scholarly journals Characterization of recombinant plasminogen activator production by primate endothelial cells transduced with retroviral vectors

Blood ◽  
1994 ◽  
Vol 84 (2) ◽  
pp. 504-516 ◽  
Author(s):  
DA Dichek ◽  
SW Lee ◽  
NH Nguyen
Keyword(s):  
Endothelial Cells ◽  
Gene Transfer ◽  
Specific Effect ◽  
Retroviral Vectors ◽  
Retroviral Vector ◽  
Wild Type ◽  
Intravascular Thrombosis ◽  
Primate System ◽  
Pai 1

Abstract Retroviral vector-mediated expression of plasminogen activators (PAs) from endothelial cells (ECs) has been proposed as a potential therapeutic approach for intravascular thrombosis. To define the potential for gene transfer to increase fibrinolytic activity in a primate system, baboon ECs were transduced with retroviral vectors expressing wild-type and glycosylphosphatidylinositol-anchored urokinase, as well as wild-type and serpin-resistant tissue PA (t-PA). Expression of either t-PA or urokinase was increased by one log over baseline levels. There was no specific effect of either t-PA or urokinase overexpression on endogenous t-PA, urokinase, or PA inhibitor 1 (PAI-1) expression. Recombinant urokinase could be anchored to the cell surface at a level eight-fold above that of receptor-bound urokinase. The majority of secreted urokinase accumulated in conditioned medium as a free proenzyme, whereas both wild-type and serpin-resistant t-PA accumulated almost exclusively in complexes with PAI-1. In most but not all of the assays, the urokinase vectors conferred PA activity above that of the t-PA vectors. These data show that PA synthesis and activity are specifically increased subsequent to retroviral vector-mediated gene transfer in primate ECs. However, definition of an optimal PA vector will require in vivo experimentation.

scholarly journals Characterization of recombinant plasminogen activator production by primate endothelial cells transduced with retroviral vectors

Blood ◽  
1994 ◽  
Vol 84 (2) ◽  
pp. 504-516 ◽  
Author(s):  
DA Dichek ◽  
SW Lee ◽  
NH Nguyen
Keyword(s):  
Endothelial Cells ◽  
Gene Transfer ◽  
Specific Effect ◽  
Retroviral Vectors ◽  
Retroviral Vector ◽  
Wild Type ◽  
Intravascular Thrombosis ◽  
Primate System ◽  
Pai 1

Retroviral vector-mediated expression of plasminogen activators (PAs) from endothelial cells (ECs) has been proposed as a potential therapeutic approach for intravascular thrombosis. To define the potential for gene transfer to increase fibrinolytic activity in a primate system, baboon ECs were transduced with retroviral vectors expressing wild-type and glycosylphosphatidylinositol-anchored urokinase, as well as wild-type and serpin-resistant tissue PA (t-PA). Expression of either t-PA or urokinase was increased by one log over baseline levels. There was no specific effect of either t-PA or urokinase overexpression on endogenous t-PA, urokinase, or PA inhibitor 1 (PAI-1) expression. Recombinant urokinase could be anchored to the cell surface at a level eight-fold above that of receptor-bound urokinase. The majority of secreted urokinase accumulated in conditioned medium as a free proenzyme, whereas both wild-type and serpin-resistant t-PA accumulated almost exclusively in complexes with PAI-1. In most but not all of the assays, the urokinase vectors conferred PA activity above that of the t-PA vectors. These data show that PA synthesis and activity are specifically increased subsequent to retroviral vector-mediated gene transfer in primate ECs. However, definition of an optimal PA vector will require in vivo experimentation.

Synthetic Vascular Grafts Seeded with Genetically Modified Endothelium in the Dog: Evaluation of the Effect of Seeding Technique and Retroviral Vector on Cell Persistence in Vivo

1995 ◽  
Vol 4 (2) ◽  
pp. 219-235 ◽  
Author(s):  
Jill E. Sackman ◽  
Michael B. Freeman ◽  
Mark G. Petersen ◽  
Zuhair Allebban ◽  
Glenn P. Niemeyer ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Gene Transfer ◽  
Gene Sequence ◽  
Genetically Modified ◽  
Vascular Grafts ◽  
Retroviral Vectors ◽  
Retroviral Vector ◽  
Marker Genes ◽  
Growth Hormone Gene

Unique characteristics of endothelium make it an attractive target cell for gene transfer. Genetically modified endothelial cells (ECs) seeded on synthetic vascular grafts offer the potential to control neointimal hyperplasia, decrease graft thrombogenicity and improve small diameter graft patency. This study addresses the issue of synthetic vascular graft colonization with endothelial cells transduced with noninducible retroviral marker genes in the dog. Autologous endothelial cells were enzymatically harvested and transduced with either the bacterial NeoR gene or human growth hormone gene using retroviral vectors. All transduced cells were positive by polymerase chain reaction (PCR) amplification for the transduced gene sequence prior to graft seeding. Transduced ECs were seeded on Dacron grafts (n = 3) pre-clotted with autologous blood. These grafts exhibited complete endothelialization at times from 250 to 360 days. Recovered DNA, however, was negative for the transduced gene sequence when analyzed by PCR and Southern blotting. Expanded polytetrafluoroethylene (ePTFE) was evaluated (n = 8) using several different cell seeding protocols. Grafts were seeded at 3 densities (ranging from 6 × 103 to 1.5 × 105 cells/cm2) and 2 different adherence times. Seeding substrate was also evaluated. Grafts were either preclotted with whole blood or incubated with 20 or 120 μg/ml fibronectin for 60 min. Graft biopsies were evaluated from 2 to 52 wk. Limited endothelialization was present in 4 dogs as early as 2 wk, but never progressed to full luminal coverage. The remaining dogs failed to ever exhibit any luminal EC adherence. Two dogs with limited EC coverage had positive DNA by PCR for the NeoR gene sequence at 2 and 3 wk. In contrast to transduced EC's, nontransduced EC colonization of ePTFE was complete at 2 wk when seeded under conditions that transduced cells had failed to persist. Neither seeding density, adherence time, seeding substrate or retroviral vector used influenced the uniformly poor graft coverage seen with transduced cells. Results of this study indicate that despite successful gene transfer using 4 different retroviral vectors, transduced endothelial cells seeded under varying conditions appear altered in their ability to stably adhere and colonize synthetic vascular grafts in vivo.

scholarly journals Augmentation of antitumor immunity by tumor cells transduced with a retroviral vector carrying the interleukin-2 and interferon-gamma cDNAs

Blood ◽  
1994 ◽  
Vol 83 (5) ◽  
pp. 1289-1298 ◽  
Author(s):  
FM Rosenthal ◽  
K Cronin ◽  
R Bannerji ◽  
DW Golde ◽  
B Gansbacher
Keyword(s):  
Gene Transfer ◽  
Tumor Cells ◽  
Interferon Gamma ◽  
Antitumor Immunity ◽  
Interleukin 2 ◽  
Retroviral Vectors ◽  
Retroviral Vector ◽  
Ifn Gamma ◽  
Gm Csf

Abstract Therapeutic models using gene transfer into tumor cells have pursued three objectives: (1) to induce rejection of the tumor transduced with therapeutic genes, (2) to induce immune-mediated regression of metastatic disease, and (3) to induce long-lasting immunity to protect against challenge with tumor cells or clinical regrowth of micrometastatic disease. Because in vivo therapy for patients with cancer using gene transfer would, as a first step, attempt to eliminate the existing tumor, we have investigated whether antitumor immunity induced by tumor cells secreting a single cytokine could be increased by cotransfer of a second cytokine gene. To test this approach, CMS-5, a murine fibrosarcoma, was transduced with retroviral vectors carrying interleukin-2 (IL-2), interferon-gamma (IFN-gamma), or granulocyte- macrophage-colony-stimulating factor (GM-CSF) cDNA alone or IL-2 cDNA in combination with IFN-gamma or GM-CSF cDNA. Single cytokine-secreting clones were selected to match levels of cytokine production by double cytokine-secreting clones so that similar amounts of cytokine were secreted. IFN-gamma- and IL-2/IFN-gamma-secreting CMS-5 cells showed increased levels of major histocompatability complex class I expression compared with IL-2- and GM-CSF-secreting or parental CMS-5 cells, IL- 2/IFN-gamma-secreting CMS-5 cells were always rejected by syngeneic mice, whereas the same number of CMS-5 cells secreting only one of these cytokines or mixtures of single cytokine-secreting CMS-5 cells were not rejected. In vivo depletion of CD4+, CD8+, or natural-killer effector cell subpopulations showed that CD8+ cytotoxic T cells were primarily responsible for rejection of IL-2/IFN-gamma-transduced tumor cells. Our data show the successful use of a single retroviral vector to stably transduce two cytokine genes into the same tumor cell, leading to an increased effect on the in vivo induction of antitumor immunity.

scholarly journals Augmentation of antitumor immunity by tumor cells transduced with a retroviral vector carrying the interleukin-2 and interferon-gamma cDNAs

Blood ◽  
1994 ◽  
Vol 83 (5) ◽  
pp. 1289-1298
Author(s):  
FM Rosenthal ◽  
K Cronin ◽  
R Bannerji ◽  
DW Golde ◽  
B Gansbacher
Keyword(s):  
Gene Transfer ◽  
Tumor Cells ◽  
Interferon Gamma ◽  
Antitumor Immunity ◽  
Interleukin 2 ◽  
Retroviral Vectors ◽  
Retroviral Vector ◽  
Ifn Gamma ◽  
Gm Csf

Therapeutic models using gene transfer into tumor cells have pursued three objectives: (1) to induce rejection of the tumor transduced with therapeutic genes, (2) to induce immune-mediated regression of metastatic disease, and (3) to induce long-lasting immunity to protect against challenge with tumor cells or clinical regrowth of micrometastatic disease. Because in vivo therapy for patients with cancer using gene transfer would, as a first step, attempt to eliminate the existing tumor, we have investigated whether antitumor immunity induced by tumor cells secreting a single cytokine could be increased by cotransfer of a second cytokine gene. To test this approach, CMS-5, a murine fibrosarcoma, was transduced with retroviral vectors carrying interleukin-2 (IL-2), interferon-gamma (IFN-gamma), or granulocyte- macrophage-colony-stimulating factor (GM-CSF) cDNA alone or IL-2 cDNA in combination with IFN-gamma or GM-CSF cDNA. Single cytokine-secreting clones were selected to match levels of cytokine production by double cytokine-secreting clones so that similar amounts of cytokine were secreted. IFN-gamma- and IL-2/IFN-gamma-secreting CMS-5 cells showed increased levels of major histocompatability complex class I expression compared with IL-2- and GM-CSF-secreting or parental CMS-5 cells, IL- 2/IFN-gamma-secreting CMS-5 cells were always rejected by syngeneic mice, whereas the same number of CMS-5 cells secreting only one of these cytokines or mixtures of single cytokine-secreting CMS-5 cells were not rejected. In vivo depletion of CD4+, CD8+, or natural-killer effector cell subpopulations showed that CD8+ cytotoxic T cells were primarily responsible for rejection of IL-2/IFN-gamma-transduced tumor cells. Our data show the successful use of a single retroviral vector to stably transduce two cytokine genes into the same tumor cell, leading to an increased effect on the in vivo induction of antitumor immunity.

scholarly journals Enhancement of the fibrinolytic activity of sheep endothelial cells by retroviral vector-mediated gene transfer

Blood ◽  
1991 ◽  
Vol 77 (3) ◽  
pp. 533-541 ◽  
Author(s):  
DA Dichek ◽  
O Nussbaum ◽  
SJ Degen ◽  
WF Anderson
Keyword(s):  
Endothelial Cells ◽  
Gene Transfer ◽  
Plasminogen Activator ◽  
Fibrinolytic Activity ◽  
Fold Increase ◽  
Retroviral Vector ◽  
Tissue Type ◽  
Plasminogen Activator Inhibitor 1 ◽  
Pai 1

Abstract In an attempt to enhance the fibrinolytic activity of endothelial cells (EC), a retroviral vector containing the human tissue-type plasminogen activator (t-PA) cDNA was constructed. Sheep EC were stably transduced with the vector, resulting in a 30-fold increase in t-PA activity over that detected in EC transduced with a control vector. Southern and Northern analyses confirmed the presence of both the vector sequence and the appropriate mRNA transcripts. Secretion of high levels of recombinant human t-PA continued in vitro for the duration of the experiments, up to 11 weeks after transduction, although the rate of t- PA secretion decreased in some of the EC lines. Zymographic analysis of conditioned medium from t-PA-transduced EC showed the presence of two new molecular species with plasminogen activator activity that could be specifically immunoprecipitated with a monoclonal antihuman t-PA antibody. The relative molecular masses of these species (60 to 80 and 110 Kd) suggest that they represent recombinant human t-PA both free and bound to sheep plasminogen activator inhibitor 1 (PAI-1). Consistent with this interpretation, the 110-Kd species could be specifically immunoprecipitated with antiserum to PAI-1. These studies demonstrate that retroviral vector-mediated gene transfer may be used to increase total EC fibrinolytic activity.

scholarly journals Enhancement of the fibrinolytic activity of sheep endothelial cells by retroviral vector-mediated gene transfer

Blood ◽  
1991 ◽  
Vol 77 (3) ◽  
pp. 533-541
Author(s):  
DA Dichek ◽  
O Nussbaum ◽  
SJ Degen ◽  
WF Anderson
Keyword(s):  
Endothelial Cells ◽  
Gene Transfer ◽  
Plasminogen Activator ◽  
Fibrinolytic Activity ◽  
Fold Increase ◽  
Retroviral Vector ◽  
Tissue Type ◽  
Plasminogen Activator Inhibitor 1 ◽  
Pai 1

In an attempt to enhance the fibrinolytic activity of endothelial cells (EC), a retroviral vector containing the human tissue-type plasminogen activator (t-PA) cDNA was constructed. Sheep EC were stably transduced with the vector, resulting in a 30-fold increase in t-PA activity over that detected in EC transduced with a control vector. Southern and Northern analyses confirmed the presence of both the vector sequence and the appropriate mRNA transcripts. Secretion of high levels of recombinant human t-PA continued in vitro for the duration of the experiments, up to 11 weeks after transduction, although the rate of t- PA secretion decreased in some of the EC lines. Zymographic analysis of conditioned medium from t-PA-transduced EC showed the presence of two new molecular species with plasminogen activator activity that could be specifically immunoprecipitated with a monoclonal antihuman t-PA antibody. The relative molecular masses of these species (60 to 80 and 110 Kd) suggest that they represent recombinant human t-PA both free and bound to sheep plasminogen activator inhibitor 1 (PAI-1). Consistent with this interpretation, the 110-Kd species could be specifically immunoprecipitated with antiserum to PAI-1. These studies demonstrate that retroviral vector-mediated gene transfer may be used to increase total EC fibrinolytic activity.

Enhanced In Vivo Antithrombotic Effects of Endothelial Cells Expressing Recombinant Plasminogen Activators Transduced With Retroviral Vectors

Circulation ◽  
1996 ◽  
Vol 93 (2) ◽  
pp. 301-309 ◽  
Author(s):  
David A. Dichek ◽  
Johanna Anderson ◽  
Andrew B. Kelly ◽  
Stephen R. Hanson ◽  
Laurence A. Harker
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activators ◽  
Retroviral Vectors ◽  
Antithrombotic Effects

Abstract 5566: Recovery of Erk Signaling Restores Defective Angiogenesis and Arteriogenesis in Synectin-Deficient Animals

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Bin Ren ◽  
Arpita Mukhopadhyay* ◽  
Anthony A Lanahan ◽  
Zhen W Zhuang ◽  
Karen L Moodie ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Branching Morphogenesis ◽  
Erk Signaling ◽  
Dependent Manner ◽  
Wild Type ◽  
Erk Activation ◽  
Arterial Development ◽  
Constitutively Active

Background : Arterial morphogenesis is an important and poorly understood process. We have previously demonstrated that disruption of synectin gene expression in mice and zebrafish results in impaired arterial development and branching morphogenesis. Synectin null endothelial cells demonstrate reduced VEGF responsiveness in terms of migration, proliferation and differentiation and ERK-1/2 activation (Chittenden et al, Dev Cell 2006). Since ERK has been established as major participants in the regulation of cell growth and differentiation and Erk activation has been previously linked to arterial morphogenesis, we evaluated whether activation of Erk signaling in synectin disrupted mice and zebrafish as well as synectin KO arterial endothelial cells (ECs) would restore defective migration, arterial differentiation, angiogenesis and arteriogenesis. To stimulate ERK signaling we used partial inhibition of PI3-K activity to reduce Akt-dependent suppression of Raf1 activation or introduction of constitutively active ERK construct. Methods : In vitro studies were conducted with primary arterial ECs isolated from synectin wild type (WT) and knock out (KO) mice. In vivo studies were carried out in WT and synectin deficient mice and synectin knockdown zebrafish embryos. Results: Exposure of synectin −/− arterial EC to two selective PI3K inhibitors GS4898 or LY294002 in vitro restored ERK activation in a dose-dependent manner and returned cell migration and in vitro branching morphogenesis to wild type levels. Transduction of a constitutively active ERK construct in vitro or in a Matrigel model in vivo had similar effect. Systemic treatment of synectin −/− mice with GS4898 fully restored impaired angiogenesis and arterial morphogenesis in adult animals in the setting of hindlimb ischemia. Similar treatment nearly completely restored arterial development defects in zebrafish treated with a synectin morpholino. Conclusions: ERK activation plays a key role in arteriogenesis both in adult tissues and during embryonic development. Activation of compromised ERK-1/2 signaling may be a novel therapeutic intervention to stimulate arteriogenesis.

Factor VIIa induces extracellular vesicles from the endothelium: A potential mechanism for its hemostatic effect

Blood ◽  
2021 ◽  
Author(s):  
Kaushik Das ◽  
Shiva Keshava ◽  
Shabbir A Ansari ◽  
Vijay Kumar Reddy Kondreddy ◽  
Charles Esmon ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Extracellular Vesicles ◽  
Factor Viia ◽  
Mutant Mice ◽  
In Vivo Studies ◽  
Cell Protein ◽  
Wild Type ◽  
Hemostatic Effect

Recombinant FVIIa (rFVIIa) is used as a hemostatic agent to treat bleeding disorders in hemophilia patients with inhibitors and other groups of patients. Our recent studies showed that FVIIa binds endothelial cell protein C receptor (EPCR) and induces protease-activated receptor 1 (PAR1)-mediated biased signaling. The importance of FVIIa-EPCR-PAR1-mediated signaling in hemostasis is unknown. In the present study, we show that FVIIa induces the release of extracellular vesicles (EVs) from endothelial cells both in vitro and in vivo. Silencing of EPCR or PAR1 in endothelial cells blocked the FVIIa-induced generation of EVs. Consistent with these data, FVIIa treatment enhanced the release of EVs from murine brain endothelial cells isolated from wild-type, EPCR overexpressors, and PAR1-R46Q mutant mice, but not EPCR-deficient or PAR1-R41Q mutant mice. In vivo studies revealed that administration of FVIIa to wild-type, EPCR overexpressors, and PAR1-R46Q mutant mice, but not EPCR-deficient or PAR1-R41Q mutant mice, increase the number of circulating EVs. EVs released in response to FVIIa treatment exhibit enhanced procoagulant activity. Infusion of FVIIa-generated EVs and not control EVs to platelet-depleted mice increased thrombin generation at the site of injury and reduced blood loss. Administration of FVIIa-generated EVs or generation of EVs endogenously by administering FVIIa augmented the hemostatic effect of FVIIa. Overall, our data reveal that FVIIa treatment, through FVIIa-EPCR-PAR1 signaling, releases EVs from the endothelium into the circulation, and these EVs contribute to the hemostatic effect of FVIIa.

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