Zhongbing Lu

Structure-activity relationship analysis of antioxidant ability and neuroprotective effect of gallic acid derivatives

Gallic acid and its derivatives are a group of naturally occurring polyphenol antioxidants which have recently been shown to have potential healthy effects. In order to understand the relationship between the structures of gallic acid derivatives, their antioxidant activities, and neuroprotective effects, we examined their free radical scavenging effects in liposome and anti-apoptotic activities in human SH-SY5Y cell induced by 6-hydrodopamine autooxidation. It was found that these polyphenol antioxidants exhibited different hydrophobicity and could cross through the liposome membrane to react with 1,1-diphenyl-2-picryl-hydrazyl (DPPH) free radical in a time and dose-dependent manner. At the same time, the structure-antioxidant activity relationship of gallic acid derivatives on scavenging DPPH free radical in the liposome was also analyzed based on theoretical investigations. Analysis of cell apoptosis, intracellular GSH levels, production of ROS and the influx of Ca(2+) indicated that the protective effects of gallic acid derivatives in cell systems under oxidative stress depend on both their antioxidant capacities and hydrophobicity. However, the neuroprotective effects of gallic acid derivatives seem to depend more on their molecular polarities rather than antioxidant activities in the human SH-SY5Y cell line. In conclusion, these results reveal that compounds with high antioxidant activity and appropriate hydrophobicity are generally more effective in preventing the injury of oxidative stress in neurodegenerative diseases.

Oxidative Stress Regulates Left Ventricular PDE5 Expression in the Failing Heart

Background— Phosphodiesterase type 5 (PDE5) inhibition has been shown to exert profound beneficial effects in the failing heart, suggesting a significant role for PDE5 in the development of congestive heart failure (CHF). The purpose of this study is to test the hypothesis that oxidative stress causes increased PDE5 expression in cardiac myocytes and that increased PDE5 contributes to the development of CHF. Methods and Results— Myocardial PDE5 expression and cellular distribution were determined in left ventricular samples from patients with end-stage CHF and normal donors and from mice after transverse aortic constriction (TAC)–induced CHF. Compared with donor human hearts, myocardial PDE5 protein was increased ≈4.5-fold in CHF samples, and the increase of myocardial PDE5 expression was significantly correlated with myocardial oxidative stress markers 3′-nitrotyrosine or 4-hydroxynonenal expression (P<0.05). Histological examination demonstrated that PDE5 was mainly expressed in vascular smooth muscle in normal donor hearts, but its expression was increased in both cardiac myocytes and vascular smooth muscle of CHF hearts. Myocardial PDE5 protein content and activity also increased in mice after TAC-induced CHF (P<0.05). When the superoxide dismutase (SOD) mimetic M40401 was administered to attenuate oxidative stress, the increased PDE5 protein and activity caused by TAC was blunted, and the hearts were protected against left ventricular hypertrophy and CHF. Conversely, increased myocardial oxidative stress in superoxide dismutase 3 knockout mice caused a greater increase of PDE5 expression and CHF after TAC. In addition, administration of sildenafil to inhibit PDE5 attenuated TAC-induced myocardial oxidative stress, PDE5 expression, and CHF. Conclusions— Myocardial oxidative stress increases PDE5 expression in the failing heart. Reducing oxidative stress by treatment with M40401 attenuated cardiomyocyte PDE5 expression. This and selective inhibition of PDE5 protected the heart against pressure overload-induced left ventricular hypertrophy and CHF.

Dimethylarginine Dimethylaminohydrolase-1 Is the Critical Enzyme for Degrading the Cardiovascular Risk Factor Asymmetrical Dimethylarginine

Objective—The objective of this study was to identify the role of dimethylarginine dimethylaminohydrolase-1 (DDAH1) in degrading the endogenous nitric oxide synthase inhibitors asymmetrical dimethylarginine (ADMA) and Ng-monomethyl-L-arginine (L-NMMA). Methods and Results—We generated a global-DDAH1 gene–deficient (DDAH1−/−) mouse strain to examine the role of DDAH1 in ADMA and L-NMMA degradation and the physiological consequences of loss of DDAH1. Plasma and tissue ADMA and L-NMMA levels in DDAH1−/− mice were several folds higher than in wild-type mice, but growth and development of these DDAH1−/− mice were similar to those of their wild-type littermates. Although the expression of DDAH2 was unaffected, DDAH activity was undetectable in all tissues tested. These findings indicate that DDAH1 is the critical enzyme for ADMA and L-NMMA degradation. Blood pressure was ≈20 mm Hg higher in the DDAH1−/− mice than in wild-type mice, but no other cardiovascular phenotype was found under unstressed conditions. Crossing DDAH1+/− male with DDAH1+/− female mice yielded DDAH1+/+, DDAH1+/−, and DDAH1−/− mice at the anticipated ratio of 1:2:1, indicating that DDAH1 is not required for embryonic development in this strain. Conclusion—Our findings indicate that DDAH1 is required for metabolizing ADMA and L-NMMA in vivo, whereas DDAH2 had no detectable role for degrading ADMA and L-NMMA.

Left Ventricular Failure Produces Profound Lung Remodeling and Pulmonary Hypertension in Mice: Heart Failure Causes Severe Lung Disease

Chronic left ventricular failure causes pulmonary congestion with increased lung weight and type 2 pulmonary hypertension. Understanding the molecular mechanisms for type 2 pulmonary hypertension and the development of novel treatments for this condition requires a robust experimental animal model and a good understanding of the nature of the resultant pulmonary remodeling. Here we demonstrate that chronic transverse aortic constriction causes massive pulmonary fibrosis and remodeling, as well as type 2 pulmonary hypertension, in mice. Thus, aortic constriction-induced left ventricular dysfunction and increased left ventricular end-diastolic pressure are associated with a ⩽5.3-fold increase in lung wet weight and dry weight, pulmonary hypertension, and right ventricular hypertrophy. Interestingly, the aortic constriction-induced increase in lung weight was not associated with pulmonary edema but resulted from profound pulmonary remodeling with a dramatic increase in the percentage of fully muscularized lung vessels, marked vascular and lung fibrosis, myofibroblast proliferation, and leukocyte infiltration. The aortic constriction-induced left ventricular dysfunction was also associated with right ventricular hypertrophy, increased right ventricular end-diastolic pressure, and right atrial hypertrophy. The massive lung fibrosis, leukocyte infiltration, and pulmonary hypertension in mice after transverse aortic constriction clearly indicate that congestive heart failure also causes severe lung disease. The lung fibrosis and leukocyte infiltration may be important mechanisms in the poor clinical outcome in patients with end-stage heart failure. Thus, the effective treatment of left ventricular failure may require additional efforts to reduce lung fibrosis and the inflammatory response.

Global Dimethylarginine Dimethylaminohydrolase-1 (DDAH1) Gene–Deficient Mice Reveal That DDAH1 Is the Critical Enzyme for Degrading the Cardiovascular Risk Factor Asymmetrical Dimethylarginine

Objective—The objective of this study was to identify the role of dimethylarginine dimethylaminohydrolase-1 (DDAH1) in degrading the endogenous nitric oxide synthase inhibitors asymmetrical dimethylarginine (ADMA) and Ng-monomethyl-L-arginine (L-NMMA). Methods and Results—We generated a global-DDAH1 gene–deficient (DDAH1−/−) mouse strain to examine the role of DDAH1 in ADMA and L-NMMA degradation and the physiological consequences of loss of DDAH1. Plasma and tissue ADMA and L-NMMA levels in DDAH1−/− mice were several folds higher than in wild-type mice, but growth and development of these DDAH1−/− mice were similar to those of their wild-type littermates. Although the expression of DDAH2 was unaffected, DDAH activity was undetectable in all tissues tested. These findings indicate that DDAH1 is the critical enzyme for ADMA and L-NMMA degradation. Blood pressure was ≈20 mm Hg higher in the DDAH1−/− mice than in wild-type mice, but no other cardiovascular phenotype was found under unstressed conditions. Crossing DDAH1+/− male with DDAH1+/− female mice yielded DDAH1+/+, DDAH1+/−, and DDAH1−/− mice at the anticipated ratio of 1:2:1, indicating that DDAH1 is not required for embryonic development in this strain. Conclusion—Our findings indicate that DDAH1 is required for metabolizing ADMA and L-NMMA in vivo, whereas DDAH2 had no detectable role for degrading ADMA and L-NMMA.

Extracellular superoxide dismutase protects the heart against oxidative stress and hypertrophy after myocardial infarction

Extracellular superoxide dismutase (EC-SOD) contributes only a small fraction to total SOD activity in the heart but is strategically located to scavenge free radicals in the extracellular compartment. EC-SOD expression is decreased in myocardial-infarction (MI)-induced heart failure, but whether EC-SOD can abrogate oxidative stress or modify MI-induced ventricular remodeling has not been previously studied. Consequently, the effects of EC-SOD gene deficiency (EC-SOD KO) on left ventricular (LV) oxidative stress, hypertrophy, and fibrosis were studied in EC-SOD KO and wild-type mice under control conditions, and at 4 and 8 weeks after permanent coronary artery ligation. EC-SOD KO had no detectable effect on LV function in normal hearts but caused small but significant increases of LV fibrosis. At 8 weeks after MI, EC-SOD KO mice developed significantly more LV hypertrophy (LV mass increased 1.64-fold in KO mice compared to 1.35-fold in wild-type mice; p<0.01) and more fibrosis and myocyte hypertrophy which was more prominent in the peri-infarct region than in the remote myocardium. EC-SOD KO mice had greater increases of nitrotyrosine in the peri-infarct myocardium, and this was associated with a greater reduction of LV ejection fraction, a greater decrease of sarcoplasmic or endoplasmic reticulum calcium2+ ATPase, and a greater increase of atrial natriuretic peptide in the peri-infarct zone compared to wild-type mice. EC-SOD KO was associated with more increases of phosphorylated p38 (p-p38(Thr180/Tyr182)), p42/44 extracellular signal-regulated kinase (p-Erk(Thr202/Tyr204)), and c-Jun N-terminal kinase (p-JNK(Thr183/Tyr185)) both under control conditions and after MI, indicating that EC-SOD KO increases activation of mitogen-activated protein kinase signaling pathways. These findings demonstrate that EC-SOD plays an important role in protecting the heart against oxidative stress and infarction-induced ventricular hypertrophy.

Extracellular Superoxide Dismutase Deficiency Exacerbates Pressure Overload–Induced Left Ventricular Hypertrophy and Dysfunction

Extracellular superoxide dismutase (SOD) contributes only a small fraction to total SOD activity in the normal heart but is strategically located to scavenge free radicals in the extracellular compartment. To examine the physiological significance of extracellular SOD in the response of the heart to hemodynamic stress, we studied the effect of extracellular SOD deficiency on transverse aortic constriction (TAC)–induced left ventricular remodeling. Under unstressed conditions extracellular SOD deficiency had no effect on myocardial total SOD activity, the ratio of glutathione:glutathione disulfide, nitrotyrosine content, or superoxide anion production but resulted in small but significant increases in myocardial fibrosis and ventricular mass. In response to TAC for 6 weeks, extracellular SOD-deficient mice developed more severe left ventricular hypertrophy (heart weight increased 2.56-fold in extracellular SOD-deficient mice as compared with 1.99-fold in wild-type mice) and pulmonary congestion (lung weight increased 2.92-fold in extracellular SOD-deficient mice as compared with 1.84-fold in wild-type mice). Extracellular SOD-deficient mice also had more ventricular fibrosis, dilation, and a greater reduction of left ventricular fractional shortening and rate of pressure development after TAC. TAC resulted in greater increases of ventricular collagen I, collagen III, matrix metalloproteinase-2, matrix metalloproteinase-9, nitrotyrosine, and superoxide anion production. TAC also resulted in a greater decrease of the ratio of glutathione:glutathione disulfide in extracellular SOD-deficient mice. The finding that extracellular SOD deficiency had minimal impact on myocardial overall SOD activity but exacerbated TAC induced myocardial oxidative stress, hypertrophy, fibrosis, and dysfunction indicates that the distribution of extracellular SOD in the extracellular space is critically important in protecting the heart against pressure overload.

Xanthine oxidase inhibition with febuxostat attenuates systolic overload-induced left ventricular hypertrophy and dysfunction in mice.

The purine analog xanthine oxidase (XO) inhibitors (XOIs), allopurinol and oxypurinol, have been reported to protect against heart failure secondary to myocardial infarction or rapid ventricular pacing. Because these agents might influence other aspects of purine metabolism that could influence their effect, this study examined the effect of the non-purine XOI, febuxostat, on pressure overload-induced left ventricular (LV) hypertrophy and dysfunction. Transverse aortic constriction (TAC) in mice caused LV hypertrophy and dysfunction and increased myocardial nitrotyrosine at 8 days. TAC also caused increased phosphorylated Akt (p-Akt(Ser473)), p42/44 extracellular signal-regulated kinase (p-Erk(Thr202/Tyr204)), and mammalian target of rapamycin (mTOR) (p-mTOR(Ser2488)). XO inhibition with febuxostat (5 mg/kg/d by gavage for 8 days) beginning approximately 60minutes after TAC attenuated the TAC-induced LV hypertrophy and dysfunction. Febuxostat blunted the TAC-induced increases in nitrotyrosine (indicating reduced myocardial oxidative stress), p-Erk(Thr202/Tyr204), and p-mTOR(Ser2488), with no effect on total Erk or total mTOR. Febuxostat had no effect on myocardial p-Akt(Ser473) or total Akt. The results suggest that XO inhibition with febuxostat reduced oxidative stress in the pressure overloaded LV, thereby diminishing the activation of pathways that result in pathologic hypertrophy and contractile dysfunction.

Nicotine attenuates β-amyloid-induced neurotoxicity by regulating metal homeostasis

Nicotine reduces β‐amyloidosis and has a beneficial effect against Alzheimers disease (AD), but the underlying mechanism is not clear. The abnormal interactions of β‐amyloid (Aβ) with metal ions such as copper and zinc are implicated in the process of Aβ deposition in AD brains. In the present study, we investigated the effect of nicotine on metal homeostasis in the hippocampus and cortex of APPV717I (London mutant form of APP) transgenic mice. A significant reduction in the metal contents of copper and zinc in senile plaques and neuropil is observed after nicotine treatment. The densities of copper and zinc distributions in a subfield of the hippocampus CA1 region are also reduced after nicotine treatment. We further studied the mechanism of nicotine‐mediated effect on metal homeostasis by using SH‐SY5Y cells overexpress‐ing the Swedish mutant form of human APP (APPsw). Nicotine treatment decreases the intracellular copper concentration and attenuates Aβ‐mediated neurotoxicity facilitated by the addition of copper, and these effects are independent of the activation of nicotinic acetylcholine‐receptor. These data suggest that the effect of nicotine on reducing β‐amyloidosis is partly mediated by regulating metal homeostasis.—Zhang, J., Liu, Q., Chen, Q., Liu, N.‐Q., Li, F.‐L., Lu, Z.‐B., Qin, C., Zhu, H., Huang, Y.‐Y., He, W., and Zhao, B.‐L. Nicotine attenuates β‐amyloid‐induced neurotoxicity by regulating metal homeostasis. FASEB J. 20, E399–E408 (2006)

Exacerbated Pulmonary Arterial Hypertension and Right Ventricular Hypertrophy in Animals With Loss of Function of Extracellular Superoxide Dismutase

Studies have demonstrated that increased oxidative stress contributes to the pathogenesis and the development of pulmonary artery hypertension (PAH). Extracellular superoxide dismutase (SOD3) is essential for removing extracellular superoxide anions, and it is highly expressed in lung tissue. However, it is not clear whether endogenous SOD3 can influence the development of PAH. Here we examined the effect of SOD3 knockout on hypoxia-induced PAH in mice and a loss-of-function SOD3 gene mutation (SOD3E124D) on monocrotaline (40 mg/kg)-induced PAH in rats. SOD3 knockout significantly exacerbated 2 weeks of hypoxia-induced right ventricular (RV) pressure and RV hypertrophy, whereas RV pressure in SOD3 knockout mice under normoxic conditions is similar to wild-type controls. In untreated control rats at age of 8 weeks, there was no significant difference between wild-type and SOD3E124D rats in RV pressure and the ratio of RV weight:left ventricular weight (0.25±0.02 in wild-type rats versus 0.25±0.01 in SOD3E124D rats). However, monocrotaline caused significantly greater increases of RV pressure in SOD3E124D rats (48.6±1.8 mm Hg in wild-type versus 57.5±3.1 mm Hg in SOD3E124D rats), of the ratio of RV weight:left ventricular weight (0.41±0.01 versus 0.50±0.09; P<0.05), and of the percentage of fully muscularized small arterioles in SOD3E124D rats (55.2±2.3% versus 69.9±2.6%; P<0.05). Together, these findings indicate that the endogenous SOD3 has no role in the development of PAH under control conditions but plays an important role in protecting the lung from the development of PAH under stress conditions.