VEGFR-1/Flt-1 inhibition increases angiogenesis and improves muscle function in a mouse model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy is characterized by structural degeneration of muscle, which is exacerbated by localized functional ischemia due to loss of nitric oxide synthase-induced vasodilation. Treatment strategies aimed at increasing vascular perfusion have been proposed. Toward this end, we have developed monoclonal antibodies (mAbs) that bind to the vascular endothelial growth factor (VEGF) receptor VEGFR-1 (Flt-1) and its soluble splice variant isoform (sFlt-1) leading to increased levels of free VEGF and proangiogenic signaling. The lead chimeric mAb, 21B3, had high affinity and specificity for both human and mouse sFlt-1 and inhibited VEGF binding to sFlt-1 in a competitive manner. Proof-of-concept studies in the mdx mouse model of Duchenne muscular dystrophy showed that intravenous administration of 21B3 led to elevated VEGF levels, increased vascularization and blood flow to muscles, and decreased fibrosis after 6-12 weeks of treatment. Greater muscle strength was also observed after 4 weeks of treatment. A humanized form of the mAb, 27H6, was engineered and demonstrated a comparable pharmacologic effect. Overall, administration of anti-Flt-1 mAbs in mdx mice inhibited the VEGF:Flt-1 interaction, promoted angiogenesis, and improved muscle function. These studies suggest a potential therapeutic benefit of Flt-1 inhibition for patients with Duchenne muscular dystrophy.

Inhibition of FLT1 ameliorates muscular dystrophy phenotype by increased vasculature in a mouse model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is an X-linked recessive genetic disease in which the dystrophin coding for a membrane stabilizing protein is mutated. Recently, the vasculature has also shown to be perturbed in DMD and DMD model mdx mice. Recent DMD transcriptomics revealed the defects were correlated to a vascular endothelial growth factor (VEGF) signaling pathway. To reveal the relationship between DMD and VEGF signaling, mdx mice were crossed with constitutive (CAGCreERTM:Flt1LoxP/LoxP) and endothelial cell-specific conditional gene knockout mice (Cdh5CreERT2:Flt1LoxP/LoxP) for Flt1 (VEGFR1) which is a decoy receptor for VEGF. Here, we showed that while constitutive deletion of Flt1 is detrimental to the skeletal muscle function, endothelial cell-specific Flt1 deletion resulted in increased vascular density, increased satellite cell number and improvement in the DMD-associated phenotype in the mdx mice. These decreases in pathology, including improved muscle histology and function, were recapitulated in mdx mice given anti-FLT1 peptides or monoclonal antibodies, which blocked VEGF-FLT1 binding. The histological and functional improvement of dystrophic muscle by FLT1 blockade provides a novel pharmacological strategy for the potential treatment of DMD.

JENNIFER M. BOSCO ANGELA CHUANG (2018) A new species of grass spider, Agelenopsis riechertii, from the Southwestern USA, with notes on its courtship behavior (Araneae: Agelenidae). Zootaxa, 4442: 579-583.

We made changes in the description of a new Agelenopsis spider species, Agelenopsis riechertae. Firstly, we declare the male holotype and a type repository to validate the species description, which was previously omitted. The original Latin name has been feminized from Agelenopsis riechertii to Agelenopsis riechertae.

A new species of grass spider, Agelenopsis riechertii, from the Southwestern USA, with notes on its courtship behavior (Araneae: Agelenidae).

We describe a new spider species of the genus Agelenopsis Giebel 1869 (Agelenidae) from adult males and females. Agelenopsis riechertii is found in dry desert scrub habitats in the southwestern United States. While A. riechertii is similar to other sympatric congeners (A. aleenae, A. aperta, and A. spatula) in morphology and courtship behavior, it can be distinguished by an examination of its genitalia. Males possess an embolus that gradually narrows into a twisted tip which displays a convex edge from the lateral view. The female epigyna are harder to distinguish from congeners, but have a flatter anterior atrial edge that lacks strong bi-lobing. The spermatheca in female A. riechertii are also more elongate and comma-shaped than the other species. We describe the sequence of courtship behaviors exhibited by males, including the use of web pulses, abdomen waggling, and web flexing, in which males with fewer pauses, longer waggling bouts, and fewer web flexes are more likely to successfully copulate.

Anti-sFlt-1 Therapy Preserves Lung Alveolar and Vascular Growth in Antenatal Models of Bronchopulmonary Dysplasia.

RATIONALE: Pregnancies complicated by antenatal stress, including preeclampsia (PE) and chorioamnionitis (CA), increase the risk for bronchopulmonary dysplasia (BPD) in preterm infants, but biologic mechanisms linking prenatal factors with BPD are uncertain. Levels of sFlt-1 (soluble fms-like tyrosine kinase 1), an endogenous antagonist to VEGF (vascular endothelial growth factor), are increased in amniotic fluid and maternal blood in PE and associated with CA. OBJECTIVES: Because impaired VEGF signaling has been implicated in the pathogenesis of BPD, we hypothesized that fetal exposure to sFlt-1 decreases lung growth and causes abnormal lung structure and pulmonary hypertension during infancy. METHODS: To test this hypothesis, we studied the effects of anti-sFlt-1 monoclonal antibody (mAb) treatment on lung growth in two established antenatal models of BPD that mimic PE and CA induced by intraamniotic (i.a.) injections of sFlt-1 or endotoxin, respectively. In experimental PE, mAb was administered by three different approaches, including antenatal treatment by either i.a. instillation or maternal uterine artery infusion, or by postnatal intraperitoneal injections. RESULTS: With each strategy, mAb therapy improved infant lung structure as assessed by radial alveolar count, vessel density, right ventricular hypertrophy, and lung function. As found in the PE model, the adverse lung effects of i.a. endotoxin were also reduced by antenatal or postnatal mAb therapy. CONCLUSIONS: We conclude that treatment with anti-sFlt-1 mAb preserves lung structure and function and prevents right ventricular hypertrophy in two rat models of BPD of antenatal stress and speculate that early mAb therapy may provide a novel strategy for the prevention of BPD.

In vitro characterization of an acetylcholine receptor-transferrin fusion protein for the treatment of myasthenia gravis.

SHG2210, a fusion protein containing the N-terminus of human nicotinic acetylcholine receptor α (AchR-α; aa1-210) and human transferrin (TF), was characterized as a potential therapeutic for myasthenia gravis (MG) caused predominately by α subunit autoantibodies. SHG2210 was shown to be able to bind to α subunit autoantibodies and the TF receptor (TFR). SHG2210 and SHG2210-anti-AchR antibody complex are internalized through TFR-mediated endocytosis. The SHG2210 and SHG2210-anti-AchR antibody complex is present in Lamp1-positive lysosomal compartments after internalization; however, neither SHG2210 nor SHG2210-antibody complex is present in Rab11-positive recycling endosomes. SHG2210 bound to α subunit of AChR autoantibodies may be cleared by the lysosome, resulting in short cellular half-life relative to SHG2210. SHG2210 is shown to have a protective effect on antigenic modulation of the AChR induced by serum from select patients with MG, suggesting that a fusion protein approach may be an effective therapeutic for treating MG.

A rapid, fluorescence-based assay for detecting antigenic modulation of the acetylcholine receptor on human cell lines.

BACKGROUND: Myasthenia gravis (MG) is an autoimmune disease affecting approximately 40,000 patients in the United States. One of the major mechanisms of disease pathology in MG is the binding, internalization, and eventual destruction of acetylcholine receptors (AChR) at the neuromuscular junction by cross-linking AChR-specific autoantibodies. This process, known as antigenic modulation, ultimately attenuates the ability of muscle cells to contract in response to signals from neurons, leading to muscle weakness and fatigue. For this reason, antigenic modulation of the AChR on cultured cells has become an important diagnostic tool for assessing the pathogenicity of AChR-specific autoantibodies. Traditionally, these assays have been done using radiolabeled AChR ligands such as (125)I alpha-bungarotoxin to determine relative AChR number. Here, we present a high-throughput immunofluorescent flow cytometry-based assay that can be used to quantify AChR levels on the cell surface and assess the efficacy of molecules designed to rescue antigenic modulation. METHODS: AChR levels were quantified on human muscle cells before and after treatment with AChR antibodies via immunofluorescent labeling with the AChR monoclonal antibodies, mAb210 and mAb B3, followed by flow cytometry of EDTA-treated cells. RESULTS: Using a novel, flow cytometry-based assay, antigenic modulation of the AChR was demonstrated on human cells using both AChR-specific monoclonal antibody and MG patient serum. The degree of antigenic modulation was dose responsive to antibody levels and could be reversed by preincubating antibodies with soluble AChR alpha subunit extracellular domain. SUMMARY: A rapid, nonradioactive assay was developed to determine the potential of AChR-specific antibodies in the serum of MG patients to bind and down-regulate the AChR. This assay can be used to assess the ability of putative therapeutics that rescue antigenic modulation and could be developed for the treatment of MG.