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Characterization of Endothelial Progenitor Cell Interactions with Human Tropoelastin.

Yu Y, Wise SG, Michael PL, Bax DV, Yuen GS, Hiob MA, Yeo GC, Filipe EC, Dunn LL, Chan KH, Hajian H, Celermajer DS, Weiss AS, Ng MK - PLoS ONE (2015)

Bottom Line: The rapid restoration of a functional endothelium is known to reduce these complications.Integrin αVβ3 blocking antibodies decreased EPC adhesion and spreading on rhTE by 39±3% and 56±10% respectively, demonstrating a large contribution from this specific integrin.In conclusion, rhTE supports EPC binding via an integrin mechanism involving αVβ3.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiology, Royal Prince Alfred Hospital, Sydney, NSW, 2050, Australia; The Heart Research Institute, Sydney, NSW, 2042, Australia; Sydney Medical School, University of Sydney, Sydney, NSW, 2006, Australia.

ABSTRACT
The deployment of endovascular implants such as stents in the treatment of cardiovascular disease damages the vascular endothelium, increasing the risk of thrombosis and promoting neointimal hyperplasia. The rapid restoration of a functional endothelium is known to reduce these complications. Circulating endothelial progenitor cells (EPCs) are increasingly recognized as important contributors to device re-endothelialization. Extracellular matrix proteins prominent in the vessel wall may enhance EPC-directed re-endothelialization. We examined attachment, spreading and proliferation on recombinant human tropoelastin (rhTE) and investigated the mechanism and site of interaction. EPCs attached and spread on rhTE in a dose dependent manner, reaching a maximal level of 56±3% and 54±3%, respectively. EPC proliferation on rhTE was comparable to vitronectin, fibronectin and collagen. EDTA, but not heparan sulfate or lactose, reduced EPC attachment by 81±3%, while full attachment was recovered after add-back of manganese, inferring a classical integrin-mediated interaction. Integrin αVβ3 blocking antibodies decreased EPC adhesion and spreading on rhTE by 39±3% and 56±10% respectively, demonstrating a large contribution from this specific integrin. Attachment of EPCs on N-terminal rhTE constructs N25 and N18 accounted for most of this interaction, accompanied by comparable spreading. In contrast, attachment and spreading on N10 was negligible. αVβ3 blocking antibodies reduced EPC spreading on both N25 and N18 by 45±4% and 42±14%, respectively. In conclusion, rhTE supports EPC binding via an integrin mechanism involving αVβ3. N25 and N18, but not N10 constructs of rhTE contribute to EPC binding. The regulation of EPC activity by rhTE may have implications for modulation of the vascular biocompatibility of endovascular implants.

No MeSH data available.


Related in: MedlinePlus

Mechanism of EPC attachment to truncated tropoelastin constructs.(A) and (B) EPCs attached to 40 μg/ml N25 and N18 respectively, in the presence of α-lactose, β-lactose, heparan sulfate or EDTA. (C) and (D) Attachment of EPCs to 40 μg/ml N25 and N18, respectively, in the presence of Ca2+, Mg2+, or Mn2+. (E) and (F), Inhibition of EPC spreading on 40 μg/ml N25 and N18 respectively, using antibody that inhibits binding to integrin αvβ3. Error bars represent S.E.M. of triplicate measurements.
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pone.0131101.g006: Mechanism of EPC attachment to truncated tropoelastin constructs.(A) and (B) EPCs attached to 40 μg/ml N25 and N18 respectively, in the presence of α-lactose, β-lactose, heparan sulfate or EDTA. (C) and (D) Attachment of EPCs to 40 μg/ml N25 and N18, respectively, in the presence of Ca2+, Mg2+, or Mn2+. (E) and (F), Inhibition of EPC spreading on 40 μg/ml N25 and N18 respectively, using antibody that inhibits binding to integrin αvβ3. Error bars represent S.E.M. of triplicate measurements.

Mentions: As for rhTE, we investigated the mechanism of interaction between N25 and N18 constructs with EPCs. No appreciable effect on cell attachment was seen in the presence of α-lactose, β-lactose, and heparan sulfate. However, the addition of EDTA led to a 65±2% (p<0.0001), and 76±5% (p<0.001) reduction in attachment to N25 and N18, respectively (Fig 6A and 6B). When divalent cations were re-introduced to cation-free buffer, a dose-dependent full recovery of EPC attachment to both N25 and N18 was seen with manganese (Fig 6C and 6D), but not for magnesium or calcium, as expected for an integrin interaction. Anti-αVβ3 antibody led to a 45±4% (p<0.001) and 42±14% (p<0.05) decrease in EPC spreading on N25 and N18, respectively (Fig 6E and 6F), while anti-α5β1 had no effect. These results collectively establish an integrin-based mode of EPC interaction with N25 and N18 that involves αVβ3.


Characterization of Endothelial Progenitor Cell Interactions with Human Tropoelastin.

Yu Y, Wise SG, Michael PL, Bax DV, Yuen GS, Hiob MA, Yeo GC, Filipe EC, Dunn LL, Chan KH, Hajian H, Celermajer DS, Weiss AS, Ng MK - PLoS ONE (2015)

Mechanism of EPC attachment to truncated tropoelastin constructs.(A) and (B) EPCs attached to 40 μg/ml N25 and N18 respectively, in the presence of α-lactose, β-lactose, heparan sulfate or EDTA. (C) and (D) Attachment of EPCs to 40 μg/ml N25 and N18, respectively, in the presence of Ca2+, Mg2+, or Mn2+. (E) and (F), Inhibition of EPC spreading on 40 μg/ml N25 and N18 respectively, using antibody that inhibits binding to integrin αvβ3. Error bars represent S.E.M. of triplicate measurements.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4482626&req=5

pone.0131101.g006: Mechanism of EPC attachment to truncated tropoelastin constructs.(A) and (B) EPCs attached to 40 μg/ml N25 and N18 respectively, in the presence of α-lactose, β-lactose, heparan sulfate or EDTA. (C) and (D) Attachment of EPCs to 40 μg/ml N25 and N18, respectively, in the presence of Ca2+, Mg2+, or Mn2+. (E) and (F), Inhibition of EPC spreading on 40 μg/ml N25 and N18 respectively, using antibody that inhibits binding to integrin αvβ3. Error bars represent S.E.M. of triplicate measurements.
Mentions: As for rhTE, we investigated the mechanism of interaction between N25 and N18 constructs with EPCs. No appreciable effect on cell attachment was seen in the presence of α-lactose, β-lactose, and heparan sulfate. However, the addition of EDTA led to a 65±2% (p<0.0001), and 76±5% (p<0.001) reduction in attachment to N25 and N18, respectively (Fig 6A and 6B). When divalent cations were re-introduced to cation-free buffer, a dose-dependent full recovery of EPC attachment to both N25 and N18 was seen with manganese (Fig 6C and 6D), but not for magnesium or calcium, as expected for an integrin interaction. Anti-αVβ3 antibody led to a 45±4% (p<0.001) and 42±14% (p<0.05) decrease in EPC spreading on N25 and N18, respectively (Fig 6E and 6F), while anti-α5β1 had no effect. These results collectively establish an integrin-based mode of EPC interaction with N25 and N18 that involves αVβ3.

Bottom Line: The rapid restoration of a functional endothelium is known to reduce these complications.Integrin αVβ3 blocking antibodies decreased EPC adhesion and spreading on rhTE by 39±3% and 56±10% respectively, demonstrating a large contribution from this specific integrin.In conclusion, rhTE supports EPC binding via an integrin mechanism involving αVβ3.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiology, Royal Prince Alfred Hospital, Sydney, NSW, 2050, Australia; The Heart Research Institute, Sydney, NSW, 2042, Australia; Sydney Medical School, University of Sydney, Sydney, NSW, 2006, Australia.

ABSTRACT
The deployment of endovascular implants such as stents in the treatment of cardiovascular disease damages the vascular endothelium, increasing the risk of thrombosis and promoting neointimal hyperplasia. The rapid restoration of a functional endothelium is known to reduce these complications. Circulating endothelial progenitor cells (EPCs) are increasingly recognized as important contributors to device re-endothelialization. Extracellular matrix proteins prominent in the vessel wall may enhance EPC-directed re-endothelialization. We examined attachment, spreading and proliferation on recombinant human tropoelastin (rhTE) and investigated the mechanism and site of interaction. EPCs attached and spread on rhTE in a dose dependent manner, reaching a maximal level of 56±3% and 54±3%, respectively. EPC proliferation on rhTE was comparable to vitronectin, fibronectin and collagen. EDTA, but not heparan sulfate or lactose, reduced EPC attachment by 81±3%, while full attachment was recovered after add-back of manganese, inferring a classical integrin-mediated interaction. Integrin αVβ3 blocking antibodies decreased EPC adhesion and spreading on rhTE by 39±3% and 56±10% respectively, demonstrating a large contribution from this specific integrin. Attachment of EPCs on N-terminal rhTE constructs N25 and N18 accounted for most of this interaction, accompanied by comparable spreading. In contrast, attachment and spreading on N10 was negligible. αVβ3 blocking antibodies reduced EPC spreading on both N25 and N18 by 45±4% and 42±14%, respectively. In conclusion, rhTE supports EPC binding via an integrin mechanism involving αVβ3. N25 and N18, but not N10 constructs of rhTE contribute to EPC binding. The regulation of EPC activity by rhTE may have implications for modulation of the vascular biocompatibility of endovascular implants.

No MeSH data available.


Related in: MedlinePlus