The dietary antioxidant quercetin reduces hallmarks of bleomycin-induced lung fibrogenesis in mice.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic, lethal disease of which the etiology is still not fully understood. Current treatment comprises two FDA-approved drugs that can slow down yet not stop or reverse the disease. As IPF pathology is associated with an altered redox balance, adding a redox modulating component to current therapy might exert beneficial effects. Quercetin is a dietary antioxidant with strong redox modulating capacities that is suggested to exert part of its antioxidative effects via activation of the redox-sensitive transcription factor Nrf2 that regulates endogenous antioxidant levels. Therefore, the aim of the present study was to investigate if the dietary antioxidant quercetin can exert anti-fibrotic effects in a mouse model of bleomycin-induced pulmonary fibrogenesis through Nrf2-dependent restoration of redox imbalance. METHODS: Homozygous Nrf2 deficient mice and their wildtype littermates were fed a control diet without or with 800 mg quercetin per kg diet from 7 days prior to a single 1 µg/2 µl per g BW bleomycin challenge until they were sacrificed 14 days afterwards. Lung tissue and plasma were collected to determine markers of fibrosis (expression of extracellular matrix genes and histopathology), inflammation (pulmonary gene expression and plasma levels of tumor necrosis factor-α (TNFα) and keratinocyte chemoattrachtant (KC)), and redox balance (pulmonary gene expression of antioxidants and malondialdehyde-dG (MDA)- DNA adducts). RESULTS: Mice fed the enriched diet for 7 days prior to the bleomycin challenge had significantly enhanced plasma and pulmonary quercetin levels (11.08 ± 0.73 µM versus 7.05 ± 0.2 µM) combined with increased expression of Nrf2 and Nrf2-responsive genes compared to mice fed the control diet in lung tissue. Upon bleomycin treatment, quercetin-fed mice displayed reduced expression of collagen (COL1A2) and fibronectin (FN1) and a tendency of reduced inflammatory lesions (2.8 ± 0.7 versus 1.9 ± 0.8). These beneficial effects were accompanied by reduced pulmonary gene expression of TNFα and KC, but not their plasma levels, and enhanced Nrf2-induced pulmonary antioxidant defences. In Nrf2 deficient mice, no effect of the dietary antioxidant on either histology or inflammatory lesions was observed. CONCLUSION: Quercetin exerts anti-fibrogenic and anti-inflammatory effects on bleomycin-induced pulmonary damage in mice possibly through modulation of the redox balance by inducing Nrf2. However, quercetin could not rescue the bleomycin-induced pulmonary damage indicating that quercetin alone cannot ameliorate the progression of IPF.

Neutrophils augment LPS-mediated pro-inflammatory signaling in human lung epithelial cells.

BACKGROUND: The role of polymorphonuclear neutrophils in pulmonary host defense is well recognized. The influence of a pre-existing inflammation driven by neutrophils (neutrophilic inflammation) on the airway epithelial response toward pro-inflammatory exogenous triggers, however, is still poorly addressed. Therefore, the aim of the present study is to investigate the effect of neutrophils on lipopolysaccharide (LPS)-induced pro-inflammatory signaling in lung epithelial cells. Additionally, underlying signaling pathways are examined. METHODS: Human bronchial epithelial cells (BEAS-2B) were co-incubated with human peripheral blood neutrophils or bone-marrow derived neutrophils from either C57BL/6J wild type or nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase deficient (p47(phox-/-)) mice. Upon stimulation with LPS, interleukin (IL)-8 production and reactive oxygen species (ROS) generation were measured. Additionally, activation of the extracellular signal-regulated kinases (ERK) 1/2 and nuclear factor (NF)-κB signaling pathways was analyzed. RESULTS: Our studies show that the presence of neutrophils synergistically increases LPS-induced IL-8 and ROS production by BEAS-2B cells without inducing cytotoxicity. The observed IL-8 response to endotoxin increases in proportion to time, LPS-concentration and the number of neutrophils present. Moreover, this synergistic IL-8 production strongly correlated with the chemotactic properties of the co-incubations and significantly depended on a functional neutrophilic NADPH oxidase. The presence of neutrophils also augments LPS-induced phosphorylation of ERK1/2 and IκBα as well as NF-κB RelA DNA binding activity in BEAS-2B cells. CONCLUSIONS: Our results indicate that the pro-inflammatory effects of LPS toward lung epithelial cells are amplified during a pre-existing neutrophilic inflammation. These findings support the concept that patients suffering from pulmonary neutrophilic inflammation are more susceptible toward exogenous pro-inflammatory triggers.

Urinary nitric oxide metabolites and individual blood pressure progression to overt hypertension.

Baseline blood pressure (BP) is the strongest known determinant of progression to hypertension, but for an individualized prediction of the incidence of hypertension, the identification of additional biomarkers is crucial. In animal models of hypertension, renal nitric oxide (NO) handling modifies the systemic BP responses prior to the development of hypertension. This study aimed to evaluate whether urinary NO metabolites (NOx) predict the progression of hypertension in normotensive subjects. Among 62 participants enrolled in the Flemish Study on Environment, Genes and Health Outcomes, we assessed progression to hypertension over 4.6 years. In a case-control design, 49 normotensive subjects including 10 subjects with high-normal blood pressure were enrolled of whom 25 remained normotensive (controls), whereas 24 'progressed' to hypertension (progressors). Thirteen hypertensive patients served as negative controls. Urinary NOx concentration, renal function and the urinary excretion of electrolytes were assessed at baseline and follow-up. At baseline, progressors showed higher BP values than controls and urinary NOx concentration was significantly lower in progressors as compared to the normotensive controls (p < 0.01). In all initially normotensive subjects baseline urinary NOx concentration was associated with follow-up BP (r = -0.55, p < 0.001) and the relative increase of BP over time (r = -0.47, p < 0.001). In progressors baseline urinary NOx was associated with follow-up BP (r = -0.52, p < 0.009) and the relative increase of BP over time (r = -0.44, p = 0.033). Baseline urinary NOx and BP were independent predictors for the relative BP increase. A urinary NOx threshold of <130.5 mg/L predicted 75% of all progressors. In context with high-normal baseline BP, 87.5% of all progressors were identified. These findings indicate that urinary NO metabolites are associated with BP development in normotensive subjects. Moreover, urinary NOx predicts the progression to hypertension independent of baseline BP suggesting urinary NOx as a biomarker for individual new-onset hypertension.

Deconjugation kinetics of glucuronidated phase II flavonoid metabolites by beta-glucuronidase from neutrophils.

Flavonoids are inactivated by phase II metabolism and occur in the body as glucuronides. Mammalian beta-glucuronidase released from neutrophils at inflammatory sites may be able to deconjugate and thus activate flavonoid glucuronides. We have studied deconjugation kinetics and pH optimum for four sources of beta-glucuronidase (human neutrophil, human recombinant, myeloid PLB-985 cells, Helix pomatia) with five flavonoid glucuronides (quercetin-3-glucuronide, quercetin-3'-glucuronide, quercetin-4'-glucuronide, quercetin-7-glucuronide, 3'-methylquercetin-3-glucuronide), 4-methylumbelliferyl-beta-D-glucuronide, and para-nitrophenol-glucuronide. All substrate-enzyme combinations tested exhibited first order kinetics. The optimum pH for hydrolysis was between 3.5-5, with appreciable hydrolysis activities up to pH 5.5. At pH 4, the K(m) ranged 44-fold from 22 microM for quercetin-4'-glucuronide with Helix pomatia beta-glucuronidase, to 981 microM for para-nitrophenol-glucuronide with recombinant beta-glucuronidase. V(max) (range: 0.735-24.012 micromol x min(-1) x unit(-1) [1 unit is defined as the release of 1 microM 4-methylumbelliferyl-beta-D-glucuronide per min]) and the reaction rate constants at low substrate concentrations (k) (range: 0.002-0.062 min(-1) x (unit/L)(-1) were similar for all substrates-enzyme combinations tested. In conclusion, we show that beta-glucuronidase from four different sources, including human neutrophils, is able to deconjugate flavonoid glucuronides and non-flavonoid substrates at fairly similar kinetic rates. At inflammatory sites in vivo the pH, neutrophil and flavonoid glucuronide concentrations seem favorable for deconjugation. However, it remains to be confirmed whether this is actually the case.

Sustained tubulo-interstitial protection in SHRs by transient losartan treatment: an effect of decelerated aging?

BACKGROUND: Hypertensive target organ damage shows characteristics of accelerated cell turnover and aging. This might have developed during the evolution of hypertension. In the kidney, high cell turnover is mainly restricted to tubular cells. It was the aim of this study to investigate whether a transient intervention in spontaneously hypertensive rats (SHRs) leads to reduced tubular cell turnover and attenuates the renal aging process and tubulo-interstitial damage in the long-term. METHODS: SHRs (i) were prehypertensively (weeks 4-8) treated with losartan (ii) or hydralazine (iii) (20 and 4 mg/kg/day, respectively) and compared to Wistar-Kyoto (WKY) rats (iv). Groups were investigated at weeks 8 and 72 (except iii). At both time points tubular cell proliferation (proliferative cell nuclear antigen) and systolic blood pressure (SBP) were evaluated. At week 72, aging parameters such as telomere length were assessed. Renal damage was semiquantitatively assessed (scale: 0-4) by measuring the parenchyma (atrophy) and vasculature (media thickness). RESULTS: Treatments equipotently reduced SBP in young SHRs (P < 0.01) but only losartan reduced renal proliferation (proliferative cell nuclear antigen: (i) 2.8 +/- 0.8, (ii) 1.3 +/- 0.3, (iii) 3.0 +/- 0.6, (iv) 0.1 +/- 0.1 cells/mm(2)). In SHRs treated with losartan(SHR-Los) tubular proliferation remained reduced and renal telomere length was significantly greater than in untreated SHRs (fold: (i) 1.0 +/- 0.1, (ii) 2.8 +/- 0.3, P < 0.01). Untreated SHRs (median 2.0, range 1-3; P < 0.007), but not SHR-Los (median 1.0, range 0-2; P = 0.06) demonstrated more tubular atrophy than WKY rats (median 0.5, range 0-1). CONCLUSIONS: Transient losartan treatment reduces cell-turnover not only acutely but also for a prolonged period after drug withdrawal. This results in the long-term in reduced aging and attenuated tubulo-interstitial damage, suggesting there exists a modulating effect of angiotensin II (ANGII)-antagonism on long-term cell turnover.

Prehypertensive renin-angiotensin-aldosterone system blockade in spontaneously hypertensive rats ameliorates the loss of long-term vascular function.

Arterial function after long-term hypertension is characterized by remodeling, endothelial dysfunction and reduction of previously enhanced contractile responses. We investigated whether transient prehypertensive renin-angiotensin-aldosterone system (RAAS) blockade modifies long-term arterial function. Wistar Kyoto rats (WKY) (i) and spontaneously hypertensive rats (SHR) (ii) were prehypertensively (week 4-8) treated with losartan (iii) or spironolactone (iv) (20 and 0.5 mg/kg/day, respectively) and investigated at 8 and 72 weeks of age. Systolic blood pressure (SBP) was measured intra-arterially. In isolated mesenteric arteries, active wall stress (AWS), relaxation in response to acetylcholine and wall-to-lumen ratio (W/L) were assessed. Western blotting and immunofluorescent staining of whole-mount arterial preparations and two photon laser scanning microscopy (TPLSM) were performed to quantify endothelial nitric oxide synthase (eNOS) and analyze its intracellular distribution. In 8-week-old SHR treatments were found to have reduced SBP. Relaxation, contractile responses and vascular morphology remained unaffected irrespective of treatment. At 72 weeks, SBP was similar in all SHR groups ((i) 129+/-6, (ii) 222 +/- 10, (iii) 210 +/- 16, (iv) 214 +/- 9 mmHg). Relaxation and maximum AWS were enhanced after treatments. W/L demonstrated hypertrophy ((i) 0.10 +/- 0.01, (ii) 0.16 +/- 0.02, (iii) 0.15 +/- 0.01, (iv) 0.17 +/- 0.01). Untreated SHR (p<0.01), SHR treated with losartan and SHR treated with spironolactone (p<0.05) showed less eNOS as compared to WKY. In treated SHR eNOS was concentrated in a perinuclear endothelial cell compartment. In conclusion, these findings demonstrate that transient prehypertensive blockade results in a long-lasting and blood pressure independent improvement of arterial contractility and endothelium-dependent vasodilatation that persists in aging SHR. This might be associated with an intracellular redistribution of eNOS in the endothelial cell layer.