Circular Intermediates of Recombinant Adeno-Associated Virus Have Defined Structural Characteristics Responsible for Long-Term Episomal Persistence in Muscle Tissue

Adeno-associated viral (AAV) vectors have demonstrated great utility for long-term gene expression in muscle tissue. However, the mechanisms by which recombinant AAV (rAAV) genomes persist in muscle tissue remain unclear. Using a recombinant shuttle vector, we have demonstrated that circularized rAAV intermediates impart episomal persistence to rAAV genomes in muscle tissue. The majority of circular intermediates had a consistent head-to-tail configuration consisting of monomer genomes which slowly converted to large multimers of >12 kbp by 80 days postinfection. Importantly, long-term transgene expression was associated with prolonged (80-day) episomal persistence of these circular intermediates. Structural features of these circular intermediates responsible for increased persistence included a DNA element encompassing two viral inverted terminal repeats (ITRs) in a head-to-tail orientation, which confers a 10-fold increase in the stability of DNA following incorporation into plasmid-based vectors and transfection into HeLa cells. These studies suggest that certain structural characteristics of AAV circular intermediates may explain long-term episomal persistence with this vector. Such information may also aid in the development of nonviral gene delivery systems with increased efficiency.     

Formation of Adeno-Associated Virus Circular Genomes Is Differentially Regulated by Adenovirus E4 ORF6 and E2a Gene Expression

A central feature of the adeno-associated virus (AAV) latent life cycle is persistence in the form of both integrated and episomal genomes. However, the molecular processes associated with episomal long-term persistence of AAV genomes are only poorly understood. To investigate these mechanisms, we have utilized a recombinant AAV (rAAV) shuttle vector to identify circular AAV intermediates from transduced HeLa cells and primary fibroblasts. The unique structural features exhibited by these transduction intermediates included circularized monomer and dimer virus genomes in a head-to-tail array, with associated specific base pair alterations in the 5′ viral D sequence. In HeLa cells, the abundance and stability of AAV circular intermediates were augmented by adenovirus expressing the E2a gene product. In the absence of E2a, adenovirus expressing the E4 open reading frame 6 gene product decreased the abundance of AAV circular intermediates, favoring instead the linear replication form monomer (Rf_m) and dimer (Rf_d) structures. In summary, the formation of AAV circular intermediates appears to represent a new pathway for AAV genome conversion, which is consistent with the head-to-tail concatemerization associated with latent-phase persistence of rAAV. A better understanding of this pathway may increase the utility of rAAV vectors for gene therapy.     

Calycosin Promotes Angiogenesis Involving Estrogen Receptor and Mitogen-Activated Protein Kinase (MAPK) Signaling Pathway in Zebrafish and HUVEC

Background

Angiogenesis plays an important role in a wide range of physiological processes, and many diseases are associated with the dysregulation of angiogenesis. Radix Astragali is a Chinese medicinal herb commonly used for treating cardiovascular disorders and has been shown to possess angiogenic effect in previous studies but its active constituent and underlying mechanism remain unclear. The present study investigates the angiogenic effects of calycosin, a major isoflavonoid isolated from Radix Astragali, in vitro and in vivo.

Methodology

Tg(fli1:EGFP) and Tg(fli1:nEGFP) transgenic zebrafish embryos were treated with different concentrations of calycosin (10, 30, 100 µM) from 72 hpf to 96 hpf prior morphological observation and angiogenesis phenotypes assessment. Zebrafish embryos were exposed to calycosin (10, 100 µM) from 72 hpf to 78 hpf before gene-expression analysis. The effects of VEGFR tyrosine kinase inhibitor on calycosin-induced angiogenesis were studied using 72 hpf Tg(fli1:EGFP) and Tg(fli1:nEGFP) zebrafish embryos. The pro-angiogenic effects of calycosin were compared with raloxifene and tamoxifen in 72 hpf Tg(fli1:EGFP) zebrafish embryos. The binding affinities of calycosin to estrogen receptors (ERs) were evaluated by cell-free and cell-based estrogen receptor binding assays. Human umbilical vein endothelial cell cultures (HUVEC) were pretreated with different concentrations of calycosin (3, 10, 30, 100 µM) for 48 h then tested for cell viability and tube formation. The role of MAPK signaling in calycosin-induced angiogenesis was evaluated using western blotting.

Conclusion

Calycosin was shown to induce angiogenesis in human umbilical vein endothelial cell cultures (HUVEC) in vitro and zebrafish embryos in vivo via the up-regulation of vascular endothelial growth factor (VEGF), VEGFR1 and VEGFR2 mRNA expression. It was demonstrated that calycosin acted similar to other selective estrogen receptor modulators (SERMs), such as raloxifene and tamoxifen, by displaying selective potency and affinity to estrogen receptors ERα and ERβ. Our results further indicated that calycosin promotes angiogenesis via activation of MAPK with the involvement of ERK1/2 and ER. Together, this study revealed, for the first time, that calycosin acts as a selective estrogen receptor modulator (SERM) to promote angiogenesis, at least in part through VEGF-VEGFR2 and MAPK signaling pathways.     

Expression,localization, and regulation of the neuregulin receptor ErbB3 in mouse heart

Neuregulins (NRG) belong to the EGF family of growth factors, which are ligands of the ErbB receptors. Their expression in the adult heart is essential, especially when the heart is submitted to cardiotoxic stress such as that produced by anthracyclines. It is considered that ErbB4 is the only NRG receptor expressed by the adult heart. Upon binding, ErbB4 may dimerize with ErbB2 to generate signals inside cells. However, here we show the presence of ErbB3 in the mouse heart from birth to adulthood by Western blotting and real‐time RT‐PCR. The expression level of ErbB3 mRNA was lower than that of ErbB2 or ErbB4, but was more stable throughout postnatal development. In isolated heart myocytes, ErbB3 localized to the Z‐lines similarly to ErbB1. Perfusion of isolated hearts with NRG‐1β induced phosphorylation of ErbB3, as well as ErbB2 and ErbB4. In adult mice, both ErbB2 and ErbB3, but not ErbB1 or ErbB4, were rapidly down‐regulated upon the induction of heart hypertrophy. In conclusion, our results demonstrate that ErbB3, in addition to ErbB4, is a receptor for neuregulin‐1β in the adult mouse heart. J. Cell. Physiol. 226: 450–455, 2011. © 2010 Wiley‐Liss, Inc.     

Carnitine worsens both injury and recovery of contractile function after transient ischemia in perfused rat heart

While carnitine overload appears to have therapeutic effects in pathological situations such as heart recovery after ischemia, its benefits as dietary supplementation for aerobic exercise have been questioned. We studied the effect of carnitine supplementation on the response of perfused rat heart to ischemia and reperfusion. Supplementation of the perfusion medium with 1 mM carnitine had no effect on cardiac performance in normoxic hearts, although it lowered lactate production by nearly 80%. Carnitine did not affect the amount of lactate accumulated during 30 min of ischemia, which was recovered in the perfusate immediately after reperfusion. However, carnitine worsened tissue injury, as shown by the 70% increase in creatine kinase release. Carnitine also worsened the recovery of contractile function, as revealed by the slower increase in heart rate and contractile force. In addition, carnitine supplementation increased contracture of the heart shortly after reperfusion. Therefore, in conditions where it does not increase glucose oxidation, carnitine supplementation worsens both injury and recovery of contractile function after transient ischemia in perfused rat heart.     

Ethanolic Extract of Fructus Alpinia oxyphylla Protects Against 6-Hydroxydopamine-Induced Damage of PC12 Cells In Vitro and Dopaminergic Neurons in Zebrafish

In an attempt to understand the neuroprotective effect of Fructus Alpinia oxyphylla (AOE) and to elucidate its underlying mechanism of action, the ethanolic extract of AOE was investigated using zebrafish and PC12 cell models. AOE prevented and restored 6-hydroxydopamine (6-OHDA)-induced dopaminergic (DA) neuron degeneration and attenuated a deficit of locomotor activity in a zebrafish (Danio rerio) model of Parkinson’s disease (PD). Treatment with AOE increased the viability of 6-OHDA-treated PC12 cells in vitro in a dose-dependent manner by attenuating cellular apoptosis. However, protocatechuic acid (PCA) and chrysin, two known polyphenol components of AOE, could not reproduce the neuroprotective activity of AOE in the PD zebrafish or PC12 cell models. A mechanistic study found that the protective effect of AOE against 6-OHDA-induced neuronal injury involved anti-inflammatory action (down-regulation of gene expression of IL-1β and TNF-α) and anti-oxidative action (inhibition of NO production and iNOS expression in PC12 cells). Moreover, the PI3K-AKT pathway might be part of the mechanism of neuroprotection of AOE. The results of this research are expected to provide a scientific rationale for the use of AOE in the treatment of PD. However, it is important that the active components that contribute to the neuroprotective action of AOE are identified and characterized.     

Epidermal growth factor protects the heart against low-flow ischemia-induced injury

The role of ErbB4 and ErbB2 in the heart of adult mammals is well established. The heart also expresses ErbB1 (the epidermal growth factor (EGF) receptor), but this receptor has received less attention. We studied the effect of EGF on the response of isolated mouse heart to low-flow ischemia and reperfusion. Reducing perfusate flow to 10% for 30 min resulted in an increase in anaerobic metabolism and the leakage of lactate dehydrogenase during reperfusion. In addition, left ventricle +dP/dt and developed pressure were depressed (20–25%) during reperfusion. The addition of EGF 5 min before and throughout the ischemic period prevented the increase in anaerobic metabolism and the leakage of intracellular lactate dehydrogenase during reperfusion. EGF improved both +dP/dt and developed pressure during ischemia and prevented the decrease in dP/dt during reperfusion. To determine whether the effect of EGF on cell integrity depends on its effect on contractility, we studied nonbeating isolated myocytes. In these cells, anoxia and reoxygenation reduced cell viability by nearly 25%. EGF prevented such a decrease. Our results indicate that, like ErbB4 and ErbB2, ErbB1 also has an important role in the heart of adult animals.     

Syndecan-2 downregulation impairs angiogenesis in human microvascular endothelial cells

The formation of new blood vessels, or angiogenesis, is a necessary process during development but also for tumour growth and other pathologies. It is promoted by different growth factors that stimulate endothelial cells to proliferate, migrate, and generate new tubular structures. Syndecans, transmembrane heparan sulphate proteoglycans, bind such growth factors through their glycosaminoglycan chains and could transduce the signal to the cytoskeleton, thus regulating cell behaviour. We demonstrated that syndecan-2, the major syndecan expressed by human microvascular endothelial cells, is regulated by growth factors and extracellular matrix proteins, in both bidimensional and tridimensional culture conditions. The role of syndecan-2 in “in vitro” tumour angiogenesis was also examined by inhibiting its core protein expression with antisense phosphorothioate oligonucleotides. Downregulation of syndecan-2 reduces spreading and adhesion of endothelial cells, enhances their migration, but also impairs the formation of capillary-like structures. These results suggest that syndecan-2 has an important function in some of the necessary steps that make up the angiogenic process. We therefore propose a pivotal role of this heparan sulphate proteoglycan in the formation of new blood vessels.     

Activated epidermal growth factor receptor (ErbB1) protects the heart against stress-induced injury in mice

Acute, high-intensity stress induces necrotic lesions in the heart. We found that restraint-and-cold (4 degrees C) exposure (RCE) raises plasma lactate dehydrogenase (LDH), creatine kinase (CK), and transaminase activity in a time-dependent manner, with a peak value 7 h after stimulus cessation. At 24 h, signs of necrotic lesions were observed in paraffin sections stained with hematoxylineosin: focal accumulation of mononuclear cells in subendocardial areas of the left ventricle wall and focal hemorrhage in papillary muscles. In contrast, intermale fighting (IF) did not increase plasma CK activity, although LDH and transaminase activities did increase. In IF, no histological evidence of heart injury was observed. Because IF, but not RCE, increased plasma epidermal growth factor (EGF) concentration by approximately 1,000-fold, we hypothesized that EGF receptor (ErbB1) activation may protect the heart against stress-induced injury. To examine this hypothesis, we injected the ErbB1 tyrosine kinase inhibitor tyrphostin AG-1478 (25 mg/kg ip) immediately before mice were exposed to IF. After 3 h, plasma activities of LDH-1 and CK increased. Plasma enzyme activities were as low in control mice (injected with vehicle alone) as in nonfighting mice. In the last experiment, we injected EGF (0.25 mg/kg ip) 20 min before exposing mice to RCE. After 7 h, plasma LDH-1 and CK activities were significantly lower in these animals than in mice injected with vehicle. The effect required ErbB1 activation, because simultaneous administration of AG-1478 completely abolished the effect of exogenous EGF. We conclude that activated ErbB1, by endogenous or exogenous ligands, may protect the heart against stress-induced injury.