Peroxynitrite causes endothelial cell monolayer barrier dysfunction.

Nitric oxide (.NO) attenuates hydrogen peroxide (H(2)O(2))-mediated barrier dysfunction in cultured porcine pulmonary artery endothelial cells (PAEC) (Gupta MP, Ober MD, Patterson C, Al-Hassani M, Natarajan V, and Hart, CM. Am J Physiol Lung Cell Mol Physiol 280: L116-L126, 2001). However,.NO rapidly combines with superoxide (O) to form the powerful oxidant peroxynitrite (ONOO(-)), which we hypothesized would cause PAEC monolayer barrier dysfunction. To test this hypothesis, we treated PAEC with ONOO(-) (500 microM) or 3-morpholinosydnonimine hydrochloride (SIN-1; 1-500 microM). SIN-1-mediated ONOO(-) formation was confirmed by monitoring the oxidation of dihydrorhodamine 123 to rhodamine. Both ONOO(-) and SIN-1 increased albumin clearance (P < 0.05) in the absence of cytotoxicity and altered the architecture of the cytoskeletal proteins actin and beta-catenin as detected by immunofluorescent confocal imaging. ONOO(-)-induced barrier dysfunction was partially reversible and was attenuated by cysteine. Both ONOO(-) and SIN-1 nitrated tyrosine residues, including those on beta-catenin and actin, and oxidized proteins in PAEC. The introduction of actin treated with ONOO(-) into PAEC monolayers via liposomes also resulted in barrier dysfunction. These results indicate that ONOO(-) directly alters endothelial cytoskeletal proteins, leading to barrier dysfunction.

Nitric oxide attenuates H(2)O(2)-induced endothelial barrier dysfunction: mechanisms of protection.

Nitric oxide (.NO) attenuates hydrogen peroxide (H(2)O(2))-mediated injury in porcine pulmonary artery endothelial cells (PAECs) and modulates intracellular levels of cGMP and cAMP. We hypothesized that.NO attenuates H(2)O(2)-induced PAEC monolayer barrier dysfunction through cyclic nucleotide-dependent signaling mechanisms. To examine this hypothesis, cultured PAEC monolayers were treated with H(2)O(2), and barrier function was measured as transmonolayer albumin clearance. H(2)O(2) caused significant PAEC barrier dysfunction that was attenuated by intracellular as well as extracellular.NO generation.NO increased PAEC cGMP and cAMP levels, but treatment with inhibitors of soluble guanylate cyclase or protein kinase G did not abrogate.NO-mediated barrier protection. In contrast, H(2)O(2) decreased protein kinase A activity, and inhibiting protein kinase A abrogated the protective effect of.NO. H(2)O(2)-induced barrier dysfunction was not associated with decreased levels of cGMP or cAMP. 3-Isobutyl-1-methylxanthine and the cGMP analog 8-bromo-cGMP had little effect on H(2)O(2)-mediated endothelial barrier dysfunction, whereas 8-bromo-cAMP plus 3-isobutyl-1-methylxanthine was protective. These results indicate that.NO modulates vascular endothelial barrier function through cAMP-dependent signaling mechanisms.

Posterior capsular opacification in phacotrabeculectomy : a long-term comparative study of silicone versus acrylic intraocular lens.

PURPOSE: To investigate the long-term outcomes of silicone versus acrylic intraocular lens (IOL) implantation in phacotrabeculectomy (PT) with special emphasis on posterior capsular opacification. DESIGN: Long-term follow-up on prior 1-year prospective, randomized study. PARTICIPANTS: A total of 200 eyes of 200 consecutive primary open-angle glaucoma patients who had undergone primary PT with capsular bag implantation of either a silicone IOL (102 eyes) or an acrylic IOL (98 eyes) according to the initial short-term prospective, randomized study protocol. INTERVENTION: The study eyes underwent primary trabeculectomy, phacoemulsification, and posterior chamber IOL implantation. Adjunctive mitomycin C was used selectively, primarily in patients with one or more risk factors for filtration failure. MAIN OUTCOME MEASURES: Incidence of posterior capsular opacification (PCO), best-corrected visual acuity (BCVA), intraocular pressure (IOP), number of pressure-lowering medications, and filtration success rates, defined as maintenance of target IOP while on one (criteria 1) or zero (criteria 2) pressure-lowering medications without further surgical intervention. RESULTS: At 3-year follow-up, the PCO rate and BCVA did not differ significantly between the two groups (P: > 0.05 for both). In addition, there were no significant differences in IOP, number of medications, and filtration success rate between the two groups (P: > 0.05 for each). CONCLUSIONS: There were no significant long-term differences between the silicone and acrylic IOL groups in PCO, BCVA, IOP, number of medications, and success of filtration surgery after PT. Both groups attained significant improvement in BCVA and IOP control after surgery.

Attenuation of oxidant-mediated endothelial cell injury with docosahexaenoic acid: the role of intracellular iron.

Previous studies have demonstrated that altering the fatty acid composition of porcine pulmonary artery endothelial cells (PAEC) significantly modulates their susceptibility to oxidative stimuli, e.g. H2O2. Based on observations that fatty acids also function to transport iron, an important catalyst for H2O2-mediated hydroxyl radical generation, we hypothesized that fatty acid-induced alterations in PAEC iron metabolism contribute to modulation of PAEC oxidant susceptibility. To test this hypothesis, PAEC were treated with culture medium supplemented with 0.1 mM oleic (18:1), linolenic (18:3) or docosahexaenoic (22:6) acids or with an equivalent volume of ethanol vehicle for 3 h. After thorough washing and incubation in unsupplemented culture medium for 24 h, PAEC monolayers were subjected to additional studies. Supplementation with 22:6 attenuated lactate dehydrogenase (LDH) release from PAEC 2 h following treatment with 100 microM H2O2 for 30 min (% LDH release: ETOH-control = 7.9 +/- 1.6, 22:6-control = 5.9 +/- 0.9, ETOH-H2O2 = 26.4 +/- 4.2, 22:6-H2O2* = 16.2 +/- 2.9; *P < 0.05 vs ETOH-H2O2). In a non-cellular system, 18:1 and 18:3 were more effective than their methyl ester derivatives or 22:6 at translocating iron from aqueous to hydrophobic environments. In contrast, only supplementation with 22:6 significantly increased PAEC uptake of 57Fe and human umbilical vein endothelial cell (HUVEC) ferritin content, whereas none of the supplementation conditions altered PAEC catalytic iron measured with bleomycin. These novel observations indicate that specific fatty acids are capable of altering PAEC iron uptake and ferritin content thereby contributing to the understanding of the mechanisms by which fatty acids modulate the oxidant susceptibility of vascular endothelial cells.