elastin and 3-nitrotyrosine

We tested the hypothesis that curcumin supplementation would reverse arterial dysfunction and vascular oxidative stress with aging. Young (Y, 4-6 months) and old (O, 26-28 months) male C57BL6/N mice were given normal or curcumin supplemented (0.2%) chow for 4 weeks (n=5-10/group/measure). Large elastic artery stiffness, assessed by aortic pulse wave velocity (aPWV), was greater in O (448±15 vs. 349±15 cm/s) and associated with greater collagen I and advanced glycation end-products and less elastin (all P<0.05). In O, curcumin restored aPWV (386±15 cm/s), collagen I and AGEs (AGEs) to levels not different vs. Y. Ex vivo carotid artery acetylcholine (ACh)-induced endothelial-dependent dilation (EDD, 79±3 vs. 94±2%), nitric oxide (NO) bioavailability and protein expression of endothelial NO synthase (eNOS) were lower in O (all P<0.05). In O, curcumin restored NO-mediated EDD (92±2%) to levels of Y. Acute ex vivo administration of the superoxide dismutase (SOD) mimetic TEMPOL normalized EDD in O control mice (93±3%), but had no effect in Y control or O curcumin treated animals. O had greater arterial nitrotyrosine abundance, superoxide production and NADPH oxidase p67 subunit expression, and lower manganese SOD (all P<0.05), all of which were reversed with curcumin. Curcumin had no effects on Y. Curcumin supplementation ameliorates age-associated large elastic artery stiffening, NO-mediated vascular endothelial dysfunction, oxidative stress and increases in collagen and AGEs in mice. Curcumin may be a novel therapy for treating arterial aging in humans.

Topics: Age Factors; Aging; Animals; Antioxidants; Aorta; Carotid Arteries; Collagen Type I; Curcumin; Dose-Response Relationship, Drug; Elastin; Endothelium, Vascular; Glycation End Products, Advanced; Male; Mice; Mice, Inbred C57BL; Nitric Oxide; Nitric Oxide Synthase Type III; Oxidative Stress; Phosphoproteins; Pulse Wave Analysis; Superoxide Dismutase; Superoxides; Tyrosine; Vascular Stiffness; Vasodilation; Vasodilator Agents

We tested the hypothesis that sodium nitrite treatment reverses large elastic artery stiffening in old mice via reductions in collagen I, increases in elastin and/or decreases in advanced glycation end products (AGEs) mediated by reduced oxidative stress. Aortic pulse wave velocity (aPWV), a measure of large elastic artery stiffness, was greater in old (26-28months) compared with young (4-6months) control animals (520±9 vs. 405±6cm/s, p<0.05), and this was reversed by 3weeks of sodium nitrite treatment (50mg/L) (435±17cm/s). Age-related increases (p<0.05) in aortic superoxide production were associated with greater total and adventitial nitrotyrosine staining, all of which were reversed by nitrite treatment. Total and adventitial transforming growth factor β and collagen I were increased, and total and medial elastin were reduced with aging (p<0.05), but were unaffected by sodium nitrite. Aorta from old mice had increased total, adventitial and medial AGEs (p<0.05 vs. young), which were normalized by sodium nitrite treatment. In aortic segments from young mice in vitro, pyrogallol (10μM), a superoxide generator, induced an "aging-like" increase in AGEs, and direct treatment with AGEs induced vascular stiffening; these effects were prevented by incubation with sodium nitrite. De-stiffening of aged large elastic arteries by short-term sodium nitrite therapy is mediated in part by normalization of AGEs secondary to amelioration of oxidative stress.

Topics: Aging; Animals; Antioxidants; Aorta; Collagen Type I; Drug Evaluation, Preclinical; Elasticity; Elastin; Glycation End Products, Advanced; Male; Mice; Oxidative Stress; Pyrogallol; Sodium Nitrite; Superoxides; Tissue Culture Techniques; Transforming Growth Factor beta; Tyrosine; Vascular Stiffness

A decrease in vascular elasticity and an increase in pulse wave velocity in hyperhomocysteinemic (HHcy) cystathionine-beta-synthase heterozygote knockout (CBS(-/+)) mice has been observed. Nitric oxide (NO) is a potential regulator of matrix metalloproteinase (MMP) activity in MMP-NO-tissue inhibitor of metalloproteinase (TIMP) inhibitory tertiary complex. However, the contribution of the nitric oxide synthase (NOS) isoforms eNOS and iNOS in the activation of latent MMP is unclear. We hypothesize that the differential production of NO contributes to oxidative stress and increased oxidative/nitrative activation of MMP, resulting in vascular remodeling in response to HHcy. The overall goal is to elucidate the contribution of the NOS isoforms, endothelial and inducible, in the collagen/elastin switch. Experiments were performed on six groups of animals [wild-type (WT), eNOS(-/-), and iNOS(-/-) with and without homocysteine (Hcy) treatment (0.67 g/l) for 8-12 wk]. In vivo echograph was performed to assess aortic timed flow velocity for indirect compliance measurement. Histological determination of collagen and elastin with trichrome and van Gieson stains, respectively, was performed. In situ measurement of superoxide generation using dihydroethidium was used. Differential expression of eNOS, iNOS, nitrotyrosine, MMP-2 and -9, and elastin were measured by quantitative PCR and Western blot analyses. The 2% gelatin zymography was used to assess MMP activity. The increase in O(2)(-) and robust activity of MMP-9 in eNOS(-/-), WT+Hcy, and eNOS(-/-)+Hcy was accompanied by the gross disorganization and thickening of the ECM along with extensive collagen deposition and elastin degradation (collagen/elastin switch) resulting in a decrease in aortic timed flow velocity. Results show that an increase in iNOS activity is a key contributor to HHcy-mediated collagen/elastin switch and resulting decline in aortic compliance.

Topics: Animals; Aorta; Blood Flow Velocity; Collagen; Compliance; Elastin; Extracellular Matrix; Hyperhomocysteinemia; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitric Oxide; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Tissue Inhibitor of Metalloproteinases; Tyrosine; Ultrasonography

Permissive hypercapnia, achieved using low tidal volume ventilation, has been an effective protective strategy in patients with acute respiratory distress syndrome. To date, no such protective effect has been demonstrated for the chronic neonatal lung injury, bronchopulmonary dysplasia. The objective of our study was to determine whether evolving chronic neonatal lung injury, using a rat model, is resistant to the beneficial effects of hypercapnia or simply requires a less conservative approach to hypercapnia than that applied clinically to date. Neonatal rats inhaled air or 60% O2 for 14 days with or without 5.5% CO2. Lung parenchymal neutrophil and macrophage numbers were significantly increased by hyperoxia alone, which was associated with interstitial thickening and reduced secondary crest formation. The phagocyte influx, interstitial thickening, and impaired alveolar formation were significantly attenuated by concurrent hypercapnia. Hyperoxic pups that received 5.5% CO2 had a significant increase in alveolar number relative to air-exposed pups. Increased tyrosine nitration, a footprint for peroxynitrite-mediated reactions, arteriolar medial wall thickening, and both reduced small peripheral pulmonary vessel number and VEGF and angiopoietin-1 (Ang-1) expression, which were observed with hyperoxia, was attenuated by concurrent hypercapnia. We conclude that evolving chronic neonatal lung injury in a rat model is responsive to the beneficial effects of hypercapnia. Inhaled 5.5% CO2 provided a significant degree of protection against parenchymal and vascular injury in an animal model of chronic neonatal lung injury likely due, at least in part, to its inhibition of a phagocyte influx.

Topics: Actins; Air; Animals; Animals, Newborn; Blood Gas Analysis; Blood Vessels; Carbon Dioxide; Elastin; Fluorescent Antibody Technique; Heart Rate; Hypercapnia; Lung; Lung Injury; Neutrophils; Organ Size; Peroxidase; Rats; Subcellular Fractions; Tyrosine; von Willebrand Factor

Previous studies have demonstrated a relationship between hyperhomocysteinemia and endothelial dysfunction, reduced bioavailability of nitric oxide, elastinolysis and, vascular muscle cell proliferation. In vivo decreased nitric oxide production is associated with increased matrix metalloproteinase (MMP) activity and formation of nitrotyrosine. To test the hypothesis that homocysteine neutralizes vascular endothelial nitric oxide, activates metalloproteinase, causes elastinolysis and vascular hypertrophy, we isolated aortas from normotensive Wistar rats and cultured them in medium containing homocysteine, and calf serum for 14 days. Homocysteine-mediated impairment of endothelial-dependent vasodilatation was reversed by co-incubation of homocysteine with nicotinamide (an inhibitor of peroxinitrite and nitrotyrosine), suggesting a role of homocysteine in redox-mediating endothelial dysfunction and nitrotyrosine formation. The Western blot analysis, using anti-nitrotyrosine antibody, on aortic tissue homogeneates demonstrated decreased nitrotyrosine in hyperhomocysteinemic vessels treated with nicotinamide. Zymographic analysis revealed increased elastinolytic gelatinase A and B (MMP-2, -9) in homocysteine treated vessels and the treatment with nicotinamide decreases the homocysteine-induced MMP activation. Morphometric analyses revealed significant medial hypertrophic thickening (1.4 +/- 0.2-fold of control, P = 0.03) and elastin disruption in homocysteine-treated vessels as compared to control. To determine whether homocysteine causes endothelial cell injury, cross-sections of aortas were analyzed for caspase activity by incubating with Ac-YVAD-AMC (substrate for apoptotic enzyme, caspase). The endothelium of homocysteine treated vessels, and endothelial cells treated with homocysteine, showed marked labeling for caspase. The length-tension relationship of homocysteine treated aortas was shifted to the left as compared to untreated aortas, indicating reduced vascular elastic compliance in homocysteine-treated vessels. Co-incubation of homocysteine and inhibitors of MMP, tissue inhibitor of metalloproteinase-4 (TIMP-4), and caspase, YVAD-CHO, improved vascular function. The results suggest that alteration in vascular elastin/collagen ratio and activation of MMP-2 are associated with decreased NO production in hyperhomocysteinemia.

Topics: Acetylcholine; Animals; Aorta; Caspases; Cysteine; Elastin; Endothelium, Vascular; Glutathione; Homocysteine; In Vitro Techniques; Metalloendopeptidases; Niacinamide; Nitric Oxide; Nitroprusside; Oxidative Stress; Rats; Rats, Wistar; Tissue Inhibitor of Metalloproteinase-4; Tissue Inhibitor of Metalloproteinases; Tyrosine; Vasodilation