Inhibition of choroidal and corneal pathologic neovascularization by Plgf1-de gene transfer.

8Ocular neovascularization (NV), the primary cause of blindness, typically is treated via inhibition of VEGF-A activity. However, besides VEGF-A, other proteins of the same family, including VEGF-B and placental growth factor (PlGF, all together VEGFs), have a crucial role in the angiogenesis process. PlGF and VEGF, which form heterodimers if co-expressed, both are required for pathologic angiogenesis. We generated a PlGF1 variant, named PlGF1-DE, which is unable to bind and activate VEGFR-1, but retains the ability to form heterodimer. PlGF1-DE acts as dominant negative of VEGF-A and PlGF1wt through heterodimerization mechanism. The purpose of our study was to explore the therapeutic potential of Plgf1-de gene in choroid and cornea NV context.In the model of laser-induced choroidal neovascularization (CNV), Plgf1-de gene, and as control Plgf1wt, LacZ, or gfp genes, were delivered using adeno-associated virus (AAV) vector by subretinal injection 14 days before the injury. After 7 days CNV volume was assessed. Corneal NV was induced by scrape or suture procedures. Expression vectors for PlGF1wt or PlGF1-DE, and as control the empty vector pCDNA3, were injected in the mouse cornea after the vascularization insults. NV was evaluated with CD31 and LYVE-1 immunostaining.The expression of Plgf1-de induced significant inhibition of choroidal and corneal NV by reducing VEGF-A homodimer production. Conversely, the delivery of Plgf1wt, despite induced similar reduction of VEGF-A production, did not affect NV.Plgf1-de gene is a new therapeutic tool for the inhibition of VEGFs driven ocular NV.nonenoneV. Tarallo;S. Bogdanovich;Y. Hirano;L. Tudisco;L. Zentilin;M. Giacca;J. Ambati;S. D. FalcoV., Tarallo; S., Bogdanovich; Y., Hirano; L., Tudisco; L., Zentilin; Giacca, Mauro; J., Ambati; S. D., Falc

Transcriptional and functional differences in stem cell populations isolated from extraocular and limb muscles

The extraocular muscles (EOMs) are a distinct muscle group that displays an array of unique contractile, structural, and regenerative properties. They also have differential sensitivity to certain diseases and are enigmatically spared in Duchenne muscular dystrophy (DMD). The EOMs are so distinct from other skeletal muscles that the term “allotype” has been coined to highlight EOM group-specific properties. We hypothesized that increased and distinct stem cells may underlie the continual myogenesis noted in EOM. The side population (SP) stem cells were isolated and studied. EOMs had 15× higher SP cell content compared with limb muscles. Expression profiling revealed 348 transcripts that define the EOM-SP transcriptome. Over 92% of transcripts were SP specific, because they were absent in previous whole muscle microarray studies. Cultured EOM-SP cells revealed superior in vitro proliferative capacity. Finally, assays of the committed progenitors or satellite cells performed on myofibers isolated from EOM and limb muscles independently validated the increased proliferative capacity of these muscles. We suggest a model in which unique EOM stem cells contribute to the continual myogenesis noted in EOM and consistent with a role for their sparing in DMD. We believe the greater numbers of stem cells, their unique transcriptome, the greater proliferative capacity of EOM stem cells, and the greater number of satellite cells also offer clues for novel cell-based therapeutic strategies

Pretreatment with a soluble activin type IIB receptor/Fc fusion protein improves hypoxia-induced muscle dysfunction

Hypoxia, or reduced oxygen, occurs in a variety of clinical and environmental situations. Hypoxic exposure is associated with decreased muscle mass and a concomitant reduction in exercise capacity, although the exact mechanisms are not completely understood. The activin type IIB receptor (ActRIIB) is a receptor for transforming growth factor-β (TGFβ) superfamily members that are involved in the negative regulation of lean tissue mass. Given that hypoxia has negative effects on muscle mass and function and that modulation of the ActRIIB has been shown to increase muscle mass, we tested the hypothesis that pharmacological targeting of the ActRIIB for 2 wk would attenuate the loss of muscle mass and function in mice after exposure to normobaric hypoxia. ActRIIB modulation was achieved using a soluble activin receptor/Fc fusion protein (sActRIIB) in mice housed in a hypoxic chamber for 1 or 2 wk. Hypoxia induced a reduction in body weight in PBS- and sActRIIB-treated mice, although sActRIIB-treated mice remained larger throughout the hypoxic exposure. The absolute forces generated by extensor digitorum longus muscles were also significantly greater in sActRIIB- than PBS-treated mice and were more resistant to eccentric contraction-induced force drop after eccentric lengthening contractions. In summary, sActRIIB pretreatment attenuated hypoxia-induced muscle dysfunction. These data suggest that targeting the ActRIIB is an effective strategy to counter hypoxia-induced muscle dysfunction and to preacclimatize to hypoxia in clinical or high-altitude settings

A Mouse Model for Dominant Collagen VI Disorders

Background: Dominant collagen VI gene mutations cause the severe Ullrich congenital muscular dystrophy (UCMD) and mild Bethlem myopathy. Results: A mutant mouse mimicking the most common molecular defect in dominant UCMD patients was generated and characterized. Conclusion: The mutant mouse displays muscle and connective tissue abnormalities. Significance: The mutant mouse provides an animal model for dominant collagen VI disorders. Dominant and recessive mutations in collagen VI genes, COL6A1, COL6A2, and COL6A3, cause a continuous spectrum of disorders characterized by muscle weakness and connective tissue abnormalities ranging from the severe Ullrich congenital muscular dystrophy to the mild Bethlem myopathy. Herein, we report the development of a mouse model for dominant collagen VI disorders by deleting exon 16 in the Col6a3 gene. The resulting heterozygous mouse, Col6a3(+/d16), produced comparable amounts of normal Col6a3 mRNA and a mutant transcript with an in-frame deletion of 54 bp of triple-helical coding sequences, thus mimicking the most common molecular defect found in dominant Ullrich congenital muscular dystrophy patients. Biosynthetic studies of mutant fibroblasts indicated that the mutant 3(VI) collagen protein was produced and exerted a dominant-negative effect on collagen VI microfibrillar assembly. The distribution of the 3(VI)-like chains of collagen VI was not altered in mutant mice during development. The Col6a3(+/d16) mice developed histopathologic signs of myopathy and showed ultrastructural alterations of mitochondria and sarcoplasmic reticulum in muscle and abnormal collagen fibrils in tendons. The Col6a3(+/d16) mice displayed compromised muscle contractile functions and thereby provide an essential preclinical platform for developing treatment strategies for dominant collagen VI disorders

Targeting latent TGFβ release in muscular dystrophy

Latent transforming growth factor-β (TGFβ) binding proteins (LTBPs) bind to inactive TGFβ in the extracellular matrix. In mice, muscular dystrophy symptoms are intensified by a genetic polymorphism that changes the hinge region of LTBP, leading to increased proteolytic susceptibility and TGFβ release. We have found that the hinge region of human LTBP4 was also readily proteolysed and that proteolysis could be blocked by an antibody to the hinge region. Transgenic mice were generated to carry a bacterial artificial chromosome encoding the human LTBP4 gene. These transgenic mice displayed larger myofibers, increased damage after muscle injury, and enhanced TGFβ signaling. In the mdx mouse model of Duchenne muscular dystrophy, the human LTBP4 transgene exacerbated muscular dystrophy symptoms and resulted in weaker muscles with an increased inflammatory infiltrate and greater LTBP4 cleavage in vivo. Blocking LTBP4 cleavage may be a therapeutic strategy to reduce TGFβ release and activity and decrease inflammation and muscle damage in muscular dystrophy

Overexpression of Latent TGFβ Binding Protein 4 in Muscle Ameliorates Muscular Dystrophy through Myostatin and TGFβ

Latent TGFβ binding proteins (LTBPs) regulate the extracellular availability of latent TGFβ. LTBP4 was identified as a genetic modifier of muscular dystrophy in mice and humans. An in-frame insertion polymorphism in the murine Ltbp4 gene associates with partial protection against muscular dystrophy. In humans, nonsynonymous single nucleotide polymorphisms in LTBP4 associate with prolonged ambulation in Duchenne muscular dystrophy. To better understand LTBP4 and its role in modifying muscular dystrophy, we created transgenic mice overexpressing the protective murine allele of LTBP4 specifically in mature myofibers using the human skeletal actin promoter. Overexpression of LTBP4 protein was associated with increased muscle mass and proportionally increased strength compared to age-matched controls. In order to assess the effects of LTBP4 in muscular dystrophy, LTBP4 overexpressing mice were bred to mdx mice, a model of Duchenne muscular dystrophy. In this model, increased LTBP4 led to greater muscle mass with proportionally increased strength, and decreased fibrosis. The increase in muscle mass and reduction in fibrosis were similar to what occurs when myostatin, a related TGFβ family member and negative regulator of muscle mass, was deleted in mdx mice. Supporting this, we found that myostatin forms a complex with LTBP4 and that overexpression of LTBP4 led to a decrease in myostatin levels. LTBP4 also interacted with TGFβ and GDF11, a protein highly related to myostatin. These data identify LTBP4 as a multi-TGFβ family ligand binding protein with the capacity to modify muscle disease through overexpression

Targeting the Activin Type IIB Receptor to Improve Muscle Mass and Function in the mdx Mouse Model of Duchenne Muscular Dystrophy

The activin receptor type IIB (ActRIIB) is a transmembrane receptor for transforming growth factor-β superfamily members, including myostatin, that are involved in the negative regulation of skeletal muscle mass. We tested the translational hypothesis that blocking ligand binding to ActRIIB for 12 weeks would stimulate skeletal muscle growth and improve muscle function in the mdx mouse. ActRIIB was targeted using a novel inhibitor comprised of the extracellular portion of the ActRIIB fused to the Fc portion of murine IgG (sActRIIB), at concentrations of 1.0 and 10.0 mg/kg−1 body weight. After 12 weeks of treatment, the 10.0 mg/kg−1 dose caused a 27% increase in body weight with a concomitant 33% increase in lean muscle mass. Absolute force production of the extensor digitorum longus muscle ex vivo was higher in mice after treatment with either dose of sActRIIB, and the specific force was significantly higher after the lower dose (1.0 mg/kg−1), indicating functional improvement in the muscle. Circulating creatine kinase levels were significantly lower in mice treated with sActRIIB, compared with control mice. These data show that targeting the ActRIIB improves skeletal muscle mass and functional strength in the mdx mouse model of DMD, providing a therapeutic rationale for use of this molecule in treating skeletal myopathies