OBJECTIVEAngiotensin converting enzyme 2 (ACE2) efficiently hydrolyses the potent vasoconstrictor angiotensin II to vasodilative angiotensin (1-7). We hypothesized that ACE2 overexpression may inhibit inflammation response in atherosclerotic plaque by degrading Ang II into Ang-(1-7).

METHODSAtherosclerosis (AS) plaques were induced in the abdominal aorta of 38 rabbits by endothelial injury and atherogenic diet for 3 months. Rabbits were then underwent injection of a recombinant adenovirus (2.5 x 10(9) pfu/ml) carrying a murine ACE2 gene (Ad-ACE2) through a catheter into the abdominal aortic segments rich in plaques (n = 19) or injection of a control vector Ad-EGFP (n = 19). One month later, all rabbits were sacrificed and plaques from aortic segments were analyzed.

RESULTSACE2 expression in aortic tissues of the Ad-ACE2 group were confirmed by immunohistochemistry. Macrophage infiltration (13.6% +/- 4.2% vs. 23.6% +/- 6.9%, P < 0.01) and MCP-1 expression (13.2% +/- 0.4% vs. 25.0% +/- 7.4%, P < 0.01) were significantly reduced in Ad-ACE2 group compared to Ad-EGFP group.

CONCLUSIONSOverexpression of ACE2 inhibited atherosclerotic plaque inflammation response in hypercholesterolemic rabbits.

Animals ; Atherosclerosis ; genetics ; metabolism ; Cells, Cultured ; Diet, Atherogenic ; Genetic Vectors ; Peptidyl-Dipeptidase A ; genetics ; Rabbits ; Transfection

Angiotensin I-converting enzyme (ACE, EC3.4.15.1) plays an important role in regulating blood pressure. The C-domain of ACE has been identified as the main catalytic site of angiotensin I cleavage in vivo. The ACE gene fragment of the C-domain was amplified by PCR and cloned into the pPIC9K secretory expression plasmid. The recombinant plasmid was transformed into Pichia pastoris strain GS115. Positive clones were selected and subject to electroporation. Antibiotic G418 was used for the screening of multicopy inserts. After optimization of the expression system, the protein yield reached 0.5 g/L by flask-shaking culture fermentation, and enzyme activity reached 7.178 U/mL in the fermentation supernatant. The purity of the target protein obtained was 97% after Ni+ affinity chromatography. Enzyme inhibitory activity assay using Captopril showed that it is promising to use ACE-C domain as new generation of target for screening ACE inhibitor antihypertensive drugs.
Angiotensin-Converting Enzyme Inhibitors ; Catalytic Domain ; genetics ; Electroporation ; Humans ; Peptidyl-Dipeptidase A ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics

OBJECTIVETo investigate the expression of angiotensin converting enzyme (ACE) and ACE2 Gene in lung of paraquat poisoning rats and the protection of sodium dimercaptopropane sulfonate (Na-DMPS).

METHODSOne hundred SD male rats were randomly equally divided into 4 groups:normal control group (10 rats), drug control group (40 rats), paraquat poisoning group (40 rats) and drug intervention group(40 rats). The paraquat poisoning and drug intervention group rats were injected intraperitoneally by paraquat (20 mg/kg). The rats in drug intervention group rats were protected by intraperitoneal injection with Na-DMPS (200 mg/kg) 15 min before exposure of paraquat. Behavioral changes of the rats and histological changes of lung tissues under light microscope were observed. And the expression of ACE and ACE2 mRNA in lung tissues of rats both in paraquat poisoned group and drug intervention group were measured by RT-PCR at different time of 6 h, 24 h, 3 and 7 d after poisoning.

RESULTSThe poisoning symptoms of shortness of breath, cramps appeared and deteriorated progressively in rats after paraquat exposure and the protection of NA-DMPS could delay and reduce these symptoms significantly. Histological appearance of disorganization of pulmonary capillary and alveolus, exudation in alveolar space, pulmonary edema, severe bleeding, and inflammatory cells infiltration were obvious in lungs of rats after paraquat poisoning, whereas the histological changes were extenuated by protection of NA-DMPS. As compared with normal control group (NC group), the expressions of ACE, ACE2 mRNA in lung tissue decreased, and the lowest level of ACE mRNA expressions appeared at 24 h (0.457 +/- 0.262), on 3 d (0.385 +/- 0.179) after Paraquat exposure (P < 0.05), while lowest level of ACE2 mRNA expressions appeared on 3 d (0.415 +/- 0.247), 7 d (0.365 +/- 0.215) (P < 0.05). As compared with paraquat poisoned group, the expressions of ACE mRNA in lung tissue of rats in NA-DMPS protected group increased significantly at 24 h (0.739 +/- 0.558) and 3 d (0.749 +/- 0.414) (P < 0.05), while the expressions of ACE2 mRNA increased markedly on 3 d (0.584 +/- 0.345) and 7 d (0.493 +/- 0.292) (P < 0.05). But the expression of ACEmRNA and ACE2 mRNA in lungs had no statistical significance between normal control group and drug intervention group (P > 0.05).

CONCLUSIONThe expressions of ACE and ACE2 mRNA in lung tissue of the rats with paraquat poisoning are decreased. Na-DMPS can effectively improve the balance of RAS in local lung tissue and reduce the pathological changes of lung tissue, delay the poisoning symptoms and show protective effects for acute lung injury induced by paraquat.

Animals ; Lung ; drug effects ; enzymology ; Male ; Paraquat ; poisoning ; Peptidyl-Dipeptidase A ; biosynthesis ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Unithiol ; pharmacology

Angiotensin converting enzyme (ACE, EC 3.4.15.1) is a membrane-bound, zinc dependent dipeptidase that catalyzes the conversion of the decapeptide angiotensin I to the potent vasopressor ocatapeptide angiotensin II, by removing two C-terminal amino acids. ACE is well known as a key part of the renin angiotenisn system that regulates blood pressure, and its inhibitors have potential for the treatment of hypertension. This paper reviewed the characteristics of ACE in aspects of its structure-function relationship, gene polymorphism and inhibitor development. In particular, the catalytic mechanisms of the two active sites of somatic ACE in the cleavage of angiotensin I and bradykin are different. Therefore, it would likely provide a new way for exploiting novel ACE inhibitors with fewer side-effects by specifically-targeting the individual active sites of somatic ACE.
Angiotensin-Converting Enzyme Inhibitors ; pharmacology ; Antihypertensive Agents ; pharmacology ; Humans ; Peptidyl-Dipeptidase A ; chemistry ; genetics ; metabolism ; Polymorphism, Genetic ; Structure-Activity Relationship

OBJECTIVETo investigate the mRNA expression of severe acute respiratory syndrome-associated coronavirus (SARS-COV) functional receptor, angiotensin-converting enzyme 2 (ACE2), in human femoral head and conjunctiva, and explore the possible entry route of SARS-COV in human femoral head.

METHODSACE2 mRNA in human femoral head was detected by nested RT-PCR with human beta actin gene as the positive control.

RESULTSThe mRNA of human beta actin gene could be amplified efficiently in all the tissue samples. The mRNA of human ACE2 was expressed efficiently in the normal lung tissue, but not in the cartilage and cancellous bone under the weight-bearing area of the femoral head.

CONCLUSIONSARS-COV can not infect the femoral head tissue and lead to avascular necrosis of the femoral head directly by the spike glycoprotein, and mechanism of the virus for causing avascular necrosis needs further investigation.

Femoral Neck Fractures ; metabolism ; Femur Head ; metabolism ; Gene Expression ; Humans ; Peptidyl-Dipeptidase A ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Receptors, Virus ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; SARS Virus ; metabolism

OBJECTIVETo investigate the effect of angiotensin II (AngII) type 1 (AT-1) receptor and angiotensin-converting enzyme (ACE) gene silencing on nuclear factor-kappaB (NF-kappaB) activity in hepatic stellate cells (HSCs).

METHODSpSilencer/AT-1 alpha receptor siRNA and pSilencer/ACE siRNA plasmids were transfected into cultured HSC-T6 cells, which were subsequently stimulated by 10(-6) mol/L AngII or ACE inhibitor (ACEI). The DNA binding activity of NF-kappaB in the transfected cells was analyzed using electrophoretic gel mobility shift assay (EMSA).

RESULTSs Gel shift studies showed that stimulation of the HSCs by AngII markedly increased the DNA-binding activity of NF-kappaB, which was inhibited by the transfection with pSilencer/ AT-1 alpha receptor siRNA plasmid or pSilencer/ACE siRNA plasmid.

CONCLUSIONAT-1 alpha receptor and ACE gene silencing result in inhibition of NF-kappaB activity in HSCs in vitro.

Cell Line ; Hepatic Stellate Cells ; cytology ; metabolism ; Humans ; NF-kappa B ; genetics ; metabolism ; Peptidyl-Dipeptidase A ; genetics ; RNA Interference ; RNA, Small Interfering ; genetics ; Receptor, Angiotensin, Type 1 ; genetics ; Transfection

Angiotensin-I converting enzyme (ACE) is a key regulator of blood pressure, electrolytes and fluid homeostasis through conversion of angiotensin I into angiotensin II. Recently, a genetic polymorphism of the ACE gene, which accounts for 47% of the variation of ACE activity in blood, has been advocated as a biomarker of athletic aptitude. Different methods of analysis and determination of ACE activity in plasma have been used in human and equine research without a consensus of a "gold standard" method. Different methods have often been used interchangeably or cited as being comparable in the existing literature; however, the actual agreement between assays has not been investigated. Therefore, in this study, we evaluated the level of agreement between three different assays using equine plasma obtained from 29 horses. Two spectrophotometric assays using Furylacryloyl-phenylalanyl-glycyl-glycine as substrate and one fluorimetric assay utilizing o-aminobenzoic acid-FRK-(Dnp)P-OH were employed. The results revealed that the measurements from the different assays were not in agreement, indicating that the methods should not be used interchangeably for measurement of equine ACE activity. Rather, a single method of analysis should be adopted to achieve comparable results and critical appraisal of the literature is needed when attempting to compare results obtained from different assays.
Animals ; Enzyme Assays/*methods ; Female ; Fluorometry/*methods ; Horses/blood/genetics/*metabolism ; Male ; Oligopeptides/pharmacology ; Peptidyl-Dipeptidase A/blood/genetics/*metabolism ; Polymorphism, Genetic ; Reference Values ; Spectrophotometry/*methods

OBJECTIVETo explore the effect of irbesartan on angiotensin-converting enzyme 2 (ACE2) mRNA expression in diabetic rat myocardium.

METHODSThirty 8-week-old male Wistar rats were randomly divided into control group (n=7), diabetic model group (n=14) and irbesartan group (n=9). Diabetes was induced by a single intraperitoneal injection of STZ (55 mg/kg), a blood glucose>16.7 mmol/L 72 h after the injection indicated successful establishment of diabetes. Four weeks after the modeling, the rats in irbesartan group were given 50 mg/kg irbesartan. ELISA was used to measure myocardial AngII content in the rats, and myocardial ACE2 mRNA expression was determined by real-time PCR.

RESULTSMyocardial AngII level in the diabetic model group was significantly higher than that in the control group (P<0.001). Irbesartan administration significantly lowered cardiac AngII levels in the diabetic rats (P<0.001). The rats in irbesartan group showed significantly increased myocardial ACE2 mRNA expression compared with those in the control and diabetic rat groups (P<0.05).

CONCLUSIONIrbesartan can increase ACE2 mRNA expression in the myocardium, which might be one of the mechanisms underlying its effect in improving the cardiac function in diabetic rats.

Animals ; Biphenyl Compounds ; pharmacology ; Diabetes Mellitus, Experimental ; enzymology ; Male ; Myocardium ; enzymology ; Peptidyl-Dipeptidase A ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Random Allocation ; Rats ; Rats, Wistar ; Tetrazoles ; pharmacology

OBJECTIVETo investigate the inhibitory effect of angiotensin-converting enzyme 2 (ACE2) on angiotensin II (Ang II)-induced down-regulation of albumin expression and enhancement of cell migration in rat hepatocytes.

METHODSCultured rat hepatocyte were treated with Ang II (10-7 mol/L) for different time lengths, and the protein expressions of vimentin and albumin and cell migration were detected. The cells transfected with lentiGFP or lentiACE2 were treated with A779 for 1 h and then with Ang II, and Western blotting and immunofluorescent cytochemistry were used to detect the protein levels; the cell migration was evaluated by Transwell assay.

RESULTAng II induced significantly increased vimentin expression and reduced albumin expression in rat hepatocytes in a time-dependent manner. Overexpression of ACE2 obviously inhibited the up-regulation of vimentin expression, reduction of albumin expression, and enhancement of cell migration induced by Ang II.

CONCLUSIONACE2 overexpression can inhibit Ang II-induced up-regulation of vimentin, reduction of albumin expression, and enhancement of cell migration in rat hepatocytes.

Albumins ; metabolism ; Angiotensin II ; pharmacology ; Animals ; Cell Movement ; Cells, Cultured ; Down-Regulation ; Hepatocytes ; cytology ; Lentivirus ; Peptidyl-Dipeptidase A ; genetics ; metabolism ; Rats ; Transfection ; Up-Regulation ; Vimentin ; metabolism

Animals ; Hypertension ; metabolism ; physiopathology ; Male ; Myocardium ; metabolism ; Peptidyl-Dipeptidase A ; genetics ; metabolism ; Proto-Oncogene Proteins ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Inbred SHR ; Rats, Inbred WKY ; Receptor, Angiotensin, Type 1 ; genetics ; metabolism ; Receptors, G-Protein-Coupled ; genetics ; metabolism