Objective-Heme oxygenase-1 (HO-1), the rate-limiting enzyme of heme degradation, has recently been considered to have protective roles against various pathophysiological conditions. Since we demonstrated that HO-1 overexpression inhibits atherosclerotic formation in animal models, we examined the effect of HO modulation on proinflammatory cytokine production, endothelial NO synthase (eNOS) expression, and endothelium-dependent vascular relaxation responses. Methods and Results-After HO-1 induction by heme arginate (HA), vascular endothelial cell cultures were exposed to oxidized low-density lipoprotein (oxLDL) or tumor necrosis factor-␣ (TNF-␣). HA pretreatment significantly attenuated the production of vascular cell adhesion molecule-1, monocyte chemotactic protein-1, and macrophage colonystimulating factor, suggesting that HO-1 induction attenuates proinflammatory responses. In addition, HO-1 overexpression also alleviated endothelial dysfunction as judged by restoration of attenuated eNOS expression after exposure to oxLDL and TNF-␣. Importantly, impaired endothelium-dependent vascular relaxation responses in thoracic aortic rings from high-fat-fed LDL receptor knockout mice were also improved. These effects were observed by treatment with bilirubin not by carbon monoxide. Key Words: heme oxygenase Ⅲ oxidized LDL Ⅲ endothelial nitric oxide synthase Ⅲ bilirubin Ⅲ carbon monoxide V ascular endothelial cell activation by oxidized LDL (oxLDL) and cytokines such as tumor necrosis factor-␣ (TNF-␣) is considered to play an essential role in the development of atherosclerotic lesions. 1 Activated endothelial cells produce adhesion molecules, chemokines, and growth factors such as vascular cell adhesion molecule-1 (VCAM-1), monocyte chemotactic protein-1 (MCP-1), and macrophage colony-stimulating factor (MCSF). [2][3][4] Numerous studies have shown that these molecules promote multiple steps in the formation of atherosclerotic lesion. 1,3,4 Endothelial dysfunction, which is associated with decreased bioavailability of NO from endothelial NO synthase (eNOS), is also considered to play an important role in atherogenesis. 1,5 NO formed by eNOS has been shown to contribute to vascular smooth muscle cell relaxation and inhibition of platelet aggregation. 1 Heme oxygenase (HO) catalyzes the rate-limiting step of heme degradation in mammals. 6 The products of the reaction are biliverdin, carbon monoxide (CO), and free iron. It has been suggested that biliverdin and CO have cytoprotective effects against various cellular stresses. [7][8][9] We demonstrated that the inducible form of HO (HO-1) is induced in cultured vascular endothelial cells, smooth muscle cells, and macrophages by oxidized low-density lipoprotein (oxLDL), and that high expression of HO-1 results in attenuation of monocyte chemotaxis by oxLDL. 9 In fact, HO-1 is expressed in atherosclerotic lesions. 10,11 We also demonstrated that overexpression of HO-1 inhibits the formation of atherosclerotic lesions by inhibiting lipid peroxidation and by affecting NO meta...

Heme oxygenase-1 (HO-1) responds to a variety of oxidative stresses. We examined whether HO-1 expression influences pro-thrombotic processes, in which the involvement of oxidative stress has been reported. Since HO-1 knockout mice with a C57/BL6J background were not viable, embryonic cells from HO-1 deficient mice (E11.5) were used. Cell viability, the level of plasminogen activator inhibitor-1 (PAI-1) expression and reactive oxygen species (ROS) generation of HO-1 deficient cells in response to the exposures to hydrogen peroxide and oxidized LDL were compared to those with wild-type cells. We also examined the effects of glutathione (GSH), desferrioxamine (DFO) and diphenyleneiodonium (DPI: an NADPH oxidase inhibitor) as well as of the HO reaction products, bilirubin (BR) and carbon monoxide (CO) on PAI-1 expression and ROS generation. PAI-1 expression and ROS generation were markedly elevated in HO-1 deficient cells compared to wild-type cells. Exposure to oxidized LDL significantly elevated PAI-1 expression and ROS production in HO-1 deficient cells. Interestingly, these increases in HO-1 deficient cells were significantly lowered by BR, CO, GSH and DPI while DFO had little effect. Furthermore, BR and CO were effective to improve viabilities of HO-1 deficient cells. These results suggest that HO-1 may be required to suppress ROS generation and the production of pro-thrombotic molecules such as PAI-1.

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Abstract-The aim of this study was to determine the effects of carbon monoxide (CO) at a nontoxic low concentration on the cardiac and vascular hypertrophic response and reactive oxygen species generation, compared with the action of a vasodilator, hydralazine. Twelve-to 16-week-old low-density lipoprotein receptor knockout mice were subjected to angiotensin II (Ang II) infusion using osmotic minipumps (Ang II group; nϭ11) for 2 weeks. Controls were administered saline (nϭ10). Animals were exposed to CO in a chamber at 60 ppm for 2 hours per day with or without Ang II infusion (Ang IIϩCO group, nϭ10; CO group, nϭ9). Hydralazine was administered with Ang II infusion (nϭ10). Animals exhibited elevated arterial carboxyhemoglobin after CO exposure. Although the CO exposure did not affect systolic blood pressure without Ang II infusion, the hypertensive response after Ang II infusion was significantly attenuated by CO. Accordingly, the mice in the Ang IIϩCO group showed lesser left ventricular hypertrophy compared with those in the Ang II group. CO treatment also attenuated aortic hypertrophy. Interestingly, these changes were accompanied by the reduction of reactive oxygen species production, p47 phox and p67 phox subunit expressions of reduced nicotinamide-adenine dinucleotide phosphate oxidase, and Akt phosphorylation. Although hydralazine showed stronger antihypertensive action, superior inhibition on cardiac hypertrophy was obtained by CO (PϽ0.05). Furthermore, Ang IIdependent myocardial reactive oxygen species generation was more effectively suppressed by CO. Low-dose exogenous CO treatment attenuates Ang II-dependent reactive oxygen species generation, suggesting that appropriate CO administration alleviates hypertension and reduces organ hypertrophy mediated by Ang II. Key Words: carbon monoxide Ⅲ angiotensin II Ⅲ reactive oxygen species Ⅲ hyperlipidemia Ⅲ hypertension C arbon monoxide (CO), a low-molecular-weight diatomic gaseous molecule, has been considered a dangerous inhalation hazard, because CO binds to hemoglobin with high affinity. 1 CO arises primarily from the large-scale environment and human activities, such as volcanic emissions, forest fires, plant metabolisms, industrial processes, and exhaust emissions.However, recent studies have revealed that CO has profound effects on intracellular signaling processes. The physiological signaling effects of CO involve modulation of soluble guanylate cyclase and subsequent upregulation of cGMP production similar to those of NO. Additional mechanisms include the modulation of several mitogen-activated protein kinase activation pathways. 2,3 CO is endogenously produced during the processes of heme catabolism via heme oxygenase (HO). 4 Because protective roles of HO under various pathophysiological processes have been reported, 5-7 the roles of CO under these processes have currently drawn attention.The vasodilating properties of CO have been investigated in the cardiovascular system. 4,8 CO also has antiapoptotic 9 and anti-inflammatory 10 potentials. However...

To elucidate the correlation between regulatory T cells (Tregs) and acute graft-versus-host disease (aGVHD) or cytomegalovirus infection following allogeneic bone marrow transplantation (allo-BMT), we evaluated either CD4⁺CD25(high) or FOXP3⁺ Treg-enriched cells in peripheral blood (PB) from 20 patients who received allo-BMT, and in biopsies of skin with aGVHD. Proportions of CD4⁺CD25(high)FOXP3⁺ cells in total lymphocytes, but not other types of T cells, were lower in patients who eventually developed grades II-IV aGVHD (n = 13) than in others (n = 7, P < 0.001). Proportions of CD62L⁺ cells in CD4⁺CD25(high) cells at day +30 were lower (P < 0.01) in patients who eventually showed cytomegalovirus viremia (n = 6) than in others (n = 14). Incidence of aGVHD (P < 0.05) or cytomegalovirus viremia (P < 0.05) was higher in patients without these complications, but with lower proportions of PB CD4⁺CD25(high)FOXP3⁺ cells at day +30 (n = 8) than in others (n = 8). However, in skin with aGVHD (n = 5), there was marked or slightly increased infiltration of CD8⁺ cells (P < 0.001) or CD3⁺FOXP3⁺ cells (P < 0.05), respectively, when compared with control (n = 5), resulting in threefold higher ratio of CD8⁺/CD3⁺FOXP3⁺ cells in aGVHD relative to controls (P < 0.05). Thus, impaired reconstitution of Tregs may be associated with aGVHD and CMV infection. Moreover, imbalance of Tregs and CD8⁺ cells may play a role in aGVHD tissue.