Uric acid stimulates proliferative pathways in vascular smooth muscle cells through the activation of p38 MAPK,p44/42 MAPK and PDGFRβ

Hyperuricemia and angiotensin II (Ang II) may have a pathogenetic role in the development of hypertension and atherosclerosis as well as cardiovascular disease (CVD) and its prognosis. The purpose of this study was to investigate whether uric acid can induce proliferative pathways of vascular smooth muscle cell (VSMC) that are thought to be responsible for the development of CVD. The phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), p44/42 mitogen-activated protein kinase (p44/42 MAPK) and platelet-derived growth factor receptor β (PDGFRβ) was measured by Elisa and Western blot techniques to determine the activation of proliferative pathways in primary cultured VSMCs from rat aorta. Results demonstrated that uric acid can stimulate p38 MAPK, p44/42 MAPK and PDGFRβ phosphorylation in a time- and concentration-dependent manner. Furthermore, treatment of VSMCs with the angiotensin II type I receptor (AT1R) inhibitor losartan suppressed p38 MAPK and p44/42 MAPK induction by uric acid. The stimulatory effect of uric acid on p38 MAPK was higher compared to that of Ang II. The results of this study show for the first time that uric acid-induced PDGFRβ phosphorylation plays a crucial role in the development of CVDs and that elevated uric acid levels could be a potential therapeutical target in CVD patients.     

Rosmarinic acid inhibits nicotine-induced C-reactive protein generation by inhibiting NLRP3 inflammasome activation in smooth muscle cells

Atherosclerosis is widely known to be a chronic inflammatory disease. C-reactive protein (CRP), an important inflammatory factor, plays an essential role in the pathogenesis of atherosclerosis. Nicotine, the main addictive component of cigarette, has been shown to induce the production of CRP. The aim of this study was to investigate the effect of rosmarinic acid (RA), a polyphenol with antiinflammatory activity, on nicotine-induced elevation of CRP in vascular smooth muscle cells (VSMCs). We found that pretreatment of VSMCs with RA attenuated nicotine-induced expression of CRP in a time- and dose-dependant manner. In addition, RA also inhibited the activation of NLR family pyrin domain containing 3 (NLRP3) inflammasome and reactive oxygen species (ROS) production resulting from nicotine treatment in VSMCs. To confirm these findings in vivo, we constructed a nicotine-induced atherosclerosis rat model. RA did not significantly reduce the serum nicotine level of the rats, whereas it significantly decreased the levels of serum lipids, including concentrations of cholesterol, triglycerides, and low-density lipoprotein cholesterol, and the serum level of CRP. RA also led to diminished nicotine-induced activation of NLRP3 inflammasome and elevation in the CRP level in the aortic tissue of the model rats. The results of this study suggested a protective role of RA in nicotine-induced atherosclerosis by inhibiting the ROS–NLRP3 inflammasome–CRP axial, and RA therefore represented a potential effective therapeutic approach to atherosclerosis, in particular for those who smoke.     

Homocysteine stimulates phosphorylation of NADPH oxidase p47phox and p67phox subunits in monocytes via protein kinase Cbeta activation

Hyperhomocysteinaemia is an independent risk factor for cardiovascular diseases due to atherosclerosis. The development of atherosclerosis involves reactive oxygen species-induced oxidative stress in vascular cells. Our previous study [Wang and O (2001) Biochem. J. 357, 233-240] demonstrated that Hcy (homocysteine) treatment caused a significant elevation of intracellular superoxide anion, leading to increased expression of chemokine receptor in monocytes. NADPH oxidase is primarily responsible for superoxide anion production in monocytes. In the present study, we investigated the molecular mechanism of Hcy-induced superoxide anion production in monocytes. Hcy treatment (20-100 microM) caused an activation of NADPH oxidase and an increase in the superoxide anion level in monocytes (THP-1, a human monocytic cell line). Transfection of cells with p47phox siRNA (small interfering RNA) abolished Hcy-induced superoxide anion production, indicating the involvement of NADPH oxidase. Hcy treatment resulted in phosphorylation and subsequently membrane translocation of p47phox and p67phox subunits leading to NADPH oxidase activation. Pretreatment of cells with PKC (protein kinase C) inhibitors Ro-32-0432 (bisindolylmaleimide XI hydrochloride) (selective for PKCalpha, PKCbeta and PKCgamma) abolished Hcy-induced phosphorylation of p47phox and p67phox subunits in monocytes. Transfection of cells with antisense PKCbeta oligonucleotide, but not antisense PKCalpha oligonucleotide, completely blocked Hcy-induced phosphorylation of p47phox and p67phox subunits as well as superoxide anion production. Pretreatment of cells with LY333531, a PKCbeta inhibitor, abolished Hcy-induced superoxide anion production. Taken together, these results indicate that Hcy-stimulated superoxide anion production in monocytes is regulated through PKC-dependent phosphorylation of p47phox and p67phox subunits of NADPH oxidase. Increased superoxide anion production via NADPH oxidase may play an important role in Hcy-induced inflammatory response during atherogenesis.     

Uric acid mediates photodynamic inactivation of caprine alpha-2-macroglobulin

Uric acid (2,6,8 trioxopurine), the end product of purine metabolism in mammalian systems, has shown a wide range of antioxidant properties including scavenging of hydroxyl radical and singlet oxygen. In this study we show that in the presence of visible light, uric acid disrupted caprine alpha-2-macroglobulin (alpha(2) M) structure and antiproteolytic function in vitro. Proteinase cleaves the bait region of caprine inhibitor inducing major conformational changes and entrapping the enzyme within its molecular cage. In contrast to native alpha(2) M, modified antiproteinase lost half of its antiproteolytic potential within 4 hours of uric acid exposure. The changes in uv-absorption spectra of the treated protein suggested possible spatial rearrangement of subunits or conformational change. Analysis of the mechanism by which alpha(2) M was inactivated revealed that the process was dependent on generation of superoxide anion and hydrogen peroxide. Our findings suggest that antiproteolytic activity of caprine alpha(2) M could be compromised via oxidative modification mediated by uric acid. Moreover, low concentrations of alpha(2) M were found to stimulate superoxide production by some unknown mechanism.     

Role of oxidative stress in high glucose-induced decreased expression of Gialpha proteins and adenylyl cyclase signaling in vascular smooth muscle cells

We have recently shown that aorta from streptozotocin (STZ)-induced diabetic rats and A10 vascular smooth muscle cells (VSMCs) exposed to high glucose exhibited decreased levels of inhibitory guanine nucleotide regulatory protein (Gi)alpha proteins. In the present studies, we investigated the implication of oxidative stress in the hyperglycemia/diabetes-induced decreased expression of the Gialpha protein and adenylyl cyclase signaling in VSMCs by using antioxidants. The levels of Gialpha proteins were significantly decreased in A10 VSMCs exposed to high glucose and in aortic VSMCs from STZ-diabetic rats compared with control cells and were restored to control levels by antioxidants. In addition, (111)Mn-tetralis(benzoic acid porphyrin) and uric acid, scavengers of peroxynitrite, and NG-nitro-L-arginine methyl ester, an inhibitor of nitric oxide synthase but not catalase, also restored the high glucose-induced decreased expression of Gialpha proteins to the control levels in A10 VSMCs. Furthermore, the enhanced production of superoxide anion (O2-) and increased activity of NADPH oxidase in these cells were also restored to control levels by diphenyleneiodonium, an inhibitor of NADPH oxidase. In addition, the diminished inhibition of adenylyl cyclase activity by inhibitory hormones and forskolin-stimulated adenylyl cyclase activity by low concentrations of GTPgammaS as well as the enhanced stimulation of adenylyl cyclase by stimulatory agonists in hyperglycemic cells were restored to control levels by antioxidant treatments. These results suggest that high glucose-induced decreased levels of Gialpha proteins and associated signaling in A10 VSMCs may be attributed to the enhanced oxidative stress due to augmented levels of peroxynitrite and not to H2O2.     

Toll-Like Receptor 4 Upregulation by Angiotensin II Contributes to Hypertension and Vascular Dysfunction through Reactive Oxygen Species Production

Hypertension is considered as a low-grade inflammatory disease, with adaptive immunity being an important mediator of this pathology. TLR4 may have a role in the development of several cardiovascular diseases; however, little is known about its participation in hypertension. We aimed to investigate whether TLR4 activation due to increased activity of the renin-angiotensin system (RAS) contributes to hypertension and its associated endothelial dysfunction. For this, we used aortic segments from Wistar rats treated with a non-specific IgG (1 µg/day) and SHRs treated with losartan (15 mg/kg·day), the non-specific IgG or the neutralizing antibody anti-TLR4 (1 µg/day), as well as cultured vascular smooth muscle cells (VSMC) from Wistar and SHRs. TLR4 mRNA levels were greater in the VSMC and aortas from SHRs compared with Wistar rats; losartan treatment reduced those levels in the SHRs. Treatment of the SHRs with the anti-TLR4 antibody: 1) reduced the increased blood pressure, heart rate and phenylephrine-induced contraction while it improved the impaired acetylcholine-induced relaxation; 2) increased the potentiation of phenylephrine contraction after endothelium removal; and 3) abolished the inhibitory effects of tiron, apocynin and catalase on the phenylephrine-induced response as well as its enhancing effect of acetylcholine-induced relaxation. In SHR VSMCs, angiotensin II increased TLR4 mRNA levels, and losartan reduced that increase. CLI-095, a TLR4 inhibitor, mitigated the increases in NAD(P)H oxidase activity, superoxide anion production, migration and proliferation that were induced by angiotensin II. In conclusion, TLR4 pathway activation due to increased RAS activity is involved in hypertension, and by inducing oxidative stress, this pathway contributes to the endothelial dysfunction associated with this pathology. These results suggest that TLR4 and innate immunity may play a role in hypertension and its associated end-organ damage.     

Uric acid mediates photodynamic inactivation of caprine alpha-2-macroglobulin

Uric acid (2,6,8 trioxopurine), the end product of purine metabolism in mammalian systems, has shown a wide range of antioxidant properties including scavenging of hydroxyl radical and singlet oxygen. In this study we show that in the presence of visible light, uric acid disrupted caprine alpha-2-macroglobulin (α₂M) structure and antiproteolytic function in vitro. Proteinase cleaves the bait region of caprine inhibitor inducing major conformational changes and entrapping the enzyme within its molecular cage. In contrast to native α₂M, modified antiproteinase lost half of its antiproteolytic potential within 4 hours of uric acid exposure. The changes in uv-absorption spectra of the treated protein suggested possible spatial rearrangement of subunits or conformational change. Analysis of the mechanism by which α₂M was inactivated revealed that the process was dependent on generation of superoxide anion and hydrogen peroxide. Our findings suggest that antiproteolytic activity of caprine α₂M could be compromised via oxidative modification mediated by uric acid. Moreover, low concentrations of α₂M were found to stimulate superoxide production by some unknown mechanism.     

Intracellular superoxide induces apoptosis in VSMCs: role of mitochondrial membrane potential,cytochrome C and caspases

Apoptosis of vascular smooth muscle cells (VSMCs) is an integral part of cardiovascular diseases including atherosclerosis, hypertension and restenosis. Here we studied the fate of VSMCs in response to intracellular superoxide stimulation. Diethyldithiocarbamic acid (DDC) was used to inhibit copper-zinc superoxide dismutase thereby increasing intracellular superoxide levels. The results show that DDC at a dose from 25–100 M is able to induce VSMC apoptosis. Superoxide was found to be responsible for DDC-induced apoptosis. In the apoptotic process mitochondrial membrane potential was decreased and caspase-3, -8 and -9 were activated. Surprisingly, neither cytochrome c release nor Bid cleavage could be observed. These data suggest a role for intracellular superoxide in the regulation of VSMCs apoptosis.     

Inhibition of xanthine oxidase by uric acid and its influence on superoxide radical production.

The inhibition of xanthine oxidase by its reaction product, uric acid, was studied by steady state kinetic analysis. Uric acid behaved as an uncompetitive inhibitor of xanthine oxidase with respect to the reducing substrate, xanthine. Under 50 microM xanthine and 210 microM oxygen, the apparent K(i) for uric acid was 70 microM. Uric acid-mediated xanthine oxidase inhibition also caused an increase in the percentage of univalent reoxidation of the enzyme (superoxide radical production). Steady-state rate equations derived by the King-Altman method support the formation of an abortive-inhibitory enzyme-uric acid complex (dead-end product inhibition). Alternatively, inhibition could also depend on the reversibility of the classical ping-pong mechanism present in xanthine oxidase-catalyzed reactions.     

Uric acid inhibition of dipeptidyl peptidase IV in vitro is dependent on the intracellular formation of triuret

Uric acid affects endothelial and adipose cell function and has been linked to diseases such as hypertension, metabolic syndrome, and cardiovascular disease. Interestingly uric acid has been shown to increase endothelial progenitor cell (EPC) mobilization, a potential mechanism to repair endothelial injury. Since EPC mobilization is dependent on activity of the enzyme CD26/dipeptidyl peptidase (DPP)IV, we examined the effect uric acid will have on CD26/DPPIV activity. Uric acid inhibited the CD26/DPPIV associated with human umbilical vein endothelial cells but not human recombinant (hr) CD26/DPPIV. However, triuret, a product of uric acid and peroxynitrite, could inhibit cell associated and hrCD26/DPPIV. Increasing or decreasing intracellular peroxynitrite levels enhanced or decreased the ability of uric acid to inhibit cell associated CD26/DPPIV, respectively. Finally, protein modeling demonstrates how triuret can act as a small molecule inhibitor of CD26/DPPIV activity. This is the first time that uric acid or a uric acid reaction product has been shown to affect enzymatic activity and suggests a novel avenue of research in the role of uric acid in the development of clinically important diseases.     

Uric Acid Promotes Apoptosis in Human Proximal Tubule Cells by Oxidative Stress and the Activation of NADPH Oxidase NOX 4

Mild hyperuricemia has been linked to the development and progression of tubulointerstitial renal damage. However the mechanisms by which uric acid may cause these effects are poorly explored. We investigated the effect of uric acid on apoptosis and the underlying mechanisms in a human proximal tubule cell line (HK-2). Increased uric acid concentration decreased tubule cell viability and increased apoptotic cells in a dose dependent manner (up to a 7-fold increase, p<0.0001). Uric acid up-regulated Bax (+60% with respect to Ctrl; p<0.05) and down regulated X-linked inhibitor of apoptosis protein. Apoptosis was blunted by Caspase-9 but not Caspase-8 inhibition. Uric acid induced changes in the mitochondrial membrane, elevations in reactive oxygen species and a pronounced up-regulation of NOX 4 mRNA and protein (p<0.05). In addition, both reactive oxygen species production and apoptosis was prevented by the NADPH oxidase inhibitor DPI as well as by Nox 4 knockdown. URAT 1 transport inhibition by probenecid and losartan and its knock down by specific siRNA, blunted apoptosis, suggesting a URAT 1 dependent cell death. In summary, our data show that uric acid increases the permissiveness of proximal tubule kidney cells to apoptosis by triggering a pathway involving NADPH oxidase signalling and URAT 1 transport. These results might explain the chronic tubulointerstitial damage observed in hyperuricaemic states and suggest that uric acid transport in tubular cells is necessary for urate-induced effects.     

Long-term activation of semicarbazide-sensitive amine oxidase lowers circulating levels of uric acid in diabetic conditions

Uric acid is involved in nitrogenous waste in animals, together with ammonia and urea. Uric acid has also antioxidant properties and is a surrogate marker of metabolic syndrome. We observed that the elevated plasma uric acid of high-fat fed mice was normalized by benzylamine treatment. Indeed, benzylamine is the reference substrate of semicarbazide-sensitive amine oxidase (SSAO), an enzyme highly expressed in fat depots and vessels, which generates ammonia when catalysing oxidative deamination. Ammonia interferes with uric acid metabolism/solubility. Our aim was therefore to investigate whether the lowering action of benzylamine on uric acid was related to an improvement of diabetic complications, or was connected with SSAO-dependent ammonia production. First, we observed that benzylamine administration lowered plasma uric acid in diabetic db/db mice while it did not modify uric acid levels in normoglycemic and lean mice. In parallel, benzylamine improved the glycemic control in diabetic but not in normoglycemic mice, while plasma urea remained unaltered. Then, uric acid plasma levels were measured in mice invalidated for AOC3 gene, encoding for SSAO. These mice were unable to oxidize benzylamine but were not diabetic and exhibited unaltered plasma uric levels. Therefore, activated or abolished ammonia production by SSAO was without influence on uric acid in the context of normoglycemia. Our observations confirm that plasma uric acid increases with diabetes and can be normalized when glucose tolerance is improved. They also show that uric acid, a multifunctional metabolite at the crossroads of nitrogen waste and of antioxidant defences, can be influenced by SSAO, in a manner apparently related to changes in glucose homeostasis.     

Effect of angiotensin II and losartan on the phagocytic activity of peritoneal macrophages from Balb/C mice

Angiotensin II (AII), a product of rennin-angiotensin system, exerts an important role on the function of immune system cells. In this study, the effect of AII on the phagocytic activity of mouse peritoneal macrophages was assessed. Mice peritoneal macrophages were cultured for 48 h and the influence of different concentrations of AII (10(-14) to 10(-7) M) and/or losartan, 10(-16) to 10(-6) M), an AT1 angiotensin receptor antagonist, on phagocytic activity and superoxide anion production was determined. Dimethylthiazoldiphenyltetrazolium bromide reduction and the nucleic acid content were used to assess the cvtotoxicity of losartan. A stimulatory effect on phagocytic activity (P < 0.05) was observed with 10(-13) M and 10(-12 M) AII concentrations. The addition of losartan (up to10(-14) M) to the cell cultures blocked (P < 0.001) the phagocytosis indicating the involvement of AT1 receptors. In contrast, superoxide anion production was not affected by AII or losartan. The existence of AT1 and AT2 receptors in peritoneal macrophages was demonstrated by immunofluorescence microscopy. These results support the hypothesis that AII receptors can modulate murine macrophage activity and phagocytosis, and suggest that AII may have a therapeutic role as an immunomodulatory agent in modifying the host resistance to infection.     

Impact of elevated uric acid on ventricular remodeling in infarcted rats with experimental hyperuricemia

Hyperuricemia is associated with cardiovascular disease, but it is usually considered a marker rather than a risk factor. Previous studies using uric acid-lowering drugs in normouricemic animals are not suitable to answer the effect of hyperuricemia on ventricular remodeling after myocardial infarction. The purpose of this study was to determine whether hyperuricemia adversely affects ventricular remodeling in infarcted rats with elevated uric acid. Male Wistar rats aged 8 wk were randomly assigned into either vehicle, oxonic acid, oxonic acid + allopurinol, oxonic acid + benzbromarone, oxonic acid + ABT-627, or oxonic acid + tempol for 4 wk starting 24 h after ligation. Postinfarction was associated with increased oxidant production, as measured by myocardial superoxide, isoprostane, xanthine oxidase activity, and dihydroethidium staining. Compared with normouricemic infarcted rats, hyperuricemic infarcted rats had a significant increase of superoxide production (1.7×) and endothelin-1 protein (1.2×) and mRNA (1.4×) expression, which was associated with increased left ventricular dysfunction and enhanced myocardial hypertrophy and fibrosis. These changes were all prevented by treatment with allopurinol. For similar levels of urate lowering, the uricosuric agent benzbromarone had no effect on ventricular remodeling. In spite of equivalent hyperuricemia, the ability of both ABT-627 and tempol to attenuate ventricular remodeling suggested involvement of endothelin-1 and redox pathways. Hyperuricemia is associated with unfavorable ventricular remodeling probably through a superoxide and endothelin-1-dependent pathway. Uric acid lowering without inhibition of superoxide and endothelin-1 may not have an effect on remodeling. Chronic administration of allopurinol, ABT-627, and tempol is associated with attenuated ventricular remodeling.     

Inactivation of alpha 1-antiproteinase by hydroxyl radicals. The effect of uric acid

The elastase-inhibitory activity of alpha 1-antiproteinase is inactivated by hydroxyl radicals (.OH) generated by pulse radiolysis or by reaction of iron ions with H2O2 in the presence of superoxide or ascorbate. Uric acid did not protect alpha 1-antiproteinase against inactivation by .OH in pulse radiolysis experiments or in the superoxide/iron/H2O2 system, whereas it did in systems containing ascorbic acid. We propose that radicals formed by attack of .OH on uric acid are themselves able to inactivate alpha 1-antiproteinase, but that these uric acid radicals can be 'repaired' by ascorbic acid.     

Hyperuricemia Causes Pancreatic β-Cell Death and Dysfunction through NF-κB Signaling Pathway

Accumulating clinical evidence suggests that hyperuricemia is associated with an increased risk of type 2 diabetes. However, it is still unclear whether elevated levels of uric acid can cause direct injury of pancreatic β-cells. In this study, we examined the effects of uric acid on β-cell viability and function. Uric acid solution or normal saline was administered intraperitoneally to mice daily for 4 weeks. Uric acid-treated mice exhibited significantly impaired glucose tolerance and lower insulin levels in response to glucose challenge than did control mice. However, there were no significant differences in insulin sensitivity between the two groups. In comparison to the islets in control mice, the islets in the uric acid–treated mice were markedly smaller in size and contained less insulin. Treatment of β-cells in vitro with uric acid activated the NF-κB signaling pathway through IκBα phosphorylation, resulting in upregulated inducible nitric oxide synthase (iNOS) expression and excessive nitric oxide (NO) production. Uric acid treatment also increased apoptosis and downregulated Bcl-2 expression in Min6 cells. In addition, a reduction in insulin secretion under glucose challenge was observed in the uric acid–treated mouse islets. These deleterious effects of uric acid on pancreatic β-cells were attenuated by benzbromarone, an inhibitor of uric acid transporters, NOS inhibitor L-NMMA, and Bay 11–7082, an NF-κB inhibitor. Further investigation indicated that uric acid suppressed levels of MafA protein through enhancing its degradation. Collectively, our data suggested that an elevated level of uric acid causes β-cell injury via the NF-κB-iNOS-NO signaling axis.     

Metformin inhibits inflammatory response via AMPK-PTEN pathway in vascular smooth muscle cells

Atherosclerosis is a chronic inflammation of the coronary arteries. Vascular smooth muscle cells (VSMCs) stimulated by cytokines and chemokines accelerate the inflammatory response and migrate to the injured endothelium during the progression of atherosclerosis. Activation of AMP activated protein kinase (AMPK), a key sensor maintaining metabolic homeostasis, suppresses the inflammatory response. However, how AMPK regulates the inflammatory response is poorly understood. To identify the mechanism of this response, we focused on phosphatase and tensin homolog (PTEN), which is a negative regulator of inflammation. We investigated that activation of AMPK-induced PTEN expression and suppression of the inflammatory response through the AMPK-PTEN pathway in VSMCs. We treated with the well-known AMPK activator metformin to induce PTEN expression. PTEN was induced by metformin (2mM) and inhibited by compound C (10μM) and AMPK siRNA. Tumor necrosis factor-alpha (TNF-α) was used to induce inflammation. The inflammatory response was confirmed by cyclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS) expression, and activation of nuclear factor (NF)-κB. Metformin suppressed COX-2 and iNOS mRNA and protein expression dose dependently. Treatment with compound C and bpv (pic) in the presence of metformin, iNOS and COX-2 protein expression increased. NF-κB activation decreased in response to metformin and was restored by inhibiting AMPK and PTEN. Inhibiting AMPK and PTEN restored ROS levels stimulated with TNF-α. Taken together, PTEN could be a possible downstream regulator of AMPK, and the AMPK-PTEN pathway might be important in the regulation of the inflammatory response in VSMCs.     

Phospholipase A2 modulates respiratory burst developed by neutrophils in patients with rheumatoid arthritis

Activated by bacterial peptides, phorbol esters, calcium ionophores and other agonists, neutrophils (PMNs) release the proinflammatory mediator, arachidonic acid (AA) via the intervention of phospholipase A(2) (PLA(2)). AA may play an essential role in activation of NADPH-oxidase, which is involved in the generation of superoxide anion by neutrophils. The present study is focused on the involvement of PLA(2) in the respiratory burst developed by PMNs isolated from patients with rheumatoid arthritis (RA). PLA(2) exists in very high levels in diseases such as rheumatoid arthritis and may cause acute inflammatory and proliferative changes in synovial structures. The respiratory burst was evaluated as superoxide anion release, using an amplified chemiluminescence method. The assays were performed using PMNs untreated or treated with different doses of stimulatory reagents (phorbol 12-myristate-13-acetate (PMA), calcium ionophore (A23187)). Our data suggested that PMA stimulated the production of superoxide anion in a dose-response manner, as compared with A23187, which did not induce a significant release of superoxide anion in PMNs-RA. The exogenous addition of AA significantly amplified the superoxide anion release by PMNs-RA stimulated with PMA and to a lesser extent, by PMNs stimulated with A23187. AA has also reversed the inhibitory effect of arachidonyl-trifluorometylketone and E-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)2H-pyran-2-one (BEL) on the superoxide anion release by PMNs-RA. In conclusion, the differential responses to these two agents suggested that different isoforms of PLA(2) were activated by A23187 or PMA, and support the idea that activation of these different PLA(2) served distinct functions of PMNs. Therefore, the inhibition of PLA(2) enzymes might be of great importance in the immunotherapy of rheumatoid arthritis.     

Pre-Treatment with Allopurinol or Uricase Attenuates Barrier Dysfunction but Not Inflammation during Murine Ventilator-Induced Lung Injury

Introduction

Uric acid released from injured tissue is considered a major endogenous danger signal and local instillation of uric acid crystals induces acute lung inflammation via activation of the NLRP3 inflammasome. Ventilator-induced lung injury (VILI) is mediated by the NLRP3 inflammasome and increased uric acid levels in lung lavage fluid are reported. We studied levels in human lung injury and the contribution of uric acid in experimental VILI.

Methods

Uric acid levels in lung lavage fluid of patients with acute lung injury (ALI) were determined. In a different cohort of cardiac surgery patients, uric acid levels were correlated with pulmonary leakage index. In a mouse model of VILI the effect of allopurinol (inhibits uric acid synthesis) and uricase (degrades uric acid) pre-treatment on neutrophil influx, up-regulation of adhesion molecules, pulmonary and systemic cytokine levels, lung pathology, and regulation of receptors involved in the recognition of uric acid was studied. In addition, total protein and immunoglobulin M in lung lavage fluid and pulmonary wet/dry ratios were measured as markers of alveolar barrier dysfunction.

Results

Uric acid levels increased in ALI patients. In cardiac surgery patients, elevated levels correlated significantly with the pulmonary leakage index. Allopurinol or uricase treatment did not reduce ventilator-induced inflammation, IκB-α degradation, or up-regulation of NLRP3, Toll-like receptor 2, and Toll-like receptor 4 gene expression in mice. Alveolar barrier dysfunction was attenuated which was most pronounced in mice pre-treated with allopurinol: both treatment strategies reduced wet/dry ratio, allopurinol also lowered total protein and immunoglobulin M levels.

Conclusions

Local uric acid levels increase in patients with ALI. In mice, allopurinol and uricase attenuate ventilator-induced alveolar barrier dysfunction.