNC-Fc. Analysis of phospho-Akt immunocytochemistry following 24-hour treatment with the purified proteins (mean ± SEM, n = 4, *P < 0.05 versus Fc, FGFR mutated and EC4 mutant). EC, extracellular; SNC, soluble N-cadherin.

Mentions: To determine which part of the SNC molecule was responsible for its antiapoptotic effect, we mutated the two active sites present in the molecule (Figure 1a). SNC can bind to N-cadherin via an interaction between an HAV binding motif in the first extracellular domain (EC1) and with INPISGQ in the EC1 domain of an adjacent molecule. To prevent this interaction, both of these motifs were mutated in SNC. SNC can also interact with the FGFR through INPDVGQ in the EC4 domain; therefore, this motif was mutated to prevent this interaction.


EC4, a truncation of soluble N-cadherin, reduces vascular smooth muscle cell apoptosis and markers of atherosclerotic plaque instability.

Lyon CA, Johnson JL, White S, Sala-Newby GB, George SJ - Mol Ther Methods Clin Dev (2014)

The effect of SNC-Fc mutation and truncation on vascular smooth muscle cell (VSMC) apoptosis. (a) Diagrammatic representation of the SNC-Fc molecule to show the locations of the fibroblast growth factor receptor (FGFR) and N-cadherin binding sites, the mutations to prevent binding, and truncation of SNC-Fc to EC4-Fc. (b,c) The percentage of apoptotic VSMCs (assessed by cleaved caspase-3 immunocytochemistry) 24 hours after induction of apoptosis with Fas-L and treatment with the purified proteins (mean ± SEM, n = 3, *P < 0.05 versus Fc, #P < 0.05 versus FGFR mutated, $P < 0.05 versus EC4 mutant). (d) EC4 activated Akt signaling to the same extent as SNC-Fc. Analysis of phospho-Akt immunocytochemistry following 24-hour treatment with the purified proteins (mean ± SEM, n = 4, *P < 0.05 versus Fc, FGFR mutated and EC4 mutant). EC, extracellular; SNC, soluble N-cadherin.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4362368&req=5

fig1: The effect of SNC-Fc mutation and truncation on vascular smooth muscle cell (VSMC) apoptosis. (a) Diagrammatic representation of the SNC-Fc molecule to show the locations of the fibroblast growth factor receptor (FGFR) and N-cadherin binding sites, the mutations to prevent binding, and truncation of SNC-Fc to EC4-Fc. (b,c) The percentage of apoptotic VSMCs (assessed by cleaved caspase-3 immunocytochemistry) 24 hours after induction of apoptosis with Fas-L and treatment with the purified proteins (mean ± SEM, n = 3, *P < 0.05 versus Fc, #P < 0.05 versus FGFR mutated, $P < 0.05 versus EC4 mutant). (d) EC4 activated Akt signaling to the same extent as SNC-Fc. Analysis of phospho-Akt immunocytochemistry following 24-hour treatment with the purified proteins (mean ± SEM, n = 4, *P < 0.05 versus Fc, FGFR mutated and EC4 mutant). EC, extracellular; SNC, soluble N-cadherin.
Mentions: To determine which part of the SNC molecule was responsible for its antiapoptotic effect, we mutated the two active sites present in the molecule (Figure 1a). SNC can bind to N-cadherin via an interaction between an HAV binding motif in the first extracellular domain (EC1) and with INPISGQ in the EC1 domain of an adjacent molecule. To prevent this interaction, both of these motifs were mutated in SNC. SNC can also interact with the FGFR through INPDVGQ in the EC4 domain; therefore, this motif was mutated to prevent this interaction.

Bottom Line: Interaction with FGFR in the extracellular (EC) 4 domain of SNC was essential for the antiapoptotic effect.EC4 significantly reduced smooth muscle cell, macrophage, and endothelial cell apoptosis in vitro by ~70%, similar to SNC.Elevation of plasma levels of EC4 in male apolipoprotein E-deficient mice with existing atherosclerosis significantly reduced apoptosis in brachiocephalic artery plaques by ~50%.

View Article: PubMed Central - PubMed

Affiliation: Bristol Heart Institute, Bristol Royal Infirmary , Bristol, UK.

ABSTRACT
Atherosclerotic plaque instability is precipitated by vascular smooth muscle cell apoptosis in the fibrous cap, weakening it and leading to plaque rupture. We previously showed that reducing smooth muscle cell apoptosis with soluble N-cadherin (SNC) increased features of plaque stability. We have now identified the active site of SNC and examined whether a truncated form containing this site retains the antiapoptotic effect. SNC was mutated to prevent interaction with N-cadherin or fibroblast growth factor receptor (FGFR). Interaction with FGFR in the extracellular (EC) 4 domain of SNC was essential for the antiapoptotic effect. Therefore, we made a truncated form consisting of the EC4 domain. EC4 significantly reduced smooth muscle cell, macrophage, and endothelial cell apoptosis in vitro by ~70%, similar to SNC. Elevation of plasma levels of EC4 in male apolipoprotein E-deficient mice with existing atherosclerosis significantly reduced apoptosis in brachiocephalic artery plaques by ~50%. EC4 reduced plaque size and the incidence of buried fibrous layers and the macrophage:smooth muscle cell ratio (surrogate markers of plaque instability). Interaction of EC4 with FGFR induced potent antiapoptotic signaling in vitro and in vivo. EC4 modulates atherosclerosis in mice demonstrating its therapeutic potential for retarding plaque size and instability.

No MeSH data available.


Related in: MedlinePlus