Cysteine and aspartic proteases possess high elastolytic activity and might contribute to the degradation of the abdominal aortic aneurysm (AAA) wall. The aim of this study was to analyze, in detail, the proteases (cathepsins B, D, K, L and S, and inhibitor cystatin C) found in human AAA and healthy aortic tissue samples. The vessel walls from AAA patients (n=36) and nonaneurysmal aortae (n=10) were retrieved using conventional surgical repair and autopsy methods. Serum samples from the same AAA patients and 10 healthy volunteers were also collected. Quantitative expression analyses were performed at the mRNA level using real-time reverse transcriptase-PCR (RT-PCR). Furthermore, analyses at the protein level included western blot and immunoprecipitation analyses. Cellular sources of cysteine/aspartic proteases and cystatin C were identified by immunohistochemistry (IHC). All cysteine/aspartic proteases and cystatin C were detected in the AAA and control samples. Using quantitative RT-PCR, a significant increase in expression was observed for cathepsins B (P=0.021) and L (P=0.018), compared with the controls. Cathepsin B and cystatin C were also detected in the serum of AAA patients. Using IHC, smooth muscle cells (SMCs) and macrophages were positive for all of the tested cathepsins, as well as cystatin C; in addition, the lymphocytes were mainly positive for cathepsin B, followed by cathepsins D and S. All cysteine/aspartic proteases analyzed in our study were detected in the AAA and healthy aorta. The highest expression was found in macrophages and SMCs. Consequently, cysteine/aspartic proteases might play a substantial role in AAA.
Aged ; Aorta/enzymology ; Aortic Aneurysm, Abdominal/*enzymology ; Aspartic Acid Proteases/genetics/*metabolism ; Case-Control Studies ; Cathepsins/genetics/metabolism ; Cysteine Proteases/genetics/*metabolism ; Humans ; Lymphocytes/enzymology ; Macrophages/enzymology ; Middle Aged ; Myocytes, Smooth Muscle/enzymology ; RNA, Messenger/genetics/metabolism

Inflammation appears to have a major role in the development of atherosclerosis. Cyclooxygenase-2 (COX-2) is involved in the inflammatory response via the generation of prostanoids that, in turn, are involved in the production of matrix metalloproteinases (MMPs). This study aimed to investigate atherosclerosis in human aortas for in situ tissue distribution of COX-2, MMPs including MMP-9 and membrane type 1 MMP (MT1-MMP), and tissue inhibitor of metalloproteinase-2 (TIMP-2). Immunohistochemical studies were performed on atherosclerotic lesions of aortas from patients with aortic aneurysms (n = 4) and dissections (n = 3) by using antibodies to COX-2, MMP-9, MT1-MMP, and TIMP-2. Control tissues were obtained from traumatically dissected aortas (n = 2). All specimens from diseased aortas had atherosclerotic lesions ranging from fatty streak to atheromatous plaques. In control, there was no expression of COX-2, MMP-9, and MT1-MMP in all aortic layers. Immunoreactivity for COX-2 was predominantly noted in macrophages and smooth muscle cells (SMCs) of the intima including atherosclerotic plaque itself and the medial layer of the plaque base, as well as in SMCs and endothelial lining of the vasa vasorum in the adventitia. Immunoreactivity for MMP-9 and MT1-MMP was found in the same distribution as that of COX-2. Additionally, the expression of TIMP-2 increased in relation to MMP-9 expression. This study demonstrates that COX-2 is coexpressed with MMP-9 and MT1-MMP, not only by macrophages and SMCs in atherosclerotic lesions, but also in endothelial lining of the vasa vasorum of human aortas. Thus, vascular inflammatory reactions may influence extracellular matrix remodeling by coactivation of MMPs in the development of atherosclerosis and, in turn, the progression of disease.
Animal ; Aorta/enzymology* ; Aortic Diseases/pathology ; Aortic Diseases/enzymology* ; Arteriosclerosis/pathology ; Arteriosclerosis/enzymology* ; Female ; Guinea Pigs ; Human ; Immunochemistry ; Isoenzymes/metabolism* ; Male ; Matrix Metalloproteinases/metabolism* ; Middle Age ; Prostaglandin-Endoperoxide Synthase/metabolism*

OBJECTIVETo investigate the therapeutic effect of Dachaihutang on the development of atherosclerosis (AS) in rabbits and its possible mechanism by detecting the expression level of carnitine patmitoyl transferase-1 (CPT-1) in vascular smooth muscle layer of atherosclerotic rabbits, and search the new way and evidence for AS cures.

METHODThirty six male New Zealand white rabbits were divided randomly into control group, model control group, simvastatin group and Chinese traditional medicine dachaihutang group. After 9 weeks and 20 weeks of treatment, serum total cholesterol (TC) and triglyceride (TG) and low-density lipoprotein (LDL) levels were examined. At the end of 25 th weeks, histological changes in ascending aorta were studied by HE staining and histomorphometric analysis. The gene expression of CPT-1 in vascular smooth muscle layer of thoracic aorta was detected by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR).

RESULTCompared with model control group, in dachaihutang group serum TC and TG and LDL levels attenuated. Pathomorphology indicated that intima and media (I + M) became thinned, and the ratios of the thickness of intima to media(I/M) and the area of intima to media (SI/SM) were decreased (P < 0.05). Aortic intimal proliferation in Dachaihutang group was associated with a marked increase in CPT-1 expression in vascular smooth muscle layer of thoracic aorta. Compared to simvastatin group, except TG value, other values were higher in Dachaihutang group, however, there were no significant differences between the two groups.

CONCLUSIONThese findings suggest that early treatment with Dachaihutang not only induces a significant regression of arterial lesions of high cholesterol diet rabbits, but also has a crucial inhibited genesis and development of atherosclerosis effect by up-regulating CPT-1 expression in vascular smooth muscle layer.

Animals ; Aorta ; cytology ; drug effects ; enzymology ; Atherosclerosis ; drug therapy ; enzymology ; genetics ; Carnitine O-Palmitoyltransferase ; genetics ; metabolism ; Disease Models, Animal ; Drugs, Chinese Herbal ; pharmacology ; Gene Expression ; drug effects ; Humans ; Male ; Myocytes, Smooth Muscle ; drug effects ; enzymology ; Rabbits ; Random Allocation

BACKGROUNDBariatric surgery offers a productive resolution of type 2 diabetes mellitus (T2DM). The development of T2DM vasculopathy is due to chronic inflammation, which increases matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) expression. This study sought to examine MMP-9 and TIMP-1 expression in the thoracic aorta after duodenal-jejunal bypass (DJB) surgery on a T2DM rat model induced by a high-fat diet and low dose streptozotocin (STZ).

METHODSTwenty-one T2DM Wistar rats induced by high-fat diet and low dose STZ were randomly divided into DJB and sham duodenal-jejunal bypass (S-DJB) groups. Ten Wistar rats were fed a normal diet as a control. Recovery of gastrointestinal function post-operation and resumption of a normal diet completed the experiment. Body weight, blood glucose, blood lipid levels, and MMP-9 and TIMP-1 expression levels in aortic endothelial cells were measured throughout.

RESULTSDJB rats showed significant weight loss 2 weeks post-operation compared with S-DJB rats. After surgery, DJB rats showed significant improvement and steady glycemic control with improved insulin sensitivity and glucose tolerance. They also exhibited improved lipid metabolism with a decrease in fasting free fatty acids (FFAs) and triglycerides (all P < 0.05). Immunohistochemistry showed decreased MMP-9 and TIMP-1 expression 12 weeks after surgery (P < 0.01).

CONCLUSIONSDJB surgery on an induced T2DM rat model improves blood glucose levels and lipids, following a high-fat diet and low dose STZ treatment. In addition, DJB decreased MMP-9 and TIMP-1 expression in vascular endothelial cells, which may play an important role in delaying the development of T2DM vascular disease.

Animals ; Aorta, Thoracic ; metabolism ; Bariatric Surgery ; Body Weight ; physiology ; Diabetes Mellitus, Type 2 ; enzymology ; Male ; Matrix Metalloproteinase 1 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Rats

The purpose of this study was to investigate the change of aortic semicarbazide-sensitive amine oxidase (SSAO) activity in diabetic rats and examine the effect of 2-bromoethylamine (2-BEA) on SSAO activity and vascular endothelium in diabetic rats. SSAO was prepared from rat aorta. For assessment of the inhibitory effect, the enzymes were preincubated in the presence of different concentrations of 2-BEA before the addition of benzylamine in vitro. Type 1 diabetic rat model was induced by a single intraperitoneal injection of streptozotocin (STZ). Sprague Dawley (SD) rats were randomly divided into normal control group (NC), diabetic model group (DM), 2-BEA 5 mg/kg group, 2-BEA 20 mg/kg group (n = 10 in each group). 2-BEA was administered daily via intraperitoneal injection for 8 weeks. At the end of 8 weeks, blood sample was collected from the abdominal aorta. Plasma nitric oxide (NO) was determined by nitrate reductase method. Plasma endothelin-1 (ET-1) was determined by radioimmunoassay. Aorta SSAO was determined by high performance liquid chromatography. The aorta was prepared to observe morphological changes and ultramicroscopic structures. The results were as follows: Compared with NC group, aortic SSAO activity and the plasma ET-1 were significantly increased (P < 0.01), and plasma NO was significantly decreased (P < 0.01) in DM group. 2-BEA decreased plasma ET-1 and elevated plasma NO by inhibiting aortic SSAO activity in diabetic rats (P < 0.01), and 2-BEA 20 mg/kg group was more significant than 2-BEA 5 mg/kg group (P < 0.05). Endothelial injury of 2-BEA group rats was less serious than DM group. These results suggest that 2-BEA protect aortic endothelium by inhibiting aortic SSAO activity.
Amine Oxidase (Copper-Containing) ; metabolism ; Animals ; Aorta, Abdominal ; enzymology ; Diabetes Mellitus, Experimental ; enzymology ; Endothelin-1 ; blood ; Endothelium, Vascular ; drug effects ; Ethylamines ; pharmacology ; Protective Agents ; pharmacology ; Rats ; Rats, Sprague-Dawley

BACKGROUNDThe most intimidatory pathological changes in patients with DM are cardiovascular illnesses, which are the major causes of death in diabetic patients and are far more prevalent than in nondiabetics because of accelerated atherosclerosis. In this study, we tried to clarify the changes in macrovascular endothelial ultrastructure and in the gene expression of endothelial nitric oxide synthase (eNOS)mRNA in diabetic rats.

METHODSThe study was conducted on 52 of 10-week old Sprague Dawley (SD) rats with body weight of (320 +/- 42) g. SD rats were divided into: experimental group treated with a single intraperitoneal injection of streptozotocin (STZ, 60 mg/kg), (male, n = 20, diabetes mellitus (DMM)); female, n = 12, diabetes mellitus female (DMF)) and control group (male, n = 10, diabetes mellitus male control (DMMC); female, n = 10, diabetes mellitus female control (DMFC)). Four weeks after treatment, half of the rats were sacrificed; the remainders were sacrificed ten weeks after treatment. One part of the abdominal aortic sample was stored under glutaraldehyde (volume fraction psiB = 2.5%). After the process of chemical fixation, chemical dehydration, drying and conductivity enhancement, all samples were observed and photographed using scanning electron microscopy (Leica-Stereoscan 260, England). The other part of the abdominal aortic sample was treated with liquid nitrogen and the expression of eNOSmRNA was assessed by semi-quantitative RT-PCR.

RESULTSThe aortic lumen of both experimental groups adsorbed much more debris than that of either control group. The endothelial surfaces of diabetic rats were coarse, wrinkled and protuberant like fingers or villi. The vascular endothelial lesions of diabetic male rats were very distinct after 4 weeks, and as obvious as those at 10 weeks. The vascular endothelial lesions of diabetic female rats were not severe at 4 weeks and only became marked after 10 weeks. In both males and females, the abdominal aortic eNOSmRNA content of 4 weeks and 10 weeks diabetic rats was very significantly lower (P < 0.01) than that of controls.

CONCLUSIONSAortic endothelial ultrastructure in DM rats is injured compared with controls. Abnormal changes of aortic endothelia in male DM rats are more obvious than those in females. Expression of abdominal aortic eNOSmRNA content of DM rats is significantly lower than that of controls.

Animals ; Aorta, Abdominal ; enzymology ; Blood Glucose ; analysis ; Diabetes Mellitus, Experimental ; enzymology ; pathology ; Endothelium, Vascular ; ultrastructure ; Female ; Male ; Nitric Oxide Synthase ; genetics ; Nitric Oxide Synthase Type III ; RNA, Messenger ; blood ; Rats ; Rats, Sprague-Dawley ; Sex Factors ; Streptozocin

OBJECTIVETo explore the effects of slenderstyle acanthopanax root-bark on cyclooxygenase in vivo and in vitro.

METHODContents of 6-keto-PGF1alpha in bovine aorta endothelial cells, PGE2 in mouse abdominal macrophages, and 6-keto-PGF1alpha in rat stomach tissue were determined. The ulcer index in rat gastric mucosa was measured.

RESULTCOX-1 and COX-2 were inhibited by slenderstyle acanthopanax root-bark, and the inhibitive rate of COX-2 was higher than that of COX-1 at the same concentration. Necrotic injuries in health gastric mucosa were not produced, but the injuries previously induced by the ethanol worsened.

CONCLUSIONOne of the antirheumatic mechanisms of slender-root-bark might probably be mediating through inhibition of cyclooxygenase. style acanthopanax

6-Ketoprostaglandin F1 alpha ; metabolism ; Animals ; Aorta ; enzymology ; Cattle ; Cyclooxygenase 1 ; metabolism ; Cyclooxygenase 2 ; metabolism ; Dinoprostone ; metabolism ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Eleutherococcus ; chemistry ; Endothelial Cells ; enzymology ; Female ; Gastric Mucosa ; enzymology ; Macrophages, Peritoneal ; enzymology ; Male ; Mice ; Plant Roots ; chemistry ; Plants, Medicinal ; chemistry ; Rats ; Rats, Sprague-Dawley

The effect of genistein on aortic atherosclerosis was studied by immunohistochemistry with RAM-11 and HHF-35 antibodies and western blotting for matrix metalloproteinase-3 (MMP-3) in New Zealand White rabbits. After provocation of atherosclerosis with hyperlipidemic diet, the rabbits were divided as hyperlipidemic diet group (HD), normal diet group (ND) and hyperlipidemic plus genistein diet group (HD+genistein) for 4 and half months. The average cross sectional area of atherosclerotic lesion was 0.269 mm2 after provocation. The lesion was progressed by continuous hyperlipidemic diet (10.06 mm2) but was increased mildly by genistein (0.997 mm2), and decreased by normal diet (0.228 mm2). The ratio of macrophages to smooth muscle cells in the lesion was not changed by genistein supplementation. The western blotting showed reduction of MMP-3 expression in HD+genistein and ND groups than HD group. The inhibition of atherogenesis by genistein was might be due to improve the endothelial dysfunction rather than direct action on macrophages and/or smooth muscle cells in the lesion, since endothelial dysfunction by lipid peroxidation was the main atherogenic factor in the hypercholesterolemicrabbits. The genistein supplementation also suggests that it helps the stabilization of the atherosclerotic lesion by inhibition of MMP-3 expression.
Animals ; Aorta/pathology ; Arteriosclerosis/*drug therapy/pathology/*prevention & control ; Blotting, Western ; Diet, Atherogenic ; Genistein/*pharmacology ; Growth Inhibitors/*pharmacology ; Hypercholesterolemia/*drug therapy/pathology ; Macrophages/pathology ; Male ; Muscle, Smooth, Vascular/enzymology/pathology ; Rabbits ; Research Support, Non-U.S. Gov't ; Stromelysin 1/metabolism

AIM: To investigate the effects of pinocembrin on angiotensin II (Ang II)-induced vascular contraction, and to explore its molecular mechanism of actions. METHODS: The isometric vascular tone was measured in rat thoracic aortic rings with denuded endothelium. Phosphorylation level of myosin phosphatase target unit 1 (MYPT1), and protein levels of Rho kinase 1 (ROCK1, ROKβ or p160ROCK) and angiotensin II type-1 receptor (AT1R) were determined by Western blot analysis. RESULTS: Pinocembrin produced a relaxant effect on endothelium-denuded aortic rings contracted by Ang II (100 nmol·L(-1)) in a dose-dependent manner. In endothelium-denuded aortic rings stimulated by Ang II, pretreatment with pinocembrin (25 and 100 μmol·L(-1)) for 20 min significantly attenuated MYPT1 phosphorylation and ROCK1 protein levels. Meanwhile, the protein level of AT1R in response to Ang II was not affected by pinocembrin in rat aortic rings. CONCLUSION These findings indicate that pinocembrin inhibits vasoconstriction induced by Ang II in rat endothelium-denuded aortic rings, and the mechanism at least in part, is due to the blockade of the RhoA/ROCK pathway.
Angiotensin II ; metabolism ; Animals ; Aorta ; drug effects ; enzymology ; metabolism ; physiopathology ; Flavanones ; pharmacology ; In Vitro Techniques ; Male ; Myocardial Contraction ; drug effects ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; drug effects ; Vasoconstriction ; drug effects ; rho-Associated Kinases ; antagonists & inhibitors ; genetics ; metabolism

The incidence of cardiovascular disease is predicted to increase as the population ages. There is accumulating evidence that arginase upregulation is associated with impaired endothelial function. Here, we demonstrate that arginase II (ArgII) is upregulated in aortic vessels of aged mice and contributes to decreased nitric oxide (NO) generation and increased reactive oxygen species (ROS) production via endothelial nitric oxide synthase (eNOS) uncoupling. Inhibiting ArgII with small interfering RNA technique restored eNOS coupling to that observed in young mice and increased NO generation and decreased ROS production. Furthermore, enhanced vasoconstrictor responses to U46619 and attenuated vasorelaxation responses to acetylcholine in aged vasculature were markedly improved following siRNA treatment against ArgII. These results might be associated with increased L-arginine bioavailability. Collectively, these results suggest that ArgII may be a valuable target in age-dependent vascular diseases.
15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid/pharmacology ; Aging ; Animals ; Aorta/enzymology/physiopathology ; Arginase/genetics/*metabolism ; Endothelium, Vascular/*enzymology/physiopathology ; Enzyme Induction ; Gene Knockdown Techniques ; Mice ; Mice, Inbred C57BL ; Nitric Oxide/metabolism ; Nitric Oxide Synthase Type III/*metabolism ; RNA, Small Interfering/genetics ; Reactive Oxygen Species/metabolism ; Up-Regulation ; Vasoconstriction/drug effects