Regulation of human heme oxygenase in endothelial cells by using sense and antisense retroviral constructs

Abstract

C ellular levels of heme are regulated by the rates of its synthesis and degradation. Heme catabolism occurs by oxidative cleavage of the ␣-methene bridge of the tetrapyrrole, eventually leading to the formation of equimolar amounts of biliverdin and CO and the release of the contained iron atom (1, 2). Biliverdin is then rapidly reduced to form bilirubin (3, 4). The heme oxygenase (HO) system controls the rate-limiting step in heme catabolism (5). To date, three HO isoforms (HO-1, HO-2, and HO-3) have been identified that catalyze this reaction (6-8). HO-1 is a 32-kDa heat shock protein (9-11) that is inducible by numerous noxious stimuli (12-17). HO-2 is a constitutively synthesized 36-kDa protein that is abundant in brain and testis (6). HO-3 is related to HO-2, but is the product of a different gene, and its ability to catalyze heme degradation is much less than that of .Overexpression of HO-1 can lead to hyperbilirubinemia in humans with certain hepatic disorders, especially patients in whom bilirubin disposition is impaired for developmental or genetic reasons, i.e., in newborns and in patients with the Crigler-Najjar type I syndrome. Pharmacological agents such as the inhibitor of HO, tin mesoporphyrin, can inhibit HO activity significantly and have been shown to be highly effective in single dose in controlling hyperbilirubinemia in newborns and other patients (19-23). However, such agents can exert only transient control of the activity of HO-1. Further, the long-term overexpression or underexpression of human HO-1 (HHO-1) would have considerable experimental value in elucidating the role of the enzyme in physiological and pathological processes.The objective of this study was to examine the feasibility of using the retrovirus-mediated transfer of an HHO-1 sense (S) and antisense (AS) orientation sequence under the control of the HHO-1 promoter to regulate endogenous HO-1 expression and function and thus permit development of a gene transfer technology to regulate the rate of heme catabolism over the long term.Our data demonstrate that selective delivery of the HHO-1 gene in AS orientation into human endothelial cells results in an attenuation of HHO-1 protein leading to a decrease in the rate of catabolism of cellular heme and that this effect is brought about without altering endogenous HO-2 protein. Thus, by the use of the retroviral HO-1 AS methodology it is possible to envisage prolonged down-regulation of the rate of heme catabolism to its degradation product bilirubin in clinical or experimental circumstances where this might prove useful. Materials and MethodsCell Culture Conditions. The amphotropic retroviral packaging cell line PA317 (American Type Culture Collection) or PT67 (CLON-TECH) was used for the generation of replication-deficient recombinant retroviruses. PA317 and PT67 cells were grown in DMEM (GIBCO͞BRL, Grand Island, NY) supplemented with 10% heatinactivated FBS. NIH 3T3 fibroblasts were cultured in DMEM with 10% calf serum. Human dermal microvessel endothelial cells (HMEC-1 cells...

Nerve Growth Factor Protects against 6-Hydroxydopamine-induced Oxidative Stress by Increasing Expression of Heme Oxygenase-1 in a Phosphatidylinositol 3-Kinase-dependent Manner

“…We also analyzed the 6-OHDA-induced HE incorporation in The antisense retroviral expression vector for HO-1 under the control of its own promoter (LSN-HOP-HHO-1-AS) has been described previously (27). In agreement with previous reports (36), cells transfected with this antisense constructs grew more slowly (data not shown), suggesting abnormalities in cell cycle progression.…”

“…PC12 cells were infected with the retroviral expression vector LSN-HHO-1, which has been described previously (27). We could not find cell clones with levels of HO-1 overexpression higher than 2-3-fold, suggesting that higher constitutive levels of HO activity may be deleterious (see "Discussion"), probably as a result of the depletion of intracellular heme reservoirs.…”

“…The amphotropic retroviral packaging cell lines PT67 and PA317 (Clontech) were used to collect replication-deficient retroviruses containing the retroviral vector (LXSN) expressing human HO-1 antisense cDNA under the control of the human HO-1 promoter (LSN-HOP-HHO-1-AS) and human HO-1 in the sense orientation (LSN-HHO-1), respectively. These retroviral constructs have been described elsewhere (27). PC12 cells infected with the pLNCX retroviral vector (a generous gift of Dr. R. Perona, Instituto de Investigaciones Biomédicas, Universidad Autónoma de Madrid) were used as controls for HO-1 infections.…”

Heme Oxygenase-1 Gene Expression Modulates Angiotensin II–Induced Increase in Blood Pressure

“…20,24 In contrast, endothelial cells infected with LSN-HO-1-AS demonstrated decreased HO-1 protein expression associated with a decrease in HO activity and an increase in heme content. [23][24][25][26] We also showed that the human HO-1 gene can be expressed in adult SHR by injecting the retroviral vector LSN-HHO-1 into newborns, 19 a method used by others, for example, to overexpress or suppress angiotensin receptors. 27 In a previous study, long-term expression of human HO-1 resulted in attenuation of hypertension in SHR.…”

Heme Oxygenase Attenuates Angiotensin II-Mediated Increase in Cyclooxygenase-2 Activity in Human Femoral Endothelial Cells

“…6 According to a recent study, prostaglandin synthesis in rabbit coronary and human endothelial cells decreases in response to interventions that upregulate the expression of heme oxygenase (HO)-1. 7,8 HO isoforms -1 and -2 catalyze the metabolisms of heme to biliverdin and carbon monoxide (CO), respectively, an antioxidant and a vasoactive mediator. 9,10 HO isoforms, particularly HO-1, play a critical role in the regulation of cellular heme levels, 11 which in turn may impact on the expression of catalytically active COX isoforms.…”