NF-Kb Inhibition in Endothelial Cells Enhances the Self-Renewal and Regeneration of the Hematopoietic System

Adult hematopoietic stem cells (HSCs) are defined by their ability to undergo self-renewal and maintain the capacity to generate all of the mature hematopoietic cell types within the blood and immune system. These unique qualities make the HSC clinically useful in bone marrow (BM) transplantation settings for a wide variety of hematological diseases. It has been demonstrated that maintenance of the HSC is dependent upon the cell-intrinsic properties of the HSC itself, as well as the extrinsic properties of the BM microenvironment. Within the hematopoietic microenvironment, we have shown that endothelial cells (ECs) are indispensable in supporting HSC self-renewal and differentiation into lineage-committed progeny during regenerative hematopoiesis. Furthermore, we have demonstrated that Akt signaling endows ECs with the capacity to instructively support HSC self-renewal through the expression of pro-hematopoietic angiocrine factors during homeostasis and hematopoietic regeneration following myelosuppressive stress. However, despite advances in the understanding of HSC biology, the exact mechanisms that regulate the balance between self-renewal and lineage-specific differentiation are still unknown.

In order to expand our understanding of the body’s vascular network in regulating HSCs and hematopoietic regeneration, we have now focused on identifying the downstream signaling pathways of Akt within ECs that are responsible for the production of the pro-hematopoietic angiocrine factors. Because of the strong supporting data demonstrating that NF-kB signaling regulates hematopoietic function, we have focused on the Akt/NF-kB signaling axis in the vascular niche and demonstrated that inhibition of NF-kB within ECs results in a significant expansion of functional HSCs. Inhibition of the NF-kB pathway by expression of an IkBa super suppressor (IkBa-SS) via lentiviral transduction in primary ECs resulted in the expansion of phenotypic HSCs, while blocking differentiation of progenitor cells in vitro with an increase in the functional potential of the expanded HSCs. Utilizing a transgenic mouse model (Tie2.IkBa-SS) in which the NF-kB signaling pathway is inhibited specifically in ECs, we found that there was a significant increase in phenotypic and functional HSCs in vivo. Endothelial-specific inhibition of NF-kB signaling resulted in an increase in HSC quiescence and serial administration of low-dose chemotherapeutic agents resulted in an increase in self-renewal activity, suggesting that suppressing NF-kB signaling in ECs controls hematopoiesis by preventing premature exhaustion of the HSC pool. Following hematopoietic insult, Tie2.IkBa-SS mice undergo a rapid recovery of hematopoiesis and the hematopoietic system is largely protected following myelosuppression when compared to controls. Gene profiling of freshly isolated BM ECs from Tie2.IkBa-SS mice suggests that the enhancement of functional hematopoiesis is, in part, due to BM ECs upregulating pro-HSC angiocrine factors, as well as suppressing the production of cytokines and growth factors responsible for eliciting inflammatory responses, forcing the differentiation of HSCs. Furthermore, transplantation of BM ECs isolated from Tie2.IkB-SS mice significantly enhanced overall hematopoietic recovery following an LD₅₀ dose of myeloablation, suggesting that transplantation of ECs could have tremendous therapeutic potential in mitigation the side effects of myeloablative injury by decreasing the morbidity and mortality associated with hematopoietic insults.

In conclusion, our data demonstrates that the IkBa-dependent NF-kB pathway in ECs can regulate the production of pro-hematopoietic angiocrine factors that promote the maintenance and expansion of the HSC pool. Additionally, we have potentially unlocked a novel therapeutic application for the transplantation of genetically modified BM ECs following myeloablative treatment. Therapeutic transplantation of BM ECs may create a more permissive microenvironment that promotes an increase in the number of engrafted HSCs following BM transplantation, accelerating the rate of hematopoietic recovery following radiation or chemotherapeutic regimens and decreasing the morbidity and mortality associated with life threatening pancytopenias.