Pignolo, Robert J.

ABSTRACT Fibrodysplasia ossificans progressiva (FOP) is an ultra-rare genetic disorder characterized by progressive heterotopic ossification (HO), often heralded by flare-ups, leading to reduced movement and life expectancy. This placebo-controlled, double-blind trial (NCT02190747) evaluated palovarotene, an orally bioavailable selective retinoic acid receptor gamma agonist, for prevention of HO in patients with FOP. Patients experiencing a flare-up were enrolled in two cohorts: (1) patients ≥15 years were randomized 3:1 to palovarotene 10/5 mg (weeks 1–2/3–6) or placebo; (2) patients ≥6 years were randomized 3:3:2 to palovarotene 10/5 mg, palovarotene 5/2.5 mg (weeks 1–2/3–6), or placebo. Cohort data were pooled. The primary endpoint was the proportion of responders (no/minimal new HO at flare-up body region by plain radiograph) at week 6. Change from baseline in HO volume and new HO incidence were assessed by computed tomography (CT) at week 12. Tissue edema was assessed by magnetic resonance imaging (MRI) or ultrasound. Forty patients (aged 7–53 years) were enrolled (placebo: n = 10; palovarotene 5/2.5 mg: n = 9; palovarotene 10/5 mg: n = 21). Disease history was similar between groups. In the per-protocol population, the proportion of responders at week 6 by plain radiograph was 100% with palovarotene 10/5 mg; 88.9% with palovarotene 5/2.5 mg; 88.9% with placebo (Cochran-Armitage trend test: p = 0.17). At week 12, the proportions were 95.0% with palovarotene 10/5 mg; 88.9% with palovarotene 5/2.5 mg; 77.8% with placebo (Cochran-Armitage trend test: p = 0.15). Week 12 least-squares mean (LSmean) new HO volume, assessed by CT, was 3.8 × 103 mm3 with palovarotene 10/5 mg; 1.3 × 103 mm3 with palovarotene 5/2.5 mg; 18.0 × 103 mm3 with placebo (pairwise tests versus placebo: p ≤ 0.12). Palovarotene was well-tolerated. No patients discontinued treatment or required dose reduction; one patient had dose interruption due to elevated lipase. Although these findings were not statistically significant, they support further evaluation of palovarotene for prevention of HO in FOP in larger studies. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Skeletal muscle atrophy is commonly associated with aging, immobilization, muscle unloading, and congenital myopathies. Generation of mature muscle cells from skeletal muscle satellite cells (SCs) is pivotal in repairing muscle tissue. Exercise therapy promotes muscle hypertrophy and strength. Primary cilium is implicated as the mechanical sensor in some mammalian cells, but its role in skeletal muscle cells remains vague. To determine mechanical sensors for exercise-induced muscle hypertrophy, we established three SC-specific cilium dysfunctional mouse models— Myogenic factor 5 ( Myf5 ) -Arf-like Protein 3 ( Arl3 ) −/− , Paired box protein Pax-7 ( Pax7 )- Intraflagellar transport protein 88 homolog ( Ift88 ) −/− , and Pax7-Arl3 −/− —by specifically deleting a ciliary protein ARL3 in MYF5-expressing SCs, or IFT88 in PAX7-expressing SCs, or ARL3 in PAX7-expressing SCs, respectively. We show that the Myf5-Arl3 −/− mice develop grossly the same as WT mice. Intriguingly, mechanical stimulation-induced muscle hypertrophy or myoblast differentiation is abrogated in Myf5-Arl3 −/− and Pax7-Arl3 −/− mice or primary isolated Myf5-Arl3 −/− and Pax7-Ift88 −/− myoblasts, likely due to defective cilia-mediated Hedgehog (Hh) signaling. Collectively, we demonstrate SC cilia serve as mechanical sensors and promote exercise-induced muscle hypertrophy via Hh signaling pathway.

Fibrodysplasia ossificans progressiva (FOP) is a rare and devastating genetic disease, in which soft connective tissue is converted into heterotopic bone through an endochondral ossification process. Patients succumb early as they gradually become trapped in a second skeleton of heterotopic bone. Although the underlying genetic defect is long known, the inherent complexity of the disease has hindered the discovery of effective preventions and treatments. New developments in the gene therapy field have motivated its consideration as an attractive therapeutic option for FOP. However, the immune system's role in FOP activation and the as-yet unknown primary causative cell, are crucial issues which must be taken into account in the therapy design. While gene therapy offers a potential therapeutic solution, more knowledge about FOP is needed to enable its optimal and safe application.

Abstract Cellular senescence, which is a major cause of tissue dysfunction with aging and multiple other conditions, is known to be triggered by p16 Ink4a or p21 Cip1 , but the relative contributions of each pathway toward inducing senescence are unclear. Here, we directly addressed this issue by first developing and validating a p21 ‐ ATTAC mouse with the p21 Cip1 promoter driving a “suicide” transgene encoding an inducible caspase‐8 which, upon induction, selectively kills p21 Cip1 ‐expressing senescent cells. Next, we used the p21 ‐ ATTAC mouse and the established p16 ‐ INK ‐ ATTAC mouse to directly compare the contributions of p21 Cip1 versus p16 Ink4a in driving cellular senescence in a condition where a tissue phenotype (bone loss and increased marrow adiposity) is clearly driven by cellular senescence—specifically, radiation‐induced osteoporosis. Using RNA in situ hybridization, we confirmed the reduction in radiation‐induced p21 Cip1 ‐ or p16 Ink4a ‐driven transcripts following senescent cell clearance in both models. However, only clearance of p21 Cip1 +, but not p16 Ink4a +, senescent cells prevented both radiation‐induced osteoporosis and increased marrow adiposity. Reduction in senescent cells with dysfunctional telomeres following clearance of p21 Cip1 +, but not p16 Ink4a +, senescent cells also reduced several of the radiation‐induced pro‐inflammatory senescence‐associated secretory phenotype factors. Thus, by directly comparing senescent cell clearance using two parallel genetic models, we demonstrate that radiation‐induced osteoporosis is driven predominantly by p21 Cip1 ‐ rather than p16 Ink4a ‐mediated cellular senescence. Further, this approach can be used to dissect the contributions of these pathways in other senescence‐associated conditions, including aging across tissues.

ABSTRACT Oxidative stress-induced reactive oxygen species, DNA damage, apoptosis, and cellular senescence have been associated with reduced osteoprogenitors in a reciprocal fashion to bone marrow adipocyte tissue (BMAT); however, a direct (causal) link between cellular senescence and BMAT is still elusive. Accumulation of senescent cells occur in naturally aged and in focally radiated bone tissue, but despite amelioration of age- and radiation-associated bone loss after senescent cell clearance, molecular events that precede BMAT accrual are largely unknown. Here we show by RNA-Sequencing data that BMAT-related genes were the most upregulated gene subset in radiated bones of C57BL/6 mice. Using focal radiation as a model to understand age-associated changes in bone, we performed a longitudinal assessment of cellular senescence and BMAT. Using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), RNA in situ hybridization of p21 transcripts and histological assessment of telomere dysfunction as a marker of senescence, we observed an increase in senescent cell burden of bone cells from day 1 postradiation, without the presence of BMAT. BMAT was significantly elevated in radiated bones at day 7, confirming the qRT-PCR data in which most BMAT-related genes were elevated by day 7, and the trend continued until day 42 postradiation. Similarly, elevation in BMAT-related genes was observed in bones of aged mice. The senolytic cocktail of Dasatinib (D) plus Quercetin (Q) (ie, D + Q), which clears senescent cells, reduced BMAT in aged and radiated bones. MicroRNAs (miRNAs or miRs) linked with senescence marker p21 were downregulated in radiated and aged bones, whereas miR-27a, a miR that is associated with increased BMAT, was elevated both in radiated and aged bones. D + Q downregulated miR-27a in radiated bones at 42 days postradiation. Overall, our study provides evidence that BMAT occurrence in oxidatively stressed bone environments, such as radiation and aging, is induced following a common pathway and is dependent on the presence of senescent cells. © 2022 American Society for Bone and Mineral Research (ASBMR). Abstract Schematic representing a senescent bone environment during aging and radiation. The schematic represents changes in an oxidatively stressed, senescent microenvironment in which mesenchymal progenitors are preferentially forming adipocytes, regulated by a common process shared between aging bone and radiation-induced bone damage, and with identical expression patterns of bone marrow adipocyte tissue (BMAT)-related genes and microRNAs (miRNAs or miRs). Increase in senescent markers p21 and p16Ink4a induce a cellular state in which production of the senescence-associated secretory phenotype (SASP) increases and influences the bone marrow environment. Secreted adipokines can promote mesenchymal stem cell (MSC) fate, switching to an adipocyte lineage. Several adipokines are also released in circulation, causing systemic effects. These changes in senescence markers and genes that regulate BMAT are in turn regulated by their corresponding miRNAs. These changes can be blocked or reversed by the clearance of senescent cells using senolytic drugs. The figure was created with BioRender.com.

ABSTRACT Fibrodysplasia ossificans progressiva (FOP) is a rare disease characterized by inflammatory flares of soft tissues, leading to heterotopic ossification and significant cumulative morbidity and early mortality. FOP minor trauma, including intramuscular medication administration, can induce ossification and should be avoided. We present a case of known FOP in a patient who presented with fevers of unknown origin and was found to have biopsy-proven ileal Crohn's disease. Crohn's disease management was complicated by concerns that intramuscular therapies would induce ossification, and oral monotherapy with methotrexate was initiated with excellent results.

ABSTRACT 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).

Abstract Circulating osteogenic progenitor (COP) cells are a population of cells in the peripheral blood with the capacity for bone formation, as well as broader differentiation into mesoderm-like cells in vitro. There are several pathologies of accelerated bone formation and physiological responses to injury in which COP cells have been theorized to play a role. These include fracture, vascular calcification, and subtypes of heterotopic ossification (HO). Overall, the available studies suggest COP cells are likely to be mobilized in response to fracture, home to the site of injury, undergo a maturation process, and contribute to the osteogenesis and angiogenesis required for fracture healing. HO is the pathological process of bone formation in nonskeletal tissue and can be acquired or hereditary. COP cells may seed sites of injury and inflammation that precede the formation of endochondral bone identified in both genetic and nongenetic forms of HO. Vascular calcification is a common occurrence in older adults and is strongly associated with poorer cardiovascular health outcomes. It appears that COP cells, particularly those expressing hematopoietic and vascular markers such as CD45 and CD34, contribute to the calcification and ossification of atherosclerotic plaques and aortic valves, and that they correlate to the severity of the calcification. Whether COP cells are attracted to sites of injury and inflammation and so are highly associated with fracture, vascular calcification/ossification and HO, or whether they underlie these processes at a more mechanistic level, remains to be more clearly demonstrated.

Abstract Although cellular senescence is increasingly recognized as driving multiple age-related co-morbidities through the senescence-associated secretory phenotype (SASP), in vivo senescent cell identification, particularly in bulk or single cell RNA-sequencing (scRNA-seq) data remains challenging. Here, we generated a novel gene set (SenMayo) and first validated its enrichment in bone biopsies from two aged human cohorts. SenMayo also identified senescent cells in aged murine brain tissue, demonstrating applicability across tissues and species. For direct validation, we demonstrated significant reductions in SenMayo in bone following genetic clearance of senescent cells in mice, with similar findings in adipose tissue from humans in a pilot study of pharmacological senescent cell clearance. In direct comparisons, SenMayo outperformed all six existing senescence/SASP gene sets in identifying senescent cells across tissues and in demonstrating responses to senescent cell clearance. We next used SenMayo to identify senescent hematopoietic or mesenchymal cells at the single cell level from publicly available human and murine bone marrow/bone scRNA-seq data and identified monocytic and osteolineage cells, respectively, as showing the highest levels of senescence/SASP genes. Using pseudotime and cellular communication patterns, we found senescent hematopoietic and mesenchymal cells communicated with other cells through common pathways, including the Macrophage Migration Inhibitory Factor (MIF) pathway, which has been implicated not only in inflammation but also in immune evasion, an important property of senescent cells. Thus, SenMayo identifies senescent cells across tissues and species with high fidelity. Moreover, using this senescence panel, we were able to characterize senescent cells at the single cell level and identify key intercellular signaling pathways associated with these cells, which may be particularly useful for evolving efforts to map senescent cells (e.g., SenNet). In addition, SenMayo represents a potentially clinically applicable panel for monitoring senescent cell burden with aging and other conditions as well as in studies of senolytic drugs.

Abstract Objective The objective was to derive a risk score that uses variables available early during the emergency department (ED) encounter to identify high‐risk geriatric patients who may benefit from delirium screening. Methods This was an observational study of older adults age ≥ 75 years who presented to an academic ED and who were screened for delirium during their ED visit. Variable selection from candidate predictors was performed through a LASSO‐penalized logistic regression. A risk score was derived from the final prediction model, and predictive accuracy characteristics were calculated with 95% confidence intervals (CIs). Results From the 967 eligible ED visits, delirium was detected in 107 (11.1%). The area under the curve for the REcognizing DElirium in Emergency Medicine (REDEEM) score was 0.901 (95% CI = 0.864–0.938). The REEDEM risk score included 10 different variables (seven based on triage information and three obtained during early history taking) with a score ranging from –3 to 66. Using an optimal cutoff of ≥11, we found a sensitivity of 84.1% (90 of 107 ED delirium patients, 95% CI = 75.5%–90.2%) and a specificity of 86.6% (745 of 860 non‐ED delirium patients, 95% CI = 84.1%–88.8%). A lower cutoff of ≥5 was found to minimize false negatives with an improved sensitivity at 91.6% (98 of 107 ED delirium patients, 95% CI = 84.2%–95.8%). Conclusion A risk stratification score was derived with the potential to augment delirium recognition in geriatric ED patients. This has the potential to assist on delirium‐targeted screening of high‐risk patients in the ED. Validation of REDEEM, however, is needed prior to implementation.