Intramuscular Injection Of Plasmid DNA Research Articles

Enhancedanti-tumor efficacy through a combination of intramuscularly expressed DNA vaccine and plasmid-encoded PD-1 antibody.

Immune check inhibitors (ICIs) have moderate response rates (~20%-30%) in some malignancies clinically, and, when used in combination with other immunotherapeutic strategies such as DNA tumor vaccines, there is evidence to suggest that they could optimize the efficacy of cancer treatment. In this study, we validated that intramuscular injection of plasmid DNA (pDNA) encoding OVA combined with pDNA encoding α-PD-1 (abbreviated as α-PD-1 in the following treatment groups) may enhance therapeutic efficacy by means of in situ gene delivery and enhanced muscle-specific potent promoter. Mice treated with pDNA-OVA or pDNA-α-PD-1 alone showed weak tumor inhibition in the MC38-OVA-bearing model. In comparison, the combined treatment of pDNA-OVA and pDNA-α-PD-1 resulted in superior tumor growth inhibition and a significantly improved survival rate of over 60% on day 45. In the B16-F10-OVA metastasis model, the addition of the DNA vaccine enhanced resistance to tumor metastasis and increased the populations of CD8+ T cells in blood and spleen. In conclusion, the current research shows that a combination of pDNA-encoded PD-1 antibody and DNA vaccine expressed in vivo is an efficient, safe, and economical strategy for tumor therapy.

Construction of Plasmid DNA Expressing Two Isoforms of Insulin-Like Growth Factor-1 and Its Effects on Skeletal Muscle Injury Models.

Insulin-like growth factor-1 (IGF-1) plays a significant role in the development of various organs, and several studies have suggested that IGF-1 isoforms, IGF-1 Ea and IGF-1 Ec, are expressed in skeletal muscle to control its growth. In this study, we designed a novel nucleotide sequence, IGF-1-X10, consisting of IGF-1 exons and introns to simultaneously express both IGF-1 Ea and IGF-1 Ec. When transfected into human cells, the expression of both isoforms was observed at the transcript and protein levels. In an animal study, intramuscular injection of plasmid DNA comprising IGF-1-X10 induced the expression of IGF-1 Ea and IGF-1 Ec, leading to the production of functional IGF-1 protein. Finally, the efficacy of this plasmid DNA was tested in a cardiotoxin (CTX)-mediated muscle injury model and age-related muscle atrophy model. We found that IGF-1-X10 increased the muscle mass and controlled several key factors involved in the muscle atrophy program in both models. Taken together, these data suggest that IGF-1-X10 may be utilized in the form of gene therapy for the treatment of various muscle diseases related to IGF-1 deficiency.

Gene therapy with B-cell maturation antigen/CD3 bispecific antibody encoding plasmid DNA for treating multiple myeloma.

The delivery of bispecific antibodies (BsAbs) targeting B-cell maturation antigen (BCMA) and CD3 using the gene therapy approach is a promising alternative for BsAb administration in patients with multiple myeloma (MM). In the present study, we evaluated the efficacy of this approach using a xenograft model. Tumour growth was significantly delayed in mice treated with single electroporation-enhanced intramuscular injection of plasmid DNA encoding BCMA/CD3 BsAb in contrast to the vehicle control-treated group. Limited toxicity was observed following treatment. This study demonstrates that the gene therapy-based approach for the delivery of BCMA/CD3 BsAb is effective and safe for the treatment of MM.

Intramuscular injection of a plasmid DNA vector expressing hepatocyte growth factor (HGF) ameliorated pain symptoms by controlling the expression of pro-inflammatory cytokines in the dorsal root ganglion

Hepatocyte growth factor (HGF) is a secretory protein that is involved in various biological activities such as angiogenesis, neuroprotection, and anti-inflammatory effects. Intramuscular injection of an HGF-encoding plasmid DNA (pCK-HGF-X7) has been shown to produce pain-relieving effects in a rodent model and patients with neuropathic pain.To further investigate the underlying mechanism, we investigated the anti-inflammatory effects of HGF in the context of neuropathic pain. Consistent with previous data, intramuscular injection of pCK-HGF-X7 showed pain relieving effects up to 8 weeks and pharmacological blockade of the c-Met receptor hindered this effect, which suggest that the analgesic effect was c-Met receptor-dependent. At the histological level, macrophage infiltration in the dorsal root ganglion (DRG) was significantly decreased in the pCK-HGF-X7 injected group. Moreover, HGF treatment significantly downregulated the LPS-mediated induction of pro-inflammatory cytokines in primary cultured DRG neurons. Taken together, these data suggest that HGF-encoding plasmid DNA attenuates neuropathic pain via controlling the expression of pro-inflammatory cytokines.

Hepatocyte growth factor is necessary for efficient outgrowth of injured peripheral axons in in vitro culture system and in vivo nerve crush mouse model

Hepatocyte growth factor (HGF) is a neurotrophic factor and its role in peripheral nerves has been relatively unknown. In this study, biological functions of HGF and its receptor c-met have been investigated in the context of regeneration of damaged peripheral nerves. Axotomy of the peripheral branch of sensory neurons from embryonic dorsal root ganglia (DRG) resulted in the increased protein levels of HGF and phosphorylated c-met. When the neuronal cultures were treated with a pharmacological inhibitor of c-met, PHA665752, the length of axotomy-induced outgrowth of neurite was significantly reduced. On the other hand, the addition of recombinant HGF proteins to the neuronal culture facilitated axon outgrowth. In the nerve crush mouse model, the protein level of HGF was increased around the injury site by almost 5.5-fold at 24 h post injury compared to control mice and was maintained at elevated levels for another 6 days. The amount of phosphorylated c-met receptor in sciatic nerve was also observed to be higher than control mice. When PHA665752 was locally applied to the injury site of sciatic nerve, axon outgrowth and injury mediated induction of cJun protein were effectively inhibited, indicating the functional involvement of HGF/c-met pathway in the nerve regeneration process. When extra HGF was exogenously provided by intramuscular injection of plasmid DNA expressing HGF, axon outgrowth from damaged sciatic nerve and cJun expression level were enhanced. Taken together, these results suggested that HGF/c-met pathway plays important roles in axon outgrowth by directly interacting with sensory neurons and thus HGF might be a useful tool for developing therapeutics for peripheral neuropathy.

Hepatocyte Growth Factor Regulates the miR-206-HDAC4 Cascade to Control Neurogenic Muscle Atrophy following Surgical Denervation in Mice

Hepatocyte growth factor (HGF) has been well characterized for its roles in the migration of muscle progenitors during embryogenesis and the differentiation of muscle stem cells, but its function in adult neurogenic muscle atrophic conditions is poorly understood. Here we investigated whether HGF/c-met signaling has any effects on muscle-atrophic conditions. It was found that HGF expression was upregulated in skeletal muscle tissue following surgical denervation and in hSOD1-G93A transgenic mice showing severe muscle loss. Pharmacological inhibition of the c-met receptor decreased the expression level of pri-miR-206, enhanced that of HDAC4 and atrogenes, and resulted in increased muscle atrophy. In C2C12 cells, HGF inhibited phosphorylation of Smad3 and relieved TGF-β-mediated suppression of miR-206 expression via JNK. When extra HGF was exogenously provided through intramuscular injection of plasmid DNA expressing HGF, the extent of muscle atrophy was reduced, and the levels of all affected biochemical markers were changed accordingly, including those of primary and mature miR-206, HDAC4, and various atrogenes. Taken together, our finding suggested that HGF might play an important role in regard to neurogenic muscle atrophy and that HGF might be used as a platform to develop therapeutic agents for neuromuscular disorders.

A suicidal strain of Listeria monocytogenes is effective as a DNA vaccine delivery system for oral administration.

In this study we determined the in vivo activity of model ovalbumin vaccines delivered by direct intramuscular delivery of plasmid DNA or oral delivery using a recombinant suicidal Listeria monocytogenes strain (rsΔ2). In a previous report we described how rsΔ2 is capable of delivering luciferase, as protein or DNA, in vitro, into non-dividing intestinal epithelial cells (Kuo et al., 2009). This is achieved by engineering a dual expression shuttle vector, pDuLX-Luc, that replicates in E. coli and rsΔ2 and drives gene expression from the Listeria promoter (Phly) as well as the eukaryotic cytomegalovirus promoter (CMV), thereby delivering both protein and plasmid DNA to the cell cytoplasm. For the current in vivo study rsΔ2 containing pDuLX-OVA was used to deliver both ovalbumin protein and the mammalian expression plasmid by the oral route. Controls were used to investigate the activity of this system versus positive and negative controls, as well as quantifying activity against direct intramuscular injection of expression plasmids. Oral administration of rsΔ2(pDuLX-OVA) produced significant titres of antibody and was effective at inducing targeted T-cell lysis (approximately 30% lysis relative to an experimental positive control, intravenous OVA-coated splenocytes+lipopolysaccharide). Intramuscular injection of plasmids pDuLX-OVA or p3L-OVA (which lacks the prokaryotic promoter) also produced significant CTL-mediated cell lysis. The delivery of the negative control rsΔ2 (pDuLX-Luc) confirmed that the observed activity was induced specifically by the ovalbumin vaccination. The data suggest that the oral activity of rsΔ2(pDuLX-OVA) is explained by delivery of OVA protein, expressed in rsΔ2 from the prokaryotic promoter present in pDuLX-OVA, but transfection of mammalian cells in vivo may also play a role. Antibody titres were also produced by oral delivery (in rsΔ2) of the p3L-OVA plasmid in which does not include a prokaryotic promoter.

Protective effect and antibody response of DNA vaccine against salmonid alphavirus 3 (SAV3) in Atlantic salmon.

This work reports the effect of two DNA vaccines against salmonid alphavirus 3 (SAV3) in Atlantic salmon. Presmolts were vaccinated by intramuscular injection of plasmids encoding the SAV3 structural polyprotein C-E3-E2-6K-E2 (pCSP), E2 only (pE2), or plasmid without insert (pcDNA3.3). E2 is expressed at the surface of cells transfected with pCSP and internally in cells transfected with pE2. A commercial vaccine based on inactivated SAV (NCPD) was used for comparison. At 10weeks post-vaccination, only fish vaccinated with pCSP showed antibody against E2 and virus-neutralizing activity. Vaccinated fish were infected with SAV3 to determine protection by virus quantitation in serum after 7days and scoring of pathological changes after 21days. Fish vaccinated with both pCSP and NCPD vaccines showed significant virus reduction in serum, while fish vaccinated with pE2 did not. All fish vaccinated with pcDNA3.3 and pE2 showed pathological changes in organs typical of PD, 60% of fish vaccinated with NCPD showed PD pathology, while fish vaccinated with pCSP did not show PD pathology. Taken together, DNA vaccination with pCSP provided strong protection for salmon against SAV3 infection, which in part may be due to production of virus-neutralizing antibodies.

Role of vascular endothelial growth factor in inducing production of angiopoetin-1 - in vivo study in Fisher rats.

The aim of the study was to investigate how an intramuscular injection of plasmids with genes coding various pro-angiogenic factors: angiopoetin-1 (ANGPT1), vascular endothelial growth factor (VEGF165) and hepatic growth factor (HGF), influences the production of ANGPT1. 40 Healthy Fisher rats received i.m. injections containing plasmids encoding pro-angiogenic genes in thigh muscles. They were divided into four equal groups. The first group received the plANGPT1 plasmid and the second group- the pIRES/ANGPT1/VEGF165 bicistronic plasmid. The pIRES/VEGF165/HGF bicistronic plasmid was administered to the third group and an empty plasmid (control group) to the fourth group. The animals were euthanized after 12 weeks. In each group, the number of vessels stained with the anti-ANGPT1 antibody was assessed under an optical microscope. The anti-ANGPT1 antibodies stained the vessels in all the groups. There were on average 14.1 ±2.3 vessels in the the plANGPT1 group, 32.5 ±10.5 in the pl/RESANGPT1/VEGF group and 30.8 ±13.3 in the plRES/HGV/VEGF group. There were on average 7.3 ±2.3 stained vessels (p < 0.0001) in the control group . The VEGF plays a role in the induction of the production of ANGPT1. The administration of plasmids only encoding ANGPT1 does not induce its production.

Plasmids encoding protein aggregation domains act as molecular adjuvants for DNA vaccines.

DNA vaccines provide high tolerability and safety but commonly suffer from suboptimal immunogenicity. We previously reported that a plasmid vector (pATRex), encoding the DNA sequence for the von Willebrand I/A domain of the tumor endothelial marker-8 (TEM8) when given in combination with plasmid-encoded tumor antigens acted as a powerful molecular adjuvant enhancing immunity against breast and melanoma tumors. In the present study we addressed two unsolved issues; would the adjuvant action of pATRex extend to a DNA vaccine against infectious disease and, if so, what is the mechanistic basis for pATRex adjuvant action? Here we show in a murine malaria vaccine model that co-administration of pATRex potentiates antibody production elicited by an intramuscular injection of plasmid encoding Plasmodium yoelii merozoite surface protein 4/5 (PyMSP4/5). pATRex enhanced the B-cell response and induced increased IgG1 production consistent with TH2 polarization of the DNA vaccine response. To explore the mechanism of adjuvant action, cells were transfected in vitro with pATRex and this resulted in formation of insoluble intracellular aggregates and apoptotic cell death. Using a structural modeling approach we identified a short peptide sequence (α3-β4) within ATRex responsible for protein aggregation and confirmed that transfection of cells with plasmid encoding this self-assembling peptide similarly triggered intracellular aggregates, caspase activation and cell death. Plasmids encoding aggregation-promoting domains induce formation of insoluble intracellular aggregates that trigger caspase activation and apoptotic cell death leading to activation of the innate immune system thereby acting as genetic adjuvants.

Pluronic L64-mediated stable HIF-1α expression in muscle for therapeutic angiogenesis in mouse hindlimb ischemia.

Intramuscular injection of plasmid DNA (pDNA) to express a therapeutic protein is a promising method for the treatment of many diseases. However, the therapeutic applications are usually hindered by gene delivery efficiency and expression level. In this study, critical factors in a pDNA-based gene therapy system, such as gene delivery materials, a therapeutic gene, and its regulatory elements, were optimized to establish an integrated system for the treatment of mouse hindlimb ischemia. The results showed that Pluronic® L64 (L64) was an efficient and safe material for gene delivery into mouse skeletal muscle. It also showed intrinsic ability to promote in vivo angiogenesis in a concentration-dependent manner, which might be through the activation of nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB)-regulated angiogenic factors. The combination of 0.1% L64 with a hybrid gene promoter (pSC) increased the gene expression level, elongated the gene expression duration, and enhanced the number of transfected muscle fibers. In mice ischemic limbs, a gene medicine (pSC-HIF1αtri/L64) composed of L64 and pSC-based expression plasmid encoding hypoxia-inducible factor 1-alpha triple mutant (HIF-1αtri), improved the expression of stable HIF-1α, and in turn, the expression of multiple angiogenic factors. As a result, the ischemic limbs showed accelerated function recovery, reduced foot necrosis, faster blood reperfusion, and higher capillary density. These results indicated that the pSC-HIF1αtri/L64 combination presented a potential and convenient venue for the treatment of peripheral vascular diseases, especially critical limb ischemia.

Protective effect of a DNA vaccine containing an open reading frame with homology to an ABC-type transporter present in the genomic island 3 of Brucella abortus in BALB/c mice

The immunogenicity of a DNA vaccine containing an open reading frame (ORF) of genomic island 3 (GI-3), specific for Brucella abortus and Brucella melitensis, has been examined. Intramuscular injection of plasmid DNA carrying the open reading frame with homology to an ABC-type transporter (pV278a) into BALB/c mice elicited both humoral and cellular immune responses. Mice injected with pV278a had a dominant immunoglobulin G2a (IgG2a) response. This DNA vaccine elicited a T-cell-proliferative response and induced significant levels of interferon gamma (INF-γ) upon restimulation with recombinant 278a protein. Upon stimulation with an appropriate recombinant protein or crude Brucella protein, the vaccine did not induce IL-4, suggesting a typical T-helper (TH1) response. Furthermore, the vaccine induced protection in BALB/c mice when challenged with the virulent strain Brucella abortus 2308. Taken together, these data suggest that DNA vaccination offers an improved delivery of the homologous of an ABC-type transporter antigen, and provides the first evidence of a protective effect of this antigen in the construction of vaccines against B. abortus.

Electro‐gene‐transfer as a new tool for cancer immunotherapy in animals

The concept of vaccines based on the direct inoculation of plasmid DNA gained initial proof-of-concept in small rodent species. Further development was hampered by the difficulty to confirm immunogenicity and efficacy in large animal species and, most importantly, in human clinical trials. These negative findings led to the search of complementary technologies which, in combination with intradermal or intramuscular plasmid DNA injection would result in more robust delivery, decreased interindividual variability, clear evidence of clinical efficacy and which would eventually lead to market approval of new vaccine products. The use of high-pressure, needleless devices as an enhancing tool for plasmid DNA delivery led to recent approval by USDA of Oncept™, a therapeutic cancer vaccine directed against tyrosinase for the therapy of melanoma in dogs. An alternative approach to improve plasmid DNA delivery is electro-gene-transfer (EGT). In this article, we briefly review the principles of DNA-EGT and the evidences for efficacy of a telomerase reverse transcriptase vaccine in a dog clinical trial, and provide perspectives for the use of this technology for broader applications in pet animals.

Evaluation of protective effect of DNA vaccines encoding the BAB1_0263 and BAB1_0278 open reading frames of Brucella abortus in BALB/c mice

The immunogenicity of two DNA vaccines encoding open reading frames (ORFs) of genomic island 3 (GI-3), specific for Brucella abortus and Brucella melitensis, has been examined. Intramuscular injection of plasmid DNA carrying the BAB1_0263 and BAB1_0278 genes (pVF263 and pVF278, respectively) into BALB/c mice elicited both humoral and cellular immune responses. Mice injected with pVF263 or pVF278 had a dominant immunoglobulin G2a (IgG2a) response. In addition, both DNA vaccines elicited a T-cell-proliferative response, but only pVF263 induced significant levels of interferon gamma (INF-γ) upon restimulation with recombinant 263 protein. Neither DNA vaccine induced interleukin (IL)-10, nor IL-4, upon stimulation with an appropriate recombinant protein or crude Brucella protein, suggesting the induction of a typical T-helper 1 (Th1)-dominated immune response. Furthermore, the pVF278 DNA vaccines induced protection in BALB/c mice against challenge with the virulent strain B. abortus 2308. Taken together, these data suggest that DNA vaccination offers an improved delivery strategy for the BAB1_0278 antigen, and provide the first evidence of a protective effect of this antigen.

Long-term and stable correction of uremic anemia by intramuscular injection of plasmids containing hypoxia-regulated system of erythropoietin expression

Relative deficiency in production of glycoprotein hormone erythropoietin (Epo) is a major cause of renal anemia. This study planned to investigate whether the hypoxia-regulated system of Epo expression, constructed by fusing Epo gene to the chimeric phosphoglycerate kinase (PGK) hypoxia response elements (HRE) in combination with cytomegalovirus immediate-early (CMV IE) basal gene promoter and delivered by plasmid intramuscular injection, might provide a long-term physiologically regulated Epo secretion expression to correct the anemia in adenine-induced uremic rats. Plasmid vectors (pHRE-Epo) were synthesized by fusing human Epo cDNA to the HRE/CMV promoter. Hypoxia-inducible activity of this promoter was evaluated first in vitro and then in vivo in healthy and uremic rats (n = 30 per group). The vectors (pCMV-Epo) in which Epo expression was directed by a constitutive CMV gene promoter served as control. ANOVA and Student's t-test were used to analyze between-group differences. A high-level expression of Epo was induced by hypoxia in vitro and in vivo. Though both pHRE-Epo and pCMV-Epo corrected anemia, the hematocrit of the pCMV-Epo-treated rats exceeded the normal (P < 0.05), but that of the pHRE-Epo-treated rats didn't. Hypoxia-regulated system of Epo gene expression constructed by fusing Epo to the HRE/CMV promoter and delivered by plasmid intramuscular injection may provide a long-term and stable Epo expression and secretion in vivo to correct the anemia in adenine-induced uremic rats.

Electroporation improves the immune response induced by a DNA vaccine against pseudorabies virus glycoprotein B in pigs

This study was performed to determine whether electroporation can be used to enhance the efficacy of a DNA vaccine against pseudorabies virus (PrV) in pigs. Immune responses to PrV were measured in pigs following a single intramuscular injection of plasmids encoding PrV glycoprotein B, with or without electroporation. Plasmid injection coupled with electroporation increased production of specific antibodies against PrV and peripheral blood mononuclear cells proliferated in response to stimulation with PrV glycoproteins. These results show that electroporation can improve the performance of a DNA vaccine against PrV in pigs. However, additional work is required to maximise the effectiveness of the vaccination protocol.

Expression levels of TRα, LAT1, and PKM2 alter the rate of metamorphic change

Event Abstract Back to Event Expression levels of TRα, LAT1, and PKM2 alter the rate of metamorphic change Jinyoung Choi1* and Daniel R. Buchholz1 1 University of Cincinnati, Department of Biological Science, United States Thyroid hormone (TH) plays a key role for most physiological and developmental changes during metamorphosis. TH exerts its function by binding thyroid hormone receptors (TRs) in the nucleus to initiate TH-dependent gene expression. Intracellular TH levels are regulated by thyroid hormone transporters (THTs) and cytosolic thyroid hormone binding proteins (CTHBPs). Intracellular TH levels and TR expression levels likely regulate the timing of transformation of different tissues during metamorphosis. We hypothesized that altered TR, LAT1 (a THT), and PKM2 (a CTHBP) expression affects the rate of metamorphosis when overexpressed. In this study, we overexpressed TR α, LAT1, and PKM2 in tail muscle cells by intramuscular plasmid DNA injection and quantified the disappearance of tail muscle cells during T3-induced metamorphosis. In cells overexpressing TR, the rate of cell death was significantly faster compared to controls.. Moreover, cell death was accelerated or delayed in muscle cells overexpressing LAT1 or PKM2, respectively, compared to controls. These results show that the expression levels of TR and proteins affecting intracellular TH levels can affect the rate of metamorphic changes. References Shi, Y., Wong, J., Puzianowska-Kuznicka, M., and Stolow, M.A. (1996). Tadpole competence and tissue-specific temporal regulation of amphibian metamorphosis: roles of thyroid hormone and its receptors. BioEssay 18(5), 391-399. Buchholz, D.R., Moskalik, C.L., Kulkarni, S.S., Hollar, A.R.,and Ng, Allison (2011).Hormone regulation and the evolution of frog metamorphic diversity. Mechanisms of Life History Evolution. Oxford publishers (in press). Hollar, A.R., Choi, J., Grimm, A.T., and Buchholz, D.R. (2011). Higher thyroid hormone receptor expression correlates with short 4 larval periods in spadefoot toads and increases metamorphic rate. Submitted to Gen. Comp. Endocrinol Friesema, E.C., Ganguly S., Abdalla, A., Fox, J.E.M., Halestrap, A.P.H. and Visser, T.J. (2003). Identification of monocarboxlyate transporter 8 as a specific thyroid hormone transporter. J. Bio. Chem. 278 (41), 40128-40315. Luze, A.D., Sachs, B., and Demeneix, B.A. (1993). Thyroid hormone-dependent transcriptional regulation of exogenous genes transfected into Xenopus tadpole muscle in vivo. Pro. Natl. Acad. USA 90 (August) 7322-7326. Kinne, A., Kleinau, G., Hoefig, G.S., Gruters, A., Kohrle, J., Krause, G., and Schweizer, U. (2010). Essential molecular determinants for thyroid hormone transport and first structural implications for monocarboxlyate transporter 8. J. Bio. Chem. 285 (36) 28054-28063 Connors, K.A., Korte, J.J., Anderson, G.W., and Degitz, S.J. (2010). Characterization of thyroid hormone transporter expression during tissue-specific metamorphic events in Xenopus tropicalis. Gen. Comp. Endocrinol. 168, 149-159. Keywords: cytosolic thyroid hormone binding protein, LAT1, metamorphosis, PKM2, Thyroid hormone, Thyroid hormone transporter, tissue asynchrony, TRα Conference: NASCE 2011: The inaugural meeting of the North American Society for Comparative Endocrinology, Ann Arbor, United States, 13 Jul - 16 Jul, 2011. Presentation Type: Poster Topic: Thyroid Citation: Choi J and Buchholz DR (2011). Expression levels of TRα, LAT1, and PKM2 alter the rate of metamorphic change. Front. Endocrinol. Conference Abstract: NASCE 2011: The inaugural meeting of the North American Society for Comparative Endocrinology. doi: 10.3389/conf.fendo.2011.04.00116 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 29 Jul 2011; Published Online: 09 Aug 2011. * Correspondence: Miss. Jinyoung Choi, University of Cincinnati, Department of Biological Science, Cincinnati, Ohio, 45221, United States, jinyoung.angela@gmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Jinyoung Choi Daniel R Buchholz Google Jinyoung Choi Daniel R Buchholz Google Scholar Jinyoung Choi Daniel R Buchholz PubMed Jinyoung Choi Daniel R Buchholz Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Neonatal intramuscular injection of plasmid encoding glucagon-like peptide-1 affects anxiety behaviour and expression of the hippocampal glucocorticoid receptor in adolescent rats

Early-life endocrine intervention may programme hippocampal glucocorticoid receptor (GR) expression and cause psychiatric disorders in later life. Glucagon-like peptide-1 (GLP-1) has been implicated in the regulation of neuroendocrine and behavioural responses, but it is yet to be determined whether and how neonatal GLP-1 overexpression may modify hippocampal GR expression and thus programme adolescent behaviour in rats. Two-day old pups were injected intramuscularly with vacant plasmid (VP) or plasmid DNA encoding secretory GLP-1 (GP). Anxiety-related behaviour was assessed in the elevated plus maze (EPM) test at 8 weeks of age. Plasma corticosterone levels were measured with enzyme immunoassay (EIA). Protein and mRNA levels were determined by western blot and real-time polymerase chain reaction (PCR), respectively. The DNA methylation status of the GR exon 1(7) promoter was determined by bisulphate sequencing PCR (BSP). GP rats exhibited anxiolytic behaviour compared with their VP counterparts. Hippocampal GLP-1 receptor (GLP-1R) and GR mRNA expression were significantly elevated in GP rats without a significant difference in plasma corticosterone. Significant reduction in DNA methyltransferase 1 (DNMT1) expression was observed in GP rats disconnected with alterations in DNA methylation of the GR exon 1(7) promoter. Nevertheless, mRNA expression of nerve growth factor-inducible protein A (NGFI-A) was significantly elevated in GP rats. These results suggest that neonatal intramuscular injection of plasmid DNA encoding GLP-1 affects anxiety behaviour in adolescent rats, probably through NGFI-A-activated upregulation of hippocampal GR expression.

Gene Therapy With the Angiogenic Cytokine Secretoneurin Induces Therapeutic Angiogenesis by a Nitric Oxide–Dependent Mechanism

The neuropeptide secretoneurin induces angiogenesis and postnatal vasculogenesis and is upregulated by hypoxia in skeletal muscle cells. We sought to investigate the effects of secretoneurin on therapeutic angiogenesis. We generated a secretoneurin gene therapy vector. In the mouse hindlimb ischemia model secretoneurin gene therapy by intramuscular plasmid injection significantly increased secretoneurin content of injected muscles, improved functional parameters, reduced tissue necrosis, and restored blood perfusion. Increased muscular density of capillaries and arterioles/arteries demonstrates the capability of secretoneurin gene therapy to induce therapeutic angiogenesis and arteriogenesis. Furthermore, recruitment of endothelial progenitor cells was enhanced by secretoneurin gene therapy consistent with induction of postnatal vasculogenesis. Additionally, secretoneurin was able to activate nitric oxide synthase in endothelial cells and inhibition of nitric oxide inhibited secretoneurin-induced effects on chemotaxis and capillary tube formation in vitro. In vivo, secretoneurin induced nitric oxide production and inhibition of nitric oxide attenuated secretoneurin-induced effects on blood perfusion, angiogenesis, arteriogenesis, and vasculogenesis. Secretoneurin also induced upregulation of basic fibroblast growth factor and platelet-derived growth factor-B in endothelial cells. In summary, our data indicate that gene therapy with secretoneurin induces therapeutic angiogenesis, arteriogenesis, and vasculogenesis in the hindlimb ischemia model by a nitric oxide-dependent mechanism.

Treatment of severe hypercholesterolemia in apolipoprotein E-deficient mice by intramuscular injection of plasmid DNA

Treatment of severe hypercholesterolemia in apolipoprotein E-deficient mice by intramuscular injection of plasmid DNA