Anti-ACVR1 antibodies exacerbate heterotopic ossification in fibrodysplasia ossificans progressiva (FOP) by activating FOP-mutant ACVR1.

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disorder whose most debilitating pathology is progressive and cumulative heterotopic ossification (HO) of skeletal muscles, ligaments, tendons, and fascia. FOP is caused by mutations in the type I BMP receptor gene ACVR1, which enable ACVR1 to utilize its natural antagonist, activin A, as an agonistic ligand. The physiological relevance of this property is underscored by the fact that HO in FOP is exquisitely dependent on activation of FOP-mutant ACVR1 by activin A, an effect countered by inhibition of anti-activin A via monoclonal antibody treatment. Hence, we surmised that anti-ACVR1 antibodies that block activation of ACVR1 by ligands should also inhibit HO in FOP and provide an additional therapeutic option for this condition. Therefore, we generated anti-ACVR1 monoclonal antibodies that block ACVR1's activation by its ligands. Surprisingly, in vivo, these anti-ACVR1 antibodies stimulated HO and activated signaling of FOP-mutant ACVR1. This property was restricted to FOP-mutant ACVR1 and resulted from anti-ACVR1 antibody-mediated dimerization of ACVR1. Conversely, wild-type ACVR1 was inhibited by anti-ACVR1 antibodies. These results uncover an additional property of FOP-mutant ACVR1 and indicate that anti-ACVR1 antibodies should not be considered as therapeutics for FOP.

Allele-Selective LNA Gapmers for the Treatment of Fibrodysplasia Ossificans Progressiva Knock Down the Pathogenic ACVR1R206H Transcript and Inhibit Osteogenic Differentiation.

Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant disorder characterized by episodic heterotopic ossification. The median life span of people with this disorder is ∼40 years, and currently, there is no effective treatment available. More than 95% of cases are caused by a recurrent mutation (c.617G>A; R206H) of Activin A receptor, type I (ACVR1)/Activin receptor-like kinase-2 (ALK2), a bone morphogenetic protein type I receptor. The mutation renders ACVR1 responsive to activin A, which does not activate wild-type ACVR1. Ectopic activation of ACVR1R206H by activin A induces heterotopic ossification. Since ACVR1R206H is a hyperactive receptor, a promising therapeutic strategy is to decrease the activity of mutated ACVR1. To accomplish this goal, we developed locked nucleic acid (LNA) gapmers. These are short DNA oligonucleotides with LNA modification at both ends. They induce targeted mRNA degradation and specific knockdown of gene expression. We demonstrated that some of these gapmers efficiently knocked down ACVR1R206H expression at RNA levels, while ACVR1WT was mostly unaffected in human FOP fibroblasts. Also, the gapmers suppressed osteogenic differentiation induced by ACVR1R206H and activin A. These gapmers may be promising drug candidates for FOP. This novel strategy will also pave the way for antisense-mediated therapy of other autosomal dominant disorders.

ActRIIB:ALK4-Fc alleviates muscle dysfunction and comorbidities in murine models of neuromuscular disorders.

Patients with neuromuscular disorders suffer from a lack of treatment options for skeletal muscle weakness and disease comorbidities. Here, we introduce as a potential therapeutic agent a heterodimeric ligand-trapping fusion protein, ActRIIB:ALK4-Fc, which comprises extracellular domains of activin-like kinase 4 (ALK4) and activin receptor type IIB (ActRIIB), a naturally occurring pair of type I and II receptors belonging to the TGF-ß superfamily. By surface plasmon resonance (SPR), ActRIIB:ALK4-Fc exhibited a ligand binding profile distinctly different from that of its homodimeric variant ActRIIB-Fc, sequestering ActRIIB ligands known to inhibit muscle growth but not trapping the vascular regulatory ligand bone morphogenetic protein 9 (BMP9). ActRIIB:ALK4-Fc and ActRIIB-Fc administered to mice exerted differential effects - concordant with SPR results - on vessel outgrowth in a retinal explant assay. ActRIIB:ALK4-Fc induced a systemic increase in muscle mass and function in wild-type mice and in murine models of Duchenne muscular dystrophy (DMD), amyotrophic lateral sclerosis (ALS), and disuse atrophy. Importantly, ActRIIB:ALK4-Fc improved neuromuscular junction abnormalities in murine models of DMD and presymptomatic ALS and alleviated acute muscle fibrosis in a DMD model. Furthermore, in combination therapy ActRIIB:ALK4-Fc increased the efficacy of antisense oligonucleotide M12-PMO on dystrophin expression and skeletal muscle endurance in an aged DMD model. ActRIIB:ALK4-Fc shows promise as a therapeutic agent, alone or in combination with dystrophin rescue therapy, to alleviate muscle weakness and comorbidities of neuromuscular disorders.

ACVR1-Fc suppresses BMP signaling and chondro-osseous differentiation in an in vitro model of Fibrodysplasia ossificans progressiva.

Fibrodysplasia ossificans progressiva (FOP) is a rare and devastating genetic disease of heterotopic endochondral ossification (HEO), and currently no effective therapies are available for this disease. A recurrent causative heterozygous mutation (c.617 G>A; R206H) for FOP was identified in activin receptor type IA (ACVR1), a bone morphogenetic protein (BMP) type I receptor. This mutation aberrantly activates the BMP-Smad1/5/8 signaling pathway and leads to HEO in FOP patients. Here we report development of a soluble recombinant ACVR1-Fc fusion protein by combining the extracellular domain of human wild type ACVR1 and the Fc portion of human immunoglobulin gamma 1 (IgG1). The ACVR1-Fc fusion protein significantly down-regulated the dysregulated BMP signaling caused by the FOP ACVR1 mutation and effectively suppressed chondro-osseous differentiation in a previously described cellular FOP model, human umbilical vein endothelial cells (HUVECs) that were infected with adenovirus-ACVR1R206H (HUVECR206H). This ACVR1-Fc fusion protein holds great promise for prevention and treatment of HEO in FOP and related diseases.

BMP9 and BMP10 are critical for postnatal retinal vascular remodeling.

ALK1 is a type I receptor of the TGF-ß family that is involved in angiogenesis. Circulating BMP9 was identified as a specific ligand for ALK1 inducing vascular quiescence. In this work, we found that blocking BMP9 with a neutralizing antibody in newborn mice significantly increased retinal vascular density. Surprisingly, Bmp9-KO mice did not show any defect in retinal vascularization. However, injection of the extracellular domain of ALK1 impaired retinal vascularization in Bmp9-KO mice, implicating another ligand for ALK1. Interestingly, we detected a high level of circulating BMP10 in WT and Bmp9-KO pups. Further, we found that injection of a neutralizing anti-BMP10 antibody to Bmp9-KO pups reduced retinal vascular expansion and increased vascular density, whereas injection of this antibody to WT pups did not affect the retinal vasculature. These data suggested that BMP9 and BMP10 are important in postnatal vascular remodeling of the retina and that BMP10 can substitute for BMP9. In vitro stimulation of endothelial cells by BMP9 and BMP10 increased the expression of genes involved in the Notch signaling pathway (Jagged1, Dll4, Hey1, Hey2, Hes1) and decreased apelin expression, suggesting a possible cross-talk between these pathways and the BMP pathway.

An inhibitor of transforming growth factor beta type I receptor ameliorates muscle atrophy in a mouse model of caveolin 3-deficient muscular dystrophy.

Skeletal muscle expressing Pro104Leu mutant caveolin 3 (CAV3(P104L)) in mouse becomes atrophied and serves as a model of autosomal dominant limb-girdle muscular dystrophy 1C. We previously found that caveolin 3-deficient muscles showed activated intramuscular transforming growth factor beta (TGF-ß) signals. However, the cellular mechanism by which loss of caveolin 3 leads to muscle atrophy is unknown. Recently, several small-molecule inhibitors of TGF-ß type I receptor (TßRI) kinase have been developed as molecular-targeting drugs for cancer therapy by suppressing intracellular TGF-ß1, -ß2, and -ß3 signaling. Here, we show that a TßRI kinase inhibitor, Ki26894, restores impaired myoblast differentiation in vitro caused by activin, myostatin, and TGF-ß1, as well as CAV3(P104L). Oral administration of Ki26894 increased muscle mass and strength in vivo in wild-type mice, and improved muscle atrophy and weakness in the CAV3(P104L) mice. The inhibitor restored the number of satellite cells, the resident stem cells of adult skeletal muscle, with suppression of the increased phosphorylation of Smad2, an effector, and the upregulation of p21 (also known as Cdkn1a), a target gene of the TGF-ß family members in muscle. These data indicate that both TGF-ß-dependent reduction in satellite cells and impairment of myoblast differentiation contribute to the cellular mechanism underlying caveolin 3-deficient muscle atrophy. TßRI kinase inhibitors could antagonize the activation of intramuscular anti-myogenic TGF-ß signals, thereby providing a novel therapeutic rationale for the alternative use of this type of anticancer drug in reversing muscle atrophy in various clinical settings.

Inhibitory effect of a TGFbeta receptor type-I inhibitor, Ki26894, on invasiveness of scirrhous gastric cancer cells.

BACKGROUND: Gastric cancer cells frequently metastasise, partly because of their highly invasive nature. Transforming growth factor-beta (TGF-beta) receptor signalling is closely associated with the invasion of cancer cells. The aim of this study was to clarify the effect of a TGF-beta receptor (TbetaR) phosphorylation inhibitor on the invasiveness of gastric cancer cells. METHODS: Four gastric cancer cell lines, including two scirrhous-type cell lines and two non-scirrhous-type cell lines, were used. A TbetaR type I (TbetaR-I) kinase inhibitor, Ki26894, inhibits the phosphorylation of Smad2 at an ATP-binding site of TbetaR-I. We investigated the expression levels of TbetaR and phospho-Smad2, and the effects of TGF-beta in the presence or absence of Ki26894 on Smad2 phosphorylation, invasion, migration, epithelial-to-mesenchymal transition (EMT), Ras homologue gene family member A (RhoA), ZO-2, myosin, and E-cadherin expression of gastric cancer cells. RESULTS: TbetaR-I, TbetaR-II, and phospho-Smad2 expressions were found in scirrhous gastric cancer cells, but not in non-scirrhous gastric cancer cells. Ki26894 decreased Smad2 phosphorylation induced by TGF-beta1 in scirrhous gastric cancer cells. Transforming growth factor-beta1 upregulated the invasion, migration, and EMT ability of scirrhous gastric cancer cells. Transforming growth factor-beta1 significantly upregulated the activity of RhoA and myosin phosphorylation, whereas TGF-beta1 decreased ZO-2 and E-cadherin expression in scirrhous gastric cancer cells. Interestingly, Ki26894 inhibited these characteristics in scirrhous gastric cancer cells. In contrast, non-scirrhous gastric cancer cells were not affected by TGF-beta1 or Ki26894 treatment. CONCLUSION: A TbetaR-I kinase inhibitor decreases the invasiveness and EMT of scirrhous gastric cancer cells. Ki26894 is therefore considered to be a promising therapeutic compound for the metastasis of scirrhous gastric carcinoma.

Genetic and pharmacological targeting of activin receptor-like kinase 1 impairs tumor growth and angiogenesis.

Members of the transforming growth factor beta (TGF-beta) family have been genetically linked to vascular formation during embryogenesis. However, contradictory studies about the role of TGF-beta and other family members with reported vascular functions, such as bone morphogenetic protein (BMP) 9, in physiological and pathological angiogenesis make the need for mechanistic studies apparent. We demonstrate, by genetic and pharmacological means, that the TGF-beta and BMP9 receptor activin receptor-like kinase (ALK) 1 represents a new therapeutic target for tumor angiogenesis. Diminution of ALK1 gene dosage or systemic treatment with the ALK1-Fc fusion protein RAP-041 retarded tumor growth and progression by inhibition of angiogenesis in a transgenic mouse model of multistep tumorigenesis. Furthermore, RAP-041 significantly impaired the in vitro and in vivo angiogenic response toward vascular endothelial growth factor A and basic fibroblast growth factor. In seeking the mechanism for the observed effects, we uncovered an unexpected signaling synergy between TGF-beta and BMP9, through which the combined action of the two factors augmented the endothelial cell response to angiogenic stimuli. We delineate a decisive role for signaling by TGF-beta family members in tumor angiogenesis and offer mechanistic insight for the forthcoming clinical development of drugs blocking ALK1 in oncology.

Sense and serendipity aid RPE generation.

Retinal pigment epithelium (RPE) is a valuable cell type for a number of blinding disorders. In this issue of Cell Stem Cell, Idelson et al. (2009) use Nicotinamide and Activin A to markedly improve RPE yield from human embryonic stem cells.

Directed differentiation of human embryonic stem cells into functional retinal pigment epithelium cells.

Dysfunction and loss of retinal pigment epithelium (RPE) leads to degeneration of photoreceptors in age-related macular degeneration and subtypes of retinitis pigmentosa. Human embryonic stem cells (hESCs) may serve as an unlimited source of RPE cells for transplantation in these blinding conditions. Here we show the directed differentiation of hESCs toward an RPE fate under defined culture conditions. We demonstrate that nicotinamide promotes the differentiation of hESCs to neural and subsequently to RPE fate. In the presence of nicotinamide, factors from the TGF-beta superfamily, which presumably pattern RPE development during embryogenesis, further direct RPE differentiation. The hESC-derived pigmented cells exhibit the morphology, marker expression, and function of authentic RPE and rescue retinal structure and function after transplantation to an animal model of retinal degeneration caused by RPE dysfunction. These results are an important step toward the future use of hESCs to replenish RPE in blinding diseases.

Mechanical evaluation of mandibular defects reconstructed using osteogenic protein-1 (rhOP-1) in a sheep model: a critical analysis.

Osteoinductive bone morphogenetic proteins (BMPs) have been used extensively in experimental and clinical orthopaedic research. It is a natural progression for these growth regulators to be tested in the craniofacial region. The aim of this investigation was to analyse the mechanical properties of the sheep mandibles reconstructed using recombinant human osteogenic protein type 1 (rhOP-1). A unilateral 35 mm osteoperiosteal continuity defect was created at the parasymphyseal region of the mandible in six adult sheep. The animals were sacrificed 3 months after surgery and mechanical properties of the regenerated bone at the operated sides (OS) were compared to the corresponding bone at the non-operated side (NOS). The regenerated tissue at the OS were then submitted for histological and histomorphometric analysis. Although all the animals achieved complete bony union, a wide range of mechanical properties was found. The rhOP-1-induced bone achieved a mean of 36% of the strength of the bone at the NOS (P < 0.05). The mean value of the stiffness of the OS was 24% of the NOS (P < 0.05). While half of the samples of the OS had 'weak' mechanical properties (9-25% strength compared to NOS) and a low stiffness (6-18%), the rest showed relatively higher strength (47-63%) and were stiffer (35-47%). Unlike the NOS, the operated sides failed under tensile stresses and cracks initiated at the superior border of the mandible. The wide mechanical variations suggest that further basic bone biology research is needed to provide better understanding of the cellular and molecular events which take place during the process of osteoinduction.

Transforming the TGFbeta pathway: convergence of distinct lead generation strategies on a novel kinase pharmacophore for TbetaRI (ALK5).

The pathological activation of the transforming growth factor beta (TGFbeta) pathway plays a critical role in the progression of fibrotic diseases and also enhances tumor invasiveness and metastasis. Due to its central role in TGFbeta signaling, the TGFbeta type I receptor (TbetaRI) is emerging as an exciting target for blockade of the TGFbeta pathway. In this review we will discuss how three independent drug discovery strategies, ie, target-hopping, high-throughput screening and virtual screening, have converged in the identification of inhibitors of TalphaRI kinase. Structural studies have provided insight into the potency and selectivity of these inhibitors and form the basis for structure-based design optimization strategies. These efforts have enabled the production of potent, selective inhibitors for dissecting the TGFalpha pathway and assessing the usefulness of TalphaRI blockade in the treatment of fibrotic diseases and cancer.

Role of TGF-beta in cancer and the potential for therapy and prevention.

Transforming growth factor (TGF)-beta is a naturally occurring potent inhibitor of cell growth. TGF-beta binds first to a Type II (TGFBR2), then a Type I receptor (TGFBR1). TGFBR1 activation results in the phosphorylation of intracellular messengers, the SMADs. Unrestricted cell growth due to decreased growth inhibitory activity is a paramount feature of a defect in TGF-beta function. There is growing evidence that common variants of the TGF-beta pathway ligand and receptors that alter TGF-beta signaling modify cancer risk. Approximately 14% of the general population carry TGFBR1*6A, a variant of the TGFBR1 gene that results in decreased TGF-beta-mediated growth inhibition. Recent studies show that overall cancer risk is increased by 70 and 19% among TGFBR1*6A homozygotes and heterozygotes, respectively. This suggests that TGFBR1*6A may contribute to the development of a large proportion of common forms of cancer and may become a target for cancer chemoprevention. While decreased TGF-beta signaling increases cancer risk, TGF-beta secretion and activated TGF-beta signaling enhances the aggressiveness of several types of tumors. The activated TGF-beta signaling pathway is emerging as an attractive target in cancer and the authors predict that assessment of functionally relevant variants of this pathway will lead to the identification of individuals with a higher cancer risk and account for some forms of familial cancer susceptibility. In addition, it is predicted that inhibitors of the TGF-beta signaling pathway will find their way into cancer clinical trials, leading to delays in tumor progression and improvements in overall survival.

Blocking transforming growth factor-beta receptor signaling down-regulates transforming growth factor-beta1 autoproduction in keloid fibroblasts.

OBJECTIVE: To study transforming growth factor-beta1 (TGF-beta1) autoproduction in keloid fibroblasts and the regulation effect of blocking TGF-beta intracellular signaling on rhTGF-beta1 autoproduction. METHODS: Keloid fibroblasts cultured in vitro were treated with either rhTGF-beta1 (5 ng/ml) or recombinant adenovirus containing a truncated type II TGF-beta receptor gene (50 pfu/cell). Their effects of regulating gene expression of TGF-beta1 and its receptor I and II were observed with Northern blot. RESULTS: rhTGF-beta1 up-regulated the gene expression of TGF-beta1 and receptor I, but not receptor II. Over-expression of the truncated receptor II down-regulated the gene expression of TGF-beta1 and its receptor I, but not receptor II. CONCLUSIONS: TGF-beta1 autoproduction was observed in keloid fibroblasts. Over-expression of the truncated TGFbeta receptor II decreased TGF-beta1 autoproduction via blocking TGF-beta receptor signaling.

[Bone regeneration in oral surgery]. / Regeneración ósea en cirugía oral.

Bone regeneration is the technique of choice in order to repair small and medium sized bone defects in maxillary. It is used as an auxiliary medical treatment for dental implants. In this work, we show the different state of the art techniques used in bone lesions repair, and we also show experimental results obtained in our research group using bone morphogenetic protein BMP-2. This protein has been expressed in an E. Coli strain previously modified with the protein nucleotide sequence. We also show a carrier developed in our laboratory that is able to bind the rhBMP-2 and to liberate it in its active form. The mechanical properties of the carrier can be chemically modified. We have tested Ti implants covered with films of this material activated with rhBMP-2. The experimental in vivo results obtained with this kind of implants has been impressive.