Effect of L-arginine on intimal proliferation and expression of related cell cycle regulatory factors after vascular injury in rats

AIM:To investigate the effect of L-arginine (L-Arg) on intimal proliferation and expression of related cell cycle regulatory factors after vascular injury in rats. METHODS:Rats were divided into three groups :sham operation group, balloon injury group(this group included balloon 48 h,7 d and 14 d subgroup) and balloon+L-Arg group. Neointima area were calculated morphologiocally. The expression of cyclin dependent kinase-2(CDK2),cyclin E and proliferation cell nuclear antigen (PCNA) were measured by means of immunohistochemical technique and computer image analyzer. RESULTS:After vascular balloon injury, the level of plasma NO decreased, CDK2、cyclin E and PCNA expressed in the media at 48 h and in the neointima at 7 d and 14 d but with low and undetected expression in the media, the expression of CDK2, cyclin E and PCNA increased with the intima thickening. Compared with balloon 14 d group, the plasma NO level increased (P＜0.01), the neointima area reduced by 59.1%(P＜0.01) and the positive expression indexes of CDK2, cyclin E and PCNA decreased by 36.1%, 46.3% and 76.2% respectively in balloon+L-Arg group (P all＜0.01). CONCLUSION:L-Arg can effectively repress intima proliferation after vascular injury, which may be associated with its inhibiting the proliferation of vascular smooth muscle cell through downregulating the excessive expression of CDK2, cyclin E and PCNA.     

Probucol prevents restenosis by regulating vascular remodeling after percutaneous transluminal angioplasty in rabbits

AIM:To investigate the relationship between the prevention of probucol on restenosis and vascular remodeling after percutaneous transluminal angioplasty(PTA) in rabbits.METHODS:New Zealand rabbit thoracic aorta atherosclerosis was induced by 3.5F ballon catheter injury following a 4-weeks feeding of high cholesterol diet, and PTA was performed by using 3.5F balloon catheter. Probucol(1g/d) or vitamin E (400 mg/d) was administrated one week before PTA. Two weeks after PTA, the bore and outside diameter (OD) of arteries, the area circumscribing by intimal elastic lamina (IEL), the area circumscribing by extral elastic lamina (EEL), medial area (MA), neointima area/medial area (NEA/MA) were analyzed by computerized digitizer system. Lipids of serum were measured by means of biochemical assay.RESULTS:After two weeks of PTA, the int ima proliferation and lumen restenosis were observed obviously.However, with probucol treatment for 3 weeks, the restenosis of aorta was inhibited significantly by increasing bore, outside diameter, and lumen area of rabbits aortas and decreasing NEA, NEA/MA.Furthermore, probucol regulated vascular remodeling by increasing the area circumscribing by IEL[(3.50 0.20)mm²υs(1.59 0.23)mm², P<0.01]and EEL[(4.61±0.29)mm²υs(2.56±0.28)mm², P<0.01]of rabbit aortas.In addition, probucol decreased lipids of serum in rabbits.CONCLUSION:Probucol prevents restenosis by regulating vascular remodeling after percutaneous transluminal angioplasty in rabbits.     

Influence of Sini decoction on the proliferation and apoptosis of artery smooth muscle cells after balloon injury

AIM: To investigate the influence of Sini decoction (SND) on the proliferation and apoptosis of rabbit abdominal aorta smooth muscle cells after ballon injury and discuss the effect of vascular smooth muscle cell's (VSMCs) proliferation and apoptosis in post-percutaneous coronary intervention (PCI) restenosis (RS) and the feasibility of SND preventing post-PCI RS. METHODS: The animal model of rabbit abdominal aorta ballon injury was set up and the therapertic group was treated with SND. The shape of proliferative and apoptotic cell were investigated by electron microscope. Immunohistochemistry staining was performed using α-actin,PCNA and Cyclin E monoclonal antibodies. In situ Cell Death Detection Kit was used to identify apoptotic cells. Abdomial aorta angiography was operated in the 84th day subgroup and the stenosis degree was evalued by quantitative angiographic analysis. RESULTS: As compared with the control group, the therapeutic group displayed a lower proliferative percentage and a higher apoptosic percentage (P<0.05). Moreover, the apoptosic peek time was on the 14th day after operation,which was longer than the control group. CONCLUSION: SND effectly inhibited the proliferation of VSMCs and iuduced apoptosis in VSMCs.     

Role of fibrinogen activity in the progress of coronary artery disease

AIM:To detect the role of fibrinogen activity (Fa) in the progress of coronary artery disease (CHD).METHODS:Fa was measured with hemorheology methods in patients with CHD stable phase (n=30) and angina pectoris (n=27).RESULTS: (1) Levels of plasmatic fibrinogen and plasmatic viscosity in patients with CHD were higher than that of control group (P＜0.01,P＜0.05).(2)Fa and platelet aggregation activity (Pt max, Pt H, Pt K) in patients with CHD angina pectoris were very much higher than that of control group (P＜0.01, respectively).(3)There was a negative correlation between PT max, Pt H and Fa(r=-0.8379,P＜0.01;r=-0.8784,P＜0.01 respectively) in patients with CHD angina pectoris.CONCLUSION: Fa may play a role in the progress of CHD.     

The alteration of urotensin-II receptors in rat aorta after balloon angioplasty injury

AIM:To observe the alteration of urotensin II (UII) receptors and contractile response to UII in rat aorta after balloon angioplasty injury. METHODS: The plasma membrane isolated from balloon injured aorta was used to study the binding of ¹²⁵-UII to the membrane and the contractile potency of UII on rat aorta was assayed. RESULTS: In contrast to the normal aorta, the contractile potency to UII enhanced in balloon injury artery and the calculated maximal number of specific binding sites (Bmax) was increased about 44% and 36% respectively in rat artery after balloon injury 3 and 21 days (P＜0.01). CONCLUSION: The density of UII receptors and the contractile response to UII had changed after balloon angioplasty injury. It was proposed that UII might play an important role in the intervention of restenosis after angioplasty.     

The change of smooth muscle cells apoptosis and apoptosis related-gene expression after balloon injury to vessel

AIM: To investigate the mechanism of vascular smooth muscle cells (VSMC) apoptosis after balloon injury. METHODS: VSMCs apoptosis, Bax protein and Bcl-2 protein was measured by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) and immunohistochemical technique after balloon injury. RESULTS:VSMCs apoptosis occurred in vascular media at 3 days after balloon injury and reached a peak in media and intima where apoptosis was mainly present at 7 days, then decreased. At 28 days after balloon injury, only a few apoptosis cells were in intima. Irbesartan significantly increased VSMCs apoptosis (P＜0.01). Expressions of Bax and Bcl-2 were significantly higher in vascular media at 3 days after balloon injury than sham(P＜0.01). At 7 days, Bax expression reached a peak which was three times as that of sham, and then decreased. After balloon injury, Bcl-2 expression was generally increased and reached its peak at 28 days. The ratio of Bax to Bcl-2 (Bax/Bcl-2) also reached its peak at 7 days and was decreased under basal level at 28 days. Irbesartan increased Bax and the ratio of Bax to Bcl-2, while decreased Bcl-2. CONCLUSION: Bcl-2 and Bax contribute to the regulation of VSMC apoptosis after balloon injury.     

Distribution and activity of calcineurin in different tissues of rat

AIM: To investigate the activity and distribution of calcineurin (CaN) in different tissues of rat. METHODS: Using western blot and immunohistochemical staining methods to measure the amount and location of CnAα, isoform of catalytic subunit of CaN in different tissues of rat. CaN activity was measured by [³²P] labelled substrate peptide. RESULTS: 1. Western blot showed that CnAα expression was detected in brain, heart, skeletal muscle and lung tissues. There was no detectable CnAα expression in kidney and aorta. 2. In immunohistochemical staining study, there was strong immunostaining of CnAα in brain. CnAα was located in cytoplasm of cardiac cell, macrophage and connective tissue of peribronchiolar in lung tissue, aorta adventitia, connective tissue around small vessels and outer wall of renal tube. 3. CaN activity was highest in brain, the following was skeletal muscle, myocardium and lung tissue. CaN activity was lowest in aorta and kidney tissue. CONCLUSION: CaN is widely distributed in rats and might be involved in functional regulation of various organs and tissues.     

Effects of astragalan on neurotransmitters and expression of c-fos mRNA in hippocampus after ischemic brain injury in rats

AIM: To explore the effects of astragalan (AG) on the neurotransmitters,acetylcholine (ACh), norepinephrine (NE) and 5-hydroxytryptamine (5-HT), and the expression of c-fos mRNA in hippocampus after ischemic brain injury in rats. METHODS: Male Wistar rats (180~220 g) were randomly divided into 10 groups (n=10): sham-operated group (SOG), 3 model groups (MG 1 d, 3 d, 7 d) and 3 low- or high-dose AG treatment groups (L/H-AGTG 1 d, 3 d, 7 d), respectively. The middle cerebral artery of the rats in MG group and AGTG group were blocked by operation to induced brain injury. The cerebral blood vessels of the animals were blocked on day 1, day 2 and day 7, respectively, after the L/H-AGTG were treated with AG (5 mg/kg and 15 mg/kg, ip). The content of ACh,5-HT and NE was determined using their respective ELISA kits, and the expression of c-fos mRNA in the hippocampus homogenate was semiquantitative analyzed by RT-PCR after neurologic impairment (NIP) was scored. RESULTS: AG attenuated the injury in hippocampus by cerebral ischemia in a dose-dependent manner. The content of ACh, 5-HT and NE in L-AGTG 7 d,H-AGTG 3 d and 7 d groups was significantly higher than that in MG group, but was lower in SOG group (P<0.05 or P<0.01). The mRNA expression of c-fos in SOG group was lower than that in MG group (P<0.05 or P<0.01), indicating that reinforcement expression of c-fos mRNA by cerebral ischemia and the expression of downstream genes may be beneficial for protecting the neurons. The mRNA expression of c-fos in H-AGTG 3 d/7 d groups was higher than that in MG group (P<0.05). CONCLUSION: AG attenuates the damage of neurons and improves the functions of hippocampus under the condition of cerebral ischemia/reperfusion by increasing the content of ACh, NA and 5-HT, and the mRNA expression of c-fos in hippocampus.     

Effect of L-Arg on plasma content of endothelin and expression of c-fos mRNA in the left ventricle of rats with renovascular hypertensive hypertrophy

AIM:To study the effect of L-Arg on plasma content of endothelin (ET) and the expression of proto-oncogene c-fos mRNA in the left ventricle of rats with renovascular hypertensive hypertrophy. METHODS: The level of c-fos mRNA were measured by in situ hybridization. The ET in plasma were measured by radioimmunoassay. RESULTS:After eight weeks of treatment with L-Arg, the expression of c-fos decreased markedly (P＜0.01). The ET content in plasma also decreased significantly by L-Arg(P＜0.01).CONCLUSION: Plasma ET content and the expression of c-fos in the left ventricle of rats with renovascular hypertensive hypertrophy could be decreased by L-Arg administration.     

Phosphorothioate-modified antisense TGF-β1 oligodeoxynucleotide inhibits neointimal hyperplasia after vascular balloon injury in rats

AIM: To evaluate the effects of antisense TGF-β1 oligodeoxynucleotide (AS TGF-β1) on the expression of TGF-β1, deposition of extracellular matrix (ECM) and the neointima formation in the arteries after balloon injury. METHODS: The unmodified and phosphorothioate-modified AS TGF-β1 which containing 15 bases and surrounding the initiation codon region (ATG) of rat TGF-β1 complementary DNA (cDNA) were designed. At the same time, sense TGF-β1 oligodeoxynucleotide (S TGF-β1) with the base sequence complement to AS TGF-β1 was synthesized as a control. The oligodeoxynucleotides were introduced into in vivo and in vitro experiments, respectively. RESULTS: The AS TGF-β1 significantly inhibited the protein expression of TGF-β1 in a concentration-dependent manner, and S TGF-β1 did not have the same effect. Furthermore, no effect of the AS TGF-β1 on the mRNA expression of TGF-β1 in injured VSMCs was observed. Moreover, for the injured VSMCs, AS TGF-β1 significantly and concentration-dependently inhibited the basal DNA synthesis. Both AS TGF-β1 and S TGF-β1 did not exhibit dose-dependent effects on DNA synthesis in uninjured VSMCs. Fibronectin (FN) mRNA expression in injured VSMCs was significantly decreased by AS TGF-β1 in a concentration (001~1 μmol/L)-dependent manner. AS TGF-β1 significantly increased the mRNA expression of contractile marker SM22α, and decreased the mRNA expression of synthetic markers osteopontin and matrix Gla, especially at the concentration of 001 μmol/L and 01 μmol/L. After treatment with AS TGF-β1 (90 μg·kg-1·d-1) for 28 d, the neointima formation was significantly inhibited, and the area ratio of intima/media was markedly decreased by 68% compared with untreated group, but S TGF-β1 had no effect on neointimal formation. CONCLUSION：The AS TGF-β1 specifically inhibits the protein expression of TGF-β1 in the VSMCs derived from injured arteries. Moreover, it significantly inhibits DNA synthesis and cell proliferation, and decreases the expression of FN. Therefore, AS TGF-β1 dramatically attenuates neointima formation after balloon njury. The effects of AS TGF-β1 on the injured VSMCs may be associated with its reverse effects on the alteration of VSMC phenotype after balloon injury.     

Expression of intercellular adhesion molecule in lung tissues of experimental acute lung injury and the effect of rhubarb

AIM:To approach the relationship between the expression of intercellular adhesion (ICAM-1 mRNA) and acute lung injury (ALI) as well as the mechanisms of rhubarb in the prevention and treatment of the lung injury. METHODS:ALI animal model was performed by Lipopolysaccharide (LPS). The rats were divided into 4 groups: LPS group, control group, rhubarb+LPS group and dexamethasone+LPS group. Histopathological examination and biological markers were measured for the lung specimens. Molecular hybridization method was used to determine the expression of ICAM-1 mRNA. RESULTS:The ICAM-1 mRNA expression in the lung tissues of LPS group significantly increased compared with control group (P＜0.01), rhubarb and dexamethasone had the action of decreasing the ICAM-1 mRNA expression (P＜0.05, P＜0.01); pathologic changes and the biological markers of ALI significantly decreased or ameliorated. CONCLUSION:The increase in the expression of ICAM-1 mRNA in the lung tissues of ALI is involved in the formation of ALI. Rhubarb and dexamethasone can ameliorate the lung damage, mechanism of which may be related to the inhibition of ICAM-1 mRNA expression.     

Retrovirus-mediated cellular repressor of E1A-stimulated genes inhibits neointima formation in the rat carotid artery after balloon injury

AIM: To evaluate the effects of over-expression of cellular repressor of E1A-stimulated genes (CREG) mediated by retrovirus on neointima formation in injured rat carotid. METHODS: The pluronic F127 containing pLNCX/CREG or pLNCX/GFP retroviral vectors was placed around the injured rat carotid.The neointima,media areas and the intima to media ratio were calculated.Expressions of CREG,SM α-actin and Ki-67 were detected. RESULTS: The GFP expression was observed at day 2 in pLNCX/GFP groups.The expression of exogenous CREG was also significantly increased in arteries at day 2 after pLNCX-CREG infection.Over-expression of CREG significantly suppressed neointima formation,attenuated the expression of Ki-67 and up-regulated SM α-actin expression. CONCLUSION: Over-expression of CREG inhibits VSMCs proliferation and promotes VSMCs differentiation after vascular injury.It suggests that modulation of CREG expression or activity may be a viable approach to treat neointimal restenosis after percutaneous coronary intervention.     

Effect of antisense oligonucleotides of c-sis on cellular cycle and proliferation of vascular smooth muscle cells in pulmonary artery

AIM:To investigate the effect of antisense oligonucleotides(ASON) of c-sis on cellular cycle and proliferation of pulmonary artery vascular smooth muscle cells(VSMC).METHODS:Tissue mass culture was done to get VSMC of pulmonary artery. Different concentrations of antisense oligonucleotides of c-sis were added into the cultures to observe the VSMC proliferation curve using MTT test. The changes of VSMC cellular cycle were also observed by flow cytometry.RESULTS:ASON with mid-to high concentrations restrained the proliferation of VSMC apparently with the peak of cell growth being attenuated or eliminated. Affected by mid-concentration ASON, PDGF-BB showed significant accelerating effect on the proliferation of VSMC. The ratio of G₀／G₁ in cellular cycle was increased significantly in VSMC culture with ASON in comparison with control. The G₀/G₁ ratio also showed significant differences among different concentration of ASON groups(P＜0.05).CONCLUSION:Mid-to high concentration of ASON was a powerful inhibitor of cellular proliferation for pulmonary artery VSMC. ASON increased the ratio of G₀／G₁ significantly and the increase seems to be ASON dosage dependent.     

Effects of aspirin on production of nitric oxide and inducible nitric oxide synthase mRNA expression under inflammatory conditions in human vascular endothelial cells

AIM: To explore the effect of aspirin on inducible nitric oxide synthesis and gene expression under inflammation in endothelial cells. METHODS:Using NADPH, Griess methods and RT-PCR, the activity of isozymes of NO synthase (NOS), nitric oxide (NO) level, and iNOS mRNA expression were examined respectively. Also, the lactate dehydrogenase (LDH) release rate, malondialdehyde (MDA) content and cell viability were measured. RESULTS: Aspirin (3 mmol/L) reduced inducible NO production and NOS activity(P＜0.05), caused a significant decrease in LDH release rate and MDA content with a further increase in cell viability. Aspirin inhibited inducible NO excretion and alleviated the damage caused by NO in a concentration-dependent manner. However,aspirin had no effect on basal NO levels in the absence of stimulation by inflammatory factor. On the other hand, under middle concentration (＜10 mmol/L), aspirin was able to reduce enzymatic activity of NOS and protein expression by increasing the stability of iNOS mRNA. In contrast, at high concentration (20 mol/L), aspirin could decrease the stability of iNOSmRNA. Sodium salicylate and indomethacin did not inhibit inducible NO production. CONCLUSION:Aspirin could significantly inhibit inducible NO production in vascular endothelial cells during inflammation.     

Effect of doxycycline on matrix metalloproteinase activity and vascular remodeling in balloon-injured rat carotid arteries

AIM:To examine the inhibition of matrix metalloproteinase (MMP) activity by doxycycline (Doxy) and its effect on vascular smooth muscle cells (SMCs) proliferation,neointimal hyperplasia and vascular remodeling.METHODS: The model of rat common carotid artery injury was established by balloon-dilatation.Doxy was administered to the animals of treatment group at dose of 30 mg·kg^-1·d^-1.The activity of MMPs in the tissue of injured carotid arteries was measured by gelatin zymography.The thickness and area of neointimal,lumen area and the proliferation of SMCs were measured by histological and morphometric analysis.RESULTS:1.After Doxy treatment,the activity of MMP-9 in the carotid arteries was reduced by 26.3% and 34.5% compared to that in rats without Doxy treatment at 24 hours and 3 days after balloon injury,respectively (P<0.01).The activity of MMP-2 was also reduced by 40.0% at 7 days after injury (P<0.01).2.The thickness of neointimal were significantly decreased by 32.0% and 38.8% (P<0.01) and the lumen area was increased by 58.0% and 90.4 % at 14 and 28 days after injury in the Doxy-treated rats compared to those in control rats,respectively (P<0.01).Doxy treatment significantly reduced intimal SMCs proliferation from 62.76%±1.02 % in the controls to 43.23%±1.06% at 7 days after injury (P<0.01).CONCLUSION: Doxy treatment inhibits the activity of MMPs,the SMCs proliferation of intimal,neointimal hyperplasia and vascular remodeling,suggesting that Doxy treatment is useful in preventing restenosis after PTCA.     

Effect of Fu-Sheng powder on NPY and its mRNA in brain of rats with hyperlipidemia after cerebral ischemia and reperfusion

AIM:To observe the changes in neuropeptide Y(NPY) and the effect of Fu-Sheng powder(FSP) on NPY in the rat brain in a steady cerebral ischemia and reperfusion(I/R) model. METHODS: The models of rat brain injury were established by repeated cerebral I/R in rats with hyperlipidemia. Radioimmunoassay was performed to determine the level of NPY, while NPY mRNA expression was observed by in situ hybridization. RESULTS: After 1 day of I/R, compared with control group, the content of NPY in the model animals were significantly increased by 51.86% (P＜0.01) and lasting 7 days after I/R, and the expression of NPY mRNA was greatly increased. FSP treatment decreased the contents of NPY (P＜0.05,P＜0.01) and its mRNA expression. CONCLUTION: There were obvious imbalances of NPY in the rat brain after cerebral I/R and the FSP might antagonize ischemia injury of brain through modulating NPY, which may be one of the mechanisms underlying FSP treatment for cerebral vascular diseases.