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

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

Introduction

Duchenne muscular dystrophy (DMD) is an X-linked muscle disease affecting one in 5,000 newborn males, in which the gene encoding the dystrophin protein is mutated. It is a progressive neurodegenerative disease with clinical symptoms manifesting at 2–3 years of age, loss of ambulation in early teen years and death by either respiratory insufficiency or cardiac failure in their 20s. A disease model for DMD is the mdx mouse, which lacks functional dystrophin expression due to a point mutation in the dystrophin gene. The mdx mouse has been extensively characterized and contributed to the understanding of the disease pathology [1].

Although the role of dystrophin in the skeletal muscle is widely appreciated, endothelium and vascular smooth muscle cells also express dystrophin [2]. The absence of dystrophin in these cells induced vessel dilation and abnormal blood flow, resulting in a state of functional ischemia, worsening the muscle pathology in mdx mice [3]. Restoration of dystrophin specifically in the smooth muscle of the vasculature rescued some aspects of the skeletal muscle pathology associated with the mdx mice [4]. Disruption of the dystrophin-associated sarcoglycan complex in vascular smooth muscle perturbed vascular function resulting in exacerbation of muscular dystrophic changes [5]. Dystrophin is responsible for anchoring neuronal nitric oxide synthase (nNOS) to the cell surface, which is crucial for exercise-induced increases in blood supply in muscle via NO-mediated vasodilation [6]. Administration of a phosphodiesterase-5 (PDE-5) inhibitors to mdx mice, which increased NO production, rescued the muscle from this state of functional ischemia, and improved muscle function in mdx mice [7, 8]. Similarly in humans, PDE-5 inhibitors given to both DMD boys and adult patients with Becker muscular dystrophy (BMD), a milder form of muscular dystrophy, alleviated functional ischemia during muscle contraction [9, 10]. More recent data showed that mdx skeletal muscle was less perfused and displayed marked microvessel alterations compared to wild-type C57BL6 mice [11, 12]. While current studies support the importance of NO-mediated vasodilation in DMD, the relationship between DMD and angiogenesis is not well understood.

Vascular endothelial growth factor (VEGF) signaling is one of the strongest modulators of angiogenesis and includes the ligands VEGFA, VEGFB, VEGFC and PlGF. VEGFA is the most well studied ligand of the system and acts through its two receptors, VEGF receptor-1 (VEGFR1/FLT1) and VEGF receptor-2 (VEGFR2/FLK1/KDR). Although FLK1 possesses stronger signaling capabilities, FLT1 has considerably higher affinity for VEGF but weaker signaling capabilities. In normal tissue, FLT1 acts as a sink trap for VEGF thereby preventing excessive pathological angiogenesis [13]. In addition, soluble FLT1 (sFLT1) functions as an endogenous VEGF trap [14]. Despite the known angiogenic defect in DMD and mdx mice, it is not known whether VEGF and its receptors are implicated in this disease process. Previous data from our laboratory demonstrated that heterozygous Flt1 gene knockout (Flt1) mice were viable and displayed developmentally increased capillary density in the skeletal muscles [15]. Importantly, when crossed Flt1 with mdx or mdx:utrophin mice, these mice displayed both histological and functional improvements of the dystrophic pathologic phenotype. However, it remained unknown whether postnatal Flt1 gene deletion could recapitulate these improvements in mdx mice. Since Flt1 is detected in several cell types including endothelial cells, myeloid cells and some neurons [16-18], it is important to know which cell type is important for the beneficial effects of FLT1-associated improvements seen in the mdx mice. Lastly, it remained to be seen whether blockage of FLT1 could be exploited pharmacologically to recapitulate the muscle improvements seen in the genetic models.

In this report, we compared adult mdx mice with a constitutive conditional knockout and an endothelial cell-specific conditional knockout of Flt1. We showed that endothelial cell-specific Flt1 deletion increased the capillary density in skeletal muscle and improved the DMD-associated muscle pathology. In addition, we showed that systemic delivery of anti-FLT1 peptides and monoclonal antibodies (MAbs) in mdx mice recapitulated the reduction in DMD-associated pathology seen after Flt1 deletion in mdx mice, validating Flt1 as a therapeutic target for the treatment of DMD.

Results

Postnatal Flt1 gene deletion in mice displays increase in capillary density

We previously found that mdx mice developmentally lacking one copy of the Flt1 allele have increased muscle angiogenesis and improved muscle pathology [14]. To investigate whether postnatal deletion of Flt1 gene could affect the vasculature density, we crossed CAG mice carrying a constitutively expressed CreER gene [19] with Flt1 mice [20] to generate conditional Flt1 gene knockout (CAG:Flt1) mice (Fig 1A). Upon treatment with tamoxifen (TMX), which leads to global Flt1 gene and FLT1 protein deletion (S1A and S1B Fig), Flt1 mice displayed significantly increased CD31+ vascular density compared to the Flt1 (Flt1) mice (Fig 1B and 1C). The increase in capillary density following TMX-mediated Flt1 gene deletion was rapid (within 8 days) and long lasting (more than 207 days) (Fig 1D). This allowed us to be confident that we were able to phenotype late term changes following deletion of Flt1 gene. Postnatal global loss of Flt1 resulted in a reduction in body mass [21] without reduction of tibialis anterior (TA) muscle mass (S1C and S1D Fig).

Fig 1

Postnatal deletion of Flt1 can increase capillary density in skeletal muscle.

(A) Experimental scheme for assessing angiogenic response from conditional Flt1 deletion. (B) Representative images of CD31-stained cryosections from the TA muscle from Flt1 (Flt1) and CAG:Flt1 (Flt1) mice following tamoxifen (TMX) induction. Scale bar indicates 100 μm. (C) Increase in capillary density is dependent on CAG as well as TMX induction in the Flt1 background. (D) Increase in capillary density is rapid and sustained following TMX induction in Flt1 mice more than 6 months following induction.

mdx:Flt1 mice display worse muscle pathology compared to control mdx:Flt1 mice

As we did not see any gross changes in the skeletal muscle except for increased vascular density in the Flt1 mice, we crossed the mdx mice to the CAG:Flt1 to obtain mdx:CAG:Flt1 mice. We obtained these mice in expected mendelian ratios (S2A Fig). Our original goal was to induce Flt1 gene deletion prior to the onset of muscle pathology, thus before postnatal day 21 (p21), by treatment with TMX or its active form, 4-hydroxy tamoxifen (4-OHT). However, perinatal loss of Flt1 resulted in lethality when TMX or 4-OHT treatment was initiated at p3 or p5 and partial lethality at p16 in mdx:CAG:Flt1 (mdx:Flt1) mice (S2B Fig), indicating that Flt1 is required in the perinatal stage for survival. The mice displayed no lethality when recombination was induced on or after p21. Similar to the Flt1 mice, body mass decreased without changing muscle mass in mdx:Flt1 mice compared with mdx:Flt1 mice (S2C and S2D Fig). The mdx:Flt1 mice displayed notable white fur, a sign of premature aging or stress (S3A Fig). Importantly, the increase in capillary density by loss of the Flt1 gene shown in Fig 1A was maintained in the mdx background in the mdx:Flt1 mice (Fig 2A and 2B). This was accompanied by a physiological increase in skeletal muscle perfusion as shown by laser Doppler imaging (Fig 2C). Next, we proceeded to look for dystrophinopathy-related muscle changes in the adult mdx:Flt1 mice. The mdx:Flt1 mice showed a shift in fiber type composition toward increases in oxidative type I slow fibers (S3B and S3C Fig). This was more pronounced in the EDL compared to the soleus, which is already predominantly type I. This large shift to type I fibers was expected as increased vascularization has been shown prior to fiber type change [22]. The type 1 fibers have been shown to be spared until late in DMD and are regarded as more injury resistant [23, 24]. Evans blue dye (EBD) accumulation [25] showed no differences, while fibrosis was markedly increased in the mdx:Flt1 mice (Fig 2A, 2D and 2E). Embryonic MHC (eMHC)+ muscle fibers were increased in the mdx:Flt1 mice (S3D and S3E Fig), suggesting increased muscle fiber regeneration. There is no improvement in grip strength generation by the mice (Fig 2F). Taken together, the mdx:Flt1 mice showed that even though there was an increase in capillary density, muscle pathology worsened.

Fig 2

Increased angiogenesis is accompanied by worsened muscle pathology in the mdx:Flt1 mice.

(A) Representative images of CD31-stained TA muscle, Evans blue dye (EBD) to measure acute damage in diaphragm and Sirius red staining to measure fibrosis in diaphragm. Scale bars indicate 100 μm. (B) Capillary density is increased in in the TA muscle mdx:Flt1 mice. (C) Skeletal muscle perfusion is increased in the TA muscle in mdx:Flt1 mice. (D) mdx:Flt1 mice show no difference in acute damage as judged by EBD. (E) mdx:Flt1 mice show increased in fibrosis as evaluated by Sirius red staining. (F) mdx:Flt1 mice show no difference in the grip strength normalized to body weight.

Endothelial cell-specific loss of Flt1 in mdx improves muscle phenotype in mdx mice

Flt1 is expressed in several cell types including endothelial cells, myeloid cells and some neurons. Thus, we hypothesized that Flt1 may be indispensable in one of these other compartments. Since endothelial cell-specific Flt1 deletion resulted in increased capillary density in heart and adipose tissue [20, 26], we hypothesized that deletion of endothelial cell-specific Flt1 would be sufficient to increase angiogenesis and improve muscle pathology in the mdx mice.

We attempted to increase the angiogenesis in skeletal muscle using an endothelial cell-specific VE-cadherin (Cdh5)-CreERT2-mediated Flt1 deletion in mice. Cdh5 is an endothelial cell-specific cadherin gene used for lineage tracing and conditional deletion of endothelial cells [27]. Goel et al. recently reported the presence of Cdh5 in satellite cells questioning the validity of using Cdh5 in skeletal muscle tissue [28]. We verified the endothelial cell specificity of the Rosa26R reporter, and Cre-mediated excision resulted in the mGFP expression in the endothelial cells but no other cell types (S4A, S4B and S4C Fig). We saw no difference in the body mass or muscle mass in endothelial cell-specific Flt1 deleted mice compared with the control mice (S4D and S4E Fig).

We crossed the mdx:Flt1 mice to the Cdh5 mice to yield the mdx:Cdh5-Flt1 mice (Fig 3A). Upon TMX treatment, capillary density was increased in the skeletal muscle in mdx:Cdh5-Flt1 mice compared with mdx:Cdh5-Flt1 mice, indicating that endothelial cell-specific deletion of Flt1 was sufficient to increase capillary density in the skeletal muscle (Fig 3B and 3C). This was accompanied by a physiological increase in skeletal muscle perfusion using laser Doppler (Fig 3D). Moreover, the mdx:Cdh5-Flt1 did not show any significant changes in body and muscle mass loss (S5 Fig). Signs of DMD-associated pathology such as increased EBD uptake and fibrosis were significantly reduced in the mdx:Cdh5-Flt1 mice (Fig 3B, 3E and 3F). The muscle fibers in the mdx:Cdh5-Flt1 mice had decreased centrally located nuclei and maintained larger muscle fibers compared with the mdx:Cdh5-Flt1 mice (Fig 4A–4C). The mdx:Cdh5-Flt1 mice showed increased grip strength compared with the mdx:Cdh5-Flt1 mice (Fig 4D). To examine whether the number of satellite cells were affected in the mdx:Cdh5-Flt1 mice, the Pax7+ satellite cells located underneath basal lamina (laminin+) was quantified in TA muscle sections (Fig 4E and 4F). Interestingly, Pax7+ satellite cell population was increased in the mdx:Cdh5-Flt1 mice compared with the control mdx:Cdh5-Flt1 mice, indicating that an increased vascular niche might promote myogenic precursor cell proliferation and/or survival, leading to improved muscle pathology in the mdx mice. Taken together, these data indicate that endothelial cell-specific Flt1 loss was sufficient to increase capillary density and result in the histological improvements correlated with a functional improvement in the mdx mice.

Fig 3

Endothelial cell-specific conditional deletion of Flt1 in mdx:Cdh5-Flt1 mice improve capillary density and muscle phenotype.

(A) Experimental scheme for assessing angiogenic response from conditional Flt1 deletion. Cdh5-Flt1 and mdx:Cdh5-Flt1 mice were used as wild-type and mdx controls, respectively. (B) Representative images of CD31 staining for capillary density, Sirius red staining to measure fibrosis and EBD to measure acute damage in the diaphragm. Scale bars indicate 100 μm. (C) Endothelial cell specific conditional deletion of Flt1 (mdx:Cdh5-Flt1) is sufficient to increase the capillary density in diaphragm. (D) Endothelial cell specific conditional deletion of Flt1 (mdx:Cdh5-Flt1) is sufficient to increase skeletal muscle perfusion. (E) Acute damage as judged by EBD is reduced in mdx:Cdh5-Flt1 mouse muscle. (F) Fibrosis is reduced in mdx:Cdh5-Flt1 mouse muscle as evaluated by Sirius red staining.

Fig 4

Endothelial cell-specific conditional deletion of Flt1 in mdx:Cdh5-Flt1 mice improve muscle histology and function.

(A) Representative images of HE staining of diaphragm. Scale bar indicates 100 μm. (B) Diaphragm muscle fiber turnover is reduced in mdx:Cdh5-Flt1 mouse muscle as evaluated by centrally located nuclei (CLN). (C) Distributions of mean fiber diameter in TA muscle of mdx:Cdh5-Flt1 mice were skewed toward the bigger fiber size compared with the control mdx:Cdh5-Flt mice. Stastical comparisons were examined in between mdx:Cdh5-Flt1 and mdx:Cdh5-Flt1 mice. (D) Grip strength is increased in mdx:Cdh5-Flt1 mice normalized to body weight. (E) Representative images from TA muscle of mdx:Cdh5-Flt1 mice stained with anti-Pax7 antibody (yellow) to detect satellite cells (arrows) and anti-laminin antibody (red) to detect basal lamina. Nuclei were counterstained with DAPI (blue). Scale bar indicates 20 μm. (F) Quantification of Pax7+ myogenic cells in a TA muscle. Relative amount of Pax7+ cells counted in a view field.

Pharmacological inhibition of FLT1 improved mdx mice

The genetic model of Flt1 deletion showed an ameliorated phenotype in the mdx mice. To translate our genetic results into therapeutic approaches for DMD model mice, we utilized a previously reported anti-FLT1 hexapeptide (Gly-Asn-Gln-Trp-Phe-Ile or GNQWFI) that inhibits VEGF-binding to FLT1. Intramuscular administration of the anti-FLT1 peptide in TA muscle of perinatal mdx mice (S6A Fig) increased capillary density and decreased muscle pathology in the treated muscle (S6B–S6D Fig). We assessed the diaphragm muscle after systemic (intraperitoneal; IP) injection of the anti-FLT1 peptide at a low (10 mg/kg body weight) and a high dose (100 mg/kg body weight) to test for therapeutic potential in mdx mice (Fig 5A). There was no significant body mass alteration following anti-FLT1 peptide treatment (S6E and S6F Fig). While treatment with a low dose of anti-FLT1 peptide had no effect on capillary density, skeletal muscle perfusion, membrane permeability or fibrosis, the high dose increased capillary density and skeletal muscle perfusion, and decreased EBD+ fibers and fibrosis (Fig 5B–5F). Importantly, the high dose of anti-FLT1 peptide also increased muscle stability since regenerating muscle fibers which have centrally located nuclei and eMHC+ fibers were decreased (S7A–S7C Fig). Consequently, anti-FLT1 peptide-treated mdx mice increased grip strength compared with the control mdx mice (Fig 5G). Since FLT1 is also detected in myeloid cells and motor neurons, systemic anti-FLT1 peptide might induce blood phenotypes and motor behavioral abnormality. However, we did not see changes in myeloid cell numbers in their peripheral blood or changes in motor coordination on the Rotarod test in mdx mice (S7A and S7D Fig).

Fig 5

Systemic anti-FLT1 peptide improves skeletal muscle pathology in mdx mice.

(A) Experimental scheme for treatment of mdx mice with systemic treatment using anti-FLT1 peptide. (B) Representative images of (top) CD31, (middle) gross whole mount and cryosections for EBD, (bottom) Sirius red staining of diaphragm in mdx mice treated with anti-FLT1 peptide. Scale bars indicate 100 μm. (C) Anti-FLT1 peptide injection increases capillary density in the mdx mouse muscle at high dose. (D) Anti-FLT1 peptide injection is sufficient to increase skeletal muscle perfusion in mdx mice at high dose. (E) Anti-FLT1 peptide injection decreases EBD+ area in the mdx mouse muscle at high dose. (F) Anti-FLT1 peptide injection decreases fibrotic area in the mdx mouse muscle at high dose. (G) Grip strength is improved by anti-FLT1 peptide injection at high dose in mdx mice normalized to body weight.

To increase stability and the hydrophobicity [29], we tested a D-isoform anti-FLT1 peptide attached to polyethylene glycol (PEG). However, systemic administration of the PEG-D-form anti-FLT1 peptide did not increase capillary density or improve muscle pathology or function in the mdx mice (S8 Fig). Taken together, these proof-of-concept experiments showed that postnatal inhibition of FLT1 by anti-FLT1 peptide could ameliorate the pathology associated with DMD in the mdx mice. However, the functional dose (100 mg/kg body weight) was orders of magnitude higher than the generally acceptable pharmacological standards of body weight dosage for small molecule drugs [30]. Therefore, a more potent or alternative strategy is required for further translational studies.

Screening for an antibody against FLT1

As postnatal gene deletion and pharmacological inhibition of FLT1 decreased the muscular dystrophy-associated pathology in the mdx mice, we next sought to examine whether it could do this in a more translational manner using biologics to block FLT1. To establish proof of principle, we screened 8 commercially available MAbs that could block VEGF-FLT1 binding. We first screened for the ability of the MAbs to block chimeric FLT1-FC binding to PlGF2, a VEGF family protein, using ELISA (S9A and S9B Fig), since both PlGF2 and VEGFA occupy the same binding sites on the extracellular domain of FLT1 [31]. Three MAbs showed higher binding affinities: EWC (Novus Biologicals), MAB0702 Mab (Angio-Proteomie) and EWC (Acris GmbH) blocked binding by 65.4%, 64.8%, and 60.6%, respectively, while the control polyclonal anti-FLT1 antibody could block 83.1% of binding (S9B Fig). We selected two MAbs (MAB0702 from Angio-Proteomie and EWC from Novus Biologicals) for further analyses based on their blocking efficiency and their availability for large in vivo studies. The MAB0702 and EWC were raised against the extracellular domains of the human FLT1, and likely target both membrane-bound (mFLT1) and sFLT1 due to similar homology of epitopes. We validated the blocking affinity to VEGFA-FLT1 binding, and found that MAB0702 and EWC blocked binding by 40.1% and 19.9%, respectively, compared to the 94.3% for the polyclonal control (S9B Fig). We further determined the antibodies’ affinities against mouse and human FLT1-FC protein using Biacore analysis (S2 Table). MAB0702 and EWC had similar association rate/binding constants (Ka) for both mouse and human FLT1-FC. By contrast, MAB0702 had significantly higher dissociation constants (Kd) and equilibrium dissociation constants (KD) for mouse FLT1-FC compared with EWC, while MAB0702 had lower dissociation constants (Kd) and equilibrium dissociation constants (KD) for human FLT1-FC compared with EWC. Based on these affinity studies, we decided to utilize both MAB0702 and EWC MAbs for in vivo experiments.

Testing Anti-FLT1 antibody treatment for mdx mice

We intravenously (IV) injected MAB0702 at a dose of 20 mg/kg body weight into mdx mice, and measured free sFLT1 and VEGFA in the serum. We found a significant decrease in free serum sFLT1 (S9C Fig) and an increase in serum VEGFA levels (S9D Fig) following MAB0702 injection, suggesting the efficient blocking of MAB0702 to sFLT1 and FLT1 in vivo. Based on our screening results, mdx mice were injected with IV MAbs or isotype control IgG dosing at 20 mg/kg body weight every 3 days for four weeks beginning at 3 weeks of age (Fig 6A). The treatment with MAB0702 at 20 mg/kg body weight but not EWC or isotype control significantly increased capillary density and skeletal muscle perfusion using laser Doppler (Fig 6B–6D). More importantly, the mice treated with MAB0702 at 20 mg/kg body weight also displayed improved histology, such as decreased number of EBD+ fibers, decreased fibrosis, decreased calcification, and decreases in CLN (Figs 6B, 6E–6G, 7A and 7B) without affecting any body weight compared with the mdx mice (S9E and S9F Fig). Treatment prevented the increase in smaller caliber muscle fibers seen in the mdx mice (Fig 7A and 7C). While the EWC had significantly lower dissociation constants to mouse FLT1 compared with MAB0702 (S2 Table), we were surprised to find that in vivo administration of the EWC did not induce changes in capillary density, skeletal muscle perfusion or muscle pathology (Figs 6B, 6C–6G, 7A, 7B). MAB0702-treated mdx mice generated increased grip strength compared with the mdx mice (Fig 7D). Skeletal muscle endurance, as assessed by treadmill running as an indicator of maximal muscle capacity, showed running duration and distance of MAB0702-treated mdx mice significantly increased compared to mdx mice (Fig 7E and 7F). Thus, anti-FLT1 antibody administration depleted sFLT1 levels and increased free serum VEGFA levels, which led to increased angiogenesis and reduced muscle pathology in mdx mice, providing a potential new pharmacological strategy for treatment of DMD. To examine whether the number of satellite cells was affected in the MAB0702-treated mdx mice, the Pax7+ cells located underneath basal lamina was quantified in TA muscle sections (Fig 7G and 7H). Importantly, Pax7+ satellite cells were increased in the MAB0702-treated mdx mice compared with the control mdx mice. Taken together, these data strongly indicate that pharmacological inhibition of FLT1 may benefit patients with DMD by either increasing VEGFA and/or decreasing sFLT1 as a therapeutic target.

Fig 6

MAbs against FLT1 for the treatment of muscular dystrophy in the mdx mice.

(A) Experimental scheme for systemic treatment of mdx mice with systemic injection of anti-FLT1 antibody. (B) Representative images of (top) CD31, (middle) EBD and (bottom) Sirius red staining of diaphragm in mdx mice treated with anti-FLT1 antibody. Scale bars indicate 100 μm. (C) Capillary destiny is increased in MAB0702 treated mice but not EWC in mdx mice compared with isotype control. (D) MAB0702 but not EWC injection is sufficient to increase skeletal muscle perfusion in mdx mice. (E) EBD+ area is decreased in MAB0702 treated mice but not EWC in mdx mice compared with isotype control. (F) Fibrotic area is decreased in MAB0702 treated mice but not EWC in mdx mice compared with isotype control. (G) Calcification is decreased in both MAB0702 and EWC treated mice in mdx mice compared with isotype control.

Fig 7

Systemic delivery of MAbs against FLT1 improves skeletal muscle pathology in mdx mice.

(A) Representative images of HE staining of diaphragm in mdx mice treated with anti-FLT1 antibodies. Scale bars indicate 100 μm. (B) Diaphragm muscle fiber turnover is reduced in MAB0702 treated mdx mouse muscle as evaluated by centrally located nuclei (CLN). (C) Distributions of mean fiber diameter in TA muscle of MAB0702 treated mdx mice were skewed toward the bigger fiber size compared with compared with isotype control. (D) Grip strength is improved in mdx mice treated with MAB0702 compared with isotype control. (E, F)Treadmill running time and distance are improved in mdx mice following MAB0702 treatment. (G) Representative images from TA muscle of MAB0702 treated mdx mice stained with anti-Pax7 antibody (yellow) to detect satellite cells (arrows) and anti-laminin antibody (red) to detect basal lamina. Nuclei were counterstained with DAPI (blue). Scale bar indicates 20 μm. (H) Quantification of Pax7+ myogenic cells in a TA muscle. Relative amount of Pax7+ cells counted in a view field.

Discussion

We have previously shown that mdx mice carrying heterozygous global deletion of Flt1 display increased capillary density, and improved muscle pathology [14]. In this report we show the effect of modulating Flt1 using several different techniques and mouse models (S1 Table). First, we show that Flt1 is important postnatally as conditional Flt1 deletion in mdx:Flt1 mice in the perinatal stage results in lethality in mice. While deletion of Flt1 in neonates increased capillary density in both C57BL6 and mdx mice, it led to the worsening of the skeletal muscle phenotype. Since Flt1 is expressed in several cell types including endothelial cells, myeloid cells and some neurons [16–18, 27], Flt1 may be indispensable in one of these other compartments when both alleles are perinatally deleted. For example, Flt1 is expressed in motor neurons, where is not simply acting as a VEGFA sink-trap. In motor neurons, tyrosine kinase activity of Flt1 is required for their survival [17]. Thus, the observed increased angiogenesis but worse pathological alterations in mdx:Flt1 muscle may be due to motor neuron–associated changes. By contrast, loss of Flt1 in endothelial cells in the postnatal stage increased the capillary density and blood perfusion in skeletal muscle without significant decreases in weight or muscle mass. Increased angiogenesis and blood perfusion were observed in the global Flt1 heterozygous knockout mice [15], indicating that loss of endothelial cell-specific Flt1 in mdx:Cdh5-Flt1 mice was sufficient to produce increased capillary density and vascular perfusion in the skeletal muscle. This also led to an improved mdx-associate muscle pathology, confirming that postnatal deletion of Flt1 is able to rescue the dystrophinopathy related muscle pathology.

Importantly, for the first time, we demonstrated that administration of both anti-FLT1 peptide and anti-FLT1 MAb increased angiogenesis, which led to an improved the pathology associated with DMD in mdx mice, and is a phenocopy of our genetic models (mdx:Cdh5-Flt1 mice). Notably, the improved DMD pathologies via systemic pharmacological inhibition of Flt1 were the phenocopy seen in mdx:Cdh5-Flt1 mice but not observed in mdx:Flt1 mice which showed markedly worsened muscle pathology. This contradiction may be partly due to the fact that anti-FLT1 peptide and MAb may be unable to target the membrane associated FLT1 in the motor neurons, which might prevent the mice from motor neuron–associated changes [32-34]. We screened commercially available MAbs for blocking ligands-FLT1, and demonstrated that administration of MAB0702 MAbs was able to phenocopy our genetic model in a manner suited for translational studies to reduce muscle pathology in mdx mice.

After MAB0702 administration, we showed a small increase in VEGFA levels in serum. It should be noted that a mere 2-fold increase of VEGFA during development is incompatible with life in transgenic mice [35]. Computation and experimental models showed that local VEGF gradients are more important than the total concentration [36, 37]. We recently demonstrated that muscle satellite cells express abundant VEGFA, which recruits endothelial cells and capillaries to the proximity of satellite cells on muscle fibers [38]. Taken together, these data strongly suggest that hot-spots with high levels of VEGFA in Flt1 knockout or anti-FLT1 treated mice may result in increased capillary density and vascular perfusion, which is predicted to result in decreased associated DMD-type pathological changes in the skeletal muscles. Improvement of dystrophic muscle function by FLT1 blockade may provide a novel pharmacological strategy for treatment of diseases associated with DMD via increased serum and tissue VEGFA levels, which induce increased vascular density and perfusion.

While VEGFA binds to both FLT1 and FLK1, VEGFB, PlGF1 and PlGF2 only bind to FLT1 [39]. This creates a scenario where PlGF1/2 and VEGFB binding can sequester FLT1, increasing serum and tissue VEGFA availability for VEGFA-FLK1 binding-mediated angiogenic induction. While PlGF is dispensable for normal development and health, and not expressed in the normal adult tissues outside the female reproductive organs [40], VEGFB is expressed in the muscle tissue and muscle fibers [41]. VEGFB may play a role in diet induced obesity and free fatty acid uptake by the endothelial cells in skeletal muscle [42], but this remains controversial [26]. However, VEGFB overexpression does not result in an angiogenic response in ischemic skeletal muscle [43], indicating that blocking VEGFB-FLT1 signaling is not likely to be responsible for the angiogenic changes seen in this study.

The present study shows that increased angiogenesis may be a novel avenue to improve some of pathology associated with loss of dystrophin, including function, and could be used in conjunction with other treatment strategies. For example, intramuscular injection of VEGF containing recombinant adeno-associated viral vectors resulted in both functional and histological improvements in both ischemic and mdx muscle [44-47]. This improvement was seen in combination with increased in angiogenesis in the muscle. Increased capillary density in the mdx muscle work through paracrine stimulation, by protecting muscle fiber damage and promoting satellite cell proliferation, survival and self-renewal in the vascular niche [38, 48]. Indeed, we demonstrated that Pax7+ satellite cell population was increased in the mdx:Cdh5-Flt1 mice and the MAB0702-treated mdx mice. Christov et al. and our group showed that satellite cells were preferentially located next to capillaries [38, 49]. Since our approaches increased the number of capillaries found in the examined muscles, it may effectively increase the amount of vascular niche that houses the satellite cell compartment in the muscle. Recently, we showed that genes encoding for endothelial and satellite cells were highly correlated across muscle groups, and endothelial cells could mediate satellite cell self-renewal via Notch activation [38]. Therefore, increase in the vascular niche may increase satellite cell self-renewal and the number of myogenic precursor cells, which we hypothesize may be responsible for the improved phenotype seen in the mdx mice with Flt1 deletion or functional blockage.

MAb-based therapeutics for the treatment of DMD have been developed to increase muscle mass via targeting myostatin and to decrease fibrosis via targeting fibroadipogenic progenitors [50]. Further optimization using with humanized antibodies are required for future translation to humans [51]. Taken together, we have gathered evidence for the first time that FLT1-targeted MAbs may be an effective therapeutic approach for the treatment of DMD.

Methods

Mice

Flt1 mice were obtained from Dr. Gua-Hua Fong [20]. Cdh5 mice were obtained from Dr. Yoshiaki Kubota [27]. B6Ros.Cg-Dmd/J (mdx; JAX stock #002379) [52], B6.Cg-Tg(CAG-cre/Esr1*)5Amc/J (CAG; JAX stock #004682) [19] and D2.129(Cg)-Gt(ROSA)26Sor/J (Rosa26R; JAX stock #007576) [53] mice were obtained from Jackson Laboratory. All the mice used in this study were listed in S1 Table. Colonies for all the mice were established in the laboratory. Cre recombination was induced using tamoxifen (TMX) (T5648, MilliporeSigma) dosed as 75 mg/kg body weight x 3 time over one week at 3–4 weeks of age unless otherwise specified. We also injected 4-hydroxy tamoxifen (4-OHT) (H6278, MilliporeSigma) dosed as 25 mg/kg body weight. Control mice contained the wild-type (WT) CreER allele or were injected with the vehicle (corn oil or 10% ethanol).

Ethical Statement

All animal studies were approved by the IACUC at University of Minnesota (190336906A).

Anti-FLT1 peptides and antibodies

The anti-FLT1 peptide was synthesized from Peptide 2.0 Inc. based on the sequence (Gly-Asn-Gln-Trp-Phe-Ile or GNQWFI) as previously described [54]. DMSO was used to dissolve the peptide. Twenty μg of peptide diluted in 2% DMSO in PBS solution was used for intramuscular injection per day in the TA muscle. Ten mg/kg body weight and 100 mg/kg body weight were used for the systemic treatment via IP injection. The second generation anti-FLT1 peptide (PEG-GDNDQDWDFDID) was synthesized with the following modifications: The polyethylene glycol moiety was attached to the peptide to improve solubility in polar solvents and the D isomeric form was used instead of the L to enhance stability of the peptide [29]. PBS was used as a vehicle. The peptides were commercially synthesized (LifeTein, LLC). Commercially available anti-FLT1 antibodies were obtained from the manufacturer in carrier free and preservative free form (AF471 from R&D Systems, D2 from Santa Cruz Biotechnology, EWC from Acris GmbH and Novus Biologicals, LS-C6855 from LifeSpan BioSciences, MAB1664 from MilliporeSigma and MAB7072 from Angio-Proteomie, Abcam and Santa Cruz Biotechnology). Isotype IgG (Santa Cruz Biotechnology) was used for control experiment. Two or 20 mg/kg body weight was used for the systemic treatment. Retro-orbital IV injections were performed for systemic treatment for the antibody.

RNA and genomic DNA isolation and qPCR

Mouse TA muscle was homogenized in TRIzol reagent (15596026, ThermoFisher Scientific) for RNA isolation. RNA was isolated using the Direct-zol RNA Microprep Kit (R2062, Zymo Research) with on-column DNase digestion, and 1 μg RNA was used for cDNA synthesis using the Transcriptor First Strand cDNA synthesis kit (04379012001, Roche Molecular Diagnostics) with random primers. Genomic DNA was isolated from mouse tail snips with lysis buffer containing Proteinase K (P2308, MilliporeSigma). Genotyping was performed by agarose gel electrophoresis-mediated detection following PCR reaction by Taq polymerase (M0273, New England Biolabs). qPCR was performed using GoTaq qPCR Master Mix (A6001, Promega). Primer sequences are listed in S3 Table. All primers were synthesized as custom DNA oligos from Integrated DNA technologies (IDT).

Muscle perfusion

RBC flux was evaluated using the moorLabTM laser Doppler flow meter as previously described [15]. with the MP7a probe that allows for collecting light from a deeper tissue level than standard probes according to the manufacturer’s instructions (Moor Instruments). The fur from the right hind leg was removed using a chemical depilatory. Readings were taken using the probe from at least 10 different spots on the TA muscle. The AU was determined as the average AU value during a plateau phase of each measurement.

Grip strength test

Forelimb grip strength test was performed following a previously published procedure [55]. Briefly, mdx mice were gently pulled by the tail after fore limb-grasping a metal bar attached to a force transducer (Columbus Instruments). Grip strength tests were performed by the same blinded examiner. Five consecutive grip strength tests were recorded, and then mice were returned to the cage for a resting period of 20 minutes. Then, three series of pulls were performed each followed by 20 min resting period. The average of the three highest values out of the 15 values collected was normalized to the body weight for comparison.

Treadmill running

Exer-3/6 Treadmill (Columbus Instruments) was used for treadmill running test as previously described [56]. Briefly, for acclimation, mice were placed in each lane and forced to run on a treadmill for 5 minutes at a speed of 10 m/min on a 0% uphill grade for 3 days. And then, mice were forced to run on a treadmill with a 10% uphill grade starting at a speed of 10 m/min for 5 minutes. Every subsequent 2 minutes, the speed was increased by 2 m/min until the mice underwent exhaustion which was defined as the inability of the mice to remain on the treadmill. The time of running as well as the distance run were recorded.

Rotarod test

Mice were trained on the rotarod (0890M-D54 Rotamex-5, Columbus Instruments) for 2 days before collecting data as previously described [57]. During each trial, mice were placed on the rod at 10 rpm for 60 seconds, and the rod accelerated from 10 to 30 rpm in 30 second intervals. The total maximum testing time was 240 seconds. Each trial was done twice a day (2-hour interval between sessions) for 3 consecutive days. The latency to fall was recorded, and the longest running time was used for analysis.

Histology and immunofluorescence

To assess the microvasculature ECs, we utilized intravital tomato-lectin (DL-1178, Vector labs) staining following retro-orbital IV injections. Tissues were frozen fresh using LiN2 chilled isopentane and stored at -80ºC. Eight μm thick transverse cryosections were used for all histological analysis. Slides were fixed using 2% PFA and washed twice using PBS + 0.01% Triton (PBST) before being used. For capillary density measurement, immunohistochemistry for CD31 was performed using anti-CD31 antibody followed by the Vectastain Elite ABC Kit (PK-6100, Vector Laboratories) according to the manufacturer’s instructions and developed using 3-amino-9-ethylcarbazole (AEC) (A5754, MilliporeSigma). For immunofluorescence, sections were fixed, washed twice in PBST, incubated with 0.2% Triton X-100 for 10 min and blocked by 10% BSA. Sections were incubated with primary antibodies overnight, washed twice in PBST and incubated in fluorescent-conjugated secondary antibody for 1 hour. Anti-FLT1 (RB-9049, NeoMarkers) and anti-Laminin (4H8-2, MilliporeSigma) antibodies followed by anti-rabbit Alexa-488 and anti-rat Alexa-568 (Molecular Probes) were used for detection of FLT1 expression in capillaries. Anti-type I slow MHC (NOQ7.5.4D, MilliporeSigma) or anti-embryonic MHC (F1.652, Developmental Study Hybridoma Bank) and anti-Laminin (4H8-2, MilliporeSigma) antibodies followed by anti-mouse Alexa-488 (A21202, ThermoFisher Scientific) and anti-rat Alexa-594 antibodies (A21471, ThermoFisher Scientific) were used for detection of slow or regenerating muscle fibers. Anti-Pax7 (MAB1675, R&D Systems) and anti-Laminin (4H8-2, MilliporeSigma) antibodies followed by anti-mouse Alexa-488 (A21202, ThermoFisher Scientific) and anti-rat Alexa-594 antibodies (A21471, ThermoFisher Scientific) were used for detection of satellite cells. One percent Evans blue dye (EBD) (E2129, MilliporeSigma) dissolved in PBS were injected IP at 1% body weight 16–20 hours prior to dissecting the mouse [21]. Sirius red, Alizarin red, and Hematoxylin & Eosin (HE) were performed as previously described [14]. All sections were co-stained using DAPI and mounted using DAKO mounting media. Myeloid cells and lymphocytes were identified by May-Grunwald’s Giemsa (660–75, Sigma-Aldrich). Microscopic images were captured by a DP-1 digital camera attached to BX51 fluorescence microscope with 10×, 20× or 40× UPlanFLN objectives (all from Olympus). Fiji was used for image processing [58].

Biacore Surface Plasmon Resonance (SPR) Binding Assay for Anti-FLT1 MAbs

The single cycle kinetics method was used for sFLT1 binding assay by Biacore (GE Healthcare Bio-Science). A CM5 series S sensor chip (GE Healthcare Bio-Science) with mouse and human soluble FLT1-FC chimeric protein (471-F1-100 and 321-FL-050/CF, R&D Systems) immobilized to about 1,000 RU was used as ligand for the analyte binding of anti-FLT1 MAbs. An analyte range of 0–5 nM was used for kinetic experiments. A 5 min association step was used for each dilution followed by a 40 min dissociation. Chip surface was regenerated using pH 2.0 glycine between experiments.

ELISA

For the initial FLT1 blocking MAb screening, 96 well ELISA plates (44-2404-21, ThermoFisher Scientific) were incubated with mouse FLT1-FC chimeric protein with His-tag (471-F1-100, R&D Systems). Plates were washed with 0.05% Tween 20 in PBS, and blocked with 5% BSA (BP1600-1, Fisher Scientific) in PBS. After washing, diluted anti-FLT1 antibodies were incubated. After washing, recombinant mouse PlGF2 (465-PL-010/CF, R&D Systems) was incubated. After washing, biotin-conjugated anti-PlGF2 antibody (BAF465, R&D Systems) was added followed by a Streptavidin-HRP (R&D Systems, DY998). For a colorimetric detection, plates were developed with 3,3′,5,5′-Tetramethylbenzidine (TMB) Liquid Substrate (T0440, MilliporeSigma) and the reaction was stopped with 0.5M H2SO4. Specs were read at 450 nm on spectramax M5 plate reader (Molecular Devices). For VEGFA-FLT1 blocking assays, 96 wells ELISA plates were incubated with recombinant mouse VEGFA (VEGF164) (493-MV-025/CF, R&D Systems,). After washing, anti-FLT1 antibodies were incubated with mouse FLT1-FC chimeric protein with His-tag (471-F1-100, R&D Systems). After incubation the antibody-FLT1-FC complex was added to the VEGFA-coated plates. A biotin conjugated anti-His-tag antibody (NB100-63172, Bio-Connect) was added followed by a Streptavidin-HRP (DY998, R&D Systems). For a colorimetric detection, TMB was used as described above. For measurement of free sFlt1 and VEGFA in the serum. Animals were IV injected with 20 mg/kg body weight either isotype control IgG or the MAB0702 twice a week for 4 weeks beginning at 4 weeks of age. Blood was collected for biomarker analysis. Serum concentrations of sFLT1 and VEGFA were measured by ELISA kits following company recommended protocols (DY471 and DY493, R&D Systems). For a colorimetric detection, TMB was used as described above.

Statistics

Statistics and graphs were calculated using GraphPad 7.1 (Prism). Students t-test or ANOVA was used to compare two or more groups. Multiple comparison adjustment was performed with comparisons of 3 groups or more. * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001.

Confirmation of conditional deletion of Flt1.

A. Immunostaining for FLT1 (green, arrows) and Laminin (red) shows effective deletion of FLT1 in TA muscle of Flt1 mice following tamoxifen (TMX) injection. DAPI staining (blue) is for all nuclei. Scale bars indicate 20 μm.

B. RT-qPCR shows deletion of the Flt1 exon 3 in the TA muscle of Flt1 mice while exons 1 and 2 are retained.

C. Flt1 male mice show no difference in the body mass during the time course

D. Flt1 male mice show no difference in TA muscle mass compared to Flt1 male mice.

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Generation of mdx:CAG mice.

A. mdx:CAG:Flt1 mice breed with mdx:Flt1 mice, and yield mice in expected ratios in a total of 92 mice genotyped. Chi-squared test shows no difference in expected.

B. Induction of CreER by TMX or 4-hydroxy-tamoxifen (4-OHT) shows that Flt1 deletion in mdx mice prior to p21 results in partial or complete lethality in the mdx:Flt1 but not control Flt1 mice.

C. mdx:Flt1 mice show reduced male body mass but no difference in (D) muscle mass.

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Phenotyping of mdx:Flt1 mice.

A. mdx:Flt1 mice show signs of premature aging such as white hair.

B. Representative image of type I slow MHC (green) and Laminin (red) in EDL and soleus muscle in mdx:Flt1 and mdx:Flt1 mice. Scale bars indicate 200 μm.

C. Type I slow MHC+ fibers are increased in the mdx:Flt1 mice in both EDL and Soleus muscle.

D. Representative image of eMHC (green) and Laminin (red) in EDL and soleus muscle in mdx:Flt1 and mdx:Flt1 mice. Scale bars indicate 200 μm.

D. eMHC staining shows decreased fiber stability in muscle fibers in the soleus but not the EDL muscle in the mdx:Flt1 mice.

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Validation of Cdh5 and mdx:Cdh5-Flt1 mice.

A. Experimental scheme for assessing angiogenic response from conditional Flt1 deletion.

B. Cdh5:Rosa26R mice reveal that Cdh5 efficiently induced mGFP expression in the capillaries (green) labeled with lectin (purple), but not in other cell types including muscle fibers (red). Scale bars indicate 20 μm.

C. Cdh5:Rosa26R mice show efficient mGFP labeling of lectin+ and CD31+ endothelial cells. Histogram indicates that more than 90% of the cells are CD31+mGFP+ or lectin+mGFP+.

D.E. Body mass and TA muscle mass are unchanged in Cdh5-Flt1 and Cdh5-Flt1 mice.

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Validation of mdx:Cdh5-Flt1 mice.

A. B. Body mass and TA muscle mass are unchanged in mdx:Cdh5-Flt1and mdx:Cdh5-Flt1 mice.

C. D. Body mass is unchanged in male or female mdx:Cdh5-Flt1and mdx:Cdh5-Flt1 mice during the time course.

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Efficacy of anti-FLT1 peptide following intramuscular injection in the TA muscle of mdx mice.

A. Experimental scheme for proof of principle study of mdx mice with intramuscular injection of anti-FLT1 peptide.

B. Representative images of CD31 (top) and EBD staining (bottom) of TA muscle injected with anti-FLT1 peptide. Scale bars indicate 50 μm.

C. Neonatal intramuscular injection of anti-FLT1 peptide increases capillary density in the TA muscle of the mdx mice.

D. Neonatal intramuscular injection of anti-FLT1 peptide decreases EBD+ area in the TA muscle of the mdx mice.

E. F. Systemic anti-FLT1 peptide injection does not change body mass in the male or female mdx mice at low or high dose.

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Systemic anti-FLT1 peptide injection improves muscle pathology without increase in leukocytes or neurologic phenotype in mdx mice.

A. Representative images of HE staining, eMHC staining of diaphragm and Giemsa staining of blood smears in mdx mice treated with anti-FLT1 peptide. Allows indicate myeloid cells and lymphocytes. Scale bars indicate 100 μm.

B. Diaphragm muscle fiber turnover is reduced in mdx mice treated with anti-FLT1 peptide as evaluated by centrally located nuclei (CLN).

C. eMHC staining shows increased fiber stability in muscle fibers in the diaphragm of mdx mice treated with anti-FLT1 peptide.

D. No difference in motor coordination or balance on the Rotarod was observed between the groups.

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Systemic PEG-anti-FLT1 peptide injection does not improve skeletal muscle pathology in mdx mice.

A. Experimental scheme for systemic treatment of mdx mice with IP injection of PEG-anti-FLT1 peptide.

B. Systemic PEG-anti-FLT1 peptide injection does not change body mass in the male mdx mice at low or high dose.

C. Systemic PEG-anti-FLT1 peptide injection does not increase capillary density in the mdx mice at low or high dose.

D. Systemic PEG-anti-FLT1 peptide injection does not decrease EBD in the mdx mice at low or high dose.

E. Systemic PEG-anti-FLT1 peptide injection does not improve grip strength in the mdx mice at low or high dose.

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Screening for commercially available antibodies against FLT1 and phenotyping of mdx mice treated with MAB0702.

A. Commercially available MAbs for anti-FLT1 screened for blocking activity against PlGF using ELISA. AF471 polyclonal anti-FLT1 antibody was used as a positive control. AP, Angio-Proteomie; SC, Santa Cruz Biotechnology.

B. Two selected MAbs screened for blocking activity against VEGFA using ELISA. AF471 polyclonal anti-FLT1 antibody was used as a positive control. AP, Angio-Proteomie.

C. Serum free sFLT1 is decreased in mice injected with MAB0702 compared to isotype control.

D. Serum free VEGFA is increased following MAB0702 treatment.

E. F. Systemic anti-FLT1 antibody injection does not change body mass in the male or female mdx mice.

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Summary of mice used for this paper.

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Biacore analysis for affinity of monoclonal antibodies against FLT1.

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DNA primer sequences.

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