Carbon Monoxide Therapy Reduces Reactive Oxygen Species Production and the Short-Term Hematopoietic Stem Cell Population In Heme-Oxygenase-1 Knockout Mice

Abstract 4767 Previous studies have demonstrated that heme-oxygenase-1 (HO-1), the rate limiting enzyme in the catabolism of heme, is a regulator of the balance between hematopoiesis and stem cell preservation under stress. HO-1 produces three by-products, carbon monoxide, biliverdin/bilirubin, and iron/ferritin, all of which have demonstrated antioxidant and anti-apoptotic properties. HO-1 -/- mice display oxidative stress, anemia, and leukocytosis with chronic inflammation. We hypothesize that therapy with inhaled carbon monoxide (CO) may reduce stress hematopoiesis in HO-1 -/- mice, restoring them to normal hematopoiesis. We have previously reported that treatment with 250 ppm CO for 1 h raises mouse carboxyhemoglobin to 14–20% which returns to baseline within 24 hours. Therefore, in order to test this hypothesis we treated 25 week old HO-1 -/- mice and HO-1 +/+ mice (n=7/strain) with 250 ppm inhaled CO for 1 h/day, 3 days/week for eight weeks and compared them to an equal number of untreated HO-1 -/- and HO-1 +/+ mice. We demonstrate that after four weeks, CO-treated HO-1 -/- mice have a total white blood cell count of 11.9 ± 3.2 K/μ L compared to 22.6 ± 3.2 K/μ L in untreated HO-1 -/- mice (p +/+ mice having no treatment effect. After 8-weeks of treatment the mice were sacrificed and methylcellulose colony-forming unit assays and flow cytometry were performed on bone marrow to assess their hematopoietic potential. Total methylcellulose colony-forming units were similar in both HO-1 -/- and HO-1 +/+ mice with CO-treatments significantly (p -/- and HO-1 +/+ mice the CFU-GM were not significantly affected. Cell cycle analysis of the bone marrow using propidium iodide demonstrates that untreated HO-1 -/- mice have a significantly decreased percent of cells in S-phase compared to untreated HO-1 +/+ mice. Treatment with CO significantly (p -/- mice but not HO-1 +/+ mice. Similarly, untreated HO-1 -/- mice have a lower frequency of cells in sub-G 1 phase compared to untreated HO-1 +/+ mice, however CO-treatments significantly (p -/- mice to levels comparable with untreated and CO-treated HO-1 +/+ mice. Reactive oxygen species (ROS) production in lineage - , c-kit + , Sca-1 + (KLS) cell population was assessed using 5-(and 6-)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate dye. HO-1 -/- mice have a significantly (p +/+ mice. Interestingly, the CO-treated HO-1 -/- mice had a significantly higher proportion of KLS cells staining positive for ROS, but the mean fluorescent intensity of this population was significantly (p -/- mice. This indicates that there is an overall decrease in ROS in the KLS cells of CO-treated HO-1 -/- mice compared to untreated HO-1 -/- mice. Analysis of long-term, short-term, and multipotent progenitor cell populations was conducted and reveals significant changes at the level of the short-term hematopoietic progenitor population (ST-HSC). Specifically, untreated HO-1 -/- mice have a significantly increased percent of ST-HSC cells compared to untreated HO-1 +/+ mice. Treatment with CO significantly (p -/- mice but not HO-1 +/+ mice. Combined this data indicates that CO therapy is able to modify the hematopoietic potential of HO-1 -/- mice. We propose a model in which CO-mediated signaling initiates a homeostatic conditioning program in stem cells to balance hematopoiesis and stem cell preservation. Disclosures: Belcher: Sangart: Research Funding. Vercellotti: Sangart: Consultancy, Research Funding.