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

Our objective was to determine whether overexpression and underexpression o f human heme oxygenase (HHO)-1 could be controlled on a long-term basis by introduction of the HO-1 gene in sense (S) and antisense (AS) orientation w ith an appropriate vector into endothelial cells. Retroviral vector (LXSN) containing viral long terminal repeat promoter-driven human HO-1 S (LSN-HHO -1) and LXSN vectors containing HHO-1 promoter (HOP)-controlled HHO-1 S and AS (LSN-HOP-HHO-1 and LSN-HOP-HHO-1-AS) sequences were constructed and use d to transfect rat lung microvessel endothelial cells (RLMV cells) and huma n dermal microvessel endothelial cells (HMEC-1 cells). RLMV cells transduce d with HHO-1 S expressed human HO-1 mRNA and HO-1 protein associated with e levation in total HO activity compared with nontransduced cells. Vector-med iated expression of HHO-1 S or AS under control of HOP resulted in effectiv e production of HO-1 or blocked induction of endogenous human HO-1 in HMEC- 1 cells, respectively. Overexpression of HO-1 AS was associated with a long -term decrease (45%) of endogenous HO-1 protein and an increase (167%) in u nmetabolized exogenous heme in HMEC-1 cells. Carbon monoxide (CO) productio n in HO-1 S- or AS-transduced HMEC-1 cells after heme treatment was increas ed (159%) or decreased (50%), respectively, compared with nontransduced cel ls. HO-2 protein levels did not change. These findings demonstrate that HHO -1 S and AS retroviral constructs are functional in enhancing and reducing HO activity, respectively, and thus can be used to regulate cellular heme l evels, the activity of heme-dependent enzymes, an the rate of heme cataboli sm to CO and bilirubin.