OBJECTIVETo study the expression of cell cycle related factor sonic hedgehog (SHH) in autogenous vein graft and its relation with neointima formation.

METHODSAutogenous vein graft model were established in 24 male Wistar rats of 8 weeks old and 140 g weight, by transplanting the left jugular vein to intra renal abdominal aorta with microsurgical technique. Graft veins were harvested at 14 d and 28 d after transplantation. The immunohistochemistry and Western blot were used to detect the SHH and PCNA expression in the vein graft. At the same time SHH mRNA was measured by quantitative real-time PCR. The opposite normal veins served as control.

RESULTSHistological staining showed that the percent of SHH+ cells was only (2.0 +/- 0.5)% in the normal vein, but was much more in the vein graft after surgery, as (39.4 +/- 0.4)% and (63.0 +/- 0.3)% respectively (P < 0.01). The expression of SHH and PCNA were both elevated in the vein graft. There was a positive correlation between them which indicated by Western blot (r = 0.808, P < 0.01). The SHH mRNA content also increased in vein graft to 9.5 and 23.8 folds of that in control.

CONCLUSIONSHH is upregulated in autogenous vein grafts and may correlated with the proliferation of vascular smooth muscle cells.

Animals ; Hedgehog Proteins ; metabolism ; Male ; Neointima ; metabolism ; Rats ; Rats, Wistar ; Transplantation, Autologous ; Tunica Intima ; metabolism ; Veins ; metabolism ; pathology ; transplantation

Aged ; Carotid Artery, Common ; pathology ; Diabetes Mellitus, Type 2 ; metabolism ; pathology ; Female ; Glucose Intolerance ; metabolism ; pathology ; Glucose Tolerance Test ; Humans ; Insulin Resistance ; Male ; Tunica Intima ; pathology

AIM AND METHODSTo determine the relationship between the nuclear envelope nucleoside triphosphatase (EC 3. 6. 1. 15, NTPase) activity and the phenotypic modulation of vascular smooth muscle cell (VSMC), the NTPase activity was detected during restenosis after de-endothelialization in vascular wall. The activities of three enzymes involved in carbohydrate and nucleic acid metabolism were also investigated by spectrophotometry.

RESULTSThe activity of NTPase increased continuously and associated with the process of intimal thickening. Western blotting showed that expression of SMalpha-actin, as the marker of contractile phenotype of VSMC, decreased continuously. Osteopontin (OPN), the marker of synthetic phenotype of VSMC, was up-regulated during the process. These suggested that intimal injury induced phenotypic modulation of VSMC. The activities of 5'-nucleotidase, adenosine deaminase and succinate dehydrogenase increased and reached their peaks on 7 days after de-endothelialization. The changes of three enzymes were associated with proliferation in VSMC.

CONCLUSIONThe efflux of mRNA and the changes of enzyme activity involved in carbohydrate or nucleic acid metabolism may be the biochemical basis in the development and progression of restenosis.

Animals ; Constriction, Pathologic ; Endothelium, Vascular ; pathology ; Female ; Hyperplasia ; enzymology ; pathology ; Male ; Muscle, Smooth, Vascular ; pathology ; Nucleoside-Triphosphatase ; metabolism ; Rats ; Rats, Sprague-Dawley ; Tunica Intima ; enzymology ; pathology

Arterial restenosis frequently develops after open or endovascular surgery due to intimal hyperplasia. Since tissue transglutaminase (TG2) is known to involve in fibrosis, wound healing, and extracellular matrix remodeling, we examined the role of TG2 in the process of intimal hyperplasia using TG2-null mice. The neointimal formation was compared between TG2-null and wild-type (C57BL/6) mice by two different injury models; carotid ligation and carotid loop injury. In ligation model, there was no difference in intimal thickness between two groups. In loop injury model, intimal hyperplasia developed in both groups and the intimal/medial area ratio was significantly reduced in TG2-null mice (P = 0.007). TG2 was intensely stained in neointimal cells in 2 weeks. In situ activity of TG2 in the injured arteries steadily increased until 4 weeks compared to uninjured arteries. Taken together, intimal hyperplasia was significantly reduced in TG2-null mice, indicating that TG2 has an important role in the development of intimal hyperplasia. This suggests that TG2 may be a novel target to prevent the arterial restenosis after vascular surgery.
Animals ; Carotid Arteries/pathology/*surgery ; Disease Models, Animal ; GTP-Binding Proteins/deficiency/genetics/*metabolism ; Hyperplasia ; Mice ; Mice, Inbred C57BL ; Transglutaminases/deficiency/genetics/*metabolism ; Tunica Intima/*pathology

OBJECTIVETo investigate the in vivo gene expression of adenovirus-mediated human tissue factor pathway inhibitor (hTFPI) and its inhibition effects on intimal proliferation in rabbit carotid arteries after balloon injury.

METHODSRabbits underwent carotid artery balloon injuries were treated with Ad-TFPI (n = 25), Ad-LacZ (n = 25) or PBS (n = 10), respectively. Sham operated rabbits (n = 10) serve as normal controls. The expressions of human TFPI at mRNA and protein levels were detected by RT-PCR and ELISA respectively on the 3rd, 7th, 10th, 14th, 28th day after operation. Intimal proliferation was detected by angiograms and morphometric analysis.

RESULTSTFPI mRNA and protein expressions were detected at 3 days and peaked at the 10th and 14th day after TFPI gene transfer. The expressions were still detectable on the 28th day. There was no TFPI expression in Ad-LacZ group. The carotid angiogram results indicated that the minimal lumen diameter in TFPI group was significantly larger and the lumina stenosis percentage was significantly lower in TFPI group compared those in Ad-LacZ and PBS groups (all P < 0.05). The morphometric analysis showed that the intimal area, the ratio of the intimal/media area, the lumina stenosis percentage in TFPI group were all significantly reduced compared with those in Ad-LacZ and PBS groups (all P < 0.01).

CONCLUSIONSThe TFPI gene could be effectively transferred by adenovirus vector to injured carotid arteries and transferred Ad-TFPI could significantly attenuate intimal proliferation in balloon injured carotid arteries in rabbits.

Adenoviridae ; genetics ; Animals ; Carotid Arteries ; metabolism ; Carotid Artery Injuries ; metabolism ; pathology ; Female ; Gene Transfer Techniques ; Genetic Vectors ; Humans ; Lipoproteins ; genetics ; Male ; Rabbits ; Transfection ; Tunica Intima ; pathology

BACKGROUNDAtherosclerosis is a chronic inflammatory disease. Accumulated evidences suggest a deep involvement of oxidative damage in the development of atherosclerosis, but little is discussed over the relationship between plasma glutathione redox status as the most important intrinsic antioxidant defensive mechanism and the atherosclerosis.

METHODSA total of 132 patients suspected with atherosclerosis were assigned to three groups by high frequency ultrasonic examination of the carotid artery. With the thickness of intima of the carotid artery as an index of degree of atherosclerosis progression, 56 were included in plaque-forming group (A), 42 in carotid artery intima-thickening group (B), and 34 in normal carotid artery intima-thickness group (C). All patients were subjected to the measurement of plasma glutathione (GSH) (reduced form GSH and oxidized form GSSG), nicotinamide adenine dinucleotide phosphate (NADP) (reduced form NADPH and oxidized form NADP(+)), oxidized low density lipoprotein (oxLDL), and malondialdehyde (MDA). The GSH/GSSG and NADPH/NADP(+) redox potentials were calculated according to the Nernst equation, and their correlation with intima thickness and oxLDL was analyzed.

RESULTSWith the thickening of artery intima (from group C to A), GSH concentration and the ratio of GSH/GSSG gradually reduced, and GSSG and GSH/GSSG redox potential gradually increased (more positive) (P < 0.05). The NADPH and NADPH/NADP(+) redox status also showed similar but milder changes. The products of oxidative stress oxLDL and MDA increased significantly along with the thickening of artery intima (P < 0.05). The analysis of the relationship between GSH/GSSG redox potential, intima thickness, and oxLDL showed positive correlations (P < 0.05). The plasma GSH/GSSG redox status was positively correlated with the intima thickness of the carotid artery and the oxidized injury of LDL. The redox status shifted to oxidizing direction along with the intima thickening and plaque-forming.

CONCLUSIONElevated peroxidative glutathione redox status was deeply implicated in atherosclerosis progressing, and it may be a sensitive and reliable index for monitoring oxidative status in atherosclerosis.

Adult ; Aged ; Atherosclerosis ; metabolism ; pathology ; Carotid Arteries ; pathology ; Female ; Glutathione ; blood ; Glutathione Disulfide ; blood ; Humans ; Lipid Peroxidation ; Male ; Middle Aged ; NADP ; blood ; Oxidative Stress ; Tunica Intima ; pathology ; Tunica Media ; pathology

The aim of this study was to investigate the relationship between obesity, insulin resistance and atherosclerosis in type 2 diabetes mellitus (T2DM) patients. Total 530 patients with T2DM were included. To evaluate the severity of atherosclerosis, we measured the coronary artery calcification (CAC) score, intima-media thickness (IMT) of the common carotid artery, and the ankle-brachial pressure index (ABPI). Subjects were classified according to body mass index (BMI), a marker of general obesity, and waist-to-hip ratio (WHR), a marker of regional obesity. The insulin sensitivity index (ISI) was measured by the short insulin tolerance test. All subjects were classified into four groups, according to BMI: the under-weight group, the normal-weight (NW) group, the over-weight (OW) group, and the obese (OB) group. WHR and systolic blood pressure, triglycerides (TG), HDL-cholesterol (HDLC), free fatty acids (FFA), fibrinogen, and fasting c-peptide levels were significantly different between BMI groups. TG, HDL-C, FFA, fibrinogen and ISI were significantly different between patients with and without abdominal obesity. In the OW group as well as in the NW group, carotid IMT, ABPI and CAC score were significantly different between patients with and without abdominal obesity. This study indicates that abdominal obesity was associated with atherosclerosis in T2DM patients.
Aged ; Atherosclerosis/complications ; Blood Pressure ; Coronary Vessels/pathology ; Diabetes Complications ; Diabetes Mellitus, Type 2/*genetics ; Female ; Humans ; Insulin Resistance ; Korea ; Male ; Middle Aged ; Obesity/*complications/*genetics ; Triglycerides/metabolism ; Tunica Intima/pathology ; Tunica Media/pathology

OBJECTIVETo evaluate the role of leptin in neointimal formation and related mechanisms.

METHODSFemoral arterial injury was induced in wild-type (Wt, n = 10), leptin-deficient (Lep(-)/-, n = 12), and leptin receptor-deficient (LepR(-)/-, n = 10) mice. Leptin treatment studies (tail vein injection of adenovirus expressing murine leptin on the RSV promoter, ad-leptin) were performed on Lep(-)/- (n = 5) and LepR(-)/- (n = 4) mice. Intimal (I) and medial (M) areas were measured and the ratio of I/M was calculated. Smooth muscle cells were detected by smooth muscle alpha-actin staining using an alpha-actin monoclonal antibody. Cellular proliferation was analyzed with BrdU Staining Kit and the number of BrdU-positive cells was counted manually. Plasma leptin level was measured by ELISA.

RESULTSThe I/M ratio of Lep(-)/- and LepR(-)/- mice was significantly lower than that in Wt separately (Lep(-)/- vs. Wt = 0.80 +/- 0.14 vs. 1.50 +/- 0.22, P < 0.01; LepR(-)/- vs. Wt = 0.55 +/- 0.20 vs. 1.50 +/- 0.22, P < 0.05). Plasma leptin level was significantly increased in Lep(-)/- and LepR(-)/- mice post leptin treatment. I/M was significantly increased in Lep(-)/- mice receiving ad-leptin compared with untreated Lep(-)/- mice (P < 0.05), while I/M was similar between LepR(-)/- mice with and without ad-leptin treatment (P > 0.05). The changes on number of positive alpha-actin and BrdU stained smooth muscle cells were consistent with the neointimal formation findings in various groups.

CONCLUSIONSMice lacking leptin or the leptin receptor were protected from neointimal formation following vascular injury. Leptin treatment increased neointimal formation in Lep(-)/- but not in LepR(-)/- mice, suggesting leptin receptor activation and vascular smooth muscle cell proliferation played a pivotal role on neointimal formation post-injury in this model, giving an evidence that high plasma leptin level is a risk factor for neointimal formation.

Actins ; analysis ; Animals ; Cell Proliferation ; Leptin ; blood ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Muscle, Smooth, Vascular ; pathology ; Receptors, Leptin ; metabolism ; Tunica Intima ; pathology

BACKGROUNDPost-stenting restenosis is a significant clinical problem, involving vascular smooth muscle cells (VSMCs) proliferation and apoptosis. It is reported that c-myc antisense oligodeoxynucleotides (ASODNs) local delivered by catheter can inhibit VSMCs proliferation. This study was designed to assess tissue distribution of c-myc ASODN local delivered using gelatin-coated platinum-iridium (Pt-Ir) stents, and its effect on apoptosis of VSMCs.

METHODSGelatin-coated Pt-Ir stents that had absorbed caroboxyfluorescein-5-succimidyl ester (FAM) labeled c-myc ASODNs (550 microg per stent) were implanted into the right carotid arteries of 6 rabbits. Tissue samples were obtained at 45 minutes, 2 hours, and 6 hours. Tissue distribution of c-myc ASODNs was assessed by fluorescence microscopy. In addition, 32 rabbits were randomly divided into two groups. Rabbits in the control group (n = 16) were implanted with gelatin-coated Pt-Ir stents, and those in the treatment group (n = 16) were implanted with gelatin-coated stents that had absorbed c-myc ASODNs. 7, 14, 30, or 90 days (n = 4, respectively, for each group) after the stenting procedure, the stented segments were harvested, and histopathological examinations were performed to calculate neointimal area and mean neointimal thickness. The expression of c-myc was assessed using in situ hybridization (ISH) and immunohistochemical methods. Apoptotic VSMCs were detected using terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) and transmission electron microscope (TEM).

RESULTSAccording to fluorescence microscopic results, FAM-labeled c-myc ASODNs were concentrated in the target vessel media at the 45 minutes time point, and then dispersed to the adventitia. Morphometric analysis showed that neointimal area and mean neointimal thickness increased continuously up to 90 days after stent implantation, but that total neointimal area and mean neointimal thickness were less in the treatment group than in the control group at all time points (P < 0.0001). At day 7 and day 14 after stenting, there were no detectable apoptotic cells in either group. However, apoptotic cells were present in the neointima 30 and 90 days after stenting, and the number of apoptotic cells was less at 30 days than at 90 days. Meanwhile, c-myc ASODNs appeared to induce apoptosis in more cells in the treatment group than that in the control group. Typical apoptotic VSMCs were observable under TEM. The expression of c-myc was positive in the control group and negative or weakly positive in the c-myc ASODN treatment group, according to both ISH and immunohistochemical examination.

CONCLUSIONGelatin-coated Pt-Ir stent mediated local delivery of c-myc ASODNs is feasible. The localization of c-myc ASODN is primarily in the target vessel walls. c-myc ASODNs can inhibit VSMCs proliferation and induce its apoptosis after local delivery in vivo.

Animals ; Apoptosis ; drug effects ; Carotid Arteries ; Female ; Gelatin ; Genes, myb ; genetics ; In Situ Hybridization ; Iridium ; Male ; Microscopy, Fluorescence ; Myocytes, Smooth Muscle ; pathology ; Oligodeoxyribonucleotides, Antisense ; administration & dosage ; metabolism ; pharmacology ; Platinum ; Rabbits ; Random Allocation ; Stents ; Tissue Distribution ; Tunica Intima ; metabolism ; Tunica Media ; metabolism

AIMOsteopontin 13-peptide(Gly158-Lys170), containing multi-function domains was used to inhibit the VSMC adhesion, migration. The mechanism of 13-peptide inhibiting neointima formation was investigated.

METHODSThe effect of 13-peptide on VSMC adhesion was tested by adhesion assay. The restenosis model was prepared balloon injury after administration of 13-peptide for 1 h, and then the 13-peptide was given by an intravenous drip for 7 days. The expression changes of OPN, FAK, ILK in vessel wall were detected by immunohistochemistry and Western blot.

RESULTSThe 13-peptide dose-dependently reduced adhesion of VSMC in OPN matrix, and the infiltration of macrophage in vessel wall also was reduced in the treatment group after balloon injury. The expression of OPN, FAK, ILK was down-regulated following with the inhibition of neointima thickening.

CONCLUSIONThe OPN 13-peptide can inhibit inflammation and neointima formation by blocking the binding of OPN to it's receptors.

Animals ; Cell Adhesion ; drug effects ; Cells, Cultured ; Disease Models, Animal ; Male ; Muscle, Smooth, Vascular ; cytology ; drug effects ; metabolism ; Osteopontin ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Tunica Intima ; pathology