Bone Marrow Progenitor Dysfunction Accelerates Retinal Injury in Bmal1 Deficient Mice

Abstract

Purpose: The clock gene BMAL1 (brain and muscle ARNT-like protein) shares a prominent position in circadian physiology by forming the positive arm of the major transcriptional auto-regulatory loop and by activating a set of the clock regulatory genes. In this study, we asked whether Bmal1 deficiency in bone marrow progenitor cells (BMPCs) is linked to an accelerated microvascular injury in an endothelial-specific knockout mouse of Bmal ( Bmal1^fx/fx ; Tek-cre)

Methods: The BMPCs from the Bmal1^fx/fx; Tek-cre and wild type (WT) mice were harvested by flushing the femurs. Lineage^- Sca 1⁺c-kit⁺ (LSK) cells were enumerated using flow cytometry. BMPC proliferation was evaluated using the methylcellulose colony forming assay. Contralateral femurs were processed for neurofilament-200 (NF-200) and tyrosine hydroxylase (TH). In parallel, the Bmal1^fx/fx ;Tek-cre and WT mice underwent retinal microvascular injury using the ischemia-reperfusion (I/R) model. One week after injury the eyes were enucleated, sectioned and processed for staining with nitrotyrosine antibodies.

Results: LSK cell numbers from the Bmal1^fx/fx;Tek-cre mice decreased by 1.5 fold (p<0.05) as compared to WT controls. The proliferative potential of BMPCs from the Bmal1^fx/fx;Tek-cre mice was profoundly reduced as evidenced by changes in colony forming units (CFUs), specifically a 3-fold decrease (P<0.05) in CFU-GM (CFU-granulocyte monocyte), a 4-fold (p<0.05) decrease in CFU-GEMM (CFU-Granulocyte, Erythrocyte, Monocyte/macrophage, Megakaryocyte) and a 6-fold decrease (p<0.05) in CFU-G (CFU granulocyte). CFU-M (CFU monocytes) remained unchanged. This defect in proliferation of BMPCs was associated with reduction in neuronal activity of the bone marrow as indicated by a 1.5-fold decrease (p<0.01) in NF-200 staining while the TH remained unchanged. The retinas of Bmal1^fx/fx;Tek-cre mice demonstrated a 1.5 fold greater nitrotyrosine staining (p<0.05) when compared to WT-injured group.

Conclusions: Our study suggests that the BMPC dysfunction due to the conditional deletion of Bmal1 is detrimental to the retina and highlights the critical role of clock gene Bmal1 in maintaining the BMPC health for contribution in vascular repair.