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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

EPC attachment (A) and spreading (B) on tropoelastin constructs N25 and N18.C) and (D) Proliferation of EPCs on N25 and N18 constructs, on days 3 and 5, respectively. Values were normalized to proliferation on collagen. Error bars represent S.E.M. of triplicate measurements. EPC attachment (E) and spreading (F) to tropoelastin construct N10. Attachment and spreading assays on N10 constructs was done on the same cell plates as for N25 and N18 constructs. Results are expressed separately to highlight higher maximal concentrations of N10 used. Error bars represent S.E.M. of triplicate measurements.
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pone.0131101.g005: EPC attachment (A) and spreading (B) on tropoelastin constructs N25 and N18.C) and (D) Proliferation of EPCs on N25 and N18 constructs, on days 3 and 5, respectively. Values were normalized to proliferation on collagen. Error bars represent S.E.M. of triplicate measurements. EPC attachment (E) and spreading (F) to tropoelastin construct N10. Attachment and spreading assays on N10 constructs was done on the same cell plates as for N25 and N18 constructs. Results are expressed separately to highlight higher maximal concentrations of N10 used. Error bars represent S.E.M. of triplicate measurements.

Mentions: The C-terminus of rhTE is known to be important for adhesion of fibroblasts [26,36]. An additional contribution from the mid-domain regions 17/18 has only been recently described [27]. In contrast, endothelial cell binding to rhTE can be supported by the first 10 N-terminal domains [28]. In this study we sought to determine a preferred binding region for EPCs to rhTE. We expressed and purified truncation constructs of rhTE, spanning regions from the N-terminus to domains 25 (N25), 18 (N18), and 10 (N10). An ELISA specific for domain 6 of tropoelastin showed no significant differences in the relative amount of each present on tissue culture plastic (S2 Fig). N25 supported 38±2% attachment of EPCs at a concentration of 20 μg/ml and up to 43±3% at 40 μg/ml. A comparable level of 38±2% attachment was seen on N18 at 20 μg/ml, and up to 42±5% at 40 μg/ml (N25 vs. N18, p = 0.09) (Fig 5A). EPC spreading was comparable on N25 and N18 at 56±2% and 57±1%, respectively (N25 vs. N18, 40μg/ml, p = 0.92) (Fig 5B). When compared to full length rhTE EPC spreading on N25 and N18 constructs was similar (rhTE vs N25 and N18, P = 0.81 and P = 0.94, respectively).


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)

EPC attachment (A) and spreading (B) on tropoelastin constructs N25 and N18.C) and (D) Proliferation of EPCs on N25 and N18 constructs, on days 3 and 5, respectively. Values were normalized to proliferation on collagen. Error bars represent S.E.M. of triplicate measurements. EPC attachment (E) and spreading (F) to tropoelastin construct N10. Attachment and spreading assays on N10 constructs was done on the same cell plates as for N25 and N18 constructs. Results are expressed separately to highlight higher maximal concentrations of N10 used. 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.g005: EPC attachment (A) and spreading (B) on tropoelastin constructs N25 and N18.C) and (D) Proliferation of EPCs on N25 and N18 constructs, on days 3 and 5, respectively. Values were normalized to proliferation on collagen. Error bars represent S.E.M. of triplicate measurements. EPC attachment (E) and spreading (F) to tropoelastin construct N10. Attachment and spreading assays on N10 constructs was done on the same cell plates as for N25 and N18 constructs. Results are expressed separately to highlight higher maximal concentrations of N10 used. Error bars represent S.E.M. of triplicate measurements.
Mentions: The C-terminus of rhTE is known to be important for adhesion of fibroblasts [26,36]. An additional contribution from the mid-domain regions 17/18 has only been recently described [27]. In contrast, endothelial cell binding to rhTE can be supported by the first 10 N-terminal domains [28]. In this study we sought to determine a preferred binding region for EPCs to rhTE. We expressed and purified truncation constructs of rhTE, spanning regions from the N-terminus to domains 25 (N25), 18 (N18), and 10 (N10). An ELISA specific for domain 6 of tropoelastin showed no significant differences in the relative amount of each present on tissue culture plastic (S2 Fig). N25 supported 38±2% attachment of EPCs at a concentration of 20 μg/ml and up to 43±3% at 40 μg/ml. A comparable level of 38±2% attachment was seen on N18 at 20 μg/ml, and up to 42±5% at 40 μg/ml (N25 vs. N18, p = 0.09) (Fig 5A). EPC spreading was comparable on N25 and N18 at 56±2% and 57±1%, respectively (N25 vs. N18, 40μg/ml, p = 0.92) (Fig 5B). When compared to full length rhTE EPC spreading on N25 and N18 constructs was similar (rhTE vs N25 and N18, P = 0.81 and P = 0.94, respectively).

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