Laminin α4 Deficiency Results in Impaired Hematopoietic Recovery Accompanied with Altered Bone Marrow Niche after Myelosuppression

Myelosuppression is a life-threatening complication of anti-cancer therapy including irradiation. Rapid and complete hematopoietic recovery after therapy-induced myelosuppression is required for a successful treatment outcome. This process relies on efficient regeneration of hematopoietic stem cells (HSCs) and is tightly controlled by bone marrow (BM) microenvironment consisting of mesenchymal stem/progenitor cells, endothelial cells as well as secreted factors including cytokines and extracellular matrix proteins (ECM). However, the extrinsic factors critical for promoting the hematopoietic recovery remain poorly understood. Laminins are heterotrimetric ECM composed of α, β, and γ chains. Laminin α4 chain (LAMA4) is an active component for laminin-411 and -421, which are located in vascular basement membrane. LAMA4 plays an important role for HSC homing after transplantation via interaction with laminin receptor integrin α6 (Qian H et al., Blood 2006). However, the role of LAMA4 in normal hematopoiesis and HSC reconstitution after irradiation-induced myelosuppression is not known.

In this study, we first detected Lama4 gene expression in BM endothelial cells (CD31⁺), mesenchymal stem cells (MSC: CD45^-Ter119^-CD31^-CD44^-Sca1⁺CD51⁺), and mesenchymal progenitor cells (MPC: CD45^-Ter119^-CD31^-CD44^-Sca1^-CD51⁺) in young adult mice. By using Lama4 deficient (Lama4^-/-) mice, we analyzed the functional role of LAMA4 on hematopoietic activity at steady state. We found the lower number of platelets (PLTs) (p = 0.03), and neutrophils (Gr1⁺CD11b⁺) (p = 0.03) in the peripheral blood (PB) of Lama4^-/- mice, but a higher frequency of common myeloid progenitor (Lin^-Sca1^-Kit⁺CD34⁺FcR^low) (p < 0.01) in the Lama4^-/- BM at steady state, indicating that LAMA4 plays a role in the maintenance of physiological hematopoiesis.

The important role of LAMA4 in hematopoietic recovery was demonstrated by delayed and incomplete recoveries of mature red blood cells, PLTs, and Gr1⁺CD11b⁺ cells in PB following sublethal irradiation (7Gy). The impaired recovery of erythropoiesis was also indicated by the higher values of mean corpuscular hemoglobin and mean corpuscular volume in PB as well as the higher frequency of megakaryocyte-erythrocyte progenitor (Lin^-Sca1^-Kit⁺CD34^-FcR^-) (p < 0.01) and colony-forming unit-erythrocyte (CFU-E) (p = 0.03) in the BM of the Lama4^-/- mice at 6 weeks after irradiation, suggesting blocked erythrocyte maturation. In keeping with the refractory neutropenia, the frequency of colony-forming unit-granulocyte-macrophage (CFU-GM) was lower in the Lama4^-/- BM compared to that in the age- and gender-matched wild type mice (p = 0.04). These data indicate that LAMA4 is critical for multiple hematopoietic lineage reconstitution post irradiation.

To investigate the cellular and molecular mechanisms underlying the critical role of LAMA4 in hematopoietic recovery after the irradiation, we characterized the BM niche by colony assay, flow cytometry immunophenotyping, quantitative real time PCR (qPCR), and histological analysis. The number of colony-forming unit-fibroblast (CFU-F) was comparable between wild type and Lama4^-/- in steady state. Interestingly, the proportion of BM MPCs, a population containing osteoblast progenitors, was significantly lower in the Lama4^-/- mice compared to that in the wild type controls at steady state (p < 0.01). qPCR analysis showed downregulation of Il6 (p < 0.05) in the MSC and Angpt1 (p = 0.02) in the MPC of the Lama4^-/-mice post irradiation. These data suggest that Lama4 deficiency alters BM stromal cell composition and gene expressions, which may be related to the impaired hematopoietic reconstitution. The recovery of BM vascular structure is essential for efficient reconstitution of hematopoiesis. We observed uniquely dilated blood vessels in Lama4^-/- BM at 6-week post irradiation. This might be caused by the lower Angpt1 expression in Lama4^-/- MPC since Angpt1/Tie2 signaling is required for vascular regeneration (Kopp HG et al., Blood 2005, Zhou BO et al., eLife 2015). The functional consequences of this phenotype are still under investigation.

Altogether, LAMA4 is required for rapid and complete hematopoietic recovery post irradiation-induced myelosuppression. Therapeutic strategies to upregulate Lama4 may facilitate the recovery of hematopoiesis following HSC transplantation under preconditioning using irradiation.