Keren Ettinger

Losartan, a therapeutic candidate in congenital muscular dystrophy: Studies in the dy2J/dy2J Mouse

Lamininα2‐deficient congenital muscular dystrophy type 1A (MDC1A) is a cureless disease associated with severe disability and shortened lifespan. Previous studies have shown reduced fibrosis and restored skeletal muscle remodeling following treatment with losartan, an angiotensin II type I receptor blocker. We therefore evaluated the effect of losartan treatment in the dy2J/dy2J mouse model of MDC1A.

Nerve growth factor stimulation of ERK1/2 phosphorylation requires both p75NTR and α9β1 integrin and confers myoprotection towards ischemia in C2C12 skeletal muscle cell model

The functions of nerve growth factor (NGF) in skeletal muscles physiology and pathology are not clear and call for an updated investigation. To achieve this goal we sought to investigate NGF-induced ERK1/2 phosphorylation and its role in the C2C12 skeletal muscle myoblasts and myotubes. RT-PCR and western blotting experiments demonstrated expression of p75(NTR), α9β1 integrin, and its regulator ADAM12, but not trkA in the cells, as also found in gastrocnemius and quadriceps mice muscles. Both proNGF and βNGF induced ERK1/2 phosphorylation, a process blocked by (a) the specific MEK inhibitor, PD98059; (b) VLO5, a MLD-disintegrin with relative selectivity towards α9β1 integrin; and (c) p75(NTR) antagonists Thx-B and LM-24, but not the inactive control molecule backbone Thx. Upon treatment for 4 days with either anti-NGF antibody or VLO5 or Thx-B, the proliferation of myoblasts was decreased by 60-70%, 85-90% and 60-80% respectively, indicative of trophic effect of NGF which was autocrinically released by the cells. Exposure of myotubes to ischemic insult in the presence of βNGF, added either 1h before oxygen-glucose-deprivation or concomitant with reoxygenation insults, resulted with about 20% and 33% myoprotection, an effect antagonized by VLO5 and Thx-B, further supporting the trophic role of NGF in C2C12 cells. Cumulatively, the present findings propose that proNGF and βNGF-induced ERK1/2 phosphorylation in C2C12 cells by functional cooperation between p75(NTR) and α9β1 integrin, which are involved in myoprotective effects of autocrine released NGF. Furthermore, the present study establishes an important trophic role of α9β1 in NGF-induced signaling in skeletal muscle model, resembling the role of trkA in neurons. Future molecular characterization of the interactions between NGF receptors in the skeletal muscle will contribute to the understanding of NGF mechanism of action and may provide novel therapeutic targets.

The Ras Antagonist, Farnesylthiosalicylic Acid (FTS), Decreases Fibrosis and Improves Muscle Strength in dy2J/dy2J Mouse Model of Muscular Dystrophy

The Ras superfamily of guanosine-triphosphate (GTP)-binding proteins regulates a diverse spectrum of intracellular processes involved in inflammation and fibrosis. Farnesythiosalicylic acid (FTS) is a unique and potent Ras inhibitor which decreased inflammation and fibrosis in experimentally induced liver cirrhosis and ameliorated inflammatory processes in systemic lupus erythematosus, neuritis and nephritis animal models. FTS effect on Ras expression and activity, muscle strength and fibrosis was evaluated in the dy2J/dy2J mouse model of merosin deficient congenital muscular dystrophy. The dy2J/dy2J mice had significantly increased RAS expression and activity compared with the wild type mice. FTS treatment significantly decreased RAS expression and activity. In addition, phosphorylation of ERK, a Ras downstream protein, was significantly decreased following FTS treatment in the dy2J/dy2J mice. Clinically, FTS treated mice showed significant improvement in hind limb muscle strength measured by electronic grip strength meter. Significant reduction of fibrosis was demonstrated in the treated group by quantitative Sirius Red staining and lower muscle collagen content. FTS effect was associated with significantly inhibition of both MMP-2 and MMP-9 activities. We conclude that active RAS inhibition by FTS was associated with attenuated fibrosis and improved muscle strength in the dy2J/dy2J mouse model of congenital muscular dystrophy.

Improved muscle strength and mobility in the dy2J/dy2J mouse with merosin deficient congenital muscular dystrophy treated with Glatiramer acetate

The therapeutic effect of Glatiramer acetate, an immune modulating agent, was evaluated in the dy(2J)/dy(2J) mouse with merosin deficient congenital muscular dystrophy, which is a milder variant of the dy/dy mouse. The treated mice showed significant improvement in hind limb muscle strength measured by electronic grip strength meter and in motor performance quantified by video detection software. Glatiramer acetate treatment was associated with significantly increased expression of regeneration transcription factors MyoD and myogenin, and attenuation of the fibrosis markers vimentin and fibronectin. No effective treatment is currently available in congenital muscular dystrophy and Glatiramer acetate may present a new potential treatment for this disorder.

Bio-Imaging of Colorectal Cancer Models Using Near Infrared Labeled Epidermal Growth Factor

Novel strategies that target the epidermal growth factor receptor (EGFR) have led to the clinical development of monoclonal antibodies, which treat metastatic colorectal cancer (mCRC) but only subgroups of patients with increased wild type KRAS and EGFR gene copy, respond to these agents. Furthermore, resistance to EGFR blockade inevitably occurred, making future therapy difficult. Novel bio-imaging (BOI) methods may assist in quantization of EGFR in mCRC tissue thus complementing the immunohistochemistry methodology, in guiding the future treatment of these patients. The aim of the present study was to explore the usefulness of near infrared-labeled EGF (EGF-NIR) for bio-imaging of CRC using in vitro and in vivo orthotopic tumor CRC models and ex vivo human CRC tissues. We describe the preparation and characterization of EGF-NIR and investigate binding, using BOI of a panel of CRC cell culture models resembling heterogeneity of human CRC tissues. EGF-NIR was specifically and selectively bound by EGFR expressing CRC cells, the intensity of EGF-NIR signal to background ratio (SBR) reflected EGFR levels, dose-response and time course imaging experiments provided optimal conditions for quantization of EGFR levels by BOI. EGF-NIR imaging of mice with HT-29 orthotopic CRC tumor indicated that EGF-NIR is more slowly cleared from the tumor and the highest SBR between tumor and normal adjacent tissue was achieved two days post-injection. Furthermore, images of dissected tissues demonstrated accumulation of EGF-NIR in the tumor and liver. EGF-NIR specifically and strongly labeled EGFR positive human CRC tissues while adjacent CRC tissue and EGFR negative tissues expressed weak NIR signals. This study emphasizes the use of EGF-NIR for preclinical studies. Combined with other methods, EGF-NIR could provide an additional bio-imaging specific tool in the standardization of measurements of EGFR expression in CRC tissues.

Association of p75NTR and α9β1 integrin modulates NGF-dependent cellular responses

Direct interaction of α9β1 integrin with nerve growth factor (NGF) has been previously reported to induce pro-proliferative and pro-survival activities of non-neuronal cells. We investigated participation of p75(NTR) in α9β1 integrin-dependent cellular response to NGF stimulation. Using selective transfection of glioma cell lines with these receptors, we showed a strong, cation-independent association of α9 integrin subunit with p75(NTR) on the cellular membrane by selective immunoprecipitation experiments. The presence of the α9/p75(NTR) complex increases NGF-dependent cell adhesion, proliferation and migration. Other integrin subunits including β1 were not found in complex with p75(NTR). FRET analysis indicated that p75(NTR) and α9 integrin subunit are not closely associated through their cytoplasmic domains, most probably because of the molecular interference with other cytoplasmic proteins such as paxillin. Interaction of α9β1 integrin with another ligand, VCAM-1 was not modulated by the p75(NTR). α9/p75(NTR) complex elevated NGF-dependent activation of MAPK Erk1/2 arty for integrin that may create active complexes with other types of receptors belonging to the TNF superfamily.

Nerve growth factor-induced myoprotection in C2C12 muscle cells is mediated by α9β1 integrin via release of PGE2.

Abstract Background: Nerve growth factor (NGF) mediates a wide range of activities in the central nervous system including neuronal differentiation, synaptic plasticity, and neuroprotection. In addition, NGF places an important role in skeletal muscle physiology by some unknown mechanisms. We recently demonstrated that NGF conferred myoprotection toward ischemia in C2C12 skeletal muscle cell model, establishing an important trophic role for NGF in skeletal muscle. Methods: In this report, using ELISA and oxygen-glucose deprivation (OGD) assays, we investigated the potential contribution of prostaglandin E2 (PGE2) to NGF myoprotective effects toward C2C12 cultures exposed to OGD insults. Vipera lebetina obtusa disintegrin 5 (VLO5), a selective antagonist of α9β1 integrin, was used as an experimental tool to clarify α9β1 integrin role in NGF action. Results: NGF-induced mitogen-activated protein kinase type 1 or 2 (ERK1/2) phosphorylation in C2C12 cells and in a dose-response fashion stimulated PGE2 release, both effects antagonized by VLO5 and PD98059. NGF-induced myoprotection of the cells exposed for 7 h to OGD, followed by 18 h of reoxygenation, was reversed by VLO5 treatment. Conclusions: These results suggest that NGF activation of α9β1 integrin induced myoprotection by stimulation of ERK phosphorylation and release of cytoprotective PGE2 mediator. This effect may be also relevant for NGF-induced pain and hyperalgesia in the skeletal muscle.

The Effects of a Chactoid Scorpion Venom and Its Purified Toxins on Rat Blood Pressure and Mast Cells Histamine Release

The effect of the venom of the Chactoid family of scorpions on blood pressure was scantly investigated and was addressed in the present study using the venom of the Israeli scorpion, Scorpio maurus palmatus. Blood pressure in rats was monitored via cannulated femoral artery, while venom and toxins were introduced into femoral vein. Venom injection elicited a biphasic effect, expressed first by a fast and transient hypotensive response, which lasted up to 10 min, followed by a hypertensive response, which lasted up to one hour. It was found that these effects resulted from different venom components. Phospholipase A2 produced the hypotensive effect, while a non-enzymatic neurotoxic polypeptide fraction produced the hypertensive effect. Surprisingly, the main neurotoxic polypeptide to mice had no effect on blood pressure. In vitro experiments indicated that the hypertensive factors caused histamine release from the peritoneal mast cells, but this effect is assumed to be not relevant to their in vivo effect. In spite of the cytotoxic activity of phospholipase A2, it did not release histamine. These findings suggest that the effects of venom and isolated fractions on blood pressure parameters are mediated by different mechanisms, which deserve further pharmacological investigation.

Cytocompatibility of novel extracellular matrix protein analogs of biodegradable polyester polymers derived from α-hydroxy amino acids

One of the challenges in regenerative medicine is the development of novel biodegradable materials to build scaffolds that will support multiple cell types for tissue engineering. Here we describe the preparation, characterization, and cytocompatibility of homo- and hetero-polyesters of α-hydroxy amino acid derivatives with or without lactic acid conjugation. The polymers were prepared by a direct condensation method and characterized using gel permeation chromatography, 1H-nuclear magnetic resonance spectroscopy, Fourier transform infrared spectroscopy, differential scanning calorimetry, optical activity, and solubility. The surface charge of the polymers was evaluated using zeta potential measurements. The polymers were coated onto glass cover slips followed by characterization using nano-surface profiler, thin film reflectometry, and atomic force microscopy (AFM). Their interaction with endothelial and neuronal cells was assessed using adhesion, proliferation, and differentiation assays. Of the characterized polymers, Poly-HOVal-LA, but not Poly-(D)HOPhe, significantly augmented nerve growth factor (NGF)-induced neuronal differentiation of the PC12 pheochromcytoma cells. In contrast, Poly-HOLeu increased by 20% the adhesion of endothelial cells, but did not affect PC12 cell differentiation. NGF-induced Erk1/2 phosphorylation in PC12 cells grown on the different polymers was similar to the effect observed for cells cultured on collagen type I. While no significant association could be established between charge and the differentiative/proliferative properties of the polymers, AFM analysis indicated augmentation of NGF-induced neuronal differentiation on smooth polymer surfaces. We conclude that overall selective cytocompatibility and bioactivity might render α-hydroxy amino acid polymers useful as extracellular matrix-mimicking materials for tissue engineering.

T.P.6 Combination anti-inflammatory and anti-fibrotic treatment in muscular dystrophy – The more the better?

Abstract Congenital muscular dystrophy is an incurable disorder with no effective treatment. Anti-inflammatory and anti-fibrotic agents have been suggested as potential therapies. As previously demonstrated by our group, Glatiramer acetate (GA), an anti-inflammatory agent and the anti-fibrotic agent Losartan exert their effect through different pathways and have distinct beneficial effects on strength, mobility and fibrosis. Thus, the aim of the present study was to evaluate the effect of combination therapy of GA with Losartan in the dy2J/dy2J mouse model of congenital muscular dystrophy. Fore and hind limb muscle strength, fibrosis and mobility parameters were assessed. dy2J/dy2J mice receiving the combination of GA/Losartan or GA alone showed mild improvement in forelimb muscle strength (11%) in contrast to treatment with Losartan alone (74%). Hind limb muscle strength was unchanged in dy2J/dy2J mice receiving the combination therapy, while the two drugs alone showed significant improvement in strength (GA; 52.7% vs. Losartan; 74%). The combined treatment showed only mild reduction in muscle fibrosis (20%) compared with Losartan alone (42.3%. No significant change for GA). GA/Losartan combination therapy or Losartan alone showed no change in mobility parameters. However, treatment with GA alone resulted in a marked improvement in mobility parameters. In conclusion, the improvements seen with GA/Losartan combination therapy were less noticeable than the improvements of each of the two agents administered as a single medication. We conclude that combination therapy should be administered with caution. Combining two medications with theoretical synergistic effect and previous positive effect may result in a reduction of the therapeutic effect compared to the use of each of these agents separately.