Classic Phytochemical Antioxidant and Lipoxygenase Inhibitor, Nordihydroguaiaretic Acid, Activates Phospholipase D through Oxidant Signaling and Tyrosine Phosphorylation Leading to Cytotoxicity in Lung Vascular Endothelial Cells.

Nordihydroguaiaretic acid (NDGA), a dicatechol and phytochemical polyphenolic antioxidant and an established inhibitor of human arachidonic acid (AA) 5-lipoxygenase (LOX) and 15-LOX, is widely used to ascertain the role of LOXs in vascular endothelial cell (EC) function. As the modulatory effect of NDGA on phospholipase D (PLD), an important lipid signaling enzyme in ECs, thus far has not been reported, here we have investigated the modulation of PLD activity and its regulation by NDGA in the bovine pulmonary artery ECs (BPAECs). NDGA induced the activation of PLD (phosphatidic acid formation) in cells in a dose- and time-dependent fashion that was significantly attenuated by iron chelator and antioxidants. NDGA induced the formation of reactive oxygen species (ROS) in cells in a dose- and time-dependent manner as evidenced from fluorescence microscopy and fluorimetry of ROS and electron paramagnetic resonance spectroscopy of oxygen radicals. Also, NDGA caused a dose-dependent loss of intracellular glutathione (GSH) in BPAECs. Protein tyrosine kinase (PTyK)-specific inhibitors significantly attenuated NDGA-induced PLD activation in BPAECs. NDGA also induced a dose- and time-dependent phosphorylation of tyrosine in proteins in cells. NDGA caused in situ translocation and relocalization of both PLD1 and PLD2 isoforms, in a time-dependent fashion. Cyclooxygenase (COX) inhibitors were ineffective in attenuating NDGA-induced PLD activation in BPAECs, thus ruling out the activation of COXs by NDGA. NDGA inhibited the AA-LOX activity and leukotriene C4 (LTC4) formation in cells. On the other hand, the 5-LOX-specific inhibitors, 5, 8, 11, 14-eicosatetraynoic acid and kaempferol, were ineffective in activating PLD in BPAECs. Antioxidants and PTyK-specific inhibitors effectively attenuated NDGA cytotoxicity in BPAECs. The PLD-specific inhibitor, 5-fluoro-2-indolyl deschlorohalopemide (FIPI), significantly attenuated and protected against the NDGA-induced PLD activation and cytotoxicity in BPAECs. For the first time, these results demonstrated that NDGA, the classic phytochemical polyphenolic antioxidant and LOX inhibitor, activated PLD causing cytotoxicity in ECs through upstream oxidant signaling and protein tyrosine phosphorylation.

Significance of HbA1c Test in Diagnosis and Prognosis of Diabetic Patients.

Diabetes is a global endemic with rapidly increasing prevalence in both developing and developed countries. The American Diabetes Association has recommended glycated hemoglobin (HbA1c) as a possible substitute to fasting blood glucose for diagnosis of diabetes. HbA1c is an important indicator of long-term glycemic control with the ability to reflect the cumulative glycemic history of the preceding two to three months. HbA1c not only provides a reliable measure of chronic hyperglycemia but also correlates well with the risk of long-term diabetes complications. Elevated HbA1c has also been regarded as an independent risk factor for coronary heart disease and stroke in subjects with or without diabetes. The valuable information provided by a single HbA1c test has rendered it as a reliable biomarker for the diagnosis and prognosis of diabetes. This review highlights the role of HbA1c in diagnosis and prognosis of diabetes patients.

Role of 5-hydroxymethylcytosine in neurodegeneration.

The recent discovery of 5-hydroxymethylcytosine (5hmC), an epigenetic modifier and oxidation product of 5-methylcytosine (5mC), has broadened the scope and understanding of neural development and neurodegenerative diseases. By virtue of their functional groups, 5mC and 5hmC exert opposite effects on gene expression; the former is generally associated with gene silencing whereas the latter is mainly involved in up-regulation of gene expression affecting the cellular processes such as differentiation, development, and aging. Although DNA methylation plays an important role in normal neural development and neuroprotection, an altered pathway due to complex interaction with environmental and genetic factors may cause severe neurodegeneration. The levels of 5hmC in brain increase progressively from birth until death, while in patients with neurodegenerative disorders, the levels are found to be highly compromised. This article discusses the recent developments in the area of hydroxymethylation, with particular emphasis on the role of 5hmC in neurodegenerative diseases including Alzheimer's disease, Parkinson's diseases and Huntington's disease. We have also included recent findings on the role of 5hmC in brain tumors (gliomas). Despite compelling evidence on the involvement of 5hmC in neurodegeneration, it is yet to be established whether this epigenetic molecule is the cause or the effect of the onset and progression of neurodegenerative diseases.

Intermittent hypoxia exacerbates pancreatic ß-cell dysfunction in a mouse model of diabetes mellitus.

STUDY OBJECTIVES: The effects of intermittent hypoxia (IH) on pancreatic function in the presence of diabetes and the underlying mechanisms are unclear. We hypothesized that IH would exacerbate pancreatic ß-cell dysfunction and alter the fatty acids in the male Tallyho/JngJ (TH) mouse, a rodent model of type 2 diabetes. DESIGN: TH mice were exposed for 14 d to either 8 h of IH or intermittent air (IA), followed by an intraperitoneal glucose tolerance test (IPGTT) and tissue harvest. The effect of IH on insulin release was determined by using a ß3-adrenergic receptor (AR) agonist. MEASUREMENTS AND RESULTS: During IH, pancreatic tissue pO2 decreased from 20.4 ± 0.9 to 5.7 ± 2.6 mm Hg, as determined by electron paramagnetic resonance oximetry. TH mice exposed to IH exhibited higher plasma glucose levels during the IPGTT (P < 0.001) while the insulin levels tended to be lower (P = 0.06). Pancreatic islets of the IH group showed an enhancement of the caspase-3 staining (P = 0.002). IH impaired the ß-AR agonist-mediated insulin release (P < 0.001). IH increased the levels of the total free fatty acids and saturated fatty acids (palmitic and stearic acids), and decreased levels of the monounsaturated fatty acids in the pancreas and plasma. Ex vivo exposure of pancreatic islets to palmitic acid suppressed insulin secretion and decreased islet cell viability. CONCLUSIONS: Intermittent hypoxia increases pancreatic apoptosis and exacerbates dysfunction in a polygenic rodent model of diabetes. An increase in free fatty acids and a shift in composition towards long chain saturated fatty acid species appear to mediate these effects.

Adiponectin protects against hyperoxic lung injury and vascular leak.

Adiponectin (Ad), an adipokine exclusively secreted by the adipose tissue, has emerged as a paracrine metabolic regulator as well as a protectant against oxidative stress. Pharmacological approaches of protecting against clinical hyperoxic lung injury during oxygen therapy/treatment are limited. We have previously reported that Ad inhibits the NADPH oxidase-catalyzed formation of superoxide from molecular oxygen in human neutrophils. Based on this premise, we conducted studies to determine whether (i) exogenous Ad would protect against the hyperoxia-induced barrier dysfunction in the lung endothelial cells (ECs) in vitro, and (ii) endogenously synthesized Ad would protect against hyperoxic lung injury in wild-type (WT) and Ad-overexpressing transgenic (AdTg) mice in vivo. The results demonstrated that exogenous Ad protected against the hyperoxia-induced oxidative stress, loss of glutathione (GSH), cytoskeletal reorganization, barrier dysfunction, and leak in the lung ECs in vitro. Furthermore, the hyperoxia-induced lung injury, vascular leak, and lipid peroxidation were significantly attenuated in AdTg mice in vivo. Also, AdTg mice exhibited elevated levels of total thiols and GSH in the lungs as compared with WT mice. For the first time, our studies demonstrated that Ad protected against the hyperoxia-induced lung damage apparently through attenuation of oxidative stress and modulation of thiol-redox status.

Eicosanoid signaling and vascular dysfunction: methylmercury-induced phospholipase D activation in vascular endothelial cells.

Mercury, especially methylmercury (MeHg), is implicated in the etiology of cardiovascular diseases. Earlier, we have reported that MeHg induces phospholipase D (PLD) activation through oxidative stress and thiol-redox alteration. Hence, we investigated the mechanism of the MeHg-induced PLD activation through the upstream regulation by phospholipase A2 (PLA2) and lipid oxygenases such as cyclooxygenase (COX) and lipoxygenase (LOX) in the bovine pulmonary artery endothelial cells (BPAECs). Our results showed that MeHg significantly activated both PLA2 (release of [(3)H]arachidonic acid, AA) and PLD (formation of [(32)P]phosphatidylbutanol) in BPAECs in dose- (0-10 µM) and time-dependent (0-60 min) fashion. The cPLA2-specific inhibitor, arachidonyl trifluoromethyl ketone (AACOCF3), significantly attenuated the MeHg-induced [(3)H]AA release in ECs. MeHg-induced PLD activation was also inhibited by AACOCF3 and the COX- and LOX-specific inhibitors. MeHg also induced the formation of COX- and LOX-catalyzed eicosanoids in ECs. MeHg-induced cytotoxicity (based on lactate dehydrogenase release) was protected by PLA2-, COX-, and LOX-specific inhibitors and 1-butanol, the PLD-generated PA quencher. For the first time, our studies showed that MeHg activated PLD in vascular ECs through the upstream action of cPLA2 and the COX- and LOX-generated eicosanoids. These results offered insights into the mechanism(s) of the MeHg-mediated vascular endothelial cell lipid signaling as an underlying cause of mercury-induced cardiovascular diseases.

Pulmonary fibrosis inducer, bleomycin, causes redox-sensitive activation of phospholipase D and cytotoxicity through formation of bioactive lipid signal mediator, phosphatidic acid, in lung microvascular endothelial cells.

The mechanisms of lung microvascular complications and pulmonary hypertension known to be associated with idiopathic pulmonary fibrosis (IPF), a debilitating lung disease, are not known. Therefore, we investigated whether bleomycin, the widely used experimental IPF inducer, would be capable of activating phospholipase D (PLD) and generating the bioactive lipid signal-mediator phosphatidic acid (PA) in our established bovine lung microvascular endothelial cell (BLMVEC) model. Our results revealed that bleomycin induced the activation of PLD and generation of PA in a dose-dependent (5, 10, and 100 µg) and time-dependent (2-12 hours) fashion that were significantly attenuated by the PLD-specific inhibitor, 5-fluoro-2-indolyl des-chlorohalopemide (FIPI). PLD activation and PA generation induced by bleomycin (5 µg) were significantly attenuated by the thiol protectant (N-acetyl-L-cysteine), antioxidants, and iron chelators suggesting the role of reactive oxygen species (ROS), lipid peroxidation, and iron therein. Furthermore, our study demonstrated the formation of ROS and loss of glutathione (GSH) in cells following bleomycin treatment, confirming oxidative stress as a key player in the bleomycin-induced PLD activation and PA generation in ECs. More noticeably, PLD activation and PA generation were observed to happen upstream of bleomycin-induced cytotoxicity in BLMVECs, which was protected by FIPI. This was also supported by our current findings that exposure of cells to exogenous PA led to internalization of PA and cytotoxicity in BLMVECs. For the first time, this study revealed novel mechanism of the bleomycin-induced redox-sensitive activation of PLD that led to the generation of PA, which was capable of inducing lung EC cytotoxicity, thus suggesting possible bioactive lipid-signaling mechanism/mechanisms of microvascular disorders encountered in IPF.

Hyperglycemic oxoaldehyde, glyoxal, causes barrier dysfunction, cytoskeletal alterations, and inhibition of angiogenesis in vascular endothelial cells: aminoguanidine protection.

Vascular endothelium is vulnerable to the attack of glucose-derived oxoaldehydes (glyoxal and methylglyoxal) during diabetes, through the formation of advanced glycation end products (AGEs). Although aminoguanidine (AG) has been shown to protect against the AGE-induced adverse effects, its protection against the glyoxal-induced alterations in vascular endothelial cells (ECs) such as cytotoxicity, barrier dysfunction, and inhibition of angiogenesis has not been reported and we investigated this in the bovine pulmonary artery ECs (BPAECs). The results showed that glyoxal (1-10 mM) significantly induced cytotoxicity and mitochondrial dysfunction in a dose- and time-dependent (4-12 h) fashion in ECs. Glyoxal was also observed to significantly inhibit EC proliferation. The study also revealed that glyoxal induced EC barrier dysfunction (loss of trans-endothelial electrical resistance), actin cytoskeletal rearrangement, and tight junction alterations in BPAECs. Furthermore, the results revealed that glyoxal significantly inhibited in vitro angiogenesis on the Matrigel. For the first time, this study demonstrated that AG significantly protected against the glyoxal-induced cytotoxicity, barrier dysfunction, cytoskeletal rearrangement, and inhibition of angiogenesis in BPAECs. Therefore, AG appears as a promising protective agent in the treatment of AGE-induced vascular endothelial alterations and dysfunction during diabetes, presumably by blocking the reactivity of the sugar-derived dicarbonyls such as glyoxal and preventing the formation of AGEs.

Lipoxygenase-catalyzed phospholipid peroxidation: preparation, purification, and characterization of phosphatidylinositol peroxides.

The importance of understanding the mechanisms of modulation of cellular signaling cascades by the peroxidized membrane phospholipids (PLs) is well recognized. The enzyme-catalyzed peroxidation of PLs, as opposed to their oxidation by air and metal catalysis, is well controlled and rapid and yields well-defined PL peroxides which are highly desirable for biological studies. Therefore, here, we chose bovine liver phosphatidylinositol (PI), a crucial membrane PL which acts as the substrate for phospholipase C in cellular signal transduction, as a model membrane PL. We successfully generated the PI peroxides with soybean type-I lipoxygenase (LOX) in the presence of deoxycholate, which facilitates the LOX-mediated peroxidation of the polyunsaturated fatty acids esterified to the PL. The LOX-peroxidized PI, after enzymatic catalysis, was separated from the unoxidized PI in the reaction mixture by normal-phase, high-performance liquid chromatography (HPLC). The extent of LOX-mediated peroxidation of PI following HPLC purification was established by the analysis of lipid phosphorus, conjugated dienes by UV spectrophotometry, peroxides, and loss of fatty acids by gas chromatography. This study established the optimal conditions yielding approximately 46% of peroxidized PI from 300 microg of neat bovine liver PI that was peroxidized by soybean type-I LOX (50 microg) for 30 min in borate buffer (0.2 M, pH 9.0) containing 10 mM deoxycholate.

Calcium and calmodulin regulate mercury-induced phospholipase D activation in vascular endothelial cells.

Earlier, we reported that mercury, the environmental risk factor for cardiovascular diseases, activates vascular endothelial cell (EC) phospholipase D (PLD). Here, we report the novel and significant finding that calcium and calmodulin regulated mercury-induced PLD activation in bovine pulmonary artery ECs (BPAECs). Mercury (mercury chloride, 25 microM; thimerosal, 25 microM; methylmercury, 10 microM) significantly activated PLD in BPAECs. Calcium chelating agents and calcium depletion of the medium completely attenuated the mercury-induced PLD activation in ECs. Calmodulin inhibitors significantly attenuated mercury-induced PLD activation in BPAECs. Despite the absence of L-type calcium channels in ECs, nifedipine, nimodipine, and diltiazem significantly attenuated mercury-induced PLD activation and cytotoxicity in BPAECs. This study demonstrated the importance of calcium and calmodulin in the regulation of mercury-induced PLD activation and the protective action of L-type calcium channel blockers against mercury cytotoxicity in vascular ECs, suggesting mechanisms of mercury vasculotoxicity and mercury-induced cardiovascular diseases.

Redox-active antioxidant modulation of lipid signaling in vascular endothelial cells: vitamin C induces activation of phospholipase D through phospholipase A2, lipoxygenase, and cyclooxygenase.

We have earlier reported that the redox-active antioxidant, vitamin C (ascorbic acid), activates the lipid signaling enzyme, phospholipase D (PLD), at pharmacological doses (mM) in the bovine lung microvascular endothelial cells (BLMVECs). However, the activation of phospholipase A(2) (PLA(2)), another signaling phospholipase, and the modulation of PLD activation by PLA(2) in the ECs treated with vitamin C at pharmacological doses have not been reported to date. Therefore, this study aimed at the regulation of PLD activation by PLA(2) in the cultured BLMVECs exposed to vitamin C at pharmacological concentrations. The results revealed that vitamin C (3-10 mM) significantly activated PLA(2) starting at 30 min; however, the activation of PLD resulted only at 120 min of treatment of cells under identical conditions. Further studies were conducted utilizing specific pharmacological agents to understand the mechanism(s) of activation of PLA(2) and PLD in BLMVECs treated with vitamin C (5 mM) for 120 min. Antioxidants, calcium chelators, iron chelators, and PLA(2) inhibitors offered attenuation of the vitamin C-induced activation of both PLA(2) and PLD in the cells. Vitamin C was also observed to significantly induce the formation and release of the cyclooxygenase (COX)- and lipoxygenase (LOX)-catalyzed arachidonic acid (AA) metabolites and to activate the AA LOX in BLMVECs. The inhibitors of PLA(2), COX, and LOX were observed to effectively and significantly attenuate the vitamin C-induced PLD activation in BLMVECs. For the first time, the results of the present study revealed that the vitamin C-induced activation of PLD in vascular ECs was regulated by the upstream activation of PLA(2), COX, and LOX through the formation of AA metabolites involving oxidative stress, calcium, and iron.