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Coronary artery disease (CAD) is a pandemic disease where up to half of the risk is explained by genetic factors. Advanced insights into the genetic basis of CAD require deeper understanding of the contributions of different cell types, molecular pathways, and genes to disease heritability. Here, we investigate the biological diversity of atherosclerosis-associated cell states and interrogate their contribution to the genetic risk of CAD by using single-cell and bulk RNA sequencing (RNA-seq) of mouse and human lesions. We identified 12 disease-associated cell states that we characterized further by gene set functional profiling, ligand-receptor prediction, and transcription factor inference. Importantly, Vcam1+ smooth muscle cell state genes contributed most to SNP-based heritability of CAD. In line with this, genetic variants near smooth muscle cell state genes and regulatory elements explained the largest fraction of CAD-risk variance between individuals. Using this information for variant prioritization, we derived a hybrid polygenic risk score (PRS) that demonstrated improved performance over a classical PRS. Our results provide insights into the biological mechanisms associated with CAD risk, which could make a promising contribution to precision medicine and tailored therapeutic interventions in the future. publishedVersion

Background: Sporadic venous malformation (VM) and angiomatosis of soft tissue (AST) are benign, congenital vascular anomalies affecting venous vasculature. Depending on the size and location of the lesion, symptoms vary from motility disturbances to pain and disfigurement. Due to high recurrence of the lesions more effective therapies are needed. Methods: As targeting stromal cells has been an emerging concept in anti-angiogenic therapies, here, by using VM/AST patient samples, RNA-sequencing, cell culture techniques and a xenograft mouse model, we investigated the crosstalk of endothelial cells (EC) and fibroblasts and its effect on vascular lesion growth. Results: We report, for the first time, expression and secretion of transforming growth factor A (TGFA) in ECs or intervascular stromal cells in AST and VM lesions. TGFA induced secretion of VEGF-A paracrinally, and regulated EC proliferation. Oncogenic PIK3CA variant in p.H1047R, a common somatic mutation found in these lesions, increased TGFA expression, enrichment of hallmark hypoxia, and in a mouse xenograft model, lesion size and vascularization. Treatment with afatinib, a pan-ErbB tyrosine-kinase inhibitor, decreased vascularization and lesion size in mouse xenograft model with ECs expressing oncogenic PIK3CA p.H1047R variant and fibroblasts. Conclusions: Based on the data, we suggest that targeting of both intervascular stromal cells and ECs is a potential treatment strategy for vascular lesions having a fibrous component. Funding: Academy of Finland, Ella and Georg Ehnrooth foundation, the ERC grants, Sigrid Jusélius Foundation, Finnish Foundation for Cardiovascular Research, Jane and Aatos Erkko Foundation, and Department of Musculosceletal and Plastic Surgery, Helsinki University Hospital.

Background Brain arteriovenous malformations (bAVM) may rupture causing disability or death. BAVM vessels are characterized by abnormally high flow that in general triggers expansive vessel remodeling mediated by cyclo-oxygenase-2 (COX2), the target of non-steroidal anti-inflammatory drugs. We investigated whether COX2 is expressed in bAVMs and whether it associates with inflammation and haemorrhage in these lesions. Methods Tissue was obtained from surgery of 139 bAVMs and 21 normal Circle of Willis samples. The samples were studied with immunohistochemistry and real-time quantitative polymerase chain reaction (RT-PCR). Clinical data was collected from patient records. Results COX2 expression was found in 78% (109/139) of the bAVMs and localized to the vessels’ lumen or medial layer in 70% (95/135) of the bAVMs. Receptors for prostaglandin E2, a COX2-derived mediator of vascular remodeling, were found in the endothelial and smooth muscle cells and perivascular inflammatory cells of bAVMs. COX2 was expressed by infiltrating inflammatory cells and correlated with the extent of inflammation (r = .231, p = .007, Spearman rank correlation). COX2 expression did not associate with haemorrhage. Conclusion COX2 is induced in bAVMs, and possibly participates in the regulation of vessel wall remodelling and ongoing inflammation. Role of COX2 signalling in the pathobiology and clinical course of bAVMs merits further studies.

Supplemental Digital Content is available in the text., Objective: Atherosclerosis is the underlying cause of most cardiovascular diseases. The main cell types associated with disease progression in the vascular wall are endothelial cells, smooth muscle cells, and macrophages. Although their role in atherogenesis has been extensively described, molecular mechanisms underlying gene expression changes remain unknown. The objective of this study was to characterize microRNA (miRNA)-related regulatory mechanisms taking place in the aorta during atherosclerosis. Approach and Results: We analyzed the miRNA expression changes in primary human aortic endothelial cells and human umbilical vein endothelial cells, human aortic smooth muscle cells, and macrophages (CD14+) under various proatherogenic stimuli by integrating GRO-seq, miRNA-seq, and RNA-seq data. Despite the highly cell-type-specific expression of multi-variant primary miRNAs, the majority of mature miRNAs were found to be common to all cell types and dominated by 2 to 5 abundant miRNA species. We demonstrate that transcription contributes significantly to the mature miRNA levels although this is dependent on miRNA stability. An analysis of miRNA effects in relation to target mRNA pools highlighted pathways and targets through which miRNAs could affect atherogenesis in a cell-type-dependent manner. Finally, we validate miR-100-5p as a cell-type specific regulator of inflammatory and HIPPO-YAP/TAZ-pathways. Conclusions: This integrative approach allowed us to characterize miRNA dynamics in response to a proatherogenic stimulus and identify potential mechanisms by which miRNAs affect atherogenesis in a cell-type-specific manner.

The BMP/TGFβ-Smad, Notch and VEGF signaling guides formation of endothelial tip and stalk cells. However, the crosstalk of bone morphogenetic proteins (BMPs) and vascular endothelial growth factor receptor 2 (VEGFR2) signaling has remained largely unknown. We demonstrate that BMP family members regulate VEGFR2 and Notch signaling, and act via TAZ-Hippo signaling pathway. BMPs were found to be regulated after VEGF gene transfer in C57/Bl6 mice and in a porcine myocardial ischemia model. BMPs 2/4/6 were identified as endothelium-specific targets of VEGF. BMP2 modulated VEGF-mediated endothelial sprouting via Delta like Canonical Notch Ligand 4 (DLL4). BMP6 modulated VEGF signaling by regulating VEGFR2 expression and acted via Hippo signaling effector TAZ, known to regulate cell survival/proliferation, and to be dysregulated in cancer. In a matrigel plug assay in nude mice BMP6 was further demonstrated to induce angiogenesis. BMP6 is the first member of BMP family found to directly regulate both Hippo signaling and neovessel formation. It may thus serve as a target in pro/anti-angiogenic therapies. Electronic supplementary material The online version of this article (10.1007/s10456-020-09748-4) contains supplementary material, which is available to authorized users.

Objective: Previous studies have demonstrated that the expression of several lysine (K)-specific demethylases (KDMs) is induced by hypoxia. Here, we sought to investigate the exact mechanisms underlying this regulation and its functional implications for endothelial cell function, such as angiogenesis. Approach and Results: We analyzed the expression changes of KDMs under hypoxia and modulation of HIF (hypoxia-inducible factor) expression using GRO-Seq and RNA-Seq in endothelial cells. We provide evidence that the majority of the KDMs are induced at the level of nascent transcription mediated by the action of HIF-1α and HIF-2α. Importantly, we show that transcriptional changes at the level of initiation represent the major mechanism of gene activation. To delineate the epigenetic effects of hypoxia and HIF activation in normoxia, we analyzed the genome-wide changes of H3K27me3 using chromosome immunoprecipitation-Seq. We discovered a redistribution of H3K27me3 at ≈2000 to 3000 transcriptionally active loci nearby genes implicated in angiogenesis. Among these, we demonstrate that vascular endothelial growth factor A ( VEGFA ) expression is partly induced by KDM4B- and KDM6B-mediated demethylation of nearby regions. Knockdown of KDM4B and KDM6B decreased cell proliferation, tube formation, and endothelial sprouting while affecting hundreds of genes associated with angiogenesis. These findings provide novel insights into the regulation of KDMs by hypoxia and the epigenetic regulation of VEGFA-mediated angiogenesis. Conclusions: Our study describes an additional level of epigenetic regulation where hypoxia induces redistribution of H3K27me3 around genes implicated in proliferation and angiogenesis. More specifically, we demonstrate that KDM4B and KDM6B play a key role in modulating the expression of the major angiogenic driver VEGFA.

Vascular endothelial growth factors regulate vascular and lymphatic growth. Dysregulation of VEGF signaling is connected to many pathological states, including hemangiomas, arteriovenous malformations and placental abnormalities. In heart, VEGF gene transfer induces myocardial angiogenesis. Besides vascular and lymphatic endothelial cells, VEGFs affect multiple other cell types. Understanding VEGF biology and its paracrine signaling properties will offer new targets for novel treatments of several diseases.

Restoring blood supply by inducing formation of neovessels is suggested as a treatment strategy for ischemic heart diseases. Growth factors can be used to guide formation of the vessels into ischemic area. As the half-life of recombinant growth factors is short and they easily diffuse from the delivery site, gene therapy has emerged as an appealing approach due to the long-lasting expression of the therapeutic compound. Gene therapy could also be used in combination with cell therapy to augment properties of cellular regenerative pathways. In this chapter, we review preclinical and clinical trials leading to current approaches for cardiac gene and protein therapies and look for future perspectives and possibilities in these areas, which could potentially influence treatments of a large number of patients suffering from severe myocardial ischemia.

Magnetic resonance imaging examinations are frequently carried out using contrast agents to improve the image quality. Practically all clinically used contrast agents are based on paramagnetic metals and lack in selectivity and specificity. A group of stable organic radicals, nitroxides, has raised interest as new metal-free contrast agents for MRI. Their structures can easily be modified to incorporate different functionalities. In the present study, a stable nitroxide TEEPO (2,2,6,6-tetraethylpiperidin-1-oxyl) was linked to a glucose moiety (Glc) to construct a water-soluble, potentially tumor-targeting compound with contrast-enhancing ability. The ability was assessed with in vivo MRI experiments. The constructed TEEPO-Glc agent proved to shorten the T1 relaxation time in tumor, while the T1 time in healthy brain tissue remained the same. The results indicate the potential of TEEPO-Glc as a valuable addition to the growing field of metal-free contrast enhancement in MRI-based diagnostics.

Vascular endothelial growth factors (VEGFs) are key mediators of endothelial cell (EC) function in angiogenesis. Emerging knowledge also supports the involvement of axon guidance-related factors in the regulation of angiogenesis and vascular patterning. In the current study, we demonstrate that fibronectin and leucine-rich transmembrane protein-3 (FLRT3), an axon guidance-related factor connected to the regulation of neuronal cell outgrowth and morphogenesis but not to VEGF-signaling, was upregulated in ECs after VEGF binding to VEGFR2. We found that FLRT3 exhibited a transcriptionally paused phenotype in non-stimulated human umbilical vein ECs. After VEGF-stimulation its nascent RNA and mRNA-levels were rapidly upregulated suggesting that the regulation of FLRT3 expression is mainly occurring at the level of transcriptional elongation. Blockage of FLRT3 by siRNA decreased survival of ECs and their arrangement into capillary-like structures but enhanced cell migration and wound closure in wound healing assay. Bifunctional role of FLRT3 in repulsive vs. adhesive cell signaling has been already detected during embryogenesis and neuronal growth, and depends on its interactions either with UNC5B or another FLRT3 expressed by adjacent cells. In conclusion, our findings demonstrate that besides regulating neuronal cell outgrowth and morphogenesis, FLRT3 has a novel role in ECs via regulating VEGF-stimulated EC-survival, migration, and tube formation. Thus, FLRT3 becomes a new member of the axon guidance-related factors which participate in the VEGF-signaling and regulation of the EC functions.

Magnetic resonance imaging examinations are frequently carried out using contrast agents to improve the image quality. Practically all clinically used contrast agents are based on paramagnetic metals and lack in selectivity and specificity. A group of stable organic radicals, nitroxides, has raised interest as new metal-free contrast agents for MRI. Their structures can easily be modified to incorporate different functionalities. In the present study, a stable nitroxide TEEPO (2,2,6,6-tetraethylpiperidin-1-oxyl) was linked to a glucose moiety (Glc) to construct a water-soluble, potentially tumor-targeting compound with contrast-enhancing ability. The ability was assessed with in vivo MRI experiments. The constructed TEEPO-Glc agent proved to shorten the T1 relaxation time in tumor, while the T1 time in healthy brain tissue remained the same. The results indicate the potential of TEEPO-Glc as a valuable addition to the growing field of metal-free contrast enhancement in MRI-based diagnostics.

Vascular endothelial growth factors (VEGFs) are key mediators of endothelial cell (EC) function in angiogenesis. Emerging knowledge also supports the involvement of axon guidance-related factors in the regulation of angiogenesis and vascular patterning. In the current study, we demonstrate that fibronectin and leucine-rich transmembrane protein-3 (FLRT3), an axon guidance-related factor connected to the regulation of neuronal cell outgrowth and morphogenesis but not to VEGF-signaling, was upregulated in ECs after VEGF binding to VEGFR2. We found that FLRT3 exhibited a transcriptionally paused phenotype in non-stimulated human umbilical vein ECs. After VEGF-stimulation its nascent RNA and mRNA-levels were rapidly upregulated suggesting that the regulation of FLRT3 expression is mainly occurring at the level of transcriptional elongation. Blockage of FLRT3 by siRNA decreased survival of ECs and their arrangement into capillary-like structures but enhanced cell migration and wound closure in wound healing assay. Bifunctional role of FLRT3 in repulsive vs. adhesive cell signaling has been already detected during embryogenesis and neuronal growth, and depends on its interactions either with UNC5B or another FLRT3 expressed by adjacent cells. In conclusion, our findings demonstrate that besides regulating neuronal cell outgrowth and morphogenesis, FLRT3 has a novel role in ECs via regulating VEGF-stimulated EC-survival, migration, and tube formation. Thus, FLRT3 becomes a new member of the axon guidance-related factors which participate in the VEGF-signaling and regulation of the EC functions.

Cardiovascular gene therapy aims to treat coronary and peripheral artery disease, heart failure, and arrhythmia. The chosen transgene, delivery method, gene therapy vector type, high-quality vector production, and dose are all determining factors of the therapeutic outcome. High-resolution vascular imaging and increased knowledge of vascular biology in physiological and pathological conditions enable the finding of novel molecular targets for cardiovascular gene therapy. Transgenic and knockout mouse models have provided researchers several powerful experimental tools for studying the effects of single genes on cardiovascular diseases. For preclinical efficacy, safety, and toxicology studies, large animal models are needed before entering into clinical testing. This review focuses on commonly used animal models in cardiovascular gene therapy and describes recent advancements in the field of vascular biology. Emphasis is also given on high-resolution imaging of microvasculature and its impact on our knowledge of vascular function.

Heart has a wide lymphatic network but the importance of cardiac lymphatic system in heart diseases has remained unclear. Vascular Endothelial Growth Factor Receptor 3 (VEGFR3) is a key molecule in the development and maintenance of cardiac lymphatic vessels. Here we characterized the role of VEGFR3 in healthy hearts and after myocardial infarction (MI) by using sVEGFR3 transgenic mice expressing a soluble decoy VEGFR3 under K14 promoter and Chy mice which have an inactivating mutation in the VEGFR3 gene. Cardiac lymphatic vessels were significantly dilated in the healthy hearts of sVEGFR3 mice when compared to controls. Lymphatic vessels formed large sheet-like structures in Chy mice. Attenuated VEGFR3 signaling led to a more severe MI predisposing to a significantly higher mortality in sVEGFR3 mice than in control mice. sVEGFR3 mice displayed intramyocardial hemorrhages in the infarcted area indicating hyperpermeability of the vasculature. Furthermore, novel MRI methods TRAFF2 and TRAFF4 and histological analysis revealed a modified structure of the fibrotic infarcted area in sVEGFR3 mice. In conclusion, the downregulation of VEGFR3 signaling modifies the structure of cardiac lymphatic network and causes vascular leakiness and increased mortality after MI.

Background A baculovirus vector is capable of efficiently transducing many nondiving and diving cell types. However, the potential of baculovirus is restricted for many gene delivery applications as a result of the transient gene expression that it mediates. The plasmid-based Sleeping Beauty (SB) transposon system integrates transgenes into target cell genome efficiently with a genomic integration pattern that is generally considered safer than the integration of many other integrating vectors; yet efficient delivery of therapeutic genes into cells of target tissues in vivo is a major challenge for nonviral gene therapy. In the present study, SB was introduced into baculovirus to obtain novel hybrid vectors that would combine the best features of the two vector systems (i.e. effective gene delivery and efficient integration into the genome), thus circumventing the major limitations of these vectors. Methods We constructed and optimized SB-baculovirus hybrid vectors that bear either SB100x transposase or SB transposon in the forward or reverse orientations with respect to the viral backbone The functionality of the novel hybrid vectors was investigated in cell cultures and in a proof-of-concept study in the mouse eye. Results The hybrid vectors showed high and sustained transgene expression that remained stable and demonstrated no signs of decline during the 2 months follow-up in vitro. These results were verified in the mouse eye where persistent transgene expression was detected two months after intravitreal injection. Conclusions Our results confirm that (i) SB-baculovirus hybrid vectors mediate long-term gene expression in vitro and in vivo, and (ii) the hybrid vectors are potential new tools for the treatment of ocular diseases. Copyright © 2014 John Wiley & Sons, Ltd.

Baculoviruses are insect-specific viruses commonly found in nature. They are not able to replicate in mammalian cells but can transduce them when equipped with an appropriate mammalian cell active expression cassette. Although the viruses have been studied in several types of mammalian cells from different origins, the receptor that baculovirus uses to enter or interact with mammalian cells has not yet been identified. Due to the wide tropism of the virus, the receptor has been suggested to be a generally found cell surface molecule. In this article, we investigated the interaction of baculovirus and mammalian cell surface heparan sulfate proteoglycans (HSPG) in more detail. Our data show that baculovirus requires HSPG sulfation, particularly N - and 6- O -sulfation, to bind to and transduce mammalian cells. According to our results, baculovirus binds specifically to syndecan-1 (SDC-1) but does not interact with SDC-2 to SDC-4 or with glypicans. Competition experiments performed with SDC-1 antibody or recombinant SDC-1 protein inhibited baculovirus binding, and SDC-1 overexpression enhanced baculovirus-mediated transduction. In conclusion, we show that SDC-1, a commonly found cell surface HSPG molecule, has a role in the binding and entry of baculovirus in vertebrate cells. The results presented here reveal important aspects of baculovirus entry and can serve as a basis for next-generation baculovirus vector development for gene delivery.

Some cell types are more susceptible to viral gene transfer or virus infection than others, irrespective of the number of viral receptors or virus binding efficacy on their surfaces. In order to characterize the cell-line-specific features contributing to efficient virus entry, we studied two cell lines (Ea.hy926 and MG-63) that are nearly nonpermissive to insect-specific baculovirus (BV) and the human enterovirus echovirus 1 (EV1) and compared their characteristics with those of a highly permissive (HepG2) cell line. All the cell lines contained high levels of viral receptors on their surfaces, and virus binding was shown to be efficient. However, in nonpermissive cells, BV and its receptor, syndecan 1, were unable to internalize in the cells and formed large aggregates near the cell surface. Accordingly, EV1 had a low infection rate in nonpermissive cells but was still able to internalize the cells, suggesting that the postinternalization step of the virus was impaired. The nonpermissive and permissive cell lines showed differential expression of syntenin, filamentous actin, vimentin, and phosphorylated protein kinase C subtype α (pPKCα). The nonpermissive nature of the cells could be modulated by the choice of culture medium. RPMI medium could partially rescue infection/transduction and concomitantly showed lower syntenin expression, a modified vimentin network, and altered activities of PKC subtypes PKCα and PKCε. The observed changes in PKCα and PKCε activation caused alterations in the vimentin organization, leading to efficient BV transduction and EV1 infection. This study identifies PKCα, PKCε, and vimentin as key factors affecting efficient infection and transduction by EV1 and BV, respectively.

Intracranial aneurysms are common but are generally untreated, and their rupture can lead to subarachnoid hemorrhage. Because of the poor prognosis associated with subarachnoid hemorrhage, preventing the progression of intracranial aneurysms is critically important. Intracranial aneurysms are caused by chronic inflammation of the arterial wall due to macrophage infiltration triggered by monocyte chemoattractant protein-1 (MCP-1), macrophage activation mediated by the transcription factor nuclear factor kappa B (NF-kappa B), and inflammatory signaling involving prostaglandin E-2 (PGE(2)) and prostaglandin E receptor subtype 2 (EP2). We correlated EP2 and cyclooxygenase-2 (COX-2) with macrophage infiltration in human intracranial aneurysm lesions. Monitoring the spatiotemporal pattern of NF-kappa B activation during intracranial aneurysm development in mice showed that NF-kappa B was first activated in macrophages in the adventitia and in endothelial cells and, subsequently, in the entire arterial wall. Mice with a macrophage-specific deletion of Ptger2 (which encodes EP2) or macrophage-specific expression of an I kappa B alpha mutant that restricts NF-kappa B activation had fewer intracranial aneurysms with reduced macrophage infiltration and NF-kappa B activation. In cultured cells, EP2 signaling cooperated with tumor necrosis factor-alpha (TNF-alpha) to activate NF-kappa B and synergistically induce the expression of proinflammatory genes, including Ptgs2 (encoding COX-2). EP2 signaling also stabilized Ccl2 (encoding MCP-1) by activating the RNA-stabilizing protein HuR. Rats administered an EP2 antagonist had reduced macrophage infiltration and intracranial aneurysm formation and progression. This signaling pathway in macrophages thus facilitates intracranial aneurysm development by amplifying inflammation in intracranial arteries. These results indicate that EP2 antagonists may therefore be a therapeutic alternative to surgery.

Vascular Endothelial Growth Factors (VEGFs) are promising molecules for the treatment of ischemic diseases by pro-angiogenic therapy. Snake venom VEGFs are a novel subgroup with unique receptor binding profiles and as such are potential new therapeutic agents. We determined the ligand-receptor interactions, gene regulation and angiogenic properties of Vipera ammodytes venom VEGF, Vammin, and compared it to the canonical angiogenic factor VEGF-A to evaluate the use of Vammin for therapeutic angiogenesis. Vammin efficiently induced VEGFR-2 mediated proliferation and expression of genes associated with proliferation, migration and angiogenesis. VEGF-A165 and especially VEGF-A109 induced less pronounced effects. Vammin regulates a number of signaling pathways by inducing the expression of NR4A family nuclear receptors and regulators of calcium signaling and MAP kinase pathways. Interestingly, MARC1, which encodes an enzyme discovered to catalyze reduction of nitrate to NO, was identified as a novel VEGFR-2 regulated gene. In rabbit skeletal muscle adenoviral delivery of Vammin induced prominent angiogenic responses. Both the vector dose and the co-receptor binding of the ligand were critical parameters controlling the type of angiogenic response from sprouting angiogenesis to vessel enlargement. Vammin induced VEGFR-2/NRP-1 mediated signaling more effectively than VEGF-A, consequently it is a promising candidate for development of pro-angiogenic therapies., published version, peerReviewed

Histamine and vascular endothelial growth factor A (VEGF) are central regulators in vascular pathologies. Their gene regulation leading to vascular remodeling has remained obscure. In this study, EC regulation mechanisms of histamine and VEGF were compared by RNA sequencing of primary endothelial cells (ECs), functional in vitro assays and in vivo permeability mice model.By RNA sequencing, similar transcriptional alterations of genes involved in activation of primary ECs, cell proliferation and adhesion were observed between histamine and VEGF. Seventy-six commonly regulated genes were found, representing similar to 53% of all VEGF-regulated transcripts and similar to 26% of all histamine-regulated transcripts. Both factors regulated tight junction formation and expression of pro-angiogenic transcription factors (TFs) affecting EC survival, migration and tube formation. Novel claudin-5 upstream regulatory genes were identified. VEGF was demonstrated to regulate expression of SNAI2, whereas pro-angiogenic TFs NR4A1, MYCN and RCAN1 were regulated by both histamine and VEGF. Claudin-5 was shown to be regulated VEGFR2/PI3K-Akt dependently by VEGF and PI3K-Akt independently by histamine. Interleukin-8 was shown to downregulate claudin-5 by histamine. Additionally, SNAI2, NR4A1 and MYCN were shown to mediate EC survival, migration and tube formation and to regulate expression of claudin-5. Further systemic delivery of VEGF and histamine was shown to induce a fast vascular hyperpermeability response in intact vasculature of C57/Bl6 mice followed by regulation of NR4A1 and MYCN.Our study identifies novel claudin-5 upstream regulatory genes of histamine and VEGF that induce cellular angiogenic processes. Our results increase knowledge of angiogenic EC phenotype and provide novel treatment targets for vascular pathologies.

The budded form of baculovirus Autographa californica multiple nucleopolyhedrovirus is used widely in biotechnological applications. In this study, we observed the morphology of baculovirus in nanometer scale by atomic force microscopy. Additionally, the correlation between transduction efficiency and virus stock storage time was evaluated. By atomic force microscopy, asymmetrical baculovirus particles with enlarged head regions were detected. Observed virus stocks contained variable-length particles, 256 ± 40 nm, along with disintegrated particles and/or cellular components. Long-term storage of stocks led to virus aggregation and decreased cellular entry and transgene expression in mammalian cells. Significantly, baculovirus vectors bearing a 33.5-kb insert induced formation of elongated nucleocapsids, indicating that viral dsDNA genome size influences particle size. Taken together, our results provide the first 3D-characterization of baculovirus morphology and insight into its stability after storage.

Baculovirus, Autographa californica multiple nucleopolyhedrovirus (AcMNPV), has the ability to transduce mammalian cell lines without replication. The general objective of this study was to detect the transcription and expression of viral immediate-early genes in human cells and to examine the interactions between viral components and subnuclear structures. Viral capsids were seen in large, discrete foci in nuclei of both dividing and non-dividing human cells. Concurrently, the transcription of viral immediate-early transregulator genes (ie-1, ie-2) and translation of IE-2 protein were detected. Quantitative microscopy imaging and analysis showed that virus transduction altered the size of promyelocytic leukaemia nuclear bodies, which are suggested to be involved in replication and transcription of various viruses. Furthermore, altered distribution of the chromatin marker Draq5 and histone core protein (H2B) in transduced cells indicated that the virus was able to induce remodelling of the host cell chromatin. To conclude, this study shows that the non-replicative insect virus, baculovirus and its proteins can induce multiple changes in the cellular machinery of human cells.

Cardiovascular gene therapy can be used to grow neovessels into ischemic tissues, reduce neointimal hyperplasia, improve cardiomyocyte function, and to treat hypertension and dyslipidemias. Human adenovirus (Ad) serotypes 2 and 5 are commonly used vectors in clinical trials because of their high gene transfer efficiency, ease of production, and well-known virus–cell biology. Ad vectors can be administrated to tissues via intramyocardial and intramuscular injections, coronary infusion, and pericardial or systemic delivery. Delivery method, serotype, and vector dosage are critical determinants of the therapeutic outcome. Animal models for cardiovascular diseases (CVDs) are essential in order to develop new therapeutic strategies. Increased understanding of the pathogenesis of CVDs enables identification of novel molecular targets for therapy. In the following chapter, methods and animal models used for gene therapy of CVDs are described in the context of Ad vector–mediated transgene delivery.

Aims Slit2 is a possible modulator of VEGF-induced angiogenesis, but its effects have not been tested on large animal models. We studied the effect of Slit2 on therapeutic angiogenesis induced by VEGF receptor 2 (VEGFR2) ligands Vammin and VEGF-DΔNΔC in vivo in rabbit skeletal muscles. The Slit2 target genes were also studied by RNA sequencing in endothelial cells. Methods and Results Adenoviral intramuscular gene transfers were performed into New Zealand White rabbit hindlimbs. Confocal and multiphoton microscopes were used for blood vessel imaging. Signaling experiments and gene expression analyses were performed to study mechanisms of Slit2 action. Slit2 decreased VEGFR2-mediated vascular permeability. Slit2 also reduced VEGFR2-mediated increase in blood perfusion and capillary enlargement, whereas sprouting of the capillaries was increased. Slit2 gene transfer alone did not have any effects on vascular functions or morphology. VEGFR2 activation was not affected by Slit2, but eNOS phosphorylation was diminished. The transcriptome profiling showed Slit2 down-regulating angiogenesis-related genes such as Nuclear receptor subfamily 4 group A member 1 (NR4A1) and Stanniocalcin-1 (STC-1) as well as genes related to endothelial cell migration and vascular permeability. Conclusion Combining Slit2 with VEGFs adjusts VEGFR2-mediated angiogenic effects into a more physiological direction. This possibly allows the use of higher VEGF vector doses to achieve a more widespread vector and VEGF distribution in the target tissues leading to a better therapeutic outcome while reducing excess vascular permeability.

(1), we investigated the interaction ofbaculovirus and mammalian cell surface heparan sulfate pro-teoglycans (HSPG). The data show that baculovirus requiresHSPG sulfation, particularly N- and 6-O-sulfation, to bind andtransduce mammalian cells. We also show that baculovirus asso-ciates specifically with syndecan-1 (SDC-1) but not with othersyndecans or glypicans.As discussed in the article, HS has previously been shown to beinvolved in glycoprotein 64 (gp64)-mediated baculovirus bindingonto mammalian cells. Heparin and heparinase I and II treatmentof cells have also been shown to prevent the virus binding (2, 3).The role of HS in baculovirus entry was further studied in ourarticle (1). Binding and baculovirus-mediated gene delivery wereseen to decrease after NaClO

“Epigenetherapy” alters epigenetic status of the targeted chromatin and modifies expression of the endogenous therapeutic gene. In this study we used lentiviral in vivo delivery of small hairpin RNA (shRNA) into hearts in a murine infarction model. shRNA complementary to the promoter of vascular endothelial growth factor (VEGF-A) was able to upregulate endogenous VEGF-A expression. Histological and multiphoton microscope analysis confirmed the therapeutic effect in the transduced hearts. Magnetic resonance imaging (MRI) showed in vivo that the infarct size was significantly reduced in the treatment group 14 days after the epigenetherapy. Importantly, we show that promoter-targeted shRNA upregulates all isoforms of endogenous VEGF-A and that an intact hairpin structure is required for the shRNA activity. In conclusion, regulation of gene expression at the promoter level is a promising new treatment strategy for myocardial infarction and also potentially useful for the upregulation of other endogenous genes.

Virus-induced alterations in cell morphology play important roles in the viral life cycle. To examine the intracellular events of coxsackievirus B3 (CVB3) infection, green monkey kidney (GMK) cells were either inoculated with the virus or transfected with the viral RNA. Various microscopic and flow cytometric approaches demonstrated the emergence of CVB3 capsid proteins at 8 h posttransfection, followed by morphological transformation of the cells. The morphological changes included formation of membranous protrusions containing viral capsids, together with microtubules and actin. Translocation of viral capsids into these protrusions was sensitive to cytochalasin D, suggesting the importance of actin in the process. Three-dimensional (3D) live-cell imaging demonstrated frequent contacts between cellular protrusions and adjacent cells. Markedly, in spite of an increase in the cellular viral protein content starting 8 h postinfection, no significant decrease in cell viability or increase in the amount of early apoptotic markers was observed by flow cytometry by 28 h postinfection. Comicroinjection of viral RNA and fluorescent dextran in the presence of neutralizing virus antibody suggested that these protrusions mediated the spread of infection from one cell to another prior to virus-induced cell lysis. Altogether, the CVB3-induced cellular protrusions could function as a hitherto-unknown nonlytic mechanism of cell-to-cell transmission exploited by enteroviruses.

The prototype baculovirus, Autographa californica multiple nucleopolyhedrovirus, an insect pathogen, holds great potential as a gene therapy vector. To develop transductional targeting and gene delivery by baculovirus, we focused on characterizing the nature and regulation of its uptake in human cancer cells. Baculovirus entered the cells along fluid-phase markers from the raft areas into smooth-surfaced vesicles devoid of clathrin. Notably, regulators associated with macropinocytosis, namely EIPA, Pak1, Rab34, and Rac1, had no significant effect on viral transduction, and the virus did not induce fluid-phase uptake. The internalization and nuclear uptake was, however, affected by mutants of RhoA, and of Arf6, a regulator of clathrin-independent entry. Furthermore, the entry of baculovirus induced ruffle formation and triggered the uptake of fluorescent E. coli bioparticles. To conclude, baculovirus enters human cells via a clathrin-independent pathway, which is able to trigger bacterial uptake. This study increases our understanding of virus entry strategies and gives new insight into baculovirus-mediated gene delivery in human cells. peerReviewed

Background Tumor-associated cells and vasculature express attractive molecular markers for site-specific vector targeting. To attain tumor-selective tropism, we recently developed a baculovirus vector displaying the lymphatic homing peptide LyP-1, originally identified by ex vivo/in vivo screening of phage display libraries, on the viral envelope by fusion to the transmembrane anchor of vesicular stomatitis virus G-protein. Methods In the present study, we explored the specificity and kinetics of viral binding and internalization as well as in vivo tumor homing of the LyP-1 displaying virus to elucidate the applicability of baculovirus for targeted therapies. Results We demonstrated that the LyP-1 peptide contributes to saturable binding of baculovirus in human MDA-MB-435 and HepG2 carcinoma cells and escalates the kinetics of viral internalization leading to earlier nuclear accumulation and enhanced transgene expression. The LyP-1 displaying virus also showed stronger competitiveness against transduction with wild-type baculovirus, suggesting involvement of a specific receptor in cellular attachment and entry. Following intravenous injections, the modified virus accumulated within the human MDA-MB-435 and MDA-MB-231 carcinoma xenografts in mice with higher specificity and efficiency than the control virus. Targeting of the modified virus was more specific in the MDA-MB-435 than in the MDA-MB-231 xenografts as demonstrated by higher tumor accumulation and lower distribution in nontarget organs. No apparent cytotoxicity was associated with the surface modification. Conclusions This first demonstration of in vivo tumor targeting of a systemically administered, tropism-modified baculoviral vector highlights the potential of baculovirus-mediated targeted therapies. Copyright © 2008 John Wiley & Sons, Ltd.