Procr+ Endothelial Progenitor Cells Modulate Adult Hematopoiesis and Microenvironment Homeostasis Via Notch Signaling

The Hematopoietic Stem Cell (HSC) microenvironment comprises diverse cell types with varied functions found in the endosteum and vasculature. Previous studies have shown that Protein C Receptor (Procr) serves as a marker for various multipotent stem cells, such as mammary epithelial progenitor cells, hematopoietic stem cells, and embryonic vascular endothelial cells. However, the precise role of Procr in the bone marrow niche and its regulatory mechanisms in adult hematopoiesis remain largely unknown.

In this study, we employed a genetically modified mouse model with specific reporter to trace Procr expression and investigate its role in both bone marrow and splenic hematopoietic tissues. We found that early-traced Procr⁺ cells were Type H-like endothelial cells (ECs) in the metaphysis. Further extended tracking revealed the proliferation of Procr-positive cells into various subtypes of vascular endothelial cells. Moreover, following irradiation injury, Procr⁺ ECs in the bone marrow and spleen rapidly expanded by around tenfold, thereby supporting vascular integrity. Single-cell RNA sequencing revealed that Procr⁺ ECs facilitated leukocyte activation and osteogenesis. Additionally, Procr⁺ ECs displayed increased levels of stemness genes, including CD34 and Kitl. Notably, the depletion of Procr in ECs or Vegfr2 in Procr⁺ ECs resulted in impairments in hematopoietic function. Procr depletion caused a significant reduction in the total number of bone marrow cells, while SLAM⁺ HSCs exhibited an abnormal expansion of approximately 21-fold. This significantly compromised the repopulation ability and self-renewal capacity of HSCs, promoting differentiation towards the myeloid lineage and negatively impacting their colony-forming abilities. Subsequently, we observed that the depletion of Procr in ECs significantly increased the number of Type H ECs while reducing the number of Type L vessels. This alteration was accompanied by impaired vascular regeneration functions and a decreased capacity for in vitro tube formation. Furthermore, the ablation of endothelial Procr leads to an increased trabecular number and bone density, along with a decrease in bone mesenchymal stem cells (BMSCs) and enhanced differentiation into adipocytes. This suggests that endothelial Procr plays a crucial role in regulating bone homeostasisFurther analysis of single-cell RNA sequencing data revealed a significant decrease in the Notch signalling pathway associated with Procr deficiency. Notably, the Delta-like 4 (Dll4)-Notch3 ligand-receptor interaction between ECs and BMSCs emerged as a critical regulatory mechanism. Dll4, identified as a key target gene, plays a pivotal role in modulating the effects of Procr deficiency on cellular signalling processes. The downregulation of Procr in HUVECs confirmed the decreased expression of Dll4. Finally, we demonstrated that a Notch pathway agonist completely alleviated the hematopoietic and microenvironmental changes caused by Procr knockout. This suggests that the upregulation of endothelial Dll4 likely mediates the maintenance of bone marrow homeostasis and hematopoiesis following Procr ablation.

In summary, we identified a novel population of endothelial progenitor cells that regulates both vascular and microenvironmental homeostasis, as well as HSC function. Our research reveals that Procr serves not only as a marker but also as a regulator of the Dll4-Notch3 pathway, which is essential for maintaining vascular integrity. These findings enhance our understanding of Procr, highlighting its significant roles in bone marrow homeostasis and hematopoiesis.