Background-We hypothesized that the inflammatory cytokine tumor necrosis factor-␣ (TNF) produces endothelial dysfunction in type 2 diabetes. Methods and Results-In m Leprdb control mice, sodium nitroprusside and acetylcholine induced dose-dependent vasodilation, and dilation to acetylcholine was blocked by the NO synthase inhibitor N G -monomethyl-L-arginine. In type 2 diabetic (Lepr db ) mice, acetylcholine-or flow-induced dilation was blunted compared with m Lepr db , but sodium nitroprusside produced comparable dilation. In Lepr db mice null for TNF (db TNFϪ /db TNFϪ ), dilation to acetylcholine or flow was greater than in diabetic Lepr db mice and comparable to that in controls. Plasma concentration of TNF was significantly increased in Lepr db versus m Lepr db mice. Real-time polymerase chain reaction and Western blotting showed that mRNA and protein expression of TNF and nuclear factor-B were higher in Lepr db mice than in controls. Administration of anti-TNF or soluble receptor of advanced glycation end products attenuated nuclear factor-B and TNF expression in the Lepr db mice. Immunostaining results show that TNF in mouse heart is localized predominantly in vascular smooth muscle cells rather than in endothelial cells and macrophages. Superoxide generation was elevated in vessels from Lepr db mice versus controls. Administration of the superoxide scavenger TEMPOL, NAD(P)H oxidase inhibitor (apocynin), or anti-TNF restored endothelium-dependent dilation in Lepr db mice. NAD(P)H oxidase activity, protein expression of nitrotyrosine, and hydrogen peroxide production were increased in Lepr db mice (compared with controls), but these variables were restored to control levels by anti-TNF. Conclusion-Advanced glycation end products/receptor of advanced glycation end products and nuclear factor-B signaling play pivotal roles in TNF expression through an increase in circulating and/or local vascular TNF production in the Lepr db mouse with type 2 diabetes. Increases in TNF expression induce activation of NAD(P)H oxidase and production of reactive oxidative species, leading to endothelial dysfunction in type 2 diabetes. (Circulation. 2007;115: 245-254.)

Rationale: Cyclic nucleotide phosphodiesterases (PDEs) through the degradation of cGMP play critical roles in maintaining cardiomyocyte homeostasis. Ca 2؉ /calmodulin (CaM)-activated cGMP-hydrolyzing PDE1 family may play a pivotal role in balancing intracellular Ca 2؉ /CaM and cGMP signaling; however, its function in cardiomyocytes is unknown. Objective: Herein, we investigate the role of Ca 2؉ /CaM-stimulated PDE1 in regulating pathological cardiomyocyte hypertrophy in neonatal and adult rat ventricular myocytes and in the heart in vivo. Methods and Results: Inhibition of PDE1 activity using a PDE1-selective inhibitor, IC86340, or downregulation of PDE1A using siRNA prevented phenylephrine induced pathological myocyte hypertrophy and hypertrophic marker expression in neonatal and adult rat ventricular myocytes. Importantly, administration of the PDE1 inhibitor IC86340 attenuated cardiac hypertrophy induced by chronic isoproterenol infusion in vivo. Both PDE1A and PDE1C mRNA and protein were detected in human hearts; however, PDE1A expression was conserved in rodent hearts. Moreover, PDE1A expression was significantly upregulated in vivo in the heart and myocytes from various pathological hypertrophy animal models and in vitro in isolated neonatal and adult rat ventricular myocytes treated with neurohumoral stimuli such as angiotensin II (Ang II) and isoproterenol. Key Words: phosphodiesterase Ⅲ cGMP Ⅲ cardiomyocyte Ⅲ cardiac hypertrophy C a 2ϩ /calmodulin (CaM)-dependent signaling has been implicated in promoting pathological gene expression involved in hypertrophy and heart failure through the activation of Ca 2ϩ /CaM-dependent kinases, phosphatases, and ion channels. 1 Recently, a number of intrinsic negative regulators of cardiac growth have been identified which activate cGMPdependent signaling. 2 Stimulation of cGMP synthesis through genetic upregulation of natriuretic peptide receptor (guanylyl cyclase-A) prevents neurohumoral or pressure overload induced hypertrophy, 3 whereas disruption of cGMP synthesis results in enhanced hypertrophy and deteriorated cardiac function. 4 Likewise, chronic inhibition of cGMP metabolism by a cyclic nucleotide phosphodiesterase (PDE)5 inhibitor prevents and reverses pressure overload induced cardiac hypertrophy. 5 PDEs, by degrading cAMP and/or cGMP, regulate the amplitude, duration, and compartmentation of intracellular cyclic nucleotide signaling. PDEs constitute a superfamily of enzymes grouped into 11 broad families based on their distinct kinetic, regulatory, and inhibitory properties. PDE family members are also differentially expressed in various tissues and present within distinct subcellular domains. Together, these properties enable PDE enzymes to regulate the spatiotemporal, intracellular cAMP and cGMP gradients in response to various external stimuli. At least 5 PDE families, PDE1 to -5, have been reported in the heart, of which PDE1 and PDE5 are most likely responsible for cGMP hydrolysis. Logically, alteration of cGMP-hydrolyzing PDE expression/ activity...

Inflammation is a hallmark of many diseases, such as atherosclerosis, chronic obstructive pulmonary disease, arthritis, infectious diseases, and cancer. Although steroids and cyclooxygenase inhibitors are effective antiinflammatory therapeutical agents, they may cause serious side effects. Therefore, developing unique antiinflammatory agents without significant adverse effects is urgently needed. Vinpocetine, a derivative of the alkaloid vincamine, has long been used for cerebrovascular disorders and cognitive impairment. Its role in inhibiting inflammation, however, remains unexplored. Here, we show that vinpocetine acts as an antiinflammatory agent in vitro and in vivo. In particular, vinpocetine inhibits TNF-α-induced NF-κB activation and the subsequent induction of proinflammatory mediators in multiple cell types, including vascular smooth muscle cells, endothelial cells, macrophages, and epithelial cells. We also show that vinpocetine inhibits monocyte adhesion and chemotaxis, which are critical processes during inflammation. Moreover, vinpocetine potently inhibits TNF-α-or LPS-induced up-regulation of proinflammatory mediators, including TNF-α, IL-1β, and macrophage inflammatory protein-2, and decreases interstitial infiltration of polymorphonuclear leukocytes in a mouse model of TNF-α-or LPSinduced lung inflammation. Interestingly, vinpocetine inhibits NF-κB-dependent inflammatory responses by directly targeting IKK, independent of its well-known inhibitory effects on phosphodiesterase and Ca 2+ regulation. These studies thus identify vinpocetine as a unique antiinflammatory agent that may be repositioned for the treatment of many inflammatory diseases.vinpocetine | inflammation | NF-κB | IKK

Melatonin acts as a crucial signaling and antioxidant molecule with multiple physiological functions in organisms. To explore effects of exogenous melatonin on postharvest browning and its possible mechanisms in litchi fruit, 'Ziniangxi' litchi fruits were treated with an aqueous solution of melatonin at 0.4 mM and then stored at 25 °C for 8 days. The results revealed that melatonin strongly suppressed pericarp browning and delayed discoloration during storage. Melatonin treatment reduced relative membrane-leakage rate and inhibited the generation of superoxide radicals (O), hydrogen peroxide (HO), and malondialdehyde (MDA). Melatonin treatment markedly promoted the accumulation of endogenous melatonin; delayed loss of total phenolics, flavonoids, and anthocyanins; and enhanced the activities of antioxidant enzymes, including superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11), and glutathione reductase (GR, EC 1.6.4.2). By contrast, the activities of browning-related enzymes including polyphenoloxidase (PPO, EC 1.10.3.1) and peroxidase (POD, EC 1.11.1.7) were reduced. In addition, melatonin treatment up-regulated the expression of four genes encoding enzymes for repair of oxidized proteins, including LcMsrA1, LcMsrA2, LcMsrB1, and LcMsB2. These findings indicate that the delay of pericarp browning and senescence by melatonin in harvested litchi fruit could be attributed to the maintenance of redox homeostasis by the improvement of the antioxidant capacity and modulation of the repair of oxidatively damaged proteins.

Background-Despite the importance of endothelial function for coronary regulation, there is little information and virtually no consensus about the causal mechanisms of endothelial dysfunction in myocardial ischemia/reperfusion (I/R) injury. Because tumor necrosis factor-␣ (TNF-␣) is reportedly expressed during ischemia and can induce vascular inflammation leading to endothelial dysfunction, we hypothesized that this inflammatory cytokine may play a pivotal role in I/R injury-induced coronary endothelial dysfunction. Methods and Results-To test this hypothesis, we used a murine model of I/R (30 minutes/90 minutes) in conjunction with neutralizing antibodies to block the actions of TNF-␣. TNF-␣ expression was increased Ͼ4-fold after I/R. To determine whether TNF-␣ abrogates endothelial function after I/R, we assessed endothelial-dependent (ACh) and endothelialindependent (SNP) vasodilation. In sham controls, ACh induced dose-dependent vasodilation that was blocked by the nitric oxide synthase (NOS) inhibitor L-NMMA (10 mol/L), suggesting a key role for NO. In the I/R group, dilation to ACh was blunted, but SNP-induced dilation was preserved. Subsequent incubation of vessels with the superoxide (O 2 ·Ϫ ) scavenger (TEMPOL), or with the inhibitors of xanthine oxidase (allopurinol, oxypurinol), or previous administration of anti-TNF-␣ restored endothelium-dependent dilation in the I/R group and reduced I/R-stimulated O 2 ·Ϫ production in arteriolar endothelial cells. Activation of xanthine oxidase with I/R was prevented by allopurinol or anti-TNF-␣. Conclusions-These results suggest that myocardial I/R initiates expression of TNF-␣, which induces activation of xanthine oxidase and production of O 2 ·Ϫ , leading to coronary endothelial dysfunction. Key Words: coronary artery disease Ⅲ endothelial function Ⅲ nitric oxide Ⅲ microcirculation Ⅲ reactive oxygen species T umor necrosis factor-␣ (TNF-␣) is an inflammatory cytokine 1 that is expressed by macrophages and cardiac tissue early during the myocardial ischemia-reperfusion (I/R) injury. 2,3 Elevations of TNF-␣ expression also appear to cause cardiomyopathy. 4,5 Interestingly, both cardiomyopathy and I/R injury are characterized by endothelial dysfunction, but, a putative role for TNF-␣ in the abnormal vasodilatory responses during I/R has not been elucidated.Studies from our laboratory 6 and others 7,8 have shown that I/R produces vascular endothelial dysfunction as defined by abrogated endothelium-dependent dilation. This adverse effect on endothelial function can occur acutely 6 or chronically (I/R injury in the pig can produce endothelial dysfunction for weeks). 9 However, endothelial dysfunction may be amplified by neutrophil-generated factors including oxygen-derived free radicals, cytokines, proteases, and lipid mediators. 10 I/R generates high levels of free radicals 11 composed of both reactive oxygen intermediates and nitric oxide (NO) via a complex sequence of events. When generated in sufficient concentrations, free radicals directly injure the myocardi...