Nitrite-generated NO circumvents dysregulated arginine/NOS signaling to protect against intimal hyperplasia in Sprague-Dawley rats.

Vascular disease, a significant cause of morbidity and mortality in the developed world, results from vascular injury. Following vascular injury, damaged or dysfunctional endothelial cells and activated SMCs engage in vasoproliferative remodeling and the formation of flow-limiting intimal hyperplasia (IH). We hypothesized that vascular injury results in decreased bioavailability of NO secondary to dysregulated arginine-dependent NO generation. Furthermore, we postulated that nitrite-dependent NO generation is augmented as an adaptive response to limit vascular injury/proliferation and can be harnessed for its protective effects. Here we report that sodium nitrite (intraperitoneal, inhaled, or oral) limited the development of IH in a rat model of vascular injury. Additionally, nitrite led to the generation of NO in vessels and SMCs, as well as limited SMC proliferation via p21Waf1/Cip1 signaling. These data demonstrate that IH is associated with increased arginase-1 levels, which leads to decreased NO production and bioavailability. Vascular injury also was associated with increased levels of xanthine oxidoreductase (XOR), a known nitrite reductase. Chronic inhibition of XOR and a diet deficient in nitrate/nitrite each exacerbated vascular injury. Moreover, established IH was reversed by dietary supplementation of nitrite. The vasoprotective effects of nitrite were counteracted by inhibition of XOR. These data illustrate the importance of nitrite-generated NO as an endogenous adaptive response and as a pathway that can be harnessed for therapeutic benefit.

Effects of cyclic flexure on endothelial permeability and apoptosis in arterial segments perfused ex vivo.

Certain arteries (e.g., coronary, femoral, etc.) are exposed to cyclic flexure due to their tethering to surrounding tissue beds. It is believed that such stimuli result in a spatially variable biomechanical stress distribution, which has been implicated as a key modulator of remodeling associated with atherosclerotic lesion localization. In this study we utilized a combined ex vivo experimental/computational methodology to address the hypothesis that local variations in shear and mural stress associated with cyclic flexure influence the distribution of early markers of atherogenesis. Bilateral porcine femoral arteries were surgically harvested and perfused ex vivo under pulsatile arterial conditions. One of the paired vessels was exposed to cyclic flexure (0-0.7 cm(-1)) at 1 Hz for 12 h. During the last hour, the perfusate was supplemented with Evan's blue dye-labeled albumin. A custom tissue processing protocol was used to determine the spatial distribution of endothelial permeability, apoptosis, and proliferation. Finite element and computational fluid dynamics techniques were used to determine the mural and shear stress distributions, respectively, for each perfused segment. Biological data obtained experimentally and mechanical stress data estimated computationally were combined in an experiment-specific manner using multiple linear regression analyses. Arterial segments exposed to cyclic flexure had significant increases in intimal and medial apoptosis (3.42+/-1.02 fold, p=0.029) with concomitant increases in permeability (1.14+/-0.04 fold, p=0.026). Regression analyses revealed specific mural stress measures including circumferential stress at systole, and longitudinal pulse stress were quantitatively correlated with the distribution of permeability and apoptosis. The results demonstrated that local variation in mechanical stress in arterial segments subjected to cyclic flexure indeed influence the extent and spatial distribution of the early atherogenic markers. In addition, the importance of including mural stresses in the investigation of vascular mechanopathobiology was highlighted. Specific example results were used to describe a potential mechanism by which systemic risk factors can lead to a heterogeneous disease.

Association of carotid intima-medial thickness and indices of arterial stiffness with cardiovascular disease outcomes in CKD.

BACKGROUND: Indices of arterial structure and stiffness are proposed as surrogate markers of cardiovascular disease in patients with chronic kidney disease (CKD), but no study examined multiple markers in the same population. STUDY DESIGN: Prospective observational study. SETTING & PARTICIPANTS: 315 subjects with stages 4 to 5 CKD, aged 24 to 79 years (mean age, 56.6 +/- 13.6 [SD] years), enrolled in the Atherosclerosis and Folic Acid Supplementation Trial. PREDICTORS: Carotid arterial intima-medial thickness (IMT; n = 315) and indices of arterial stiffness (n = 207), including aortofemoral pulse wave velocity (PWV[a-f]), systemic arterial compliance (SAC), and carotid-derived augmentation index. OUTCOMES: The primary outcome was a composite of all fatal and nonfatal cardiovascular events. RESULTS: During follow-up (median, 3.6 years), 95 cardiovascular events occurred. On Cox proportional-hazard modeling, mean maximum IMT, PWV(a-f), and SAC were predictive of the composite clinical end point of all cardiovascular events, but carotid-derived augmentation index was not (hazard ratio [HR] for every 0.01-mm increase in IMT, 1.09; P = 0.001; 95% confidence interval [CI], 1.03 to 1.14; HR for every 1-m/s increase in PWV(a-f), 1.18; P < 0.001; 95% CI, 1.12 to 1.25; HR for every 0.01-U/mm Hg decrease in SAC, 0.98; P = 0.01; 95% CI, 0.97 to 0.99). After adjustment for age, sex, blood pressure, diabetes, past cardiovascular disease, cholesterol level, and smoking, PWV(a-f) remained a significant independent predictor of cardiovascular events (adjusted HR, 1.12; P = 0.001; 95% CI, 1.05 to 1.20), but IMT and SAC did not. LIMITATIONS: Study power to analyze differences between predialysis and dialysis stages of CKD. CONCLUSIONS: PWV(a-f) is the only arterial index independently associated with cardiovascular outcome in patients with CKD.

Rapamycin inhibits release of tumor necrosis factor-alpha from human vascular smooth muscle cells.

Neointimal proliferation with plaque formation is the principal cause of coronary artery disease. In the neointima, inflammatory cytokines like tumor necrosis factor-alpha (TNF-alpha) are expressed by vascular smooth muscle cells (VSMCs). These cytokines stimulate proliferation and migration of VSMCs, events that are crucial to neointima formation. Stents, liberating rapamycin, have been shown to reduce neointima formation in human coronary arteries. The purpose of this study was to determine if rapamycin could inhibit the production of TNF-alpha by VSMCs. With institutional review board approval, VSMCs were cultured from saphenous vein segments obtained from five patients. Cells were identified as VSMC by immunostaining for smooth muscle alpha-actin. Cells were exposed to bacterial lipopolysaccharide (LPS), LPS plus rapamycin, or LPS plus isoproterenol for 24 hours. Cells with no treatment served as controls. The culture medium was then removed and analyzed for TNF-alpha. Additionally, the effect of treatment on viability was determined by assay of mitochondrial activity. TNF-alpha released into the culture medium is expressed as pg TNF-alpha/mg cell protein. Statistical analysis was by ANOVA. In control cells, TNF-alpha was undetectable in the culture medium. The addition of LPS (10 microg/mL) increased TNF-alpha release to 4312 +/- 705 pg/mg at 24 hours. The addition of 1 ng/mL rapamycin with LPS reduced TNF-alpha production 50 per cent (P < 0.01 vs LPS alone). A similar reduction of TNF-alpha release was seen with 1 microM isoproterenol. LPS, rapamycin, or isoproterenol did not affect cell viability. These data show that rapamycin effectively inhibits the release of TNF-alpha from VSMCs stimulated with inflammatory mediators like LPS. Rapamycin is as effective as agents that raise intracellular cyclic AMP (e.g., isoproterenol). Therefore, a potential mechanism for the effectiveness of rapamycin-releasing stents is reduction of inflammatory cytokine expression by VSMCs.

[Effect of vitamin E on the endothelial function of the regenerated aorta in rats following direct arterial injury]. / Influence de la vitamine E sur la fonction endothéliale de l'aorte de rat régénérée après traumatisme artériel direct.

Fibromuscular intimal injury is frequent after coronary or peripheral angioplasty. Peroxidation of circulating and membrane lipids have been implicated in intimal hyperplasia and endothelial dysfunction following direct arterial trauma. The aim of this study was to evaluate the effects of natural membrane antioxidant, vitamin E (VE), on the vascular reactivity of the regenerated endothelium. A first group of rats (n = 10) was pretreated with VE 100 IU/kg/day for a week before undergoing aortic (thoracic) endothelial denudation with a Fogarty catheter. Rats were then fed with the same VE supplemented diet for a 2-month period. A second group (n = 10), was similarly denudated and fed with soya oil (SO), VE vehicle, for the same period. A third group (n = 10) was denuded and used as control (CL). Endothelial-dependent and independent relaxations were assessed in organ chambers with acetylcholine (ACH), adenosine diphosphate (ADP), histamine (HIS), 5-hydroxytryptamine (5-HT) and sodium nitroprusside (SNP). Endothelial regeneration was evaluated with Evans blue staining. Vascular relaxation to SNP was not affected either by the regeneration process or the VE supplementation. However, endothelial-dependent relaxation to ACH and HIS were significantly impeded in the regenerated endothelium compared to control but was not influenced by the VE or the SO supplementation. Vascular contraction to 5-HT was significantly decreased in VE group compared to the other groups. Morphometric studies with Evans blue staining showed over 95% regeneration of the endothelial surface of the denuded aortas. Our results suggest that in spite of a complete anatomical regeneration, endothelial cells did not resume predenudation function. Endothelial-independent relaxation was preserved in all groups indicating that smooth muscle function was not altered by the regenerating process. The presence of dietary supplement of VE (up to 20 fold the daily requirement) did not prevent the endothelial dysfunction to ACH and HIS but attenuated the vaso-contraction to 5-HT and then could contribute to decreased vascular tone in endothelium-regenerated vessels.

Reduction of experimental vein graft intimal hyperplasia and preservation of nitric oxide-mediated relaxation by the nitric oxide precursor L-arginine.

BACKGROUND: Previous studies in animals and human beings have shown that vein bypass grafts exhibit diminished endothelium-dependent relaxation and the development of intimal hyperplasia. This study examines the effect of L-arginine on experimental vein graft endothelial cell function and the development of intimal hyperplasia. METHODS: Common carotid vein bypass grafts were performed in 24 New Zealand White rabbits: 12 were controls and 12 received L-arginine (2.25%) orally 7 days before operation and thereafter until harvest 28 days after operation. Intimal and medial dimensions were determined by planimetry on pressure-fixed vessels. Relaxation to acetylcholine, serotonin, calcium ionophore (A23187), and sodium nitroprusside was performed on precontracted vessel rings. RESULTS: Arginine-treated vein grafts showed a 47% reduction in mean intimal thickness (p < 0.001) compared with controls. By scanning and transmission electron microscopy, all vein grafts showed a confluent endothelium. In contrast to control grafts, which do not relax to acetylcholine and serotonin, arginine-treated vein grafts relaxed in response to both agonists. There was a significant increase (p < 0.05) in the maximal relaxation to calcium ionophore (A23187) in arginine-treated vein grafts compared with control grafts. Non-endothelium-dependent responses to sodium nitroprusside were equivalent in all vein grafts. CONCLUSIONS: This study shows that oral L-arginine supplementation significantly reduces intimal hyperplasia and preserves nitric oxide-mediated relaxation in experimental vein grafts, suggesting a role for nitric oxide in the regulation of the cellular events that lead to intimal hyperplasia.