Expression of exogenous tissue factor pathway inhibitor in vivo suppresses thrombus formation in injured rabbit carotid arteries
Paolo Golino, MD, PhD*,
Plinio Cirillo, MD*,
Paolo Calabro’, MD*,
Massimo Ragni, MD*,
Davide D’Andrea, MD,
Enrico V. Avvedimento, MD ,
Francesco Vigorito, MD*,
Nicola Corcione, MD*,
Francesco Loffredo, BS* and
Massimo Chiariello, MD*
* Department of Internal Medicine, Division of Cardiology, University of Naples "Federico II," Naples, Italy
Department of Experimental Medicine, School of Medicine at Catanzaro, University of Reggio Calabria, Italy
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Figure 1 Time course of recombinant tissue factor pathway inhibitor (TFPI) expression in rabbit smooth muscle cells (SMCs) transfected in vitro with a recombinant retrovirus containing the gene of rabbit TFPI. The SMCs were probed with a monoclonal antibody directed against a 6-histidine tag present only on the recombinant protein, thus allowing differentiation of rTFPI from the wild-type protein. After induction of rTFPI with the addition of doxycycline in the culture medium, a progressive increase in rTFPI expression was observed, peaking at 12 to 24 h, although no significant expression was observed at baseline (i.e., uninduced conditions) or in SMCs transfected with a retrovirus containing the gene of GFP or a control retrovirus (pRETRO). See text for details.
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Figure 2 Time course of green fluorescent protein (GFP) expression in rabbit smooth muscle cells (SMCs) transfected in vitro with a recombinant retrovirus containing the gene of GFP. Because GFP is spontaneously fluorescent when observed under ultraviolet light, the SMCs were directly observed under an ultraviolet microscope. After induction of GFP with the addition of doxycycline in the culture medium, a progressive increase in GFP expression was observed, peaking at 12 to 24 h, although no significant expression was observed at baseline (i.e., uninduced conditions). See text for details.
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Figure 3 Ex vivo secretion of recombinant tissue factor pathway inhibitor (TFPI) by rabbit smooth muscle cells (SMCs). The rTFPI levels were measured by a two-antibody sandwich assay, using a monoclonal antibody against a 6-histidine tag present only in the recombinant protein, thus excluding the contribution of the wild-type TFPI. A progressive increase in rTFPI levels was observed, peaking at 12 to 24 h after doxycycline induction.
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Figure 4 Representative carotid Doppler flow recordings obtained from a rabbit after local treatment of one carotid artery with retro-tissue factor pathway inhibitor (TFPI) and the other one with retro-green fluorescent protein (GFP). At baseline (i.e., before induction of the recombinant proteins), cyclic flow variations (CFVs) developed similarly in both carotid arteries. In contrast, 2 h after induction with doxycycline (3 mg/kg intravenous bolus plus 300 µg/kg per min), CFVs were completely inhibited only in the carotid artery previously transfected with retro-TFPI, although they remained unchanged in the vessel previously transfected with retro-GFP.
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Figure 5 Frequency of cyclic flow variations (CFVs) (cycles/hour) in carotid arteries transfected with retro-tissue factor pathway inhibitor (TFPI) (A) and carotid arteries transfected with retro-green fluorescent protein (GFP) (B). After induction of the recombinant proteins with doxycycline, CFVs were completely inhibited only in retro-TFPI transfected vessels, although they remained unchanged in the contralateral arteries transfected with retro-GFP. Each dot represents one animal.
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Figure 6 The prothrombin times (PTs) and partial thromboplastin times (PTTs) measured at baseline (i.e., uninduced conditions) and at the time of inhibition of cyclic flow variations (CFVs) after administration of doxycycline. No significant changes in PTs and PTTs were observed when induction of recombinant human tissue factor pathway inhibitor (rTFPI) exerted its antithrombotic effects.
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Figure 7 Immunohistochemical staining for recombinant human tissue factor pathway inhibitor (rTFPI) in arterial sections obtained from carotid arteries transfected with retro-TFPI under uninduced conditions (i.e., no infusion of doxycycline) (A) and after administration of doxycycline to an animal subjected to a previous injury (B) or after administration of doxycycline to an animal not subjected to a previous arterial injury (C). After doxycycline infusion, a diffuse and intense reactivity was present across the arterial wall, although no staining was present under uninduced conditions. No significant differences in rTFPI expression were observed in the arterial wall between the two groups of animals. Recombinant TFPI was probed with a monoclonal antibody against a 6-histidine tag present only in the recombinant protein, thus allowing detection of rTFPI from the wild-type protein. (D) Histologic section of an artery obtained during doxycycline infusion and probed with an unrelated mouse monoclonal antibody (Anti-Xpress, Invitrogen). No cross reactivity is present in this section (see Methods for further details).
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Figure 8 Detection of green fluorescent protein (GFP) in the arterial wall after transfection with retro-GFP and induction with doxycycline. (A) Section stained with hematoxylin-eosin. (B) Adjacent section not stained, observed under the ultraviolet microscope equipped with a filter specific for GFP fluorescence.
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