Angiotensin-converting Enzyme In Endothelial Cells Research Articles

Epigenetic control of the angiotensin-converting enzyme in endothelial cells during inflammation.

The angiotensin-converting enzyme (ACE) plays a central role in the renin-angiotensin system, which is involved in the regulation of blood pressure. Alterations in ACE expression or activity are associated with various pathological phenotypes, particularly cardiovascular diseases. In human endothelial cells, ACE was shown to be negatively regulated by tumor necrosis factor (TNF) α. To examine, whether or not, epigenetic factors were involved in ACE expression regulation, methylated DNA immunoprecipitation and RNA interference experiments directed against regulators of DNA methylation homeostasis i.e., DNA methyltransferases (DNMTs) and ten-eleven translocation methylcytosine dioxygenases (TETs), were performed. TNFα stimulation enhanced DNA methylation in two distinct regions within the ACE promoter via a mechanism linked to DNMT3a and DNMT3b, but not to DNMT1. At the same time, TET1 protein expression was downregulated. In addition, DNA methylation decreased the binding affinity of the transcription factor MYC associated factor X to the ACE promoter. In conclusion, DNA methylation determines the TNFα-dependent regulation of ACE gene transcription and thus protein expression in human endothelial cells.

Abstract 105: 20-HETE: An Inducer of ACE in Human Microvascular Endothelial Cells (HMVEC).

20-hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P450 derived arachidonic acid metabolite, is manufactured in the microcirculation and shown to sensitize the smooth muscle to constrictor stimuli and cause endothelial activation and dysfunction. Increased vascular production of 20-HETE promotes hypertension that is dependent, in part, on the renin angiotensin system. Microarray analysis of endothelial cells treated with 20-HETE revealed a five-fold upregulation of angiotensin converting enzyme (ACE). In cultured human microvascular endothelial cells (HMVEC), 20-HETE (5 nM) induced ACE mRNA by 3.1-fold (± 0.16 p<0.05), increased ACE protein levels by 4.2-fold (±0.99 p<0.05) and ACE activity by 2-fold (±0.23 p<0.05). These effects were abrogated by co-treatment with 20-HEDE, a 20-HETE antagonist. 20-HETE induction of ACE mRNA was abolished by inhibitors of EGFR-tyrosine kinase, MAPK and IKKβ activation but was not affected by PKC inhibition. Moreover, transfection of cells with IKKβ siRNA prevented 20-HETE from inducing ACE mRNA. Collectively, theses results suggest a role for 20-HETE as a regulator of endothelial ACE. This regulatory activity impacts vascular function since downregulation of ACE in endothelial cells treated with siRNA exogenous 20-HETE from inhibiting NO synthesis and stimulating O 2 - production. This study demonstrates that induction of ACE expression and activity is critical to the actions of 20-HETE on endothelial cell function, further implicating the induction of ACE and activation of RAS as a key mechanism by which excessive production of 20-HETE within the vasculature leads to hypertension.

Exercise training normalizes ACE and ACE2 in the brain of rabbits with pacing-induced heart failure

Exercise training (EX) normalizes sympathetic outflow and plasma ANG II in chronic heart failure (CHF). The central mechanisms by which EX reduces this sympathoexcitatory state are unclear, but EX may alter components of the brain renin-angiotensin system (RAS). Angiotensin-converting enzyme (ACE) may mediate an increase in sympathetic nerve activity (SNA). ACE2 metabolizes ANG II to ANG-(1-7), which may have antagonistic effects to ANG II. Little is known concerning the regulation of ACE and ACE2 in the brain and the effect of EX on these enzymes, especially in the CHF state. This study aimed to investigate the effects of EX on the regulation of ACE and ACE2 in the brain in an animal model of CHF. We hypothesized that the ratio of ACE to ACE2 would increase in CHF and would be reduced by EX. Experiments were performed on New Zealand White rabbits divided into the following groups: sham, sham + EX, CHF, and CHF + EX (n = 5 rabbits/group). The cortex, cerebellum, medulla, hypothalamus, paraventricular nucleus (PVN), nucleus tractus solitarii (NTS), and rostral ventrolateral medulla (RVLM) were analyzed. ACE protein and mRNA expression in the cerebellum, medulla, hypothalamus, PVN, NTS, and RVLM were significantly upregulated in CHF rabbits (ratio of ACE to GAPDH: 0.3 +/- 0.03 to 0.8 +/- 0.10 in the RVLM, P < 0.05). EX normalized this upregulation compared with CHF (0.8 +/- 0.1 to 0.4 +/- 0.1 in the RVLM). ACE2 protein and mRNA expression decreased in CHF (ratio of ACE2 to GAPDH: 0.3 +/- 0.02 to 0.1 +/- 0.01 in the RVLM). EX increased ACE2 expression compared with CHF (0.1 +/- 0.01 to 0.8 +/- 0.1 in the RVLM). ACE2 was present in the cytoplasm of neurons and ACE in endothelial cells. These data suggest that the activation of the central RAS in animals with CHF involves an imbalance of ACE and ACE2 in regions of the brain that regulate autonomic function and that EX can reverse this imbalance.

Exercise training normalizes ACE and ACE2 in the brain of rabbits with pacing induced chronic heart failure

Activation of the brain renin‐angiotensin system (RAS) and elevated Angiotensin II (Ang II) increase sympathetic nerve activity (SNA) in chronic heart failure (CHF). Exercise training (EX) normalizes SNA and plasma Ang II. The mechanisms are unclear, but EX may modulate components of the RAS such as Angiotensin‐converting enzyme (ACE) and ACE2. This study investigated the effect of EX on the regulation of ACE and ACE2 in the brain with a pacing CHF+EX model. We hypothesized that the ratio of ACE to ACE2 would increase in CHF to mediate increased SNA and be normalized by EX. Experiments were performed on four groups of rabbits: normal, normal+EX, CHF, and CHF+EX (n=4‐5/group). We analyzed the cortex, cerebellum, medulla, hypothalamus, PVN, NTS, and RVLM by Western blotting, PCR, and double immunofluorescence (IF). ACE protein and mRNA expression in the cerebellum, medulla, hypothalamus, PVN, NTS, and RVLM were significantly upregulated in CHF (0.3±0.03 to 0.8±0.1, RVLM, P&lt;0.05). EX normalized this in all areas (0.8±0.1 to 0.4±0.1, RVLM). ACE2 protein and mRNA significantly decreased in CHF and also normalized with EX. IF showed ACE2 is present in the cytoplasm of neurons and ACE in endothelial cells. This suggests RAS activation involves an imbalance of ACE and ACE2 and may mediate changes in Ang II and Ang 1‐7 in regions of the brain that regulate autonomic function and suggests EX as a therapy to normalize SNA in CHF.

Signaling via the Angiotensin-Converting Enzyme Enhances the Expression of Cyclooxygenase-2 in Endothelial Cells

Angiotensin-converting enzyme (ACE) inhibitors elicit outside-in signaling via ACE in endothelial cells. This involves the CK2-mediated phosphorylation of ACE on Ser1270 and the activation of the c-Jun N-terminal kinase (JNK)/c-Jun pathway, resulting in an enhanced endothelial ACE expression. Because cyclooxygenase-2 (COX-2) expression is reported to be increased in subjects treated with ACE inhibitors, we determined the role of ACE signaling in this phenomenon and the transcription factors involved. In lungs from mice treated with the ACE inhibitor ramipril for 5 days, COX-2 expression was increased. A similar (1.5- to 2-fold) increase in COX-2 protein was detected in primary cultures of human endothelial cells treated with ramiprilat. In an endothelial cell line stably expressing human somatic ACE, ramiprilat increased COX-2 promoter activity, an effect not observed in ACE-deficient cells or cells expressing a nonphosphorylatable ACE mutant (S1270A). The ramiprilat-induced, ACE-dependent increase in COX-2 expression and promoter activity (both 1.5- to 2-fold greater than control) was prevented by the inhibition of JNK. Ramiprilat significantly enhanced the DNA binding activity of activator protein-1 in cells expressing ACE but not S1270A ACE. Activator protein-1 decoy oligonucleotides prevented the ACE inhibitor-induced increase in COX-2 promoter activity and protein expression. As a consequence of the ramiprilat-induced increase in COX-2 expression, prostacyclin and prostaglandin E2, but not thromboxane A2, production was increased and was inhibited by the COX-2 inhibitor celecoxib. These results indicate that ACE signaling may underlie the increase in COX-2 and prostacyclin levels in patients treated with ACE inhibitors.

Upregulation of angiotensin-converting enzyme by vascular endothelial growth factor.

The role of vascular endothelial growth factor (VEGF), a potent endothelium-specific angiogenic factor, in the regulation of angiotensin-converting enzyme (ACE) in cultured human umbilical vein endothelial cells (HUVECs) was studied. VEGF (0.07-1.2 x 10(-6) mmol/l) caused a dose-dependent increase in ACE measured in intact endothelial cells and increased the expression of ACE mRNA. The stimulatory effect of VEGF was inhibited by pretreatment of endothelial cells with the tyrosine kinase inhibitor herbimycin (4.35 x 10(-5) mmol/l). The stimulatory effect of VEGF was potentiated by the selective cGMP phosphodiesterase inhibitor zaprinast (0.1 mmol/l). The nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME; 5.4 mmol/l) suppressed the stimulatory effect of VEGF. The nonselective cyclooxygenase (COX) inhibitor indomethacin (5 microM) and the selective COX-2 inhibitor NS-398 (5 microM) potentiated the stimulatory effect of VEGF, whereas the selective COX-1 inhibitor resveratrol (5 microM) was without effect. ACE induction by VEGF was inhibited by the selective protein kinase C (PKC) inhibitor GF109203X (2.5 x 10(-3) mmol/l) and by downregulating PKC with phorbol 12-myristate 13-acetate. In summary, VEGF induced ACE in cultured HUVECs. Intracellular events such as tyrosine kinase activation, PKC activation, and increase of cGMP were probably involved in ACE induction by VEGF. Nitric oxide may partially contribute to ACE induction by VEGF. The powerful capacity of VEGF to increase ACE in endothelial cells shown here suggests a synergistic relation between VEGF and the renin-angiotensin system in vascular biology and pathophysiology.

Angiotensin converting enzyme protects acetylcholine-induced relaxation from its attenuation by formyl-methionyl-leucyl-phenylalanine in rat aorta.

A chemotactic tripeptide formyl-methionyl-leucyl-phenylalanine (fMLP) attenuated acetylcholine (ACh)-induced relaxation. Because angiotensin converting enzyme (ACE), which inactivates fMLP, is rich in vascular endothelial cells, we examined whether endothelial cell ACE inhibits the attenuating effect of fMLP on ACh-induced relaxation. ACh-induced relaxation was evaluated in aortic rings from 9-week-old Sprague-Dawley rats. We examined the effects of ACE, the ACE inhibitor captopril and/or fMLP on ACh-induced relaxation in aortas from rats with or without dexamethasone treatment, which enhances ACE activity. Pre-treatment with ACE did not alter ACh-induced relaxation in control aortas but abolished the inhibitory effect of fMLP on ACh-induced relaxation [maximal relaxation (Emax): 95.4 +/- 1.2 versus 75.5 +/- 1.9%, P < 0.05]. Conversely, captopril enhanced the attenuation of ACh-induced relaxation by fMLP (Emax: 62.5 +/- 3.3 versus 74.0 +/- 2.2%, P < 0.05), although captopril did not affect ACh-induced relaxation in control aortas. In addition, fMLP did not attenuate ACh-induced relaxation in aortas from dexamethasone-treated rats (Emax: 89.7 +/- 3.7 versus 85.2 +/- 3.8%, NS), which enhanced ACE activity of aortas (3.37 +/- 0.25 versus 2.70 +/- 0.20 IU/mg protein, P < 0.05). Endothelial-cell ACE attenuates the effect of fMLP on ACh-induced relaxation, possibly by its cleavage.

Enhanced reduction of atherosclerosis in hamsters treated with pravastatin and captopril: ACE in atheromas provides cellular targets for captopril.

Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase and angiotensin-converting enzyme (ACE) reduce experimental atherosclerosis by different mechanisms. To determine whether dual-drug therapy additively retards the progression of early lesions, control hyperlipidemic hamsters were compared with those treated with pravastatin, captopril, and pravastatin plus captopril. After 8 weeks of treatment, pravastatin (34 mg/kg/day) reduced plasma total cholesterol and triglycerides by 41 and 84%, respectively, whereas captopril (100 mg/kg/day) reduced normal blood pressure by 21%. The combination of pravastatin and captopril (33 and 100 mg/kg/day) decreased plasma total cholesterol and triglycerides by 44 and 84%, and blood pressure was decreased by 14%. In the aortic arch, pravastatin reduced macrophage-foam cell size and fatty streak area by 21 and 31%, respectively, whereas captopril decreased macrophage-foam cell number and fatty streak area by 34 and 35%. Pravastatin plus captopril decreased macrophage-foam cell number, foam cell size, and fatty streak area by 38, 24, and 67%. ACE inhibitors were previously reported to retard atherosclerosis without affecting blood pressure, suggesting that these agents acted on the artery wall. Therefore the expression of arterial ACE was determined in normal and atherosclerotic hamster aortas. ACE messenger RNA (mRNA) and protein were detected in endothelial cells, intimal macrophage-foam cells and medial smooth-muscle cells of atherosclerotic arteries indicating an upregulation of ACE expression with hyperlipidemia and atherosclerosis. In conclusion, dual-therapy with pravastatin and captopril produced an additive reduction in fatty streak area compared with either drug alone and suggested that atherogenesis can be retarded beyond the level achieved with monotherapy. The presence of ACE in endothelial cells and intimal macrophage-foam cells provides cellular targets for captopril to directly modify the formation of early atherosclerotic lesions.

Monocyte- and cytokine-induced downregulation of angiotensin-converting enzyme in cultured human and porcine endothelial cells.

We investigated the effects of monocytes on endothelial cell (EC) ectoenzyme activity. Coculture of human aortic ECs with human monocytes (2 x 10(5) monocytes per 2-cm2 well) led to a decrease in EC angiotensin-converting enzyme (ACE) activity (64.5 +/- 3.5% of control) but not aminopeptidase N, aminopeptidase P, and 5'-nucleotidase activities. Similar results were obtained using human umbilical vein EC-human monocyte and porcine aortic EC-porcine monocyte cocultures. The decrease in ACE activity was monocyte concentration and coculture time dependent, reaching a maximum of 65% decrease in activity at 120 hours. Monocyte-mediated reduction in ACE activity did not require cell to cell contact, since exposure of ECs to conditioned medium from cocultures (CCCM) or from monocyte cultures (MCM) produced a decrease in ACE activity similar to that observed in EC-monocyte cocultures. Exogenously added tumor necrosis factor (TNF)-alpha and interleukin (IL)-1 alpha, two known secretory products of monocytes, simulated the effects of monocytes on ACE activity. Western blot analysis revealed a decrease in the amount of ACE protein in TNF-alpha-treated and CCCM-treated ECs compared with control ECs. Both TNF-alpha and IL-1 alpha were present in CCCM and MCM but not EC-conditioned medium. Incubation of the cocultures with a mixture of neutralizing antibodies against TNF-alpha and IL-1 totally abolished the monocyte-induced decrease in ACE activity. In conclusion, monocytes decrease ACE activity in cultured ECs through the release of cytokines such as TNF-alpha and IL-1.

Monocrotaline pyrrole-induced changes in angiotensin-converting enzyme activity of cultured pulmonary artery endothelial cells.

1. Changes in the structural and functional integrity of endothelium have been recognized as relatively early features of delayed and progressive pulmonary vascular injury caused by the pyrrolizidine alkaloid, monocrotaline (MCT). Although a number of investigators have evaluated angiotensin-converting enzyme (ACE) activity in the lungs of rats treated with MCT, the exact nature of changes in activity of this enzyme and the role they may play in MCT pneumotoxicity remain controversial. 2. We examined the direct effects of monocrotaline pyrrole (MCTP), a toxic metabolite of MCT, on cultured endothelial cell ACE activity. Post-confluent monolayers of porcine or bovine pulmonary artery endothelial cells (PECs or BECs, respectively) were treated with a single administration of MCTP at time 0; then they were examined for their ability to degrade the synthetic peptide, [3H]-benzoyl-Phe-Ala-Pro. 3. In PECs, which are relatively insensitive to the direct cytolytic effects of MCTP, monolayer ACE activity was unchanged initially but gradually decreased within 4 days after treatment with a high concentration of MCTP (150 microM). This decrease was transient, and PEC monolayer ACE activity returned to the control value by 10 days post treatment. 4. BEC monolayer ACE activity was also unchanged initially but rapidly declined within 4 days after MCTP treatment and remained depressed throughout the post treatment period. BECs were quite sensitive to the cytolytic effects of MCTP and the decline in ACE activity occurred coincident with the decrease in monolayer cellularity and appearance of marked cytotoxicity. 5. We conclude that high concentrations of MCTP decrease endothelial ACE activity. The decline in ACE activity is delayed and the magnitude and duration of the decrease corresponds to the degree ofMCTP-induced cytotoxicity. This suggests that altered endothelial ACE activity is unlikely to be a direct effect of MCTP on the enzyme but may reflect the delayed cell injury which results from exposure to this compound.

Stimulation of bovine pulmonary artery endothelial cell ACE by dexamethasone: involvement of steroid receptors.

After exposure of bovine pulmonary artery endothelial cells in culture to 1 microM dexamethasone for 24-48 h, angiotensin-converting enzyme (ACE) activity of these cells was elevated severalfold. The increase in ACE activity was preceded by an increase in ACE mRNA, which could be detected after treatment of cells with dexamethasone for 4 h. When the increase in ACE mRNA produced by dexamethasone at 4 h was blocked by alpha-amanitin, an RNA polymerase II inhibitor, the increase in ACE activity detected at 48 h was inhibited. RU 38486, a steroid receptor antagonist, inhibited the elevation of both ACE activity and mRNA produced by dexamethasone. Among other steroids tested, only hydrocortisone, aldosterone and corticosterone-21-acetate had a stimulatory effect on ACE activity. RU 38486 effectively blocked the elevation in ACE activity produced by both aldosterone and dexamethasone, but had no effect on the elevation of ACE activity produced by other agents (3-isobutyl-1-methylxanthine, A23187, and dibutyryl adenosine 3',5' cyclic monophosphate). From these data we conclude that dexamethasone and certain other steroids with an hydroxyl group in the 11th carbon position regulate ACE gene expression of bovine endothelial cells at the transcriptional level via a steroid receptor-mediated mechanism.

Lipopolysaccharides decrease angiotensin converting enzyme activity expressed by cultured human endothelial cells.

Angiotensin converting enzyme (ACE) is present on endothelial cells and plays a role in regulating blood pressure in vivo by converting angiotensin I to angiotensin II and metabolizing bradykinin. Since ACE activity is decreased in vivo in sepsis, the ability of lipopolysaccharide (LPS) to suppress endothelial cell ACE activity was tested by culturing human umbilical vein endothelial cells (HUVEC) for 0-72 hr with or without LPS and then measuring ACE activity. ACE activity in intact HUVEC monolayers incubated with LPS (10 micrograms/ml) decreased markedly with time and was inhibited by 33%, 71%, and 76% after 24 hr, 48 hr, and 72 hr, respectively, when compared with control, untreated cells. The inhibitory effect of LPS was partially reversible upon removal of the LPS and further incubation in the absence of LPS. The LPS-induced decrease in ACE activity was dependent on the concentrations of LPS (IC50 = 15 ng/ml at 24 hr) and was detectable at LPS concentrations as low as 1 ng/ml. That LPS decreased the Vmax of ACE in the absence of cytotoxicity and without a change in Km suggests that LPS decreased the amount of ACE present on the HUVEC cell membrane. While some LPS serotypes (Escherichia coli 0111:B4 and 055:B5, S. minnesota) were more potent inhibitors of ACE activity than others (E. coli 026:B6 and S. marcescens), all LPS serotypes tested were inhibitory. These finding suggest that LPS decreases endothelial ACE activity in septic patients; in turn, this decrease in ACE activity may decrease angiotensin II production and bradykinin catabolism and thus play a role in the pathogenesis of septic shock.

Effect of atrial natriuretic factor on angiotensin converting enzyme

We studied the effects of atrial natriuretic factor (ANF) on the conversion of angiotensin I to angiotensin II. Pulmonary artery endothelial cells converted 1.22 nmol/min per dish [125I]angiotensin I to angiotensin II, but ANF suppressed the conversion by 0.475 nmol/min per dish. The maximum rate of inhibition of angiotensin converting enzyme (ACE) activity by ANF was 60% at 10(-6) mol/l ANF compared with control conditions. The amino acid fragments of ANF were studied to determine whether its specific structure was necessary for inhibiting ACE in endothelial cells. We found that atriopeptin II (fragment 5-27) inhibited ACE activity as much as ANF. Fragment 7-25 had the same rate of inhibition, but fragment 13-27 had no effect on the conversion of angiotensin I to angiotensin II. These results suggest that the amino acid sequence of Cys (7)-Cys (23) in ANF is important for the inhibition of ACE in pulmonary endothelial cells.

Elevation of bovine endothelial cell angiotensin converting enzyme by cationophores and inhibition by ouabain

We recently reported that calcium ionophore A23187 causes a several-fold elevation of angiotensin converting enzyme (ACE) activity of bovine pulmonary artery endothelial cells in culture and that this elevation is dependent upon extracellular calcium. Now we have observed that monensin, a sodium ionophore, also elevates the ACE activity of these cells. This elevation in ACE was not inhibited by 0.2 mM EGTA or the calcium channel inhibitor nifedipine, and monensin did not alter intracellular calcium as measured by fluorimetric assessment of fura-2 AM-loaded cells. When confluent endothelial cells were incubated with monensin or A23187 in the presence of 10–20 nM ouabain, a specific inhibitor of Na + K +- ATPase , the elevation in ACE produced by both of the ionophores was totally eliminated. Concentrations of ouabain greater than 10 nM also inhibited baseline levels of ACE activity. Ca 2+ measurements of fura-2 AM-loaded cells showed that ouabain had no effect on the influx of Ca 2+ produced by A23187. The elevation of ACE seemed to require new protein synthesis, since 0.1 μg/ml cycloheximide inhibited the elevation produced by monensin and A23187. Other sodium transport inhibitors such as amiloride or bumetanide had no effect on ACE elevation caused by monensin. These results suggest that ACE levels of bovine endothelial cells in culture are under cation regulation and may be modulated by ouabain-sensitive Na + K +- ATPase .

The effect of alphanapthylthiourea (ANTU)-induced acute injury on lung binding of antibody to angiotensin converting enzyme (ACE).

The effects of ANTU-induced acute pulmonary capillary injury on lung and serum ACE functional activity and the specific accumulation of radio-labelled anti-ACE in lung were explored. Rats were injected either with ANTU or the solvent and sacrificed at various intervals up to one week after injection. All ANTU-injected animals developed pulmonary edema and bilateral pleural effusions which resolved by the one week time point. At no time was there any significant change in serum ACE levels. The specific activity of total lung ACE however rose from 11.0 +/- .95 (mean +/- SEM) to 18.4 +/- 1.1 by two hours after ANTU; by 24 hours, however, solubilized lung ACE had fallen significantly to 6.9 +/- .79 (p less than .01). Total lung ACE had returned to control values by one week. In parallel groups of animals the accumulation of 125I-labelled anti-ACE (AA) or normal sheep immunoglobulin (NSG) was compared in control and ANTU-treated rats. The ratio of the radioactivity in the lungs of AA--injected animals to that in NSG--injected animals fell significantly after ANTU administration (5.0 +/- .88 to 1.2 +/- .28 at 2 hours) suggesting that immunoreactive ACE had fallen despite an increase in ACE functional activity. The decreased binding of AA at the early time points perhaps reflects internalization of endothelial cell ACE in response to injury and an inability of the antibody to interact with the enzyme. The reduction in binding at 24 hours (1.38 +/- .47) correlates with a reduction in total lung ACE. ANTI-ACE may be a useful reagent for quantitating endothelial cell damage following lung injury.