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

Abstract

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-kappaB) 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.

Fig. 1

After 8 weeks, HO-1 protein expression is increased in the livers of sickle mice injected with SB-wt-HO-1, SB-ns-HO-1, or hemin. S+S-Antilles sickle mice were injected hydrodynamically with an albumin promoter-driven SB transposase vector with either a wild-type rat hmox-1 gene (wt-HO-1) or a nonsense rat hmox-1 gene (ns-HO-1) containing an early stop codon. Control sickle mice were either not injected (control), injected hydrodynamically with lactated Ringer's solution (LRS), or injected three consecutive days with hemin (hemin). Seven to 8 weeks after hydrodynamic injection or 18 h after the third hemin injection, the livers were removed and flash frozen. Microsomal membranes (n=2–4 mice per treatment group) were isolated from the livers, and 30 μg of microsomal protein from each liver was run on a western blot and immunostained for HO-1 and GAPDH protein expression (a). HO-1 protein bands at 32 kDa were quantified by densitometry (b). HO enzyme activity is increased in the livers of sickle mice injected with SB-wt-HO-1 or hemin, but not in control, LRS- or SB-ns-HO-1-treated mice (c). HO enzymatic activity was measured using 2 mg of liver microsomes per reaction (n=4 mice per treatment group) by measuring bilirubin production. Values are means±SEM, *p<0.05 and **p<0.01 compared to LRS controls using one-way ANOVA

Fig. 2

After 8 weeks, rat hmox-1 mRNA is present in mouse livers. Hmox-1 mRNA was measured by qRT-PCR using total RNA isolated from livers. HO-1 to GAPDH mRNA ratios were higher in the livers of sickle mice injected with SB-wt-HO-1, SB-ns-HO-1, or hemin compared to LRS controls (a). Values are mean±SEM, *p<0.05 compared to LRS controls using one-way ANOVA. Rat and mouse hmox-1 RT-PCR products from mouse livers were differentiated by RFLP analysis using ApaI restriction enzyme digestion. The rat hmox-1 sequence has an ApaI restriction site, and the mouse hmox-1 sequence does not. The mouse fragment is 212 bp, and the rat fragments are 92 and 120 bp. Rat hmox-1 mRNA was present in the livers of sickle mice injected with SB-wt-HO-1 or SB-ns-HO-1, but not in control, LRS-, or hemin-treated mice (b). Endogenous mouse hmox-1 mRNA was present in all mouse livers

Fig. 3

Immunohistochemistry demonstrates HO-1 overexpression in hepatocytes. Formalin-fixed paraffin embedded liver tissues were sectioned and stained with anti-HO-1. Sites of primary antibody binding were visualized with HRP-conjugated secondary IgG. HO-1/HRP immunoconjugates were detected with diaminobenzidine and H₂O₂. Nuclei were counter-stained with hematoxylin

Fig. 4

After 8 weeks, cytoprotective pathways are activated in sickle mice overexpressing wt-HO-1. Nuclear phospho-p38 MAPK (a) and phospho-Akt (b) were increased in mice injected with SB-wt-HO-1 or hemin, but not in control, LRS-, or SB-ns-HO-1-treated mice. Total nuclear p38 MAPK (a) and Akt (b) were not different between treatment groups. NF-κB p65 was decreased in liver nuclear extracts of sickle mice injected with SB-wt-HO-1 or hemin, but not in control, LRS-, or SB-ns-HO-1-treated mice (c). sVCAM-1 was lower in serum from sickle mice injected with SB-wt-HO-1 (p<0.05) or hemin (p< 0.05), but not in control or SB-ns-HO-1-treated mice compared to LRS mice (d). Nuclear extracts were isolated from livers, and 30 μg of nuclear extract protein from each liver was run on a western blot and immunostained for phospho- and total p38 (a) and Akt (b), and NF-κB p65. NF-κB p65 protein bands at 65 kDa were quantified by densitometry (c). Serum levels of sVCAM-1 were measured by ELISA (d). Values are mean±SEM; n=2–4 mice per treatment group; *p<0.05 compared to LRS controls using one-way ANOVA

Fig. 5

After 8 weeks, hypoxia-induced vascular stasis is inhibited in the skin of sickle mice injected with SB-wt-HO-1 or hemin, but not in control, LRS-, or SB-ns-HO-1-treated mice. Vascular stasis was measured in a DSFC model after hypoxia–reoxygenation. At baseline in room air, the mice were placed under a microscope, and flowing venules were selected inside the DSFC. The mice were then subjected to 1 h of hypoxia (7% O₂/93% N₂) followed by 1 h of reoxygenation in room air. After 1 h of reoxygenation, the same venules were re-examined for blood flow. The number of static venules exhibiting no blood flow were counted and expressed as a percentage of the total number of venules examined. There were seven mice and 403 venules in the control group, five mice and 243 venules in the LRS group, five mice and 347 venules in the wt-HO-1 group, six mice and 325 venules in the ns-HO-1 group, and five mice and 227 venules in the hemin group. There was a minimum of 26 venules per mouse. Values are mean %stasis±SEM. The proportions of venules exhibiting stasis in each treatment group were compared using a Z test; *p<0.05 compared to LRS controls

Fig. 6

Rat HO-1 mRNA is not expressed in dorsal skin. The dorsal skin of sickle mice was examined for the presence of rat hmox-1 mRNA by qRT-PCR RFLP analysis. The rat hmox-1 ApaI fragments at 92 and 120 bp were absent in the dorsal skin samples. The mouse product at 212 bp was seen in the skin of all mice. Rat hepatocytes were analyzed by qRT-PCR RFLP as controls and show the rat hmox-1 fragments at 92 and 120 bp but not the mouse product at 212 bp. The lane marked L designates DNA ladder