PRMT4-mediated arginine methylation promotes tyrosine phosphorylation of VEGFR-2 and regulates filopodia protrusions.

Through tightly controlled multilayer mechanisms, vascular endothelial growth factor receptor-2 (VEGFR-2) activation and its downstream signal transduction govern vasculogenesis and pathological angiogenesis, such as tumor angiogenesis. Therefore, it is critical to understand the molecular mechanisms governing VEGFR-2 signal transduction. We report that protein arginine methyltransferase 4 (PRMT4) via its highly conserved EVH1 and PH domain-like N-terminal domain binds to VEGFR-2 and mediates methylation of the juxtamembrane arginine 817 (R817) on VEGFR-2. Methylation of R817 selectively increases phosphorylation of tyrosine 820 (Y820). Phosphorylation of Y820 facilitates the c-Src binding with VEGFR-2 via Src homology domain 2 (SH2). Interfering with the methylation of R817 or phosphorylation of Y820 inhibits VEGFR-2-induced filopodia protrusions, a process that is critical for the core angiogenic responses of VEGFR-2. Methylation of R817 is an important previously unrecognized mechanism of the angiogenic signaling of VEGFR-2, with implications for the development of novel-targeted VEGFR-2 inhibitors.

Higher Seroprevalence of Anti-Adeno-Associated Viral Vector Neutralizing Antibodies Among Racial Minorities in the United States.

Anti-adeno-associated viral vector (AAV) neutralizing antibodies (NAbs) can ablate efficacy of transgene expression following intravenous vector administration. This observation in both preclinical and clinical trials has led to exclusion of NAb-positive patients from receiving AAV gene therapy. AAV drug development includes selection of capsids with lower NAb seroprevalence that also possess other favorable traits. Often a limited number of healthy volunteers are screened to gauge NAb seroprevalence. However, limited data sets can be biased leading to inaccurate estimates of NAb incidence. In this study, we evaluated AAV NAbs against a panel of vectors among healthy donors within the United States. While the overall seroprevalence against most AAVs was lower, we did observe increased NAb incidence among black and Hispanic donors. These findings of increased NAb seroprevalence among the minority races were confirmed in a second set of donors who also demonstrated higher seroprevalence among these races. Interracial- and intraracial differences within genders were also observed among donors. The increased incidence of AAV NAb among racial minorities was unexpected. Our findings underscore the need for removing bias in sample data sets and evaluating seroprevalence within the patient population while selecting capsids.

Collagen VII deficient mice show morphologic and histologic corneal changes that phenotypically mimic human dystrophic epidermolysis bullosa of the eye.

BACKGROUND: Absence of collagen VII causes blistering of the skin, eyes and many other tissues. This disease is termed dystrophic epidermolysis bullosa (DEB). Corneal fibrosis occurs in up to 41% and vision loss in up to 64% of patients. Standard treatments are supportive and there is no cure. The hypomorphic mouse model for DEB shows production of collagen VII at 10% of wild type levels in skin and spleen, but the eyes have not been described. Our purpose is to characterize the corneas to determine if this is an appropriate model for study of ocular therapeutics. METHODS: Western blot analysis (WB) and immunohistochemistry (IHC) were performed to assess presence and location of collagen VII protein within the hypomorphic mouse cornea. Additional IHC for inflammatory and fibrotic biomarkers transforming growth factor-beta-1 (TGF-ß1), alpha-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF), proteinase 3, tenascin C and collagen III were performed. Clinical photographs documenting corneal opacification were assessed and scored independently by 2 examiners. Histology was then used to investigate morphologic changes. RESULTS: IHC and WB confirmed that hypomorphic mice produce less collagen VII production at the level of the basement membrane when compared with wild-types. IHC showed anomalous deposition of collagen III throughout the stroma. Of the 5 biomarkers tested, TGF-ß1 showed the strongest and most consistently staining. Photographs documented corneal opacities only in mice older than 10 weeks, opacities were not seen in younger animals. Histology showed multiple abnormalities, including epithelial hyperplasia, ulceration, fibrosis, edema, dysplasia, neovascularization and bullae formation. CONCLUSIONS: The collagen VII hypomorphic mouse shows reduced collagen VII production at the level of the corneal basement membrane. Corneal changes are similar to pathology seen in humans with this disease. The presence of anomalous stromal collagen III and TGF-ß1 appear to be the most consistent and strongest staining biomarkers in diseased mice. This mouse appears to mimic human corneal disease. It is an appropriate model for testing of therapeutics to treat EB ocular disease.

AAV-ID: A Rapid and Robust Assay for Batch-to-Batch Consistency Evaluation of AAV Preparations.

Adeno-associated virus (AAV) vectors are promising clinical candidates for therapeutic gene transfer, and a number of AAV-based drugs may emerge on the market over the coming years. To insure the consistency in efficacy and safety of any drug vial that reaches the patient, regulatory agencies require extensive characterization of the final product. Identity is a key characteristic of a therapeutic product, as it ensures its proper labeling and batch-to-batch consistency. Currently, there is no facile, fast, and robust characterization assay enabling to probe the identity of AAV products at the protein level. Here, we investigated whether the thermostability of AAV particles could inform us on the composition of vector preparations. AAV-ID, an assay based on differential scanning fluorimetry (DSF), was evaluated in two AAV research laboratories for specificity, sensitivity, and reproducibility, for six different serotypes (AAV1, 2, 5, 6.2, 8, and 9), using 67 randomly selected AAV preparations. In addition to enabling discrimination of AAV serotypes based on their melting temperatures, the obtained fluorescent fingerprints also provided information on sample homogeneity, particle concentration, and buffer composition. Our data support the use of AAV-ID as a reproducible, fast, and low-cost method to ensure batch-to-batch consistency in manufacturing facilities and academic laboratories.

In Silico Reconstruction of the Viral Evolutionary Lineage Yields a Potent Gene Therapy Vector.

Adeno-associated virus (AAV) vectors have emerged as a gene-delivery platform with demonstrated safety and efficacy in a handful of clinical trials for monogenic disorders. However, limitations of the current generation vectors often prevent broader application of AAV gene therapy. Efforts to engineer AAV vectors have been hampered by a limited understanding of the structure-function relationship of the complex multimeric icosahedral architecture of the particle. To develop additional reagents pertinent to further our insight into AAVs, we inferred evolutionary intermediates of the viral capsid using ancestral sequence reconstruction. In-silico-derived sequences were synthesized de novo and characterized for biological properties relevant to clinical applications. This effort led to the generation of nine functional putative ancestral AAVs and the identification of Anc80, the predicted ancestor of the widely studied AAV serotypes 1, 2, 8, and 9, as a highly potent in vivo gene therapy vector for targeting liver, muscle, and retina.

A critical role for the E3-ligase activity of c-Cbl in VEGFR-2-mediated PLCgamma1 activation and angiogenesis.

Activation of phospholipase Cgamma1 (PLCgamma1) by vascular endothelial growth factor receptor-2 (VEGFR-2) in endothelial cells in part is responsible for angiogenesis in vivo. The cellular mechanisms exerting negative control over PLCgamma1 activation, however, remain unaddressed. Here by using in vitro and in vivo binding assays, we show that the Casitas B-lineage lymphoma (c-Cbl) E3 ubiquitin ligase constitutively associates with PLCgamma1 via its C-terminal domain and conditionally interacts with VEGFR-2 via the N-terminal/TKB domain. Site-directed mutagenesis of VEGFR-2 showed that full activation of c-Cbl requires its direct association with phospho-tyrosines 1052 and 1057 of VEGFR-2 via its TKB domain and indirect association with phospho-tyrosine 1173 of VEGFR-2 via PLCgamma1. The tertiary complex formation between VEGFR-2, PLCgamma1 and c-Cbl selectively promotes ubiquitylation and suppression of tyrosine phosphorylation of PLCgamma1 by a proteolysis-independent mechanism. Further analysis showed that association of c-Cbl with VEGFR-2 does not impact ubiquitylation, down-regulation, or tyrosine phosphorylation of VEGFR-2. Silencing of c-Cbl by siRNA revealed that endogenous c-Cbl plays an inhibitory role in angiogenesis. Our data demonstrate that corecruitment of c-Cbl and PLCgamma1 to VEGFR-2 serves as a mechanism to fine-tune the angiogenic signal relay of VEGFR-2.

Lifelong caloric restriction increases expression of apoptosis repressor with a caspase recruitment domain (ARC) in the brain.

Aging may increase apoptotic events and the susceptibility of the central nervous system to apoptosis. Calorie restriction has been shown to have neuroprotective effects, but the mechanisms in vivo are unknown. We investigated apoptosis and apoptotic regulatory proteins in the brain frontal cortex of 12-month-old ad libitum fed, 26-month-old ad libitum fed, and 26-month-old calorie-restricted (CR) male Fischer 344 rats (CR = 40% restricted compared to ad libitum). We found that specific DNA fragmentation indicative of apoptosis was increased with age (+124%) in the cortices of the brain and that calorie restriction attenuated this increase significantly (-36%). We determined levels of ARC (apoptosis repressor with a caspase recruitment domain), which inhibits caspase-2 activity and also attenuates cytochrome c release from the mitochondria. We found a significant age-associated decline in ARC level, which was attenuated in the brains of the CR rats. In accordance with the changes in ARC expression observed, calorie restriction attenuated the increases in cytosolic cytochrome c and caspase-2 activity with age and suppressed the age-associated rise in cleaved caspase-9 and cleaved caspase-3. However, neither age nor calorie restriction had any effect on caspase-3 and caspase-9 activities. This data provides evidence for an increased incidence of apoptosis in rat brain with age and evidence that calorie restriction has the ability to attenuate this. Furthermore, our data suggest that calorie restriction provides neuroprotection through ARC by suppressing cytochrome c release and caspase-2 activity.