Michal Laniado-Schwartzman

Regulation of Cyclooxygenase-2 by Hypoxia and Peroxisome Proliferators in the Corneal Epithelium

Hypoxic injury provokes inflammation of many tissues including the ocular surface. In rabbit corneal epithelial cells, both peroxisome proliferator-activated receptor (PPAR)-inducible cytochrome P450 4B1 and cyclooxygenase-2 (COX-2) mRNAs were increased by hypoxia. PPAR α and β but not γ mRNAs were detected in these cells. The PPAR activator, WY-14,643 increased COX-2 expression. Similarly, non-steroidal anti-inflammatory drugs with the ability to activate PPARs induced COX-2 independently of prostaglandin synthesis inhibition. COX-2 protein overexpression by hypoxia and PPAR activation was not associated with a parallel increase in prostaglandin E2 accumulation. However, the enzyme regained full catalytic activity when: 1) hypoxic cells were re-exposed to normoxic conditions in the presence of heme and arachidonic acid, and 2) WY-14,643-treated cells were depleted of intracellular GSH. Consistent with previous observations showing that the corneal production of cytochrome P450-derived inflammatory eicosanoids is elevated by hypoxia and inflammation, the current data suggest that hypoxic injury is a model of inflammation in which molecules other than COX-derived arachidonic acid metabolites play a major proinflammatory role. This study also suggests that increased cellular GSH may be the mechanism responsible for the characteristic dissociation of PPAR-induced COX-2 expression and activity. Moreover, we provide new insights into the commonly observed lack of efficacy of classical non-steroidal anti-inflammatory drugs in the treatment of hypoxia-related ocular surface inflammation.

20-Hydroxyeicosatetraenoic acid stimulates nuclear factor-κB activation and the production of inflammatory cytokines in human endothelial cells

Endothelial dysfunction is associated with endothelial cell activation, i.e., up-regulation of surface cell adhesion molecules and the release of proinflammatory cytokines. 20-Hydroxyeicosatetraenoic acid (HETE), a major vasoactive eicosanoid in the microcirculation, has been implicated in the regulation of endothelial cell function through its angiogenic and pro-oxidative properties. We examined the effects of 20-HETE on endothelial cell activation in vitro. Cells transduced with adenovirus containing either CYP4A1 or CYP4A2 produced higher levels of 20-HETE, and they demonstrated increased expression levels of the adhesion molecule intercellular adhesion molecule (ICAM) (4–7-fold) and the oxidative stress marker 3-nitrotyrosine (2–3-fold) compared with cells transduced with control adenovirus. Treatment of cells with 20-HETE markedly increased levels of prostaglandin (PG) E2 and 8-epi-isoprostane PGF2α, commonly used markers of activation and oxidative stress, and most prominently, interleukin-8, a potent neutrophil chemotactic factor whose overproduction by the endothelium is a key feature of vascular injury. 20-HETE at nanomolar concentrations increased inhibitor of nuclear factor-κB phosphorylation by 2 to 5-fold within 5 min, which was followed with increased nuclear translocation of nuclear factor-κB (NF-κB). Likewise, 20-HETE activated the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway by stimulating phosphorylation of ERK1/2. Inhibition of NF-κB activation and inhibition of ERK1/2 phosphorylation inhibited 20-HETE-induced ICAM expression. It seems that 20-HETE triggers NF-κB and MAPK/ERK activation and that both signaling pathways participate in the cellular mechanisms by which 20-HETE activates vascular endothelial cells.

Endothelial Dysfunction and Hypertension in Rats Transduced With CYP4A2 Adenovirus

Vascular cytochrome P450 (CYP) 4A enzymes catalyze the synthesis of 20-hydroxyeicosatetraenoic acid (20-HETE), an eicosanoid which participates in the regulation of vascular tone by sensitizing the smooth muscle cells to constrictor and myogenic stimuli. This study was undertaken to investigate the consequences of CYP4A overexpression on blood pressure and endothelial function in rats treated with adenoviral vectors carrying the CYP4A2 construct. Intravenous injection of Adv-CYP4A2 increased blood pressure (from 114±1 to 133±1 mm Hg, P<0.001), and interlobar renal arteries from these rats displayed decreased relaxing responsiveness to acetylcholine, which was offset by treatment with an inhibitor of CYP4A. Relative to data in control rats, arteries from Adv-CYP4A2–transduced rats produced more 20-HETE (129±10 versus 97±7 pmol/mg protein, P<0.01) and less nitric oxide (NO; 4.2±1.6 versus 8.4±1 nmol nitrite+nitrate/mg; P<0.05). They also displayed higher levels of oxidative stress as measured by increased generation of superoxide anion and increased expression of nitrotyrosine and gp91phox. Collectively, these findings demonstrate that augmentation in vascular 20-HETE promotes the development of hypertension and causes endothelial dysfunction, a condition characterized by decreased NO synthesis and/or bioavailability, imbalance in the relative contribution of endothelium-derived relaxing and contracting factors, and enhanced endothelial activation.

Differential Regulation of Natriuresis by 20-Hydroxyeicosatetraenoic Acid in Human Salt-Sensitive Versus Salt-Resistant Hypertension

Background—Twenty-hydroxyeicosatetraenoic acid (20-HETE) is a cytochrome P450 metabolite of arachidonic acid that produces vasoconstriction and inhibition of renal tubular sodium transport. In Dahl rats, a 20-HETE deficiency plays a role in salt-sensitive (SS) hypertension. In humans, there are no data on regulation of 20-HETE by salt intake or on a role for this compound in SS hypertension. Methods and Results—Thirteen salt-resistant (SR) and 13 SS hypertensive subjects had urine 20-HETE excretion measured during salt-loading and depletion. In all patients, 20-HETE was 66.6% higher in the salt-replete (1.75±0.25 &mgr;g/h) than in the salt-depleted state (1.05±0.16, P <0.003). There was no difference in 20-HETE excretion between SR and SS patients in either state of salt balance. In SR patients, sodium excretion during salt-loading correlated with 20-HETE (r =0.61, P <0.03) but not with blood pressure. In contrast, in SS patients, sodium excretion did not correlate with 20-HETE but did correlate with blood pressure (r =0.66, P <0.02). Finally, in the SS group only, there was a negative correlation between body mass index and 20-HETE excretion (r =−0.79, P <0.002) that was present during both salt-loading and depletion. Conclusions—We demonstrate for the first time that 20-HETE excretion is regulated by salt intake in hypertension. We find a disrupted relationship between sodium excretion and 20-HETE in SS patients, which results in dependence of their salt excretion on blood pressure and may be related to the magnitude of their obesity. We conclude that salt-sensitivity of blood pressure in essential hypertension may result from impairment of a natriuretic mechanism dependent on 20-HETE.

Contribution of cytochrome P-450 4A1 and 4A2 to vascular 20-hydroxyeicosatetraenoic acid synthesis in rat kidneys

20-Hydroxyeicosatetraenoic acids (20-HETE), a biologically active cytochrome P-450 (CYP) metabolite of arachidonic acid in the rat kidney, can be catalyzed by CYP4A isoforms including CYP4A1, CYP4A2, and CYP4A3. To determine the contribution of CYP4A isoforms to renal 20-HETE synthesis, specific antisense oligonucleotides (ODNs) were developed, and their specificity was examined in vitro in Sf9 cells expressing CYP4A isoforms and in vivo in Sprague-Dawley rats. Administration of CYP4A2 antisense ODNs (167 nmol ⋅ kg body wt-1 ⋅ day-1iv for 5 days) decreased vascular 20-HETE synthesis by 48% with no effect on tubular synthesis, whereas administration of CYP4A1 antisense ODNs inhibited vascular and tubular 20-HETE synthesis by 52 and 40%, respectively. RT-PCR of microdissected renal microvessel RNA indicated the presence of CYP4A1, CYP4A2, and CYP4A3 mRNAs, and a CYP4A1-immunoreactive protein was detected by Western analysis of microvessel homogenates. Blood pressure measurements revealed a reduction of 17 ± 6 and 16 ± 4 mmHg in groups receiving CYP4A1 and CYP4A2 antisense ODNs, respectively. These studies implicate CYP4A1 as a major 20-HETE synthesizing activity in the rat kidney and further document the feasibility of using antisense ODNs to specifically inhibit 20-HETE synthesis and thereby investigate its role in the regulation of renal function and blood pressure.

Interdependence of lipoxin A4 and heme-oxygenase in counter-regulating inflammation during corneal wound healing

In the immune‐privileged cornea, epithelial wounds heal rapidly with almost no scarring and, unlike in most other tissues, acute inflammation in the absence of infection is beneficial to healing. Molecular mechanisms, which account for this striking property, remain to be clearly defined, but they likely include autacoids that control leukocyte activation. Two prominent enzymes, 12/15‐lipoxygenase (LOX), which generates antiinflammatory lipid autacoids, and heme‐oxy‐genase (HO), which generates antioxidants and carbon monoxide, are highly expressed in human and mouse corneas. LXA4, an endogenous 12/15‐LOX product, proved to be a potent inhibitor of exacerbated inflammation and significantly increased re‐epithelialization in corneal wounds. In vivo deletion of 12/15‐LOX correlated with exacerbated inflammation and impaired wound healing in 12/15‐LOX−/− mice, a phe‐notype that was rescued by treatment with LXA4. More importantly, 12/15‐LOX−/− mice demonstrated impaired induction of HO‐1 in both acute and exacerbated inflammation. Topical LXA4 restored HO‐1 expression in 12/15‐LOX−/− mice and amplified HO‐1 gene expression in human corneal epithelial cells. HO‐2−/− mice, which fail to induce HO‐1, also demonstrated exacerbated inflammation in response to injury, a phenotype that, notably, correlated with a 50% reduction in endogenous LXA4 formation. Collectively, results demonstrate a critical role for LXA4 in inflammatory/reparative responses and provide the first evidence that 12/15‐LOX and HO systems function in concert to control inflammation.–Biteman, B., Hassan, I. R., Walker, E., Leedom, A. J., Dunn, M., Seta, F., Laniado‐Schwartzman, M., Gronert, K. Interdependence of lipoxin A4 and heme‐oxygenase in counter‐regulating inflammation during corneal wound healing. FASEB J. 21, 2257–2266 (2007)

Heme oxygenase induction with attenuation of experimentally induced corneal inflammation

Heme oxygenase (HO), by catabolizing heme to bile pigments, down-regulates cellular levels of heme and hemeproteins; certain of the latter, i.e. cytochrome P450s, generate pro-inflammatory products from endogenous substrates. Two HO isozymes, the products of distinct genes, have been described; HO-1 is the inducible one, whereas HO-2 is believed to be constitutively expressed. We studied the inducing effects of several metal compounds [CoCl2, SnCl2, ZnCl2, heme, and cobalt protoporphyrin (CoPP)] on HO-1 mRNA content and enzyme activity in cultures of rabbit corneal epithelial (RCE) cells; these metal compounds are known to induce HO in other tissues. Additionally, we studied HO-1 expression in an experimental model of ocular inflammation produced in rabbit corneas by extended contact lens wear, and the relation of HO expression to the induced inflammatory process. SnCl2 added to RCE cells in vitro produced marked time- and concentration-dependent increases in HO-1 mRNA and HO-1 enzyme activity; CoCl2, ZnCl2, and CoPP were inducers of HO as well, though to a lesser degree than SnCl2. Corneas treated for 6 days with contact lenses impregnated with SnCl2 displayed substantially less corneal inflammation, swelling, and new vessel invasion than did controls; attenuation of ocular inflammation was paralleled by SnCl2-induced increases in HO mRNA and HO activity in corneal epithelial cells from treated eyes. It is suggested that amelioration of the inflammatory response produced by extended contact lens wear is due, in part, to the induction of high levels of HO-1 activity by SnCl2, which results in diminished production of pro-inflammatory mediators generated through heme-dependent metabolic processes. Regulation of HO activity in this manner may have clinical applications.

Chronic treatment with tin normalizes blood pressure in spontaneously hypertensive rats.

We have reported that short-term treatment of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats with stannous chloride (SnCl2), which selectively depletes renal cytochrome P4S0, restores blood pressure to normal in young but not in adult SHR, and is without effect on blood pressure of either young or adult WKY rats. We report in the present study that chronic treatment with SnCl2, begun at age 5 weeks, prevented the development of hypertension in SHR over a period of 15 weeks at which time they were killed. Suspension of SnCl2 treatment after 8 weeks (i.e., at age 13 weeks) did not result in return of blood pressure to hypertensive levels in SHR. Age-matched WKY rats were not affected by tin treatment These findings provide additional evidence that administration of tin, which stimulates heme oxygenase, thereby producing depletion of cytochrome P450, restores blood pressure to normal levels in SHR.

19(S)-hydroxyeicosatetraenoic acid is a potent stimulator of renal Na+-K+ATPase

Omega- and omega-1 hydroxylations are the major pathways by which arachidonic acid is metabolized in cortical and outer medullary microsomes of rat and rabbit kidneys. It is a cytochrome P450-dependent oxidation leading to the formation of 20-hydroxy- and 19-hydroxyeicosatetraenoic acids. In this study, we compared the effects of the synthetically prepared omega- and omega-1 metabolites of arachidonic acid on the activity of the renal Na+-K+-ATPase partially purified from rat renal cortical microsomes. 19(S)-hydroxyeicosatetraenoic acid caused a dose related stimulation of Na+-K+-ATPase activity with an EC50 of 3 x 10(-7) M. In contrast, neither 19(R)-hydroxyeicosatetraenoic acid, 20-hydroxyeicosatetraenoic acid nor arachidonic acid at 10(-6) M had any effect on Na+-K+-ATPase activity. In the same preparation, ouabain at 10(-3) M and 12(R)-hydroxyeicosatetraenoic acid at 10(-6) M inhibited the enzyme activity by 75% and 60%, respectively. We conclude that 19(S)-hydroxyeicosatetraenoic acid is a specific stimulator of renal Na+-K+-ATPase. Therefore, the formation of 19(S)-hydroxyeicosatetraenoic acid by renal cortical cytochrome P450 omega-1-hydroxylase may contribute to the regulation of renal function by regulating Na+-K+-ATPase which is essential for transtubular transport processes.

The T8590C Polymorphism of CYP4A11 and 20-Hydroxyeicosatetraenoic Acid in Essential Hypertension

A role for a deficit in transport actions of 20-hydroxyeicosatetraenoic acid (20-HETE) in hypertension is supported by the following: (1) diminished renal 20-HETE in Dahl-S rats; (2) altered salt- and furosemide-induced 20-HETE responses in salt-sensitive hypertensive subjects; and (3) increased population risk for hypertension in C allele carriers of the T8590C polymorphism of CYP4A11, which encodes an enzyme with reduced catalytic activity. We determined T8590C genotypes in 32 hypertensive subjects, 25 of whom were phenotyped for salt sensitivity of blood pressure and insulin sensitivity. Urine 20-HETE was lowest in insulin-resistant, salt-sensitive subjects (F=5.56; P<0.02). Genotypes were 13 TT, 2 CC, and 17 CT. C allele frequency was 32.8% (blacks: 38.9%; whites: 25.0%). C carriers (CC+CT) and TT subjects were similarly distributed among salt- and insulin-sensitivity phenotypes. C carriers had higher diastolic blood pressures and aldosterone:renin and waist:hip ratios but lower furosemide-induced fractional excretions of Na and K than TT. The T8590C genotype did not relate to sodium balance or pressure natriuresis. However, C carriers, compared with TT, had diminished 20-HETE responses to salt loading after adjustment for serum insulin concentration and resetting of the negative relationship between serum insulin and urine 20-HETE to a 1-&mgr;g/h lower level of 20-HETE. The effect of C was insulin independent and equipotent to 18 &mgr;U/mL of insulin (&Dgr;20-HETE= 2.84−0.054×insulin−0.98×C; r2=0.53; F=11.1; P<0.001). Hence, genetic (T8590C) and environmental (insulin) factors impair 20-HETE responses to salt in human hypertension. We propose that genotype analyses with sufficient homozygous CC will establish definitive relationships among 20-HETE, salt sensitivity of blood pressure, and insulin resistance.