Increases in heme oxygenase-1 (HO-1) and administration of heme degradation products CO and biliverdin inhibit vascular inflammation and vasoocclusion in mouse models of sickle cell disease (SCD). In this study, an albumin (alb) promoter-driven Sleeping Beauty (SB) transposase plasmid with a wild-type rat hmox-1 (wt-HO-1) transposable element was delivered by hydrodynamic tail vein injections to SCD mice. Eight weeks after injection, SCD mice had three- to five-fold increases in HO-1 activity and protein expression in liver, similar to hemin-treated mice. Immunohistochemistry demonstrated increased perinuclear HO-1 staining in hepatocytes. Messenger RNA transcription of the hmox-1 transgene in liver was confirmed by quantitative real-time polymerase chain reaction restriction fragment length polymorphism (qRT-PCR RFLP) with no detectible transgene expression in other organs. The livers of all HO-1 overexpressing mice had activation of nuclear phospho-p38 mitogen-activated protein kinase (MAPK) and phospho-Akt, decreased nuclear expression of nuclear factor-kappa B (NF-κB) p65, and decreased soluble vascular cell adhesion molecule-1 (sVCAM-1) in serum. Hypoxia-induced stasis, a characteristic of SCD, but not normal mice, was inhibited in dorsal skin fold chambers in wt-HO-1 SCD mice despite the absence of hmox-1 transgene expression in the skin suggesting distal effects of HO activity on the vasculature. No protective effects were seen in SCD mice injected with nonsense (ns-) rat hmox-1 that encodes carboxy-truncated HO-1 with little or no enzyme activity. We speculate that HO-1 gene delivery to the liver is beneficial in SCD mice by degrading pro-oxidative heme, releasing anti-inflammatory heme degradation products CO and biliverdin/bilirubin into circulation, activating cytoprotective pathways and inhibiting vascular stasis at sites distal to transgene expression.

The vascular pathobiology of sickle cell anemia involves inflammation, coagulation, vascular stasis, reperfusion injury, iron-based oxidative biochemistry, deficient nitric oxide (NO) bioavailability, and red cell sickling. These disparate pathobiologies intersect and overlap, so it is probable that multimodality therapy will be necessary for this disease. We have, therefore, tested a histone deacetylase (HDAC) inhibitor, trichostatin A (TSA), for efficacy in reducing endothelial activation. We found that pulmonary vascular endothelial VCAM-1 and tissue factor (TF) expression (both are indicators of endothelial activation) are powerfully and significantly inhibited by TSA. This is seen both with pretreatment before the inducing stress of hypoxia/reoxygenation (NY1DD sickle transgenic mouse), and upon longer-term therapy after endothelial activation has already occurred (hBERK1 sickle mouse at ambient air). In addition, TSA prevented vascular stasis in sickle mice, it exhibited activity as an iron chelator, and it induced expression of the antisickling hemoglobin, hemoglobin F. Notably, the TSA analog SAHA (suberoylanilide hydroxaminc acid) that is already approved for human clinical use exhibits the same spectrum of biologic effects as TSA. We suggest that SAHA possibly could provide true, multimodality, salubrious effects for prevention and treatment of the chronic vasculopathy of sickle cell anemia. (Blood. 2010; 115:2483-2490)

GM. Inhaled carbon monoxide reduces leukocytosis in a murine model of sickle cell disease.

Evaluation of the transfer efficiency of a rat heme oxygenase-1 (HO-1) transgene into mice requires differentiation of rat and mouse HO-1. However, rat and mouse HO-1 have 94% homology; antibodies and enzyme activity cannot adequately distinguish HO-1. We designed a qRT-PCR method to monitor HO-1 transcription relative to a housekeeping gene, GAPDH. The ratio of rat and mouse HO-1 mRNA could be estimated through restriction fragment length polymorphism (RFLP) analysis of the PCR products. In vitro, murine AML12 hepatocytes were transfected with rat HO-1. After 40 h, total HO-1 mRNA was enriched 2-fold relative to control cells and rat HO-1 comprised 84% of HO-1 cDNA. In vivo, the rat HO-1 transgene was cloned into a Sleeping Beauty transposase (SB-Tn) construct and injected hydrodynamically into a mouse model of sickle cell disease (SCD). After 21 days, there was a 32% enrichment of HO-1 mRNA relative to control mice and the rat transgene comprised 88% of HO-1 cDNA. After 21 days, HO-1 protein expression in liver was increased 2.5-fold. In summary, qRT-PCR RFLP is a useful and reliable method to differentiate transgene from host gene transcription, especially when the host and transgene protein are identical or highly homologous. This method has translational applications to the design, delivery and monitoring of gene therapy vectors.

Elevated white blood cell counts (WBC) in sickle cell disease (SCD) are risk factors for morbid vaso-occlusive events including acute chest syndrome and stroke. The mechanisms of leukocytosis in SCD include increased inflammation, oxidative stress, increased hematopoiesis, and hyposplenism. Heme oxygenase-1 (HO-1) plays critical roles in metabolizing the excess heme generated during hemolysis and in modulating vaso-occlusion in murine models of sickle cell disease (SCD). The products of HO-1 activity, carbon monoxide (CO), Fe2+/ferritin, and biliverdin/bilirubin have demonstrable anti-oxidant and anti-inflammatory effects. We have previously demonstrated that inhaled CO treatments decrease white blood cell counts (WBC), as well as liver redox-active iron and heme content in heterozygous BERK mice, a mouse model for sickle cell trait. To dissect the mechanism of this decrease in leukocytosis, we hypothesize that prolonged treatment with inhaled CO significantly decreases granulopoiesis in SCD mice. For this study, we exposed S+S-Antilles mice to 250 ppm inhaled CO for 1h 3X/week for 8–10 weeks. Upon completion of the treatment period animals were euthanized, blood was removed by cardiac puncture, and bone marrow and organs were harvested for analysis. Treatment for 10 weeks with 250 ppm CO significantly decreased total WBC (19.19±1.29 × 1000/ul (untreated) to 12.8±1.30 × 1000/ul (CO treated), p<0.05). The decrement in total WBC count was primarily due to a significant decrease in neutrophils (p<0.05) and lymphocytes (p<0.005), There was no significant change in the reticulocyte count, hematocrit, or bilirubin in CO-treated animals compared to controls. The spleens of the control and CO-treated animals had similar weights but no differences in their histopathology, ruling out the possibility of increased sequestration as a cause of the decrease in total WBC. Bone marrow staining reveals that CO-treated mice have a significant decrease in polymorphonuclear (PMN) precursors in bone marrow (p<0.05). Flow cytometry on bone marrow stained for hematopoietic markers CD117, CD45R/B220, SCA1, CD90.1, and CD135 demonstrates a significant (p<0.005) decrease in common lymphoid progenitor (CLP) and common myeloid progenitor (CMP) cells in CO-treated animals compared to controls. Flow cytometry for myeloperoxidase (MPO) and the granulocyte differentiation marker GR-1 (Ly-6G/6C) also demonstrates a significant (p<0.05) decrease in mature granulocytes cells in CO-treated mice. Colony-forming cell (CFC) assays verify the flow cytometry data, with a significant decrease (p<0.05) in CFU-GM in 250 ppm treated bone marrow compared to controls. Consistent with the lack of effect on hematocrit, CO had minimal effects on the erythroid marrow compartment since total CD71 positive erythroid cells were unchanged. In summary, we conclude that inhaled CO treatments decrease total WBC by decreasing granulopoiesis. We speculate that inhaled CO treatments may be a potential therapy for patients with SCD by acting as a modulator of oxidative stress and inflammation.