Shuo Quan

D-4F Induces Heme Oxygenase-1 and Extracellular Superoxide Dismutase, Decreases Endothelial Cell Sloughing, and Improves Vascular Reactivity in Rat Model of Diabetes

Background—Apolipoprotein A1 mimetic peptide, synthesized from D-amino acid (D-4F), enhances the ability of HDL to protect LDL against oxidation in atherosclerotic animals. Methods and Results—We investigated the mechanisms by which D-4F provides antioxidant effects in a diabetic model. Sprague-Dawley rats developed diabetes with administration of streptozotocin (STZ). We examined the effects of daily D-4F (100 &mgr;g/100 g of body weight, intraperitoneal injection) on superoxide (O2−), extracellular superoxide dismutase (EC-SOD), vascular heme oxygenase (HO-1 and HO-2) levels, and circulating endothelial cells in diabetic rats. In response to D-4F, both the quantity and activity of HO-1 were increased. O2− levels were elevated in diabetic rats (74.8±8×103 cpm/10 mg protein) compared with controls (38.1±8×103 cpm/10 mg protein; P<0.01). D-4F decreased O2− levels to 13.23±1×103 (P<0.05 compared with untreated diabetics). The average number of circulating endothelial cells was higher in diabetics (50±6 cells/mL) than in controls (5±1 cells/mL) and was significantly decreased in diabetics treated with D-4F (20±3 cells/mL; P<0.005). D-4F also decreased endothelial cell fragmentation in diabetic rats. The impaired relaxation typical of blood vessels in diabetic rats was prevented by administration of D-4F (85.0±2.0% relaxation). Western blot analysis showed decreased EC-SOD in the diabetic rats, whereas D-4F restored the EC-SOD level. Conclusions—We conclude that an increase in circulating endothelial cell sloughing, superoxide anion, and vasoconstriction in diabetic rats can be prevented by administration of D-4F, which is associated with an increase in 2 antioxidant proteins, HO-1 and EC-SOD.

Heme Oxygenase-1 Attenuates Glucose-Mediated Cell Growth Arrest and Apoptosis in Human Microvessel Endothelial Cells

Abstract— Heme oxygenase-1 (HO-1) is a stress protein that has been suggested to participate in defense mechanisms against agents that may induce oxidative injury, such as heme and inflammatory molecules. Incubation of endothelial cells in a high-glucose (33 mmol/L) medium for 7 days resulted in a decrease of HO activity by 34% and a decrease in HO-1 and HO-2 proteins compared with cells exposed to low glucose (5 mmol/L) (P <0.05) or cells exposed to mannitol (33 mmol/L). Overexpression of HO-1 was coupled with an increase in HO activity and carbon monoxide synthesis, decreased cellular heme, and acceleration in all phases of the cell cycle (P <0.001). The rate of cell cycle or cell birth rate was increased by 29% (P <0.05) in cells overexpressing HO-1 but decreased by 23% (P <0.05) in cells underexpressing HO-1 compared with control cells. Exposure to high glucose significantly decreased cell-cycle progression in control cells and in cells underexpressing HO-1 but did not decrease cell-cycle progression in cells overexpressing HO-1. High glucose induced p21 and p27 in control cells but not in cells overexpressing HO-1. The addition of tin-mesoporphyrin (SnMP), an inhibitor of HO activity, reversed the HO-1–mediated decrease of p21 and p27 in cells overexpressing HO-1. These findings identify a novel effect of HO-1 on endothelial cell growth and indicate that heme metabolism and HO-1 expression regulate signaling systems in cells exposed to high glucose, which controls cell-cycle progression.

Retrovirus-mediated HO gene transfer into endothelial cells protects against oxidant-induced injury

Heme oxygenase (HO)-1 is a stress protein that has been implicated in defense mechanisms against agents that may induce oxidative injury, such as endotoxins, heme, and cytokines. Overexpression of HO-1 in cells might, therefore, protect against oxidative stress produced by certain agents, specifically heme, by catalyzing its degradation to bilirubin, which by itself has antioxidant properties. We report for the first time the successful transduction of human HO-1 gene into rat lung microvessel endothelium using replication-defective retroviral vector. Cells transduced with human HO-1 gene exhibited a 2.1-fold increase in HO-1 protein level, which was associated with a 2.3-fold elevation in enzyme activity compared with that in nontransduced cells. The cGMP content in transduced endothelial cells was increased by 2.9-fold relative to that in nontransduced cells. Moreover, human HO-1 gene-transduced endothelial cells acquired substantial resistance to toxicity produced by exposure to heme and H(2)O(2) compared with that in nontransduced cells. The protective effect of enhancement of HO-1 activity against heme and H(2)O(2) was reversed by pretreatment with stannic mesoporphyrin, a competitive inhibitor of HO. These data demonstrate that the induction of HO-1 in response to injurious stimuli represents an important mechanism for moderating the severity of cell damage. Regulation of HO activity in this manner may have clinical applications.Heme oxygenase (HO)-1 is a stress protein that has been implicated in defense mechanisms against agents that may induce oxidative injury, such as endotoxins, heme, and cytokines. Overexpression of HO-1 in cells might, therefore, protect against oxidative stress produced by certain agents, specifically heme, by catalyzing its degradation to bilirubin, which by itself has antioxidant properties. We report for the first time the successful transduction of human HO-1 gene into rat lung microvessel endothelium using replication-defective retroviral vector. Cells transduced with human HO-1 gene exhibited a 2.1-fold increase in HO-1 protein level, which was associated with a 2.3-fold elevation in enzyme activity compared with that in nontransduced cells. The cGMP content in transduced endothelial cells was increased by 2.9-fold relative to that in nontransduced cells. Moreover, human HO-1 gene-transduced endothelial cells acquired substantial resistance to toxicity produced by exposure to heme and H2O2compared with that in nontransduced cells. The protective effect of enhancement of HO-1 activity against heme and H2O2was reversed by pretreatment with stannic mesoporphyrin, a competitive inhibitor of HO. These data demonstrate that the induction of HO-1 in response to injurious stimuli represents an important mechanism for moderating the severity of cell damage. Regulation of HO activity in this manner may have clinical applications.

Heme Oxygenase-1 Enhances Renal Mitochondrial Transport Carriers and Cytochrome c Oxidase Activity in Experimental Diabetes *

Up-regulation of heme oxygenase (HO-1) by either cobalt protoporphyrin (CoPP) or human gene transfer improves vascular and renal function by several mechanisms, including increases in antioxidant levels and decreases in reactive oxygen species (ROS) in vascular and renal tissue. The purpose of the present study was to determine the effect of HO-1 overexpression on mitochondrial transporters, cytochrome c oxidase, and anti-apoptotic proteins in diabetic rats (streptozotocin, (STZ)-induced type 1 diabetes). Renal mitochondrial carnitine, deoxynucleotide, and ADP/ATP carriers were significantly reduced in diabetic compared with nondiabetic rats (p < 0.05). The citrate carrier was not significantly decreased in diabetic tissue. CoPP administration produced a robust increase in carnitine, citrate, deoxynucleotide, dicarboxylate, and ADP/ATP carriers and no significant change in oxoglutarate and aspartate/glutamate carriers. The increase in mitochondrial carriers (MCs) was associated with a significant increase in cytochrome c oxidase activity. The administration of tin mesoporphyrin (SnMP), an inhibitor of HO-1 activity, prevented the restoration of MCs in diabetic rats. Human HO-1 cDNA transfer into diabetic rats increased both HO-1 protein and activity, and restored mitochondrial ADP/ATP and deoxynucleotide carriers. The increase in HO-1 by CoPP administration was associated with a significant increase in the phosphorylation of AKT and levels of BcL-XL proteins. These observations in experimental diabetes suggest that the cytoprotective mechanism of HO-1 against oxidative stress involves an increase in the levels of MCs and anti-apoptotic proteins as well as in cytochrome c oxidase activity.

Role of human heme oxygenase‐1 in attenuating TNF‐α‐mediated inflammation injury in endothelial cells

Heme oxygenase (HO) is the rate‐limiting enzyme in the formation of bilirubin, an antioxidant, and carbon monoxide (CO), a cell cycle modulator and a vasodilator. Cyclooxygenase (COX) is a hemeprotein that catalyzes the conversion of arachidonic acid (AA) to various prostanoids, which play an important role in the regulation of vascular endothelial function in normal and disease states. The influence of suppression or overexpression of HO isoforms on COX expression and synthesis of prostanoids is of considerable physiological importance. Consequently, the goal of the present study was to determine whether the heme‐HO system regulates COX enzyme expression and activity in vascular endothelial cells in the absence and presence of TNF‐α (100 ng/ml). Endothelial cells stably transfected with the retrovirus containing the human HO‐1 gene exhibited a several‐fold increase in HO‐1 protein levels, which was accompanied by an increase in HO activity and a marked decrease in PGE2 and 6‐keto PGF1α levels. We also assessed the effect of retrovirus‐mediated HO‐1 gene transfer in the sense and antisense orientation on HO‐1 expression and cell cycle progression in human endothelial cells. The levels of CO and HO activity were increased in cells transduced with the HO‐1 sense and were greatly suppressed in cells transduced with HO‐1 antisense as compared to control sham‐transduced cells (P < 0.05). The percentage of the G1‐phase in cells transduced with HO‐1 significantly increased (41.4% ± 9.1) compared with control endothelial cells (34.8% ± 4.9). We measured COX activity by determining the levels of PGI2 and PGE2. The levels of PGI2 decreased in cells transduced with HO‐1 sense and increased in cells transduced with HO‐1 in antisense orientation. The expression of p27 was also studied and showed a marked decrease in cells transduced with HO‐1 sense and a marked increase in the HO‐1 antisense transduced cells. Cell cycle analysis of endothelial cell DNA distributions indicated that the TNF‐α‐induced decrease in the proportion of G1‐phase cells and increase in apoptotic cells in control cultures could be abrogated by transfection with HO‐1 in the sense orientation. Tin mesoporphyrin (SnMP) reversed the protective effect of HO‐1. These results demonstrate that overexpressing HO‐1 mitigated the TNF‐α‐mediated changes in cell cycle progression and apoptosis, perhaps by a decrease in the levels of COX activity. J. Cell. Biochem. 87: 377–385, 2002.

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

Our objective was to determine whether overexpression and underexpression of 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 with 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 used to transfect rat lung microvessel endothelial cells (RLMV cells) and human dermal microvessel endothelial cells (HMEC-1 cells). RLMV cells transduced with HHO-1 S expressed human HO-1 mRNA and HO-1 protein associated with elevation in total HO activity compared with nontransduced cells. Vector-mediated expression of HHO-1 S or AS under control of HOP resulted in effective 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 unmetabolized exogenous heme in HMEC-1 cells. Carbon monoxide (CO) production in HO-1 S- or AS-transduced HMEC-1 cells after heme treatment was increased (159%) or decreased (50%), respectively, compared with nontransduced cells. 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 levels, the activity of heme-dependent enzymes, and the rate of heme catabolism to CO and bilirubin.

Impact of silencing HO‐2 on EC‐SOD and the mitochondrial signaling pathway

The contribution of heme oxygenase HO‐2, the primary source of bilirubin and carbon monoxide (CO) under physiological conditions, to the regulation of vascular function has remained largely unexplored. Using siRNA HO‐2, we examined the effect of suppressed levels of HO‐2 on vascular antioxidant and survival proteins. In vivo HO‐2 siRNA treatment decreased the basal levels of EC‐SOD, pAKT proteins (serine‐473 and threonine‐308), without changing Akt protein expression. HO‐2 siRNA treatment increased 3‐nitrotyrosine (3‐NT) and apoptotic signaling kinase‐1 (ASK‐1) (P < 0.01). HO activity was decreased by the use of siRNA HO‐2. We extended these studies to the mitochondria, examining for the presence of HO‐1 and its role in the regulation of pro‐ and anti‐apoptotic proteins. HO activity was increased by the administration of CoPP resulting in the translocation of HO‐1 into the mitochondria, mainly to the inner face of the mitochondrial inner membrane. These findings suggest that HO‐2 is critical in the maintenance of heme homeostasis and also the regulation of apoptosis by controlling levels of EC‐SOD, Akt, 3‐NT, and ASK‐1. In addition, localization of HO‐1 in the mitochondrial compartment plays a critical role in mitochondria‐mediated apoptosis. J. Cell. Biochem. 100: 815–823, 2007.

Functional expression of human heme oxygenase‐1 (HO‐1) driven by HO‐1 promoter in vitro and in vivo

We developed a retrovirus‐mediated human heme oxygenase‐1 (HO‐1) gene expression system and assessed the impact of heme on the inducibility of the HO‐1 gene in rat lung microvessel (RLMV) endothelial cells and in newborn Sprague‐Dawley (SD) rats. Overexpression of the HO‐1 gene driven by HO‐1 promoter (HOP) resulted in an increase in HO‐1 protein and HO activity by 4.8‐ and 1.3‐fold, respectively, compared to the viral LTR promoter. The increased HO‐1 gene expression was associated with the enhancement of CO production. In cells transduced by HOP‐driven HO‐1 gene, there was a decrease in basal cyclooxygenase (COX) activity as measured by PGE2. The degree of HO‐1 expression and, consequently, the levels of cellular heme were directly related to COX activity. Supplementation with heme markedly increased PGE2 and cGMP synthesis. In all (6/6) of newborn SD rats injected with retrovirus LSN‐HOP‐HO‐1, both HO‐1 and neor transcripts were expressed in tissues. We hypothesize that degree of HO‐1 gene expression resulted in a differential rate of cellular heme‐dependent enzyme gene expression, which may play a vital role in maintaining cellular homeostasis. J. Cell. Biochem. 85: 410–421, 2002.

Influence of Heme and Heme Oxygenase-1 Transfection of Pulmonary Microvascular Endothelium on Oxidant Generation and cGMP

Heme is a co-factor required for the stimulation of soluble guanylate cyclase (sGC) by nitric oxide (NO) and carbon monoxide, and sGC activation by these agents is inhibited by superoxide. Because heme promotes oxidant generation, we examined the influence of rat pulmonary microvascular endothelial cells (PMECs) with a stable human heme oxygenase-1 (HO-1) transfection and heme on oxidant generation and cGMP. Culture of PMEC with low serum heme decreased cGMP and the detection of peroxide with 10 μM 2′,7′-dichlorofluorescin diacetate and increased HO-1 further decreased cGMP without altering the peroxide detection under these conditions. Under conditions where heme (30 μM) has been shown to stimulate cGMP production in PMECsby mechanisms involving NO and CO, heme increased the detection of peroxide in a PMEC-dependent manner and HO-1 transfection did not markedly alter the effects heme on peroxide detection. The addition of 1 μM catalase markedly inhibited the effects of heme on peroxide detection whereas increasing (0.1 mM ebselen) or decreasing (depleting glutathione with 7 mM diethylmaleate) rates of intracellular peroxide metabolism or inhibiting the biosynthesis of oxidants (with 10 μM diphenyliodonium or 0.1 mM nitro-L-arginine) had only modest effects. The detection of superoxide by 10 μM dihydroethidium from PMECs was not increased by exposure to heme. These actions of oxidant probes suggest that intracellular oxidants have a minimal influence on the response to heme. Thus, exposure of PMECs to heme causes a complex response involving an extracellular generation of peroxide-derived oxidant species, which do not appear to originate from increases in intracellular superoxide or peroxide. This enables heme and HO to regulate sGC through mechanisms involving NO and CO, which are normally inhibited by superoxide.

Diminished heme oxygenase potentiates cell death: Pyrrolidinedithiocarbamate mediates oxidative stress

Pyrrolidinedithiocarbamate (PDTC) is a metal-chelating compound that exerts both pro-oxidant and antioxidant effects and is widely used as an antitumor and anti-inflammatory agent. Heme oxygenase-1 (HO-1) is a redox-sensitive-inducible protein that provides efficient cytoprotection against oxidative stress. Because it has been reported that several angiogenic stimulating factors upregulating HO-1 in endothelial cells cause a significant increase in angiogenesis, we investigated the effect of PDTC on cell proliferation and angiogenesis and the effect of overexpression and underexpression of HO-1. The evaluation of PDTC (20 or 50 μM) in endothelial cells resulted in significant increase in HO-1 mRNA and protein (P < 0.001), but a decrease in cell proliferation. Pretreatment of endothelial cells with SnCl2 (10 μM), an inducer of HO-1 attenuated the PDTC-mediated decrease in cell proliferation (P < 0.05). In contrast, pretreatment with SnMP, an inhibitor of HO activity, magnified the inhibiting effect of PDTC on cell proliferation. Upregulation of HO-1 gene expression by retrovirus-mediated delivery of the human HO-1 gene also attenuated the PDTC-induced decrease in cell proliferation. Underexpression of HO-1, by delivery of the human HO-1 in antisense orientation, enhanced the PDTC-mediated decrease in cell proliferation. The decrease, by PDTC, in proliferation of cells underexpressing HO-1 is related to an increase in O− 2 production. Collectively, these results demonstrate that upregulation of HO-1 was able to attenuate the PDTC-mediated cell proliferation, but was unable to reverse the high concentration of PDTC-induced decrease in angiogenesis.