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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

Cell binding to recombinant human tropoelastin.(A) Relative attachment of human dermal fibrolasts, EPCs and human coronary artery smooth muscle cells (SMC) to increasing concentrations of tropoelastin. (B) The percentage of spread EPCs on increasing concentrations of tropoelastin. (C) Phase contrast microscopy of spreading EPCs on BSA-blocked wells, tropoelastin (rhTE) collagen (CN) fibronectin (FN). Images were taken at 10x magnification. (D) EPC proliferation on days 3 and 5, respectively. Error bars represent S.E.M. of triplicate measurements.
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pone.0131101.g003: Cell binding to recombinant human tropoelastin.(A) Relative attachment of human dermal fibrolasts, EPCs and human coronary artery smooth muscle cells (SMC) to increasing concentrations of tropoelastin. (B) The percentage of spread EPCs on increasing concentrations of tropoelastin. (C) Phase contrast microscopy of spreading EPCs on BSA-blocked wells, tropoelastin (rhTE) collagen (CN) fibronectin (FN). Images were taken at 10x magnification. (D) EPC proliferation on days 3 and 5, respectively. Error bars represent S.E.M. of triplicate measurements.

Mentions: EPC attachment to rhTE was concentration dependent, reaching 57±1% at 10 μg/ml, but a maximum of 67±1% at 40 μg/ml, the highest concentration tested (Fig 3A). For context, attachment of human dermal fibroblasts and human coronary artery smooth muscle cells (SMC) to rhTE was also examined in the same assay. Fibroblast attachment was greatest, reaching 83±2% at the highest tropoelastin concentration, in contrast to SMCs which only had 39±1% of cells attached. rhTE also enhanced EPC spreading in a concentration-dependent manner reaching 54±3% at 25 μg/ml and 58±2% at 40 μg/ml. Spreading was not significantly different between 25 and 40 μg/ml coating concentrations (p = 0.92, Fig 3B).


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)

Cell binding to recombinant human tropoelastin.(A) Relative attachment of human dermal fibrolasts, EPCs and human coronary artery smooth muscle cells (SMC) to increasing concentrations of tropoelastin. (B) The percentage of spread EPCs on increasing concentrations of tropoelastin. (C) Phase contrast microscopy of spreading EPCs on BSA-blocked wells, tropoelastin (rhTE) collagen (CN) fibronectin (FN). Images were taken at 10x magnification. (D) EPC proliferation on days 3 and 5, respectively. 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.g003: Cell binding to recombinant human tropoelastin.(A) Relative attachment of human dermal fibrolasts, EPCs and human coronary artery smooth muscle cells (SMC) to increasing concentrations of tropoelastin. (B) The percentage of spread EPCs on increasing concentrations of tropoelastin. (C) Phase contrast microscopy of spreading EPCs on BSA-blocked wells, tropoelastin (rhTE) collagen (CN) fibronectin (FN). Images were taken at 10x magnification. (D) EPC proliferation on days 3 and 5, respectively. Error bars represent S.E.M. of triplicate measurements.
Mentions: EPC attachment to rhTE was concentration dependent, reaching 57±1% at 10 μg/ml, but a maximum of 67±1% at 40 μg/ml, the highest concentration tested (Fig 3A). For context, attachment of human dermal fibroblasts and human coronary artery smooth muscle cells (SMC) to rhTE was also examined in the same assay. Fibroblast attachment was greatest, reaching 83±2% at the highest tropoelastin concentration, in contrast to SMCs which only had 39±1% of cells attached. rhTE also enhanced EPC spreading in a concentration-dependent manner reaching 54±3% at 25 μg/ml and 58±2% at 40 μg/ml. Spreading was not significantly different between 25 and 40 μg/ml coating concentrations (p = 0.92, Fig 3B).

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