Plasma-Soluble Biomarkers for Fibrodysplasia Ossificans Progressiva (FOP) Reflect Acute and Chronic Inflammatory States.

Fibrodysplasia ossificans progressiva (FOP) is a progressive, debilitating genetic disease in which skeletal muscle and connective tissue is episodically replaced by heterotopic bone. Discovery of surrogate biomarkers of disease (genotype)-related and flare-up-associated activity of FOP in a readily accessible matrix, such as plasma, would facilitate an understanding of the complex pathophysiology of FOP, aid patient care, and provide a valuable tool for the development and monitoring of potential therapeutics. In a case-control study, using a carefully collected and curated set of plasma samples from 40 FOP patients with the classic ACVR1R206H mutation and 40 age- and sex-matched controls, we report the identification of disease-related and flare-up-associated biomarkers of FOP using a multiplex analysis of 113 plasma-soluble analytes. Adiponectin (implicated in hypoxia, inflammation, and heterotopic ossification) as well as tenascin-C (an endogenous activator of innate immune signaling through the TLR4 pathway and a substrate for kallikrein-7) were highly correlated with FOP genotype, while kallikrein-7 was highly correlated with acute flare-up status. Plasma-soluble biomarkers for FOP support a flare-up-related acute inflammatory phase of disease activity superimposed on a genotypic background of chronic inflammation. © 2021 American Society for Bone and Mineral Research (ASBMR).

Heterogenetic parabiosis between healthy and dystrophic mice improve the histopathology in muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a progressive muscle disease, characterized by mutations in the X-linked dystrophin, that has several therapeutic options but no curative treatment. Transplantation of muscle progenitor cells for treatment of DMD has been widely investigated; however, its application is hindered by limited cell survival due to the harmful dystrophic microenvironment. An alternative approach to utilize progenitor cells and circulatory factors and to improve the dystrophic muscle pathology and microenvironment is through parabiotic pairing, where mice are surgically sutured to create a joint circulatory system. Parabiotic mice were generated by surgically joining wild type (WT) mice expressing green fluorescent protein (GFP) with mdx mice. These mice developed a common circulation (approximately 50% green cells in the blood of mdx mice) 2-weeks after parabiotic pairing. We observed significantly improved dystrophic muscle pathology, including decreased inflammation, necrotic fibers and fibrosis in heterogenetic parabionts. Importantly, the GFP + cells isolated from the mdx mice (paired with GFP mice) underwent myogenic differentiation in vitro and expressed markers of mesenchymal stem cells and macrophages, which may potentially be involved in the improvement of dystrophic muscle pathology. These observations suggest that changing the dystrophic microenvironment can be a new approach to treat DMD.

Cellular Hypoxia Promotes Heterotopic Ossification by Amplifying BMP Signaling.

Hypoxia and inflammation are implicated in the episodic induction of heterotopic endochondral ossification (HEO); however, the molecular mechanisms are unknown. HIF-1α integrates the cellular response to both hypoxia and inflammation and is a prime candidate for regulating HEO. We investigated the role of hypoxia and HIF-1α in fibrodysplasia ossificans progressiva (FOP), the most catastrophic form of HEO in humans. We found that HIF-1α increases the intensity and duration of canonical bone morphogenetic protein (BMP) signaling through Rabaptin 5 (RABEP1)-mediated retention of Activin A receptor, type I (ACVR1), a BMP receptor, in the endosomal compartment of hypoxic connective tissue progenitor cells from patients with FOP. We further show that early inflammatory FOP lesions in humans and in a mouse model are markedly hypoxic, and inhibition of HIF-1α by genetic or pharmacologic means restores canonical BMP signaling to normoxic levels in human FOP cells and profoundly reduces HEO in a constitutively active Acvr1(Q207D/+) mouse model of FOP. Thus, an inflammation and cellular oxygen-sensing mechanism that modulates intracellular retention of a mutant BMP receptor determines, in part, its pathologic activity in FOP. Our study provides critical insight into a previously unrecognized role of HIF-1α in the hypoxic amplification of BMP signaling and in the episodic induction of HEO in FOP and further identifies HIF-1α as a therapeutic target for FOP and perhaps nongenetic forms of HEO. © 2016 American Society for Bone and Mineral Research.

Circulating osteogenic precursor cells in heterotopic bone formation.

Cells with osteogenic potential can be found in a variety of tissues. Here we show that circulating osteogenic precursor (COP) cells, a bone marrow-derived type I collagen+/CD45+ subpopulation of mononuclear adherent cells, are present in early preosseous fibroproliferative lesions in patients with fibrodysplasia ossificans progressiva (FOP) and nucleate heterotopic ossification (HO) in a murine in vivo implantation assay. Blood samples from patients with FOP with active episodes of HO contain significantly higher numbers of clonally derived COP cell colonies than patients with stable disease or unaffected individuals. The highest level of COP cells was found in a patient just before the clinical onset of an HO exacerbation. Our studies show that even COP cells derived from an unaffected individual can contribute to HO in genetically susceptible host tissue. The possibility that circulating, hematopoietic-derived cells with osteogenic potential can seed inflammatory sites has tremendous implications and, to our knowledge, represents the first example of their involvement in clinical HO. Thus, bone formation is not limited to cells of the mesenchymal lineage, and circulating cells of hematopoietic origin can also serve as osteogenic precursors at remote sites of tissue inflammation.

Inhibition of p300/CBP by early B-cell factor.

Early B-cell factor (EBF) is a DNA binding protein required for early B-cell development. It activates transcription of several B-cell-specific genes, including the lambda5 gene, which encodes a protein necessary for signaling by the pre-B-cell receptor. In an effort to understand the mechanism by which EBF activates transcription, we examined its interaction with the coactivator protein p300/CBP. We found that two domains of EBF each bind the histone acetyltransferase (HAT)/CH3 domain of p300/CBP both in vitro and in vivo. Surprisingly, transcriptional activation by EBF was not sensitive to E1A, a potent p300/CBP inhibitor. In fact, overexpressed EBF mimicked E1A by severely repressing the activity of several other transcription factors, including E47, a protein that acts cooperatively with EBF to promote transcription of the lambda5 gene. This broad inhibitory profile correlated with EBF's ability to repress the HAT activity of p300/CBP in vivo and in vitro. However, such a repressed complex is not likely to form at the lambda5 promoter in vivo since (i) EBF could not bind p300/CBP and DNA simultaneously and (ii) the cooperativity imparted by E47 was sensitive to E1A. Our data reveal an intriguing inhibitory property of EBF-a property shared only by E1A, Twist, Pu.1, and the Hox family of homeodomain proteins-and suggest that E47 and EBF play distinct roles during lambda5 promoter activation.