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1328 Vascular Cell Adhesion Molecule-1 Regulates Bone Marrow Mesenchymal Stem Cell Maintenance and Niche Function

Program: Oral and Poster Abstracts
Session: 506. Bone Marrow Microenvironment: Poster I
Hematology Disease Topics & Pathways:
Research, Translational Research, hematopoiesis, Biological Processes
Saturday, December 9, 2023, 5:30 PM-7:30 PM

Anna M Di Staulo1*, Charles E Ayemoba1*, Aydolun Petenkaya2*, Sen Zhang, PhD1*, Maria Maryanovich, PhD3*, Konstantinos Chronis, PhD2* and Sandra Pinho, PhD1

1Department of Pharmacology & Regenerative Medicine, University of Illinois at Chicago, Chicago, IL
2Department of Biochemistry & Molecular Genetics, University of Illinois at Chicago, Chicago, IL
3Ruth L. and David S. Gottesman Institute for Stem Cell Biology and Regenerative Medicine, Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY

Bone marrow (BM) mesenchymal stem cells (MSCs) are rare non-hematopoietic perivascular stromal cells characterized by their unique ability to self-renew and differentiate into bone, cartilage, and fat, ensuring proper skeletal development and maintenance. Different subsets of BM MSCs associate with arteriolar and sinusoidal vessels to form specialized niches that regulate hematopoietic stem cell (HSC) function by secreting high levels of niche factors such as CXC-chemokine ligand 12 (CXCL12), stem cell factor (SCF), and Vascular Cell Adhesion Molecule-1 (VCAM1). VCAM1 is classically expressed on endothelial and stromal cells where it acts as an adhesion molecule that preferentially binds to α4β1 integrin on HSCs and progenitor cells (HSPCs). Deletion of Vcam1 in endothelial and hematopoietic cells induces HSPC mobilization into the peripheral blood without affecting endothelial cells homeostasis. Recent work from our lab further demonstrated that VCAM1 expression on HSCs and leukemic stem cells confers innate immune tolerance (Nat Cell Biol 2022). While the contribution of HSC and endothelial-derived VCAM1 to homeostasis has been well studied, the role of MSC-derived VCAM1 on HSC and MSC maintenance and multilineage potency remains unknown. Our supporting data suggest that MSCs are the BM’s main source of Vcam1 (p<0.0001) and that VCAM1 is critical for the maintenance, survival, and function of MSCs. Using a mouse model in which Vcam1 can be conditionally deleted in BM MSCs by a tamoxifen-inducible N-Cadherin-CreER line (referred to as Vcam1N-CadCreER), we found that deletion of Vcam1 significantly reduces the number (4-fold, p=0.0030), frequency (4-fold, p=0.0042), and viability (2-fold, p=0.0039) of BM MSCs. Previous findings in breast cancer revealed that VCAM1 promotes PI3K/Akt survival signaling via Akt phosphorylation. Accordingly, Vcam1 deletion in N-Cadherin-CreER MSCs, demonstrated a significant reduction in phospho-Akt (Ser473) (p<0.05), supporting the role of VCAM1 in mediating PI3K/Akt survival signaling in MSCs. Intriguingly, the remaining viable VCAM1-null MSCs isolated from Vcam1N-CadCreER mice exhibited reduced clonogenic potential (determined by CFU-F assays; 3-fold, p=0.0001). Furthermore, micro-CT analysis of 6-month-old Vcam1N-CadCreER mice presented an altered bone phenotype compared to the control as evidenced by their significantly increased bone thickness and volume (p<0.05), yet significantly decreased tissue mineral density and medullary cavity area (p<0.05). Bulk RNA sequencing (RNAseq) of sorted viable MSCs revealed distinct grouping between Control and Vcam1N-CadCreER MSCs. Gene ontology analysis demonstrated that deletion of Vcam1 caused an upregulation of pathways associated with inflammation and cytokine release while pathways associated with ossification, extracellular matrix organization, and skeletal development were all downregulated. Surprisingly, both bulk RNAseq and qPCR analysis confirmed a significant reduction in the expression of HSC niche factors (Vcam1, Cxcl12, and Scf) following Vcam1 deletion, indicative of reduced niche activity. As expected, Vcam1 deletion in N-Cadherin-CreER MSCs, caused significant mobilization of HSCs into the blood (p<0.0001) and BM homing defects (p<0.05), however the number and repopulation capacity of BM HSCs remained unaltered, as seen by BM transplantation experiments. Surprisingly, imaging analysis of whole-mount bone marrow sternum of phenotypic HSCs revealed no differences in HSC niche localization between Vcam1-null and Control mice. Accordingly, ELISA analysis of BM extracellular fluid revealed no differences in overall CXCL12 and SCF levels suggesting that there may be some local or systemic niche compensation at play. Single-cell (sc)RNAseq of pooled BM and compact bone sorted non-hematopoietic stromal cells (DAPI-Lineage-CD45-Ter119-CD19-CD71-) from Vcam1N-CadCreERand Control mice further confirmed the downregulation of HSC niche factors in MSCs, and overall remodeling of the BM stromal cell composition following Vcam1 deletion. Interestingly, our scRNAseq analysis revealed increased expression of Cxcl12 and Scf in specific vascular and stromal cell populations, supporting the notion of HSC niche compensation. Overall, these findings demonstrate that MSC-derived Vcam1 is critical for BM MSC survival and their HSC niche function.

Disclosures: No relevant conflicts of interest to declare.

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