Regulation of angiotensin-converting enzyme production by nicotine in human endothelial cells

Nicotine, a component of cigarette smoke, has been implicated in the pathogenesis of cardiovascular disease. We examined whether nicotine regulates angiotensin-converting enzyme (ACE), an enzyme that plays an important role in the pathophysiology of atherosclerosis and hypertension. Human umbilical cord vein endothelial cells were treated with nicotine (0.1-1 microM) alone or in combination with vascular endothelial growth factor (VEGF; 0.5 nM) or GF-109203X (GFX; 2.5 microM). The amount of ACE in intact endothelial cells was measured by an inhibitor-binding assay method, and ACE mRNA levels were quantified using LightCycler technology. Phosphorylated PKC levels were measured by Western immunoblotting. Nicotine did not modulate basal ACE production but significantly potentiated VEGF-induced ACE upregulation. Treatment of endothelial cells with the PKC inhibitor GFX totally blocked VEGF- and nicotine-induced ACE upregulation. VEGF induced PKC phosphorylation, which was potentiated by cotreatment with nicotine. We conclude that nicotine significantly potentiated VEGF-induced ACE upregulation. This effect was probably mediated by PKC phosphorylation. The interaction of nicotine with VEGF in ACE induction may contribute to the pathogenesis of smoking-related cardiovascular disease.     

Modulation of angiotensin-converting enzyme in cultured human vascular endothelial cells

Summary Previous work has suggested that not all immunoreactive angiotensin-converting enzyme (ACE) in tissues or cells is in a biologically active state. We have explored this possibility in cultured human umbilical vein endothelial cells (HUVEC), one of the most widely studied in vitro endothelial cell systems. Our approach included characterization of the effect of increasing passage number on ACE activity and expression of immunoreactive ACE at the single cell level, the subcellular compartmentalization of active ACE, and the effect of phorbol ester (PMA) treatment. We found that both ACE activity and expression of ACE antigen were downregulated by cultivation (30% of ACE-positive cells at seventh passage vs. 90% in primary culture). ACE downregulation is specific (number of CD31-positive cells did not change with cultivation) and correlated with downregulation of factor VIII-antigen. The percentage of ACE-positive cells in permeabilized HUVEC at third passage was almost twice that in nonpermeabilized HUVEC (90% vs. 50%), indicating that HUVEC contain intracellular immunoreactive ACE. ACE activity, however, was similar when measured in intact cells and in cell lysates. Moreover, diazonium salt of sulfanilic acid (DASA), a membrane-impermeable ACE inhibitor, inhibited ACE activity in intact cells and in cell lysates at the same extent, thus implying that intracellular ACE is inactive. PMA (100 nM) treatment increased the percentage of ACE-positive cells at third passage from 57 to 96%. ACE activity was increased 3-fold in cell and 1.5-fold in the culture medium of PMA-treated cells. Analysis of ACE activity in intact monolayers and cell lysates of control and PMA-treated cells revealed that all enzymatically active ACE in PMA-treated cells is localized on the plasma membrane and acts as an ectoenzyme. We conclude that expression of ACE by HUVEC is downregulated by repeated passage in culture but can be restored by PMA treatment. In addition, ACE expression is heterogeneous between neighboring cells, and total immunoreactive ACE protein associated with HUVEC includes an inactive pool of the enzyme.     

Expression of angiotensin-converting enzyme activity in cultured pulmonary artery endothelial cells

Angiotensin-converting enzyme (EC 3.4.51.1) is a carboxyterminal dipeptidyl peptidase. The enzyme catalyzes the conversion of the decapeptide angiotensin I to the octapeptide angiotensin II. In addition, the enzyme catabolizes bradykinin. Because of these actions, the enzyme is of pivotal importance in blood pressure homeostasis. Numerous investigators have demonstrated the presence of the enzyme in association with endothelial cells but relatively little is known concerning the factors controlling the expression enzyme activity by endothelial cells in culture. We have demonstrated that endothelial cells in culture do not express significant amounts of enzyme activity until several days after growth ceases due to high cell density. This is important because it demonstrates a change in function with stage of growth in culture and a possible difference in functional capabilities between nondividing endothelial cells and cells that are dividing in response to injury. Since density-dependent expression of differentiated traits does not appear to be unique to endothelial cells an understanding of the mechanisms underlying this phenomenon may provide a general explanation for the expression of differentiated traits by cultured cells.     

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.     

Induction of SPARC by VEGF in human vascular endothelial cells

SPARC/osteonectin/BM-40 is a matricellular protein that is thought to be involved in angiogenesis and endothelial barrier function. Previously, we have detected high levels of SPARC expression in endothelial cells (ECs) adjacent to carcinomas of kidney and tongue. Although SPARC-derived peptide showed an angiogenic effect, intact SPARC itself inhibited the mitogenic activity of vascular endothelial growth factor (VEGF) for ECs by the inhibiting phosphorylation of flt-1 (VEGF receptor 1) and subsequent ERK activation. Thus, the role of SPARC in tumor angiogenesis, stimulation or inhibition, is still unclear. To clarify the role of SPARC in tumor growth and progression, we determined the effect of VEGF on the expression of SPARC in human microvascular EC line, HMEC-1, and human umbilical vein ECs. VEGF increased the levels of SPARC protein and steady-state levels of SPARC mRNA in serum-starved HMEC-1 cells. Inhibitors (SB202190 and SB203580) of p38, a mitogen-activated protein (MAP) kinase, attenuated VEGF-stimulated SPARC production in ECs. Since intact SPARC inhibits phosphorylation ERK MAP kinase in VEGF signaling, it was suggested that SPARC plays a dual role in the VEGF functions, tumor angiogenesis, and extravasation of tumors mediated by the increased permeability of endothelial barrier function.     

Calcium ionophore A23187 elevates angiotensin-converting enzyme in cultured bovine endothelial cells

Calcium ionophore A23187 (0.3-0.4 microM) elevated cellular angiotensin-converting enzyme activity (ACE) 2-7-fold after 48 h incubation with bovine pulmonary artery endothelial cells in culture. Cycloheximide (0.1 micrograms/ml) blocked the elevation in ACE produced by A23187. The increase in ACE was inhibited by 0.2 mM EGTA, 50 microM verapamil and 50 microM nifedipine, and was not associated with changes in cellular cAMP. Melittin, a phospholipase A2 activator, or addition of exogenous arachidonic acid failed to reproduce the elevation, and indomethacin only partially blocked the A23187 effect. The elevation of ACE was also inhibited by the calcium-calmodulin inhibitor, calmidazolium. Thus, we postulate that the ionophore A23187 elevates ACE in endothelial cells through a calcium-dependent mechanism other than phospholipase A2 activation. The elevation depends on new protein synthesis and involves calcium-calmodulin-dependent cellular mechanisms.     

Isolation of human liver angiotensin-converting enzyme by chromatofocusing

Angiotensin-converting enzyme (EC 3.4.15.1) has been isolated from human liver by chromatofocusing. The isolation procedure permitted us to obtain a 9000-fold purified enzyme with a 22% yield. Specific activity of the angiotensin-converting enzyme was 10 units/mg of protein. The molecular mass of enzyme determined by polyacrylamide gel electrophoresis under denaturing conditions was 150,000. The isoelectric point (4.2-4.3) was also determined by chromatofocusing. The Km values of the enzyme for hippuryl-L-histidyl-L-leucine and N-benzyloxycarbonyl-L-phenylalanyl-L-histidyl-L-leucine are 5000 and 125 microM, respectively. The human liver angiotensin-converting enzyme is inhibited by bradykinin-potentiating factor SQ 20881 (IC50 = 18 nM).     

Induction by fibroblast growth factor of angiotensin converting enzyme in vascular endothelial cells in vitro

Induction of vascular endothelial cells with pituitary fibroblast growth factor (FGF) provoked an increase in angiotensin converting enzyme activity. The stimulatory effect of FGF on ACE activity was dose-dependent (ED50 = 1.0 ng/ml). Our results suggest a possible role for pituitary FGF in regulation of ACE production in vascular endothelial cells.     

Kinin- and angiotensin-converting enzyme (ACE) inhibitor-mediated nitric oxide production in endothelial cells

Carboxypeptidase cleavage of the C-terminal Arg of kinins generates specific agonists of the B1 receptor. Activation of B1 receptors produces nitric oxide via eNOS in bovine endothelial cells and iNOS in cytokine-stimulated human endothelial cells. Angiotensin-converting enzyme (ACE) inhibitors are direct agonists of B1 receptors in endothelial cells, although they release NO via a different signaling pathway than peptide ligands in bovine cells. This brief review discusses carboxypeptidase M as a required processing enzyme for generating B1 agonists, how ACE inhibitors and peptide ligands stimulate NO production and the evidence for, as well as some consequences of, the direct activation of B1 receptors by ACE inhibitors.     

Induction of monocyte chemoattractant protein-1 by Porphyromonas gingivalis in human endothelial cells

The association between periodontal and cardiovascular diseases could be mediated by direct interaction of periodontal pathogens with cardiac tissue. In order to explore this possibility, the effect of the periodontal pathogen Porphyromonas gingivalis on monocyte chemoattractant protein-1 (MCP-1) production by endothelial cells was investigated. When incubated with live P. gingivalis 381, MCP-1 production by human umbilical vein endothelial cells (HUVEC) was potently increased. Compared to the type strain 381, non-adhesive/invasive strains (W50 and DPG3) did not increase MCP-1 production, which was also demonstrated at the mRNA level. Killed P. gingivalis 381 was much less effective than live bacteria for MCP-1 induction. Treatment of HUVEC with cytochalasin D, an inhibitor of endocytosis, prevented MCP-1 mRNA up-regulation by P. gingivalis 381, suggesting that internalization of P. gingivalis is necessary for MCP-1 induction. In conclusion, the secretion of high levels of MCP-1 resulting from interactions of P. gingivalis with endothelial cells could enhance atherosclerosis progression by contributing to the recruitment of monocytes.     

Induction of adrenomedullin by hypoxia in cultured human coronary artery endothelial cells.

To explore the role of adrenomedullin (ADM) in pathophysiology of ischemic heart disease, we investigated the effects of hypoxia on the production and secretion of ADM in cultured human coronary artery endothelial cells. Treatment with hypoxia (5% CO2/94% N2/1% O2) for 6 and 12 h increased expression levels of ADM mRNA 2.2-fold and fivefold compared with the normoxia control, respectively. The levels of immunoreactive ADM in the media were increased by 12-h hypoxia about fivefold compared with the control (39.0+/-1.1 fmol/10(5) cells per 12 h under hypoxia and 7.9+/-0.4 fmol/10(5) cells per 12 h under normoxia; P<0.01, n = 4, mean +/- SEM). Reverse-phase high-performance liquid chromatography of the extracts of culture media under normoxia and hypoxia showed one major peak eluting in the position of human ADM standard. The production and secretion of ADM were increased in cultured human coronary artery endothelial cells under hypoxia. ADM may therefore play an important pathophysiological role in ischemic heart disease.     

Induction of thioredoxin reductase gene expression by peroxynitrite in human umbilical vein endothelial cells

Thioredoxin reductase (TR), a flavoprotein, catalyzes the reduction of oxidized thioredoxin in a NADPH-dependent manner, and contains a selenocysteine residue near the C-terminus. TR plays an important role in protecting against oxidative stress and in regulating cell growth and cell death. Constitutive TR expression has been observed in several cell types of the mammalian body, including endothelial cells. The latter are continually exposed to both exogenous and endogenous sources of nitric oxide (NO) and NO-derived species. Reactive nitrogen species (RNS) are associated with pathological events, contributing to the cell and tissue damage accompanying inflammation, atherogenesis and autoimmune diseases. In this study, we report on the effect of peroxynitrite on TR in human umbilical vein endothelial cells (HUVECs). Exposure to the peroxynitrite donor SIN-1 for 1 h resulted in a decrease in TR activity. Interestingly, the activity was completely restored within 24 h. To further examine this mechanism, the expression of TR at the mRNA and protein level was examined. TR mRNA levels were markedly increased by treatment of SIN-1 within 6 h, and TR protein level was also increased after the treatment in HUVECs. These results suggest that the inactivation of TR by peroxynitrite might be involved in the upregulation of the TR gene in HUVECs. Therefore, HUVECs have a unique protective mechanism that allows the maintenance of balance in intracellular redox status via TR induction as an adaptive response to nitrooxidative stress.     

Inactivation of bradykinin by angiotensin-converting enzyme and by carboxypeptidase N in human plasma

Because bradykinin (BK) appears to have cardioprotective effects ranging from improved hemodynamics to antiproliferative effects, inhibition of BK-degrading enzymes should potentiate such actions. The purpose of this study was to find out which enzymes are responsible for the degradation of BK in human plasma. Human plasma from healthy donors (n = 10) was incubated with BK in the presence or absence of specific enzyme inhibitors. At high (micromolar) concentrations, BK was mostly (>90%) degraded by carboxypeptidase N (CPN)-like activity. In contrast, at low (nanomolar) substrate concentrations, at which the velocity of the catalytic reaction is equivalent to that under physiological conditions, BK was mostly (>90%) converted into an inactive metabolite, BK-(1-7), by angiotensin-converting enzyme (ACE). BK-(1-7) was further converted by ACE into BK-(1-5), with accumulation of this active peptide. A minor fraction (<10%) of the BK was converted into another active metabolite, BK-(1-8), by CPN-like activity. The present study shows that the most critical step in plasma kinin metabolism, i.e., inactivation of BK, is mediated by ACE. Thus inhibition of plasma ACE activity would be cardioprotective by elevating the concentration of BK in the circulation.     

Monoclonal antibody to human lung angiotensin-converting enzyme

The hybridoma producing monoclonal antibody (IgG1) to human angiotensin-converting enzyme (ACE) has been prepared by fusion of murine myeloma P3O1 with spleen cells of BALB/c mice immunized with a purified human lung ACE preparation. A high specificity of monoclonal antibody (MAb) binding to immobilized ACE has been demonstrated by enzyme-linked immunosorbent assay and that of soluble ACE by an immunoadsorption test. The latter technique permits the use of impure ACE preparations for the screening procedure. This MAb did not affect ACE activity. We believe this antibody will be useful not only for immunoassay and immunopurification of ACE, but also as a tool for the investigation of the tissue distribution of the enzyme as well as for the study of the structure and mechanism of action of ACE.     

Induction of angiotensin-converting enzyme inhibitory activity by acid-limited proteolysis of glyceraldehyde 3-phosphate dehydrogenase

Angiotensin-converting enzyme (ACE) inhibitors were excised from glyceraldehyde 3-phosphate dehydrogenase (GAPDH) preparations of tuna and porcine muscles by heating at 120 degrees C for 5 min in 1 M AcOH-20 mM HCl. The inhibitors were then purified by successive chromatographies. The final product from tuna was identified as Pro-Thr-His-Ile-Lys-Trp-Gly-Asp, which was the ACE inhibitor obtained from tuna muscle [Kohama et al. (1988) Biochem. Biophys. Res. Commun. 155, 332-337]. The porcine ACE inhibitor was found to be Pro-Ala-Asn-Ile-Lys-Trp-Gly-Asp, which was identical to the porcine muscle GAPDH peptide 79-86. These results strongly suggested that the ACE inhibitory octapeptides derived from GAPDH proteins by acid-limited proteolysis at Asp-Pro and Asp-Ala peptide bonds.