Joan D. Beckman

Heme oxygenase-1 gene delivery by Sleeping Beauty inhibits vascular stasis in a murine model of sickle cell disease

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.

Regulation of Heme Oxygenase-1 Protein Expression by miR-377 in Combination with miR-217

Heme oxygenase-1 (HO-1) enzyme plays a critical role in metabolizing the excess heme generated during hemolysis. Our previous studies suggested that during intravascular hemolysis the expression of HO-1 protein is not sufficient to reduce the oxidative burden of free heme in the vasculature. This led us to hypothesize that a post-translational mechanism of control exists for HO-1 expression. Micro-RNAs (miRNA) affect gene expression by post-transcriptional gene regulation of transcripts. We performed in silico analysis for the human HMOX1–3′ untranslated region (3′ UTR) and identified candidate miRNA binding sites. Two candidate miRNAs, miR-377 and miR-217, were cloned and co-transfected with a luciferase vector containing the human HMOX1-3′UTR region. The combination of miR-377 and miR-217 produced a 58% reduction in HMOX1–3′UTR luciferase reporter expression compared with controls. The same constructs were then used to assess how overexpression of miR-217 and miR-377 affected HO-1 levels after induction with hemin. Cells transfected with the combination of miR-377 and miR-217 exhibited no change in HMOX1 mRNA levels, but a significant reduction in HMOX1 (HO-1) protein expression and enzyme activity compared with non-transfected hemin-stimulated controls. Transfection with either miR-377 or miR-217 alone did not produce a significant decrease in HO-1 protein expression or enzyme activity. Knockdown of miR-217 and miR-377 in combination leads to up-regulation of HO-1 protein. Exposure to hemin induced a significant reduction in miR-217 expression and a trend toward decreased miR-377 expression in two different cells lines. In summary, these data suggests that the combination of miR-377 and miR-217 help regulate HO-1 protein expression in the presence of hemin.

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

Carbon monoxide (CO) has anti-inflammatory properties. We previously reported that acute treatments with inhaled CO inhibit vascular inflammation and hypoxia-induced vasoocclusion in sickle cell disease mouse models. Therefore, we hypothesized that chronic CO inhalation would decrease vascular inflammation and organ pathology in a sickle cell disease mouse model. The treatment of sickle cell disease mice with 25 or 250 parts/million inhaled CO for 1 h/day, 3 days/wk for 8-10 wk significantly decreased the total mean white blood cell, neutrophil, and lymphocyte counts in peripheral blood. Eight weeks of 250 parts/million CO treatments reduced staining for myeloid and lymphoid markers in the bone marrow of sickle mice. Bone marrow from treated sickle mice exhibited a significant decrease in colony-forming unit granulocyte-macrophage during colony-forming cell assays. Anti-inflammatory signaling pathways phospho-Akt and phospho-p38 MAPK were markedly increased in CO-treated sickle livers. Importantly, CO-treated sickle mice had a significant reduction in liver parenchymal necrosis, reflecting the anti-inflammatory benefits of CO. We conclude that inhaled CO may be a beneficial anti-inflammatory therapy for sickle cell disease.

Isolation and characterization of ovine luteal pericytes and effects of nitric oxide on pericyte expression of angiogenic factors

We have demonstrated that vascular endothelial growth factor (VEGF) is expressed in capillary pericytes of the developing corpus luteum (CL) and other have shown that basic fibroblast growth factor (FGF2) and angio-poietins (ANGPT) are present in the CL. VEGF and FGF2 target endothelial cells to initiate angiogenesis and stimulate nitric oxide (NO) production. Conversely, NO may increase VEGF expression by vascular smooth muscle cells and pericytes. To investigate the relationship between these angiogenic factors and NO in the CL, microvascular perricytes and endothelial cell were isolated from CL collected from superovulated ewes (n=5) on d 9 of the estrous cycle. Pericytes were identified by their morphology in culture and by immunofluorescent staining for smooth muscle cell actin. Pericytes were incubated with or without varying doses of the NO-donor DETA-NO for 8 h. Then, total cellular RNA was extracted from the cells and evaluated for expression of mRNA for VEGF, FGF2, ANGPT1, ANGPT2, and NO receptor, guanylate cyclase 1, soluble β3 (GUCY1B3), using renal-time quantitative RT-PCR. NO caused a dose-dependent increase in VEGF (p<0.001), FGF2 (p<0.001), 0.001), ANGTP2 (p<0.06), and GUCY1B3 (p<0.03) mRNA expression. Expression of mRNA for ANGPT1 in luteal pericytes was not affected by the NO treatment. These data provide further evidence of the role of the luteal pericyte and NO in angiogenic factor expression, and of the potential interactions of pericytes with endothelial cells via NO production.

Quantitative real-time polymerase chain reaction (qRT-PCR) restriction fragment length polymorphism (RFLP) method for monitoring highly conserved transgene expression during gene therapy

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 quantitative real-time polymerase chain reaction (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, the 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 was 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 the 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.