BMP2 gene transfer induces pericardial effusion and inflammatory response in the ischemic porcine myocardium

Pro-angiogenic gene therapy is being developed to treat coronary artery disease (CAD). We recently showed that bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor-A synergistically regulate endothelial cell sprouting in vitro. BMP2 was also shown to induce endocardial angiogenesis in neonatal mice post-myocardial infarction. In this study, we investigated the potential of BMP2 gene transfer to improve cardiomyocyte function and neovessel formation in a pig chronic myocardial infarction model. Ischemia was induced in domestic pigs by placing a bottleneck stent in the proximal part of the left anterior descending artery 14 days before gene transfer. Intramyocardial gene transfers with adenovirus vectors (1 × 1012 viral particles/pig) containing either human BMP2 (AdBMP2) or beta-galactosidase (AdLacZ) control gene were performed using a needle injection catheter. BMP2 transgene expression in the myocardium was detected with immunofluorescence staining in the gene transfer area 6 days after AdBMP2 administration. BMP2 gene transfer did not induce angiogenesis or cardiomyocyte proliferation in the ischemic pig myocardium as determined by the quantitations of CD31 or Ki-67 stainings, respectively. Accordingly, no changes in heart contractility were detected in left ventricular ejection fraction and strain measurements. However, BMP2 gene transfer induced pericardial effusion (AdBMP2: 9.41 ± 3.17 mm; AdLacZ: 3.07 ± 1.33 mm) that was measured by echocardiography. Furthermore, an increase in the number of immune cells and CD3+ T cells was found in the BMP2 gene transfer area. No changes were detected in the clinical chemistry analysis of pig serum or histology of the major organs, implicating that the gene transfer did not induce general toxicity, myocardial injury, or off-target effects. Finally, the levels of fibrosis and cardiomyocyte apoptosis detected by Sirius red or caspase 3 stainings, respectively, remained unaltered between the groups. Our results demonstrate that BMP2 gene transfer causes inflammatory changes and pericardial effusion in the adult ischemic myocardium, which thus does not support its therapeutic use in chronic CAD

Comparison of Efficiency and Function of Vascular Endothelial Growth Factor Adenovirus Vectors in Endothelial Cells for Gene Therapy of Placental Insufficiency

Introduction: Severe fetal growth restriction (FGR) affects 1:500 pregnancies, is untreatable and causes serious neonatal morbidity and death. Reduced uterine blood flow (UBF) and lack of bioavailable VEGF due to placental insufficiency is a major cause. Transduction of uterine arteries in normal or FGR sheep and guinea pigs using an adenovirus (Ad) encoding VEGF isoforms A (Ad.VEGF-A165) and a FLAG-tagged pre-processed short form D (DΔNΔC, Ad.VEGF-DΔNΔC-FLAG) increases endothelial nitric oxide expression, enhances relaxation and reduces constriction of the uterine arteries and their branches. UBF and angiogenesis are increased long term, improving fetal growth in utero. For clinical trial development we compared Ad.VEGF vector transduction efficiency and function in endothelial cells (ECs) derived from different species. Objective: To compare the transduction efficiency and function of the pre-clinical study Ad. constructs (Ad.VEGF-A165, Ad.VEGF-DΔNΔC-FLAG) with the intended clinical trial construct (Ad.VEGF-DΔNΔC) where the FLAG tag is removed. Methods: We infected ECs from human umbilical vein, pregnant sheep uterine artery, pregnant guinea pig aorta and non-pregnant rabbit aorta, with increasing multiplicity of infection (MOI) for 24 or 48 hours of three Ad.VEGF vectors, compared to control Ad. containing the LacZ gene (Ad.LacZ). VEGF supernatant expression was analysed by ELISA. Functional assessment used tube formation assay and Erk-Akt phosphorylation by ELISA. Results: VEGF expression was higher after Ad.VEGF-DΔNΔC-FLAG and Ad.VEGF-DΔNΔC transduction compared to Ad.VEGF-A165 in all EC types (*p<0.001). Tube formation was higher in ECs transduced with Ad.VEGF-DΔNΔC in all species compared to other constructs (***p<0.001, *p<0.05 with rabbit aortic ECs). Phospho-Erk and phospho-Akt assays displayed no differences between the three vector constructs, whose effect was, as in other experiments, higher than Ad.LacZ (***p<0.001). Conclusion: We observed high transduction efficiency and functional effects of Ad.VEGF-DΔNΔC vector with comparability in major pathway activation to constructs used in pre-clinical studies, supporting its use in a clinical trial

Uptake of 68gallium in atherosclerotic plaques in LDLR-/- ApoB100/100 mice

Background: Atherosclerosis is a chronic inflammatory disease of artery wall characterized by infiltration of monocytes into subendothelial space and their differentiation into macrophages. Since rupture-prone plaques commonly contain high amounts of activated macrophages, imaging of the macrophage content may provide a useful tool for the evaluation of plaque vulnerability. The purpose of this study was to explore the uptake of 68gallium (68Ga) in atherosclerotic plaques in mice.Methods: Uptake of ionic 68Ga was investigated in atherosclerotic LDLR-/-ApoB100/100 and C57BL/6N control mice at 3 h after injection. The ex vivo biodistribution of the 68Ga was assessed and autoradiography of aortic cryosections was defined. In vivo imaging of 68Ga was performed using a small animal positron emission tomography PET/CT scanner.Results: Our results revealed that the uptake of 68Ga-radioactivity was higher in atherosclerotic plaques than in healthy vessel wall (ratio 1.8 +/- 0.2, p = 0.0002) and adventitia (ratio 1.3 +/- 0.2, p = 0.0011). The autoradiography signal co-localized with macrophages prominently as demonstrated by Mac-3 staining. In both mice strains, the highest level of radioactivity was found in the blood.Conclusions: We observed a moderate but significantly elevated 68Ga-radioactivity uptake in the aortic plaques of atherosclerotic mice, especially at the sites rich in macrophages. While the uptake of 68Ga was promising in this animal model, the slow blood clearance may limit the usability of 68Ga as a PET tracer for clinical imaging of atherosclerotic plaques.</p

Nuclear microRNA-466c regulates Vegfa expression in response to hypoxia

MicroRNAs are well characterized in their role in silencing gene expression by targeting 3´-UTR of mRNAs in cytoplasm. However, recent studies have shown that miRNAs have a role in the regulation of genes in the nucleus, where they are abundantly located. We show here that in mouse endothelial cell line (C166), nuclear microRNA miR-466c participates in the regulation of vascular endothelial growth factor a (Vegfa) gene expression in hypoxia. Upregulation of Vegfa expression in response to hypoxia was significantly compromised after removal of miR-466c with CRISPR-Cas9 genomic deletion. We identified a promoter-associated long non-coding RNA on mouse Vegfa promoter and show that miR-466c directly binds to this transcript to modulate Vegfa expression. Collectively, these observations suggest that miR-466c regulates Vegfa gene transcription in the nucleus by targeting the promoter, and expands on our understanding of the role of miRNAs well beyond their canonical role

A multipurpose vector system for the screening of libraries in bacteria, insect and mammalian cells and expression in vivo

We have constructed a novel tetra-promoter vector (pBVboostFG) system that enables screening of gene/cDNA libraries for functional genomic studies. The vector enables an all-in-one strategy for gene expression in mammalian, bacterial and insect cells and is also suitable for direct use in vivo. Virus preparation is based on an improved mini Tn7 transpositional system allowing easy and fast production of recombinant baculoviruses with high diversity and negligible background. Cloning of the desired DNA fragments or libraries is based on the recombination system of bacteriophage lambda. As an example of the utility of the vector, genes or cDNAs of 18 different proteins were cloned into pBVboostFG and expressed in different hosts. As a proof-of-principle of using the vector for library screening, a chromophoric Thr(65)-Tyr-Gly(67)-stretch of enhanced green fluorescent protein was destroyed and subsequently restored by novel PCR strategy and library screening. The pBVboostFG enables screening of genome-wide libraries, thus making it an efficient new platform technology for functional genomics

18-kDa translocator protein ligand 18F-FEMPA: Biodistribution and uptake into atherosclerotic plaques in mice

18F-FEMPA shows rapid blood clearance and uptake in the mouse aorta. Uptake in atherosclerotic plaques correlated with the amount of macrophages, but did not exceed that in the normal vessel wall.</p

Imaging of αvβ3 integrin expression in experimental myocardial ischemia with [68Ga]NODAGA-RGD positron emission tomography

BACKGROUND: Radiolabeled RGD peptides detect αvβ3 integrin expression associated with angiogenesis and extracellular matrix remodeling after myocardial infarction. We studied whether cardiac positron emission tomography (PET) with [68Ga]NODAGA-RGD detects increased αvβ3 integrin expression after induction of flow-limiting coronary stenosis in pigs, and whether αvβ3 integrin is expressed in viable ischemic or injured myocardium.METHODS: We studied 8 Finnish landrace pigs 13 ± 4 days after percutaneous implantation of a bottleneck stent in the proximal left anterior descending coronary artery. Antithrombotic therapy was used to prevent stent occlusion. Myocardial uptake of [68Ga]NODAGA-RGD (290 ± 31 MBq) was evaluated by a 62 min dynamic PET scan. The ischemic area was defined as the regional perfusion abnormality during adenosine-induced stress by [15O]water PET. Guided by triphenyltetrazolium chloride staining, tissue samples from viable and injured myocardial areas were obtained for autoradiography and histology.RESULTS: Stent implantation resulted in a partly reversible myocardial perfusion abnormality. Compared with remote myocardium, [68Ga]NODAGA-RGD PET showed increased tracer uptake in the ischemic area (ischemic-to-remote ratio 1.3 ± 0.20, p = 0.0034). Tissue samples from the injured areas, but not from the viable ischemic areas, showed higher [68Ga]NODAGA-RGD uptake than the remote non-ischemic myocardium. Uptake of [68Ga]NODAGA-RGD correlated with immunohistochemical detection of αvβ3 integrin that was expressed in the injured myocardial areas.CONCLUSIONS: Cardiac [68Ga]NODAGA-RGD PET demonstrates increased myocardial αvβ3 integrin expression after induction of flow-limiting coronary stenosis in pigs. Localization of [68Ga]NODAGA-RGD uptake indicates that it reflects αvβ3 integrin expression associated with repair of recent myocardial injury.</p

Regulatory evaluation of Glybera in Europe-two committees, one mission

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