Nevertheless, some studies have documented important additional biochemical actions of nitroxides to facilitate metabolism of a wide range of ROS and reactive nitrogen species. Indeed, a comparison of the efficacy of tempol in metabolizing cellular O₂^{. −} or H₂O₂ or in protecting cells from damaging effects of hydroxyl radical (·OH) demonstrated that the sensitivity of tempol was greatest for ·OH, intermediate for H₂O₂ and least for O₂^{. −}. Tempol is effective as a catalase-like agent and in preventing the generation of ·OH from H₂O₂ in the presence of transition metals in the Fenton reaction (Soule et al., 2007; Wilcox & Pearlman, 2008). Thus, tempol is best considered a general purpose redox cycling agent rather than a specific SOD mimetic (Wilcox & Pearlman, 2008). Tempol is an ampholyte with a high capacity to permeate cell membranes, the gastrointestinal tract (GIT) or the blood-brain barrier which accounts for its effectiveness after oral administration and its central nervous system actions.

The reaction between O₂^{. −} and nitric oxide (NO) yields peroxynitrite (ONOO^-) which is highly oxidant. Carroll et al demonstrated that tempol inhibited nitration of test compounds by a large molar excess of ONOO^- and was recovered intact as tempol thereafter, leading to the conclusion that tempol acted catalytically (Carroll et al., 2000). The reaction entailed oxidation of tempol by ONOO^- derived radicals such as ·OH, carbonate or nitrogen dioxide radicals to the oxammonium cation which itself was reduced back to tempol while oxidizing ONOO^- to oxygen (O₂) and NO. The details of these interactions were given by Carroll et al (Carroll et al., 2000) and were confirmed by Bonini et al (Bonini et al., 2002). Since peroxynitrite is implicated in many pathophysiologic conditions of oxidative stress, this is likely an important mechanism of tissue protection by tempol as, for example, in traumatic brain injury (Deng-Bryant et al., 2008; Xiong & Hall, 2009).

Nishiyama and colleagues demonstrated that tempol protected the kidneys from glomerulosclerosis in aldosterone-and salt-dependent models of hypertension (Nishiyama & Abe, 2004; Nishiyama et al., 2004a; Nishiyama et al., 2004b) and protected the heart in models of cardiac hypertrophy and cardiac failure (Zhang et al., 2005a; Guo et al., 2006). They demonstrated the importance of the mitogen-activated protein kinase pathway as a target for the tissue protective effects of tempol (Zhang et al., 2004; Nishiyama & Abe, 2004; Nishiyama et al., 2004a; Nishiyama et al., 2004b; Zhang et al., 2005a). Fujita and colleagues demonstrated that a reduction of ROS with tempol in models of mineralocorticosteroid excess and salt prevented the mineralocorticosteroid receptor signalling and preserved podocyte function in the glomerulus. They demonstrated that tempol had an anti-proteinuric action and preserved cardiac diastolic relaxation and thereby prevented diastolic heart failure (Nagase et al., 2007; Matsui et al., 2008; Nagase & Fujita, 2008; Wang et al., 2008a) in models of the metabolic syndrome (Furukawa et al., 2004; Nagase et al., 2006; Ando & Fujita, 2009) and diabetes mellitus (DM) (Asaba et al., 2007).

Schnackenberg, Welch, Wilcox et al related the biological effects of acute intravenous nitroxides in hypertensive models to their in vitro SOD mimetic activity (Patel et al., 2006), to the generation of vascular H₂O₂ (Chen et al., 2007c), to enhancement of NO signaling (Schnackenberg et al., 1998; Welch & Wilcox, 2001), to inhibition of the sympathetic nervous system and to activation of adenosine triphosphate-dependent potassium channels (Chen et al., 2007b). The effects of more prolonged oral tempol administration were not accompanied by increased vascular H₂O₂ (Chen et al., 2007c) but were related to correction of oxidative stress (Schnackenberg & Wilcox, 1999) and salt sensitivity (Welch et al., 2005b). They reported that tempol improved the efficiency with which the hypertensive rat kidney used oxygen for tubular sodium transport and thereby improved the oxygen tension in the renal cortex and medulla (Welch et al., 2003; Welch et al., 2005a). They showed further that tempol restored the endothelium dependent relaxation/nitric oxide (EDRF/NO) responses and diminished the endothelium dependent contraction factor (EDCF) responses in the renal afferent arterioles and mesenteric resistance vessels in models of oxidative stress and restored NO signalling between renal tubular epithelial cells in the macula densa and the renal afferent arteriole (Wilcox & Welch, 2000; Welch et al., 2000; Schnackenberg et al., 2000; Schnackenberg & Wilcox, 2001; Welch & Wilcox, 2001; Wang et al., 2003a; Wang et al., 2004; Wang et al., 2006). This restoration by tempol of NO signalling within the juxtaglomerular apparatus may maintain renal blood flow and protect against salt-dependent increases in BP.

In an extensive set of studies of the protective roles of tempol, Thiemermann et al reported that pretreatment with tempol prevented ischemia/reperfusion damage in the kidney (Chatterjee et al., 2000; Patel et al., 2002; Thiemermann, 2003), heart (McDonald et al., 1999; McCormick et al., 2006), liver (Sepodes et al., 2004) and brain (Cuzzocrea et al., 2000c), prevented experimental colitis (Cuzzocrea et al., 2000b), arthritis (Cuzzocrea et al., 2000d) and pleurisy (Cuzzocrea et al., 2000a) and provided protection against shock from hemorrhage (Mota-Filipe et al., 1999), gram-positive (Zacharowski et al., 2000) or gram-negative (Leach et al., 1998) bacteria and multiple organ failure (Cuzzocrea et al., 2001).

PON-1 is a circulating protein that has been associated with reduced rates of coronary events and atherosclerosis (Mackness et al., 2003). Tempol blocked the effect of leptin infusion to reduce PON-1 activity in the kidneys and aorta (Beltowski et al., 2005b). Thus, increased leptin generation in the metabolic syndrome may contribute to oxidative stress and a reduction in PON-1, high-density lipoproteins and NO generation that can be corrected by tempol.

Adiponectin is an adipokine that counters inflammation, atherosclerosis and insulin resistance (Ouchi et al., 2000; Ouchi et al., 2001). Circulating levels of adiponectin were reduced in patients or animal models of obesity, diabetes or acute coronary syndromes (Kumada et al., 2003). However, subsequent studies reported that plasma levels of adiponectin were increased in patients with stable congestive heart failure (CHF) (George et al., 2006) in proportion to the severity of the CHF (Nakamura et al., 2006). In patients with CHF, serum adiponectin was positively correlated with high density lipoprotein cholesterol and negatively with plasma triglycerides (von Eynatten et al., 2006). The authors concluded that adiponectin may mediate part of its proposed anti-atherosclerotic properties by influencing high density lipoprotein cholesterol concentrations and might be a therapeutic target for atherogenic dyslipidemia. Oral tempol (2 mmol · l^-1) or tetrahydrobiopterin given to rats prevented the effects of Ang II infusion to reduce the plasma levels and adipose tissue expression of adiponectin (Hattori et al., 2005). However, until a clearer picture emerges about the benefits or adverse effects of adiponectin, it is hard to predict whether a suppression of adiponectin by tempol would be of benefit or harm.

The oral administration of tempol (1 mmol · l^-1 of drinking water for ten weeks) to obese/Zucker rats fed a high fat diet reduced the abdominal fat and the weight gain, without changing the food intake (Ebenezer et al., 2009). This suggests that tempol may modify fat metabolism to limit the abdominal obesity that occurs with excessive caloric intake and which contributes to insulin resistance. Mice fed tempol from birth had a reduced weight gain, but appeared fully healthy (Mitchell et al., 2003; Schubert et al., 2004).

Tempol blocked the cardiac fibrosis, myofibroblast proliferation and cardiac collagen accumulation produced by an infusion of Ang II (Zhao et al., 2008b). The administration of tempol and vitamin C to rats after NOS blockade moderated the cardiac hypertrophy and the adrenomedullin signalling without reducing the hypertension (Bell et al., 2007). These studies established the efficacy of tempol in preventing cardiac ROS, fibrosis, and apoptosis in several rodent models associated with lipopolysaccharide, cytokines, reduced NO or prolonged Ang II.

In other models, ROS promoted apoptosis and decreased cardiac contractility. Thus, tempol reduced ROS production and apoptosis in cardiac cells exposed to high glucose concentrations (Fiordaliso et al., 2007), blocked apoptotic responses of cardiomyocytes to aldosterone signaling via a non-genomic pathway (Hayashi et al., 2008) and inhibited the Ca⁺⁺ transient within cardiac myosites stimulated by pressure-flow stress (Belmonte & Morad, 2008).

These different effects of tempol may relate to preservation of NO which has complicated effects on cardiomyocytes because of spatial confinement and differential effects of NO generation by specific NOS isoforms (Barouch et al., 2002). For example, Gonzales et al (Gonzalez et al., 2008) studied the acute effect of tempol on the ionotropic state of the rat isolated heart. The addition of 100 μM tempol did not affect contractility but tempol did prevent the increased contractility caused by a modest concentration of a NO donor (Paolocci et al., 2000; Espey et al., 2002; Schrammel et al., 2003; Koshiishi et al., 2007) yet did not prevent the increased contractility with higher concentrations of NO (Gonzalez et al., 2008).

Tempol has been effective in protecting the heart against injury and dysfunction following ischemia and reperfusion or myocardial infarction. Tempol prevented post-ischemic activation of NADPH oxidase and xanthine oxidase in the guinea pig (Duda et al., 2007) or rat heart or coronary vessels (Zhang et al., 2006). Tempol given to rats or rabbits after coronary ligation and reperfusion reduced the infarct size of the myocardium by up to 60% when given during the reperfusion period (McDonald et al., 1999; Zacharowski et al., 2007), protected the heart against oxidative damage due to ischemia-reperfusion injury (Zeltcer et al., 1997; McDonald et al., 1999; Zeltcer et al., 2002; Li et al., 2002; Hoffman et al., 2003; Kutala et al., 2006; McCormick et al., 2006), and prevented reperfusion arrhythmias in one study (Guo et al., 2005) but did not change, or increased, the frequency of ventricular arrhythmias in another (Neckar et al., 2008). Long term administration of oral tempol (2 mmol · l^-1) for six weeks after coronary artery ligation prevented the signs of cardiac dysfunction and failure manifest as an increase in left ventricular end diastolic pressure and volume, reduced ejection fracton and enhanced renal sympathetic nerve activity and plasma norepinephrine (Shi et al., 2009).

A note of caution derives from the report of Kimura et al in the rat (Kimura et al., 2005b) that a 30 minute pressor infusion of Ang II prior to acute coronary occlusion diminished the infarct size. This pharmacologic preconditioning depended on the generation of ROS and was prevented by tempol.

Diastolic heart failure which accounts for almost one half of the cases of chronic congestive cardiac failure is a complication of hypertensive or ischemic cardiac disease and remodeling that leads to impaired diastolic relaxation. Presently, the therapy for diastolic heart failure is unsatisfactory. Tempol has been effective in preventing or reversing cardiac hypertrophy and diastolic dysfunction in several rat models of salt sensitive hypertension. Tempol (3 mmol · l^-1) added to the drinking water of DSS rats fed a high salt diet for 10 weeks reduced the BP moderately, but normalized the left ventricular hypertrophy and cardiac relaxation and reduced the cardiac expression of brain natriuretic peptide, TGFβ, connective tissue growth factor, collagen types I and III, the p22^phox and neutrophil oxidase (Nox)-2 components of NADPH oxidase and mitochondrial uncoupling protein 2 (Guo et al., 2006). Thus, inhibition of cardiac ROS by tempol prevented the cardiac fibrosis, remodeling and defective relaxation that underlie diastolic heart failure. Indeed, four weeks of oral tempol (1 mmol · l^-1) or eplerenone to SHR given a high salt diet, which is a model of the metabolic syndrome, moderated the diastolic dysfunction when assessed directly by echocardiography and cardiac catheterization, and prevented the cardiac perivascular fibrosis and upregulation of the mineralocorticosteroid signaling pathways (Matsui et al., 2008) (Figure 6). Likewise, oral tempol or eplerenone given for six weeks to rats infused with Ang II and fed a high salt diet prevented the diastolic dysfunction and cardiac oxidative stress in this model also (Wang et al., 2008a). Oral tempol given to DSS rats fed a high salt diet prevented the development of left ventricular dilation and cardiac failure (Hasegawa et al., 2006) and, when given to rats infused with aldosterone and fed a high salt diet, reduced the collagen accumulation in the kidney and the fibrosis in the heart and aorta substantially (Iglarz et al., 2004). Thus tempol can prevent the development of left ventricular hypertrophy, cardiac inflammation and fibrosis and diastolic dysfunction and cardiac failure in salt sensitive rodent models by actions that may entail interruption of mineralocorticosteroid receptor signaling pathways.

High sugar diets increase mortality and left ventricular dysfunction in models of pressure overload. Tempol given to fructose-fed mice with pressure overload prevented the cardiac ROS accumulation, hypertrophy and impaired left ventricular ejection fraction whereas these effects were not seen in mice fed a low sugar chow (Chess et al., 2008). Another study of fructose-fed mice with pressure overload reported that ROS were generated in the heart from uncoupled NOS (Moens et al., 2008). Tetrahydrobiopterin recoupled NOS and diminished the late development of cardiac fibrosis and hypertrophy whereas oral tempol did not recouple NOS was not effective in preventing the adverse cardiac changes. Thus, in this model, the main problem appears to be ROS generated from an uncoupled NOS and preventing this was more effective than a general antioxidant strategy with tempol.

The administration of tempol to several rat models of hypertension not associated with salt loading has not shown benefit in preventing cardiac hypertrophy or dysfunction. The administration of tempol (3 mmol · l^-1) for 2 weeks to rats infused with aldosterone but maintained on a normal salt diet prevented hypertension but failed to prevent the myocardial changes, in contrast to treatment with another antioxidant NAC (Yoshida et al., 2005). Tempol infused into rats with the beta-adrenergic agonist, isoproterenol prevented the increased cardiac collagen accumulation but not the cardiac hypertrophy (Zhang et al., 2005a). Likewise, oral tempol given to rats infused with thyroxine for 6 weeks did not prevent the cardiac hypertrophy, despite a substantial reduction in the MAP and indices of oxidative stress (Moreno et al., 2005). It is not clear presently why the beneficial cardiac effects of tempol in models of hypertension appear specific to those associated with high salt intake but this may relate to interruption of MR signaling pathways that are activated in these salt-sensitive models (Nagase & Fujita, 2008).

As reviewed under “3.1. Metabolic effects of tempol – Insulin resistance, metabolic syndrome and diabetes mellitus”, tempol protected the heart from some of the adverse effects of diabetes type 1 or 2. For example, an infusion of tempol (550 μmol · kg^-1) into hyperglycemic dogs normalized their coronary endothelial dysfunction and coronary wall oscillatory shear stress (Gross et al., 2003). Tempol given to SHR made diabetic with STZ reduced the cardiac indices of ROS and the cardiac apoptosis (Fiordaliso et al., 2007).

Repeated exposure of rat isolated aortic segments to nitrates led to tolerance as shown by diminishing relaxation that was corrected by tempol. This was ascribed to a reduction in excessive H₂O₂ accumulation, since the beneficial effects of tempol were prevented by catalase (Ghatta et al., 2007). Pretreatment of rat aortic rings with tempol, vitamin C, uric acid, or the PKC inhibitor, chelerythrine all prevented the development of tolerance to the vasodilating actions of nitroglycerin (bou-Mohamed et al., 2004). Thus, the prevention of nitrate tolerance by tempol likely involves a prevention of H₂O₂-induced activation of PKC signalling.

Many investigators have studied the effects of tempol on central sympathetic stimulation, since excitation of the SNS during heart failure predisposes to bad outcomes. A rabbit model of chronic heart failure from paced ventricular tachycardia had enhanced renal sympathetic nerve activity which was reduced by intracerebroventricular (icv) administration of an ARB, an NADPH oxidase inhibitor, apocynin or tempol (Gao et al., 2004). The addition of tempol or apocynin to Ang II-stimulated cultured neuronal cells reversed the increased expression of the AT1-R, and NADPH oxidase activity (Liu et al., 2008). The rostral ventrolateral medulla (RVLM) and paraventricular nucleus (PVN) are brain stem centres that regulate the sympathetic neural discharge. The 2K,1C Goldblatt model of Ang II excess had increased expression of the AT1-R and NADPH oxidase subunits in these nuclei (Oliveira-Sales et al., 2009). Injection of tempol (1 to 5 mmol · l^-1) into the RVLM of these rats reduced their BP and renal sympathetic nerve discharge (Oliveira-Sales et al., 2009). Moreover, prolonged Ang II infusion into rats increased the expression of NFκB and AT1-R in the PVN, and increased the BP, plasma cytokines, renal sympathetic nerve activity and plasma norepinephrine (Kang et al., 2009). The infusion of tempol (80 μg · h^-1) into the PVN reduced these parameters which was blocked by the NFκB antagonist pyrrolidine dithiocarbamate. The authors concluded that tempol prevented Ang II-induced superoxide formation in these brain stem centers that activated NFκB-induced sympathoexcitation (Kang et al., 2009). Indeed, intravenous tempol (120 μmol · kg^-1) given to normal or hypertensive rats reduced their BP, heart rate and renal sympathetic nerve activity concomitant with a significant reduction in the spontaneous discharge of neurons in the PVN and RVLM (Wei et al., 2009a). These effects of tempol were independ of NO since they occurred after nitric oxide synthase blockade and were ascribed to prevention of the formation of ·OH by tempol since they were prevented by systemic administration of ·OH scavenger, dimethyl sulfoxide (Wei et al., 2009a). Rats with heart failure due to coronary artery ligation had increased expression in the hypothalamic PVN of the AT1-R, pro-inflammatory cytokines, NADPH oxidase components and NFκB, and increased plasma levels of norepinephrine. Icv infusion of the ARB, losartan or of tempol (80 μg · h^-1) corrected these parameters and decreased plasma levels of Ang II and left ventricular end diastolic pressure (Kang et al., 2008). The authors concluded that O₂^{. −} stimulates NFκB in the PVN to sustain the neurohumoral activation that worsened heart failure. Similarly, an icv infusion of tempol for seven days into rabbits with congestive cardiac failure due to chronic ventricular tachycardia decreased the expression of AT1-Rs in the RVLM (Liu et al., 2008). Six weeks of oral tempol (2 mmol · l^-1) given to rats with a prior myocardial infarction caused by coronary artery ligation normalized renal sympathetic nerve discharge, plasma norepinephrine, left ventricular end diastolic pressure and contraction (Shi et al., 2009). Administration of tempol and apocynin to rabbits with chronic heart failure corrected the increased expression in the RVLM of the AT1-R (Shi et al., 2009) and the cytosolic phosphorylation of c-JUN N-terminal kinases protein and the activator protein-1 DNA binding activity (Liu et al., 2008).

Tempol also diminishes reflex activation of the SNS. The cardiac sympathetic afferent reflex which was activated by epicardial bradykinin or Ang II was enhanced in rats with chronic heart failure and led to enhanced renal sympathetic nerve activity (Ding et al., 2009). This was prevented by microinjection of tempol into the PVN (Han et al., 2007). Rabbits with chronic heart failure had increased peripheral chemoreceptor sensitivity that was corrected by local application of tempol to the carotid body chemoreceptor nerve (Li et al., 2007). These studies have established that ROS generated in brain stem cardiovascular nuclei can lead to sustained angiotensin-dependent activation of the SNS. Tempol, whether given systemically or into these nuclei, can prevent the activation of the SNS which might contribute to beneficial effects in models of heart failure where ongoing SNS drive in the heart limits cardiac function and in the kidney where the sympathic drive may contribute to azotemia and renal NaCl and fluid retention. The inhibition of the SNS by tempol during heart failure can involve reduced afferent input and reduced reflex activation as well as reduced brain-stem SNS drive.

Tempol has also been effective in reducing some manifestation of right heart failure. Tempol (500 μmol · kg^-1 · 24h^-1) normalized the right ventricular systolic pressure and reduced the right ventricular hypertrophy in a rat model of hypoxic right heart failure and pulmonary hypertension (Elmedal et al., 2004) and prevented hypoxic vasoconstriction of rat pulmonary arteries (Knock et al., 2009). Administration of tempol to neonatal rats subjected to hypoxia from birth limited pulmonary oxidative stress and attenuateal right ventricular remodeling. However, tempol slowed cellular differentiation in the distal air spaces of these hypoxic neonatal rats (Jankov et al., 2008). Tempol administration to transgenic Ren2 rats limited the increased pulmonary artery pressure and the hypertrophy of the pulmonary artery and right ventricle (DeMarco et al., 2008). Adrenomedullin is a vasodilator that activates antioxidant pathways whose expression in pulmonary vessels is upregulated by hypoxia (Matsui et al., 2004). Adrenomedullin +/- mice subjected to 3 weeks of hypoxia had enhanced pulmonary artery remodeling and ROS, both of which were normalized by the administration of tempol (Matsui et al., 2004). Tempol prevented the hypoxic pulmonary vasoconstriction, independent of NO, in isolated rat lungs (Hodyc et al., 2007). Thus, tempol may improve manifestations of right heart failure due to prolonged hypoxia, but experience in neonatal animals suggest that it might have adverse effects in reducing pulmonary cellular differentiation.

On the other hand, ROS generated in response to hyperglycemia (Zhang et al., 2008b) accelerated the senescence of stem and progenitor cells and inhibited angiogenesis (Dernbach et al., 2004). Tempol largely prevented the inhibition of proliferation of progenitor cells by high glucose concentrations (Zhang et al., 2008b). Other studies have documented a pro-angiogenic action of tempol that may relate to a decrease in O₂^.- and thereby to increased NO bioactivity. Ischemia of the hind-limb of mice increased oxidative stress, collateral vessel formation, capillary density and tissue blood flow and decreased NO bioactivity, apoptosis and the expression of extracellular SOD (Kim et al., 2007). Since these effects of ischemia were diminished in EC-SOD (-/-) mice, but were rescued by infusion of tempol, they were related to a reduction in vascular O₂^{. −} (Kim et al., 2007). An impaired collateral blood vessel growth after ileal artery occlusion in the SHR model of oxidative stress was restored by oral tempol (1 and 5 mmol · l^-1) or apocynin (NADPH oxidase inhibitor) but not by an angiotensin converting enzyme inhibitor or an ARB (Miller et al., 2007). Tempol promoted the healing response of capillary ECs after a scrape wound (Braunhut et al., 1996). Two weeks of treatment of salt-loaded stroke-prone spontaneously hypertensive rats (SHRsp) with an ARB (candesartan 1 mg · kg^-1 · day^-1) or tempol (5 mg · kg^-1 · day^-1) but not a diuretic (trichlormethiazide 1.6 mg · kg^-1 · day^-1) reduced markers of ROS and markedly increased circulating endothelial progenitor cells (Yu et al., 2008).

These conflicting effects with tempol on angiogenesis are not presently resolved. It may be that an inhibitory effect of tempol on angiogenesis is mediated by reduced tissue levels of H₂O₂ in some circumstances but a stimulant effect of tempol on angiogenesis is mediated by reduced tissue levels of O₂^{. −} and enhanced NO in others.

Vascular injury, diabetes, hyperperfusion or hypertension cause vascular remodeling that enhances contractility and resistance to flow (Folkow, 1978; Schiffrin, 2004) and contributes to accelerated neointimal growth and atherosclerosis (Tong et al., 2008). Vascular remodeling predicted poor cardiovascular outcomes in hypertensive patients and therefore is a target for therapeutic intervention (Schiffrin, 2001). Tempol diminished the enhanced VSMC medial migration and vascular remodeling in the injured arteries (Jagadeesha et al., 2009). Ligation of branches of the mesenteric artery caused high flow-induced vascular remodeling in surviving arteries that was diminished in obese Zucker diabetic rats but restored by three weeks of oral tempol (Belin de Chantemele et al., 2009). However, the administration of hydralazine or tempol for two weeks prevented the flow-induced remodeling of the mesenteric arteries of old rats (Dumont et al., 2008). The administration of tempol or apocynin to the adrenomedullin +/-mouse blocked the exaggerated generation of ROS and the intimal hyperplasia after femoral artery damage (Kawai et al., 2004). Oral tempol (1 mmol · l^-1) for six weeks given to salt-fed, SHRsp reduced the media:lumen ratio of small mesenteric resistance vessels (Park et al., 2002a). Oral administration of tempol (18 mg/kg/day) for 8 weeks to rats with 2K,1C Goldblatt hypertension corrected the increased media:lumen ratio of the aorta (vascular remodeling) whereas apocynin was ineffective despite a similar reduction in hypertension (Castro et al., 2009). Tempol corrected the effects of high medium glucose to increase migration of cultured VSMCs by preventing the oxidation and inactivation of the sarcoplasmic/endoplasmic reticulum calcium adenosine triphosphatase that mediates the inhibitory effects of NO on cell migration (Tong et al., 2008). Thus, tempol has proved effective in preventing or reversing the vascular remodeling accompanying vascular repair or prolonged hyperglycemia or hypertension in several models.

Ischemia-reperfusion injury of the brain produced by ligation and release of a cerebral artery, induced oxidative stress and increased cell [Ca⁺⁺] that contributed to the neuronal cell death (Cuzzocrea et al., 2000c; Schild & Reiser, 2005) and defective vascular regulation (Sun et al., 2008). Tempol diminished the cerebral oxidative stress that followed ischemia-reperfusion or hypoxia-reoxygenation injury to the brain and preserved neuronal viability (Rak et al., 2000; Cuzzocrea et al., 2000c; Leker et al., 2002; Behringer et al., 2002; Hu et al., 2003; Kato et al., 2003; Mehta et al., 2004; Schild & Reiser, 2005). Tempol infused one hour after middle cerebral arterial occlusion in SHR improved subsequent motor performance and cognitive function and reduced the cerebral infarct size (Leker et al., 2002). Tempol given intravenously to dogs prior to induced cardiac arrest provided substantial protection against the subsequent impairment of brain function (Behringer et al., 2002). Tempol was as effective as blockade of N-methyl-D-aspartate (NMDA) receptors with dexanabinol in neuroprotection after middle cerebral artery occlusion in the rat. This suggests that the neural protection provided by tempol entailed preservation of bioactive NO and/or a reduction in peroxinitrate. Indeed, tempol blocked the enhanced NMDA-induced neurotoxicity in rat cultured cortical brain cells following induction of inducible NOS, suggesting that ONOO^- was the damaging species (Hewett et al., 1994; Teichner et al., 2003).

A reduction in BP after an acute ischemic stroke can worsen the neurologic deficit because the blood flow to the ischemic brain tissue at the margin of the infarcted region is no longer autoregulated. Therefore, the benefits of tempol to protect the brain from ischemia-reperfusion injury might be limited by the associated hypotension. The nitroxide 3-carbamoyl-PROXYL given at the time of reperfusion caused dose-dependent cerebral protection without reducing the BP (Hu et al., 2003). As with other protective treatments, the time at which tempol was given was critical. Thus, iv tempol given at the time of reperfusion reduced lipid peroxidation in the brain and reduced the infract size, but if given 15 minutes after reperfusion, it was not effective (Kato et al., 2003).

Tempol protected cultured neuronal cells against hypoxia-reoxygenation damage (Tabakman et al., 2002; Yamada et al., 2003; Lang-Rollin et al., 2003) and prevented phosphorylation of ERK-1 and -2 in ischemic brain tissue (Wakade et al., 2008). Tanycytes cultured from the hypothalamus supported axonal regeneration but did not survive grafting to the brain because of sensitivity to ROS which was prevented by tempol and vitamin C (Prieto & Alonso, 1999). This suggests possible roles for tempol in protecting hypoxic neuronal cells and aiding regeneration in the brain.

ARBs may have a special protective role against stroke in patients with hypertension (Dahlöf et al., 2002). Tempol also had neuroprotective effects in a model of hypertension in the SHRsp infused with Ang II where tempol prevented cerebral neuronal cell loss and preserved the blood-brain barrier (Kim-Mitsuyama et al., 2005).

Tempol protected the brain or spinal cord from oxidative damage in several models of traumatic injury (Zhang et al., 1998; Trembovler et al., 1999; Hillard et al., 2004; Hillard et al., 2007). Tempol (300 mg · kg^-1) given to rats at the time of traumatic injury to the spinal cord (Xiong & Hall, 2009) or to the cerebral cortex (Deng-Bryant et al., 2008) reduced protein nitration and mitochondrial respiratory dysfunction and reduced calpain-mediated protein degradation. These effects were attributed to prevention of mitochondrial oxidative damage in the injured spinal cord (Patel et al., 2009) but were confined to a therapeutic window of thirty to sixty minutes after cord damage (Patel et al., 2009; Xiong & Hall, 2009). However, repeated doses of tempol after cerebral injury improved recovery of motor function (Merenda et al., 2008; Deng-Bryant et al., 2008). Tempol also was protective in transient focal cerebral ischemia (Rak et al., 2000) and prevented constriction of rat epineurial arterioles during exposure to high levels of oxygen (Sakai et al., 2007). A subdural hematoma induced cerebral oxidative stress and cerebral infarction that were reduced in rats given tempol (55 μmol · kg^-1 iv) (Kwon et al., 2003).

Tempol protected the eye against retinal apoptosis, loss of retinal neurones and ONOO^- accumulation after injection of NMDA (el-Remessy et al., 2003) and protected retinal blood vessels of rats from adverse effects of infused Ang II or from DM (Chen et al., 2007a). Oxidative damage of retinal pigment epithelial cells may underlie macula dengeneration (Zhou et al., 2008). The reduced form of tempol, tempol-H was more effective than Trolox or α-tocopherol in protecting these cells from photooxidation (Zhou et al., 2008). The tempol derivative 1-hydroxy-4-cyclopropanecarbonyloxy-2,2,6,6-tetramethylpiperidine hydrochloride given to albino rats protected their eyes from photodamage (Tanito et al., 2007). Tempol prevented the development of autoimmune uveitis in a rat model (Zamir et al., 1999) and protected the lens from cataract caused by H₂O₂ (Reddan et al., 1999; Zigler, Jr. et al., 2003; Akiyama et al., 2009) or radiation (Sasaki et al., 1998). Tempol protected retinal ganglion cells from the toxic effects of hypoxia or from TNFα (Tezel & Yang, 2004). Thus, a number of studies have demonstrated protective effects of tempol on retinal blood vessels, the retinal pigment cells and the lens in rodent models of ocular damage.

Tempol protected the liver from ethanol-induced damage and the formation of megamitochondria (Matsuhashi et al., 1998). Tempol conjugated to amino acids protected the liver from ischemia-reperfusion injury (Bi et al., 2008). Tempol prevented hepatocyte toxicity from hypoxia, cyanide or antimycin (Niknahad et al., 1995) and reduced hepatic reperfusion damage (Blonder et al., 2000; Sepodes et al., 2004). Tempol reduced hepatic ROS, steatosis, fibrosis and hepatic mitochondrial damage in rats transgenic for the ren-2 gene. Thus, tempol was effective in models of both alcoholic and non-alcoholic liver disease (Wei et al., 2009b).

Tumors and tumor cells produced excess ROS, often from NADPH oxidase (Szatrowski & Nathan, 1991; Burdon, 1995; Burdon, 1996; Droge, 2002; Chamulitrat et al., 2003; Lambeth, 2007). Malignant tumor cells proliferated despite increased ROS production in most (Toyokuni et al., 1995; Kondo et al., 1999; Martin et al., 2007) but not all assay systems (Ko et al., 2007). Therefore, a reduction in tumor ROS by tempol might not be anticipated to have anti-tumor actions. However, the situation is complicated since preservation of bioactive NO by tempol can cause apoptosis, depending on the concentration and underlying state of proliferation (Muhl et al., 1996; Shami et al., 1998; Hajri et al., 1998), whereas ROS can cause DNA damage and thereby become mutagenic or cytotoxic at higher concentrations (Schubert et al., 2004).

Several studies have demonstrated that tempol reduced tumor growth or incidence. Important insights derived from studies by Gariboldi, Monti and colleagues of apparently paradoxical oxidant-like effects of tempol in neoplastic cells that underlied its antiproliferative and antitumor actions (Gariboldi et al., 1998; Gariboldi et al., 2000; Gariboldi et al., 2003; Gariboldi et al., 2006). Thus, whereas tempol diminished apoptosis in normal lymphocytes (Kobayashi & Schmid-Schonbein, 2006), it induced apoptosis in a human promyelocytic leukemic cell line (Gariboldi et al., 2000; Monti et al., 2001). Nitroxides caused oxidative stress, cell death (Gariboldi et al., 2000), decreased mitochondrial membrane potential and increased mitochandrial ROS generation in neoplastic human promyelocytic leukemic cells (Gariboldi et al., 2000; Monti et al., 2001). The cytotoxic effect of tempol on tumor cells was related to glutathione depletion and hypoxia (Metodiewa et al., 2000; Samuni et al., 2004b) via reactions of tempol with glutathione (Glebska et al., 2003) or with heme (Yin et al., 2004). Tempol elicited distinct signalling mechanisms in human breast cancer cells culminating in apoptotic cell death (Suy et al., 1998).

Further insight came from studies of mice deficient in the ataxia-telangiectasia gene with a high incidence of lymphoid tumors and neurodegeneration (Reliene et al., 2008). Although tempol prevented these effects and doubled their lifespan (Schubert et al., 2004; Reliene & Schiestl, 2007; Reliene et al., 2008), this remarkable effect was dissociated from its antioxidant actions. Thus tumor formation was unaffected when the tissue levels of ROS were enhanced by deletion of the genes for SOD-1 or -2 or reduced by α-tocopherol (Erker et al., 2006). Rather, the ability of nitroxides to inhibit tumor growth was ascribed to an increase in tumor cell ROS (Gariboldi et al., 1998; Gariboldi et al., 2000; Monti et al., 2001) which explained the observation that the anti-oxidant drug NAC blocked the anti-tumor effect of tempol in breast cancer cells (Gariboldi et al., 2006).

The observation that the incidence of cancer was lower in those consuming a diet rich in naturally occurring antioxidants and that ROS were increased in cancerous tissues suggested that antioxidants might prevent tumor formation (Schubert et al., 2004). Indeed, normal mice fed tempol from birth had a 4-fold reduction in spontaneous tumorgenesis (Mitchell et al., 2003). Tempol reduced glioma formation in rats (Gariboldi et al., 2003) and delayed the development of tumors (Gariboldi et al., 2003; Zhang et al., 2008a), and prolonged the lifespan, of cancer-prone mice harboring a p53 gene deletion (Erker et al., 2005). These differential effect of tempol on tumor cells suggested that tempol could be a novel agent for cancer prevention or chemotherapy (May et al., 1998; Schor et al., 2004).

A note of caution comes from some studies which have demonstrated potential tumorgenic effects of tempol. Tempol caused post-transcriptional activation of the urokinase receptor (Lejeune et al., 2006) which is associated with progression of human prostate cancer (Rabbani & Xing, 1998). Rats subjected to severe exercise showed DNA strand breaks that were enhanced by oral tempol (200 μmol · kg^-1 · d^-1) (Wierzba et al., 2006).

Tempol interacts with many chemotherapeutic agents. Tempol potentiated the cell death and/or apoptosis, and upregulated p22 protein expression (Ravizza et al., 2004b) in Jurkat tumor cells (Sugimoto et al., 2002), human Burkitt lymphoma cells (Shacter et al., 2000) and colon cancer cells (Ravizza et al., 2004b). Tempol reduced ROS and diminished toxicity of the chemotherapeutic agents doxorubicin, cisplatin, AraC or VP-16 (Czepas et al., 2008). Tempol reduced the lethality of 6-mercaptopurine and enhanced its anti-tumor effect (Konovalova et al., 1996a; Konovalova et al., 1996b). Tempol reduced glutathione levels and synergetically enhanced the cytotoxic effects of the methylating agent, temozolomide in human glioblastoma cells (Ravizza et al., 2004a).

A limitation of chemotherapy with anthracycline agents such as doxorubicin has been the development of multidrug resistance (Longley & Johnston, 2005). Tempol synergized with doxorubicin in causing cytotoxicity of human breast adenocarcinoma cells by inhibiting the multidrug resistance pathway and thereby enhanced the cellular uptake of doxorubicin (Gariboldi et al., 2006).

An enhancement of chemotherapeutic efficacy by tempol has not been a universal finding. Apoptosis induced by the chemotherapeutic agents VP-16 and cisplatin in human B leukemic cells (Senturker et al., 2002) or by antimycin A in Hela cancer cells was unaffected by tempol (Han et al., 2008b). Tirapazamine becomes an active anti-tumor agent after reduction to nitrogen oxide anion radical which was prevented by tempol (Khan & O'Brien, 1995). The cytotoxicity of mitomycin C, or its quinolone redox-cycling analogues, were inhibited by tempol (Samuni et al., 2002).

Tempol protected normal cells from the cytotoxic effect of some chemotherapeutic agents. It prevented the cardiotoxicity of adriamycin (DeGraff et al., 1994; Monti et al., 1996), the hepatotoxicity of antimycin (Niknahad et al., 1995), the cytotoxicity of the semiquinone redox cycling agents, such as streptonigrin (DeGraff et al., 1994), the neurotoxicity of 6-hydroxydopamine (Purpura et al., 1996; Weinberg et al., 2004) and the spinal cord toxicity of etanidazole (Palayoor et al., 1994).

Tempol can prevent the effects of free iron to catalyze the production of ·OH from H₂O₂ by the Fenton reaction (Goralska et al., 2000). However, tempol has complicated effects on iron metabolism that can affect the outcome. Thus, high concentrations of tempol added to lens epithelial cells reduced the incorporation of iron into ferritin and increased the pool of free iron (Goralska et al., 2000) whereas lower concentrations of tempol protected against iron release during hypoxia (Borisenko et al., 2000). Tempol given to mice with deletion of the gene for the iron regulatory protein-2 restored iron homeostasis in the brain and attenuated the associated progressive neuromuscular dysfunction (Ghosh et al., 2008). The profound endothelial dysfunction that occurs the day after injection of iron gluconate into rats was prevented by co-administration of tempol or NAC, but not by ascorbate (Nouri et al., 2007).

Tempol prevented cuprous ions from forming a superoxide generating complex with glutathione (Speisky et al., 2009). Tempol protected human lymphocytes from toxic effects of chromium and cadmium salts (Lewinska et al., 2008), protected arterial endothelium from the dysfunction caused by nanomolar concentrations of mercury chloride (Wiggers et al., 2008) and protected cultured juxtaglomerular (Han et al., 2008c) and lung cells (Han et al., 2008a) from apotosis by arsenic trioxide.

The aortas from Apo E gene deleted mice exposed to gasoline engine exhaust had enhanced endothelin-1 expression and upregulation of membrane metalloprotease-2 and -9 and TIMP-2 which were corrected by oral tempol (41 mg · kg^-1 · day^-1) (Lund et al., 2009a).

The pathogenicity of malaria parasites involves the generation of ·OH following release of iron within erythrocytes (Schwartz et al., 1999). Tempol inhibited parasite growth in the erythrocytes (Schwartz et al., 1999) and protected erythrocytes from oxidative hemolysis (Wu et al., 1997; Li et al., 2006) likely by limiting ·OH formation by iron-catalyzed metabolism of H₂O₂. Erythrocytes from mice infected with the malaria parasite Plasmodium berghei reduced nitroxides at an accelerated rate (imply a hypoxic or reducing environment) that was corrected by chloroquine (Deslauriers et al., 1987).

Tempol given with fluid resuscitation has been effective in alleviating many of the adverse consequences of hemorrhagic shock. Tempol improved the survival of rats following hemorrhage (Kentner et al., 2002) and diminished the associated hemodynamic instability, the multi-organ dysfunction (Mota-Filipe et al., 1999) and the hepatic damage (Paxian et al., 2002; Paxian et al., 2003). Tempol (170 μmol · kg^-1 · h^-1 iv) given to rats during reperfusion four hours after hypotensive hemorrhage prevented the delayed circulatory failure (Mota-Filipe et al., 1999) but when given without adequate volume resuscitation, tempol enhanced the mortality (Kentner et al., 2007).

Tempol also has been effective in mitigating the consequences of severe circulatory failure. Tempol protected the brain following cardiac arrest (Behringer et al., 2002) and ameliorated the multiorgan failure, DNA damage and impaired mitochondrial respiration in rats after zymosan (Cuzzocrea et al., 2001). Tempol given to swine one hour after chest injury reduced their requirements for fluid infusion and reduced pulmonary inflammation, but did not modify mortality (Maxwell et al., 2000). Hind limb ischemia and reperfusion in the rat led to multiorgan dysfunction with edema, inflammation and the release of lactate dehydrogenase and creatine phosphokinase from unaffected muscle which were ameliorated by tempol given at the time of reperfusion (Arieli et al., 2008).

The author is grateful to Ms Emily Wing Kam Chan for preparing and editing the manuscript.

Sources of funding: Work in the author's laboratory is supported by grants from the NIDDK (DK-049870 and DK-036079) and the NHLBI (HL-68686), by funds from the George E. Schreiner Chair of Nephrology and the Georgetown University Hypertension, Kidney and Vascular Disease Center.