Angiogenic Therapy in a Fibrotic Murine Model of Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a devastating neuromuscular disorder that affects approximately 1 in 5000 males. Vascular-targeted therapy has been proposed as a treatment for DMD to reduce ischemia and enhance endogenous repair. Additionally, a more vascularized environment may enhance regenerative approaches currently under investigation. Vascular endothelial growth factor (VEGF) and angiopoietin-1 (ANG1) are two of the most studied pro-angiogenic factors for this approach. To date, little is known regarding the effect of these pro-angiogenic factors on muscle function and whether they may exacerbate fibrosis in a relevant murine model of DMD. The first aim of this thesis was to determine the murine model that is best suited for assessing vascular therapy. We demonstrate the dystrophin null, utrophin heterozygous mouse (mdx/utrn+/-) develops more collagen deposition at an earlier age than the commonly used mdx mouse and is therefore a superior choice for assessing therapeutic effects on fibrosis. Next, we investigated the effect of exogenous VEGF treatment on fibroblasts derived from severely affected diaphragm and mildly affected gastrocnemius muscles of mdx/utrn+/- mice. VEGF treatment induced differentiation into myofibroblasts in both cell types, suggesting induction of a fibrotic response. The final aim of the thesis was to assess the effect of VEGF alone or in combination with ANG1 on functional perfusion as assessed non-invasively using dynamic contrast-enhanced computed tomography. A combination of VEGF and ANG1, but not VEGF alone, slowed progression of ischemia in the mdx/utrn+/- hind limb. Increased vessel maturation, as assessed histologically, validated the imaging findings. The combination treatment also decreased fibrosis and leukocyte infiltration, consistent with decreased vascular permeability following ANG1 treatment. Overall, the research in this thesis highlights the drawbacks to use of VEGF as a treatment for ischemia associated with DMD and reveals considerations for future use of vascular therapy in murine models of the disease

Molecular Imaging to Target Transplanted Muscle Progenitor Cells

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle degeneration(1, 2). In patients, the ability of resident muscle satellite cells (SCs) to regenerate damaged myofibers becomes increasingly inefficient(4). Therefore, transplantation of muscle progenitor cells (MPCs)/myoblasts from healthy subjects is a promising therapeutic approach to DMD. A major limitation to the use of stem cell therapy, however, is a lack of reliable imaging technologies for long-term monitoring of implanted cells, and for evaluating its effectiveness. Here, we describe a non-invasive, real-time approach to evaluate the success of myoblast transplantation. This method takes advantage of a unified fusion reporter gene composed of genes (firefly luciferase [fluc], monomeric red fluorescent protein [mrfp] and sr39 thymidine kinase [sr39tk]) whose expression can be imaged with different imaging modalities(9, 10). A variety of imaging modalities, including positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), optical imaging, and high frequency 3D-ultrasound are now available, each with unique advantages and limitations(11). Bioluminescence imaging (BLI) studies, for example, have the advantage of being relatively low cost and high-throughput. It is for this reason that, in this study, we make use of the firefly luciferase (fluc) reporter gene sequence contained within the fusion gene and bioluminescence imaging (BLI) for the short-term localization of viable C2C12 myoblasts following implantation into a mouse model of DMD (muscular dystrophy on the X chromosome [mdx] mouse) (12-14). Importantly, BLI provides us with a means to examine the kinetics of labeled MPCs post-implantation, and will be useful to track cells repeatedly over time and following migration. Our reporter gene approach further allows us to merge multiple imaging modalities in a single living subject; given the tomographic nature, fine spatial resolution and ability to scale up to larger animals and humans(10,11), PET will form the basis of future work that we suggest may facilitate rapid translation of methods developed in cells to preclinical models and to clinical applications

Skeletal Muscle Fibrosis in the mdx/utrn plus /- Mouse Validates Its Suitability as a Murine Model of Duchenne Muscular Dystrophy

Various therapeutic approaches have been studied for the treatment of Duchenne muscular dystrophy (DMD), but none of these approaches have led to significant long-term effects in patients. One reason for this observed inefficacy may be the use of inappropriate animal models for the testing of therapeutic agents. The mdx mouse is the most widely used murine model of DMD, yet it does not model the fibrotic progression observed in patients. Other murine models of DMD are available that lack one or both alleles of utrophin, a functional analog of dystrophin. The aim of this study was to compare fibrosis and myofiber damage in the mdx, mdx/utrn+/- and double knockout (dko) mouse models. We used Masson\u27s trichrome stain and percentage of centrally-nucleated myofibers as indicators of fibrosis and myofiber regeneration, respectively, to assess disease progression in diaphragm and gastrocnemius muscles harvested from young and aged wild-type, mdx, mdx/utrn+/- and dko mice. Our results indicated that eight week-old gastrocnemius muscles of both mdx/utrn+/- and dko hind limb developed fibrosis whereas age-matched mdx gastrocnemius muscle did not (p = 0.002). The amount of collagen found in the mdx/utrn+/- diaphragm was significantly higher than that found in the corresponding diaphragm muscles of wild-type animals, but not of mdx animals (p = 0.0003). Aged mdx/utrn+/- mice developed fibrosis in both diaphragm and gastrocnemius muscles compared to wild-type controls (p = 0.003). Mdx diaphragm was fibrotic in aged mice as well (p = 0.0235), whereas the gastrocnemius muscle in these animals was not fibrotic. We did not measure a significant difference in collagen staining between wild-type and mdx gastrocnemius muscles. The results of this study support previous reports that the moderately-affected mdx/utrn+/- mouse is a better model of DMD, and we show here that this difference is apparent by 2 months of age

Skeletal Muscle Fibrosis in the mdx/utrn+/- Mouse Validates Its Suitability as a Murine Model of Duchenne Muscular Dystrophy

Various therapeutic approaches have been studied for the treatment of Duchenne muscular dystrophy (DMD), but none of these approaches have led to significant long-term effects in patients. One reason for this observed inefficacy may be the use of inappropriate animal models for the testing of therapeutic agents. The mdx mouse is the most widely used murine model of DMD, yet it does not model the fibrotic progression observed in patients. Other murine models of DMD are available that lack one or both alleles of utrophin, a functional analog of dystrophin. The aim of this study was to compare fibrosis and myofiber damage in the mdx, mdx/utrn+/- and double knockout (dko) mouse models.We used Masson’s trichrome stain and percentage of centrally-nucleated myofibers as indicators of fibrosis and myofiber regeneration, respectively, to assess disease progression in diaphragm and gastrocnemius muscles harvested from young and aged wild-type, mdx, mdx/utrn+/- and dko mice. Our results indicated that eight week-old gastrocnemius muscles of both mdx/utrn+/- and dko hind limb developed fibrosis whereas age-matched mdx gastrocnemius muscle did not (p = 0.002). The amount of collagen found in the mdx/utrn+/- diaphragm was significantly higher than that found in the corresponding diaphragm muscles of wild-type animals, but not of mdx animals (p = 0.0003). Aged mdx/utrn+/- mice developed fibrosis in both diaphragm and gastrocnemius muscles compared to wild-type controls (p = 0.003). Mdx diaphragm was fibrotic in aged mice as well (p = 0.0235), whereas the gastrocnemius muscle in these animals was not fibrotic. We did not measure a significant difference in collagen staining between wild-type and mdx gastrocnemius muscles. The results of this study support previous reports that the moderately-affected mdx/utrn+/- mouse is a better model of DMD, and we show here that this difference is apparent by 2 months of age

ANG1 treatment reduces muscle pathology and prevents a decline in perfusion in DMD mice

Vascular endothelial growth factor (VEGF) and other pro-angiogenic growth factors have been investigated to enhance muscle tissue perfusion and repair in Duchenne muscular dystrophy (DMD). Current understanding is limited by a lack of functional data following in vivo delivery of these growth factors. We previously used dynamic contrast-enhanced computed tomography to monitor disease progression in murine models of DMD, but no study to date has utilized this imaging technique to assess vascular therapy in a preclinical model of DMD. In the current study, we locally delivered VEGF and ANG1 alone or in combination to dystrophic hind limb skeletal muscle. Using functional imaging, we found the combination treatment as well as ANG1 alone prevented decline in muscle perfusion whereas VEGF alone had no effect compared to controls. These findings were validated histologically as demonstrated by increased alpha-smooth muscle actin-positive vessels in muscles that received either VEGF+ANG1 or ANG1 alone compared to the sham group. We further show that ANG1 alone slows progression of fibrosis compared to either sham or VEGF treatment. The findings from this study shed new light on the functional effects of vascular therapy and suggest that ANG1 alone may be a candidate therapy in the treatment of DMD

The misnomer of the ‘oral contraceptive pill’

Oral contraception is one of the most commonly prescribed medications globally. Despite the vast body of literature describing the non-contraceptive health benefits of the combined oral contraceptive pill, widespread knowledge of these benefits in both medical education and the community is still lacking. Some of the most problematic gynaecological health issues may be treated with the combined oral contraceptive pill, including: endometrial and ovarian cancers; endometriosis; ovarian cyst accidents; and, menstrual cycle abnormalities. Thus, the incorporation of evidence-based information in standard medical education, and in turn in patient communication, could significantly impact gynaecological health. The goal of this review is to increase awareness of the non-contraceptive benefits of the combined oral contraceptive pill by providing a summary of current clinical observations </p

VEGF induces stress fiber formation in fibroblasts isolated from dystrophic muscle

© 2015, The International CCN Society. Treatment with vascular endothelial growth factor (VEGF) to reduce ischemia and enhance both endogenous muscle repair and regenerative cell therapy in Duchenne muscular dystrophy (DMD) has been widely proposed in recent years. However, the interaction between angiogenesis and fibrosis, a hallmark feature of DMD, remains unclear. To date, it has not been determined whether VEGF exerts a pro-fibrotic effect on DMD-derived fibroblasts, which may contribute to further disease progression. Thus, the purpose of this study was to investigate the effect of exogenous VEGF on fibroblast cultures established from a murine model of DMD. Primary fibroblast cultures were established from gastrocnemius and diaphragm muscles of 10 week-old mdx/utrn+/- mice. Quantitative polymerase chain reaction (qPCR) was employed to assess changes in transcript expression of alpha-smooth muscle actin (Acta2), type-1 collagen (Col1a1), connective tissue growth factor (Ctgf/ccn2) and fibronectin (Fn1). Immunofluorescence and Western blot analysis was further employed to visualize changes in protein expression of alpha-smooth muscle actin (α-SMA), CTGF/CCN2 and fibronectin. mRNA levels of Col1a1, Ctgf/ccn2, and FN did not increase following treatment with VEGF in fibroblasts derived from either diaphragm or gastrocnemius muscles. Acta2 expression increased significantly in diaphragm-derived fibroblasts following treatment with VEGF. Morphological assessment revealed increased stress fiber formation in VEGF-treated fibroblasts compared to the untreated control fibroblasts. The findings from this study suggest that further investigation into the effect of VEGF on fibroblast function is required prior to the utilization of the growth factor as a treatment for DMD

Muscle pathology in 8 week-old GM muscle of wild-type, mdx, mdx/utrn+/- and dko mice.

<p>Extent of total collagen staining (blue) in 8 week-old wild-type (A), mdx (B), mdx/utrn+/- (C) and dko (D) GM muscles was used as a marker of fibrosis. Proportion of centrally nucleated fibers in the same tissues (E, F, G, and H) were measured to assess extent of regeneration. Quantification of total collagen staining (I) and proportion of centrally-nucleated myofibers (J) is represented as the mean +SD. * represents p<0.05, ** represents p<0.01, and *** represents p<0.001 (scale bar = 100μm).</p

Muscle pathology in 8 week-old diaphragm muscle of wild-type, mdx, mdx/utrn+/- and dko mice.

<p>Extent of total collagen staining (blue) in 8 week-old wild-type (A), mdx (B), mdx/utrn+/- (C) and dko (D) diaphragms was used as a marker of fibrosis. Proportion of centrally nucleated fibers in the same tissues (E, F, G, and H) were measured to assess extent of regeneration. Quantification of total collagen staining (I) and proportion of centrally-nucleated myofibers (J) is represented as the mean +SD. * represents p<0.05, ** represents p<0.01, and *** represents p<0.001 (scale bar = 100μm)</p