Targeting Photoreceptors via Intravitreal Delivery Using Novel,Capsid-Mutated AAV Vectors

Development of viral vectors capable of transducing photoreceptors by less invasive methods than subretinal injection would provide a major advancement in retinal gene therapy. We sought to develop novel AAV vectors optimized for photoreceptor transduction following intravitreal delivery and to develop methodology for quantifying this transduction in vivo. Surface exposed tyrosine (Y) and threonine (T) residues on the capsids of AAV2, AAV5 and AAV8 were changed to phenylalanine (F) and valine (V), respectively. Transduction efficiencies of self-complimentary, capsid-mutant and unmodified AAV vectors containing the smCBA promoter and mCherry cDNA were initially scored in vitro using a cone photoreceptor cell line. Capsid mutants exhibiting the highest transduction efficiencies relative to unmodified vectors were then injected intravitreally into transgenic mice constitutively expressing a Rhodopsin-GFP fusion protein in rod photoreceptors (Rho-GFP mice). Photoreceptor transduction was quantified by fluorescent activated cell sorting (FACS) by counting cells positive for both GFP and mCherry. To explore the utility of the capsid mutants, standard, (non-self-complementary) AAV vectors containing the human rhodopsin kinase promoter (hGRK1) were made. Vectors were intravitreally injected in wildtype mice to assess whether efficient expression exclusive to photoreceptors was achievable. To restrict off-target expression in cells of the inner and middle retina, subsequent vectors incorporated multiple target sequences for miR181, an miRNA endogenously expressed in the inner and middle retina. Results showed that AAV2 containing four Y to F mutations combined with a single T to V mutation (quadY−F+T−V) transduced photoreceptors most efficiently. Robust photoreceptor expression was mediated by AAV2(quadY−F+T−V) −hGRK1−GFP. Observed off-target expression was reduced by incorporating target sequence for a miRNA highly expressed in inner/middle retina, miR181c. Thus we have identified a novel AAV vector capable of transducing photoreceptors following intravitreal delivery to mouse. Furthermore, we describe a robust methodology for quantifying photoreceptor transduction from intravitreally delivered AAV vectors.     

Peroxiredoxin 2 deficiency reduces white adipogenesis due to the excessive ROS generation

Reactive oxygen species (ROS) act as signaling molecules to regulate various cell functions. Numerous studies have demonstrated ROS to be essential for the differentiation of adipocytes. Peroxiredoxins (Prxs) are a ubiquitous family of antioxidant enzymes in mammalian cells. Prx2 is present in the cytoplasm and cell membranes and demonstrates ROS scavenging activity. We focused on Prx2 involvement in regulating adipogenesis and lipid accumulation and demonstrated that Prx2 expression was upregulated during adipocyte differentiation. In addition, the silencing of Prx2 (shPrx2) inhibited adipogenesis by modulating adipogenic gene expression, and cell death was enhanced via increased ROS production in shPrx2‐3T3‐L1 cells. These results demonstrate that shPrx2 triggers adipocyte cell death and weakens adipocyte function via ROS production. Taken together, our data suggest the participation of Prx2 in adipocyte function and differentiation. Our results also imply that the downregulation of Prx2 activity could help prevent obesity. Overall, findings support the development of ROS‐based therapeutic solutions for the treatment of obesity and obesity‐related metabolic disorders.     

Angiopoietin-1 elicits pro-inflammatory responses in monocytes and differentiating macrophages

The angiopoietin/Tie2 system is an important regulator of angiogenesis and inflammation. In addition to its functions in endothelial cells, Tie2 expression on non-endothelial cells allows for angiopoietin ligands to stimulate the cells. Although Ang1 is a strong Tie2 receptor agonist, little is known regarding the effect of Ang1 on non-endothelial cells, such as monocytes and macrophages. In this study, we found that Ang1 functionally binds to and stimulates monocytes via p38 and Erk1/2 phosphorylation. Ang1-mediated monocyte stimulation is associated with proinflammatory cytokine TNF-α expression. We also determined that Ang1 switched macrophage differentiation toward a pro-inflammatory phenotype, even in the presence of an anti-inflammatory mediator. These findings suggest that Ang1 plays a role in stimulating pro-inflammatory responses and could provide a new strategy by which to manage inflammatory responses.     

The Effect of Static Stretch on Elastin Degradation in Arteries

Previously we have shown that gradual changes in the structure of elastin during an elastase treatment can lead to important transition stages in the mechanical behavior of arteries [1]. However, in vivo arteries are constantly being loaded due to systolic and diastolic pressures and so understanding the effects of loading on the enzymatic degradation of elastin in arteries is important. With biaxial tensile testing, we measured the mechanical behavior of porcine thoracic aortas digested with a mild solution of purified elastase (5 U/mL) in the presence of a static stretch. Arterial mechanical properties and biochemical composition were analyzed to assess the effects of mechanical stretch on elastin degradation. As elastin is being removed, the dimensions of the artery increase by more than 20% in both the longitude and circumference directions. Elastin assays indicate a faster rate of degradation when stretch was present during the digestion. A simple exponential decay fitting confirms the time constant for digestion with stretch (0.11±0.04 h⁻¹) is almost twice that of digestion without stretch (0.069±0.028 h⁻¹). The transition from J-shaped to S-shaped stress vs. strain behavior in the longitudinal direction generally occurs when elastin content is reduced by about 60%. Multiphoton image analysis confirms the removal/fragmentation of elastin and also shows that the collagen fibers are closely intertwined with the elastin lamellae in the medial layer. After removal of elastin, the collagen fibers are no longer constrained and become disordered. Release of amorphous elastin during the fragmentation of the lamellae layers is observed and provides insights into the process of elastin degradation. Overall this study reveals several interesting microstructural changes in the extracellular matrix that could explain the resulting mechanical behavior of arteries with elastin degradation.     

Expressed Sequence Tags for Bovine Muscle Satellite Cells,Myotube Formed-Cells and Adipocyte-Like Cells

Background

Muscle satellite cells (MSCs) represent a devoted stem cell population that is responsible for postnatal muscle growth and skeletal muscle regeneration. An important characteristic of MSCs is that they encompass multi potential mesenchymal stem cell activity and are able to differentiate into myocytes and adipocytes. To achieve a global view of the genes differentially expressed in MSCs, myotube formed-cells (MFCs) and adipocyte-like cells (ALCs), we performed large-scale EST sequencing of normalized cDNA libraries developed from bovine MSCs.

Results

A total of 24,192 clones were assembled into 3,333 clusters, 5,517 singletons and 3,842contigs. Functional annotation of these unigenes revealed that a large portion of the differentially expressed genes are involved in cellular and signaling processes. Database for Annotation, Visualization and Integrated Discovery (DAVID) functional analysis of three subsets of highly expressed gene lists (MSC233, MFC258, and ALC248) highlighted some common and unique biological processes among MSC, MFC and ALC. Additionally, genes that may be specific to MSC, MFC and ALC are reported here, and the role of dimethylarginine dimethylaminohydrolase2 (DDAH2) during myogenesis and hemoglobin subunit alpha2 (HBA2) during transdifferentiation in C2C12 were assayed as a case study. DDAH2 was up-regulated during myognesis and knockdown of DDAH2 by siRNA significantly decreased myogenin (MYOG) expression corresponding with the slight change in cell morphology. In contrast, HBA2 was up-regulated during ALC formation and resulted in decreased intracellular lipid accumulation and CD36 mRNA expression upon knockdown assay.

Conclusion

In this study, a large number of EST sequences were generated from the MSC, MFC and ALC. Overall, the collection of ESTs generated in this study provides a starting point for the identification of novel genes involved in MFC and ALC formation, which in turn offers a fundamental resource to enable better understanding of the mechanism of muscle differentiation and transdifferentiation.     

Clinical Benefit of Liver Stiffness Measurement at 3 Months after Kasai Hepatoportoenterostomy to Predict the Liver Related Events in Biliary Atresia

Background

The progression of hepatic fibrosis may result in decompensated hepatic failure with cirrhosis, liver related events (LRE) such as ascites, variceal bleeding, and death after successful and timely Kasai hepatoportoenterostomy (HPE) in biliary atresia. The aim of this study is to suggest clinical benefit of the liver stiffness measurement (LSM) using transient elastography at 3 months after the Kasai operation to predict LRE.

Methods

Between January 2007 and December 2011, 69 eligible biliary atresia patients who underwent Kasai HPE and performed transient elastography before and 3 months after HPE were included. The occurrences of LRE were analyzed for all patients. All patients were divided into 2 groups (with and without LRE) for comparison. Multivariate analysis was used to detect the independent risk factors of LRE. The area under the receiver operation characteristics curve (AUROC) was used to establish the LSM optimal cutoff value of 3 months after Kasai operation in predicting LRE.

Results

LSM value, aminotransferase, albumin, bilirubin, and PT-INR significantly differed among the two groups. Multivariate analysis demonstrated LSM value as the most powerful independent factor of the development of LRE. The cut-off value of 19.9 kPa was calculated to be optimal for predicting LRE development with total sensitivity and specificity of 1.804. AUROC resulted in 0.943, with sensitivity of 85.3% and specificity of 95.2%.

Conclusions

The LSM value of 3 months after Kasai HPE can be a useful predictor of LRE development.