Nguyen, Julia

Abstract Abstract 1318 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−/− mice display oxidative stress, anemia, and leukocytosis with chronic inflammation. We hypothesize that therapy with inhaled carbon monoxide (CO), a by-product of HO-1 activity with antioxidant and anti-apoptotic properties; will reduce stress hematopoiesis in HO-1−/− mice, reducing inflammation and redistributing hematopoietic potential. 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. After 8-weeks of treatment the mice were sacrificed and flow cytometry was performed on bone marrow to assess hematopoietic potential. Cell cycle analysis of the bone marrow 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<0.05) increases the percent of cells in S-phase in HO-1−/− mice but not 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<0.001) increased proportion of ROS positive KLS cells compared with HO-1+/+ mice. Analysis of long-term (LT), short-term, and multipotent (MPP) progenitor cell populations was conducted. Treatment with CO significantly (p<0.05) increases the percent of LT-HSC and MPP progenitor cells in HO-1−/− mice but not HO-1+/+ mice. Concordantly, the total white blood cell count of the CO-treated mice increased significantly. The differential of the mature blood cells demonstrates significant shift in cell maturation, with significant increase in red blood cells, platelets, and lymphocytes and a significant decrease in monocytes. Combined this data indicates that CO therapy is able to modify the hematopoietic potential of HO-1−/− mice leading to a change in mature cell populations. We propose a model in which CO-mediated signaling initiates a homeostatic conditioning program in stem cells to balance hematopoiesis and stem cell preservation, ultimately leading to a change in the inflammatory milieu of the mice. Disclosures: Belcher: Sangart, Inc: Research Funding. Vercellotti:Sangart, Inc: Research Funding.

Abstract 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<0.05), with HO-1+/+ 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<0.05) increasing the total number of colonies per animal. Of note, in both the CO-treated HO-1-/- 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<0.05) increases the percent of cells in S-phase in HO-1-/- mice but not HO-1+/+ mice. Similarly, untreated HO-1-/- mice have a lower frequency of cells in sub-G1 phase compared to untreated HO-1+/+ mice, however CO-treatments significantly (p<0.01) increase this frequency in HO-1-/- 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<0.001) increased proportion of ROS positive KLS cells compared with HO-1+/+ 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<0.05) decreased compared to untreated HO-1-/- 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<0.05) decreases the percent of ST-HSC cells in HO-1-/- 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.

Aims: Heme derived from hemolysis is pro-oxidative and proinflammatory and promotes vaso-occlusion in murine models of sickle cell disease (SCD), suggesting that enhanced detoxification of heme may be beneficial. Nuclear factor erythroid-2-related factor-2 (Nrf2) transcription pathway is the principal cellular defense system responding to pro-oxidative and proinflammatory stress. Dimethyl fumarate (DMF), a drug approved for treatment of multiple sclerosis, provides neuroprotection by activating Nrf2-responsive genes. We hypothesized that induction of Nrf2 with DMF would be beneficial in murine SCD models. Results: DMF (30 mg/kg/day) or vehicle (0.08% methyl cellulose) was administered for 3–7 days to NY1DD and HbSS-Townes SCD mice. Vaso-occlusion, a hallmark of SCD, measured in sickle mice with dorsal skinfold chambers, was inhibited by DMF. The inhibitory effect of DMF was abrogated by the heme oxygenase-1 (HO-1) inhibitor tin protoporphyrin. DMF increased nuclear Nrf2 and cellular mRNA of Nrf2-responsive genes in livers and kidneys. DMF increased heme defenses, including HO-1, haptoglobin, hemopexin, and ferritin heavy chain, although plasma hemoglobin and heme levels were unchanged. DMF decreased markers of inflammation, including nuclear factor-kappa B phospho-p65, adhesion molecules, and toll-like receptor 4. DMF administered for 24 weeks to HbSS-Townes mice decreased hepatic necrosis, inflammatory cytokines, and irregularly shaped erythrocytes and increased hemoglobin F, but did not alter hematocrits, reticulocyte counts, lactate dehydrogenase, plasma heme, or spleen weights, indicating that the beneficial effects of DMF were not attributable to decreased hemolysis. Innovation: These studies identify Nrf2 activation as a new therapeutic target for the treatment of SCD. Conclusion: DMF activates Nrf2, enhances antioxidant defenses, and inhibits inflammation and vaso-occlusion in SCD mice. Antioxid. Redox Signal . 26, 748–762.

Key Points Heme, released from hemoglobin, elicits vaso-occlusion in transgenic sickle mice via endothelial TLR4 signaling. Heme/TLR4 signaling activates NF-κB and triggers vaso-occlusion through Weibel-Palade body degranulation and adhesion molecule expression.