In vitro functional testing of endothelial progenitor cells that overexpress thrombomodulin

Abstract

This study investigated the augmentation of endothelial progenitor cell (EPC) thromboresistance by using gene therapy to overexpress thrombomodulin (TM), an endothelial cell membrane glycoprotein that has potent anti-coagulant properties. Late outgrowth EPCs were isolated from peripheral blood of patients with documented coronary artery disease and transfected with an adenoviral vector containing human TM. EPC transfection conditions for maximizing TM expression, transfection efficiency, and cell viability were employed. TM-overexpressing EPCs had a fivefold increase in the rate of activated protein C production over native EPCs and EPCs transfected with an adenoviral control vector expressing β-galactosidase (p<0.05). TM upregulation caused a significant threefold reduction in platelet adhesion compared to native EPCs, and a 12-fold reduction compared to collagen I-coated wells. Additionally, the clotting time of TM-transfected EPCs incubated with whole blood was significantly extended by 19% over native cells (p<0.05). These data indicate that TM-overexpression has the potential to improve the antithrombotic performance of patient-derived EPCs for endothelialization applications.

FIG. 1.

Optimization of adenoviral transfection of EPCs. EPCs were assessed for transfection efficiency after AdTM by flow cytometry for CD141-PE. Representative image of native EPCs (black line) and EPCs transfected 4 h with 100 MOI (bold line) (A). The percentage of EPC expressing CD141 higher than native EPCs increased with viral concentration (white bars: 4-h transfection time; gray bars: 24-h transfection time) (B). Four days after transfecting EPCs with AdTM, EPC numbers were decreased versus native EPCs (all cultures initially had 1.9×10⁵ cells at transfection) (C). EPC, endothelial progenitor cell; AdTM, adenoviral expressing human thrombomodulin; MOI, multiplicity of infection.

FIG. 2.

Long-term TM expression measured through TM mRNA (A) and TM surface (B) expression in native and AdTM-transfected EPCs (*p<0.05 vs. native EPCs).

FIG. 3.

Transfected EPCs maintain firm adhesion and reorient in the direction of flow. Before exposure to flow, AdTM EPCs had random orientation and stained for PECAM (green), F-actin (red), and cell nuclei (blue) (A). After 48 h of laminar flow, AdTM-transfected EPCs remained adherent and aligned in the direction of flow (white arrow) (B) (scale bar 100 μm). EPC angle of orientation pre- and postexposure to flow (*p<0.0001 vs. static condition) (C). AdCV, adenovirus control vector. Color images available online at www.liebertonline.com/tea

FIG. 4.

Activated protein C production rate of bare wells, EPCs, EPCs + AdCV, and EPCs + AdTM (*p<0.05 vs. bare well, ^#p<0.05 vs. native EPC and EPC + AdCV).

FIG. 5.

Platelet area coverage was determined after 30-min incubation with platelet-rich plasma (A). Representative phase-contrast and fluorescent images showing EPCs and CD41-labeled platelets (green). Collagen I-coated wells had highest coverage (A), followed by EPCs (B), EPCs + AdCV (C), and EPCs + AdTM (D). Percent platelet area coverage was quantified (E). (*p<0.0001 vs. collagen coated well, ^#p<0.05 vs. native EPC or EPC + AdCV). Color images available online at www.liebertonline.com/tea

FIG. 6.

The clotting time of whole blood was measured on tissue culture polystyrene, and confluent layers of EPCs, EPC + AdCV, and EPC + AdTM (*p<0.05 vs. bare well, ^#p<0.05 vs. native EPC or EPC + AdCV).