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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

OEC characterization by flow cytometry.A) Stained cells are shown as blue histograms, while unstained controls are shown in black. The percentage of positive cells is shown in the top right of each graph. The OECs are CD34/31/54/VEGFR2 positive and CD45/14 negative. B) Representative images of the binding of isothiocyanate-Ulex europaeus agglutinin I lectin binding (ULEX), uptake of acetylated low density lipoprotein (AcLDL) and staining for CD31 by EPCs (bottom row of panel) but not by fibroblasts (top row of panel). Together, these results are indicative of a positive endothelial cell phenotype.
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pone.0131101.g002: OEC characterization by flow cytometry.A) Stained cells are shown as blue histograms, while unstained controls are shown in black. The percentage of positive cells is shown in the top right of each graph. The OECs are CD34/31/54/VEGFR2 positive and CD45/14 negative. B) Representative images of the binding of isothiocyanate-Ulex europaeus agglutinin I lectin binding (ULEX), uptake of acetylated low density lipoprotein (AcLDL) and staining for CD31 by EPCs (bottom row of panel) but not by fibroblasts (top row of panel). Together, these results are indicative of a positive endothelial cell phenotype.

Mentions: Consistent with previous studies [31], colonies of EPCs with a cobblestone morphology emerged from peripheral MNCs between days 14–21. Characterization by flow cytometry suggests that the OECs used in these experiments are of endothelial lineage (CD31 99.7±0.1%; CD54 95.4±0.1%; VEGFR2 96.3±0.1% positive) with no hematopoietic cells present (CD45 0.43±0.03%; CD14 0.20±0.03% negative). A subset of cells also still express CD34 (25.2±0.5%), a progenitor cell marker. In contrast to human dermal fibroblasts, EPCs displayed characteristic Ulex binding and incorporation of acLDL, and stained positively for CD31 (Fig 2).


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)

OEC characterization by flow cytometry.A) Stained cells are shown as blue histograms, while unstained controls are shown in black. The percentage of positive cells is shown in the top right of each graph. The OECs are CD34/31/54/VEGFR2 positive and CD45/14 negative. B) Representative images of the binding of isothiocyanate-Ulex europaeus agglutinin I lectin binding (ULEX), uptake of acetylated low density lipoprotein (AcLDL) and staining for CD31 by EPCs (bottom row of panel) but not by fibroblasts (top row of panel). Together, these results are indicative of a positive endothelial cell phenotype.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131101.g002: OEC characterization by flow cytometry.A) Stained cells are shown as blue histograms, while unstained controls are shown in black. The percentage of positive cells is shown in the top right of each graph. The OECs are CD34/31/54/VEGFR2 positive and CD45/14 negative. B) Representative images of the binding of isothiocyanate-Ulex europaeus agglutinin I lectin binding (ULEX), uptake of acetylated low density lipoprotein (AcLDL) and staining for CD31 by EPCs (bottom row of panel) but not by fibroblasts (top row of panel). Together, these results are indicative of a positive endothelial cell phenotype.
Mentions: Consistent with previous studies [31], colonies of EPCs with a cobblestone morphology emerged from peripheral MNCs between days 14–21. Characterization by flow cytometry suggests that the OECs used in these experiments are of endothelial lineage (CD31 99.7±0.1%; CD54 95.4±0.1%; VEGFR2 96.3±0.1% positive) with no hematopoietic cells present (CD45 0.43±0.03%; CD14 0.20±0.03% negative). A subset of cells also still express CD34 (25.2±0.5%), a progenitor cell marker. In contrast to human dermal fibroblasts, EPCs displayed characteristic Ulex binding and incorporation of acLDL, and stained positively for CD31 (Fig 2).

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