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Vascular-targeted therapies for Duchenne muscular dystrophy

Vascular-targeted therapies for Duchenne muscular dystrophy Duchenne muscular dystrophy (DMD) is the most common muscular dystrophy and an X-linked recessive, progressive muscle wasting disease caused by the absence of a functional dystrophin protein. Dystrophin has a structural role as a cytoskeletal stabilization protein and protects cells against contraction-induced damage. Dystrophin also serves a signaling role through mechanotransduction of forces and localization of neuronal nitric oxide synthase (nNOS), which produces nitric oxide (NO) to facilitate vasorelaxation. In DMD, the signaling defects produce inadequate tissue perfusion caused by functional ischemia due to a diminished ability to respond to shear stress induced endothelium- dependent dilation. Additionally, the structural defects seen in DMD render myocytes with an increased susceptibility to mechanical stress. The combination of both defects is necessary to generate myocyte damage, which induces successive rounds of myofiber degeneration and regeneration, loss of calcium homeostasis, chronic inflammatory response, fibrosis, and myonecrosis. In individuals with DMD, these processes inevitably cause loss of ambulation shortly after the first decade and an abbreviated life with death in the third or fourth decade due to cardio-respiratory anomalies. There is no known cure for DMD, and although the culpable gene has been identified for more than twenty years, research on treatments has produced few clinically relevant results. Several recent studies on novel DMD therapeutics are vascular targeted and focused on attenuating the inherent functional ischemia. One approach improves vasorelaxation capacity through pharmaceutical inhibition of either phosphodiesterase 5 (PDE5) or angiotensin- converting enzyme (ACE). Another approach increases the density of the underlying vascular network by inducing angiogenesis, and this has been accomplished through either direct delivery of vascular endothelial growth factor (VEGF) or by downregulating the VEGF decoy-receptor type 1 (VEGFR-1 or Flt-1). The pro-angiogenic approaches also seem to be pro-myogenic and could resolve the age-related decline in satellite cell (SC) quantity seen in mdx models through expansion of the SC juxtavascular niche. Here we review these four vascular targeted treatment strategies for DMD and discuss mechanisms, proof of concept, and the potential for clinical relevance associated with each therapy. Keywords: Duchenne muscular dystrophy, VEGF, Flt-1, Flk-1, Nitric oxide, PDE5 inhibitor, ACE inhibitor, Satellite cell, Muscle regeneration, Myofiber damage Review and has an annual incidence affecting one in every 3600– Duchenne muscular dystrophy (DMD) is an X-linked re- 6000 newborn males [3]. Normally, dystrophin serves as cessive, progressive muscle wasting disease caused by muta- the bridge in the dystrophin-associated glycoprotein com- tions in the DMD gene that lead to absence of a functional plex (DAPC), connecting the cytoskeleton, via attachments dystrophin protein [1,2]. Both fatal and devastating, DMD to subsarcolemmal F-actin, to the extracellular matrix is the most common muscular dystrophy seen in children through an association with plasma membrane bound β- dystroglycan [4]. In the DAPC, dystrophin has a structural role as a cytoskeletal stabilization protein and protects * Correspondence: asakura@umn.edu cells against contraction-induced damage. Dystrophin also Stem Cell Institute, University of Minnesota Medical School, McGuire Translational Research Facility, Room 4-220, 2001 6th Street SE, Minneapolis, serves signaling roles, including mechanotransduction of MN 55455, USA forces and localization of signaling proteins, such as neu- Paul and Shelia Wellstone Muscular Dystrophy Center, University of ronal nitric oxide synthase (nNOS), which synthesizes ni- Minnesota Medical School, Wallin Medical Biosciences Building, 2101 6th Steet SE, Minneapolis, MN 55455, USA tric oxide (NO) to facilitate vasorelaxation [5-7]. Without Full list of author information is available at the end of the article © 2013 Ennen et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Ennen et al. Skeletal Muscle 2013, 3:9 Page 2 of 12 http://www.skeletalmusclejournal.com/content/3/1/9 dystrophin, the DAPC cannot completely assemble, and the supportive link between the cytoskeleton and the extracellular matrix becomes destabilized [8]. Despite nor- Functional ischemia Greater cellular Combined effect from reduced NO- susceptibility of both factors mal development, the membrane in dystrophin-deficient mediated protection to metabolic stress cells is easily damaged. Membrane microlesions facilitate an influx of calcium ions, which activate proteases to begin auto-digestion of the musculature sarcoplasm Myofiber damage [9-11]. Macrophages later arrive at the tissue to remove cellular debris, and satellite cells (SCs) are activated and proliferate to induce myofiber regeneration. This causes Reduced net Greater cellular Reduced damage from Vascular Therapy susceptibility successive rounds of myofiber degeneration and regener- functional Treatment Target combining to metabolic stress ischemia both factors ation that is exacerbated by continual membrane damage and ensuing myonecrosis. In addition, cytokines released in the process of myonecrosis recruit inflammatory cells, Reduced myofiber damage which release inflammatory cytokines to activate fibro- blasts that lay down extracellular matrix proteins and lead Figure 1 The two-hit hypothesis for myocyte damage and the to fibrosis [12]. Skeletal muscle regenerative capacity later proposed outcome of functional ischemia attenuation in Duchenne muscular dystrophy (DMD). (A) The combined effects diminishes with advancing age and decreasing numbers of from functional ischemia due to reduced nitric oxide (NO)-mediated SCs, and muscle tissue is steadily replaced by adipose and protection and greater cellular susceptibility to metabolic stress are connective tissues [13]. necessary to produce the myofiber damage observed in DMD [17]. The previously described cellular events manifest them- (B) Attenuating functional ischemia by administering a vascular selves clinically in a devastating and progressive manner. targeted treatment can reduce the net-combined effect of both two-hit factors and consequently curtail myofiber damage. Despite continuous contractions by the myocardium, the skeletal muscles deteriorate first in individuals with DMD, and most permanently lose ambulatory abilities shortly after the first decade [14]. Myocardial problems present or increasing the underlying vascular density in order to later, and clinically relevant cardiomyopathy is seen in 90% reduce the functional ischemia and improve the DMD of patients over 18 years old, namely due to the onset of phenotype. cardiac fibrosis in addition to rhythm and conduction ab- normalities [14]. Respiratory problems are also inevitable due to muscle wasting in the diaphragm and the onset of Defect of nitric oxide-mediated vasodilation contributes scoliosis [14]. Even with improvements in treatment, to Duchenne muscular dystrophy phenotype notably multidisciplinary care, the combined cardio- The DMD pathogenesis is partially explained by the lack of respiratory anomalies mean that most individuals with the signaling role of dystrophin, which normally localizes DMD die in their third or fourth decade of life [15,16]. nNOS to the sarcolemma through binding to the C- Despite knowledge of the responsible gene for over twenty terminal region of dystrophin [6]. The nNOS is responsible years, a DMD cure remains to be found, and research on for NO production to facilitate smooth muscle vasodila- treatments has produced few clinically relevant results. tion in response to increased metabolic demands. During Current treatment options, such as corticosteroid admin- muscle contraction, NO-mediated vasodilation is import- istration, physical therapy, nocturnal ventilation, and sur- ant to help offset the α-adrenergic vasoconstriction in re- gical interventions aim for symptomatic management and sponse to sympathetic activation, which optimizes muscle have been shown to improve lifespan and quality of life perfusion [18]. This functional response is intact in healthy [16]. The clinical utility and feasibility of gene therapy and children, but in children with DMD the sympathetic vaso- cell therapy remain to be elucidated, and other treatment constriction in skeletal muscle is unopposed due to lack of areas must be sought. Our current, more holistic under- NO-mediated vasodilation [18]. standing of DMD pathogenesis, especially with more The nNOS is absent from the sarcolemma and is recent knowledge of the vascular role of dystrophin, im- greatly downregulated in the cytoplasm of dystrophin- plies that vascular-targeted therapies are strong candi- deficient muscle, which results in muscle vasoconstric- dates for future investigation. Specifically, attenuating tion and abnormal blood flow during skeletal muscle functional ischemia could reduce myocyte damage, in- contraction [6,18,19]. Specifically, loss of dystrophin in crease tissue perfusion, reduce cardiac workload, and pre- the smooth muscle results in a decreased capacity of vent cardiac and skeletal muscle remodeling (Figure 1B). the vasculature to respond to shear stress induced This review will focus on vascular-targeted treatment endothelium-dependent dilation, probably related to the avenues aimed at either improving vasorelaxation capacity signaling defects seen in both force transduction and Ennen et al. Skeletal Muscle 2013, 3:9 Page 3 of 12 http://www.skeletalmusclejournal.com/content/3/1/9 inadequate NO production [19]. Furthermore, shear stress Improved vasorelaxation capacity at the endothelial cell surface is a known catalyst for Angiotensin-converting enzyme inhibitors angiogenesis [20-23], so new blood vessel formation could The renin-angiotensin-aldosterone system plays a vital be downregulated and mismatched to metabolic need in role in regulating both systemic vascular resistance and the absence of dystrophin due to defects in mechano- total blood volume, which together impact arterial pres- transduction. Lack of the signaling and structural roles sure and myocardial function. A key component is the of dystrophin in DMD pathogenesis have led to a two- angiotensin-converting enzyme (ACE), which transforms hit hypothesis, whereby the combination of functional the peptide hormone angiotensin I into angiotensin II; cir- ischemia due to reduced capacity to benefit from NO- culating angiotensin II then stimulates vascular smooth mediated protection and an increased susceptibility to muscle contraction, increasing vascular resistance and ar- metabolic stress are both required to cause myocyte da- terial pressure. Angiotensin II also induces the release of mage (Figure 1A) [17]. So although not completely culpable aldosterone, which increases sodium and water retention, for the observed pathogenesis, impaired vascular function- and vasopressin, which increases water retention. ing seems to be both inherent to DMD and an accelerant Preventing angiotensin II production through pharmaco- to tissue damage in the skeletal and cardiac muscles. logical ACE inhibition has been shown to reduce high Recent studies suggest that the two-hit hypothesis blood pressure and cardiac workload through enhanced should migrate away from simply observational specula- vasorelaxation and prevention of downstream hormone tion towards a more widely accepted, evidence-based release, and ACE inhibitors (ACEIs) are currently used to DMD pathogenic theory. One functional study using treat congestive heart failure and hypertension [28-30]. As model DMD mdx mice showed quantitative evidence such, improving cardiac function and enhancing systemic supporting the two-hit hypothesis, where inhibition of vasorelaxation capacity through ACE inhibition in DMD NO/EDHF (EDHF is endothelium-derived hyperpolariz- patients could have prophylactic benefit by mitigating the ing factor, another vasodilator) alone in wild-type mice functional ischemia and consequently diminishing myo- caused similar functional ischemia (one-hit) to that seen necrosis. In mdx mice, the ACEI captopril administered in mdx mice [24]. But, the forced functional ischemia over an 8-week period and prior to the onset of cardio- alone in the wild-type mice did not induce similar levels myopathy was shown to reduce cardiac afterload, of muscle cell death seen in mdx mice [24]. Two-hits, increase myocardial contractility, and improve cardiac consisting of severe ischemia and strenuous tetanic stim- hemodynamics compared to mdx control mice [31]. In uli, were necessary to produce the same contraction- clinical studies, administration of the ACEI perindopril dependent myofiber damage in wild-type mice to that of has shown that early treatment in 9.5- to 13-year-old mdx mice [24]. So, mdx myofibers exhibit enhanced DMD patients with normal cardiac functioning (as mea- vulnerability to metabolic and mechanical stress inde- sured by normal left ventricular ejection fraction or LVEF), pendently of the altered vasodilatory response, yet the is capable of delaying both the onset and progression of combinatory effect of both factors (two-hits) is necessary left ventricular dysfunction and significantly lowering to mediate the cell death numbers seen in mdx myo- mortality rates compared to patients starting treatment 3 fibers. This could also explain why nNOS knockout mice, years later [32,33]. Results are less clear in DMD cases in- a one-hit model, do not develop muscular dystrophy, yet volving established cardiomyopathy where administration myocardial specific nNOS expression prevents cardiomy- of the ACEI enalapril showed functional normalization in opathy in mdx mice by increasing the capacity to benefit just 43% of cases, but this positive functional effect was from NO-mediated protection [25,26]. Interestingly, mdx maintained by most subjects for up to four years [34]. mice that only express dystrophin in smooth muscle Combination therapy using ACEIs and β-adrenergic (SMTg/mdx), which has a prominent role in regulation of receptor antagonists or β-blockers (BBs) has also been vascular tone and blood flow, have an intermediate investigated for use in DMD treatment. Catecholamines phenotype between wild-type and mdx mice [27]. The increase heart rate and myocardial contractility through SMTg/mdx mice showed some, albeit not total, recovery the β-adrenergic receptors, and thus targeted BBs cause of the NO-dependent vasorelaxation mechanism in ac- the heart to beat slower and with less force and are typ- tive skeletal muscle [27]. In the SMTg/mdx model, lack ically given to patients with arrhythmia or disordered of dystrophin at the myofiber level and the consequent automaticity. One study that utilized a combination of lack of complete force mechanotransduction in response BBs and ACEIs in DMD patients with established cardio- to functional demands were perhaps partially responsible myopathy found a positive effect on long-term survival, for the insufficient phenotypic recovery. These data over- especially for individuals that showed no overt symptoms whelmingly reveal the important role the vasculature of heart failure despite documented left ventricular dys- plays in DMD pathogenesis and highlight a novel arena function [35]. A more recent study also investigated DMD for therapeutic intervention. patients with established cardiomyopathy but broke Ennen et al. Skeletal Muscle 2013, 3:9 Page 4 of 12 http://www.skeletalmusclejournal.com/content/3/1/9 treatment groups down into ACEI (lisinopril) alone or angiotensin II dependent stimulation of pro-oxidant and ACEI plus BB (metoprolol) [36]. Both treatment groups pro-inflammatory pathways [53]. Overall, ACEIs and/or displayed improvements in cardiac function compared to BBs appear to be excellent candidates for DMD therapy pre-therapy measurements, but no significant difference based on current clinical availability and demonstrated in cardiac function was seen between groups [36]. Future ability to reduce many of the negative outcomes normally studies should address treatment using ACEI alone or associated with the DMD pathogenic process. Still, a ACEI plus BB in DMD cases where cardiomyopathy has broader investigation regarding the potential prophylactic not been fully established to assess the potential for benefit of ACEIs should be conducted to determine an op- prophylactic benefit as this could definitively rule out the timal age of initiation. need for a BB. Additionally, with regard to the two-hit hy- pothesis, studies that address functional ischemia attenu- Phosphodiesterase 5 inhibitors ation to mitigate myonecrosis through enhanced tissue In the NO-cGMP signaling pathway, nitric oxide synthase perfusion by ACEI-mediated vasorelaxation have not yet (NOS) produces NO to activate soluble guanylyl cyclase been performed. (sGC) to synthesize cyclic guanosine monophosphate Another therapeutic strategy that targets the renin- (cGMP), and cGMP activates protein kinase G to induce angiotensin-aldosterone system to improve vasorelax- vasodilation. The cGMP-specific phosphodiesterases are ation capacity utilizes the antihypertensive drug losartan, responsible for cGMP degradation, so vasoconstriction be- which is an angiotensin II type I receptor antagonist or gins as concentrations of cGMP diminish. This pathway is angiotensin receptor blocker (ARB). Long-term adminis- disrupted in dystrophin deficient membranes, as nNOS is tration of losartan in mdx mice showed improvements absent from the sarcolemma and greatly downregulated, in myocardial function, but not skeletal muscle function, which contributes to the observed functional ischemia and reductions in mortality compared to control [37,38]. [6,19]. Additionally, studies have shown greater cGMP- Explanations as to why losartan could only ameliorate specific phosphodiesterase 5 (PDE5) activity in mdx skel- the function of cardiac muscle remain limited, but the etal muscle samples and decreased cGMP production primary mechanism could be the significant reduction in compared with controls [54,55]. Recently, cGMP-specific ™ ™ afterload seen in the hearts of losartan treated mdx mice PDE5 inhibitors, specifically tadalafil (Cialis or Adcirca ) ™ ™ [38]. Decreased afterload certainly reduces cardiac work- and sildenafil (Viagra or Revatio ) have been investi- load, and this may minimize mechanical injury and sub- gated for their potential in ameliorating the functional is- sequent fibrosis to the sensitive cardiomyocytes in mdx chemia in DMD by increasing intracellular levels of cGMP hearts. Still, the use of losartan as a prophylactic treatment to prolong vasodilation and increase blood flow to tissues. against DMD-related cardiomyopathy seems promising Asai et al. elegantly showed that tadalafil administra- based on these pre-clinical studies and the current clinical tion prior to progressive myofiber damage was able to availability of losartan (COZAAR ) for its use in hyper- significantly lower the net quantity of myofiber damage tension. Future investigation should be directed at evaluat- in mdx mice compared to placebo [24]. Essentially, at- ing losartan in DMD patients but also, owing to pathway tenuation of functional ischemia using tadalafil was similarity, at comparing the effectiveness of losartan to the shown to reduce the extent of contraction-induced dam- many FDA-approved ACEIs. age [24]. Additionally, early treatment utilizing PDE5 The definitive mechanism behind reducing cardiomyop- inhibitors could have clinical prophylactic benefit, for athy via ARBs, ACEIs, and/or BBs in DMD patients is not mdx mice treated with tadalafil from conception showed completely established, but reduced aldosterone signaling improved histology [24]. Khairallah et al. showed that car- through ACE inhibition could prevent fibrotic tissue diac mRNA expression levels of atrial natriuretic factor development, as previous use of aldosterone-specific (ANF), an early indicator for initiation of cardiomyopathic blockers has shown benefit in cases of heart failure remodeling, was significantly reduced in mdx mice treated [39-42]. Additionally, angiotensin II can directly induce with sildenafil [56,57]. This implies that sildenafil is cap- vasoconstriction, pro-fibrotic Smad signaling, pro-fibrotic able of inhibiting the advancement of cardiomyopathic transforming growth factor beta (TGF-β) production, and remodeling at early stages of DMD [57]. Sildenafil has also the ubiquitin-proteasome pathway that has a role in the been shown to have positive functional effects in the proteolysis happening in dystrophic tissues [43-46]. hearts of mdx mice, notably by avoidance of cardio- Angiotensin II is also known to enhance NADPH-oxidase myocyte damage produced in vivo via cardiac workload activity, which leads to overproduction of superoxide augmentation and maintenance of an elevated heart rate anion and accounts for the oxidative stress in cardiac and response for a significantly longer period of time com- skeletal muscle of the mdx mouse [47-52]. ACE inhibition pared with placebo [57]. Interestingly, administration of can reduce these adverse effects, and a study with mdx sildenafil is even capable of reversing cardiac dysfunction mice demonstrated that the ACEI enalapril can prevent in mdx mice with established cardiomyopathy [58]. Ennen et al. Skeletal Muscle 2013, 3:9 Page 5 of 12 http://www.skeletalmusclejournal.com/content/3/1/9 Nevertheless, the target cell and mechanism behind the an intracellular signaling domain and thus only serves in a reversal are still unclear. regulatory capacity by sequestering VEGF-A [63]. Flt-1 Additionally, PDE5 inhibitors have also shown im- and Flk-1 contain an extracellular VEGF-A-binding do- provement in muscle tissue from other vertebrate models main and an intracellular tyrosine kinase domain, and of DMD, namely two dystrophin deficient zebrafish both show expression during the developmental stage and models known as sapje and sapje-like mutants [59,60]. tissue regeneration in hemangioblasts and endothelial cell Dystrophin-null zebrafish treated with aminophylline, a lineages [63-65]. Flt-1 has a 10 times greater binding affin- nonselective phosphodiesterase inhibitor, were able to ity for VEGF-A (K approximately 2 to 10 pM) compared survive significantly longer compared to controls and to Flk-1, but the weaker tyrosine kinase domain indicates had restored skeletal muscle structure similar to wild- that angiogenic signal transduction following VEGF-A type zebrafish [61]. Furthermore, analysis of sapje mu- binding to Flt-1 is comparably weaker than the Flk-1 sig- tants treated 1 to 4 days postfertilization with different nal (Figure 2A) [63]. As such, homozygous Flt-1 gene phosphodiesterase inhibitors revealed that treatment with knockout mice die in the embryonic stage from endothe- aminophylline or sildenafil citrate resulted in the lowest lial cell overproduction and blood vessel disorganization percentage of fish showing abnormal muscle structure (Figure 2B) [64-66]. Inversely, homozygous Flk-1 gene [61]. These data suggest that aminophylline and sildenafil knockout mice die from defects in the development of or- citrate are capable of preventing the onset of aberrant ganized blood vessels due to lack of yolk-sac blood island muscle architecture in dystrophin-null zebrafish. These formation during embryogenesis (Figure 2C) [67]. Both findings from DMD model zebrafish are analogous to the the Flt-1 and Flk-1 receptors are needed for normal devel- results seen from mdx mice treated with PDE5 inhibitors, opment, but selective augmentation in VEGF-A concen- and the consistency of these results across several DMD tration should allow for greater binding to the Flk-1 models leaves hope that these compounds could benefit receptor and induce a pro-angiogenic effect that increases individuals living with DMD. Clinical trials assessing capillary density. PDE5 inhibitors for DMD patients are currently under- Several studies have demonstrated that administration of way, and future use of tadalafil or sildenafil for these indi- VEGF using exogenous expression mediated through viduals seems promising based on preclinical studies and engineered myoblasts, direct systemic injections, or adeno- current clinical availability of these drugs for their use in associated viral (AAV) vectors are capable of initiating an treating erectile dysfunction and pulmonary hypertension. angiogenic signal in the myocardium and skeletal muscle in both ischemic and non-ischemic conditions [69-72]. Increased vascular density However, these same studies do highlight the importance Vascular endothelial growth factor administration of precisely regulating VEGF delivery quantities for future The central paradigm behind the previously discussed clinical use, as overadministration has been shown to have PDE5 and ACEI therapies for DMD was that increasing deleterious effects in animal models, such as hemangioma the vasorelaxation capacity of the vasculature would be formation [70]. Unfortunately, and to the best of our able to increase perfusion, diminish the effects from knowledge, functional studies that assess blood flow in functional ischemia, and decrease myocyte damage. How- DMD model organisms following VEGF-induced angio- ever, another technique to increase tissue perfusion would genesis have not yet been conducted. However, one study be to increase the density of the underlying vascular archi- found that four weeks following intramuscular adminis- tecture that nourishes the skeletal and cardiac muscles. tration of rAAV-VEGF vectors in the bicep and tibialis One method of increasing vascular density is to augment anterior (TA) muscles of 4-week-old mdx mice, the angiogenesis, which regulates the production of new rAAV-VEGF-treated mdx mice showed significantly vasculatures from the existing framework. The vascular greater forelimb strength compared to pretreatment endothelial growth factor (VEGF) family of signal glyco- levels and AAV-LacZ-treated control mdx mice [73]. The proteins acts as potent promoters of angiogenesis during same study confirmed the feasibility of VEGF-mediated embryogenesis and postnatal growth. Specifically, the angiogenic induction in mdx mice and showed greater binding of the VEGF-A ligand with the VEGF receptors capillary density, particularly in the area of regenerating has been shown to promote vascular permeability and also fibers, as well as reduced necrotic fiber area in biceps trigger endothelial cell migration, proliferation, and sur- muscle compared to AAV-LacZ-treated control mdx vival, and the newly formed endothelial cells provide the mice [73]. So, although this study did not directly assess basic structure of new vasculatures [62]. The dominant reduction of functional ischemia via enhanced vascular VEGF signal molecule for angiogenesis, VEGF-A, mediates density through VEGF-induced angiogenesis, it was able its signal through VEGF receptor-1 (VEGFR-1, hereafter to demonstrate similar outcomes from the PDE5 inhibi- Flt-1) and VEGF receptor-2 (VEGFR-2, hereafter Flk-1) tor studies, especially the decrease seen in necrotic fiber [63]. A soluble form of Flt-1 (sFlt-1) also exists, but lacks area and improvement in muscle function. Ennen et al. Skeletal Muscle 2013, 3:9 Page 6 of 12 http://www.skeletalmusclejournal.com/content/3/1/9 AC B Heterozygous Wild Type Flk-1gene KO Flt-1gene KO Flt-1gene KO sFlt-1 sFlt-1 sFlt-1 sFlt-1 VEGF = VEGF Affinity Flt-1 Flk-1 Flt-1 Flk-1 Flt-1 Flk-1 Flt-1 Flk-1 Membrane Angiogenic Signal Strength Enhanced Normal Embryonic Embryonic Angiogenesis and Lethal: Excessive Lethal: Signaling Angiogenesis Improved DMD Angiogenesis Defect Phenotype Figure 2 Flt-1 is a decoy receptor for vascular endothelial growth factor (VEGF) pro-angiogenic signaling. (A) In the wild-type scenario, VEGF induces a pro-angiogenic signal by binding the Flt-1 or Flk-1 receptors [63]. Flt-1 has a higher binding affinity for VEGF but transmits a weaker angiogenic signal compared to Flk-1, which implies that Flt-1 acts a negative regulator of angiogenesis [63]. The soluble form of Flt-1 (sFlt-1) lacks the transmembrane and intracellular signaling domains of Flt-1 and only serves a regulatory role by sequestering VEGF [63]. (B) Flt-1 −/− homozygous knockout (Flt-1 ) mice die in the early embryonic stage from endothelial cell overproduction and blood vessel disorganization, −/− indicating that Flt-1 is a decoy regulator for endothelial growth/differentiation [64-66]. (C) Flk-1 homozygous knockout (Flk-1 ) mice die in the early embryonic stage from defects in the development of organized blood vessels, indicating that Flk-1 is a positive regulator for endothelial +/− growth/differentiation [67]. (D) Developmental reduction of the Flt-1 receptor through haploinsufficiency of the Flt-1 gene (Flt-1 ) has been +/− shown to increase capillary density in skeletal muscle, and this same phenomenon has been demonstrated in mdx mice (mdx:Flt-1 ) [79]. The +/− mdx:Flt-1 mice also showed improved histological and functional parameters normally associated with the Duchenne muscular dystrophy (DMD) pathology [68]. But apart from the documented pro-angiogenic effect, pro-myogenic effects, are capable of improving both the VEGF delivery also has a powerful pro-myogenic effect. histological and functional parameters normally associated In normal skeletal muscle tissues, VEGF administration with mdx muscle pathophysiology. induces muscle fiber regeneration and promotes muscle These data seem logical because developmentally redu- recovery after ischemic and chemical damage [74]. In cing angiogenesis in mdx mice through ablation of matrix in vitro studies using C2C12 myoblast cell line and pri- metalloproteinase-2 impairs the growth of regenerated mary mouse myoblasts derived from cultured SCs, VEGF myofibers and decreases VEGF expression, further com- was shown to promote growth and protect cells from plementing current theories about the close developmen- apoptosis [74]. Similar effects have been documented tal relationship between angiogenesis and myogenesis in dystrophin deficient muscle tissues, where rAAV- [76]. But what is the pro-myogenic mechanism of VEGF VEGF-treated mdx mice showed an increase in the area delivery? SCs are clearly the dominant muscle-specific occupied by regenerating fibers and an increased number stem cells utilized for muscle growth, repair, and regener- of activated SCs and developmental myosin-heavy chain- ation, and the number of SCs parallels muscle capillary positive fibers in skeletal muscles [73]. In vivo transplant- quantity, largely because SCs reside in a juxtavascular ation of muscle-derived stem cells (MDSCs) engineered to niche [77,78]. In fact, most SCs maintain tight locality to overexpress VEGF into dystrophic skeletal muscle results capillaries regardless of character, including quiescent SCs, in an increase in angiogenesis and endogenous muscle re- proliferating SCs (myogenic precursor cells), and differen- generation along with reduction in fibrosis both two and tiating SCs (myocytes), and differentiating myogenin- four weeks following transplantation [75]. Thus, the dual positive myocytes assessed from DMD muscle biopsies functionalities of VEGF, especially the pro-angiogenic and show spatiotemporal association with new capillary Ennen et al. Skeletal Muscle 2013, 3:9 Page 7 of 12 http://www.skeletalmusclejournal.com/content/3/1/9 growth [78]. A recent in vitro study specifically showed VEGF receptor regulation that endothelial cells augment myogenic precursor cell A different pro-angiogenic approach that also increases growth while differentiating SCs display pro-angiogenic vascular density to ameliorate the functional ischemia in characteristics, thus demonstrating a complementary DMD would be to modulate the VEGF receptors. As angio-myogenesis signaling system [78]. Specifically, VEGF previously described, Flt-1 acts as a decoy receptor and stimulated in vitro myogenic precursor cell growth, which modulates angiogenesis through its ability to sequester supports the notion that VEGF is a co-regulatory substance VEGF-A, which reduces signaling through Flk-1. So, al- for both angiogenesis and myogenesis [78,79]. Recent work though both Flt-1 and Flk-1 are inherently pro-angiogenic, demonstrates that myofibers, SCs or myogenic precursor due to the high affinity and low tyrosine kinase activity of cells are negative for both Flt-1 and Flk-1, indicating that Flt-1 over Flk-1 with respect to VEGF-A, Flt-1 acts as a the effects of VEGF on these cells may be mediated VEGF-A sink preventing Flk-1 access to VEGF-A and through other VEGF receptors, such as neuropilin-1 thereby functioning as a negative regulator of angiogen- (NRP1) and neuropilin-2 (NRP2) [68]. NRP1 and NRP2 esis. This phenomenon has been previously discussed in can bind to VEGF with a high affinity and act as co- more detail, and it implies that reduced levels of Flt-1 receptors for Flk-1 and Flt-3, respectively [80]. However, and/or sFlt-1 could increase the serum concentration of other studies have shown that in some developmental free VEGF-A available to bind Flk-1 and induce a pro- cases, the cellular proliferation functions of VEGF via angiogenic response [86]. binding to NRP1 are independent of Flk-1 [81]. Thus, the Interestingly, Flt-1 reduction through haploinsufficiency +/− exact mechanism of Flt-1/Flk-1 independent VEGF sig- of Flt-1 (Flt-1 ) produced increased capillary density naling remains to be elucidated. in skeletal and cardiac muscle compared with control +/+ An additional element of pro-angiogenic induction is (Flt-1 ) in a murine model (Figure 2D) [68]. More sig- that the expansion of the juxtavascular niche of SCs also nificant to this discussion, developmentally reducing Flt-1 serves to increase the basal number of SCs, and this is through the same genetic method was also shown to something not seen in the previously described vasorelax- further enhance capillary density in the skeletal muscle +/− ation strategies [68]. Normally, the successive rounds of of mdx mice (mdx:Flt-1 ) compared to controls (mdx: +/+ regeneration and degeneration seen in mdx tissue serves Flt-1 ) [68]. The increased capillary density seen in mdx: +/− to exhaust the SC pool and reduce the regenerative cap- Flt-1 mice serve as a proof of concept that regulating acity of the muscle tissue, which decreases SC quantity VEGF-A receptors can stimulate a similar pro-angiogenic over time [82,83]. This predetermined decline can be effect to that seen with direct VEGF-A administration in attenuated by improving the regenerative capacity of the dystrophin-deficient organisms. More importantly, the +/− muscle through an increase in the number of SCs present, changes seen in the mdx:Flt-1 mice persist into adult- which can be accomplished through expansion of the SC hood, yet it remains unclear if this effect must be initiated juxtavascular niche, including microcapillaries [78,84,85]. during developmental stages or if it can be recapitulated in As for the future of VEGF therapies in DMD patients, postnatal models. +/− more data is certainly needed before clinical benefit can Analysis of the mdx:Flt-1 mice with increased capil- be realized. Most important is deciphering effective dos- lary density demonstrated improved skeletal muscle hist- ing levels and delivery vehicles, which are both extraor- ology with a reduction in both myocyte damage and dinarily difficult owing to the fact that the body of fibers with centrally located nuclei, which strongly sug- +/− pharmacology knowledge has shown us that inhibition is gests that mdx:Flt-1 fibers display less fiber turnover +/+ easier than introduction. Similarly, there is no agreed compared to the mdx:Flt-1 controls [68]. Addition- +/− upon clinical standard for what constitutes a therapeutic ally, mdx:Flt-1 fibers show less calcification, fibrosis, or pathologic increase in the density of the vasculatures. and membrane permeability, all of which are downstream Also, growth factors like VEGF need to be closely mo- effects of dystrophin deficiency [68]. These histological nitored due to carcinogenic properties. Similar to the improvements also translated to improved functional pa- PDE5 inhibitor studies, the effects of VEGF-induced rameters such that increased capillary density resulted in angiogenesis to enhance capillary quantity and mitigate increased muscle tissue perfusion and improved skeletal contraction-induced muscle damage via functional ische- muscle contractile function [68]. Furthermore, Flt-1 has mia reduction should be further investigated to validate also been assessed in another DMD mouse model: mdx: −/− this hypothesis. Moreover, data regarding the functional utrophin (utrn) mice. These mice, deficient for both aspects of the myocardium in dystrophin deficient tissues, dystrophin and the dystrophin-related protein utrophin, the developmental effects of earlier VEGF administration, display a more severe, progressive form of muscular dys- and the basal quantity of SCs following VEGF-induced trophy as compared with the mdx mice [87,88]. Long term −/− +/− angiogenesis in organisms lacking dystrophin all could studies using the mdx:utrn :Flt-1 mice with increased help ready this therapy for clinical trials in DMD patients. capillary density showed significant increases in body mass Ennen et al. Skeletal Muscle 2013, 3:9 Page 8 of 12 http://www.skeletalmusclejournal.com/content/3/1/9 Table 1 Summary of vascular targeted therapies for Duchenne muscular dystrophy Treatment Outcome Physiologic effects (mdx mice) Physiologic effects (DMD patients) Potential pharmaceuticals Future directions Delayed onset and progression of LV dysfunction and lower mortality rates FDA approved ACEIs to treat heart in 9.5 to 13 year olds with normal failure and hypertension include: Improved myocardial function (prior to cardiac functioning [32,33]. benazepril (Lotensin™), captopril Decide effective pharmaceutical agent Angiotensin- Improved onset of cardiomyopathy) [31]. Myocardial functional improvements (Capoten™), enalapril (Vasotec™), and use clinical trial to assess potential converting enzyme vasorelaxation Prevented angiotensin II dependent in some cases with established fosinopril (Monopril™), lisinopril prophylactic benefit and/or ability to (ACE) Inhibitors capacity stimulation of pro-oxidant and pro- cardiomyopathy [34,36]. Given in (Prinivil™, Zestril™) moexipril attenuate functional ischemia. inflammatory pathways [51]. combination with BBs, patients with (Univasc™), perindopril (Aceon™), established cardiomyopathy saw quinapril (Accupril™), ramipril positive effect on long term survival (Altace™), and trandolapril (Mavik™). [35]. Decreased myofiber damage after myofiber injury [24]. Improved muscle PDE5 inhibitors that are FDA Complete the in-progress phase 1 histology with treatment started at approved for treating erectile Improved clinical trial (NCT01580501) assessing Phosphodiesterase conception [24]. Reduced dysfunction or hypertension include: Vasorelaxation N/D the ability of tadalafil and sildenafil to 5 (PDE5) Inhibitors cardiomyopathy remodeling signals tadalafil (Cialis™ or Adcirca™), sildenafil Capacity attenuate functional ischemia in boys [54,55]. Reversed myocardial (Viagra™ or Revatio™), and vardenafil with DMD. dysfunction in models with established (Levitra™ or STAXYN™). cardiomyopathy [56]. Determine best strategy and dosing Increased forelimb strength and schedule for delivery, acquire more safety Vascular reduced necrotic fiber area [67]. Pro- Engineered myoblasts expressing Increased data, and agree on values that constitute Endothelial Growth myogenic effects, including increased VEGF , VEGF protein systemic Vascular N/D therapeutic increases in vascular density. Factor (VEGF) regenerating fiber area and number of injections , adeno-associated viral Density Assess potential prophylactic benefit Administration activated satellite cells in skeletal (AAV) VEGF vectors and/or ability to attenuate functional muscles [67]. ischemia in mdx mice. a b N/D, not determined. These are not Food and Drug Administration (FDA) approved and are undergoing pre-clinical investigation in mdx models. Ennen et al. Skeletal Muscle 2013, 3:9 Page 9 of 12 http://www.skeletalmusclejournal.com/content/3/1/9 −/− +/+ and survival compared to the mdx:utrn :Flt-1 control functional ischemia (see Table 1 for summary of therapies). mice [68]. One therapy improves systemic vasorelaxation capacity Absolute mechanisms that explain the improved using ACEIs with or without BBs, and this method has +/− phenotype and survival seen in mdx:Flt-1 mice and shown clinical utility in both preventing and improving the −/− +/− mdx:utrn :Flt-1 mice remain to be elucidated. But adverse cardiac events normally associated with the DMD owing to pathway similarity, the explanation is probably phenotype. Treatment using PDE5 inhibitors also improves similar to descriptions of VEGF-induced angiogenesis in systemic vasorelaxation capacity, and preclinical evidence mdx mice, namely the close developmental relationship from DMD murine models demonstrates the ability of between myogenesis and angiogenesis. Increasing tissue PDE5 inhibitors to prevent skeletal and cardiac muscle perfusion may compensate for lack of NO-mediated damage and even reverse the functional parameters associ- vasodilation, which would attenuate one of the proposed ated with established cardiomyopathy. Both PDE5 and ‘two-hits’ required for myocyte damage. Another theory ACEI therapies have a clear practical advantage as they behind the progressive nature of the DMD pathology is have extensive clinical safety records and many of the the SC exhaustion model [83]. This theory states that drugs are clinically available. There are a wide variety of easily damaged DMD myofibers are constantly replaced ACEIs that are FDA approved to treat heart failure and by endogenous SCs, yet the constant SC cycling leads hypertension, including benazepril (Lotensin ), captopril ™ ™ ™ to rapid shortening of telomerase length and eventual (Capoten ), enalapril (Vasotec ), fosinopril (Monopril ), ™ ™ ™ exhaustion of the SC pool [82,83]. Interestingly, mdx: lisinopril (Prinivil , Zestril ) moexipril (Univasc ), perin- +/− ™ ™ Flt-1 were shown to have developmentally increased dopril (Aceon ), quinapril (Accupril ), ramipril ™ ™ numbers of SCs, perhaps mediated through an expanded (Altace ), and trandolapril (Mavik ). There are also sev- SC vascular niche. Thus, enhancement in the basal num- eral PDE5 inhibitors that received FDA approval for ber of SCs could mitigate the accelerated age-related de- treating erectile dysfunction or hypertension, including ™ ™ ™ cline seen among SCs from dystrophin-deficient muscle tadalafil (Cialis or Adcirca ), sildenafil (Viagra or ™ ™ ™ tissue [13]. Revatio ), and vardenafil (Levitra or STAXYN ). Add- Overall, Flt-1 is a novel target for pro-angiogenic ther- itionally, sildenafil has already been extensively studied in apy in DMD. Greater blood perfusion alone seems to a pediatric population and was found to be safe for pul- compensate for the functional ischemic phenotype in monary hypertension treatment [91]. Both tadalafil and mdx mice, but definitive studies showing attenuation of sildenafil are currently in a phase 1 clinical trial functional ischemia through Flt-1 signal mitigation to re- (NCT01580501) that will assess these drugs ability to at- duce the effects of contraction induced damage have not tenuate functional ischemia in boys with DMD, and other been shown. Flt-1 has also been investigated for its role future clinical studies could address the ability of ACEIs to in cancer where it acts as a positive regulator of the mitigate the same effect. pathological angiogenesis seen with tumor formation Therapies that enhance the underlying vascular archi- [89], which opposes the physiological role of Flt-1 as a tecture through pro-angiogenic induction include VEGF negative angiogenic regulator. Thus, numerous small administration and also VEGF receptor modulation. The molecules have already been investigated and verified pro-angiogenic therapies have shown exciting preclinical (in vivo and in vitro) that can antagonize Flt-1 binding proof of concept evidence in DMD murine models, espe- to VEGF, reduce angiogenesis, and prevent tumor cially the expansion in the basal number of SCs mediated growth [90]. These same small molecules could be used through a larger juxtavascular SC niche and the docu- to selectively block Flt-1 function and promote angio- mented pro-myogenic effects. Still, the pro-angiogenic genesis in dystrophin-deficient tissue. Still, more screen- strategies are in early stages and both methods need de- ing studies could be needed to decipher substances that finitive means of achieving their desired result and more are viable in vivo and can reduce Flt-1 function in order safety information before clinical trial initiation. In all, the to fully translate the results seen from the developmental hope is that at least some or combinations of these +/− −/− +/− studies with mdx:Flt-1 and mdx:utrn :Flt-1 mice vascular-targeted therapies will soon have clinical utility using a pharmaceutical agent. and provide current and future human beings living with DMD enhanced control over their own destiny. Conclusions Abbreviations The role of the vasculature in DMD can no longer be ig- AAV: Adeno-associated virus; ACE: Angiotensin-converting enzyme; nored in light of the mounting evidence for its role in the ACEI: Angiotensin-converting enzyme inhibitor; ANF: Atrial natriuretic factor; pathogenic process. With this new knowledge in mind and ARB: Angiotensin receptor blocker; BB: β-blocker; BMD: Becker muscular dystrophy; cGMP: Cyclic guanosine monophosphate; CNS: Central nervous with the dearth of current treatments, this review focused system; DAPC: Dystrophin-associated glycoprotein complex; DMD: Duchenne on a variety of new therapeutic options that specifically tar- muscular dystrophy; EDHF: Endothelium-derived hyperpolarizing factor; get these DMD vascular defects, namely attenuation of the LVEF: Left ventricular ejection fraction; MDSCs: Muscle-derived stem cells; Ennen et al. Skeletal Muscle 2013, 3:9 Page 10 of 12 http://www.skeletalmusclejournal.com/content/3/1/9 nNOS: Neuronal nitric oxide synthase; NO: Nitric oxide; NOS: Nitric oxide 8. Rybakova IN, Patel JR, Ervasti JM: The dystrophin complex forms a synthase; NRP1: Neuropilin-1; NRP2: Neuropilin-2; PDE5: Phosphodiesterase 5; mechanically strong link between the sarcolemma and costameric actin. sGC: Soluble guanylyl cyclase; SC: Satellite cell; rAAV: Recombinant adeno- J Cell Biol 2000, 150:1209–1214. associated virus; TA: tibialis anterior; TGF- β: Transforming growth factor-β; 9. Duncan CJ: Role of intracellular calcium in promoting muscle damage: VEGF: Vascular endothelial growth factor; VEGFR: Vascular endothelial growth a strategy for controlling the dystrophic condition. Experientia 1978, factor receptor. 34:1531–1535. 10. Spencer MJ, Croall DE, Tidball JG: Calpains are activated in necrotic fibers from mdx dystrophic mice. J Biol Chem 1995, 270:10909–10914. Competing interests 11. Hopf FW, Turner PR, Steinhardt RA: Calcium misregulation and the The authors have no financial competing interests. pathogenesis of muscular dystrophy. Subcell Biochem 2007, 45:429–464. 12. Spencer MJ, Montecino-Rodriguez E, Dorshkind K, Tidball JG: Helper Authors’ contributions (CD4(+)) and cytotoxic (CD8(+)) T cells promote the pathology of JPE completed the literature review, developed the figures, and prepared the dystrophin-deficient muscle. Clin Immunol 2001, 98:235–243. review. MV revised the manuscript. AA advised the literature review process 13. Jejurikar SS, Kuzon WM Jr: Satellite cell depletion in degenerative skeletal and revised the manuscript. All authors read and approved the final muscle. Apoptosis 2003, 8:573–578. manuscript. 14. Townsend D, Yasuda S, Metzger J: Cardiomyopathy of Duchenne muscular dystrophy: pathogenesis and prospect of membrane sealants as a new therapeutic approach. Expert Rev Cardiovasc Ther 2007, 5:99–109. Authors’ information 15. Eagle M, Baudouin SV, Chandler C, Giddings DR, Bullock R, Bushby K: JPE is a staff researcher in the laboratory of Dr. Atsushi Asakura at the Survival in Duchenne muscular dystrophy: improvements in life University of Minnesota Stem Cell Institute. expectancy since 1967 and the impact of home nocturnal ventilation. MV is an MD/PhD candidate through the Medical Scientist Training Program Neuromuscul Disord 2002, 12:926–929. at the University of Minnesota Medical School. 16. Eagle M, Bourke J, Bullock R, Gibson M, Mehta J, Giddings D, Straub V, AA is an Assistant Professor of Neurology and a faculty member of the Stem Bushby K: Managing Duchenne muscular dystrophy–the additive effect Cell Institute in the University of Minnesota Medical School. He also belongs of spinal surgery and home nocturnal ventilation in improving survival. to the Paul & Sheila Wellstone Muscular Dystrophy Center in the University Neuromuscul Disord 2007, 17:470–475. of Minnesota Medical School. 17. 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Clin Cancer Res 2005, 11:2651–2661. 91. Huddleston AJ, Knoderer CA, Morris JL, Ebenroth ES: Sildenafil for the treatment of pulmonary hypertension in pediatric patients. Pediatr • Convenient online submission Cardiol 2009, 30:871–882. • Thorough peer review • No space constraints or color figure charges doi:10.1186/2044-5040-3-9 Cite this article as: Ennen et al.: Vascular-targeted therapies for • Immediate publication on acceptance Duchenne muscular dystrophy. Skeletal Muscle 2013 3:9. • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Skeletal Muscle Springer Journals

Vascular-targeted therapies for Duchenne muscular dystrophy

Skeletal Muscle , Volume 3 (1) – Apr 23, 2013

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Copyright © 2013 by Ennen et al.; licensee BioMed Central Ltd.
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Life Sciences; Cell Biology; Developmental Biology; Biochemistry, general; Systems Biology; Biotechnology
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23618411
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Abstract

Duchenne muscular dystrophy (DMD) is the most common muscular dystrophy and an X-linked recessive, progressive muscle wasting disease caused by the absence of a functional dystrophin protein. Dystrophin has a structural role as a cytoskeletal stabilization protein and protects cells against contraction-induced damage. Dystrophin also serves a signaling role through mechanotransduction of forces and localization of neuronal nitric oxide synthase (nNOS), which produces nitric oxide (NO) to facilitate vasorelaxation. In DMD, the signaling defects produce inadequate tissue perfusion caused by functional ischemia due to a diminished ability to respond to shear stress induced endothelium- dependent dilation. Additionally, the structural defects seen in DMD render myocytes with an increased susceptibility to mechanical stress. The combination of both defects is necessary to generate myocyte damage, which induces successive rounds of myofiber degeneration and regeneration, loss of calcium homeostasis, chronic inflammatory response, fibrosis, and myonecrosis. In individuals with DMD, these processes inevitably cause loss of ambulation shortly after the first decade and an abbreviated life with death in the third or fourth decade due to cardio-respiratory anomalies. There is no known cure for DMD, and although the culpable gene has been identified for more than twenty years, research on treatments has produced few clinically relevant results. Several recent studies on novel DMD therapeutics are vascular targeted and focused on attenuating the inherent functional ischemia. One approach improves vasorelaxation capacity through pharmaceutical inhibition of either phosphodiesterase 5 (PDE5) or angiotensin- converting enzyme (ACE). Another approach increases the density of the underlying vascular network by inducing angiogenesis, and this has been accomplished through either direct delivery of vascular endothelial growth factor (VEGF) or by downregulating the VEGF decoy-receptor type 1 (VEGFR-1 or Flt-1). The pro-angiogenic approaches also seem to be pro-myogenic and could resolve the age-related decline in satellite cell (SC) quantity seen in mdx models through expansion of the SC juxtavascular niche. Here we review these four vascular targeted treatment strategies for DMD and discuss mechanisms, proof of concept, and the potential for clinical relevance associated with each therapy. Keywords: Duchenne muscular dystrophy, VEGF, Flt-1, Flk-1, Nitric oxide, PDE5 inhibitor, ACE inhibitor, Satellite cell, Muscle regeneration, Myofiber damage Review and has an annual incidence affecting one in every 3600– Duchenne muscular dystrophy (DMD) is an X-linked re- 6000 newborn males [3]. Normally, dystrophin serves as cessive, progressive muscle wasting disease caused by muta- the bridge in the dystrophin-associated glycoprotein com- tions in the DMD gene that lead to absence of a functional plex (DAPC), connecting the cytoskeleton, via attachments dystrophin protein [1,2]. Both fatal and devastating, DMD to subsarcolemmal F-actin, to the extracellular matrix is the most common muscular dystrophy seen in children through an association with plasma membrane bound β- dystroglycan [4]. In the DAPC, dystrophin has a structural role as a cytoskeletal stabilization protein and protects * Correspondence: asakura@umn.edu cells against contraction-induced damage. Dystrophin also Stem Cell Institute, University of Minnesota Medical School, McGuire Translational Research Facility, Room 4-220, 2001 6th Street SE, Minneapolis, serves signaling roles, including mechanotransduction of MN 55455, USA forces and localization of signaling proteins, such as neu- Paul and Shelia Wellstone Muscular Dystrophy Center, University of ronal nitric oxide synthase (nNOS), which synthesizes ni- Minnesota Medical School, Wallin Medical Biosciences Building, 2101 6th Steet SE, Minneapolis, MN 55455, USA tric oxide (NO) to facilitate vasorelaxation [5-7]. Without Full list of author information is available at the end of the article © 2013 Ennen et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Ennen et al. Skeletal Muscle 2013, 3:9 Page 2 of 12 http://www.skeletalmusclejournal.com/content/3/1/9 dystrophin, the DAPC cannot completely assemble, and the supportive link between the cytoskeleton and the extracellular matrix becomes destabilized [8]. Despite nor- Functional ischemia Greater cellular Combined effect from reduced NO- susceptibility of both factors mal development, the membrane in dystrophin-deficient mediated protection to metabolic stress cells is easily damaged. Membrane microlesions facilitate an influx of calcium ions, which activate proteases to begin auto-digestion of the musculature sarcoplasm Myofiber damage [9-11]. Macrophages later arrive at the tissue to remove cellular debris, and satellite cells (SCs) are activated and proliferate to induce myofiber regeneration. This causes Reduced net Greater cellular Reduced damage from Vascular Therapy susceptibility successive rounds of myofiber degeneration and regener- functional Treatment Target combining to metabolic stress ischemia both factors ation that is exacerbated by continual membrane damage and ensuing myonecrosis. In addition, cytokines released in the process of myonecrosis recruit inflammatory cells, Reduced myofiber damage which release inflammatory cytokines to activate fibro- blasts that lay down extracellular matrix proteins and lead Figure 1 The two-hit hypothesis for myocyte damage and the to fibrosis [12]. Skeletal muscle regenerative capacity later proposed outcome of functional ischemia attenuation in Duchenne muscular dystrophy (DMD). (A) The combined effects diminishes with advancing age and decreasing numbers of from functional ischemia due to reduced nitric oxide (NO)-mediated SCs, and muscle tissue is steadily replaced by adipose and protection and greater cellular susceptibility to metabolic stress are connective tissues [13]. necessary to produce the myofiber damage observed in DMD [17]. The previously described cellular events manifest them- (B) Attenuating functional ischemia by administering a vascular selves clinically in a devastating and progressive manner. targeted treatment can reduce the net-combined effect of both two-hit factors and consequently curtail myofiber damage. Despite continuous contractions by the myocardium, the skeletal muscles deteriorate first in individuals with DMD, and most permanently lose ambulatory abilities shortly after the first decade [14]. Myocardial problems present or increasing the underlying vascular density in order to later, and clinically relevant cardiomyopathy is seen in 90% reduce the functional ischemia and improve the DMD of patients over 18 years old, namely due to the onset of phenotype. cardiac fibrosis in addition to rhythm and conduction ab- normalities [14]. Respiratory problems are also inevitable due to muscle wasting in the diaphragm and the onset of Defect of nitric oxide-mediated vasodilation contributes scoliosis [14]. Even with improvements in treatment, to Duchenne muscular dystrophy phenotype notably multidisciplinary care, the combined cardio- The DMD pathogenesis is partially explained by the lack of respiratory anomalies mean that most individuals with the signaling role of dystrophin, which normally localizes DMD die in their third or fourth decade of life [15,16]. nNOS to the sarcolemma through binding to the C- Despite knowledge of the responsible gene for over twenty terminal region of dystrophin [6]. The nNOS is responsible years, a DMD cure remains to be found, and research on for NO production to facilitate smooth muscle vasodila- treatments has produced few clinically relevant results. tion in response to increased metabolic demands. During Current treatment options, such as corticosteroid admin- muscle contraction, NO-mediated vasodilation is import- istration, physical therapy, nocturnal ventilation, and sur- ant to help offset the α-adrenergic vasoconstriction in re- gical interventions aim for symptomatic management and sponse to sympathetic activation, which optimizes muscle have been shown to improve lifespan and quality of life perfusion [18]. This functional response is intact in healthy [16]. The clinical utility and feasibility of gene therapy and children, but in children with DMD the sympathetic vaso- cell therapy remain to be elucidated, and other treatment constriction in skeletal muscle is unopposed due to lack of areas must be sought. Our current, more holistic under- NO-mediated vasodilation [18]. standing of DMD pathogenesis, especially with more The nNOS is absent from the sarcolemma and is recent knowledge of the vascular role of dystrophin, im- greatly downregulated in the cytoplasm of dystrophin- plies that vascular-targeted therapies are strong candi- deficient muscle, which results in muscle vasoconstric- dates for future investigation. Specifically, attenuating tion and abnormal blood flow during skeletal muscle functional ischemia could reduce myocyte damage, in- contraction [6,18,19]. Specifically, loss of dystrophin in crease tissue perfusion, reduce cardiac workload, and pre- the smooth muscle results in a decreased capacity of vent cardiac and skeletal muscle remodeling (Figure 1B). the vasculature to respond to shear stress induced This review will focus on vascular-targeted treatment endothelium-dependent dilation, probably related to the avenues aimed at either improving vasorelaxation capacity signaling defects seen in both force transduction and Ennen et al. Skeletal Muscle 2013, 3:9 Page 3 of 12 http://www.skeletalmusclejournal.com/content/3/1/9 inadequate NO production [19]. Furthermore, shear stress Improved vasorelaxation capacity at the endothelial cell surface is a known catalyst for Angiotensin-converting enzyme inhibitors angiogenesis [20-23], so new blood vessel formation could The renin-angiotensin-aldosterone system plays a vital be downregulated and mismatched to metabolic need in role in regulating both systemic vascular resistance and the absence of dystrophin due to defects in mechano- total blood volume, which together impact arterial pres- transduction. Lack of the signaling and structural roles sure and myocardial function. A key component is the of dystrophin in DMD pathogenesis have led to a two- angiotensin-converting enzyme (ACE), which transforms hit hypothesis, whereby the combination of functional the peptide hormone angiotensin I into angiotensin II; cir- ischemia due to reduced capacity to benefit from NO- culating angiotensin II then stimulates vascular smooth mediated protection and an increased susceptibility to muscle contraction, increasing vascular resistance and ar- metabolic stress are both required to cause myocyte da- terial pressure. Angiotensin II also induces the release of mage (Figure 1A) [17]. So although not completely culpable aldosterone, which increases sodium and water retention, for the observed pathogenesis, impaired vascular function- and vasopressin, which increases water retention. ing seems to be both inherent to DMD and an accelerant Preventing angiotensin II production through pharmaco- to tissue damage in the skeletal and cardiac muscles. logical ACE inhibition has been shown to reduce high Recent studies suggest that the two-hit hypothesis blood pressure and cardiac workload through enhanced should migrate away from simply observational specula- vasorelaxation and prevention of downstream hormone tion towards a more widely accepted, evidence-based release, and ACE inhibitors (ACEIs) are currently used to DMD pathogenic theory. One functional study using treat congestive heart failure and hypertension [28-30]. As model DMD mdx mice showed quantitative evidence such, improving cardiac function and enhancing systemic supporting the two-hit hypothesis, where inhibition of vasorelaxation capacity through ACE inhibition in DMD NO/EDHF (EDHF is endothelium-derived hyperpolariz- patients could have prophylactic benefit by mitigating the ing factor, another vasodilator) alone in wild-type mice functional ischemia and consequently diminishing myo- caused similar functional ischemia (one-hit) to that seen necrosis. In mdx mice, the ACEI captopril administered in mdx mice [24]. But, the forced functional ischemia over an 8-week period and prior to the onset of cardio- alone in the wild-type mice did not induce similar levels myopathy was shown to reduce cardiac afterload, of muscle cell death seen in mdx mice [24]. Two-hits, increase myocardial contractility, and improve cardiac consisting of severe ischemia and strenuous tetanic stim- hemodynamics compared to mdx control mice [31]. In uli, were necessary to produce the same contraction- clinical studies, administration of the ACEI perindopril dependent myofiber damage in wild-type mice to that of has shown that early treatment in 9.5- to 13-year-old mdx mice [24]. So, mdx myofibers exhibit enhanced DMD patients with normal cardiac functioning (as mea- vulnerability to metabolic and mechanical stress inde- sured by normal left ventricular ejection fraction or LVEF), pendently of the altered vasodilatory response, yet the is capable of delaying both the onset and progression of combinatory effect of both factors (two-hits) is necessary left ventricular dysfunction and significantly lowering to mediate the cell death numbers seen in mdx myo- mortality rates compared to patients starting treatment 3 fibers. This could also explain why nNOS knockout mice, years later [32,33]. Results are less clear in DMD cases in- a one-hit model, do not develop muscular dystrophy, yet volving established cardiomyopathy where administration myocardial specific nNOS expression prevents cardiomy- of the ACEI enalapril showed functional normalization in opathy in mdx mice by increasing the capacity to benefit just 43% of cases, but this positive functional effect was from NO-mediated protection [25,26]. Interestingly, mdx maintained by most subjects for up to four years [34]. mice that only express dystrophin in smooth muscle Combination therapy using ACEIs and β-adrenergic (SMTg/mdx), which has a prominent role in regulation of receptor antagonists or β-blockers (BBs) has also been vascular tone and blood flow, have an intermediate investigated for use in DMD treatment. Catecholamines phenotype between wild-type and mdx mice [27]. The increase heart rate and myocardial contractility through SMTg/mdx mice showed some, albeit not total, recovery the β-adrenergic receptors, and thus targeted BBs cause of the NO-dependent vasorelaxation mechanism in ac- the heart to beat slower and with less force and are typ- tive skeletal muscle [27]. In the SMTg/mdx model, lack ically given to patients with arrhythmia or disordered of dystrophin at the myofiber level and the consequent automaticity. One study that utilized a combination of lack of complete force mechanotransduction in response BBs and ACEIs in DMD patients with established cardio- to functional demands were perhaps partially responsible myopathy found a positive effect on long-term survival, for the insufficient phenotypic recovery. These data over- especially for individuals that showed no overt symptoms whelmingly reveal the important role the vasculature of heart failure despite documented left ventricular dys- plays in DMD pathogenesis and highlight a novel arena function [35]. A more recent study also investigated DMD for therapeutic intervention. patients with established cardiomyopathy but broke Ennen et al. Skeletal Muscle 2013, 3:9 Page 4 of 12 http://www.skeletalmusclejournal.com/content/3/1/9 treatment groups down into ACEI (lisinopril) alone or angiotensin II dependent stimulation of pro-oxidant and ACEI plus BB (metoprolol) [36]. Both treatment groups pro-inflammatory pathways [53]. Overall, ACEIs and/or displayed improvements in cardiac function compared to BBs appear to be excellent candidates for DMD therapy pre-therapy measurements, but no significant difference based on current clinical availability and demonstrated in cardiac function was seen between groups [36]. Future ability to reduce many of the negative outcomes normally studies should address treatment using ACEI alone or associated with the DMD pathogenic process. Still, a ACEI plus BB in DMD cases where cardiomyopathy has broader investigation regarding the potential prophylactic not been fully established to assess the potential for benefit of ACEIs should be conducted to determine an op- prophylactic benefit as this could definitively rule out the timal age of initiation. need for a BB. Additionally, with regard to the two-hit hy- pothesis, studies that address functional ischemia attenu- Phosphodiesterase 5 inhibitors ation to mitigate myonecrosis through enhanced tissue In the NO-cGMP signaling pathway, nitric oxide synthase perfusion by ACEI-mediated vasorelaxation have not yet (NOS) produces NO to activate soluble guanylyl cyclase been performed. (sGC) to synthesize cyclic guanosine monophosphate Another therapeutic strategy that targets the renin- (cGMP), and cGMP activates protein kinase G to induce angiotensin-aldosterone system to improve vasorelax- vasodilation. The cGMP-specific phosphodiesterases are ation capacity utilizes the antihypertensive drug losartan, responsible for cGMP degradation, so vasoconstriction be- which is an angiotensin II type I receptor antagonist or gins as concentrations of cGMP diminish. This pathway is angiotensin receptor blocker (ARB). Long-term adminis- disrupted in dystrophin deficient membranes, as nNOS is tration of losartan in mdx mice showed improvements absent from the sarcolemma and greatly downregulated, in myocardial function, but not skeletal muscle function, which contributes to the observed functional ischemia and reductions in mortality compared to control [37,38]. [6,19]. Additionally, studies have shown greater cGMP- Explanations as to why losartan could only ameliorate specific phosphodiesterase 5 (PDE5) activity in mdx skel- the function of cardiac muscle remain limited, but the etal muscle samples and decreased cGMP production primary mechanism could be the significant reduction in compared with controls [54,55]. Recently, cGMP-specific ™ ™ afterload seen in the hearts of losartan treated mdx mice PDE5 inhibitors, specifically tadalafil (Cialis or Adcirca ) ™ ™ [38]. Decreased afterload certainly reduces cardiac work- and sildenafil (Viagra or Revatio ) have been investi- load, and this may minimize mechanical injury and sub- gated for their potential in ameliorating the functional is- sequent fibrosis to the sensitive cardiomyocytes in mdx chemia in DMD by increasing intracellular levels of cGMP hearts. Still, the use of losartan as a prophylactic treatment to prolong vasodilation and increase blood flow to tissues. against DMD-related cardiomyopathy seems promising Asai et al. elegantly showed that tadalafil administra- based on these pre-clinical studies and the current clinical tion prior to progressive myofiber damage was able to availability of losartan (COZAAR ) for its use in hyper- significantly lower the net quantity of myofiber damage tension. Future investigation should be directed at evaluat- in mdx mice compared to placebo [24]. Essentially, at- ing losartan in DMD patients but also, owing to pathway tenuation of functional ischemia using tadalafil was similarity, at comparing the effectiveness of losartan to the shown to reduce the extent of contraction-induced dam- many FDA-approved ACEIs. age [24]. Additionally, early treatment utilizing PDE5 The definitive mechanism behind reducing cardiomyop- inhibitors could have clinical prophylactic benefit, for athy via ARBs, ACEIs, and/or BBs in DMD patients is not mdx mice treated with tadalafil from conception showed completely established, but reduced aldosterone signaling improved histology [24]. Khairallah et al. showed that car- through ACE inhibition could prevent fibrotic tissue diac mRNA expression levels of atrial natriuretic factor development, as previous use of aldosterone-specific (ANF), an early indicator for initiation of cardiomyopathic blockers has shown benefit in cases of heart failure remodeling, was significantly reduced in mdx mice treated [39-42]. Additionally, angiotensin II can directly induce with sildenafil [56,57]. This implies that sildenafil is cap- vasoconstriction, pro-fibrotic Smad signaling, pro-fibrotic able of inhibiting the advancement of cardiomyopathic transforming growth factor beta (TGF-β) production, and remodeling at early stages of DMD [57]. Sildenafil has also the ubiquitin-proteasome pathway that has a role in the been shown to have positive functional effects in the proteolysis happening in dystrophic tissues [43-46]. hearts of mdx mice, notably by avoidance of cardio- Angiotensin II is also known to enhance NADPH-oxidase myocyte damage produced in vivo via cardiac workload activity, which leads to overproduction of superoxide augmentation and maintenance of an elevated heart rate anion and accounts for the oxidative stress in cardiac and response for a significantly longer period of time com- skeletal muscle of the mdx mouse [47-52]. ACE inhibition pared with placebo [57]. Interestingly, administration of can reduce these adverse effects, and a study with mdx sildenafil is even capable of reversing cardiac dysfunction mice demonstrated that the ACEI enalapril can prevent in mdx mice with established cardiomyopathy [58]. Ennen et al. Skeletal Muscle 2013, 3:9 Page 5 of 12 http://www.skeletalmusclejournal.com/content/3/1/9 Nevertheless, the target cell and mechanism behind the an intracellular signaling domain and thus only serves in a reversal are still unclear. regulatory capacity by sequestering VEGF-A [63]. Flt-1 Additionally, PDE5 inhibitors have also shown im- and Flk-1 contain an extracellular VEGF-A-binding do- provement in muscle tissue from other vertebrate models main and an intracellular tyrosine kinase domain, and of DMD, namely two dystrophin deficient zebrafish both show expression during the developmental stage and models known as sapje and sapje-like mutants [59,60]. tissue regeneration in hemangioblasts and endothelial cell Dystrophin-null zebrafish treated with aminophylline, a lineages [63-65]. Flt-1 has a 10 times greater binding affin- nonselective phosphodiesterase inhibitor, were able to ity for VEGF-A (K approximately 2 to 10 pM) compared survive significantly longer compared to controls and to Flk-1, but the weaker tyrosine kinase domain indicates had restored skeletal muscle structure similar to wild- that angiogenic signal transduction following VEGF-A type zebrafish [61]. Furthermore, analysis of sapje mu- binding to Flt-1 is comparably weaker than the Flk-1 sig- tants treated 1 to 4 days postfertilization with different nal (Figure 2A) [63]. As such, homozygous Flt-1 gene phosphodiesterase inhibitors revealed that treatment with knockout mice die in the embryonic stage from endothe- aminophylline or sildenafil citrate resulted in the lowest lial cell overproduction and blood vessel disorganization percentage of fish showing abnormal muscle structure (Figure 2B) [64-66]. Inversely, homozygous Flk-1 gene [61]. These data suggest that aminophylline and sildenafil knockout mice die from defects in the development of or- citrate are capable of preventing the onset of aberrant ganized blood vessels due to lack of yolk-sac blood island muscle architecture in dystrophin-null zebrafish. These formation during embryogenesis (Figure 2C) [67]. Both findings from DMD model zebrafish are analogous to the the Flt-1 and Flk-1 receptors are needed for normal devel- results seen from mdx mice treated with PDE5 inhibitors, opment, but selective augmentation in VEGF-A concen- and the consistency of these results across several DMD tration should allow for greater binding to the Flk-1 models leaves hope that these compounds could benefit receptor and induce a pro-angiogenic effect that increases individuals living with DMD. Clinical trials assessing capillary density. PDE5 inhibitors for DMD patients are currently under- Several studies have demonstrated that administration of way, and future use of tadalafil or sildenafil for these indi- VEGF using exogenous expression mediated through viduals seems promising based on preclinical studies and engineered myoblasts, direct systemic injections, or adeno- current clinical availability of these drugs for their use in associated viral (AAV) vectors are capable of initiating an treating erectile dysfunction and pulmonary hypertension. angiogenic signal in the myocardium and skeletal muscle in both ischemic and non-ischemic conditions [69-72]. Increased vascular density However, these same studies do highlight the importance Vascular endothelial growth factor administration of precisely regulating VEGF delivery quantities for future The central paradigm behind the previously discussed clinical use, as overadministration has been shown to have PDE5 and ACEI therapies for DMD was that increasing deleterious effects in animal models, such as hemangioma the vasorelaxation capacity of the vasculature would be formation [70]. Unfortunately, and to the best of our able to increase perfusion, diminish the effects from knowledge, functional studies that assess blood flow in functional ischemia, and decrease myocyte damage. How- DMD model organisms following VEGF-induced angio- ever, another technique to increase tissue perfusion would genesis have not yet been conducted. However, one study be to increase the density of the underlying vascular archi- found that four weeks following intramuscular adminis- tecture that nourishes the skeletal and cardiac muscles. tration of rAAV-VEGF vectors in the bicep and tibialis One method of increasing vascular density is to augment anterior (TA) muscles of 4-week-old mdx mice, the angiogenesis, which regulates the production of new rAAV-VEGF-treated mdx mice showed significantly vasculatures from the existing framework. The vascular greater forelimb strength compared to pretreatment endothelial growth factor (VEGF) family of signal glyco- levels and AAV-LacZ-treated control mdx mice [73]. The proteins acts as potent promoters of angiogenesis during same study confirmed the feasibility of VEGF-mediated embryogenesis and postnatal growth. Specifically, the angiogenic induction in mdx mice and showed greater binding of the VEGF-A ligand with the VEGF receptors capillary density, particularly in the area of regenerating has been shown to promote vascular permeability and also fibers, as well as reduced necrotic fiber area in biceps trigger endothelial cell migration, proliferation, and sur- muscle compared to AAV-LacZ-treated control mdx vival, and the newly formed endothelial cells provide the mice [73]. So, although this study did not directly assess basic structure of new vasculatures [62]. The dominant reduction of functional ischemia via enhanced vascular VEGF signal molecule for angiogenesis, VEGF-A, mediates density through VEGF-induced angiogenesis, it was able its signal through VEGF receptor-1 (VEGFR-1, hereafter to demonstrate similar outcomes from the PDE5 inhibi- Flt-1) and VEGF receptor-2 (VEGFR-2, hereafter Flk-1) tor studies, especially the decrease seen in necrotic fiber [63]. A soluble form of Flt-1 (sFlt-1) also exists, but lacks area and improvement in muscle function. Ennen et al. Skeletal Muscle 2013, 3:9 Page 6 of 12 http://www.skeletalmusclejournal.com/content/3/1/9 AC B Heterozygous Wild Type Flk-1gene KO Flt-1gene KO Flt-1gene KO sFlt-1 sFlt-1 sFlt-1 sFlt-1 VEGF = VEGF Affinity Flt-1 Flk-1 Flt-1 Flk-1 Flt-1 Flk-1 Flt-1 Flk-1 Membrane Angiogenic Signal Strength Enhanced Normal Embryonic Embryonic Angiogenesis and Lethal: Excessive Lethal: Signaling Angiogenesis Improved DMD Angiogenesis Defect Phenotype Figure 2 Flt-1 is a decoy receptor for vascular endothelial growth factor (VEGF) pro-angiogenic signaling. (A) In the wild-type scenario, VEGF induces a pro-angiogenic signal by binding the Flt-1 or Flk-1 receptors [63]. Flt-1 has a higher binding affinity for VEGF but transmits a weaker angiogenic signal compared to Flk-1, which implies that Flt-1 acts a negative regulator of angiogenesis [63]. The soluble form of Flt-1 (sFlt-1) lacks the transmembrane and intracellular signaling domains of Flt-1 and only serves a regulatory role by sequestering VEGF [63]. (B) Flt-1 −/− homozygous knockout (Flt-1 ) mice die in the early embryonic stage from endothelial cell overproduction and blood vessel disorganization, −/− indicating that Flt-1 is a decoy regulator for endothelial growth/differentiation [64-66]. (C) Flk-1 homozygous knockout (Flk-1 ) mice die in the early embryonic stage from defects in the development of organized blood vessels, indicating that Flk-1 is a positive regulator for endothelial +/− growth/differentiation [67]. (D) Developmental reduction of the Flt-1 receptor through haploinsufficiency of the Flt-1 gene (Flt-1 ) has been +/− shown to increase capillary density in skeletal muscle, and this same phenomenon has been demonstrated in mdx mice (mdx:Flt-1 ) [79]. The +/− mdx:Flt-1 mice also showed improved histological and functional parameters normally associated with the Duchenne muscular dystrophy (DMD) pathology [68]. But apart from the documented pro-angiogenic effect, pro-myogenic effects, are capable of improving both the VEGF delivery also has a powerful pro-myogenic effect. histological and functional parameters normally associated In normal skeletal muscle tissues, VEGF administration with mdx muscle pathophysiology. induces muscle fiber regeneration and promotes muscle These data seem logical because developmentally redu- recovery after ischemic and chemical damage [74]. In cing angiogenesis in mdx mice through ablation of matrix in vitro studies using C2C12 myoblast cell line and pri- metalloproteinase-2 impairs the growth of regenerated mary mouse myoblasts derived from cultured SCs, VEGF myofibers and decreases VEGF expression, further com- was shown to promote growth and protect cells from plementing current theories about the close developmen- apoptosis [74]. Similar effects have been documented tal relationship between angiogenesis and myogenesis in dystrophin deficient muscle tissues, where rAAV- [76]. But what is the pro-myogenic mechanism of VEGF VEGF-treated mdx mice showed an increase in the area delivery? SCs are clearly the dominant muscle-specific occupied by regenerating fibers and an increased number stem cells utilized for muscle growth, repair, and regener- of activated SCs and developmental myosin-heavy chain- ation, and the number of SCs parallels muscle capillary positive fibers in skeletal muscles [73]. In vivo transplant- quantity, largely because SCs reside in a juxtavascular ation of muscle-derived stem cells (MDSCs) engineered to niche [77,78]. In fact, most SCs maintain tight locality to overexpress VEGF into dystrophic skeletal muscle results capillaries regardless of character, including quiescent SCs, in an increase in angiogenesis and endogenous muscle re- proliferating SCs (myogenic precursor cells), and differen- generation along with reduction in fibrosis both two and tiating SCs (myocytes), and differentiating myogenin- four weeks following transplantation [75]. Thus, the dual positive myocytes assessed from DMD muscle biopsies functionalities of VEGF, especially the pro-angiogenic and show spatiotemporal association with new capillary Ennen et al. Skeletal Muscle 2013, 3:9 Page 7 of 12 http://www.skeletalmusclejournal.com/content/3/1/9 growth [78]. A recent in vitro study specifically showed VEGF receptor regulation that endothelial cells augment myogenic precursor cell A different pro-angiogenic approach that also increases growth while differentiating SCs display pro-angiogenic vascular density to ameliorate the functional ischemia in characteristics, thus demonstrating a complementary DMD would be to modulate the VEGF receptors. As angio-myogenesis signaling system [78]. Specifically, VEGF previously described, Flt-1 acts as a decoy receptor and stimulated in vitro myogenic precursor cell growth, which modulates angiogenesis through its ability to sequester supports the notion that VEGF is a co-regulatory substance VEGF-A, which reduces signaling through Flk-1. So, al- for both angiogenesis and myogenesis [78,79]. Recent work though both Flt-1 and Flk-1 are inherently pro-angiogenic, demonstrates that myofibers, SCs or myogenic precursor due to the high affinity and low tyrosine kinase activity of cells are negative for both Flt-1 and Flk-1, indicating that Flt-1 over Flk-1 with respect to VEGF-A, Flt-1 acts as a the effects of VEGF on these cells may be mediated VEGF-A sink preventing Flk-1 access to VEGF-A and through other VEGF receptors, such as neuropilin-1 thereby functioning as a negative regulator of angiogen- (NRP1) and neuropilin-2 (NRP2) [68]. NRP1 and NRP2 esis. This phenomenon has been previously discussed in can bind to VEGF with a high affinity and act as co- more detail, and it implies that reduced levels of Flt-1 receptors for Flk-1 and Flt-3, respectively [80]. However, and/or sFlt-1 could increase the serum concentration of other studies have shown that in some developmental free VEGF-A available to bind Flk-1 and induce a pro- cases, the cellular proliferation functions of VEGF via angiogenic response [86]. binding to NRP1 are independent of Flk-1 [81]. Thus, the Interestingly, Flt-1 reduction through haploinsufficiency +/− exact mechanism of Flt-1/Flk-1 independent VEGF sig- of Flt-1 (Flt-1 ) produced increased capillary density naling remains to be elucidated. in skeletal and cardiac muscle compared with control +/+ An additional element of pro-angiogenic induction is (Flt-1 ) in a murine model (Figure 2D) [68]. More sig- that the expansion of the juxtavascular niche of SCs also nificant to this discussion, developmentally reducing Flt-1 serves to increase the basal number of SCs, and this is through the same genetic method was also shown to something not seen in the previously described vasorelax- further enhance capillary density in the skeletal muscle +/− ation strategies [68]. Normally, the successive rounds of of mdx mice (mdx:Flt-1 ) compared to controls (mdx: +/+ regeneration and degeneration seen in mdx tissue serves Flt-1 ) [68]. The increased capillary density seen in mdx: +/− to exhaust the SC pool and reduce the regenerative cap- Flt-1 mice serve as a proof of concept that regulating acity of the muscle tissue, which decreases SC quantity VEGF-A receptors can stimulate a similar pro-angiogenic over time [82,83]. This predetermined decline can be effect to that seen with direct VEGF-A administration in attenuated by improving the regenerative capacity of the dystrophin-deficient organisms. More importantly, the +/− muscle through an increase in the number of SCs present, changes seen in the mdx:Flt-1 mice persist into adult- which can be accomplished through expansion of the SC hood, yet it remains unclear if this effect must be initiated juxtavascular niche, including microcapillaries [78,84,85]. during developmental stages or if it can be recapitulated in As for the future of VEGF therapies in DMD patients, postnatal models. +/− more data is certainly needed before clinical benefit can Analysis of the mdx:Flt-1 mice with increased capil- be realized. Most important is deciphering effective dos- lary density demonstrated improved skeletal muscle hist- ing levels and delivery vehicles, which are both extraor- ology with a reduction in both myocyte damage and dinarily difficult owing to the fact that the body of fibers with centrally located nuclei, which strongly sug- +/− pharmacology knowledge has shown us that inhibition is gests that mdx:Flt-1 fibers display less fiber turnover +/+ easier than introduction. Similarly, there is no agreed compared to the mdx:Flt-1 controls [68]. Addition- +/− upon clinical standard for what constitutes a therapeutic ally, mdx:Flt-1 fibers show less calcification, fibrosis, or pathologic increase in the density of the vasculatures. and membrane permeability, all of which are downstream Also, growth factors like VEGF need to be closely mo- effects of dystrophin deficiency [68]. These histological nitored due to carcinogenic properties. Similar to the improvements also translated to improved functional pa- PDE5 inhibitor studies, the effects of VEGF-induced rameters such that increased capillary density resulted in angiogenesis to enhance capillary quantity and mitigate increased muscle tissue perfusion and improved skeletal contraction-induced muscle damage via functional ische- muscle contractile function [68]. Furthermore, Flt-1 has mia reduction should be further investigated to validate also been assessed in another DMD mouse model: mdx: −/− this hypothesis. Moreover, data regarding the functional utrophin (utrn) mice. These mice, deficient for both aspects of the myocardium in dystrophin deficient tissues, dystrophin and the dystrophin-related protein utrophin, the developmental effects of earlier VEGF administration, display a more severe, progressive form of muscular dys- and the basal quantity of SCs following VEGF-induced trophy as compared with the mdx mice [87,88]. Long term −/− +/− angiogenesis in organisms lacking dystrophin all could studies using the mdx:utrn :Flt-1 mice with increased help ready this therapy for clinical trials in DMD patients. capillary density showed significant increases in body mass Ennen et al. Skeletal Muscle 2013, 3:9 Page 8 of 12 http://www.skeletalmusclejournal.com/content/3/1/9 Table 1 Summary of vascular targeted therapies for Duchenne muscular dystrophy Treatment Outcome Physiologic effects (mdx mice) Physiologic effects (DMD patients) Potential pharmaceuticals Future directions Delayed onset and progression of LV dysfunction and lower mortality rates FDA approved ACEIs to treat heart in 9.5 to 13 year olds with normal failure and hypertension include: Improved myocardial function (prior to cardiac functioning [32,33]. benazepril (Lotensin™), captopril Decide effective pharmaceutical agent Angiotensin- Improved onset of cardiomyopathy) [31]. Myocardial functional improvements (Capoten™), enalapril (Vasotec™), and use clinical trial to assess potential converting enzyme vasorelaxation Prevented angiotensin II dependent in some cases with established fosinopril (Monopril™), lisinopril prophylactic benefit and/or ability to (ACE) Inhibitors capacity stimulation of pro-oxidant and pro- cardiomyopathy [34,36]. Given in (Prinivil™, Zestril™) moexipril attenuate functional ischemia. inflammatory pathways [51]. combination with BBs, patients with (Univasc™), perindopril (Aceon™), established cardiomyopathy saw quinapril (Accupril™), ramipril positive effect on long term survival (Altace™), and trandolapril (Mavik™). [35]. Decreased myofiber damage after myofiber injury [24]. Improved muscle PDE5 inhibitors that are FDA Complete the in-progress phase 1 histology with treatment started at approved for treating erectile Improved clinical trial (NCT01580501) assessing Phosphodiesterase conception [24]. Reduced dysfunction or hypertension include: Vasorelaxation N/D the ability of tadalafil and sildenafil to 5 (PDE5) Inhibitors cardiomyopathy remodeling signals tadalafil (Cialis™ or Adcirca™), sildenafil Capacity attenuate functional ischemia in boys [54,55]. Reversed myocardial (Viagra™ or Revatio™), and vardenafil with DMD. dysfunction in models with established (Levitra™ or STAXYN™). cardiomyopathy [56]. Determine best strategy and dosing Increased forelimb strength and schedule for delivery, acquire more safety Vascular reduced necrotic fiber area [67]. Pro- Engineered myoblasts expressing Increased data, and agree on values that constitute Endothelial Growth myogenic effects, including increased VEGF , VEGF protein systemic Vascular N/D therapeutic increases in vascular density. Factor (VEGF) regenerating fiber area and number of injections , adeno-associated viral Density Assess potential prophylactic benefit Administration activated satellite cells in skeletal (AAV) VEGF vectors and/or ability to attenuate functional muscles [67]. ischemia in mdx mice. a b N/D, not determined. These are not Food and Drug Administration (FDA) approved and are undergoing pre-clinical investigation in mdx models. Ennen et al. Skeletal Muscle 2013, 3:9 Page 9 of 12 http://www.skeletalmusclejournal.com/content/3/1/9 −/− +/+ and survival compared to the mdx:utrn :Flt-1 control functional ischemia (see Table 1 for summary of therapies). mice [68]. One therapy improves systemic vasorelaxation capacity Absolute mechanisms that explain the improved using ACEIs with or without BBs, and this method has +/− phenotype and survival seen in mdx:Flt-1 mice and shown clinical utility in both preventing and improving the −/− +/− mdx:utrn :Flt-1 mice remain to be elucidated. But adverse cardiac events normally associated with the DMD owing to pathway similarity, the explanation is probably phenotype. Treatment using PDE5 inhibitors also improves similar to descriptions of VEGF-induced angiogenesis in systemic vasorelaxation capacity, and preclinical evidence mdx mice, namely the close developmental relationship from DMD murine models demonstrates the ability of between myogenesis and angiogenesis. Increasing tissue PDE5 inhibitors to prevent skeletal and cardiac muscle perfusion may compensate for lack of NO-mediated damage and even reverse the functional parameters associ- vasodilation, which would attenuate one of the proposed ated with established cardiomyopathy. Both PDE5 and ‘two-hits’ required for myocyte damage. Another theory ACEI therapies have a clear practical advantage as they behind the progressive nature of the DMD pathology is have extensive clinical safety records and many of the the SC exhaustion model [83]. This theory states that drugs are clinically available. There are a wide variety of easily damaged DMD myofibers are constantly replaced ACEIs that are FDA approved to treat heart failure and by endogenous SCs, yet the constant SC cycling leads hypertension, including benazepril (Lotensin ), captopril ™ ™ ™ to rapid shortening of telomerase length and eventual (Capoten ), enalapril (Vasotec ), fosinopril (Monopril ), ™ ™ ™ exhaustion of the SC pool [82,83]. Interestingly, mdx: lisinopril (Prinivil , Zestril ) moexipril (Univasc ), perin- +/− ™ ™ Flt-1 were shown to have developmentally increased dopril (Aceon ), quinapril (Accupril ), ramipril ™ ™ numbers of SCs, perhaps mediated through an expanded (Altace ), and trandolapril (Mavik ). There are also sev- SC vascular niche. Thus, enhancement in the basal num- eral PDE5 inhibitors that received FDA approval for ber of SCs could mitigate the accelerated age-related de- treating erectile dysfunction or hypertension, including ™ ™ ™ cline seen among SCs from dystrophin-deficient muscle tadalafil (Cialis or Adcirca ), sildenafil (Viagra or ™ ™ ™ tissue [13]. Revatio ), and vardenafil (Levitra or STAXYN ). Add- Overall, Flt-1 is a novel target for pro-angiogenic ther- itionally, sildenafil has already been extensively studied in apy in DMD. Greater blood perfusion alone seems to a pediatric population and was found to be safe for pul- compensate for the functional ischemic phenotype in monary hypertension treatment [91]. Both tadalafil and mdx mice, but definitive studies showing attenuation of sildenafil are currently in a phase 1 clinical trial functional ischemia through Flt-1 signal mitigation to re- (NCT01580501) that will assess these drugs ability to at- duce the effects of contraction induced damage have not tenuate functional ischemia in boys with DMD, and other been shown. Flt-1 has also been investigated for its role future clinical studies could address the ability of ACEIs to in cancer where it acts as a positive regulator of the mitigate the same effect. pathological angiogenesis seen with tumor formation Therapies that enhance the underlying vascular archi- [89], which opposes the physiological role of Flt-1 as a tecture through pro-angiogenic induction include VEGF negative angiogenic regulator. Thus, numerous small administration and also VEGF receptor modulation. The molecules have already been investigated and verified pro-angiogenic therapies have shown exciting preclinical (in vivo and in vitro) that can antagonize Flt-1 binding proof of concept evidence in DMD murine models, espe- to VEGF, reduce angiogenesis, and prevent tumor cially the expansion in the basal number of SCs mediated growth [90]. These same small molecules could be used through a larger juxtavascular SC niche and the docu- to selectively block Flt-1 function and promote angio- mented pro-myogenic effects. Still, the pro-angiogenic genesis in dystrophin-deficient tissue. Still, more screen- strategies are in early stages and both methods need de- ing studies could be needed to decipher substances that finitive means of achieving their desired result and more are viable in vivo and can reduce Flt-1 function in order safety information before clinical trial initiation. In all, the to fully translate the results seen from the developmental hope is that at least some or combinations of these +/− −/− +/− studies with mdx:Flt-1 and mdx:utrn :Flt-1 mice vascular-targeted therapies will soon have clinical utility using a pharmaceutical agent. and provide current and future human beings living with DMD enhanced control over their own destiny. Conclusions Abbreviations The role of the vasculature in DMD can no longer be ig- AAV: Adeno-associated virus; ACE: Angiotensin-converting enzyme; nored in light of the mounting evidence for its role in the ACEI: Angiotensin-converting enzyme inhibitor; ANF: Atrial natriuretic factor; pathogenic process. With this new knowledge in mind and ARB: Angiotensin receptor blocker; BB: β-blocker; BMD: Becker muscular dystrophy; cGMP: Cyclic guanosine monophosphate; CNS: Central nervous with the dearth of current treatments, this review focused system; DAPC: Dystrophin-associated glycoprotein complex; DMD: Duchenne on a variety of new therapeutic options that specifically tar- muscular dystrophy; EDHF: Endothelium-derived hyperpolarizing factor; get these DMD vascular defects, namely attenuation of the LVEF: Left ventricular ejection fraction; MDSCs: Muscle-derived stem cells; Ennen et al. Skeletal Muscle 2013, 3:9 Page 10 of 12 http://www.skeletalmusclejournal.com/content/3/1/9 nNOS: Neuronal nitric oxide synthase; NO: Nitric oxide; NOS: Nitric oxide 8. Rybakova IN, Patel JR, Ervasti JM: The dystrophin complex forms a synthase; NRP1: Neuropilin-1; NRP2: Neuropilin-2; PDE5: Phosphodiesterase 5; mechanically strong link between the sarcolemma and costameric actin. sGC: Soluble guanylyl cyclase; SC: Satellite cell; rAAV: Recombinant adeno- J Cell Biol 2000, 150:1209–1214. associated virus; TA: tibialis anterior; TGF- β: Transforming growth factor-β; 9. Duncan CJ: Role of intracellular calcium in promoting muscle damage: VEGF: Vascular endothelial growth factor; VEGFR: Vascular endothelial growth a strategy for controlling the dystrophic condition. Experientia 1978, factor receptor. 34:1531–1535. 10. Spencer MJ, Croall DE, Tidball JG: Calpains are activated in necrotic fibers from mdx dystrophic mice. J Biol Chem 1995, 270:10909–10914. Competing interests 11. Hopf FW, Turner PR, Steinhardt RA: Calcium misregulation and the The authors have no financial competing interests. pathogenesis of muscular dystrophy. Subcell Biochem 2007, 45:429–464. 12. Spencer MJ, Montecino-Rodriguez E, Dorshkind K, Tidball JG: Helper Authors’ contributions (CD4(+)) and cytotoxic (CD8(+)) T cells promote the pathology of JPE completed the literature review, developed the figures, and prepared the dystrophin-deficient muscle. Clin Immunol 2001, 98:235–243. review. MV revised the manuscript. AA advised the literature review process 13. Jejurikar SS, Kuzon WM Jr: Satellite cell depletion in degenerative skeletal and revised the manuscript. All authors read and approved the final muscle. Apoptosis 2003, 8:573–578. manuscript. 14. Townsend D, Yasuda S, Metzger J: Cardiomyopathy of Duchenne muscular dystrophy: pathogenesis and prospect of membrane sealants as a new therapeutic approach. Expert Rev Cardiovasc Ther 2007, 5:99–109. Authors’ information 15. Eagle M, Baudouin SV, Chandler C, Giddings DR, Bullock R, Bushby K: JPE is a staff researcher in the laboratory of Dr. Atsushi Asakura at the Survival in Duchenne muscular dystrophy: improvements in life University of Minnesota Stem Cell Institute. expectancy since 1967 and the impact of home nocturnal ventilation. MV is an MD/PhD candidate through the Medical Scientist Training Program Neuromuscul Disord 2002, 12:926–929. at the University of Minnesota Medical School. 16. Eagle M, Bourke J, Bullock R, Gibson M, Mehta J, Giddings D, Straub V, AA is an Assistant Professor of Neurology and a faculty member of the Stem Bushby K: Managing Duchenne muscular dystrophy–the additive effect Cell Institute in the University of Minnesota Medical School. He also belongs of spinal surgery and home nocturnal ventilation in improving survival. to the Paul & Sheila Wellstone Muscular Dystrophy Center in the University Neuromuscul Disord 2007, 17:470–475. of Minnesota Medical School. 17. 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Clin Cancer Res 2005, 11:2651–2661. 91. Huddleston AJ, Knoderer CA, Morris JL, Ebenroth ES: Sildenafil for the treatment of pulmonary hypertension in pediatric patients. Pediatr • Convenient online submission Cardiol 2009, 30:871–882. • Thorough peer review • No space constraints or color figure charges doi:10.1186/2044-5040-3-9 Cite this article as: Ennen et al.: Vascular-targeted therapies for • Immediate publication on acceptance Duchenne muscular dystrophy. Skeletal Muscle 2013 3:9. • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit

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Skeletal MuscleSpringer Journals

Published: Apr 23, 2013

References