Lee-Young Chau

Heme oxygenase is a rate-limiting enzyme in heme degradation, leading to the generation of free iron, biliverdin, and carbon monoxide. Induction of heme oxygenase-1 is implicated in the antioxidant defense mechanism and can modulate vascular function. To test the association of microsatellite polymorphism in the promoter region of human HO-1 gene with the risk of coronary artery disease (CAD) in type 2 diabetic patients, we examined the allele frequencies of (GT) _n repeats in HO-1 gene in 474 patients with CAD and in 322 controls. A transient-transfection assay with HO-1 promoter/luciferase fusion constructs carrying various lengths of (GT) _n repeats was performed to explore the regulatory effect of (GT) _n repeats on HO-1 gene expression in cultured rat aortic smooth muscle cells. Serum thiobarbituric acid-reactive substances (TBARs), a measure of lipid peroxidation, was significantly higher in subjects carrying the L/L genotype (≥32 repeats). Among type 2 diabetic subjects, the frequencies of the L alleles and proportion of genotypes with L alleles were significantly higher in those with CAD than in those without CAD. The adjusted odds ratio for CAD in type 2 diabetic patients with L alleles was 4.7 (95% confidence interval, 1.9–12.0, P=0.001). Transfection experiments in aortic smooth muscle cells revealed that HO-1 promoter/luciferase fusion constructs containing longer (GT) _n repeats exhibited lower transcriptional activity. These results imply that the length polymorphism in the HO-1 gene promoter modulate the transcription of the gene in vascular cells. Type 2 diabetics carrying longer (GT) _n repeats might have higher oxidative stress and increased susceptibility to the development of CAD.

Background Increasing evidence supports the role of heme oxygenase-1 (HO-1) in cytoprotective response and iron homeostasis. The object of this study was to investigate whether adenovirus-mediated gene transfer of HO-1 in arteries reduces iron overload and inhibits lesion formation in apolipoprotein E (apoE)–deficient mice. Methods and Results Infection of rat aortic smooth muscle cells with adenovirus carrying the human HO-1 gene (Adv-HO-1) resulted in a high-level expression of HO-1 protein, which effectively reduced the hemin-induced iron overload in these cells. Adenovirus-mediated gene transfer in arteries in vivo was achieved by direct injection of Adv-HO-1 into the left ventricles of anesthetized animals. Transgene was expressed in the endothelium and aortic lesion of apoE-deficient mice after they had received recombinant adenovirus for 1 week and gradually decayed during the next 5 weeks. When young apoE-deficient mice (14 weeks old) received Adv-HO-1 (2.5×10 ⁹ pfu) for 6 weeks, lesions that developed in the aortic root or aortic arch were significantly smaller than those in control littermates receiving empty viral vector. Furthermore, the iron deposition as well as tissue iron content was much less in aortic tissue of Adv-HO-1–treated mice. The inhibitory effect of HO-1 gene transfer on the progression of advanced lesions was also observed in older apoE-deficient mice (20 weeks old) receiving Adv-HO-1 intraventricularly. Conclusions Overexpression of HO-1 in vascular cells facilitates iron metabolism and attenuates development of atherosclerosis in apoE-deficient mice.

Background—Iron deposition is evident in human atherosclerotic lesions, suggesting that iron may play a role in the development of atherosclerosis. To test this idea, the correlation between the extent of iron deposition and the severity of atherosclerosis in apolipoprotein E (apoE)-deficient mice was investigated. Furthermore, the effect of a low-iron diet on the progression of atherosclerotic lesions in these animals was evaluated.Methods and Results—Iron deposition in tissues of apoE-deficient mice was examined by Perls’ staining method. The results clearly demonstrated that iron deposits are present in atherosclerotic lesions and tissue sections of heart and liver in an age-dependent manner. When the young mice received a low-iron diet for 3 months, the hematocrit, serum iron, hemoglobin, and cholesterol concentrations were not significantly altered compared with those of littermates placed on a chow diet. However, the serum ferritin level of animals in the iron-restricted group was 27% to 30% lower than that of the control group in either sex. Furthermore, the lipoproteins isolated from the iron-restricted group exhibited greater resistance to copper-induced oxidation. Histological examination revealed that atherosclerotic lesions developed in mice fed a low-iron diet were significantly smaller than those found in control littermates. Likewise, the iron deposition as well as tissue iron content was much less in aortic tissues of the iron-restricted animals. Circulating autoantibodies to oxidized LDL and immunostains for epitopes of malondialdehyde-modified LDL detected on lesions were also significantly lower in mice fed a low-iron diet.Conclusions—Iron deposition is closely associated with the progression of atherosclerosis in apoE-deficient mice. Restriction in dietary iron intake leads to significant inhibition of lesion formation in these animals. These results suggest that the beneficial effect of a low-iron diet may be mediated, at least in part, by the reduction of iron deposition as well as LDL oxidation in vascular lesions.

—The cytokine profile of atherosclerotic aortas from apoE-deficient mice was assessed by reverse transcriptase-polymerase chain reaction. The results clearly showed that the expression of mRNA for IL-12p40 was evident in aortas from 3-month-old apoE-deficient mice. The mRNA for IL-10 was detected in aorta from these mice at the age of 6 months, indicating that expression of IL-12 is earlier than that of IL-10 in these animals. Concurrent with IL-12p40, the mRNA for the T-cell cytokine IFN-γ, but not IL-4, was detected in aortas of mice at young and old ages. Both in situ hybridization and immunostaining further demonstrated the localization of IL-12 in macrophages of atherosclerotic lesions. Immunohistochemistry also demonstrated the expression of costimulatory molecules B7–1 and B7–2 in macrophages, suggesting that activation of T lymphocytes by macrophages may occur via surface antigens in lesions. When the immunoglobulin isotype of the antioxidized LDL antibodies in sera of apoE-deficient mice was determined, it revealed that both IgM and IgG were present. Furthermore, IgG2a is predominant and comprises ≈50% of the antioxidized LDL IgG in sera from young mice (3 months), but decreased to lower levels (35%) in older mice (6 months). Daily administration of IL-12 led to an increase in serum levels of antioxidized LDL antibodies and accelerated atherosclerosis in young apoE-deficient mice compared with control mice injected with PBS alone. Taken together, these data suggest that IL-12 plays an active role in regulating the immune response during the early phase of atherosclerosis in apoE-deficient mice.