RhoA Expression Is Controlled by Nitric Oxide through cGMP-dependent Protein Kinase Activation

To investigate the role of RhoA and Rho kinase system in the onset of labor. Forty term pregnant women, who delivered through cesarean section at the Xiangya Second Hospital of Central South University from February 2007 to November 2007, were selected and divided into 2 groups: 20 in labor group and 20 in non-labor group. Another 20 non-pregnant women undergoing hysterectomy due to cervical intraepithelial neoplasia were chosen as the control. Real-time fluorescence quantitative RT-PCR and western blot were applied to detect the expression RhoA and ROCK I mRNA and protein in uterine smooth muscle tissue and the correlation between the mRNA and protein expression of RhoA and ROCK I were analyzed. (1) The mRNA expressions of both RhoA and ROCK I were detected in all groups, and higher levels were found in the labor group than in the non-labor group and the control [RhoA mRNA: (3.51 +/- 0.56) x 10(-3) vs. (2.75 +/- 0.52) x 10(-3) and (2.11 +/- 0.54) x 10(-3); ROCK I mRNA: (4.07 +/- 0.66) x 10(-3) vs. (2.71 +/- 0.52) x 10(-3) and (2.01 +/- 0.23) x 10(-3), P < 0.01]. (2) RhoA and ROCK I proteins were also identified in all three groups, and the expressions in the labor and non-labor group were higher than those of the control (RhoA protein: 0.72 +/- 0.23 and 0.64 +/- 0.17 vs. 0.46 +/- 0.15; ROCK I protein: 0.56 +/- 0.14 and 0.42 +/- 0.16 vs. 0.29 +/- 0.08, P < 0.01). (3) The expression of RhoA mRNA and ROCK I mRNA were positively correlated in each of the three groups (r = 0.73, P < 0.01), and the same was found in the expression of RhoA protein and ROCKI protein (r = 0.37, P < 0.01). The increased expression of RhoA and Rho kinase may play an important role in the initiation of labor.

To observe the effects of electroacupuncture (EA) at "Jiaji" (EX-B 2) points combined with nerve mobilization on protein and mRNA expression of RhoA in rabbits with sciatic nerve injury, and to provide theoretical basis for the treatment of peripheral nerve injury by EA at "Jiaji" (EX-B 2) points combined with nerve mobilization. A total of 180 New Zealand rabbits were randomly divided into a normal control group, a model control group, a nerve mobilization group, an EA group, an EA plus nerve mobilization group, 36 rabbits in each group. Each group was further divided into a 1-week subgroup, 2-week subgroup and 4-week subgroup, 12 rabbits in each subgroup. The sciatic nerve injury model was made by clamping method. The rabbits in the normal control group did not receive any intervention. The rabbits in the model control group was normally fed after operation. The rabbits in the nerve mobilization group were treated with nerve mobilization; the manipulation lasted for 1 s and relaxed for 5 s, 10 times per day, 6 days per week. The rabbits in the EA group were treated with EA at "Jiaji" (EX-B 2) points (L4-L6), once a day, 30 min each time, 6 times per week. The rabbits in the EA plus nerve mobilization group were treated with EA at "Jiaji" (EX-B 2) points, followed by nerve mobilization. The function of sciatic nerve on the injured side was evaluated by toe tension reflex and modified Tarlov score; the tissues of corresponding segments of spinal cord L4-L6 and sciatic nerve were taken; the expression of RhoA gene was detected by real-time PCR and the expression of RhoA protein was detected by Western Blot. ① Toe tension reflex and modified Tarlov score: at 1, 2 and 4 weeks, the scores in the model control group were lower than those in the normal control group (all P<0.01). The scores in the subgroup of nerve mobilization group, EA group and EA plus nerve mobilization group were higher than those in the model control group (all P<0.01), and the scores in the subgroup of EA plus nerve mobilization group were higher than those in the nerve mobilization group and the EA group (all P<0.01); the recovery was the best at 4 weeks. ② The mRNA and protein expression of RhoA: in segment of spinal cord, at 1, 2 and 4 weeks, the expression in the model control group was higher than that in the normal control group (all P<0.01). The expression in the subgroup of nerve mobilization group, EA group and EA plus nerve mobilization group was lower than that in the model control group (all P<0.01), and the expression in the subgroup of EA plus nerve mobilization group was lower than that in the nerve mobilization group and the EA group (all P<0.01); at 1 week and 4 weeks, the expression in the nerve mobilization group was lower than that in the EA group (all P<0.01); at 2 weeks, the expression in the nerve mobilization group was higher than that in the EA group (all P<0.01). In the sciatic nerve, at 1, 2 and 4 weeks, the expression in the model control group was higher than that in the normal control group (all P<0.01). The expression in the subgroup of nerve mobilization group, EA group and EA plus nerve mobilization group was lower than that in the model control group (all P<0.01); at 2 weeks and 4 weeks, the expression in the EA plus nerve mobilization group was lower than that in the nerve mobilization group and EA group (all P<0.01); at 1 week, the expression in the nerve mobilization group was lower than that in the EA group and EA plus nerve mobilization group (all P<0.01), but the differences between the EA group and the EA plus nerve mobilization group were not significant (P>0.05); at 2 weeks, the expression in the nerve mobilization group was higher than that in the EA group (all P<0.01); at 4 weeks, the expression in the nerve mobilization group was lower than that in the EA group (all P<0.01). The nerve mobilization and EA at "Jiaji" (EX-B 2) points could both promote the repair of injured sciatic nerve, which may be related to the down-regulation of RhoA expression, and the combination of the two methods has better effects.

To investigate the expression of RhoA and Rho kinase in junctional zone (JZ) of adenomyosis and normal myometrium and explore its relationship with severity of dysmenorrheal. From Mar. to Dec. 2012, 32 cases with adenomyosis undergoing hysterectomy were enrolled as adenomyosis group including 18 cases with proliferative endometrium and 14 cases with secretory endometrium matched with 29 cases with hysterectomy due to cervical disease and ovarian tumor as control group including 12 cases with proliferative endometrium and 17 cases with secretory endometrium in Beijing Obstetrics and Gynecology Hospital Affiliated to Capital Medical University. JZ smooth muscle cells were isolated and cultured immediately after the operation. The expression of mRNA and protein of RhoA and ROCK1 in JZ in two groups were measured by real-time fluorescence quantitative RT-PCR and western blot. (1) The mRNA expression of RhoA and ROCK1 in JZ of adenomyosis group did not show cyclic change. In proliferative phase, the expression of RhoA and ROCK1 (1.41 ± 0.16, 1.05 ± 0.15) was not significantly higher than that in secretory phase (1.17 ± 0.25, 0.98 ± 0.10) (P > 0.05). While JZ in control group, it showed obviously cyclic change. The expression level of them in proliferative phase (0.93 ± 0.10, 1.00 ± 0.18) was significantly higher than that in secretory phase (0.48 ± 0.03, 0.55 ± 0.05) (P < 0.05); It also showed that expressions of RhoA and ROCK1 in adenomysis group were significant higher than those in the control (P < 0.05). (2) The mRNA and protein expression of RhoA and ROCK1 was positively correlated in each of two groups(r = 0.48, P < 0.01;r = 0.67, P < 0.01). (3)The expression of RhoA and ROCK1 were 1.66 ± 0.19, 1.32 ± 0.11 in severe dysmenorrheal, 1.28 ± 0.12, 1.09 ± 0.08 in moderate dysmenorrheal and 0.93 ± 0.09,0.81 ± 0.06 in mild dysmenorrheal, it all reached statistical difference when compared the other group. (All P < 0.05). The expressions of RhoA and ROCK1 in JZ in adenomyosis group were higher than those in control group, and positively correlated with the severity of dysmenorrheal in adenomysis group, but it does not change with the menstrual cycle. High expression of RhoA and ROCK1 might be involved in abnormal contraction of uterine myometrium and correlated with the dysmenorrheal in adenomysis.

Objective To explore the expression and clinical significance of RhoA in oesophageal cancer.Methods The expression of RhoA protein in 140 specimens of primary oesophageal cancer tissues was detected by using immunohistochemical strept avidin-biotin complex (SABC) staining,and its correlation with clinicopathological parameters and prognosis of patients was analyzed.The expression of RhoA mRNA in 30 cases of primary oesophageal cancer tissues and their paired adjacent non-cancer tissues was evaluated by using reverse transcription-polymerase chain reaction (RT-PCR).Results The positive rate of RhoA protein was 60.7％,and the high expression of RhoA protein was significantly associated with depth of invasion (T),lymph node metastasis (N),and clinical TNM stage respectively (P ＜ 0.05).Kaplan-Meier univariate survival analysis revealed that five-year disease-free survival and overall survival rate for patients with high expression of RhoA protein were 6.0％ and 18.1％ respectively,which were significantly lower than 22.6％ and 39.6％ in patients with low expression of RhoA protein (P ＜ 0.05).Multivariate Cox regression analysis revealed that RhoA expression was an independent prognostic factor in disease-free survival and overall survival for patients with oesophageal cancer (Hazard ratio was 1.669 and 1.638 respectively,P ＜ 0.05).The expression of RhoA mRNA was detectable in 30 cases of primary oesophageal cancer tissues (0.49 ±0.21) and their paired adjacent non-cancer tissues (0.28 ±0.19),but the expression in oesophageal cancer tissues was significantly higher than that in paired adjacent non-cancer tissues (P ＜ 0.01).Conclusion High expression of RhoA was detectably in oesophageal cancer,suggesting its important role in the malignant tumorigenesis and progression of oesophageal cancer,and its close correlation with invasion and metastasis of oesophageal cancer. Key words: RhoA ; Oesophageal cancer; Invasion; Metastasis ; Prognosis

See related article, pages 1483–1491 The Notch family of receptors, Notch1 to -4, are heterodimer transmembrane proteins, consisting of an extracellular domain and a noncovalently linked intracellular domain (ICD). Upon interaction with the DSL family of proteins (Jagged, Delta-like) on neighboring cells, Notch undergoes proteolytic cleavage, which frees the ICD from the plasma membrane. This results in translocation of the ICD into the nucleus, where it forms a complex with the CSL family of transcriptional repressors (CBF1/RBP-Jk), removing the repression and allowing for target gene (Hes, Hey) transcription.1,2 Tissue distribution of the Notch proteins varies widely. Notch1 and -4 are predominantly endothelial, prominent in both arteries and veins, and present in all stages of development (embryonic to adult); the expression of Notch2 is typically confined to pulmonary endothelium, but Notch3 is primarily expressed in adult arterial vascular smooth muscle cells (VSMCs) in large conduit, pulmonary, and systemic resistance arteries.3 This specific pattern of temporal and spatial distribution correlate to diverse functions of the Notch family in vascular development and physiology in vertebrates reported to date.4 In the cardiovascular system, Notch signaling plays a role in several aspects of vascular development, including vasculogenesis, angiogenesis, differentiation, vascular remodeling, and VSMC maturation. Notch1 and -4 signaling appears critical in vasculogenesis and angiogenesis during early development, when it interacts with vascular endothelial growth factor signaling to specify artery–vein differentiation of endothelial cells (ECs). Transgenic mice deficient for Notch1 fail to undergo embryonic angiogenic remodeling, and vascular development is arrested at the primitive undifferentiated plexus, resulting in an embryonic lethal phenotype. Notch2-null mutations likewise result in embryonic lethality characterized by multiple large and small vessel aneurysms. Notch4 deletion results in no obvious phenotypic alteration. Notch3−/− mice are viable; however, they fail to develop proper arterial VSMC phenotype, resulting in vein-like …

p57 and Ras homology A (RhoA) have been implicated in the growth and metastasis of several types of human cancers. This study aimed to detect their expression in hepatocellular carcinoma (HCC) tissue specimens and to determine a possible association with clinicopathological data and patient survival. A total of 80 HCC and corresponding distant normal tissue specimens were processed for immunohistochemical and qPCR analyses of p57 and RhoA expression. The data showed that expression of p57 mRNA and protein was reduced in HCC tissues when compared to that in distant non-cancer tissues (P<0.05), while expression of RhoA mRNA and protein was significantly higher in HCC tissue specimens when compared to that of the distant normal tissues. Loss of p57 expression was associated with HCC with higher α-fetoprotein (AFP) levels (>400 ng/ml; P=0.044), larger tumor size (>5 cm, P=0.004), poor tumor differentiation (P=0.020), advanced TNM stage (P=0.027), capsule invasion (P=0.018) and tumor thrombosis (P=0.008), whereas expression of RhoA protein was significantly associated with poor tumor differentiation (P=0.042), capsule invasion (P=0.022), and tumor thrombosis (P=0.002). Furthermore, there was a strong inverse relationship between p57 and RhoA expression in HCC tissues, indicating that loss of p57 expression may contribute to RhoA overexpression in HCC tissues. The median survival time of HCC patients with p57+ and p57- expression was 13.0 and 9.0 months, respectively, whereas the median survival time of HCC patients with RhoA+ and RhoA- was 9.0 and 15.0 months. Univariate analysis revealed that the levels of AFP, tumor size, TNM stage, histological grade, capsule invasion, tumor thrombosis, p57, RhoA and co-expression of p57 and RhoA were all significant prognostic indicators for overall survival of HCC patients. Multivariate analysis showed that tumor size, TNM stage, p57, RhoA and combined loss of p57 with RhoA were risk factors for poor survival of HCC patients. This study indicates that the abnormal expression of p57 and RhoA contributes to progression of HCC and poor survival of patients.

To investigate the influence of the Rho/Rho-associated kinase (ROCK) signaling pathway in rats with pulmonary arterial hypertension (PAH) under the intervention with transforming growth factor-beta 1 (TGF-β1). A total of 30 rats were divided into three groups using a random number table, including control group (healthy rats, n=10), model group (PAH rats, n=10), and TGF group (PAH rats injected with 5 ng/mL TGF-β1 recombinant protein, n=10). The systolic blood pressure, ventricular hypertrophy index, pathological changes in lung tissues, TGF-β1 level, protein, and messenger ribonucleic acid (mRNA) expressions of RhoA and ROCK, as well as concentrations of serum nitric oxide (NO) and endothelin-1 (ET-1) were detected via hemodynamics test, hematoxylin and eosin (HE) staining, immunohistochemical method, reverse transcription-polymerase chain reaction (RT-PCR), and enzyme-linked immunosorbent assay (ELISA). The results of hemodynamics test showed that the right ventricular systolic pressure was increased markedly in model group (46.53±8.81) and TGF group (56.79±9.12) compared with that in control group (26.03±4.21) (p<0.05). The mean pulmonary systolic pressure in model group (25.89±1.92) and TGF group (29.41±1.91) was evidently higher than that in control group (15.77±2.71) (p<0.05). According to the results of heart weight measurement, model group (0.5118±0.1635) exhibited a higher ventricular hypertrophy index than control group (0.2908±0.0313) (p<0.05) but a lower ventricular hypertrophy index than TGF group (0.7231±0.1004) (p<0.05). The medial thickness of the pulmonary artery of the rats was observed through the HE staining. It was found that compared with control group, the medial thickness of the pulmonary artery was increased significantly in model group (p<0.05), while it was raised more prominently in TGF group, higher than that in model group, suggesting that TGF-β1 expression can increase the medial thickness of the pulmonary artery. It was manifested in immunohistochemical results that the protein expression of RhoA in the left lung tissues rose notably in model group compared with that in control group (p<0.05), and it was also raised remarkably in TGF group in comparison with that in model group (p<0.05), illustrating that the protein expression of TGF can activate the activity of RhoA and ROCK. The results of RT-PCR indicated that the mRNA expressions of RhoA and ROCK in the left lung tissues were elevated distinctly in model group and TGF group compared with those in control group (p<0.05), and the increases were more apparent in TGF group than those in model group (p<0.05). It was revealed in ELISA results that in comparison with control group, model group, and TGF group had markedly increased concentrations of serum NO and ET-1 (p<0.05), while the rises of serum NO and ET-1 concentrations in TGF group were the most prominent compared with those in model group (p<0.05). Overexpressed TGF-β1 can activate the RhoA/ROCK signaling pathway, thus promoting the occurrence and development of PAH.

The small G protein RhoA plays a major role in several vascular processes and cardiovascular disorders. Here we analyze the mechanisms of RhoA regulation by serotonin (5-HT) in arterial smooth muscle. 5-HT (0.1-10 microM) induced activation of RhoA followed by RhoA depletion at 24-72 h. Inhibition of 5-HT1 receptors reduced the early phase of RhoA activation but had no effect on 5-HT-induced delayed RhoA activation and depletion, which were suppressed by the 5-HT transporter inhibitor fluoxetine and the transglutaminase inhibitor monodansylcadaverin and in type 2 transglutaminase-deficient smooth muscle cells. Coimmunoprecipitations demonstrated that 5-HT associated with RhoA both in vitro and in vivo. This association was calcium-dependent and inhibited by fluoxetine and monodansylcadaverin. 5-HT promotes the association of RhoA with the E3 ubiquitin ligase Smurf1, and 5-HT-induced RhoA depletion was inhibited by the proteasome inhibitor MG132 and the RhoA inhibitor Tat-C3. Simvastatin, the Rho kinase inhibitor Y-27632, small interfering RNA-mediated RhoA gene silencing, and long-term 5-HT stimulation induced Akt activation. In contrast, inhibition of 5-HT-mediated RhoA degradation by MG132 prevented 5-HT-induced Akt activation. Long-term 5-HT stimulation also led to the inhibition of the RhoA/Rho kinase component of arterial contraction. Our data provide evidence that 5-HT, internalized through the 5-HT transporter, is transamidated to RhoA by transglutaminase. Transamidation of RhoA leads to RhoA activation and enhanced proteasomal degradation, which in turn is responsible for Akt activation and contraction inhibition. The observation of transamidation of 5-HT to RhoA in pulmonary artery of hypoxic rats suggests that this process could participate in pulmonary artery remodeling and hypertension.

The purpose of the present study was to examine the effects of portal hypertension on agonist-induced myosin phosphorylation and RhoA expression in vascular smooth muscle. A possible link to cAMP-dependent events was also examined. Portal hypertension was produced by stenosis of the portal vein. Vessel segments were treated with or without 50 microM of the PKA inhibitor Rp-cAMPS for 30 min and subsequently stimulated with 10(-4) M phenylephrine. Myosin regulatory light-chain phosphorylation was detected by immunoblotting. Total RNA from first-order mesenteric arteries and portal veins was isolated and amplified by RT-PCR using RhoA and GAPDH primers. RhoA protein expression was also measured in first-order mesenteric arteries using Western blot analysis. Myosin phosphorylation in maximally stimulated first-order mesenteric arteries was significantly lower in portal hypertensive animals (19.9 +/- 2.86%) when compared with sham-operated control (43.8 +/- 3.53%). Inhibition of PKA selectively increased myosin phosphorylation to 34.7 +/- 4.18%. Rp-cAMPS did not affect the phosphorylation of the portal veins or superior mesenteric arteries. RhoA mRNA and membrane-associated RhoA protein expression in portal hypertensive first-order mesenteric arteries were significantly lower when compared with controls. Acute inhibition of PKA had no effect on RhoA mRNA expression. However, it restored membrane-associated RhoA protein expression in portal hypertensive vessels to control levels. The results suggest that reductions in membrane-associated RhoA expression, which appear to be regulated by cAMP-dependent events, lead to reduced myosin phosphorylation and may underlie the reduced vasoconstrictor effectiveness in the resistance vasculature of portal hypertensive intestine.

Large-conductance Ca 2+-activated K + currents blocked and impaired by homocysteine in human and rat mesenteric artery smooth muscle cells

The present study aimed to observe the effect of rat bone marrow mesenchymal stem cells (MSCs) in vitro on hepatic stellate cell (HSC) RhoA signaling factors and the expression of the cell cycle regulators P27 and cyclin D1. Rat HSC-T6 and fibroblast cells were divided into control, negative control and MSC experimental groups. The cell proliferation rate was examined using the WST8 assay. The cell cycle was analyzed using flow cytometry. RT-PCR and western blot analysis were used to examine cyclin in D1 (cyclin D1), RhoA and P27 mRNA and protein expression in HSCs. After 12 h of co-culture, transition of the MSCs from the G0/G1 to S phase was blocked by HSCs. In the MSC experimental group, the RhoA mRNA and RhoA protein expression showed a decreasing trend with time, which was statistically significant compared with that in the control and negative control groups. MSC P27 protein expression showed an increasing trend with time. RhoA and P27 expression were significantly negatively correlated. After 24 h of co-culture, MSCs inhibited cyclin D1 expression. The difference was statistically significant in the experimental and control groups as well as in the negative control group (P<0.01). In conclusion, co-culture of HSCs with MSCs is capable of inhibiting HSC proliferation, promoting apoptosis and inhibiting RhoA expression. Reduced RhoA activity may induce an upregulation in P27 protein expression in HSCs, which promotes the inhibition of cyclin D1 by MSCs and induces cell cycle arrest at the G0/G1 phase, indicating a role in inhibiting rat HSC proliferation.

Breast cancer has a poor prognosis owing to tumor cell invasion and metastasis. Although Ras homolog (Rho) A is involved in tumor cell invasion, its role in breast carcinoma is unclear. Here, RhoA expression was examined in invasive ductal carcinoma (IDC), with a focus on its relationships with epidermal-mesenchymal transition (EMT) and collective cell invasion. Forty-four surgical IDC tissue samples and two normal breast tissue samples were obtained. RhoA, E-cadherin, vimentin, and F-actin protein expression were analyzed by immunohistochemistry. RhoA, ROCK, mTOR, AKT1, and PIK3CA mRNA expression were conducted using laser microdissection and semi-nested quantitative reverse transcription-polymerase chain reaction. RhoA expression was stronger on the tumor interface of IDCs than the tumor center (P<0.001). RhoA expression was correlated with ROCK expression only in HER2-subtype IDC (P<0.05). In IDCs co-expressing RhoA and ROCK, F-actin expression was stronger on the tumor interface, particularly at the edges of tumor cells, than it was in ROCK-negative IDCs (P<0.0001). In conclusion, RhoA expression was not correlated with EMT in IDC, but enhanced F-actin expression was localized on the edge of tumor cells that co-expressed ROCK. RhoA/ROCK signaling may be associated with collective cell invasion, particularly in HER2-subtype IDC.

We investigated whether GKN1, a gastric tumor suppressor, contributes to the progression of gastric cancer by regulating RhoA expression. We analyzed the expression of GKN1, RhoA, miR-185, and miR-34a in 35 gastric cancer tissues, and compared their expression with T category and TNM stage. Cell migration and invasion, as well as the expression of epithelial-to-mesenchymal transition (EMT)-related proteins, were assessed in GKN1- and RhoA small interfering RNA (siRhoA)-transfected and recombinant-GKN1-treated AGS and MKN1 gastric cancer cells. Expression of RhoA protein and messenger RNA (mRNA) was increased in 15 (42.9%) and 17 (48.6%) of 35 gastric cancer tissues respectively, and was associated with higher T category and TNM stage. GKN1 expression was significantly decreased in 27 gastric cancers (77.1%) with a higher T category, and was inversely correlated with RhoA mRNA expression. In AGS and MKN1 cells, GKN1 expression increased miR-185 and miR-34a expression and reduced RhoA mRNA and protein expression. A positive relationship between GKN1 and miR-34a and miR-185 expression and an inverse relationship between miR-34a and RhoA expression were observed in gastric cancer tissues. Cell migration and invasiveness were markedly decreased in GKN1- and siRhoA-transfected cells. GKN1 expression and silencing of RhoA decreased the expression of the proteins Snail, Slug, and vimentin. Furthermore, miR-185 and miR-34a silencing in MKN1 cells transfected with GKN1 stimulated cell migration and invasion, and increased the expression of EMT-related proteins. Our data suggest that GKN1 may inhibit gastric cancer cell migration and invasion by downregulating RhoA expression in a miR-185- and miR-34a-dependent manner.

Electroacupuncture is beneficial for the recovery of spinal cord injury, but the underlying mechanism is unclear. The Rho/Rho-associated kinase (ROCK) signaling pathway regulates the actin cytoskeleton by controlling the adhesive and migratory behaviors of cells that could inhibit neurite regrowth after neural injury and consequently hinder the recovery from spinal cord injury. Therefore, we hypothesized electroacupuncture could affect the Rho/ROCK signaling pathway to promote the recovery of spinal cord injury. In our experiments, the spinal cord injury in adult Sprague-Dawley rats was caused by an impact device. Those rats were subjected to electroacupuncture at Yaoyangguan (GV3), Dazhui (GV14), Zusanli (ST36) and Ciliao (BL32) and/or monosialoganglioside treatment. Behavioral scores revealed that the hindlimb motor functions improved with those treatments. Real-time quantitative polymerase chain reaction, fluorescence in situ hybridization and western blot assay showed that electroacupuncture suppressed the mRNA and protein expression of Rho-A and Rho-associated kinase II (ROCKII) of injured spinal cord. Although monosialoganglioside promoted the recovery of hindlimb motor function, monosialoganglioside did not affect the expression of Rho-A and ROCKII. However, electroacupuncture combined with monosialoganglioside did not further improve the motor function or suppress the expression of Rho-A and ROCKII. Our data suggested that the electroacupuncture could specifically inhibit the activation of the Rho/ROCK signaling pathway thus partially contributing to the repair of injured spinal cord. Monosialoganglioside could promote the motor function but did not suppress expression of RhoA and ROCKII. There was no synergistic effect of electroacupuncture combined with monosialoganglioside.

Vitamin C and E are suggested to play a preventive role in the pathogenesis of atherosclerosis. They reduce oxidation of low density lipoproteins (oxLDL), thereby protecting human vascular arterial endothelial and smooth muscle cells from oxLDL induced damages. Since vascular arterial endothelial and smooth muscle cells are both involved in atherosclerotic plaque formation, we simultaneously examined the effect of vitamin C, E and oxLDL on their DNA synthesis and proliferation to further elucidate their joint role in this process. Human umbilical arterial endothelial cells (HUAEC) and human arterial smooth muscle cells (HUASMC) were incubated with "preventive concentrations" of vitamin C (60 microM) and E (30 microM) and with LDL (60 microg/ml) of increasing oxidation grade. Cell proliferation and DNA synthesis were determined by cell count and [3H]-thymidine uptake, respectively. Vitamin C alone or in combination with E increased significantly cell number and [3H]-thymidine uptake in HUAEC. The combination exhibited the strongest effect. In contrast, cell number and [3H]-thymidine uptake in HUASMC were significantly decreased in the presence of vitamin C, vitamin E or its combination. OxLDL (60 microg/ml) inhibited cell number and [3H]-thymidine uptake in HUAECs, the latter in an oxidation-grade dependent manner. In HUASMC oxLDL promoted a higher cell number and [3H]-thymidine uptake. If induced by minimally oxLDL, this reduction or increase could be partially reversed by vitamin C alone or in combination with vitamin E. Vitamin C and E, alone or in combination, modulate proliferation and DNA synthesis of human arterial endothelial and muscle cells and this modulation is antagonistic. Thus, vitamin C and E may act "preventive" on atherosclerotic plaque formation in two steps: first reendothelialisation is promoted, then HUASMC growth is inhibited.