Role of ROCK2 in CD4+ cells in allergic airways responses in mice.

BACKGROUND: Rho kinases (ROCKs) contribute to allergic airways disease. ROCKs also play a role in lymphocyte proliferation and migration. OBJECTIVE: To determine the role of ROCK2 acting within CD4+ cells in allergic airways responses. METHODS: ROCK2-haploinsufficient (ROCK2+/- ) and wild-type mice were sensitized with ovalbumin (OVA). ROCK2+/- mice then received either CD4+ cells from ROCK2-sufficient OVA TCR transgenic (OT-II) mice or saline i.v. 48 h before challenge with aerosolized OVA. Wild-type mice received saline before challenge. Allergic airways responses were measured 48 h after the last challenge. Allergic airways responses were also assessed in mice lacking ROCK2 only in CD4+ cells (ROCK2CD4Cre mice) vs. control (CD4-Cre and ROCK2flox/flox ) mice. RESULTS: OVA-induced increases in bronchoalveolar lavage lymphocytes, eosinophils, IL-13, IL-5, and eotaxin were reduced in ROCK2+/- vs. wild-type mice, as were airway hyperresponsiveness and mucous hypersecretion. In ROCK2+/- mice, adoptive transfer with CD4+ cells from OT-II mice restored effects of OVA on lymphocytes, eosinophils, IL-13, IL-5, and mucous hypersecretion to wild-type levels, whereas eotaxin and airway hyperresponsiveness were not affected. ROCK2 inhibitors reduced IL-13-induced release of eotaxin from airway smooth muscle (ASM), similar to effects of these inhibitors on ASM contractility. Despite the ability of adoptive transfer to restore allergic airways inflammation in ROCK2-insufficient mice, allergic inflammation was not different in ROCK2CD4Cre vs. control mice. CONCLUSION: ROCK2 contributes to allergic airways responses likely via effects within ASM cells and within non-lymphocyte cells involved in lymphocyte activation and migration into the airways.

Role of Rho kinase isoforms in murine allergic airway responses.

Inhibition of Rho-associated coiled-coil forming kinases (ROCKs) reduces allergic airway responses in mice. The purpose of this study was to determine the roles of the two ROCK isoforms, ROCK1 and ROCK2, in these responses. Wildtype (WT) mice and heterozygous ROCK1 and ROCK2 knockout mice (ROCK1(+/-) and ROCK2(+/-), respectively) were sensitised and challenged with ovalbumin. ROCK expression and activation were assessed by western blotting. Airway responsiveness was measured by forced oscillation. Bronchoalveolar lavage was performed and the lungs were fixed for histological assessment. Compared with WT mice, ROCK1 and ROCK2 expression were 50% lower in lungs of ROCK1(+/-) and ROCK2(+/-) mice, respectively, without changes in the other isoform. In WT lungs, ROCK activation increased after ovalbumin challenge and was sustained for several hours. This activation was reduced in ROCK1(+/-) and ROCK2(+/-) lungs. Airway responsiveness was comparable in WT, ROCK1(+/-), and ROCK2(+/-) mice challenged with PBS. Ovalbumin challenge caused airway hyperresponsiveness in WT, but not ROCK1(+/-) or ROCK2(+/-) mice. Lavage eosinophils and goblet cell hyperplasia were significantly reduced in ovalbumin-challenged ROCK1(+/-) and ROCK2(+/-) versus WT mice. Ovalbumin-induced changes in lavage interleukin-13, interleukin-5 and lymphocytes were also reduced in ROCK1(+/-) mice. In conclusion, both ROCK1 and ROCK2 are important in regulating allergic airway responses.

Anti-inflammatory properties of cytochrome P450 epoxygenase-derived eicosanoids.

The epoxyeicosatrienoic acids (EETs) are products of cytochrome P450 epoxygenases that have vasodilatory properties similar to that of endothelium-derived hyperpolarizing factor. The cytochrome P450 isoform CYP2J2 was cloned and identified as a potential source of EETs in human endothelial cells. Physiological concentrations of EETs or overexpression of CYP2J2 decreased cytokine-induced endothelial cell adhesion molecule expression, and EETs prevented leukocyte adhesion to the vascular wall by a mechanism involving inhibition of transcription factor NF-kappaB and IkappaB kinase. The inhibitory effects of EETs were independent of their membrane-hyperpolarizing effects, suggesting that these molecules play an important nonvasodilatory role in vascular inflammation.

The G proteins of the G alpha i and G alpha q family couple the bradykinin receptor to the release of endothelium-derived relaxing factor.

Bradykinin stimulates diverse functions in endothelial cells including the release of endothelium-derived relaxing factor (EDRF). Little is known, however, regarding the identity of the G protein(s) involved. Here we demonstrate that G proteins of the G alpha i and G alpha q family are coupled to the bradykinin receptor (BKR) in bovine aortic endothelial cells by using specific antisera directed against the COOH-terminal region of G alpha i2 (P4), G alpha i3 (EC), and G alpha q (QL). These antisera are specific since their effects are blocked by the decapeptides from which they were derived. The degree of receptor-G protein coupling was assessed by the formation of high affinity agonist binding sites (HABS) and GTP hydrolysis. In a concentration-dependent manner, the QL antisera reduced HABS and GTPase activity by 65 and 60%, respectively, and effectively abolished them in membranes from pertussis toxin-treated cells. The combination of P4 and EC antisera produced a loss of HABS (41%) and GTPase activity (40%) comparable to the effects of pertussis toxin. These findings indicate that G alpha i and G alpha q proteins mediate the cellular responses to bradykinin in bovine aortic endothelial cells and support the observation that bradykinin-stimulated EDRF release is relatively insensitive to pertussis toxin.

Regulation of G-protein alpha i2 subunit expression by oxidized low-density lipoprotein.

Oxidized low-density lipoprotein (LDL) inhibits signalling pathways mediated by pertussis toxin-sensitive guanine nucleotide-binding proteins (Gi proteins). To determine whether this inhibition is due to altered G protein alpha i subunit expression, mRNA and protein levels of alpha i isoforms were assessed in bovine aortic endothelial cells treated with oxidized LDL (0-100 micrograms/ml, 0-72 h). Oxidized LDL did not affect the expression of alpha i3, but did cause time- and concentration-dependent decrease in alpha i2 mRNA and protein resulting in a 3.2- and 3.5-fold reduction, respectively, after 72 h. This decrease in alpha i2 coincided with a 86% decrease in alpha i2 GTPase activity. Nuclear run-off studies did not show any significant effect of oxidized LDL on alpha i2 or alpha i3 transcription. In the presence of actinomycin D, oxidized LDL shortened the t1/2 of alpha i2 mRNA from 16 h to 8 h which was attenuated by cycloheximide. In addition, pulse-chase labelling with [35S]methionine revealed that oxidized LDL reduced the t1/2 of alpha i2 protein from 27 to 14 h. Our results indicate that oxidized LDL can modulate receptor-Gi coupling by downregulating the expression of alpha i2, but not alpha i3. The mechanism involves both mRNA destabilization and protein degradation.

Regulation of bovine endothelial constitutive nitric oxide synthase by oxygen.

Oxygen (O2) may regulate pulmonary vascular resistance through changes in endothelial nitric oxide (NO) production. To determine whether constitutive NO synthase (cNOS) is regulated by O2, we assessed cNOS expression and activity in bovine pulmonary artery endothelial cells exposed to different concentrations of O2. In a time-dependent manner, changes in O2 concentration from 95 to 3% produced a progressive decrease in cNOS mRNA and protein levels resulting in 4.8- and 4.3-fold reductions after 24h, respectively. This correlated with changes in cNOS activity as determined by nitrite measurements. Compared with 20% O2, cNOS activity was increased 1.5-fold in 95% O2 and decreased 1.9-fold in 3% O2. A decrease in O2 concentration from 94 to 3% shortened cNOS mRNA half-life from 46 to 24 h and caused a 20-fold repression of cNOS gene transcription. Treatment with cycloheximide produced a threefold increase in cNOS mRNA at all O2 concentrations, suggesting that cNOS mRNA expression is negatively regulated under basal condition. We conclude that O2 upregulates cNOS expression through transcriptional and post-transcriptional mechanisms. A decrease in cNOS activity in the presence of low O2 levels, therefore, may contribute to hypoxia-induced vasoconstriction in the pulmonary circulation.

Statins as antioxidant therapy for preventing cardiac myocyte hypertrophy.

Cardiac hypertrophy is a major cause of morbidity and mortality worldwide. The hypertrophic process is mediated, in part, by small G proteins of the Rho family. We hypothesized that statins, inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase, inhibit cardiac hypertrophy by blocking Rho isoprenylation. We treated neonatal rat cardiac myocytes with angiotensin II (AngII) with and without simvastatin (Sim) and found that Sim decreased AngII-induced protein content, [3H] leucine uptake, and atrial natriuretic factor (ANF) promoter activity. These effects were associated with decreases in cell size, membrane Rho activity, superoxide anion (O2*-) production, and intracellular oxidation, and were reversed with L-mevalonate or geranylgeranylpyrophosphate, but not with farnesylpyrophosphate or cholesterol. Treatments with the Rho inhibitor C3 exotoxin and with cell-permeable superoxide dismutase also decreased AngII-induced O2*- production and myocyte hypertrophy. Overexpression of the dominant-negative Rho mutant N17Rac1 completely inhibited AngII-induced intracellular oxidation and ANF promoter activity, while N19RhoA partially inhibited it, and N17Cdc42 had no effect. Indeed, Sim inhibited cardiac hypertrophy and decreased myocardial Rac1 activity and O2*- production in rats treated with AngII infusion or subjected to transaortic constriction. These findings suggest that statins prevent the development of cardiac hypertrophy through an antioxidant mechanism involving inhibition of Rac1.

Nitric oxide decreases cytokine-induced endothelial activation. Nitric oxide selectively reduces endothelial expression of adhesion molecules and proinflammatory cytokines.

To test the hypothesis that nitric oxide (NO) limits endothelial activation, we treated cytokine-stimulated human saphenous vein endothelial cells with several NO donors and assessed their effects on the inducible expression of vascular cell adhesion molecule-1 (VCAM-1). In a concentration-dependent manner, NO inhibited interleukin (IL)-1 alpha-stimulated VCAM-1 expression by 35-55% as determined by cell surface enzyme immunoassays and flow cytometry. This inhibition was paralleled by reduced monocyte adhesion to endothelial monolayers in nonstatic assays, was unaffected by cGMP analogues, and was quantitatively similar after stimulation by either IL-1 alpha, IL-1 beta, IL-4, tumor necrosis factor (TNF alpha), or bacterial lipopolysaccharide. NO also decreased the endothelial expression of other leukocyte adhesion molecules (E-selectin and to a lesser extent, intercellular adhesion molecule-1) and secretable cytokines (IL-6 and IL-8). Inhibition of endogenous NO production by L-N-monomethyl-arginine also induced the expression of VCAM-1, but did not augment cytokine-induced VCAM-1 expression. Nuclear run-on assays, transfection studies using various VCAM-1 promoter reporter gene constructs, and electrophoretic mobility shift assays indicated that NO represses VCAM-1 gene transcription, in part, by inhibiting NF-kappa B. We propose that NO's ability to limit endothelial activation and inhibit monocyte adhesion may contribute to some of its antiatherogenic and antiinflammatory properties within the vessel wall.

Neuroprotection mediated by changes in the endothelial actin cytoskeleton.

Cerebral blood flow is regulated by endothelium-derived nitric oxide (NO), and endothelial NO synthase-deficient (eNOS-deficient; eNOS(-/-)) mice develop larger cerebral infarctions following middle cerebral artery (MCA) occlusion. We report that disruption of Rho-mediated endothelial actin cytoskeleton leads to the upregulation of eNOS expression and reduces the severity of cerebral ischemia following MCA occlusion. Mice treated with the Rho inhibitor Clostridium botulinum C3 transferase (10 microgram/d) or the actin cytoskeleton disrupter cytochalasin D (1 mg/kg) showed a two- to fourfold increase in vascular eNOS expression and activity. This increase in eNOS expression was not due to increases in eNOS gene transcription, but to prolongation of eNOS mRNA half-life from 10 +/- 3 hours to 24 +/- 4 hours. Indeed, endothelial cells overexpressing a dominant-negative Rho mutant (N19RhoA) exhibited decreased actin stress fiber formation and increased eNOS expression. Inhibition of vascular Rho guanosine-5'-triphosphate binding activity by the 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor simvastatin increased cerebral blood flow to ischemic regions of the brain, and mice treated with simvastatin, C3 transferase, or cytochalasin D showed smaller cerebral infarctions following MCA occlusion. No neuroprotection was observed with these agents in eNOS(-/-) mice. These findings suggest that therapies which target the endothelial actin cytoskeleton may have beneficial effects in ischemic stroke.

Simvastatin decreases IL-6 and IL-8 production in epithelial cells.

Many cardiovascular studies have suggested that 3-hydroxy-3-methylglutaryl co-enzyme A reductase inhibitors (statins) have anti-inflammatory effects independent of cholesterol lowering. As a chronic inflammatory disease, periodontitis shares some mechanisms with atherosclerosis. Since oral epithelial cells participate importantly in periodontal inflammation, we measured simvastatin effects on interleukin-6 and interleukin-8 production by cultured human epithelial cell line (KB cells) in response to interleukin-1alpha. Simvastatin decreased production, an effect reversed by adding mevalonate or geranylgeranyl pyrophosphate, but not farnesyl pyrophosphate. Simvastatin was found to reduce NF-kappaB and AP-1 promoter activity in KB cells. Dominant-negative Rac1 severely inhibited interleukin-1alpha-induced NF-kappaB and AP-1 promoter activity. Our results may indicate an anti-inflammatory effect of simvastatin on human oral epithelial cells, apparently involving Rac1 GTPase inhibition.

Inhibition of vascular smooth muscle cell proliferation by sodium salicylate mediated by upregulation of p21(Waf1) and p27(Kip1).

BACKGROUND: Salicylates may have direct vascular effects by mechanisms that are independent of platelet inhibition. METHODS AND RESULTS: We investigated the effect of salicylates on vascular smooth muscle cell (SMC) proliferation in response to platelet-derived growth factor (PDGF) in vitro. Salicylate concentrations of 5 and 10 mmol/L inhibited serum- or PDGF-induced SMC cell count and [(3)H]thymidine incorporation by 62% and 81%, respectively. There was no evidence of cellular toxicity or apoptosis as determined by trypan blue exclusion and FACS analyses. Because cell cycle progression is regulated by hyperphosphorylation of the retinoblastoma (Rb) protein, we examined the effects of salicylate on Rb hyperphosphorylation. Treatment with salicylate, but not indomethacin, inhibited nuclear factor-kappaB activation and completely abolished Rb hyperphosphorylation in PDGF-treated SMCs. This effect was associated with a decrease in cyclin-dependent kinase (Cdk)-2 and, to a lesser extent, Cdk-6, but not Cdk-4 activity, without changes in Cdk-2, -4, and -6 and cyclin D and E protein levels. Because Cdk-2 activity is regulated by the Cdk inhibitors p21(Waf1) and p27(Kip1), we studied the effects of salicylate on p21(Waf1) and p27(Kip1) expression. Treatment with salicylate prevented PDGF-induced downregulation of p21(Waf1) and p27(Kip1) but not of the Cdk-4/-6 inhibitor p16(Ink4). CONCLUSIONS: These findings indicate that high doses of salicylates inhibit SMC proliferation by cell cycle arrest at the G(1)-S phase and suggest a beneficial role for high-dose salicylates in the treatment of vascular proliferative disorders.

Suppression of endothelial nitric oxide production after withdrawal of statin treatment is mediated by negative feedback regulation of rho GTPase gene transcription.

BACKGROUND: Statins improve endothelial function by upregulating endothelial nitric oxide (NO) production that is mediated by inhibiting the isoprenylation of rho GTPase. Withdrawal of statin treatment could suppress endothelial NO production and may impair vascular function. METHODS AND RESULTS: To test this hypothesis, mice were treated for 14 days with 10 mg/kg atorvastatin per day; this led to the upregulation of endothelial NO synthase expression and activity by 2.3- and 3-fold, respectively. Withdrawal of statins resulted in a dramatic, 90% decrease of NO production after 2 days. In mouse aortas and cultured endothelial cells, statins upregulated the expression of rho GTPase in the cytosol, but statins blocked isoprenoid-dependent rho membrane translocation and GTP-binding activity. Inhibiting the downstream targets of rho showed that rho expression is controlled by a negative feedback mechanism mediated by the actin cytoskeleton. Measuring rho mRNA half-life and nuclear run-on assays demonstrated that statins or disruption of actin stress fibers increased rho gene transcription but not rho mRNA stability. Therefore, treatment with statins leads to the accumulation of nonisoprenylated rho in the cytosol. Withdrawing statin treatment restored the availability of isoprenoids and resulted in a massive membrane translocation and activation of rho, causing downregulation of endothelial NO production. CONCLUSIONS: Withdrawal of statin therapy in normocholesterolemic mice results in a transient increase of rho activity, causing a suppression of endothelial NO production. The underlying molecular mechanism is a negative feedback regulation of rho gene transcription mediated by the actin cytoskeleton.

Pleiotropic effects of 3-hydroxy-3-methylglutaryl coenzyme a reductase inhibitors.

The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors or statins are potent inhibitors of cholesterol biosynthesis. Several large clinical trials have demonstrated the beneficial effects of statins in the primary and secondary prevention of coronary heart disease. However, the overall clinical benefits observed with statin therapy appear to be greater than what might be expected from changes in lipid profile alone, suggesting that the beneficial effects of statins may extend beyond their effects on serum cholesterol levels. Indeed, recent experimental and clinical evidence indicates that some of the cholesterol-independent or "pleiotropic" effects of statins involve improving or restoring endothelial function, enhancing the stability of atherosclerotic plaques, and decreasing oxidative stress and vascular inflammation. Many of these pleiotropic effects of statins are mediated by their ability to block the synthesis of important isoprenoid intermediates, which serve as lipid attachments for a variety of intracellular signaling molecules. In particular, the inhibition of small GTP-binding proteins, Rho, Ras, and Rac, whose proper membrane localization and function are dependent on isoprenylation, may play an important role in mediating the direct cellular effects of statins on the vascular wall.

Induction of endothelial-leukocyte interaction by interferon-gamma requires coactivation of nuclear factor-kappaB.

To determine whether nuclear factor (NF)-kappaB is necessary to confer endothelial cell responsiveness to interferon (INF)-gamma in terms of vascular cell adhesion molecule (VCAM)-1 expression and leukocyte adhesion, human endothelial cells were treated with IFN-gamma in the presence of low concentrations (LCs) of interleukin (IL)-1alpha (

Differential regulation of endothelial cell adhesion molecule expression by nitric oxide donors and antioxidants.

Although nitric oxide (NO) and antioxidants inhibit adhesion molecule expression, their inhibitory effects on nuclear factor kappaB (NF-kappaB) activation may differ. The NO donors, but not 8-bromo-cGMP, decreased tumor necrosis factor alpha (TNF-alpha)-induced VCAM-1, ICAM-1, and E-selectin expression by 11-70%. In contrast, NAC completely abolished VCAM-1 and E-selectin expression and decreased ICAM-1 expression by 56%. Gel shift assays demonstrate that NF-kappaB activation was inhibited by both NO and antioxidants. The activation of NF-kappaB involves the phosphorylation and degradation of its cytoplasmic inhibitor IkappaB-alpha by 26S proteasomes. The 26S proteasome inhibitor MG132 prevented the degradation of phosphorylated IkappaB-alpha. NAC inhibited IkappaB kinase (IKK) activity and prevented IkappaB-alpha phosphorylation and degradation. In contrast, NO did not inhibit IKK activity, IkappaB-alpha phosphorylation, or IkappaB-alpha degradation. However, NO, but not antioxidants, induced IkappaB-alpha promoter activity. The inhibitory effects of NO on adhesion molecule expression, therefore, differs from that of antioxidants in terms of the mechanism by which NF-kappaB is inactivated.

Direct vascular effects of HMG-CoA reductase inhibitors.

The 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors, or statins, are potent inhibitors of cholesterol synthesis and large clinical trials have demonstrated that these agents reduce cholesterol and the incidence of cardiovascular diseases. Recent evidence, however, suggests that the beneficial effects of statins may extend beyond their effects on serum cholesterol levels. Because statins also inhibit the synthesis of isoprenoid intermediates in the cholesterol biosynthetic pathway, they may have pleiotropic effects on vascular wall cells. In particular, the small GTP-binding protein, Rho, whose membrane localization and activity are affected by post-translational isoprenylation, may play an important role in mediating the direct vascular effects of statins.

Salicylates and vascular smooth muscle cell proliferation: molecular mechanisms for cell cycle arrest.

Salicylates are effective prophylactic treatment strategies for myocardial infarction and ischemic strokes. Recent evidence suggests that high doses of salicylates may exert direct, platelet-independent effects on the vascular wall. Salicylate and aspirin, in concentrations between 1 and 5 mM, effectively inhibit vascular smooth muscle cell proliferation and DNA synthesis without inducing cellular toxicity or apoptosis. This inhibition is associated with effects on specific cell-cycle regulatory molecules, and may proceed via downregulation of the transcription factor, nuclear factor (NF)-kappaB. High-dose salicylates and selective NF-kappaB inhibitors may, therefore, play an important role in the management of vascular proliferative disorders.

Mevastatin, an HMG-CoA reductase inhibitor, reduces stroke damage and upregulates endothelial nitric oxide synthase in mice.

BACKGROUND AND PURPOSE: The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) lower serum cholesterol and decrease the incidence of stroke and cardiovascular disease. There is growing evidence that statins exert some of their beneficial effects independent of cholesterol lowering. Indeed, we have previously demonstrated that chronic simvastatin administration upregulates endothelial nitric oxide synthase (eNOS), resulting in more functional protein, augmentation of cerebral blood flow, and neuroprotection in a murine model of cerebral ischemia. In this report we examined whether another member of the statin family shared these effects and whether eNOS upregulation is sustained with longer treatment. METHODS: Mevastatin (2 mg/kg or 20 mg/kg per day) was administered to 18- to 22-g male mice for 7, 14, or 28 days before 2-hour middle cerebral artery occlusion with the use of the filament model (n=9 to 12). Neurological deficits and cerebral infarct volumes were assessed at 24 hours. Arterial blood pressure and gases, relative cerebral blood flow, and blood cholesterol levels were monitored in a subset of animals (n=5). Absolute cerebral blood flow was measured by the [(14)C]iodoamphetamine indicator fractionation technique (n=6). eNOS mRNA and protein levels were determined. RESULTS: Mevastatin increased levels of eNOS mRNA and protein, reduced infarct size, and improved neurological deficits in a dose- and time-dependent manner. Greatest protection was seen with 14- and 28-day high-dose treatment (26% and 37% infarct reduction, respectively). Cholesterol levels were reduced only after 28 days of treatment and did not correlate with infarct reduction. Baseline absolute cerebral blood flow was 30% higher after 14-day high-dose treatment. CONCLUSIONS: Chronic prophylactic treatment with mevastatin upregulated eNOS and augmented cerebral blood flow. These changes occurred in the absence of changes in serum cholesterol levels, were sustained for up to 1 month of treatment, and resulted in neuroprotection after middle cerebral artery occlusion.

Endothelial nitric oxide synthase-dependent cerebral blood flow augmentation by L-arginine after chronic statin treatment.

Nitric oxide, a product of nitric oxide synthase activity, relaxes vascular smooth muscle and elevates brain blood flow. We evaluated the importance of eNOS to cerebral blood flow augmentation after L-arginine infusion and increases in flow after eNOS upregulation in SV-129 mice. Blood flow was measured by laser-Doppler flowmetry before and after L-arginine infusion (450 mg/kg during a 15-minute period) or measured by 14C-iodoamphetamine indicator fractionation or 14C-iodoantipyrine tissue equilibration techniques. rCBF increased by 26% (laser Doppler flowmetry) after L-arginine infusion but did not change in mutant mice deficient in eNOS expression. After eNOS upregulation by chronic simvastatin treatment (2 mg/kg subcutaneously, daily for 14 days), L-arginine amplified and sustained the hyperemia (38%) and increased absolute brain blood flow from 86 +/- 7 to 119 +/- 10 mL/100 g per minute. Furthermore, pretreatment with simvastatin enhanced blood flow within ischemic brain tissue after middle cerebral artery occlusion. Together, these findings suggest that eNOS activity is critical for blood flow augmentation during acute L-arginine infusion, and chronic eNOS upregulation combined with L-arginine administration provides a novel strategy to elevate cerebral blood flow in the normal and ischemic brain.

Fatty acid modulation of endothelial activation.

Dietary balance of long-chain fatty acids may influence processes involving leukocyte-endothelial interactions, such as atherogenesis and inflammation, that involve increased endothelial expression of leukocyte adhesion molecules, or endothelial activation. We compared the ability of various saturated, monounsaturated, and polyunsaturated fatty acids to modulate endothelial activation. Consumption of the n-3 fatty acid docosahexaenoic acid (DHA; 22:6n-3) reduced endothelial expression of vascular cell adhesion molecule 1 (VCAM-1), E-selectin, intercellular adhesion molecule 1 (ICAM-1), interleukin 6 (IL-6), and IL-8 in response to IL-1, IL-4, tumor necrosis factor, or bacterial endotoxin, with a half-maximal inhibitory concentration (IC(50)) of 1-25 micromol, ie, in the range of nutritionally achievable plasma concentrations. The magnitude of this effect paralleled its incorporation into cellular phospholipids. DHA also reduced the adhesion of human monocytes and monocytic U937 cells to cytokine-stimulated endothelial cells. These effects were accompanied by a reduction in VCAM-1 messenger RNA, indicating a pretranslational effect. To assess structural fatty acid determinants of VCAM-1 inhibitory activity, we compared various saturated, monounsaturated, and n-6 and n-3 polyunsaturated fatty acids for their VCAM-1 inhibitory activity. Saturated fatty acids did not inhibit cytokine-induced expression of adhesion molecules. However, a progressive increase in inhibitory activity was observed with dietary intake of fatty acids with the same chain length but increasing double bonds, ie, from monounsaturated to n-6 and, further, to n-3 fatty acids. Thus, the greater number of double bonds seems critical for the greater activity of n-3 compared with n-6 fatty acids in inhibiting endothelial activation. These properties are likely to be relevant to the antiatherogenic and antiinflammatory properties of n-3 fatty acids.