The lymphatic system, composed of capillary lymphatics, collecting vessels, and ducts lined by lymphatic endothelial cells (LECs), performs fluid homeostasis and immune surveillance. Although lymphatics are abundant in organs such as skin, heart, lung, and intestine, whether they exist in bone has been debated for decades. The traditional view holds that healthy bone lacks lymphatics, yet a recent study using whole-bone immunolabeling with LEC markers and light-sheet microscopy claimed their presence and involvement in bone regeneration.
In a study published in Cell, the research team led by Prof. ZHOU Bin from the Center for Excellence in Molecular Cell Science (Shanghai Institute of Biochemistry and Cell Biology) of the Chinese Academy of Sciences, in collaboration with Prof. Ralf H. Adams from the Max Planck Institute for Molecular Biomedicine, Germany, reported the absence of lymphatic vessels in homeostasis and regenerating bone and their abnormal invasion under pathological conditions by using dual-recombinase-mediated genetic strategies.
Previous studies relied on single markers such as LYVE1(lymphatic vessel endothelial hyaluronan receptor 1), PDPN (podoplanin), and PROX1 (prospero-related homeobox 1) to identify LECs in bone. However, these markers lack cell lineage specificity: LYVE1 also labels immune cells, PDPN labels mesenchymal cells, and PROX1 labels neurons, cardiomyocytes, hepatocytes, and skeletal myocytes. In this study, the researchers found that conventional single-recombinase-mediated lineage tracing tools suffer from non-specific labeling, which greatly interferes with lineage tracing results. Re-analysis of single-cell RNA sequencing data further confirmed that most PROX1+ cells lacked Cdh5 expression, indicating contamination from non-endothelial lineages.
To overcome these limitations, the researchers developed a dual-recombinase (Cre-loxP and Dre-rox) system, LEC-iCre (Cdh5-Dre;Prox1-RSR-CreER), which restricts labeling to cells co-expressing Cdh5 (vascular endothelial marker) and PROX1, greatly improving labeling accuracy and specificity. Using this system, they found that under homeostasis, LECs were exclusively localized to the periosteal fibrous layer and surrounding connective tissues, and were absent from trabecular bone, cortical bone, and bone marrow.
Following irradiation injury, LECs remained restricted to the periosteum and did not invade bone at various stages after transplantation. After fracture injury, although LECs expanded significantly in the periosteum and callus, they never invaded bone tissue. These results demonstrate that during post-injury bone regeneration, lymphatic vessels do not enter bone to participate in repair.
In contrast, in GLA/GSD mouse models carrying the Pik3caH1047R activating mutation, the researchers captured the entire process of lymphatic invasion. Using the dual-recombinase system for LEC-specific expression of the mutant gene, they observed that periosteal LECs proliferated, sequentially breached the periosteum, eroded cortical bone, and ultimately invaded the bone marrow. This finding demonstrates that intraosseous lymphatics in GLA/GSD originate from active invasion of extraosseous lymphatics, providing direct genetic evidence for the pathogenesis of these diseases.
This study resolves the long-standing controversy regarding the existence of lymphatic vessels in bone and provides a clear pathological mechanism for GLA and GSD, and the newly developed lineage tracing strategies provide new technical methods for studying lymphatic vessel function, related regulatory mechanisms, and lymphatic-associated diseases.
Reference: https://www.cell.com/cell/fulltext/S0092-8674(26)00639-2
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