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Loss of Notch signaling in skeletal stem cells enhances bone formation with aging

Remark, Lindsey H; Leclerc, Kevin; Ramsukh, Malissa; Lin, Ziyan; Lee, Sooyeon; Dharmalingam, Backialakshmi; Gillinov, Lauren; Nayak, Vasudev V; El Parente, Paulo; Sambon, Margaux; Atria, Pablo J; Ali, Mohamed A E; Witek, Lukasz; Castillo, Alesha B; Park, Christopher Y; Adams, Ralf H; Tsirigos, Aristotelis; Morgani, Sophie M; Leucht, Philipp
Skeletal stem and progenitor cells (SSPCs) perform bone maintenance and repair. With age, they produce fewer osteoblasts and more adipocytes leading to a loss of skeletal integrity. The molecular mechanisms that underlie this detrimental transformation are largely unknown. Single-cell RNA sequencing revealed that Notch signaling becomes elevated in SSPCs during aging. To examine the role of increased Notch activity, we deleted Nicastrin, an essential Notch pathway component, in SSPCs in vivo. Middle-aged conditional knockout mice displayed elevated SSPC osteo-lineage gene expression, increased trabecular bone mass, reduced bone marrow adiposity, and enhanced bone repair. Thus, Notch regulates SSPC cell fate decisions, and moderating Notch signaling ameliorates the skeletal aging phenotype, increasing bone mass even beyond that of young mice. Finally, we identified the transcription factor Ebf3 as a downstream mediator of Notch signaling in SSPCs that is dysregulated with aging, highlighting it as a promising therapeutic target to rejuvenate the aged skeleton.
PMID: 37752132
ISSN: 2095-4700
CID: 5608842

Skeletal adaptation to mechanical cues during homeostasis and repair: the niche, cells, and molecular signaling

Atria, Pablo J; Castillo, Alesha B
Bones constantly change and adapt to physical stress throughout a person's life. Mechanical signals are important regulators of bone remodeling and repair by activating skeletal stem and progenitor cells (SSPCs) to proliferate and differentiate into bone-forming osteoblasts using molecular signaling mechanisms not yet fully understood. SSPCs reside in a dynamic specialized microenvironment called the niche, where external signals integrate to influence cell maintenance, behavior and fate determination. The nature of the niche in bone, including its cellular and extracellular makeup and regulatory molecular signals, is not completely understood. The mechanisms by which the niche, with all of its components and complexity, is modulated by mechanical signals during homeostasis and repair are virtually unknown. This review summarizes the current view of the cells and signals involved in mechanical adaptation of bone during homeostasis and repair, with an emphasis on identifying novel targets for the prevention and treatment of age-related bone loss and hard-to-heal fractures.
PMID: 37916223
ISSN: 1664-042x
CID: 5606872

Bioactive, full-length parathyroid hormone delivered using an adeno-associated viral vector

Burr, Alexandra M; Zuckerman, Pamela Cabahug; Castillo, Alesha B; Partridge, Nicola C; Parekkadan, Biju
Delivering the parathyroid hormone (PTH) gene has been attempted preclinically in a handful of studies, but delivering full-length PTH (1-84) using adeno-associated viral (AAV) vectors has not. Given the difficulty in achieving therapeutic levels of secreted proteins using gene therapy, this study seeks to determine the feasibility of doing so with PTH. An AAV vector was used to deliver human PTH driven by a strong promoter. We demonstrate the ability to secrete full-length PTH from various cell types in vitro. PTH secretion from hepatocytes was measured over time and a fluorescent marker was used to compare the secretion rate of PTH in various cell types. Potency was measured by the ability of PTH to act on the PTH receptors of osteosarcoma cells and induced proliferation. PTH showed potency in vitro by inducing proliferation in two osteosarcoma cell lines. In vivo, AAV was administered systemically in immunocompromised mice which received xenografts of osteosarcoma cells. Animals that received the highest dose of AAV-PTH had higher liver and plasma concentrations of PTH. All dosing groups achieved measurable plasma concentrations of human PTH that were above the normal range. The high-dose group also had significantly larger tumors compared to control groups on the final day of the study. The tumors also showed dose-dependent differences in morphology. When looking at endocrine signaling and endogenous bone turnover, we observed a significant difference in tibial growth plate width in animals that received the high-dose AAV as well as dose-dependent changes in blood biomarkers related to PTH. This proof-of-concept study shows promise for further exploration of an AAV gene therapy to deliver full-length PTH for hypoparathyroidism. Additional investigation will determine efficacy in a disease model, but data shown establish bioactivity in well-established models of osteosarcoma.
PMID: 35666091
ISSN: 1535-3699
CID: 5248202

Mechanically-regulated bone repair

Anani, Tareq; Castillo, Alesha B
Fracture healing is a complex, multistep process that is highly sensitive to mechanical signaling. To optimize repair, surgeons prescribe immediate weight-bearing as-tolerated within 24 hours after surgical fixation; however, this recommendation is based on anecdotal evidence and assessment of bulk healing outcomes (e.g., callus size, bone volume, etc.). Given challenges in accurately characterizing the mechanical environment and the ever-changing properties of the regenerate, the principles governing mechanical regulation of repair, including their cell and molecular basis, are not yet well defined. However, the use of mechanobiological rodent models, and their relatively large genetic toolbox, combined with recent advances in imaging approaches and single-cell analyses is improving our understanding of the bone microenvironment in response to loading. This review describes the identification and characterization of distinct cell populations involved in bone healing and highlights the most recent findings on mechanical regulation of bone homeostasis and repair with an emphasis on osteo-angio coupling. A discussion on aging and its impact on bone mechanoresponsiveness emphasizes the need for novel mechanotherapeutics that can re-sensitize skeletal stem and progenitor cells to physical rehabilitation protocols.
PMID: 34624558
ISSN: 1873-2763
CID: 5110752

Single-cell RNA sequencing of CXCL12+ osteoprogenitors in mechanically loaded mouse tibiae reveals osteogenic mechanosensitive pathways [Meeting Abstract]

Cabahug-Zuckerman, P; Anani, T; Atria, P; Tikhonova, A; Khodadadi-Jamayran, A; Segal, N; Aifantis, I; Castillo, A
Mechanical loading (ML) is a potent anabolic stimulus in healthy adult bone [1]. A better understanding of cell and molecular processes in load-induced osteogenesis, including underlying mechanosensitive pathways, could yield effective treatment strategies for aged and diseased bone. Cortical bone (CB) osteocytes (OCYs) originate from LepR+ cells, of which 98.8% co-express CXCL12 [2]. Given that skeletal stem cells (SSCs) express an array of overlapping markers, including LepR and CXCL12 [2-6], we sought to determine how these distinct SSC populations respond to ML with regard to their number and gene expression profiles at the single cell level. We hypothesized that ML leads to an expansion of CXCL12+ and LepR+ cell populations and regulates their fate. Following NYU IACUC approval, adult Cxcl12tm2.1Sjm/J dsRed reporter mice (N=19) and C57BL/6 (C57, N=6) mice (Jackson Labs) were subjected to 4 daily bouts of tibial axial compressive loading (L) (6N,2Hz,120cycles) with appropriate non-loaded (NL) controls. Bone marrow (BM) and CB cell suspensions from L and NL tibiae were prepared for FACS and single-cell RNAseq (10XGenomics) as previously described [5]. FACS data are presented as %change and significance determined by a Student's T-test at alpha=0.05; expression data are presented as normalized fold-change. The enriched CXCL12+ cell population was shown to highly express LepR. ML led to a significant increase in the number of LepR+ cells (+114%, p=0.022) in the BM. Differentially expressed genes in the L versus NL CXCL12-dsRed+ cells included upregulated osteogenic genes Wnt4 (1.32X, p<0.0001) and BMP4 (1.72X, p<0.0001), and downregulated adipo-associated genes PPARgamma (0.79X, p=0.037) and Apoe (0.92X, p<0.001). Unbiased principle component analysis (PCA) yielded 11 cell clusters, including reticular cells [2,5,6] and pre-osteoblasts [5,6]. Loading resulted in a significant increase in BMP4 (2.7X, p=0.002) and a significant decrease in sFRP expression (0.55X, p<0.0001), 10 a negative regulator of Wnt signaling [7], in reticular cells. A significant increase in Wnt4 (1.9X, p<0.0001) was also observed in pre-osteoblasts. Our data demonstrate that loading promotes osteogenic differentiation via promotion of Wnt signaling, consistent with previous reports [8-10] while attenuating pro-adipogenic genes in cells expressing both LepR and CXCL12, which are known osteoprogenitors [2]; that is, loading effectively pushes progenitors towards an osteogenic fate
ISSN: 1523-4681
CID: 4774352

Cells involved in mechanotransduction including mesenchymal stem cells

Chapter by: Cabahug-Zuckerman, Pamela; Liu, Chao; Castillo, Alesha B.
in: Encyclopedia of Bone Biology by
[S.l.] : Elsevier, 2020
pp. 311-332
ISBN: 9780128140826
CID: 5057062

Site-Specific Load-Induced Expansion of Sca-1+Prrx1+ and Sca-1-Prrx1+ Cells in Adult Mouse Long Bone Is Attenuated With Age

Cabahug-Zuckerman, Pamela; Liu, Chao; Cai, Cinyee; Mahaffey, Ian; Norman, Stephanie C; Cole, Whitney; Castillo, Alesha B
Aging is associated with significant bone loss and increased fracture risk, which has been attributed to a diminished response to anabolic mechanical loading. In adults, skeletal progenitors proliferate and differentiate into bone-forming osteoblasts in response to increasing mechanical stimuli, though the effects of aging on this response are not well-understood. Here we show that both adult and aged mice exhibit load-induced periosteal bone formation, though the response is significantly attenuated with age. We also show that the acute response of adult bone to loading involves expansion of Sca-1+Prrx1+ and Sca-1-Prrx1+ cells in the periosteum. On the endosteal surface, loading enhances proliferation of both these cell populations, though the response is delayed by 2 days relative to the periosteal surface. In contrast to the periosteum and endosteum, the marrow does not exhibit increased proliferation of Sca-1+Prrx1+ cells, but only of Sca-1-Prrx1+ cells, underscoring fundamental differences in how the stem cell niche in distinct bone envelopes respond to mechanical stimuli. Notably, the proliferative response to loading is absent in aged bone even though there are similar baseline numbers of Prrx1 + cells in the periosteum and endosteum, suggesting that the proliferative capacity of progenitors is attenuated with age, and proliferation of the Sca-1+Prrx1+ population is critical for load-induced periosteal bone formation. These findings provide a basis for the development of novel therapeutics targeting these cell populations to enhance osteogenesis for overcoming age-related bone loss. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.
PMID: 31667455
ISSN: 2473-4039
CID: 4163372

Mechanical loading promotes the expansion of primitive osteoprogenitors and organizes matrix and vascular morphology in long bone defects

Liu, Chao; Cabahug-Zuckerman, Pamela; Stubbs, Christopher; Pendola, Martin; Cai, Cinyee; Mann, Kenneth A; Castillo, Alesha B
Elucidating the effects of mechanical stimulation on bone repair is crucial for optimization of the healing process. Specifically, the regulatory role that mechanical loading exerts on the osteogenic stem cell pool and vascular morphology during healing is incompletely understood. As dynamic loading has been shown to enhance osteogenesis and repair, we hypothesized that loading induces the expansion of the osteoprogenitor cell population within a healing bone defect, leading to an increased presence of osteogenic cells. We further hypothesized that loading during the repair process regulates vascular and collagen matrix morphology and spatial interactions between vessels and osteogenic cells. To address these hypotheses, we used a mechanobiological bone repair model, which produces a consistent and reproducible intramembranous repair response confined in time and space. Bilateral tibial defects were created in adult C57BL/6 mice, which were subjected to axial compressive dynamic loading either during the early cellular invasion phase on post-surgical days (PSD) 2-5 or during the matrix deposition phase on PSD 5-8. Confocal and two-photon microscopy was used to generate high-resolution 3D renderings of longitudinal thick sections of the defect on PSD 10. Endomucin (EMCN)-positive vessels, Prrx1+ Sca-1+ primitive osteoprogenitors (OPCs), and Osx+ preosteoblasts were visualized and quantified using deep tissue immunohistochemistry. New bone matrix was visualized with second harmonic generation autofluorescence of collagen fibers. We found that mechanical loading during the matrix deposition phase (PSD 5-8) increased vessel volume and number, and aligned vessels and collagen fibers to the load-bearing direction of bone. Furthermore, loading led to a significant increase in the proliferation and number of Prrx1+ Sca-1+ primitive OPCs, but not Osx+ preosteoblasts within the defect. Together, these data illustrate the adaptation of both collagen matrix and vascular morphology to better withstand mechanical load during bone repair, and that the mechanoresponsive cell population consists of the primitive osteogenic progenitors.
PMID: 30645780
ISSN: 1523-4681
CID: 3627192

Author Correction: WNT-activated bone grafts repair osteonecrotic lesions in aged animals [Correction]

Salmon, B; Liu, B; Shen, E; Chen, T; Li, J; Gillette, M; Ransom, R C; Ezran, M; Johnson, C A; Castillo, A B; Shen, W J; Kraemer, F B; Smith, A A; Helms, J A
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.
PMID: 29662149
ISSN: 2045-2322
CID: 3041102

Targeting Osteogenesis-Angiogenesis Coupling for Bone Repair

Liu, Chao; Castillo, Alesha B
PMID: 29489598
ISSN: 1940-5480
CID: 2965932