Leptin receptor (LepR)-positive cells are key components of the bone marrow hematopoietic microenvironment, and highly enrich skeletal stem and progenitor cells that maintain homeostasis of the adult skeleton. However, the heterogeneity and lineage hierarchy within this population has been elusive. Using genetic lineage tracing and single-cell RNA sequencing, we found that Lepr-Cre labels most bone marrow stromal cells and osteogenic lineage cells in adult long bones. Integrated analysis of Lepr-Cre-traced cells under homeostatic and stress conditions revealed dynamic changes of the adipogenic, osteogenic, and periosteal lineages. Importantly, we discovered a Notch3 + bone marrow sub-population that is slow-cycling and closely associated with the vasculatures, as well as key transcriptional networks promoting osteo-chondrogenic differentiation. We also identified a Sca-1 + periosteal sub-population with high clonogenic activity but limited osteo-chondrogenic potential. Together, we mapped the transcriptomic landscape of adult LepR + stem and progenitor cells and uncovered cellular and molecular mechanisms underlying their maintenance and lineage specification.

Highlights d Osteolectin interacts with Fap and inhibits its protease activity d Genetic deletion of Fap ameliorates bone loss in aged mice d Pharmacological inhibition of Fap attenuates ovariectomyinduced osteoporosis d Osteolectin inhibits Fap to promote mineralization during zebrafish development

RATIONALE: Myocardial infarction (MI) elicits cardiac fibroblast activation and ECM (extracellular matrix) deposition to maintain the structural integrity of the heart. Recent studies demonstrate that Fap (fibroblast activation protein)—a prolyl-specific serine protease—is an important marker of activated cardiac fibroblasts after MI. OBJECTIVE: This study aims to test whether Fap is a critical regulator of cardiac repair after MI and to uncover the underlying cellular and molecular mechanisms for better treatment of MI. METHODS AND RESULTS: We found that Fap is upregulated in patient cardiac fibroblasts after cardiac injuries, while plasma Fap is downregulated and functions as a prognostic marker for cardiac repair. Genetic or pharmacological inhibition of Fap in mice significantly improved cardiac function after MI. Histological and transcriptomic analyses showed that Fap inhibition leads to increased angiogenesis in the peri-infarct zone, which promotes ECM deposition and alignment by cardiac fibroblasts and prevents their overactivation, thereby limiting scar expansion. Mechanistically, we found that BNP (brain natriuretic peptide) is a novel substrate of Fap that mediates postischemic angiogenesis. Fap degrades BNP to inhibit vascular endothelial cell migration and tube formation. Pharmacological inhibition of Fap in Nppb (encoding pre-proBNP) or Npr1 (encoding the BNP receptor)-deficient mice showed no cardioprotective effects, suggesting that BNP is a physiological substrate of Fap. CONCLUSIONS: This study identifies Fap as a negative regulator of cardiac repair and a potential drug target to treat MI. Inhibition of Fap stabilizes BNP to promote angiogenesis and cardiac repair.

Unlike long bones, jawbone development is mainly accomplished by intramembranous ossification resulting from the differentiation of periosteal progenitor cells. However, the spatiotemporal ontogeny of periosteal progenitor cells during jawbone development and repair remains elusive. In this study, we mapped the transcriptional landscape of the human jawbone periosteum at single-cell resolution and identified a cathepsin K (Ctsk)+ periosteal subset. Lineage tracing analysis indicated that Ctsk-Cre–labeled periosteal cells could make contributions to jawbone development. However, different from the periosteal-specific location of Ctsk+ cells in long bone, we also identified Ctsk+ stromal cells in jawbone marrow and implied the heterogeneity of jawbone Ctsk+ hierarchy. In further analysis of the periosteal progenitor cell subset of heterogeneous Ctsk+ hierarchy, we identified a unique Ctsk+Ly6a+ subset of cells. The additional marker Ly6a helped to further confine the progenitor subset to the jawbone periosteum and was nearly undetectable in the bone marrow. Defects in the jawbone could activate the migration and osteogenic differentiation of Ctsk+Ly6a+ cells. Local ablation of Ctsk+ cells by diphtheria reduced the number of Ctsk+Ly6a+ cells and delayed the repair of the bone defect. Taken together, we identify a novel periosteal osteogenic progenitor subset that is active in jawbone osteogenesis and healing.

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Fibroblast activation protein (Fap) is a serine protease that degrades denatured type I collagen, α2-antiplasmin and FGF21. Fap is highly expressed in bone marrow stromal cells and functions as an osteogenic suppressor and can be inhibited by the bone growth factor Osteolectin (Oln). Fap is also expressed in synovial fibroblasts and positively correlated with the severity of rheumatoid arthritis (RA). However, whether Fap plays a critical role in osteoarthritis (OA) remains poorly understood. Here, we found that Fap is significantly elevated in osteoarthritic synovium, while the genetic deletion or pharmacological inhibition of Fap significantly ameliorated posttraumatic OA in mice. Mechanistically, we found that Fap degrades denatured type II collagen (Col II) and Mmp13-cleaved native Col II. Intra-articular injection of rFap significantly accelerated Col II degradation and OA progression. In contrast, Oln is expressed in the superficial layer of articular cartilage and is significantly downregulated in OA. Genetic deletion of Oln significantly exacerbated OA progression, which was partially rescued by Fap deletion or inhibition. Intra-articular injection of rOln significantly ameliorated OA progression. Taken together, these findings identify Fap as a critical pathogenic factor in OA that could be targeted by both synthetic and endogenous inhibitors to ameliorate articular cartilage degradation.

A distinct population of skeletal stem/progenitor cells (SSPCs) has been identified that is indispensable for the maintenance and remodeling of the adult skeleton. However, the cell types that are responsible for age-related bone loss and the characteristic changes in these cells during aging remain to be determined. Here, we established models of premature aging by conditional depletion of Zmpste24 (Z24) in mice and found that Prx1-dependent Z24 deletion, but not Osx-dependent Z24 deletion, caused significant bone loss. However, Acan-associated Z24 depletion caused only trabecular bone loss. Single-cell RNA sequencing (scRNA-seq) revealed that two populations of SSPCs, one that differentiates into trabecular bone cells and another that differentiates into cortical bone cells, were significantly decreased in Prx1-Cre; Z24f/f mice. Both premature SSPC populations exhibited apoptotic signaling pathway activation and decreased mechanosensation. Physical exercise reversed the effects of Z24 depletion on cellular apoptosis, extracellular matrix expression and bone mass. This study identified two populations of SSPCs that are responsible for premature aging-related bone loss. The impairment of mechanosensation in Z24-deficient SSPCs provides new insight into how physical exercise can be used to prevent bone aging.