Biomedical subjects

Beate Fisslthaler

Publications and source records attributed to Beate Fisslthaler.

At least 19 recordsLinked to original sources

PubMed2007

Fluid shear stress and NO decrease the activity of the hydroxy-methylglutaryl coenzyme A reductase in endothelial cells via the AMP-activated protein kinase and FoxO1.

The rate-limiting enzyme for cholesterol synthesis, the hydroxy-methylglutaryl coenzyme A reductase (HCR), is phosphorylated by the AMP-activated protein kinase (AMPK). As shear stress activates the AMPK in endothelial cells, we determined whether it affects HCR activity and subsequent HCR-dependent signaling. Shear stress (12 dynes cm(-2)) rapidly increased the phosphorylation and activity (6.5- and 4-fold, respectively) of the AMPK in cultured endothelial cells and the activated AMPK phosphorylated the HCR in vitro. Moreover, shear stress and the AMPK activator 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) attenuated endothelial HCR activity by 37% and 33%, respectively. Inhibition of NO production attenuated the acute shear stress-induced phosphorylation of the AMPK and the decrease in HCR activity. Prolonged shear stress (18 hours) led to a significant (50%) decrease in HCR mRNA expression that was dependent on NO, AMPK, and the subsequent phosphorylation and degradation of FoxO1a. Correspondingly, the downregulation of FoxO (small interfering RNA) decreased HCR expression. Prolonged shear stress also attenuated the bradykinin-induced activation of Ras and extracellular signal-regulated kinase 1/2, a phenomenon that was comparable to the effects of cerivastatin and that was reversed by mevalonate and thus attributed to HCR inhibition. A decrease (35%) in HCR expression was also detected in femoral arteries from mice following voluntary exercise, and the bradykinin-induced vasodilatation of the mouse hindlimb was attenuated by both exercise and the HCR inhibitor cerivastatin. These data indicate that fluid shear stress regulates the activity and expression of the HCR in endothelial cells and determines responsiveness to stimuli, such as bradykinin via a mechanism involving NO, AMPK, FoxO1a, and p21Ras.

AMP-Activated Protein Kinases↗

PubMed2006

Cytochrome P450 epoxygenase gene function in hypoxic pulmonary vasoconstriction and pulmonary vascular remodeling.

We assessed pulmonary cytochrome P450 (CYP) epoxygenase expression and activity during hypoxia and explored the effects of modulating epoxygenase activity on pulmonary hypertension. The acute hypoxic vasoconstrictor response was studied in Swiss Webster mice, who express CYP2C29 in their lungs. Animals were pretreated with vehicle, the epoxygenase inhibitor (N-methylsulfonyl-6-[2-propargyloxyphenyl] hexanamide) or an inhibitor of the soluble epoxide hydrolase. Whereas the epoxygenase inhibitor attenuated hypoxic pulmonary constriction (by 52%), the soluble epoxide hydrolase inhibitor enhanced the response (by 39%), indicating that CYP epoxygenase-derived epoxyeicosatrienoic acids elicit pulmonary vasoconstriction. Aerosol gene transfer of recombinant adenovirus containing the human CYP2C9 significantly elevated mean pulmonary artery pressure and total pulmonary resistance indices, both of which were sensitive to the inhibitor sulfaphenazole. The prolonged exposure of mice to hypoxia increased CYP2C29 expression, and transcript levels increased 5-fold after exposure to normobaric hypoxia (FIO2 0.07) for 2 hours. This was followed by a 2-fold increase in protein expression and by a significant increase in epoxyeicosatrienoic acid production after 24 hours. Chronic hypoxia (7 days) elicited pulmonary hypertension and pulmonary vascular remodeling, effects that were significantly attenuated in animals continually treated with N-methylsulfonyl-6-[2-propargyloxyphenyl] hexanamide (-46% and -55%, respectively). Our results indicate that endogenously generated epoxygenase products are associated with hypoxic pulmonary hypertension in mice and that selective epoxygenase inhibition significantly reduces acute hypoxic pulmonary vasoconstriction and chronic hypoxia-induced pulmonary vascular remodeling. These observations indicate potential novel targets for the treatment of pulmonary hypertension and highlight a pivotal role for CYP epoxygenases in pulmonary responses to hypoxia.

Adenoviridae↗

PubMed2005

Cytochrome P450 epoxygenases 2C8 and 2C9 are implicated in hypoxia-induced endothelial cell migration and angiogenesis.

Recent studies suggest that cytochrome P450 (CYP) epoxygenase-derived epoxyeicosatrienoic acids (EETs) elicit cell proliferation and promote angiogenesis. The aim of this study was to determine the role of CYP 2C8/9-derived EETs in the process of angiogenesis under hypoxic conditions. In human endothelial cells, hypoxia enhanced the activity of the CYP 2C9 promoter, increased the expression of CYP 2C mRNA and protein and augmented 11,12-EET production. In Transwell assays, the migration of endothelial cells pre-exposed to hypoxia to increase CYP expression was abolished by CYP 2C antisense oligonucleotides as well as by the CYP inhibitor MS-PPOH and the EET antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (EEZE). Similar findings were obtained in porcine coronary artery endothelial cells. CYP 2C9 overexpression in endothelial cells increased the association of PAK-1 with Rac, a response also elicited by the CYP 2C9 product 11,12-EET. Matrix metalloprotease (MMP) activity was increased in CYP-2C9-overexpressing cells and correlated with increased invasion through Matrigel-coated Transwell chambers: an effect sensitive to the CYP 2C9 inhibitor sulfaphenazole as well as to EEZE and the MMP inhibitor GM6001. In in vitro angiogenesis models, the EET antagonist inhibited tube formation induced by CYP 2C9 overexpression as well as that in endothelial cells exposed to hypoxia to increase CYP 2C expression. Furthermore, in the chick chorioallantoic membrane assay, EEZE abolished hypoxia-induced angiogenesis. Taken together, these data indicate that CYP 2C-derived EETs significantly affect the sequence of angiogenic events under hypoxic conditions.

8,11,14-Eicosatrienoic Acid↗

PubMed2005

Gab1, SHP2, and protein kinase A are crucial for the activation of the endothelial NO synthase by fluid shear stress.

Fluid shear stress enhances NO production in endothelial cells by a mechanism involving the activation of the phosphatidylinositol 3-kinase and the phosphorylation of the endothelial NO synthase (eNOS). We investigated the role of the scaffolding protein Gab1 and the tyrosine phosphatase SHP2 in this signal transduction cascade in cultured and native endothelial cells. Fluid shear stress elicited the phosphorylation and activation of Akt and eNOS as well as the tyrosine phosphorylation of Gab1 and its association with the p85 subunit of phosphatidylinositol 3-kinase and SHP2. Overexpression of a Gab1 mutant lacking the pleckstrin homology domain abrogated the shear stress-induced phosphorylation of Akt but failed to affect the phosphorylation or activity of eNOS. The latter response, however, was sensitive to a protein kinase A (PKA) inhibitor. Mutation of Gab1 Tyr627 to phenylalanine (YF-Gab1) to prevent the binding of SHP2 completely prevented the shear stress-induced phosphorylation of eNOS, leaving the Akt response intact. A dominant-negative SHP2 mutant prevented the activation of PKA and phosphorylation of eNOS without affecting that of Akt. Moreover, shear stress elicited the formation of a signalosome complex including eNOS, Gab1, SHP2 and the catalytic subunit of PKA. In isolated murine carotid arteries, flow-induced vasodilatation was prevented by a PKA inhibitor as well as by overexpression of either the YF-Gab1 or the dominant-negative SHP2 mutant. Thus, the shear stress-induced activation of eNOS depends on Gab1 and SHP2, which, in turn, regulate the phosphorylation and activity of eNOS by a PKA-dependent but Akt-independent mechanism.

Acetylcholine↗

PubMed2005

Role of PECAM-1 in the shear-stress-induced activation of Akt and the endothelial nitric oxide synthase (eNOS) in endothelial cells.

The application of fluid shear stress to endothelial cells elicits the formation of nitric oxide (NO) and phosphorylation of the endothelial NO synthase (eNOS). Shear stress also elicits the enhanced tyrosine phosphorylation of endothelial proteins, especially of those situated in the vicinity of cell-cell contacts. Since a major constituent of these endothelial cell-cell contacts is the platelet endothelial cell adhesion molecule-1 (PECAM-1) we assessed the role of PECAM-1 in the activation of eNOS. In human endothelial cells, shear stress induced the tyrosine phosphorylation of PECAM-1 and enhanced the association of PECAM-1 with eNOS. Endothelial cell stimulation with shear stress elicited the phosphorylation of Akt and eNOS as well as of the AMP-activated protein kinase (AMPK). While the shear-stress-induced tyrosine phosphorylation of PECAM-1 as well as the serine phosphorylation of Akt and eNOS were abolished by the pre-treatment of cells with the tyrosine kinase inhibitor PP1 the phosphorylation of AMPK was unaffected. Down-regulation of PECAM-1 using a siRNA approach attenuated the shear-stress-induced phosphorylation of Akt and eNOS, as well as the shear-stress-induced accumulation of cyclic GMP levels while the shear-stress-induced phosphorylation of AMPK remained intact. A comparable attenuation of Akt and eNOS (but not AMPK) phosphorylation and NO production was also observed in endothelial cells generated from PECAM-1-deficient mice. These data indicate that the shear-stress-induced activation of Akt and eNOS in endothelial cells is modulated by the tyrosine phosphorylation of PECAM-1 whereas the shear-stress-induced phosphorylation of AMPK is controlled by an alternative signaling pathway.

AMP-Activated Protein Kinases↗

PubMed2005

Oxidized low-density lipoprotein increases superoxide production by endothelial nitric oxide synthase by inhibiting PKCalpha.

OBJECTIVE: Oxidized low-density lipoprotein (ox-LDL) increases superoxide anion (O(2)(-)) production by the endothelial nitric oxide (NO) synthase (eNOS). We assessed whether the uncoupling of eNOS was associated with alterations in eNOS phosphorylation and/or the assembly of the eNOS signaling complex. METHODS AND RESULTS: In unstimulated human endothelial cells, eNOS Thr(495) was constitutively phosphorylated. ox-LDL, but not native LDL, enhanced the production of O(2)(-) by endothelial cells, an effect that was partially sensitive to NOS inhibition. ox-LDL, but not native LDL, induced a time- and concentration-dependent decrease in the phosphorylation of eNOS on Thr(495). Protein kinase C (PKC) has been reported to phosphorylate this residue, and the increase in the phosphorylation of Thr(495) induced by phorbol 12-myristate 13-acetate was attenuated in cells pretreated with ox-LDL. Moreover, the phosphorylation and activity of PKCalpha was attenuated by ox-LDL and paralleled the changes in eNOS phosphorylation. ox-LDL also induced the dissociation of eNOS from the plasma and Golgi membranes. In COS-7 cells, a T495A eNOS mutant generated significantly more O(2)(-) than a T495D mutant did, indicating that the dephosphorylation of Thr(495) alone can increase O(2)(-) production by eNOS. However, although the dephosphorylation of Thr(495) in histamine-stimulated endothelial cells enhanced the binding of calmodulin to eNOS, calmodulin no longer bound to eNOS from ox-LDL-treated endothelial cells. CONCLUSIONS: These results indicate that a decrease in the activity of PKCalpha in ox-LDL-treated endothelial cells is associated with the dephosphorylation of eNOS, dissociation of the eNOS signaling complex, and the enhanced production of eNOS-derived O(2)(-).

Animals↗

PubMed2003

Aged spontaneously hypertensive rats exhibit a selective loss of EDHF-mediated relaxation in the renal artery.

Endothelium-dependent relaxation is frequently attenuated in hypertension. We hypothesized that the contribution of the endothelium-derived hyperpolarizing factor (EDHF) to the acetylcholine (ACh)-induced, endothelium-dependent relaxation is attenuated with aging in the renal artery of spontaneously hypertensive rats (SHR) compared with age-matched Wistar-Kyoto (WKY) rats. ACh-induced, NO-mediated relaxation was identical in young (8-week-old) WKY and SHR, whereas EDHF-mediated relaxations (assessed in the presence of Nomega-nitro-l-arginine and diclofenac) were much more pronounced in SHR than WKY. KCl-induced relaxations were more pronounced in vessels from young WKY rats than from young SHR. The cytochrome P450 inhibitor sulfaphenazole significantly inhibited EDHF-mediated relaxation in vessels from young SHR but not WKY. Vessels from old (22 months) SHR exhibited a slightly reduced NO-mediated relaxation but a complete loss of EDHF-mediated responses. In contrast, aging did not affect EDHF-mediated responses in WKY. Moreover, ACh-induced hyperpolarization and resting membrane potential were decreased in old SHR but not in WKY. KCl-induced relaxation increased with age in WKY, whereas no response to KCl was recorded in arteries from aged SHR. In vessels from old WKY but not old SHR, mRNA expression of the Na-K-ATPase subunit alpha2 was increased by 2-fold compared with young animals. These data indicate that the increase in EDHF responses in renal arteries from aged WKY can be attributed to the release of K+ ions from the endothelium, whereas increased EDHF responses in renal arteries from young SHR can be attributed to a sulfaphenazole-sensitive cytochrome P450-dependent EDHF.

Acetylcholine↗

PubMed2003

11,12-Epoxyeicosatrienoic acid-induced inhibition of FOXO factors promotes endothelial proliferation by down-regulating p27Kip1.

Cytochrome P450-derived epoxyeicosatrienoic acids (EETs) stimulate endothelial cell proliferation and angiogenesis. In this study, we investigated the involvement of the forkhead box, class O (FOXO) family of transcription factors and their downstream target p27Kip1 in EET-induced endothelial cell proliferation. Incubation of human umbilical vein endothelial cells with 11,12-EET induced a time- and dose-dependent decrease in p27Kip1 protein expression, whereas p21Cip1 was not significantly affected. This effect on p27Kip1 protein was associated with decreased mRNA levels as well as p27Kip1 promoter activity. 11,12-EET also stimulated the time-dependent phosphorylation of Akt and of the forkhead factors FOXO1 and FOXO3a, effects prevented by the phosphatidylinositol 3-kinase inhibitor LY 294002. Transfection of endothelial cells with either a dominant-negative or an "Akt-resistant"/constitutively active FOXO3a mutant reversed the 11,12-EET-induced down-regulation of p27Kip1, whereas transfection of a constitutive active Akt decreased p27Kip1 expression independently of the presence or absence of 11,12-EET. To determine whether these effects are involved in EET-induced proliferation, endothelial cells were transfected with the 11,12-EET-generating epoxygenase CYP2C9. Transfection of CYP2C9 elicited endothelial cell proliferation and this effect was inhibited in cells co-transfected with CYP2C9 and either a dominant-negative Akt or constitutively active FOXO3a. Reducing FOXO expression using RNA interference, on the other hand, attenuated p27Kip1 expression and stimulated endothelial cell proliferation. These results indicate that EET-induced endothelial cell proliferation is associated with the phosphatidylinositol 3-kinase/Akt-dependent phosphorylation and inactivation of FOXO factors and the subsequent decrease in expression of the cyclin-dependent kinase inhibitor p27Kip1.

8,11,14-Eicosatrienoic Acid↗

PubMed2003

Cytochrome P450 epoxygenases and vascular tone: novel role for HMG-CoA reductase inhibitors in the regulation of CYP 2C expression.

Over the last 10 years it has become increasingly clear that cytochrome P450 (CYP) enzymes expressed within endothelial and vascular smooth muscle cells play a crucial role in the modulation of vascular homeostasis. There is strong evidence suggesting that the activation of a CYP 2C epoxygenase in endothelial cells is an essential step in nitric oxide (NO)- and prostacyclin (PGI(2))-independent vasodilatation of several vascular beds, particularly in the heart and kidney. Moreover, CYP epoxygenase products as well as CYP-derived reactive oxygen species are intracellular signal transduction molecules involved in several signaling cascades affecting numerous cellular processes, including vascular cell proliferation and angiogenesis. Various pharmacological compounds enhance vascular CYP 2C expression. One group of substances which highlight the possible effects of CYP induction in endothelial cells on vascular function are the HMG-CoA reductase inhibitors (statins). Cerivastatin and fluvastatin increase CYP 2C mRNA and protein in native and cultured endothelial cells, and enhance the bradykinin-induced NO/PGI(2)-independent relaxation of arterial segments as well as the generation of reactive oxygen species. However, statins also increase the expression of the endothelial NO synthase by approximately twofold. As a consequence, the probability that NO and reactive oxygen species react to generate peroxynitrite is increased and the treatment of vascular segments with statins resulted in enhanced protein tyrosine nitration. These data highlight the role played by CYP 2C in vascular homeostasis and its potential regulation by cardiovascular drugs.

Animals↗

PubMed2003

The N-terminal domain of mammalian soluble epoxide hydrolase is a phosphatase.

The mammalian soluble epoxide hydrolase (sEH) is an enzyme with multiple functions, being implicated in detoxification of xenobiotic epoxides as well as in regulation of physiological processes such as blood pressure. The enzyme is a homodimer, in which each subunit is composed of two domains. The 35-kDa C-terminal domain has an alpha/beta hydrolase fold and harbors the catalytic center for the EH activity. The 25-kDa N-terminal domain has a different alpha/beta fold and belongs to the haloacid dehalogenase superfamily of enzymes. The catalytic properties of the enzyme reported so far can all be explained by the action of the C-terminal domain alone. The function of the N-terminal domain, other than in structural stabilization of the dimer, has therefore remained unclear. By structural comparison of this domain to other haloacid dehalogenase family members, we identified a putative active site containing all necessary components for phosphatase activity. Subsequently, we found rat sEH hydrolyzed 4-nitrophenyl phosphate with a rate constant of 0.8 s(-1) and a K(m) of 0.24 mM. Recombinant human sEH lacking the C-terminal domain also displayed phosphatase activity. Presence of a phosphatase substrate did not affect epoxide turnover nor did epoxides affect dephosphorylation by the intact enzyme, indicating both catalytic sites act independently. The enzyme was unable to hydrolyze 4-nitrophenyl sulfate, suggesting its role in xenobiotic metabolism does not extend beyond phosphates. Thus, we propose this domain participates instead in the regulation of the physiological functions associated with sEH.

Amino Acid Sequence↗

PubMed2003

Cytochrome P450 2C9-derived epoxyeicosatrienoic acids induce angiogenesis via cross-talk with the epidermal growth factor receptor (EGFR).

Cytochrome P450 (CYP) epoxygenase products, such as 11,12-epoxyeicosatrienoic acid (EET), stimulate endothelial cell proliferation. We set out to identify the signal transduction cascade linking EET generation to enhanced proliferation and angiogenesis. In human endothelial cells overexpressing CYP 2C9, cell number was increased compared with control cells and was inhibited by the CYP 2C9 inhibitor, sulfaphenazole. CYP 2C9 overexpression was associated with the activation of Akt and an increase in cyclin D1 expression, effects that were abolished by the epidermal growth factor (EGF) receptor inhibitor, AG1478, which also prevented the CYP 2C9-induced increase in cell proliferation. Stimulation of EGF receptor overexpressing cells with 11,12-EET or transfection of these cells with CYP 2C9 enhanced the tyrosine phosphorylation of the EGF receptor. Endothelial tube formation in a fibrin gel was significantly enhanced (6-fold) in CYP 2C9 overexpressing cells and was comparable with the tube formation induced by EGF. In the chick chorioallantoic membrane, 11,12-EET stimulated vessel formation (3.5-fold) and induced vessel convergence, an effect that was abolished by cotreatment with either an EGF receptor-neutralizing antibody or AG1478. These results indicate that CYP 2C9-derived EETs stimulate angiogenesis by a mechanism involving the activation of the EGF receptor.

8,11,14-Eicosatrienoic Acid↗

PubMed2003

Insulin enhances the expression of the endothelial nitric oxide synthase in native endothelial cells: a dual role for Akt and AP-1.

Insulin-induced vasodilatation in vivo has been attributed to the activation of the endothelial nitric oxide (NO) synthase (eNOS). The present study addressed the effects of insulin on the activity and expression of eNOS in native and cultured endothelial cells. Insulin applied to native porcine aortic endothelial cells elicited the tyrosine phosphorylation of the insulin receptor and receptor substrate, the subsequent activation of phosphatidylinositol 3-kinase (PI 3-K), Akt (protein kinase B), and ERK1/2. Insulin did not activate eNOS in cultured endothelial cells nor relax endothelium-intact arterial segments. However, 4h after application of insulin to native endothelial cells eNOS mRNA was increased 2-fold. A comparable increase in eNOS protein was detected after 18-24h and associated with an increase in intracellular cyclic GMP. In native endothelial cells, insulin enhanced the DNA-binding activity of Sp1 and AP-1, but not that of NF-kappaB. The insulin-induced increase in eNOS expression was prevented by wortmannin as well as by AP-1 decoy oligonucleotides. The MEK1 inhibitor, PD 98059, also enhanced eNOS expression in native and cultured endothelial cells, an effect which was independent of ERK1/2 and associated with an increase in the DNA-binding activity of AP-1 and Sp1. These results demonstrate that insulin activates multiple signalling pathways in endothelial cells but does not acutely activate eNOS. Insulin however enhances eNOS mRNA and protein by a mechanism involving the combined activation of a PI 3-K- and AP-1-dependent pathway.

Animals↗

PubMed2003

Identification of a cis-element regulating transcriptional activity in response to fluid shear stress in bovine aortic endothelial cells.

Fluid shear stress exerts numerous effects on gene expression in endothelial cells. To investigate the regulatory mechanisms involved, we designed oligonucleotides composed of a 20-bp core containing the classical shear stress response element (SSRE+) or of a 20-bp core, in which base pairs flanking the SSRE were mutated (SSRE-). Hexamers of the oligonucleotides were cloned in front of reporter genes, transfected in bovine aortic endothelial cells (BAECs), and subjected to either a continuous low shear stress (3 dynes cm(-2)) or to a stepwise increase in shear stress from 3 dynes cm(-2) to 12 dynes cm(-2) (16/4 h). Shear stress increased reporter gene activity in cells transfected with pSSRE-, but not with pSSRE+. Cyclic strain (6%, 1 Hz, 4 h) did not significantly affect reporter gene activity. In gel retardation assays, more proteins bound to SSRE- than to SSRE+ in response to high shear stress. In competition experiments, a cAMP response element-binding (CREB) protein-specific oligonucleotide suppressed protein binding to the SSRE-; however, an antibody directed against CREB itself did not affect protein binding to the SSRE+. Our results indicate that the sequence ACC(G)/(T)AGACCAG represents a novel SSRE and that a protein that binds a CREB-specific oligonucleotide is part of the complex implicated in response to shear stress.

Animals↗

PubMed2003

AMP-activated protein kinase (AMPK) regulates the insulin-induced activation of the nitric oxide synthase in human platelets.

Little is known about the signaling cascades that eventually regulate the activity of the endothelial nitric oxide synthase (eNOS) in platelets. Here, we investigated the effects of insulin on the phosphorylation and activation of eNOS in washed human platelets and in endothelial cells. Insulin activated the protein kinase Akt in cultured endothelial cells and increased the phosphorylation of eNOS on Ser(1177) but failed to increase endothelial cyclic GMP levels or to elicit the relaxation of endothelium-intact porcine coronary arteries. In platelets, insulin also elicited the activation of Akt as well as the phosphorylation of eNOS and initiated NO production which was associated with increased cyclic GMP levels and the inhibition of thrombin-induced aggregation. The insulin-induced inhibition of aggregation was accompanied by a decreased Ca(2+) response to thrombin and was also prevented by N(omega) nitro-L-arginine. In platelets, but not in endothelial cells, insulin induced the activation of the AMP-activated protein kinase (AMPK), a metabolic stress-sensing kinase which was sensitive to the phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin and the AMPK inhibitor iodotubercidin. Moreover, the insulin-mediated inhibition of thrombin-induced aggregation was prevented by iodotubercidin. Insulin-independent activation of the AMPK using 5-aminoimidazole-4-carboxamide ribonucleoside, increased platelet eNOS phosphorylation, increased cyclic GMP levels and attenuated platelet aggregation. These results highlight the differences in the signal transduction cascade activated by insulin in endothelial cells and platelets, and demonstrate that insulin stimulates the formation of NO in human platelets, in the absence of an increase in Ca(2+), by acti-vating PI3-K and AMPK which phosphorylates eNOS on Ser(1177).

AMP-Activated Protein Kinases↗

PubMed2002

Inhibitors of histone deacetylation downregulate the expression of endothelial nitric oxide synthase and compromise endothelial cell function in vasorelaxation and angiogenesis.

The histone deacetylase (HDAC) inhibitor trichostatin A (TSA) inhibits hypoxia-stimulated angiogenesis. Endothelial nitric oxide synthase (eNOS)-derived NO is central to angiogenesis signaling in endothelial cells (ECs). We hypothesized that the HDAC-dependent regulation of angiogenesis may involve a modulatory effect on eNOS expression. The HDAC inhibitors TSA, butyric acid (BuA), and MS-275 time- and concentration-dependently suppressed eNOS protein levels to 41+/-2%, 46+/-12%, and 40+/-12% of control, respectively. In parallel, TSA and BuA also downregulated eNOS mRNA expression to 21+/-4% and 37+/-4% of control. TSA also attenuated the NO-dependent relaxation of porcine coronary arteries (P<0.0001, TSA 1 micromol/L) and prevented tube formation in a human angiogenesis assay. Although vascular endothelial growth factor substitution did not compensate for the inhibitory effect of TSA, exogenous NO reversed the inhibition of angiogenesis by TSA. To address the underlying signaling mechanism, we characterized the effect of TSA on eNOS gene transcription and mRNA half-life. Although TSA decreased both eNOS protein and mRNA levels, TSA paradoxically enhanced the activity of the eNOS promoter, and did not alter the eNOS transcription rate in nuclear run-on experiments, suggesting that TSA posttranscriptionally targets eNOS mRNA. These data indicate that HDAC-dependent mechanisms contribute to the regulation of eNOS expression in ECs.

Animals↗

PubMed2002

Epoxygenase-driven angiogenesis in human lung microvascular endothelial cells.

Angiogenesis is one of the most recent physiological functions attributed to products of cytochrome P-450 (CYP450) enymes. To test this at a molecular level in human cells, we used a cloned cDNA for the human endothelial enzyme CYP450 2C9 (CYP2C9) to study growth as well as differentiation of human microvascular endothelial cells from the lung (HMVEC-L). Using adenoviral vectors overexpressing mRNA for CYP2C9, we show that the presence of CYP2C9 doubles thymidine incorporation and stimulates proliferation of primary cultures of endothelial cells compared with Ad5-GFP (control) in 24 h. In addition, there is a significant increase of tube formation in Matrigel after infection of HMVEC-L with Ad5-2C9 than with Ad5-GFP. More interestingly, Ad5-2C9 expressing the antisense product of CYP2C9 (2C9AS) inhibited tube formation compared with both Ad5-GFP as well as the Ad5-2C9 constructs. Finally, we tested the most abundant arachidonic acid metabolite of CYP2C9, 14,15-epoxyeicosatrienoic acid, which induced angiogenesis in vivo when embedded in Matrigel plugs and implanted in adult rats. These data support an important role for CYP2C9 in promoting angiogenesis.

8,11,14-Eicosatrienoic Acid↗

PubMed2002

Vascular gene transfer of phosphomimetic endothelial nitric oxide synthase (S1177D) using ultrasound-enhanced destruction of plasmid-loaded microbubbles improves vasoreactivity.

BACKGROUND: Local gene therapy has enormous potential for the treatment of vascular disease. We determined whether diagnostic ultrasound-mediated destruction of plasmid-loaded albumin microbubbles is a feasible and efficient technique for local vascular gene delivery. For gene transfer, we used a phosphomimetic, active endothelial nitric oxide synthase (eNOS) construct in which Ser1177 was replaced by aspartic acid (S1177D) and exhibits a 2-fold higher basal activity than the wild-type enzyme. METHODS AND RESULTS: Gas-filled microbubbles (3.0 +/- 1.2 microm) were created by sonication of 5% human albumin in the presence of plasmid DNA encoding for LacZ or eNOS S1177D. Porcine coronary arteries were perfused with DNA-loaded albumin microbubbles in vitro, exposed to diagnostic ultrasound (5 seconds), and incubated for a further 24 hours. Detection of the beta-galactosidase in LacZ-transfected vessels revealed a predominant staining of endothelial cells without any functional impairment of vasoreactivity. Western blotting demonstrated the expression of the eNOS S1177D construct in extracts from the transfected segments. Vascular responsiveness was tested with prostaglandin F(2alpha) and the NOS inhibitor N(omega)nitro-L-arginine. Compared with segments treated with the expression plasmid alone, the contractile response to prostaglandin F(2alpha) was impaired in segments transfected with eNOS S1177D, whereas the contractile response to the administration of N(omega)nitro-L-arginine was markedly enhanced. CONCLUSIONS: Ultrasound-mediated destruction of eNOS S1177D DNA-loaded albumin microbubbles is a feasible and efficient method for vascular gene transfection. Transfection resulted in significant protein expression and enhanced NO-mediated relaxation of bradykinin-stimulated porcine coronary arteries.

Amino Acid Substitution↗

PubMed2002

Cytochrome P450 2C9-induced endothelial cell proliferation involves induction of mitogen-activated protein (MAP) kinase phosphatase-1, inhibition of the c-Jun N-terminal kinase, and up-regulation of cyclin D1.

Cytochrome P450 (CYP)-derived epoxyeicosatrienoic acids (EETs) are important modulators of endothelial cell homeostasis. We investigated the signaling pathway linking the activation of CYP 2C9 to enhanced endothelial cell proliferation. Overexpression of CYP 2C9 in cultured human endothelial cells markedly increased proliferation. This effect was paralleled by an up-regulation of the G(1) phase regulatory protein, cyclin D1. The specific CYP 2C9 inhibitor, sulfaphenazole, prevented both the enhanced cell proliferation and up-regulation of cyclin D1. CYP 2C9 overexpression also decreased the activity of the c-Jun N-terminal kinase (JNK). Coexpression of wild type JNK with CYP 2C9 attenuated the CYP 2C9-induced increase in cyclin D1 expression and abolished the CYP 2C9-induced proliferation response. In contrast, cotransfecting dominant negative JNK with CYP 2C9 restored the CYP 2C9-mediated up-regulation of cyclin D1 and proliferation. The inactivation of JNK is linked to its dephosphorylation by dual specificity mitogen-activated protein (MAP) kinase phosphatases (MKPs). Overexpression of CYP 2C9 significantly increased the expression of MKP-1, as did incubation with 11,12-EET. These data demonstrate that the mitogenic effect of CYP 2C9 is due to the generation of EETs, which promote the MKP-1-mediated dephosphorylation and inactivation of JNK, effects ultimately culminating in the expression of cyclin D1 and endothelial cell proliferation.

8,11,14-Eicosatrienoic Acid↗