625 Green Fluorescent Protein Selectively Induces HSP70 Mediated Upregulation of COX 2 Expression in Endothelial Cells Molecular Therapy �������� ��� ���� ���������������� �������� ��� ������© �������[.]
CARDIOVASCULAR 623 Gene Delivery of Bone Morphogenetic Proteins and Type Receptors Using Adenoviral Vectors Ann M Reynolds,1 Katherine M Finan,1 Nicholas W Morrell,2 David T Curiel,3 Mark D Holmes,1 Paul N Reynolds.1 Department of Thoracic Medicine, Royal Adelaide Hospital, Adelaide, South Australia, Australia; 2Respiratory Medicine Unit, Department of Medicine, University of Cambridge, Cambridge, England, United Kingdom; 3Gene Therapy Center at UAB, University of Alabama at Birmingham, Birmingham, AL, United States Primary pulmonary hypertension (PPH) is a fatal disease characterised by abnormal proliferation of pulmonary vascular endothelial and smooth muscle cells Recent evidence indicates defects in the bone morphogenetic protein receptor type (BMPR2) pathway in this disease These mutations are predicted to lead to a downregulation in BMPR2 signalling, a pathway which involves a SMAD-protein signal cascade Thus, gene replacement therapy using a normal BMPR2 gene could potentially be useful in this disease Previously, we developed a technique to achieve selective gene expression in pulmonary vascular endothelium using adenoviral (Ad) vectors, based on transductional targeting and transcriptional control using the endothelial-specific flt-1 promoter Hypothesis: A targeted Ad vector system could be developed to deliver functional BMPR2 receptors and BMPs to pulmonary vascular endothelium Aims: In this phase of the study we sought to develop Ad vectors containing the BMPR2 gene, then evaluate gene delivery in vitro using immunohistochemistry and a functional assay using a SMADsensitive reporter construct (p3GC2wt-Lux) whereby increased SMAD levels drive the expression of the luciferase reporter gene Methods: The cDNAs for the human BMPR2 receptor, incorporating a c-terminal myc or FLAG tag (for ease of later immunohistochemical detection), or for the BMPR2 ligands (BMP-2 or BMP-7) were cloned into Ad vectors by standard techniques Cells in culture were infected with BMPR2 Ads then stained with an anti-myc or antiFLAG antibody, with signal detected with immunofluorescence Evidence of receptor function was sought by transfecting cells first with p3GC2wt-Lux, then co-infecting with AdBMPR2 and AdBMP2 Results: Positive fluorescent signal was detected in AdBMPR2 infected cells in a distribution consistent with membrane localisation Analysis of luciferase activity in cells infected with BMPR2/BMP2 Ads revealed 10-fold greater luciferase activity than cells transfected with p3GC2wt-Lux alone or with control vectors Conclusion: Functional BMPR2 and BMPs can be delivered by Ad vectors, at least in vitro In vivo studies are currently underway 624 Identification and Evaluation of Plaque Targeting Peptides by Phage Display Emmanuel D Papadakis,1 Stuart A Nicklin,2 Andrew H Baker,2 Andrew C Newby,1 Stephen J White.1 Bristol Heart Institute, University of Bristol, Bristol, United Kingdom; 2Division of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow, United Kingdom Cardiovascular disease is the greatest single cause of morbidity and mortality in the western world Current and potential drug and gene therapies would benefit from the ability to be specifically directed to unstable intravascular plaques in vivo Endothelium overlying atherosclerotic plaques exhibits upregulated expression of receptors such as VCAM-1, ICAM-1, E&P Selectin and LOX-1 Ligands binding to receptors specifically expressed on atherosclerotic endothelium could represent a means of targeting gene therapy vectors to these sites We have utilized two phage display approaches to identify short peptide ligands which could support binding to atherosclerotic plaques The first approach involved in vivo phage Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright © The American Society of Gene Therapy biopanning using a random 12 amino acid library in 3-month hi-fat fed apoE -/- mice to identify peptides that home to diseased vasculature in vivo Phage were isolated from the braciocephalic artery in these mice as it offers a consistent and reproducible site of plaque formation, also being a site exhibiting frequent plaque rupture Triplicate independent panning experiments were performed with an initial input titer of 4x1012 pfu phage, which was decreased to 5x109 pfu and 1x109 pfu over the following rounds Recovery of phage from the braciocephalic artery increased between and logs over four rounds 110 phage were sequenced from the third round of biopanning revealing 79 unique sequences, in addition eight peptides bore identical motifs of at least amino acids In contrast, a repeat experiment using a cyclic amino acid library failed to show any increases in target-specific phage recovery over five successive rounds of in vivo panning To supplement the in vivo biopanning, in vitro phage display was performed against immobilized vascular cell adhesion molecule (VCAM-1) as this has been shown to be selectively upregulated on endothelium overlying plaques in the high fat-fed apoE-/- mouse model Panning was performed using both 12mer and cyclic 7mer libraries in triplicate, with a fixed input titer of 4x1010 pfu against microtiter wells coated with 1.5 μg VCAM-1 An increased recovery of phage of between and logs was observed over three rounds for both libraries, following which individual phage were then screened for their ability to selectively bind to VCAM-1 by an ELISA-based assay Phage selected from both the in vivo panning and those with affinity for VCAM-1 were screened for their ability to bind to TNF-stimulated primary human endothelial cells in vitro Several individual phage isolated by each of these methods showed up to five-fold higher binding to stimulated endothelial cells over quiescent cells in vitro, and also demonstrated reduced binding of non-endothelial cell types indicating selectivity for endothelial cell receptors This work demonstrates the potential utility of using phage display to identify novel targeting ligands that may have potential in targeting gene therapy vectors to atherosclerotic plaques in vivo 625 Green Fluorescent Protein Selectively Induces HSP70-Mediated Upregulation of COX-2 Expression in Endothelial Cells Fan Zhang,1 Neil R Hackett,1 George Lam,1 Sergey Shmelkov,1 Robert G Pergolizzi,1 Lan Luo,1 Joseph Cheng,1 Jay Edelberg,1 Ronald G Crystal,1 Shahin Rafii.1 Weill Medical College of Cornell Universtiy, New York, NY Reporter genes, such as green fluorescent protein (GFP), have been used extensively to follow the expression of transgenes introduced into vascular cells by gene transfer vectors This study is designed to test the hypothesis that overexpression of the GFP reporter gene might impact the normal physiology of endothelial cells Microarray studies comparing gene expression in human umbilical vein endothelial cells (HUVEC) infected with an adenovirus vector expressing GFP (AdGFP) to gene expression in naive cells showed that over 200 genes were induced by greater than 4-fold The most highly induced gene was HSP70 which increased in expression level 80-fold (mean of n=2 measurements) in response to AdGFP but not in response to a control Ad vector with no transgene (AdNull) This effect was specific to endothelial cells and AdGFP expression was shown not to induce HSP70 in HeLa, vascular cells from muscle and A549 adenocarcinoma cells AdGFP induced expression of HSP70 at both messenger RNA and protein level in a dose-dependent manner To further distinguish the effects of GFP from those of Ad vector, a lentiviral vector expressing GFP was used and shown to also induce HSP70 expression Upregulation of HSP70, whether by infection by AdGFP or by addition of HSP70 protein to HUVEC, resulted in induction of cyclooxygenase-2 (COX2) followed by increased prostaglandin E2 (PGE2) production (1150 S243 CARDIOVASCULAR ± 160 pg/ml for AdGFP treated HUVEC versus 290 ± 190 pg/ml for naive cells, p