[Show abstract][Hide abstract] ABSTRACT: Heme oxygenase-1 (HO-1) has been viewed as a cytoprotective protein, ameliorating the effects of inflammatory cellular damage, and as beneficial in allograft protection from acute and chronic rejection, suggesting important functions in both innate and adaptive immune responses. Mice deficient in HO-1 exhibit defective immune regulation characterized by a proinflammatory phenotype. We examined if impaired regulatory T cell (Treg) function contributes to the immunoregulatory defects observed in HO-1(-/-) mice. HO-1(-/-) mice exhibited a significantly higher proportion of Foxp3-expressing cells among total CD4(+) and CD4(+)CD25(+) cells in comparison to HO-1(+/+) mice, and HO-1(-/-) Treg cells were at least as effective as HO-1(+/+) Treg cells in suppressing proliferation of effector T cells in vitro from either HO-1(+/+) or HO-1(-/-) mice. However, the absence of HO-1 in antigen-presenting cells abolished the suppressive activity of Treg cells on effector T cells. These findings demonstrate that HO-1 activity in antigen-presenting cells is important for Treg-mediated suppression, providing an explanation for the apparent defect in immune regulation in HO-1(-/-) mice.
Preview · Article · Aug 2008 · American Journal Of Pathology
[Show abstract][Hide abstract] ABSTRACT: Heme oxygenase-1, an enzyme degrading heme to carbon monoxide, iron, and biliverdin, has been recognized as playing a crucial role in cellular defense against stressful conditions, not only related to heme release. HO-1 protects endothelial cells from apoptosis, is involved in blood-vessel relaxation regulating vascular tone, attenuates inflammatory response in the vessel wall, and participates in blood-vessel formation by means of angiogenesis and vasculogenesis. The latter functions link HO-1 not only to cardiovascular ischemia but also to many other conditions that, like development, wound healing, or cancer, are dependent on neovascularization. The aim of this comprehensive review is to address the mechanisms of HO-1 regulation and function in cardiovascular physiology and pathology and to demonstrate some possible applications of the vast knowledge generated so far. Recent data provide powerful evidence for the involvement of HO-1 in the therapeutic effect of drugs used in cardiovascular diseases. Novel studies open the possibilities of application of HO-1 for gene and cell therapy. Therefore, research in forthcoming years should help to elucidate both the real role of HO-1 in the effect of drugs and the clinical feasibility of HO-1-based cell and gene therapy, creating the effective therapeutic avenues for this refined antioxidant system.
[Show abstract][Hide abstract] ABSTRACT: We examined the effect of the vasoactive agents carbon monoxide (CO) and nitric oxide (NO) : n the phosphorylation and intracellular redistribution of vasodilator-stimulated phosphoprotein (VASP), a critical actin motor protein required for cell migration that also controls vasodilation and platelet aggregation.
We examined the effect of donor-released CO and NO in endothelial progenitor cells (EPCs) and platelets from nondiabetic and diabetic subjects and in human microvascular endothelial cells (HMECs) cultured under low (5.5 mmol/l) or high (25 mmol/l) glucose conditions. VASP phosphorylation was evaluated using phosphorylation site-specific antibodies.
In control platelets, CO selectively promotes phosphorylation at VASP Ser-157, whereas NO promotes phosphorylation primarily at Ser-157 and also at Ser-239, with maximal responses at 1 min with both agents on Ser-157 and at 15 min on Ser-239 with NO treatment. In diabetic platelets, neither agent resulted in VASP phosphorylation. In nondiabetic EPCs, NO and CO increased phosphorylation at Ser-239 and Ser-157, respectively, but this response was markedly reduced in diabetic EPCs. In endothelial cells cultured under low glucose conditions, both CO and NO induced phosphorylation at Ser-157 and Ser-239; however, this response was completely lost when cells were cultured under high glucose conditions. In control EPCs and in HMECs exposed to low glucose, VASP was redistributed to filopodia-like structures following CO or NO exposure; however, redistribution was dramatically attenuated under high glucose conditions.
Vasoactive gases CO and NO promote cytoskeletal changes through site- and cell type-specific VASP phosphorylation, and in diabetes, blunted responses to these agents may lead to reduced vascular repair and tissue perfusion.
[Show abstract][Hide abstract] ABSTRACT: The mechanisms that allow Mycobacterium tuberculosis (Mtb) to persist in human tissue for decades and to then abruptly cause disease are not clearly understood. Regulatory elements
thought to assist Mtb to enter such a state include the heme two-component sensor kinases DosS and DosT and the cognate response regulator DosR.
We have demonstrated previously that O2, nitric oxide (NO), and carbon monoxide (CO) are regulatory ligands of DosS and DosT. Here, we show that in addition to O2 and NO, CO induces the complete Mtb dormancy (Dos) regulon. Notably, we demonstrate that CO is primarily sensed through DosS to induce the Dos regulon, whereas
DosT plays a less prominent role. We also show that Mtb infection of macrophage cells significantly increases the expression, protein levels, and enzymatic activity of heme oxygenase-1
(HO-1, the enzyme that produces CO), in an NO-independent manner. Furthermore, exploiting HO-1+/+ and HO-1-/- bone marrow-derived macrophages, we demonstrate that physiologically relevant levels of CO induce the Dos regulon. Finally,
we demonstrate that increased HO-1 mRNA and protein levels are produced in the lungs of Mtb-infected mice. Our data suggest that during infection, O2, NO, and CO are being sensed concurrently rather than independently via DosS and DosT. We conclude that CO, a previously
unrecognized host factor, is a physiologically relevant Mtb signal capable of inducing the Dos regulon, which introduces a new paradigm for understanding the molecular basis of Mtb persistence.
[Show abstract][Hide abstract] ABSTRACT: Induction of heme oxygenase-1 (HO-1) is associated with potential antifibrogenic effects. The effects of HO-1 expression on epithelial-mesenchymal transition (EMT), which plays a critical role in the development of renal fibrosis, are unknown. In this study, HO-1(-/-) mice demonstrated significantly more fibrosis after 7 d of unilateral ureteral obstruction compared with wild-type mice, despite similar degrees of hydronephrosis. The obstructed kidneys of HO-1(-/-) mice also had greater macrophage infiltration and renal tubular TGF-beta1 expression than wild-type mice. In addition, the degree of EMT was more extensive in obstructed HO-1(-/-) kidneys, as assessed by alpha-smooth muscle actin and expression of S100A4 in proximal tubular epithelial cells. In vitro studies using proximal tubular cells isolated from HO-1(-/-) and wild-type kidneys confirmed these observations. In conclusion, HO-1 deficiency is associated with increased fibrosis, tubular TGF-beta1 expression, inflammation, and enhanced EMT in obstructive kidney disease. Modulation of the HO-1 pathway may provide a new therapeutic approach to progressive renal diseases.
Preview · Article · Jun 2008 · Journal of the American Society of Nephrology
[Show abstract][Hide abstract] ABSTRACT: Angiogenesis involves the formation of new blood vessels and is critical for fundamental events such as development and repair after injury. Perturbances in angiogenesis contribute to the pathogenesis of diverse clinical conditions including cancer, complications of diabetes mellitus, ischemia/reperfusion injury of the heart and other organs, and preeclampsia, as well as a number of inflammatory disorders. Recent work has identified heme oxygenase-1 and its gaseous product, carbon monoxide, to possess potent proangiogenic properties in addition to well-recognized antiinflammatory, antioxidant, and antiapoptotic effects. Angiogenic factors, such as vascular endothelial growth factor and stromal cell-derived factor-1, mediate their proangiogenic effects through induction of heme oxygenase-1, making it an attractive target for therapeutic intervention. This review will provide an overview of the role of heme oxygenase-1 and carbon monoxide in angiogenesis.
[Show abstract][Hide abstract] ABSTRACT: Growth factors are mediators of both normal homeostasis and pathophysiology through their effects on various cellular processes. Similarly, heme oxygenase-1 (HO-1) has a role in maintaining physiologic equilibrium, by which it can either alleviate or exacerbate disease, depending on several considerations, including amount, timing, and location of expression, as well as the disease setting. Thus, the synthesis and activities of growth factors and HO-1 are intricately regulated. Interestingly, several growth factors induce HO-1, and, conversely, HO-1 can regulate the expression of some growth factors. This review focuses on the influence of growth factors and HO-1 and potential physiologic effects of the growth factor(s)-HO-1 interaction.
No preview · Article · Jan 2008 · Antioxidants and Redox Signaling
[Show abstract][Hide abstract] ABSTRACT: Accurate determination of renal function in mice is a major impediment to the use of murine models in acute kidney injury. The purpose of this study was to determine whether early changes in renal function could be detected using dynamic gamma camera imaging in a mouse model of ischemia-reperfusion (I/R) injury. C57BL/6 mice (n = 5/group) underwent a right nephrectomy, followed by either 30 min of I/R injury or sham surgery of the remaining kidney. Dynamic renal studies (21 min, 10 s/frame) were conducted before surgery (baseline) and at 5, 24, and 48 h by injection of (99m)Tc-mercaptoacetyltriglycine (MAG3; approximately 1.0 mCi/mouse) via the tail vein. The percentage of injected dose (%ID) in the kidney was calculated for each 10-s interval after MAG3 injection, using standard region of interest analyses. A defect in renal function in I/R-treated mice was detected as early as 5 h after surgery compared with sham-treated mice, identified by the increased %ID (at peak) in the I/R-treated kidneys at 100 s (P < 0.01) that remained significantly higher than sham-treated mice for the duration of the scan until 600 s (P < 0.05). At 48 h, the renal scan demonstrated functional renal recovery of the I/R mice and was comparable to sham-treated mice. Our study shows that using dynamic imaging, renal dysfunction can be detected and quantified reliably as early as 5 h after I/R insult, allowing for evaluation of early treatment interventions.
Full-text · Article · Nov 2007 · American journal of physiology. Renal physiology
[Show abstract][Hide abstract] ABSTRACT: Recent studies in our laboratory using the Zucker obese (ZO) and Zucker diabetic fatty (ZDF) rat models resulted in unexpectedly high mortality rates in all genotypes including healthy homozygous lean Zucker rats, possibly because of renal dysfunction. Therefore, we evaluated left ventricular (LV) and kidney morphology and function in young ZO, Zucker diabetic fatty obese (ZDFO), homozygous Zucker/ZDF lean (ZL), and Sprague-Dawley (SD) rats. Hydronephrosis was evident in ZL, ZO, and ZDFO but not SD kidneys. ZDFO rats exhibited impaired LV shortening and relaxation with increased arterial stiffness. LV wall thickness was lower and LV end-systolic wall stress was higher in ZDFO compared with SD rats. Plasma ANG II was lower in ZO and ZDFO rats, which may be a result of reduced renal parenchyma with hydronephrosis; norepinephrine was higher in ZDFO rats than SD controls. Covariate analysis indicated that LV end-systolic wall stress was associated with renal dysfunction. The presence of hydronephrosis and its association with LV dysfunction potentially limits the ZDF model for study of the effects of diabetes on renal and cardiovascular function.
No preview · Article · Aug 2007 · AJP Heart and Circulatory Physiology
[Show abstract][Hide abstract] ABSTRACT: Heme oxygenase (HO-1) is the rate-limiting enzyme in the catabolism of heme, which leads to the generation of biliverdin, iron, and carbon monoxide. It has been shown to have important antioxidant and antiinflammatory properties that result in a vascular antiatherogenic effect. To determine whether HO-1 expression in macrophages constitutes a significant component of the protective role in atherosclerosis, we evaluated the effect of decreased or absent HO-1 expression in peritoneal macrophages on oxidative stress and inflammation in vitro, and the effect of complete deficiency of HO-1 expression in macrophages in atherosclerotic lesion formation in vivo. We found that compared with HO-1(+/+) controls, peritoneal macrophages from HO-1(-/-) and HO-1(+/-) mice exhibited (1) increased reactive oxygen species (ROS) generation, (2) increased proinflammatory cytokines such as monocyte chemotactic protein 1 (MCP-1) and interleukin 6 (IL-6), and (3) increased foam cell formation when treated with oxLDL, attributable in part to increased expression of scavenger receptor A (SR-A). Bone marrow transplantation experiments performed in lethally irradiated LDL-R null female mice, reconstituted with bone marrow from HO-1(-/-) versus HO-1(+/+) mice, revealed that HO-1(-/-) reconstituted animals exhibited atherosclerotic lesions with a greater macrophage content as evaluated by immunohistochemistry and planimetric assessment. We conclude that HO-1 expression in macrophages constitutes an important component of the antiatherogenic effect by increasing antioxidant protection and decreasing the inflammatory component of atherosclerotic lesions.
Full-text · Article · Jul 2007 · Circulation Research
[Show abstract][Hide abstract] ABSTRACT: Heme oxygenase-1 is a highly inducible gene, the product of which catalyzes breakdown of the prooxidant heme. The purpose of this study was to investigate the regulation of the human heme oxygenase-1 gene in renal epithelial cells. DNase I hyper-sensitivity studies identified three distal sites (HS-2, -3, and -4) corresponding to approximately -4.0, -7.2, and -9.2 kb, respectively, of the heme oxygenase-1 promoter in addition to one proximal region, HS-1, which we have shown previously to be an E box. In vivo dimethyl sulfate footprinting of the HS-2 region revealed six individual protected guanines. Two mutations within HS-2 combined with a third mutation of the proximal E box abolished hemin- and cadmium-driven heme oxygenase-1 promoter activation, suggesting that these three sites synergized for maximal heme oxygenase-1 induction. Jun proteins bound to the antioxidant response element in the HS-2 region in vitro and associated with the heme oxygenase-1 promoter in vivo. JunB and JunD contribute opposing effects; JunB activated whereas JunD repressed heme oxygenase-1 expression in human renal epithelial cells, results that were corroborated in junB(-)(/)(-) and junD(-)(/)(-) cells. We propose that heme oxygenase-1 induction is controlled by a dynamic interplay of regulatory proteins, and we provide new insights into the molecular control of the human heme oxygenase-1 gene.
[Show abstract][Hide abstract] ABSTRACT: Stromal cell-derived factor 1 (SDF-1) plays a major role in the migration, recruitment, and retention of endothelial progenitor cells to sites of ischemic injury and contributes to neovascularization. We provide direct evidence demonstrating an important role for heme oxygenase 1 (HO-1) in mediating the proangiogenic effects of SDF-1. Nanomolar concentrations of SDF-1 induced HO-1 in endothelial cells through a protein kinase C zeta-dependent and vascular endothelial growth factor-independent mechanism. SDF-1-induced endothelial tube formation and migration was impaired in HO-1-deficient cells. Aortic rings from HO-1(-/-) mice were unable to form capillary sprouts in response to SDF-1, a defect reversed by CO, a byproduct of the HO-1 reaction. Phosphorylation of vasodilator-stimulated phosphoprotein was impaired in HO-1(-/-) cells, an event that was restored by CO. The functional significance of HO-1 in the proangiogenic effects of SDF-1 was confirmed in Matrigel plug, wound healing, and retinal ischemia models in vivo. The absence of HO-1 was associated with impaired wound healing. Intravitreal adoptive transfer of HO-1-deficient endothelial precursors showed defective homing and reendothelialization of the retinal vasculature compared with HO-1 wild-type cells following ischemia. These findings demonstrate a mechanistic role for HO-1 in SDF-1-mediated angiogenesis and provide new avenues for therapeutic approaches in vascular repair.
Full-text · Article · Apr 2007 · Journal of Experimental Medicine
[Show abstract][Hide abstract] ABSTRACT: Exogenous bilirubin (BR) substitutes for the protective effects of heme oxygenase (HO) in several organ systems. Our objective was to investigate the effects of exogenous BR in an in vivo model of ischemia-reperfusion injury (IRI) in the rat kidney. Four groups of male Sprague-Dawley rats were anesthetized using isoflurane in oxygen and treated with 1) 5 mg/kg intravenous (iv) BR, 1 h before ischemia and 6-h reperfusion; 2) vehicle 1 h before ischemia and 6-h reperfusion; 3) 20 mg/kg iv BR, 1 h before and during ischemia; and 4) vehicle 1 h before and during ischemia. Bilateral renal clamping (30 min) was followed by 6-h reperfusion. Infusion of 5 mg/kg iv BR achieved target levels in the serum at 6 h postischemia (31 +/- 9 micromol/l). Infusion of 20 mg/kg BR reached 50 +/- 22 micromol/l at the end of ischemia, and a significant improvement was seen in serum creatinine at 6 h (1.07 +/- 28 vs. 1.38 +/- 0.18 mg/dl, P = 0.043). Glomerular filtration rate, estimated renal plasma flow, fractional excretion of electrolytes, and renal vascular resistance were not significantly improved in BR-treated groups. Histological grading demonstrated a trend toward preservation of cortical proximal tubules in rats receiving 20 mg/kg iv BR compared with control; however, neither BR dose provided protection against injury to the renal medulla. At the doses administered, iv BR did not provide complete protection against IRI in vivo. Combined supplementation of both BR and carbon monoxide may be required to preserve renal blood flow and adequately substitute for the protective effects of HO in vivo.
Preview · Article · Mar 2007 · American journal of physiology. Renal physiology
[Show abstract][Hide abstract] ABSTRACT: We describe a 59-year-old patient who developed acute renal failure because of rhabdomyolysis after extensive red fire ant bites. This case illustrates a serious systemic reaction that may occur from fire ant bites. Consistent with the clinical presentation in rhabdomyolysis associated with non-traumatic causes, hyperkalemia, hypophosphatemia, hypocalcemia, and high anion gap acidosis were not observed in this patient. While local allergic reactions to fire ant bites are described in the literature, serious systemic complications with rhabdomyolysis and renal failure have not been previously reported. It is our effort to alert the medical community of the possibility of such a complication that can occur in the victims of fire ant bites.
Preview · Article · Feb 2007 · Journal of General Internal Medicine
[Show abstract][Hide abstract] ABSTRACT: Nitroalkene derivatives of linoleic acid (LNO2) and oleic acid (OA-NO2) are present; however, their biological functions remain to be fully defined. Herein, we report that LNO2 and OA-NO2 inhibit lipopolysaccharide-induced secretion of proinflammatory cytokines in macrophages independent of nitric oxide formation, peroxisome proliferator-activated receptor-gamma activation, or induction of heme oxygenase-1 expression. The electrophilic nature of fatty acid nitroalkene derivatives resulted in alkylation of recombinant NF-kappaB p65 protein in vitro and a similar reaction with p65 in intact macrophages. The nitroalkylation of p65 by fatty acid nitroalkene derivatives inhibited DNA binding activity and repressed NF-kappaB-dependent target gene expression. Moreover, nitroalkenes inhibited endothelial tumor necrosis factor-alpha-induced vascular cell adhesion molecule 1 expression and monocyte rolling and adhesion. These observations indicate that nitroalkenes such as LNO2 and OA-NO2, derived from reactions of unsaturated fatty acids and oxides of nitrogen, are a class of endogenous anti-inflammatory mediators.
Full-text · Article · Dec 2006 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Induction of heme oxygenase-1 (HO-1) in renal tubules occurs as an adaptive and beneficial response in acute renal failure (ARF) following ischemia and nephrotoxins. Using an in vitro model of polarized Madin-Darby canine kidney (MDCK) epithelial cells, we examined apical and basolateral cell surface sensitivity to HO-1 induction by heme. Basolateral exposure to 5 microM hemin (heme chloride) resulted in higher HO-1 induction than did apical exposure. The peak induction of HO-1 by basolateral application of hemin occurred between 12 and 18 h of exposure and was dose dependent. Similar cell surface sensitivity to hemin-induced HO-1 expression was observed using a mouse cortical collecting duct cell line (94D cells). Hepatocyte growth factor (HGF) is known to decrease cell polarity of MDCK cells. Following pretreatment with HGF, apically applied hemin gave greater stimulation of HO-1 expression, whereas HGF alone did not induce HO-1. We also examined the effect of hypoxia on hemin-mediated HO-1 induction. MDCK cells were subjected to hypoxia (1% O(2)) for 24 h to simulate the effects of ischemic ARF. Under hypoxic conditions, both apical as well as basolateral surfaces of MDCK were more sensitive to HO-1 induction by hemin. Hypoxia alone did not induce HO-1 but appeared to potentiate both apical and basolateral sensitivity to hemin-mediated induction. These data demonstrate that the induction of HO-1 expression in polarized renal epithelia by heme is achieved primarily via basolateral exposure. However, under conditions of altered renal epithelial cell polarity and hypoxia, increased HO-1 induction occurs following apical exposure to heme.
No preview · Article · Nov 2006 · American journal of physiology. Renal physiology
[Show abstract][Hide abstract] ABSTRACT: Renal tubular cells elicit adaptive responses following exposure to nephrotoxins, such as cadmium. One response is the up-regulation of the 32-kDa redox-sensitive protein, heme oxygenase-1. Exposure of renal proximal tubular epithelial cells to 10 mum cadmium demonstrated induction ( approximately 20-fold) of heme oxygenase-1 mRNA and protein. Using a 4.5-kb human heme oxygenase-1 promoter construct, the importance of a previously identified cadmium response element (TGCTAGAT) in HeLa cells was verified in renal epithelial cells. Specific protein-DNA interaction with this sequence was demonstrated using nuclear extracts from cadmium-treated cells. Yeast one-hybrid screen of a human kidney cDNA library resulted in the identification of pescadillo, a unique nucleolar, developmental protein, as an interacting protein with the cadmium response element and was confirmed by chromatin immunoprecipitation in vivo and gel shift assays with purified glutathione S-transferase-pescadillo protein in vitro. The specificity of the DNA-protein interaction was verified by the absence of a binding complex when the core sequence of the cadmium response element was mutated or deleted. In addition, B23/nucleophosmin, another nucleolar protein, did not interact with the cadmium response sequence. Overexpression of pescadillo resulted in increased activity of the 4.5-kb human heme oxygenase-1 promoter construct but failed to activate this construct when the cadmium response sequence was mutated. The findings demonstrate the important and previously unrecognized role of pescadillo as a DNA-binding protein interacting specifically with the cadmium response element of the human heme oxygenase-1 gene.
No preview · Article · Sep 2006 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: A combination of gene and cell-based therapies may provide significant advantages over existing treatments in terms of their effectiveness. However, long-term efficient gene delivery has been difficult to achieve in many cell types, including endothelial cells. We developed a freeze-thaw technique which significantly increases the transduction efficiency of recombinant adeno-associated virus vectors in human aortic endothelial cells (23-fold) and in human renal proximal tubular epithelial cells (128-fold) in comparison to current methods for transduction. Freeze-thaw resulted in a transient but significant increase in cell surface area by 1,174 +/- 69.8 microM2 per cell. Reduction of cryogenic medium volume and repeated freeze-thaw further increased transduction efficiency by 2.8- and 2.4-fold, respectively. Trypsinization, dimethylsulfoxide, and cold temperatures, which are also involved in cell preservation, had no significant impact on transduction efficiency. Increased transduction was also observed in mesenchymal stem cells (42-fold) by the freeze-thaw method. The potential mechanism of this novel technique likely involves an increase in the net permeable area of biological membranes caused by water crystallization. These findings provide a new approach for gene delivery in various cell types, particularly in those resistant to transduction by conventional methods.