Nitric Oxide Promotes Human Hematopoietic Stem Cell Homing and Engraftment Via cGMP-Pkg Signaling
Authors: Huang Xinxin , Guo Bin , Capitano Maegan L. , Broxmeyer Hal E. 
Published: October 2019

Abstract
The success of hematopoietic stem cell (HSC) transplantation relies on adequate homing and long-term engraftment of HSC into the bone marrow (BM). The free radical nitric oxide (NO) is a gaseous molecule that plays important roles in a variety of physiological regulations. NO can freely diffuse across cellular membranes and activate an enzyme, soluble guanylyl cyclase, to produce cyclic guanosine monophosphate (cGMP) from guanosine triphosphate (GTP). cGMP binding activates cGMP-dependent protein kinase (PKG) and other proteins to regulate many biological processes. However, the roles of NO and cGMP in regulating HSC function still remain poorly understood.
To explore the importance of NO signaling in HSC, we first evaluated the effects of NO on human cord blood (CB) HSC chemotaxis in an in vitro transwell migration assay. We found that treatment of human CB CD34+ cells for 16 hours with an NO synthesis inhibitor, L-NAME, did not affect chemotaxis towards CXCL12. However, treatment of human CB CD34+ cells 16 hours with NO donor compound sodium nitroprusside (SNP) resulted in 55% more migration toward CXCL12 compared to vehicle control. Enhanced chemotaxis by SNP was also observed in a more primitive HSC cell population (Lin-CD34+CD38-CD45RA-CD49f+CD90+, 71% more migration) suggesting NO donor treatment promote HSC migration. The other two NO donors, SNAP and NOC5 showed similar effects on promoting CD34+ cells and HSC migration. NO activates soluble guanylyl cyclase in target cells, so next we explored the relationship between HSC migration and soluble guanylyl cyclase. By treating human CB CD34+ cells with riociguat, a soluble guanylyl cyclase stimulator, we found that riociguat treatment also resulted in 78% more HSC migration toward CXCL12. The other soluble guanylyl cyclase activator BAY412272 showed a similar effect as riociguat by promoting HSC chemotaxis (68% more migration compared with vehicle control). Inside the cell cGMP is degraded by phosphodiesterase 5 (PDE5), so a PDE5 inhibitor would suppress cGMP breakdown and activate cGMP signaling. Consistently, we found that CD34+ cells with PDE5 inhibitor avanafil or sildenafil treatment showed increased HSC chemotaxis compared with vehicle control (57% and 62% more migration respectively). Next we used PKG inhibitor KT5823 to test whether PKG is involved in HSC migration and found that KT5823 totally blocked the effects of SNP, riociguat and avanafil on enhanced HSC migration, suggesting that NO promote HSC migration through cGMP-PKG signaling.
To directly evaluate in vivo homing, vehicle, SNP or riociguat treated CB CD34+ cells were injected into sublethally irradiated NSG (NOD.Cg-PrkdcscidIL2rgtm1Wjl/Sz) mice, and human cell homing to mouse BM, as indicated by human CD45 percentage, was analyzed 24 hours after transplantation. Consistently, SNP or riociguat treatment resulted in a 2.1 fold and 2.3 fold increase of human cell homing in NSG mice respectively compared with vehicle control treatment. Next, we performed a limiting dilution assay to compare the frequency of SCID-repopulating cells (SRCs) in vehicle and riociguat treated CB CD34+ cells. Engraftment of riociguat-treated CB CD34+ cells was significantly increased in primary NSG recipient mice compared with that of vehicle control treated group four months after transplantation (47.6% vs 23.7%). Both human myeloid and lymphoid chimerisms were also increased. Poisson distribution analysis revealed an SRC frequency of 1/2977 in vehicle control treated group and 1/512 in Riociguat treatment, resulting in the presence of 335.9 SRCs and 1953.1 SRCs in 1×106 cells from vehicle control and riociguat-treated cultures. We are currently performing RNA-seq and quantitative proteomic analysis in riociguat-treated CB CD34+ cells to reveal specific downstream targets in regulating HSC homing.
Taken together, our study suggests that human HSC homing and engraftment can be enhanced by modulating the NO/cGMP signaling pathway. Some compounds tested in our study, such as SNP, riociguat, avanafil are FDA approved medications broadly used for myocardial infarction, pulmonary hypertension and erectile dysfunction. So utilization of these drugs in HSC transplantation should be practical. Our work offers a new and simple approach to bolster the effectiveness of HSC transplantation.

Disclosures
No relevant conflicts of interest to declare.