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.

Lipid raft-associated cholesterol has been identified as a pivotal player among membrane lipids in regulating cellular functions. Cholesterol of the vascular endothelial cell (EC) membranes is also being recognized as an important element in the vascular EC signaling. However, methods utilized in studying the important role of lipid raft-associated cholesterol in cell signaling involve removal of the raft cholesterol with the aid of chemical agents called cyclodextrins. Caution should be exercised in using cyclodextrins to remove the cellular lipid raft-associated cholesterol as the cyclodextrins cause adverse effects on cells such as loss of cell viability or induction of cytotoxicity. Therefore, the choice of a cyclodextrin to remove the cellular lipid raft-associated cholesterol is extremely important in order to ensure effective and safe removal of cholesterol from the cellular lipid rafts. In order to achieve this, here, we have selected the bovine pulmonary artery endothelial cells (BPAECs) and subjected them to the removal of cholesterol using two different beta-cyclodextrin compounds, methyl-beta-cyclodextrin (MbetaCD) and hydroxypropyl-beta-cyclodextrin (HPCD). Phospholipase D (PLD), which generates one of the most potent bioactive lipid signal mediators (phosphatidic acid), is activated by oxidants. Therefore, we examined the effects of cholesterol removal by utilizing our current methods on the hydrogen peroxide (H(2)O(2))-activated PLD in BPAECs. Differences in the loss of cholesterol and the resulting effects on the cell membrane, cell viability, morphology, and the extent of oxidant-induced PLD activation were determined. The results revealed that both MbetaCD and HPCD caused loss of cholesterol, loss of cell viability, and altered cell morphology in the chosen EC system. It was also determined that the HPCD compound caused far less extensive damage to the cells than the MbetaCD, therefore making the HPCD compound a safer tool for EC cholesterol removal.

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.

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.