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John F Keaney

Publications and source records attributed to John F Keaney.

At least 19 recordsLinked to original sources
PubMed2006

Hydrogen peroxide restrains endothelium-derived nitric oxide bioactivity -- role for iron-dependent oxidative stress.

Vascular diseases are characterized by impairment of endothelial-derived nitric oxide (NO) bioactivity and increased vascular levels of hydrogen peroxide (H(2)O(2)). Here we examined the implications of H(2)O(2) for agonist-stimulated endothelial NO bioactivity in rabbit aortic rings and cultured porcine aortic endothelial cells (PAEC). Vessels pre-treated with H(2)O(2) exhibited impaired endothelial-dependent relaxation induced by acetylcholine or calcium ionophore. In contrast, H(2)O(2) had no effect on endothelium-independent relaxation induced by a NO donor, indicating a defect in endothelium-derived NO. This defect was not related to eNOS catalytic activity; treatment of PAEC with H(2)O(2) enhanced agonist-stimulated eNOS activity indicated by increased eNOS phosphorylation at Ser-1177 and de-phosphorylation at Thr-495 and enhanced conversion of [(3)H]-L-arginine to [(3)H]-L-citrulline that was prevented by inhibitors of Src and phosphatidylinositol-3 kinases. Despite activating eNOS, H(2)O(2) impaired endothelial NO bioactivity indicated by attenuation of the increase in intracellular cGMP in PAEC stimulated with calcium ionophore or NO. The decrease in cGMP was not due to impaired guanylyl cyclase as H(2)O(2) treatment increased cGMP accumulation in response to BAY 41-2272, a NO-independent activator of soluble guanylyl cyclase. At concentrations that impaired endothelial NO bioactivity H(2)O(2) increased intracellular oxidative stress and size of the labile iron pool in PAEC. The increase in oxidative stress was prevented by the free radical scavenger's tempol or tiron and the iron chelator desferrioxamine and these antioxidants reversed the H(2)O(2)-induced impairment of NO bioactivity in PAEC. This study shows that despite promoting eNOS activity, H(2)O(2) impairs endothelial NO bioactivity by promoting oxidative inactivation of synthesized NO. The study highlights another way in which oxidative stress may impair NO bioactivity during vascular disease.

Animals↗
PubMed2006

Relations of inflammation and novel risk factors to valvular calcification.

Investigators have suggested that inflammation may play a role in the pathogenesis of valve calcium. Participants in the Framingham Heart Study's offspring cohort had systemic levels of C-reactive protein, intercellular adhesion molecule-1, interleukin-6, and monocyte chemoattractant protein-1 measured at examination cycle 7. Mitral annular calcium, aortic annular calcium, aortic sclerosis, and aortic stenosis were assessed by echocardiography at examination cycle 6. Logistic regression was used to examine the odds of valvular calcium per 1 unit increase in inflammation (ISUM), a summary statistic of all normalized deviates of the individual markers. Two thousand six hundred eighty-three participants (mean age 61 +/- 10 years; 52% women) were analyzed: 8.2% (n = 216) had > or = 1 calcified valve or annulus; 89 had mitral annular calcium, 78 had aortic annular calcium, 135 had aortic sclerosis, and 33 had aortic stenosis. Participants with valvular calcium were older and were more likely to have hypertension and diabetes mellitus. Participants with valve calcium had higher median levels of all markers. For each log unit increase in ISUM, after adjustment for age and gender, there was an associated 1.1-fold increased odds of > or = 1 calcified valve (p = 0.02); the odds ratios were no longer significant after adjustment for cardiovascular disease risk factors (odds ratio 1.0, 95% confidence interval 0.9 to 1.1). Similar results were obtained for the individual markers and the odds of > or = 1 calcified valve. In conclusion, inflammatory markers were elevated in patients with valvular calcium. Our findings suggest that much of the observed association between systemic inflammatory markers and valvular calcium may be due to shared risk factors.

Aged↗
PubMed2006

Contribution of clinical correlates and 13 C-reactive protein gene polymorphisms to interindividual variability in serum C-reactive protein level.

BACKGROUND: Serum C-reactive protein (CRP) level is a heritable complex trait that predicts incident cardiovascular disease. We investigated the clinical and genetic sources of interindividual variability in serum CRP. METHODS AND RESULTS: We studied serum CRP in 3301 Framingham Heart Study (FHS) participants (mean age 61 years, 53% women). Twelve clinical covariates explained 26% of the variability in CRP level, with body mass index alone explaining 15% (P<0.0001) of the variance. To investigate the influence of genetic variation at the CRP gene on CRP levels, we first constructed a dense linkage disequilibrium map for common single-nucleotide polymorphisms (SNPs) spanning the CRP locus (1 SNP every 850 bases, 26 kilobase [kb] genomic region). Thirteen CRP SNPs were genotyped in 1640 unrelated FHS participants with measured CRP levels. After adjustment for clinical covariates, 9 of 13 SNPs were associated with CRP level (P<0.05). To account for correlation among SNPs, we conducted forward stepwise selection among all 13 SNPs; a triallelic SNP (rs3091244) remained associated with CRP level (stepwise P<0.0001). The triallelic SNP (C-->T-->A; allele frequencies 62%, 31%, and 7%), located in the promoter sequence, explained 1.4% of total serum CRP variation; haplotypes harboring the minor T and A alleles of this SNP were associated with higher CRP level (haplotype P=0.0002 and 0.004). CONCLUSIONS: In our community-based sample, clinical variables explained 26% of the interindividual variation in CRP, whereas a common triallelic CRP SNP contributed modestly. Studies of larger samples are warranted to assess the association of genetic variation in CRP and risk of cardiovascular disease.

Age Factors↗
PubMed2006

Endoscopic versus conventional radial artery harvest for coronary artery bypass grafting: functional and histologic assessment of the conduit.

BACKGROUND: The radial artery's propensity for vasospasm and vulnerability to surgical trauma are well known. A less invasive endoscopic method to harvest the radial artery was recently introduced, but its effect on radial artery integrity is unknown. METHODS: To compare the effects of harvest method on radial artery function, we prospectively randomized 54 patients undergoing coronary artery bypass grafting with the radial artery into 3 groups on the basis of harvest techniques: endoscopic, conventional with cautery, and conventional with harmonic scalpel. We assessed endothelium-dependent and endothelium-independent relaxation of radial artery segments to sequential doses of acetylcholine and nitroglycerin, respectively, using standard organ-chamber methodology. Vasospasm was assessed as the vasoconstrictor response to the thromboxane analog U46619. We assessed endothelial integrity using light and electron microscopy and by rating intercellular adhesion molecule 1, vascular cell adhesion molecule 1, and P-selectin expression by means of immunohistochemistry on a semiquantitative 0- to 3-point scale. Harvest procedures were performed by a single surgeon, and data analyses were blinded to the harvesting method. RESULTS: Maximal relaxation-contraction responses to acetylcholine, nitroglycerin, and U46619 and effective drug concentration yielding 50% response were similar in the 3 groups. Adhesion molecule expression and histologic changes, as assessed by means of light and electron microscopy, were similar in the 3 groups. CONCLUSIONS: Endoscopic harvest does not alter radial artery vasoreactivity or endothelial integrity compared with conventional harvest techniques. Because the endoscopic technique is less invasive, it might prove to be the technique of choice to harvest the radial artery.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed2006

Cross-sectional correlates of serum heat shock protein 70 in the community.

BACKGROUND: Recent studies of referral samples suggest that heat shock proteins play a key role in the pathogenesis of high BP and cardiovascular diseases (CVD) including heart failure. It is unclear whether circulating heat shock protein 70 (HSP70) levels are related to CVD risk factors, echocardiographic indexes of left ventricular (LV) remodeling, and prevalent CVD in the population. METHODS: We evaluated the cross-sectional relations of serum HSP70 to established CVD risk factors (including hypertension), markers of oxidative stress (urinary 8-epi-PGF(2alpha)) and inflammation (plasma interleukin-6, C-reactive protein, monocyte chemoattractant protein-1 MCP-1, and soluble intercellular adhesion molecule sICAM-1), echocardiographic LV dimensions and prevalent CVD in 456 Framingham Offspring Study participants (mean age 61 years, 42% women). RESULTS: In multivariable analyses, serum HSP70 was not associated with age, sex, vascular risk factors (including hypertension), echocardiographic LV mass or prevalent CVD. Also, serum HSP70 was not related to any of the biomarkers evaluated (p> or = 0.10 for all). CONCLUSIONS: In our community-based sample, serum HSP70 was similar in men and women, and not significantly related to traditional or novel risk factors, to LV mass or to prevalent CVD. Our data suggest that blood levels may not adequately reflect the important role of heat shock proteins in prevalent CVD.

Biomarkers↗
PubMed2006

The relationship between aldosterone, oxidative stress, and inflammation in chronic, stable human heart failure.

BACKGROUND: Aldosterone antagonists reduce morbidity and mortality in patients with severe heart failure, but the mechanisms responsible are not fully understood. Observations in animal models suggest that elevated levels of aldosterone promote oxidative stress and inflammation in the myocardium. It is unknown if these findings are relevant to heart failure patients who may have much lower aldosterone levels. METHODS AND RESULTS: We therefore examined the relationship of plasma aldosterone levels to markers of oxidative stress, inflammation and matrix turnover in 58 patients with chronic, stable heart failure from systolic dysfunction (LV ejection fraction <0.40) who were not receiving aldosterone antagonists. Chronic, stable heart failure patients had modestly elevated levels of aldosterone. Additionally, these patients had elevated levels of 8-isoprostaglandin F(2alpha), C-reactive protein, soluble intercellular adhesion molecule-1, osteopontin, brain natriuretic peptide, procollagen type III aminoterminal peptide, and tissue inhibitor of metalloproteinase-1. Among these patients with heart failure, aldosterone levels correlated with 8-iso-PGF(2alpha) (P = .003), ICAM-1 (P = .008), and TIMP-1 (P = .006) after adjustment for age, gender, race, diabetes, smoking, heart rate, left ventricular mass, and body mass index. CONCLUSION: In chronic, stable heart failure patients on standard therapy, higher aldosterone levels are associated with systemic evidence of oxidative stress, inflammation, and matrix turnover.

Aldosterone↗
PubMed2005

CCL2 polymorphisms are associated with serum monocyte chemoattractant protein-1 levels and myocardial infarction in the Framingham Heart Study.

BACKGROUND: Monocyte chemoattractant protein-1 (MCP-1) is a chemokine strongly implicated in promoting atherosclerosis in animal models, but human genetic evidence is contradictory. METHODS AND RESULTS: We analyzed the association of genetic variation in the MCP-1 gene (CCL2) with prevalent myocardial infarction and serum MCP-1 levels in the community-based Framingham Heart Study Offspring Cohort (50% women; mean age, 62 years). MCP-1 levels and CCL2 genotypes were determined in 3236 and 1797 individuals, respectively. Significant clinical correlates of MCP-1 levels were age, cigarette smoking, triglycerides, body mass index, and waist-to-hip ratio. The MCP-1-2578G allele located in the CCL2 regulatory region was significantly associated with both higher serum MCP-1 levels in a recessive genetic model (358+/-10 versus 328+/-3 pg/mL; P=0.002) and higher prevalence of myocardial infarction in a dominant genetic model (adjusted odds ratio, 2.0; 95% CI, 1.2 to 3.3; P=0.005). We also defined the linkage disequilibrium structure at the CCL2 locus and observed 6 common haplotypes in whites. We performed haplotype-based association analysis and found that only the most frequent haplotype, defined by the MCP-1-2578G allele, was associated with prevalent MI. CONCLUSIONS: Our data are consistent with the hypothesis that MCP-1 is involved in the pathogenesis of human atherosclerosis and myocardial infarction.

Aged↗
PubMed2005

Increased susceptibility to pulmonary hypertension in heterozygous BMPR2-mutant mice.

BACKGROUND: Bone morphogenetic protein receptor-2 (BMPR2)-heterozygous, mutant (BMPR2(+/-)) mice have a genetic trait similar to that of certain patients with idiopathic pulmonary arterial hypertension (IPAH). To understand the role of BMPR2 in the development of IPAH, we examined the phenotype of BMPR2(+/-) mice and their response to inflammatory stress. METHODS AND RESULTS: BMPR2(+/-) mice were found to have the same life span, right ventricular systolic pressure (RVSP), and lung histology as those of wild-type mice under unstressed conditions. However, when treated with recombinant adenovirus expressing 5-lipoxygenase (Ad5LO), BMPR2(+/-) mice exhibited significantly higher RVSP than wild-type mice. The increase of RVSP occurred in the first 2 weeks after Ad5LO delivery. Modest but significant muscularization of distal pulmonary arterioles appeared in BMPR2(+/-) mice 4 weeks after Ad5LO treatment. Measurement of urinary metabolites of vasoactive molecules showed that cysteinyl leukotrienes, prostacyclin metabolites, and PGE2 were all increased to a similar degree in both BMPR2(+/-) and wild-type mice during 5LO transgene expression, whereas urinary endothelin-1 remained undetectable. Urinary thromboxane A2 metabolites, in contrast, were significantly higher in BMPR2(+/-) than in wild-type mice and paralleled the increase in RVSP. Platelet activation markers, serotonin, and soluble P-selectin showed a trend toward higher concentrations in BMPR2(+/-) than wild-type mice. Cell culture studies found that BMP treatment reduced interleukin-1beta-stimulated thromboxane A2 production in the pulmonary epithelial cell line A549. CONCLUSIONS: BMPR2(+/-) mice do not develop pulmonary hypertension spontaneously; however, under inflammatory stress, they are more susceptible to an increase in RVSP, thromboxane A2 production, and vascular remodeling than wild-type mice.

Adenoviridae↗
PubMed2005

Estradiol-mediated endothelial nitric oxide synthase association with heat shock protein 90 requires adenosine monophosphate-dependent protein kinase.

BACKGROUND: Estradiol activates endothelial nitric oxide synthase (eNOS) by mechanisms that involve estrogen receptor-alpha (ERalpha), protein kinase B/Akt, mitogen-activated protein kinases, and heat shock protein 90 (HSP90). Recently, AMP-activated protein kinase (AMPK), an enzyme that plays a crucial role in cellular adaptation to metabolic stress, has been implicated in physiological eNOS activation by the hormones adiponectin and insulin. We therefore investigated whether AMPK is activated by estradiol in endothelial cells and plays a role in estradiol-induced eNOS activation. METHODS AND RESULTS: Porcine aortic endothelial cells exhibited time- and concentration-dependent AMPK activation as determined by phosphorylation of AMPK and its downstream target acetyl coenzyme A carboxylase in response to estradiol (1 nmol/L to 10 micromol/L, 1 to 30 minutes). AMPK activation by estradiol was independent of both AMP levels and ERalpha but required estradiol conversion to its catechol metabolites. Estradiol treatment increased eNOS catalytic activity, an effect that was largely reversed when endothelial cells were infected with an AMPK dominant-negative adenovirus. However, inhibition of AMPK did not alter estradiol-induced eNOS phosphorylation at serine 1177 or threonine 495 but decreased eNOS interaction with HSP90. Consistent with this observation, blood vessels from alpha1-AMPK-null mice exhibited defective eNOS-mediated NO production in response to estradiol. CONCLUSIONS: Taken together, these data indicate that AMPK activity is essential for estradiol-induced eNOS activation via the promotion of eNOS interaction with HSP90. These data point to a novel role for AMPK in modulating endothelial cell NO bioactivity and HSP90 function.

Adenylate Kinase↗
PubMed2005

p38 mitogen-activated protein kinase activates eNOS in endothelial cells by an estrogen receptor alpha-dependent pathway in response to black tea polyphenols.

Black tea has been shown to improve endothelial function in patients with coronary artery disease and recent data indicate the polyphenol fraction of black tea enhances endothelial nitric oxide synthase (eNOS) activity through p38 MAP kinase (p38 MAPK) activation. Because the mechanisms for this phenomenon are not yet clear, we sought to elucidate the signaling events in response to black tea polyphenols. Bovine aortic endothelial cells (BAECs) exposed to black tea polyphenols demonstrated eNOS activation that was inhibited by the estrogen receptor (ER) antagonist ICI 182,780, and siRNA-mediated silencing of ER expression. Consistent with this observation, black tea polyphenols induced time-dependent phosphorylation of ERalpha on Ser-118 that was inhibited by ICI 182,780. Phosphorylation of ERalpha on Ser-118 was due to p38 MAP kinase (p38 MAPK) as, it was inhibited by SB203580 and overexpression of dominant-negative p38alpha MAPK. Conversely, constitutively active MKK6 induced p38 MAPK activation that recapitulated the effects of polyphenols by inducing ERalpha phosphorylation and downstream activation of Akt, and eNOS. The key role of ERalpha Ser-118 phosphorylation was confirmed in eNOS-transfected COS-7 cells, as polyphenol-induced eNOS activation required cotransfection with ERalpha subject to phosphorylation at Ser-118. This residue appeared critical for functional association of ERalpha with p38 MAPK as ERalpha with Ser-118 mutated to alanine could not form a complex with p38 MAPK. These findings suggest p38 MAP kinase-mediated eNOS activation requires ERalpha and these data uncover a new mechanism of ERalpha activation that has broad implications for NO bioactivity and endothelial cell phenotype.

Animals↗
PubMed2005

Differential effects of diabetes on the expression of the gp91phox homologues nox1 and nox4.

The nox2-dependent NADPH oxidase was shown to be a major superoxide source in vascular disease, including diabetes. Smooth muscle cells of large arteries lack the phagocytic gp91phox subunit of the enzyme; however, two homologues have been identified in these cells, nox1 and nox4. It remained to be established whether also increases in protein levels of the nonphagocytic NADPH oxidase contribute to increased superoxide formation in diabetic vessels. To investigate changes in the expression of these homologues, we measured their expression in aortic vessels of type I diabetic rats. Eight weeks after streptozotocin treatment, we found a doubling in nox1 protein expression, while the expression of nox4 remained unchanged. This was associated with a significant increase in the NADPH oxidase activity in membrane fractions of diabetic heart and aortic tissue. Furthermore, we observed a decreased sensitivity of diabetic vessels to acetylcholine and nitroglycerin and a decrease in both acetylcholine-stimulated NO production and phosphorylation of VASP, despite an increase in endothelial NO synthase (NOSIII) expression. In addition, xanthine oxidase activity was markedly increased in plasma and 100,000 g supernatant of cardiac tissue of diabetic rats, while myocardial mitochondrial superoxide formation was only weakly enhanced. We conclude that in addition to phagocytic NADPH oxidase, also nonphagocytic, vascular NADPH oxidase subunit nox1, uncoupled NOSIII, and plasma xanthine oxidase contribute to endothelial dysfunction in the setting of diabetes mellitus.

Acetylcholine↗
PubMed2005

Genome scan of systemic biomarkers of vascular inflammation in the Framingham Heart Study: evidence for susceptibility loci on 1q.

Vascular inflammation plays a central role in atherosclerosis and inflammatory biomarkers predict risk of cardiovascular disease (CVD). Thus, finding genes that influence systemic levels of inflammatory biomarkers may provide insights into genetic determinants of vascular inflammation and CVD. We conducted variance-component linkage analyses of blood levels of four biomarkers of vascular inflammation [C-reactive protein (CRP), interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), soluble intercellular adhesion molecule-1 (sICAM-1)] in 304 extended families from the Framingham Heart Study, using data from a 10cM genome scan. We computed p-values by a permutation approach. Heritability estimates ranged from 14% (IL-6) to 44% (MCP-1) after log transforming and adjusting for covariates. Significant linkage to MCP-1 was found on chromosome 1 (LOD=4.27 at 186cM; genome-wide p=0.005), in a region containing inflammatory candidate genes such as SELE, SELP (E- and P-selectin) and CRP. Other linkage peaks with LOD scores >2 were found for MCP-1 on chromosome 1 (LOD=2.04 at 16cM; LOD=2.34 at 70cM) and chromosome 17 (LOD=2.44 at 22cM) and for sICAM-1 on chromosome 1 at 229cM (LOD=2.09) less than 5cM from the interleukin-10 (IL10) gene. Multiple genes on chromosome 1 may influence inflammatory biomarker levels and may have a potential role in development of CVD.

Aged↗
PubMed2005

EGF receptor-dependent JNK activation is involved in arsenite-induced p21Cip1/Waf1 upregulation and endothelial apoptosis.

Arsenic exposure is associated with an increased risk of atherosclerosis and vascular diseases. Although endothelial cells have long been considered to be the primary targets of arsenic toxicity, the underlying molecular mechanism remains largely unknown. In this study, we sought to explore the signaling pathway triggered by sodium arsenite and its implication for endothelial phenotype. We found that sodium arsenite produced time- and dose-dependent decreases in human umbilical vein endothelial cell viability. This effect correlated with the induction of p21Cip1/Waf1 (up to 10-fold), a regulatory protein of cell cycle and apoptosis. We also found that arsenite-stimulated EGF (ErbB1) and ErbB2 receptor transactivation, manifest as receptor tyrosine phosphorylation, appeared to be a proximal signaling event leading to p21Cip1/Waf1 induction, because both pharmacological inhibitors and knockdown of receptors by RNA interference blocked arsenite-induced p21Cip1/Waf1 upregulation. Arsenite-induced activation of JNK and p38 MAPK was distinct, with only JNK as a downstream target of the EGF receptor. Moreover, inhibition of JNK with SP-600125 or dominant negative MKK7 inhibited only p21Cip1/Waf1 induction, whereas the p38 MAPK inhibitor SB-203580 or dominant negative MKK4 inhibited both p21Cip1/Waf1 and p53 induction. Functionally, inhibition of p21Cip1/Waf1 induction prevented endothelial apoptosis due to arsenite treatment. Insofar as endothelial dysfunction promotes vascular disease, these data provide a mechanism for the increased incidence of cardiovascular disease due to arsenite exposure.

Apoptosis↗
PubMed2005

Effects of black tea consumption on plasma catechins and markers of oxidative stress and inflammation in patients with coronary artery disease.

We previously demonstrated that black tea consumption reverses endothelial dysfunction in patients with coronary artery disease. To investigate potential mechanisms of this effect, we examined plasma catechins and systemic markers of oxidation, inflammation, and antioxidant protection from 66 subjects enrolled in that study. We collected samples at baseline, 2 h after 450 ml of black tea (acute), after 4 weeks of 900 ml of black tea per day (chronic), and after acute and chronic consumption of water. Total catechins increased 33% after acute tea (P < 0.05) and 29% after chronic tea (P < 0.05). Of individual catechins, plasma epicatechin gallate (ECG) concentration significantly increased with acute tea consumption, and plasma epicatechin (EC) increased with chronic tea consumption. Tea consumption did not improve plasma antioxidant capacity and did not reduce urinary 8-hydroxy-2'-deoxyguanosine, or urinary 8-isoprostane levels. Changes in catechin levels did not correlate with changes in endothelial function, plasma markers of oxidative stress, or C-reactive protein. In contrast, endothelial function at baseline correlated with dietary flavonoid intake (beta = 0.32, P = 0.02) and with baseline plasma EC concentration after adjusting for confounding variables (beta = 0.39, P = 0.03). These findings suggest that the benefits of black tea consumption on endothelial function may not be attributable to tea catechins or a systemic antioxidant or anti-inflammatory effect. Chronic dietary flavonoid status appears to relate to endothelial function, possibly suggesting that other flavonoids or polyphenolic components of tea favorably influence vascular health and risk for cardiovascular disease.

Catechin↗
PubMed2005

Effect of medical and surgical weight loss on endothelial vasomotor function in obese patients.

We prospectively examined brachial artery endothelial function using vascular ultrasound in 41 obese subjects treated with medical or surgical (gastric bypass) weight loss interventions. Surgical intervention produced greater weight loss and more pronounced improvement in endothelial function than medical treatment alone. Improved endothelial function with weight loss correlated strongly with fasting glucose but not with alteration in blood pressure, lipids, degree of weight loss, or plasma resistin concentrations. These data demonstrate that weight loss in markedly obese patients improves endothelial function and glycemic control that may represent important mechanisms of the cardiovascular benefit associated with weight reduction.

Adult↗
PubMed2004

Cytokine-stimulated GTP cyclohydrolase I expression in endothelial cells requires coordinated activation of nuclear factor-kappaB and Stat1/Stat3.

Endothelial production of nitric oxide (NO) is dependent on adequate cellular levels of tetrahydrobiopterin (BH4), an important cofactor for the nitric oxide synthases. Vascular diseases are often characterized by vessel wall inflammation and cytokine treatment of endothelial cells increases BH4 levels, in part through the induction of GTP cyclohydrolase I (GTPCH I), the rate-limiting enzyme for BH4 biosynthesis. However, the molecular mechanisms of cytokine-mediated GTPCH I induction in the endothelium are not entirely clear. We sought to investigate the signaling pathways whereby cytokines induce GTPCH I expression in human umbilical vein endothelial cells (HUVECs). Interferon-gamma (IFN-gamma) induced endothelial cell GTPCH I protein and BH4 modestly, whereas high-level induction required combinations of IFN-gamma and tumor necrosis factor-alpha (TNF-alpha). In the presence of IFN-gamma, TNF-alpha increased GTPCH I mRNA in a manner dependent on nuclear factor-kappaB (NF-kappaB), as this effect was abrogated by overexpression of a dominant-negative IkappaB construct. HUVEC IFN-gamma treatment resulted in signal transducer and activator of transcription 1 (Stat1) activation and DNA binding in a Jak2-dependent manner, as this was inhibited by AG490. Conversely, overexpression of Jak2 effectively substituted for IFN-gamma in supporting TNF-alpha-mediated GTPCH I induction. The role of IFN-gamma was also Stat1-dependent as Stat1-null cells exhibited no GTPCH I induction in response to cytokines. However, Stat1 activation with oncostatin M failed to support TNF-alpha-mediated GTPCH I induction because of concomitant Stat3 activation. Consistent with this notion, siRNA-mediated Stat3 gene silencing allowed oncostatin M to substitute for IFN-gamma in this system. These data implicate both NF-kappaB and Stat1 in endothelial cell cytokine-stimulated GTPCH I induction and highlight the role of Stat3 in modulating Stat1-supported gene transcription. Thus, IFN-gamma and TNF-alpha exert distinct but cooperative roles for BH4 biosynthesis in endothelium that may have important implications for vascular function during vascular inflammation.

Animals↗